Title of Invention

NOVEL GEL-MICROBEADS BASED ON NATURAL POLYSACCHARIDE

Abstract

A novel gel-microbeads system based on the natural polysaccharide, xanthomonas campestris, is provided. Native polysaccharide was chemically modified to make it ionic and suitable for producing gel-microbeads under simple and mild process conditions. Gel-microbeads were found suitable as carrier system for therapeutic agents by maintaining the gel integrity throughout the dissolution studies (Figure 4).

Full Text

PRIOR ART AND DEFECTS In the past decades, microparticles/microbeads have received a growing scientific and industrial interest because of the several advantages offered by them over the single - unit dosage forms. In Pharmaceuticals, multiunit dosage forms have been mainly used as drug delivery systems, as they offer advantages like controlled and targeted drug delivery of drugs, predictable and uniform drug release avoiding the vagaries of gastric emptying and different transit rates through the gastrointestinal tract, and avoidance of local irritation. And many other specific applications of microparticles/microbeads are given in the literatures. In the above context, oil-in-oil-emulsion-soivent (O/O) evaporation technique was developed to prepare microparticles/microbeads. Previously, we have prepared microparticles by emulsifying colloidal dispersion of ethylcellulose/acetone (Sa. B., et al., Drug Development and Industrial Pharmacy, Vol.16 (1990)1153), eudragit/ acetone (PurkaitK., et al., S.T.P.Pharma Sciences, Vol.4 (1994) 264), and cellulose acetate butyrate/acetone by other workers (Torres. D.,et al., International Journal of Pharmaceutics,Vol.173(1998)171) in liquid paraffin followed by rigidization of the formed microparticles using large volume of organic solvents like petroleum ether, n- hexane etc. Use of large volume of organic solvents have been criticized by several Governmental agencies and environmentalists as large scale use of these solvents may give rise to toxicity to the working personnel, environmental pollution and are uneconomical. These literatures did not discuss on the disadvantages associated with the use of large amount of organic solvents. In order to overcome the problems associated with oil in oil-emulsion-solvent evaporation technique, oil-in-water-emulsion-solvent evaporation (O/W) technique was developed. In examples based on oil-in-water-emulsion solvent evaporation techniques, microparticles were prepared by emulsifying colloidal dispersion of polymethyl methacrylate/dichloromethane (Sa. B., et al., Pharmaceutical Sciences.Vol.2 (1996) 209), polystyrene/dichloromethane (Sa. B.,et al., Bolletino Chimico Farmaceutico,Vol .138 (1999)47l)cellulose butyrate/acetone in gelatin solution(Torres. D.,et al., International Journal of Pharmaceutics, Vol.173(1998)171) in water containing suitable hydrocolloids like gelatin, methylcellulose. Although, in O/W emulsion-solvent evaporation technique, large amount of organic solvent was replaced by aqueous phase, use of organic solvent in the internal phase could not be eliminated. The absence of trace amount of organic solvent in the final product is yet questionable. An alternative and promising research deals with microparticfes/microbeads obtained from hydrophilic polymers. Most of the hydrophilic polymers are available as natural polymers at affordable price. Among the natural polymers, polyanionic polymers such as sodium alginate, sodium pectinate and carrageenan have been exploited for preparing microparitcles/mierobeads by ionotropic in-situ gelation technique which can be carried out under mild process conditions. The references related to the use of sodium alginate for preparing microparticles/ microbeads include the following: Kierstan.M., et.al,, Biotechnology and Bioengineering. Vol..26(1982)1507, describes the use of sodium alginate gels for immobilization, solid separation and diffusion chromatography techniques; US patent 6,432,449 discloses the use of alginate for sustained release of proteins; Tomida.H.,et al., Chemical and Pharmaceutical Bulletin.Vol.41(1993)1475; El-Kamel., et al, Journal of Microencapsulation. Vol. 20 (2003)21. Use of pectin has been cited in the following examples: Pillay.V., et al., Journal of Controlled Release.Vol.59( 1999) 229; Aydun.Z., et al., International Journal of Pharmaceutics. Vol. 137( 1996) 133; Sriamornsak.P. European Journal of Pharmaceutical Sciences. Vol.8, 1999,221. Another polyanionic natural polymer tried in this area is k-carrageenan: Garcia A.M., et al, Journal of controlled Release, 40(1996) 179; An-Lac Nguyen., et al., Biotechnology and Bioengineering. Vol.28, 1986,1261. These reports teach us that, using in-situ gelation technique, polyanions can be easily tailored to formulate into carriers for active agents and other materials. It is also well known that unless specially treated, none of these gel beads remain intact and maintain its integrity throughout the dissolution studies in intestinal fluids. Hence it would have been appreciated if any of the gel beads prepared by these polyanionic polymers had maintained gel-integrity even after dissolution studies. Thus, there is still a need to develop gel-bead system that influences the release of incorporated active agents by maintaining the gel-integrity till all the core material is released from the beads. Subsequent studies in prior art were related to improve the strength of gel beads, and comprise the following examples. Poly-L-lysine, a polycation was used to modulate the permeability and strength of alginate beads in the following references: Gugerli.R., et al., Journal of Microencapsulation. Vol. 19(2002)571; Goosen.M.F.A., et al.,Biotechnology and Bioengineering.Vol.27(1985)146;O'SheaG.M. et al., Biochimica et Biophysica Acta.Vol.804(1984)113;Leung.Y.F.,et al., Journal of ann.Diabetology.Vol.20( 1982)161; Thu.B., et al., Biomaterials. 17(1996) 1069. In the following reference polyethyleneimine was used as polycation to increase the strength and stability of the alginate beads: Lim.F.,et al., Science.Vol.210(1980)908. Chitosan, a natural polycation polysaccharide, was also used in the following references to increase the strength of the alginate beads: U.S.Pat.6, 165, 503, (2000) discloses the use of chitosan to increase the strength of alginate beads. Hari.P.R., Journal of Applied Polymer Science. Vol.59(1996)1795; Huguet.M.L.,et al., Journal of Applied Polymer Science.Vol. 51( 1994) 1427; Miyazaki.S.,et al., Biological and Pharmaceutical Bulletin.Vol. 17(1995)745 The prior art mentioned above contains reports mainly based on alginate gel matrix to prepare microparticles/microbeads and modifications of gel strength through the use of other poloycations (poly-L-lysine,chitosan,etc.,). It would have been a novel and economical approach, if any natural polysaccharide other than sodium alginate, was exploited for preparing gel-microbead system. In this view, the present invention seeks to provide a novel gel-microbead system based on natural polysaccharide, which promises to retain its structural integrity even after the dissolution studies. The objectives of this invention are as follows: 1. The main objective of this invention is to provide a gel-microbeads system based on natural polysaccharide and produced under simple and mild process conditions. 2. Another objective of the invention is to establish the gel-microbeads suitable as carrier for therapeutic agents and as drug delivery system. 3. Yet another objective of the invention is to prove that gel-microbeads maintain their gel-integrity at different pH values encountered in gastro-intestinal tract. SUMMARY OF THE INVENTION This invention presents a new gel-microbeads as a carrier for bioactive agents and other materials used in pharmaceutical and biomedical applications. The gel- microbeads of the invention are produced by an interaction between a new polyanion, sodium carboxyalkyl polysaccharide and metallic cation(s), using ionotropic in-situ gelation technique. This invention also presents a procedure for chemical modification of polysaccharide from native polysaccharide. Another embodiment of the invention includes the optimization of concentration of polymer and gelation medium to improve the shape and strength of the gel- microbeads. In yet another embodiment of the invention, gel microbeads have been presented as carrier for the model drugs theophylline anhydrous and furosemide, and in vitro release studies were carried out to assess release profiles and stability /integrity of the gel-microbeads after dissolution studies. DETAILED DESCRIPTION Gel-microbead system based on the chemically modified bacterial polysaccharide is provided. The gel-microbeads according to the invention are compulsorily made from alkali metal salt of carboxy alkyl polysaccharide. Chemical modification of bacterial polysaccharide is known from the prior art and do not form main part of this invention. The chemical modification of bacterial polysaccharide, xanthomonas campestris, include substitution of alkali metal carboxy alkyl groups through ether linkage to at least one of the hydroxyl groups on the back bone of the polysaccharide. Composition of the gel microbeads and process for the production of these gel-microbeads comprise the main part of this invention. The process for the production of gel-microbeads can be any of the methods known as interionic/polyelectrolyte interaction, chemical gelation, ionic-cross linking or ionotropic in-situ gelation technique but is preferably the latter. The present invention provides a process for gel-microbead production comprising the following steps, a) Preparation of aqueous solutions (sol) of chemically modified polysaccharide, b) Dropping the sol (step a) to metal salt solution known as as gelation medium, c) Curing of gel beads (step b) for different periods in metal salt solution to provide gelation or crosslinking of polysaccharide, d) Harvesting the gel-microbeads (step c) from the gelation medium and e) Drying of gel-microbeads (step d). Methods for preparing sol (step a) are well known in the art and do not comprise the part of this invention. The metal salt solutions (step b) used can be known as gelation medium, curing agent or crosslinking agent. The gelation medium used in the invention comprises both divalent and trivalent type of metal cations. Metal cations used include but are not limited to Ca2+, Ba2+, Mg2+, Sn2+, Al3+ and Fe3+. The transition of sol to a firm gel state was dependent on the type of cation. Addition of sol (step b) for the production of gel-microbeads can make use of hypodermic syringe with needle, peristaltic pump, spraying apparatus which can atomize the sol in to an appropriate mean particle size or any container with or without spout on it to let the droplets onto the gelation bath. The droplets, as soon as they come in contact with the gelation medium, should completely enter into the gelation medium for more effective cross-linking. Gel formation (step c) is an instantaneous process at the interlace of sol and gelation medium; however, complete gelation occurs over a period of 24 h. Gel beads can be harvested (step d) from gelation bath by conventional technique. i.e., filtration. Gel beads can be dried in hot-air oven, vacuum oven or rotary dryer at 60°C, or overnight in open air at room temperature. Mean size of the gel-microbeads depends on bore size of the hypodermic needle or droplet of sol entering the gelation medium. As an example, needle with bore number 16 produced gel-microbeads with mean size range 941.55(±35.71) micrometer. Reproducibility of the shape and size depends upon, the flow rate of sol droplets from the needle, distance of the needle from gelation bath surface and direction (vertical/horizontal) of the needle. Although sol-gel transition is independent of concentration of sol and gelation medium, formation of gel-microbeads of appropriate size and shape are dependent on the solution viscosity, which is dependent on the concentration of sol, that in turn affects the mobility of sol, and hence shape and size of the droplets of sol entering the gelation medium. Gel-microbeads produced in this invention are free-flowing provided the beads are symmetric in shape. Other factors considered in gel-microbead production include, variations in length of curing time and strength of gelation medium. To produce isolable gels beads, gelation step (step c) proceeds at least 5-30 min, while for almost completely gelled beads, prefers 24 h and more optimally 2-4 h. The strength of gelling medium was varied between 0.25-5%w/v, preferably 1-2% concentration was found to be effective enough in producing perfect gel-microbeads. Gel-microbeads can be stored in moisture-free sealed container or vacuum desiccator at room temperature. Both polymer solution and gelation medium are formed using water as solvent and water is the only solvent used throughout the invention, which makes the device safe and economic. Gel-microbeads: as carrier gystem:- Many other microbeads/microparticles have been used in the art as carriers for bioactive agents or other materials. Gel-microbeads produced from chemically modified polysaccharide polymer provided herein can also be used as carrier for bioactive agents(s), or any other agent which is compatible, on-gelling or non-dehydrating. Such agent(s) include drug, enzyme, protein, plant or animal cells, fine particles, powder and/or water miscible agent. In other preferred embodiment of this invention, the process further comprises incorporating drugs such as, theophylline anhydrous, furosemide. indomethacin, diltiazem hydrochloride, propranolol hydrochloride, ibuprofen, and mtrofutsntoln. The invention can be appreciated from the following examples, which are intended as further illustration of the invention, but not as a limitation on the scope of the invention, hi addition, with respect to the above description or examples below, one skilled in the art will be able to make the necessary changes to the disclosure for a large scale production of gel-microbeads. EXAMPLE 1: This example describes the preparation of chemically modified polysaccharide (i.e. sodium salt of carboxymethyl xanthan gum). Preparation and standardization of alkali metal carboxy alkyl polysaccharide (i.e. sodium salt of carboxymethyl xanthan gum) was carried out with a batch size of 10 G of xanthomonas campestris polysaccharide/xanthan gum. Initially, polysaccharide was slowly dispersed in ice-cold alkali metal hydroxide solution (45%w/v) for lh. Alkali metal hydroxide preferred is sodium hydroxide. A solution (75%w/v) of hallo fatty acid, preferably with smallest chain length (chloroacetic acid), was added under constant blending and mixing. The temperature of the reaction mixture was raised slowly and held at 75°C for lh. The reaction mixture was cooled to room temperature, subdivided on a flat surface and dried in a vacuum oven at 50°C. The product was ground to a 65 mesh size(British Standard Sieve) and washed in 80% methanol to get products with different degree of metal ion substitution ranging from 2.2 - 2.8 ppm (analyzed by atomic absorption spectrophotometer, Perkin-Elmer, Model No.2380). EXAMPLES 2: Formation of gel beads. A solution (1-3%) of chemically modified polysaccharide was prepared in deionized water. Bubble free solution was loaded in a hypodermic syringe (with a needle of bore size. 16) and dripped into 100ml of gelation medium (0.25-5%). The gel beads were harvested from the gelation medium after 24 h, and washed with deionized water (3x100ml) for 30 min. After blotting excess surface water with a tissue paper, gel beads were spread on petri-dish and dried in open-air for 12 h followed by drying in vacuum- oven at 60°C for 4 h. EXAMPLE 3: Formulation of drug loaded gel-microbeads and in vitro dissolution studies: (A) Theophylline loaded gel-microbeads. Theophylline (30%w/\v)- loaded gel-microbeads were prepared by dispersing known amount of theophylline in 2.0%w/v modified polysaccharide and dropping the solution to a gelation medium (l%w/v, 100ml) containing same concentration of theophylline. Gel beads were cured for 4 h, filtered, and dried for 12h in open-air followed by dxying in vacuum-oven at 60°C for 4 h. Release of theophylline from gel-microbeads was assessed using dissolution rate test equipment USP XXI (Campbell Electronics, Bombay, India) in 500ml (37±0.5°C, l00rpm) of both hydrochloric acid buffer solution (pH 1.2, 0AM) and phosphate buffer (pH 6.8). (3) Furosemide loaded gel-microbeads. Furoseraide loaded (30%w/w) gel-microbeads were prepared by dispersing weighed amount of furosemide in 2%w/v modified polysaccharide solution and dropping the solution into a gelation medium(l%w/v, 100ml). Gel beads were cured for 4 h, filtered, and dried for 12h in open-air followed by drying in vacuum-oven at 60°C for 4 h. Release of furosemide from gel-microbeads was assessed using dissolution rate test equipment USP XXI (Campbell Electronics, Bombay, India) in 500ml (37±0.5°C, 50 rpm) phosphate buffer (pH 6.8). The following figures are used to substantiate the accompanying drawings of the invention. FIGURE 1. FTIR spectra shows chemical modification of native polysaccharide. FIGURE 2. Scanning electron micrographs of gel-microbeads depict the effect of variables such as polymer concentration and gelation medium concentration on the morphology of beads. FIGURE 3. shows in vitro release profiles of the model drugs theophylline (A) in buffer- pH 1.2 (•), pH 6.8 (o); and furosemide (B) in buffer pH 6.8. FIGURE 4. Scanning electron micrographs of gel-microbeads indicating the gel-integrity of the gel-microbeads before (A) and after (B) dissolution studies. Figure 1 shows that, after chemical modification of the native polysaccharide, the bands of carboxylate anion at 1613 cm"1 (asymmetrical) and 1406cm-1 (symmetrical) of native polysaccharide (Figure 1A), were shifted and sharpened at 1608 cm-1 and 141 lcm- 1 as seen in modified polysaccharide (Figure 1B), and a sharp peak appeared at 1328cm-1 (Figure 1B) confirms C-0 stretching bond. Loss of band at 1726 cm"1 from native polysaccharide (in Figure 1 A) was due to the hydrolysis of O-6-acetyl group by sodium hydroxide addition. Figure 2 shows the effect of process variables (such as gelation medium concentration and polymer concentration) on the morphology of dried gel-microbeads. It is clear from the figure 2 that, beads developed wavy surface at lowest gelation medium concentration (0.25%)(Figure 2A) and any gelation medium above 1% concentration had no influence on the surface structure of gel-microbeads(Figure 2B).Gel-microbeads were spherical in shape at 2% polymer concentration (Figure 2C) and at polymer concentration above 3% beads were elongated (Figure 2D). Figure 3 shows that, theophylline (Figure 3A) was released from the gel- microbeads over a period of 3 h while furosemide (Figure 3B) was released over an extended period of 5 h. Figures 4A and 4B respectively show the gel-microbead structures before and after dissolution study in pH 6.8, and demonstrate the stability of the gel-microbeads in terms of gel-integrity. We claim: 1. Gel microbeads, comprising: a known modified natural polysaccharide crosslinked with polyvalent metal- cations; wherein modified natural polysaccharide is sodium salt of carboxymethyl xanthan gum; and the microbeads optionally containing atleast one therapeutic agent. 2. Gel microbeads of claim 1, wherein sodium salt of carboxymethyl xanthan gum is obtained from the modification of natural polysaccharide, i.e. xanthan gum. 3. Gel microbeads of claim 1, wherein sodium salt of carboxymethyl xanthan gum comprises sodium content of 2.2 to 2.8 ppm. 4. A process for preparing gel microbeads of claim 1, comprising: (i) preparation of sodium salt of carboxymethyl xanthan gum; (ii) dropping the aqueous solution of sodium salt of carboxymethyl xanthan optionally containing atleast one therapeutic agent, into aqueous polyvalent metal salt solutions; (iii) curing of gelled beads in same polyvalent metal salt solutions; (iv) harvesting and washing of gelled beads in water; (v) drying of gelled beads. 5. Gel microbeads of claim 1, wherein the polyvalent metal cation is selected from the group of polyvalent metal salts consisting of calcium chloride, barium chloride, stannous chloride, magnesium chloride, aluminium chloride and ferrous chloride, preferably wherein the metal salt is aluminium chloride. 6. The process of claim 4, wherein the aqueous solution containing the sodium salt of carboxymethyl xanthan gum at a level of 1% to 3% by weight. 7. The process of claim 4, wherein the aqueous polyvalent metal salt solutions containing the polyvalent metal salts at a level of 0.5% to 5% by weight. 8. The process of claim 4, wherein the gelled beads are cured in aqueous polyvalent metal salt solutions for 0.5 hour to 24 hour. 9. Gel microbeads of claim 1, wherein atleast one therapeutic agent from a group of theophylline and furosemide, is entrapped within the gel microbeads. A novel gel-microbeads system based on the natural polysaccharide, xanthomonas campestris, is provided. Native polysaccharide was chemically modified to make it ionic and suitable for producing gel-microbeads under simple and mild process conditions. Gel-microbeads were found suitable as carrier system for therapeutic agents by maintaining the gel integrity throughout the dissolution studies (Figure 4).

Documents:

578-kol-2004-granted-abstract.pdf

578-kol-2004-granted-claims.pdf

578-kol-2004-granted-correspondence.pdf

578-kol-2004-granted-description (complete).pdf

578-kol-2004-granted-drawings.pdf

578-kol-2004-granted-examination report.pdf

578-kol-2004-granted-form 1.pdf

578-kol-2004-granted-form 18.pdf

578-kol-2004-granted-form 2.pdf

578-kol-2004-granted-form 3.pdf

578-kol-2004-granted-form 5.pdf

578-kol-2004-granted-reply to examination report.pdf

578-kol-2004-granted-specification.pdf