Purified forms, methods of preparation and uses of rofecoxib

文档序号：260713 发布日期：2021-11-16 浏览：16次中文

阅读说明：本技术 罗非昔布的纯化形式、制备方法和用途 (Purified forms, methods of preparation and uses of rofecoxib ) 是由 B.C.西皮 R.斯科维尔钦斯基 J-M.施奈德 S.H.德劳因 C.盖林于 2019-11-13 设计创作，主要内容包括：本文所公开的主题涉及罗非昔布(也称为TRM-201或RXB-201)、其制备方法和用途。在某些方面,如本文所提供的高纯度或大体上纯的罗非昔布具有有利的纯度特征,并且是为了治疗或预防多种病症(包括与出血障碍引起的病症相关的疼痛)而被施用的药物组合物中的活性成分。(The subject matter disclosed herein relates to rofecoxib (also known as TRM-201 or RXB-201), methods of making, and uses thereof. In certain aspects, high purity or substantially pure rofecoxib as provided herein has advantageous purity characteristics and is an active ingredient in a pharmaceutical composition that is administered for the treatment or prevention of a variety of conditions, including pain associated with conditions caused by bleeding disorders.)

1. A pharmaceutical composition comprising high purity rofecoxib or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1, wherein the high purity rofecoxib comprises less than about 0.10%, about 0.075%, about 0.05%, about 0.025%, about 0.02%, about 0.01%, or 0.001% of total impurities.

3. The pharmaceutical composition of claim 1, wherein the high purity rofecoxib is substantially free or free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione.

4. The pharmaceutical composition of claim 1, wherein the high purity rofecoxib comprises less than about 0.10%, about 0.05%, about 0.02%, or about 0.01% 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone.

5. The pharmaceutical composition of claim 1, wherein the high purity rofecoxib comprises less than about 0.10%, about 0.05%, about 0.02%, or about 0.01% 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone.

6. A pharmaceutical composition comprising substantially pure rofecoxib or an acceptable salt thereof and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, wherein the substantially pure rofecoxib comprises less than about 0.40%, about 0.30%, about 0.25%, about 0.20%, or about 0.15% total impurities.

8. The pharmaceutical composition of claim 6, wherein the substantially pure rofecoxib is substantially free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione.

9. The pharmaceutical composition of claim 6, wherein the substantially pure rofecoxib comprises less than about 0.25%, about 0.20%, or about 0.15% 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone.

10. The pharmaceutical composition according to claim 6, wherein the substantially pure rofecoxib comprises less than about 0.25%, about 0.20%, or about 0.15% 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone.

11. The pharmaceutical composition of claim 1, wherein the high purity rofecoxib comprises less than about 0.10%, about 0.05%, about 0.02%, about 0.01%, or is free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one.

12. The pharmaceutical composition of claim 1, wherein the high purity rofecoxib comprises less than about 0.10%, about 0.05%, about 0.02%, about 0.01%, or is free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione.

13. A method of treating pain, fever, or inflammation in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of high purity rofecoxib and a pharmaceutically acceptable carrier.

14. The method of claim 13, wherein the pain, fever, or inflammation is caused by one or more conditions selected from the group consisting of: hemophiliac arthropathy, osteoarthritis, rheumatoid arthritis, Juvenile Rheumatoid Arthritis (JRA) of the oligoarticular or polyarthritic course, juvenile idiopathic arthritis, acute pain, primary dysmenorrhea, migraine attack, or migraine associated with von willebrand disease.

15. A method of treating pain, fever, or inflammation in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of substantially pure rofecoxib and a pharmaceutically acceptable carrier.

16. The method of claim 15, wherein the pain, fever, or inflammation is caused by one or more conditions selected from the group consisting of: hemophiliac arthropathy, osteoarthritis, rheumatoid arthritis, Juvenile Rheumatoid Arthritis (JRA) of the oligoarticular or polyarthritic course, juvenile idiopathic arthritis, acute pain, primary dysmenorrhea, migraine attack, or migraine associated with von willebrand disease.

17. A method of treating pain or migraine associated with a condition caused by a bleeding disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of high purity rofecoxib and a pharmaceutically acceptable carrier.

18. A method of treating pain or migraine associated with a condition caused by a bleeding disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of substantially pure rofecoxib and a pharmaceutically acceptable carrier.

19. The method of any one of claims 13-18, wherein the subject is from 12 to 75 years old.

20. The method of any one of claims 13-19, wherein the subject does not have a history or current symptoms of cardiovascular disease.

21. The method of any one of claims 13-20, wherein the subject does not have a history or current symptoms of gastrointestinal bleeding, ulceration, or perforation.

22. The method of any one of claims 13-21, wherein the pharmaceutical composition is administered once daily.

23. The method of any one of claims 13-22, wherein the pharmaceutical composition is administered two or more times per day.

24. The method of any one of claims 17-18, wherein the condition is caused by a bleeding disorder.

25. The method of claim 24, wherein the bleeding disorder is hemophilia a or b, hemophilia c, von willebrand disease, or drug-induced bleeding disorder.

26. The method of any one of claims 17-18, wherein the disorder is hemophilia arthropathy, juvenile idiopathic arthritis, or migraine associated with von willebrand disease.

27. The method of any one of claims 13-26, wherein the treatment is effective to treat pain without co-administration of an analgesic.

28. The method of any one of claims 13-27, wherein the treatment results in the subject reducing or ceasing use of pain medications or rescue medications during the treatment as compared to before the treatment is initiated.

29. The method of any one of claims 13-28, wherein the treatment results in the subject reducing or discontinuing use of opioids during the treatment.

30. The method of any one of claims 13-29, wherein the method further comprises administering a gastroprotective agent.

31. The method of any one of claims 13-30, wherein the gastroprotective agent is co-administered with the pharmaceutical composition.

32. The method of any of claims 13-29, wherein the treatment does not include administration of a gastroprotective agent.

33. The method of any one of claims 13-32, wherein the treatment achieves a reduction of at least 1 from baseline on a numerical rating scale for pain level.

34. The method of any one of claims 13-33, wherein the treatment achieves a reduction of at least 2 relative to baseline on a numerical rating scale for pain level.

35. The method of any one of claims 13-34, wherein the treatment achieves a reduction of at least 3 relative to baseline on a numerical rating scale for pain level.

36. The method of any one of claims 13-35, wherein the treatment achieves a reduction of at least 4 relative to baseline on the numerical rating for pain level scale.

37. The method of any one of claims 13-36, wherein the treatment achieves a reduction of at least 5 relative to baseline on a numerical rating scale for pain level.

38. The method of any one of claims 33-37, wherein the reduction in the numerical rating scale of pain level is achieved within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, or 2 weeks of administration of the pharmaceutical composition.

39. The method according to any one of claims 13-38, wherein the effective amount of rofecoxib administered to the subject is 12.5 mg.

40. The method according to any one of claims 13-38, wherein the effective amount of rofecoxib administered to the subject is 17.5 mg.

41. The method according to any one of claims 13-38, wherein the effective amount of rofecoxib administered to the subject is 20 mg.

42. The method according to any one of claims 13-38, wherein the effective amount of rofecoxib administered to the subject is 25 mg.

43. The method according to any one of claims 13-38, wherein the effective amount of rofecoxib administered to the subject is selected from 1mg, 2mg, 3mg, 5mg, 6.25mg, 7.5mg, 10mg, 10.5mg, 11mg, 11.5mg, 12mg, 12.5mg, 13mg, 13.5mg, 14mg, 14.5mg, 15mg, 15.5mg, 16mg, 16.5mg, 17mg, 17.5mg, 20mg, 20.5mg, 21mg, 21.5mg, 22.5mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, and 70 mg.

44. The method according to any one of claims 13-38, wherein the effective amount of rofecoxib administered to the subject is selected from 0.10mg/kg, 0.15mg/kg, 0.20mg/kg, 0.25mg/kg, 0.30mg/kg, 0.35mg/kg, 0.40mg/kg, 0.45mg/kg, 0.50mg/kg, 0.55mg/kg, 0.60mg/kg, 0.65mg/kg, or 0.70 mg/kg.

45. A method of treating pain associated with a condition caused by a bleeding disorder in a subject in need thereof, the method comprising administering to the subject once daily a pharmaceutical composition comprising 12.5mg of substantially pure rofecoxib or 12.5mg of high purity rofecoxib as the sole active ingredient, wherein the treatment achieves a reduction of at least 1 relative to baseline in a numerical rating scale for the degree of pain.

46. A method of treating pain associated with a condition caused by a bleeding disorder in a subject in need thereof, the method comprising administering to the subject once daily a pharmaceutical composition comprising 17.5mg of substantially pure rofecoxib or 17.5mg of high purity rofecoxib as the sole active ingredient, wherein the treatment achieves a reduction of at least 1 relative to baseline in a numerical rating scale for the degree of pain.

47. A method of treating pain associated with a condition caused by a bleeding disorder in a subject in need thereof, the method comprising administering to the subject once daily a pharmaceutical composition comprising 20mg of substantially pure rofecoxib or 20mg of high purity rofecoxib as the sole active ingredient, wherein the treatment achieves a reduction of at least 1 relative to baseline in a numerical rating scale for the degree of pain.

48. The method according to any one of claims 45-47, wherein the reduction achieved by the pharmaceutical composition is equal to or greater than once daily administration of a pharmaceutical composition comprising 25mg of non-high purity rofecoxib on a numerical rating scale of pain levels.

49. The method of any one of claims 45-48, wherein the disorder is hemophilia arthropathy.

50. A method of treating pain associated with hemophiliac arthropathy in a subject in need thereof, wherein the subject is 12 years of age or older, the method comprising administering once daily a pharmaceutical composition comprising 12.5mg of substantially pure rofecoxib or 12.5mg of high purity rofecoxib, wherein the treatment achieves a reduction of at least 1 relative to baseline in a numerical rating scale for the degree of pain.

51. A method of treating pain associated with hemophiliac arthropathy in a subject in need thereof, wherein the subject is 12 years of age or older, the method comprising administering once daily a pharmaceutical composition comprising 17.5mg of substantially pure rofecoxib or 17.5mg of high purity rofecoxib, wherein the treatment achieves a reduction of at least 1 relative to baseline in a numerical rating scale for the degree of pain.

52. A method of treating pain associated with hemophiliac arthropathy in a subject in need thereof, wherein the subject is 12 years of age or greater, the method comprising administering once daily a pharmaceutical composition comprising 20mg of substantially pure rofecoxib or 20mg of high purity rofecoxib, wherein the treatment achieves a reduction of at least 1 relative to baseline in a numerical rating scale for the degree of pain.

53. A method of treating pain associated with a condition caused by a bleeding disorder in a subject in need thereof, the method comprising:

determining whether the subject has a history or current symptoms of cardiovascular disease;

administering to the subject a pharmaceutical composition comprising at least 12.5mg of substantially pure rofecoxib once daily if the subject is determined not to have a history or current symptoms of cardiovascular disease,

wherein within 1 week of the first administration of the pharmaceutical composition to the subject, the treatment achieves a reduction in the numerical rating scale of pain degree of at least 1 relative to baseline.

54. The method according to claim 53, wherein the pharmaceutical composition comprises 17.5mg, 20mg, 20.5mg, 21mg, 21.5mg, 22.5mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg or 70mg of substantially pure rofecoxib.

55. A method of treating pain in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of high purity rofecoxib, wherein the treatment achieves a greater reduction in the numerical grade scale of the degree of pain compared to administering the same amount of a pharmaceutical composition comprising non-high purity rofecoxib.

56. The method of claim 55, wherein the pain is caused by one or more conditions selected from the group consisting of: hemophiliac arthropathy, osteoarthritis, rheumatoid arthritis, Juvenile Rheumatoid Arthritis (JRA) of the oligoarticular or polyarthritic course, juvenile idiopathic arthritis including juvenile idiopathic arthritis of general type, acute pain, primary dysmenorrhea, migraine attack, or migraine associated with von willebrand disease.

57. A method of treating pain, fever, or inflammation in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of high purity rofecoxib, wherein the treatment results in fewer side effects compared to administering the same amount of a pharmaceutical composition comprising non-high purity rofecoxib.

58. The method of claim 57, wherein the pain, fever, or inflammation is caused by one or more conditions selected from the group consisting of: hemophiliac arthropathy, osteoarthritis, rheumatoid arthritis, Juvenile Rheumatoid Arthritis (JRA) of the oligoarticular or polyarthritic course, juvenile idiopathic arthritis, acute pain, primary dysmenorrhea, migraine attack, or migraine associated with von willebrand disease.

59. A method of treating pain, fever, or inflammation caused by one or more conditions, the method comprising administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of substantially pure rofecoxib and a pharmaceutically acceptable carrier, or high purity rofecoxib and a pharmaceutically acceptable carrier, wherein the treatment results in a reduction in pain, further wherein the treatment results in a reduction in an effect associated with administration of non-high purity rofecoxib.

60. The method of claim 59, wherein the one or more disorders are selected from hemophiliac arthropathy, osteoarthritis, rheumatoid arthritis, Juvenile Rheumatoid Arthritis (JRA) with oligoarticular or polyarticular course, acute pain, primary dysmenorrhea, migraine headache, or migraine associated with von Willebrand disease.

61. A process for preparing substantially pure rofecoxib or high purity rofecoxib as shown in figure 1, the process comprising the steps of:

at TBABr₃Brominating 4' - (methylthio) acetophenone (RSM1) in the presence of to obtain the intermediate RXB-bromoacetone product in stage 1;

subjecting the intermediate product to a nucleophilic substitution reaction with phenylacetic acid (RSM2) in the presence of sodium hydroxide to obtain RXB-phenylacetate product at stage 2;

Crystallizing the product of stage 2 in isopropanol and obtaining filterable crystals;

intramolecular cyclization of the product of stage 2 in DMSO at 70 ℃ in the presence of diisopropylamine;

converting 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone to high purity rofecoxib or substantially pure rofecoxib by using hydrogen peroxide and a catalytic amount of sodium tungstate dihydrate in acetonitrile; and

recrystallizing the rofecoxib in a mixture of DMSO and water.

62. A pharmaceutical composition comprising rofecoxib comprising less than about 0.05% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione and a pharmaceutically acceptable carrier.

63. A pharmaceutical composition comprising high purity or substantially pure rofecoxib having at least one impurity that is a prodrug of rofecoxib, and a pharmaceutically acceptable carrier.

64. The pharmaceutical composition according to claim 63, wherein the rofecoxib is substantially free or free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one and/or 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione.

65. The pharmaceutical composition according to claim 63, wherein the rofecoxib comprises less than 0.02% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one and/or 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione.

66. A method for treating pain, fever, or inflammation in a subject, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of rofecoxib comprising less than 0.05% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione and a pharmaceutically acceptable carrier, wherein the subject is in a population of subjects with reduced risk of a serious cardiovascular thrombotic event.

67. The method of claim 66, wherein the effective amount of rofecoxib is 12.5 mg.

68. The method of claim 66, wherein the effective amount of rofecoxib is 17.5 mg.

69. The method of claim 66, wherein the effective amount of rofecoxib is 20 mg.

70. The method of any one of claims 66-69, wherein the rofecoxib comprises less than about 0.02% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione.

71. A method for reducing one or more side effects associated with administration of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione while treating pain, fever, or inflammation in a subject, comprising administering to the subject a pharmaceutical composition comprising an effective amount of rofecoxib comprising less than about 0.05% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione and a pharmaceutically acceptable carrier.

72. The method of claim 71, wherein the effective amount of rofecoxib is 12.5 mg.

73. The method of claim 71, wherein the effective amount of rofecoxib is 17.5 mg.

74. The method of claim 71, wherein the effective amount of rofecoxib is 20 mg.

75. The method of any one of claims 71-74, wherein the rofecoxib comprises less than about 0.05% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione.

76. A pharmaceutical composition comprising 12.5mg of high purity rofecoxib or an acceptable salt thereof and a pharmaceutically acceptable carrier.

77. A pharmaceutical composition comprising 17.5mg of high purity rofecoxib or an acceptable salt thereof and a pharmaceutically acceptable carrier.

78. A pharmaceutical composition comprising 20mg of high purity rofecoxib or an acceptable salt thereof and a pharmaceutically acceptable carrier.

79. A pharmaceutical composition comprising 12.5mg of substantially pure rofecoxib or an acceptable salt thereof and a pharmaceutically acceptable carrier.

80. A pharmaceutical composition comprising 17.5mg of substantially pure rofecoxib or an acceptable salt thereof and a pharmaceutically acceptable carrier.

81. A pharmaceutical composition comprising 20mg of substantially pure rofecoxib or an acceptable salt thereof and a pharmaceutically acceptable carrier.

82. A method of treating pain, fever, or inflammation in a subject in need thereof, comprising administering an effective amount of high purity rofecoxib.

83. The method of claim 82, wherein said effective amount of high purity rofecoxib is 12.5 mg.

84. The method of claim 82, wherein the effective amount of high purity rofecoxib is 17.5 mg.

85. The method of claim 82, wherein the effective amount of high purity rofecoxib is 20 mg.

86. A method of treating pain, fever, or inflammation in a subject in need thereof, the method comprising administering an effective amount of substantially pure rofecoxib.

87. The method of claim 86, wherein the effective amount of substantially pure rofecoxib is 12.5 mg.

88. The method of claim 86, wherein the effective amount of substantially pure rofecoxib is 17.5 mg.

89. The method of claim 86, wherein the effective amount of substantially pure rofecoxib is 20 mg.

90. A pharmaceutical composition comprising an effective amount of rofecoxib having less than about 0.10%, about 0.05%, about 0.02%, or about 0.01% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione, and a pharmaceutically acceptable carrier.

91. The pharmaceutical composition according to claim 90, wherein the effective amount of rofecoxib is 17.5 mg.

92. The pharmaceutical composition according to claim 90, wherein the effective amount of rofecoxib is 20 mg.

93. A pharmaceutical composition comprising an effective amount of rofecoxib having less than about 0.10%, about 0.05%, about 0.02%, or about 0.01% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one, and a pharmaceutically acceptable carrier.

94. The pharmaceutical composition of claim 93, wherein the effective amount of rofecoxib is 10 mg.

95. The pharmaceutical composition of claim 93, wherein the effective amount of rofecoxib is 12.5 mg.

96. The pharmaceutical composition of claim 93, wherein the effective amount of rofecoxib is 17.5 mg.

97. The pharmaceutical composition of claim 93, wherein the effective amount of rofecoxib is 20 mg.

98. The pharmaceutical composition of claim 93, wherein the effective amount of rofecoxib is 25 mg.

Background

Rofecoxib is a selective COX-2 inhibitor, a non-steroidal anti-inflammatory drug (NSAID) sold under the trade name "VIOXX", which was withdrawn from the market until 2004 for safety reasons. Prior to market withdrawal, "VIOXX" was approved in the united states for the following indications: signs and symptoms of Osteoarthritis (OA); signs and symptoms of adult Rheumatoid Arthritis (RA); signs and symptoms of juvenile rheumatoid arthritis of the oligoarticular or polyarticular course; management of acute pain in adults; treatment of primary dysmenorrhea; and migraine attacks with and without precursor in adults.

There is a long felt but unmet need in the art for new drugs for the treatment of pain, fever and inflammation. This is particularly true for individuals with pain that is co-morbid or associated with diseases or conditions caused by bleeding disorders, including hemophiliac arthropathy and von willebrand disease. Hemophilia is a bleeding disorder caused by genetic or spontaneous mutations in the gene encoding a coagulation factor. The most common form of hemophilia is the result of a deficiency in coagulation factor VIII (hemophilia a) or coagulation factor IX (hemophilia b). In the united states, the prevalence of individuals with hemophilia (PWH) is estimated to be 20,000.

Blood-induced joint diseases can occur as a result of acute joint injury and are associated with hemophilia, with intra-articular bleeding (hemarthrosis) accounting for more than 90% of all severe bleeding events in individuals with severe hemophilia (baseline factor (F) VII or FIX activity < 1%). Over time, repeated bleeding of the same joint leads to progressive injury and the development of hemophilic arthropathy. Despite advances in specialized central treatment and providing comprehensive care, joint bleeding and arthropathy remain the only largest causes of PWH morbidity.

Although the pathogenesis of blood-induced joint injury of hemophilia arthropathy has not been fully elucidated, it appears to have similarities to degenerative joint injury occurring in mechanically-induced joint injury such as OA and inflammatory processes associated with RA.

Arthritis as a result of hemophilia is the most common comorbidity in adult PWH, with 44-55% of individuals reporting hemophilic arthropathy. Pain/discomfort is the most commonly reported defect in PWH, with 75% of PWH reporting this defect. 89% of PWH reports that pain has prevented their daily lives in the past 4 weeks, and 50% reports sustained pain.

In addition to its impact on quality of life, hemophilic arthropathy has a significant impact on the costs associated with hemophilia treatment. Since acute pain associated with hemarthrosis and chronic pain associated with arthritis are largely indistinguishable in PWH, factor replacement has been reported to be significantly over-utilized to address pain symptoms, and 58% of PWH reports the use of factor replacement to treat chronic pain. In addition to being medically ineffective, such misuse also has a significant economic impact, as factor replacement is one of the most expensive known pharmaceutical interventions.

Currently, management of pain and inflammation associated with hemophiliac arthropathy is difficult due to lack of approved therapies and limitations on off-label options available. Acetaminophen is generally recommended as a first-line treatment, however, its utility is limited due to its lack of anti-inflammatory effects and increased risk of liver adverse events in a population of individuals with significant comorbid chronic hepatitis c incidence.

Opioids are useful in alleviating the pain associated with hemophilia arthropathy, and have less anti-inflammatory effects, and chronic use of this analgesic treatment modality can lead to tachyphylaxis, dependence and potential for abuse. In addition, the 2010 observational studies by Solomon et al showed that chronic use of opioids in the general population of arthritis led to an increased risk of (cardiovascular) CV events, bone fractures, hospitalization and all-cause mortality, relative to non-selective NSAIDs or a combination group of COX-2 selective NSAIDs at therapeutic and supratherapeutic doses.

Due to the effect of NSAIDs on platelet function, NSAIDs, in particular acetylsalicylic acid (ASA), are not recommended for use in PWH. Furthermore, even other mild NSAID-induced ulcers may be expected to lead to more severe or prolonged bleeding in the case of underlying coagulation disorders, and NSAIDs have been shown to be associated with an increased risk of Upper Gastrointestinal (UGI) complications, including ulcers, bleeding and perforation, in PWH. Accordingly, there is a long felt but unmet need in the art for a drug for the treatment of pain in PWH.

Von willebrand disease (vWD) is an inherited disorder caused by defective von willebrand factor, and the coagulation protein vWD is the most common form of bleeding disorder, affecting about 1% of the us population, although mild cases are usually undiagnosed. Mild is usually characterized by severe mucosal bleeding, and more severe forms can manifest as significant extensive internal bleeding. There is evidence that vWD individuals are more susceptible to migraine. NSAIDs are not recommended for pain management in individuals with vWD due to the risk of bleeding exacerbations.

Juvenile idiopathic arthritis, including juvenile idiopathic arthritis of all-body type (SJIA), is one of several rheumatic diseases affecting children. Sjisa affects the entire body, including the joints. Sjisa typically occurs at exacerbations, with a healthy phase between some individual exacerbations. The diagnosis can be delayed within the course of the disease by these fluctuations. During exacerbations, sjisa is generally easier to diagnose. The most common symptoms of sjisa are recurrent fever with daily high fever peaks, rashes, and painful, strong joints.

Disclosure of Invention

In certain aspects, the subject matter disclosed herein provides a pharmaceutical composition comprising high purity rofecoxib or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In some embodiments, the high purity rofecoxib comprises less than about 0.10%, about 0.075%, about 0.05%, about 0.025%, about 0.02%, about 0.01%, or about 0.001% of total impurities. In some embodiments, the high purity rofecoxib is substantially free or free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione. In some embodiments, the high purity rofecoxib comprises less than about 0.10%, about 0.05%, about 0.02%, or about 0.01% 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone. In some embodiments, the high purity rofecoxib comprises less than about 0.10%, about 0.05%, about 0.02%, or about 0.01% 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone.

In certain aspects, the subject matter disclosed herein provides a pharmaceutical composition comprising substantially pure rofecoxib or an acceptable salt thereof, and a pharmaceutically acceptable carrier.

In some embodiments, the substantially pure rofecoxib comprises less than about 0.40%, about 0.30%, about 0.25%, about 0.20%, or about 0.15% total impurities. In some embodiments, the substantially pure rofecoxib is substantially free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione. In some embodiments, the substantially pure rofecoxib comprises less than about 0.25%, about 0.20%, or about 0.15% 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone. In some embodiments, the substantially pure rofecoxib comprises less than about 0.25%, about 0.20%, or about 0.15% 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone.

In some embodiments, the high purity rofecoxib comprises less than about 0.10%, about 0.05%, about 0.02%, about 0.01%, or is free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one. In some embodiments, the high purity rofecoxib comprises less than about 0.10%, about 0.05%, about 0.02%, about 0.01%, or is free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione.

In some embodiments, the pain, fever, or inflammation is caused by one or more conditions selected from the group consisting of: hemophiliac arthropathy, osteoarthritis, rheumatoid arthritis, Juvenile Rheumatoid Arthritis (JRA) of the oligoarticular or polyarthritic course, juvenile idiopathic arthritis, acute pain, primary dysmenorrhea, migraine attack, or migraine associated with von willebrand disease. In other embodiments, the pain or inflammation is caused by psoriatic arthritis or fibromyalgia.

In certain aspects, the subject matter disclosed herein provides a method of treating pain or migraine associated with a condition caused by a bleeding disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of high purity rofecoxib and a pharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides a method of treating pain or migraine associated with a condition caused by a bleeding disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of substantially pure rofecoxib and a pharmaceutically acceptable carrier.

In some embodiments, the subject is 2 years of age or older. In some embodiments, the subject is 12 to 75 years old. In some embodiments, the subject does not have a history or current symptoms of cardiovascular disease. In some embodiments, the subject does not have a history or current symptoms of gastrointestinal bleeding, ulceration, or perforation. In some embodiments, the pharmaceutical composition is administered once daily. In some embodiments, the pharmaceutical composition is administered two or more times per day. In some embodiments, the disorder is caused by a bleeding disorder. In some embodiments, the bleeding disorder is hemophilia a or b, hemophilia c, von willebrand disease, or drug-induced bleeding disorder. In some embodiments, the disorder is hemophiliac arthropathy, juvenile idiopathic arthritis, or a migraine associated with von willebrand disease.

In some embodiments, the treatment is effective to treat pain without co-administration of an analgesic. In some embodiments, the treatment results in the subject reducing or discontinuing use of the analgesic or rescue medication during the treatment as compared to before the treatment is initiated. In some embodiments, the treatment results in the subject reducing or discontinuing use of opioids during the treatment period.

In some embodiments, the method further comprises administering a gastroprotective agent. In some embodiments, the gastroprotective agent is co-administered with a pharmaceutical composition. In some embodiments, the treatment does not include administration of a gastroprotective agent.

In some embodiments, the treatment achieves a reduction of at least 1 from baseline on a numerical rating scale for pain levels. In some embodiments, the treatment achieves a reduction of at least 2 from baseline on the numerical rating scale for pain level. In some embodiments, the treatment achieves a reduction of at least 3 from baseline on the numerical rating scale for pain level. In some embodiments, the treatment achieves a reduction of at least 4 from baseline on the numerical rating scale for pain level. In some embodiments, the treatment achieves a reduction of at least 5 from baseline on a numerical rating scale for pain level. In some embodiments, the reduction in the numerical rating scale of pain level is achieved within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, or 2 weeks of administration of the pharmaceutical composition.

In some embodiments, the effective amount of rofecoxib administered to the subject is 12.5 mg. In some embodiments, the effective amount of rofecoxib administered to the subject is 17.5 mg. In some embodiments, the effective amount of rofecoxib administered to the subject is 20 mg. In some embodiments, the effective amount of rofecoxib administered to the subject is 25 mg.

In some embodiments, the effective amount of rofecoxib administered to the subject is selected from 1mg, 2mg, 3mg, 5mg, 6.25mg, 7.5mg, 10mg, 10.5mg, 11mg, 11.5mg, 12mg, 12.5mg, 13mg, 13.5mg, 14mg, 14.5mg, 15mg, 15.5mg, 16mg, 16.5mg, 17mg, 17.5mg, 18mg, 18.5mg, 19mg, 19.5mg, 20mg, 20.5mg, 21mg, 21.5mg, 22.5mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, and 70 mg. In some embodiments, the effective amount of rofecoxib administered to the subject is selected from 0.10mg/kg, 0.15mg/kg, 0.20mg/kg, 0.25mg/kg, 0.30mg/kg, 0.35mg/kg, 0.40mg/kg, 0.45mg/kg, 0.50mg/kg, 0.55mg/kg, 0.60mg/kg, 0.65mg/kg, or 0.70 mg/kg.

In certain aspects, the subject matter disclosed herein provides a method of treating pain associated with a condition caused by a bleeding disorder in a subject in need thereof, the method comprising administering to the subject once daily a pharmaceutical composition comprising 12.5mg of substantially pure rofecoxib or 12.5mg of high purity rofecoxib as the sole active ingredient, wherein the treatment achieves a reduction of at least 1 relative to baseline in a numerical rating scale for the degree of pain.

In certain aspects, the subject matter disclosed herein provides a method of treating pain associated with a condition caused by a bleeding disorder in a subject in need thereof, the method comprising administering to the subject once daily a pharmaceutical composition comprising 17.5mg of substantially pure rofecoxib or 17.5mg of high purity rofecoxib as the sole active ingredient, wherein the treatment achieves a reduction of at least 1 relative to baseline in a numerical rating scale for the degree of pain.

In certain aspects, the presently disclosed subject matter provides a method of treating pain associated with a condition caused by a bleeding disorder in a subject in need thereof, the method comprising administering to the subject once daily a pharmaceutical composition comprising 20mg of substantially pure rofecoxib or 20mg of high purity rofecoxib as the sole active ingredient, wherein the treatment achieves a reduction of at least 1 relative to baseline in a numerical rating scale for the degree of pain.

In some embodiments, the reduction achieved by the pharmaceutical composition in the numerical rating scale of pain levels is equal to or greater than once daily administration of a pharmaceutical composition comprising 25mg of non-highly pure rofecoxib. In some embodiments, the disorder is hemophiliac arthropathy.

In certain aspects, the subject matter disclosed herein provides a method of treating pain associated with hemophiliac arthropathy in a subject in need thereof, wherein the subject is 12 years of age or greater, the method comprising administering once daily a pharmaceutical composition comprising 12.5mg of substantially pure rofecoxib or 12.5mg of high purity rofecoxib, wherein the treatment achieves a reduction of at least 1 relative to baseline in the numerical rating scale for the degree of pain.

In certain aspects, the subject matter disclosed herein provides a method of treating pain associated with hemophiliac arthropathy in a subject in need thereof, wherein the subject is 12 years of age or greater, the method comprising administering once daily a pharmaceutical composition comprising 17.5mg of substantially pure rofecoxib or 17.5mg of high purity rofecoxib, wherein the treatment achieves a reduction of at least 1 relative to baseline in the numerical rating scale for the degree of pain.

In certain aspects, the subject matter disclosed herein provides a method of treating pain associated with hemophilic arthropathy in a subject in need thereof, wherein the subject is 12 years of age or older, the method comprising administering once daily a pharmaceutical composition comprising 20mg of substantially pure rofecoxib or 20mg of high purity rofecoxib, wherein the treatment achieves a reduction of at least 1 relative to baseline in a numerical rating scale for the degree of pain.

In certain aspects, the subject matter disclosed herein provides a method of treating pain associated with a condition caused by a bleeding disorder in a subject in need thereof, the method comprising: determining whether the subject has a history or current symptoms of cardiovascular disease; administering to the subject a pharmaceutical composition comprising at least 12.5mg of substantially pure rofecoxib once daily if the subject is determined not to have a history or current symptoms of cardiovascular disease, wherein the treatment achieves a reduction in the numerical rating scale of pain levels of at least 1 relative to baseline within 1 week of the first administration of the pharmaceutical composition to the subject.

In some embodiments, the pharmaceutical composition comprises 1mg, 2mg, 3mg, 5mg, 6.25mg, 7.5mg, 10mg, 10.5mg, 11mg, 11.5mg, 12mg, 12.5mg, 13mg, 13.5mg, 14mg, 14.5mg, 15mg, 15.5mg, 16mg, 16.5mg, 17mg, 17.5mg, 20mg, 20.5mg, 21mg, 21.5mg, 22.5mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, or 70mg of substantially pure rofecoxib.

In certain aspects, the subject matter disclosed herein provides a method of treating pain in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of high purity rofecoxib, wherein the treatment achieves a greater reduction in the numerical rating scale of the degree of pain compared to administering the same amount of a pharmaceutical composition comprising non-high purity rofecoxib.

In some embodiments, the pain is caused by one or more conditions selected from the group consisting of: hemophiliac arthropathy, osteoarthritis, rheumatoid arthritis, Juvenile Rheumatoid Arthritis (JRA) of the oligoarticular or polyarthritic course, juvenile idiopathic arthritis including juvenile idiopathic arthritis of general type, acute pain, primary dysmenorrhea, migraine attack, or migraine associated with von willebrand disease. In other embodiments, the pain is caused by psoriatic arthritis or fibromyalgia.

In certain aspects, the subject matter disclosed herein provides a method of treating pain, fever, or inflammation in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of high purity rofecoxib, wherein the treatment results in fewer side effects compared to administering the same amount of a pharmaceutical composition comprising non-high purity rofecoxib. In some embodiments, the pain, fever, or inflammation is caused by one or more conditions selected from the group consisting of: hemophiliac arthropathy, osteoarthritis, rheumatoid arthritis, Juvenile Rheumatoid Arthritis (JRA) of the oligoarticular or polyarthritic course, juvenile idiopathic arthritis, acute pain, primary dysmenorrhea, migraine attack, or migraine associated with von willebrand disease. In other embodiments, the pain or inflammation is caused by psoriatic arthritis or fibromyalgia.

In certain aspects, the presently disclosed subject matter provides a method of treating pain, fever, or inflammation caused by one or more conditions, the method comprising administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of substantially pure rofecoxib and a pharmaceutically acceptable carrier or high purity rofecoxib and a pharmaceutically acceptable carrier, wherein the treatment results in a reduction in pain, fever, or inflammation, further wherein the treatment results in a reduction in an effect associated with administration of rofecoxib that is not of high purity.

In some embodiments, the one or more conditions are selected from hemophiliac arthropathy, osteoarthritis, rheumatoid arthritis, Juvenile Rheumatoid Arthritis (JRA) with oligoarticular or polyarticular disease (JRA), acute pain, primary dysmenorrhea, migraine attacks, or migraine associated with von willebrand disease. In other embodiments, the one or more conditions is psoriatic arthritis or fibromyalgia.

In some casesIn one aspect, the subject matter disclosed herein provides a method of preparing substantially pure rofecoxib or high purity rofecoxib as shown in fig. 1, the method comprising the steps of: at TBABr ₃Brominating 4' - (methylthio) acetophenone (RSM1) in the presence of to obtain the intermediate RXB-bromoacetone product in stage 1; subjecting the intermediate product to nucleophilic substitution reaction with phenylacetic acid (RSM2) in the presence of sodium hydroxide to obtain RXB-phenylacetate product in stage 2; crystallizing the product of stage 2 in isopropanol and achieving filterable crystals; intramolecular cyclisation of the product of stage 2 in DMSO at 70 ℃ in the presence of diisopropylamine; 4- [4- (methylthio) phenyl ] acetic acid by using hydrogen peroxide and catalytic amount of sodium tungstate dihydrate in acetonitrile]-conversion of 3-phenyl-2 (5H) -furanone to high purity rofecoxib or substantially pure rofecoxib; and recrystallizing the rofecoxib in a mixture of DMSO and water.

In certain aspects, the subject matter disclosed herein provides a pharmaceutical composition comprising rofecoxib comprising less than 0.05% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione, and a pharmaceutically acceptable carrier.

In certain aspects, the presently disclosed subject matter provides a pharmaceutical composition comprising high purity or substantially pure rofecoxib having at least one impurity that is a prodrug of rofecoxib, and a pharmaceutically acceptable carrier.

In some embodiments, rofecoxib is substantially free or free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one and/or 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione. In some embodiments, rofecoxib comprises less than 0.02% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one and/or 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione.

In certain aspects, the subject matter disclosed herein provides a method for treating pain, fever, or inflammation in a subject, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of rofecoxib comprising less than 0.05% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione and a pharmaceutically acceptable carrier, wherein the subject is in a population of subjects with reduced risk of a serious cardiovascular thrombotic event.

In some embodiments, the effective amount of rofecoxib is 12.5 mg. In some embodiments, the effective amount of rofecoxib is 17.5 mg. In some embodiments, the effective amount of rofecoxib is 20 mg. In some embodiments, rofecoxib comprises less than 0.02% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione.

In certain aspects, the subject matter disclosed herein provides a method for reducing one or more side effects associated with administration of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione while treating pain, fever, or inflammation in a subject, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of rofecoxib comprising less than 0.05% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione and a pharmaceutically acceptable carrier.

In some embodiments, the effective amount of rofecoxib is 12.5 mg. In some embodiments, the effective amount of rofecoxib is 17.5 mg. In some embodiments, the effective amount of rofecoxib is 20 mg. In some embodiments, rofecoxib comprises less than 0.05% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione.

In certain aspects, the subject matter disclosed herein provides a pharmaceutical composition comprising 12.5mg of high purity rofecoxib or an acceptable salt thereof, and a pharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides a pharmaceutical composition comprising 17.5mg of high purity rofecoxib or an acceptable salt thereof, and a pharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides a pharmaceutical composition comprising 20mg of high purity rofecoxib or an acceptable salt thereof, and a pharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides a pharmaceutical composition comprising 12.5mg of substantially pure rofecoxib or an acceptable salt thereof, and a pharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides a pharmaceutical composition comprising 17.5mg of substantially pure rofecoxib or an acceptable salt thereof, and a pharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides a pharmaceutical composition comprising 20mg of substantially pure rofecoxib or an acceptable salt thereof, and a pharmaceutically acceptable carrier.

In some embodiments, the effective amount of high purity rofecoxib is 12.5 mg. In some embodiments, the effective amount of high purity rofecoxib is 17.5 mg. In some embodiments, the effective amount of high purity rofecoxib is 20 mg.

In some embodiments, an effective amount of substantially pure rofecoxib is 12.5 mg. In some embodiments, an effective amount of substantially pure rofecoxib is 17.5 mg. In some embodiments, the effective amount of substantially pure rofecoxib is 20 mg.

In certain aspects, the subject matter disclosed herein provides a pharmaceutical composition comprising an effective amount of rofecoxib having less than 0.10%, 0.05%, 0.02%, or 0.01% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione, and a pharmaceutically acceptable carrier.

In some embodiments, the effective amount of rofecoxib is 17.5 mg. In some embodiments, the effective amount of rofecoxib is 20 mg.

In certain aspects, the subject matter disclosed herein provides a pharmaceutical composition comprising an effective amount of rofecoxib having less than 0.10%, 0.05%, 0.02%, or 0.01% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one, and a pharmaceutically acceptable carrier.

In some embodiments, the effective amount is 10 mg. In some embodiments, the effective amount is 12.5 mg. In some embodiments, the effective amount of rofecoxib is 17.5 mg. In some embodiments, the effective amount of rofecoxib is 20 mg. In some embodiments, the effective amount of rofecoxib is 25 mg.

Drawings

This patent application contains at least one drawing executed in color.

FIG. 1 shows the preparation of rofecoxib A₁Which avoids the production of one or more impurities present in the previously obtained rofecoxib starting drug product: 4- [4- (methylsulfonyl) phenyl]-3-phenyl-5-hydroxyfuran-2-one; and 4- [4- (methylsulfonyl) phenyl]-3-phenyl-2, 5-furandione.

Figure 2 shows that after the recrystallization step, rofecoxib has a purity of about 99.9% with a total impurity of about 0.1% or less. Rofecoxib prepared according to the subject matter described herein is also referred to as compound identifier TRM-201 or RXB-201.

Figure 4 shows the mass spectrum of rofecoxib in acetonitrile.

Figures 5A-D show Nuclear Magnetic Resonance (NMR) spectroscopic analysis of rofecoxib. FIG. 5A shows rofecoxib in DMSO-d₆600MHz of¹H-NMR spectrum. FIG. 5B shows rofecoxib in DMSO-d₆Extended 600MHz in¹H-NMR spectrum. FIG. 5C shows rofecoxib in DMSO-d₆125MHz of¹³C-NMR spectrum. FIG. 5D shows rofecoxib in DMSO-D₆Extended 125MHz in¹³C-NMR spectrum.

Fig. 6A-C show two-dimensional spectra of rofecoxib. FIG. 6A shows rofecoxib in DMSO-d₆H-H COSYNMR spectra in (1). FIG. 6B shows rofecoxib in DMSO-d₆HSQC in (iii) multiple edit NMR spectra. FIG. 6C shows rofecoxib in DMSO-d₆HMBC NMR spectrum of (1).

Fig. 7 shows an Infrared (IR) absorption spectrum of solid rofecoxib taken using an Attenuated Total Reflectance (ATR) cell.

Fig. 8 shows the single crystal structure of rofecoxib in the cambridge structure database.

Fig. 9 shows superimposed graphs showing that the X-ray powder diffraction (XRPD) pattern of rofecoxib reference standard compares favorably with the XRPD pattern calculated from the crystal structure recorded in the cambridge structure database.

Fig. 10A-B show rofecoxib characteristics. Fig. 10A shows Differential Scanning Calorimetry (DSC) analysis of rofecoxib. Fig. 10B shows thermogravimetric analysis (TGA) of rofecoxib.

Figure 11 shows the solubilization and nucleation curves of rofecoxib in DMSO.

Figure 12 shows the solubilization and nucleation curves of rofecoxib in DMSO.

FIG. 13 shows UHPLC results of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone oxidation under biphasic conditions.

FIG. 14 shows UHPLC results for the oxidation of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone in acetonitrile/sulfolane mixtures.

FIGS. 15A-B show the oxidation trace of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone. FIG. 15A shows the oxidation trace of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone for CHG P059-074. FIG. 15B shows the oxidation trace of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone for CHG P059-078.

FIG. 16 shows the expected structure of the 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one (RXB-hydroxy) impurity.

Fig. 17 shows a general scheme of the oxidation reaction.

FIG. 18 shows effort H₂O₂Is added.

Fig. 19 shows the maximum temperature achievable for the synthesis reaction.

Figure 20 shows the conversion to product.

Fig. 21 shows the oxidation reaction force.

FIG. 22 shows a reaction sorting system.

Figure 23 shows a summary of the computer mutagenicity findings.

Detailed Description

Definition of

The following are definitions of terms used in this specification. Unless otherwise indicated, the initial definitions provided herein for a group or term apply to that group or term throughout this specification, either alone or as part of another group. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

As used herein, "rofecoxib" refers to the active ingredient 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2 (5H) -furanone, or a pharmaceutically acceptable salt or solvate thereof. Rofecoxib and methods of preparing rofecoxib are described in U.S. patent No.5,474,995, which is incorporated herein by reference in its entirety. Rofecoxib as provided herein is produced in compliance with and in accordance with GMP requirements and is suitable for use in humans. Rofecoxib as described herein can be in amorphous or crystalline form.

Purity of rofecoxib obtained by the preparation methods as described herein is determined on an area percent (%) basis, typically quantified by analytical chromatography, such as using HPLC, UHPLC, or UPLC.

In some embodiments, the high purity rofecoxib resulting from the preparation process as described herein comprises less than or equal to about 0.10%, 0.075%, 0.050%, 0.025%, 0.020%, or 0.001% total impurities on an area basis. In some embodiments, the high purity rofecoxib is substantially free or free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione. In some embodiments, the high purity rofecoxib is substantially free or free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one. In some embodiments, the high purity rofecoxib comprises less than or equal to about 0.10%, 0.05%, 0.02%, or 0.01% on an area basis of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone. In some embodiments, the high purity rofecoxib comprises less than or equal to about 0.10%, 0.05%, 0.02%, or 0.01% on an area basis of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone.

As used herein, "co-administering" means administering two agents in any manner (e.g., simultaneously or sequentially) in which the pharmacological effects of both are manifested simultaneously in the subject. Common administration does not require that the two agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents need not be manifested simultaneously. These effects need only overlap for a period of time and do not require the range to be completely uniform.

As used herein, "substantially free" with respect to impurities means having less than about 0.10% impurities on an area basis.

As used herein, "free" with respect to impurities means having an amount of impurities below the detection limit, i.e., less than 0.02% impurities on an area basis.

As used herein, "high purity" with respect to an active ingredient means having less than or equal to about 0.10% total impurities on an area basis.

As used herein, "substantially free" with respect to impurities means having less than or equal to about 0.50% of impurities on an area basis.

As used herein, "substantially pure" with respect to an active ingredient means having less than or equal to about 0.50% total impurities on an area basis.

As used herein, "detection limit" with respect to an impurity means having at least 0.02% of the impurity on an area basis.

Also contemplated herein are prodrugs and solvates of the compounds of the inventive subject matter. The term "prodrug" as used herein denotes a compound that undergoes a chemical transformation upon administration to a subject by metabolic or chemical processes to yield a compound of the inventive subject matter, or a salt and/or solvate thereof. Solvates of the compounds of the inventive subject matter include, for example, hydrates.

As used herein, "effective amount" refers to any amount necessary or sufficient to achieve or facilitate a desired result. In certain instances, an effective amount is a therapeutically effective amount. A therapeutically effective amount is any amount necessary or sufficient to promote or achieve a desired biological response in a subject. An effective amount for any particular application may vary depending on factors such as the disease or disorder being treated, the particular agent being administered, the size of the subject, or the severity of the disease or disorder. One of ordinary skill in the art can empirically determine an effective amount of a particular agent without undue experimentation.

As used herein, the term "subject" refers to a vertebrate. In one embodiment, the subject is a mammal or mammalian species. In one embodiment, the subject is a human. In other embodiments, the subject is a non-human vertebrate, including but not limited to, a non-human primate, laboratory animal, livestock, race horse, domesticated animal, and non-domesticated animal.

As used herein, the term "patient" refers to a human or an animal.

The term "mammal" includes, but is not limited to, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or a non-human primate such as a monkey, chimpanzee, baboon, or rhesus monkey. In one embodiment, the mammal is a human.

As used herein, "gastroprotectants" include, but are not limited to, antacid therapy, proton pump inhibitors, H2 receptor antagonists, or misoprostol.

As used herein, "bleeding disorders" include, but are not limited to, hemophilia a (factor VIII deficiency), hemophilia b (factor IX deficiency), von willebrand disease, rare factor deficiencies (including I, II, V, VII, X, XI, XII, and XIII), and drug-induced bleeding disorders.

Compositions of the present subject matter

Rofecoxib (also known as 4- [4- (methylsulfonyl) phenyl)]-3-phenyl-2 (5H) -furanone) is a non-steroidal anti-inflammatory drug that exhibits anti-inflammatory, analgesic, and antipyretic activities. Without being bound by theory, it is believed that rofecoxibThe mechanism of action is due to inhibition of prostaglandin synthesis via inhibition of cyclooxygenase-2 (COX-2). Additionally, rofecoxib does not inhibit cyclooxygenase-1 (COX-1) isozymes at therapeutic concentrations in humans. The chemical structure of rofecoxib is shown below. Rofecoxib has no chiral center and has 314.355g moL ^-1Molecular weight of (2).

Despite years of market withdrawal and the thought of unsafe for human use, the surprising findings show that rofecoxib is safe in the treatment of many conditions and diseases. For example, there is a long felt but unmet need for new non-opioid based analgesic drugs for subjects suffering from pain associated with or co-morbid with diseases or conditions caused by bleeding disorders, including but not limited to hemophiliac arthropathy and von willebrand disease.

In addition to the discovery that rofecoxib can be used to safely treat a number of diseases, it has also been surprisingly discovered that the safety and efficacy profile of rofecoxib can be enhanced by administering a pharmaceutical composition comprising rofecoxib as described herein, which is substantially pure or highly pure, or substantially free or free of one or more impurities present in previously obtained rofecoxib medicaments.

The current process for preparing rofecoxib produces a rofecoxib drug containing certain impurities, some of which are associated with safety concerns that prompted the withdrawal from the market in 2004 of the previously obtained "VIOXX" products. Without being bound by theory, oxidation of the conjugate base of rofecoxib is a process thought to introduce impurities that can survive long enough to react with biomolecules, nucleophilic groups of tissues, and amino groups when entering the body of a subject. Thus, it is believed that these impurities can lead to low levels of chronic toxicity that accumulate and are dangerous over a period of months. It is believed for this reason that cardiotoxicity of VIOXX is not readily observed during short-term (one year or less) studies. Impurities resulting from the oxidation of rofecoxib include 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one and 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione. See Reddy et al, front air oxidation of the conjugate base of rofecoxib (Vioxx), a reactive regulator to cyclic human sensitivity, Tetrahedron letter 46:927-929 (2005).

The novel preparation process described herein surprisingly produces high yields of substantially pure or highly pure rofecoxib, or rofecoxib substantially free or free of those undesirable impurities. As further described herein, it has also been surprisingly found that the novel preparation process produces one or more prodrugs of rofecoxib having beneficial therapeutic properties.

In some embodiments, the pain, fever, or inflammation is caused by one or more conditions selected from the group consisting of: hemophiliac arthropathy, osteoarthritis, rheumatoid arthritis, Juvenile Rheumatoid Arthritis (JRA) of the oligoarticular or polyarthritic course, juvenile idiopathic arthritis, acute pain, primary dysmenorrhea, migraine attack, or migraine associated with von willebrand disease. In other embodiments, the one or more conditions is psoriatic arthritis or fibromyalgia.

In certain aspects, the subject matter disclosed herein provides a method of treating pain or migraine associated with a condition caused by a bleeding disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of substantially pure rofecoxib and a pharmaceutically acceptable carrier.

In some embodiments, the subject is 2 years of age or older. In some embodiments, the subject is 12 years of age or older. In some embodiments, the subject is 12 to 75 years old. In some embodiments, the subject does not have a history or current symptoms of cardiovascular disease. In some embodiments, the subject does not have a history or current symptoms of gastrointestinal bleeding, ulceration, or perforation. In some embodiments, the pharmaceutical composition is administered once daily. In some embodiments, the pharmaceutical composition is administered two or more times per day. In some embodiments, the disorder is caused by a bleeding disorder. In some embodiments, the bleeding disorder is hemophilia a or b, hemophilia c, von willebrand disease, or drug-induced bleeding disorder. In some embodiments, the disorder is hemophiliac arthropathy, juvenile idiopathic arthritis, or a migraine associated with von willebrand disease.

In some embodiments, the treatment is effective to treat pain without co-administration of an analgesic. In some embodiments, the treatment results in the subject having reduced or discontinued use of an analgesic or rescue medication during the treatment as compared to before the treatment is initiated. In some embodiments, the treatment results in the subject reducing or discontinuing opioid use during the treatment.

In some embodiments, the effective amount of rofecoxib administered to the subject is selected from the group consisting of 1mg, 2mg, 3mg, 5mg, 6.25mg, 7.5mg, 10mg, 10.5mg, 11mg, 11.5mg,12mg, 12.5mg, 13mg, 13.5mg, 14mg, 14.5mg, 15mg, 15.5mg, 16mg, 16.5mg, 17mg, 17.5mg, 20mg, 20.5mg, 21mg, 21.5mg, 22.5mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, and 70 mg. In some embodiments, the effective amount of rofecoxib administered to the subject is selected from 0.10mg/kg, 0.15mg/kg, 0.20mg/kg, 0.25mg/kg, 0.30mg/kg, 0.35mg/kg, 0.40mg/kg, 0.45mg/kg, 0.50mg/kg, 0.55mg/kg, 0.60mg/kg, 0.65mg/kg, or 0.70 mg/kg.

In certain aspects, the subject matter disclosed herein provides a method of treating pain associated with a condition caused by a bleeding disorder in a subject in need thereof, the method comprising administering to the subject once daily a pharmaceutical composition comprising 17.5mg of substantially pure rofecoxib or 17.5mg of high purity rofecoxib as the sole active ingredient, wherein the treatment achieves a reduction of at least 1 relative to baseline in a numerical rating scale for the degree of pain.

In certain aspects, the subject matter disclosed herein provides a method of treating pain associated with hemophiliac arthropathy in a subject in need thereof, wherein the subject is 12 years of age or greater, the method comprising administering once daily a pharmaceutical composition comprising 17.5mg of substantially pure rofecoxib or 17.5mg of high purity rofecoxib, wherein the treatment achieves a reduction of at least 1 relative to baseline in the numerical rating scale for the degree of pain.

In certain aspects, the subject matter disclosed herein provides a method of treating pain associated with a condition caused by a bleeding disorder in a subject in need thereof, the method comprising: determining whether the subject has a history or current symptoms of cardiovascular disease; administering to the subject a pharmaceutical composition comprising at least 12.5mg of substantially pure rofecoxib once daily if the subject is determined not to have a history or current symptoms of cardiovascular disease, wherein the treatment achieves a reduction in the numerical rating scale of pain levels of at least 1 relative to baseline within 1 week of the first administration of the pharmaceutical composition to the subject.

In certain aspects, the subject matter disclosed herein provides a method of treating pain, fever, or inflammation caused by one or more conditions, the method comprising administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of substantially pure rofecoxib and a pharmaceutically acceptable carrier or high purity rofecoxib and a pharmaceutically acceptable carrier, wherein the treatment results in a reduction in pain, further wherein the treatment results in a reduction in an effect associated with administration of rofecoxib that is not high purity.

In certain aspects, the subject matter disclosed herein provides a method of preparing substantially pure rofecoxib or high purity rofecoxib as shown in fig. 1, the method comprising the steps of: at TBABr₃Brominating 4' - (methylthio) acetophenone (RSM1) in the presence of to obtain the intermediate RXB-bromoacetone product in stage 1; subjecting the intermediate product to nucleophilic substitution reaction with phenylacetic acid (RSM2) in the presence of sodium hydroxide to obtain RXB-phenylacetate product in stage 2; crystallizing the product of stage 2 in isopropanol and achieving filterable crystals; intramolecular cyclisation of the product of stage 2 in DMSO at 70 ℃ in the presence of diisopropylamine; 4- [4- (methylthio) phenyl ] acetic acid by using hydrogen peroxide and catalytic amount of sodium tungstate dihydrate in acetonitrile ]-conversion of 3-phenyl-2 (5H) -furanone to high purity rofecoxib or substantially pure rofecoxib; and recrystallizing the rofecoxib in a mixture of DMSO and water.

In some embodiments, the effective amount of rofecoxib is 12.5 mg. In some embodiments, the effective amount of rofecoxib is 17.5 mg. In some embodiments, the effective amount of rofecoxib is 20 mg. In some embodiments, rofecoxib comprises less than 0.05% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione.

In some embodiments, the effective amount of rofecoxib is 17.5 mg. In some embodiments, the effective amount of rofecoxib is 20 mg.

In certain aspects, the subject matter disclosed herein provides a pharmaceutical composition comprising an effective amount of rofecoxib having less than 0.10%, 0.05%, 0.02%, or 0.01% 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one, and a pharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides a pharmaceutical composition comprising rofecoxib or an acceptable salt thereof, substantially free of one or more impurities selected from the group consisting of: 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione, 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one, 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone and 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone.

In some aspects, the subject matter disclosed herein provides a method of treating pain, fever, or inflammation caused by one or more conditions in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising rofecoxib or an acceptable salt thereof, substantially free of one or more impurities selected from the group consisting of: 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione, 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one, 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone and 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone.

In certain aspects, the subject matter disclosed herein provides a method of treating pain associated with a condition caused by a bleeding disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of rofecoxib, or an acceptable salt thereof, substantially free of one or more impurities selected from: 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione, 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one, 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone and 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone.

The subject matter disclosed herein relates to substantially pure or high purity rofecoxib (also referred to as TRM-201 or RXB-201) or pharmaceutically acceptable salts or solvates thereof having favorable impurity profiles, methods of preparing rofecoxib, and the use of pharmaceutical compositions comprising rofecoxib for the treatment or prevention of various conditions and diseases.

In one embodiment, the rofecoxib as provided herein is substantially pure or highly pure with respect to all impurities. In another embodiment, rofecoxib as provided herein is substantially free, or free of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone. In one aspect, rofecoxib as provided herein is substantially free, or free of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone.

As described herein, it has been found that despite being previously considered unsafe for human use, rofecoxib can be prepared in a substantially pure or highly pure form that is substantially free, or free of impurities, including those present in the previously obtained pharmaceutical product of raw material of rofecoxib, and can be safely administered to humans as an active ingredient in a pharmaceutical composition for a variety of diseases or conditions, including but not limited to diseases or conditions caused by or co-morbid with bleeding disorders, such as hemophilic arthropathy. In one aspect, the subject matter described herein addresses a long felt but unmet need for new treatments for hemophilia arthropathy by safely administering rofecoxib or a pharmaceutically acceptable salt or solvate thereof.

In another aspect, the subject matter described herein relates to the treatment of juvenile idiopathic arthritis, including juvenile idiopathic arthritis of the whole body type (SJIA), by safe administration of rofecoxib or a pharmaceutically acceptable salt or solvate thereof. In yet another aspect, the subject matter described herein relates to treating migraine associated with von willebrand disease by safely administering rofecoxib or a pharmaceutically acceptable salt or solvate thereof, wherein the subject being treated expresses von willebrand factor at a level about 50% lower than normal.

The subject matter disclosed herein includes, but is not limited to, the treatment of various diseases or conditions by administering a pharmaceutical composition comprising rofecoxib or a pharmaceutically acceptable salt or solvate thereof, which is substantially free or free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one and/or 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione, wherein the treatment results in greater efficacy and/or reduced side effects (including those reported as adverse events or serious adverse events) as compared to a previously obtained raw material pharmaceutical product of rofecoxib, thereby facilitating safe, long-term use of the pharmaceutical composition.

Purity of rofecoxib obtained from the preparation methods described herein is determined on an area percent (%) basis, typically quantified by analytical chromatography, such as using HPLC, UHPLC, or UPLC, or other analytical means in the art.

Preparation of rofecoxib

In certain aspects, rofecoxib as provided herein is prepared in a manner that results in rofecoxib that is substantially free or free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one and/or 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione (both of which are impurities present in the previously obtained rofecoxib starting drug product).

In one aspect, rofecoxib as provided herein can be prepared according to the process as shown in figure 1. In one aspect, the process relies on two feedstocks. In one aspect, rofecoxib is prepared via a four-step continuous process, followed by a crystallization step and a micronization step. The first step may include at TBABr₃Brominating 4' - (methylthio) acetophenone (SM1) in the presence of (a) to result in the formation of step 1 intermediate RXB-bromoacetone. In one embodiment, 4' - (methylthio) acetophenone is dissolved in a mixture of methanol and dichloromethane, to which is added tetrabutylammonium Tribromide (TBAB) dissolved in dichloromethane₃) To form RXB-bromoacetone. After completion, water may be added to quench the reaction. The layers can then be separated and the aqueous layer decanted. In one embodiment, no separation is performed on RXB-bromoacetone.

In one embodiment, the bromination reaction of step 1 is followed by the esterification reaction in step 2 of the rofecoxib preparation process. In step 2, phenylacetic acid and sodium hydroxide may be dissolved in water. The phenylacetate solution can then be added to the RXB-bromoacetone solution of step 1 to form the step 2 product RXB-phenylacetate. In step 2, distillation and crystallization may be performed in isopropanol. The solvent volume can be reduced by distillation at about 55 ℃ at atmospheric pressure, and isopropanol can be added to the mixture, which is then cooled to about 0 ℃ to produce solid RXB-phenylacetate. Seeds may be added during the cooling process. In one embodiment, RXB-phenylacetate is isolated from the resulting slurry by centrifugation at about 0 ℃ and the collected solids are then washed with isopropanol. The resulting filter cake can be dried under vacuum at about 60 ℃.

In one embodiment, step 3 of the rofecoxib preparation process shown in figure 1 involves the preparation of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone (referred to as "RXB-furanone" in figure 1). In one embodiment, RXB-phenylacetate is placed in dimethyl sulfoxide (DMSO) and heated to about 70 ℃. Diisopropylamine (DIPA) and additional DMSO may be added to the solution and the resulting solution maintained at that temperature until the reaction is complete. Isopropanol may be added to the mixture, which is then cooled to about 0 ℃ to give 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone (referred to as "RXB-furanone" in figure 1) as a solid. Seeds may be added during the cooling process. 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone (referred to as "RXB-furanone" in FIG. 1) can be isolated from the resulting slurry by centrifugation at about 0 deg.C, and the collected solid can then be washed with isopropanol. In one embodiment, the isolated 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone (referred to as "RXB-furanone" in figure 1) is not dried.

In one embodiment, step 4 of the process involves the preparation of rofecoxib. In one embodiment, step 4 involves the formation of a sulfoxide. In step 4 of the process, 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone (referred to as "RXB-furanone" in fig. 1) and a catalytic amount of sodium tungstate dihydrate may be suspended in acetonitrile and heated to about 65 ℃. An aqueous hydrogen peroxide solution may be added to the suspension. In one embodiment, the resulting slurry is maintained at this temperature until the reaction is complete, and then allowed to cool to ambient temperature. An aqueous sodium sulfite solution may be added to the slurry and mixed. In one embodiment, the crude rofecoxib is separated from the slurry by centrifugation at about 0 ℃ and the collected solids are then washed with water and isopropanol. In one embodiment, the isolated rofecoxib is not dried.

In one embodiment, step 5 involves crystallization of rofecoxib. The crude rofecoxib can be dissolved in DMSO and filtered at about 40 ℃. The filtrate may then be heated to about 50 ℃, and purified water may be added to induce crystallization. The suspension may then be slowly cooled to about 20 ℃. The resulting slurry can be filtered and the collected solids can be washed with DMSO-water mixture, water, and isopropanol. In one embodiment, the resulting filter cake is dried under vacuum at about 60 ℃. In one embodiment, step 6 of the process comprises micronizing the crystalline rofecoxib using a jet mill. In some embodiments, the d90 particle size of the micronized rofecoxib is less than about 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, or 4 μm. In some embodiments, the d50 particle size of the micronized rofecoxib is less than about 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, or 1 μm. In some embodiments, the d10 particle size of the micronized rofecoxib is less than about 4 μm, 3 μm, 2 μm, 1 μm, 0.9 μm, 0.8 μm, 0.7 μm, 0.6 μm, or 0.5 μm. In some embodiments, the particle size distribution of the micronized rofecoxib is as follows: a) d90 particle size is about 10-12 μm; b) d50 particle size is about 3-4 μm; and c) d10 having a particle size of about 0.5 to 1.0 μm.

According to the process shown in fig. 1, the final intermediate 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone (CAS number 162012-30-8) (referred to as "RXB-furanone" in fig. 2) can be converted to rofecoxib via an oxidation step, for example, by using an oxidizing agent. In one aspect, the oxidizing agent is hydrogen peroxide containing a catalytic amount of sodium tungstate dihydrate in acetonitrile. In another aspect, the oxidizing agent is not potassium monopersulfate (potassium monopersulfate). The oxidation reaction converts the sulfide function of step 3 to the corresponding sulfone using hydrogen peroxide containing a catalytic amount of sodium tungstate dihydrate in acetonitrile. It has been surprisingly found that raising the reaction temperature to at least 50 ℃, preferably at least 60 ℃ and more preferably 65 ℃, and increasing the amount of solvent (acetonitrile) can reduce the amount of impurities, i.e. result in substantially pure rofecoxib prior to any recrystallization step. In one aspect, prior to any recrystallization step, the oxidation step can produce rofecoxib that is at least 99.7% pure, i.e., contains less than 0.30% impurities. In another aspect, the oxidation step produces rofecoxib comprising less than 0.15% or 0.10% of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone prior to any recrystallization step. In one aspect, the oxidation step produces rofecoxib comprising less than 0.15%, 0.10%, 0.075%, or 0.05% of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone (referred to as "RXB-sulfoxide" in figure 2) prior to any recrystallization step. In another aspect, the oxidation step in acetonitrile may produce rofecoxib, which does not require any drying prior to recrystallization.

In one aspect, the oxidation product is recrystallized in a mixture of DMSO and water, which can remove pale yellow and residual impurities, and can provide the desired active pharmaceutical ingredient, such as rofecoxib, described herein. It has surprisingly been found that DMSO can be used as solvent in the recrystallization step, resulting in similar yields to those in which Dimethylformamide (DMF) is used as the recrystallization solvent, while avoiding the safety issues associated with the use of DMF in the final recrystallization step. In one aspect, rofecoxib as described herein does not comprise detectable DMF.

It has also been surprisingly found that the recrystallization process described herein produces rofecoxib substantially free or free of 6-methanesulfonyl-phenanthro- [9,10-C ] furan-1(3H) -one, which is described as a photocyclization degradation product of rofecoxib, and which may be present "without proper control of the recrystallization process. See Dean PM, structural extension of 6-Methylsulfonylphenylanthro- [9,10-C ] -furan-1(3H) -one-A Rofecoxib Degradation product, pharmaceuticals (base). 2010; 3(2), published in 2 months and 1 day in 369-378.2010, doi:10.3390/ph 3020369.

In one aspect, the process shown in fig. 1 produces rofecoxib as described herein in substantially pure or highly pure form. In another aspect, the rofecoxib produced from the final intermediate is recrystallized, e.g., in DMSO and water, to produce substantially pure or highly pure rofecoxib as described herein.

It was surprisingly found that the oxidation step (stage 3 to stage 4) resulted in the formation of rofecoxib as provided herein, which contained only two impurities, 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone and 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone, at or above the detection limit. In one aspect, the oxidizing step results in the formation of rofecoxib comprising less than 0.25%, 0.20%, 0.15%, or 0.10% of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone and/or 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone. In one aspect, the oxidizing step results in the formation of rofecoxib as provided herein that is substantially free or free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one and/or 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione, or otherwise contains either or both of those impurities in an amount below detection limits. Thus, the preparation method described herein avoids the production of the compound 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione, which has been identified in the literature as a "possible contributor to chronic human toxicity". See Reddy et al, front air oxidation of the conjugate base of rofecoxib (Vioxx), a reactive regulator to cyclic human sensitivity, Tetrahedron letter 46:927-929 (2005).

It has also been surprisingly found that the preparation process described herein produces at least one impurity having advantageous properties, in particular 4- [4- (methylsulfinyl) phenyl]3-phenyl-2 (5H) -furanone, a prodrug of rofecoxib, and has been described as having "slightly improved pharmacokinetic profile in arthritis model and better pharmacological activity … … when compared to rofecoxib". See Caturla, Francisco et al, "Racemic and cardiac sulfoxides as potential precursors of the COX-2 inhibitorsandBioorganic&Medicinal Chemistry Letters,16:3209-3212(2006)。

In one embodiment, the evaluation of the potential toxicity of the starting materials and intermediates is performed in silico. Each compound can be evaluated using rule-based DEREK software that utilizes all 51 endpoints, encompassing all major organ systems as well as mutagenicity and skin sensitization. In addition, to predict potential mutagenicity, a statistical-based leader Model applicator can be used to evaluate the structure. The use of two computer-complementary methods is sufficient for the overall determination of mutagenicity, according to ICH M7. Computer evaluation has shown that both starting materials and all intermediates in the preparation process of rofecoxib are predicted to be negative for mutagenicity in both complementary processes. Thus, it is not necessary to employ special controls for the mutagenic compound in the preparation of rofecoxib.

In one embodiment, acetic acid is an excellent solvent (>1000g/L) for 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone. In one embodiment, the starting material may comprise admixed 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone.

The purity of the resulting rofecoxib as described herein is determined on an area percent (%) basis, typically quantified by analytical chromatography, such as using HPLC, UHPLC or UPLC or other analytical means in the art.

Characteristics of impurities

It has been found that the process shown in figure 1 avoids the production of one or more impurities present in the previously obtained rofecoxib starting drug product: 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one; and 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione. See Ahuja et al, Rofecoxib: an update on physicochogic, pharmacological and pharmacological assays, Journal of Pharmacy and Pharmacology,2003,55: 859-.

In addition, two potential impurities may appear during the last bond formation step (step 4) of the rofecoxib preparation process: 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone and 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone. 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone is the product of step 3, and 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone is a partially oxidized intermediate.

FIG. 3 shows a representative chromatogram of a mixture comprising 1mg/mL rofecoxib, 1.5 μ g/mL 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone, and 1.5 μ g/mL 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone, and a blank solution. Table 1 shows that the components of the mixture are well resolved. The quantification limit was < 0.05% on an area basis, which is the reporting threshold. Other potential impurities that may be generated during the preparation are residual solvents (acetonitrile, dichloromethane, dimethyl sulfoxide, isopropanol and methanol) and inorganic materials.

TABLE 1 specificity results for UHPLC purification method

RT ═ retention time; RRT is relative retention time; r_sDegree of separation

Thus, in one aspect of the subject matter disclosed herein, rofecoxib incorporated into a pharmaceutical composition can be substantially free, or free, of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one and/or 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione.

In addition to reducing or eliminating impurities known in existing rofecoxib formulations, it has also been found that the process shown in fig. 1 can produce substantially pure or highly pure rofecoxib. Specifically, after the above-described recrystallization step, rofecoxib as described herein can have a purity of about 99.9%, containing about 0.1% or less of total impurities (as detailed in fig. 2). It has also been found that the method shown in figure 1 can produce substantially pure or highly pure rofecoxib comprising at least one impurity having a beneficial therapeutic effect in an amount above the detection limit. In one embodiment, the impurity having a beneficial therapeutic effect is a prodrug of rofecoxib, such as 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone.

In one aspect of the subject matter disclosed herein, the substantially pure rofecoxib comprises less than about 0.40%, 0.30%, 0.25%, 0.20%, or 0.15% total impurities.

In another aspect, a high purity rofecoxib as provided herein comprises less than about 0.075%, 0.050%, 0.025%, 0.020%, or 0.001% total impurities.

In one aspect, rofecoxib as provided herein comprises less than about 0.25%, 0.20%, 0.15%, 0.10%, 0.05%, 0.02% or 0.01% 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone and/or 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone as an impurity. In other aspects, rofecoxib as provided herein comprises greater than or equal to about 0.001%, 0.005%, 0.01%, 0.02%, 0.05% or 0.10%, but in all cases less than or equal to about 0.15% of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone and/or 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone as an impurity.

In other aspects, rofecoxib as provided herein comprises less than about 0.10%, 0.05%, 0.02%, 0.01% or is free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one. In another aspect, rofecoxib as provided herein comprises less than about 0.10%, 0.05%, 0.02%, 0.01% or is free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione.

In one embodiment, representative sampling and analysis of the suspension in a stirred suspension showed that complete conversion of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone could be achieved, wherein no 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone could be detected. In one embodiment, sampling 1mL of the reaction medium by cooling to room temperature, filtering and analyzing the filter cake and mother liquor shows the presence of 0.02% 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone on an area basis in the filter cake. In one embodiment, 1mL of the reaction medium is sampled by cooling to room temperature, and the addition of 2V of water, filtration and analysis of the filter cake and mother liquor shows 0.02% of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone on an area basis in the filter cake. In one embodiment, analysis of the entire product of the experiment obtained after cooling of the reaction medium to room temperature, addition of 2V water, cooling to 0 ℃, filtration and standard filter cake washing shows a 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone content of 0.02% on an area basis in the filter cake.

Without being bound by theory, in one embodiment, it is believed that the presence of a basic salt such as sodium sulfite, sodium sulfide, sodium acetate, sodium bicarbonate, calcium carbonate, or potassium cyanide in the crude or final product contributes to the production of the 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one impurity. In one embodiment, controlling the rate of production of basic salts, or avoiding the use or formation of basic salts in the oxidation or recrystallization step, in the preparation of crude rofecoxib is critical to the UHPLC characteristics of the recrystallized compound. Clarifying filtration can effectively avoid the increase in the level of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one during UHPLC analysis. In one embodiment, quenching of alkaline salts such as sodium sulfite, sodium sulfide, sodium acetate, sodium bicarbonate, calcium carbonate, or potassium cyanide is inhibited during the oxidation of step 4. In another embodiment, the basic salt may be removed from the crude or final product by polish filtration. In yet another embodiment, performing the preparation process in an inert atmosphere, alone or in combination with alkaline salt quenching, can effectively avoid an increase in the level of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one during UHPLC analysis. In some embodiments, performing the preparation process in an inert atmosphere, alone or in combination with alkaline salt quenching, forms rofecoxib substantially free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one levels during UHPLC analysis. In some embodiments, the crude rofecoxib is formed by sulfoxide formation. In some embodiments, the crude rofecoxib is formed by suspending 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone and a catalytic amount of sodium tungstate dihydrate in acetonitrile. In some embodiments, the crude rofecoxib is formed by heating a suspension of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone and tungstate to about 65 ℃. In some embodiments, the crude rofecoxib is formed by maintaining the slurry at a temperature until the reaction is complete, and then allowing it to cool to ambient temperature. In some embodiments, the crude rofecoxib is formed by adding an aqueous solution of sodium sulfite to the slurry. In some embodiments, the crude rofecoxib is formed by separating it from a slurry by centrifugation at 0 ℃. In some embodiments, the crude rofecoxib is obtained by washing the collected solids with water and isopropanol. In some embodiments, the rofecoxib is formed by: brominating 4' - (methylthio) acetophenone in the presence of TBABr3 to obtain intermediate RXB-bromoacetone product; subjecting the intermediate product to a nucleophilic substitution reaction with phenylacetic acid (RSM2) in the presence of sodium hydroxide to obtain RXB-phenylacetate product; crystallizing the product in isopropanol and achieving filterable crystals; intramolecular cyclisation of the product of stage 2 in DMSO at 70 ℃ in the presence of diisopropylamine; converting 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone to high purity rofecoxib or substantially pure rofecoxib by using hydrogen peroxide and a catalytic amount of sodium tungstate dihydrate in acetonitrile; and recrystallizing the rofecoxib in a mixture of DMSO and water.

The purity of the resulting rofecoxib as described herein is determined on an area percent basis, typically quantified by analytical chromatography, such as using HPLC, UHPLC, or UPLC, or other analytical means in the art.

Development of preparation method

Several routes to rofecoxib synthesis are described in the literature and patent references. See international patent application publication No. wo 98/00416, which is incorporated herein in its entirety. The following summarizes the rofecoxib preparation process A₁Some key results of laboratory scale development.

Some early laboratory tests involved the oxidation of 4' - (methylthio) acetophenone in the first step of the synthesis of rofecoxib. In order to reduce the impurity levels in the intermediate and rofecoxib, the process may be abandoned in favor of performing the oxidation of the sulphide in the final bond formation step.

Dimethylformamide (DMF) was used in the previous preparation method. The high boiling point class 2 solvent may be replaced with acetonitrile (low boiling point class 2 solvent) or DMSO (class 3 solvent).

It may be desirable for bromine (Br)₂) A safer alternative to (2). Therefore, alternative reagents were explored, and tetrabutylammonium bromide (TBAB)₃) May be a useful alternative.

In one embodiment, TBAB is used ₃In an amount of less than 1 equivalent relative to the starting 4' - (methylthio) acetophenone, thereby preventing the formation of dibromoacetone impurities.

In some embodiments, the product RXB-bromoacetone of step 1 is not isolated as the intermediate may be a sensitizer and a lachrymator.

The solvent system used for the RXB-phenylacetate crystallization in step 2 is important to reduce the impurity level and to increase the yield. In one embodiment, a solvent system comprising isopropanol and dichloromethane provides the best results at laboratory scale.

The yield and purity of the cyclization reaction in step 3 is solvent dependent. In one embodiment, the solvent used for the reaction (DMSO) and crystallization (DMSO and isopropanol) provides the best results in purity and yield of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone on a laboratory scale. Furthermore, a drying step may not be required.

Although step 4 (oxidation) and step 5 (crystallization) resulted in high purity rofecoxib, in one embodiment, the reaction conditions may need to be optimized to minimize the amount of the incomplete oxidation product 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone observed during pilot scale-up.

In one embodiment, during the lab scale development, only one crystalline form is observed in the crude rofecoxib of step 4 and the crystalline rofecoxib of step 5.

Characterization of rofecoxib

The structural and physical properties of rofecoxib were established by analyzing the spectra and solid state characterization of reference standard lot number SHD 390-187. Molecular formula C₁₇H₁₄O₄S and structure were confirmed by elemental analysis, high resolution mass spectrometry, nuclear magnetic resonance spectroscopy, infrared spectroscopy, and X-ray powder diffraction. Solid state characterization data supports anhydrous crystalline forms.

In one embodiment, the results of the elemental analysis shown in table 2 are correlated with empirical formula C of rofecoxib₁₇H₁₄O₄And S is consistent.

TABLE 2 elemental analysis of rofecoxib

^aTheoretical value according to the expected molecular formula C of rofecoxib₁₇H₁₄O₄And S, determining.

^bThe experimental percentage of oxygen is calculated as follows: 100% -% C-% H-% S.

In one embodiment, high resolution mass spectral data of rofecoxib in electrospray ionization positive ion mode (ESI-Pos) is obtained. The mass spectrum of rofecoxib in acetonitrile is shown in figure 4. [ M + H ]]⁺The exact mass of the ion was 315.0687 daltons, with a difference of 0.3ppm from the theoretical mass of 315.0686 daltons.

Nuclear Magnetic Resonance (NMR) spectroscopic data also supported the structure of rofecoxib, as shown by the analysis in fig. 5A-D. FIG. 5A shows rofecoxib in DMSO-d₆600MHz of¹H-NMR spectrum. FIG. 5B shows rofecoxib in DMSO-d₆Extended 600MHz in ¹H-NMR spectrum. FIG. 5C shows rofecoxib in DMSO-d₆125MHz of¹³C-NMR spectrum. FIG. 5D shows rofecoxib in DMSO-D₆Extended 125MHz in¹³C-NMR spectrum.

The following is a numbering scheme for assigning the NMR resonance of rofecoxib

The proton and carbon resonances listed in table 2 were assigned by 2-dimensional (2D) correlation spectroscopy (COSY), Heteronuclear Single Quantum Coherence (HSQC), and Heteronuclear Multiple Bond Correlation (HMBC) experiments. The 2-dimensional spectra are shown in FIGS. 6A-C. FIG. 6A shows rofecoxib in DMSO-d₆H-H COSYNMR spectra in (1). FIG. 6B shows rofecoxib in DMSO-d₆HSQC in (iii) multiple edit NMR spectra. FIG. 6C shows rofecoxib in DMSO-d₆HMBC NMR spectrum of (1).

In one embodiment, an Infrared (IR) absorption spectrum of solid rofecoxib is acquired using an Attenuated Total Reflectance (ATR) cell and is shown in fig. 7. The absorbance analysis is provided in table 3. The results of the infrared spectroscopy support the structure of the assignments.

TABLE 3 proposed IR Absorbance analysis of rofecoxib

In one embodiment, rofecoxib is in the single crystalline form (form a), which is the only structure recorded in the cambridge structure database shown in fig. 8. See Groom c.r., Bruno MP, Lightfoot SC et al The Cambridge Structural database.acta cryst.2016; b72: 171-. The superimposed graphs shown in fig. 9 show that the prepared rofecoxib reference standard X-ray powder diffraction (XRPD) pattern compares favorably with the XRPD pattern calculated from the crystal structure recorded in the cambridge structure database.

In one embodiment, the analytical process employed herein uses reversed phase ultra performance liquid chromatography with gradient elution and UV detection at 290nm to determine the identity, assay and impurities of rofecoxib drug. The separation was performed on a phenyl-hexyl column using an acid modified polar mobile phase. The assay was calculated using external reference standards. Impurities are reported as area percent. Identity is confirmed by comparing the sample retention time to a reference standard.

The physical properties of rofecoxib are provided in table 4.

TABLE 4 Rofexib technical parameters

The thermal properties of rofecoxib were evaluated using Differential Scanning Calorimetry (DSC). In one embodiment, the rofecoxib is administered at 4 ℃ min^-1The rate of temperature rise of (2) is from 30 ℃ to 400 ℃. The DSC thermogram in FIG. 10A shows an onset temperature of 209 ℃ andan endothermic event with a peak maximum at 201 ℃ corresponds to melting, and a broad exothermic event with an onset temperature of 371 ℃ and a peak maximum at 385 ℃ corresponds to decomposition. The absence of an endotherm at a temperature below the melting temperature is consistent with the anhydrous crystal structure of rofecoxib.

Thermogravimetric analysis (TGA) by subjecting rofecoxib to 10 ℃ min from ambient temperature^-1Heating to 500 c is performed at the ramp rate of (2). TGA data as shown in figure 10B has no weight loss before or during melting; the results are consistent with the anhydrous crystal structure of rofecoxib. The weight loss at temperatures above 370 ℃ corresponds to the decomposition of rofecoxib.

Residual DMSO in the rofecoxib drug was quantified relative to an external standard using direct injection gas chromatography and FID detection. The separation was performed using a dimethyl polysiloxane column and helium carrier gas. Residual acetonitrile, dichloromethane, isopropanol and methanol in the rofecoxib drug were quantified relative to external standards using headspace gas chromatography and FID detection. The separation was performed using a wax bond column and a helium carrier gas.

Preparation

In certain aspects, substantially pure or high purity rofecoxib as provided herein can be formulated as a pharmaceutically acceptable salt or solvate thereof to produce a pharmaceutical composition. In certain aspects, substantially pure or high purity rofecoxib, or a pharmaceutically acceptable salt or solvate thereof, as provided herein can be formulated with a pharmaceutically acceptable carrier to produce a pharmaceutical composition. Pharmaceutical compositions comprising substantially pure or high purity rofecoxib, or pharmaceutically acceptable salts or solvates thereof, as provided herein, can comprise excipients and can be otherwise formulated, as described in U.S. patent No.6,063,811, which is incorporated herein by reference in its entirety, including but not limited to the formulations specified in examples 2, 2a, 2b, and 2c of U.S. patent No.6,063,811. In this regard, the term "pharmaceutically acceptable salts" refers to the relatively non-toxic inorganic and organic acid salts of the compounds of the subject matter described herein.

In some embodiments, the compounds of the subject matter described herein may contain one or more acidic functional groups and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. In these instances, the term "pharmaceutically acceptable salts" refers to the relatively non-toxic inorganic and organic base addition salts of the compounds of the subject matter described herein. These salts can also be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified free acid form of the compound with a suitable base (such as a hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation), with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. See, e.g., Berge et al, supra.

The formulations of the subject matter described herein include, but are not limited to, those suitable for oral, nasal, inhalation, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Typically, this amount will range from about 1% to about 99% of the active ingredient in 100%, preferably from about 5% to about 70%, most preferably from about 10% to about 30%.

Methods of making these formulations or compositions include the step of bringing into association a compound of the subject matter described herein with a carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the subject matter described herein with liquid carriers or finely divided solid carriers or both, and then shaping the product as necessary.

Formulations of the subject matter described herein suitable for oral administration may be in the form of capsules, cachets, chewable gels, pills, tablets, lozenges (using a flavored base, typically sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as lozenges (using an inert base such as gelatin and glycerin, or sucrose and acacia), and/or as mouthwashes and the like, each containing a predetermined amount of the compound of the subject matter described herein as an active ingredient. The compounds of the subject matter described herein can also be administered as a bolus, electuary or paste.

In the solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like) of the subject matter described herein, the active ingredient is admixed with one or more pharmaceutically acceptable carriers, such as microcrystalline cellulose, lactose or dicalcium phosphate and/or any of the following: fillers or extenders such as starch, lactose, microcrystalline cellulose, sucrose, glucose, mannitol, and/or silicic acid; binders such as carboxymethyl cellulose, hydroxypropyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerin; disintegrating agents such as agar-agar, calcium carbonate, croscarmellose sodium, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate and sodium starch glycolate; solution retarders, such as paraffin; absorption promoters, such as quaternary ammonium compounds; wetting agents such as cetyl alcohol, glyceryl monostearate, sodium lauryl sulfate and polyethylene oxide-polybutylene oxide copolymer; absorbents such as kaolin and bentonite clay; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols and mixtures thereof; and a colorant. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or sugar pastes, as well as high molecular weight polyethylene glycols and the like.

Tablets may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (for example, gelatin or hydroxybutyl methyl cellulose), lubricants, inert diluents, preservatives, disintegrating agents (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agents. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

Tablets and other solid dosage forms of the subject pharmaceutical compositions described herein (such as dragees, capsules, pills, and granules) can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may also be formulated using, for example, hydroxybutyl methyl cellulose in varying proportions (to provide the desired release characteristics), other polymer matrices, liposomes and/or microspheres to provide sustained or controlled release of the active ingredient therein. They may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water or some other sterile injectable medium just prior to use. These compositions may also optionally contain opacifying agents and may be of a composition that releases the active ingredient or ingredients only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, with one or more of the above-mentioned excipients where appropriate.

Liquid dosage forms for oral administration of the compounds of the subject matter disclosed herein include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain, for example, inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isobutyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, butylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Additionally, cyclodextrins, such as hydroxybutyl- β -cyclodextrin, can be used to solubilize compounds.

In addition to inert diluents, oral compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain, for example, suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Dosage forms for topical or transdermal administration of the compounds of the subject matter disclosed herein include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants which may be required.

Ointments, pastes, creams and gels may contain, in addition to an active compound of the subject matter disclosed herein, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of the subject matter disclosed herein, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances. Sprays can additionally contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane.

An additional advantage of transdermal patches is that they provide controlled delivery of the compounds of the subject matter disclosed herein to the body. Such dosage forms may be prepared by dissolving or dispersing the agent in the appropriate medium. Absorption enhancers can also be used to increase the flux of the agents of the presently disclosed subject matter through the skin. Such flux rates can be controlled by providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions, and the like are also contemplated within the scope of the subject matter disclosed herein.

Pharmaceutical compositions of the presently disclosed subject matter suitable for parenteral administration comprise one or more compounds of the presently disclosed subject matter in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions; or a sterile powder that can be reconstituted into a sterile injectable solution or dispersion immediately prior to use, which can contain antioxidants, buffers, bacteriostats, or solutes that render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection, which can be achieved by using liquid suspensions of crystalline or amorphous materials with poor water solubility. The rate of absorption of the drug then depends on its rate of dissolution, which in turn may depend on the crystallite size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oily vehicle. One strategy for depot injection involves the use of polyethylene oxide-polypropylene oxide copolymers, where the vehicle is fluid at room temperature and cures at body temperature.

Injectable depot forms are prepared by forming a microcapsule matrix of the subject compounds in a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

When the compounds of the subject matter disclosed herein are administered to a subject as a medicament, they may be administered per se, or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (more preferably 0.5% to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

The compounds and pharmaceutical compositions of the subject matter disclosed herein may be used in combination therapy, i.e., the compounds and pharmaceutical compositions may be administered simultaneously with, before, or after one or more other desired therapeutic agents or medical procedures. The particular combination of therapies (therapeutic agents or protocols) employed in a combination regimen will take into account the compatibility of the desired therapeutic agent and/or protocol with the desired therapeutic effect to be achieved. It is also understood that the therapy employed may achieve the desired effect on the same disorder (e.g., the compound of the subject matter disclosed herein may be administered concurrently with another anti-cancer agent).

The compounds of the subject matter disclosed herein may be administered intravenously, intramuscularly, intraperitoneally, subcutaneously, topically, orally, or by other acceptable means. The compounds are useful for treating arthritic disorders in mammals (e.g., humans, livestock and domestic animals), racehorses, birds, lizards, and any other organism that can tolerate the compounds).

The subject matter disclosed herein also provides a pharmaceutical package or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical compositions of the subject matter disclosed herein. Optionally associated with such containers may be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate, magnesium stearate, and polyethylene oxide-polybutylene oxide copolymers, as well as coloring, releasing, coating, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the pharmaceutical compositions as described herein.

In one embodiment, a pharmaceutical composition useful according to the methods of the subject matter described herein can be formulated in any manner suitable for pharmaceutical use.

In one embodiment, the formulations of the subject matter disclosed herein can be administered in a pharmaceutically acceptable solution, which may conventionally comprise pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants and optionally other therapeutic ingredients.

Administration of

Some aspects of the subject matter disclosed herein relate to administering to a subject a pharmaceutical composition comprising an effective amount of an active agent to achieve a particular result.

For use in therapy, an effective amount of the compound may be administered to a subject by any mode that enables the compound to be taken up by the appropriate target cells. "administering" the subject pharmaceutical compositions described herein can be accomplished by any means known to the skilled artisan. Specific routes of administration include, but are not limited to, oral, transdermal (e.g., via a patch), parenteral injection (subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, intrathecal, etc.), or mucosal (intranasal, intratracheal, inhalation, intrarectal, intravaginal, etc.). The injection may be a bolus injection or a continuous infusion.

For example, pharmaceutical compositions according to the subject matter disclosed herein may be administered by intravenous, intramuscular, or other parenteral means. They may also be administered intranasally, by inhalation, topically, orally, or as an implant; even rectal or vaginal use is possible. Suitable liquid or solid pharmaceutical dosage forms are, for example, aqueous or saline solutions for injection or inhalation, microencapsulation, encapsulation (encochleeted), coating on microscopic gold particles, inclusion in liposomes, nebulization, aerosols, pellets for implantation into the skin, or drying onto sharp objects to lacerate into the skin. Pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, drops, or extended-release preparations of the active compounds in which excipients and additives and/or auxiliaries such as disintegrants, binders, coatings, swelling agents, lubricants, flavorings, sweeteners or solubilizers are usually used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of the drug delivery methods of the present invention, see Langer R (1990) Science 249:1527-33, which is incorporated herein by reference in its entirety.

The pharmaceutical compositions disclosed herein can be prepared and administered in dosage units. Liquid dosage units are vials or ampoules for injection or other parenteral administration. Solid dosage units are tablets, capsules, powders and suppositories. For treatment of a subject, different dosages may be required depending on the activity of the compound, the mode of administration, the purpose of administration (i.e., prophylactic or therapeutic), the nature and severity of the disorder, the age and weight of the subject. Administration of a given dose can be carried out by a single administration in the form of a single dosage unit or several smaller dosage units. The subject matter described herein also contemplates repeated and multiple administrations of doses at specific intervals of days, weeks, or months.

The pharmaceutical compositions described herein may be administered as such (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may also be conveniently used in the preparation of pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulfonic, tartaric, citric, methanesulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzenesulfonic acids. Moreover, such salts can be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium or calcium salts of carboxylic acid groups.

Compositions suitable for parenteral administration conveniently include sterile aqueous preparations which are isotonic with the blood of the recipient. Acceptable vehicles and solvents are water, ringer's solution, phosphate buffered saline, 5 wt/wt% dextrose, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed mineral or non-mineral oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Suitable carrier formulations for subcutaneous, intramuscular, intraperitoneal, intravenous administration and the like can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.

Compounds useful in the subject matter disclosed herein can be delivered as a mixture of more than two such compounds. In addition to the combination of compounds, the mixture may also include one or more adjuvants.

A variety of routes of administration are available. The particular mode selected will, of course, depend upon the particular compound selected, the age and general health of the subject, the particular condition being treated, and the dosage required for therapeutic efficacy. In general, the methods of the subject matter described herein can be practiced using any mode of administration that is medically acceptable (meaning any mode that produces an effective level of response without causing clinically unacceptable side effects). Preferred modes of administration are as discussed above.

The compositions may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the compound with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and intimately bringing into association the compound with liquid carriers, finely divided solid carriers, or both, and then shaping the product as necessary.

Other delivery systems may include timed release, delayed release or sustained release delivery systems. Such systems can avoid repeated administration of the compound, thereby increasing convenience to the subject and the physician. Many types of delivery systems are available and known to those of ordinary skill in the art. They include polymer binder systems such as poly (lactide-co-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of drugs containing the aforementioned polymers are described, for example, in U.S. Pat. No.5,075,109. The delivery system also includes non-polymeric systems, i.e.: lipids including sterols such as cholesterol, cholesterol esters and fatty acids, or neutral fats such as monoglycerides, diglycerides and triglycerides; a hydrogel release system; a silicone rubber system; a peptide-based system; a wax coating; compressed tablets using conventional binders and excipients; a partially fused implant; and the like. Specific examples include, but are not limited to: (a) eroding systems in which the subject agents described herein are contained in a matrix, such as those described in U.S. Pat. nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusing systems in which the active component permeates from the polymer at a controlled rate, such as described in U.S. Pat. nos. 3,854,480, 5,133,974, and 5,407,686. In addition, pump-based hardware delivery systems may be used, some of which are adapted for implantation.

In one aspect, the pharmaceutical composition comprising rofecoxib as provided herein can be administered in various ways, including but not limited to orally, by subcutaneous or intravenous or other parenteral injection. The form in which the drug is administered (e.g., tablet, capsule, solution, suspension, emulsion) will depend on its route of administration. In one aspect, the subject matter disclosed herein includes a pharmaceutical composition comprising substantially pure or high purity rofecoxib as provided herein and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is in the form of a tablet, and wherein the amount of rofecoxib as provided herein is 12.5mg or 25 mg. In another aspect, the subject matter disclosed herein includes a pharmaceutical composition comprising substantially pure or high purity rofecoxib as provided herein and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is in the form of a tablet, and wherein the amount of rofecoxib as provided herein is about 1mg, 2mg, 3mg, 5mg, 6.25mg, 7.5mg, 10mg, 10.5mg, 11mg, 11.5mg, 12mg, 12.5mg, 13mg, 13.5mg, 14mg, 14.5mg, 15mg, 15.5mg, 16mg, 16.5mg, 17mg, 17.5mg, 20mg, 20.5mg, 21mg, 21.5mg, 22.5mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, or 70 mg.

In another aspect, the subject matter disclosed herein includes a pharmaceutical composition comprising substantially pure or high purity rofecoxib as provided herein and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is in the form of a tablet, and wherein the amount of rofecoxib as provided herein is about 0.10mg/kg, 0.15mg/kg, 0.20mg/kg, 0.25mg/kg, 0.30mg/kg, 0.35mg/kg, 0.40mg/kg, 0.45mg/kg, 0.50mg/kg, 0.55mg/kg, 0.60mg/kg, 0.65mg/kg, or 0.70 mg/kg.

In one aspect, a pharmaceutical composition comprising rofecoxib as provided herein can be packaged with a set of instructions to alert a subject to cardiovascular and/or gastrointestinal risks associated with administration of the composition.

Formulations for both human and veterinary use of compounds according to the subject matter described herein typically comprise such compounds in combination with a pharmaceutically acceptable carrier.

As used herein, the phrase "pharmaceutically acceptable carrier" includes, but is not limited to, pharmaceutically acceptable materials, compositions, or vehicles, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, which are involved in carrying or transporting the subject agent from one organ or portion of the body to another organ or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, microcrystalline cellulose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered gum tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as butanediol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; phosphate buffer; and other non-toxic compatible materials used in pharmaceutical formulations. The term "carrier" denotes a natural or synthetic organic or inorganic ingredient with which the active ingredient is combined to facilitate administration. The components of the pharmaceutical composition can also be admixed with the compounds of the inventive subject matter and with one another in such a way that there are no interactions that would substantially impair the desired pharmaceutical efficacy.

The carrier should be "acceptable" in the sense of being compatible with the compounds of the subject matter described herein and not deleterious to the recipient. In this regard, pharmaceutically acceptable carriers are intended to include any and all solvents, dispersion media, coatings, absorption delaying agents and the like that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds (determined or designed according to the subject matter disclosed herein and/or known in the art) may also be incorporated into the compositions. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. In general, some formulations are prepared by combining the compound with a liquid carrier or a finely divided solid carrier, or both, and then shaping the product into the desired formulation, as desired. The pharmaceutical compositions of the subject matter disclosed herein should be formulated to be compatible with their intended route of administration. The solution or suspension may comprise the following components: sterile diluents such as water, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjusting tonicity such as sodium chloride or dextrose. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide.

A wide variety of Formulations and methods of administration, including, for example, intravenous Formulations and methods of administration can be found in S.K. Niazi editors, Handbook of Pharmaceutical Formulations, volumes 1-6 [ Compressed Solid Products, volume 2 Uncomp Compressed Drug Products, volume 3 Liquid Products, volume 4 Semi-Solid Products, volume 5 over the Counter Products and volume 6 Steriles Products ], CRC Press,2004, 4 months 27.

Useful solutions for oral administration can be prepared by any method well known in the Pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences, 18 th edition (Mack Publishing Company, 1990). The formulations of the subject matter described herein suitable for oral administration may be in the form of: discrete units, such as capsules, gelatin capsules, sachets, tablets, lozenges, or troches, each containing a predetermined amount of a drug; a powder or granular composition; solutions or suspensions in aqueous or non-aqueous liquids; or an oil-in-water or water-in-oil emulsion. The medicament can also be administered in the form of a bolus, electuary or paste, or in the form of a topical composition comprising, for example, a cream or gel. Tablets may be prepared by compressing or molding the drug substance, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the drug in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surfactant or dispersing agent. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered medicament and a suitable carrier moistened with an inert liquid diluent.

Oral compositions typically comprise an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound may be incorporated into an excipient. Oral compositions prepared using a fluid carrier for use as a mouthwash comprise the compound in a fluid carrier and are administered orally and rinsed and expectorated or swallowed. Pharmaceutically compatible binders and/or excipients may be included as part of the composition. Tablets, pills, capsules, lozenges, and the like may contain any of the following ingredients or compounds of similar properties: a binder such as hydroxypropyl cellulose, gum tragacanth or gelatin; excipients, such as starch or lactose; disintegrants, such as croscarmellose sodium, alginic acid, pullulan (Primogel), or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ), or Phosphate Buffered Saline (PBS), which should be stable under the conditions of preparation and storage, and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. In many cases it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions can be formulated in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage form of the subject matter disclosed herein are determined by and directly depend on the following factors: the unique characteristics of the active compounds and the therapeutic effects to be achieved, as well as limitations inherent in the art of formulating such active compounds for the treatment of individuals. Further, administration may be a periodic bolus injection, or may be more continuous by intravenous, intramuscular, or intraperitoneal administration from an external reservoir (e.g., an intravenous bag).

The topical composition can be formulated as a cream, ointment, gel, solution, suspension, or the like.

Application method

Pharmaceutical compositions comprising rofecoxib as provided herein are useful for treating or preventing a disorder or disease in a subject (including a human).

In one aspect, the subject matter disclosed herein includes administering to a subject a pharmaceutical composition comprising rofecoxib having favorable impurity characteristics to treat or prevent a disease or disorder, including but not limited to one of: osteoarthritis, rheumatoid arthritis, analgesia, juvenile idiopathic arthritis (including systemic juvenile idiopathic arthritis), migraine or headache, juvenile rheumatoid arthritis, ankylosing spondylitis, acute pain, primary dysmenorrhea, psoriatic arthritis, and fibromyalgia.

In other aspects, the disease or condition is pain associated with conditions caused by bleeding disorders, including migraine headache associated with von willebrand disease. In another aspect, a subject receiving treatment for von willebrand disease-associated migraine expresses von willebrand factor at a level that is about 50% lower than normal.

In one aspect, the treatment described herein can be administered to a subject of any age. In another aspect, the subject is 2 years of age or older, or 12 years of age or older. In another aspect, the subject is 12 to 75 years of age (inclusive).

In one aspect, a subject is screened for inclusion or exclusion criteria for all or some of the study protocols described below as part of a treatment.

In another aspect, the subject is in a population of subjects with reduced risk of arterial thrombosis, cardiovascular thrombotic events, or other serious cardiovascular disease or event, e.g., subjects with hereditary bleeding disorders or coagulation disorders such as hemophilia or von willebrand disease, or subjects with medically induced bleeding disorders or coagulation disorders.

In one aspect, the subject is screened for a history or current symptoms of cardiovascular disease. In one aspect, if the subject is determined to have a history or current symptoms of cardiovascular disease, the pharmaceutical composition is not administered to the subject. In another aspect, if the subject is determined to have no history or current symptoms of cardiovascular disease, the pharmaceutical composition as further set forth herein is administered to the subject. In yet another aspect, a subject is screened for one or more risk factors that will increase the likelihood that the subject will have a serious cardiovascular thrombotic event following administration of a pharmaceutical composition as set forth further herein. In one aspect, a pharmaceutical composition as further set forth herein is administered to a subject if it is determined that the subject can safely be administered the pharmaceutical composition as further set forth herein without increasing the likelihood that the subject has a serious cardiovascular thrombotic event.

In another aspect, the subject is screened for a history or current symptoms of gastrointestinal bleeding, ulceration and perforation. In one aspect, if the subject is determined to have a history or current symptoms of gastrointestinal bleeding, ulceration and perforation, the pharmaceutical composition is not administered to the subject. In another aspect, if the subject is determined to have no history or current symptoms of gastrointestinal bleeding, ulceration, and perforation, the pharmaceutical composition as further set forth herein is administered to the subject.

In addition to any of the study protocol inclusion or exclusion criteria listed below, subjects may be screened for a history or current symptoms of cardiovascular disease or gastrointestinal bleeding, ulceration and perforation.

The pharmaceutical composition comprising rofecoxib administered for any of the diseases or conditions described herein can be substantially pure or highly pure, or can be substantially free or free of one or more of the impurities described herein.

In another aspect, a pharmaceutical composition comprising rofecoxib as provided herein is administered to a subject suffering from mild, moderate or severe pain associated with a condition caused by a bleeding disorder. Pain can be measured by any clinically validated pain assessment measure. In one aspect, pain is measured by a pain level numerical scale. In another aspect, Pain associated with a particular condition caused by a bleeding disorder, hemophilic Arthropathy is measured by a numerical rating scale for Pain levels or a Patient's arthritic Pain Assessment (visual analogue scale; VAS).

In one aspect, a pharmaceutical composition comprising rofecoxib as provided herein is administered to a subject suffering from pain associated with SJIA. In another aspect, a pharmaceutical composition comprising rofecoxib as provided herein is administered to a subject having migraine associated with von willebrand disease, wherein the subject being treated expresses von willebrand factor at a level about 50% lower than normal.

In one aspect, the treatment of the subject matter disclosed herein comprises administering a pharmaceutical composition comprising about 12.5mg of rofecoxib as provided herein per day. In another aspect, the treatment comprises administering a pharmaceutical composition comprising about 25mg of rofecoxib as provided herein per day. In another aspect, the treatment comprises daily administration of a pharmaceutical composition comprising about 1mg, 2mg, 3mg, 5mg, 6.25mg, 7.5mg, 10mg, 10.5mg, 11mg, 11.5mg, 12mg, 12.5mg, 13mg, 13.5mg, 14mg, 14.5mg, 15mg, 15.5mg, 16mg, 16.5mg, 17mg, 17.5mg, 20mg, 20.5mg, 21mg, 21.5mg, 22.5mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg or 70mg of rofecoxib as provided herein. The treatment can be administered once daily in the form of one or more tablets. In other aspects, the pharmaceutical composition comprising rofecoxib as provided herein is administered two or more times per day.

In one aspect, a treatment regimen is provided for the safe treatment of pain, inflammation, migraine and/or arthritis. Pain, inflammation, migraine and/or arthritis may be associated with a disease or condition caused by a bleeding disorder. In one aspect, the subject is a human patient of any age. In another aspect, the patient is 12 years of age or older.

The treatment regimen may comprise administering an initial (or first) dose of the pharmaceutical composition comprising 10mg, 10.5mg, 11mg, 11.5mg, 12mg, or 12.5mg of rofecoxib once daily, as further described herein. The treatment regimen can further comprise evaluating the subject after administration of the initial dose to determine whether the initial dose is completely, partially effective, or ineffective for treating pain, inflammation, migraine, and/or arthritis. In another aspect, the treatment regimen can comprise determining whether the subject would benefit from administration of a higher dose of rofecoxib. The evaluating and determining steps can occur after a single administration of the initial dose, or after multiple administrations of the initial dose (e.g., two days, three days, one week, two weeks, or more after the first administration of the initial dose), and can be performed by a physician, physician's assistant, nurse, or other health care provider. In one aspect, the evaluating and determining steps may be based on results reported by the subject and may include an assessment of the beneficial effects of a higher dose of rofecoxib as compared to any potential safety risks associated with a higher dose. For example, if a subject experiences a clinically meaningful pain relief after administration of an initial dose, it can be determined that the subject should continue on the initial dose throughout the duration of the bleeding event that caused the pain.

If it is determined that the initial dose is ineffective or only partially effective in treating pain, inflammation, migraine and/or arthritis, or if it is determined that the subject can benefit from a higher daily dose of rofecoxib to treat pain, inflammation, migraine and/or arthritis (e.g., the higher dose can achieve greater reduction in pain in the subject), the treatment regimen can further comprise administering a subsequent (or second) dose of a pharmaceutical composition comprising 17.5mg, 20mg or 25mg of rofecoxib once daily. In one aspect, if it is determined that the initial dose does not achieve a clinically meaningful reduction in pain, inflammation, migraine, and/or arthritis in the subject, a subsequent dose is administered. In another aspect, a subsequent dose is administered if it is determined that the subsequent dose can increase the effectiveness of the treatment without increasing the risk of adverse events or other side effects. In another aspect, if the initial dose is determined to be effective to treat pain, inflammation, migraine, and/or arthritis, then no higher dose is administered. In another aspect, a higher dose is not administered if it is determined that a higher dose will increase the risk of an adverse event or other side effect in the subject. In another aspect, if it is determined that the risk (e.g., in terms of adverse events or side effects) of administering a higher dose outweighs the beneficial effect (e.g., in terms of effectiveness in treating pain, inflammation, migraine, and/or arthritis), the higher dose is not administered. In another aspect, the step of not administering the higher dose comprises instructing the subject not to take the higher dose of the pharmaceutical composition (e.g., not to take 12.5mg of the pharmaceutical composition once per day).

In one aspect, treatment comprises administering a pharmaceutical composition comprising about 0.10mg/kg, 0.15mg/kg, 0.20mg/kg, 0.25mg/kg, 0.30mg/kg, 0.35mg/kg, 0.40mg/kg, 0.45mg/kg, 0.50mg/kg, 0.55mg/kg, 0.60mg/kg, 0.65mg/kg, or 0.70 mg/kg.

In one aspect, an effective amount of rofecoxib as provided herein for treating pain associated with a disease or condition caused by a bleeding disorder is about 12.5mg once daily, and on the other hand results in less side effects or pain relief, which is equal to or better than using a pharmaceutical composition comprising about 25mg of rofecoxib, which is not substantially pure or highly pure, or is substantially free or free of one or more of the impurities described herein present in a previously obtained rofecoxib raw drug product. In one aspect, an effective amount of rofecoxib as provided herein for treating pain associated with a disease or condition caused by a bleeding disorder is about 17.5mg once daily, and on the other hand results in less side effects or pain relief, which is equal to or better than using a pharmaceutical composition comprising about 25mg of rofecoxib, which is not substantially pure or highly pure, or is substantially free or free of one or more of the impurities described herein present in a previously obtained rofecoxib raw drug product. In one aspect, an effective amount of rofecoxib as provided herein for treating pain associated with a disease or condition caused by a bleeding disorder is about 20mg once daily, and in another aspect results in less side effects or pain relief, which is equal to or better than using a pharmaceutical composition comprising about 25mg of rofecoxib that is not substantially pure or highly pure, or is substantially free or free of one or more of the impurities described herein present in a previously obtained pharmaceutical product of rofecoxib raw material. In one aspect, an effective amount of rofecoxib as provided herein for treating pain associated with a disease or condition caused by a bleeding disorder is about 1mg, 2mg, 3mg, 5mg, 6.25mg, 7.5mg, 10mg, 10.5mg, 11mg, 11.5mg, 12mg, 12.5mg, 13mg, 13.5mg, 14mg, 14.5mg, 15mg, 15.5mg, 16mg, 16.5mg, 17mg, 17.5mg, 20mg, 20.5mg, 21mg, 21.5mg, 22.5mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, or 70mg once daily. Thus, the subject may not need to administer higher amounts of the active ingredient to experience pain relief.

In one aspect, an effective amount of rofecoxib as provided herein for treating pain associated with a disease or condition caused by a bleeding disorder, pain associated with juvenile idiopathic arthritis (including systemic juvenile idiopathic arthritis), or migraine associated with von willebrand disease is about 0.10mg/kg, 0.15mg/kg, 0.20mg/kg, 0.25mg/kg, 0.30mg/kg, 0.35mg/kg, 0.40mg/kg, 0.45mg/kg, 0.50mg/kg, 0.55mg/kg, 0.60mg/kg, 0.65mg/kg, or 0.70 mg/kg.

In one aspect, the treatment described herein is effective to treat mild, moderate, or severe pain in a subject without co-administration of another analgesic drug or analgesic.

In another aspect, the treatment described herein results in the subject reducing or ceasing the use of another analgesic drug or painkiller, including rescue medication, during the course of treatment as compared to before the start of the treatment. In yet another aspect, the treatment results in the subject reducing or ceasing the use of acetaminophen and/or opioid during the course of treatment as compared to before the start of treatment.

In one aspect, a pharmaceutical composition comprising rofecoxib as provided herein is co-administered with factor replacement therapy to a subject having a bleeding disorder. In another aspect, the treatment described herein is administered to a subject with a bleeding disorder who is being administered prophylactically or is taking factor replacement therapy. In one aspect, a pharmaceutical composition comprising 12.5mg, 17.5mg, 20mg, or 25mg of rofecoxib as provided herein is administered once daily to a subject who is also being prophylactically administered or taking factor replacement therapy.

In one aspect, the pharmaceutical composition comprising rofecoxib as provided herein is administered daily and does not increase the risk of cardiovascular disease and/or gastrointestinal bleeding, ulceration or perforation during the course of treatment, as determined at 2 weeks, 4 weeks, 8 weeks, 12 weeks, 24 weeks, 52 weeks and/or two or more years. In another aspect, rofecoxib as provided herein can be administered during a course of treatment without or with concomitant administration of a gastroprotective agent, including but not limited to antacid therapy, H2 antagonists, proton pump inhibitors, or misoprostol.

In another aspect, a pharmaceutical composition comprising rofecoxib as provided herein is administered only on an as-needed basis, e.g., when a subject experiences pain "exacerbation," the pain "exacerbation" is described as an increase in pain rating of >1 or a pain rating of ≧ 4 to ≦ 9 based on the pain degree numerical rating scale. In yet another aspect, the pharmaceutical composition comprising rofecoxib as further set forth herein is not administered as a maintenance therapy, prophylactically, or for extended periods of time (e.g., >1 year). In one aspect, a pharmaceutical composition comprising rofecoxib as provided herein is administered on an as-needed basis and used for a short period of time, e.g., less than one, two, three or four weeks, or until the pain, migraine, arthritis, inflammation or other condition or symptom subsides or is eliminated, e.g., until there is a clinically significant improvement in the pain level based on a numerical rating scale of the extent of pain.

In one aspect, the subject uses or co-administers a gastroprotectant, which prevents or treats gastrointestinal bleeding, ulceration and perforation in the subject, during a course of treatment with a pharmaceutical composition comprising rofecoxib as provided herein. In another aspect, the subject uses or co-administers antiviral therapy such as famciclovir or penciclovir during a course of treatment with a pharmaceutical composition comprising rofecoxib as provided herein to treat or prevent fibromyalgia.

In one aspect, the treatment described herein achieves a reduction of at least 1 from baseline on the numerical rating scale for pain level. In another aspect, the treatment described herein achieves a reduction of at least 2, 3, 4, or 5 from baseline in the numerical rating scale for pain level.

In one aspect, the reduction in the numerical rating scale of pain level is achieved within 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days, or 1 week or 2 weeks of the first administration of the pharmaceutical composition.

In one aspect, treatment of a disease or condition by administration of a pharmaceutical composition comprising substantially pure or high purity rofecoxib does not result in one or more of the following adverse events: upper respiratory tract infections, headaches, nausea, vomiting, and coughing, or one or more of the following serious adverse events: bleeding and hypotension. In one aspect, treating a disease or condition caused by a bleeding disorder by administering a pharmaceutical composition comprising substantially pure or high purity rofecoxib does not result in an increase in the number of joint bleeding events. In another aspect, treating a disease or condition caused by a bleeding disorder by administering a pharmaceutical composition comprising substantially pure or high purity rofecoxib does not increase the risk of a joint bleeding event. In one aspect, treating a disease or condition caused by a bleeding disorder by administering a pharmaceutical composition comprising substantially pure or high purity rofecoxib does not result in an increase in the amount of factor used in the subject. In another aspect, treatment of a disease or condition by administration of a pharmaceutical composition comprising substantially pure or high purity rofecoxib does not result in an increased risk of side effects, including but not limited to bleeding, hypotension, or serious cardiovascular thrombotic events, as compared to when the previously sold "VIOXX" product is used for the disease or condition. In another aspect, a pharmaceutical composition comprising substantially pure or high purity rofecoxib as provided herein results in greater efficacy in a disease or condition (as measured by clinically validated measures, such as a pain level numerical scale) than when the previously sold VIOXX "product is used in the disease or condition.

In another aspect, treatment of a disease or condition by administration of a pharmaceutical composition comprising high purity rofecoxib (i.e., substantially free or free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one and/or 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione) does not result in one or more of the following adverse events: upper respiratory tract infections, headaches, nausea, vomiting, and coughing, or one or more of the following serious adverse events: bleeding and hypotension. In another aspect, a pharmaceutical composition comprising high purity rofecoxib as provided herein (i.e., substantially free or free of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one and/or 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-2, 5-furandione) results in greater efficacy or reduced side effects (e.g., bleeding, hypotension, or severe cardiovascular thrombotic events) in a disease or condition (as measured by clinically validated measures, such as a numerical rating scale for pain level) as compared to when the previously sold VIOXX "product is used in the disease or condition. The purity of the resulting rofecoxib as described herein is determined on an area percent basis, typically quantified by analytical chromatography, such as using HPLC, UHPLC, or UPLC, or other analytical means in the art.

Examples

Example 1 protocol for treatment of Hemophiliac Arthropathy (HA)

The research aims are as follows: the efficacy of a pharmaceutical composition comprising rofecoxib as provided herein (study drug) was evaluated relative to placebo in subjects with hemophilic arthropathy.

The secondary purposes include:

to evaluate the effect on sleep disturbance due to pain, subject global impression and quality of life in subjects treated with rofecoxib versus placebo as provided herein.

To evaluate the safety and tolerability of rofecoxib as provided herein in subjects with hemophilic arthropathy.

To evaluate the long-term safety and efficacy of 2 dosing regimens of rofecoxib in subjects with hemophilic arthropathy as provided herein.

Study endpoint:

primary end point: the weekly mean of the daily pain scores as measured using the pain level numerical rating scale [ PI-NRS ], i.e., the 11 point value scale, varied from baseline to week 12, where 0 is no pain and 10 is as poor as predicted.

Secondary endpoint: change from baseline to month 15 in the weekly average of the daily pain score as measured using the pain level numerical rating scale [ PI-NRS ].

Secondary endpoints at week 12 and month 15:

30% and 50% responders, week average according to daily pain score

Change in weekly average of daily sleep disturbance scores from baseline.

Percentage of subjects who were more or greatly improved using the impression of global change (PGIC) of the patient

European quality of life Scale (EQ-5D-5L)

·SF-36

Safety endpoints (during double-blind and open label periods, respectively):

incidence of thrombotic events

Incidence of GI bleeding episodes

Incidence of any bleeding event

Adverse events, laboratory safety tests (hematology, coagulation, clinical chemistry), blood pressure, pulse rate, ECG, C-SSRS

Factor usage

The percentage of subjects who were discontinued due to an adverse event.

Other end values:

percentage of subjects who discontinued due to lack of efficacy

The average acetaminophen usage and the percentage of subjects using rescue medication per day.

Research and design: a multicenter double-blind, randomized placebo-controlled, parallel-group study to evaluate the efficacy and safety of rofecoxib as provided herein in subjects with hemophilic arthropathy. Eligible subjects must be diagnosed with hemophilia a, hemophilia b (factor VIII or factor IX deficiency with or without inhibitors) or von willebrand disease (von willebrand factor levels ≦ 30IU/dL), a history of joint bleeding, chronic symptomatic pain in one or more joints 20 days prior to screening, and hemo-friendly arthropathy at least 6 months prior to screening, with a major source of pain or disability in the hip, knee, ankle or elbow.

At screening, pain in the last week will be assessed using 11-point PI-NRS (0-10), and pain intensity needs to be at least 3. After screening, subjects meeting eligibility criteria will need to stop on-going NSAID and Cox-2 inhibitor drugs for at least 7 days prior to randomization. Subjects who used weaker or low doses of opioid or other non-NSAID analgesics were allowed to continue those at stable doses during the study.

Subjects will record their average pain intensity over the last 24 hours daily in an electronic log using the 11-point PI NRS for the duration of the study treatment period. If desired, rescue medication (paracetamol/paracetamol) may be used up to 3 g/day. If the rescue medication is used for more than 2 consecutive days, the maximum dose is 2.5 g. Subjects who required a dose of rescue medication of 2.5 g/day over 7 consecutive days needed to stop study medication. The dose of any emergency medication must be recorded in an electronic log.

The weekly average of the PI-NRS collected on day 7 before randomization was defined as baseline. Subjects with significant pain intensity indicated by a week-averaged increase in daily pain score of at least 1.5 points and with at least moderate pain intensity (week-averaged baseline arthritis pain score ≧ 4 and ≦ 9) will be eligible for entry into the double-blind treatment period.

On day 1, eligible subjects will be randomized at a 1:1:1 ratio to receive 25mg QD of rofecoxib as provided herein, 12.5mg QD of rofecoxib as provided herein, or a matched placebo. Double blind treatment will last 12 weeks. After the initial double-blind treatment period, subjects will re-randomize at a 1:1 ratio to receive either 25mg QDs or 12.5mg QDs for 12 months of rofecoxib supplementation as provided herein. Subjects will be enrolled in a follow-up visit about 1 week after the last dose of study treatment and will receive a call back visit 4 weeks after the last dose.

Study population:

inclusion criteria

In order to be eligible to participate in the study and to receive study medication, the candidates must meet the following eligibility criteria:

1. subjects were able to understand the purpose and risk of the study and provided informed consent signing and noting the date and authorization to use confidential health information according to state and local subject privacy regulations.

2. Age 12 to 75 years (inclusive) with informed consent.

3. All women and all men with fertility potential must practice effective contraception during the study, and after the final dose of their study treatment, women will practice for 5 weeks and men for 14 weeks.

4. Diagnosis of haemophilia A, haemophilia B (factor VIII or IX deficiency, with or without inhibitors) or von Willebrand disease (von Willebrand factor level ≦ 30IU/dL)

5. History of joint bleeding

6. Hemo-friendly arthropathy was diagnosed at least 6 months prior to screening, with a history of joint bleeding and a major source of pain or disability in the hip, knee, ankle or elbow.

7. Chronic symptomatic pain in one or more joints 20 days prior to screening.

8. At screening, an intensity of 3 or more and 9 or less on a numerical rating scale based on a paper-answer question asking for the average pain intensity of arthritic pain caused by hemophilic arthropathy in the last week.

9. If pain due to hemophilia arthropathy is managed by analgesic drugs, the subject must take a stable analgesic drug a minimum of 30 days prior to screening.

10. Having a significant pain intensity indicated by a week-averaged increase of at least 1.5 points in the daily pain score

11. A baseline weekly average of daily pain scores of ≥ 4 and ≤ 9 on the electronic log PI-NRS; baseline was defined as 7 days prior to randomization (day 1).

Exclusion criteria

Candidates will be excluded from the study at the beginning and from receiving study medication if any of the following exclusion criteria are present:

Medical history

1. Is pregnant or lactating (female subject only).

2. Known to be allergic to rofecoxib or any other component of the formulation

3. A history of asthma, urticaria or allergic type reactions following aspirin or other NSAID administration

4. Has a history of end stage renal disease.

5. There was a history of any liver disease over the past 6 months, with the exception of the known gilbert disease.

6. With a history of alcohol or substance abuse.

7. Currently, there is uncontrolled or poorly controlled hypertension.

8. Has a history of severe cardiac ischemic symptoms, events or interventions, such as angina, myocardial infarction, acute coronary syndrome, decompensated congestive heart failure, coronary stents or bypass grafts.

9. With a history of ischemic cerebrovascular events (TIA or stroke). Subjects with a history of intracerebral or extracerebral hemorrhage may be eligible if their condition is stable.

10. Have a history of severe vascular ischemic symptoms, such as intermittent claudication, or vascular bypass or replacement surgery.

11. Have a significant history or existence of cardiovascular, gastrointestinal or renal diseases or other conditions known to interfere with absorption, distribution, metabolism or excretion of drugs.

12. Have a history of or presence of any clinically significant abnormalities in vital signs, ECG or laboratory tests, or have any medical or psychiatric disorder that, to the knowledge of the researcher, may interfere with the study process or compromise subject safety.

13. The first 6 months were screened for a history of severe upper GI events (upper GI perforation, obstruction, or severe upper GI bleeding).

14. There were major depressive episodes already within 6 months prior to screening.

15. The patients had a history of suicidal hemiplegia within 6 months before the screening.

Vital signs and laboratory protocols

1. After repeated measurement, the BP has the systolic pressure more than or equal to 160mmHg and/or the diastolic pressure more than or equal to 100mmHg when screened.

2. QT intervals corrected using Fridericia's formula (QTcF) at screening were > 450msec (male) or > 470msec (female) [ average of 3 measurements taken at least 5 minutes apart and over 15 minutes ].

3. Pregnancy tests were positive at screening (only women with fertility potential).

4. Estimated creatine clearance (using the Cockroft-Gault equation) <30 ml/min.

5. The screening has AST or ALT more than or equal to 2 times the upper limit of normal value (ULN) or has alkaline phosphatase or bilirubin more than or equal to 1.5 times ULN.

6. There is a history of Human Immunodeficiency Virus (HIV) or a positive test result at the time of screening.

7. There is a history of, or positive test results at the time of screening for, antibodies to Hepatitis C Virus (HCV) or hepatitis b virus (defined as positive for hepatitis b surface antigen [ HBsAg ] or hepatitis b core antibody [ hbcabs ]).

8. Positive drug screening was performed for drugs of abuse (amphetamines, barbiturates, benzodiazepines, cocaine, opioids, tetrahydrocannabinol) except as explained by the use of approved prescription drugs.

Other screening evaluation

Positive response to C-SSRS at item 4 or 5 at screening.

General principles

1. Mental or legal disabilities.

2. The limitations associated with the prohibited concurrent therapy limitations cannot be observed.

3. There were previous studies in this study that registered or used rofecoxib.

4. The study was already involved in the interventional study and was treated within 3 months prior to screening.

5. Blood or blood products were donated within 30 days prior to screening.

6. The research requirements cannot be complied with.

7. To the investigator's knowledge, other unspecified reasons for making the subject unsuitable for participation.

Medicine

1. Simultaneous use of rifampicin

2. No simultaneous NSAID or Cox-2 analgesic drug discontinuation 7 days before randomization

3. A rescue medication at a dose of 2.5g paracetamol/paracetamol was used for more than 5 consecutive days prior to randomization.

Baseline pain score

1. Over 2 of the 7 day pain score entries have been missed during the last 7 days of treatment before randomization.

2. During the last 7 days of treatment before randomization, there was a daily pain score of ≦ 2 for 1 or more days.

3. The difference between the lowest and highest daily pain scores was ≧ 4 during the last 7 days of treatment before randomization.

Treatment groups:

subjects will receive double blind study drugs at a 1:1:1 ratio at random:

rofecoxib (25mg QD oral) as provided herein

Rofecoxib (12.5mg QD oral) as provided herein

Placebo (matching tablet QD oral)

The combined use of the medicines:

allowing the drug to be administered

PPI therapy will be provided to all study participants for gastric protection

Stable low opioid or other non-NSAID analgesic

Forbidden medicine

NSAID or Cox-2 inhibitors

Visiting the schedule: there will be 7 visits: screening visits up to 28 days before dosing began; randomization (day 1); double blind treatment visits (weeks 4, 8 and 12); open label treatment visits (months 4, 6, 9, 12) and follow-up 7 to 10 days after the last dose. In addition, there will be a call back 28 days after the last dose.

Stopping treatment: subjects had to stop study treatment permanently for any of the following reasons:

pregnancy of the subject.

Subject withdraws consent to continue study treatment.

Subjects experienced a medical emergency requiring permanent cessation of study treatment.

The subject experienced a medical emergency requiring blinding of the subject's treatment assignment.

Subject unwilling or unable to comply with the protocol.

Subject compliance with individual liver chemistry, vital signs or ECG, C-SSRS or adverse event discontinuation criteria.

The investigator decided it for medical reasons.

And (3) evaluating the efficacy:

eleven points of average daily pain PI-NRS (evening daily assessment) [ PINRS score will be collected in electronic logs. ]

Eleven points of daily sleep disturbance (DSIS, morning daily assessment) S-NRS score S NRS score will be collected in the electronic log. ]

·PGIC

·EQ-5D-5L

·SF-36

And (3) safety evaluation:

incidence of thrombotic events

Incidence of GI bleeding episodes

Adverse event

Laboratory safety tests (hematology, coagulation clinical chemistry)

Blood pressure

Pulse rate

·ECG

·C-SSRS

Example 2 batch analysis

Batch analysis of two rofecoxib batches (batch SHD390-187 and batch 16P3140F851) are summarized in table 5. If the individual impurity is 0.05% area or more, the result is reported to the nearest 0.01%. If the individual impurity is < 0.02% (LOD), the result is reported as "not detected" (ND). If the individual impurities are <0.05 area% but ≧ 0.02 area%, the results are reported as <0.05 area%.

TABLE 5 batch analysis data for rofecoxib drug

Example 3 stability data for rofecoxib

Stability data for rofecoxib drug (batch 16P3140F851) are provided in tables 6A-B. If the individual impurity is 0.05% area or more, the result is reported to the nearest 0.01%. If the individual impurity is < 0.02% (LOD), the result is reported as "not detected" (ND). If the individual impurities are <0.05 area% but ≧ 0.02 area%, the results are reported as <0.05 area%.

TABLE 6A. stability of Rofexib drug (batch 16P3140F851) at 40 deg.C/75% RH

TABLE 6B stability of rofecoxib drug (batch 16P3140F851) at 25 deg.C/60% RH

Example 4 Oxidation reaction

TABLE 7 influence of solvent on kinetics.

As shown in table 7, the effect of the solvents (ACN, IPA, water) on the completion of the oxidation reaction and the effect of the solvents on the kinetics were investigated in several experiments. The use of 0.25V deionized water allows the reaction to begin from the beginning of the addition of hydrogen peroxide. The first run (VLA P075-176) was carried out using standard methods (at 65 ℃) with the addition of 0.25V of pure water. The addition of hydrogen peroxide was effected within 2h 20 min. A slightly turbid reaction medium is obtained with very little solids in suspension. At H₂O₂At the end of the addition, 4- [4- (methylthio) phenyl group was never detected]-3-phenyl-2 (5H) -furanone and this test only detected 2.5% of 4- [4- (methylsulphine) Acyl) phenyl]-3-phenyl-2 (5H) -furanone. After stirring for a further 1 h at 65 ℃ no 4- [4- (methylsulfinyl) phenyl group could be detected in UHPLC]-3-phenyl-2 (5H) -furanone.

This test results in a very rapid conversion of the species to rofecoxib. At the end of the reaction, 1VACN was added to the reaction in an attempt to obtain complete solubilization of the reaction medium. In fact, the reaction medium immediately becomes heterogeneous as a white suspension. This indicates supersaturation of the reaction medium. Rofecoxib crystallizes out under the perturbation generated by the addition of acetonitrile.

The effect of isopropanol content was then investigated in a second trial (VLA P075-178). The reaction is performed without IPA, which should have a negative effect on the solubility of the rofecoxib species. The reaction medium is already in suspension at the end of the addition of hydrogen peroxide before seeding of rofecoxib. Co-crystallization of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone and rofecoxib may result in lower conversion of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone, with longer time to reach the IPC specification.

Finally, in comparison with VLA P075-176, H is added₂O₂At the end, better conversion was observed. Furthermore, H ₂O₂The addition was performed faster than the first trial (2h instead of 2h 20 min). The absence of IPA shows faster oxidation conditions than with 36% IPA. This can be attributed to the somewhat higher concentration of the reaction medium. Crystallization of the reaction medium appeared to be on 4- [4- (methylsulfinyl) phenyl]The oxidation kinetics of (E) -3-phenyl-2 (5H) -furanone had no effect.

Test CHG P059-084 started with 7VACN containing normal amounts of IPA. This higher dilution appears to have no effect on the reaction medium at the start of oxidation. At H₂O₂Seeding was performed twice during the addition to obtain a white suspension at the time of addition. The reaction medium is similar to VLA P075-178. No 4- [4- (methylsulfinyl) phenyl group was detected 45 minutes after the completion of the addition of hydrogen peroxide]-3-phenyl-2 (5H) -furanone. After quenching with 2V water, cooling to 0 ℃, filtering and washing the filter cake, the solid obtained contains 0.02% of 4- [4- (methylsulphinylyl) sulphinylRadical) phenyl]-3-phenyl-2 (5H) -furanone. Thus, the reaction medium may be slightly turbid or a heterogeneous white suspension, the IPC specification being achieved after only 1h of heating after the hydrogen peroxide addition is complete. Supersaturation of the reaction medium with 6VACN does not appear to be a problem for the conversion of the rofecoxib species to rofecoxib.

Example 5 Process optimization-Oxidation of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone

Process optimization leads to the following oxidation process. A sample batch (CHG P059-092) was performed on 24g of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone in a jacketed reactor equipped with mechanical agitation. The method is performed as shown in table 8 below.

TABLE 8 Oxidation Process

Example 6 optimization of recrystallization of Rofexib (RXB-201)

The solubility and nucleation of rofecoxib were measured at four different concentrations: 3V, 4V, 5V and 6V. As shown in fig. 11, the solubilization curve is reported in blue and nucleation is reported in red. The process for the initial solubilization of rofecoxib is reported as a purple dot (5.5V at 40 ℃). In one embodiment, the yield (green dot) can be improved by increasing the concentration to 4.5V by heating at 50 ℃. These conditions allow a good solubilization of rofecoxib with a large safety margin compared to the nucleation temperature (at least 30 ℃). This margin is needed to avoid spontaneous crystallization during clarification filtration. Additional amounts of DMSO (+0.5V) were used to flush the filtration system.

The tests VLA P075-180 were carried out with rofecoxib containing a large amount of ash (1.5%). This resulted in high levels of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one impurity. At higher DMSO ratios (6V) and lower temperatures (50 ℃ C.), test VLA P075-184 produced rofecoxib in good yield, satisfactorily eliminating the 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one impurity. These good results were obtained even at high ash ratios when runs 180 and 184 were run with the same feedstock. Test CHG P059-090 was conducted using the procedure described in the recrystallization flow chart below. The product was obtained in good yield with a slightly lower clearance of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone. The formation of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one is avoided due to the use of clarification filtration.

The tests are presented in tables 9A-B below:

TABLE 9A study test

TABLE 9B study test (continuation)

The recrystallization procedure is shown in table 10 below.

TABLE 10 recrystallization procedure

Example 7 further optimization of recrystallization of Rofexib (RXB-201)

Background of the study

Scheme 1 below shows the process flow for the oxidation of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone.

Scheme 1

The optimization is performed with respect to the following factors:

the clearance factor for 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone is low. The recrystallization process halves the amount of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone (clearance factor ═ 2). This poor efficiency of impurity removal results in the need to oxidize a lower specification for IPC (4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone < 0.20% area) to obtain the API specification.

Use of high volume solvents. 7 volumes of DMSO and 7.5 volumes of purified water were required to perform the recrystallization. A total of 14.5 volumes may be problematic for throughput during scale-up.

The first part of the optimization work was directed to solvent screening to find the optimal solvent for solubilization and crystallization of rofecoxib (RXB-201). The scavenger factor for 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone was then studied in various solvents. The formation of another impurity, 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one, was also investigated. Finally, optimization efforts have focused on increasing recrystallization yield.

Solvent screening aided by Dynochem software

A tool called "Early phase solvent selection" solvent prediction "is used. The tool allows prediction of the solubility of materials in a wide range of solvents, the solubility of which in some of them is known. Solubility can also be predicted based on temperature and solvent combination. Solubility predictions are based on similar structural moieties between solvents. Thus, measurement of solubility in 16 different solvents allows for estimation of solubility in up to 106 solvents. Table 11 below reports the solubility measurements of rofecoxib (RXB-201) performed in 14 solvents and the predicted solubilities of anisole and isopropanol. Solubility was obtained by UHPLC assay. Of these 106 solvents, 27 appear to be of interest as solvents or anti-solvents. DMF, NMP and DMSO are the most soluble solvents of rofecoxib (RXB-201) (> 100g/L at 30 ℃). However, ICH Q3C, DMF and NMP are not as suitable as DMSO, which is still the best choice. Thus, no change was achieved with respect to the solvent.

TABLE 11 solubility of rofecoxib (RXB-201) in various solvents

With respect to the anti-solvent, rofecoxib (RXB-201) is very poorly soluble in water (insoluble with respect to the united states pharmacopeia). This means that rapid crystallisation occurs upon addition of water and anti-solvent. Which may be the origin of poor impurity clearance. In the solvents studied for solubility measurement, IPA seems to be a good candidate, similar to the previous stage. Indeed, rofecoxib (RXB-201) is slightly more soluble in IPA than in water, which can result in smoother crystallization.

Since the main objective of recrystallization was to reduce the ratio of the single identified impurities, the solubility of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone was measured in several possible anti-solvents identified in Table 11. According to International patent application No. WO/2005/120584, incorporated herein in its entirety, this study was carried out in the laboratory with 8g of a reference (CHG P059-038) for the synthesis of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone, having a purity of 98%. Table 12 below reports the solubility of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone in various anti-solvent candidates. Notably, 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone is very soluble in acetic acid. Since acetic acid is a potential anti-solvent (solubility ═ 5g/L) for the crystallization of rofecoxib (RXB-201) and appears to be a good candidate-it should allow good crystallization of rofecoxib (RXB-201) while keeping 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone in solution. The alcohol may alternatively be used as an anti-solvent.

TABLE 12 solubility of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone in various solvents

Solvent(s)	Temperature (. degree.C.)	Solubility (g/L)	Data of
				Acetic acid	30	1540	Measuring
N-butanol	30	14	Measuring
				Ethanol	30	28	Measuring
IPA	30	0.2	Measuring
				Water (W)	30	0.7	Measuring

Recrystallization test of different anti-solvents

Isopropanol was first tested as an anti-solvent followed by the treatments shown in tables 13A-B below (LMC P045-157). The recrystallization yield is poor and is only 64 percent. The clearance factor for 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone is almost unchanged compared to the use of water. The low yield is surprising given the low solubility of rofecoxib (RXB-201) in IPA. The mixture between IPA and water was tested as an anti-solvent (LMC P045-165). This test results in a much better yield of 93% while the clearance of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone remains around 2. The extremely high solubility of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone in acetic acid indicates that this solvent would be a good choice for improving the elimination of impurities. Furthermore, the low solubility of rofecoxib (RXB-201) should result in high yields. LMC P045-076 proposes to replace water with acetic acid. This experiment gave a low yield of 68% in which the clearance of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone increased slightly to 3. This clearance factor is far from what would be expected from solubility data. Both final experiments were carried out in the same way, but the initially incorporated 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone reached about 1% and 2% impurities before recrystallization. This resulted in the same yield as LMC P045-076, with no practical effect on the removal efficiency. These experiments with the new anti-solvent did not contribute to a significant increase in the efficiency of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone clearance. Thus, water is used as an anti-solvent.

TABLE 13A. recrystallization experiments of rofecoxib (RXB-201) using various antisolvents

TABLE 13B rofecoxib (RXB-201) recrystallization test (continue) with various antisolvents

TABLE 14A. optimization of recrystallization of rofecoxib (RXB-201) with DMSO/water

TABLE 14B optimization of recrystallization of rofecoxib (RXB-201) with DMSO/water

Study on formation of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one (RXB-hydroxy) impurity

It has been reported that 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one is synthesized in air. This impurity was not detected in pilot batch F801, but was always observed in the experiment. In test VLA P075-180, the ratio of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one rises to 2.66% at 60 ℃ before the addition of water. Sodium sulfite is suspected to produce this impurity as depicted in scheme 2 below. In pilot scale, polish filtration removed trace amounts of salt, while the filtration was not performed in laboratory tests. Furthermore, the inert atmosphere is better controlled in pilot trials than in the laboratory.

Scheme 2.4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one (RXB-hydroxy) formation of an impurity

An experiment was performed to examine both temperature and sodium sulfite for p-4- [4- (methylsulfonyl) phenyl ]Influence of the ratio of 3-phenyl-5-hydroxyfuran-2-one. Pure rofecoxib (RXB-201) (F851) was dissolved in 5.5V DMSO, to which Na2SO3 was added. Test threeAmount of Na₂SO₃：

0%: filtered rofecoxib (RXB-201)

0.5%: ash results obtained on pilot plant lots (crude rofecoxib (RXB-201) F801)

1.5%: ash results obtained on APG P052-110 batches

The samples were subjected to 2 different temperatures under an air atmosphere:

40 ℃ C: current heating temperature for polishing filtration

60 ℃: possible future temperature for polish filtration (if DMSO volume is reduced for better yield)

The UHPLC results are reported in table 15 below.

TABLE 15.UHPLC ratios of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one (RXB-hydroxy) as a function of RoI and temperature

The UHPLC results clearly show that the recrystallization medium (rofecoxib (RXB-201), in 5.5V DMSO) is almost stable at 40 ℃ and 60 ℃ for 6h, where 4- [4- (methylsulfonyl) phenyl]-3-phenyl-5-hydroxyfuran-2-one in an amount of up to 0.17%. However, the introduction of sodium sulfite and elevated temperatures have a negative effect on the purity profile. The worst case gives 4- [4- (methylsulfonyl) phenyl ]The amount of-3-phenyl-5-hydroxyfuran-2-one was 9 times higher than at the beginning. Control of the ratio of sodium sulfite in crude rofecoxib (RXB-201) is critical to UHPLC characterization of recrystallized compounds. In order to solve the problem, an optimized oxidation process without sodium sulfite quenching is designed. For Na₂SO₃Inhibition of (oxidation of CHG P059-092 followed by recrystallization of CHG P059-098) was tested and successfully gave specification-compliant APIs, as can be seen in Table 14A-B above.

Optimization of recrystallization in DMSO/Water

A. Preliminary test

With DMSO and waterAll recrystallization tests are reported in tables 14A-B. Column three "4- [4- (methylsulfinyl) phenyl]-3-phenyl-2 (5H) -furanone "means 4- [4- (methylsulfinyl) phenyl group prior to recrystallization of crude rofecoxib (RXB-201)]-amount of 3-phenyl-2 (5H) -furanone. Since most of the crude rofecoxib (RXB-201) subjected to recrystallization is pure, 4- [4- (methylsulfinyl) phenyl group is added]Evaluation of the scavenging efficiency was carried out with 3-phenyl-2 (5H) -furanone (CHG P059-038). Test LMC P045-186 was carried out to evaluate 4- [4- (methylsulfinyl) phenyl]The clearance factor for 3-phenyl-2 (5H) -furanone, at the current recrystallization process, is much higher than usual (up to 1.8% a/a). Tests R1 and R2 gave the same clearance factors as usual (2.4 and 2.3). The recrystallization process has the same purification efficiency (from 0.10% to 1.8% of 4- [4- (methylsulfinyl) phenyl group) ]-3-phenyl-2 (5H) -furanone). The study then focused on reducing the total amount of solvent to improve yield. VLA P075-180 was performed with reduced amounts of DMSO (-1.5V) and water (-2.5V) compared to the current procedure. After standard recrystallization procedures with these reduced amounts of solvent, the product was obtained in 89% yield, 2.3 also being obtained as 4- [4- (methylsulfinyl) phenyl]-3-phenyl-2 (5H) -furanone. However, the purity of rofecoxib (RXB-201) is due to the formation of 4- [4- (methylsulfonyl) phenyl]-3-phenyl-5-hydroxyfuran-2-one (0.47%) but not satisfactory (99.43%). The test was started with crude rofecoxib (RXB-201) containing high levels of RoI (APG P052-110, 1.5% RoI). Na (Na)₂SO₃The presence of (b) explains the higher amount of RXB-hydroxy groups. At higher DMSO ratios (6V) and lower temperatures (50 ℃), test VLA P075-184 allows rofecoxib (RXB-201) with 4- [4- (methylsulfonyl) phenyl ] to be obtained in good yields after filtration at 0 ℃]-3-phenyl-5-hydroxyfuran-2-one and 4- [4- (methylsulfinyl) phenyl]Mean level of clearance of-3-phenyl-2 (5H) -furanone.

B. Solubility study of rofecoxib (RXB-201)

In order to accurately observe the solvent volume reduction ability, it is necessary to properly know the solubility of rofecoxib (RXB-201) according to the DMSO volume. Solubility and nucleation of rofecoxib (RXB-201) were measured at four different concentrations due to the Crystal 16 equipment: 3V, 4V, 5V and 6V. The results are shown in FIG. 12. The solubilization curve is reported in blue and the nucleation is reported in orange. The process for solubilization of RXB-201 or recrystallization process batch F851 at the start is reported as the purple point (5.5V at 40 ℃). To improve the yield, the concentration can be increased to 4.5V (red dots) with heating at 50 ℃. These conditions allow good solubilization of rofecoxib (RXB-201), with a large safety margin compared to the nucleation temperature (at least 30 ℃). This margin is needed to avoid spontaneous crystallization during clarification filtration. An additional amount of DMSO (+0.5V) was used to flush the filtration system, resulting in a total of 5V DMSO.

C. Optimization of recrystallization yield

The recrystallization assay was started with 20g of crude rofecoxib (RXB-201) (CHG P059-098) in total 5V DMSO. 5.5V water was used to ensure crystallization and resulted in very high yields (97%) with satisfactory purity. However, the crude product subjected to recrystallization is already very pure. In this case a lower clearance of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone was observed (clearance factor ═ 1.6). It has been shown previously that the clearance factor for 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone is almost constant starting from a crude containing 0.1% to 2% of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone (clearance factor ═ 2). It is unknown whether the low clearance factor is due to the lower amount of impurities to be removed (disrupting the linearity of clearance), the recrystallization conditions (solvent amount and ratio), reproducibility problems or measurement uncertainty. The final experiment (CHG P059-104) was performed with lower amounts of water (3V) in an attempt to improve the removal efficiency while maintaining high yield. The yield remained high (96%) with a clearance factor of 1.8 (on average) for 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone. A lower ratio (0.05%) of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone was detected in the batch. The crude rofecoxib (RXB-201) used in this test has specificity derived from calorimetry studies of oxidation of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone. The crude contained traces of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone and 0.22% of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone. Thus, the presence of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone in the sample batch should not be considered as a warning. The solvent ratio (5V DMSO/3V water) was chosen to optimize the recrystallization process. The process has less solvent consumption and increases the yield of the recrystallization step by + 45% compared to prior art processes without affecting the removal efficiency. The yellowish color of the crude rofecoxib (RXB-201) was effectively removed to give a white recrystallized solid of rofecoxib (RXB-201).

The optimized recrystallization procedure for rofecoxib (RXB-201) is shown in Table 16 below.

TABLE 16 recrystallization of RXB-201

Solvent screening aided by the Dynochem software only emphasized 3 candidates as good solvents and 6 candidates as anti-solvents. DMSO remains as a good solvent for recrystallization. Isopropanol and acetic acid were tried as anti-solvents without successfully improving 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone clearance. The combination of DMSO and water remains the best choice for recrystallization of crude rofecoxib (RXB-201). Optimization resulted in a 45% reduction in total volume, which had a significant impact on process yield. The ratio of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone after recrystallization was almost only half.

To address this problem, the IPC and crude rofecoxib (RXB-201) specifications are shown in table 17 below:

TABLE 17 Oxidation of IPC

The formation 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one is now also better understood and should be significantly reduced due to the inhibition of sodium sulfite used for oxidation quenching. 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one was substantially eliminated during recrystallization.

EXAMPLE 8 further Process optimization of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone Oxidation

Background of the study

Scheme 3 below shows the current scheme for the oxidation of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone (RXB-furanone).

Scheme 3.

The optimization is performed with respect to the following factors:

oxidation is classified as 5/5 according to Stoessel scale, with higher heat build-up observed at the start of oxidation.

The heating time to reach the IPC specification (4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone ≦ 0.2% area UHPLC) was 14H instead of 6H. This long reaction time may be due to a slower reaction or difficulty in obtaining a representative IPC sample of the batch composition. Even after this long heating time, the impurity characteristics are not affected.

Higher amounts of RoI were obtained in isolated crude rofecoxib (RXB-201) (0.5%, assay # CQ 18-0487). This material resulted in filter plugging during clarification prior to recrystallization.

The presence of insoluble material may be attributed to sodium sulfite as difficulties were encountered during its dissolution prior to quenching. The first part of the optimization work is directed to the study of the solvents of the oxidation process. The second section addresses the suppression of energy build-up to improve process safety. The conversion of traces of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone (RXB-sulfoxide) at the end of the oxidation was then investigated, as well as the preparation of IPC samples. Finally, the need for sodium sulfite quenching was evaluated.

Investigation of solvents for oxidation processes

In the current oxidation process, the solvent used is acetonitrile as depicted in scheme 3. Under the process conditions, 6 volumes of ACN permit the preparation of 4- [4- (methylthio) phenyl]-3-phenyl-2 (5H) -furanone is completely dissolved at 65 ℃ at the start of the process. 6h after the completion of the addition of hydrogen peroxide, the reaction medium was heterogeneous, containing a higher amount of white solid in suspension. The solid is mainly prepared from rofecoxib (RXB-201) ((RXB-201))>99%) with traces of 4- [4- (methylsulfinyl) phenyl]-3-phenyl-2 (5H) -furanone (f)<1%). This shows that the solubility of rofecoxib (RXB-201) in ACN is lower than that of 4- [4- (methylthio) phenyl group]-3-phenyl-2 (5H) -furanone. 4- [4- (methylsulfinyl) phenyl ] although rofecoxib (RXB-201) crystallized out]-3-phenyl-2 (5H) -furanone may also accumulate in the crystals. Thus, better solubility of RXB species was investigated to allow for 4- [4- (methylsulfinyl) phenyl]Complete oxidation of traces of (E) -3-phenyl-2 (5H) -furanone. The solubility of rofecoxib (RXB-201) in a large group of solvents was determined with the goal of improving recrystallization. Before measuring these solubilities, several tests were performed. Scheme 4 below shows 4- [4- (methylthio) phenyl ]-3-phenyl-2 (5H) -furanone in DCM/H₂Oxidation in O.

Scheme 4.4- [4- (methylthio) phenyl]-3-phenyl-2 (5H) -furanone in DCM/H₂Oxidation in O

In this experiment (CHG P059-058), 6VACN was substituted with 5V DCM, which is expected to be a good solvent for polar organic molecules such as rofecoxib derivatives. Five conditions were investigated, as shown in table 18 below.

TABLE 18 in DCM/H₂4- [4- (methylthio) phenyl ] in O]Conditional screening of (E) -3-phenyl-2 (5H) -furanone oxidation

Tests A and B allow the study of 4- [4- (methylthio) phenyl group]-3-benzeneInfluence of residual IPA in the radical-2 (5H) -furanone on the kinetics. Runs C, D and E used the phase transfer catalyst tetrabutylammonium chloride (TBACl) to improve mixing between the organic and aqueous layers. All tests were performed at room temperature. For practical purposes, 0.5V catalyst (Na) was introduced₂WO₄.2H₂O) pure water solution. Fig. 13 shows the results of the oxidation test.

Experiment A shows that the conversion of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone is very low under UHPLC. After 3H at room temperature, only 10% of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone was observed. The effect of the IPA content of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone is evident, and a slightly better conversion (22% 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone after 3H at room temperature) is obtained in test B. Significant differences were observed using TBACl as a phase transfer catalyst. Runs C, D and E show the formation of rofecoxib (RXB-201) as the major product even after 30min of reaction. The three experiments did not show true kinetic differences. Crystallization occurred during oxidation, indicating that rofecoxib (RXB-201) is poorly soluble in DCM. In view of the insufficient conversion and solubility, the results obtained in DCM are inferior to the current process with ACN. Scheme 5 below shows oxidation in acetonitrile/sulfolane.

Scheme 5 Oxidation in acetonitrile/sulfolane

The following tests were carried out in 6VACN as the main solvent. Additional additions of sulfolane (1V or 2V) were performed to examine the effect on solubility (CHG P059-062). Sulfolane was chosen because of its structure similarity to rofecoxib (RXB-201). The reaction was started with wet 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone, in which the catalyst was pre-dissolved in water, as in the biphasic test shown in Table 19 below. The results of the oxidation test are reported in fig. 14.

TABLE 19 screening of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone Oxidation Using sulfolane

For the two-phase test, 0.5V of catalyst (Na) was introduced₂WO₄.2H₂O) to bring 1 mol% of catalyst into the reaction medium. Then, only 0.25 equivalent of H is introduced first₂O₂To observe potential energy build-up. Adding the first drop of H to the mixture at 65 ℃₂O₂In time, an exotherm was observed in the 3 reaction media. After 5min in each experiment, UHPLC confirmed the formation of ≈ 30% area 4- [4- (methylsulfinyl) phenyl]-3-phenyl-2 (5H) -furanone (see FIG. 14).

Experiment a is equivalent to the current method. Thus, the addition of 0.5V water appears to be effective in suppressing the energy accumulation phenomenon. The remaining hydrogen peroxide was then introduced and the reaction medium was stirred at 65 ℃. In the presence of H ₂O₂After 30min UHPLC analysis was performed and 4- [4- (methylthio) phenyl ] indicated]-total conversion of 3-phenyl-2 (5H) -furanone. However, sulfolane p-4- [4- (methylsulfinyl) phenyl]The conversion of 3-phenyl-2 (5H) -furanone has a negative effect, with 1.48% of residual intermediate in test C and 0.98% in test B, whereas the standard method (test A) shows only 0.23% of 4- [4- (methylsulfinyl) phenyl group]-3-phenyl-2 (5H) -furanone. Dilution effects may be the cause of such a lack of conversion: a total of 8V for C and a total of 6V for a. The use of sulfolane does not appear to be very effective in improving the oxidation of RXB species. In addition, sulfolane has a low PDE limit of 160ppm in ICH Q3C. Thus, the risk benefit is less favorable for the use of this solvent. The following 3 solvents provided rofecoxib (RXB-201) having a solubility greater than 100g/L at 30 ℃:

·DMSO：107g/L

·DMF：133g/L

·NMP：140g/L

DMSO is reactive towards the oxidation process and cannot be used as a solvent for the oxidation step. DMF is a solvent that has been widely used heretofore. For safety reasons, the solvent is replaced in all steps. Therefore, DMF cannot be considered as an alternative. Finally, NMP was shown to be the best solvent with up to 140g/L of rofecoxib (RXB-201) solubility at 30 ℃. However, the literature mentions the oxidation of NMP to N-methylsuccinimide by hydrogen peroxide under metal catalysis and at 0 ℃. See Dong, j.j. et al, ChemSusChem,2013,6, 1-6, which is incorporated herein in its entirety. The process conditions at 65 ℃ allow to envisage the risk of using NMP without checking the potential formation of 5-hydroxy-N-methylpyrrolidone and N-methylsuccinimide. Finally, NMP was withdrawn from the list of candidate solvents for the oxidation process. ACN remains as a solvent for the oxidation process. The addition of water appears to have a positive effect on the reaction kinetics, as shown in figure 14, experiment a, which shows only 0.23% of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone 30min after the completion of the hydrogen peroxide addition. Kinetic models were constructed to better understand the evolution of RXB species over time. The heat accumulation phenomenon of the current process requires precise knowledge of the reaction kinetics.

Kinetic model for RXB species oxidation

Previous experiments have shown that the use of DCM and sulfolane does not allow to obtain better conversion. Dissolving the catalyst prior to addition of the hydrogen peroxide avoids build up. Kinetic modeling was established to better understand the oxidation reactions that occur in the reaction medium with the current process. Use ofThe software uses the tool "Simple metadata action" to achieve this. Two experiments were performed using the parameters described in table 20, with reaction tracking by UHPLC.

Table 20. experiments performed on the kinetic modeling.

To allow the reaction to be followed over a reasonable amount of time, the amount of catalyst divided by 100 (0.012 mole%) was usedTest CHG P059-074. To achieve all kinetic parameters of the reaction, a second test, CHG P059-078, was performed at a lower temperature (45 ℃). To increase the solubility of RXB species in the reaction medium, the amount of acetonitrile was multiplied by 4 (28.2V). By increasing the amount of catalyst to 0.18 mole%, a balance of lower temperature and higher dilution is achieved. FIGS. 15A-B show the ratio of species in the reaction medium over time. FIG. 15A shows 4- [4- (methylthio) phenyl ] of CHG P059-074]Oxidation trace of (E) -3-phenyl-2 (5H) -furanone. FIG. 15B shows 4- [4- (methylthio) phenyl ] of CHG P059-078 ]Oxidation trace of (E) -3-phenyl-2 (5H) -furanone. Hydrogen peroxide was added in 4 batches (10%, 20%, 30% and 40%). Each vertical line belonging to an instantaneously added H₂O₂. The instantaneous addition of the reagent is mandatory in order to avoid the limitation of the reaction kinetics by the rate of addition of the reagent. Three reactions are believed to occur in the reaction medium:

·Na₂WO₄+H₂O₂→Na₂WO₅+H₂O

4- [4- (methylthio) phenyl group]-3-phenyl-2 (5H) -furanone + Na₂WO₅→ 4- [4- (methylsulfinyl) phenyl group]-3-phenyl-2 (5H) -furanone + Na₂WO₄

4- [4- (methylsulfinyl) phenyl]-3-phenyl-2 (5H) -furanone + Na₂WO₅→RXB-201+Na₂WO₄

The fit between the model (line) and the experimental points (points) was found to be satisfactory. The reaction rate coefficient and activation energy of 3 reactions were measured.

First oxide (4- [4- (methylthio) phenyl)]-3-phenyl-2 (5H) -furanone → 4- [4- (methylsulfinyl) phenyl]-3-phenyl-2 (5H) -furanone: k 10L/mol. s) showed ten times faster than the second oxidation (4- [4- (methylsulfinyl) phenyl)]-3-phenyl-2 (5H) -furanone → RXB-201: k ≈ 1L/mol. s). Regeneration of the catalyst (Na)₂WO₄→Na₂WO₅) With a reaction rate constant of k ≈ 8.5L/mol. s. These data reflect the difficulty in rendering 4- [4- (methylsulfinyl) phenyl group when the second oxidation is ten times slower]Complete conversion of (E) -3-phenyl-2 (5H) -furanone to rofil Coxib (RXB-201). However, these kinetic data must be carefully considered with respect to reaction completion. In the formation of rofecoxib (RXB-201), both oxidations proceed with little excess. This is to avoid crystallization of rofecoxib (RXB-201) at the end of the process. Both processes were performed with fully soluble RXB species throughout the data acquisition process, which simplifies the model. The model also shows oxidation with addition of H₂O₂Immediately thereafter, indicating a highly reduced energy accumulation. Both experiments were performed with the catalyst with the addition of 0.5V water. This addition results in a decrease in the solubility of RXB species in the oxidizing medium, as RXB species are hardly soluble in water. Therefore, the volume of water was investigated to introduce the minimum amount required for energy accumulation inhibition and to minimize the effect on RXB solubility. The kinetic parameters of the reaction that occurred during the oxidation process are shown in table 21 below.

TABLE 21 kinetic parameters of the reactions taking place during the oxidation process

Solvent optimization

A. Study of optimal Water and IPA content for inhibition of accumulation phenomena

An experiment was performed to evaluate the amount of water introduced at the beginning of the reaction, which is suitable for dissolving the catalyst and suppressing the accumulation phenomenon.

The following experiments were performed in 6V ACN using the current method, using dried 4- [4- (methylthio) phenyl-]-3-phenyl-2 (5H) -furanone, catalyst, addition of IPA to represent different LoD values, and addition of water. The experiment was started with continuous introduction of catalyst and water into the reaction medium. The mixture was heated at 65 ℃ and then 0.125 equivalents of H were added instantaneously₂O₂. IPC was performed after 2 minutes of contact to check the reaction. The IPC results reported in Table 22 below are for 4- [4- (methylsulfinyl) phenyl]-3-phenyl-2 (5H) -furanone.

TABLE 22 screening of IPA and water content for inhibition of accumulation.

Note that 0.25V H was used₂O allows an optimum conversion to 4- [4- (methylsulfinyl) phenyl group only after 2 minutes]-3-phenyl-2 (5H) -furanone, up to 14% area UHPLC. When the water volume was reduced to 0.15V, the reaction was still started, but the conversion was almost halved. All experiments showed slightly turbid reaction media, except the reaction medium with the largest amount of IPA and the smallest amount of water (VLA P075-170). This indicates the positive effect of IPA and the water on 4- [4- (methylthio) phenyl group]Negative effect of solubility of 3-phenyl-2 (5H) -furanone. However, the combination of a high ratio of IPA with a low ratio of water produces a different suspension (VLA P075-170) containing a high density colorless solid, possibly an insoluble catalyst. It can be assumed that 0.25V of pure water allows partial dissolution of the catalyst, sufficient to avoid the accumulation effect. Therefore, this water volume is selected. The water content is combined with the inhibition of the build-up effect, with wetting of the 4- [4- (methylthio) phenyl group ]Acceptable solubility (mean LoD) of 3-phenyl-2 (5H) -furanone. The use of purified water is mandatory. Passing through 12 Na₂WO₄.2H₂The dissolution of O in pure water, and 12 samples in tap water were tested. The 24 samples were prepared at the same concentration (about the working concentration) during the oxidative synthesis. All samples dissolved rapidly after stirring at room temperature. After a few hours, 12 samples in tap water showed a white precipitate, while 12 samples in pure water remained as clear solutions. Calcium cation (Ca 2) in tap water is considered⁺) With WO₄ ^2-Formation of calcium tungstate by anionic association₄I.e. a salt which is practically insoluble in water (0.02 g/L).

B. Investigation of the effect of solvent on the completion of the oxidation reaction.

Previous experiments have shown optimal conditions to suppress the accumulation phenomenon. The use of 0.25V deionized water allows the reaction to begin as soon as the addition of hydrogen peroxide begins. This is indicated by the different values of IPA content in 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone (LoD ═ 36% or 50%). After working on the start of the reaction, the effect of the solvent on the completion of the reaction was then investigated as shown in table 23 below.

TABLE 23 influence of solvent on the completion of the reaction.

The first run (VLA P075-176) was carried out using standard methods (at 65 ℃) with the addition of 0.25V of pure water. The addition of hydrogen peroxide was effected within 2 h. A slightly turbid reaction medium is obtained with very little solids in suspension. At H ₂O₂At the end of the addition, no 4- [4- (methylthio) phenyl group was detected]3-phenyl-2 (5H) -furanone, and this test (IPC 1) detected only 2.5% of 4- [4- (methylsulfinyl) phenyl]-3-phenyl-2 (5H) -furanone. After stirring at 65 ℃ for a further 1 h, no 4- [4- (methylsulfinyl) phenyl group was detected in UHPLC (IPC 2)]-3-phenyl-2 (5H) -furanone. IPC was performed by sampling of heterogeneous mixtures. This test is very satisfactory in view of the extremely rapid conversion of the species to rofecoxib (RXB-201). It is reminded here that the current process without addition of water requires at least 6h to reach the IPC specification. At the end of the reaction, 1VACN was added to the reaction in an attempt to achieve complete solubilization of the reaction medium. In fact, the reaction medium immediately becomes heterogeneous as a white suspension. This indicates supersaturation of the reaction medium. Rofecoxib (RXB-201) crystallizes out under the perturbation generated by the addition of acetonitrile.

The effect of isopropanol content was then investigated in a second trial (VLA P075-178). This reaction was performed without IPA, which should have a negative impact on the solubility of RXB species, considering table 22. In this test, which was carried out under the same conditions, the reaction medium was already in suspension at the end of the addition of hydrogen peroxide. In contrast to VLA P075-176, in the presence of H ₂O₂At the end, better conversion was observed. Furthermore, H₂O₂The addition was performed faster than the first trial (2h instead of 2h 20). The absence of IPA showed faster than 36% IPAThe oxidation conditions of (1). This may be due to the slightly higher concentration of reaction medium due to the absence of IPA. Crystallization of the reaction medium appeared to be on 4- [4- (methylsulfinyl) phenyl]The oxidation kinetics of (E) -3-phenyl-2 (5H) -furanone had no effect.

A third trial was performed to confirm this. Test CHG P059-084 started with 7V ACN containing normal amounts of IPA. This higher dilution shows no effect on the reaction medium at the start of oxidation. In the presence of H₂O₂Seeding is performed twice in the meantime to promote suspension of the reaction medium upon addition. The reaction medium is similar to VLA P075-178. At 45 minutes after completion of the addition of hydrogen peroxide, no 4- [4- (methylsulfinyl) phenyl group was detected]-3-phenyl-2 (5H) -furanone. After quenching by addition of 2V water, cooling to 0 ℃, filtering and washing the filter cake, the solid obtained contains 0.02% of 4- [4- (methylsulfinyl) phenyl]-3-phenyl-2 (5H) -furanone. This experiment confirmed the satisfactory results obtained in VLA P075-178.

Thus, the reaction medium may be a slightly turbid or heterogeneous white suspension, the IPC specification being achieved after only 1h of heating after the hydrogen peroxide addition is complete. Supersaturation of the reaction medium by 6V ACN does not appear to be a problem for the conversion of RXB species to rofecoxib (RXB-201). The addition of 0.25V pure water had a significant effect on the improvement of the oxidation kinetics. Supersaturation of RXB species in the oxidizing medium results in crystallization of the sample dedicated to IPC. A study was conducted to establish optimal sample preparation.

Optimization of IPC sample preparation

IPC was performed by directly sampling the stirred reaction mixture. Such non-uniform sampling is difficult to perform representatively. Therefore, the stirred suspension sample was replaced. The suspended aliquot was filtered and analyzed for solids. Due to the low solubility of RXB species, it is assumed that the representativeness of the filter cake analysis is satisfactory, in particular with respect to the 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone ratio. This assumption is verified in this work. A study of IPC preparation was performed during the runs CHG P059-084 and is shown in Table 24 below.

TABLE 24 screening of IPC sample preparation conditions.

Two first IPCs (IPC1 and IPC2) were performed under representative sampling (sampling in stirred suspension). IPC2 indicated complete conversion: 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone ═ n.d. IPC3a was performed by sampling ≈ 1mL of reaction medium, cooling to room temperature and filtering. The filter cake and mother liquor were analyzed. 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone in the filter cake was 0.02%. IPC3b was performed by sampling ≈ 1mL of reaction medium, cooling to room temperature and adding ≈ 2V water and filtering. The filter cake and mother liquor were analyzed. 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone in the filter cake was 0.02%. The entire product of the experiment was obtained after cooling the reaction medium to room temperature, adding 2V water, cooling to 0 ℃, filtering and standard cake washing. 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone in the filter cake was 0.02%.

These experiments show that the IPC3a and IPC 3b techniques give results for the same ratio of 4- [4- (methylsulfinyl) phenyl ] -3-phenyl-2 (5H) -furanone as the isolated compound of experiment CHG P059-084. If we consider all RXB derivatives, IPC3a technique gives the closest results. Another impurity, designated 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one (RXB-hydroxy), is reported in Table 24. The proposed structure is shown in fig. 16. The impurity was detected at RRT 0.63. LCMS analysis performed during the pre-degradation test showed a mass equal to [ RXB-201+16 ]. This hydroxylation is reported in the literature, in particular with oxygen. See d.a. nicoll-Griffith et al bioorg.med.chem.lett.2000,10,2683 and e.j.corey et al Tetrahedron lett.2005,46,927, which are incorporated herein in their entirety. This impurity was always observed in the rofecoxib (RXB-201) sample, where no nitrogen flow was used during the oxidation process. The ratio of 4- [4- (methylsulfonyl) phenyl ] -3-phenyl-5-hydroxyfuran-2-one was only 0.03% in crude rofecoxib (RXB-201) (batch F801). This lower ratio can be explained by the nitrogen atmosphere used.

Inhibition of sodium sulfite quenching

In the current synthesis process described in scheme 3, sodium sulfite is used as an aqueous solution to quench the excess hydrogen peroxide added in the reaction medium. Crude rofecoxib (RXB-201) F801 having 0.5% of residue on ignition was obtained in advance. It is believed that this material caused filter plugging during clarification prior to recrystallization. The effect of ash during the recrystallization process was also investigated. Inhibition of sodium sulfite quenching was envisioned and use tests were performed. The quenching was replaced by the addition of 2V pure water (CHG P059-092). This test shows a number of advantages:

better yields were obtained without Na2SO 3: 92% instead of 86%

No effect was encountered on the crude rofecoxib (RXB-201) UHPLC characteristics.

No particles in suspension during the hot filtration when recrystallization is carried out (CHG P059-098)

Several verifications were performed to confirm the feasibility of hydrogen peroxide removal:

stainless steel 316L in contact with crude rofecoxib (RXB-201) mother liquor (enriched with excess hydrogen peroxide) after 10 days at 20 ℃ is corrosion free (CHG P059-084).

Crude rofecoxib (RXB-201) does not require drying.

The test strip peroxide test was negative for both crude and recrystallized rofecoxib (RXB-201) (CHG P059-098). The crude wash appeared to be sufficient to effectively remove traces of peroxide in the cake wash.

The stability of the isolated crude rofecoxib (RXB-201) towards hydrogen peroxide is very satisfactory. The crude unwashed rofecoxib (RXB-201) was stored at room temperature for 17 days. No evolution of the UHPLC characteristics was observed (CHG P059-104).

In view of all these advantages, it was decided to quench by adding 2V pure water instead of sodium sulfite.

4- [4- (methylthio) phenyl]Scheme for the optimized oxidation of 3-phenyl-2 (5H) -furanone.

Scheme 6 shows an optimized scheme for the oxidation of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone.

Scheme 6.4 optimization scheme for Oxidation of- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone

The information relates to: quality, HSE, Process

Calorimetry study

The optimized process depicted in scheme 6 was subjected to calorimetry studies. A thermal study was performed on 40g of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone on an AlgochemARLARF reactor unit. The results of the calorimetry directed to the oxidation of 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone are summarized in Table 25 below.

TABLE 25 calorimetric data for 4- [4- (methylthio) phenyl ] -3-phenyl-2 (5H) -furanone oxidation under optimized process.

The reaction enthalpy was measured at Δ Hr-187 kJ/kg, where Cp is 2.3J/g/° c. This results in a potential temperature increase at adiabatic conditions Δ Tad-82 ℃. The exothermic nature of the reaction is already known, but the main improvement of the new process is the maximum build-up, now measured as 6% after addition of 20% hydrogen peroxide. This accumulation is divided by 3 compared to the previous process. Thus, the exotherm is well controlled by the addition of hydrogen peroxide. After all calorimetric parameters have been considered, the reaction is rated 4 within 5 according to the Stoessel scale.

This optimization resulted in the 0.25V pure water addition being performed at the start of the process. Due to this water addition, the heat build-up phenomenon is significantly reduced. Furthermore, this addition appears to have a very positive effect on the oxidation of RXB species. At this point the IPC specification was reached after 1 or 2 hours, rather than 6 hours, after the hydrogen peroxide addition was complete. In order to make the IPC results more reproducible, new IPC preparation techniques were also tested. The kinetic model allows a better understanding of the evolution of the oxidation. The oxidation starts with the beginning of the addition of hydrogen peroxide, wherein the first oxidation is ten times faster than the second oxidation. After examining all the advantages it brings, the removal of the sodium sulfite quench was verified.

Report on thermal research

The reaction scheme is shown in scheme 7 below:

scheme 7 reaction scheme

The raw materials are shown in table 26.

TABLE 26 raw materials

The flow of the calorimetric test is shown in scheme 8 below.

Scheme 8.

The calorimetric data are shown in table 27 below.

TABLE 27 calorimetric data

The thermal stability data are shown in table 28 below (DSC).

TABLE 28 thermal stability

H₂O₂Is exothermic and energy is released from H₂O₂The addition rate of (2) is well controlled (maximum heat build up is 6% after about 20% addition). The reaction mixture is homogeneous and remains readily stirrable. In DSC, H ₂O₂An important exotherm (-981J/g) was exhibited from 47 deg.C. Mixture acetonitrile/H₂O₂It is unstable from 272 ℃ (492J/g). The reaction mixture before and after addition was more stable with a very small exotherm starting at 284 ℃.

Under normal synthesis conditions, the semi-batch process is safe. The MSTR is 70 ℃ and TD24 can be calculated at 144 ℃. The risk of initiating decomposition is low, but the rate of addition must be well controlled and the reaction must be properly initiated to avoid H₂O₂And (4) accumulating.

TABLE 29 additional data

(symbol)	Description of the invention	Unit of	Results

Tf batch size	Maximum final temperature, with 100% accumulation	℃	146

Tdec	Minimum decomposition temperature by DSC	℃	47
				TD₂₄	TMR is 24h	℃	Is not available
TD₈	TMR 8 temperature	℃	Is not available

Eb	Boiling point of reaction mixture	℃	82
				Tp	Temperature of the process	℃	65

					Heat potential of main reaction		Average level
	Thermal potential of secondary reaction		Height of
					Step grading		4

The energy of the main reaction can increase the mass temperature to 146 ℃. At this temperature, the decomposition to H may be initiated₂O₂Decompose to give H₂0₂981J/g additional energy. If we consider half H₂O₂The decomposition and the other half used for the reaction, the adiabatic temperature rise would be 24 ℃ and the final temperature 170 ℃. Decomposition of the reaction mixture should not be initiated. If a closed vessel is used, the pressure can reach 9 bar. If an open vessel is used without a condenser, the boiling point will be reached. The total energy was 72g evaporated, i.e. 38% of the initial amount of acetonitrile. The reaction force can be estimated at 82 ℃ under lSOW/kg. The vapor rate is about 230m ³Calculated at ACN/h of (1). There may be a risk of flooding. The safety of the process depends on the ratio of H₂O₂Temperature and time of addition. Additional data are shown in table 29.

Fig. 17 shows a general scheme of the oxidation reaction. FIG. 18 shows effort H₂O₂Is added. FIG. 19 shows a graph showing the maximum Temperature (TSR) achievable by the synthesis reaction. Figure 20 shows the conversion of the reaction product. Fig. 21 shows the oxidation reaction force.

Table 30 shows the values for the reaction classifications. The reaction classifications are also shown in FIG. 22.

TABLE 30 values for Classification reaction

Example 9 computational mutagenicity analysis of Rofexib (TRM-201) -related impurities

Introduction to the design reside in

The subject matter described herein also relates to the evaluation of the potential mutagenicity of a rofecoxib (TRM-201) impurity. The evaluation is performed by testing the structure in several different computer software programs and then performing an expert review of the computer data.

The use of computer (computational) tools to predict toxicity has increased dramatically over the past few years and is now mature not only in the pharmaceutical industry, but also in the chemical and cosmetic fields. This is reflected in particular in the International Council for standardization of Technical Requirements for Human medicine (ICH) M7 guidelines in the field of potentially genotoxic impurities and typing in 2015, the first regulatory document (ICH M7 — Step 5,2015) supporting the Use of computer tools as a primary substitute for in vitro or in vivo testing. The purpose of the M7 guidelines is to help identify and characterize impurities at risk of mutagenicity, and to outline control strategies for various classes (classes 1-5) of compounds to limit potential carcinogenic risk to subjects. According to ICH M7, "the sensitivity of structural alerts from two structural activity relationships [ (Q) SAR ] methods from insufficient to complete the impedance of the electronic content, and no to complete the impedance of the electronic content (Class 5in FIG. 23)".

Computer software programs to predict toxicity combine biology and chemistry with modeling and computational science to improve the predictive power in the field of toxicology. Computer technology employing knowledge-based Expert systems such as DEREK Nexus (Lhasa, Ltd.), Leadscope Expert Alerts (Leadscope, Inc.) and GT _ Expert (Multicase, Inc.) is based on the existence of structural rules. Expert knowledge based on toxicity data and mechanisms is used to create rules about the likelihood of structural toxicity, with information from all applicable rules being used to make an overall prediction. The rules are typically encoded as one or more substructures that match the test chemical. The prediction is made when the predicted alerts match. These rules generally indicate the basis of a mechanism for any positive prediction. Statistical-based systems, such as Model Applier (Leadscope, Inc.), Case Ultra (Multicase, Inc.), and epat.e.s.t. (US EPA), are commonly referred to as quantitative structure-activity relationship (QSAR) models and are used to predict various toxicity endpoints based on chemical structure. These models are constructed from historical laboratory data (training sets) in which chemical substructures and molecular properties (descriptors) are generated from chemical lists. These descriptors are used to construct a statistical-based mathematical model to predict the target toxicology effects.

The primary endpoint involved in the ICH M7 guidelines was DNA reactive mutagenicity, for which the Ames bacterial mutagenicity assay was the preferred assay. The structure-based computer evaluation has a good ability to distinguish mutagens from non-mutagens, with generally higher agreement when compared to Ames assay results (Sutter et al, 2013). Non-mutagenic genotoxins generally have a threshold mechanism and generally do not pose a carcinogenic risk to a subject at levels that are normally present as Impurities (EMEA guidelines on the Limits of genomic imprints, 2006).

Materials and methods

The current ICH M7 guidelines (ICH M7(R1),2018) indicate that certain drug impurities should be characterized as non-mutagenic using two complementary computational methods. To meet the ICH M7 guidelines, the report covers computer analysis using a rule-based system (derekexus) and a statistics-based system (Leadscope Model applicator). Predictive data from each computer software program was reviewed in order to provide additional supporting evidence about any positive or negative predictive relevance and to clarify any potential cause of conflicting results. Dobo et al (2012) have shown that predicting negative results using computer methods has a negative predictive value of 94% throughout the industry, and that this value increases to 99% when additionally expert review is performed. After data review, a global mutagenicity prediction (positive or negative) was performed.

For the recommended control behavior, the results were structurally categorized from 1-5 according to the ICH M7 control strategy originally developed by Muller et al (2006). For example, if an impurity is found to have no structural warning or a warning summarizing sufficient data to justify a lack of mutagenicity, the impurity can be considered as class 5 (non-mutagenic). An impurity may also be considered non-mutagenic if it has a warning but the warning is the same as the non-mutagenic parent, or if the warning can be scientifically dismissed (class 4). If an impurity has a warning structure that does not preclude potential mutagenicity, the impurity will be considered as class 3 and should be controlled at or below the universal toxicological threshold of interest (TTC) or adjusted TTC of 1.5 μ g/day based on the duration of administration.

Computer software-DEREK Nexus

DEREK is a knowledge and rule based predictive toxicology software program that makes qualitative estimates of endpoint risk. The knowledge-based system is a computer program that: it contains expert knowledge rules of toxicology and is typically applied to predict chemical toxicity when no experimental data is available. Qualitative estimates of risk are categorized as "specific", "likely", "plausible", "equivocal", "suspected", "unlikely", or "inactive" in descending order of probability.

DEREK Nexus contains expert-derived functions to provide a negative (inactive) prediction of bacterial in vitro mutagenicity. The Lhasa Ames reference set consists of various datasets (e.g., NTP, FDA CFSAN, issty, Kirkland, bursti, Benchmark, Acid Halide Data, Member Data, etc.) and consists of 9,900 compounds with 132 mutagenic alerts (Lhasa Knowledge Suite, Nexus 2.2Release Notes).

Non-alert compounds were evaluated to identify unclassified and misclassified features. Misclassification features in the molecule were derived from non-alert mutagens in the Lhasa reference set. Features not present in the molecules in the Lhasa reference set are considered unclassified. Negative prediction (no activity) was shown for compounds in which all features in the molecule were present in the exact classification of compounds from the reference set. Predictions for compounds with misclassified or unclassified features remain negative and these features are highlighted to enable expert evaluation of the predictions. Negative predictability was reported to be high for compounds without misclassified or unclassified features (86-94%), comparable to the Ames assay.

The DEREK analysis was performed specifically using a mutagenic endpoint.

Computer software-CASE Ultra

CASE Ultra (CASE) software is a statistical (GT1 BMut model) and rule (GT Expert) based system designed to reveal the relationship between the structure of chemicals and their activity in specific biological assays. It has been designed to handle "non-homogeneous" databases, i.e., databases composed of structurally unrelated molecules that are generally unsuitable for processing by conventional Quantitative Structure Activity Relationship (QSAR) type techniques. Therefore, its main objective is to find structural entities that distinguish between active and inactive molecules, and its success depends on the validity of the working hypothesis that the relationship between chemical structure and activity does exist. As noted, the program selects its own descriptor from a plurality of possible sub-building units and creates a dictionary of molecular descriptors without human intervention. The selected descriptors were characterized as activated (mycoplasma) or inactivated (non-biologically active fragments). The program also takes into account several other factors such as molecular weight, octanol/water LogP, water solubility, Lipinski penta-rule and intestinal absorption. All alert contributions are taken into account and the scaled alert weights and regression coefficients are used for the final total probability calculation. Unknown fragments, positive (activation) and inactivation alerts are highlighted in the procedure, and also provide overall positive probability. The ability of CASE Ultra to select alerts that are readily recognized as part of the molecule is a major advantage of this approach. Indeed, the identification of structural components embedded in molecules by CASE Ultra provides a foothold for the development of possible structural sites for human intelligence to metabolize or to bind to receptors. These modules were developed through a collaborative collaboration between the FDA and Multicase, Inc. The database (version 3.0) contained 13,514 unique structures (6982 positives, 6532 negatives).

Konsolidator is a knowledge-driven algorithm that generates useful supporting evidence to help perform expert review. It generates computer predictions of supporting evidence and regulatory submissions required for expert review. It takes test results from multiple statistical and expert rule models and re-evaluates alerts by running queries using a large chemical database. Konsolidator has been introduced with CASE Ultra version 1.6.0.0 and currently supports only a bacterial mutagenicity model.

Model Applier

FDA Model applicator (LSMA) is a statistical-based system that uses a (Q) SAR Model to provide quantitative predictive probabilities for potential toxicity of chemicals. All Model applicator (Q) SARs were constructed at the FDA by Information and Computational Safety Analysts (ICSAS) Staff. A complete document of evidence weight methods and data sources for a training set of preparatory models has been published by the ICSAS group (Matthews et al, 2008).

LSMA assessment was performed using domain analysis using the ICH M7 setting. The arrangement of the present application designates a prediction probability below 0.4 as negative and a probability greater than or equal to 0.6 as positive. If the prediction indicates "uncertain", the probabilities fall between those cutoffs. For analytical purposes in this report, predictive probability scores of 0.61-0.79 or greater were considered moderately positive, and 0.80-1.0 or greater were considered strongly positive.

The Bacterial Mut model was newly developed in the 2018 software. It is configured to integrate the data of the SAR Genetox database, the Bacterial Mutation alert reference set, and the existing FDA RCA model (Salmonella Mut and E Coli-TA 102A-T Mut) to improve performance overall. The training set contained 9109 training compounds (4710 positives/3752 negatives). When the model (version 1.0) is tested against itself and cross-validated, its performance metrics are as follows:

practice of		Interactive verification (5% LMO)
			83.4	Accuracy of	84.6
82.9	Sensitivity of the probe	84.1
			84.3	Specificity of	85.3
86.6	Positive prediction	87.8
			79.9	Negative prediction	81.0

For all structures, the Model applicator assay was performed specifically using mutagenic endpoints.

EPA T.E.S.T.

The EPA toxicity assessment software tool (TEST) was developed by the Environmental Protection Agency (Environmental Protection Agency) to allow users to easily estimate toxicity using various QSAR methods. TEST provides a variety of prediction methods (hierarchical, FDA, single model, group contribution, nearest neighbor, consistency, and random forest) so that there can be greater confidence in the predicted toxicity. Several endpoints are available for evaluation; however, only Ames mutagenic endpoints were used in this evaluation. The predicted toxicity from the consensus method represents the average of the predicted toxicity from all the different QSAR methods incorporated into the TEST software. The consensus method achieves the best prediction accuracy (consistency) and prediction coverage for Ames assay, and this is the method used for current evaluations. In TEST, a prediction value ≧ 0.50 is considered positive. For analytical purposes, predictive probability scores of ≧ 0.61-0.79 are considered moderate positives, and ≧ 0.80-1.0 are considered strong positives.

The molecular descriptors (physical characteristics of the structure) are computed using computer code written in Java. The basis for molecular computing is the Chemistry Development suite (Steinbeck et al, 2003). The descriptor values were verified using MDL QSAR, Dragon, and Molconn-z. Descriptor values generally fit better (except for minor differences defined for the descriptor of the descriptor, such as the number of hydrogen bond acceptors). The final dataset consisted of 5743 chemicals, which were based on the dataset compiled by Hansen et al (2009).

Rofecoxib (TRM-201)

According to NDA21,042(1999) andthe drug label (rofecoxib tablet and oral suspension) (2016):

rofecoxib (L-748,731) was tested in a series of genotoxicity assays and was not found to be mutagenic or chromosomally disrupted. When tested up to 6000 μ g/plate, L-748,731 was negative for mutagenicity in Salmonella typhimurium (TA98, TA100, TA1535, TA97a) and escherichia coli (e.coli) (WP2, WP2uvrA, WP2uvrA pKM101) in the presence and absence of an exogenous metabolic activation system (S9). L-748,731 was negative for mutations in Chinese hamster lung cells in an in vitro assay at all concentrations tested with and without S-9. In chromosome aberration studies using CHO cells, the percentage of abnormal cells did not increase significantly at 25 to 125 μ M with S-9 or 25 to 100 μ M without S-9 activation.

Rofecoxib is also not carcinogenic in mice or rats with dose levels up to 60mg/kg and 8mg/kg, respectively.

Structure of evaluation

Results

1.A-RSM1-00

In rule-based DEREK, A-RSM1-00 is predicted to be "mutagenic" inactive (negative) against "in vitro bacteria (Salmonella typhimurium and E.coli) and to have no misclassified or unclassified characteristics.

In the statistics-based leader Model applicator, A-RSM1-00 predicted negative in the Bacterial Mut Model with a probability score of 0.399. Most of the main features of the structure are covered in this model, and several analogues are shown to support prediction.

2.A-RSM1-01

In rule-based DEREK, A-RSM1-01 is predicted to be "mutagenic" inactive (negative) against "in vitro bacteria (Salmonella typhimurium and E.coli) and to have no misclassified or unclassified characteristics.

In the statistics-based leader Model applicator, the A-RSM1-01 was not predicted because the predicted value is 0.472, which is in a gray area that cannot be reliably predicted by software. However, non-mutagenic 1, 2-bis (phenylthio) ethane (LS-1491) was shown to be an analogue in the training set with 79% similarity. The A-RSM1-01 was also evaluated using Case Ultra due to uncertainty prediction in the Model Appler. The prediction is negative in a GT1_ BMut model, and the calculation probability is 22.9%; no alert is identified.

3.A-RSM1-02

In rule-based DEREK, A-RSM1-02 is predicted to be "mutagenic" inactive (negative) against "in vitro bacteria (Salmonella typhimurium and E.coli) and to have no misclassified or unclassified characteristics.

In the statistics-based Leadscope Model Appler, the A-RSM1-02 was not predicted because the predicted value is 0.4, which is in a gray area that cannot be reliably predicted by software. The A-RSM1-02 was also evaluated using Case Ultra due to uncertainty prediction in the Model Applier. It is predicted uncertain in the GT1_ BMut model with a calculated probability of 48.3%, which is in the 40-60% gray region. A positive alert was identified, but the software excluded it because the analogs were negative and all positive analogs contained reactive groups not present in a-RSM 1-02. The Konsolidator total result call was negative because all the identified alerts/features were found to be unrelated to mutagenic activity.

For further confidence in the statistics-based prediction, TEST was used to analyze a-RSM1-02 and predict that its mutagenicity was negative with a predictive value of 0.09. The similarity coefficient for the analogues in the outer and training sets was as high as 0.80, and related analogues were shown to support predictions, including non-mutagenic CASRN123-09-1 (p-chloroanisole) (Leber et al, 1993).

4.A-RSM2-00

In rule-based DEREK, A-RSM2-00 is predicted to be "mutagenic" inactive (negative) against "in vitro bacteria (Salmonella typhimurium and E.coli) and to have no misclassified or unclassified characteristics.

In the statistics-based leader Model applicator, A-RSM2-00 predicted negative in the Bacterial Mut Model with a probability score of 0.072. The main features of the structure were covered in this model and relevant analogs were shown to support prediction, including LS-7536(α -naphthaleneacetic acid) with 72% similarity.

5.A-RSM2-01

In rule-based DEREK, A-RSM2-01 is predicted to be "mutagenic" inactive (negative) against "in vitro bacteria (Salmonella typhimurium and E.coli) and to have no misclassified or unclassified characteristics.

In the statistics-based leader Model applicator, A-RSM2-01 predicted negative in the Bacterial Mut Model with a probability score of 0.145. The major features of the structure are covered in this model and a-RSM2-01 matches exactly LS-817 (phenylacetonitrile) in the Leadscope database, which is test negative for mutagenicity.

6.A-RSM2-02

In rule-based DEREK, A-RSM2-02 is predicted to be "mutagenic" inactive (negative) against "in vitro bacteria (Salmonella typhimurium and E.coli) and to have no misclassified or unclassified characteristics.

In the statistics-based leader Model applicator, A-RSM2-02 was predicted to be negative in the Bacterial Mut Model with a probability score of 0.170. The main features of the structure are covered in this model, and several analogues are shown to support prediction.

7.A-CRM1-00

In rule-based DEREK, A-CRM1-00 was predicted to be "mutagenic" inactive (negative) against in vitro bacteria (Salmonella typhimurium and E.coli) and to have no misclassified or unclassified characteristics. Br^[III]Salt features are considered unclassified features. Unclassified features are those not present in the Lhasa Ames test reference set and do not match any structural warning or example of (in vitro bacterial) mutagenicity in Derek. It was predicted to be inactive in an in vitro bacterial (Ames) mutagenicity test.

In the statistics-based leader Model applicator, A-CRM1-00 predicted negative in the Bacterial Mut Model with a probability score of 0.055. The major features of the structure were covered in this model and relevant analogs were shown to support prediction, including non-mutagenic LS-190530 (tetrapropylammonium bromide; CASRN 1941-30-6).

8.A-STG1-00

In rule-based DEREK, A-STG1-00 is predicted to be "mutagenic" inactive (negative) against "in vitro bacteria (Salmonella typhimurium and E.coli) and to have no misclassified or unclassified characteristics. Although containing alkyl bromides, 027 in DEREK alerts that several exclusion criteria are contained, including exclusion criteria for primary alpha-halo ketones. Studies have shown that some phenacyl bromides can be oxidized by DMSO solvents to give mutagenized benzoylformaldehydes, which produce a positive response not observed when acetone is used as the solvent (Azuma et al, 1997).

In the statistics-based leader Model applicator, A-STG1-00 predicted positive in the Bacterial Mut Model with a probability score of 0.770. Positive predictions are mainly due to alkyl bromide character. While several analogs with alkyl halide characteristics were shown to support prediction, the analog LS-188087 (2-bromoacetophenone) with 62% similarity was shown and experimentally negative for mutagenicity, and the analog LS-394467 (2-bromo-1- [4- (methylsulfonyl) phenyl ] -1-ethanone) with 54% similarity was experimentally positive for mutagenicity.

For further confidence in the statistics-based prediction, TEST was used to analyze a-STG1-00 and its mutagenicity was predicted to be negative with a predictive value of 0.40. The similarity coefficient for the analogs in the outer and training sets was as high as 0.66, and the relevant alkyl halide analogs were shown to support prediction.

9.A-STG1-01

In rule-based DEREK, it is reasonable to predict the "mutagenicity of A-STG1-01 against" in vitro bacteria (Salmonella typhimurium and E.coli) "in DEREK due to the match alert 326 (alert pharmacophore highlighted in grey below) for gem-dihalide. Such compounds show mutagenicity in the Ames test both in the presence and in the absence of metabolic activation, and have generally shown activity of terminal gem-dihalides, gem-dibromides and gem-mixed dihalides which are alpha-unsaturated carbon atoms, e.g.alpha, alpha-dichlorotoluene (Zeiger et al, 1992).

In the statistics-based leader Model applicator, A-STG1-01 predicted positive in the Bacterial Mut Model with a probability score of 0.870. Positive predictions are mainly due to alkyl dibromide characteristics, and several analogues with alkyl bromide characteristics are shown to support the predictions. Although not a dihalide, 2-bromoacetophenone (LS-188087) was shown to be an analog and was experimentally negative for mutagenicity.

The mechanism by which gem-dihalides exert their mutagenic effect is not known, but may involve direct reaction of these compounds with DNA, since they are inherently electrophilic species. The reactivity of a given gem-dihalide will depend on many factors, including the steric and electronic environment surrounding the reaction center and the nature of the halogen atom forming the functional group, with dichloro compounds being less reactive than dibromo compounds.

10.A-STG2-00

In rule-based DEREK, A-STG2-00 is predicted to be "mutagenic" inactive (negative) against "in vitro bacteria (Salmonella typhimurium and E.coli) and to have no misclassified or unclassified characteristics.

In the statistics-based leader Model applicator, A-STG2-00 predicted negative in the Bacterial Mut Model with a probability score of 0.235. The main features of the structure are covered in this model and relevant analogues are shown to support prediction.

11.A-STG2-01

In rule-based DEREK, it is plausible to predict the "mutagenicity of bacteria in vitro (salmonella typhimurium and escherichia coli)" in DEREK of a-STG2-01 due to the matching alert 027 (alert pharmacophore highlighted in grey below) of the alkylating agent. 027 Warning encompasses alkylating agents in which the carbon bearing the functional group is a primary or secondary alkyl carbon atom, and which include alkylsulfenylene esters, sulfonates, and sulfates that lack a hydroxyl group directly bonded to sulfur.

In the statistics-based leader Model applicator, A-STG2-01 predicted positive in the Bacterial Mut Model with a probability score of 0.739. Positive predictions are mainly due to alkyl bromide characteristics, and several analogues with alkyl bromide characteristics are shown to support the predictions.

Alkyl halides are electrophilic species that are capable of directly alkylating DNA. Shorter chain alkyl chlorides (e.g., methyl chloride) are known to be mutagenic (Andrews et al, 1976).

12.A-STG3-00

In rule-based DEREK, A-STG3-00 is predicted to be "mutagenic" inactive (negative) against "in vitro bacteria (Salmonella typhimurium and E.coli) and to have no misclassified or unclassified characteristics.

In the statistics-based Leadscope Model applicator, the A-STG3-00 was not predicted because the predicted value was 0.488, which is in a gray area that cannot be reliably predicted by software. However, rofecoxib is shown as an analog in the training set with 59% similarity.

Due to uncertainty prediction in the Model Applier, A-STG3-00 was also evaluated using Case Ultra. The prediction in the GT1_ BMut model is negative, and the calculation probability is 17.8%; 2 alerts and 1 inactive feature were identified. The software eliminated all warnings, as most of the analogs were negative, and the positive analogs contained reactive groups not present in a-STG 3-00. Rofecoxib is also shown as an analog in the database, with 90.4% similarity.

13.A-STG3-01

In rule-based DEREK, A-STG3-01 was predicted to be "mutagenic" inactive (negative) against "in vitro bacteria (Salmonella typhimurium and E.coli) and to have no misclassified or unclassified characteristics.

In the statistics-based Leadscope Model applicator, the A-STG3-01 was not predicted because the predicted value was 0.486 in the gray region. However, rofecoxib is shown as an analog in the training set, with 43% similarity.

A-STG3-01 was also evaluated using Case Ultra due to uncertainty prediction in the Model Applier. The prediction of the model in a GT1_ BMut model is uncertainty, and the calculation probability is 42.4%; 2 alerts are identified. The software eliminated all warnings, as most analogs were negative, and most positive analogs contained reactive groups not present in a-STG 3-00. Rofecoxib is also shown in the database as an analog, with 50% similarity. The result proposed by Konsolidator is a negative call because the alert/feature was found to be independent of mutagenic activity.

14.A-STG4-01

In rule-based DEREK, A-STG4-01 was predicted to be "mutagenic" inactive (negative) against "in vitro bacteria (Salmonella typhimurium and E.coli) and to have no misclassified or unclassified characteristics.

In the statistics-based leader Model applicator, A-STG4-01 predicted negative in the Bacterial Mut Model with a probability score of 0.353. Most of the major features of the structure are covered in this model, and relevant analogs (including non-mutagenic rofecoxib) are shown to support prediction.

15.A-STG4-02

In rule-based DEREK, A-STG4-02 was predicted to be "mutagenic" inactive (negative) against "in vitro bacteria (Salmonella typhimurium and E.coli) and to have no misclassified or unclassified characteristics.

In the statistics-based leader Model applicator, A-STG4-02 was predicted to be negative in the Bacterial Mut Model with a probability score of 0.162. The major features of the structure were covered in this model, and relevant analogs (including non-mutagenic rofecoxib) were shown to support prediction.

16.A-STG4-03

In rule-based DEREK, A-STG4-03 is predicted to be "mutagenic" inactive (negative) against "in vitro bacteria (Salmonella typhimurium and E.coli) and to have no misclassified or unclassified characteristics.

In the statistics-based leader Model applicator, A-STG4-01 predicted negative in the Bacterial Mut Model with a probability score of 0.182. The major features of the structure were covered in this model, and relevant analogs (including non-mutagenic rofecoxib) were shown to support prediction.

The overall computational assessment of toxicity focuses on the potential for DNA reactive mutagenicity. A summary of the computer results and overall mutagenicity predictions is set forth below in fig. 23.

Conclusion

The potential mutagenicity of the TRM-201 impurity was evaluated in silico in at least 2 complementary software programs according to the ICH M7 standard, and the data were then subjected to expert review. The model results alone and the overall mutagenicity prediction are shown in figure 23.

In both the rule-based DEREK and the statistic-based Model appliers, A-RSM1-00, A-CRM1-00, A-RSM2-00, A-RSM2-02, and A-STG2-00 were predicted to be significantly negative. No alerts are identified and relevant analogs are shown to support the prediction. Thus, they can be considered non-mutagenic (class 5) according to ICH M7.

A-RSM1-01 was predicted to be significantly negative in rule-based DEREK and in statistics-based Case Ultra. No alerts are identified and relevant analogs are shown to support the prediction. Thus, it can be considered non-mutagenic (class 5) according to ICH M7.

In both rule-based DEREK and statistical-based methods, the mutagenicity of A-RSM1-02 was predicted to be negative, which was considered uncertain in both Model Applier and Case Ultra, but was predicted to be negative in TEST. Related analogs are shown to support prediction. Negative prediction and lack of structural warning indicate that A-RSM1-02 is non-mutagenic. Therefore, it is considered as a type 5 impurity.

A-RSM2-01 was predicted to be significantly negative in rule-based DEREK and in a statistical-based Model applicator. It was matched exactly with negative mutagenic data in the Leadscope database. Therefore, it is considered as a type 5 impurity.

A-STG1-00 was predicted to be negative in rule-based DEREK and positive in a statistical-based Model applicator due to the warning of alkyl bromide characteristics. Since phenacyl bromide was excluded, the alkyl halide character was not alerted in DEREK, phenacyl bromide may be oxidized by DMSO used in the Ames assay, leading to false positive results. In the case of a-STG1-00, sulfur will attract electrons due to its ability to possess some double bond character, supporting the reduced reactivity of the structure. A-STG1-00 was also predicted to be negative in the statistics-based TEST. Thus, evidence suggests that A-STG1-00 is non-mutagenic (class 4).

A-STG1-01 was predicted to be positive in rule-based DEREK and statistical-based Model Applier due to the warning of alkyl dibromide characteristics. Although the mechanism by which gem-dihalides exert their mutagenic effect is not known, it may involve direct interaction with DNA due to the intrinsic electrophilicity of these species. The actual reactivity of any given gem-dihalide depends on many factors and, considering that a-STG1-01 does not offer much steric hindrance and that it contains a dibromide character, which is generally more reactive than the dichloro character, a-STG1-01 was considered potentially mutagenic until tested in the Ames assay (category 3).

A-STG2-01 was predicted to be positive in rule-based DEREK and statistical-based Model Applier due to the warning of alkyl bromide characteristics. Alkyl halides are electrophilic species capable of directly alkylating DNA and have a positive prediction in two complementary computer systems, a-STG2-01 is considered potentially mutagenic (class 3).

In both rule-based DEREK and statistics-based Case Ultra, the A-STG3-00 was predicted to be negative for mutagenicity. Although no prediction was made in the Model applicator, the non-mutagenic rofecoxib showed close analogs. A-STG3-00 was predicted to be negative in two complementary computer systems and was therefore considered non-mutagenic (class 5).

A-STG3-01 differs from A-STG3-00 (above) only in the presence of a hydroxyl group on the furan ring and is therefore predicted to be similar. In both the rule-based DEREK and the statistical-based methods, the mutagenicity of A-STG3-01 was predicted to be negative, which was considered uncertain in both Model Applier and Case Ultra. Although no prediction was made in the statistical model, non-mutagenic rofecoxib showed close analogs. Negative prediction and similarity to the non-mutagenic parent compound indicate that A-STG3-01 is non-mutagenic. Therefore, it is considered as a type 5 impurity.

In both the rule-based DEREK and the statistic-based Model applicator, A-STG4-01, A-STG4-02 and A-STG4-03 were predicted to be significantly negative. No alert was identified and relevant analogs (including rofecoxib) were shown to support the prediction. These structures are very similar to the non-mutagenic parent compound, rofecoxib. Thus, they can be considered non-mutagenic (class 5) according to ICH M7.

Reference to example 9

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Case Ultra, 1.7.0.5 edition, Multicase, Inc (Beachwood, OH).

DEREK Nexus, 6.0.1 th edition (2018), Nexus v.2.2.1, Lhasa Limited (Leeds, UK).

Dobo K, Greene N, Fred C et al (2012), In silica methods combined with extra knowledge of pharmaceutical activities, An industry surveiy, Regul Toxicol Pharmcol, 62(3) 449-55.

EMEA/CHMP/QWP/251344/2006(2006).Guideline on the Limits of Genotoxic Impurities.

EPA t.e.s.t., version 4.2.1 (2016.) a Program to Estimate sensitivity from Molecular structure.u.s.epa.

Hansen K, Mika S, Schroeter T et al (2009), Benchmark Data Set for in silicon Prediction of the amines music, journal of Chemical Information and Modeling,49(9): 2077-.

ICH M7 Guideline (2015), Association and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit patent scientific Risk.2015, 5 months.

ICH M7(R1) Guideline (2018), Association and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit positional Carcinogenic Risk.2018, 3 months.

Leadscope(2018).Model Applier v2.4.1-36,Enterprise v3.7.1-36；Personal v4.7.1-36.Inc.(Columbus,OH).

Leber A, Dacre J, Thake D et al (1993), p-chlorophenylmethyl sulfate, and p-chlorophenylmethyl sulfate-acid reactivity and bacterial mutagenity sulfates J.am.col.reactivity, 12(4): 369-.

Matthews E,Kruhlak N,Benz D,Contrera J.(2008).Combined Use of MC4PC,MDL-QSAR,BioEpisteme,Leadscope PDM,and Derek for Windows Software to Achieve High-Performance,High-Confidence,Mode of Action–Based Predictions of Chemical Carcinogenesis in Rodents.Toxicology Mechanisms and Methods,18:189-206.

Muller L, Mauthe R, Riley C et al (2006), aromatic for determining, testing, and controlling 724 specific electronic activities in pharmaceutical that stress for genetic oxidizing pharmacy.44: 198. beta. 211.

NDA 21,042(1999).VIOXX(rofecoxib).Merck&Co.

Steinbeck C, Han Y, Kuhn S et al (2003), The Chemistry Development Kit (CDK), An Open-Source Java Library for Chemo-and bioinformatics. journal of Chemical Information and Computer Sciences,43:493 500.

SutterA, Amberg A, Boyer S, Brigo A et al (2013), Use of in silicon systems and expert knowledge for structural-based assessment of connectivity multigenic inputs, Reg.

US Food and Drug administration.vioxx (Rofecoxib) u.s.prediscription Information,2016, 5 months and 09 days.

Zeiger E, Anderson B, Haworth S, Lawlor T and Mortelmans K (1992). Salmonella mutagenetics tests V.results from the testing of 311chemicals, Environmental and Molecular Mutagenesis,19(supplement 21),2-141.

Equivalents of

The subject matter described herein can be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are, therefore, to be considered in all respects illustrative rather than limiting of the subject matter described herein. The scope of the subject matter described herein is, therefore, indicated by the appended claims rather than by the foregoing detailed description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

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