Novel formulations of digitalis glycosides for treating cell-proliferative and other diseases

• US 20040082521A1
• Filed: 03/31/2003
• Published: 04/29/2004
• Est. Priority Date: 03/29/2002
• Status: Abandoned Application

• Current Assignee: Azaya Therapeutics, Inc. (Plus Therapeutics, Inc.)
• Original Assignee: Azaya Therapeutics, Inc. (Plus Therapeutics, Inc.)
• Inventors: Singh, Chandra U.
• Application Number:
  US10/404,902
• Publication Number:
  US 20040082521A1
• Time in Patent Office: Days
• US Class Current: 514/26
• CPC Class Codes: A61K 31/704 attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin digitoxin A61K31/7048
• CPC Class Codes: A61K 31/7048 having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin , digitoxin or digoxin
• CPC Class Codes: A61K 9/1271 Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers liposome as conjugate A61K47/6911
• CPC Class Codes: A61K 9/1272 with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids with cholesterol as the only non-phosphatidyl surfactant A61K9/127; lipids as modifying agent A61K47/543
• CPC Class Codes: A61K 9/148 with compounds of unknown constitution, e.g. material from plants or animals with oils, fats, waxes, shellac A61K9/145

Abstract

The present invention provides methods, preparations, and uses of a variety of liposomal-digitalis glycoside compositions. The present invention also provides protein-stabilized nanoparticle formulations containing liposomal-digitalis glycosides such as Oleandrin, digitoxin, and digoxin with reduced toxicity, high drug to lipid ratio, long circulating time in the bloodstream and the ability to deliver the drug to tumor sites. In another aspect, the present invention provides an effective method to reduce the growth of cancers or reduce the incidence of metastases.

Claims

1. A method for the preparation of a variety of liposomal digitalis glycosides compositions for the use of treating cell-proliferative diseases in humans and mammals, the method comprising:

a) obtaining in a hydrophobic solvent a liposome forming mixture comprising a phosphalipid and cholesterol;

b) adding a hydrophobic solution of digitalis glycoside to the mixture to form a drug;

lipid ratio of about 1;

10;

c) hydrating the mixture with high speed mixing to form a suspension in aqueous solution;

d) subjecting the suspension to high pressure microfluidization;

e) evaporating the organic solvent under reduced pressure to form a lipid suspension in aqueous solution;

f) filtering the suspension to remove any precipitated digitalis glycoside.

2. A method for the preparation of a variety of liposomal digitalis glycosides compositions for the use of treating cell-proliferative diseases in humans and mammals, the method comprising:

a) obtaining a hydrophobic solvent comprising dissolved HSPC;

cholesterol and PEG 2000-DSPE at a 55;

40;

5 molar ratio;

b) adding a hydrophobic solution of digitalis glycoside to the mixture to form a drug;

lipid ratio of about 1;

10;

c) hydrating the mixture with high speed mixing to form a suspension in aqueous solution;

d) subjecting the suspension to high pressure microfluidization;

e) evaporating the organic solvent under reduced pressure to form a lipid suspension in aqueous solution;

f) filtering the suspension to remove any precipitated digitalis glycoside.

3. The liposomal digitalis glycoside compositions prepared by the method of claim 1 or 2 defined further as having reduced toxicity, high drug to lipid ratio, long-circulating time in the bloodstream and the ability to deliver the drug to the tumor sites.

4. The liposomal digitalis glycoside composition prepared by the method of claim 1 or 2, defined further as being effective in reducing the growth of cancers or the incidence of metastases.

5. The liposomal digitalis glycoside prepared by the method of claim 1 or 2 defined further as being effective in treating cell-proliferative and other diseases in a warm-blooded animals.

6. The liposomal digitalis glycoside composition prepared by the method of claim 1 defined further as being prepared using lipids including at least one hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC, phosphatidylethanolamine (PE), phosphatidylglycerol(PG), phosphatidylinsitol (PI), monosialoganglioside sphingomyelin (SPM);

derivatized vesicle forming lipids such as poly(ethylene glycol)-derivatized distearoylphosphatidylethanolamine (PEG-DSPE) distearoylphosphatidylcholine (DSPC), dimyristoylphospha-tidylcholine (DMPC), and dimyristoylphosphatidylglycerol (DMPG), poly(ethylene glycol)-derivatized ceramides (PEG-CER) and mixtures thereof.

7. The liposomal digitalis glycoside composition made by the method of claim 1 or 2 wherein the phospholipids are synthetic or derived from natural services.

8. The liposomal digitalis glycoside composition prepared by the method of claim 1 or 2, as defined further as having a drug to lipid weight ratio between 0.01 and 1, preferably between 0.1 and 1.

9. The liposomal composition prepared by the method of claim 1 or 2 wherein digitalis glycoside is derived from plants or animals or by synthetic routes.

10. The liposomal digitalis glycoside composition prepared by the method of claim 1 or 2 defined further as being prepared by solvent evaporation of digitalis glycosides, cholesterol, and lipids followed by hydration and high shear stress to reduce particle size of the liposomal-digitalis glycosides.

11. The method of claim 11 where the ratio by weight of digitalis glycosides to cyclodextrin is 0.01 to 10.

12. The method of claim 1 defined further as yielding liposomal digitalis glycoside having a size less than 220 nm, preferably 80-160 nm, most preferably between about 100-120 nm.

13. The liposomal digitalis glycoside composition prepared by the method of claim 1 or 2 defined further as being sterile filtered through a 0.22 μ

m filtered and lyophilized.

14. The liposomal digitalis glycoside composition prepared by the method of claim 13 defined further as being lyophilized in the form of a cake in vials using cryoprotectants such as sucrose, mannitol, trehalose or the like.

15. The composition prepared by the method of claim 14 wherein the lyophized cake is defined further as being reconstitutable to the original liposomes, without modifying liposome particle size.

16. The liposomal digitalis glycoside composition prepared by the method of claim 1 or 2 defined further as being deliverable in the form of nanoparticles or suspensions, by slow infusions or by bolus injection or by other parenteral or oral delivery routes.

17. A pharmaceutical formulation of digitalis glycosides active for in vivo delivery, said formulation is prepared by subjecting a mixture comprising:

a) an organic phase containing digitalis glycoside dispersed therein, and

b) aqueous medium containing biocompatible polymer,

wherein said mixture contains substantially no surfactants, to high shear conditions in a high pressure homogenizer at a pressure in the range of about 3,000 up to 30,000 psi.

18. The pharmaceutical formulation according to claim 17 further comprising removing said organic phase from said mixture.

19. The pharmaceutical formulation according to claim 17 further comprising removing said aqueous phase from said mixture.

20. The pharmaceutical formulation to claim 17 wherein said organic phase has a boiling point of no greater than about 200.degree. C.

21. The pharmaceutical formulation according to claim 20 wherein said organic phase is selected from soybean oil, coconut oil, olive oil, safflower oil, cotton seed oil, sesame oil, orange oil, limonene oil, aliphatic, cycloaliphatic or aromatic hydrocarbons having 4-30 carbon atoms, aliphatic or aromatic alcohols having 2-30 carbon atoms, aliphatic or aromatic esters having 2-30 carbon atoms, alkyl, aryl, or cyclic ethers having 2-30 carbon atoms, alkyl or aryl halides having 1-30 carbon atoms, optionally having more than one halogen substituent, ketones having 3-30 carbon atoms, polyalkylene glycol, or combinations of any two or more thereof.

22. The pharmaceutical formulation according to claim 20 wherein said organic phase comprises a mixture of a substantially water immiscible organic solvent and a water soluble organic solvent.

23. The pharmaceutical formulation according to claim 17 wherein said biocompatible polymer is a naturally occurring polymer, a synthetic polymer, or a combination thereof.

24. The pharmaceutical formulation according to claim 17 wherein said biocompatible polymer is capable of being crosslinked by disulfide bonds.

25. The pharmaceutical formulation according to claim 23 wherein said naturally occurring polymers are selected from proteins, peptides, polynucleic acids, polysaccharides, proteoglycans or lipoproteins.

26. The pharmaceutical formulation according to claim 23 wherein said synthetic polymers are selected from synthetic polyamino acids containing cysteine residues and/or disulfide groups;

polyvinyl alcohol modified to contain free sulfhydryl groups and/or disulfide groups;

polyhydroxyethyl methacrylate modified to contain free sulfhydryl groups and/or disulfide groups;

polyacrylic acid modified to contain free sulfhydryl groups and/or disulfide groups;

polyethyloxazoline modified to contain free sulfhydryl groups and/or disulfide groups;

polyacrylamide modified to contain free sulfhydryl groups and/or disulfide groups;

polyvinyl pyrrolidinone modified to contain free sulfhydryl groups and/or disulfide groups;

polyalkylene glycols modified to contain free sulfhydryl groups and/or disulfide groups;

polylactides, polyglycolides, polycaprolactones, or copolymers thereof, modified to contain free sulfhydryl groups and/or disulfide groups;

as well as mixtures of any two or more thereof.

27. The pharmaceutical formulation according to claim 17 wherein said high shear conditions comprise contacting said organic phase and said aqueous medium in a high pressure homogenizer at a pressure in the range of about 6,000 up to 25,000 psi.

28. The pharmaceutical formulation according to claim 17 wherein said biocompatible polymer is the protein albumin.

29. The pharmaceutical formulation according to claim 17 wherein said aqueous medium is selected from water, buffered aqueous media, saline, buffered saline, solutions of amino acids, solutions of sugars, solutions of vitamins, solutions of carbohydrates, or combinations of any two or more thereof.

30. The pharmaceutical formulation according to claim 17 wherein said high shear conditions produce particles comprising said pharmacologically active agent coated with said biocompatible polymer.

31. The pharmaceutical formulation according to claim 30 wherein said particles have an average diameter of less than 1 micron.

32. The pharmaceutical formulation according to claim 30 wherein said particles have an average diameter of less than 200 nm.

33. The pharmaceutical formulation according to claim 32 wherein said mixture is sterile filtered.

34. The pharmaceutical formulation according to claim 30 wherein said particles are amorphous, crystalline, or a mixture thereof.

35. The pharmaceutical formulation according to claim 34 wherein said particles are substantially amorphous.

36. The pharmaceutical formulation for the preparation of digitalis glycosides for in vivo delivery in the form of sterile-filterable particles, said formulation is prepared by subjecting a mixture comprising:

(a) an organic phase containing digitalis glycoside dispersed therein, wherein said organic phase comprises a mixture of a substantially water immiscible organic solvent and a water soluble organic solvent, and

(b) aqueous medium containing biocompatible polymer,

wherein said mixture contains substantially no surfactants, to high shear conditions in a high pressure homogenizer at a pressure in the range of about 3,000 up to 30,000 psi.

37. The pharmaceutical formulation according to claim 36 further comprising removing said organic phase from said mixture.

38. The pharmaceutical formulation according to claim 36 further comprising filtering said mixture through a 0.22 micron filter.

39. The pharmaceutical formulation according to claim 36 further comprising removing said aqueous phase from said mixture.

40. The pharmaceutical formulation according to claim 36 wherein said high shear conditions produce amorphous particles, crystalline particles, or a mixture thereof.

41. The pharmaceutical formulation according to claim 40 wherein said particles are substantially amorphous.

42. A pharmaceutical composition comprising:

a protein-stabilized liposome containing digitalis glycosides, the pharmaceutical composition is prepared by the method comprising;

a) preparing an organic solution of liposome-forming phospholipids, cholesterol and PEG-phospholipid;

b) adding at least one digitalis glycoside dissolved in an organic solvent;

c) mixing the phospholipid and pharmaceutical agent mixture at a ratio of about one part drug to ten parts lipid;

d) subjecting said mixture to infusion into a one to ten percent aqueous solution of a protein in an aqueous medium a pH between about 7.2 and 7.6;

e) after agitating said mixture at between about 3000 and about 10,000 revolutions per minute, said mixture is subjected to high pressure microfluidization or homogenization at 20,000 to 30,000 PSI, which results in a fine oil and water emulsion;

f) evaporating the organic solvent under reduced pressure to form a lipid suspension in aqueous solution;

g) filtering the suspension to remove any precipitated digitalis glycoside.

43. The method of claim 42 defined further as having a final step of lyophilization to produce a powder suitable for reconstituting a protein stabilized liposomal formulation in an aqueous suspension.

44. The protein-stabilized liposomal (PSL) formulations containing digitalis glycosides as nanoparticle suspensions or lyophilized powders for the use in treating cell-proliferative and other diseases in humans and mammals, said formulation prepared by the method of claims 42 or 43.

45. The protein-stabilized liposomal (PSL) formulation prepared according to claim 42 defined further as having reduced toxicity, high drug to lipid ratio, long-circulating time in the bloodstream and ability to deliver the drug to the target sites.

46. The protein-stabilized liposomal (PSL) formulations containing digitalis glycosides prepared according to claim 42 defined further as effective in reducing the growth of cancers or reducing the incidence of metastases.

47. The protein-stabilized liposomal (PSL) formulations containing digitalis glycosides prepared according to claim 42 defined further as effective in treating cell-proliferative and other diseases in a warm-blooded animals.

48. The protein-stabilized liposomal (PSL) formulations prepared according to claim 42 defined further as prepared using lipids such as hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC, phosphatidylethanolamine (PE), phosphatidylglycerol(PG), phosphatidylinsitol (PI), monosialogangolioside and spingomyelin (SPM);

the derivatized vesicle forming lipids such as poly(ethylene glycol)-derivatized distearoylphos-phatidylethanolamine (PEG-DSPE) and poly(ethylene glycol)-derivatized ceramides (PEG-CER). The phospholipid can be either synthetic or derived from natural sources such as egg or soy. The phospholipids can also be distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), and dimyristoylphosphatidylglycerol (DMPG) and proteins such as human serum albumin, bovine serum albumin, and erythroprotein.

49. The protein-stabilized liposomal (PSL) formulations prepared according to claim 42, wherein the cardiac glycoside to lipid-protein weight ratio varies between 0.01 and 1, preferably between 0.05 and 1.

50. The Pharmaceutical formulations prepared according to claim 42 wherein the liposome-forming materials prepared from plants or by synthetic routes.

51. The PSL formulations prepared according to claim 42 defined further as prepared by solvent evaporation of digitalis glycoside, cholesterol, and lipids followed by hydration and high shear stress to reduce the particle size of the PSL nanoparticles.

52. The method of claim 42 defined further as yielding PSL nanoparticles having size less than 220 nm, preferably 80-160 nm, most preferably between about 100-120 nm.

53. The PSL formulations prepared according to claim 42 defined further as being sterile filtered through a 0.22 μ

m filtered and lyophilized.

54. The PSL formulations prepared according to claim 53 defined further as being sterile-filtered and lyophilized in the form of a cake in vials using cryoprotectants such as sucrose, mannitol, trehalose or the like.

55. The formulations of claim 54 wherein the lyophized cake can be reconstituted to the original PSL formulations, without modifying the particle size of PSL nanoparticles.

56. The PSL formulations prepared according to claim 42 defined further as being deliverable in the form of nanoparticles or suspensions by slow infusions or by bolus injection or by other parenteral or oral delivery routes.

57. The pharmaceutical formulation according to claim 42 wherein said protein is selected from albumins, immunoglobulins, caseins, insulins, hemoglobins, lysozymes, immunoglobulins, α

-2-macroglobulin, fibronectins, vitronectins, fibrinogens, lipases, enzymes and antibodies.

58. The pharmaceutical formulation according to claim 42 wherein said protein is albumin.

59. The digitalis glycoside in claims 1, 2, 17, 36, 42 is selected from the group consisting of oleandrin, neriifolin, odoroside A and H, ouabain (G-strophantin), cymarin, sarmentocymarin, periplocymarin, K-strophantin, thevetin A, cerberin, peruvoside, thevetosin, thevetin B, tanghinin, deacetyltanghinin, echujin, hongheloside G, honghelin, periplocin, strophantidol, nigrescin. uzarin, calotropin, cheiroside A, cheirotoxin, euonoside, euobioside, euomonoside, lancetoxin A and B, kalanchoside, bryotoxin A-C, bryophyllin B, cotiledoside, tyledoside A-D, F and G, orbicuside A-C, alloglaucotoxin, corotoxin, coroglaucin, glaucorin, scillarene A and B, scilliroside, scilliacinoside, scilliglaucoside, scilliglaucosidin, scillirosidin, scillirubrosidin, scillirubroside, proscillaridin A, rubelin, convalloside, convallatoxin, bovoside A, glucobovoside A, bovoruboside, antiarin A, helleborin, hellebrin, adonidin, adonin, adonitoxin, thesiuside, digitoxin, gitoxin, gitalin, digoxin, F-gitonin, digitonin, lanatoside A-C, bufotalin, bufotalinin, bufotalidin, pseudobufotalin, acetyl-digitoxin, acetyl-oleandrin, beta-methyldigoxin and alpha-methyldigoxin.

60. The pharmaceutical formulation according to claim 42 wherein said organic phase is selected from soybean oil, coconut oil, olive oil, safflower oil, cotton seed oil, sesame oil, orange oil, limonene oil, aliphatic, cycloaliphatic or aromatic hydrocarbons having 4-30 carbon atoms, aliphatic or aromatic alcohols having 2-30 carbon atoms, aliphatic or aromatic esters having 2-30 carbon atoms, alkyl, aryl, or cyclic ethers having 2-30 carbon atoms, alkyl or aryl halides having 1-30 carbon atoms, optionally having more than one halogen substituent, ketones having 3-30 carbon atoms, polyalkylene glycol, or combinations of any two or more thereof.

61. The digitalis glycoside in claims 1, 2, 17, 36 and 42 is Oleandrin

62. The digitalis glycoside in claims 1, 2, 17, 36 and 42 is Neriifolin.

63. The digitalis glycoside in claims 1, 2, 17, 36 and 42 is Odoroside A.

64. The digitalis glycoside in claims 1, 2, 17, 36 and 42 is Odoroside H.

65. The digitalis glycoside in claims 1, 2, 17, 36 and 42 is Digoxin.

66. The digitalis glycoside in claims 1, 2, 17, 36 and 42 is Digitoxin.

67. The digitalis glycoside in claims 1, 2, 17, 36 and 42 is Proscillaridin A.