阅读说明：本技术 控制猫糖尿病的化合物 (Compounds for the control of feline diabetes ) 是由 M·哈德 A·柯林森 B·锡德 于 2020-03-13 设计创作，主要内容包括：本文提供了控制猫糖尿病的方法；所述方法包括向有需要的猫每天施用总剂量约为5-50mg的化合物1,或其药学上可接受的形式,其结构式如下：本文还提供了控制猫糖尿病的方法,包括向有需要的猫施用有效量的SGLT抑制剂,其中所述有效量不超过导致健康猫腹泻或稀便频率升高所需剂量的10％至30％。(Provided herein are methods of controlling diabetes in cats; the method comprises administering to a cat in need thereof a total daily dose of about 5mg to about 50mg of compound 1, or a pharmaceutically acceptable form thereof, having the formula: also provided herein are methods of controlling diabetes in cats comprising administering to a cat in need thereof an effective amount of an SGLT inhibitor, wherein the effective amount is no more than 10% to 30% of the dose required to cause an increase in the frequency of diarrhea or loose stools in healthy cats.)

1. A method for managing diabetes in cats, comprising administering to a cat in need thereof a total daily dose of about 5mg to about 50mg of compound 1, or a pharmaceutically acceptable form thereof, having the formula:

2. a method for reducing hyperglycemia-related clinical signs in a cat having diabetes, comprising administering to a cat in need thereof a total daily dose of about 5mg to about 50mg of compound 1, or a pharmaceutically acceptable form thereof.

3. A method for ameliorating hyperglycemia-related clinical signs in a cat having diabetes, comprising administering to a cat in need thereof a total daily dose of about 5mg to about 50mg of compound 1, or a pharmaceutically acceptable form thereof.

4. A method for inducing clinical remission of diabetes in a cat, which comprises administering to a cat in need thereof a total daily dose of about 5 to 50mg of compound 1 or a pharmaceutically acceptable form thereof.

5. A method of reducing the serum fructosamine level in diabetic cats to below the upper normal limit of the test laboratory reference range, said method comprising administering to a cat in need thereof a total daily dose of about 5-50mg of compound 1, or a pharmaceutically acceptable form thereof.

6. A method for improving glycemic control in diabetic cats having a blood glucose profile in which all blood glucose measurements fell to a peak of 10mmol/L (180mg dL)^-1) And a minimum value of 4.5mmol/L (80mg dL)^-1) In a mammal in need thereof, comprising administering to the cat a total daily dose of about 5 to about 50mg of compound 1 or a pharmaceutically acceptable form thereof.

7. A method for preventing weight loss in a diabetic cat, comprising administering to a cat in need thereof a total daily dose of about 5-50mg of compound 1 or a pharmaceutically acceptable form thereof.

8. A method of managing feline diabetes mellitus exhibiting an IGF-1 concentration greater than the upper normal limit of a test laboratory reference range comprising administering to a cat in need thereof a total daily dose of about 5-50mg of compound 1 or a pharmaceutically acceptable form thereof.

9. The method of claim 8, wherein the upper normal limit of the test laboratory reference range for IGF-1 is 92 nmol/L.

10. The method of any one of claims 1-9, comprising administering to a cat in need thereof a low carbohydrate diet and a total daily dose comprising about 5-50mg of compound 1.

11. The method of claim 10, wherein the low carbohydrate diet is a canned diet.

12. The method of claim 10, wherein the low carbohydrate diet is a diabetic diet.

13. The method of claim 10, wherein the low carbohydrate diet is a ketogenic diet.

14. The method of claim 10, wherein the low carbohydrate diet is a grain-free diet.

15. The method of claim 10, wherein the low carbohydrate diet contains less than 40% calories in the form of carbohydrates.

16. The method of claim 10, wherein the low carbohydrate diet contains less than 35% calories in the form of carbohydrates.

17. The method of claim 10, wherein the low carbohydrate diet contains less than 26% calories in the form of carbohydrates.

18. The method of claim 10, wherein the low carbohydrate diet contains less than 12% calories in the form of carbohydrates.

19. A method for managing diabetes in cats, comprising administering to a cat in need thereof a total daily dose of compound 1 from about 5mg to about 50mg, or a pharmaceutically acceptable form thereof,

wherein, the compound 1 or the pharmaceutically acceptable form thereof is a tablet, a capsule or a solid dosage form.

20. A method for managing diabetes in cats, comprising administering to a cat in need thereof a total daily dose of about 5-50mg of Compound 1 or a pharmaceutically acceptable form thereof,

wherein, the compound 1 or the pharmaceutically acceptable form thereof is in the form of an oral solution.

21. A method for managing diabetes in cats, comprising administering to a cat in need thereof a total daily dose of about 5-50mg of Compound 1 or a pharmaceutically acceptable form thereof,

wherein the compound 1 or a pharmaceutically acceptable form thereof is in a drug-fed form.

22. The method of any one of claims 1-21, wherein compound 1 is the bisproline complex of (2S, 3R, 4R, 5S, 6R) -2- (4-chloro-3- (4- (2-cyclopropoxyethoxy) benzyl) phenyl) -6- (hydroxymethyl) tetrahydro-2H-pyran-3, 4, 5-triol having the structure shown below

23. The method of any one of claims 1-21, wherein compound 1 is a crystalline form of (2S, 3R, 4R, 5S, 6R) -2- (4-chloro-3- (4- (2-cyclopropoxyethoxy) benzyl) phenyl) -6- (hydroxymethyl) tetrahydro-2H-pyran-3, 4, 5-triol having the structure shown below

24. The method of any one of claims 1 to 23, wherein the total daily dose is from about 10 mg to about 20 mg.

25. The method of any one of claims 1 to 23, wherein the total daily dose is about 15 mg.

26. The method of any one of claims 1-25, wherein compound 1 is administered orally.

27. The method of any one of claims 1-26, wherein compound 1 is administered once daily.

28. The method of any one of claims 1-27, wherein compound 1 is administered in a control regimen that lasts at least 1 day.

29. The method of any one of claims 1-27, wherein compound 1 is administered in a control regimen that lasts at least 3 days.

30. The method of any one of claims 1-27, wherein compound 1 is administered in a control regimen that lasts at least 7 days.

31. The method of any one of claims 1-27, wherein compound 1 is administered in a control regimen that lasts at least 14 days.

32. The method of any one of claims 1-27, wherein compound 1 is administered in a control regimen that lasts at least 28 days.

33. The method of any one of claims 1-27, wherein compound 1 is administered in a control regimen that lasts from 1 day to 2 months.

34. The method of any one of claims 1-33, wherein compound 1 is the only antidiabetic agent administered to the cat.

35. The method of any one of claims 1-34, wherein administration of compound 1 results in clinical remission in the cat.

36. The method of any one of claims 1-35, wherein compound 1 is administered to the cat having a serum fructosamine level greater than the upper normal limit of a test laboratory reference range prior to the commencement of control.

37. The method of claim 36, wherein the upper limit of the test laboratory reference is about 356 μmol L^-1Or 275. mu. mol L^-1。

38. The method of any one of claims 1 to 35, wherein the fructosamine level is 450 μmol L to serum before control is initiated^-1Or higher in the cat administering compound 1.

39. The method of any one of claims 1-35, wherein the blood or serum glucose level is 170mg dL before control is initiated^-1Or higher in the cat administering compound 1.

40. The method of any one of claims 28-39, wherein the serum fructosamine level measured after completion of said control regimen is reduced in said cat.

41. The method of claim 40, wherein the serum fructosamine level of said cat is reduced by at least about 20% after completion of said control regimen.

42. The method of claim 40, wherein the serum fructosamine level of said cat is reduced by at least about 30% after completion of said control regimen.

43. The method of claim 40, wherein the serum fructosamine level of said cat is reduced by at least about 40% after completion of said control regimen.

44. The method of claim 40, wherein the serum fructosamine level of said cat is reduced by at least about 50% after completion of said control regimen.

45. The method of claim 40, wherein the serum fructosamine level of said cat is less than 450 μmol L after completion of said control regimen^-1。

46. The method of claim 40, wherein the serum fructosamine level of said cat is less than 400 μmol L after completion of said control regimen^-1。

47. The method of claim 40, wherein the serum fructosamine level of said cat is less than 350 μmol L after completion of said control regimen^-1。

48. The method of claim 40, wherein the serum fructosamine level of said cat is below the upper normal limit of the test laboratory reference range after completion of said control regimen.

49. The method of claim 48, wherein the upper limit of the test laboratory reference is about 356 μmol L^-1Or 275. mu. mol L^-1。

50. The method of any one of claims 30-49, wherein the blood or serum glucose level measured after completion of a control regimen is reduced in the cat.

51. The method of claim 50, wherein the blood or serum glucose level of the cat is reduced by at least about 20% after the control regimen is completed.

52. The method of claim 50, wherein the blood or serum glucose level of the cat is reduced by at least about 30% after completion of the management regimen.

53. The method of claim 50, wherein the blood or serum glucose level of the cat is reduced by at least about 40% after completion of the management regimen.

54. The method of claim 50, wherein the blood or serum glucose level of the cat is reduced by at least about 50% after completion of the management regimen.

55. The method of claim 50, wherein the cat has a blood or serum glucose level of less than 250mg dL after the control regimen is complete^-1。

56. The method of claim 50, wherein the cat has a blood or serum glucose level of less than 200mg dL after the control regimen is complete^-1。

57. The method of claim 50, wherein the cat has a blood or serum glucose level below 170mg dL after the control regimen is complete^-1。

58. The method of claim 50, wherein the controlling scheme is performedThereafter, the cat has a blood or serum glucose level below 150mg dL^-1。

59. The method of any one of claims 1-58, wherein the feline diabetes is type 1 diabetes.

60. The method of any one of claims 1-58, wherein the feline diabetes is type 1 diabetes.

61. The method of any one of claims 35 to 60, wherein the serum fructosamine level of cats in clinical remission is maintained at or below 450 μmol L^-1。

62. The method of any one of claims 35 to 60, wherein the serum fructosamine level of cats in clinical remission is maintained at or below 350 μmol L¹。

63. The method of any one of claims 35-62, wherein the cat remains in clinical remission for at least one month.

64. The method of any one of claims 35-62, wherein the cat remains in clinical remission for at least three months.

65. A method for managing diabetes in cats comprising administering to a cat in need thereof an effective amount of an SGLT inhibitor or a pharmaceutically acceptable form thereof,

wherein the effective amount is no more than 10% to 30% of the dose required to cause diarrhea or loose stool frequency in a healthy cat.

66. The method of claim 65, wherein the effective amount is no more than 30% of the dose required to exacerbate or dilute diarrhea in a healthy cat.

67. The method of claim 65, wherein the effective amount is no more than 20% of the dose required to exacerbate or dilute diarrhea in a healthy cat.

68. The method of claim 65, wherein the effective amount is no more than 10% of the dose required to exacerbate or dilute diarrhea in a healthy cat.

69. The method of any one of claims 65-69, wherein the healthy cat feeds on commercial ration.

70. The method of any one of claims 65-69, wherein the effective amount is a dose that produces about 90% of the maximal effect of the SGLT inhibitor.

Background

Diabetes mellitus is a disease with sustained high blood glucose (hyperglycemia) concentrations that adversely affect multiple organ systems and, in severe cases, can lead to death. Diabetes has been observed in companion animals such as cats, dogs and horses, as well as in humans. Like humans, diabetes in cats is an increasingly serious health problem and is associated with age and obesity.

In veterinary medicine, the goal of treatment takes into account the belief and needs of the animal owner or the person responsible for caring for the animal. Disease treatment strategies that increase the objective health of an animal but have an adverse effect on the owner, such as increasing the owner's anxiety about the animal's health, would be considered less than treatment strategies that have less objective efficacy but reduced owner anxiety. Thus, veterinary goals emphasize disease control, as opposed to simple disease treatment.

There are two main types of human diabetes: type 1 (T1DM), also known as Insulin Dependent Diabetes Mellitus (IDDM) and type 2 (T2DM), also known as insulin independent or non-insulin dependent diabetes mellitus (NIDDM).

Type 1 diabetes is due to the body's inability to produce insulin, most commonly due to the lack of endocrine cells, i.e., beta cells, which, in many organisms, cluster within the exocrine pancreas, called islets. Type 2 diabetes is an insulin resistant disease, insulin is produced by the beta cells of the islets of langerhans, but the tissues responsible for the absorption of glucose from the blood do not respond adequately to the insulin produced. During sustained T2DM, the beta cells have reduced ability to produce insulin and/or may yield to sustained high insulin production pressures. In this case, exogenous insulin may be required to maintain normal blood glucose levels (euglycemia or euglycemia). However, the risk of taking too much insulin is hypoglycemia, which can lead to coma and death.

Insulin lowers the concentration of glucose in the blood by stimulating the uptake and metabolism of glucose by the liver, muscle and adipose tissue. Insulin stimulates the storage of glucose in the form of glycogen in the liver and muscle and triglycerides in adipose tissue. Insulin also promotes glucose in the muscle for energy. Thus, insufficient levels of insulin in the blood, or reduced sensitivity to insulin, result in excessive levels of glucose in the blood. When the glucose concentration in the blood exceeds a certain critical level, the urine glucose renal threshold, glucose begins to appear in the urine. The presence of glucose in urine is often the first sign of the disease before reliable blood-based diabetes detection occurs.

Under normal conditions, the kidneys allow low molecular weight compounds in the blood, including glucose, to pass out of the body through the glomerular filtrate. The kidney selectively recovers from the filtrate almost all of the water, sodium, potassium and chloride ions, as well as important metabolites including vitamins, glucose and other sugars, and amino acids. Discarding almost all plasma components and then selectively re-absorbing only those relevant components is a costly process underlying the large unpredictable toxin output by the kidneys. The glucose reabsorption mechanism also provides a steady-state capability when blood glucose levels rise too high. When the glucose concentration in the blood exceeds the renal threshold for urine glucose, some of the excess glucose is excreted through the urine, somewhat alleviating the adverse consequences of elevated blood glucose.

With increasing degree of diabetes, an increase in urine output is observed, which is the result of the phenomenon of osmotic diuresis. Glucose in the filtrate prevents concentration of urine by reabsorption of water, resulting in increased water loss and hence dehydration. Dehydration can lead to thirst and increased water consumption. The inability to utilize glucose energy ultimately leads to weight loss despite increased appetite. Excessive drinking (polydipsia), excessive eating (polyphagia or polyphagia), and excessive urine (polyuria) are common symptoms of late-stage diabetes.

The toxic effects of excess blood glucose include non-enzymatic (spontaneous) glycosylation of cells and tissues. The glycosylation products accumulate in the tissue, eventually forming cross-linked proteins, known as advanced glycosylation endproducts. Although the detailed mechanisms are largely unclear, it is well known that diabetes increases the likelihood or severity of a variety of conditions and can lead to painful neuropathy, circulatory disturbances, gangrene, amputation, renal failure, blindness, myocardial infarction, and stroke.

Determination of the overall degree of glycemic control is an important part of a diabetes control program. The assessment of blood glucose status is facilitated by measuring serum glucose in an analytical laboratory or by measuring blood glucose using point-of-care devices such as a home-use blood glucose meter. Glucometers measure glucose in whole blood, while analytical laboratories typically measure glucose in serum, the liquid phase resulting from blood coagulation. Serum or blood glucose levels of individuals vary greatly throughout the day, and generally rise after meals and fall after long periods of fasting. Such daily fluctuations may reduce the utility of serum or blood samples for determining the degree of glycemic control produced by a given therapeutic intervention.

To better assess the degree of control of feline diabetes, multiple blood samples are typically taken during the day, usually as a hospitalization study, during which the cat is placed in a veterinary clinic. The resulting curve of blood glucose over time, referred to as a blood glucose curve, is typically used to confirm an initial diagnosis, or to assess the effectiveness of a control plan.

The housing of cats in veterinary clinics can put stress on the cat, and one of the consequences of stress is hyperglycemia. Thus, the clinically obtained blood glucose profile, while less disturbed by technical limitations affecting blood draw or measurement accuracy, may contain unreliable high values reflecting the effect of the major hormone cortisol released upon stress rather than the natural fluctuating course of daily blood glucose concentrations in cats. Therefore, clinical measurements always need to assess their likelihood of underestimating the degree of glycemic control.

The primary goal of feline diabetes control, as measured by the blood glucose curve criteria, is to maintain blood glucose at a peak of 10-14 mmol/L (180-; 252mg dL;, 252 mg) as measured by guidelines issued by the International Felidae medical Association (Sparkes et al, 2015; J. Cat. Med. and surgery 17:235)^-1) And lowest point 4.5-8.0 mmol/L (80-144 mg dL)^-1) In the meantime. The peak and nadir are assigned because the only approved diabetes medication is insulin, which if used in excess can lead to dangerous hypoglycemia. According to this guideline, the blood glucose curve of a cat with diabetes measures between 80-252mg dL^-1And then fully controlling the same.

Relevant guidelines issued by the American Hospital Association of animals (Rucinsky et al, 2010J Am Anim Hosp Assoc 46: 215) recommend home blood glucose curve testing with an average blood glucose below 250mg dL^-1Single blood glucose measurements did not exceed 300mg dL^-1The lowest point is 80-150mg dL^-1。

The plotting of blood glucose curves is both inconvenient and expensive. Alternatively, glycemic control substitutes that reflect long-term (e.g., weeks to months) average blood glucose concentrations, as well as glycemic control substitutes that can be measured in a single blood sample, can be employed.

In humans, non-enzymatically glycated hemoglobin provides a convenient method for determining long-term glycemic control. The N-terminal valine residue of hemoglobin A1 undergoes a spontaneous chemical reaction with reducing sugars, with glucose being the highest in blood. The first step is the formation of an enamine (schiff base) between the glucuronal tautomer and the N-terminal amino group. The second step is called the glucosamine rearrangement reaction (Amadori) rearrangement), leading to tautomerization to produce β -ketoamines, commonly referred to as Amadori adducts. Since the average life span of human erythrocytes is about 120 days, the degree of non-enzymatic glycosylation represents the average half-time glycosylation productAverage accumulation of (2). The content of the glycated hemoglobin is measured as hemoglobin A_1c(HbA_1c) The basis of the assay. HbA_1cBlood samples with a percentage below 6.5 are generally considered to reflect good or adequate glycemic control, while higher percentages are generally interpreted as indicating the presence of diabetes.

Feline erythrocytes have a shorter half-life than human erythrocytes and HbA_1cThe levels are much lower and the measurement is less accurate to the contrary, the preferred method of measuring the continuous glycemic state of cats is the serum fructosamine assay, which also measures the non-enzymatic adducts produced by the reaction of reducing sugars with primary amines. Mechanistically, these adducts are formed by the same reaction sequence as the N-terminal amino group of hemoglobin, but can be formed on the lysine epsilon-amino side chain, in the case of glucose, forming a structure known as fructosyl lysine. The fructosamine assay measures serum total ketoamines by reversing amadori rearrangement. Under basic conditions, amadori product was reduced to the original enamine and nitro blue tetrazolium to a colored formazan dye, which was spectrophotometrically measured at 540 nm. The fructosamine method measures total β -ketoamines, but the largest component is serum albumin, which is the most abundant protein in plasma. Albumin has a half-life of about 20 days, so fructosamine measurement effectively measures a three-week history of glycemic control.

According to the guidelines issued by the International Felidae medical Association (Sparkes et al 2015; "J. Cat. Med. surgery 17:235), serum fructosamine levels are below 350 μmol L^-1Indicating good glycemic control, insulin overdose, or diabetes remission; the serum fructosamine level is 350-^-1Indicating good glycemic control; the serum fructosamine level is 550 mu mol L at 450-^-1Indicating proper glycemic control; the serum fructosamine level is greater than 550 μmol L^-1Indicating poor glycemic control. These numerical ranges are predicted according to the laboratory test method which places an upper normal limit of about 350 μmol L for serum fructosamine^-1。

There are currently no approved oral hypoglycemic agents for treating feline diabetes. The standard of care for feline diabetes requires twice daily injections of insulin to achieve the desired effect after titration. Cats exhibit significant individual variation in insulin sensitivity and must be carefully observed to ensure that fatal or neurologically catastrophic hypoglycemia does not occur. While the use of insulin helps control diabetes and slow the progression of the disease, providing an appropriate insulin dose and time can be a challenge. For example, it is recommended to administer insulin before and after a meal, but continuous administration of insulin at the meal can be difficult to schedule and often results in lower compliance.

Accordingly, there is a need in the art for improved methods and compositions for reducing the hyperglycemia and hyperglycemia-related clinical symptoms of diabetes in cats, particularly for methods that do not require injections and do not require careful dose adjustments to maintain the health of the cat. The present invention addresses these needs and provides related advantages. In particular, the present invention provides methods and compositions for controlling diabetes in cats comprising compounds that inhibit the glucose transporters known as SGLT1 and SGLT 2.

Much of the known information about these proteins comes from studies in rodents and humans, and is not necessarily applicable to cats. Cats are obligate carnivores and typically consume very little carbohydrate. Cats lack the sweet taste receptors present in rodents and humans and cannot assume that the receptors and transporters responsible for carbohydrate perception and movement function in the same way as their homologues in rodents or humans. Thus, the following is a description of the general nature of carbohydrate transport, which may differ in matter from the description of transport for cats, as reflected by the present best understanding of subjects.

Because glucose does not spontaneously diffuse across the cell membrane, there are two classes of integral membrane proteins, known as transporters, in rodents and humans to facilitate the entry of glucose from the extracellular medium into the cell. One class is called "equilibrated" which does not favor internal or external, but rather allows glucose to move from a region of high concentration to a region of low concentration (toward equilibrium). Since the cells consume glucose, in most cases this results in a net flux into the cells. Another type is called "concentration", relying on the self-assembly of sodium ions from outside to inside the cellBut the gradient. This gradient is maintained by an energy demand (ATP depletion) mechanism that drives intracellular Na⁺Exchange for extracellular K⁺Thereby increasing extracellular Na⁺Concentration and intracellular K⁺And (4) concentration. Sodium-glucose linked transporters (SGLTs) transport one glucose and one Na across membranes in one action⁺Ions (in the case of SGLT2) or one glucose and two Na⁺Ion (in the case of SGLT 1). Na (Na)⁺The ions effectively carry the glucose into the cell. Thus, the concentrated transporter allows the diluted extracellular glucose to be concentrated inside the cell.

Most cells show only equilibrium transport. The intestines and kidneys rely on the intensive transport to take glucose from the diet or to recover glucose from the urine. In the species studied so far, SGLT1 is present in the intestine and kidney, while SGLT2 is present in the kidney, anatomically upstream of SGLT1 in the renal tubules. Na of glucose is obtained by SGLT2 under low-sodium condition⁺At a lower cost than SGLT1, about 90% of the glucose in the filtrate is reabsorbed under normal conditions. In the absence of SGLT2, SGLT1 is partially compensated, retaining 40-50% of glucose. The remainder was lost to the urine. Genetic defects in SGLT2 are known in both mice and humans and are generally benign, latent syndrome that can only be detected in humans by random urine testing. The SGLT1 gene deficiency is a potentially fatal disease in humans because severe diarrhea can only be controlled by a severely carbohydrate-restricted diet. Both SGLT1 and SGLT2 can react with Na⁺Co-delivering large amounts of water with glucose. SGLT1 can transport glucose and galactose in humans and rodents. It is currently unclear whether galactose is a substrate for feline SGLT 1.

The identification of phlorizin, a natural product isolated from apple tree bark, has prompted many early studies of the physiology of renal reuptake of glucose, and was finally found to be an inhibitor of SGLT1 and SGLT2 in various species.

Phlorizin (also known in the literature as phlorizin, phloridzin), phlorizin and phlorizin) was found to promote diabetes in the 19 th century. At that time, since diabetes was mainly characterized by the presence of glucose in urine, phlorizin was considered to be a causative factor of diabetes, and was referred to as "phlorizin diabetes" in the early literature. However, it was soon recognized that phlorizin-induced diabetes was different from pancreatic injury or resection-induced diabetes, and that E.H edon (Compt Red Soc Biol 4: 601897) reported the corrective effect of phlorizin on experimental diabetes in dogs prior to the era of the century. His opinions were translated from french as follows: "to my knowledge, another fact that has not been observed is the disappearance of hyperglycemia when phlorizin is administered to a completely diabetic pancreas-resected animal; then we see an inverse relationship between diabetes and glucose; while the former grows to a large extent (as seen with minkowski), the second falls all the way down before returning to normal. By the 20 th century, the action of phlorizin on the kidney was elucidated, and a comprehensive review of its action (Nash physiol. review 7: 3851927) was reported.

Lukens and colleagues reported in 1943 the use of phlorizin to reverse hyperglycemia in cats caused by experimental diabetes (Lukens et al, Endocrinology 32: 4751943), and published supplementary observations in 1961 (Lukens et al, diabetes 10: 1821961). The beneficial effects of phlorizin on feline diabetes include a reduction in the stress of insulin-producing islet cells in the pancreas and a significant reduction in hyperglycemia. Lukens et al have demonstrated many effects that are believed to be characteristic of the action of hypoglycemic agents on diabetic cats, for example, they have shown that phlorizin can prevent animals from developing diabetes, can restore normal glucose tolerance in diabetic animals, and can prevent langerhans islet failure in experimental diabetes (Lukens et al, 1943, 1961). One key factor in the study of Lukens and colleagues is the administration of phlorizin by subcutaneous injection of a suspension of olive oil containing the compound. This effectively acts as a slow release formulation, releasing the active agent over several days. Without this strategy, it is likely that no results will be available. Phlorizin is an O-glucoside, susceptible to β -glucosidase metabolism, with a short half-life in most species.

Recently, us 2015/0164856AL teaches the use of one or more SGLT2 inhibitors to treat feline diabetes. This publication teaches that SGLT2 inhibitors are versatile in use and can, for example, prevent loss of pancreatic β -cell mass or prevent β -cell degeneration, prevent or treat diabetes, and treat various diabetes-related diseases or symptoms. US 2015/0164856Al is distinguished from the very similar demonstrations and claims of Lukens and co-workers by emphasizing SGLT2 inhibition. Phlorizin, a mixed SGLT1/2 inhibitor, has an effect on both transporters in most species, whereas us 2015/0164856AL only teaches inhibition of SGLT 2. Indeed, us 2015/0164856AL does not mention SGLT 1. Notably, the selectivity of phlorizin for feline SGLT1 and SGLT2 is currently unknown, as is the relative contribution of these two transporters to feline renal glucose reuptake.

US 2015/0164856Al also does not disclose that predicting the effect of a compound in one species based on experience with another species is one of the most dangerous predictions in drug development, as is well known in the art. Due to evolutionary variation in the structure of the target protein, it is difficult to accurately predict cross-species effects. Furthermore, species-related off-target effects are well known in the art. The rate and type of exogenous metabolism is an important determinant of drug exposure and is known to exhibit large differences between different species. This may be due, in part, to the strong genetic selection for heterologous metabolic pathways, which enables some species to consume food sources that are toxic to other species.

Summary of The Invention

Provided herein are methods of controlling diabetes in cats; the method comprises administering to a cat in need thereof a total daily dose of about 5 to about 50mg of compound 1 (besiflozin), or a pharmaceutically acceptable form thereof, having the formula:

other objects, features and advantages of the present invention will be apparent to those skilled in the art from the following detailed description and the accompanying drawings.

Brief description of the drawings

FIG. 1 provides an X-ray powder diffraction (XRPD) spectrum of (2S, 3R, 4R, 5S, 6R) -2- (4-chloro-3- (4- (2-cyclopropoxyethoxy) benzyl) phenyl) -6- (hydroxymethyl) tetrahydro-2H-pyran-3, 4, 5-triol.

FIG. 2 provides a table of XRPD data for the XRPD spectrum of FIG. 1.

Figure 3 provides a raman spectrum of crystalline (2S, 3R, 4R, 5S, 6R) -2- (4-chloro-3- (4- (2-cyclopropoxyethoxy) benzyl) phenyl) -6- (hydroxymethyl) tetrahydro-2H-pyran-3, 4, 5-triol.

Fig. 4 provides a list of raman peaks of the raman spectrum of fig. 1.

Fig. 5 shows the urine glucose assay for healthy cats administered besagliflozin in a hospitalized setting.

Figure 6 shows weight loss over time in healthy cats following daily administration of high doses of besiflozin.

Figure 7 shows five clinical blood glucose curve measurements of model-adjusted least squares means, with a follow-up visit of 8 hours or more for each curve with a confidence interval of 95%. The mean blood glucose concentration observed clinically decreases with treatment time and is statistically of high significance.

Figure 8 shows the average of the various follow-up measurements making up the blood glucose curve as a function of sampling time. The blood glucose curves at each visit did not differ significantly after the initial measurement. Mean of 5 blood glucose determinations at week 8 was 114.9mg dL^-1(95% confidence interval 102.8128.4).

Figure 9 shows the mean values of fructosamine in the sera of the follow-up cats. The graph shows the model adjusted least squares means with 95% confidence intervals. The embedded value Δ represents the difference in least squares means from baseline to week 8, expressed as a percentage of the upper normal limit, and the corresponding 95% confidence interval. At week 8, the mean serum fructosamine value in the population was 86.7% of the upper normal limit (95% confidence interval 80.0%, 93.9%) with very significant changes in fructosamine concentration.

Figure 10 shows a model adjusted least squares mean of cat serum glucose concentrations at 95% confidence intervals. The inserted text gives the difference in least squares means from baseline to week 8, and the corresponding 95% confidence interval. At week 8, mean blood glucose was 144 (95% confidence interval 127, 163), and blood glucose concentration changes were highly significant.

Figure 11 shows the least squares mean cat body mass adjusted for the follow-up model with 95% confidence intervals. The inset gives the difference in least squares means from baseline to week 8, as a percentage of initial body weight, with a 95% confidence interval. Despite the wide confidence interval, since the population is heterogeneous in weight, the effect of treatment on weight gain in each cat was very significant.

Figure 12 shows the average of the owner's reduced score for polydipsia (overfeeding) during the visit. The data were not normally distributed, but rather were highly significant by non-parametric tests (indicating significant differences between values, but no specific pairs were identified).

Figure 13 shows the average of owner scores for reductions in polyuria (excessive urination) during the visit. The data were not normally distributed, but rather were highly significant by non-parametric tests (indicating significant differences between values, but no specific pairs were identified).

Figure 14 shows the average of the owner's score for reduced polyphagia (overfeeding) during the visit. The data were not normally distributed, but rather significant with a non-parametric test (indicating significant differences between values, but no specific pairs were identified).

Figure 15 shows the mean serum β -hydroxybutyrate concentration with 95% confidence interval following diagnosis. The breadth of the first visit reflects the large difference in value between cats participating in the study. At week 8, the mean serum β -hydroxybutyrate concentration was 1.76mg dL^-1(95% confidence interval 1.40, 2.20), lower than the upper limit of the laboratory's normal value (1.9mg dL)^-1) And the variation from the beginning of the study is highly significant.

Detailed Description

I. Overview

The surprising species-dependent potency and selectivity possessed by the compound besagliflozin originally developed for the treatment of type 2 diabetes in humans is disclosed.

Balaglitazone is a C-aryl glucoside, a highly selective inhibitor of human sodium-glucose junction transporter 2(SGLT2), and SGLT2 is an intact membrane protein expressed on apical plasma membranes of renal tubular epithelium of the proximal tubule segments S1 and S2. Under normal physiological conditions, it is responsible for re-uptake of most of the glucose in the filtrate. Besiflozin is 2400 times selective for human SGLT2 as human SGLT 1. Besiflozin produces significant saturated diabetes in mice, rats, cats, dogs, rabbits, monkeys, and humans. Experiments using a rodent genetic model of diabetes show that besiflozin can lower blood glucose levels, partially alleviating the disease, even in the presence of large amounts of pre-existing diabetes. Thus, the presence of diabetes does not preclude the use of besagliflozin to control diabetes.

In the present invention, besiflozin has been found to be 5 times as potent at cat SGLT2 as human SGLT2 and 235 times as potent at cat SGLT1 as human SGLT 1. As a dual inhibitor of cat SGLT1/2, besiflozin has both desirable and undesirable effects in its mechanism of action. Notably, in diabetic cats, it caused significant rapid relief from diabetes and hyperglycemia, while also exhibiting a tendency to induce loose stools and diarrhea at high doses, which are typical consequences of SGLT1 inhibition. Thus, in vivo observations confirm the low selectivity of in vitro predictions.

In humans, null or subtype mutations in the genes encoding SGLT1, SLC5a1 cause severe neonatal diarrhea, which is believed to reflect increased luminal water abundance and microbial overgrowth. However, partial inhibition of SGLT1 will reduce glucose (and, in at least some species, galactose) absorption, and thus may reduce the effect of dietary carbohydrates on blood glucose concentration, thereby achieving a desirable therapeutic effect. Since the site of action is the intestinal tract, rather than the kidney, the benefits of the dual SGLT1 and SGLT2 inhibitors should not decrease with the decline of renal function, and therefore diabetic cats with renal disease or renal impairment may benefit from the effects of besagliflozin. When the inhibitor acts simultaneously on SGLT1 and SGLT2, it can be described as an SGLT inhibitor.

Therefore, in order to benefit from the effects of the dual SGLT1 and SGLT2 inhibitors, a balance must be struck. If the SGLT inhibitor is too active on SGLT1, the intestinal effects will dominate compared to SGLT2, and the accompanying diarrhea, while not necessarily detrimental to hyperglycemia, will adversely affect the cat owner and his cognition on cat health. The adverse consequences of SGLT1 inhibition are not limited to diarrhea in humans and other species, but may include other intestinal effects such as flatulence, abdominal pain, and abdominal distension.

Therefore, it is advantageous that the activity to SGLT1 is lower than the activity to SGLT 2. The optimal ratio cannot be estimated from the limited data available, but one factor to remember is the local concentration of the compound. When administered by the oral route, the concentration of a drug in the intestinal lumen may be many times higher than in plasma. Thus, a relatively weak SGLT1 inhibitor may still have relatively potent in vivo effects. Although in vitro data is helpful in guiding the selection of appropriate mixed inhibitors of SGLT1 and SGLT2 to some extent, the best guidance is still an assessment of the actual efficacy of the cat. The dose threshold for an adverse intestinal reaction should be several times higher than the minimum dose that produces 90% of the maximum effect.

The frequency and severity of diarrhea may vary depending on the diet or weight of the cat, breed, medical history, or other specific factors. The threshold at which diarrhea or loose stools cause discomfort to the owner is also multiply affected. For purposes of this specification, elevated diarrhea frequency refers to any frequency of defecation that exceeds 10% in an unadministered cat. Thus, preferred dual inhibitors produce diarrhea or loose stools in healthy cats at a frequency of no less than three times, more preferably five times, with the minimum dose producing 90% of the maximum urinary glucose excretion for healthy cats on an over-the-counter normal diet (e.g., a dry diet).

Diarrhea caused by SGLT1 inhibition can be reduced by providing a low carbohydrate diet. As noted above, cats are obligate carnivores and consume little carbohydrate in a non-domestic environment. In contrast, the commercial diet of non-diabetic cats may provide up to 50% of the caloric value in the form of carbohydrates.

In some countries, a prescribed diet containing low carbohydrates may be used to control feline diabetes. These diets typically include wet (canned) foods, although there are also some dry-grain diets that have a low carbohydrate content. The recommendations of the ISFM panel indicate that, although the preferred carbohydrate content is not yet established, a diet with caloric value from carbohydrates of less than 12% may be suitable for diabetic cats (Sparkes et al, 2015; Felidae. Med. 17: 235). Several over-the-counter canned diets also had the lowest carbohydrate content.

The majority of carbohydrates in a diet of a dry food are derived from cereals or cereal derivatives, which are usually present in negligible proportions in the natural diet. Some cat food vendors offer low grain diets as a healthier alternative to traditional diets. Since these diets provide less metabolic energy in the form of carbohydrates, they can be a beneficial supplement to diabetes management programs.

Besiflozin has been studied in diabetic mice, rats and humans. In addition to the following information, this compound is now being studied in cats. In every organism, this compound has been found to lower blood glucose levels and improve long term glycemic control measures, such as HbA in rodents or humans_1cOr fructosamine from cats. However, for cats, the effect of besiflozin is exceptionally strong and qualitatively superior to that observed in other organisms. In a high proportion of diabetic cats, besagliflozin produced clinical remission of the disease, resulting in serum fructosamine concentrations falling within the reference range for healthy cats. Despite the high potency of besagliflozin in cats, no clinically significant signs of hypoglycemia have been observed to date. The combination of high potency and low risk makes besagliflozin the best choice for controlling diabetes in cats. Of particular interest and usefulness is that, in a field study in which the drug is administered to cats by the owner in an unsupervised (home) environment, besiflozin as a monotherapy has been shown to reduce the fructosamine levels in most catsAnd returning to the normal reference range of the test laboratory.

A surprising and important therapeutic benefit is the weight gain of the treated cats, which is quite different from the expected effect of the compound in healthy and diabetic animals. For example, weight loss is a common and consistent observation in diabetic patients taking besiflozin, and us 2015/0164856 teaches weight loss in cats taking SGLT2 inhibitors.

It will be apparent to those skilled in the art that the effectiveness of a drug will depend on a number of factors, including the severity and duration of the disease, the metabolic rate of the active ingredient or its active metabolites, and the regularity of administration. Errors in administration, especially omissions, can have a significant impact on the apparent utility of the drug. In practice, omissions are common and the degree of omission may be a determining factor in the effectiveness of a drug.

Co-morbidities may also affect the effectiveness of the drug. For example, because dual inhibitors of SGLT1 and SGLT2 block the reuptake of glucose by the kidney, they may lose efficacy as the renal filtration function declines, or as a natural consequence of aging, or as renal disease develops. The SGLT1 and 2 dual inhibitors of the invention are considered to be not attractive control options for cats with severe kidney disease, although they may be effective in cases of mild to moderate kidney damage, and they have been found to be effective in clinical studies in cats of different ages.

When humans initially develop T2DM, they are rarely in distress at the diabetic crisis. Instead, the disease progresses gradually, with diagnosis being a side result of regular examinations, or due to patient complaints such as thirst or frequent urination, indicating a more severe disease. However, cats with diabetes are often presented to veterinarians in acute illness, with very high blood glucose levels, urine glucose, and weight loss. Clinical symptoms associated with hyperglycemia characteristic of cats include polydipsia (excessive drinking), polyphagia (excessive eating), polyuria (excessive urination), and weight loss. Often weight loss and physical discomfort cause concerns to the owner. Surprisingly, despite malnutrition of the diabetic cats due to massive loss of glucose in the urine (via the renal mechanisms detailed above), paradoxically, administration of besiflozin to increase urine glucose secretion prevented weight loss and resulted in weight gain in many cats. In a similar manner, due to its mechanism of action, it is expected that the administration of besagliflozin to diabetic cats will exacerbate the hyperglycemic-related clinical symptoms of polydipsia, polyphagia, and polyuria, as these symptoms are all thought to be a mechanistic consequence of diabetes caused by the disease. However, contrary to this expectation, the administration of besiflozin to diabetic cats reduces the clinical symptoms of polydipsia, polyphagia, and polyuria, even when the cat is administered a drug that is expected to exacerbate these symptoms. Of course, due to the mechanism of action of the drug, they do continue to exhibit severe glucose urination. Thus, while besagliflozin may exacerbate the clinical symptoms associated with hyperglycemia by its mechanism, in clinical studies it reverses weight loss and allows the cat to resume normal or near normal behavior.

There is no clear explanation as to the mechanism by which besiflozin improves clinical symptoms associated with hyperglycemia in diabetic cats. However, by inhibiting SGLT1 and SGLT2, sufficient additional glucose may be excreted in the urine to cause a decrease in plasma glucose concentration. As blood glucose concentration decreases, the proportion of diabetes caused by drug action increases, contrary to disease, further improving glycemic control. Eventually, the rate of glucose excretion and excess glucose production are balanced and the clinical symptoms associated with hyperglycemia are improved.

Diabetes in cats may be associated with acromegaly, a disease caused by the hyperplasia of the pituitary growth hormone compartment, resulting in inappropriate secretion of growth hormone. The increase in growth hormone in turn causes an increase in insulin-like growth factor 1(IGF-1), which antagonizes the action of insulin. Insulin resistance in diabetic cats is often associated with acromegaly (Scott-Moncriff, JC, Vet Clin North Am Small Anim practice (2010)40:241), and it is estimated that up to one-fourth of diabetic cats in Europe may have potential acromegaly (Niessen PLoS one. 201510: e 0127794). Very high doses of insulin are often required to control diabetic cats with acromegaly. An effective insulin-independent antidiabetic agent would be an important supplement in the treatment of feline diabetes.

Described herein are methods and compositions for reducing hyperglycemia and hyperglycemia-related clinical symptoms of feline diabetes by administering compound 1 (besagliflozin). The methods described herein include specific amounts and frequencies of administration. Surprisingly, administration of compound 1 as the sole therapeutic agent provided a therapeutic benefit to diabetic cats that resulted in serum fructosamine levels in most animals that were below the upper normal limit of the reference range for the testing laboratory, i.e., within the normal range for healthy cats. Compound 1 is effective in treating diabetes in felines that are not receiving any other antidiabetic agent treatment. In contrast, when compound 1 was administered to diabetic rats and mice, there was no way to achieve blood glucose levels or HbA_1cReturning to the normal range. Similarly, when compound 1 is administered to a T2DM patient, in many cases, additional drugs need to be administered to bring blood glucose levels or HbA_1cReturning to the normal range.

Advantageously, compound 1 does not produce hypoglycemia. Thus, compound 1 was administered without careful control of the dose or the time of dosing.

Moreover, in certain instances, the methods of controlling diabetes in a cat described herein result in clinical remission of the cat.

Definition of

"Compound 1" refers to the compound (2S, 3R, 4R, 5S, 6R) -2- (4-chloro-3- (4- (2-cyclopropoxyethoxy) benzyl) phenyl) -6- (hydroxymethyl) tetrahydro-2H-pyran-3, 4, 5-triol, having the formula:

as used herein, "pharmaceutically acceptable form" refers to pharmaceutically acceptable salt, polymorphic, cocrystal, and single crystal forms of a given compound.

As used herein, "clinical remission" refers to a sustained reduction, alleviation, or attenuation of one or more clinical measures of a disease such that the indicator falls within an acceptable range of test values obtained from a healthy population. Such test values fall within the "normal range". "clinical remission" does not mean cessation of treatment. As used herein, "clinical remission" does not require that all clinical indicators of the disease fall within the normal range. For example, a cat with fructosamine in the normal range but fasting glucose above the upper limit of the normal range is said to be in clinical remission.

As used herein, the "normal range" depends on the equipment and procedures of the testing laboratory and thus may vary from laboratory to laboratory. As used herein, when a value falls within a "normal range," the phrase refers to a range that is determined by the particular laboratory that provided the result at the time of measurement.

As used herein, "no therapeutic relief" refers to a state of relief that persists after cessation of administration of a therapeutic agent.

As used herein, "hyperglycemia-associated clinical symptoms" refers to one or more characteristic markers in polydipsia, polyphagia, polyuria, or weight loss. Clinical signs of weight loss can be quantified by directly measuring the weight of the cat during the interrogation. Recording other indicia often requires the owner to observe the behavior of the cat or its results.

As used herein, the words "cat (cat)" and the word "cat (feline)" when used as adjectives are used interchangeably to refer to or relate to animals from the family Felidae, including particularly members of the family raised as pets or companion animals, typically belonging to the genus feline (Felis), species feline (silvestris catus) or species feline (catus), and are commonly referred to as domestic cats or domestic cats.

As used herein, the word cat, when used as a noun, refers to a feline.

As used herein, "controlling diabetes" or "management of diabetes" refers to the process by which an owner or other person responsible for the care of an animal treats a disease by specific measures aimed at alleviating or curing the disease or providing symptomatic relief or altering the perceived health of the animal by various means. These measures include changes in the diet of the animal, including providing a special or prescribed diet, or changing the type or amount of food provided, or encouraging or providing activities that increase physical or metabolic energy expenditure, or providing herbal preparations, dietary supplements or pharmaceuticals.

As used herein, "blood glucose," "blood glucose concentration," or "blood glucose level" refers to a measurement of glucose in whole blood. Typically, the sample drawn is capillary blood and glucose is measured by a point-of-care device such as a blood glucose meter.

As used herein, a "blood glucose curve" refers to the result of measuring the concentration of glucose in a sample of a whole blood series obtained over a period of time, typically 8 to 24 hours, as a means of assessing the degree of disease control and the appropriateness of control, e.g., determining whether the amount of therapeutic agent is too large, and adverse hypoglycemia is observed.

As used herein, "serum glucose," "serum glucose concentration," or "serum glucose level" refers to the glucose concentration measured in whole blood in the fluid phase (typically venous blood) that is allowed to clot. Serum glucose concentrations are typically determined in clinical practice by automated procedures conducted by diagnostic testing laboratories.

As used herein, "serum fructosamine", "serum fructosamine concentration" or "serum fructosamine level" refers to the fructosamine concentration measured in whole blood in the liquid phase (typically venous blood) which is allowed to clot. Serum fructosamine concentrations are typically determined in clinical practice by automated procedures conducted at diagnostic testing laboratories.

As used herein, "plasma glucose" or "plasma glucose concentration" refers to the glucose concentration obtained by measuring whole blood (typically a vein) in a liquid phase, which is blood that is separated from the cellular components of the blood in a non-clotting manner.

As used herein, the term "fasting," when used in the context of collecting specimens for testing, indicates that the animal from which the specimen is taken has been deprived of food for an extended period of time, typically 6 hours or more, and typically overnight if the specimen is taken in the morning. Fasting samples can be used to measure analytes that are more affected by feeding, such as glucose or lipids.

As used herein, "reduction in hyperglycemia-related clinical signs" and "improvement in related clinical signs" refer to improvement in one or more of polydipsia, polyphagia, polyuria, or prevention of weight loss from the onset of control.

As used herein, an improvement in polydipsia means that a decrease in the frequency or volume of water or liquid consumption is observed, or a decrease in the frequency of searching for unusual water sources that cats do not consume often.

As used herein, an improvement in polyphagia means a reduction in food consumption, or a reduction in the frequency of discussing or asking for an abnormal amount of food, or discussing or asking for food in an abnormal situation, e.g., discussing or asking for food immediately after feeding.

As used herein, improvement in polyuria means a reduction in the frequency or amount of urination, or a reduction in abnormal behavior associated with urination, including urination outside of a box provided for urination or overflow of a urination box.

As used herein, "preventing weight loss" means that body weight is not reduced by more than 5% from the start of control. For the avoidance of doubt, "preventing weight loss" includes any increase in weight from the onset of control.

As used herein, the definition of "hypoglycemia" refers to a clinical condition in which the measured blood glucose concentration is below the blood glucose ISFM definition (Sparkes et al 2015; J Feline Med Surg 17:235)<3.0–.5mM(53–63mg dL^-1) The upper limit of the range of (1). For the avoidance of doubt, hypoglycemia refers to blood glucose<63mg dL^-1。

Clinical markers of diabetes include, but are not limited to, serum fructosamine levels, blood or serum glucose levels, or glycated hemoglobin levels. The control regimen may last for at least 1, 3, 7, 14, 28 or more days; or 1, 2,3, 4 or more months, or for the remainder of life. In some embodiments, the control regimen is 2 months. In some embodiments, the clinical remission is permanent, i.e., persists through the remainder of the life of the cat. In some embodiments, uncontrolled mitigation is achieved. In some embodiments, the uncontrolled remission duration is at least 1, 3, 7, 14, 28 or more days; or 1, 2,3, 4 or moreFor many months, or for the remainder of life. The duration of uncontrolled remission will depend on a number of factors, including the feline, its diet and the amount of exercise on a daily basis. As a non-limiting example, clinical remission may be identified by serum fructosamine levels at or below the upper normal limit of a test laboratory reference range. As another non-limiting example, by fasting blood glucose levels equal to or below 170mg dL^-1To identify clinical remission.

As used herein, the "upper normal limit" or "ULN" of a test laboratory reference range refers to the minimum upper limit of the range of laboratory test values that is considered to be found in normal variations of samples taken from a healthy population. The upper limit of the normal value is typically provided by the testing laboratory, is relevant to communicating the test results to the practitioner, and may vary from laboratory to laboratory or from time to time within the laboratory, depending on the test calibration, test procedure, or specimen preparation. For example, during the field studies reported below, changes in central laboratory test methods resulted in an upper normal limit of serum fructosamine of 356 μmol L^-1It was changed to 275. mu. mol L^-1。

As used herein, "antidiabetic agent" refers to compositions comprising a drug, medicament, or medicament commonly used to treat diabetes in humans or animals. Common antidiabetic agents used in the treatment of human T2DM include, but are not limited to, alpha-glucosidase inhibitors, amylin analogs, biguanides, dipeptidyl peptidase 4 inhibitors, incretins or incretin analogs, insulin like, meglitinide, non-sulfonylurea secretagogues, SGLT2 inhibitors, sulfonylureas, and thiazolidinediones. It is generally accepted that oral medications used to treat human T2DM have little effect in the treatment of feline diabetes. To date, the regulatory agencies of the united states, european union, or japan have not approved any oral drugs for the treatment of feline diabetes.

As used herein, "low carbohydrate diet" refers to the food intake of the cat from the beginning of control. In particular, a low carbohydrate diet refers to a diet in which the relative intake of carbohydrates does not exceed a certain threshold level. A low carbohydrate diet typically includes a percentage of calories from carbohydrates of less than 40%, 35%, 30%, 26%, 20%, 15%, 12%, or less.

As used herein, "diabetic diet" refers to the food intake of a cat from the beginning of control. In particular, a diabetic diet refers to a diet having relatively high protein and low carbohydrate. High amounts of protein include protein with a percentage of calories greater than 60%, 65%, 70%, 75%, 80% or more, while low amounts of carbohydrate are as described above. In particular embodiments, the diabetic diet does not include dry cat food.

As used herein, "diarrhea or loose stool frequency increase" means that the number of diarrhea or loose stool exceeds more than 10% of the number of bowel movements in an unadministered animal. As used herein, diarrhea does not include incidental causes such as infection by bacteria, viruses, coccidia or intestinal worms, but refers to diarrhea caused by SGLT1 inhibition. In some embodiments, "diarrhea" refers to at least one loose or liquid stool occurring daily for at least three days during the course of treatment.

As used herein, "SGLT inhibitor" refers to compounds that are active on both SGLT1 and SGLT2, particularly those that have a better SGLT2 activity compared to SGLT1 activity, e.g., glucosuria and the benefits of impaired absorption of intestinal carbohydrates are accompanied by a lower risk of adverse gastrointestinal symptoms such as diarrhea, loose stools, flatulence and bloating.

As used herein, the term "administering" refers to by oral, buccal, nasal, rectal, vaginal or cutaneous route or other external contact, or by intravenous, intraperitoneal, intramuscular, intralesional or subcutaneous route, or by implantation of a slow release device or formulation, such as a pump, gel, reservoir or erodible substance, into a subject. Administration can be by any route, including parenteral and transmucosal administration (e.g., buccal, nasal, vaginal, rectal, or transdermal). Parenteral administration includes intravenous injection, intramuscular injection, intraarterial administration, intradermal administration, subcutaneous administration, intraperitoneal administration, intracerebroventricular administration, intrathecal administration and intracranial administration. Other modes of administration include, but are not limited to, liposomal formulations, intravenous infusion, transdermal patches, and the like.

As used herein, a method of controlling, inducing, reducing, ameliorating, or preventing a disorder, disease, or condition, comprising administering a compound or composition, may also refer to the use of a compound or composition to control, induce, reduce, ameliorate, or prevent a disorder, disease, or condition, and the use of a compound or composition in the manufacture of a medicament for controlling, inducing, reducing, ameliorating, or preventing a disorder, disease, or condition.

Methods for treating feline diabetes

In some aspects of the present invention, provided herein are methods for controlling diabetes in a cat, comprising administering to a cat in need thereof a total daily dose of about 5mg to about 50mg of compound 1, or a pharmaceutically acceptable form thereof, having the formula:

in some aspects of the invention, there is provided a method for reducing hyperglycemia-related clinical signs in a cat having diabetes, the method comprising administering to a cat in need thereof a total dose of about 5-50mg per day of compound 1, or a pharmaceutically acceptable form thereof.

In some aspects of the invention, there is provided a method for improving hyperglycemia-related clinical signs in a cat having diabetes, the method comprising administering to a cat in need thereof a total dose of about 5 to 50mg per day of compound 1, or a pharmaceutically acceptable form thereof.

In some aspects of the invention, there is provided a method for inducing clinical remission of diabetes in a cat, which comprises administering to a cat in need thereof a total dose of about 5-50mg per day of compound 1 or a pharmaceutically acceptable form thereof.

In some aspects of the invention, there is provided a method of reducing the serum fructosamine level in diabetic cats to below the upper normal limit of the reference range for the testing laboratory, said method comprising administering to a cat in need thereof a total dose of about 5-50mg per day of compound 1 or a pharmaceutically acceptable form thereof. In some embodiments, the test laboratory is referenced aboveThe limit is about 356. mu. mol L^-1Or 275. mu. mol L^-1。

In some aspects of the invention, methods are provided for improving glycemic control in diabetic cats having a blood glucose curve in which all blood glucose measurements fall at a peak of 10mmol/L (180mg dL)^-1) And minimum 4.5mmol/L (80mg dL)^-1) In a mammal in need thereof, the method comprises administering to the cat a total daily dose of about 5 to about 50mg of compound 1 or a pharmaceutically acceptable form thereof.

In some aspects of the invention, there is provided a method for preventing weight loss in a diabetic cat, the method comprising administering to a cat in need thereof a total dose of about 5-50mg per day of compound 1 or a pharmaceutically acceptable form thereof.

In some aspects of the invention, there is provided a method for controlling feline diabetes mellitus exhibiting an IGF-1 concentration greater than the upper normal limit of a test laboratory reference range, comprising administering to a cat in need thereof a total dose of about 5-50mg per day of compound 1 or a pharmaceutically acceptable form thereof. In some embodiments, the upper normal limit for the test laboratory reference is 92 nmol/L.

In some embodiments, the methods provided herein comprise administering to a cat in need thereof a low carbohydrate diet and a total daily dose comprising about 5-50mg of compound 1. Carbohydrates are typically present in commercial cat foods, and maintaining a low carbohydrate diet will improve the clinical pathology of the cat. In some embodiments, the low carbohydrate diet is a canned diet. Cats fed the cans will not be fed any dry cat food. In some embodiments, the low carbohydrate diet is a diabetic diet. The diabetic diet is typically a high protein, low carbohydrate diet. In some embodiments, the diabetic diet comprises little or no dry cat food. In some embodiments, the low carbohydrate diet is a ketogenic diet. Ketogenic diets include diets rich in seafood, meat, poultry and eggs. In some embodiments, the low carbohydrate diet is a grain-free diet.

In some embodiments, the low carbohydrate diet contains less than 40% calories in the form of carbohydrates. In some embodiments, the low carbohydrate diet contains less than 35% calories in the form of carbohydrates. In some embodiments, the low carbohydrate diet contains less than 30% calories in the form of carbohydrates. In some embodiments, the low carbohydrate diet contains less than 26% calories in the form of carbohydrates.

In some embodiments, compound 1 is the bisproline complex of (2S, 3R, 4R, 5S, 6R) -2- (4-chloro-3- (4- (2-cyclopropoxyethoxy) benzyl) phenyl) -6- (hydroxymethyl) tetrahydro-2H-pyran-3, 4, 5-triol, having the structure shown below

Administering the bisproline complex of claim 1 to a cat in need thereof. For more information on the bisproline complex see WO2010/022313, the contents of which are incorporated herein by reference for all purposes.

In some embodiments, compound 1 is a crystalline form of (2S, 3R, 4R, 5S, 6R) -2- (4-chloro-3- (4- (2-cyclopropoxyethoxy) benzyl) phenyl) -6- (hydroxymethyl) tetrahydro-2H-pyran-3, 4, 5-triol having the structure shown below

Administering said crystalline form of compound 1 to a cat in need thereof.

The crystalline form of the compound is characterized by an X-ray powder diffraction pattern as shown in figure 1. In some embodiments, the X-ray powder diffraction (XRPD) pattern comprises one or more peaks selected from the group consisting of: 5.4 °, 11.2 °, 11.3 °, 11.9 °, 12.9 °, 15.5 °, 16.3 °, 17.8 °, 19.1 °, 20.0 °, 20.6 °, 20.7 °, 21.2 °, 22.8 °, 23.0 °, 23.4 °, 23.6 °, 23.9 °, 24.7 °, 25.4 °, 25.8 °, 27.8 °, and 28.2 ° θ (± 0.1 ° 2 θ), wherein the XRPD is with CuK_α1And (4) radiation manufacturing. In a further embodiment of the process of the present invention,a crystalline form of the compound is characterized by XRPD comprising two or more, three or more, four or more or five or more peaks selected from the group consisting of: 5.4 °, 11.2 °, 11.3 °, 11.9 °, 12.9 °, 15.5 °, 16.3 °, 17.8 °, 19.1 °, 20.0 °, 20.6 °, 20.7 °, 21.2 °, 22.8 °, 23.0 °, 23.4 °, 23.6 °, 23.9 °, 24.7 °, 25.4 °, 25.8 °, 27.8 °, and 28.2 ° θ (± 0.1 ° 2 θ). In other embodiments, a crystalline form of the compound is characterized by XRPD, comprising peaks at 12.9 °, 19.1 °, and 20.7 ° θ (± 0.1 ° 2 θ). In other embodiments, the crystalline form of the compound is characterized by an XRPD pattern comprising peaks selected from the group consisting of: 11.2 °, 12.9 °, 15.5 °, 17.8 °, 19.1 °, 20.0 °, and 20.7 ° θ (± 0.1 ° 2 θ). In other embodiments, the crystalline form of the compound is characterized by an XRPD pattern comprising peaks selected from the group consisting of: 5.4 °, 11.2 °, 11.9 °, 12.9 °, 15.5 °, 16.3 °, 17.8 °, and 19.1 ° θ (± 0.1 ° 2 θ). In other embodiments, the crystalline form of the compound is characterized by an XRPD pattern comprising peaks selected from the group consisting of: 5.4 °, 11.2 °, 11.9 °, and 12.9 ° θ (± 0.1 ° 2 θ). In another embodiment, the crystalline form of the compound is characterized by an XRPD pattern comprising peaks selected from the group consisting of: 11.2 ° and 12.9 ° θ (± 0.1 ° 2 θ). In other embodiments, the crystalline form of the compound is characterized by an XRPD pattern having XRPD peaks substantially as in figure 2.

The crystalline compound of the present invention is also characterized by a raman spectrum substantially as shown in fig. 3 and peaks substantially as shown in fig. 4. In some embodiments, the crystalline form of the compound is characterized by raman spectroscopy, comprising one or more peaks selected from the group consisting of: 353. 688, 825, 1178, 1205, 1212, 1608, 2945, 3010 and 3063cm^-1. In another embodiment, the crystalline form of the compound is characterized by raman spectroscopy, comprising two or more, three or more, four or more, or five or more peaks. In other embodiments, the crystalline form of the compound is characterized by raman spectroscopy, comprising peaks selected from the group consisting of: about 353, 688 and 825cm^-1. In other embodiments, the crystalline form of the compound is characterized by a raman spectrum peak substantially as shown in figure 4。

In some embodiments, the therapeutically effective amount of compound 1 is a total daily dose of about 5mg to 50mg (e.g., about 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 30, 35, 40, 45, or 50mg for a day)^-1). In some embodiments, the total daily dose of compound 1 is about 10-20 mg. In some embodiments, the total daily dose of compound 1 is about 15 mg.

Compound 1 can be administered to cats by a number of suitable routes. In some embodiments, compound 1 is administered orally. Further control methods will be discussed below.

In some embodiments, compound 1 is administered in combination with an additional therapeutic agent. In some embodiments, compound 1 is administered as a monotherapy. That is, when compound 1 is the only antidiabetic agent administered to cats, it has a positive therapeutic benefit in treating cat diabetes.

Advantageously, administration of compound 1 need not be simultaneous with feeding or other events. In some embodiments, the total daily dose is administered once daily, independent of other activities (including meal time). In some embodiments, the total daily dose is administered twice daily, independent of other activities (including meal time). In some embodiments, the dose is mixed with the food of the cat. In some embodiments, the dose is delivered to the cat as a single solid dosage form. In some embodiments, the dose is delivered in the form of an oral solution or oral suspension. In some embodiments, the maximum fluid volume delivered is 1 mL. In some embodiments, the maximum volume delivered is 0.5 mL. In some embodiments, the dosage is adjusted according to the weight of the cat. In some embodiments, a single dose intensity is provided for all cats.

Serum fructosamine levels and/or blood or serum glucose levels can be monitored during treatment as evidence of glycemic control. Clinical symptoms such as polyuria, polydipsia, polyphagia, or weight loss can also be monitored. If symptoms of diabetes persist, the control plan can be altered to incorporate other features, including other medications.

The cats maintain the positive therapeutic benefits obtained from the accepted treatment regimen while they remain in clinical remission. In some embodiments, clinical remission is maintained when the cat does not exhibit one or more clinical symptoms of feline diabetes. As noted above, symptoms of feline diabetes include elevated serum fructosamine levels, elevated blood or serum glucose levels, polyuria, polydipsia, and polyphagia.

In some embodiments, the cat maintaining clinical remission is determined by the serum fructosamine level of the cat. In some embodiments, the serum fructosamine level of the cat is compared to the upper normal limit of the test laboratory reference range. In some embodiments, the upper limit of the test laboratory reference is about 356. mu. mol L^-1Or 275. mu. mol L^-1. In some embodiments, cats in clinical remission exhibit serum fructosamine levels at or below the upper normal limit of the test laboratory reference range. In some embodiments, cats in clinical remission exhibit serum fructosamine levels at or below 360 μmol L^-1. In some embodiments, cats in clinical remission exhibit serum fructosamine levels at or below 350 μmol L^-1. In some embodiments, cats in clinical remission exhibit serum fructosamine levels at or below the upper normal limit of the testing laboratory to which the specimen was submitted.

In some embodiments, the cat maintaining clinical remission is determined by the blood or serum glucose level of the cat. In some embodiments, the blood or serum glucose level of a cat in clinical remission is maintained at less than 250mg dL^-1. In some embodiments, the blood or serum glucose level of a cat in clinical remission is maintained at less than 200mg dL^-1. In some embodiments, the blood or serum glucose level of a cat in clinical remission is maintained at less than 190mg dL^-1. In some embodiments, the blood or serum glucose level of a cat in clinical remission is maintained at less than 180mg dL^-1. In some embodiments, the blood or serum glucose level of a cat in clinical remission is maintained below 170mg dL^-1. At one endIn some embodiments, the blood or serum glucose level of a cat in clinical remission is maintained below 160mg dL^-1. In some embodiments, the blood or serum glucose level of a cat in clinical remission is maintained at less than 150mg dL^-1。

The methods described herein reduce, alleviate, or eliminate the symptoms of diabetes in cats. For example, in some embodiments, the serum fructosamine level of the cat measured after completion of the control regimen is reduced in said cat. In some embodiments, the blood or serum glucose level of the cat measured after completion of the control regimen is reduced in said cat.

In some embodiments, the serum fructosamine level of the cat is reduced by at least about 20% after completion of said control regimen. In some embodiments, the serum fructosamine level of the cat is reduced by at least about 30% after completion of said control regimen. In some embodiments, the serum fructosamine level of the cat is reduced by at least about 40% after completion of said control regimen. In some embodiments, the serum fructosamine level of the cat is reduced by at least about 50% after completion of said control regimen.

In some embodiments, the serum fructosamine level of the cat is below the upper normal limit of the test laboratory reference range after completion of said control regimen. In some embodiments, the upper limit of the test laboratory reference is about 356. mu. mol L^-1Or 275. mu. mol L^-1. In some embodiments, the serum fructosamine level of the cat is less than 500 μmol L after completion of said control regimen^-1. In some embodiments, the serum fructosamine level of the cat is less than 450 μmol L after completion of said control regimen^-1. In some embodiments, the serum fructosamine level of the cat is less than 400 μmol L after completion of said control regimen^-1. In some embodiments, the serum fructosamine level of the cat is less than 350 μmol L after completion of said control regimen^-1。

In some embodiments, the blood or serum glucose level of the cat is reduced by at least about 20% after completion of the control regimen. In some embodiments, the blood or serum glucose level of the cat is reduced by at least about 30% after completion of the control regimen. In some embodiments, the blood or serum glucose level of the cat is reduced by at least about 40% after completion of the control regimen. In some embodiments, the blood or serum glucose level of the cat is reduced by at least about 50% after completion of the control regimen.

In some embodiments, the blood or serum glucose level of the cat is less than 250mg dL after completion of the management regimen^-1. In some embodiments, the blood or serum glucose level of the cat is less than 200mg dL after completion of the management regimen^-1. In some embodiments, the blood or serum glucose level of the cat is less than 190mg dL after completion of the management regimen^-1. In some embodiments, the blood or serum glucose level of the cat is less than 180mg dL after completion of the management regimen^-1. In some embodiments, the blood or serum glucose level of the cat is less than 170mg dL after completion of the management regimen^-1. In some embodiments, the blood or serum glucose level of the cat is less than 160mg dL after completion of the management regimen^-1. In some embodiments, the blood or serum glucose level of the cat is less than 150mg dL after completion of the management regimen^-1。

As mentioned above, it is advantageous that the activity against SGLT1 is lower than the activity against SGLT2, SGLT1 inhibition may cause diarrhea. Thus, the SGLT inhibitors of the present invention provide a pharmacodynamic effect in the treatment of feline diabetes at dosage levels below the threshold level for adverse intestinal effects. Thus, in another aspect, the invention provides a method for controlling diabetes in cats comprising administering to a cat in need thereof an effective amount of an SGLT inhibitor wherein the effective amount is no more than 10% to 30% of the dose required to cause diarrhea or increased frequency of loose stools in healthy cats. In some embodiments, the healthy cats are on commercial dry food. In some embodiments, the healthy cat refers to a cat that did not exhibit diarrhea or increased frequency of loose stools prior to administration of the SGLT inhibitor. In some embodiments, the healthy cat is not diabetic.

In some embodiments, the effective amount is no more than 10, 12, 16, 18, 20, 22, 24, 26, 28, or 30% of the dose required to cause diarrhea in a healthy cat to exacerbate or loose stool. In some embodiments, the effective amount is no more than 30% of the dose required to cause diarrhea in a healthy cat. In some embodiments, the effective amount is no more than 20% of the dose required to cause diarrhea in a healthy cat. In some embodiments, the effective amount is no more than 10% of the dose required to cause diarrhea in a healthy cat.

The effective amount includes a dose that produces at least 90% of the maximum pharmacodynamic effect of the SGLT inhibitor.

Also provided herein is a method of controlling diabetes in a cat comprising administering to a cat in need thereof an effective amount of an SGLT inhibitor, wherein the SGLT inhibitor causes an increase in the frequency of diarrhea or loose stools in healthy cats at a dose not less than 3 to 10 times the effective amount. In some embodiments, the healthy cats are on commercial dry food. In some embodiments, the healthy cat is a cat that did not exhibit diarrhea or increased frequency of loose stools prior to treatment. In some embodiments, the healthy cat is not diabetic.

In some embodiments, the SGLT inhibitor causes an increase in the frequency of diarrhea or loose stools in healthy cats at a dose of no less than 3,4,5, 6, 7,8, 9, or 10 times the effective amount. In some embodiments, the SGLT inhibitor causes an increase in the frequency of diarrhea or loose stools in healthy cats at a dose not less than 3 times the effective amount. In some embodiments, the SGLT inhibitor causes an increase in the frequency of diarrhea or loose stools in healthy cats at a dose not less than 5 times the effective amount. In some embodiments, the SGLT inhibitor causes an increase in the frequency of diarrhea or loose stools in healthy cats at a dose not less than 10 times the effective amount.

The effective amount includes a dose that produces at least 90% of the maximum pharmacodynamic effect of the SGLT inhibitor.

The methods described herein can be used to control all forms of feline diabetes. In some embodiments, the feline diabetes is type 1 diabetes. In some embodiments, the feline diabetes is type 2 diabetes.

Pharmaceutical compositions

Compound 1 can be prepared in a variety of compositions suitable for delivery to a subject. Compositions suitable for administration to a subject typically include compound 1 (or a pharmaceutically acceptable form thereof) and a pharmaceutically acceptable carrier.

Compound 1 can be incorporated into a variety of formulations for therapeutic administration. More specifically, compound 1 can be formulated into a pharmaceutical composition by together or separately with an appropriate pharmaceutically acceptable carrier or diluent, and can be formulated into preparations in solid, semisolid, liquid or gaseous form, such as tablets, capsules, pills, powders, granules, preparations, gels, slurries, ointments, solutions, suppositories, injections, inhalants and aerosols. Thus, administration of the compounds of the invention can be accomplished in a variety of ways, including orally, buccally, parenterally, intravenously, intradermally (e.g., subcutaneously, intramuscularly), transdermally, and the like. In addition, compound 1 can be administered locally rather than systemically, e.g., by administration in a depot or sustained release formulation.

Pharmaceutical compositions for administering compound 1 may conveniently be presented in unit dosage form and may be prepared by any of the methods known in the art of pharmacy and drug delivery. All methods include the step of bringing into association the active ingredient with the carrier which contains one or more accessory ingredients. In general, pharmaceutical compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired formulation.

Formulations suitable for use in the present invention are described in remington: pharmaceutical sciences and practices, 21 st edition, edited by Gennaro, lipak, williams & wilkins (LWW) (2003), is now incorporated by reference. The pharmaceutical compositions described herein may be manufactured in a manner known to those skilled in the art, i.e., by means of conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. The following methods and excipients are exemplary only and are not limiting in any way.

In some embodiments, compound 1 is formulated as a sustained release, controlled release, extended release, timed release, or delayed release formulation, for example, delivered in a semipermeable matrix of a solid hydrophobic polymer containing the therapeutic agent. Various types of sustained release materials have been established and are well known to those skilled in the art. Current sustained release formulations include film coated tablets, multiparticulate or pellet systems, matrix technologies using hydrophilic or lipophilic materials, and wax based tablets containing pore forming excipients (see, e.g., Huang et al, drug development and industry, 29: 79(2003), pierce et al, drug development and industry, 29: 925(2003), Maggi et al, european journal of pharmacy and biopharmaceutical, 55: 99(2003), khavilkar et al, drug development and industry, 216216228: 601(2002), and schmidt et al, international journal of pharmacy, 2169 (2001)). Depending on its design, a sustained release delivery system may release the compound over hours or days, for example, over 4,6, 8, 10, 12, 16, 20, 24 hours or more. In general, sustained release formulations may be prepared using natural or synthetic polymers, for example, the polymers vinylpyrrolidone, such as polyvinylpyrrolidone (PVP); a carboxyvinyl hydrophilic polymer; hydrophobic and/or hydrophilic hydrocolloids, such as methyl cellulose, ethyl cellulose, hydroxypropyl cellulose and hydroxypropylmethyl cellulose; and carboxypolymethylene.

The sustained or extended release formulations can also be prepared using natural ingredients, such as minerals, including titanium dioxide, silicon dioxide, zinc oxide, and clays (see U.S. patent 6,638,521, incorporated herein by reference). Exemplary sustained release formulations (in any of the forms described herein) that can be used to deliver compound 1 include U.S. patent 6,635,680; 6,624,200, respectively; 6,613,361, respectively; 6,613,358,6,596,308; 6,589,563, respectively; 6,562,375, respectively; 6,548,084, respectively; 6,541,020; 6,537,579; 6,528,080 and 6,524,621, each of which is incorporated herein by reference. Particularly relevant controlled release formulations include U.S. patent 6,607,7516,599,529; 6,569,463, respectively; 6,565,883, respectively; 6,482,440, respectively; 6,403,597; 6,3199,919, respectively; 6,150,354, respectively; 6,080,736, respectively; 5,672,356, respectively; 5,472,704, respectively; 5,445,829, respectively; 5,312,817 and 5,296,483, each of which is incorporated herein by reference. One skilled in the art will readily recognize other applicable sustained release formulations to work with.

For oral administration, compound 1 can be readily formulated by combining with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions and the like, for oral administration to a patient in need thereof. Oral pharmaceutical preparations can be obtained by mixing the compounds with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, if desired with the addition of suitable auxiliaries, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, for example cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate.

The tablets of the invention contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, inert diluents, such as cellulose, silica, alumina, calcium carbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example PVP, cellulose, PEG, starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated, enteric coated or otherwise, by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated to form osmotic therapeutic tablets for controlled release.

Pharmaceutical preparations for oral use include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a layer of a plasticizer, such as glycerol or sorbitol. Push-fit capsules may contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, for example fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may also be added. All dosages for oral formulations should be suitable for such administration.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example microcrystalline cellulose, lactose, starch, pregelatinized starch, or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil. Additionally, emulsions may be prepared with non-water miscible ingredients such as oils and stabilized with surfactants such as monoglycerides, PEG esters and the like.

In some cases, compound 1 may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. For injection, the compound may be formulated into preparations by dissolving, suspending or emulsifying it in an aqueous or non-aqueous solvent, such as vegetable oil or other similar oils, synthetic fatty acid glycerides, higher fatty acid esters or propylene glycol; if desired, conventional additives such as solubilizers, isotonizing agents, suspending agents, emulsifiers, stabilizers and preservatives can be used. Preferably, compound 1 can be formulated in aqueous solution, preferably in a physiologically compatible buffer, such as Hank's solution, ringer's solution, or physiological saline buffer. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain agents such as suspending, stabilizing, and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the water-soluble forms of compound 1 (any form described herein). In addition, suspensions of compound 1 can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds to prepare highly concentrated solutions. Alternatively, compound 1 may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. For external administration, compound 1 can be formulated into ointments, creams, salves, powders, and gels. In one embodiment, the transdermal delivery agent is DMSO. Transdermal drug delivery systems may include, for example, patches. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Transdermal formulations useful in the present invention include those described in U.S. patents 6,589,549, 6,544,548, 6,517,864, 6,512,010, 6,465,006, 6,379,696, 6,312,717, and 6,310,177, each of which is incorporated herein by reference.

In addition to the formulations described previously, compound 1 can also be formulated as a long acting formulation. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, compound 1 can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble complex or salt.

The pharmaceutical composition may also include a suitable solid or gel phase carrier or excipient. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, and polymers, such as polyethylene glycol.

V. pharmaceutical preparation

The present disclosure includes novel pharmaceutical dosage forms of compound 1, or pharmaceutically acceptable forms thereof. The dosage forms described herein are suitable for oral administration to a subject. The dosage form may be in any form suitable for oral administration, including but not limited to capsules or tablets.

In some embodiments, the present invention provides a single unit dose capsule or tablet form containing 5-50mg of compound 1 (besartan), or a crystalline form thereof, having the structural formula:

in some embodiments, the amount of compound 1 is about 10-20 mg. In some embodiments, the amount of compound 1 is about 15 mg.

In some embodiments, the single unit dosage form of compound 1 is a capsule. In some embodiments, the single unit dosage form of compound 1 is a tablet.

In some embodiments, the single unit dosage form is a capsule having a size selected from the group consisting of: #0, #1, #2, #3, #4, or # 5. In some embodiments, the single unit dosage form is a capsule size # 4. In some embodiments, the single unit dosage form is a capsule of size # 5.

VI. kit

Also provided herein are kits comprising pharmaceutical compositions and dosage forms of compound 1 or formulations thereof.

In certain aspects, the invention provides a kit comprising compound 1. Some of the kits described herein include a label describing a method of administering compound 1. Some of the kits described herein include a label describing a method for treating diabetes in a cat. In some embodiments, the kits described herein include a label describing a method of reducing serum fructosamine and/or blood or serum glucose levels in a cat.

Compositions of the invention, including, but not limited to, a bottle, jar, vial, ampoule, tube, or other container closure system approved by the U.S. Food and Drug Administration (FDA) or other regulatory agency, comprising compound 1, can provide one or more doses containing compound 1. The package or dispenser may also be accompanied by a notice associated with the container in a format prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice indicates approval by the agency. In some aspects of the invention, the kit comprises a formulation or composition as described herein, a container closure system comprising the formulation, or a dosage unit form comprising the formulation, and a notice or instruction describing a method of use as described herein.

Examples

The invention will be further illustrated with reference to the following specific examples. It is to be understood that these examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention. Those skilled in the art will readily recognize various non-critical parameters that may be altered or modified to produce substantially the same result.

Example 1 Effect of Bexagliptin on diabetic mice

In study 350, the effect of besiflozin (as a 2:1 proline: besiflozin co-crystal) was examined in hereditary diabetic db/db mice. Blood glucose was measured with a glucometer (a one-touch ultra-intensive (LifeScan) blood glucose monitoring system). Typically, blood is obtained from a wound on the tail. When the blood glucose level was above the upper limit of glucometer quantification by 33.3mmol/L, 2 drops of blood were taken from the orbital plexus, collected in an anticoagulant microcentrifuge tube containing heparin, and then diluted and blood glucose measured. The mass of beslagrangian in table 1 is about 2/3 for the mass of the 2:1 component.

By gavage, animals were dosed daily with either vehicle or proline: besiflozin co-crystals, lasting 28 days. Animals were dosed daily between 10:00 am and 12:00 pm. Body weight was measured every 4 days and the dose was adjusted according to the last body weight. The dose was 10mL/kg body weight. All animals were observed daily and any abnormal findings were recorded. Blood glucose concentration measurements were performed 6h after dosing on days-3, 0,1, 7, 14, 21 and 28, respectively.

As shown in Table 1, except for 0.1mg kg^-1In all treatment groups, the non-fasting plasma glucose group was significantly lower than the control group on days 1, 7, 14, 21, and 28. Bexaglipizine at the dose 6 hours after administrationThe non-fasting blood glucose levels in db/db mice were reduced in a dependent manner. 0.067, 0.2, 0.67 and 2mg kg were administered respectively^-1After 28 days of besiflozin, the blood glucose values of the animals were 79.74%, 56.33%, 51.81% and 53.25% of the control group, respectively. Although these data indicate that besiflozin is an effective antidiabetic agent in diabetic mice, none of the dose groups normalized blood glucose concentrations within the non-diabetic range. For fasting mice of the C57/BL6 line (a non-diabetic reference strain of db/db mice), an average fasting plasma glucose of 7.3mM (131mg dL) was reported^-1(ii) a Andrikopoulos et al, 2005; endocrine magazine (2005) 187: 45). Typically, blood glucose levels are greater than 180mg dL^-1Are considered to be markers of diabetes. However, in db/db mice, besagliflozin as the sole drug failed to lower blood glucose to 300mg dL^-1The following.

TABLE 1 Effect of Bexagliptin treatment on non-fasting blood glucose concentration (mg dL-1) in db/db mice

P <0.05, P <0.01vs. control group

Compound or vehicle was taken orally once daily for 4 weeks. Blood glucose was measured without fasting using a glucometer 6 hours after the administration.

TABLE 1 Effect of Bexagliptin treatment on non-fasting blood glucose concentration (mg dL-1) in db/db mice

The values represent the mean ± SD (n ═ 8). The besiflozin/proline co-crystal was administered separately to each group at a dose of more than 1.5 times the mass of besiflozin (as shown).

Example 2 Effect of Bexagliptin on diabetic rats

In study 338, the effect of besagliflozin (as a 2:1 proline: besagliflozin co-crystal) was examined in hereditary diabetic ZDF rats. Blood was collected from the saphenous vein of male ZDF rats, placed in Capijet tubes containing sodium fluoride and potassium oxalate (batch number: HA 0931; Telumo healthcare Co.) and used to determine non-fasting blood glucose levels. Plasma glucose levels were analyzed by a colorimetric method based on the hexokinase methodology (glucose-SL method: diagnostic chemistry, Inc.). Prior to analysis, plasma samples were taken in 0.9% saline for 1: and 2, diluting.

The oral vehicle (10% PEG400) or four dosage levels (0.067, 0.2, 0.67 and 2.0mg kg) were administered to the animals once daily for 28 consecutive days at 9:00-11:00 am^-1) One dose of test compound. Body weight was measured 3 times per week and the dose adjusted accordingly. Food (prairia 5008; formula diet) and water consumption were measured 3 times per week and daily values were estimated. Food and water consumption was recorded for 24 hours as urine was collected in the metabolism cage. All animals were observed daily and any abnormal findings were recorded.

Blood samples were collected from the great saphenous vein (Capject batch # HA 0931; Telumo healthcare) on days 7, 14, and 21 for determination of plasma glucose levels, followed by oral administration of the compound or vehicle.

Daily oral treatment with besagliflozin significantly reduced blood glucose levels within 7 days. There was no significant difference in blood glucose between groups at the start of dosing, since the composition of the study group was to minimize the difference between groups for this variable (F (4,45) ═ 0.04; p ═ 0.99). A significant drop in blood glucose levels was observed on day 7 of treatment (F (4,45) ═ 3.99; p ═ 0.007); two-by-two comparisons showed lower blood glucose levels (0.2 and 0.67mg kg) in the groups receiving bexagliptin, except for the lowest dose group, compared to the vehicle control group (table 2)^-1P of group<0.05，2.0mg kg^-1P of group<0.01). The decrease from baseline was still significant (F (4,45) ═ 4.05; p ═ 0.007), and subsequent comparisons again indicated 0.067mg kg of bestatin-except for bestatin^-1The reduction rate of all other groups was significantly higher than that of the group (0.3 and 1.0mg kg)^-1P of group<0.05；3.0mg kg^-1P of group<0.01) the increase in blood glucose levels observed in the control group (Table 2). Changes in blood glucose levels observed after 7 daysThe following were used: + 2.8%, -16.8%, -17.1% and-25.3% respectively for besalaglipizine 0.067, 0.2, 0.67 and 2.0mg kg^-1The vehicle control group was + 9.6%.

After 2 weeks of treatment, a decrease in blood glucose levels was observed (F (4,45) ═ 12.3; p < 0.0001); two-by-two comparison showed p <0.01 compared to vehicle control, except 0.067mg kg^-1The blood sugar level of the besiflozin group is obviously lower, namely the besiflozin is 0.2, 067 and 2.0mg kg^-1Time (fig. 1 and table 1). Similarly, the decrease from baseline treatment was also significant after 2 weeks of treatment (F (4,45) ═ 5.13; p ═ 0.002) and besagliflozin at 2.0mg kg^-1The reduction in size produced was significantly greater than that of the vector group (p)<0.01) (table 2). After 14 days, besiflozin was observed at 0.067, 0.2, 0.67, 2.0mg kg^-1Then, the blood glucose changes were + 9.5%, -5.6%, -8.1%, and-25.1%, respectively, and the vehicle control was + 17.0%.

Similarly, blood glucose levels decreased significantly after 3 weeks of treatment (F (4,44) ═ 7.77; p < 0.0001); after receiving 0.3, 1.0 and 3.0mg kg^-1The group of EGT0001474 had significantly lower blood glucose levels (P <0.05, <0.01 and <0.01, respectively) (Table 2). The decrease from baseline was still significant (F (4,44) ═ 3.36; p ═ 0.017) and besagliflozin at 2.0mg kg^-1The reduction in size produced was significantly greater than that of the vector group (p)<0.01) (table 2). The changes in blood glucose levels observed after 21 days were as follows: + 15.5%, + 4.3%, + 1.5% and-15.4% respectively for besalaglipizine 0.067, 0.2, 0.67 and 2.0mg kg^-1The vehicle control group was + 25.5%. One received 0.67mg kg before blood draw on day 21^-1The rats of besiflozin were euthanized by moribund. Necropsy results indicated that the animal was likely to have inhaled some of the dosing solution during the first few weeks of dosing. This effect is considered to be independent of the test article. Data from euthanized animals were retained on days 7 and 14, but no analysis was retained on day 21 (thus, the degrees of freedom in the F statistic denominator were reduced).

Although these data indicate that besiflozin is an effective antidiabetic drug in diabetic rats, none of the dose groups normalized blood glucose concentrations within the non-diabetic range. Generally, bloodSugar level greater than 180mg dL^-1Are considered to be markers of diabetes. However, in ZDF rats, bessaxagliptin as a single drug failed to lower blood glucose to 287mg dL^-1The following (Table 2).

Table 2 long-term effect of oral beslagritazine on blood glucose levels in male Zucker diabetic obese rats.

a. Bespagcage/proline was administered at a dose 1.5 times greater than the mass of bespagcage in each group (as shown).

b. Euthanasia was performed before blood collection on day 21; on day 0, n is 10, but on days 21 and 21 δ, n is 9 (thus, subtracting the average value yields a value different from the value shown in the table: -10.2 g).

Previous studies have shown that administration of besalaglipizide to diabetic rodents can significantly improve the severity of the disease, but does not restore the animal to a normoglycemic state or produce blood glucose levels in the normal range.

Example 3 Effect of besiflozin on feline SGLT1 and SGLT2 transporters

In study 5, the DNA fragment encoding feline SGLT2 was inserted downstream of the cytomegalovirus immediate early protein enhancer/chicken β -actin promoter, and the rabbit β -globin intron (CAG promoter) (pPB-CAG-SGLT2 Cat-IRES-EGFP; Eru.s.P.M. No. 1118 Bailu pharmaceutical Shanghai, Zhang Jiang, high tech., park, China, zip code 201203) was inserted between SalI and Hind III sites of the mammalian expression vector pNL 715. The plasmid expression cassette is flanked by inverted terminal repeats of the PiggyBac transposon, contains an Internal Ribosome Entry Site (IRES), and is located in an enhanced upstream GFP open reading frame and a bovine growth hormone polyadenylation signal. Plasmids containing the desired cDNA insert were identified by restriction endonuclease cleavage analysis. Similarly, a plasmid encoding feline SGLT1 (pNL717) was inserted into SalI and Hind III sites to create pPB-CAG-SGLT1Cat-IRES-EGFP (Shanghai Egret pharmaceutical Co., Ltd.). The C-to-T transition mutation at position 891 in the coding region of cDNA clone fSGLT1 (creating a stop codon at amino acid residue 297) was restored to the wild type sequence by polymerase chain reaction. Reversion restored the activity of the expression plasmid.

Feline SGLT expression plasmid DNA was transfected into Cos-7 cells using Lipofectamine 3000 (Seimerlife, Waltham, Mass.) according to manufacturer's suggested protocol. Cells were plated at approximately 3X 10 hours prior to transfection⁶Individual cells/well were seeded in 100mm dishes containing 10mL of medium per well and reached a confluency of 95% or more at the time of transfection. 24 hours after transfection, transfected cells were harvested with trypsin and seeded into 96-well poly-D-lysine coated scintillation plates (Perkin Elmer) supplemented with 10% FBS, 2mM glutamine, 100. mu.L DMMEM/well, then 5% CO at 37 ℃ C₂For 48 hours. Transfected cells were stored in DMEM containing 10% dimethyl sulfoxide and cryopreserved at-195 ℃ or by methyl-alpha-D- [ U-¹⁴C]The glucopyranoside (AMG) uptake assay assesses transporter activity.

150 μ L of sodium buffer (137mM NaCl, 5.4mM KCl, 2.8mM CaCl)₂、1.2mM MgCl₂10mM Tris/HEPES, pH 7.2) or sodium-free buffer (137mM N-methylglucamine, 5.4mM KCl, 2.8mM CaCl₂、1.2mM MgCl₂10mM Tris/HEPES, pH 7.2) washing of transfected cells expressing SGLT1 or SGLT2 (4X 10 per well⁴One cell) twice. mu.L of sodium-free buffer containing 40. mu. Ci/mL α -methyl-D-glucopyranoside (AMG; Perkin Elmer) or 50. mu.L of sodium buffer containing 8. mu. Ci/mL AMG, 10% cat plasma and the desired concentration of besiflozin was added to each well of the plate and incubated at 37 ℃ for 1h with shaking. Cells were washed twice with 150 μ L phosphate buffered saline and plates covered with TopSeal (perkinelmer) and AMG uptake were quantitated using a model 1450 micro β -well plate scintillation counter (perkinelmer). AMG uptake results were analyzed using GraphPad Prism (scientific visual software). Calculating IC using non-linear regression with variable slope₅₀。

Bexagliptin exhibits high efficacy, IC, in the presence of 10% cat plasma in cats SGLT1 and SGLT2₅₀Value division23.8nM and 412pM, respectively. The activity of besiflozin on feline SGLT2 was 5 times higher and on feline SGLT1 235 times higher than its activity on homologous human transporters (Zhang et al, pharmacological study 63: 2842011).

Example 4 Effect of Bexagliptin on non-diabetic cats

In study 1 and study 2, gelatin-encapsulated bessaxagliptin was administered to healthy cats and urine glucose excretion was recorded within 24 hours after administration. Either the cats were dosed once (qd) or twice (bid) (the latter at hours 0 and 12). As shown in FIG. 5, at about 3mg kg^-1At the dose of (a), maximum glucosuria was observed, with administration twice a day being only slightly better than once a day. The severity of loose stools and/or diarrhea in cats exposed to high doses of besiflozin correlates positively with the dose-course, and this effect is consistent with (and due to) the inhibitory effect of this drug on intestinal SGLT 1. In view of the lower in vitro selectivity to feline SGLT2, the pharmacological effects of besiflozin in cats may be the result of inhibition of SGLT1 and SGLT 2.

The dose tested for the adverse effects produced by SGLT1 inhibition was 5 to 10 times higher than the dose that produced the maximum efficacy, and thus the efficacy of besiflozin on cat SGLT1 appeared to be near optimal. If the efficacy against SGLT1 is higher, an overlap between the maximum pharmacodynamic effect and the unwanted side effects will ensue.

Further evidence of the ideal efficacy of besiflozin in cats at SGLT1 was found in study 3, in which healthy cats were dosed with 15mg kg twice daily^-1Bexaglipizine (30mg kg)^-1d^-1) For 21 days. As shown in fig. 6, the cats lost weight throughout the study due to the combined caloric loss of glucosuria and diarrhea. After dosing was complete, all cats gained weight. Because diabetic cats often experience weight loss as a result, the degree of diarrhea is clearly undesirable and incompatible with treatment. Thus, a dosage ten times higher than that which produces the maximum pharmacodynamic effect cannot be used continuously. Fortunately, the effect of besagliflozin on SGLT1 is not sufficient to cause diarrhea at the doses that produce the most potent effect, but is still sufficient to do so at the mostAt relatively low multiples of large effective doses, evidence of significant secondary intestinal inhibition is provided.

Example 5 Effect of Bexagliptin on diabetic cats

In one on-site efficacy study, cats kept by customers were diagnosed with diabetes based on: i) two separate fasting (≧ 6h) blood glucose measurements>250mg dL^-1(ii) a ii) diabetes mellitus; iii) fructosamines>450μmol L^-1(since the laboratory method of testing changes later on>360μmol L^-1) And iv) one or more of the following: polyuria/polydipsia, polyphagia, and/or weight loss (recorded in the medical records of cats). During visit 1 (7 days before day 0), cats suspected of being diabetic were evaluated. Eligible cats were enrolled during visit 2 (day 0) and started to be treated with oral besagliflozin once daily. The cats were given diabetic diets (Purina DM, dry or wet breed). Cats were returned to the clinic for glycemic control assessment during visit 3 (day 14 + -3), visit 4 (day 28 + -3), and visit 5 (day 56 + -3). Treatment periods ranged from visit 2 (day 0) to visit 5 (day 56 ± 3). If the owner or researcher deems it necessary, the cat can be brought back to the clinic during any unplanned visit. From visit 2 (day 0), an 8 hour blood glucose curve was performed for each visit (blood samples were collected every 2 hours ± 15 minutes for 8 hours) and blood glucose was measured using an AlphaTRAK 2 glucometer (yapei laboratory). Blood samples for hematology and serum chemistry were collected at screening and during each scheduled visit after the start of dosing. All clinical pathology samples (blood, serum and urine) were evaluated in a central laboratory and were not clinically analyzed for blood glucose curves. Serum chemistry includes the evaluation of fructosamine. Changes in the central laboratory measurement method resulted in a Upper Normal Limit (ULN) of 356. mu. mol L during the course of the study^-1It was changed to 275. mu. mol L^-1. To accommodate for changes in the reference range, data are expressed as a percentage of the upper limit of normal values, analyzed with unstructured covariance repeated measures ANCOVA, and visits were a fixed effect. The upper normal limit for detecting fasting blood glucose in the laboratory is 155mg dL^-1. In thatAfter the 32 cats completed the day 56 visit, the following table was prepared.

In this study, owners considered that cats exposed to besiflozin had improvements in polydipsia, polyphagia, and polyuria. Of the three host-assessed signs, the likelihood of finding improvement in polyphagia was minimal. Although besiflozin causes heat loss, weight gain is also often observed.

By measuring the normalization of fructosamine serum, glycemic control was significantly better. Of the 32 cats completed the study, 26 had fructosamine concentrations below the normal upper limit of the reference laboratory test range. None of the cats exhibited symptomatic hypoglycemia, consistent with the results of studies in healthy animals showing that many times higher than the expected clinical dose of besagliflozin did not produce hypoglycemia.

It was found that mice and humans deficient in the SGLT2 gene are euglycemic, which also indicates that hypoglycemia does not occur.

Three cats showed elevated serum insulin-like growth factor-1 (IGF-1), and given the known association of acromegaly with insulin resistance and insulin refractory diabetes, it is reasonable to believe that acromegaly has a potential contribution to its disease etiology. All three of the IGF-1 elevated cats reached normal fructosamine concentrations and completed the study and extended periods of safety assessment for four months. Subsequently, cats with the highest IGF-1 concentration were identified as acromegaly. After the study was completed, cats required 11 units of insulin per day and had poor clinical performance.

Improvements in cat health or physical condition, either as assessed by the owner or by the veterinarian, were recorded, with many of the changes reaching statistical significance. Ketonemia is usually corrected during the course of the study if its performance is evident. The study employed a validated investigational tool aimed at measuring the effect of feline diabetes on the owner's quality of life and detected a statistically significant improvement in owner's quality of life.

Results of detailed study

On day 56 ± 3, each cat was classified as achieving (or not achieving) glycemic control (success/failure). Will succeed in the treatmentDefined as the improvement in at least one glycemic variable (BG mean value)<250mg dL^-1(ii) a Or fructosamine<450 μmol/L or<360 μmol/L, depending on the assay reference range at the time of analysis), and a veterinarian's assessment of adequate glycemic control at the time of final assessment. As shown in table 3, 32 out of 40 cats were considered successful treatment (80%). All cats continuing to study V5 were considered successful in treatment. Of the 8 cats considered treatment failure, 6 were removed from the study after SAE, 1 should be removed only on demand from the donor, and 1 removed only for treatment of AE with study-prohibited drugs. Table 3 also reports that fructosamine concentrations below the upper normal limit (initially 356. mu. mol L) were achieved^-1Then 275. mu. mol L^-1) The cat of (4).

TABLE 3 treatment success evaluation

Clinical signs of acute diabetes in cats include weight loss, polyuria, polydipsia, and polyphagia. All this is thought to be secondary to caloric waste due to glucose urination, which manifests itself after blood glucose concentrations exceed the renal threshold for glucose diabetes. Since besiflozin lowers the diabetic threshold of the kidney, it is expected to exacerbate the clinical symptoms of hyperglycemia. However, if besiflozin raises glycuria to a level that can significantly reduce blood glucose concentration, the net effect may be to reduce glomerular total glucose flux once blood glucose concentration returns to normal. Following normalization, the severity of clinical signs of hyperglycemia may be reduced. Data collected from the study cohort indicate that the latter effect may exist.

Responsibility for assessing clinical signs of diabetes is shared by the host and the treating veterinarian. Veterinarians record weight at each visit, owners record signs of polydipsia, polyuria and polyphagia at each visit, using a four-point (0 to 3) integer score, with low scores representing good ratings.

In addition to providing a measure of cat status as a function of time, quantitative assessments are also used to produce a binary outcome of success or failure at the completion of the study. Success was scored by body weight, with the body weight at the 5 th visit exceeding the body weight at the 2 nd visit (treatment was initiated). Successful scoring with other markers, owner's score was lower at visit 5 than at visit 2.

Table 4 shows the binary results for each cat in the quantitative assessment. Any cat that exited the study was scored as failed in all categories (0). The last row of table 4 gives the sum of each column, or the total work count as calculated by the column criteria. Improvement in at least one clinical symptom ("any success" column) was observed in 31 cats, with less than one found achieving glycemic control. Example 5 glycemic control was successful, but clinical symptoms failed. For this cat, the owner scored equally for each category for each visit, with the weight dropping from 5.4 kg to 5.1 kg. Excluding body weight as a criterion (the "non-weight success" column), 30 cats were scored as successful. Additional cats not meeting the non-weight criteria are example 4. The final and initial scores for each category were the same for this cat. However, the cats of examples 4 and 5 were rated by their owners as having improved polydipsia, polyuria or polyphagia when compared qualitatively to the start of the study, as described in the following section.

TABLE 4 binary success results for quantitative assessment of clinical symptoms

Blood was collected at 0, 2,4, 6, and 8 hours after administration, and a blood glucose curve was plotted by glucometer measurement. Mixed model repeated measures ANCOVA with first order autoregressive covariance Structure for analysis of logarithmically transformed data for interviews, hours, and hourly runsThe visit is a fixed effect and the hour is a random effect. Figure 7 below shows the model adjusted least squares means with five measurements per curve shown by visit with 95% confidence intervals. The mean data from each visit (second panel below) was analyzed using repeated measures ANCOVA, with a fixed effect in hours, and using a first-order autoregressive covariance structure. Data for 40 enrolled cats is shown in figure 8. Mean of 5 blood glucose determinations at week 8 was 114.9mg dL^-1(95% confidence intervals 102.8, 128.4).

The central laboratory measured serum fructosamine concentrations at screening (V1) and at each visit post-enrollment (V3, V4 and V5). Changes in the central laboratory measurement procedure resulted in a Upper Normal Limit (ULN) of from 356. mu. mol L^-1It was changed to 275. mu. mol L^-1. Data are expressed as a percentage of the upper limit of normal values, analyzed by unstructured covariance repeated measures ANCOVA, and interviews were a fixed effect. Figure 9 shows model adjusted least squares means with 95% confidence intervals. The embedded value Δ represents the difference in least squares means from baseline to week 8, expressed as a percentage of ULN, and the corresponding 95% confidence interval. At week 8, the mean serum fructosamine value in the population was 86.7% of the upper normal limit (95% confidence interval 80.0%, 93.9%).

The central laboratory measured serum glucose concentrations at screening (V1) and at each visit after enrollment (V3, V4 and V5). Log-transformed data were analyzed for unstructured repeated measures ANCOVA using covariance as a fixed effect. Figure 10 shows model adjusted least squares means with 95% confidence intervals. The inserted text gives the difference in least squares means from baseline to week 8, and the corresponding 95% confidence interval. At week 8, mean blood glucose was 144 (95% confidence interval 127, 163).

Cats often participated in this study because their owners observed that their weight was losing, even though they ate more than usual. As shown in fig. 11, the average weight gain of the cats during the study was determined. By day 56, 82% of cats maintained or gained weight, and none lost more than 5% of the weight at the beginning of the study.

Body weight was measured at each study visit. The analysis of the log transformed data is as described above. Figure 11 shows model adjusted least squares means with 95% confidence intervals. The inset gives the difference in least squares means from baseline to week 8, as a percentage of initial body weight, with a 95% confidence interval. Although the confidence interval was broad, since the population was heterogeneous in body weight at the beginning of the study, the effect of treatment on body weight gain per cat was very significant (p < 0.0001). The effect of weight gain was surprising, contrary to the expectation that the consumption of calories from diabetes mellitus in grapes by besagliflozin would lead to weight loss in cats.

Throughout the study, owners were asked to provide assessments of cats of hyperglycemia-related clinical symptoms based on a four-point qualitative score, with 0 points representing excellent and 3 points representing poor. Assessments of polyphagia, polydipsia and polyuria were recorded separately. Significant differences were detected in all three measurements (figures 12, 13 and 14) with minimal effect of host-assessed polyphagia. Although owners believe that their cats show a reduction in signs of polyphagia, the objective body weight of their cats increases. Thus, although the mechanism of action of besiflozin includes the consumption of calories by glucose excretion, the effects on hyperglycemia-related clinical symptoms are more profound than the effects of besiflozin-induced caloric loss.

Feline acromegaly-associated diabetes is a unique etiology and presents special challenges for its treatment. Very high doses of insulin are often required to overcome the severe insulin resistance typically encountered in such situations. In addition to morphological changes associated with long-term disease, elevated IGF-1 concentrations are also a pathological feature of acromegaly in cats. The IGF-1 concentration in the three cats admitted the study was above the upper normal limit (92 nmol/L): case 7(172nmol/L), case 28(100nmol/L) and case 30(120 nmol/L). All three cases were considered treatment successful at V5 and completed a 6 month extended safety study.

Ketonemia and ketoacidosis are two manifestations of severe loss of control of blood glucose. Because available insulin does not lower blood glucose sufficiently, the glucose load is not fully absorbed by adipocytes leading to ketosis. Measurement ofThe most reliable analyte for ketonemia is beta-hydroxybutyrate. The central laboratory measured serum β -hydroxybutyrate (β -OHB) concentrations at screening (V1) and at each visit post enrollment (V3, V4 and V5). The log-transformed data were analyzed using a mixed model with covariance as unstructured repeated measures, ANCOVA, and accessed as a fixed effect. Figure 15 shows model adjusted least squares means with 95% confidence intervals. The inserted text gives the difference in least squares means from baseline to week 8, and the corresponding 95% confidence interval. At week 8, the mean serum β -hydroxybutyrate concentration was 1.76mg dL^-1(95% confidence interval 1.40, 2.20), lower than the upper limit of the laboratory's normal value (1.9mg dL)^-1). The confidence interval of the greatest initial visit value reflects the extreme change in the degree of feline ketonemia observed at the beginning of the study.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference were individually incorporated by reference. In the event of a conflict between the present application and a reference provided herein, the present application controls.