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Details for Patent: 5,770,222

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Details for Patent: 5,770,222

Title: Therapeutic drug delivery systems
Abstract:Therapeutic drug delivery systems comprising gas-filled microspheres comprising a therapeutic are described. Methods for employing such microspheres in therapeutic drug delivery applications are also provided. Drug delivery systems comprising gas-filled liposomes having encapsulated therein a drug are preferred. Methods of and apparatus for preparing such liposomes and methods for employing such liposomes in drug delivery applications are also disclosed.
Inventor(s): Unger; Evan C. (Tucson, AZ), Fritz; Thomas A. (Tucson, AZ), Matsunaga; Terry (Tucson, AZ), Ramaswami; VaradaRajan (Tucson, AZ), Yellowhair; David (Tucson, AZ), Wu; Guanli (Tucson, AZ)
Assignee: ImaRx Pharmaceutical Corp. (Tucson, AZ)
Filing Date:Jun 07, 1995
Application Number:08/472,305
Claims:1. A targeted therapeutic delivery system comprising a gas-filled lipid-containing microsphere wherein said gas-filled microsphere has an interior volume of at least about 50% gas, and comprises a therapeutic compound wherein said therapeutic compound is selected from the group consisting of antineoplastic agents; blood products; biological response modifiers; anti-fungal agents; hormones; vitamins; peptides; anti-tuberculars; enzymes; anti-allergic agents; anti-coagulation agents; circulatory drugs; metabolic potentiators; antivirals; antianginals; antibiotics; antiinflammatories; antiprotozoans; antirheumatics; narcotics; opiates; cardiac glycosides; neuromuscular blockers; sedatives; local anesthetics; general anesthetics; radioactive compounds; monoclonal antibody; genetic material; prodrugs; and combinations thereof.

2. A therapeutic delivery system of claim 1 wherein the microsphere comprises a material having a phase transition temperature greater than about 20.degree. C.

3. A therapeutic delivery system of claim 1 wherein the microsphere is less than about 100 .mu.m in outside diameter.

4. A therapeutic delivery system of claim 3 wherein the microsphere is less than about 10 .mu.m in outside diameter.

5. A therapeutic delivery system of claim 1 wherein the therapeutic compound comprises genetic material.

6. A therapeutic delivery system of claim 5 wherein the genetic material is deoxyribonucleic acid.

7. A therapeutic delivery system of claim 5 wherein the genetic material is ribonucleic acid.

8. A therapeutic delivery system of claim 5 comprising an antisense ribonucleic acid or an antisense deoxyribonucleic acid.

9. A therapeutic delivery system of claim 5 wherein the microsphere comprises a cationic lipid material.

10. A therapeutic delivery system of claim 9 wherein the cationic lipid comprises N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoium chloride.

11. A therapeutic delivery system of claim 1 wherein the lipid is selected from the group consisting of distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine and egg phosphatidylcholine.

12. A therapeutic delivery system of claim 6 wherein the therapeutic compound comprises a deoxyribonucleic acid encoding at least a portion of a gene selected from the group consisting of a human major histocompatibility gene, dystrophin, Cystic Fibrosis transmembrane conductance regulator, Interleukin-2 and Tumor Necrosis Factor.

13. A therapeutic delivery system of claim 8 wherein the therapeutic compound comprises an antisense oligonucleotide capable of binding at least a portion of a deoxyribonucleotide encoding Ras.

14. A therapeutic delivery system of claim 1 wherein the therapeutic comprises a monoclonal antibody.

15. A therapeutic delivery system of claim 14 wherein the monoclonal antibody is capable of binding to melanoma antigen.

16. A therapeutic delivery system of claim 1 wherein the microspheres comprise gas-filled liposomes substantially devoid of liquid in the interior thereof and having encapsulated therein a therapeutic compound.

17. A therapeutic delivery system of claim 1 wherein the microspheres comprise gas-filled liposomes prepared by a vacuum drying gas instillation method and having encapsulated therein a therapeutic compound.

18. A therapeutic delivery system of claim 1 wherein the microspheres comprise gas-filled liposomes prepared by a gel state shaking gas instillation method.

19. A drug delivery system comprising a gas-filled liposome prepared by a vacuum drying gas instillation method and having encapsulated therein a drug, wherein said gas-filled liposome has an interior volume of at least about 50% gas, and wherein said drug is selected from the group consisting of antineoplastic agents; blood products; biological response modifiers; anti-fungal agents; hormones; vitamins; peptides; anti-tuberculars; enzymes; anti-allergic agents; anti-coagulation agents; circulatory drugs; metabolic potentiators; antivirals; antianginals; antibiotics; antiinflammatories; antiprotozoans; antirheumatics; narcotics; opiates; cardiac glycosides; neuromuscular blockers; sedatives; local anesthetics; general anesthetics; radioactive compounds; monoclonal antibody; genetic material; prodrugs and combinations thereof.

20. A drug delivery system of claim 19 wherein said liposomes are comprised of lipid materials selected from the group consisting of fatty acids, lysolipids, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, phosphatidylcholine, phosphatidic acid, sphingomyelin, cholesterol, cholesterol sulfate, cholesterol hemisuccinate, tocopherol hemisuccinate, phosphatidylethanolamine, phosphatidylinositol, glycosphingolipids, glucolipids, glycolipids, sulphatides, lipids bearing sulfonated mono-, di-, oligo- or polysaccharides, lipids with ether and ester-linked fatty acids, and polymerized lipids.

21. A drug delivery system of claim 20 wherein said liposomes are comprised of dipalmitoylphosphatidylcholine.

22. A drug delivery system of claim 19 wherein said liposomes are filled with a gas selected from the group consisting of air, nitrogen, carbon dioxide, oxygen, argon, xenon, helium, and neon.

23. A drug delivery system of claim 22 wherein said liposomes are filled with nitrogen gas.

24. A drug delivery system of claim 19 wherein said liposomes are stored suspended in an aqueous medium.

25. A drug delivery system of claim 19 wherein said liposomes are stored dry.

26. A drug delivery system of claim 19 wherein said liposomes have a stability of at least about three weeks.

27. A drug delivery system of claim 19 wherein said liposomes have a reflectivity of at least about 2 dB.

28. A drug delivery system of claim 27 wherein said liposomes have a reflectivity of between about 2 dB and about 20 dB.

29. A drug delivery system comprising a gas-filled liposome wherein said gas-filled liposome has an interior volume of at least about 50% gas and comprises a drug encapsulated therein, wherein said drug is selected from the group consisting of antineoplastic agents; blood products; biological response modifiers; anti-fungal agents; hormones; vitamins; peptides; anti-tuberculars; enzymes; anti-allergic agents; anti-coagulation agents; circulatory drugs; metabolic potentiators; antivirals; antianginals; antibiotics; antiinflammatories; antiprotozoans; antirheumatics; narcotics; opiates; cardiac glycosides; neuromuscular blockers; sedatives; local anesthetics; general anesthetics; radioactive compounds; monoclonal antibody; genetic material; prodrugs; and combinations thereof.

30. A drug delivery system of claim 29 wherein said liposomes are comprised of lipid materials selected from the group consisting of fatty acids, lysolipids, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, phosphatidylcholine, phosphatidic acid, sphingomyelin, cholesterol, cholesterol hemisuccinate, tocopherol hemisuccinate, phosphatidylethanolamine, phosphatidylinositol, glycosphingolipids, glucolipids, glycolipids, sulphatides, lipids bearing sulfonated mono-, di-, oligo- or polysaccharides, lipids with ether and ester-linked fatty acids, and polymerized lipids.

31. A drug delivery system of claim 30 wherein said liposomes are comprised of dipalmitoylphosphatidylcholine.

32. A drug delivery system of claim 29 wherein said liposomes are filled with a gas selected from the group consisting of air, nitrogen, carbon dioxide, oxygen, argon, xenon, helium, and neon.

33. A drug delivery system of claim 32 wherein said liposomes are filled with nitrogen gas.

34. A drug delivery system of claim 29 wherein said liposomes are stored suspended in an aqueous medium.

35. A drug delivery system of claim 29 wherein said liposomes are stored dry.

36. A drug delivery system of claim 29 wherein said liposomes have a shelf life stability of at least about three weeks.

37. A drug delivery system of claim 29 wherein said liposomes have a reflectivity of at least about 2 dB.

38. A drug delivery system of claim 37 wherein said liposomes have a reflectivity of between about 2 dB and about 20 dB.

39. A drug delivery system prepared by a method comprising the steps of:

(i) placing under negative pressure liposomes having encapsulated therein a drug, wherein said drug is selected from the group consisting of antineoplastic agents; blood products; biological response modifiers; anti-fungal agents; hormones; vitamins; peptides; anti-tuberculars; enzymes; anti-allergic agents; anti-coagulation agents; circulatory drugs; metabolic potentiators; antivirals; antianginals; antibiotics; antiinflammatories; antiprotozoans; antirheumatics; narcotics; opiates; cardiac glycosides; neuromuscular blockers; sedatives; local anesthetics; general anesthetics; radioactive compounds; monoclonal antibody; genetic material; prodrugs; and combinations thereof;

(ii) incubating said liposomes under the negative pressure for a time sufficient to remove substantially all liquid from said liposomes; and

(iii) instilling gas into said liposomes until ambient pressures are achieved to produce a drug delivery system comprising gas-filled liposomes having an interior volume of at least about 50% gas.

40. The therapeutic delivery system of claim 1 wherein said lipid-containing microsphere comprises at least one lipid selected from the group consisting of dipalmitoylphosphotdylcholine, dipalmitoylphosphatidylethanolamine, and a phosphalidic acid, and said liposome further comprising polyethylene glycol.

41. The therapeutic delivery system of claim 1 wherein said lipid-contaning microsphere comprises at least one dipalmitoyl lipid.

42. The drug delivery system of claim 19 comprising at least one lipid selected from the group consisting of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, phosphastidic acid, and a lipid bearing polyethylene glycol.

43. The drug delivery system of claim 19 comprising at least one dipalmitoyl lipid.

44. The drug delivery system of claim 19 comprising a lipid having a phase transition temperature of greater than about 20.degree. C.

45. The therapeutic delivery system of claim 1 wherein said therapeutic is selected from the group consisting of peptides, glycopeptides, and lectins, said therapeutic incorporated into the surface of said microsphere.

46. The drug delivery system of claim 19 comprising dipalmitoylphosphatidylethanolamine, said liposome further comprising polyethylene glycol.

47. A therapeutic delivery system of claim 1 wherein said gas-filled microsphere is a lipid-containing unilamellar microsphere.

48. A drug delivery system of claim 19 wherein said gas-filled liposome is unilamellar.

49. A drug delivery system of claim 29 wherein said gas-filled liposome is unilamellar.

50. A drug delivery system of claim 39 wherein said gas-filled liposomes are unilamellar.

51. A therapeutic delivery system of claim 47 wherein said unilamellar microsphere comprises a phospholipid.

52. A drug delivery system of claim 48 wherein said unilamellar liposome comprises a phospholipid.

53. A drug delivery system of claim 49 wherein said unilamellar liposome comprises a phospholipid.

54. A drug delivery system of claim 50 wherein said unilamellar liposomes comprise a phospholipid.

55. A delivery system of claim 1 wherein said microsphere further comprises a polymer.

56. A delivery system of claim 55 wherein said polymer is selected from the group consisting of polyethyleneglycol, chitin, polyglutamic acid, hyaluronic acid, polyvinylpyrrolidone, polylysine, polyarginine, alginic acid, dextran, starch, HETA starch, polylactide, polyethyleneimines, polyionenes, and polyiminocarboxylates.

57. A delivery system of claim 55 wherein said gas is air.

58. A delivery system of claim 1 wherein said microsphere further comprises a polysaccharide.

59. A delivery system of claim 58 wherein said polysaccharide comprises polymerized galactose.

60. A delivery system of claim 58 wherein said gas is nitrogen.

61. A therapeutic delivery system of claim 47 wherein said lipid is a polymerized lipid.

62. A drug delivery system of claim 48 wherein said liposome comprises polymerized lipids.

63. A drug delivery system of claim 49 wherein said liposome comprises polymerized lipids.

64. A drug delivery system of claim 50 wherein said liposomes comprise polymerized lipids.

65. A therapeutic delivery system of claim 47 wherein said microsphere further comprises polyethylene glycol.

66. A drug delivery system of claim 48 wherein said liposome further comprises polyethylene glycol.

67. A drug delivery system of claim 49 wherein said liposome further comprises polyethylene glycol.

68. A drug delivery system of claim 50 wherein said liposomes further comprise polyethylene glycol.

69. A therapeutic delivery system of claim 1 wherein said microsphere has been rehydrated from a lyophilized microsphere.

70. A therapeutic delivery system of claim 1 wherein said gas-filled microsphere further comprises a protein.

71. A therapeutic delivery system of claim 70 wherein said microsphere has been rehydrated from a lyophilized microsphere.

72. A therapeutic delivery system of claim 1 wherein the antineoplastic agents are selected from the group consisting of platinum compounds, methotrexate, adriamycin, mitomycin, ansamitocin, bleomycin, cytosine arabinoside, arabinosyl adenine, mercaptopolylysine, vincristine, busulfan, chlorambucil, melphalan, mercaptopurine, mitotane, procarbazine hydrochloride dactinomycin, daunorubicin hydrochloride, doxorubicin hydrochloride, taxol, mitomycin, plicamycin, arminoglutethimide, estramustine phosphate sodium, flutamide, leuprolide acetate, megestrol acetate, tamoxifen citrate, testolactone, trilostane, amsacrine, asparaginase, etoposide, interferon .alpha.-2a, interferon .alpha.-2b, teniposide, vinblastine sulfate, vincristine sulfate, bleomycin, bleomycin sulfate, methotrexate, adriamycin, and arabinosyl; wherein the blood products are selected from the group consisting of parenteral iron, hemin, hematoporphyrins and derivatives thereof; wherein the biological response modifiers are selected from the group consisting of muramyldipeptide, muramyltripeptide, microbial cell wall components, lymphokines, sub-units of bacteria, and synthetic dipeptide N-acetyl-muramyl-L-alanyl-D-isoglutamine; wherein the anti-fungal agents are selected from the group consisting of ketoconazole, nystatin, griseofulvin, flucytosine, miconazole, amphotericin B, ricin, and .beta.-lactam antibiotics; and wherein the hormones are selected from the group consisting of growth hormone, melanocyte stimulating hormone, estradiol, beclomethasone dipropionate, betamethasone, betamethasone acetate, betamethasone sodium phosphate, vetamethasone disodium phosphate, vetamethasone sodium phosphate, cortisone acetate, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, flunisolide, hydrocortisone, hydrocortisione acetate, hydrocortisone cypionate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, prednisone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate, triamicinolone hexacetonide and fludrocortisone acetate; wherein the vitamins are selected from the group consisting of cyanocobalamin neinoic acid, retinoids and derivatives thereof; wherein the peptides are manganese super oxide dismutase; wherein the enzymes are alkaline phosphatase; wherein the anti-allergic agents are amelexanox; wherein the anti-coagulation agents are selected from the group consisting of phenprocoumon and heparin; wherein the circulatory drugs are propranolol; wherein the metabolic potentiators are glutathione; wherein the antituberculars are selected from the group consisting of para-aminosalicylic acid, isoniazid, capreomycin sulfate cycloserine, ethambutol hydrochloride ethionamide, pyrazinamide, rifampin, and streptomycin sulfate; wherein the antivirals are selected from the group consisting of acyclovir, amantadine azidothymidine, ribavirin and vidarabine monohydrate; wherein the antianginals are selected from the group consisting of diltiazem, nifedipine, verapamil, crythritol tetranitrate, isosorbidc dinitrate, nitroglycerin and pentaerythritol tetranitrate; wherein the antibiotics are selected from the group consisting of dapsone, chloramphenicol, neomycin, cefaclor, cefadroxil, cephalexin, cephradine erythromycin, clindamycin, lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, ticarcillin rifampin and tetracycline; wherein the antiinflammitories are selected from the group consisting of diflunisal, ibuprofen, indomethacin, meclofenamate, mefenamic acid, naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac, tolmetin, aspirin and salicylates; wherein the antiprotozoans are selected from the group consisting of chloroquine, hydroxychloroquine, metronidazole, quinine and meglumine antimonate; wherein the antirheumatics are penicillamine; wherein the narcotics are paregoric; wherein the opiates are selected from the group consisting of codeine, heroin, methadone, morphine and opium; wherein the cardiac glycosides are selected from the group consisting of deslanoside, digitoxin, digoxin, digitalin and digitalis; wherein the neuromuscular blockers are selected from the group consisting of atracurium mesylate, gallamine triethiodide, hexafluorenium bromide, metocurine iodide, pancuronium bromide, succinylcholine chloride, tubocurarine chloride and vecuronium bromide; wherein the sedatives are selected from the group consisting of amobarbital, amobarbital sodium, aprobarbital, butabarbital sodium, chloral hydrate, ethchlorvynol, ethinamate, flurazepam hydrochloride, glutethimide, methotrimeprazine hydrochloride, methyprylon, midazolam hydrochloride, paraldehyde, pentobarbital, pentobarbital sodium, phenobarbital sodium, secobarbital sodium, talbutal, temazepam and triazolam; wherein the local anesthetics are selected from the group consisting of bupivacaine hydrochloride, chloroprocaine hydrochloride, etidocaine hydrochloride, lidocaine hydrochloride, mepivacaine hydrochloride, procaine hydrochloride and tetracaine hydrochloride; wherein the general anesthetics are selected from the group consisting of droperidol, etomidate, fentanyl citrate with droperidol, ketamine hydrochloride, methohexital sodium and thiopental sodium; and wherein the radioactive compounds are selected from the group consisting of strontium, iodide rhenium and yttrium.

73. A therapeutic delivery system of claim 1 wherein said lipid microsphere has been rehydrated from a lyophilized microsphere.

74. A therapeutic delivery system of claim 29 wherein the antineoplastic agents are selected from the group consisting of platinum compounds, methotrexate, adriamycin, mitomycin, ansarnitocin, bleomycin, cytosine arabinoside, arabinosyl adenine, mercaptopolylysine, vincristine, busulfan, chlorambucil, melphalan, mercaptopurine, mitotane, procarbazine hydrochloride dactinomycin, daunorubicin hydrochloride, doxorubicin hydrochloride, taxol, mitomycin, plicamycin, aminoglutethimide, estramustine phosphate sodium, flutamide, leuprolide acetate, megestrol acetate, tamoxifen citrate, testolactone, trilostane, amsacrine, asparaginase, etoposide, interferon .alpha.-2a, interferon .alpha.-2b, teniposide, vinblastine sulfate, vincristine sulfate, bleomycin, bleomycin sulfate, methotrexate, adriamycin, and arabinosyl; wherein the blood products are selected from the group consisting of parenteral iron, hemin, hematoporphyrins and derivatives thereof; wherein the biological response modifiers are selected from the group consisting of muramyldipeptide, muramyltripeptide, microbial cell wall components, lymphokines, sub-units of bacteria, and synthetic dipeptide N-acetyl-muramyl-L-alanyl-D-isoglutamine; wherein the anti-fungal agents are selected from the group consisting of ketoconazole, nystatin, griseofulvin, flucytosine, miconazol, amphotericin B, ricin, and .beta.-lactam antibiotics; and wherein the hormones are selected from the group consisting of growth hormone, melanocyte stimulating hormone, estradiol, beclomethasone dipropionate, betamethasone, betamethasone acetate, betamethasone sodium phosphate, vetamethasone disodium phosphate, vetamethasone sodium phosphate, cortisone acetate, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, flunisolide, hydrocortisone, hydrocortisone acetate, hydrocortisone cypionate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, prednisone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate, triamcinolone hexacetonide and fludrocortisone acetate; wherein the vitamins are selected from the group consisting of cyanocobalamin neinoic acid, retinoids and derivatives thereof; wherein the peptides are manganese super oxide dismutase; wherein the enzymes are alkaline phosphatase; wherein the anti-allergic agents are amelexanox; wherein the anti-coagulation agents are selected from the group consisting of phenprocoumon and heparin; wherein the circulatory drugs are propranolol; wherein the metabolic potentiators are glutathione; wherein the antituberculars are selected from the group consisting of para-aminosalicylic acid, isoniazid, capreomycin sulfate cycloserine, ethambutol hydrochloride ethionamide, pyrazinamide, rifampin, and streptomycin sulfate; wherein the antivirals are selected from the group consisting of acyclovir, amantadine azidothymidine, ribavirin and vidarabine monohydrate; wherein the antianginals are selected from the group consisting of diltiazem, nifedipine, verapamil, erythritol tetranitrate, isosorbide dinitrate, nitroglycerin and pentaerythritol tetranitrate; wherein the antibiotics are selected from the group consisting of dapsone, chloramphenicol, neomycin, cefaclor, cefadroxil, cephalexin, cephradine erythromycin, clindamycin, lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, ticarcillin rifampin and tetracycline; wherein the antiinflammatories are selected from the group consisting of diflunisal, ibuprofen, indomethacin, meclofenamate, mefenamic acid, naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac, tolmetin, aspirin and salicylates; wherein the antiprotozoans are selected from the group consisting of chloroquine, hydroxychloroquine, metronidazole, quinine and meglumine antimonate; wherein the antirheumatics are penicillamine; wherein the narcotics are paregoric; wherein the opiates are selected from the group consisting of codeine, heroin, methadone, morphine and opium; wherein the cardiac glycosides are selected from the group consisting of deslanoside, digitoxin, digoxin, digitalin and digitalis; wherein the neuromuscular blockers are selected from the group consisting of atracuriurn mesylate, galamine triethiodide, hexafluorenium bromide, metocurine iodide, pancuronium bromide, succinylcholine chloride, tubocurarine chloride and vecuronium bromide; wherein the sedatives are selected from the group consisting of amobarbital, amobarbital sodium, aprobarbital, butabarbital sodium, chloral hydrate, ethchlorvynol, ethinamate, flurazepam hydrochloride, glutethimide, methotrimeprazine hydrochloride, methyprylon, midazolam hydrochloride, paraldehyde, pentobarbital, pentobarbital sodium, phenobarbital sodium, secobarbital sodium, talbutal, temazepam and triazolam; wherein the local anesthetics are selected from the group consisting of bupivacaine hydrochloride, chloroprocaine hydrochloride, etidocaine hydrochloride, lidocaine hydrochloride, mepivacaine hydrochloride, procaine hydrochloride and tetracaine hydrochloride; wherein the general anesthetics are selected from the group consisting of droperidol, etomidate, fentanyl citrate with droperidol, ketamine hydrochloride, methohexital sodium and thiopental sodium; and wherein the radioactive compounds are selected from the group consisting of strontium, iodide rhenium and yttrium.

75. A drug delivery system of claim 39 wherein prior to said placing said liposomes under the negative pressure, said liposomes are allowed to cool to a temperature between about -10.degree. C. and about -20.degree. C., wherein the negative pressure is between about 700 mm Hg to about 760 mm Hg, wherein said incubating step is for about 24 to about 72 hours, wherein during said incubating step said liposomes are allowed to warm to a temperature between about 10.degree. C. and about 20.degree. C., wherein said instilling step occurs over a period of about 4 to about 8 hours, and wherein during said instilling step said liposomes are allowed to warm to ambient temperature.
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