WO2006028996A2 - Emulsan-alginate microspheres and methods of use thereof - Google Patents

Abstract

The present invention relates generally to emulsan-alginate compositions and to methods of use. Methods are provided for use of emulsan-alginate compositions as drug delivery vehicles. In addition, methods are provided for use of emulsan-alginate compositions in applications requiring protein adsorption (e.g removal of protein toxins from food products or other products and solutions). The compositions of the invention avoid some of the problems seen in the art with alginate microspheres or particles.

Description

EMULSAN-ALGINATE MICROSPHERES AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This Application claims the benefit of under 35 U.S.C. §119(e) of U.S. provisional Patent Application No. 60/607,005, filed September 03, 2004; U.S. provisional Patent Application No. 60/607,611, filed September 07, 2004; and U.S. provisional Patent Application No. 60/643,807, filed January 14, 2005.

GOVERNMENT SUPPORT

[002] This invention was supported by USDA Grant number 99-355504-7915 and

USDA-MASR-2002-01549, the government of the United States has certain rights thereto.

FIELD OF THE INVENTION

[003] The present invention relates generally to emulsan-alginate compositions and to methods of use. Methods are provided for use of emulsan-alginate compositions as drug delivery vehicles. In addition, methods are provided for use of emulsan-alginate compositions in applications requiring protein adsorption (e.g. removal of protein toxins from food products or other products and solutions).

BACKGROUND OF THE INVENTION

[004] Protein adsorption has found extensive scientific and technological utility in many areas of science and engineering. For example, in downstream processing in many industrial processes, such as the purification of recombinant proteins and antibodies, it is critical to remove toxins or other factors present in cellular extracts (Walsh and Headon, 1994). Therapeutically, protein adsorption is studied for extracorporeal treatment of patients to remove serum proteins to improve hemodynamics and to restore leukocyte responsiveness in septic shock (Hanasawa, 2002). In human and animal nutrition, sequestering protein toxins in food processing is critical to health and safety (Walsh and Headon, 1994). [005] In recent years, the development of systems able to transport, capture or deliver biologically active molecules has accelerated. For example, biocompatible hydrogels have become an active area of research for encapsulation of living cells, drug delivery, and implants (Dornish et al., 2001).

[006] Hydrogels and polymer-based systems have gained particular interest as providing a means for sustained release of therapeutic agents. For example, Great Britain Patent No, 1,388,580 discloses the use of hydrogels for sustained-release of insulin. Sustained-release formulations have also included the use of a variety of biodegradable and non-biodegradable polymers (e.g. poly(lactide-co-glycolide)) (see e.g., Wise et al., Contraception, 1 :227-234 (1973); and Hutchinson et al., Biochem. Soc. Trans., 13:520- 523 (1985)), and a variety of techniques are known by which therapeutic agents, e.g. proteins, can be incorporated into polymeric microspheres (see e.g., U.S. Patent No. 4,675,189 and references cited therein).

[007] Alginate gels have also been used in sustained release formulations. In general, alginates are well known, naturally occurring, anionic, polysaccharides comprised of 1 ,4-linked-β-D-mannuronic acid and α-L-guluronic acid (Smidsrod, et al, Trends in Biotechnology, 8:71-78 (1990) and Aslani, P. et al., J. Microencapsulation, 13/5: 601-614 (1996)). Alginates typically vary from 70% mannuronic acid and 30% guluronic acid, to 30% mannuronic acid and 70% guluronic acid (Smidsrod, supra). Alginic acid is water insoluble whereas salts formed with monovalent ions like sodium, potassium and ammonium are water soluble (McDowell, R. H., "Properties of Alginates" (London, Alginate Industries Ltd, 4th edition 1977). Polyvalent cations are known to react with alginates and to spontaneously form gels.

[008] Alginates have a wide variety of applications such as food additives, adhesives, pharmaceutical tablets and wound dressings. Alginates have also been recommended for protein separation techniques. For example, Gray, CJ. et al., in Biotechnology and Bioengineering, 31 : 607-612 (1988), entrapped insulin in zinc/calcium alginate gels for separation of insulin from other serum proteins. [009] Alginate gels have been used in a variety of drug delivery systems. U.S. Patent No. 4,789,550 discloses the use of polylysine coated alginate microcapsules for delivery of protein by encapsulating living cells. U.S. Patent No. 4,695,463 discloses an alginate based chewing gum delivery system and pharmaceutical preparations. Further, alginate beads have been used for controlled release of various proteins such as: tumor necrosis factor receptor in cation-alginate beads coated with polycations (Wee, S, F, Proceed. Intern. Symp. Control, ReI. Bioact. Mater., 21 : 730-31 (1994)); transforming growth factor encapsulated in alginate beads (Puolakkainen, P.A. et al, Gastroenterology, 107: 1319-1326 (1994)); angiogenic factors entrapped in calcium-alginate beads (Downs, E. C, et al, J. of Cellular Physiology, 152: 422-429 (1992)); albumin entrapped in chitosan-alginate microcapsules (Polk, A. et al, J. Pharmaceutical Sciences, 83/2: 178-185 (1994)), or chitosan-calcium alginate beads coated with polymers (Okhamafe, A. 0, et al., J. Microencapsulation, 13/5: 497-508 (1996)); hemoglobulin encapsulated with chitosan- calcium (Huguet, M. L. et al., J, Applied Polymer Science, 51 1427.1432 (1994) and Huguet, M. L, et al., Process Biochemistry, 31 : 745-751 (1996)); and interleukin-2 encapsulated in alginate-chitosan microspheres (Liu, L.S, et al., Proceed. Intern. Symp. Control. ReI. Bioact. Mater, 22: 542-543 (1995)). In addition, the use of alginates alone, or alginates coated with other biodegradable polymers, for controlled release of polypeptide compositions or cation precipitates thereof are described in PCT publications WO 96/00081 , PCT WO 95/29664, PCT WO 96/03116 and PCT WO 98/46211. [010] Systems using alginate gel beads, or alginate/calcium gel beads, to entrap proteins suffer from lack of any sustained-release effect due to rapid release of the protein from the alginate beads Liu, L. et al., J. Control. ReL, 43: 65-74 (1997). To avoid such rapid release, a number of the above systems attempted to use polycation polymer coatings (e.g., polylysine, chitosan) to retard the release of the protein alginate beads. See, e.g., Wheatley, M. A. et al, J. Applied Polymer Science, 43: 2123-2135 (1991); Wee, S.F, et al. supra; Liu, L.S. et al. supra; Wee, S.F. et al, Controlled Release Society, 22: 566- 567 (1995) and Lim, et al. supra.

[Oi l] Unfortunately, such coated alginate beads have had limited success. Often the formulations are cytotoxic due to the polycations (Huguet, M,L, et al,, supra; Zimmermann, Ulrich, Electrophoresis, 13: 269.(1992); Bergmann, P. et al., Clincial Science, 67: 35.(1984)). In addition, polycations are prone to oxidation and ionic interactions between the protein and polycations can result in loss of protein activity or can cause protein instability. Further, beads with polycation coatings tend not to be erodible and build up in the body.

[012] Accordingly, there is a need in the art to develop drug delivery formulations which achieve a better means of delivery or sustained-release for clinical application. SUMMARY OF THE INVENTION

[013] The present invention provides a composition which comprises emulsan and alginate. The composition is preferably in the form of a microsphere (e.g. beads of 1 μm to 5 mm in diameter, preferably 10 to 1 ,000 micron diameter beads wherein the size range is dependent upon application). The composition can adsorb, carry, and deliver molecules and at the same time avoids some of the problems seen in the art with alginate microspheres or particles. It has been discovered that the emulsion composition of the present invention can carry non-activated proteins and enzymes, enzyme inhibitors and other molecules without altering the biological activation of the molecules. In one embodiment, release from the emulsan carrier can be the result of enzymatic processes.

[014] In one embodiment the composition comprising emulsan and alginate further comprises an agent. Agents of the composition can be, for example, a protein, peptide, enzymes, nucleic acid, PNA, aptamer, antibody, small molecule (e.g., drugs), or dyes.

[015] In another embodiment, the composition of the invention further comprises a pharmaceutically acceptable carrier, dilutent or adjuvant.

[016] The invention further provides for a method of treating an indication comprising administering to a patient in need thereof a composition comprising emulsan, alginate and an agent, i.e., a therapeutic agent.

[017] A method for removing protein contaminants from a solution suspected of containing protein contaminants comprising contacting said solution with a composition comprising emulsan and alginate is also provided.

[018] In one embodiment, the contaminant to be removed is a bacterial toxin.

[019] In one embodiment, the solution suspected of containing the protein contaminant is a food product.

[020] The invention further provides for a method for producing a pharmaceutical formulation for controlled release of at least one therapeutic agent, the method comprising: contacting a microsphere comprising emulsan and alginate with at least one therapeutic agent.

[021] In one embodiment, the therapeutic agent used in the method for producing a pharmaceutical formulation is selected from the group consisting of a protein (e.g., enzyme), a peptide, nucleic acid, PNA, aptamer, antibody, and small molecule (e.g., drug).

[022] In one embodiment, the pharmaceutical formulation is biodegradable. [023] In one embodiment, the pharmaceutical formulation further comprises a targeting agent that specifically targets said device to a specific cell or tissue type. The targeting agent can be a sugar, peptide, and fatty acid.

BRIEF DESCRIPTION OF THE DRAWINGS

[024] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the objects, advantages, and principles of the invention. [025] Figures IA to ID show environmental scanning electron microscopy images of alginate (Fig. IA and Fig. 1C) and emulsan-alginate microspheres (Fig. IB and Fig ID).

[026] Figure 2 shows the kinetics of BSA adsorption by emulsan-alginate (•) and alginate (τ)microspheres.

[027] Figures 3 A to 3C show the effect of temperature (Fig. 3A), pH (Fig. 3B), and ionic strength (Fig. 3C) on BSA adsorption by emulsan-alginate (•, cross bars) and alginate (T , empty bars) microspheres.

[028] Figure 4 shows the relative protein adsorption of bacterial cell supernatants to emulsan-alginate (open bars) and alginate (cross bars) microspheres. [029] Figure 5 shows a table depicting predicted protein adsorption on the emulsan- alginate (Emulsan) or alginate microspheres in equilibrium using the Langmuir and Freundlich models.

[030] Figure 6 shows a_ table depicting the dynamic absorption of BSA on emulsan- alginate (Emulsan) or alginate microspheres using 2^nd order Lagergren and Intraparticle diffusion models.

[031] Figure 7 shows a table indicating the standard free energy changes of emulsan- alginate (Emulsan) or alginate microsphere BSA adsorption with temperature. [032] Figure 8 shows a table indicating changes of entropy contribution of BSA adsorption on emulsan-alginate (Emulsan) or alginate microspheres associated with increase of temperature.

[033] Figure 9 shows a table depicting a comparison of Langmuir constants of BSA adsorption on emulsan-alginate, alginate, BRX-Q or PHEMA found in the literature. [034] Figures 1OA & 1OB show emulsan (10A), and alginate (10B) microspheres containing adsorbed azo-BSA. [035] Figure 1 1 shows release of azo-BSA and sulfanilic acid adsorbed by microspheres using Candida rugosa lipase. Symbols: D and O, sulfanilic acid and azo- BSA release from ECM and ACM respectively; O and V, sulfanilic acid release from ECM and ACM respectively, Δ and sulfanilic acid and azo-BSA release from ECM and ACM incubated without lipase respectively.

[036] Figure 12 shows cleavage of sulfanilic acid from azo-BSA adsorbed on microspheres. Symbols: O, ECM; V, ACM; D and O, ECM and ACM incubated with subtilisin previously inhibited with DFP respectively; Δ and , ECM and ACM incubated with subtilisin.

[037] Figure 13 shows lanes: 1, molecular weight markers; 2, subtilisin inhibited with DFP; 3, substrate, casein, at zero time; 4, 6 and 8, ACM supernatant incubated with subtilisin at 10, 30 and 50 minutes respectively; 5, 7 and 9, ECM supernatant incubated at 10, 30 and 50 minutes.

[038] Figure 14 shows Stability of lipase adsorbed on ECM (O), and ACM (V) respectively.

[039] Figure 14 shows Activity of subtilisin activity adsorbed on ECM (O), and ACM (V) respectively.

DETAILED DESCRIPTION OF THE INVENTION

[040] Alginate is a linear polysaccharide of β-D-mannuronic and α-L-guluronic acids, which can form hydrogels in the presence of calcium and other bivalent cations. Alginate gels are considered safe and currently used in many biotechnology applications (Dornish et al., 2001). However, alginate gels are unstable in the presence of cation chelating agents such as citrate, lactate, phosphate, or tartrate and/or competing cations such as sodium or potassium that are commonly present in biological fluids (Smidsrød and Skjak-Brask, 1990). In order to prevent microsphere swelling, polymers such as chitosan, poly-L-lysine, polyacrylates, and others have been added to stabilize calcium- alginate gels (Takahashi et al., 1989; Thu et al., 1996). Recently, several studies reported the use of chitosan in alginate gels. Chitosan, a cationic copolymer of N- acetylglucosamine and glucosamine, is a water-soluble and biodegradable polymer often used in pharmaceutical industries as an excipient, because of is biocompatibility (Dornish et al., 2001). However, the degree of acetylation of chitosan, which correlates with its biological and chemical properties, depends on the chemical treatment of chitin by alkaline N-deacetylation. In addition, the major source of chitin is the exoskeleton of crustaceans, and when this source is combined with variable storage and treatment of the material prior to processing, variable material properties are often an issue (Dornish et al, 2001).

[041] Emulsan is an amphipathic lipoheteropolysaccharide of IxIO⁶ Da produced by Acinetobater venetianus strain RAG-I . This polymer is released into the medium in large amounts during stationary phase growth. The main chain consists of three amino sugars: D-galactosamine, D-galactosamine uronic acid, and 2,4-diamino-6-deoxy-D- glucosamine, and the amphipathic properties of the polymer are conferred by fatty acid side chains appended via N- and O-acyl bonds to the sugar backbone (Belsky et al, 1979). In our recent studies we have demonstrated that emulsans provide unique and important attributes in terms of macrophage activation responses related to proinflammatory cytokines and as delivery agents for vaccines (Panilaitis et al, 2002). Furthermore, we have recently shown that both physiological and genetic manipulation of the biosynthetic pathway the structure and function of these complex polymers can be altered and controlled for specific properties (Gorkovenko et al., 1999; Johri et al., 2002; Blank et al., 2002). Specific properties that can be controlled include solution behavior such as emulsifying and surface tension features (Zhang et al., 1997) and biological functions such as cell activation (Panilaitis et al., 2002). These types of structure- function controls depend on the nature of the fatty acids present on the polysaccharide backbone and their degree of substitution.

[042] Of specific interest relevant to the present study was the observation that when emulsan is produced and secreted by the bacterium, in the native state is contains or carries with it up to 23% by weight protein (Zuckerberg et al, 1979). This protein is non- covalently bound and thus normally a hot phenol extraction or proteolytic digestion process is required to remove these 'contaminants' in order to purify the emulsan for subsequent analytical characterization or for solution and cellular studies. The proteins bound to emulsan include an esterase (Bach et al, 2003), while the other components have not yet been identified. This same capacity to adsorb proteins was considered a potential benefit that has been explored in the present study.

[043] Based on the history of use of alginates in coacervates, structures defined as heterogeneous macromolecular structures without precise stoichiometric relationship between components (Oparin et al, 1963), and the innate ability of emulsans to adsorb proteins, the potential for combining these two polymers into protein adsorption vesicles was studied in the present work. In addition, emulsans have innate biological activation features correlated to structure that impart additional value in some therapeutic applications.

[044] Accordingly, the present invention provides for compositions comprising emulsan and alginate that can be used in a variety of applications such as drug delivery systems or purification techniques (e.g. use in filter like devices to remove contaminants and/or toxins from food products or biological products).

[045] Emulsan-alginate microspheres can be made by mixing emulsan and alginate at various concentrations including, but not limited to, the concentrations set forth in the examples below. Alginate from 10 to 99 weight percent and emulsan from 90 to 1 weight percent can be used. The microsphere mechanical integrity improves as alginate content increases while a higher content of emulsan leads to improved adsorption or carrying capcity of the microspheres.

[046] Emulsan production and purification are described in Johri et. al., 2002 and

Blank et al. 2002, which are herein incorporated by reference. Methods for making alginate gels and alginate beads are described in PCT publication WO 98/46211, which is also herein incorporated by reference.

[047] In one preferred embodiment, emulsan-alginate beads are prepared as described in Example 1. For example, for the formation of microspheres, a solution of alginate and emulsan can be pumped into an aqueous solution of CaCl₂ under conditions of continuous stirring. After incubation in the calcium solution at room temperature for 1 to 48 hours, the microspheres can be filtered on filter paper (e.g. Whatman #1) then stored in 70% ethanol until use. . .

[048] In one preferred embodiment, the emulsan-alginate compositions of the invention are used in methods of drug delivery.

[049] Therapeutic agents can be adsorbed to emulsan-alginate microspheres as described herein (Example 1 and Example 2) or by any means known to those skilled in the art.

[050] The variety of different therapeutic agents that can be used in conjunction with the formulations of the present invention is vast and includes small molecules, proteins, peptides and nucleic acids. In general, therapeutic agents which may be administered via the invention include, without limitation: anti-infectives such as antibiotics and antiviral agents; chemotherapeutic agents (i.e. anticancer agents); anti-rejection agents; analgesics and analgesic combinations; anti-inflammatory agents; hormones such as steroids; growth factors (bone morphogenic proteins (i.e. BMP's 1-7), bone morphogenic-like proteins (i.e. GFD-5, GFD-7 and GFD-8), epidermal growth factor (EGF), fibroblast growth factor (i.e. FGF 1-9), platelet derived growth factor (PDGF), insulin like growth factor (IGF-I and IGF-II), transforming growth factors (i.e. TGF-β-III), vascular endothelial growth factor (VEGF)); anti-angiogenic proteins such as endostatin, and other naturally derived or genetically engineered proteins, polysaccharides, glycoproteins, or lipoproteins. Growth factors are described in The Cellular and Molecular Basis of Bone Formation and Repair by Vicki Rosen and R. Scott Thies, published by R. G. Landes Company, hereby incorporated herein by reference.

[051] Additionally, the emulsan-alginate compositions of the present invention can be used to deliver any type of molecular compound, such as, pharmacological materials, vitamins, sedatives, steroids, hypnotics, antibiotics, chemotherapeutic agents, prostaglandins, and radiopharmaceuticals. The delivery system of the present invention is suitable for delivery of the above materials and others including but not limited to proteins, peptides, nucleotides, carbohydrates, simple sugars, cells, genes, anti¬ thrombotics, anti-metabolics, growth factor inhibitor, growth promoters, anticoagulants, antimitotics, fibrinolytics, enzymes and proenzymes, anti-inflammatory steroids, and monoclonal antibodies.

[052] In one embodiment, the therapeutic agent can be bound to the microspheres such that the agent can be activated and/or released by specific enzymes. Such enzymes include, for example, oxidoredutases, transferases, lyases, isomerases, ligases and hydrolases (e.g. subtilisin and lipase), peroxidase and glycosidases. [053] The pharmaceutical formulation of the present invention may also have a targeting ligand. Targeting ligand refers to any material or substance which may promote targeting of the pharmaceutical formulation to tissues and/or receptors in vivo and/or in vitro with the formulations of the present invention. The targeting ligand may be synthetic, semi-synthetic, or naturally-occurring. Materials or substances which may serve as targeting ligands include, for example, proteins, including antibodies, antibody fragments, hormones, hormone analogues, glycoproteins and lectins, peptides, polypeptides, amino acids, sugars, saccharides, including monosaccharides and polysaccharides, carbohydrates, vitamins, steroids, steroid analogs, hormones, cofactors, and genetic material, including nucleosides, nucleotides, nucleotide acid constructs, peptide nucleic acids (PNA), aptamers, and polynucleotides. Other targeting ligands in the present invention include cell adhesion molecules (CAM), among which are, for example, cytokines, integrins, cadherins, immunoglobulins and selectin. The pharmaceutical formulations of the present invention may also encompass precursor targeting ligands. A precursor to a targeting ligand refers to any material or substance which may be converted to a targeting ligand. Such conversion may involve, for example, anchoring a precursor to a targeting ligand. Exemplary targeting precursor moieties include maleimide groups, disulfide groups, such as ortho-pyridyl disulfide, vinylsulfone groups, azide groups, and iodo acetyl groups.

[054] The emulsan-alginate compositions of the present invention, e.g., microspheres, may be used in controlled release systems. The amount of therapeutic agent adsorbed to emulsan-alginate compositions can be controlled by temperature and/or ionic strength. Release of adsorbed drug can be controlled by, for example, pH and or enzymatic activity.

[055] Controlled release permits dosages to be administered over time, with controlled release kinetics. In some instances, delivery of the therapeutic agent is continuous to the site where treatment is needed, for example, over several weeks. Controlled release over time, for example, over several days or weeks, or longer, permits continuous delivery of the therapeutic agent to obtain optimal treatment. The controlled delivery vehicle is advantageous because it protects the therapeutic agent from degradation in vivo in body fluids and tissue, for example, by proteases. [056] Controlled release from the pharmaceutical formulation may be designed to occur over time, for example, for greater than about 12 or 24 hours. The time of release may be selected, for example, to occur over a time period of about 12 hours to 24 hours; about 12 hours to 42 hours; or, e. g., about 12 to 72 hours. In another embodiment, release may occur for example on the order of about 2 to 90 days, for example, about 3 to 60 days. In one embodiment, the therapeutic agent is delivered locally over a time period of about 7-21 days, or about 3 to 10 days. In other instances, the therapeutic agent is administered over 1 ,2,3 or more weeks in a controlled dosage. The controlled release time may be selected based on the condition treated. For example, longer times may be more effective for wound healing, whereas shorter delivery times may be more useful for some cardiovascular applications.

[057] Controlled release of the therapeutic agent from the emulsan-alginate composition in vivo may occur, for example, in the amount of about 1 ng to 1 mg/day, for example, about 50 ng to 500 pg/day, or, in one embodiment, about 100 ng/day. Delivery systems comprising therapeutic agent and a carrier may be formulated that include, for example, 10 ng to 1 mg therapeutic agent, or in another embodiment, about 1 ug to 500 ug, or, for example, about 10 ug to 100 ug, depending on the therapeutic application. [058] The emulsan-alginate delivery vehicle (e.g. microsphere ) may be administered by a variety of routes known in the art including topical, oral, parenteral (including intravenous, intraperitoneal, intramuscular and subcutaneous injection as well as intranasal or inhalation administration) and implantation. The delivery may be systemic, regional, or local. Additionally, the delivery may be intrathecal, e. g., for CNS delivery. For example, administration of the pharmaceutical formulation for the treatment of wounds may be by topical application, systemic administration by enteral or parenteral routes, or local or regional injection or implantation. The emulsan-alginate vehicle may be formulated into appropriate forms for different routes of administration as described in the art, for example, in "Remington: The Science and Practice of Pharmacy", Mack Publishing Company, Pennsylvania, 1995, the disclosure of which is incorporated herein by reference.

[059] The controlled release vehicle may include excipients available in the art, such as diluents, solvents, buffers, solubilizers, suspending agents, viscosity controlling agents, binders, lubricants, surfactants, preservatives and stabilizers. The formulations may include bulking agents, chelating agents, and antioxidants. Where parenteral formulations are used, the formulation may additionally or alternately include sugars, amino acids, or electrolytes.

[060] Excipients include polyols, for example of a molecular weight less than about 70,000 kD, such as trehalose, mannitol, and polyethylene glycol. See for example, U. S. Patent No. 5,589,167, the disclosure of which is incorporated herein. Exemplary surfactants include nonionic surfactants, such as Tween surfactants, polysorbates, such as polysorbate 20 to 85, etc., and the poloxamers, such as poloxamer 184 or 188, Pluronic (r) polyols, and other ethyl ene/polypropylene block polymers, etc. Buffers include Tris, citrate, succinate, acetate, or histidine buffers. Preservatives include phenol, benzyl alcohol, metacresol, methyl paraben, propyl paraben, benzalconium chloride, and benzethonium chloride. Other additives include carboxymethylcellulose, dextran, and gelatin. Stabilizing agents include heparin, pentosan polysulfate and other heparinoids, and polyvalent cations such as magnesium and zinc.

[061] The pharmaceutical formulation of the present invention may be sterilized using conventional sterilization process such as radiation based sterilization (i.e. gamma- ray), chemical based sterilization (ethylene oxide), autoclaving, or other appropriate procedures. Preferably the sterilization process will be with ethylene oxide at a temperature between 52 - 55° C for a time of 8 or less hours. After sterilization the formulation may be packaged in an appropriate sterilize moisture resistant package for shipment.

[062] Therapeutic uses depend on the biologically active agent used. One skilled in the art will readily be able to adapt a desired biologically active agent to the present invention for its intended therapeutic use. Therapeutic uses for such agents are set forth in greater detail in the following publications hereby incorporated by reference including drawings. Therapeutic uses include but are not limited to uses for proteins like interferons (see, U.S. Patent Nos. 5,372,808; 5,541,293; 4,897,471; and 4,695,623 hereby incorporated by reference including drawings), interleukins (see, U.S. Patent No. 5,075,222, hereby incorporated by reference including drawings), erythropoietins (see, U.S. Patent Nos. 4,703,008, 5,441,868, 5,618,698 5,547,933, and 5,621,080 hereby incorporated by reference including drawings), granulocyte-colony stimulating factors (see, U.S. Patent Nos. 4,999,291, 5,581,476, 5,582,823, 4,810,643 and PCT Publication No WO 94/17185, hereby incorporated by reference including drawings), stem cell factor (PCT Publication Nos. 91/05795, 92/17505 and 95/17206, hereby incorporated by reference including drawings), and the OB protein(see PCT publication Nos. 96/40912, 96/053091 97/00128, 97/01010 and 97/06816 hereby incorporated by reference including figures).

[063] In addition, therapeutic uses of the present invention include uses of biologically active agents including but not limited to anti-obesity related products, insulin, gastrin, prolactin, adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG), motilin, interferons (alpha, beta, gamma), interluekins (IL-I to IL- 12), tumor necrosis factor (TNF), tumor.necrosis factor-binding protein (TNF- bp), brain derived neurotrophic factor (EDNF), glial derived neurotrophic factor (GDNF), neurotrophic factor 3 (M), fibroblast growth factors (FGF), neurotrophic growth factor (NGF), bone growth factors such as osteoprotegerin (OPG), insulin-like growth factors (IGFs), macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GMCSF), megakeratinocyte derived growth factor (MGDF), thrombopoietin, platelet-derived growth factor (PGDF), colony simulating growth factors (CSFs), bone morphogenetic protein (BMP), superoxide dismutase (SOD), tissue plasminogen activator (TPA), urokinase, streptokinase and kallikrein. The term proteins, as used herein, includes peptides, polypeptides, consensus molecules, analogs, derivatives or combinations thereof. In addition, the present compositions may also be used for manufacture of one or more medicaments for treatment or amelioration of the conditions the biologically active agent is intended to treat.

[064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference. In addition, the materials, methods and examples are illustrative only and not intended to be limiting. In case of conflict, the present specification, including definitions, controls.

[065] The invention will be further characterized by the following examples which are intended to be exemplary of the invention.

EXAMPLES Summary

[066] Emulsan-alginate coacervate microspheres were prepared, characterized, and studied for controlled release function. Environmental scanning electron microscopy images of the microspheres revealed homogeneous "cloudy" surfaces in contrast to the smooth surface of pure alginate microspheres. Surface analysis of the microspheres by X- ray photon spectrometry determined 8% differences in oxygen/carbon ratios on the emulsan-alginate microspheres and an increase in calcium content when compared with pure alginate microspheres. BSA binding to alginate-emulsan microspheres improved two-fold over alginate microspheres alone due to protein adsorption, functional results confirmed by XPS showing increases in nitrogen and sulfur. Increased temperature, 24 to 37°C, resulted in a relative increase of ~40 % in BSA adsorption for both emulsan- alginate and alginate microspheres which implies total BSA adsorption by emulsan coacervates. An increase in ionic strength favored BSA adsorption by both microspheres systems, while the emulsan-alginate systems were less sensitive to these changes. BSA adsorption of -70% in alkaline pH shifted to 100% between pH 5.5 to 5.0 for the emulsan-alginate microspheres, compared to 30% to 100% between pH 5.0 to 4.0 for the alginate microspheres. Equilibrium and dynamic kinetic studies with BSA adsorption showed 2 to 3 times improved adsorption for the emulsan-alginate microspheres compared to alginate alone. Gibbs free energy calculations of BSA adsorption showed a positive trend correlated with increase in temperature. In the case of the alginate microspheres, below 303 K the BSA adsorption process was unfavorable. BSA adsorption by both types of microspheres is an exothermic process estimated by Van't Hoff plot. Entropic contributions to the free energy were about twice as high for the emulsan-alginate microspheres compared to the alginate system. The data suggest that the protein-binding capacity of emulsan enhances the potential utility of gel systems in which this amphophilic polymer is included and suggests new opportunities to bind and deliver proteins using these types of systems. In addition, the biological activation features innate with emulsans can provide additional benefit to these structures in certain therapeutic applications.

[067] The aim of the present work was to characterize protein adsorption by these novel emulsan-alginate coacervate-based biogel systems using BSA as the model protein. The effects of temperature, pH, and ionic strength on BSA adsorption were determined both on the emulsan-alginate systems and a control system consisting of alginate alone. Classical isotherm adsorption models of Freundlich and Langmuir, as well as dynamic kinetic studies using intraparticle diffusion and Lagergren were tested and thermodynamic parameters of the adsorption process are discussed. In addition, mixed microbial culture medium from bacteria known to produce toxic products was also studied for adsorption to complement the model BSA studies.

EXAMPLE 1 : Emulsion - alginate microspheres and protein adsorption Material and Methods Chemicals & Reagents

[068] Chemicals and solvents were analytical grade and other reagents and microbiological media were of highest available grade obtained form Aldrich (St. Louis, MO), Difco (Franklin Lakes, NJ), and Sigma (St. Louis, MO) and used as provided. Alginate was obtained from Sigma. A. venetianus strain RAG-I (ATCC 31012) was maintained on Luria Bertram (LB) agar slants covered with LB medium to prevent cell dehydration at 5°C. Ethanol Saline (ES) medium and culture conditions for emulsan production and purification were described previously (Johri et al., 2002; Blank et al., 2002). Bacillus subtilis BGSC 1 Al , Bacillus cereus BGSC 6A5 (ATCC 14579), Escherichia coli BLR (Novagen, Wisconsin), Staphylococcus epidermidis (ATCC 12228) and Salmonella typhimiurum TA98 (Xenometrix, CA) were cultivated in 250 cm3 flasks containing 100 cm³ of LB or nutrient broth (Difco) at 30 or 37°C.

Microsphere Formation.

[069] Two grams of alginate sodium salt was dissolved in distilled water or in the following buffers at 25 to 100 mM Tris-HCl (pH= 7.5 to 8.5). Alginate solution was pumped into an aqueous solution Of CaCl₂ (25.0 to 50.0 mM) with or without emulsan (125 to 3,000 μg/cm3) in 25 mM Tris-HCl (pH=7.5) or sodium acetate (pH=5.0) buffers at 0.30 to 1.0 cnrVminute under continuous stirring. Fresh microspheres were incubated in the calcium solutions for 1 to 48 hours in a shaker at 50 rpm and room temperature, followed by filtration on paper (Whatman #1). Filtered microspheres were kept at 5°C in 70% ethanol until use. Emulsan content in the microspheres was quantified by a phenol sulfuric acid technique (Taylor, 1995).

Environmental Scanning Electron Microscopy (ESEM).

[070] Microsphere samples were mounted on a microscope plate without treatment for imaging with a FEl, Quanta 200 Scanning Electronic Microscope consisting of a Falcon System running Genesis 1.1 software and a super ultra thin window (SUTW)) (FEI Company, Peabody, MA). The chamber was saturated with water, and the pressure was maintained between 4.3 to 6.75 Torr to avoid extensive sample dehydration.

Adsorption experiments.

[071 ] In order to characterize the protein adsorption process, bovine serum albumin (BSA) was selected as the model protein for the present studies because it is well characterized and is responsible for 99% of free fatty acid transport in mammals, with equilibrium constants ~10⁷ M^"1 (Peters, 1996). Alginate and emulsan-alginate microspheres (50.0 to 300.0 ± 5.0 mg) were placed in 1.5 ml tubes and filled with 1.0 ml of a protein solution. The tubes were incubated 10 to 120 minutes at 24 to 37 °C, followed by centrifugation at 10,000 x g for 2 minutes at room temperature. Aliquots of 0.50 ml of supernatant were purified through 100 kDa filters (Microcon, Millipore, Billerica, MA) and the BSA content quantified using Coomassie Brilliant Blue dye binding with BSA, Fraction V, as standard (Bradford, 1976). Kinetics of BSA adsorption were determined in a reaction medium containing 185 mM NaCl, 25 mM acetate buffer (pH= 5.0) with 50 μg/ml of BSA, and incubated for 10 to 60 minutes at 30°C. Assays to determine the effects of ionic strength, temperature, and pH on the kinetics of BSA adsorption were performed using NaCl concentrations from 10.0 mM to 1.0 M (3O⁰C, pH 5.0), temperatures from 24 to 37 °C (100 mM NaCl, pH 5.0), and pH from 3.3 to 8.5 (100 mM NaCl at 30°C). Adsorption was determined on solutions containing 20.0 to 45.0 μg/ml of BSA using sodium acetate (pH 3.3 to 5.6), Bis-tris (pH 5.8 to 7.2), Tris-HCl (7.5 to 8.50), or Bis-tris-propane (7.0 to 9.5) at 25 mM.

Adsorption Isotherm Models.

[072] Langmuir isotherms (Langmuir, 1918), where Qm, and KL are the Langmuir constants, and Ce the equilibrium concentration of BSA left in the aqueous solution expressed in μg/ml, can also be expressed in dimensionless mode where KL is the Langmuir constant and Co is the highest initial solute concentration tested. The RL value indicates the type of isotherm, irreversible if RL is equal zero, favorable if RL is higher than 0 but less than 1, or unfavorable if RL is higher than 1. The Freundlich adsorption (Freundlich, 1926), where Qf, and n are the empirical Freundlich parameter, and exponent respectively, were also applied in the studies. If n is higher than 1 this implies that adsorption is favorable, whereas if n is less than 1 adsorption is unfavorable.

Adsorption Kinetics.

[073] First and second order Lagergren and Interparticle diffusion models were tested (Lagergren 1898), where qe, and q are the amount of BSA adsorbed at equilibrium and at time t expressed in mg/μg; kl , and k2 are the pseudo-first and pseudo-second order compound adsorption rate constants in min-1 and mg/μg. min respectively, and t time in minutes. The slopes and intercepts of plots t/q vs. t were used to estimate the pseudo- order rate constants and the amount of BSA adsorbed in equilibrium (qe calc). The intraparticle diffusion model, where ki is the intraparticle diffusion rate constant expressed in μg/mg min-0.50 (Weber and Morris, 1963) was also applied to the data.

Thermodynamic Calculations.

[074] Standard Gibbs free energy (ΔG°), Enthalpy (ΔH°), and Entropy (ΔS°) were determined assuming an adsorption equilibrium constant (Ka) where Cad is the amount of BSA adsorbed per L of solution in equilibrium, and Ce is the equilibrium concentration of BSA in solution. The Gibbs free energy was calculated where T is the solution temperature in Kelvin and R is the universal gas constant. ΔH° was evaluated using a Van't Hoff plot and assuming that ΔH° is equal to ΔH.

Bacterial Supernatant Adsorption.

[075] Alginate and emulsan microspheres (50.0 to 300.0±5.0 mg) were placed in 1.5 ml tubes and filled with 1.0 ml solutions. The tubes were incubated 10 to 120 minutes at 24 to 37°C, followed by centrifugation at 10,000xg for 2 min at room temperature. Aliquots of 0.5 ml of the supernatant were filtered through an ultrafiltration device with a molecular weight cut-off of 100 kDa (Microcon, Millipore, Billerica, MA), and then assayed for total protein content. Analysis of microspheres made in 1.0 mg/ml solution of the polymer rendered 0.84 mg/ml of emulsan by phenol-sulfuric technique.

Statistics.

[076] Experiments were performed in duplicate or triplicate.

Results

[077] ESEM imaging of microspheres in a water-saturated chamber showed the significantly different surface morphological differences between the alginate and emulsan-alginate (Figure 1). The alginate beads displayed a smooth surface in contrast to the irregular and mottled surface on the emulsan-alginate beads. The presence of the fatty acid acyl chains on the emulsan main chain, and the lack of these pendent groups on alginate, are the likely the reason for the observed difference in surface morphology. Molecules larger than 4000 MW (PEG model) can not diffuse into the alginate core, as it was published many years ago. The presence of BSA on the surface of the emulsan- alginate microspheres was also confirmed (data not shown) by XPS. [078] Isothermal kinetic analysis of BSA adsorption on the two types of microspheres at room temperature is shown in Figure 2. Adsorption of BSA occurs in two steps in both types of microspheres; first fast adsorption involving 51.8% and 26.4% of total protein for the emulsan-alginate and alginate microspheres, respectively, in about 10 minutes. However, one-hour incubations were required to reach the equilibrium adsorption in both systems, reflecting the slower second phase or step. While different amounts of BSA were adsorbed in both types of microspheres in the first 10 minutes, adsorption from 10 minutes to 60 minutes increased 47.3 and 48.9% for the emulsan- alginate and alginate microspheres, respectively. [079] The effect of temperature, pH and salt concentration on BSA adsorption by microspheres is displayed in Figure 3. An increase of temperature between 24 to 37°C induced increased adsorption of BSA by more than 40% in the emulsan-alginate and alginate microspheres, and the difference was almost constant over the range of temperatures studied. The effect of temperature on BSA binding showed an increase in adsorption that could be attributed to an increase in chain dynamics flexibility on the surface of the microspheres, likely exposing more binding groups to the solution. In addition, considering Fick's laws, diffusion coefficients of macromolecules increase with temperature, thereby favoring the adsorption of BSA. In addition, secondary adsorption related to BSA-BSA interactions and cooperative effects, rather than BSA-microsphere surface interactions, motivated by changes of BSA topology associated with binding (Peters, 1996), may also play a role. Changes in pH from 4 to 5 resulted in significant reduction in BSA adsorption, from 100 to about 40% in the emulsan-alginate microspheres. On the contrary, BSA adsorption by alginate microspheres showed less of a decrease, from 100 to about 75% of the BSA between pH 5.0 to 5.5. BSA adsorption on emulsan-alginate microspheres was about 10 times more resistant to these changes in pH compared to the alginate samples. The reduction in adsorption with the increase in pH can be attributed to an increase in electrostatic repulsion between the ionized state of the carboxylic acid groups present in the microspheres and BSA, which has an isoelectric point of 5.15 (Peters, 1996). The higher sensitivity of protein adsorption by the alginate microspheres to changes in pH could be related to the ionization of free β-D-mannuronic and α-L-guluronic acids, which convertjo the anionic form

[080] In the case of the emulsan-alginate microspheres, charge complexes through the interaction of free amino groups of emulsan with free carboxylic groups of alginate, reduces the impact of pH changes on protein adsorption. In addition, the higher content of calcium detected by EDX (data not shown) in the emulsan-alginate systems compared to alginate system probably reduces sensitivity to changes in pH changes of the emulsan- alginate microspheres, following the Gulberg-Waage law. Similar to previous one, if EDX is not showed I will delete this paragraph. Also, the Gulberg-waage law is the basis of chemical equilibrium, is too elemental to be referenced.

[081] The influence of ionic strength from 0 to 100 mM on BSA adsorption is shown in Figure 3. BSA adsorption increased from 70 to 100% as the ionic strength increased with the emulsan-alginate microspheres, and a greater increase was observed with the emulsan-alginate microspheres from 13 to 68%. Ionic strength can influenced BSA adsorption mediated via conformational changes of BSA upon binding to the surface, rather than an effect on the microsphere structure. There are two process in this case: 1st is the competition between sodium with calcium (which is complexed inside the gel, and can cause gel disruption, and the other one is to increase the strength of the ionic interaction between ionic parts and also hydrophobic portions of the molecules. The increase in ionic strength likely exposed otherwise inaccessible polar domains in order to keep the molecule in solution (Peters, 1996).

[082] In order to evaluate the protein adsorption on the microspheres in equilibrium, Langmuir and Freundlich models were assessed (Figure 5). The correlation factors for the linear regression of the experimental data using Langmuir and Freundlich models were higher than 0.9, indicating reasonable fit to the models. For the Langmuir model, BSA adsorption from aqueous solution follows monolayer adsorption in the available sites of the microspheres (Langmuir, 1918). Analysis of RL, a dimensionless Langmuir equilibrium parameter, predicts that the adsorption of BSA by the microspheres is favorable; value is between 0 and 1. This conclusion is also supported by the n parameter the Freundlich model, with values between to 1 and 10 (Freundlich, 1926). A comparison between the maximum adsorption for the emulsan-alginate and alginate microspheres showed significantly higher adsorption of BSA by the emulsan-alginate system by a factor of about 2.5 and 3.0, based on comparisons of the Langmuir and Freundlich isotherm constants, respectively (Figure 5).

[083] Dynamic kinetic models of BSA adsorption by the two types of microspheres were also investigated using Lagergren and the Intraparticle Diffusion models (Lagergren, 1898; Weber and Morris, 1963). The first order rate expression of Lagergren does not fit well to the full range of the adsorption data, with correlation coefficients lower than 0.6. These results indicate that the adsorption is not pseudo first-order reaction (data not shown). However, experimental data of BSA adsorption by both microspheres correlated well with a pseudo second-order Lagergren kinetic model (Figure 6). The kinetic values at initial times and at equilibrium for BSA on emulsan-alginate microspheres were higher than for the alginate microspheres. The error calculated for the amount of BSA adsorbed was about 12 and 6 % for the emulsan-alginate and alginate microspheres, respectively. These studies of BSA adsorption suggest the presence of two different BSA binding sites, one with high affinity and other with low affinity, which are also independent of the adsorbent. A previous study of the interaction between free fatty acids and BSA reported two binding sites with different affinities (Ricchieri et al., 1993). In the case of emulsan-alginate microspheres, the presence of the fatty acids in the microspheres is essential for high BSA adsorption.

[084] Equilibrium and dynamic kinetic models assessed for BSA adsorption revealed higher values for the emulsan-alginate vs. the alginate microspheres in all cases. The low correlation coefficient for the Lagergren pseudo-first order reaction indicates a lack of predictability of the first-order model, probably because of high kinetic constants. In this model it is interesting to mention that the equilibrium pseudo-second order constant of alginate is approximately 3 times higher than for the emulsan-alginate microspheres, however the qe is about half of that observed with the emulsan-alginate microspheres, suggesting an energy dependent process. Also, the low correlation factor of the intraparticle diffusion kinetic model compared to Lagergren pseudo second order model validated that hypothesis.

[085] Thermodynamic calculations based on equilibrium adsorption constants were estimated (Figure 7). For emulsan-alginate systems, ΔG° values were negative over the range of temperatures, while in the case of the alginate systems ΔG° changed with BSA adsorption. ΔH° was calculated from the slope of the Van't Hoff isochore plot of log Ka vs. 1/T and values for the adsorption of BSA on emulsan-alginate and alginate systems were -892 kJ/g and -367 kJ/g, respectively. Values of ΔG° suggest that the adsorption process is spontaneous in the case of emulsan-alginate but ranged from non-spontaneous to some spontaneity in the case of the alginate. In all cases the decrease of ΔG° with increase in temperature indicates that the energy barrier for the adsorption the process of BSA on the microspheres is small at high temperature, in agreement wi^fh results of - Figure 4. However, differences between ΔG° of emulsan-alginate and alginate microspheres increased about twofold in the range of 297.15 to 307.15 K, indicating that the BSA adsorption process is more favorable for the emulsan-alginate microspheres (Figure 7). The negative ΔH° values indicate an exothermic adsorption reaction according to Le Chatelier's principle which favors BSA adsorption.

[086] The dependence of the entropic parameter (T . ΔS°) on BSA adsorption by the microspheres with the temperature is displayed in Table 4. The entropic contribution to Gibbs energy observed in both types of microspheres increases with temperature, from a negative to a positive value. However, the entropic contribution to Gibbs free energy for BSA adsorption by alginate microspheres decreases with the increase in temperature while with the emulsan-alginate micropheres the contribution increases (Table 4). Considering that the entropic contribution to the adsorption is associated with hydrophobic interactions between macromolecules, and the enthalpy to the electrostatic interactions between molecules, the process of BSA adsorption on the microspheres can be described in terms of multiple interactions between the BSA and the microspheres. In the case of emulsan-alginate microspheres at 307.15 K, the major contribution to the adsorption process is based on hydrophobic interaction, probably between the acyl-fatty acids on the emulsan and the BSA. In addition, positive values of T.ΔS⁰ indicate that during the process of BSA adsorption onto the microspheres the protein losses degrees of freedom, which raises the entropy. On the contrary, the process of binding between macromolecules in aqueous phases involves some dehydration of BSA and the microspheres, considered an endothermic process involving a reduction of entropy. The overall process is exothermic suggesting that the dehydration process is not the major driving force for BSA adsorption in the microspheres.

[087] Bacterial protein toxins are generated by Gram-positive microorganisms, such as Bacillus species, and Staphylococcus species, and by Gram-negative bacteria, such as Escherichia and Salmonella species (Alouf and Freer, 1999). Bacillus subtilis, a non¬ pathogenic microorganism considered as GRAS (Generally Regarded As Safe) is an important producer of extracellular enzymes, some of which are aggressive In the same genus, Bacillus cereus produces a strong extracellular food-poisoning multi -component protein toxin with the same type of multimeric structure of B. anthracis toxin (Alouf and Freer, 1999). Staphylococus species produces a large amount of exoproteins some of which are cytotoxic, including lipases, collagenases, and pyrogenic toxins (Alouf and Freer, 1999). Gram-negative E. coli and Salmonella ; species generate enterotoxin and cause food poisoning (Alouf and Freer, 1999). The effect of toxins on cells is mediated by adsorption to cell lipid surface described as the first stage of intracellular translocation (Bakas et al., 1996; Nordera et al, 1997).

[088] To assess the removal of toxins from bacterial supernatants with the alginate and emulsan-alginate microspheres, cell-free supernatants of five selected toxin producing microorganisms were analyzed in combination with the microspheres. Alginate and emulsan-alginate coacervate microspheres were selected for the adsorption experiments based on preliminary screening with BSA. Emulsan-alginate microspheres bound more than 70% of total protein content in all the cell extracts tested. Additionally, the emulsan-alginate microspheres exhibited higher binding capacity then the alginate microspheres, about 38% higher on average. The adsorption of extracts by emulsan- alginate microspheres of S. epidermidis, E. coli and B. cereus were between 80 to 90 %. This is of particular interest as B. cereus is closely related species to B. anthracis (Mock and Fouet, 2001). In contrast, protein adsorption of extracelullar extracts of S. epidermidis and E. coli were less than 25% by the alginate microspheres. In the case of S. typhimurium, the emulsan-alginate microspheres have -30% higher binding capacity then the alginate microspheres. The crucial role of fatty acids on the emulsan in this adsorption process was demonstrated when the fatty acids were stripped off of the emulsan polysaccharide backbone, resulting in similar adsorption capacities for of E. coli and B. subtilis extracellular extracts between the alginate and emulsan(-fa)-alginate microspheres.

[089] The present work demonstrates that emulsan-alginate microspheres display different morphological properties compared to alginates microspheres. High adsorption of BSA as well as mixed extracellular microbial proteins by the emulsan-alginate microspheres offers new possibilities for the use of these microspheres both due to the carrying capacity of these systems as well as the unique structural tailorability and biological interactions of the emulsan polymers. Applications for these systems include controlled release drug delivery systems with high ligand binding capacity which would allow for decreased dosage to compensate for low solubility and stability of several important pharmaceutical compounds. Since no toxicity has been observed with emulsan-mediated cytotoxicity to bacterial or mammalian cells (Panilaitis et al., 2002), the emulsan-alginate microspheres can be utilized in various biomedical applications.

EXAMPLE 2: Emulsion - alginate microspheres and controlled release

Chemicals & Reagents.

[090] Chemicals and solvents were of analytical grade, and other reagents and microbiological media were of highest available grade. They were obtained from Aldrich (Milwaukee, WI), Difco (Franklin Lakes, NJ), or Sigma (St. Louis, MO).

Bacterial Cultures and Emulsan Purification.

[091 ] Emulsan synthesis by Acinetobacter venetianus strain RAG- 1 (ATCC 31012) was in saline medium supplemented with ethanol, and purified according to previously reported techniques (Johri et al., 2002). Microsphere Formation.

[092] Two grams of alginate sodium salt was dissolved in 100 ml of distilled water. Alginate solution was pumped at 1.0 ml/minute to an aqueous solution containing 25 raM CaCl₂ with or without 0.125 to 3.00 mg/ml emulsan under continuous stirring. Fresh microspheres were incubated in the calcium solutions for 72 hours in a shaker at 1.0 Hertz at room temperature. The microspheres were then filtered out of calcium solution on Whatman #1 filter paper (Whatman, Clifton, NJ). Filtered microspheres were kept in 70% ethanol at 5°C until use.

Sugar Determination.

[093] Thirteen mg of microspheres were placed in test tubes containing 1.0 ml of distilled water and 3.2 ml of concentrated sulfuric acid was added; the tubes were kept at room temperature for 1 minute and then cooled in a water bath. Fifty μl of 90% phenol was added to the vials, and spectrophotometric readings were taken at 480 nm after 30 minutes incubation (Taylor, 1995).

Protein Quantification.

[094] Determination of proteins was carried out using Coomassie Brilliant Blue dye binding assay and Bovine Serum Albumin (BSA, Fraction V) as a standard (Bradford,

1976).

Lipase Activity.

[095] Candida rugosa lipase was assayed by incubating the sample in the presence of 150 μM p-nitrophenylacetate, 20 mM Tris-HCl buffer (pH=7.2) at 37⁰C for 30 minutes. One lipase unit is defined as the amount of enzyme able to produce one μmol of p- nitrophenol per minute at 400 nm in a 1 -cm light path cuvette.

Protease Activity.

[096] Subtilisin Carlsberg activity was determined by incubating a 1 -ml reaction vial containing 400 μg/ml azoalbumin (azo-BSA)/50 mM Tris-HCl buffer (pH=7.7) solution at 37°C for 10 minutes. Free dye in the supernatant was determined at 334 nm using an appropriate standard. Alternatively, protease activity was assayed in the presence of 10 μg/ml casein as previously reported (Ferrero et al., 1996). The reaction was stopped by adding 50 μl of 5% tricholoroacetic acid, and then centrifuged (2 minutes at 10,000xg). Analysis of supernatant proteins was quantified using Coomassie Brilliant Blue assay as described above. Controls without protease, and with protease previously inhibited with 1.0 mM diisopropyl fiuorphosphate (DFP), were performed.

Adsorption Experiments.

[097] Three hundred mg of alginate or emulsan microspheres were placed in 1.5 ml tubes and filled with 1.0 ml of experimental solutions. Tubes were incubated at 37°C for 60 minutes, and then centrifuged at 10,000xg at room temperature for 2 minutes. Aliquots of 0.50 ml of supernatant were filtered through a 100 kDa ultrafiltration device. (Microcon, Millipore, Billerica, MA), and the filtered products were analyzed.

Results

[098] Microspheres generated in this study exhibited an average weight of 220±20 μg of polymeric material per bead, with dimensions of 400±80. The emulsan content in the microspheres, as determined by the sulfuric acid-phenol assay, was 24.0±4.3 μg per bead.

[099] In order to determine the stability of the microspheres, analysis of carbohydrate leaching from the emulsan/alginate microspheres and the alginate microspheres was performed in distilled water and pH 7.7 buffer solutions in the presence or absence of bound subtilisin. Samples were incubated at 37°C for one hour and liquid phase analyzed with the sulfuric acid-phenol assay (Taylor, 1995). Only residual quantities of polymer in solution were detected (data not shown). [0100] In Example 1, the higher protein adsorption capacity of emulsan/alginate microspheres when compared to alginate microspheres was attributed to the presence of the fatty acid esters in emulsan. The difference in azo-BSA content adsorbed by each type of microsphere was clear when visualized by light microscopy (Figure 10). In the absence of dye both types of microspheres were colorless. Spectrophotometric quantification of azo-BSA adsorbed in the microspheres was performed by dissolving the microspheres incubated for one hour in the presence of 10 mM EDTA and 100 mM buffer phosphate (pH=7.0) at 37°C. These results confirmed the visual differences: 0.637 μg/mg and 0.170 μg/mg of azo-BSA was adsorbed per emulsan/alginate and alginate microspheres, respectively.

[0101] In order to examine the induced release of bound proteins by the cleavage of the emulsan-based fatty acid esters, a set of experiments was performed using C. rugosa lipase to release bound protein from the microspheres. Azo-BSA adsorbed to the microspheres was treated with soluble lipase for 20 minutes at 37°C. Due to the potential for non-specific and/or protease-like activity in the lipase preparations, the release of free sulfanilic acid from the azo-BSA was estimated by precipitation with 5% trifiuoroacetic acid at O⁰C. The cleavage of sulfanilic acid from azo-BSA directly by lipase was negligible (data not shown). In the absence of lipase the release of azo-BSA was negligible in both types of microspheres. Additionally, release of azo-BSA from the alginate microspheres was not significantly increased in the presence of lipase. In contrast, release of azo-BSA from emulsan/alginate microspheres by soluble lipase was nearly complete at 10 minutes of incubation (Figure 11).

[0102] With consideration for binding pre-drugs to the microspheres to be activated and/or released by specific enzymes, the microspheres were loaded with azo-BSA, followed by treatment with subtilisin. Subtilisin is a serine protease that belongs to the same group as trypsin and chymotrypsin, which are present in significant concentrations in the mammalian gastro-intestinal tract. Sulfanilic acid release from azo-BSA adsorbed to the microspheres via enzymatic hydrolysis was characterized. Enzymatic release of sulfanilic acid from the azo-BSA adsorbed to the microsphere surface was performed with and without subtilisin, and with subtilisin previously treated with DFP, an irreversible protease inhibitor (Figure 3). The rate of sulfanilic acid release from emulsan/alginate and alginate alone microspheres without protease, and with previously DFP-inhibited subtilisin, was negligible. Kinetic analysis of dye release from the microspheres in the linear range showed reaction rate constants of 1 1.34 xl0-3 min-1 and 4.02 xlO-3 min-1 for the emulsan/alginate and alginate alone microspheres, respectively.

[0103] Studies of supernatant samples by SDS-PAGE at several times were monitored in order to detect possible changes in cleavage of adsorbed BSA on microsphere surfaces or differences in protease activity. No differences in the pattern of bands of microsphere free supernatants by gel electrophoresis were observed in the two systems throughout the period of treatment (Figure 12).

[0104] As an alternative, subtilisin adsorption by the two types of microspheres was characterized in order to test the potential of a biologically active delivery system. For these experiments, soluble azo-BSA was used to quantify protease activity adsorbed to the microspheres. It was necessary, however, to address the potential problem of azo- BSA adsorption onto the microsphere surface, which may reduce the availability of azo- BSA for subtilisin, thereby leading to an underestimate of subtilisin activity. After protease adsorption, a blocking step utilizing BSA (without azo dye) was performed. BSA may act as a competing substrate for the active site of subtilisin, and could therefore reduce the observed cleavage of azo-BSA. The strong pH-dependence of subtilisin activity was taken advantage of to allow blocking by BSA at pH 6.0 where subtilisin Carlsberg activity is reversibly reduced to almost zero (Philip et al., 1979). This shift in pH also allowed for an increase in BSA adsorption by the microspheres as was previously described above. The blocking step was followed by detection of subtilisin activity in standard conditions (pH= 7.7). In both microsphere systems the enzymatic activity reached a plateau after 5 minutes of incubation, and no differences in subtilisin activities were detected in the two types of microspheres at 37⁰C for 20 minutes (Figure 13). [0105] The hydrolysis rate of azo-BSA by subtilisin adsorbed to each microsphere type exhibited similar values: 8.645 xlO-6 and 8.997 xlO-6 mM azo-BSA per nM of subtilisin per min for the emulsan/alginate and alginate alone systems, respectively. These rates were significantly lower than that found for soluble subtilisin which exhibited a rate of azo-BSA hydrolysis of 3.967 xlO-2 mM azo-BSA per nM of subtilisin per min under the same experimental conditions. Of particular interest however, the activity of subtilisin adsorbed in either microsphere preparation remained constant after 4 hours of incubation (data not shown), while the half-life of soluble subtilisin was lower than 30 minutes (Castro, 1999).

[0106] In another set of experiments, lipase stability on microsphere surfaces was examined. C. rugosa lipase was first adsorbed to microspheres, followed by a blocking step with BSA. Lipase-bound microspheres were incubated.at 37°C for 40 minutes and lipase activity was monitored with p-nitrophenylacetate conversion to p-nitrophenol. Release of p-nitrophenol by lipase adsorbed in ECM and ACM surfaces showed similar and stable activities (Figure 14).

[0107] These studies utilizing azo-BSA adsorption confirmed our previous findings (Example 1) indicating high protein adsorption of proteins by microspheres coated with emulsan when compared to those coated with alginate. The emulsan/alginate to alginate alone adsorption ratio was 3.7 times higher with azo-BSA as a model and kinetic experiments of sulfanilic acid release from azo-BSA by subtilisin showed a rate ratio of 3.33 for the emulsan/alginate vs. alginate alone microspheres. The similar adsorption ratio and dye release ratio of the two types of microspheres suggests that little inhibition of soluble subtilisin can be attributed to the emulsan in the microspheres. Moreover, analysis of subtilisin supernatants incubated with casein by SDS-PAGE indicated that protease remains active for at least 50 minutes (Figure 12). Analysis of azo-BSA microsphere supernatants treated with subtilisin by SDS-PAGE showed the presence of a strong band close to 66.0 kDa., indicating the release of free BSA (66.4-66.7 kDa, Peters, 1996) from both types of microspheres. Also, SDS-PAGE analysis of each microsphere supernatant showed a similar profile after 10, 30, or 50 minutes, suggesting no changes in the subtilisin activity in the presence of either microsphere (Figure 12).

[0108] Protease autolysis is a significant drawback in the use of serine proteases. In particular, subtilisin Carlsberg, one of the most active enzymes of this family, has a half- life of approximately 25 minutes in aqueous medium at 30⁰C and the autolysis rate increases about 10-fold at 37°C (Castro, 1999). Reaction rates of subtilisin adsorbed on the two types of microspheres were not significantly different, indicating that the biological activity of subtilisin is unaffected by adsorption on the surfaces of these microspheres. However, soluble subtilisin showed 4,588 and 4,409 times higher rates compared to adsorbed subtilisin in the emulsan/alginate and alginate alone microspheres, respectively. A decrease of enzyme activity is a common result of enzyme immobilization processes, due to enzyme structure rigidifϊcation and steric hindrance, which drastically reduces the rate of molecular transfer in the catalytic triad center of the enzyme. As a result, the significant decrease in subtilisin activity when adsorbed onto the microsphere surface is somewhat compensated by the more than 8-fold increase in enzyme stability when compared to the soluble enzyme. In addition, the increased stability of adsorbed subtilisin by the microspheres .can also be attributed to the calcium ions present in both microsphere preparations which inhibits the process of autolysis.

[0109] In a separate set of experiments lipase was utilized to induce release of bound protein from emulsan microspheres. Lipase treatment of the emulsan/alginate microspheres containing adsorbed azo-BSA showed high release of dye coupled BSA, and little conversion of azo-BSA into sulfanilic acid and BSA (Figure 14). This specific release induced by lipase can likely be attributed to the release of fatty acids from the emulsan, and therefore, decreased binding capacity of the ECM.

[01 10] As was described with subtilisin, lipase bound to the two types of microspheres maintained its enzymatic activity. The decrease of lipase activity adsorbed in both microspheres systems was low after 40 minutes, on the order of 10-4 μmol/min. In the case of the alginate microspheres, lipase inactivation was practically negligible, but the reduction of lipase activity was 3.75 times higher in the emulsan/alginate system, which could be considered due to inhibition by product by free fatty acids released from emulsan, a competitor with p-nitrophenyl acetate substrate for the active site of the enzyme rather than biocatalyst inactivation by experimental conditions (e.g. temperature, pH).

[01 11] In summary, proteins bound to emulsan/alginate microspheres can be specifically released by treatment with lipase which presumably cleaves the fatty esters from the emulsan structure, thereby releasing the bound protein. Similarly, bound protein can be enzymatically activated while bound to the emulsan/alginate microspheres. Finally, the emulsan/alginate microsphere preparations allowed lipase and subtilisin to maintain activity while bound, albeit at a lower level, and also extended the half-life of the bound enzyme. The results presented here further establish the versatility and utility of emulsan coacervate microspheres for protein binding and delivery.

[0112] The references cited herein and throughout the specification are incorporated by reference.

References

1. Alouf J.E., Freer J. H. (ed.) 1999. The comprehensive sourcebook of bacterial protein toxins. Second editions. Academic Press, London.

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Claims

CLAIMSWhat is claimed is:

1. A composition comprising emulsan and alginate.

2. The composition of claim 1, wherein said composition is in the form of a microsphere.

3. The composition of claim 1 or 2, further comprising an agent.

4. The composition of claim 3, wherein the agent is selected from the group consisting of a protein, peptide, nucleic acid, PNA, aptamer, antibody and small molecule.

5. The composition of claim 3, further comprising a pharmaceutically acceptable carrier, dilutent or adjuvant.

6. The composition of claim 3, further comprising a linker between the microphere and the agent, wherein the linker is cleavable by a target enzyme.

7. The method of claim 6, wherein the target enzyme is selected from the group consisting of oxidoredutases, transferases, lyases, isomerases, ligases and hydrolases (e.g. subtilisin and lipase), peroxidase and glycosidases.

8. A method for treating an indication comprising administering to a patient in need thereof a composition of claims 4 - 7.

9. A method for removing protein contaminants from a solution suspected of containing protein contaminants comprising contacting said solution with a composition comprising emulsan and alginate.

10. The method of claim 9, wherein the contaminant is a bacterial toxin.

1 1. The method of claim 9, wherein the solution suspected of containing the protein contaminant is a food product.

12. A method for producing a pharmaceutical formulation for controlled release of at least one therapeutic agent, the method comprising: contacting a microsphere comprising emulsan and alginate with at least one therapeutic agent.

13. The method of claim 12, wherein the therapeutic agent is selected from the group consisting of a protein, a peptide, nucleic acid, PNA, aptamer, antibody, and small molecule.

14. The method of claim 12, wherein said pharmaceutical formulation is biodegradable.

15. The method of claim 12, wherein said pharmaceutical formulation further comprises a targeting agent that specifically targets said device to a specific cell or tissue type.

16. The method of claim 12, wherein said targeting agent is selected from the group consisting of a sugar, peptide, and fatty acid.

17. The method of claim 12, wherein said pharmaceutical formulation comprises multiple therapeutic agents.

18. The method of claim 12, wherein the microsphere further comprises a linker between the microsphere and the agent, wherein the linker is cleavable by a^' target^' enzyme.