WO2000067779A2

WO2000067779A2 - Polymerized liposomes for the delivery of mammalian growth hormone

Info

Publication number: WO2000067779A2
Application number: PCT/US2000/012902
Authority: WO
Inventors: Hansi J. Dean; Kyle Bucher; Elijah Bolotin
Original assignee: Endorex Corporation
Priority date: 1999-05-12
Filing date: 2000-05-11
Publication date: 2000-11-16
Also published as: AU5004000A; WO2000067779A3

Abstract

The present invention encompasses polymerized liposomes systems for oral and/or mucosal delivery of protein drugs. In a preferred embodiment, the polymerized liposome is modified to contain mammalian growth hormone for the delivery of biologically active hormone after mucosal administration. The present invention further relates to the synthesis, preparation and use of the polymerized liposomes of the present invention as, or in, pharmaceutical compositions for oral delivery of bioactive molecules, in particular, mammalian growth hormone.

Description

POLYMERIZED LIPOSOMES FOR THE DELIVERY OF MAMMALIAN GROWTH HORMONE

1. INTRODUCTION

The present invention relates to liposomes for oral and/or mucosal delivery of protein drugs. In particular, the present invention relates to the use of polymerized liposomes modified to contain mammalian growth hormone for the delivery of biologically active hormone after mucosal administration. The present invention further relates to the synthesis, preparation and use of the polymerized liposomes of the present invention as, or in, pharmaceutical compositions for oral delivery of bioactive molecules, in particular, mammalian growth hormone.

¹⁵ 2. BACKGROUND OF THE INVENTION

2.1. DRUG DELIVERY

Drug delivery takes a variety of forms, depending on the agent to be delivered and the administration route. The most convenient way to administer drugs into the body is by oral administration. However, many drugs, in particular proteins and peptides, are poorly

20 absorbed and unstable during passage through the gastrointestinal (GI) tract. The administration of these protein drugs is generally performed through parenteral injection.

Controlled release systems for drug delivery are often designed to administer drugs to specific areas of the body. In the gastrointestinal tract it is important that the drug not be eliminated before it has had a chance to exert a localized effect or to pass into the

? ^^J5 bloodstream.

Enteric coated formulations have been widely used for many years to protect drugs administered orally, as well as to delay release. Several microsphere formulations have been proposed as a means for oral drug delivery. For example, PCT/US90/0643 and

PCT/US90/06433 by Enzytech discloses the use of a hydrophobic protein, such as zein, to form microparticles; U.S. Patent No. 4,976,968 to Steiner et al. discloses the use of

"proteinoids" to form microparticles; and European Patent Application 0,333,523 by the

UAB Research Foundation and Southern Research Institute discloses the use of synthetic polymers such as polylactic acid-glycolic acid to form in microspheres.

Entrapping a protein in a microparticulate system can protect the protein from acidic

35 and enzymatic degradation, yet still allow the protein to be administered orally. The particulate can be designed to enhance uptake by cells lining the GI tract, and to release the entrapped material at the desired location. When the entrapped material is a protein, elimination of the first-pass effect (metabolism by the liver) is also highly advantageous.

2.2. LIPOSOMES Liposomes have been proposed for use as an oral drug delivery system, for example, by Patel and Ryman, FEBS Letters 62(1), 60-63 (1976). Liposomes also have some features that should be advantageous for a particulate system for oral protein drug delivery. The phospholipid bilayer membrane of liposomes separates and protects entrapped materials in the inner aqueous core from the outside. Both water-soluble and -insoluble substances can be entrapped in different compartments, the aqueous core and bilayer membrane, respectively, of the same liposome. Liposomes can be formed which are less than 1 micron in diameter, enhancing their ability to be taken up across mucosal surfaces. The surface of liposomes can be modified with respect to charge, hydrophobicity, and specific targeting agents to maximize interaction with the surface of cells lining the gastrointestinal tract. Further, liposomes are easy to prepare.

However, conventional liposomes are chemically and physically unstable and therefore are unsuitable for oral delivery as they rapidly degrade upon introduction into the harsh environment of the gastrointestinal tract (GI tract). The entrapped protein is released into this environment, which in the case of mammalian GH would lead to destruction and lack of therapeutic effect. Likewise, the premature breakdown of the liposome in the GI tract will result in the inability of the entrapped protein to be taken up by absorptive cells in the intestine and elsewhere in the GI tract. In order to be useful for mucosal delivery, liposomes must be stabilized to permit survival in the presence of degradataive enzymes, bile salts, and acids present in the intestine and stomach. Several methods have been tried to enhance liposome stability. Some methods involved intercalating cholesterol into the bilayer membrane or generating the liposomes in the presence of polysaccharides. These methods are not useful in making liposome for oral delivery since during oral delivery liposomes are exposed to an acidic pH in the stomach and bile salts and phospholipases in intestine. Under these conditions cholesterol- containing liposomes are still subjected to degradation.

Investigators have explored the improved stability of polymerized liposomes, however in the area of drug delivery their ultimate utility remains uncertain (Regen, 1987 in Liposomes From Biophysics to Therapeutics, edit. Ostro, Marcel Dekker, N.Y.). Most methods for polymerization of liposomes are too harsh and result in destruction of protein drugs. Polymerized liposomes have been developed in attempts to improve oral delivery of encapsulated drugs (Chen et al. WO 9503035). Furthermore, polymerized liposomes can be engineered successfully to release contents in a controlled fashion, to contain surface ligands capable of targeting specific receptors, and for stability to environmental degradation. Because they can essentially entrap any drug, hydrophilic or hydrophobic, they have great potential as carriers for drug delivery. However, to the inventors' knowledge, to date the utility of conventional or polymerized liposomes for oral delivery of mammalian growth hormone has not been described. Similarly, whether previously described polymerized liposomes are more advantageous than conventional or unpolymerized liposomes for oral drug delivery is still unclear since improved stability alone may not be sufficient for oral drug delivery. Thus, there remains a need for drug delivery systems that can be prepared using the mild conditions required for protein drugs, survive the harsh conditions in the GI tract, enhance uptake from the GI tract and effectively deliver the drug.

2.3. GROWTH HORMONE Human growth hormone (hGH) is used to treat children who have growth failure due to lack of adequate endogenous GH secretion, Turner's syndrome, chronic renal insufficiency (prior to renal transplantation), GH deficiency in adults, and wasting associated with AIDS. There is also evidence that hGH may be efficacious in other indications related to metabolic activity, including children with idiopathic short stature, defined by a persistently low growth rate in the absence of GH deficiency, systemic disease, malnutrition, hypothyroidism, wasting due to chronic and acute respiratory insufficiency, or aging. Growth hormone from other mammalian species including, but not limited to, cattle, swine, and dogs, has similar properties in enhancing metabolic activity.

Oral delivery systems must overcome several technical challenges, including protection of mammalian GH through the harsh environment in the gastrointestinal tract, and enhancing permeability of the intestinal epithelium to uptake of mammalian GH. These two major factors combined, result in extremely poor bioavailability of orally administered mammalian GH. Increased stability and bioactivity of orally delivered hGH has been achieved by complexation of hGH with N-acetylated non-a aromatic amino acids (Leone- Bay et al. 1996 J. Med. Chem 39:2571-2578). Intranasal delivery of hGH has been achieved in combination with a membrane permeation enhancer (Olsson et. al. 1993. J.Clin. Endocrinol. Metab. 76: 962-967). Both of these approaches are fundamentally different from entrapment of mammalian growth hormone in a liposome for enhanced uptake across mucosal surfaces and protection during transit in the GI tract. The present invention relates to the development of polymerized liposomes with increased stability characteristics combined with optimal size and surface characteristics for intestinal cell uptake. These techniques were applied specifically to development of polymerized liposomes containing mammalian GH and demonstration of unexpected high oral bioavailability of the mammalian GH delivered in these polymerized liposomes.

3. SUMMARY OF THE INVENTION

The present invention encompasses the use of delivery systems for the oral delivery of protein drugs. In a preferred embodiment, the delivery system is a polymerized liposome and the polymerized liposomes of the present invention contain mammalian growth hormone. The polymerized liposomes of the present invention may be utilized for the delivery of a wide variety of compounds, including, but not limited to growth hormone. In other embodiments, the delivery system is a polymer microsphere, wherein the polymer microsphere matrix contains a mammalian growth hormone.

The present invention is based on, inter alia, Applicants' discovery that the polymerized liposomes of the present invention have enhanced stability against the harsh environment of the gastrointestinal tract. In addition, the polymerized liposome delivery system resulted in unexpected high bioavailability of mammalian GH in the blood. That the polymerized liposome delivery system of the present invention is especially effective for the oral and/or mucosal delivery of protein drugs and therapeutics is demonstrated by the working examples described infra. The present invention relates to the formulation of mammalian GH in a polymerized lioposome bilayer structure. Particles were designed to be less than 1 μm in size to enhance the probability of uptake by cells of the GI tract. The present invention encompasses the use of mammalian growth hormone in assaying the efficacy oft!"-: liposomes of the present invention. The present invention also relates to synthesis, preparation and use of the polymerized liposomes. The liposomes of the present invention are composed of phospholipids which are polymerized by covalent bonding to each other. Covalent bonding the layers adds strength, resulting in a less fluid bilayer than unpolymerized liposome. The less fluid bilayer membrane suppresses leakage. Further, the detergent-like bile salts in the intestine cannot solvate the phospholipid molecules. These cross-linked membranes are strong enough to maintain their structure even if the phospholipids undergo hydrolysis at low pH and enzymatic degradation by phospholipases. Thus, polymerized liposomes reach the ileum of the GI tract as intact particulates, and are absorbed.

3.1. DEFINITIONS As used herein, the term "liposome" is defined as an aqueous compartment enclosed by a lipid bilayer. (Stryer, Biochemistry, 2d Edition, W.H. Freeman & Co., p. 213 (1981)). The liposomes can be prepared by a thin film hydration technique followed by a few freeze- thaw cycles. Liposomal suspensions can also be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811, incorporated herein by references in its entirety.

As used herein, the term "polymerized liposome" is defined as a liposome in which the constituent phospholipids are covalently bonded to each other by inter and intra molecular interactions. The phospholipids can be bound together within a single layer of the phospholipid bilayer (the leaflets) and/or bound together between the two layers of the bilayer.

The degree of crosslinking in the polymerized liposomes can range from 30 to 100 percent, i.e., up to 100 percent of the available bonds are made. The size range of polymerized liposomes is between approximately 15 ran to 10 μm. The polymerized liposomes can be loaded with up to 100% of the material to be delivered, when the material is hydrophobic and attracted by the phospholipid layers. In general, about 5 to about 40 percent of the material is encapsulated when the material is hydrophilic.

As used herein, the term "trap ratio" is defined as the ratio of inner aqueous phase volume to total aqueous phase volume used.

As used herein, the term "radical initiator" is defined as a chemical which initiates free-radical polymerization.

As used herein, the term "reverse phase evaporation technique" is defined as a method involving dissolving a lipid in an organic solvent, adding a buffer solution, and evaporating the organic solvent at reduced pressure, as described by Skoza, F. Jr., and Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA. Volume 75, No. 9, pp. 4194-4198 (1978).

As used herein, the term "freeze-thaw technique," or "F-T," is defined as freezing a suspension in liquid nitrogen, and subsequently thawing the suspension in a roughly 30°C water bath.

As used herein, the terms "mucosa" or "mucosal" refers to a mucous tissue such as epithelium, lamina propria, and a layer of smooth muscle in the digestive tract. Mucosal delivery as used herein is meant to include delivery through bronchi, gingival, lingual, nasal, oral, and intestinal mucosal tissue.

As used herein, the term "buffer solution" is defined as an aqueous solution or aqueous solution containing less than 25% of a miscible organic solvent, in which a buffer has been added to control the pH of the solution. Examples of suitable buffers include but are not limited to PBS (phosphate buffered saline), TRIS (tris- (hydroxymethyl)aminomethane), HEPES (hydroxyethylpiperidine ethane sulfonic acid), and TES 2-[(tris-hydroxymethyl)methyl]amino-l-ethanesulfonic acid.

As used herein, the term "leaflets" is defined as a single layer of phospholipid in the bilayer forming the liposome.

4. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to polymerized liposomes for oral and/or mucosal delivery of protein drugs. In particular, the present invention relates to polymerized liposomes for mucosal delivery of mammalian growth hormone. In a preferred embodiment of the invention, the liposome comprises a phospholipid bilayer having covalently bonded polymerizable phospholipids and an aqueous core containing mammalian growth hormone.

The present invention is based in part on Applicants' discovery that the polymerized liposomes of the present invention have surprisingly enhanced stability against the harsh environment of the gastrointestinal tract. The present invention relates to polymerized liposomes which have been modified to contain mammalian growth hormone. The use growth hormone containing liposomes provides a method for assaying the efficacy of the liposomes of the present invention. The polymerized liposomes of the present invention may be modified to include a variety of molecules and moieties to target the liposomes, including but not limited to glycoproteins, peptides, carbohydrates, lectins i.e., ulex europeas agglutinin I, monoclonal antibodies, cholera toxin B subunit, bacterial adhesotopes or phage display hybrid peptides encompassing binding domains and magnetic particles.

The polymerized liposomes of the present invention have utility for the oral and/or mucosal delivery of antigens, allergens, vaccines and therapeutics. The polymerized liposomes of the present invention are designed to deliver a wide variety of therapeutics including RNA and DNA nucleotides as used, for example, in gene therapy, peptides and small molecules. These therapeutics include but are not limited to antiviral agents, antibacterial agents, attenuated viruses, antifungal agents, cytokines, hormones, insulin, calcitonin, fertility drugs, antibiotics and chemotherapy agents.

The present invention also relates to the synthesis, preparation and the use of the modified polymerized liposome. The liposomes of the present invention are generally prepared by polymerization of double and triple bond-containing monomeric phospholipids. In a preferred embodiment, examples of polymerizable functional groups, include but are not limited to olefins, acetylenes, and thiols. The liposomes of the present invention may be polymerized by a variety of techniques including but not limited to free radical initiation and radiation. The polymerized liposomes of the present invention may be prepared by a variety of techniques as described infra.

4.1 TARGETING MOLECULES

A variety of molecules and ligands may be used to modify the polymerized liposomes of the present invention in order to target them to a specific site cell type, including, but not limited to, glycoproteins, carbohydrates, lectins, monoclonal antibodies, antibody fragments, viral proteins, bacterial proteins, e., cholera toxin B subunit, phage display hybrid peptides and magnetic particles.

In a preferred embodiment of the present invention carbohydrates or lectins are used to target the polymerized liposomes of the present invention to M cells and Peyer's Patch cells of the small intestine. In another preferred embodiment of the present invention, lectins which bind to fucosyl sugars are used to modify the polymerized liposomes. Lectins are a heterogenous group of proteins or glycoproteins that recognize carbohydrate residues on cell surface glycoconjugates with a high degree of specificity. Examples of lectins that may be used to modify the polymerized liposomes of the present invention, include but are not limited to, lectins specific for fucosyl glycoconjugates, such as Ulex Europeas Agglutinin I (UEA); lectins specific for galactose/N-acetylgalactoseamine, such as Phaseolus vulgaris haemagglutinin (PHA), tomato lectin (Lycopersicon esculentum) (TL), wheat germ agglutinin (WGA); lectins specific for mannose, such as, Galanthus nivalis agglutinin (GNA); lectins specific for mannose/glucose, such as, con A/concavalan A. (See e.g.. Lehr et al, 1995, in Lectins Biomedical Perspectives, pp. 117-140). These targeting molecules can be derivatized if desired. (See e.g., Chen et al., 1995, Proceed. Internat. Symp. Control. Rel. Bioact. Mater. 22, Cohen WO 9503035).

In another embodiment of the invention, polymerized liposomes may be modified with viral proteins or bacterial proteins that have an affinity for a particular residue expressed on a cell surface or that have an affinity for a cell surface protein or receptor. Examples of such proteins include, but are not limited to, cholera toxin B subunit and bacterial adhesotopes.

In yet another embodiment of the present invention, polymerized liposomes may be modified with monoclonal antibodies or fragments of antibodies which target the polymerized liposome to a particular cell type. The polymerized liposomes of the present invention may be modified with ligands for specific mucosal cell surface receptors and proteins. As used herein, the term "ligand" refers to a ligand attached to the polymerized liposomes which adheres to the mucosa in the intestine or can be used to target the liposomes to a specific cell type in the G-I tract or following absorption. These can range from ligands for specific cell surface proteins and antibodies or antibody fragments immunoreactive with specific surface molecules, to less specific targeting such as coatings of materials which are bioadhesive, such as alginate and polyacrylate. In general, ligands are bound to or inserted within the polymerized phospholipids; adhesive polymers are applied as a coating to the particles.

As noted above, the liposomes can be modified, for example, by attaching to the surface of the particle specific ligands for given cells in a mixture of cells. When magnetic particles are also incorporated, the particles can be targeted using the ligands, such as tissue specific surface proteins, then maintained as the targeted cells using a magnetic field while the particles are imaged or a compound to be delivered is released. Such magnetic particles are known in the art and include aqueous-based ferro fluid EMB 807 (Ferrofluids, NH).

4.2. MATERIALS TO BE ENCAPSULATED

In a preferred embodiment, the polymerized liposomes of the present invention have utility for the oral and/or mucosal delivery of mammalian growth hormone, including, but not limited, to ovine growth hormone, bovine growth hormone, porcine growth hormone, canine growth hormone, human growth hormone, in addition to ovine growth hormone releasing hormone, bovine growth hormone releasing hormone, porcine growth hormone releasing hormone and human growth hormone releasing hormone.

The mammalian growth hormone can be prepared by many procedures, including, but not limited to, recombinant DNA methods, solid phase peptide synthesis techniques, or solution phase peptide synthesis techniques. The present invention encompasses sequences coding growth hormone or a functionally active analog or fragment disclosed for any species (e.g., the growth hormone disclosed in U.S. Patent Nos. 4,446,235, 4,670,393, 4,665,180, and 5,849,535, which are incorporated herein by reference in their entirety). Using known techniques of DNA recombination, the polynucleotide sequence encoding growth hormone or a functionally active analog or fragment can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. A variety of host- expression vector systems may be utilized to express the target gene coding sequences of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing target gene product coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing the target gene product coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculo virus) containing the target gene product coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV); and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing target gene product coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

Polypeptides coding growth hormone can be conveniently synthesized according to the usual methods of peptide chemistry, such as by solid phase peptide synthesis, as described by E. Atherton and R. C. Sheppard in "Solid Phase Peptide Synthesis" IRL Press at Oxford University Press 1989, by solution phase synthesis as described by J. Jones in "The Chemical Synthesis of Peptides", Clarendon Press, Oxford 1994, or by both solid- and solution-phase methods, as known in the art. Polypeptides of the present invention can be purified by techniques known to those of skill in the art (e.g., preparative high performance liquid chromatography (HPLC)).

The polymerized liposomes of the present invention can be used for the oral and/or mucosal delivery of a wide variety of therapeutics, including but limited to, chemotherapy agents, antibiotics, insulin, cytokines, interferon, hormones, calcitonin, hormones, fertility drugs, antiviral agents (ddl, AZT, ddC, acyclovir and the like), antibacterial agents, antifungal agents, DNA and RNA nucleotides, i.e., useful for gene therapy.

4.3. PREPARATION OF POLYMERIZED PHOSPHOLIPIDS The polymerized liposomes of the present invention may be prepared by a variety of techniques as described infra. For example, and not by way of limitation, polymerized liposomes are prepared by polymerizing double and triple bond-containing olefinic and acetylenic phospholipids. In addition, polymerized liposomes can be prepared by chemical oxidation of thiol groups in the phospholipids to disulfide linkages. The polymerization can take place in a solution containing a biologically active substance, such as a drug or antigen, in which case the substance is encapsulated during the polymerization. Alternatively, the liposomes can be polymerized first, and the biologically active substance can be added later by resuspending the polymerized liposomes in a solution of a biologically active substance, and entrapping the substance by sonication of the suspension. Another method of entrapping a biologically active substance in polymerized liposomes is to dry the polymerized liposomes to form a film, and hydrate the film in a solution of the biologically active substance. The above conditions are typically mild enough to entrap biologically active substances without denaturing them.

The polymerized liposomes are generally prepared by polymerization of double bond-containing monomenc phospholipids. These phospholipids may contain any unsaturated functional group, including polymerizable functional group double or triple bonds, any may contain more than one polymerizable functional group double or triple bonded. Suitable monomenc phospholipids are known to those skilled in the art, and include, but are not limited to, phosphatidylaholines DODPC (l,2-di(2,4-Octadecadienoyl)- 3-phosphatidylcholine), 2,4-diene phospholipids, di-yne phospholipids, see e.g., U.S. Patent No. 4,485,045, U.S. Patent No. 4,861,521.

The liposomes of the present invention may be polymerized by free radical initiation. The monomeric double bond-containing phospholipids can be polymerized using a hydrophobic free radical initiator, such as AIBN (azo-bis-isobutyronitrile), or a hydrophilic free radical initiator such as AAPD (azo-bis-amidinopropane dihydrochloride). The latter is particularly useful for initiating polymerization between layers of the bilayer. The present invention also encompasses the use of other mild redox initiators, such as Na₂S₂O₅ and K₂S₂O₈. Alternatively, polymerization can be initiated by using a radiation source, such as ultraviolet or gamma radiation.

The ratio between the phospholipid and crosslinker and aqueous phase all affect the percent of crosslinking. In general, the percent crosslinking increases as the amount of crosslinker or time or temperature of reaction are increased. As the percent crosslinking increases, the release rate of the materials from the liposomes decreases and the stability increases.

The liposomes of the present invention may be polymerized by radiation including, polymerization with ultraviolet and/or gamma radiation, provided the biologically active substance can survive exposure to the radiation. Typical conditions for initiating the polymerization with ultraviolet radiation include but are not limited to irradiating the solution at 302 nm, 4 W, for 3 - 12 hours at room temperature. Typical conditions for initiating the polymerization with gamma radiation include but are not limited to irradiating the solution at 0.3 mRad per hour for 3 hours at room temperature.

In addition, the growth hormone of the present invention may be encapsulated or formulated in a polymeric particle. Polymeric particles in this case should therefore be of an appropriate size to be maximally taken up by cells of the GI tract. Such microparticles can be formulated as a liquid suspension or a dry powder and can be administered orally or via inhalation (intranasal or mtrapulmonary). The particles are useful for delivery of drugs, hormones, cytokines, nucleic acids, and human growth hormone for which slow release in the gastrointestinal tract or at a mucosal surface is desired. The rate of degradation, and consequently of release, varies with the polymeric formulation.

Composition of Polymeric Particles

Polymeric material is obtained from commercial sources or can be prepared by known methods. For example, polymers of lactic and glycolic acid can be generated as described in U.S. Pat. No. 4,293,539 or purchased form Aldrich. Alternatively, or in addition, the polymeric matrix can include polylactide, polyglcolide, poly(lactide-co- glycolide), polyanhydride, polyorthoester, polycaprolactone, polyphosphazene, proteinaceous polymer, polypeptide, polyester, or polyorthoester. Preferred controlled release substances which are useful in the formulations of the invention include the polyanhydrides, co-polymers of lactic acid and glycolic acid wherein the weight ratio of lactic acid to glycolic acid is no more than 4: 1, and polyorthoesters containing a degradation-enhancing catalyst, such as an anhydride, e.g., 1% maleic anhydride.

4.4. ENCAPSULATION OF BIOLOGICALLY ACTIVE MATERIAL Materials are generally incorporated into the liposomes at the time of formation, following polymerization using physical disruption by sonication, freeze-thaw, etc. of a solution of the material which contains the liposomes, dehydration and rehydration of the liposomes with a solution containing the drug to be loaded.

The following is a general method for the preparation of polymerized liposomes wherein a biologically active substance is entrapped prior to the polymerization of the monomeric double bond-containing liposome. First, the monomeric liposome is prepared by the hydration of a monomeric double bond-containing phospholipid. The monomeric phospholipid is dissolved, and the solution is then dried by lyophilization. A solution containing the substance to be entrapped is added, together with appropriate stabilizing agents. The solution is thoroughly mixed. If necessary, the solution can be frozen and thawed one or more time to increase the loading. The material is then subjected to extrusion through the appropriate size filter, to provide liposomes of the desired size, in this case 100 or 200 nm. This extrusion is continued until the liposomes are the appropriate size. Polymerization is initiated preferably with a catalytic amount (2 - 100 mol%) of free radical initiator or by exposure to UV light. Next, the polymerization is carried out at a low temperature, from 20 - 37 °C, preferably at around 25 °C, for 15 minutes to 20 hours, preferably about 3 hours, or until the polymerization has reached the desired level. This step is particularly important, as higher temperature or extended reaction time would be expected to inactivate the protein, in particular, mammalian GH. The desired degree of crosslinking is from 30 to 100 percent. In a preferred embodiment of the present invention, the diameter of the polymerized liposome is between 15 nm and 1 μm.

Unentrapped biologically active substance can be removed by several means, preferably ultrafiltration, centrifugation or column chromatography. The polymerized liposomes are then suspended in a buffer solution. The buffer solution has a pH preferably between pH 4.5 and pH 9.5, in the case of mammalian GH, between 5.0 and 7.0.

This method of entrapping biologically active substances is preferred because it does not involve the use of organic solvents. Use of organic solvents can denature biologically active substances. Materials can be entrapped within the liposomes, as well as or alternatively in one or more of the lipid layers of the phospholipid bilayer. This is typically determined by the hydrophobicity/hydrophilicity of the material to be incorporated as well as the method of preparation.

4.5. MODES OF ADMINISTERING THE

POLYMERIZED LIPOSOMES TO A PATIENT The polymerized liposomes of the present invention are administered by those routes which optimize uptake by mucosa. For example, oral, sublingual, buccal, vaginal, rectal and intranasal are preferred routes of administration. However, topical, transdermal and parenteral delivery may also be used. The most preferred route is oral. Further, the polymerized liposomes are particularly suitable for delivery through mucosal tissue or epithelia. The polymerized liposomes of the invention can be delivered orally in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions and the like. When the dosage unit form is a capsule, it can contain, in addition to the material of the above type, a liquid. If administered topically the liposomes will typically be administered in the form of an ointment or transdermal patch. If administered intranasally the liposomes will typically be administered in an aerosol form, or in the form of drops. Suitable formulations can be found in Remington's Pharmaceutical Sciences, 16th and 18th Eds., Mack Publishing, Easton, PA (1980 and 1990), and Introduction to Pharmaceutical Dosage Forms, 4th Edition, Lea & Febiger, Philadelphia (1985), each of which is incorporated herein by reference. The polymerized liposomes of the present invention are suitable for administration to mammals, including humans, as well as other animals and birds. For example, domestic animals such as dogs and cats, as well as domesticated herds, cattle, sheep, pigs and the like may be treated with the polymerized liposomes of the present invention.

4.5.1. PACKAGING

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

The present invention will be further understood by reference to the following non- limiting examples.

5. EXAMPLE 1 : PREPARATION OF POLYMERIZED LIPOSOMES

Liposomes preparation: 10 mg hGH in phosphate buffer and sucrose stabilizer was rehydrated in 1M Tris, pH 8.5 to a concentration of 2.5 mg/ml of hGH. 3.6 ml hGH solution was used to reconstitute 300 μmoles lyophilized DODPC. This solution was then extruded through 200 run filters, until the mean particle size was 187 ran. 1.1 ml of this solution was removed and labeled as "Unpolymerized". To the remaining 200 μmoles of the mixture were added 94.5 mg of sodium bisulfite and 54.4 mg of potassium persulfate to initiate the polymerization reaction. A 1.1 ml aliquot was removed from the reaction when polymerization reached the 41% level, after 45 minutes at 25 °C. This sample was labeled "Low polymerized". This sample along with the unpolymerized sample were then centrifuged for 15 minutes at 140,000 x g to remove the initiators of the polymerization reaction and any free hGH from the liposomes. The supernatants were removed and the samples resuspended in cold Tris buffer and washed by a second centrifugation. The samples were resuspended to the original volume in 1M Tris, pH 8.5. The polymerization reaction was carried out to completion for the remaining volume (14 hours x 25 °C). This sample, labeled "high polymerized" was washed as described above for the other samples. Mean particle diameter and % polymerization were determined for each sample (Table 1). To verify increased stability conferred by polymerization, each sample was tested for stability to Triton X- 100.

Table 1 : Properties of Unpolymerized, Low, and High Polymerized

Liposome Particles Sample Mean % Stability to Triton X- 100 diameter polymerization²

(ran)¹ l mM 50 mM 100 nM

Unpolymerized 194 7 Yes no No

Low polymerized 199 43 Yes yes No

High polymerized 204 79 Yes yes Yes

1 Determined by laser light scattering (Coulter NS4)

2 Determined by absorbance at 254 ran

3 Samples were treated with the indicated concentration and then mean particle diameter determined by laser light scattering

Most of the liposomes were small unilamellar vesciles, as observed by negative staining electron microscopy. In the unpolymerized samples, some multilamellar clumps were observed. The low and high polymerized samples consisted of primarily small unilamellar vesicles.

Each sample was tested for the presence of hGH by Western blot analysis. The hGH level in each sample was semi-quantitated using densitometric scanning of the bands (Table 2). The lipid concentration was determined in the samples by the method of Bartlett. The percent loading and hGH ipid ratios were calculated using the Western blot data.

Table 2: hGH and Lipid Composition of Polymerized Liposomes

6. EXAMPLE: ORAL DELIVERY OF MAMMALIAN GROWTH HORMONE

Female ICR mice, 6-8 weeks old, were dosed at time 0 with a single 200 μl dose of samples or control solutions as described in Table 3. Bioavailability of hGH was determined by quantitating serum hGH in the mice. Approximately 70 μl of blood were taken at time 0, 0.5, 1, 2, 3, and 4 hours from methoxyfuran anesthetized mice. These blood samples were spun down in a micro-centrifugefuge for approximately 5 minutes to obtain the serum. The serum samples were analyzed for hGH level by an ELISA specific for human GH and not mouse GH. Bioactivity was tested by ability of the serum sample to stimulate proliferation of rat Nb2 cells.

Table 3: Study Groups

No elevation of serum hGH was observed in mice dosed orally with empty polymerized liposomes, Tris buffer, or 100 μg free hGH (groups 4, 6, 7; Table 4). The mean serum hGH level was elevated from 0.5 - 3 h in mice receiving 3 μg hGH subcutaneously (group 5). In mice receiving 40 μg hGH in low polymerized liposomes (group 2), mean serum hGH was elevated from 0.5 - 3 h. The kinetics and magnitude of the response was similar in mice receiving 3 μg subcutaneously and 40 μg orally in low polymerized liposomes. In mice receiving 44 μg hGH in high polymerized liposomes (group 3), the kinetics of serum hGH bioavailability were similar, but the magnitude of the response was lower. In mice receiving 6 μg hGH in unpolymerized liposomes (group 1), a low level of hGH was detected in the serum at 0.5 h only. Peak hGH level was observed at 0.5 h for all groups which responded.

Table 4: Kinetics of hGH Bioavailability After Oral Dosing in Polymerized Liposomes

The results of these studies demonstrate the efficacy of modified polymerized liposomes to provide protection of human growth hormone, which retained its biological activity after the encapsulation and polymerization process. When polymerized liposomes containing human growth hormone were orally administered to mice, human growth hormone displayed its desired biological activity, thus demonstrating the successful application of polymerized liposomes to orally administer biologically active molecules.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

What is claimed is:

1. A liposome comprising a phospholipid bilayer having covalently bonded polymerizable phospholipids, an aqueous core, and mammalian growth hormone.

2. The liposome of claim 1 wherein the polymerizable phospholipid is DODPC.

10 3. The liposome of claim 2 having a degree of crosslinking between 30 and 100 percent.

4. The liposome of claim 1 comprising phospholipids selected from the group consisting of double bond-containing olefinic and acetylenic phospholipids and

15 phospholipids containing thiol groups.

5. The liposome of claim 1 wherein the growth hormone is human growth hormone.

0 6. A method of delivering mammalian growth hormone to an animal which comprises administering to said animal mammalian growth hormone in a polymeric particulate of less than 1 micron in size.

7. The method of claim 6 wherein the polymeric particulate is a polymerized liposome.

8. A method of delivering mammalian growth hormone to an animal which comprises administering to said animal polymerized liposomes comprising a phospholipid

_ „ bilayer having covalently bonded phospholipids, an aqueous core and mammalian growth hormone.

9. The method of claim 8 in which the phopholipid is DODPC.

35 10. The method of claim 8 in which the liposomes has a degree of crosslinking between 30 and 100 present.

11. The method of claim 8 wherein said administration is by an oral, intranasal, vaginal, rectal, sublingual or buccal route.