阅读说明：本技术 包封萜类化合物和/或大麻素的治疗性纳米颗粒 (Therapeutic nanoparticles encapsulating terpenoids and/or cannabinoids ) 是由 A·斯马尔-霍华德 L·马丁·班德拉斯 M·埃尔-哈马迪 M·费尔南德斯·阿雷瓦洛 于 2019-11-08 设计创作，主要内容包括：本文提供包封萜类化合物和大麻素的PEGA纳米颗粒和包含所述纳米颗粒的药物组合物。还提供了制备和使用包封萜类化合物和大麻素的PEGA纳米颗粒用于治疗目的方法。(Provided herein are PEGA nanoparticles encapsulating terpenoids and cannabinoids and pharmaceutical compositions comprising the nanoparticles. Methods of making and using PEGA nanoparticles encapsulating terpenoids and cannabinoids for therapeutic purposes are also provided.)

1. A PLGA nanoparticle encapsulating a terpenoid, comprising: a PLGA nanoparticle and a first terpenoid encapsulated in the PLGA nanoparticle.

2. The terpenoid encapsulated PLGA nanoparticle according to claim 1, wherein the PLGA nanoparticle comprises a PLGA copolymer and the weight ratio of the first terpenoid to the PLGA copolymer is from 1:50 to 1: 4.

3. The terpenoid encapsulated PLGA nanoparticle according to claim 2, wherein the weight ratio of the first terpenoid to the PLGA copolymer is from 1:25 to 1: 5.

4. The PLGA nanoparticle encapsulating terpenoids according to any one of claims 1-3, wherein the first terpenoid is selected from the group consisting of myrcene, beta-caryophyllene and nerolidol.

5. The terpenoid encapsulated PLGA nanoparticle according to claim 4, wherein the first terpenoid is myrcene, and the weight ratio of the first terpenoid to the PLGA copolymer is about 1: 22.

6. The terpenoid encapsulated PLGA nanoparticle according to claim 4, wherein the first terpenoid is β -caryophyllene and the weight ratio of the first terpenoid to the PLGA copolymer is 1:5 to 1:7, optionally wherein the nanoparticle further comprises PEG.

7. The terpenoid encapsulated PLGA nanoparticle according to claim 4, wherein the first terpenoid is nerolidol and the weight ratio of the first terpenoid to the PLGA copolymer is 1:5 to 1:7, optionally wherein the nanoparticle further comprises PEG.

8. The terpenoid encapsulated PLGA nanoparticle according to any one of claims 1-7, wherein said nanoparticle further encapsulates at least a second compound, wherein said second encapsulated compound is (i) a cannabinoid or (ii) a second terpenoid different from said first terpenoid.

9. The terpenoid encapsulated PLGA nanoparticle according to claim 4, wherein the second encapsulated compound is cannabigerolic acid (CBGA).

10. The terpenoid encapsulated PLGA nanoparticle according to claim 4, wherein the second encapsulated compound is Cannabidiol (CBD).

11. The PLGA nanoparticle encapsulating terpenoids according to claim 4, wherein the second encapsulating compound is Cannabinol (CBN).

12. The terpenoid encapsulated PLGA nanoparticle according to claim 4, wherein the second encapsulated compound is Cannabidivarin (CBDV).

13. The terpenoid encapsulated PLGA nanoparticle according to claim 4, wherein the second encapsulating compound is cannabichromene (CBC).

14. The terpenoid encapsulated PLGA nanoparticle according to claim 4, wherein the second encapsulating compound is cannabidiolic acid (CBDA).

15. The terpenoid encapsulated PLGA nanoparticle according to claim 4, wherein the second encapsulated compound is Cannabigerol (CBG).

16. The terpenoid encapsulated PLGA nanoparticle according to claim 4, wherein the second encapsulating compound is myrcene, beta-caryophyllene or nerolidol.

17. The terpenoid encapsulated PLGA nanoparticle according to claim 4, wherein the second encapsulating compound is limonene.

18. The terpenoid encapsulated PLGA nanoparticle according to claim 4, wherein the second encapsulating compound is phytol.

19. The terpenoid encapsulated PLGA nanoparticle according to claim 4, wherein the second encapsulating compound is pinene.

20. The terpenoid encapsulated PLGA nanoparticle according to claim 4, wherein the second encapsulating compound is linalool.

21. The terpenoid encapsulated PLGA nanoparticle according to any one of the preceding claims, wherein the PLGA nanoparticle has an average diameter of 200-350 nm.

22. The terpenoid encapsulated PLGA nanoparticle of any of the preceding claims, wherein the PLGA nanoparticle comprises a PLGA copolymer having a ratio of lactic acid to glycolic acid of about 10-90% lactic acid and about 90-10% glycolic acid.

23. The terpenoid encapsulated PLGA nanoparticle of claim 22, wherein the ratio of lactic acid to glycolic acid is about 10% lactic acid to about 90% glycolic acid.

24. The terpenoid encapsulated PLGA nanoparticle of claim 22, wherein the ratio of lactic acid to glycolic acid is about 25% lactic acid to about 75% glycolic acid.

25. The terpenoid encapsulated PLGA nanoparticle of claim 22, wherein the ratio of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid.

26. The terpenoid encapsulated PLGA nanoparticle of claim 22, wherein the ratio of lactic acid to glycolic acid is about 75% lactic acid to about 25% glycolic acid.

27. The terpenoid encapsulated PLGA nanoparticle of claim 22, wherein the ratio of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

28. A pharmaceutical composition comprising a first population of PLGA nanoparticles encapsulating a terpenoid according to any one of the preceding claims, and a pharmaceutically acceptable carrier or diluent.

29. The pharmaceutical composition of claim 28, further comprising trehalose.

30. The pharmaceutical composition of any one of claims 28-29, wherein the composition is a lyophilized composition.

31. The pharmaceutical composition of any one of claims 28-30, wherein the composition further comprises a second population of PLGA nanoparticles, wherein the second population of nanoparticles encapsulates a third compound, wherein the third compound is a different cannabinoid or terpenoid than the cannabinoid or terpenoid encapsulated in the first population of nanoparticles.

32. The pharmaceutical composition of claim 31, wherein the third encapsulating compound is cannabigerolic acid (CBGA).

33. The pharmaceutical composition of claim 31, wherein the third encapsulating compound is Cannabidiol (CBD).

34. The pharmaceutical composition of claim 31, wherein the third encapsulating compound is Cannabinol (CBN).

35. The pharmaceutical composition of claim 31, wherein the third encapsulating compound is Cannabidivarin (CBDV).

36. The pharmaceutical composition of claim 31, wherein the third encapsulating compound is cannabichromene (CBC).

37. The pharmaceutical composition of claim 31, wherein the third encapsulating compound is cannabidiolic acid (CBDA).

38. The pharmaceutical composition of claim 31, wherein the third encapsulating compound is Cannabigerol (CBG).

39. The pharmaceutical composition of claim 31, wherein the third encapsulating compound is myrcene, beta-caryophyllene, or nerolidol.

40. The pharmaceutical composition of claim 31, wherein the third encapsulating compound is limonene.

41. The pharmaceutical composition of claim 31, wherein the third encapsulating compound is phytol.

42. The pharmaceutical composition of claim 31, wherein the third encapsulating compound is pinene.

43. The pharmaceutical composition of claim 31, wherein the third encapsulating compound is linalool.

44. The pharmaceutical composition of any one of claims 31-43, wherein the composition further comprises a third population of PLGA nanoparticles, wherein the third population of nanoparticles encapsulates a fourth compound, wherein the fourth compound is a different cannabinoid or terpenoid than the cannabinoid or terpenoid encapsulated in the first population of nanoparticles or the second population of nanoparticles.

45. The pharmaceutical composition of claim 44, wherein the composition further comprises a fourth population of PLGA nanoparticles, wherein the fourth population of nanoparticles encapsulates a fifth compound, wherein the fifth compound is a different cannabinoid or terpenoid than the cannabinoid or terpenoid encapsulated in the first population of nanoparticles, the second population of nanoparticles, or the third population of nanoparticles.

46. A method for obtaining PLGA nanoparticles encapsulating terpenoids, wherein the method comprises the steps of:

(a) providing an organic solution comprising a terpenoid, a PLGA copolymer, and a solvent, and an aqueous solution comprising a surfactant;

(b) emulsifying the two solutions to form a suspension of PLGA nanoparticles encapsulating terpenoids;

(c) evaporating solvent from the emulsion; and

(d) obtaining PLGA nano-particles encapsulating terpenoids.

47. The method of claim 46, wherein the weight ratio of first terpenoid to PLGA copolymer in the solution of step (a) is about 1:5 to about 1: 1.

48. The method of claim 47, wherein the weight ratio of first terpenoid to PLGA copolymer in the solution of step (a) is about 1: 5.

49. The method of claim 47, wherein the weight ratio of first terpenoid to PLGA copolymer in the solution of step (a) is about 1: 4.

50. The method of claim 47, wherein the weight ratio of first terpenoid to PLGA copolymer in the solution of step (a) is about 1: 3.

51. The method of claim 47, wherein the weight ratio of first terpenoid to PLGA copolymer in the solution of step (a) is about 1: 2.

52. The method of claim 47, wherein the weight ratio of first terpenoid to PLGA copolymer in the solution of step (a) is about 1: 1.

53. The method of any one of claims 46-52, wherein the encapsulation efficiency of the first terpenoid in step (b) is about 4% to about 10%.

54. The method of claim 53, wherein the encapsulation efficiency is at least 4%.

55. The method of claim 53, wherein the encapsulation efficiency is at least 5%.

56. The method of claim 53, wherein the encapsulation efficiency is at least 6%.

57. The method of claim 53, wherein the encapsulation efficiency is at least 7%.

58. The method of claim 53, wherein the encapsulation efficiency is at least 8%.

59. The method of claim 53, wherein the encapsulation efficiency is at least 9%.

60. The method of claim 53, wherein the encapsulation efficiency is at least 10%.

61. The method of any one of claims 46-60, wherein the average weight ratio of encapsulated terpenoid and PLGA copolymer in the terpenoid-encapsulated PLGA nanoparticles of step (d) is between about 1:50 and about 1: 10.

62. The method of claim 61, wherein the average weight ratio of encapsulated terpenoid and PLGA copolymer in the terpenoid encapsulated PLGA nanoparticles is at least about 1: 50.

63. The method of claim 61, wherein the average weight ratio of encapsulated terpenoid and PLGA copolymer in the terpenoid encapsulated PLGA nanoparticles is at least about 1: 40.

64. The method of claim 61, wherein the average weight ratio of encapsulated terpenoid to PLGA copolymer in the terpenoid encapsulated PLGA nanoparticles is at least about 1: 30.

65. The method of claim 61, wherein the average weight ratio of encapsulated terpenoid to PLGA copolymer in the terpenoid encapsulated PLGA nanoparticles is at least about 1: 20.

66. The method of claim 61, wherein the average weight ratio of encapsulated terpenoid and PLGA copolymer in the terpenoid encapsulated PLGA nanoparticles is at least about 1: 10.

67. The method of any one of claims 46-66, wherein the PLGA copolymer has a lactic acid to glycolic acid ratio of about 10-90% lactic acid and about 90-10% glycolic acid.

68. The method of claim 67, wherein the ratio of lactic acid to glycolic acid is about 10% lactic acid to about 90% glycolic acid.

69. The method of claim 67, wherein the ratio of lactic acid to glycolic acid is about 25% lactic acid to about 75% glycolic acid.

70. The method of claim 67, wherein the ratio of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid.

71. The method of claim 67, wherein the ratio of lactic acid to glycolic acid is about 75% lactic acid to about 25% glycolic acid.

72. The method of claim 67, wherein the ratio of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

73. The method of any one of claims 46-72, wherein the solution of step (a) further comprises at least one cannabinoid or a terpenoid different from the first terpenoid.

74. The method of claim 73, wherein said at least one cannabinoid or terpenoid is selected from the group consisting of: cannabidiol (CBD), Cannabinol (CBN), Cannabidiol (CBDV), cannabidiol (CBC), cannabidiolic acid (CBDA) and Cannabinol (CBG).

75. The method of claim 73, wherein said at least one cannabinoid or terpenoid is selected from the group consisting of: myrcene, beta-caryophyllene, nerolidol, phytol, limonene, linalool and pinene.

76. The process of any one of claims 46-75, wherein the solvent is acetone, dichloromethane, or ethyl acetate.

77. The method of any one of claims 46-76, wherein the surfactant is a polyethylene glycol, a poloxamer, or a polyvinyl alcohol (PVA).

78. The method of claim 77, wherein the surfactant is polyvinyl alcohol (PVA).

79. The method of any one of claims 46-78, wherein the emulsifying step comprises homogenization or sonication.

80. The method of claim 79, wherein the emulsifying step is homogenization.

81. The method of claim 80, wherein homogenizing is performed at 20,000 to 30,000 rpm.

82. The method of claim 80, wherein homogenizing is performed at 24,000 rpm.

83. The method of any one of claims 80-82, wherein the solution is homogenized for 30 seconds to 10 minutes.

84. The method of claim 83, wherein the solution is homogenized for 1 minute.

85. The method of any one of claims 46-84, wherein the step of evaporating the solvent comprises at least one of stirring the solvent, applying a gas flow, applying heat, maintaining a low temperature of 10 ℃, or generating a vacuum.

86. The method of claim 85, wherein the step of evaporating the solvent comprises stirring the suspension at room temperature.

87. The method of claim 86, wherein the suspension is stirred for 5 minutes to 120 minutes to evaporate the solvent.

88. The method of claim 87, wherein the suspension is stirred for 60 minutes.

89. The method of any one of claims 46-88, wherein the step of obtaining PLGA nanoparticles encapsulating terpenoids comprises centrifugation, filtration, or centrifugation and filtration.

90. The method of claim 89, wherein the obtaining step comprises centrifugation.

91. The method of claim 90, wherein centrifugation is performed at 2,000x g to 15,000x g.

92. The method of claim 91, wherein the centrifugation is performed at 4,000 Xg.

93. The method of any one of claims 46-92, further comprising the subsequent step of adding a cryoprotectant to the PLGA nanoparticles encapsulating terpenoids.

94. The method of claim 93, wherein the cryoprotectant is trehalose.

95. The method of any one of claims 93-94, wherein the cryoprotectant is added in an amount of 1-10% (w/v) of the PLGA nanoparticles encapsulating a terpenoid.

96. The method of claim 95, wherein the cryoprotectant is added in an amount of 5% (w/v) of the PLGA nanoparticles encapsulating terpenoids.

97. The method of any one of claims 44-96, further comprising lyophilizing the PLGA nanoparticles encapsulating a terpenoid.

98. A PLGA nanoparticle encapsulating terpenoids obtained by the method of any one of claims 46-97.

99. The pharmaceutical composition of any one of claims 28-45 for desensitization of TRPV1 in cells of a mammalian subject, wherein the pharmaceutical composition is administered to the mammalian subject by a route of administration in an amount and for a time sufficient to cause TRPV1 desensitization in cells of the mammalian subject.

100. The pharmaceutical composition for use according to claim 99, wherein the cell is a nociceptor.

101. The pharmaceutical composition for use according to claim 100, wherein the nociceptors are peripheral nociceptors.

102. The pharmaceutical composition for use according to claim 100, wherein the nociceptors are visceral nociceptors.

103. The pharmaceutical composition for use according to any one of claims 99-102, wherein the pharmaceutical composition is for oral administration.

104. The pharmaceutical composition for use according to any one of claims 99-102, wherein the pharmaceutical composition is for topical administration.

105. The pharmaceutical composition for use according to any one of claims 99-102, wherein the pharmaceutical composition is administered systemically.

106. The pharmaceutical composition for use according to any one of claims 99-102, wherein the pharmaceutical composition is administered intravenously.

107. The pharmaceutical composition for use according to any one of claims 99-102, wherein the pharmaceutical composition is administered subcutaneously.

108. The pharmaceutical composition for use according to any one of claims 99-102, wherein the pharmaceutical composition is administered by inhalation.

109. The pharmaceutical composition of any one of claims 28-45 for use in treating pain in a mammalian subject, wherein the pharmaceutical composition is administered to the subject by a route of administration in an amount and for a time sufficient to cause desensitization of TRPV1 at nociceptors in the subject.

110. The pharmaceutical composition for use according to claim 109, wherein the nociceptors are peripheral nociceptors and the pharmaceutical composition is administered topically.

111. The pharmaceutical composition for use according to claim 109, wherein the nociceptors are visceral nociceptors and the pharmaceutical composition is administered systemically.

112. The pharmaceutical composition for use according to any one of claims 109-111, wherein the pain is neuropathic pain.

113. The pharmaceutical composition for use according to claim 112, wherein the neuropathic pain is diabetic peripheral neuropathic pain.

114. The pharmaceutical composition for use according to any one of claims 109-111, wherein the pain is postherpetic neuralgia.

115. The pharmaceutical composition for use according to any one of claims 109-114, wherein the pharmaceutical composition is administered at least once daily for at least 3 days.

116. The pharmaceutical composition for use according to claim 115, wherein said pharmaceutical composition is administered at least once daily for at least 5 days.

117. The pharmaceutical composition for use according to claim 115, wherein said pharmaceutical composition is administered at least once daily for at least 7 days.

118. The pharmaceutical composition for use according to any one of claims 109-117, wherein the pharmaceutical combination is administered by a route and schedule of administration at a dose sufficient to maintain the first terpenoid at the nociceptor at a level effective to desensitize TRPV1 at the nociceptor for at least 3 days.

119. The pharmaceutical composition for use according to claim 118, wherein the pharmaceutical combination is administered by a route and schedule of administration at a dose sufficient to maintain the first terpenoid at the nociceptor at a level effective to desensitize TRPV1 at the nociceptor for at least 5 days.

120. The pharmaceutical composition for use according to claim 118, wherein the pharmaceutical combination is administered by a route and schedule of administration at a dose sufficient to maintain the first terpenoid at the nociceptor at a level effective to desensitize TRPV1 at the nociceptor for at least 7 days.

121. The pharmaceutical composition according to any one of claims 28-45, for use in the treatment of cardiac hypertrophy in a mammalian subject wherein an anti-hypertrophy effective amount of said pharmaceutical composition is administered to said subject.

122. The pharmaceutical composition for use according to claim 121, wherein the pharmaceutical composition is for oral administration.

123. The pharmaceutical composition for use according to claim 121, wherein the pharmaceutical composition is administered systemically.

124. The pharmaceutical composition for use according to claim 121, wherein the pharmaceutical composition is administered intravenously.

125. The pharmaceutical composition for use according to claim 121, wherein the pharmaceutical composition is administered subcutaneously.

126. The pharmaceutical composition for use according to claim 121, wherein the pharmaceutical composition is administered by inhalation.

127. The pharmaceutical composition for use of claim 121, wherein the pharmaceutical composition is administered orally.

128. The pharmaceutical composition of any one of claims 28-45 for use in the prophylactic treatment of cardiac hypertrophy in a mammalian subject wherein an anti-hypertrophy effective amount of the pharmaceutical composition is administered to a subject at risk of cardiac hypertrophy.

129. The pharmaceutical composition of any one of claims 28-45, for use in treating overactive bladder in a mammalian subject, wherein a therapeutically effective amount of the pharmaceutical composition is administered to the subject.

130. The pharmaceutical composition for use according to claim 129, wherein the pharmaceutical composition is administered systemically.

131. The pharmaceutical composition for use according to claim 129, wherein the pharmaceutical composition is administered by bladder irrigation.

132. The pharmaceutical composition of any one of claims 28-45 for use in treating a refractory chronic cough in a mammalian subject, wherein a therapeutically effective amount of the pharmaceutical composition is administered to the subject.

133. The pharmaceutical composition for use according to claim 132, wherein the pharmaceutical composition is administered systemically.

134. The pharmaceutical composition for use according to claim 132, wherein the pharmaceutical composition is administered by inhalation.

135. A cannabinoid-encapsulating PLGA nanoparticle comprising: a PLGA nanoparticle and a first cannabinoid encapsulated in the PLGA nanoparticle.

136. The cannabinoid-encapsulating PLGA nanoparticle of claim 135, wherein the PLGA nanoparticle comprises a PLGA copolymer, and the weight ratio of the first cannabinoid to the PLGA copolymer is from 1:50 to 1: 4.

137. The cannabinoid-encapsulated PLGA nanoparticle of claim 136, wherein the weight ratio of the first cannabinoid to the PLGA copolymer is from 1:25 to 1: 5.

138. The cannabinoid-encapsulating PLGA nanoparticle of any of claims 135-137, wherein the first cannabinoid is selected from the group consisting of cannabidiol, cannabidivarin, cannabigerol, and cannabichromene.

139. The cannabinoid-encapsulated PLGA nanoparticle of claim 138, wherein the first cannabinoid is cannabidiol and the weight ratio of the first cannabinoid to the PLGA copolymer is about 1: 14.

140. The cannabinoid-encapsulated PLGA nanoparticle of claim 138, wherein the first cannabinoid is cannabidiol and the weight ratio of the first cannabinoid to the PLGA copolymer is from 1:5 to 1: 7.

141. The cannabinoid-encapsulated PLGA nanoparticle of claim 138, wherein the first cannabinoid is cannabidiol and the weight ratio of the first cannabinoid to the PLGA copolymer is from 1:5 to 1: 7.

142. The cannabinoid-encapsulating PLGA nanoparticle of any of claims 135-141, wherein the nanoparticle further encapsulates at least one second compound, wherein the second encapsulated compound is (i) a terpenoid or (ii) a second cannabinoid that is different from the first cannabinoid.

143. The cannabinoid-encapsulating PLGA nanoparticle of claim 142, wherein the second encapsulating compound is cannabigerolic acid (CBGA).

144. The cannabinoid-encapsulating PLGA nanoparticle of claim 142, wherein the second encapsulating compound is Cannabivarinol (CBV).

145. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulated compound is Cannabinol (CBN).

146. The cannabinoid-encapsulating PLGA nanoparticle of claim 142, wherein the second encapsulating compound is Cannabidivarin (CBDV).

147. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulating compound is cannabichromene (CBC).

148. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulating compound is cannabidiolic acid (CBDA).

149. The cannabinoid-encapsulating PLGA nanoparticle recited in claim 142, wherein the second encapsulating compound is Cannabigerol (CBG).

150. The cannabinoid-encapsulating PLGA nanoparticle recited in claim 142, wherein the second encapsulating compound is myrcene, β -caryophyllene, or nerolidol.

151. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulating compound is limonene.

152. The cannabinoid-encapsulating PLGA nanoparticle recited in claim 142, wherein the second encapsulating compound is phytol.

153. The cannabinoid-encapsulating PLGA nanoparticle of claim 142, wherein the second encapsulating compound is pinene.

154. The cannabinoid-encapsulated PLGA nanoparticle of claim 142, wherein the second encapsulating compound is linalool.

155. The cannabinoid-encapsulated PLGA nanoparticle according to claim 142, wherein the second encapsulated compound is myrcene.

156. The cannabinoid-encapsulating PLGA nanoparticle of any of claims 135-155, wherein the PLGA nanoparticle has an average diameter of 200-350 nm.

157. The cannabinoid-encapsulating PLGA nanoparticle of any of claims 135-156, wherein the PLGA nanoparticle comprises a PLGA copolymer having a ratio of lactic acid to glycolic acid of between about 10-90% lactic acid and about 90-10% glycolic acid.

158. The cannabinoid-encapsulated PLGA nanoparticle of claim 157, wherein the ratio of lactic acid to glycolic acid is about 10% lactic acid to about 90% glycolic acid.

159. The cannabinoid-encapsulated PLGA nanoparticle of claim 157, wherein the ratio of lactic acid to glycolic acid is about 25% lactic acid to about 75% glycolic acid.

160. The cannabinoid-encapsulated PLGA nanoparticle of claim 157, wherein the ratio of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid.

161. The cannabinoid-encapsulated PLGA nanoparticle of claim 157, wherein the ratio of lactic acid to glycolic acid is about 75% lactic acid to about 25% glycolic acid.

162. The cannabinoid-encapsulated PLGA nanoparticle of claim 157, wherein the ratio of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

163. A pharmaceutical composition comprising a first population of cannabinoid-encapsulating PLGA nanoparticles according to any of claims 135 and 162, and a pharmaceutically acceptable carrier or diluent.

164. The pharmaceutical composition of claim 163, further comprising trehalose.

165. The pharmaceutical composition of any one of claims 163-164, wherein the composition is a lyophilized composition.

166. The pharmaceutical composition of any one of claims 163-165, wherein the composition further comprises a second population of PLGA nanoparticles, wherein the second population of nanoparticles encapsulate a third compound, wherein the third compound is a terpenoid or cannabinoid that is different from the terpenoid and cannabinoid encapsulated in the first population of nanoparticles.

167. The pharmaceutical composition of claim 166, wherein the third encapsulating compound is cannabigerolic acid (CBGA).

168. The pharmaceutical composition of claim 166, wherein the third encapsulating compound is Cannabidiol (CBD).

169. The pharmaceutical composition of claim 166, wherein the third encapsulating compound is Cannabinol (CBN).

170. The pharmaceutical composition of claim 166, wherein the third encapsulating compound is Cannabidivarin (CBDV).

171. The pharmaceutical composition of claim 166, wherein the third encapsulating compound is cannabichromene (CBC).

172. The pharmaceutical composition of claim 166, wherein the third encapsulating compound is cannabidiolic acid (CBDA).

173. The pharmaceutical composition of claim 166, wherein the third encapsulating compound is Cannabigerol (CBG).

174. The pharmaceutical composition of claim 166, wherein the third encapsulating compound is myrcene, beta-caryophyllene, or nerolidol.

175. The pharmaceutical composition of claim 166, wherein the third encapsulating compound is limonene.

176. The pharmaceutical composition of claim 166, wherein the third encapsulating compound is phytol.

177. The pharmaceutical composition of claim 166, wherein the third encapsulating compound is pinene.

178. The pharmaceutical composition of claim 166, wherein the third encapsulating compound is linalool.

179. The pharmaceutical composition of claim 166, wherein the third encapsulating compound is myrcene.

180. The pharmaceutical composition of any one of claims 163-179, wherein the composition further comprises a third population of PLGA nanoparticles, wherein the third population of nanoparticles encapsulates a fourth compound, wherein the fourth compound is a cannabinoid or terpenoid different from the cannabinoids and terpenoids encapsulated in the first population of nanoparticles or the second population of nanoparticles.

181. The pharmaceutical composition of claim 180, wherein the composition further comprises a fourth population of PLGA nanoparticles, wherein the fourth population of nanoparticles encapsulates a fifth compound, wherein the fifth compound is a cannabinoid or a terpenoid different from the cannabinoids and terpenoids encapsulated in the first population of nanoparticles, the second population of nanoparticles, or the third population of nanoparticles.

182. A method for obtaining cannabinoid-encapsulating PLGA nanoparticles, wherein the method comprises the steps of:

(a) providing an organic solution comprising a first cannabinoid, a PLGA copolymer, and a solvent, and an aqueous solution comprising a surfactant;

(b) emulsifying the two solutions to form a suspension of cannabinoid-encapsulating PLGA nanoparticles;

(c) evaporating the solvent from the emulsion; and

(d) obtaining cannabinoid-encapsulating PLGA nanoparticles.

183. The method of claim 182, wherein the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is from about 1:5 to about 1: 1.

184. The method of claim 182, wherein the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1: 5.

185. The method of claim 182, wherein the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1: 4.

186. The method of claim 182, wherein the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1: 3.

187. The method of claim 182, wherein the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1: 2.

188. The method of claim 182, wherein the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1: 1.

189. The method of any of claims 182-188 wherein the encapsulation efficiency of the first cannabinoid in step (b) is from about 4% to about 10%.

190. The method of claim 189, wherein the encapsulation efficiency is at least 4%.

191. The method of claim 189, wherein the encapsulation efficiency is at least 5%.

192. The method of claim 189, wherein the encapsulation efficiency is at least 6%.

193. The method of claim 189, wherein the encapsulation efficiency is at least 7%.

194. The method of claim 189, wherein the encapsulation efficiency is at least 8%.

195. The method of claim 189, wherein the encapsulation efficiency is at least 9%.

196. The method of claim 189, wherein the encapsulation efficiency is at least 10%.

197. The method of claim 182, 196, wherein the average weight ratio of cannabinoid to PLGA copolymer encapsulated in the cannabinoid-encapsulating PLGA nanoparticles of step (d) is from about 1:50 to about 1: 10.

198. The method of claim 197, wherein the average weight ratio of cannabinoid encapsulated and PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticle is at least about 1: 50.

199. The method of claim 197, wherein the average weight ratio of cannabinoid encapsulated and PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticle is at least about 1: 40.

200. The method of claim 197, wherein the average weight ratio of cannabinoid encapsulated to PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles is at least about 1: 30.

201. The method of claim 197, wherein the average weight ratio of cannabinoid encapsulated and PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticle is at least about 1: 20.

202. The method of claim 197, wherein the average weight ratio of cannabinoid encapsulated and PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticle is at least about 1: 10.

203. The method of any one of claims 182 to 202 wherein the PLGA copolymer has a lactic acid to glycolic acid ratio of about 10-90% lactic acid and about 90-10% glycolic acid.

204. The method of claim 203, wherein the ratio of lactic acid to glycolic acid is about 10% lactic acid to about 90% glycolic acid.

205. The method of claim 203, wherein the ratio of lactic acid to glycolic acid is about 25% lactic acid to about 75% glycolic acid.

206. The method of claim 203, wherein the ratio of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid.

207. The method of claim 203, wherein the ratio of lactic acid to glycolic acid is about 75% lactic acid to about 25% glycolic acid.

208. The method of claim 203, wherein the ratio of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

209. The method of any of claims 182-208 wherein the solution of step (a) further comprises at least one cannabinoid or terpenoid different from the first cannabinoid.

210. The method of claim 209, wherein said at least one cannabinoid or terpenoid is selected from the group consisting of: cannabinol (CBN), sub-Cannabinol (CBDV), cannabichromene (CBC), cannabidiolic acid (CBDA) and Cannabigerol (CBG).

211. The method of any one of claims 208-209, wherein the at least one cannabinoid or terpenoid is selected from the group consisting of: myrcene, beta-caryophyllene, nerolidol, phytol, limonene, linalool and pinene.

212. The process of any one of claims 182-211, wherein the solvent is acetone, dichloromethane, or ethyl acetate.

213. The method of any one of claims 182-212, wherein the surfactant is a polyethylene glycol, a poloxamer or a polyvinyl alcohol (PVA).

214. The method of claim 213, wherein the surfactant is polyvinyl alcohol (PVA).

215. The method of any one of claims 182-214, wherein the emulsifying step comprises homogenization or sonication.

216. The method of claim 215, wherein the emulsifying step is homogenization.

217. The method of claim 216, wherein homogenizing is performed at 20,000 to 30,000 rpm.

218. The method of claim 217, wherein the homogenizing is performed at 24,000 rpm.

219. The method of any one of claims 216-218 wherein the solution is homogenized for 30 seconds to 10 minutes.

220. The method of claim 219, wherein the solution is homogenized for 1 minute.

221. The method of any one of claims 182-220, wherein the step of evaporating the solvent comprises at least one of stirring the solvent, applying a gas flow, applying heat, maintaining a low temperature of 10 ℃, or generating a vacuum.

222. The method of claim 221, wherein the step of evaporating the solvent comprises stirring the suspension at room temperature.

223. The process of claim 222, wherein the suspension is stirred for 5 minutes to 120 minutes to evaporate the solvent.

224. The method of claim 223, wherein the suspension is stirred for 60 minutes.

225. The method of any one of claims 182-224, wherein the step of obtaining the cannabinoid-encapsulated PLGA nanoparticles comprises centrifugation, filtration, or centrifugation and filtration.

226. The method of claim 225, wherein the obtaining step comprises centrifugation.

227. The method of claim 226, wherein the centrifugation is performed at 2,000x g to 15,000x g.

228. The method of claim 224, wherein the centrifugation is performed at 4,000 xg.

229. The method as recited in any one of claims 182-228, further comprising the subsequent step of adding a cryoprotectant to the cannabinoid-encapsulating PLGA nanoparticles.

230. The method of claim 229, wherein said cryoprotectant is trehalose.

231. The method of any one of claims 229-230 wherein the cryoprotectant is added in an amount of 1-10% (w/v) of the cannabinoid-encapsulating PLGA nanoparticles.

232. The method of claim 231, wherein the cryoprotectant is added in an amount of 5% (w/v) of the cannabinoid-encapsulated PLGA nanoparticles.

233. The method of any one of claims 182-232, further comprising lyophilizing the cannabinoid-encapsulated PLGA nanoparticles.

234. Cannabinoid-encapsulating PLGA nanoparticles obtained by the method of any of claims 182-233.

235. The pharmaceutical composition of any one of claims 163-181 for use in desensitization of TRPV1 in a cell of a mammalian subject, wherein the pharmaceutical composition is administered to the mammalian subject by a route of administration in an amount and for a time sufficient to cause desensitization of TRPV1 in a cell within the mammalian subject.

236. The pharmaceutical composition for use according to claim 235, wherein the cell is a nociceptor.

237. A pharmaceutical composition for use according to claim 236, wherein the nociceptors are peripheral nociceptors.

238. A pharmaceutical composition for use according to claim 236, wherein the nociceptors are visceral nociceptors.

239. The pharmaceutical composition for use according to any one of claims 235-238, wherein the pharmaceutical composition is for oral administration.

240. The pharmaceutical composition for use according to any one of claims 235-238, wherein the pharmaceutical composition is for topical administration.

241. The pharmaceutical composition for use according to any one of claims 235-238, wherein the pharmaceutical composition is administered systemically.

242. The pharmaceutical composition for use according to any one of claims 235-238, wherein the pharmaceutical composition is for intravenous administration.

243. The pharmaceutical composition for use according to any one of claims 235-238, wherein the pharmaceutical composition is administered subcutaneously.

244. The pharmaceutical composition for use according to any one of claims 235-238, wherein the pharmaceutical composition is administered by inhalation.

245. The pharmaceutical composition of any one of claims 163-181 for use in treating pain in a mammalian subject, wherein the pharmaceutical composition is administered to the subject by a route of administration in an amount and for a time sufficient to cause desensitization of TRPV1 of nociceptors in the subject.

246. The pharmaceutical composition for use according to claim 245, wherein the nociceptors are peripheral nociceptors and the pharmaceutical composition is administered topically.

247. The pharmaceutical composition for use according to claim 245, wherein the nociceptors are visceral nociceptors and the pharmaceutical composition is administered systemically.

248. The pharmaceutical composition for use according to any one of claims 245-247, wherein the pain is neuropathic pain.

249. The pharmaceutical composition for use according to claim 248, wherein said neuropathic pain is diabetic peripheral neuropathic pain.

250. The pharmaceutical composition for use according to any one of claims 245-247, wherein the pain is post-herpetic neuralgia.

251. The pharmaceutical composition for use according to any one of claims 245-250, wherein the pharmaceutical composition is administered at least once daily for at least 3 days.

252. The pharmaceutical composition for use according to claim 251, wherein the pharmaceutical composition is administered at least once daily for at least 5 days.

253. The pharmaceutical composition for use according to claim 252, wherein said pharmaceutical composition is administered at least once daily for at least 7 days.

254. The pharmaceutical composition for use according to claim 253, wherein said pharmaceutical composition is administered at least once daily for more than 7 days.

255. The pharmaceutical composition for use according to any one of claims 245-254, wherein the pharmaceutical composition is administered by a route and schedule of administration at a dose sufficient to maintain cannabinoid levels at nociceptors at a level effective to desensitize TRPV1 at nociceptors for at least 3 days.

256. The pharmaceutical composition for use according to claim 255, wherein the pharmaceutical composition is administered by a route and schedule of administration at a dose sufficient to maintain cannabinoid levels at nociceptors at a level effective to desensitize TRPV1 at nociceptors for at least 5 days.

257. The pharmaceutical composition for use according to claim 256, wherein the pharmaceutical composition is administered by a route and schedule of administration at a dose sufficient to maintain cannabinoid levels at nociceptors at a level effective to desensitize TRPV1 at nociceptors for at least 7 days.

258. The pharmaceutical composition of any one of claims 163-181 for use in treating cardiac hypertrophy in a mammalian subject wherein an anti-hypertrophy effective amount of the pharmaceutical composition is administered to the subject.

259. The pharmaceutical composition for use according to claim 258, wherein the pharmaceutical composition is administered orally.

260. The pharmaceutical composition for use according to claim 258, wherein the pharmaceutical composition is administered systemically.

261. The pharmaceutical composition for use according to claim 258, wherein the pharmaceutical composition is administered intravenously.

262. The pharmaceutical composition for use according to claim 258, wherein the pharmaceutical composition is administered subcutaneously.

263. The pharmaceutical composition for use according to claim 258, wherein the pharmaceutical composition is administered by inhalation.

264. The pharmaceutical composition for use according to claim 258, wherein the pharmaceutical composition is administered orally.

265. The pharmaceutical composition according to any one of claims 163-181 for use in the prophylactic treatment of cardiac hypertrophy in a mammalian subject wherein an anti-hypertrophy effective amount of the pharmaceutical composition is administered to a subject at risk of cardiac hypertrophy.

266. The pharmaceutical composition of any one of claims 163-181 for use in treating overactive bladder in a mammalian subject, wherein a therapeutically effective amount of the pharmaceutical composition is administered to the subject.

267. The pharmaceutical composition for use according to claim 266, wherein said pharmaceutical composition is administered systemically.

268. The pharmaceutical composition for use according to claim 266, wherein the pharmaceutical composition is administered by bladder irrigation.

269. The pharmaceutical composition of any one of claims 163-181 for use in treating intractable chronic cough in a mammalian subject, wherein a therapeutically effective amount of the pharmaceutical composition is administered to the subject.

270. The pharmaceutical composition for use according to claim 269, wherein the pharmaceutical composition is administered systemically.

271. The pharmaceutical composition for use according to claim 269, wherein the pharmaceutical composition is administered by inhalation.

Background

Transient receptor superfamily (TRP) channels, such as TRPV1, TRPM8, and TRPA1 are non-selective cation channels that conduct calcium and sodium to a range of cells in mammals. They are present on sensory neurons and were originally identified as playing a role in nociception due to their reactivity at the molecular level to nociceptive plant secondary metabolites (e.g., capsaicin) and compounds that otherwise stimulate and mimic the burning or cooling sensation (e.g., allicin, cinnamaldehyde, menthol).

Because of their role in nociception, TRP channels have been identified as targets for the treatment of pain disorders. Both antagonism and agonism of TRP channels have been used for pain management. For example, TRPV1 antagonists may be useful for acute analgesia. For chronic pain management TRPV1 agonists are commonly used. The latter strategy takes advantage of the fact that: sustained TRPV1 receptor agonism leads to desensitization of the cell surface (receptor internalization, degradation and recycling). Prolonged agonism of TRPV1 also leads to calcium and sodium cation overload of TRPV 1-containing sensory neurons, leading to cell death.

In practice, achieving desensitization using TRPV1 agonists involves repeated local application of high levels of the well-known TRPV1 agonist capsaicin to the affected area over time. This treatment is effective and relatively inexpensive. However, it also has weaknesses.

First, high affinity and high specificity TRPV1 agonists target only the nociceptor-containing TRPV1 leaving other sensory neurons and TRP channels involved in pain unaffected. Second, the use of high affinity and high specificity TRPV1 agonists such as capsaicin causes high discomfort during the initial stages of treatment prior to desensitization. For this reason, post-herpetic pain cannot currently be addressed using TRPV 1-mediated desensitization due to the highly irritating nature of treatments to sensitive areas such as the gastric and genital mucosa. Third, capsaicin-mediated desensitization therapy is limited to topical use; visceral pain, headache and certain musculoskeletal pain conditions cannot be addressed by this therapy.

Thus, there is a need for therapeutic TRPV1 ligands, such as TRPV1 agonists, that have lower affinity than capsaicin and can be administered orally for systemic relief. Such lower affinity ligands may reduce pain during desensitization, thereby allowing local treatment of sensitive body regions. There is a need for TRPV1 ligands with broader target specificity that are capable of targeting multiple nociceptor-bearing TRP types, thereby improving the degree of tissue desensitization. Furthermore, there is also a need for pharmaceutical compositions comprising such TRPV1 ligands that are suitable for systemic administration in addition to local administration and long-term release of such TRPV1 ligands to induce long-term down-regulation of TRPV 1. Oral administration is highly desirable for analgesics that are used systemically.

Such novel oral drugs can also be used for the treatment of various diseases related to TRPV1 other than pain. Although TRP channels were first shown to be involved in pain and nociception, they are now known to have various other physiological roles, suggesting that they may be targets for the treatment of other diseases. For example, TRP channels have been identified as targets for the treatment of cardiovascular diseases; targeted pharmacological inhibition of TRPV1 has been shown to significantly reduce cardiac hypertrophy in mouse models. See U.S. patent No. 9,084,786. Thus, chronic downregulation of TRPV1 levels by receptor desensitization with TRPV1 agonists is expected to similarly protect and potentially rescue cardiac hypertrophy and its associated symptoms and consequences (cardiac remodeling, cardiac fibrosis, apoptosis, hypertension or heart failure).

However, there are currently no oral or other pharmacological compositions of TRP agonists suitable for systemic administration and long-term down-regulation of TRP in internal organs. Therefore, there is a need to develop such methods in a manner similar to the chronic pain methods described above.

The generation of nanoparticles is an effective way to alter the surface properties of active molecules to make them more bioavailable and to alter their kinetics of systemic release. Oral nanoparticles can be produced that provide effective, time-release formulations of various TRP channel modulators (e.g., terpenoids and cannabinoids). Oral formulations of such TRP ligands and pharmacological compositions would provide a new and more effective means of treating various diseases associated with the TRP channel including pain disorders and cardiovascular disease

Disclosure of Invention

Applicants have recently demonstrated that myrcene is a novel TRPV1 agonist that causes TRPV1 desensitization after prolonged exposure. Applicants further indicate that other terpenoids and cannabinoids, including those that do not themselves exhibit significant TRPV1 agonist activity, are used in combination to increase the efficacy of myrcene. Furthermore, multiple TRP channels other than TRPV1 were identified as targets for myrcene, suggesting that myrcene efficacy may extend beyond TRPV1 to other nociceptive neurons, with the primary pain-transmitting channel being a distinct TRP receptor. These findings are described in PCT application PCT/US2018/033956, which is incorporated herein by reference in its entirety.

New findings indicate that myrcene, as well as other terpenoids and cannabinoids, may be alternative therapeutic TRP receptor ligands, which may affect multiple TRP channels in addition to TRPV1, cause less discomfort during initial treatment compared to capsaicin, and may be suitable for systemic administration for chronic treatment. See PCT/US 2018/033956. For the therapeutic use of new TRPV1 agonists and agonist mixtures, there is a need to further develop pharmaceutical compositions comprising myrcene and other terpenoids and cannabinoids, in particular compositions that allow systemic administration as well as chronic and long-term release of myrcene, other terpenoids or cannabinoids. This is particularly important because terpenoids and cannabinoids are highly lipophilic and volatile, having low boiling points, and thus administration of terpenoids and cannabinoids is limited to rapid release pharmaceutical or natural product applications.

The present invention addresses the need to stabilize terpenoids and cannabinoids and make them more bioavailable under sustained release by providing novel therapeutic compositions for systemic administration (e.g., oral administration) and for long-term release of terpenoids and cannabinoids, as well as methods of making and using the same. In particular, the present invention employs PLGA nanoparticles encapsulating terpenoids and cannabinoids for delivery of the terpenoids and cannabinoids to a mammalian subject. In addition, the present invention provides therapeutic compositions containing more than one terpenoid or cannabinoid. The therapeutic composition may contain more than one population of nanoparticles, each population encapsulating a separate terpenoid or cannabinoid, or a population of nanoparticles, each nanoparticle encapsulating more than one terpenoid or cannabinoid. The terpenoid or cannabinoid encapsulated in the nanoparticle may be a synthetic compound or a compound isolated from a chemical or natural mixture. In some embodiments, the compound is isolated from a cannabis extract. In some embodiments, the delta-9 tetrahydrocannabinol is additionally or separately encapsulated in a nanoparticle.

Accordingly, in a first aspect, provided herein is a PLGA nanoparticle encapsulating terpenoids, comprising one PLGA nanoparticle and a first terpenoid encapsulated in the PLGA nanoparticle.

In some embodiments, wherein the PLGA nanoparticle comprises a PLGA copolymer, and the weight ratio of the first terpenoid to the PLGA copolymer is 1:50 to 1: 10. In one embodiment, the weight ratio of the first terpenoid to the PLGA copolymer is about 1: 14.

In some embodiments, the first terpenoid is selected from myrcene, beta-caryophyllene, and nerolidol. In some embodiments, wherein the first terpenoid is myrcene, and the weight ratio of the first terpenoid to the PLGA copolymer is about 1: 22. In some embodiments, wherein the first terpenoid is β -caryophyllene, and the weight ratio of the first terpenoid to the PLGA copolymer is 1:5 to 1: 7. in some embodiments, the first terpenoid is nerolidol and the weight ratio of the first terpenoid to the PLGA copolymer is in the range of 1:5 and 1: 7.

in some embodiments, the PLGA nanoparticles are surface-modified with PEG. The molecular weight of PEG can be in the range of 2,000-20,000 Da.

In some embodiments, the nanoparticle further encapsulates at least a second compound, wherein the second encapsulated compound is (i) a cannabinoid or (ii) a second terpenoid different from the first terpenoid. In some embodiments, the second encapsulating compound is cannabigerolic acid (CBGA). In some embodiments, the second encapsulating compound is Cannabidiol (CBD). In some embodiments, the second encapsulating compound is Cannabinol (CBN). In some embodiments, the second encapsulating compound is Cannabidivarin (CBDV). In some embodiments, the second encapsulating compound is cannabichromene (CBC). In some embodiments, the second encapsulating compound is cannabidiolic acid (CBDA). In some embodiments, the second encapsulating compound is Cannabigerol (CBG). In some embodiments, the second encapsulating compound is myrcene, beta-caryophyllene, or nerolidol. In some embodiments, the second encapsulating compound is limonene. In some embodiments, the second encapsulated compound is phytol. In some embodiments, the second encapsulated compound is pinene. In some embodiments, the second encapsulating compound is linalool.

In some embodiments, the PLGA nanoparticles have an average diameter of 200-350 nm.

In some embodiments, the PLGA nanoparticles comprise a PLGA copolymer having a ratio of lactic acid to glycolic acid of about 10-90% lactic acid and about 90-10% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 10% lactic acid to about 90% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 25% lactic acid to about 75% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 75% lactic acid to about 25% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

In another aspect, the present invention provides a pharmaceutical composition comprising a first population of PLGA nanoparticles encapsulating a terpenoid described herein and a pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical composition further comprises trehalose. In some embodiments, the composition is lyophilized. In some embodiments, the composition further comprises a second population of PLGA nanoparticles, wherein the second population of nanoparticles encapsulates a third compound, wherein the third compound is a different cannabinoid or terpenoid than the cannabinoid or terpenoid encapsulated in the first population of nanoparticles.

In some embodiments, the third encapsulating compound is cannabigerolic acid (CBGA). In some embodiments, the third encapsulating compound is Cannabidiol (CBD). In some embodiments, the third encapsulating compound is Cannabinol (CBN). In some embodiments, the third encapsulating compound is Cannabidivarin (CBDV). In some embodiments, the third encapsulating compound is cannabichromene (CBC). In some embodiments, the third encapsulating compound is cannabidiolic acid (CBDA). In some embodiments, the third encapsulating compound is Cannabigerol (CBG). In some embodiments, the third encapsulated compound is myrcene, beta-caryophyllene, or nerolidol. In some embodiments, the third encapsulating compound is limonene. In some embodiments, the third encapsulated compound is phytol. In some embodiments, the third encapsulated compound is pinene. In some embodiments, the third encapsulated compound is linalool.

In some embodiments, the composition further comprises a third population of PLGA nanoparticles, wherein the third population of nanoparticles encapsulates a fourth compound, wherein the fourth compound is a different cannabinoid or terpenoid than the cannabinoid or terpenoid encapsulated in the first or second population of nanoparticles. In some embodiments, the composition further comprises a fourth population of PLGA nanoparticles, wherein the fourth population of nanoparticles encapsulates a fifth compound, wherein the fifth compound is a different cannabinoid or terpenoid than the cannabinoid or terpenoid encapsulated in the first, second, or third population of nanoparticles.

In yet another aspect, the present invention provides a method for obtaining PLGA nanoparticles encapsulating terpenoids, wherein the method comprises the steps of: (a) providing an organic solution comprising a terpenoid, a PLGA copolymer, and a solvent, and an aqueous solution comprising a surfactant; (b) emulsifying the two solutions to form a suspension of PLGA nanoparticles encapsulating terpenoids; (c) evaporating the solvent from the emulsion; and (d) obtaining PLGA nanoparticles encapsulating terpenoids.

In some embodiments, the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is about 1:5 to about 1: 1. in some embodiments, the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is about 1: 5. in some embodiments, the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is about 1: 4. in some embodiments, the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is about 1: 3. in some embodiments, the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is about 1: 2. in some embodiments, the weight ratio of the first terpenoid to the PLGA copolymer in the solution of step (a) is about 1: 1.

in some embodiments, the encapsulation efficiency of the first terpene in step (b) is from about 4% to about 10%. In some embodiments, the encapsulation efficiency is at least 4%. In some embodiments, the encapsulation efficiency is at least 5%. In some embodiments, the encapsulation efficiency is at least 6%. In some embodiments, the encapsulation efficiency is at least 7%. In some embodiments, the encapsulation efficiency is at least 8%. In some embodiments, the encapsulation efficiency is at least 9%. In some embodiments, the encapsulation efficiency is at least 10%.

In some embodiments, the average weight ratio of encapsulated terpenoid and PLGA copolymer in the PLGA nanoparticles encapsulating terpenoid of step (d) is about 1:50 to about 1: 10. In some embodiments, the average weight ratio of encapsulated terpenoid and PLGA copolymer in the terpenoid encapsulated PLGA nanoparticles is at least about 1: 50. In some embodiments, the average weight ratio of encapsulated terpenoid and PLGA copolymer in the terpenoid encapsulated PLGA nanoparticles is at least about 1: 40. In some embodiments, the average weight ratio of encapsulated terpenoid and PLGA copolymer in the terpenoid encapsulated PLGA nanoparticles is at least about 1: 30. In some embodiments, the average weight ratio of encapsulated terpenoid and PLGA copolymer in the terpenoid encapsulated PLGA nanoparticles is at least about 1: 20. In some embodiments, the average weight ratio of encapsulated terpenoid and PLGA copolymer in the terpenoid encapsulated PLGA nanoparticles is at least about 1: 10.

In some embodiments, the PLGA copolymer has a ratio of lactic acid to glycolic acid of about 10-90% lactic acid and about 90-10% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 10% lactic acid to about 90% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 25% lactic acid to about 75% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 75% lactic acid to about 25% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

In some embodiments, the solution of step (a) further comprises at least one cannabinoid or terpenoid other than the first terpenoid. In some embodiments, the at least one cannabinoid or terpenoid is selected from: cannabidiol (CBD), Cannabinol (CBN), Cannabidivarin (CBDV), cannabichromene (CBC), cannabidiolic acid (CBDA) and Cannabigerol (CBG). In some embodiments, the at least one cannabinoid or terpenoid is selected from the group consisting of myrcene, beta-caryophyllene, nerolidol, phytol, limonene, linalool, and pinene.

In some embodiments, the solvent is acetone, dichloromethane, or ethyl acetate. In some embodiments, the surfactant is polyethylene glycol, poloxamer, or polyvinyl alcohol (PVA). In some embodiments, the surfactant is polyvinyl alcohol (PVA).

In some embodiments, the emulsifying step comprises homogenization or sonication. In some embodiments, the emulsifying step is homogenization. In some embodiments, homogenization is performed at 20,000 to 30,000 rpm. In some embodiments, homogenization is performed at 24,000 rpm. In some embodiments, the solution is homogenized for 30 seconds to 10 minutes. In some embodiments, the solution is homogenized for 1 minute.

In some embodiments, the step of evaporating the solvent comprises at least one of stirring the solvent, applying a gas stream, applying heat, maintaining a low temperature of 10 ℃, or creating a vacuum. In some embodiments, the step of evaporating the solvent comprises stirring the suspension at room temperature. In some embodiments, the suspension is stirred for 5min to 120min to evaporate the solvent. In some embodiments, the suspension is stirred for 60 minutes.

In some embodiments, the step of obtaining PLGA nanoparticles encapsulating terpenoids comprises centrifugation, filtration, or centrifugation and filtration. In some embodiments, the obtaining step comprises centrifugation. In some embodiments, centrifugation is performed at 2,000x g to 15,000x g. In some embodiments, centrifugation is performed at 4,000x g.

In some embodiments, the method further comprises the subsequent step of adding a cryoprotectant to the PLGA nanoparticles encapsulating the terpenoid. In some embodiments, the cryoprotectant is trehalose. In some embodiments, the cryoprotectant is added in an amount of 1-10% (w/v) of the PLGA nanoparticles encapsulating terpenoids. In some embodiments, the cryoprotectant is added in an amount of 5% (w/v) of the PLGA nanoparticles encapsulating terpenoids.

In some embodiments, the method further comprises lyophilizing the PLGA nanoparticles encapsulating the terpenoid.

In one aspect, the present invention provides a terpenoid encapsulated PLGA nanoparticle obtained by the method described herein.

In another aspect, the present invention provides a method of achieving TRPV1 desensitization in a cell of a mammalian subject comprising: the pharmaceutical compositions described herein are administered to a mammalian subject by route and time of administration in an amount sufficient to cause desensitization of TRPV1 in cells of the mammalian subject. In some embodiments, the cell is a nociceptor. In some embodiments, the nociceptor is a peripheral nociceptor. In some embodiments, the nociceptor is a visceral nociceptor.

In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered topically. In some embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered by inhalation.

In one aspect, the present invention provides a method of treating pain in a mammalian subject, the method comprising the steps of: administering to the subject by a route of administration a pharmaceutical composition described herein in an amount and for a time sufficient to cause desensitization of TRPV1 at nociceptors of the subject.

In some embodiments, the nociceptors are peripheral nociceptors and the pharmaceutical composition is administered topically. In some embodiments, the nociceptor is a visceral nociceptor and the pharmaceutical composition is administered systemically.

In some embodiments, the pain is neuropathic pain. In some embodiments, the neuropathic pain is diabetic peripheral neuropathic pain. In some embodiments, the pain is post-herpetic neuralgia.

In some embodiments, the pharmaceutical composition is administered at least once daily for at least 3 days. In some embodiments, the pharmaceutical composition is administered at least once daily for at least 5 days. In some embodiments, the pharmaceutical composition is administered at least once daily for at least 7 days.

In some embodiments, the pharmaceutical composition is administered by the route and schedule of administration at a dose sufficient to maintain the level of the first terpene at the nociceptor for at least 3 days at which TRPV1 at the nociceptor is effectively desensitized. In some embodiments, the pharmaceutical composition is administered by the route and schedule of administration at a dose sufficient to maintain the level of the first terpene at the nociceptor for at least 5 days at which TRPV1 at the nociceptor is effectively desensitized. In some embodiments, the pharmaceutical composition is administered by the route and schedule of administration at a dose sufficient to maintain the level of the first terpene at the nociceptor for at least 7 days at which TRPV1 at the nociceptor is effectively desensitized.

In one aspect, the present invention provides a method of treating cardiac hypertrophy in a mammalian subject comprising the steps of: administering to the subject an anti-hypertrophic effective amount of a pharmaceutical composition described herein. In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered by inhalation. In some embodiments, the pharmaceutical composition is administered orally.

In one aspect, the present invention provides a method of prophylactic treatment of cardiac hypertrophy in a mammalian subject comprising the steps of: administering to a subject at risk of cardiac hypertrophy an anti-hypertrophy effective amount of a pharmaceutical composition described herein.

In one aspect, the present invention provides a method of treating overactive bladder in a mammalian subject, comprising the steps of: administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein. In some embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered by bladder irrigation.

In one aspect, the present invention provides a method of treating refractory chronic cough in a mammalian subject, comprising the steps of: administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. In some embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered by inhalation.

In another aspect, the present invention provides a cannabinoid-encapsulating PLGA nanoparticle comprising: a PLGA nanoparticle and a first cannabinoid encapsulated in the PLGA nanoparticle.

In some embodiments, the PLGA nanoparticles comprise a PLGA copolymer, and the weight ratio of the first cannabinoid to the PLGA copolymer is from 1:50 to 1: 4. in some embodiments, the weight ratio of the first cannabinoid to the PLGA copolymer is in the range of 1:25 to 1: 5. in some embodiments, the first cannabinoid is selected from the group consisting of cannabidiol, cannabidivarin, cannabinol, cannabigerol, and cannabichromene.

In some embodiments, the first cannabinoid is cannabidiol and the weight ratio of the first cannabinoid to the PLGA copolymer is about 1: 14. In some embodiments, the first cannabinoid is cannabidiol and the weight ratio of the first cannabinoid to the PLGA copolymer is in a range of 1:5 to 1: 7. in some embodiments, the first cannabinoid is cannabidiol and the weight ratio of the first cannabinoid to the PLGA copolymer is in a range of 1:5 and 1: 7.

in some embodiments, the nanoparticle further encapsulates at least a second compound, wherein the second encapsulated compound is (i) a terpenoid or (ii) a second cannabinoid other than the first cannabinoid. In some embodiments, the second encapsulating compound is cannabigerolic acid (CBGA). In some embodiments, the second encapsulating compound is Cannabidivarin (CBV). In some embodiments, the second encapsulating compound is Cannabinol (CBN). In some embodiments, the second encapsulating compound is Cannabidivarin (CBDV). In some embodiments, the second encapsulating compound is cannabichromene (CBC). In some embodiments, the second encapsulating compound is cannabidiolic acid (CBDA). In some embodiments, the second encapsulating compound is Cannabigerol (CBG). In some embodiments, the second encapsulating compound is myrcene, beta-caryophyllene, or nerolidol. In some embodiments, the second encapsulating compound is limonene. In some embodiments, the second encapsulated compound is phytol. In some embodiments, the second encapsulated compound is pinene. In some embodiments, the second encapsulating compound is linalool. In some embodiments, the second encapsulating compound is myrcene.

In some embodiments, the PLGA nanoparticles have an average diameter of 200-350 nm. In some embodiments, the PLGA nanoparticles comprise a PLGA copolymer having a ratio of lactic acid to glycolic acid of about 10-90% lactic acid and about 90-10% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 10% lactic acid to about 90% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 25% lactic acid to about 75% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 75% lactic acid to about 25% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

One aspect of the present invention provides a pharmaceutical composition comprising a first population of cannabinoid-encapsulating PLGA nanoparticles provided herein and a pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical composition further comprises trehalose.

In some embodiments, the composition is lyophilized. In some embodiments, the composition further comprises a second population of PLGA nanoparticles, wherein the second population of nanoparticles encapsulates a third compound, wherein the third compound is a different terpenoid or cannabinoid than the terpenoid and cannabinoid encapsulated in the first population of nanoparticles. In some embodiments, the third encapsulating compound is cannabigerolic acid (CBGA). In some embodiments, the third encapsulating compound is Cannabidiol (CBD). In some embodiments, the third encapsulating compound is Cannabinol (CBN). In some embodiments, the third encapsulating compound is Cannabidivarin (CBDV). In some embodiments, the third encapsulating compound is cannabichromene (CBC). In some embodiments, the third encapsulating compound is cannabidiolic acid (CBDA). In some embodiments, the third encapsulating compound is Cannabigerol (CBG). In some embodiments, the third encapsulating compound is myrcene, beta-caryophyllene, or nerolidol. In some embodiments, the third encapsulating compound is limonene. In some embodiments, the third encapsulating compound is phytol. In some embodiments, the third encapsulating compound is pinene. In some embodiments, the third encapsulating compound is linalool. In some embodiments, the third encapsulated compound is myrcene.

In some embodiments, the composition further comprises a third population of PLGA nanoparticles, wherein the third population of nanoparticles encapsulates a fourth compound, wherein the fourth compound is a different cannabinoid or terpenoid than the cannabinoid or terpenoid encapsulated in the first or second population of nanoparticles. In some embodiments, the composition further comprises a fourth population of PLGA nanoparticles, wherein the fourth population of nanoparticles encapsulates a fifth compound, wherein the fifth compound is a different cannabinoid or terpenoid than the cannabinoid or terpenoid encapsulated in the first, second, or third population of nanoparticles.

Another aspect of the invention provides a method for obtaining cannabinoid-encapsulating PLGA nanoparticles, wherein the method comprises the steps of: (a) providing an organic solution comprising a terpenoid, a PLGA copolymer, and a solvent, and an aqueous solution comprising a surfactant; (b) emulsifying the two solutions to form a suspension of cannabinoid-encapsulating PLGA nanoparticles; (c) evaporating the solvent from the emulsion; and (d) obtaining cannabinoid-encapsulating PLGA nanoparticles.

In some embodiments, the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1:5 to about 1: 1. in some embodiments, the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1: 5. in some embodiments, the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1: 4. in some embodiments, the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1: 3. in some embodiments, the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1: 2. in some embodiments, the weight ratio of the first cannabinoid to the PLGA copolymer in the solution of step (a) is about 1: 1.

in some embodiments, the encapsulation of the first cannabinoid in step (b) is from about 4% to about 10%. In some embodiments, the encapsulation efficiency is at least 4%. In some embodiments, the encapsulation efficiency is at least 5%. In some embodiments, the encapsulation efficiency is at least 6%. In some embodiments, the encapsulation efficiency is at least 7%. In some embodiments, the encapsulation efficiency is at least 8%. In some embodiments, the encapsulation efficiency is at least 9%. In some embodiments, the encapsulation efficiency is at least 10%.

In some embodiments, the average weight ratio of encapsulated cannabinoid to PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles of step (d) is from about 1:50 to about 1: 10. In some embodiments, the average weight ratio of encapsulated cannabinoid to PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles is at least about 1: 50. In some embodiments, the average weight ratio of encapsulated cannabinoid to PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles is at least about 1: 40. In some embodiments, the average weight ratio of encapsulated cannabinoid to PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles is at least about 1: 30. In some embodiments, the average weight ratio of encapsulated cannabinoid to PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles is at least about 1: 20. In some embodiments, the average weight ratio of encapsulated cannabinoid to PLGA copolymer in the cannabinoid-encapsulated PLGA nanoparticles is at least about 1: 10.

In some embodiments, the PLGA copolymer has a ratio of lactic acid to glycolic acid of about 10-90% lactic acid and about 90-10% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 10% lactic acid to about 90% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 25% lactic acid to about 75% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 75% lactic acid to about 25% glycolic acid. In some embodiments, the ratio of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

In some embodiments, the solution of step (a) further comprises at least one cannabinoid or terpenoid other than the first cannabinoid. In some embodiments, the at least one cannabinoid or terpenoid is selected from: cannabinol (CBN), Cannabidivarin (CBDV), cannabichromene (CBC), cannabidiolic acid (CBDA) and Cannabigerol (CBG). In some embodiments, the at least one cannabinoid or terpene is selected from: myrcene, beta-caryophyllene, nerolidol, phytol, limonene, linalool and pinene.

In some embodiments, the solvent is acetone, dichloromethane, or ethyl acetate. In some embodiments, the surfactant is polyethylene glycol, poloxamer, or polyvinyl alcohol (PVA). In some embodiments, the surfactant is polyvinyl alcohol (PVA).

In some embodiments, the emulsifying step comprises homogenization or sonication. In some embodiments, the emulsifying step is homogenization. In some embodiments, homogenization is performed at 20,000 to 30,000 rpm. In some embodiments, homogenization is performed at 24,000 rpm. In some embodiments, the solution is homogenized for 30 seconds to 10 minutes. In some embodiments, the solution is homogenized for 1 minute.

In some embodiments, the step of evaporating the solvent comprises at least one of stirring the solvent, applying a gas stream, applying heat, maintaining a low temperature of 10 ℃, or creating a vacuum. In some embodiments, the step of evaporating the solvent comprises stirring the suspension at room temperature. In some embodiments, the suspension is stirred for 5min to 120min to evaporate the solvent. In some embodiments, the suspension is stirred for 60 minutes.

In some embodiments, the step of obtaining cannabinoid-encapsulating PLGA nanoparticles comprises centrifugation, filtration, or centrifugation and filtration. In some embodiments, the obtaining step comprises centrifugation. In some embodiments, centrifugation is performed at 2,000x g to 15,000x g. In some embodiments, centrifugation is performed at 4,000x g.

In some embodiments, the method further comprises the subsequent step of adding a cryoprotectant to the cannabinoid-encapsulated PLGA nanoparticles. In some embodiments, the cryoprotectant is trehalose. In some embodiments, the cryoprotectant is added in an amount of 1-10% (w/v) of the cannabinoid-encapsulating PLGA nanoparticles. In some embodiments, the cryoprotectant is added in an amount of 5% (w/v) of the cannabinoid-encapsulating PLGA nanoparticles.

In some embodiments, the method further comprises lyophilizing the cannabinoid-encapsulating PLGA nanoparticles.

In one aspect, the present invention provides cannabinoid-encapsulating PLGA nanoparticles obtained by the methods described herein.

In another aspect, the present invention provides a method of achieving TRPV1 desensitization in a cell of a mammalian subject comprising: administering to the mammalian subject by a route of administration the pharmaceutical composition provided herein in an amount and for a time sufficient to cause desensitization of TRPV1 in cells of the mammalian subject. In some embodiments, the cell is a nociceptor. In some embodiments, the nociceptor is a peripheral nociceptor. In some embodiments, the nociceptor is a visceral nociceptor.

In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered topically. In some embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered by inhalation.

In another aspect, the present invention provides a method of treating pain in a mammalian subject, the method comprising the steps of: administering to the subject by a route of administration a pharmaceutical composition provided herein in an amount and for a time sufficient to cause desensitization of TRPV1 at nociceptors of the subject.

In some embodiments, the nociceptors are peripheral nociceptors and the pharmaceutical composition is administered topically. In some embodiments, the nociceptor is a visceral nociceptor and the pharmaceutical composition is administered systemically.

In some embodiments, the pain is neuropathic pain. In some embodiments, the neuropathic pain is diabetic peripheral neuropathic pain. In some embodiments, the pain is post-herpetic neuralgia.

In some embodiments, the pharmaceutical composition is administered at least once daily for at least 3 days. In some embodiments, the pharmaceutical composition is administered at least once daily for at least 5 days. In some embodiments, the pharmaceutical composition is administered at least once daily for at least 7 days. In some embodiments, the pharmaceutical composition is administered at least once daily for more than 7 days. In some embodiments, the pharmaceutical composition is administered by the route and schedule of administration at a dose sufficient to maintain cannabinoid levels at the nociceptors for at least 3 days effective to desensitize TRPV1 at the nociceptors. In some embodiments, the pharmaceutical composition is administered by a route and schedule of administration at a dose sufficient to maintain cannabinoid levels at nociceptors for at least 5 days effective to desensitize TRPV1 at nociceptors, and the pharmaceutical composition is administered by a route and schedule of administration at a dose sufficient to maintain cannabinoid levels at nociceptors for at least 7 days effective to desensitize TRPV1 at nociceptors.

One aspect of the present invention provides a method of treating cardiac hypertrophy in a mammalian subject comprising the steps of: administering to the subject an anti-hypertrophic effective amount of a pharmaceutical composition provided herein.

In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered by inhalation. In some embodiments, the pharmaceutical composition is administered orally.

Another aspect of the invention provides a method of prophylactic treatment of cardiac hypertrophy in a mammalian subject comprising the steps of: administering to a subject at risk of cardiac hypertrophy an anti-hypertrophy effective amount of a pharmaceutical composition provided herein.

In another aspect, the present invention provides a method of treating overactive bladder in a mammalian subject, comprising the steps of: administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein.

In some embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered by bladder irrigation.

In one aspect, the present invention provides a method of treating refractory chronic cough in a mammalian subject, comprising the steps of: administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein.

In some embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered by inhalation.

Drawings

These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and accompanying drawings where:

fig. 1 provides a scheme of the nanoprecipitation method.

Fig. 2 provides a schematic of PLGA nanocapsules obtained using a microemulsion system.

Figure 3 provides a scheme of the emulsification (high speed homogenizer) process for solvent evaporation.

Fig. 4A shows a gas chromatogram, fig. 4B shows a mass spectrum, and fig. 4C shows a calibration curve of myrcene at a concentration range of 1-60ppm as measured and analyzed by a GC-MS Instrument of Thermo Scientific Instrument Delta V.

Fig. 5A shows a gas chromatogram of β -caryophyllene, fig. 5B a mass spectrum of β -caryophyllene, and fig. 5C shows a calibration curve of β -caryophyllene at a concentration range of 1-60ppm measured and analyzed by a GC-MS Instrument of Thermo Scientific Instrument Delta V.

FIG. 6 shows gas chromatograms of cis-and trans-nerolidol measured and analyzed by a GC-MS Instrument Thermo Scientific Instrument Delta V.

FIG. 7A shows a mass spectrum of cis-nerolidol and FIG. 7B shows a calibration curve of cis-nerolidol at 1-60ppm concentration measured and analyzed by a GC-MS Instrument at Thermo Scientific Instrument Delta V. Similarly, fig. 7C and 7D show the mass spectrum and calibration curve, respectively, of trans-nerolidol measured and analyzed under the same conditions as the cis isomer.

Fig. 8A-8F show the average diameter (fig. 8A, 8C, and 8E) and zeta potential (fig. 8B, 8D, and 8F) measurements of Nanoparticles (NPs) prepared using nanoprecipitation.

FIGS. 9A-9B show the mean diameter (FIG. 9A) and zeta potential (FIG. 9B) measurements of NPs prepared using microemulsions.

FIGS. 10A-10B show the mean diameter (FIG. 10A) and zeta potential (FIG. 10B) measurements of NPs prepared using homogenizer emulsification.

FIGS. 11A-11D show the mean diameter (FIGS. 11A and 11C) and zeta potential (FIGS. 11B and 11D) measurements of NPs prepared by emulsification (FIGS. 11A and 11B) and nano-precipitation (FIGS. 11C and 11D).

Fig. 12A-12B show the average diameter (fig. 12A) and zeta potential (fig. 12B) measurements of emulsions prepared by adding myrcene dissolved in acetone to a 0.5% (w/w) PVA solution.

Fig. 13A and 13B show Scanning Electron Microscope (SEM) images of myrcene-containing Nanoparticles (NPs).

Fig. 14A and 14B show Scanning Electron Microscope (SEM) images of β -caryophyllene-containing Nanoparticles (NPs).

Fig. 15A and 15B show Scanning Electron Microscope (SEM) images of nerolidol-containing Nanoparticles (NPs).

Fig. 16 shows a schematic of a nanocapsule.

Fig. 17 shows the change in fluorescence measured using a calcium signaling assay after treatment of HEK TRPV1 cells with free and encapsulated terpenoids including myrcene, nerolidol, and β -caryophyllene and ionomycin, as shown in the single figure.

Figures 18A-18C show separate representations of fluorescence changes measured using the calcium signaling assay after treatment of HEK TRPV1 cells with unencapsulated and encapsulated myrcene (figure 18A), nerolidol (figure 18B), and β -caryophyllene (figure 18C).

Figures 19A-19D show the change in fluorescence measured using a calcium signaling assay after treatment of HEK TRPV1 cells with unencapsulated terpenoids alone compared to their corresponding combinations, i.e., figure 19A shows unencapsulated myrcene, nerolidol, and combinations thereof; fig. 19B shows unencapsulated myrcene, beta-caryophyllene, and combinations thereof; figure 19C shows unencapsulated β -caryophyllene, nerolidol, and combinations thereof; and fig. 19D shows unencapsulated myrcene, nerolidol, beta-caryophyllene, and combinations thereof.

Figures 20A-20D show the change in fluorescence measured using a calcium signaling assay after treatment of HEK TRPV1 cells with single encapsulated terpenoid Nanoparticles (NPs) compared to their corresponding combinations, i.e., figure 20A shows the results for myrcene NPs, nerolidol NPs, and combinations thereof; FIG. 20B shows results for myrcene NPs, beta-caryophyllene NPs, and combinations thereof; FIG. 20C shows results of nerolidol NPs, β -caryophyllene NPs, and combinations thereof; and fig. 20D shows the results of NPs containing myrcene, nerolidol, and β -caryophyllene, NPs containing myrcene and nerolidol, NPs containing myrcene and β -caryophyllene, and NPs containing nerolidol and β -caryophyllene.

Figures 21A-21D show changes in fluorescence measured using a calcium signaling assay after treatment of HEK TRPV1 cells with nanoparticles of encapsulated terpenoids alone, compared to their corresponding unencapsulated terpenoid combinations. Figure 21A shows results from combinations of unencapsulated and encapsulated myrcene and nerolidol; figure 21B shows results from combinations of unencapsulated and encapsulated myrcene and beta-caryophyllene; figure 21C shows results from combinations of unencapsulated and encapsulated nerolidol and β -caryophyllene; and figure 21D shows results from combinations of unencapsulated and encapsulated myrcene, nerolidol, and β -caryophyllene.

Detailed Description

Definition of

Unless otherwise indicated, the terms used in the claims and the specification are defined as follows.

The term "myrcene" or "beta-myrcene" as used herein refers to 7-methyl-3-methyleneocta-1, 6-diene, and is represented by the structural formula

As used herein, the term "alpha-ocimene" refers to cis-3, 7-dimethyl-1, 3, 7-octatriene, as shown in the structural formula

The term "cis- β -ocimene" as used herein refers to (Z) -3, 7-dimethyl-1, 3, 6-octatriene, represented by the structural formula

The term "trans-beta-ocimene" as used herein refers to (E) -3, 7-dimethyl-1, 3, 6-octatriene, represented by the structural formula

The term "linalool" as used herein refers to 3, 7-dimethyl-1, 6-octadien-3-ol, represented by the structural formula

The term "nerolidol" as used herein refers to 3,7, 11-trimethyl-1, 6, 10-dodecatrien-3-ol, represented by the structural formula

As used herein, the term "cis-nerolidol" refers to the cis isomer of nerolidol, represented by the structural formula

The term "trans-nerolidol" as used herein refers to the trans isomer of nerolidol, represented by the structural formula

The term "bisabolol" as used herein refers to 6-methyl-2- (4-methylcyclohex-3-en-1-yl) hept-5-en-2-ol, represented by the structural formula

As used herein, the term "β -caryophyllene" refers to (1R,4E,9S) -4,11, 11-trimethyl-8-methylenebicyclo [7.2.0] undec-4-ene, represented by the structural formula

The term "dimethylallyl" group refers to an unsaturated C represented by the formula₅H₉Alkyl substituent

The term "terpenoids" as used herein refers to a class of organic compounds produced by plants, including α -bisabolol (α -bisabolol), α -lupalene (α -humulene), α -pinene (α -pinene), β -caryophyllene (β -caryophyllene), myrcene, (+) - β -pinene (β -pinene), camphene, limonene, linalool, phytol, and nerolidol.

The term "cannabinoid" as used herein refers to a class of compounds comprising cannabigerolic acid (CBGA), Cannabidiol (CBD), Cannabidivarin (CBDV), cannabichromene (CBC), cannabidiolic acid (CBDA) and Cannabigerol (CBG).

By "pharmaceutically active ingredient" (synonymously, active pharmaceutical ingredient) is meant any substance or mixture of substances intended for use in the manufacture of a pharmaceutical product and which becomes the active ingredient in the pharmaceutical product when used in the manufacture of a medicament. These substances are intended to provide pharmacological activity or other direct effects in the diagnosis, cure, mitigation, treatment or prevention of disease, or to affect the structure and function of the body. These substances or mixtures of substances are preferably produced according to Current Good Manufacturing Practice (CGMP) regulations in section 501(a) (2) (B) of the Federal food, drug and cosmetic Act.

A pharmaceutically active ingredient is "substantially free of THC" if the ingredient contains less than 0.3% (w/w) delta-9 tetrahydrocannabinol. A pharmaceutical composition is "substantially free of THC" if it contains less than 0.3% (w/v) delta-9 tetrahydrocannabinol.

"cannabis extract" is a composition obtained from cannabis plant material by fluid and/or gas extraction, for example by Supercritical Fluid Extraction (SFE) with CO 2. Cannabis extracts typically contain myrcene, cannabinoids and terpenoids, and may also contain phytocannabinoids and other secondary metabolites.

The terms "treatment", "treating" and the like are used herein generally to mean obtaining a desired pharmacological and/or physiological effect. The effect can be prophylactic in terms of completely or partially preventing the disease, disorder, or symptom thereof, and/or therapeutic in terms of a partial or complete cure for the disease or disorder and/or adverse effects (e.g., symptoms) attributable to the disease or disorder. As used herein, "treatment" encompasses any treatment of a disease or condition in a mammal, particularly a human, and includes: (a) preventing the disease or disorder from occurring in a subject that may be predisposed to the disease or disorder but has not yet been diagnosed as having the disease or disorder; (b) inhibiting a disease or disorder (e.g., arresting its development); or (c) ameliorating the disease or disorder (e.g., causing regression of the disease or disorder, providing an improvement in one or more symptoms). The amelioration of any condition can be readily assessed according to standard methods and techniques known in the art. The population of subjects treated by this method includes subjects with an undesirable condition or disease, as well as subjects at risk of developing a condition or disease.

The term "therapeutically effective dose" or "therapeutically effective amount" refers to a dose or amount that produces the desired effect for administration. The exact dose or amount will depend on The purpose of The treatment and will be determined by one of skill in The Art using known techniques (see, e.g., Lloyd (2012) The Art, Science and Technology of Pharmaceutical Compounding, fourth edition). A therapeutically effective amount may be a "prophylactically effective amount" since prophylaxis may be considered treatment.

The term "sufficient amount" refers to an amount sufficient to produce the desired effect.

The term "ameliorating" refers to any therapeutically beneficial outcome in the treatment of a disease state (e.g., an immune disorder), including its prevention, lessening of severity or progression, alleviation or cure.

The term "mammal" as used herein includes humans and non-humans, and includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

The term "PLGA nanoparticle" as used herein refers to a nanoparticle comprising PLGA copolymer and includes PLGA nanocapsules and PLGA nanospheres. PLGA nanocapsules are characterized by an outer PLGA polymer film (shell) surrounding an inner core of a substance, e.g. a pharmaceutical compound such as myrcene, other terpenoids or cannabinoids. PLGA nanospheres are characterized by a spherical matrix of PLGA polymer in which a substance or drug compound is dispersed or embedded.

The term "myrcene-encapsulated PLGA nanoparticles" as used herein refers to nanoparticles made of a myrcene-encapsulated or containing PLGA copolymer. The nanoparticles may be PLGA nanospheres or PLGA nanocapsules. Myrcene can be used as a liquid inner core to be encapsulated in a PLGA outer membrane (shell) to form a nano capsule; or myrcene may be dispersed or embedded in a PLGA matrix to form nanospheres.

The term "cannabinoid-encapsulating PLGA nanoparticles" as used herein refers to nanoparticles made of PLGA copolymers that encapsulate or contain cannabinoids. The nanoparticles may be PLGA nanospheres or PLGA nanocapsules. Cannabinoids can be encapsulated as a liquid core within the outer PLGA membrane (shell) to form nanocapsules; or the cannabinoids may be dispersed or embedded in a PLGA matrix to form nanospheres.

The term "terpenoid encapsulated PLGA nanoparticles" as used herein refers to nanoparticles made of PLGA copolymers, which encapsulate or contain terpenoids. The nanoparticles may be PLGA nanospheres or PLGA nanocapsules. Terpenoids can be encapsulated as a liquid core in a PLGA outer membrane (shell) to form nanocapsules; or the terpenoid may be dispersed or embedded in a PLGA matrix to form nanospheres.

The term "terpene/cannabinoid encapsulated PLGA nanoparticles" as used herein refers to either terpene encapsulated PLGA nanoparticles or cannabinoid encapsulated PLGA nanoparticles.

The terms "free terpenoid" and "unencapsulated terpenoid" as used herein refer to terpenoids that are not encapsulated within nanoparticles and are in bulk solution. These terms are used interchangeably throughout the specification.

The term "PLGA polymer" as used herein refers to poly (lactic-co-glycolic acid).

The term "lyophilization" as used herein refers to a low temperature dehydration process in which water is removed from a material by freezing the material at low pressure or in a vacuum. The water freezes and is then removed from the material by sublimation. Alternative terms to this method include freeze-drying.

The term "encapsulation efficiency" or "EE%" as used herein refers to the percentage of the mass of a compound in a nanoparticle compared to the starting mass of the compound. The EE percentage was calculated using the following equation. "CPD" is an abbreviation for "compound" and refers to a pharmaceutical compound, such as myrcene or other terpenoid or cannabinoid, encapsulated in nanoparticles.

The term "drug loading" or "DL%" as used herein refers to the mass of a compound incorporated into a nanoparticle compared to the total mass of the nanoparticle comprising the compound. The DL percentage was calculated using the following equation. "CPD" means a pharmaceutical compound, such as myrcene or other terpenoid or cannabinoid, encapsulated in a nanoparticle.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Ranges described herein are to be understood as shorthand for all values within the range, including the endpoints. For example, a range of 1 to 50 should be understood to include any number, combination of numbers, or subrange selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

Unless otherwise indicated, reference to a compound having one or more stereocenters means each stereoisomer thereof and all combinations of its stereoisomers.

PLGA nanoparticles encapsulating terpenoids/cannabinoids

Terpenoid encapsulated PLGA nanoparticles

In one aspect, the present invention provides a PLGA nanoparticle encapsulating terpenoids, comprising a PLGA nanoparticle and a first terpenoid encapsulated in the PLGA nanoparticle. The terpenoid encapsulated PLGA nanoparticles may encapsulate compounds other than the first terpenoid, such as other terpenoids and/or cannabinoids.

The first terpenoid encapsulated in the PLGA nanoparticles may be a terpenoid extractable from cannabis.

The first terpenoid may be myrcene, beta-caryophyllene or nerolidol.

In various such embodiments, the first terpenoid, the optional cannabinoid, and the optional terpenoid other than the first terpenoid collectively comprise at least 75% by weight, but less than 100% by weight, of the total compounds encapsulated in the PLGA nanoparticle. In specific embodiments, the first terpenoid, the optional cannabinoid, and the optional terpenoid other than the first terpenoid collectively comprise at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, but less than 100%, by weight of the total compound encapsulated in the PLGA nanoparticle. In specific embodiments, the first terpenoid, the optional cannabinoid, and the optional terpenoid other than the first terpenoid collectively comprise at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, by weight of the total compound encapsulated in the PLGA nanoparticle.

In embodiments where the first terpenoid, the optional cannabinoid, and the optional terpenoid other than the first terpenoid together comprise less than 100% by weight (wt%) of the total compound encapsulated in the PLGA nanoparticle, the PLGA nanoparticle may further encapsulate a compound other than the first terpenoid, the optional cannabinoid, or the optional terpenoid. In such embodiments, the additional compounds may be extracted from cannabis. In a particular embodiment, all or some of the compounds encapsulated in the PLGA nanoparticles are present in the extract made from cannabis.

The amount of the first terpenoid encapsulated in the PLGA nanoparticle may be quantified as the weight ratio of the weight of the PLGA copolymer in the nanoparticle to the amount of the first terpenoid encapsulated within the nanoparticle. The weight ratio of the first terpenoid to the PLGA copolymer may be 1:50 to 1: 1. the weight ratio of the first terpenoid to the PLGA copolymer may be 1:100 to 1:5, 1:75 to 1:10, 1:50 to 1:10, 1:40 to 1:10, 1:30 to 1:10, 1:20 to 1:10, 1:50 to 1:40, 1:40 to 1:30, 1:30 to 1:20, 1:20 to 1:10, 1:10 to 1:5 and 1:5 to 1: 1. the weight ratio of the first terpenoid to the PLGA copolymer may be 1:50, 1:45, 1:40, 1:35, 1:30, 1:25, 1:20, 1:19, 1:17, 1:18, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1: 1. in a specific embodiment, the ratio is about 1: 14. In a specific embodiment, the ratio is about 1: 22.

In some embodiments, the weight ratio of the first terpenoid to the PLGA copolymer is from 1:50 to 1: 10. In one embodiment, the weight ratio of the first terpenoid to the PLGA copolymer is about 1: 14. In one embodiment, the weight ratio of the first terpenoid to the PLGA copolymer is about 1: 22.

In one embodiment, the terpenoid encapsulated in the nanoparticle is myrcene. The amount of myrcene encapsulated in the PLGA nanoparticles may be quantified as the weight ratio of the weight of the PLGA copolymer in the PLGA nanoparticles to the amount of myrcene encapsulated within the nanoparticles. The weight ratio of myrcene to PLGA copolymer may be in the range of 1:50 to 1: 1. the weight ratio of myrcene to PLGA copolymer may be in the range of 1:100 to 1:5, 1:75 to 1:10, 1:50 to 1:10, 1:40 to 1:10, 1:30 to 1:10, 1:20 to 1:10, 1:50 to 1:40, 1:40 to 1:30, 1:30 to 1:20, 1:20 to 1:10, 1:10 to 1:5 and 1:5 to 1: 1. the weight ratio of myrcene to PLGA copolymer may be 1:50, 1:45, 1:40, 1:35, 1:30, 1:25, 1:20, 1:19, 1:17, 1:18, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2 or 1: 1. in a specific embodiment, the ratio is about 1: 22.

In some embodiments, the weight ratio of myrcene to PLGA copolymer is 1:50 to 1: 10. In one embodiment, the weight ratio of myrcene to PLGA copolymer is about 1:14 or about 1: 22.

Compounds other than the first terpenoid ("second encapsulated compounds") may also be encapsulated in the PLGA nanoparticles. In various embodiments, the second encapsulated compound is a cannabinoid and/or a terpenoid other than the first terpenoid. In one embodiment, the second encapsulating compound is cannabigerolic acid (CBGA). In one embodiment, the second encapsulated compound is Cannabidiol (CBD). In one embodiment, the second encapsulating compound is Cannabinol (CBN). In one embodiment, the second encapsulating compound is Cannabidivarin (CBDV). In one embodiment, the second encapsulating compound is cannabichromene (CBC). In one embodiment, the second encapsulating compound is cannabidiolic acid (CBDA). In one embodiment, the second encapsulating compound is Cannabigerol (CBG). In one embodiment, the second encapsulating compound is myrcene. In one embodiment, the second encapsulating compound is caryophyllene. In one embodiment, the second encapsulating compound is nerolidol. In one embodiment, the second encapsulating compound is limonene. In one embodiment, the second encapsulated compound is phytol. In one embodiment, the second encapsulated compound is pinene. In one embodiment, the second encapsulating compound is linalool.

Cannabinoid-encapsulating PLGA nanoparticles

In another aspect, the present invention provides cannabinoid-encapsulating PLGA nanoparticles comprising a PLGA nanoparticle and a first cannabinoid encapsulated in the PLGA nanoparticle. The cannabinoid-encapsulating PLGA nanoparticles may encapsulate compounds other than the first cannabinoid, such as other terpenoids and/or cannabinoids.

The first terpenoid encapsulated in the PLGA nanoparticles may be a cannabinoid extractable from cannabis.

The first cannabinoid is cannabidiol (cannabidivarin), cannabidivarin (cannabidivarin), cannabinol (cannabibinol), cannabigerol (cannabibergol) or cannabichromene (cannabidichroromene).

In various such embodiments, the first cannabinoid, the optional cannabinoid, and the optional terpenoid other than the first cannabinoid together comprise at least 75% by weight, but less than 100% by weight, of the total compound encapsulated in the PLGA nanoparticle. In specific embodiments, the first cannabinoid, the optional cannabinoid, and the optional terpenoid other than the first cannabinoid together comprise at least 80 wt.%, at least 85 wt.%, at least 90 wt.%, at least 91 wt.%, at least 92 wt.%, at least 93 wt.%, at least 94 wt.%, or at least 95 wt.% but less than 100 wt.% of the total compound encapsulated in the PLGA nanoparticle. In specific embodiments, the first cannabinoid, the optional cannabinoid, and the optional terpenoid other than the first cannabinoid together comprise at least 96%, at least 97%, at least 98%, or at least 99% by weight, but less than 100% by weight, of the total compound encapsulated in the PLGA nanoparticle.

In embodiments where the first cannabinoid, the optional cannabinoid, and the optional terpenoid other than the first cannabinoid together comprise less than 100% by weight (wt%) of the total compound encapsulated in the PLGA nanoparticle, the PLGA nanoparticle may further encapsulate a compound other than the first cannabinoid, the optional cannabinoid, or the optional terpenoid. In such embodiments, the additional compounds may be extracted from cannabis. In a particular embodiment, all or some of the compounds encapsulated in the PLGA nanoparticles are present in the extract made from cannabis.

The amount of first cannabinoid encapsulated in the PLGA nanoparticle can be quantified as the weight ratio of the weight of the PLGA copolymer in the PLGA nanoparticle shell to the amount of first cannabinoid encapsulated within the nanoparticle. The weight ratio of the first cannabinoid to the PLGA copolymer can be from 1:50 to 1: 1. the weight ratio of the first cannabinoid to the PLGA copolymer can be 1:100 to 1:5, 1:75 to 1:10, 1:50 to 1:10, 1:40 to 1:10, 1:30 to 1:10, 1:20 to 1:10, 1:50 to 1:40, 1:40 to 1:30, 1:30 to 1:20, 1:20 to 1:10, 1:10 to 1:5 and 1:5 to 1: 1. the weight ratio of the first cannabinoid to the PLGA copolymer can be 1:50, 1:45, 1:40, 1:35, 1:30, 1:25, 1:20, 1:19, 1:17, 1:18, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1: 1. in a specific embodiment, the ratio is about 1: 14. In a specific embodiment, the ratio is about 1: 22.

In some embodiments, the weight ratio of the first cannabinoid to the PLGA copolymer is from 1:50 to 1: 10. In one embodiment, the weight ratio of the first cannabinoid to the PLGA copolymer is about 1: 14.

Compounds other than the first cannabinoid ("second encapsulating compounds") may also be encapsulated in the PLGA nanoparticles. In various embodiments, the second encapsulated compound is a cannabinoid and/or a terpenoid other than the first cannabinoid. In one embodiment, the second encapsulating compound is cannabigerolic acid (CBGA). In one embodiment, the second encapsulating compound is Cannabidiol (CBD). In one embodiment, the second encapsulating compound is Cannabinol (CBN). In one embodiment, the second encapsulating compound is Cannabidivarin (CBDV). In one embodiment, the second encapsulating compound is cannabichromene (CBC). In one embodiment, the second encapsulating compound is cannabidiolic acid (CBDA). In one embodiment, the second encapsulating compound is Cannabigerol (CBG). In one embodiment, the second encapsulating compound is myrcene. In one embodiment, the second encapsulating compound is caryophyllene. In one embodiment, the second encapsulating compound is nerolidol. In one embodiment, the second encapsulated compound is limonene. In one embodiment, the second encapsulated compound is phytol. In one embodiment, the second encapsulated compound is pinene. In one embodiment, the second encapsulated compound is linalool.

Delta-9 Tetrahydrocannabinol (THC) content

In typical embodiments, the terpenoid/cannabinoid-encapsulating PLGA nanoparticles are completely or substantially free of delta-9 Tetrahydrocannabinol (THC), and thus lack psychoactive effects, which provide certain regulatory and other physiological advantages.

In certain embodiments, the terpene/cannabinoid encapsulated PLGA nanoparticles are substantially free of delta-9 THC. In certain such embodiments, the terpene/cannabinoid encapsulated PLGA nanoparticles comprise 1-10 weight percent (wt%) THC. In a specific embodiment, the terpene/cannabinoid encapsulated PLGA nanoparticles comprise 2-9 wt.% THC, 3-8 wt.% THC, 4-7 wt.% THC. In certain embodiments, the terpene/cannabinoid-encapsulating PLGA nanoparticles comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 wt.% THC.

PLGA nanoparticles

Nanoparticles comprise the copolymer poly (lactic-co-glycolic acid) (PLGA), which is known to have high biocompatibility, low toxicity, and high control over drug delivery. PLGA copolymers suitable for use in the nanoparticles described herein are commercially available, such as those available under the name of Resomer from commercial sources including Sigma-Aldrich.

The amount of each component in the PLGA polymer and PLGA nanoparticle composition can be varied by adjusting the ratio of lactic acid and glycolic acid in the PLGA copolymer. The weight percentage (w/w) of lactic acid in the PLGA polymer may be about 10-90%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, or 80-90%. The weight percentage (w/w) of lactic acid in the PLGA polymer may be about 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. The weight percentage (w/w) of glycolic acid in the PLGA polymer may be about 10-90%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, or 80-90%. The weight percentage (w/w) of glycolic acid in the PLGA polymer may be about 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%. The weight ratio (w/w) of lactic acid to glycolic acid in the polymer can be from about 10% lactic acid and about 90% glycolic acid to about 90% lactic acid and about 10% glycolic acid. The weight ratio (w/w) of lactic acid to glycolic acid in the polymer can be about 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% lactic acid and about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10% glycolic acid.

In various embodiments, the weight ratio (w/w) of lactic acid to glycolic acid is about 10-90% lactic acid and about 90-10% glycolic acid. In some embodiments, the weight ratio of lactic acid to glycolic acid (w/w) is about 10% lactic acid to about 90% glycolic acid. In some embodiments, the weight ratio of lactic acid to glycolic acid (w/w) is about 25% lactic acid to about 75% glycolic acid. In some embodiments, the weight ratio (w/w) of lactic acid to glycolic acid is about 50% lactic acid to about 50% glycolic acid. In some embodiments, the weight ratio of lactic acid to glycolic acid (w/w) is about 75% lactic acid to about 25% glycolic acid. In some embodiments, the weight ratio (w/w) of lactic acid to glycolic acid is about 90% lactic acid to about 10% glycolic acid.

Furthermore, PLGA nanoparticles may be surface modified or functionalized with additional polymers such as polyethylene glycol (PEG) or copolymers such as poloxamers. Various PEG and poloxamer reagents commercially available from various sources including Sigma-Aldrich and Thermo Fisher Scientific are suitable for this use.

The size and diameter of the PLGA nanoparticles may depend on the method used to make the nanoparticles, such as nano-precipitation or emulsion. In all cases, the size and surface charge of the nanoparticles can be quantified by Dynamic Light Scattering (DLS) and zeta potential (zeta potential) measurements, respectively. A common instrument for making these measurements is Malvern Zetasizer Nano ZS; all measurements described herein are measurements obtained using the apparatus, unless otherwise specified. These measurements can be used to determine the average diameter of the particles, the surface charge (zeta potential) of the particles, and the polydispersity (polydispersity index, PdI) of the particles in the population.

In various embodiments, the average diameter of the myrcene-encapsulating PLGA nanoparticles is about 150-500nm, 150-200nm, 200-250nm, 200-350nm, 250-300nm, 300-350nm, 350-400nm, 400-450nm or 450-500 nm. In some embodiments, the average diameter of the nanoparticles is about 150nm, 175nm, 200nm, 225nm, 250nm, 275nm, 300nm, 325nm, 350nm, 375nm, 400nm, 450nm, 475nm, or 500 nm. In some embodiments, the average diameter of the nanoparticles is no greater than about 150-. In some embodiments, the nanoparticles have an average diameter of no more than about 150nm, 175nm, 200nm, 225nm, 250nm, 275nm, 300nm, 325nm, 350nm, 375nm, 400nm, 450nm, 475nm, or 500 nm.

The diameter of the nanoparticles in the population can also be described using the particle size distribution (D value). The D value reflects the mass of the nanoparticles in the population as a percentage when the particles are arranged on an ascending mass basis. For example, the D10 value is a diameter where 10% of the nanoparticle population mass consists of particles smaller than the indicated diameter value. In this case, the nanoparticle population consists essentially of particles larger than the indicated diameter value. The D50 value is the diameter at which 50% of the nanoparticle population mass consists of particles smaller than the indicated diameter value and 50% of the nanoparticle population mass consists of particles larger than the indicated value. In this case, the nanoparticle population is also composed of particles larger than the indicated diameter value and smaller than the indicated diameter. The D90 value is the diameter at which 90% of the nanoparticle population mass is composed of particles smaller than the indicated diameter value. In this case, the nanoparticle population consists essentially of particles smaller than the indicated diameter value.

In various embodiments, the terpene/cannabinoid encapsulated PLGA nanoparticles have a D10 diameter value of about 150nm, 175nm, 200nm, 225nm, 250nm, 275nm, 300nm, 325nm, 350nm, 375nm, 400nm, 450nm, 475nm, or 500 nm. The D50 diameter value for the terpene/cannabinoid encapsulated PLGA nanoparticles may be about 150nm, 175nm, 200nm, 225nm, 250nm, 275nm, 300nm, 325nm, 350nm, 375nm, 400nm, 450nm, 475nm, or 500 nm. The D90 diameter value for the terpene/cannabinoid encapsulated PLGA nanoparticles may be about 150nm, 175nm, 200nm, 225nm, 250nm, 275nm, 300nm, 325nm, 350nm, 375nm, 400nm, 450nm, 475nm, or 500 nm.

In certain embodiments, the nanoparticle comprises additional polymers known in the art. Such polymers include poly (lactic acid), poly (glycolic acid), gelatin, dextran, chitosan, lipids, phospholipids, polycyanoacrylates, polyesters, and poly (epsilon-caprolactone). The polymers may be selected based on their degradation characteristics, resulting in the formulation of pharmaceutical compositions for prolonged drug delivery. The polymer may be selected according to its ability to affect persistence in the subject, thereby allowing for timed release of the drug during treatment.

Pharmaceutical composition

In another aspect, provided herein is a pharmaceutical composition comprising a population of terpene/cannabinoid encapsulated PLGA nanoparticles as described herein, and a pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical composition is a lyophilized composition. In some embodiments, the pharmaceutical composition is lyophilized in the presence of trehalose.

In some embodiments, the pharmaceutical composition further comprises another population of PLGA nanoparticles (i.e., a "second population of PLGA nanoparticles"), wherein the second population of nanoparticles encapsulates at least one compound (i.e., a "third encapsulated compound"). The compound encapsulated in the second nanoparticle population may be a cannabinoid or a terpenoid. In some embodiments, the cannabinoid or terpene in the second nanoparticle population is different from the cannabinoid and terpene encapsulated in the first terpenoid/cannabinoid-encapsulated PLGA nanoparticle population.

In one embodiment, the third encapsulating compound is cannabigerolic acid (CBGA). In one embodiment, the third encapsulating compound is Cannabidiol (CBD). In one embodiment, the third encapsulating compound is Cannabinol (CBN). In one embodiment, the third encapsulating compound is Cannabidivarin (CBDV). In one embodiment, the third encapsulating compound is cannabichromene (CBC). In one embodiment, the third encapsulating compound is cannabidiolic acid (CBDA). In one embodiment, the third encapsulating compound is Cannabigerol (CBG). In one embodiment, the third encapsulated compound is myrcene. In one embodiment, the third encapsulated compound is β -caryophyllene. In one embodiment, the third encapsulating compound is nerolidol. In one embodiment, the third encapsulating compound is limonene. In one embodiment, the third encapsulated compound is phytol. In one embodiment, the third encapsulated compound is pinene. In one embodiment, the third encapsulated compound is linalool. In some embodiments, the second population of nanoparticles encapsulates compounds other than cannabinoids or terpenoids.

In some embodiments, the pharmaceutical composition further comprises an additional population of PLGA nanoparticles (i.e., "a third population of PLGA nanoparticles"), wherein the third population of nanoparticles encapsulates at least one compound (i.e., "a fourth encapsulated compound"). The compound encapsulated in the third nanoparticle population may be a cannabinoid or a terpenoid. In some embodiments, the cannabinoid or terpene in the third nanoparticle population is different from the cannabinoid or terpene encapsulated in the first or second terpene/cannabinoid encapsulated PLGA nanoparticle population.

In some embodiments, the pharmaceutical composition further comprises one or more additional populations of PLGA nanoparticles (i.e., "fourth population of PLGA nanoparticles," "fifth population of PLGA nanoparticles," etc.), wherein the one or more additional populations of nanoparticles encapsulate the at least one compound. The compound encapsulated in the one or more additional populations of nanoparticles may be a cannabinoid or a terpenoid. In some embodiments, the cannabinoids or terpenes in one or more additional nanoparticle populations are different from the cannabinoids and terpenes in the other terpene encapsulated/cannabinoid encapsulated PLGA nanoparticle populations.

In some embodiments, the pharmaceutical composition comprises more than one nanoparticle population, wherein each nanoparticle population comprises a unique terpenoid or cannabinoid. In one embodiment, a pharmaceutical composition comprises a first nanoparticle population encapsulating a terpenoid and a second nanoparticle population encapsulating a cannabinoid. In one embodiment, the pharmaceutical composition comprises a first nanoparticle population encapsulating a first terpenoid and a second nanoparticle population encapsulating a second terpenoid. The pharmaceutical composition may further comprise a third population of nanoparticles encapsulating a third terpenoid, a fourth population of nanoparticles encapsulating a fourth terpenoid, and the like. In another embodiment, a pharmaceutical composition comprises a first population of nanoparticles encapsulating a first cannabinoid and a second population of nanoparticles encapsulating a second cannabinoid. The pharmaceutical composition can further comprise a third population of nanoparticles encapsulating a third cannabinoid, a fourth population of nanoparticles encapsulating a fourth cannabinoid, and the like.

In a particular embodiment, the pharmaceutical composition comprises nanoparticles encapsulating beta-myrcene. In one embodiment, a pharmaceutical composition comprises a nanoparticle encapsulating beta-caryophyllene. In one embodiment, the pharmaceutical composition comprises nanoparticles encapsulating nerolidol. In one embodiment, the pharmaceutical composition comprises a first nanoparticle population encapsulating beta-myrcene, a second nanoparticle population encapsulating beta-caryophyllene, and/or a third nanoparticle population encapsulating nerolidol.

In one embodiment, the pharmaceutical composition comprises nanoparticles encapsulating cannabidiol. In one embodiment, the pharmaceutical composition comprises a first nanoparticle population encapsulating cannabidiol and a second nanoparticle population encapsulating terpenoids such as beta-myrcene, beta-caryophyllene, nerolidol, and/or other terpenoids. In one embodiment, each terpenoid is encapsulated in a separate population of nanoparticles.

General dosage range

In vivo and/or in vitro assays may optionally be employed to help determine the optimal dosage range for use. The precise dose to be employed in the formulation will also depend on the route of administration and the severity of the condition, and should be determined at the discretion of the practitioner and in the individual subject's circumstances. Effective doses can be extrapolated from dose response curves in vitro or in animal model test systems.

Unit dosage form

The pharmaceutical composition comprising terpenoid/cannabinoid-containing PLGA nanoparticles may conveniently be presented in unit dosage form.

The unit dosage form will generally be adapted to the particular route or routes of administration of the nanoparticle pharmaceutical composition.

In various embodiments, the unit dosage form is suitable for administration by inhalation. In certain of these embodiments, the unit dosage form is suitable for administration by a vaporizer. In certain such embodiments, the unit dosage form is suitable for administration by nebulizer. In certain such embodiments, the unit dosage form is suitable for administration by nebulizer.

In various embodiments, the unit dosage form is suitable for oral, buccal, or sublingual administration.

In some embodiments, the unit dosage form is suitable for intravenous, intramuscular, or subcutaneous administration.

In some embodiments, the unit dosage form is suitable for intrathecal or intracerebroventricular administration.

In some embodiments, the unit dosage form is suitable for topical administration, including, for example, transdermal administration.

Method for obtaining PLGA nanoparticles encapsulating terpenoids/cannabinoids

In another aspect, a method for obtaining PLGA nanoparticles encapsulating terpenoids/cannabinoids is provided. In various embodiments, the method for obtaining PLGA nanoparticles encapsulating terpenoids/cannabinoids comprises the steps of: (a) providing an organic solution comprising a first terpenoid or a first cannabinoid, a PLGA copolymer and a solvent, and an aqueous solution comprising a surfactant, (b) emulsifying the two solutions to form a suspension of terpenoid/cannabinoid encapsulated PLGA nanoparticles, (c) evaporating the solvent from the emulsion, and (d) obtaining terpenoid/cannabinoid encapsulated PLGA nanoparticles.

Solution for obtaining PLGA nanoparticles encapsulating terpenoids/cannabinoids

Different ratios of terpenoid/cannabinoid and PLGA copolymers may be used in the solution of step (a). These ratios can be equal amounts of the first terpenoid or cannabinoid and PLGA copolymer (e.g., a 1:1 weight ratio, or a 100% (w/w) ratio of the first terpenoid/cannabinoid to the PLGA copolymer), a lower content of the first terpenoid/cannabinoid than the PLGA copolymer (e.g., a 1:2, 1:3, or 1:5 weight ratio, or a 50%, 33%, 20% (w/w) ratio of the first terpenoid/cannabinoid to the PLGA copolymer), or a higher content of the first terpenoid/cannabinoid than the PLGA copolymer (e.g., a 2:1, 3:1, or 5: 1 weight ratio, or a 200%, 300%, 500% (w/w) ratio of the first terpenoid/cannabinoid to the PLGA copolymer). The weight ratio of terpenoid/cannabinoid to PLGA copolymer may be about 1:1 to 1:20, 1:1 to 1:10, 1:1 to 1:5, 1:5 to 1:10, 1:10 to 1:15, or 1:15 to 1: 20. The weight ratio of the first terpenoid/cannabinoid to the PLGA copolymer can be at least about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1: 20. In various embodiments, the weight ratio of the first terpenoid/cannabinoid and PLGA copolymer in the solution is about 1:5 to about 1: 1. in various embodiments, the weight ratio of the first terpenoid/cannabinoid and PLGA copolymer in the solution is about 1: 5. in various embodiments, the weight ratio of the first terpenoid/cannabinoid and PLGA copolymer in the solution is about 1: 4. in various embodiments, the weight ratio of the first terpenoid/cannabinoid and PLGA copolymer in the solution is about 1: 3. in various embodiments, the weight ratio of the first terpenoid/cannabinoid and PLGA copolymer in the solution is about 1: 2. in various embodiments, the weight ratio of the first terpenoid/cannabinoid and PLGA copolymer in the solution is about 1: 1.

the solution may comprise additional terpenoids and/or cannabinoids in addition to the first terpenoid or cannabinoid. In one embodiment, the solution comprises at least one cannabinoid or terpenoid other than the first terpenoid or cannabinoid, and is substantially free of THC. In some embodiments, the at least one cannabinoid or terpenoid other than the first terpenoid or cannabinoid is selected from the group consisting of: cannabidiol (CBD), Cannabinol (CBN), Cannabidivarin (CBDV), cannabichromene (CBC), cannabidiolic acid (CBDA) and Cannabigerol (CBG). In other embodiments, the at least one cannabinoid or terpenoid other than the first terpenoid or cannabinoid is selected from the group consisting of: myrcene, beta-caryophyllene, nerolidol, phytol, limonene, linalool and pinene.

The solution used in step (a) of the method of preparing the PLGA nanoparticles encapsulating terpenoid/cannabinoid further comprises a solvent and a surfactant.

Various solvents are known in the art. Any suitable solvent may be used to make the PLGA nanoparticles encapsulating terpenoid/cannabinoid. Exemplary solvents include, but are not limited to, acetone, ethyl acetate, ethanol, methanol, diethyl ether, toluene, hexane, benzene, dichloromethane, tetrahydrofuran, acetonitrile, dimethylformamide, dimethyl sulfoxide, acetic acid, n-butanol, isopropanol, n-propanol, and formic acid, or any combination thereof. In one embodiment, the solvent is acetone, dichloromethane or ethyl acetate. In another embodiment, the solvent is ethyl acetate.

Various surfactants are known in the art, and any suitable surfactant may be used to make the PLGA nanoparticles encapsulating terpenoids/cannabinoids. Exemplary surfactants include, but are not limited to, anionic surfactants such as ammonium lauryl sulfate, sodium lauryl sulfate or sodium dodecyl sulfate, sodium lauryl ether sulfate, sodium myristyl polyether sulfate, sodium stearate, and other carboxylic acid salts; cationic surfactants, such as compounds having primary, secondary, tertiary or quaternary amines, such as octenidine dihydrochloride; cetrimide, benzalkonium chloride and benzethonium chloride; or nonionic surfactants, such as ethoxylates, including fatty acid ethoxylates, alkylphenol ethoxylates, such as nonoxynol, poloxamers, fatty acid esters of glycerol, such as glycerol monostearate and glycerol monolaurate, polyethylene glycol (PEG), and polyvinyl alcohol (PVA), or any combination thereof. The surfactant may be hydrophilic or lyophilic. In one embodiment, the surfactant is polyethylene glycol, poloxamer or polyvinyl alcohol (PVA). In one embodiment, the surfactant is polyvinyl alcohol (PVA).

Emulsification

The terpene/cannabinoid encapsulated nanoparticles are formed in step (b) by mixing the two solvent phases, resulting in nanocapsules or nanospheres, depending on the method chosen. Methods such as nanoprecipitation form nanospheres, while microemulsions and emulsification with high speed homogenizers produce nanocapsules.

In various embodiments, the emulsification process is homogenization or sonication. In certain embodiments, the emulsifying step is performed using homogenization.

Homogenization may be performed using a high speed homogenizer, such as a Polytron homogenizer available from Thomas Scientific. Homogenization may be performed at about 10,000 to 50,000 rpm. In some embodiments, homogenization is performed at about 10,000 to 50,000rpm, 10,000 to 15,000rpm, 15,000 to 20,000rpm, 20,000 to 25,000rpm, 25,000 to 30,000rpm, 30,000 to 35,000rpm, 35,000 to 40,000rpm, 40,000 to 45,000rpm, or 45,000 to 50,000 rpm. In some embodiments, homogenization is performed at about 10,000rpm, 15,000rpm, 20,000rpm, 21,000rpm, 22,000rpm, 23,000rpm, 24,000rpm, 25,000rpm, 26,000rpm, 27,000rpm, 28,000rpm, 29,000rpm, 30,000rpm, 35,000rpm, 40,000rpm, 45,000rpm, or 50,000 rpm. In one embodiment, homogenization is performed at 20,000 to 30,000 rpm. In another embodiment, homogenization is performed at 24,000 rpm.

In some embodiments, the solution is homogenized for at least about 10 seconds to 10 minutes. In some embodiments, the solution is homogenized for at least about 10s, 15s, 20s, 30s, 40s, 45s, 50s, 55s, 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, 5 minutes, 5.6 minutes, 6 minutes, 6.5 minutes, 7 minutes, 7.5 minutes, 8 minutes, 8.5 minutes, 9 minutes, 9.5 minutes, or 10 minutes. In one embodiment, the solution is homogenized for about 30 seconds to 10 minutes. In one embodiment, the solution is homogenized for 1 minute.

Evaporation of the solvent

After the emulsification step, the solution comprises a suspension of PLGA nanoparticles encapsulating terpenoid/cannabinoid. In step (c), the solvent in the nanoparticle suspension is then removed by evaporation. Evaporation techniques include, but are not limited to, stirring the suspension and solvent, applying a gas stream, applying heat, maintaining the temperature at 10 ℃, and creating a vacuum. A rotary evaporator can be used to evaporate the solvent in the nanoparticle suspension at room temperature or at a cooler temperature. In this case, the sample may be placed on ice in a refrigerator or cold room during evaporation, or the rotary evaporator may have a cooler unit attached. The solvent may also be evaporated by stirring the nanoparticle suspension. In some embodiments, evaporating the solvent comprises stirring the suspension at room temperature. The suspension may be stirred for about 5min to 120 min. In some embodiments, the suspension is stirred for about 5-10 minutes, 10-15 minutes, 15-30 minutes, 30-45 minutes, 45-60 minutes, 60-75 minutes, 75-90 minutes, 90-105 minutes, or 105-120 minutes. In some embodiments, the suspension is stirred for about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, or 120 minutes. In one embodiment, the suspension is stirred for 5min to 120min to evaporate the solvent. In one embodiment, the suspension is stirred for 60 minutes.

Centrifugation

In some embodiments, evaporation of the solvent in step (c) allows obtaining PLGA nanoparticles. In one embodiment, centrifugation is performed at 10 ℃.

In some embodiments, after evaporation of the solvent, the terpene/cannabinoid encapsulated PLGA nanoparticles are obtained by further centrifugation, filtration, or centrifugation and filtration. In a specific embodiment, obtaining the nanoparticles comprises centrifugation. The nanoparticle suspension may be centrifuged at 2,000_ x g to 15,000_ x g. In some embodiments, the nanoparticle suspension is centrifuged at 2,000 to 4,000_ x g, 2,000 to 10,000_ x g, 2,000 to 15,000_ x g, 4,000 to 10,000_ x g, 4,000 to 15,000_ x g, 7,000 to 10,000_ x g, 7,000 to 15,000_ x g, or 10,000 to 15,000_ x g. The nanoparticle suspension may be centrifuged at 2,000rpm to 15,000 rpm. In some embodiments, the nanoparticle suspension is centrifuged at 2,000 to 4,000rpm, 2,000 to 10,000rpm, 2,000 to 15,000rpm, 4,000 to 10,000rpm, 4,000 to 15,000rpm, 7,000 to 10,000rpm, 7,000 to 15,000rpm, or 10,000 to 15,000 rpm. In one embodiment, centrifugation is performed at 4,000x g.

The suspension may be centrifuged for about 5 to 60 minutes. The suspension may be centrifuged for about 5 to 10 minutes, 10 to 15 minutes, 15 to 20 minutes, 20 to 25 minutes, 25 to 30 minutes, 30 to 35 minutes, 35 to 40 minutes, 40 to 45 minutes, 45 to 50 minutes, 50 to 55 minutes, or 55 to 60 minutes. The suspension may be centrifuged for about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes. In some embodiments, centrifugation is performed for 10 minutes, 15 minutes, 30 minutes, 45 minutes, or 10-45 minutes. In one embodiment, centrifugation is performed for 30 minutes. In some embodiments, the suspension may be centrifuged for a longer period of time. For example, the suspension may be centrifuged for about 30 minutes to 1 hour, 30 minutes to 2 hours, or 3 minutes to 3 hours.

Freeze-drying

After obtaining the terpenoid/cannabinoid encapsulated nanoparticles, the nanoparticles may optionally be lyophilized. In this case, a cryoprotectant or a cryopreservative may be added to the terpene/cannabinoid encapsulated PLGA nanoparticles. Any suitable cryoprotectant known in the art may be used, including but not limited to trehalose, ethylene glycol, propylene glycol, glycerol, and dimethyl sulfoxide, and any combination thereof. In one embodiment, the cryoprotectant is trehalose. The cryoprotectant may be added at a concentration of about 1% (w/v) to 25% (w/v) of the PLGA nanoparticles encapsulating the terpenoid/cannabinoid. The cryoprotectant may be added to the nanoparticle at a concentration of about 1-2% (w/v), 2-5% (w/v), 5-10% (w/v), 10-15% (w/v), 15-20% (w/v), or 20-25% (w/v). The cryoprotectant may be added to the nanoparticle at a concentration of at least about 1% (w/v), 2% (w/v), 3% (w/v), 4% (w/v), 5% (w/v), 6% (w/v), 7% (w/v), 8% (w/v), 9% (w/v), 10% (w/v), 12% (w/v), 15% (w/v), 17% (w/v), 20% (w/v), 22% (w/v), or 25% (w/v). In some embodiments, the cryoprotectant is added at a concentration of 1-10% (w/v) of the PLGA nanoparticles encapsulating terpenes/cannabinoids. In one embodiment, the cryoprotectant is added at a concentration of 5% (w/v) of PLGA nanoparticles encapsulating terpenoids/cannabinoids.

The nanoparticles may be lyophilized for about 10 to 240min, 10 to 30min, 30 to 45min, 45 to 60min, 60 to 75min, 75 to 90min, 90 to 105min, 105 to 120min, 120 to 135min, 135 to 150min, 150 to 165min, 165 to 180min, 180 to 195min, 195 to 210min, 210 to 225min, 225 to 240min, or more than 240min, e.g., overnight. The nanoparticles can be lyophilized for about 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 135 minutes, 150 minutes, 165 minutes, 180 minutes, 195 minutes, 210 minutes, 225 minutes, 240 minutes, or overnight. In some embodiments, the terpenoid/cannabinoid-encapsulating PLGA nanoparticles are lyophilized for 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, or 30-180 minutes. In one embodiment, the terpenoid/cannabinoid encapsulated PLGA nanoparticles are lyophilized for 120 minutes.

In some embodiments, the nanoparticles are lyophilized for a longer period of time. For example, depending on the volume of water used, the nanoparticles are lyophilized for about 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or more (e.g., 24-72 hours).

Drug loading and encapsulation efficiency

The amount of terpenoid/cannabinoid or other compound encapsulated in the nanoparticle can be quantified. Drug compound content can be expressed as encapsulation efficiency (EE,%) and drug loading (DL,%), as defined above in section 4.1.

In some embodiments, the method has an encapsulation efficiency (EE,%) of about 1% to 100%. In some embodiments, the encapsulation efficiency is about 1-5%, 5-10%, 4-7%, 4-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, or 95-100%. In some embodiments, the encapsulation efficiency is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. In some embodiments, the encapsulation efficiency is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. In one embodiment, the encapsulation efficiency is at least 4%. In one embodiment, the encapsulation efficiency is at least 5%. In one embodiment, the encapsulation efficiency is at least 6%. In one embodiment, the encapsulation efficiency is at least 7%. In one embodiment, the encapsulation efficiency is at least 8%. In one embodiment, the encapsulation efficiency is at least 9%. In one embodiment, the encapsulation efficiency is at least 10%.

In some embodiments, the method has a drug loading (DL%) of about 1% to 40%. In some embodiments, the drug load is about 1-5%, 5-10%, 4-7%, 4-10%, 10-15%, 15-20%, 20-25%, or 25-30%. In some embodiments, the drug loading is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, or 30%. In some embodiments, the drug load is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, or 30%.

In some embodiments, drug loading is expressed as the average weight ratio of terpenoid/cannabinoid and PLGA copolymer encapsulated in the nanoparticles. In some embodiments, the average weight ratio of encapsulated terpenoid/cannabinoid and PLGA copolymer in the terpenoid/cannabinoid encapsulated PLGA nanoparticles is from about 1:50 to about 1: 10. In some embodiments, the average weight ratio is about 1:50 to 1:40, 1:40 to 1:30, 1:30 to 1:20, and 1:20 to 1: 10. In some embodiments, the average weight ratio is at least about 1:50, 1:45, 1:40, 1:35, 1:30, 1:25, 1:20, 1:15, 1:14, 1:13, 1:12, 1:11, 1: 10. In some embodiments, the average weight ratio is at least about 1: 50. In some embodiments, the average weight ratio is at least about 1: 40. In some embodiments, the average weight ratio is at least about 1: 30. In some embodiments, the average weight ratio is at least about 1: 20. In some embodiments, the average weight ratio is at least about 1: 10.

Therapeutic methods using PLGA nanoparticles encapsulating terpenoids/cannabinoids

In another aspect, methods of treating various symptoms or diseases in a mammalian subject are provided. The method comprises administering a terpenoid/cannabinoid-containing PLGA nanoparticle described herein. These methods are particularly directed to the therapeutic and prophylactic treatment of mammals, particularly humans.

The actual amount administered, as well as the rate and schedule of administration, will depend on the nature and severity of the condition or disease being treated. The prescription of treatment, e.g., determination of dosages, etc., is within the skill of general practitioners and other medical professionals, and generally takes into account the condition to be treated, the individual condition of the patient, the route of administration, the site to be treated, and other factors known to practitioners.

In vivo and/or in vitro assays may optionally be employed to help determine the optimal dosage range to use, as well as the route and time of administration. The precise dose employed in the formulation will also depend on the route of administration and the severity of the condition, and should be decided according to the judgment of the practitioner and each subject's circumstances. Effective doses and methods of administration can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

In some embodiments, the encapsulated terpenoid or cannabinoid is administered in an amount of less than 1g, less than 500mg, less than 100mg, less than 10mg per dose.

In the methods of treatment described herein, the pharmaceutical composition comprising the terpenoid/cannabinoid-containing PLGA nanoparticles may be administered alone or in combination with other treatments administered simultaneously or sequentially with the terpenoid/cannabinoid-containing composition.

Cells in mammalian subjectsMethods of effecting TRPV1 desensitization

Methods for achieving TRPV1 desensitization in a cell of a mammalian subject are provided, the methods comprising administering to the subject a terpenoid/cannabinoid-containing pharmaceutical composition described herein by route and time of administration in an amount sufficient to cause TRPV1 desensitization in the cell of the subject. In some embodiments, the cell to be subjected to TRPV1 desensitization is a nociceptor, such as a peripheral nociceptor and a visceral nociceptor.

In various embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered orally, buccally or sublingually.

In various embodiments, the pharmaceutical composition is administered topically. In particular embodiments, the pharmaceutical composition is administered topically to achieve transdermal delivery.

In some embodiments, the pharmaceutical composition is administered parenterally. In certain embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered by inhalation.

Method of treating pain

In some embodiments, the receptor to be subjected to TRPV1 desensitization is nociceptive, and the method comprises administering to the subject by route of administration a terpene/cannabinoid-containing pharmaceutical composition described herein in an amount and for a time sufficient to cause TRPV1 desensitization at nociceptors in the subject.

In some embodiments, the nociceptor is a peripheral nociceptor. In certain such embodiments, the pharmaceutical composition is administered topically. In some embodiments, the pain neuron is visceral. In certain such embodiments, the pharmaceutical composition is administered systemically.

In a related aspect, methods for treating pain in a mammalian subject are provided. The method comprises administering to the subject a pharmaceutical composition described herein by route and time of administration in an amount sufficient to reduce pain.

In certain embodiments, the pain is neuropathic pain. In a particular embodiment, the neuropathic pain is diabetic peripheral neuropathic pain. In a particular embodiment, the pain is post-herpetic neuralgia. In a particular embodiment, the pain is trigeminal neuralgia.

In some embodiments, the subject has pain associated with or caused by: strain, sprain, arthritis or other joint pain, bruising, back pain, fibromyalgia, endometriosis, surgery, migraine, cluster headache, psoriasis, irritable bowel syndrome, chronic interstitial cystitis, vulvodynia, trauma, musculoskeletal disease, shingles, sickle cell disease, heart disease, cancer, stroke, or an oral ulcer or ulcer due to chemotherapy or radiation.

In some embodiments, the pharmaceutical composition is administered at least once daily for at least 3 days. In some embodiments, the pharmaceutical composition is administered at least once daily for at least 5 days. In some embodiments, the pharmaceutical composition is administered at least once daily for at least 7 days. In some embodiments, the pharmaceutical composition is administered at least once daily for more than 7 days.

In various embodiments, the pharmaceutical composition is administered at a dose, route of administration, and schedule sufficient to maintain an effective level of terpenoid or cannabinoid at the nociceptor for at least 3 days, at least 5 days, or at least 7 days.

Method for treating cardiac hypertrophy

In another aspect, a method of treating cardiac hypertrophy in a mammalian subject is provided. The method comprises administering to the subject an anti-hypertrophic effective amount of a pharmaceutical composition described herein.

In typical embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered by inhalation.

Method for prophylactic treatment of cardiac hypertrophy

In another aspect, a method of prophylactically treating cardiac hypertrophy in a mammalian subject is provided. The method comprises administering to a subject at risk of cardiac hypertrophy an anti-hypertrophy effective amount of a terpenoid/cannabinoid-containing pharmaceutical composition described herein.

Method of treating overactive bladder

In another aspect, a method of treating overactive bladder in a mammalian subject is provided. The method comprises administering to the subject a therapeutically effective amount of a terpenoid/cannabinoid-containing pharmaceutical composition described herein.

In typical embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered by bladder irrigation.

Method for treating intractable chronic cough

In another aspect, there is provided a method of treating refractory chronic cough, the method comprising administering to a subject a therapeutically effective amount of a terpenoid/cannabinoid-containing pharmaceutical composition described herein.

In some embodiments, the pharmaceutical composition is administered systemically.

In some embodiments, the pharmaceutical composition is administered by inhalation.

Methods of treating disorders having a TRPV1 etiology

In another aspect, the diseases or conditions treated with the terpenoid/cannabinoid-containing pharmaceutical compositions described herein include diseases associated with abnormal function of TRPV 1. The disease may be associated with abnormal activation, inhibition or dysregulation of TRPV 1. In some embodiments, the disease is associated with aberrant expression or mutation of a gene encoding TRPV 1.

In some embodiments, the disease treated with the terpenoid/cannabinoid-containing pharmaceutical compositions described herein is a disease associated with abnormal synthesis of an endogenous TRPV1 agonist.

Examples

The following are examples of specific embodiments for practicing the invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology, within the skill of the art. These techniques are explained fully in the literature.

Abbreviations

DCM dichloromethane

DI drug Loading

EE encapsulation efficiency

NP nanoparticles

PEG polyethylene glycol

Copolymers of PLGA lactic acid and glycolic acid

PVA polyvinyl alcohol

Zeta potential

Method

Characterization method of nanoparticles containing terpenoids

The mean diameter and size distribution of the synthesized and purified nanoparticles containing terpenoid compounds were measured in triplicate at 25.0 ± 0.5 ℃ by dynamic light scattering (Nanosizer ZS, Malvern Instruments ltd., UK).

Zeta potential of PLGA-PEG based NPs (terpenoid-containing and terpenoid-free) was characterized by laser doppler electrophoresis (Nanosizer ZS, Malvern Instruments ltd., UK) and triplicate zeta measurements of NPs were performed after final washing with MQ water at 25 ℃.

Morphological characterization of the nanoparticles was performed by image analysis by scanning electron microscopy in a FEI TENEO microscope. Samples were prepared by diluting the terpenoid-containing nanoparticle suspension (without trehalose) to about 0.8mg/ml and covering with 8-9nm Pd/Pt shell (Leica EM SCD500) under vacuum.

The amount of myrcene, beta-caryophyllene, or nerolidol encapsulated in the nanoparticles was measured by OC-MS spectroscopy. Analysis was performed using a Trace 1300GC gas chromatography system equipped with a ZB-1MS capillary column (30 m.times.0.25 mm.times.0.25 μm, Thermo Scientific) in series with a mass spectrometer TSQ8000(Thermo Scientific), and data acquisition and analysis was performed using an Xcalibur.

For mass spectrometry, ionization was performed by Electron Impact (EI) in positive ion mode at a voltage of 70eV, full scan mode in the m/z range of 20-300, and ion source temperature of 200 ℃. To quantify myrcene, caryophyllene, and nerolidol, the characteristic ions of terpenoids at m/z 69, 93, and 133, respectively, were monitored by a Selective Ion Monitoring (SIM) mode. To generate a calibration curve, myrcene, caryophyllene, or nerolidol was prepared in Dichloromethane (DCM) with a range of concentrations ranging from 1-60ppm (1, 2,5, 10, 20, 40, and 60ppm) and measured in a GC-MS instrument.

The drug encapsulation capacity of PLGA-PEG nanoparticles can be expressed as an encapsulation efficiency (EE,%) and a drug loading capacity (DL,%) according to equations 1 and 2, respectively.

Cells

HEK TRPV1 cells were supplied by Helen Turner laboratories of Hamine University of Honolulu and maintained in MEM essential medium supplemented with 10% fetal bovine serum, 50 units/ml-50. mu.g/ml penicillin-streptomycin, and 0.6mg/ml geneticin.

Calcium signal determination method

HEK TRPV1 cells were harvested with trypsin, inactivated with media and counted. Cells were centrifuged at 1000rpm for 5 minutes at room temperature. Cells were washed twice with 1mM Ca assay buffer (Na ringer [140mM NaCl, 2.8mM KCl, 2mM MgCl2, 11mM glucose, 10mM HEPES ], 2mM probenecid, 1mM CaCl 2; pH 7.4) and harvested by centrifugation at 1000rpm for 5 minutes at room temperature. Cells were resuspended in 1. mu.M Fluo-4 (1. mu.l of 5mM Fluo-4 mixed with 1. mu.l of 20% Pluronic F-127 in DMSO, then 5mL of 1mM Ca assay buffer was added and the mixture incubated in 37 ℃ dark for 10 min) and in 37 ℃ dark for 30 min. The cells were then washed twice with 1mM Ca assay buffer, then resuspended in assay buffer, and pipetted into opaque-walled 96-well plates at a density of 150,000 cells/180 μ Ι/well. Fluorescence was measured using a plate reader (Synergy HTX, BioTek, USA) at an excitation wavelength of 485nm and an emission wavelength of 528 nm. Once a baseline was established (3 measurements), 20 μ Ι of stimulus (free terpenoid, terpenoid-loaded NP, and control) solution was added to each well and the measurements were continued for 1 hour, reading every 40 seconds.

Preparation of irritant solutions

All solutions were prepared in 1mM Ca assay buffer. The free terpenoids are first dissolved in DMSO and then diluted to a specific volume with assay buffer to give the desired concentration. Terpenoid-loaded PLGA-PEG or poly (ethylene glycol) methyl ether-block-poly (lactide-co-glycolide) Nanoparticles (NPs) were prepared by an emulsification method and diluted directly using assay buffer. Negative controls of assay buffer containing DMSO and blank NPs were also prepared. Ionomycin 4 μ M was used as a positive control. Free and encapsulated terpenoids were prepared at a concentration of 400 μ g/ml (resulting in a final concentration of 40 μ g/ml after addition to the cells) alone or in combination, for example, myrcene plus nerolidol, myrcene plus caryophyllene, nerolidol plus caryophyllene, and myrcene plus nerolidol plus β -caryophyllene.

Experiments were performed in triplicate, reactions of negative controls were removed, and the average was calculated and used to plot a calcium response curve.

Example 1-Nanopacipitation method preparation of myrcene-encapsulated nanoparticles

The solvent displacement-based nano-precipitation method (fig. 1) was tested for the production of myrcene-containing PLGA nanoparticles. The preparation of particles by the nano-precipitation method is determined by the so-called marangoni effect, which is subject to interfacial turbulence occurring at the interface of solvent and non-solvent and is caused by complex and cumulative phenomena such as flow, diffusion and surface tension variations. The presence of a stabilizer is very important to avoid aggregate formation and to impart stability to the nanoparticles during the stage of the nanoprecipitation technique.

Preparation

PLGA-based Nanoparticles (NPs) were prepared using a nanoprecipitation method according to the formulation in Table 1 (F1-F11). A mixture of hydrophilic and lipophilic surfactants (pluronic and Span) was used to ensure system stability. Different experimental conditions were used-acetone was evaporated at room temperature or using a rotary evaporator in the absence or presence of Tocopherol Polyethylene Glycol Succinate (TPGS).

% w/w based on PLGA weight

Preparation method using evaporation of organic solvent at room temperature (F1-F6)

PLGA (22.5mg) and spanDissolved in 1.5mL of acetone. Myrcene (10-100% w/w) was added to the organic solution. The organic phase was added dropwise to 4.5mL of an aqueous solution of pluronic F-68 (0.5% w/v) at a rate of 5mL/h using a syringe pump with continuous stirring. Stirring was continued at room temperature for 2 hours to evaporate the acetone. To remove unencapsulated compounds as well as excess surfactant, the suspension was adjusted to 45mL with MQ water and centrifuged at 10,000rpm for 30 minutes at 4 ℃. The pellet was resuspended in 45mL MQ water and centrifuged again using the same conditions. The NP pellet was resuspended in MQ water to prepare 1mL NP suspension. The suspension was then dried in an oven to measure the supported myrcene.

To measure the weight and yield of the NPs produced, NPs were prepared using the same method. After final centrifugation, the NP pellet was resuspended in 2mL MQ water and the formulation was freeze dried. The lyophilized NPs were collected as a white cotton-like material. The supernatant was also collected and lyophilized to measure unencapsulated myrcene.

Samples were prepared for OC-MS analysis. 100 μ L of freshly prepared NPs were completely dried (water removed) in an oven at 40 ℃ for 30-40 minutes. DCM (2ml) was added to dissolve the dried residue. Next, 1.5mL of DCM solution was transferred to a suitable vial and sealed. The lyophilized supernatants stored at-20 ℃ were weighed and samples prepared for GC-MS analysis as described for NPs. Briefly, a 5mg sample of np was dissolved in DCM and 1.5mL of the solution was transferred to a suitable vial and sealed.

Preparation of organic solvent by evaporation using a rotary evaporator in the absence of TPGS (F7-8)

PLGA (22.5mg) and spanDissolved in 1.5mL of acetone. Myrcene (40 or 60% w/w) was added to the organic solution. To 4.5mL of pluronicTo an aqueous solution (0.5% w/v), the organic phase was added dropwise at a rate of 5mL/h using a syringe pump. During mixing, the mixture was kept in a cold water bath to reduce evaporation of the drug. Acetone was removed using a rotary evaporator without heating over 20 minutes. To remove unencapsulated compounds as well as excess surfactant, the suspension was adjusted to 45mL with MQ water and centrifuged at 10,000rpm for 30 minutes at 4 ℃. The pellet was resuspended in 45ml mqmqmq water and centrifuged again using the same conditions. The NP pellet was resuspended in 2mL MQ water and the formulation was freeze dried. The lyophilized NPs were collected as a white cotton-like material. The supernatant was also collected and lyophilized to measure unencapsulated myrcene.

Samples were prepared for GC-MS analysis. The lyophilized NPs were dissolved in DCM. To facilitate dissolution, the samples were sonicated in a bath sonicator for 5 minutes. The sample was filtered using a1 μm syringe filter (compatible with DCM). 1.5mL of the sample solution was transferred to a suitable vial and sealed. The lyophilized supernatants stored at-20 ℃ were weighed and samples prepared for GC-MS analysis as described for NPs. Briefly, a 5mg sample of NP was dissolved in DCM and 1.5mL of the solution was transferred to a suitable vial and sealed.

Preparation method of evaporating organic solvent using rotary evaporator in the Presence of TPGS (F9-11)

Tocopheryl Polyethylene Glycol Succinate (TPGS), vitamin E TPGS, is a water-soluble derivative of natural vitamin E. It is chosen as an emulsifier in the formulation due to its several advantages, including its bulky structure and large surface area characteristics, making it an excellent emulsifier. PLGA-based NPs were prepared using a nanoprecipitation method in the presence of Tocopherol Polyethylene Glycol Succinate (TPGS) according to formulation F9-F11 (table 1).

Mixing PLGA (22.5mg),(5, 2.5 or 0mg) and TPGS (2.5, 5 or 7.5mg) were dissolved in 1.5mL of acetone. Myrcene (9 mg; 40% w/w) was added to the organic solution. To 4.5mL of pluronicTo an aqueous solution (0.5% w/v), the organic phase was added dropwise at a rate of 5mL/h using a syringe pump. During mixing, the mixture was kept in a cold water bath to reduce evaporation of the drug. Acetone was removed using a rotary evaporator without heating over 20 minutes. To remove unencapsulated compounds as well as excess surfactant, the suspension was adjusted to 45mL with MQ water and centrifuged at 10,000rpm for 30 minutes at 4 ℃. The pellet was resuspended in 45mL MQ water and the rotation repeated using the same conditions. The NPs pellet was resuspended in 2ml mqmq water and the formulation was freeze-dried. The lyophilized NPs were collected as a white cotton-like material. The supernatant was also collected and lyophilized to measure unencapsulated myrcene.

Samples were prepared for GC-MS analysis. NPs lyophilisate was dissolved in DCM. To facilitate dissolution, the samples were sonicated in a bath sonicator for 5 minutes. The sample was filtered using a1 μm syringe filter (compatible with DCM). 1.5mL of the sample solution was transferred to a suitable vial and sealed. The lyophilized supernatants stored at-20 ℃ were weighed and samples prepared for GC-MS analysis as described for NPs. Briefly, a 5mg sample of NP was dissolved in DCM and 1.5mL of the solution was transferred to a suitable vial and sealed.

Characterization of nanoparticles prepared by Nanopacipitation

Table 2 presents the average diameter, size distribution, zeta potential, weight, yield, amount of myrcene loaded, EE% and dI% of NPs prepared by nanoprecipitation.

% w/w based on PLGA weight

The NPs produced by nanoprecipitation have a particle size in the range of 200-240nm, a good polydispersity (PdI) of 0.2 or less and a negative zeta potential of about-30 (Table 2). Fig. 8A and 8B show the intensity size distribution and zeta potential distribution, respectively, of sample F6. Fig. 8C and 8D show the intensity size distribution and zeta potential distribution, respectively, of sample F8. Fig. 8E and 8F show the intensity size distribution and zeta potential distribution, respectively, of sample F9.

Stable NPs with concentrations of myrcene of up to 60% were successfully prepared, wherein at concentrations of myrcene above 60% (w/w) the NPs were not physically stable and formed aggregates during the initial mixing step.

Evaporation of acetone using a rotary evaporator without heating increased the amount of myrcene loaded significantly by 5-fold or more, from 0.036mg and 0.042mg in F5 and F6 to 0.155mg and 0.275mg in F7 and F8, respectively (table 2).

TPGS is a synthetic amphiphile for vitamin E and is approved by the FDA as a water-soluble vitamin E nutritional supplement and drug delivery vehicle. It acts as a surfactant, solubilizer, and has the potential to improve drug loading in NPs. Furthermore, alpha-tocopherol derivatives can enhance the oral absorption of hydrophobic materials. The use of TPGS did not appear to improve the amount of myrcene loaded in PLGA NPs, but rather it was found that as the percentage of TPGS increased, the amount of myrcene loaded decreased (F9 and F10, table 2).

Example 2-microemulsion Process for preparing myrcene encapsulated nanoparticles

The formation of microemulsions is also used to obtain PLGA nanoparticles. Microemulsions are systems of water, oil and amphiphiles, which are single optically isotropic and thermodynamically stable liquid solutions. Its formation is spontaneous (fig. 2). Soy lecithin is a natural lipid containing a mixture of phospholipids that has previously been used as amphiphiles for the preparation of various delivery nanosystems, such as nanoemulsions, liposomes, micelles and nanoparticles. This method allows the formation of spherical nanocapsules in which an oily core consisting of myrcene is embedded and retained in a thin dense wall formed by a PLGA polymer and phosphatidylcholine.

Preparation

PLGA-based NPs were prepared using a microemulsion method according to the formulation in table 3.

Myrcene-loaded NPs were prepared according to the method previously described by Iannitelli et al (int. J. mol. Sci.2011,12, 5019-5051). Briefly, PLGA (11mg) and Epikuron 200(13mg) were dissolved in 2.5ml acetone. Myrcene (48mg) was added to the organic solution. The drug-containing organic phase was immediately added to the aqueous phase (5mL of pluronic) with continuous stirring(0.5% w/v or 5% w/v) or polyvinyl alcohol (PVA) (0.5% w/v)). The beaker containing the mixture was covered with parafilm to prevent evaporation of myrcene and stirring was continued for 10 minutes. Acetone was removed using a rotary evaporator without heating over 40 minutes. To remove unencapsulated compounds as well as excess surfactant, the suspension was adjusted to 45mL with MQ water and centrifuged at 10,000rpm for 30 minutes at 4 ℃. The pellet was resuspended in 45mL MQ water,and centrifuged again using the same conditions. The NPs pellet was resuspended in 2mL MQ water and the formulation was freeze-dried. The lyophilized NPs were collected as a white cotton-like material. The supernatant was also collected and lyophilized to measure unencapsulated myrcene.

Samples were prepared for GC-MS analysis. The lyophilized NPs were dissolved in DCM. To facilitate dissolution, the samples were sonicated in a bath sonicator for 5 minutes. The sample was filtered using a1 μm syringe filter (compatible with DCM). 1.5mL of the sample solution was transferred to a suitable vial and sealed. The lyophilized supernatants stored at-20 ℃ were weighed and samples prepared for GC-MS analysis as described for NPs. Briefly, a 5mg sample of NP was dissolved in DCM and 1.5mL of the solution was transferred to a suitable vial and sealed.

Characterization of micro-emulsification method for preparing nano-particles

The microemulsion process for preparing NPs involves preparing an intermediate microemulsion when high concentrations and the correct combination of surfactants (usually surfactants and co-surfactants, here pluronic) are usedAnd Epikuron 200), the intermediate microemulsion forms spontaneously, and then the solvent is diffused from the droplets of the emulsion and evaporated, leaving the NPs.

The mean diameter, size distribution, zeta potential, weight, yield, amount of myrcene loaded, EE% and DL% of NPs prepared by microemulsion are presented in table 4. Fig. 9A shows the intensity size distribution of sample F17, and fig. 9B shows the zeta potential distribution of sample F17.

NPs prepared using PVA 0.5% (as the aqueous phase) were not physically stable and formed aggregates during preparation (F12). The particle size of the blank NPs was about 150nm (F13, F14), while the myrcene-loaded NPs were about 100nm, with a diameter of about 250 nm. All NPs showed good polydispersity of less than 0.2 and negative zeta potentials of-30 to-35.

In all cases, particles in the nanometer range with narrow size distribution (PdI <0.2) and zeta potential of about-32 mV were obtained. No difference between the loaded and blank (-30mV) nanoparticles was observed. Thus, little drug remains on the surface.

Example 3-emulsification (high speed homogenizer) Process for the preparation of myrcene encapsulated nanoparticles

A method based on emulsification of a polymer organic solution into an aqueous phase followed by organic solvent evaporation (fig. 3) was used to prepare myrcene encapsulated nanoparticles. The organic phase is poured into the continuous (aqueous) phase, where the surfactant is dissolved to impart stability to the emulsion. Emulsification is performed under high shear to reduce the size of the emulsion droplets. This process will largely determine the final particle size.

Preparation

PLGA-based NPs were prepared using a single emulsion method according to the formulation in table 5. By usingThe homogenizer performs emulsification.

% w/w based on PLGA weight

PLGA (40mg) was dissolved in 1mL ethyl acetate. Myrcene (10-100% w/w based on PLGA) was added to the organic solution. Preparation of NPs using a high speed homogenizer). The probe of the homogenizer was immersed in a falcon tube containing 5mL of PVA (0.5% w/v). The organic phase containing the drug was added dropwise using a pipette, with the homogenizer set at 24,000rpm, and homogenization continued for 1 min. Homogenization was performed on ice to reduce the temperature generated during the process. The organic solvent was removed using a rotary evaporator without heating over 1 hour. To remove unencapsulated compounds as well as excess surfactant, the suspension was adjusted to 45mL with MQ water and centrifuged at 10,000rpm for 30 minutes at 4 ℃. The pellet was resuspended in 45mL MQ water and centrifuged again using the same conditions. The NPs pellet was resuspended in 2mL MQ water and the formulation was freeze-dried. The lyophilized NPs were collected as a white cotton-like material. The supernatant was also collected and lyophilized to measure unencapsulated myrcene.

Samples were prepared for GC-MS analysis. The lyophilized NPs were dissolved in DCM. To facilitate dissolution, the samples were sonicated in a bath sonicator for 5 minutes. The sample was filtered using a1 μm syringe filter (compatible with DCM). 1.5mL of the sample solution was transferred to a suitable vial and sealed. The lyophilized supernatants stored at-20 ℃ were weighed and samples prepared for GC-MS analysis as described for NPs. Briefly, a 5mg sample of NP was dissolved in DCM and 1.5mL of the solution was transferred to a suitable vial and sealed.

Characterization of nanoparticles produced by emulsification

The average diameter, size distribution, zeta potential, weight, yield, amount of myrcene loaded, EE% and DL% of NPs prepared by the emulsification method are presented in table 6. Fig. 10A shows the intensity size distribution of sample F20, and fig. 10B shows the zeta potential distribution of sample F20.

% w/w based on PLGA weight

The particle size of blank NPs was 225nm, that of-265 nm for myrcene-loaded NPs at drug concentrations of 10-40%, and that of 350-400nm for myrcene-loaded NPs at drug concentrations of 80-100%. However, NPs in F22, F23 and F24 appeared to be damaged during centrifugation, resulting in turbidity of the supernatant. All NPs had good polydispersity (0.2 or less) and a slightly lower negative zeta potential than the other processes (about-25 mV). No difference between the loaded and blank (-25mV) nanoparticles was observed. Thus, little drug is retained on the surface.

The emulsification method showed higher drug loading efficiency compared to other methods used in this study; the highest DI% values were obtained for F23 (1.29%) and F20 (1.20%). However, there was evidence of NPs damage or explosion during centrifugation as indicated by the cloudy supernatant and a portion of the polymer adhering to the walls of the falcon tube near the top of the supernatant. By performing centrifugation at lower rotational speeds or replacing centrifugation with another purification method, such as dialysis, damage to NPs can be reduced and higher drug loading efficiency can result.

Example 4 solvent optimization

Next, the volume of solvent used is optimized to reduce the time of the evaporation step, thereby minimizing potential evaporation of myrcene during the solvent evaporation step. Two preparation methods were used: nano-precipitation (acetone) and emulsification (ethyl acetate) explore the effect of reducing solvent volume.

Emulsification process

The effect of changing the volume of organic solvent using the single emulsion method was determined. Nanoparticles were prepared and lyophilized as previously described in example 3.

% w/w based on PLGA weight

Nano precipitation method

The effect of changing the volume of the organic solvent using the nanoprecipitation method was determined. Nanoparticles were prepared and lyophilized as previously described in example 1.

% w/w based on PLGA weight

Characterization of nanoparticles produced using lower amounts of solvent

We start with the smallest volume of solvent to dissolve the organic phase components first. In general, particle size was found to decrease with increasing solvent volume (table 9). Fig. 11A shows the intensity size distribution of sample F28, and fig. 11B shows the zeta potential distribution of sample F28. Fig. 11C shows the intensity size distribution of sample F31, and fig. 11D shows the zeta potential distribution of sample F31.

In the single emulsion process, 0.125mL (F25) and 0.25mL (F26) of low volume ethyl acetate produced NPs with larger diameters that were not reusable after centrifugation, and the NPs produced using 0.5mL of ethyl acetate were physically stable. F27 showed the largest diameter in stable NPs, but also the highest drug loading efficiency of 0.486mg (EE%: 6.08, DL%: 2.02). It is important to note that this formulation was centrifuged at 6,000rpm and, due to its large diameter ≈ 670nm, can be collected at this speed, while all other formulations were centrifuged at 10,000 rpm. It is possible that higher centrifugation speeds may lead to deformation of the NPs, leading to escape of the drug. Although myrcene is insoluble in water, it can be dissolved by the surfactant PVA or Pluronic.

Similarly, in the nanoprecipitation method, the F31 formulation with the larger diameter (. apprxeq.400 nm) showed a higher myrcene loading efficiency in F31 (EE%: 2.09, DL%: 1.04) of up to 0.188 mg. The use of a cold bath during addition of the organic phase seems to increase the particle size, but it cannot be concluded that it can increase the drug loading (by preventing/reducing drug evaporation during preparation).

Example 5 measurement of myrcene in supernatant

Since the amount of myrcene loaded in NP was relatively low, we attempted to measure the drug in the supernatant to see how the drug was lost. Table 10 shows the total amount of myrcene detected, including the amount of myrcene loaded in the NPs and the amount of free myrcene present in the supernatant, at 0.06-3.44% of the initial amount of drug. The results shown in table 10 indicate that most of the myrcene is lost at some stage; during preparation of NPs, during preparation of samples for analysis, or during storage.

Myrcene loss may occur during production rather than during storage by evaporation, as the freeze-dried sample is stored in a closed container in a refrigerator, which should prevent evaporation of the drug.

Example 6 factors leading to loss of myrcene

Filtration during sample precipitation for GC-MS analysis

The effect of filtration on possible myrcene loss was investigated using myrcene-loaded NPs prepared using emulsification methods (F27, F28, F29 and F30). During the preparation of samples for GC-MS analysis, filtration was used to remove undissolved components of NPs, such as PVA, in DCM. Insoluble impurities in the sample can cause clogging of the syringe during analysis and can interfere with the measurement. 5mg of lyophilized NPs were dissolved in DCM. To facilitate dissolution, the samples were sonicated in a bath sonicator for 5 minutes. The sample was filtered using a1 μm syringe filter (compatible with DCM). The sample solutions (1.5 mL each) were transferred to appropriate vials and sealed. Another set of samples (each weighing 5mg NP) was prepared in DCM without filtration.

The amount of myrcene loaded in the NPs, EE% and dI% before and after filtration using a1 μm syringe filter are shown in table 11.

Filtration results in a relative decrease in the measured amount of myrcene. Most samples showed little concentration reduction, however this effect was more pronounced in F27 with the highest concentration of loaded myrcene. The amount of myrcene was reduced by about 25% from 0.486mg before filtration to 0.368mg after filtration. This may be due to the interaction between the drug and the filter resulting in the entrapment of a proportion of the drug. Based on these findings, filtration is not the main cause of myrcene loss during preparation.

Evaporation of organic solvents

To investigate the effect of evaporation using a rotary evaporator on myrcene loss, two different samples were prepared, as shown in the following table. In the first sample, 40mg of Resomer 502 was dissolved in 1ml of ethyl acetate, followed by the addition of 8mg of myrcene. In a second sample, 8mg of myrcene was mixed with 1ml of ethyl acetate without polymer. Both samples were evaporated under pressure for 1 hour using a rotary evaporator without heating. The formulation is shown in table 12.

The amount of myrcene retained and the percent myrcene loss in the samples are shown in table 13.

The rotary evaporation of myrcene and ethyl acetate samples without PLGA (F38) resulted in complete loss of myrcene. However, when PLGA was added to myrcene (F37), about 40% of the starting drug was retained, probably due to adsorption of the drug onto the polymer. This result may importantly indicate that myrcene trapped in the polymer (in the NP) may be protected from evaporation, while any free drug may be lost during the evaporation step.

Evaporation, centrifugation and freeze drying

To investigate the possible loss of myrcene during the different steps of NPs production, samples were prepared using the nano-precipitation method as described in example 1 and according to the formulation in table 14.

F39 was centrifuged at 10,000rpm for 30 minutes at 4 ℃. The NPs pellet was resuspended in 2mL MQ water and the formulation was freeze-dried. F40 and F41 were freeze dried immediately after evaporation of the solvent without centrifugation. The amount of myrcene in the sample was measured using GC-MS spectroscopy. 5mg of each sample was dissolved in 2mL of DCM and the solution was filtered using a1 μm syringe filter, filled in a suitable vial and sealed.

% w/w based on PLGA weight

The mean diameter, size distribution, zeta potential, weight of lyophilizate, amount of myrcene retained in the sample and percentage of myrcene loss for the different formulations are shown in table 15. Fig. 9A shows the intensity size distribution of sample F41, and fig. 9B shows the zeta potential distribution of sample F41.

F41 was prepared by adding myrcene (without polymer) dissolved in acetone to the aqueous phase. The measurement size of 357nm indicates the formation of a mimic of myrcene in the presence of PVA as a surfactant. These are not polymeric nanoparticles because no PLGA is used, only micelles of PVA are used. F39 was prepared to explore whether centrifugation might be the cause of drug loss from NPs during centrifugation. However, F40 from NPs prepared even without centrifugation showed low retention of drug. Similarly, F3 as a myrcene emulsion (without PLGA addition) also showed similar drug retention after lyophilization. These findings indicate that most of the drug may be lost during lyophilization. There is a possibility that some drug escapes from the NPs during centrifugation, but this cannot be detected with current manufacturing methods, since the measurement of the drug should be performed immediately after centrifugation, rather than after freeze-drying.

Myrcene is encapsulated in PLGA NPs using several methods, including: (i) nano-precipitation, (ii) emulsification using a high-speed homogenizer, and (iii) micro-emulsification. To improve loading efficiency, we also developed and studied several formulations. To reduce the possible evaporation of myrcene, the preparation process was optimized with respect to the temperature during the production of NPs and using a rotary evaporator without heating the sample, which would result in an improved drug loading capacity.

We obtained particles in the nanometer range with narrow size distribution (PdI <0.2) and negative zeta potential (from-25 to-35 mV). The highest loading capacity was achieved with a single emulsion process with encapsulation rates of up to 6% of the starting drug (F27). Small amounts of myrcene measured in the supernatant collected after NPs centrifugation indicate loss of drug during production, not due to the failure of PLGA to encapsulate myrcene.

Myrcene has a low molecular weight (136.23g/mol), a low vapor pressure (2.09 mm Hg at 25 ℃), and although it has a boiling point of 167 ℃, it is a volatile liquid at room temperature, which makes it difficult to keep it within NPs and prevent it from evaporating. Although myrcene can be evaporated during the evaporation step of the production process, the absence of heat in the rotary evaporator significantly reduces the evaporation of PLGA. Thus, loading of drugs in NPs can protect myrcene from substantial evaporation. The amount of solvent and evaporation time were also optimized and decreased from 1 hour to 25 minutes to minimize possible evaporation of the drug. Myrcene is also lost during freeze-drying. It is unclear whether the loss is due to evaporation or sublimation. However, myrcene does not freeze even at-80 ℃.

Example 7 optimization of lyophilization and centrifugation of nanoparticles

Freeze-drying

To investigate whether myrcene was lost during lyophilization, 3 samples of free myrcene, a mixture of myrcene and water, or a mixture of myrcene, water and PLGA were prepared in duplicate, as shown in table 16. Samples were lyophilized for 24 h. Vials containing the samples were weighed before and after lyophilization to calculate the weight of the dried sample.

The results of the lyophilization assay are shown in table 17.

The results show that most of the myrcene is lost during the 24 hour lyophilization process, whether the myrcene is free or combined with a polymer and water. Myrcene does not freeze at the lyophilization temperature (about-80 ℃), which may indicate that it is lost by evaporation under vacuum in a freeze-dryer, rather than by sublimation.

Since lyophilization is a critical step in the loss of myrcene from NPs, we hypothesize that a reduction in lyophilization time may reduce the loss of myrcene in the process. Aliquots of NPs suspensions of 100. mu.L of formulations F49 (myrcene: 100% w/w; centrifugation: Falcon tube, 10,000rpm, 30min, 4 ℃) and F54 (myrcene: 100% w/w; centrifugation: Amicon tube, 4,000Xg, 30min, 12 ℃) were transferred to Eppendorf, frozen and lyophilized for only 2 hours; this time was sufficient to remove 100. mu.L of water. The dried product was then dissolved in DCM and analyzed for myrcene content using a GC-MS instrument as previously described. Samples were prepared in duplicate.

Centrifugation

After confirming the loss of myrcene during lyophilization, we measured the amount of myrcene loaded in NPs prior to the lyophilization step. Two different centrifugation protocols were compared to determine optimal conditions. PLGA NPs were prepared using the single emulsion homogenization method described in example 3. Sample formulations are shown in tables 18 and 19. Formulations F45-F49 were centrifuged at 10,000rpm for 30 minutes at 4 ℃ in a Falcon tube to collect NPs. Formulations F50-F54 were spun at 4,000Xg in Amicon tubes at 12 ℃ for 30 minutes. Samples were prepared in duplicate.

% w/w based on PLGA weight

% w/w based on PLGA weight

The results of increasing the initial amount of myrcene are shown in Table 20. When 100% (w/w, myrcene with PLGA; 40mg myrcene and 40mg PLGA) myrcene was used, it was found that increasing the amount of drug loaded in the initial amount of myrcene NPs increased the drug loading to about 5%. The amount of myrcene loaded was slightly higher when the nanoparticles were collected by centrifugation at 10,000rpm in a Falcon tube, compared to those collected at 4,000 × g by an Amicon tube.

% w/w based on PLGA weight

Comparison of production methods

Once the loss of myrcene during lyophilization was confirmed, we repeated the preparation of NPs using the three preparation methods previously described herein to select the optimal method for drug loading. Myrcene content in NPs was measured after centrifugation but before lyophilization to select the method that provided the highest myrcene loading efficiency and amount.

NPs were prepared using emulsification induced by a high speed homogenizer as described in example 3; nanoprecipitation, as described in example 1; and microemulsions as described in example 2; according to the formulation presented in table 21. After centrifugation of the preparation, NPs were resuspended in MQ water in a total volume of 1 mL. Samples were prepared for GC-MS analysis as previously described. 100 μ L of each NP suspension was transferred to an Eppendorf tube and centrifuged at 10,000rpm for 30 minutes at 4 ℃. The supernatant was discarded and 1mL of DCM was added to dissolve NPs and myrcene. The DCM solution was then diluted and analyzed for myrcene content using a GC-MS instrument as previously described. Samples were prepared in duplicate.

As shown in table 22, emulsification using a homogenizer, particularly after centrifugation with falcon tubes, resulted in the highest drug loading, 9% EE. Nanoprecipitation also produces NPs with relatively good loading, although the initial maximum concentration of myrcene is 60%, since higher concentrations promote aggregation of NPs. In contrast, NPs produced by microemulsions have poor drug loading. In emulsification and microemulsion methods using homogenizers, it is expected that liquid drugs will be entrapped within NPs, resulting in so-called nanocapsules, which are nanoparticles having a liquid core surrounded by a polymer shell. In the nanoprecipitation method, solid NPs, called nanospheres, a polymer matrix in which the drug is dispersed, are produced. In the case of liquid myrcene, it can be expected that there is more drug loading when it is loaded in nanocapsules than nanospheres. This may explain the higher drug loading achieved by emulsification using a homogenizer and suggest that the formulation used in the microemulsion formulation may need to be optimized to produce stable particles with the ability to entrap the drug.

% w/w based on PLGA weight

Stabilization of nanoparticles using cryoprotectants

Myrcene-loaded NPs appear to be damaged during freezing and freeze-drying, resulting in increased drug loss during the lyophilization process. To test the effect of the cryoprotectant trehalose on stabilized NPs, samples were prepared using the emulsification method as previously described using two centrifugation methods described in section 5.8.2. mu.L of the NPs suspension was mixed with 50. mu.L of 10% trehalose solution (to obtain a final mixture of 5% trehalose). The product was frozen and then lyophilized for 2 hours. The dried product was then dissolved in DCM and analyzed for myrcene content using a GC-MS instrument as previously described. Samples were prepared in duplicate. The measurement results are shown in Table 23.

% w/w based on PLGA weight

Conclusion

Myrcene was found to be lost during lyophilization. Measurements of myrcene in NPs suspensions before lyophilization showed that the best preparation method was emulsification with a high speed homogenizer. The method may produce nanocapsules that entrap liquid myrcene in their core. Optimization of lyophilization conditions includes duration and addition of cryoprotectants to create optimal conditions to enhance drug loading. Factors such as centrifugation speed and freezing cause damage to NPs and may contribute significantly to the loss of myrcene during lyophilization. The duration of lyophilization also affected myrcene loss.

NPs prepared by emulsification with a homogenizer, centrifugation at 4,000 × g in an amicon tube, cryoprotection with 5% trehalose, and lyophilization for 2 hours successfully showed the highest drug load of about 7% in the lyophilized NPs. These conditions allow for the production of lyophilized NPs with relatively high drug loadings of up to 8% when 100% (w/w, myrcene with PLGA; 40mg myrcene and 40mg PLGA) of myrcene are used.

Example 8 Synthesis and characterization of nanoparticles containing beta-myrcene, beta-caryophyllene, or nerolidol

This example shows the synthesis and characterization of PLGA-PEG nanoparticles containing beta-myrcene, beta-caryophyllene, or nerolidol.

Using a high speed homogenizer (Kinematica Polytron)^TMPT 2500E homogenizer, Fisher Scientific) PLGA-PEG or poly (ethylene glycol) methyl ether-block-poly (lactide-co-glycolide) nanoparticles containing β -myrcene, β -caryophyllene and nerolidol were prepared by emulsification and solvent evaporation methods. The process is based on emulsifying an organic solution of the polymer into an aqueous phase, followed by evaporation of the organic solvent, as shown in figure 3. The organic phase is poured into a continuous or aqueous phase in which a surfactant is dissolved to impart stability to the emulsion. Emulsification is performed under high shear to reduce the size of the emulsion droplets. This process will largely determine the final particle size of the nanoparticles. This emulsification step is followed by evaporation of the solvent under reduced pressure to yield the desired nanoparticles.

Synthesis of terpenoid-containing PLGA-PEG nanoparticles

Table 24 summarizes the formulations of blank and terpenoid-containing PLGA-PEG nanoparticles prepared using the emulsification method.

In a vessel, 40mg of PLGA-PEG (PEG average Mn2,000, PLGA average Mn 11,500; lactide: glycolide 50: 50) was dissolved in 1mL of ethyl acetate, followed by the addition of 10mg of myrcene, beta-caryophyllene, or nerolidol (based on 12.5% w/w PLGA weight) to give an organic phase solution. The organic solution containing the terpenoid compound was added dropwise over 1 minute using a pipette under homogenization conditions operating at 24,000rpm and 0 ℃ in a separate falcon tube containing 5ml PVA (0.5% w/v) and a homogenizer probe, using a pipette. The organic solvent is then removed without heating using a rotary evaporator for at least 30 minutes to yield a mixture comprising nanoparticles comprising terpenoid. To remove unencapsulated terpenoids and remaining surfactant (PVA), the mixture was diluted with 10mL deionized and purified water, transferred to Amicon tubes (Ultracel-100kDa regenerated cellulose membrane, 15mL sample volume) and evaporated under centrifugation conditions (4,000 × g, 30min at 12 ℃); this procedure was repeated a total of three times. The resulting nanoparticle concentrated suspension was further mixed as a mixture with at least 1mL deionized water and purified water with 1mL trehalose solution (10w/v) and the resulting mixture was lyophilized or freeze dried at-80 ℃ and <0.100 mbar (TESLAR Cryodos). The lyophilized nanoparticles containing terpenoids were collected as a white cotton-like solid. For each terpenoid, three nanoparticle samples were prepared in triplicate for further testing and study.

Sample preparation for GC-MS analysis

For GC-MS analysis and characterization, the terpenoid-containing nanoparticle lyophilizates were dissolved in 1mL of Dichloromethane (DCM) and the resulting mixtures were sonicated for at least 5 minutes. The sample was then centrifuged for 4 minutes to pellet undissolved material, such as trehalose and trace amounts of PVA surfactant. Then 0.25 or 0.5mL of this solution was diluted from the supernatant with Dichloromethane (DCM) to a final volume of 2mL, wherein 1.5mL of the final solution was transferred to a vial suitable for GC-MS analysis.

Mean diameter and size distribution

The mean diameter, size distribution, zeta potential, weight, yield of terpenoid-loaded PLGA-PEG NPs prepared by emulsification are presented in table 25. Encapsulation of terpenoids within nanoparticles results in an increase in particle size. This increase in particle size is more pronounced for nanoparticles containing β -caryophyllene and nerolidol, which have particle sizes of about 350nm and 323nm, respectively, compared to nanoparticles containing myrcene, which have a diameter of 266 nm.

Although encapsulation of terpenoids increases the polydispersity of the nanoparticles, all nanoparticles have a polydispersity index (PDI) < 0.3. The nanoparticles also exhibited a negative surface charge, with a measured zeta potential of about-35 mV. In addition, 77-92 wt% of the starting material was recovered.

Scanning Electron Microscope (SEM) images

SEM images confirmed the spherical structure of terpenoid-containing nanoparticles, as shown in FIGS. 13A-B (myrcene), 14A-B (β -caryophyllene), and 15A-B (nerolidol). Furthermore, a similar structure and size distribution of the nanoparticles was observed regardless of which terpenoid was encapsulated. As shown in fig. 13A and 15B, large nanoparticles that broke were detected and evidence that the nanoparticles containing terpenoids are of the core-shell nanocapsule type as shown in fig. 16 can be provided. One possible explanation is that the nanoparticles break under vacuum during sample preparation for SEM imaging.

Gas chromatography-Mass Spectrometry (GC-MS) analysis

Using the GC-MS method and conditions as described herein, the retention times of myrcene (fig. 4A), β -caryophyllene (fig. 5A), cis-nerolidol (fig. 6), and trans-nerolidol (fig. 6) were measured as 6.93, 5.72, respectively6.77 and 7.16 minutes, and their mass spectra are shown in fig. 4B, 5B, 6A and 6C, respectively. Calibration curves for myrcene (FIG. 4C) and beta-caryophyllene (FIG. 5C) at concentrations ranging from 1-60ppm, respectively, with r of 0.9990 and 0.9993²The values are linear. It was found that the ratio of cis to trans isomers of nerolidol was 1.02:0.98 or about 1:1 and that the calibration curves for the cis (FIG. 7B) and trans (FIG. 7D) isomers of nerolidol at concentrations ranging from 0.5 to 30ppm were correlated with r of 0.9998 and 0.9997, respectively²The values are linear.

Terpenoid encapsulation

The results of terpenoid encapsulation are shown in table 26. The starting mass of all terpenoids was 10 mg. The amount of encapsulated myrcene was 1.8mg, EE ═ 18.1% and DL ═ 4.7%, possibly due to its low boiling point and volatile nature. Both β -caryophyllene and nerolidol showed higher encapsulation amounts of 6.3mg (EE 64.9% and DL 15.1%) and 5.7mg (EE 55.7% and DL 13.9%), respectively. The increase in encapsulation efficiency of β -caryophyllene and nerolidol compared to myrcene may be due to the increase in diameter of the corresponding terpenoid-containing nanoparticles, as summarized in table 25.

Example 9 calcium response in HEK TRPV1 cells induced by terpenoid encapsulated PLGA nanoparticles

This example shows a study of the ability of PLGA-PEG or poly (ethylene glycol) methyl ether-block-poly (lactide-co-glycolide) Nanoparticles (NPs) containing terpenoids to improve the interaction of encapsulated substances with TRPV1 receptors as an in vitro assessment of their potential application in pain management.

Ion influx into HEK TRPV1 cells was assessed using a calcium signaling assay. Cells were dispersed in 1mM calcium assay buffer and intracellular calcium levels were measured using a plate reader in the presence of Fluo-4 as the fluorophore. Fluo-4 showed an increase in fluorescence upon Ca2+ ion binding and was used to measure intracellular Ca2+ concentration in living cells. The experiment was carried out for 1 hour to provide sufficient time for the encapsulated terpenoid to be released from the nanoparticles. Changes in fluorescence in response to free terpenoids, encapsulated terpenoids, and combinations of free and encapsulated terpenoids are detected and compared.

Unencapsulated versus encapsulated individual terpenoids

As shown in figure 17, all unencapsulated terpenoids induced a calcium response, with free myrcene producing the most intense response followed by free nerolidol and free β -caryophyllene. Surprisingly, all nanoparticle formulations containing terpenoids significantly increased fluorescence intensity compared to unencapsulated terpenoids in bulk solution. While both free and encapsulated β -caryophyllene showed the lowest calcium response compared to the rest (fig. 18C), the calcium response of nerolidol at TRPV1 showed a significant but delayed increase when encapsulated within nanoparticles compared to in bulk solution (fig. 18B). The calcium response of free and encapsulated myrcene is shown in fig. 18A.

Combinations of unencapsulated terpenoids

The calcium response to combinations of unencapsulated terpenoids in HEK TRPV1 cells was also investigated. A total of 4 combinations were made of myrcene plus nerolidol (FIG. 19A), myrcene plus beta-caryophyllene (FIG. 19B), nerolidol plus beta-caryophyllene (FIG. 19C) and myrcene plus nerolidol plus beta-caryophyllene (FIG. 19D), each terpenoid at a concentration of 40. mu.g/ml. Surprisingly, all combinations of unencapsulated terpenoids had improved calcium influx compared to the use of terpenoids alone in bulk solution.

Combination of different nanoparticles encapsulating terpenes

Combinations of various nanoparticle populations were also tested, each encapsulating a different terpenoid, and each terpenoid was at a concentration of 40 μ g/ml compared to treatment with a single nanoparticle population. As shown in fig. 20A-20D, the combination of different nanoparticle populations resulted in higher and improved calcium responses compared to a single nanoparticle population containing an individual terpenoid.

Combinations of encapsulated terpenoids as compared to combinations of unencapsulated terpenoids

Finally, nanoparticles encapsulating combinations of terpenoids were tested and compared to combinations of terpenoids not encapsulated, each terpenoid being at a concentration of 40 μ g/ml. In all combinations tested, namely myrcene plus nerolidol (fig. 21A), myrcene plus β -caryophyllene (fig. 21B), nerolidol plus β -caryophyllene (fig. 21C), and myrcene plus β -caryophyllene plus nerolidol (fig. 21D), nanoparticles encapsulating combinations of terpenoids resulted in a greatly improved calcium response compared to combinations of non-encapsulated terpenoids.

Conclusion

The calcium response of HEK TRPV1 cells in the presence of free terpenoids, PLGA-PEG nanoparticles containing terpenoids, and a combination of unencapsulated terpenoids and nanoparticle encapsulated terpenoids in bulk solution was investigated using the calcium signaling assay described herein. As shown in FIGS. 17, 18-18C, 19A-19D, 20A-20D, 21A-21D and described above, terpenoid combinations improve calcium response regardless of encapsulation within the nanoparticles. More importantly, whether administered alone or in combination, the terpenoid-containing nanoparticles were found to produce higher and improved calcium cell influx compared to unencapsulated terpenoids in the bulk solution. These data and results provide strong evidence that encapsulation of terpenoids within nanoparticles significantly improves the in vitro pharmacological activity of the terpenoids. Furthermore, these results highlight the advantages of formulations and pharmaceutical compositions comprising terpenoid combinations for future in vivo studies and as potential therapeutic agents.

While the present invention has been particularly shown and described with reference to a preferred embodiment and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

All references, issued patents and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

It will be understood that when an aspect of the invention is formulated as a method of treating a disease or condition, comprising administering to a subject a pharmaceutical composition of the invention, it is also intended to encompass a pharmaceutical composition of the invention for use in treating the disease or condition.