Title:  Multilamellar coalescence vesicles (MLCV) containing biologically active compounds

United States Patent:  6,544,549

Issued:  April 8, 2003

Inventors:  Boni; Lawrence T. (Monmouth Junction, NJ); Batenjany; Michael M. (Hamilton, NJ); Gevantmakher; Stella (Plainsboro, NJ); Popescu; Mircea C. (Plainsboro, NJ)

Assignee:  Biomira USA Inc. (Cranbury, NJ)

Appl. No.:  164350

Filed:  October 1, 1998

A method for producing multilamellar coalescence vesicles (MLCVs) containing increased amounts of biologically active compound is disclosed. The method involves hydrating at least one powdered lipid in an aqueous buffer at a temperature above the phase transition temperature of the highest melting lipid to form multilamellar vesicles, reducing the size of the multilamellar vesicles to about 20-400 nm to produce small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs) or a mixture thereof; and incubating the SUVs, LUVs or mixture thereof with a biologically active compound in an aqueous solution under sufficient conditions to form MLCVs containing the biologically active compound without the use of an organic solvent, a freeze-thawing step or a dehydration step. MLCVs produced by this method contain increased amounts of biologically active compound over prior art liposomes produced with an organic solvent, a freeze-thawing step or a dehydration step and fewer vesicles are substantially free of biologically active compound

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a novel method of producing MLCVs containing increased amounts of biologically active compounds. The method of the present invention produces MLCVs without the use of steps involving freeze-thawing, organic solvents or dehydration.

The first step of the present method involves direct hydration of a powdered lipid or mixture of lipids with the appropriate buffer in a temperature jacketed mixing vessel. Examples of appropriate buffers are phosphate buffered saline (PBS), acetate, or citrate with a pH between 2 and 12, preferably between 5 and 9. The hydration is preferably performed above the phase transition temperature of the highest melting lipid, and the hydrated suspension is mixed well. The next step involves size reduction, preferably to about 20-70 nm comprising SUVs and/or LUVs, but in any case, below about 400 nm, preferably below about 200 nm. This size reduction is performed using standard means, such as sonication, including bath or probe sonication, homogenization or extrusion. The size reduction is performed above the phase transition temperature of the highest melting lipid employed. If a mixture of lipids is used, the phase transition temperature of the highest melting lipid would be used. The resultant SUVs and/or LUVs are then sterile filtered (0.22 micron filter) into a sterile reaction vessel equipped with a mixing device and a temperature jacket to maintain the temperature above the phase transition temperature of the highest melting lipid. These LUVs are sufficiently deformable that larger sizes can squeeze through a sterile filter. To this vessel, one or more pharmaceutical(s), along with any other necessary excipients, such as, HSA, mannitol, and glycerol, are added through a sterilizing filter. This mixture is continually mixed while dropping the temperature to the phase transition temperature of the highest melting lipid, or preferably between its pretransition and main transition temperature. The incubation temperature may be below the pretransition temperature, below the subtransition temperature or above the main transition temperature. This mixture is then incubated for an extended period of time, but it can be incubated from minutes to hours, to possibly days, with optional continuous or intermittent mixing. During this time the SUVs and/or LUVs coalesce to form large vesicles, MLCVs, typically between 1000 and 5000 nm, but the MLCVs can be as small as about 100 nm in average diameter, and are multilamellar, with entrapped pharmaceuticals. The MLCVs of the present invention are unique structurally in that they possess a varying degree of partially coalesced vesicles in addition to numerous lamellae.

The preferred lipids are saturated lecithins, such as dimyristoyl phosphatidylcholine (DMPC); dipalmitoyl phosphatidylcholine (DPPC); distearoyl phosphatidylcholine (DSPC); saturated phosphatidylglycerols, such as dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearoyl phosphatidylglycerol (DSPG); saturated phosphatidic acids, saturated phosphatidylethanolamines or mixtures of the above lipids. Unsaturated lipids may also be employed, such as egg phosphatidylcholine (EPC) and dioleoyl phosphatidylcholine (DOPC).

The temperature of incubation may be at the pretransition temperature or the main transition temperature of the highest melting lipid used, although it could also be below the pretransition temperature or above the main transition temperature. The incubation can also be performed by cycling through a temperature range, such as between the pretransition and main transition temperatures.

Prior literature indicates that fusion of pure saturated phosphatidylcholines will occur below the phase transition of that lipid, and specifically below the pretransition. See Schmidt, C. F., Lichtenberg, D., and Thompson, T. E., Biochemistry 20:4792-4797 (1981); Larrabee, A. L. Biochemistry 18:3321-3326 (1979); Schullery, S. E., Schmidt, C. F., Felper, Tillack, T. W., and Thompson, T. E. Biochemisty 19:3919-3923 (1980); Petersen, N. O. and Chan 51 S. I., Biochim. Biophys. Acta 509:111-128 (1978); Wong, M., Anthony, F. H., Tillack, T. W., and Thompson, T. E. Biochemistry 21:4126-4132 (1982); McConnell, D. S. and Schullery, S. E., Biochim. Biophys. Acta 818:13-22 (1985); Gaber, B. P. and Sheridan, J. P. Biochim. Biophys. Acta 685:87-93 (1982). Fusion is typically to unilamellar vesicles of 70-95 nm. The fusion product is also capable of entrapping solute. See McConnell et al. Fusion at the phase transition was observed in one case to be similar to that obtained below the pretransition temperature. See Gaber and Sheridan (1982). The entrapment was not established and the kinetics of fusion was quite slow, taking up to weeks to occur.

The lipid used in the present method preferably should be saturated and may have any head group, such as phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, or phosphatidylserine. In addition, mixtures of lipids with respect to both head group and chain length can be used. Mixtures with lipids that alone do not form liposomes, such as cholesterol or fatty acids, can be employed in this process. The lipid concentration should be between 1 mg/ml to 400 mg/mL, preferably for most applications between 100 mg/mL and 250 mg/mL. Saturated lecithins are preferred lipids for use in the present method.

The biologically active compound useful in the present invention can be any of the known biologically active compounds that can be entrapped in the liposomes and whose rate of diffusion out of the liposomes is not significantly greater than the rate of deterioration of liposomes in the body of the recipient. The biologically active compounds may have properties that enhance vesicle coalescence. These substances would act directly on the SUVs and/or LUVs by destabilizing their bilayer structure. A simple measurement of increased turbidity, such as illustrated in Example 1, permits ready identification of substances having this property of enhancing vesicle coalescence. The biologically active compounds can be selected from proteins, peptides, antigens, antibiotics, hormones, immunological activators, cytokines, lymphokines, polynucleotides, and other drugs. Specific examples of such compounds are IL-2, interferon, granulocyte-macrophage colony stimulating factor (GMCSF), insulin, growth hormone, epidermal growth factor, calcitonin, gentamicin, and antigens derived from bacteria, parasites, viruses or rickettsia, tumor antigens, allergens, poison or venom.

IL-2 is commercially available as recombinant T-cell growth factor (human IL-2, recombinant; T3267) or as a preparation derived from cultured rat splenocytes (TO892) from Sigma Chemical Co. (St. Louis, Mo.). Recombinant IL-2 may also be obtained from Genzyme (Boston, Mass.) or R & D Systems (Minneapolis, Minn.). Other lymphokines known and available in the art also can be used in the present invention. These include interleukin-4 (IL-4), interleukin-6 (IL-6), interferon alpha and interferon gamma. It is envisioned that these lymphokines can be used alone, in sequence, or in combination, such as co-entrapment in the liposome (e.g., IL-2 and IL-6).

MLCVs according to the present invention are characterized by several features that distinguish them from MLVs made by prior art processes. One particularly salient feature is the highly uniform distribution of biologically active compound among the MLCVs. In many prior art MLV preparations, a high proportion, often about 50%, of the liposomes are substantially free of biologically active compound. By contrast, the MLCVs of the invention, when seen in freeze-fracture electron micrographs, normally have a low percentage of vesicles that are substantially free of biologically active compound, generally less than 30%, preferably less than 20%, often less than 10% and even less than 5% or even less than 2% of vesicles that are substantially free of biologically active compound as indicated by the absence of bulges. This property, in turn, permits a higher total entrapment of biologically active compounds by the MLCVs of the invention compared to prior art MLVs. A further feature, illustrated in Example 12 below, is that MLCVs according to the invention have a higher proportion of the biologically active material on the vesicle surface than prior art MLVs. In addition, the MLCVs of the invention permit a higher proportion of the entrapped biologically active compound to retain its biological activity compared to MLVs made by other methods.

The MLCVs of the invention contain in the range of 10-100% greater amount of biologically active compound than liposomes produced by the prior art methods using an organic solvent, a freeze-thawing step or a dehydration step. The MLCVs preferably contain at least 10% greater amount of biologically active compound than the prior art liposomes, preferably at least 20% greater amount biologically active compound, more preferably at least 30% greater amount biologically active compound, more preferably at least 40% greater amount biologically active compound, more preferably at least 50% greater amount biologically active compound, more preferably at least 80% greater amount biologically active compound, and more preferably at least 100% greater amount biologically active compound and even more than 100% greater amount biologically active compound.

Claim 1 of 20 Claims

What is claimed is:

1. A method for producing multilamellar coalescence vesicles (MLCVs) containing a biologically active compound, said method comprising:

incubating small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs) or mixture thereof with at least one biologically active compound in an aqueous solution at a temperature above the temperature of the pretransition of the lipid component for a time sufficient to form MLCVs containing said at least one biologically active compound;

wherein said method is performed without the use of an organic solvent, a freeze-thawing step or a dehydration step.
 

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