WO2006028996A2 - Microspheres d'emulsan-alginate et procedes d'utilisation de celles-ci - Google Patents

Microspheres d'emulsan-alginate et procedes d'utilisation de celles-ci Download PDF

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WO2006028996A2
WO2006028996A2 PCT/US2005/031372 US2005031372W WO2006028996A2 WO 2006028996 A2 WO2006028996 A2 WO 2006028996A2 US 2005031372 W US2005031372 W US 2005031372W WO 2006028996 A2 WO2006028996 A2 WO 2006028996A2
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alginate
emulsan
microspheres
bsa
adsorption
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WO2006028996A3 (fr
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David L. Kaplan
Guillermo R. Castro
Bruce Panilaitis
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Tufts University
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Tufts University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/167—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction with an outer layer or coating comprising drug; with chemically bound drugs or non-active substances on their surface

Definitions

  • 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).
  • 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).
  • Hydrogels and polymer-based systems have gained particular interest as providing a means for sustained release of therapeutic agents.
  • 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).
  • Alginate gels have also been used in sustained release formulations.
  • alginates are well known, naturally occurring, anionic, polysaccharides comprised of 1 ,4-linked- ⁇ -D-mannuronic acid and ⁇ -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.
  • 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.
  • 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.
  • 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 ⁇ m 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.
  • 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.
  • composition of the invention further comprises a pharmaceutically acceptable carrier, dilutent or adjuvant.
  • 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.
  • 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.
  • the contaminant to be removed is a bacterial toxin.
  • the solution suspected of containing the protein contaminant is a food product.
  • 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.
  • 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).
  • the pharmaceutical formulation is biodegradable.
  • 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.
  • 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).
  • Figure 2 shows the kinetics of BSA adsorption by emulsan-alginate (•) and alginate ( ⁇ )microspheres.
  • 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.
  • Figure 4 shows the relative protein adsorption of bacterial cell supernatants to emulsan-alginate (open bars) and alginate (cross bars) microspheres.
  • 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.
  • 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.
  • Figure 7 shows a table indicating the standard free energy changes of emulsan- alginate (Emulsan) or alginate microsphere BSA adsorption with temperature.
  • 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.
  • 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.
  • Figures 1OA & 1OB show emulsan (10A), and alginate (10B) microspheres containing adsorbed azo-BSA.
  • Figure 1 1 shows release of azo-BSA and sulfanilic acid adsorbed by microspheres using Candida rugosa lipase.
  • 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
  • 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; ⁇ and , ECM and ACM incubated with subtilisin.
  • 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.
  • Figure 14 shows Stability of lipase adsorbed on ECM (O), and ACM (V) respectively.
  • Figure 14 shows Activity of subtilisin activity adsorbed on ECM (O), and ACM (V) respectively.
  • Alginate is a linear polysaccharide of ⁇ -D-mannuronic and ⁇ -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 (Smidsr ⁇ d and Skjak-Brask, 1990).
  • cation chelating agents such as citrate, lactate, phosphate, or tartrate
  • competing cations such as sodium or potassium that are commonly present in biological fluids (Smidsr ⁇ d and Skjak-Brask, 1990).
  • 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).
  • acetylation of chitosan which correlates with its biological and chemical properties, depends on the chemical treatment of chitin by alkaline N-deacetylation.
  • 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).
  • Emulsan is an amphipathic lipoheteropolysaccharide of IxIO 6 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).
  • 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).
  • 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.
  • 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).
  • 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.
  • emulsan-alginate beads are prepared as described in Example 1.
  • a solution of alginate and emulsan can be pumped into an aqueous solution of CaCl 2 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. . .
  • the emulsan-alginate compositions of the invention are used in methods of drug delivery.
  • 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.
  • 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- ⁇ -III), vascular endothelial growth factor (VEGF)); anti-angiogenic proteins such as endostatin, and other naturally derived or genetically engineered proteins, polysaccharides, glycoproteins, or lipoproteins.
  • PDGF platelet derived growth factor
  • IGF-I and IGF-II insulin like growth factor
  • TGF- ⁇ -III transforming growth factors
  • VEGF vascular endothelial growth factor
  • anti-angiogenic proteins such as endostatin, and other naturally derived or genetically engineered proteins, polysaccharides, glycoproteins, or lipoproteins.
  • 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.
  • the therapeutic agent can be bound to the microspheres such that the agent can be activated and/or released by specific enzymes.
  • enzymes include, for example, oxidoredutases, transferases, lyases, isomerases, ligases and hydrolases (e.g. subtilisin and lipase), peroxidase and glycosidases.
  • 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.
  • 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,
  • targeting ligands in the present invention include cell adhesion molecules (CAM), among which are, for example, cytokines, integrins, cadherins, immunoglobulins and selectin.
  • CAM cell adhesion molecules
  • 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.
  • the emulsan-alginate compositions of the present invention 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.
  • 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.
  • 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.
  • release may occur for example on the order of about 2 to 90 days, for example, about 3 to 60 days.
  • the therapeutic agent is delivered locally over a time period of about 7-21 days, or about 3 to 10 days.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • sterilization process will be with ethylene oxide at a temperature between 52 - 55° C for a time of 8 or less hours.
  • the formulation may be packaged in an appropriate sterilize moisture resistant package for shipment.
  • 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.
  • 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
  • biologically active agents
  • proteins includes peptides, polypeptides, consensus molecules, analogs, derivatives or combinations thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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 cm 3 of LB or nutrient broth (Difco) at 30 or 37°C.
  • 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.
  • 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 ⁇ 10 7 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.
  • Standard Gibbs free energy ( ⁇ G°), Enthalpy ( ⁇ H°), and Entropy ( ⁇ S°) 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.
  • Ka adsorption equilibrium constant
  • the Gibbs free energy was calculated where T is the solution temperature in Kelvin and R is the universal gas constant.
  • ⁇ H° was evaluated using a Van't Hoff plot and assuming that ⁇ H° is equal to ⁇ H.
  • 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.
  • 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 ⁇ -D-mannuronic and ⁇ -L-guluronic acids, which convertjo the anionic form
  • 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).
  • 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.
  • the process of BSA adsorption on the microspheres can be described in terms of multiple interactions between the BSA and the microspheres.
  • the major contribution to the adsorption process is based on hydrophobic interaction, probably between the acyl-fatty acids on the emulsan and the BSA.
  • positive values of T. ⁇ S 0 indicate that during the process of BSA adsorption onto the microspheres the protein losses degrees of freedom, which raises the entropy.
  • 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).
  • 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.
  • the emulsan-alginate microspheres can be utilized in various biomedical applications.
  • EXAMPLE 2 Emulsion - alginate microspheres and controlled release
  • Emulsan synthesis by Acinetobacter venetianus strain RAG- 1 was in saline medium supplemented with ethanol, and purified according to previously reported techniques (Johri et al., 2002). Microsphere Formation.
  • One lipase unit is defined as the amount of enzyme able to produce one ⁇ mol of p- nitrophenol per minute at 400 nm in a 1 -cm light path cuvette.
  • protease activity was assayed in the presence of 10 ⁇ g/ml casein as previously reported (Ferrero et al., 1996). The reaction was stopped by adding 50 ⁇ l of 5% tricholoroacetic acid, and then centrifuged (2 minutes at 10,000xg).
  • Microspheres generated in this study exhibited an average weight of 220 ⁇ 20 ⁇ g of polymeric material per bead, with dimensions of 400 ⁇ 80.
  • 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.
  • subtilisin adsorption by the two types of microspheres was characterized in order to test the potential of a biologically active delivery system.
  • 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.
  • a blocking step utilizing BSA 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.
  • 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.
  • 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).
  • subtilisin Carlsberg one of the most active enzymes of this family, has a half- life of approximately 25 minutes in aqueous medium at 30 0 C 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 rigidif ⁇ cation and steric hindrance, which drastically reduces the rate of molecular transfer in the catalytic triad center of the enzyme.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ mol/min.
  • 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).
  • 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.
  • bound protein can be enzymatically activated while bound to the emulsan/alginate microspheres.
  • 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.

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Abstract

L'invention concerne, en général, des compositions d'émulsan-alginate et des procédés d'utilisation de celles-ci. Les procédés permettent d'utiliser les compositions d'émulsan-alginate comme véhicules d'administration de médicaments. De plus, l'invention concerne des procédés d'utilisation des compositions d'émulsan-alginate dans des applications nécessitant l'adsorption de protéines (par exemple, le retrait de toxines protéiques de produits alimentaires ou d'autres produits et solutions). Les compositions selon l'invention permettent de contourner les problèmes de l'art antérieur relatifs aux microsphères ou particules d'alginate.
PCT/US2005/031372 2004-09-03 2005-09-02 Microspheres d'emulsan-alginate et procedes d'utilisation de celles-ci Ceased WO2006028996A2 (fr)

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