United States Patent:   6,001,395

Assignee:  Danbiosyst UK Limited (Nottingham, GB)

Filed:  March 30, 1998

The invention provides a composition for delivery of an active agent comprising a plurality of lamellar particles of a biodegradable polymer which is at least in part crystalline, and an active agent adsorbed to at least most of the particles. Preferably the biodegradablepolymer is at least 5% by weight crystalline. Preferred biodegradable polymers are poly(L-lactide) (L.PLA) or copolymers or blends of L.PLA. The particles are especially useful for the immobilization of antigens or allergens for vaccines.

DETAILED DESCRIPTION OF THE INVENTION

The term "biodegradable polymer" includes polymeric systems at least a part of which can degrade into low molecular weight compounds which are known to be involved normally in metabolic pathways. The term also includes polymer systems which can be attacked in the biological milieu so that the integrity of the system, and in some cases of the macromolecules themselves is affected and gives fragments or other degradation by-products which can move away from their site of action, but not necessarily from the body.

The biodegradable polymer used is preferably at least 5 percent by weight crystallisable.

The biodegradable polymer in the particle is preferably at least 5 percent by weight crystalline, more preferably at least 30%, more preferably at least 50%, at least 70% and most preferably at least 90% crystalline.

Whether or not a polymer is crystalline, and the degree of crystallinity, can be determined by methods well known in the art, for example X-ray diffraction methods as applied to polymers or by differential scanning calorimetry.

A preferred polymer is poly(L-lactide) (L.PLA) which is semi-crystalline in nature. The molecular weight of the L.PLA polymer is preferably in the range 2,000 to 100,000.

The polymer may a mixture of L.PLA with another biodegradable polymer or with a biocompatible but non-degradable polymer, either as a copolymer or as a blend of polymers. In either case, the resulting mixture should still be at least in part crystalline and preferably at least 5% by weight crystalline. The content of a non-crystallisable or non-crystalline polymer component should therefore be limited as necessary.

Suitable copolymers are copolymers of L.PLA and other poly(.alpha.-hydroxy acids) such as DL lactide or glycolide (eg. PLG), crystallisable copolymers of lactic acid and lactone, copolymers of L-lactide and poly(ethylene glycol) [PEG], copolymers of L-lactide and .alpha.-amino acids (polydepsipeptides), polyanhydrides, and polyorthoesters.

Suitable blends of L.PLA with other polymers include other poly(.alpha.-hydroxy acids) such as poly(DL lactide co-glycolide), PEG, copolymers of polyethylene oxide and polypropylene oxide (PEO-PPO), polydepsipeptides, polyorthoesters, polyanhydrides, polyphosphazene and copolymers of acrylic and methacrylic acid esters (EUDRAGIT.TM.).

Other biodegradable synthetic polymers potentially useful for preparing lamellar substrates include copolymers of .alpha.-hydroxy acids, .alpha.-amino acids (polydepsipeptides), polyhydroxybutyric acid, copolymers of lactic acid and lactone, copolymers of lactic acid and PEG, copolymers of hydroxybutyrate and hydroxyvalerate, polyethylene terephthalate, polyphosphazenes, polycaprolactone, polyorthoesters, polyanhydrides and copolymers thereof or blends of such polymers.

By "lamellar" it is meant that the particles comprise thin plates or layers; lipsomes are not lamellar particles of the invention.

It is preferred if the lamellar particles are irregularly shaped as may be formed using some of the methods in the Examples.

The lamellar particles are often "lozenge-shaped", and may be present in the composition as discrete lamellar particles, or as sheave-like, polyhedral particles formed by lamellae which are coalesced together along a common plane. The term "lamellar particle" is used to include both possibilities. The lamella particle thickness is in the range 50 nm to 80 .mu.m, but is preferably in the range 50 to 500 nm. The lower end of the range corresponds to single lamellar particles that have substantially flat surfaces and the upper end corresponds to stepped or coalesced lamellar particles. The surface of the lamella often exhibits a stepped topography which is typical of polymer crystal growth.

The plan dimensions of the lamellar particles, both width and length, are typically in the range 0.5 .mu.m to 80 .mu.m, preferably 1 .mu.m to 40 .mu.m, more preferably 1 .mu.m to 10 .mu.m and most preferably 3 .mu.m to 5 .mu.m. The aspect ratio, the ratio of length to width, is in the range 160:1 to 1:1, more preferably 2.5:1 to 3:2.

The particle morphology can be measured using scanning electron microscopy and atomic force microscopy.

The term "active agent" is used herein to include any agent which it may be desired to administer to the human or animal body for any purpose, including therapeutic, pharmaceutical, pharmacological, diagnostic, cosmetic and prophylactic agents and immunomodulators.

Active agents include growth hormone such as bone morphogenic protein (BMP), insulin, interferons (alpha, beta, gamma), erythropoietin, colony stimulating factor such as granulocyte macrophage colony stimulating factor (GM-CSF), interleukin 2 and 12, parathyroid hormone, leutenising hormone releasing hormone, calcitonin, heparin, somatropin and various analogues thereof. Nucleic acid, which includes oligonucleotides as small as 10 nucleotides in length, is also included in the term "active agent".

The active agent is preferably a vaccine, antigen or allergen or DNA.

Tetanus toxoid and influenza virus are especially preferred.

Antigens include polypeptides, proteins, glycoproteins and polysacchraides that are obtained from animal, plant, bacterial, viral and parasitic sources or produced by synthetic methods. The term antigen includes any material which will cause an antibody reaction of any sort when administered. Such antigens can be administered by injection or to various mucosal sites (nasal, oral, vaginal, rectal, colonic).

Vaccines for the treatment of allergens and for auto immune diseases are well described in the prior art. For example in autoimmune disease it has been suggested that the slow administration of essential factors can be beneficial. Such factors can include insulin for the treatment of diabetes and collagen for treating rheumatoid arthritis.

The active agent may also be a polypeptide, peptide or protein, a carbohydrate or an oligonucleotide such as DNA, including growth hormone, insulin, interferons (alpha, beta, gamma), interleukins erythropoietin, colony stimulating factors, growth factors, parathyroid hormone, leutenizing hormone releasing hormone, calcitonin, heparin, somatostatin and various analogues thereof.

The active agent is adsorbed onto or into the lamellar particles after preparation thereof by admixing the agent with the particles.

If the active agent is a peptide or protein drug, the lamellar particle, with the adsorbed active agent, is preferably encapsulated or enteric coated with polymer such as poly (D,L-lactide co-glycolide) (PLG) or a EUDRAGIT.TM. polymer, prior to oral administration.

Adjuvants immobilised on lamellae may be co-administered with immobilised antigens by mixing two populations of lamellae. Adjuvants include dextran sulphate, synthetic analogies of mycobacterial fragments (muramyl dipeptide, lauroyltetrapeptide), muramyl tripeptide phosphatidylethanolamine (MTP-PE), monophosphoryl lipid derived from bacterial endotoxin (MPL A), chitosan and its oligomers.

The lamellar surface may be modified to improve interaction of the substrate with bioactive agents.

The lamellar surface may be modified to improve interaction of the substrate with host cells and tissue.

The lamellar surface may be modified to improve interaction of the substrate with bioactive agents.

Modification of the surface characteristics of the lamellae may be desirable so as to attract particular molecules, ligands or to modulate the interaction of the lamellae with host cells and tissues.

In one instance, chances to the immonomodulatory character of the lamellae may be desirable to stimulate a particular type of response. TH1 lymphocytes are predominantly associated with cell mediated immunity (essential for recognition and killing of virus or bacteria infected cells) producing cytokines such as IL-2 and IFN-.gamma.. TH2 cells are associated with antibody production and humoral immune responses producing cytokines such as IL-4 and IL-10. Co-administration of cytokine-modified lamellae and antigens may be useful for conferring protection against infectious agents of viral and bacterial origin.

The lamellae may be modified by a molecule recognised by, and having an affinity for, a particular cellular receptor in the human or animal body to provide a targeting potential. The attachment of lectins or monoclonal antibodies to the surface of lamellae could be useful for improving interaction with the mucosal surface of the gastrointestinal tract.

Polymeric surface modifiers may be attached to the lamellae by adsorption, by physical chain entanglement and interpenetration with the surface or by chemical grafting.

The surface modifier may be added to the non-solvent used for precipitation of the lamellae in process stage (b) (see below). Alternatively, the solid substrate may be treated after production.

Surface modifying polymers include the block copolymers based on polyethylene oxide and polypropylene oxide (POLOXAMERS.TM., POLOXAMINES.TM.) and tetra-functional block copolymers derived from the sequential addition of propylene oxide and ethylene oxide to ethylene diamine (POLOXAMINES.TM.), polyvinylalcohol, polyvinylpyrrolidone, sorbitan esters such as sorbitan monostearate (SPAN 60.TM.), polysorbates (TWEEN.TM.), polyoxyethylene fatty esters, phospholipids such as lecithin, lysophosphatidylcholine (LPC), fatty acids, stearic acid, stearates and their derivatives with for example, polyoxyethylene.

Other polymers potentially useful for surface modification include the polyamidoamines, polymalic acid, polyamino acids, poly(L-lysine), poly(L-glutamic acid), poly(L-aspartic acid) and their copolymers, polymers of acrylic acid (CARBOPOL.TM.) and copolymers based on maleic anhydride.

Other categories of surface modifying agents include the anionic detergents, sodium salts of deoxycholic acid, glycocholic acid or sodium lauryl sulfate (SDS), cationic detergents and non-ionic detergents such as polyoxyethylene ether, which is sold under the trademark TRITON.TM. (Union Carbide Ltd.).

Surface modifying polymers may also be selected from the group comprising protein and polysaccharides whether natural or synthetically made and their derivatives such as conjugates with polyethylene glycol PEG.

The term protein is intended to include peptides, polypeptides glycoproteins, metalloproteins, lipoproteins and sub-units or fragments thereof. Proteinaceous materials include albumin, gelatin, collagen, glycoproteins and their derivatives with, for example, polyethylene glycol. Methods for preparing conjugates of PEG and protein have been described by Nucci et al in Advances in Drug Delivery Reviews, 6 113-151 (1991).

The term polysaccharides includes polymers of amino sugars.

Examples of polysaccharides useful as surface modifiers include dextran, xanthan, chitosan, chitosan lactate, chitosan glutamate, pectin, dextrin, maltodextrin, hyaluronic acid, cellulose, starch, hydroxyethyl starch, pullulan, inulin, alginates, heparin and heparin-like synthetic polymers and their respective derivatives. Conjugates of PEG and polysaccharides for example have been described by Duval et al in Carbohydrate Polymers, 15 233-242 (1991).

The surface modifier may be a mixture of two or more of the types of polymer listed above.

The lamellae may be adapted for injection either intramuscularly, intravenously, subcutaneously, intraarticularly or intraperitoneally. They will generally be sterile and pyrogen-free. The lamellae may be adapted for administration to the dermal or epidermal layer of the skin by injection or needleless injector system. The lamellae may also be adapted for administration to mucosa such as the nose, the gastrointestinal tract, the colon, the vagina and the rectum.

The lamellar particles of the invention can be formulated in ways well known in the art.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the lamellar particles to which the active agent has been adsorbed with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the said lamellar particles with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose container, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question.

The amount of the lamellar particle composition of the invention to be administered to a patient may be determined in relation to the amount of active agent to be administered and to the amount of active agent adsorbed on the said particle and to the way in which the active agent becomes available in the patient following administration of the composition.

Suitably, the amount of the composition administered would be that which contains between 1% and 1000% of the normal amount of the active agent administered to the patient when administered in a conventional way. Preferably, the amount contains between 10% and 500% of the normal amount of the active agent; more preferably between 20% and 80%.

For nasal administration, the vaccines can be administered as a fine suspension using a spray device or if in the form of a powder using a powder device or nasal insufflator. Such devices are well familiar to those skilled in the art. Formulations for the gastrointestinal tract can be administered as suspensions or formulated as tablets and capsules or into compressed or extruded pellets.

For surface adsorbed antigens that are sensitive to the acid conditions in the stomach the delivery system can be protected by an enteric polymer familiar to those skilled in the art of formulation. The enteric polymer can be used to coat the dosage form. Vaginal systems suitable for delivery include gels and vaginal suppositories. Rectally administrated vaccines can be given as enemas or incorporated into suppositories.

A method for making the compositions described herein includes the following steps:

a) dissolving the polymer in a solvent;

b) stirring the polymer solution vigorously and adding a non-solvent for the polymer;

c) evaporating the solvent from the mixture of step (b); and

d) admixing an active agent with the thus formed particles.

It has been found that by using crystallisable polymers, the above precipitation method will form lamellar particles, in contrast to the prior art spherical particles formed using amorphous polymers.

The solvent used, which is a "poor solvent" for the biodegradable polymer, is preferably acetone, ethyl acetate, xylene or dioxane, although any other suitable solvent may be used. Heat may need to be applied to dissolve the polymer in the solvent. The non-solvent is preferably water, methanol or ethanol.

By "poor" solvent we mean a solvent in which the polymer has a low or negligible solubility so that the polymer will come out of solution as a (partly) crystalline material (precipitation process). The solvents and non-solvents for polymers can be found in standard texts (eg. see Fuchs, in Polymer Handbook, 3rd Edition) and Deasy, Microencapsulation and Related Drug Processes, 1984, Marcel Dekker, Inc., New York.

The ability of a polymer to dissolve in a solvent can be estimated using the Cohesive Energy Density Concept (CED) and related solubility parameter values as discussed by Deasy and to be found in detail in the article by Grulke in Polymer Handbook.

Thus a person skilled in the art will be able to select a "poor" solvent to give the required precipitation of the lamellar material.

The lamellar particles may also be made by a crystallization method in which the polymer is dissolved in the solvent as before, cooled and left to crystallize. The particles can then be harvested by filtration. The thus-formed particles are then admixed with the active agent to form a composition according to Claims 1 to 12.

It has been found that the lamellar particles of the invention provide a greatly increased adsorption of active agents compared with prior art spherical particles formed from amorphous polymers. The adsorption of active agents onto lamellar particles also avoids the disadvantages found with prior art microencapsulated vaccines based on PLG. These include avoidance of exposure to high shear forces and solvents and acid degradation products produced by PLG which may denature certain antigens. Furthermore, the lamellar particles have been found to have much greater retention of the antigen over long time periods in vitro. It is thought that the irregular lamellar form of the particles may function as an immunomodulator and stimulate the immune system.

When used in animal studies to measure the immune response to absorbed influenza virus, the lamellar particles of the invention resulted in 60% of challenged mice being protected.

Claim 1 of 18 Claims

1. A composition for delivery of an active agent comprising

a plurality of lamellar particles comprising a biodegradable polymer which is at least partially crystalline, and

an active agent adsorbed to at least a majority of the particles.

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