Title of Invention	HIGH STRENGHT GEL-MICROCAPSULES BASED ON NATURAL POLYSACCHARIDE
Abstract	Novel polymeric gel-microcapsules based on the sodium salt of carboxymehtyl xanthan gum, for drug delivery, is provided. Polyelectrolyte complex membrane was developed over the gel-microbeads of sodium salt of carboxymehtyl xanthan gum and the resulting high strength gel-microcapsules are provided as release controlling devices.

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Title: High strength gel-microcapsules based on natural polysaccharide PRIOR ART and DRAW BACKS Although, there are some cases where pharmacokinetic and/or pharmacodynamic features of drug therapy demand frequent administration of doses. The repeated administration either orally or parenterally may result in fluctuating medication levels in blood as well as significant physical and monetary burden on the patients. Rational drug therapy requires sustained blood levels of drugs/ therapeutic agents to achieve the most effective therapeutic results. Moreover, many conditions respond better to controlled levels of therapeutic agent. Thus, a need exists for controlled release medicament to provide prolonged drug concentration in blood.. Such controlled release medication would provide the patients with not only enhanced prophylactic, therapeutic or diagnostic effects and reduced adverse effects but also a decrease in the frequency of administration as well as overall cost. Oral solid dosage forms can be prepared in the form of single-unit and multi-unit microparticulate dosage forms. The advantages of the microparticulates (like microcapsules, microbeads, etc.) over single-unit (tablets and capsules, etc.,) have been recognized (Davis, et al., (1984), Int.J.Pharm, 21,167-177; Follonier et al., (1992), STP Pharma Science, 2,141-158). In this context, many methods to formulate microparticulates have been developed. Of these, both oil in oil and oil in water solvent evaporation methods (both oil/oil and oil/water) were widely studied (Sa, (1991), Drug Dev.Ind.Pharm, 27, 893-900; Barkai et al.,(1990), Drug Dev.Ind.Pharm, 16, 2057-2075). However, these methods are debated for the use of organic solvents because of toxicity factors, and during the course of solvent evaporation, apart from the partitioning of organic solvent of the dispersed phase, the active principle may also partition to some extent at the interface. Moreover, quantities of organic solvents employed at industrial scales are very expensive (prices being quoted for high-purity solvents, recovery costs, and solvent destructions), and trace residues of organic solvents are often difficult to eliminate from the dosage forms. International health authorities and scientific bodies have recently focused their attention towards the investigation of toxic residual solvents in active ingredients and pharmaceutical formulations, and they have imposed maximum limits on particular solvent (Gachon, (1991), STP-Pharma Pratiques. 1, 531-536). As result of these new directives, microparticle manufacturers are trying to concentrate their efforts on organic solvent-free technologies. The only way to overcome such technological and regulatory restrictions is the use of hydrophilic polymers and mild aqueous processing conditions. One such promising method is ionotropic gelation method. The concept of ionotropic gelation is the interaction between a polyelectrolyte and either oppositely charged polyelectrolyte or polyvalent ion and both leads to the formation of gel networks. Using this concept, many polyanionic polyelectrolytes (sodium alginate, pectinate, carrageenan, kojakmanan, carboxymethyl cellulose) have been cross-linked with polycations and converted to microparticles. These systems are used both in biomedical and pharmaceutical applications because of aqueous, ecofriendly and simple preparative Conditions and economical reasons (Kierstan and Coughlan, (1985)Kierstan, M.P.J. & Coughlan, MP. (1985). Immobilization ofcells and enzymes by gel entrapment. In: Woodward. J (Ed.)., Immobilized cells and enzymes: Apractical approach. Oxford: IRL Press, pp. 39-47). In the above context, Indian patent application no. 578/KOL/2004 discloses that by ionotropic gelation technique, sodium salt of carboxymethyl xanthan (NaCMX) gum can be cross-linked by polyvalent metal ions to form gel-microbead system, and also discloses that these gel-microbeads can be used as carrier as well as drug delivery devices. However, from the industrial point of view and protecting the encapsulated therapeutic agents from harsh process conditions, process and formulation conditions were not optimized. Also, the polyelectrolyte complexation between NaCMX gum (a polyanion) and a polycation such as chitosan that increases the strength of the gel- microbeads was not explored. It is well established that, in addition to the ability to form cation-induced gels, anionic polysaccharides can cross-link with high molecular weight polycations to form water-insoluble polyelectrolyte complexes (PECs).The application of PEC membrane in the preparation of durable and semi-permeable membrane capsules for the immobilization of living cells, enzymes and controlled release of drugs have been described in several studies. Many microspheres/microcapsules based on the alginate and chitosan polyelectrolyte complexes have been widely investigated with respect to drug delivery applications. For example: Huguet et al., (1994). J. Appl. Polym. Sci. 51: 1427-1432.; Polk et al., J. Pharm. Sci. 83: 178-185.1994; Ribeiro et al., Int.J.Pharm.187: 115-123.1999; Sezer et al., J.Microencaps.16: 195-203. 1999; Gaserod et al., Biomaterials. 20: 773-783.1999; Elcin et al., Artif. Cells Blood Subst. Biotech. 28:95-111. 2000; Vandenberg et al., J. Control. Rel.77: 297-307. 2001; Shu et al., Eur. J. Pharm.Biopharm.53: 193-201. 2002; Coppi et al., J. Microencapsul. 232: 225-234.2002; Gonzalez-Rodriguez et al., Int. J.Pharm.232: 225-234.2002). In view of the above, the present invention seeks to provide drug-loaded gel- microbead system based on sodium carboxymethyl xanthan gum and a method for preparing polyelectrolyte complex (CMXG gum-chitosan i.e. CMXG/CS) gel- microcapsules of higher strength for controlled drug delivery. BRIEF SUMMARY OF THE PRESENT INVENTION The present invention grew out of the studies using sodium carboxymethyl xanthan (NaCMX) gum (a modified xanthan gum), a polyanionic polysaccharide, for the preparation of gel-microbeads (see co-pending Indian patent application no. 578/KOL/2004) by ionotropic cross-linking with polyvalent cations. These beads were claimed as carrier systems for bioactive agents. However, the utility of these gel- microbeads in the drug delivery was not explored. The present invention thus relates to a novel polymeric gel-microbead/microcapsule system based on the NaCMX gum and is concerned - a) with the formulation of drug-loaded microbeads from NaCMX gum by ionotropic gelation with polyvalent cation solution, b) with the effect of variables on the release profiles of the drug from the beads, c) with interionic-polyelectrolyte complexation between two oppositely charged polyelectrolytes; NaCMX gum (a polyanion) and chitosan (a polycation), and d) with the effect of variables on the release from carboxymethyl xanthan gum/chitosan(CMXG/CS) gel-microcapsules. DETAILED DESCRIPTION OF THE INVENTION Recently, the preparations of microspheres and microcapsules using water based techniques have been encouraged because of regulatory restrictions to limit the use of organic solvents and also cost considerations. In preferred embodiment of the invention, polyanionic polysaccharide is sodium carboxymethyl xanthan (NaCMX) gum, which can be cross-linked and / or gelled by means of polyvalent cation. The knowledge of its polyanionic nature, cross-linking with polyvalent cations, and as carrier device for active agents has been drawn from the previous invention (Indian patent application no. 578/KOL/2004). Also in the preferred embodiment of the invention, polyvalent cation in step (a) is selected from trivalent metal ions such as aluminium(III), ferric(IIII), etc, and is most preferably aluminium(III) and is obtained from aluminium chloride solutions. Although ferric (III) ions can form beads it gives unpleasant dark coloured beads and may lead to unstable formulations because of its redox characteristics. The successes or acceptance of any new technique for the formulation of delivery device is based on the optimization of the both process and formulation variables which not only protect the encapsulated active therapeutic agents but also provide a cost effective production technology. In the previous application 578/KOL/2004, we have reported a gel-microbead system as bioactive carrier. However, to protect the active agents such as cells, enzymes, proteins, vaccines, drugs, etc., from harsher process conditions and keep their viability longer or reduce the time and production cost with drug based formulations, it requires a thorough optimization of the process variables. The furosemide was used in this invention as a model active agent and various variables were optimized. In the previous application 578/KOL/2004, we have reported on the production of gel-microbead system based on the ionotropic gelation method. However, in that invention, various preparative variables (such as concentration of cross-linking agent/counter ion, time of cross-linking/incubation, and drug loading) which would be used in the manufacture of the gel-microbeads were not optimized. It is well known that, gel formation depends on the concentration of cross-linking agent that induces gelation. The use of unlimited amount of cross-linking agents may lead to the exposure of the active agents to the harsh effects and also increase the cost of manufacturing. Therefore, in this invention, concentration of AICI3 was varied from a lower level (1%) to a higher level (5%), but gelation is not limited in this range (1- 5%w/v) and may occur at any level. The effect of concentration of Al ions on the gelation was studied using gelation, swelling behavior, differential scanning calorimetric (DSC) and in vitro drug release studies. The increase in the time of incubation (cross-linking) should generally increase the degree of cross-linking; however, there exists a time where entire matrix will be cross- linked as soon as the number of active groups (Na) in the matrix of NaCMX gum are no more available for exchange with A1JT ions of the cross-linking agent. It can be said that preparation of gel-microbeads by exposing to an optimum period not only saves the time but also reduces the leaching of active agent to the cross-linking agent (provided the active agent is soluble in the solutions of cross-linking agent, i.e. AlCl3).The cross- linking (or incubation) time was optimized between 0.5hr to 24hr. However, gelation is not limited to this level studied, since the gelation is an instantaneous reaction and forms the gel-front as soon as the solution of the NaCMX gum contacts the AICI3 solution and cross-linking goes on for about five days. An optimum amount of drug must be released to reach the drug plasma concentration in the therapeutic level. Since the release rate of drug from the formulation will influence the therapeutic effect of drug, various release rates can be obtained by varying the formulation variables. The nature of the drug release profiles may also depend on the concentration of drug present inside the matrix. The presence of drug may affect the strength of the gel matrix and influence the drug release profile. So by varying the concentration of the drug in the matrix, desired release rate/profile can be obtained. In this embodiment, the concentration of the drug was varied between 30 to 70% w/w of the polymer. In the second embodiment of this invention, in an attempt to control/retard the release of drug from beads, the strength of the beads was increased by preparing polyelectrolyte complex (PEC) beads. To achieve this, the polyanionic nature of the NaCMX gum has been exploited. It is well known that, in addition to the ability to form cation-induced gels, anionic polysaccharides can also cross-link with high molecular weight polycations to form water-insoluble polyelectrolyte complexes (PECs) (Fukuda, Bull. Chem. Soc. Jpn. 53, 1980, 837; Takahashi et al., Int J Pharm. 61, 1990, 35-41). Of all the polycations, chitosan, a deacetylated chitin, received more attention as it is obtained from natural sources, biodegradable, least toxic with good medicinal functionality, and has been used in wound dressings (Denuziere, et al 1998), and controlled-release drug delivery systems (Ilium, L et al 1994). EXPERIMANTAL 1. Preparation of furosemide-loaded beads The drug-loaded gel-microbeads were prepared following the method described elsewhere (Indian patent application no. 578/KOL/2004). In 30ml aqueous solution of NaCMX gum, required amount of furosemide was dispersed and homogenized for 15 min. Bubble free dispersion was dropped through a 16 bore glass syringe in gently agitated AlCl3 solution. After incubating for predetermined times the gelled beads were separated by filtration, washed with 3 x 100 ml de-ionized water, air dried overnight and finally dried at 60°C for 4 h. The following experimental parameters were varied : (i) Gelation time : 0.5. 1,4, 8 and 24 h, (ii) Concentration of AlCl3 solution : 1, 3 and 5% w/v, and (iii) Amount of furosemide : 30, 50 and 70% w/w of the polymer. 2. In vitro release study In vitro release of furosemide from beads were monitored using about 20 mg beads, accurately weighed, in 900mL of simulated intestinal fluid (SIF) (USP phosphate buffer solution, pH 6.8) at 37±0.5 °C and 50 rpm using programmable dissolution tester (Paddle type, Electrolab, model TDT-06P (USP), India). Aliquots were withdrawn at predetermined intervals and were replenished immediately with the same volume of fresh buffer solution. Aliquots, following suitable dilutions, were assayed spectrophotometrically at 277 nm, 3. Preparation of microcapsules from NaCMX gum The gel-microbeads were prepared following the method described elsewhere (Indian patent application no. 578/KOL/2004). In brief, solution (2.25%w/v) of NaCMX gum in deionized water was extruded through 16 bore glass syringe into gently agitated 100ml of l%w/v AlCl3 solutions. The gel beads were harvested after 30min, washed with deionized water (3x100ml) and dried at room temperature overnight followed by drying in vacuum oven at 60° C. In another set of formulations, solutions of the NaCMX gum were extruded into 100ml of l%w/v AlCl3 solutions each containing chitosan (0.1 or 0.3%w/v in 1% w/v glacial acetic acid) solutions. The gel capsules were then harvested after 15 or 30 min, and washed with deionized water (3x100 ml) and dried in room temperature for 12 h followed by vacuum oven at 60°C. 4. FTIR IR spectra for finely powdered A1CMX beads and, A1CMX/CS beads were recorded in a Fourier Transform Infrared (FTIR) spectrophotometer (FT-IR,410 JASCO, Japan) with KBr pellets. 5. Swelling studies Swelling ratios of the dried microbeads of A1CMX and A1CMX/CS microcapsules without drug were determined gravimetrically in slightly agitated USP phosphate buffer solutions pH 6.8. The beads were removed periodically from the buffer solution, blotted to remove excess surface liquid and weighed on electronic balance. Swelling ratio (%w/w) was determined from the following relationship and plotted against time. Swelling ratio = (WrWo)/Wox 100 where, Wo and Wt are respectively initial weight of the beads and weight of the beads at time 't' 6. Preparation of furosemide loaded microcapsules'. In 30ml aqueous solution of NaCMX gum, required amount of furosemide(30%w/w) was dispersed and homogenized for 30min. Bubble free dispersion was extruded through a 16 bore glass syringe into a gently agitated 1%AlCl3 solution. After incubating for 30 min, the gelled beads were separated by filtration, washed with 3 xl00 ml de-ionized water, dried at room temperature overnight and finally dried at 60 °C for 4 h. In another set of formulations, the microcapsules were prepared by extruding into gently agitated 100 ml of chitosan solutions (0.1 or 0.3% w/v) containing glacial acetic acid (0.5% w/v) and incubated for 10 or 30 min. Beads were washed with deionized water, dried in air followed by drying in vacuum oven drying for 4h at 60°C. 7. SEM Dried A1CMX beads and, A1CMX/CS (coated with chitosan at different variables) were mounted onto stubs, using double sided adhesive tape and vacuum coated with gold film using sputter coater (Edward S-150, UK). Coated particles were observed under scanning electron microscope (Jeol, ISM-5200, Japan) for surface characteristics. BRIEF EXPAINATION OF DRAWINGS Figure 1: The effect of incubation time in A1C13 (l%w/v) solution on furosemide release profiles As accompanying in Figure 1, at lower incubation times (0.5 and 1h), the release of furosemide from the beads was faster compared to that at higher incubation times (4,8, and 24 h). Figure 2: The effect of concentration of AlCl3 solution on furosemide release profiles As accompanying in Figure 2, increasing concentrations of cross-linking agent (1- 5%w/v AlCl3) has decreased the both t50% and t8o% (time to release 50% and 80% of the release) values. Figure 3: The effect of initial drug loading on furosemide release profiles As accompanying in Figure 3, increase in the amount of furosemide increases the release of furosemide. Figure. 4: FT-IR spectroscopy IR spectra of CMXG beads, chitosan and CMXG/CS beads as accompanying in figure 4 (a), 4(b) and 4(c) respectively. In comparision with CMXG(3434.7 cm-1) and chitosan(3368 cm-1), the -OH stretching vibration bands of the microspheres were broadened and shifted to a lower wave number(3423cm-1), also -COO -gropus( 1623.3cm" 1) for CMXG beads shifted to 1634.3cm-1, suggesting the ionic interaction between - COO- groups of CMX and -NH3+ groups of chitosan. In CMXG/CS beads(figure 4c), appearance of a new peak at 1479.1cm-1 for primary amides will further confirm the formation PEC membrane by the interaction between -COO- groups of CMXG beads and -NH3+ of the chitosan. Figure. 5: Scanning electron microscopy As accompanying in Figure.5, surface characteristics of the drug loaded CMXG beads and CMXG/CS microcapsules. The CMXG beads (Figure5a) found to be smoother compared to CMXG/CS microcapsules. In addition, the surface of the CS treated microcapsules appears to have bigger depressions at lower chitosan treatment intervals (Figure.5c and 5e, at 15 min exposure in CS solutions). The reason may attributed to the collapse of the gel structure due to the formation of PEC membrane that recovered shape with increasing incubation times(Figure 5d and 5f, at 30 min exposure in CS solutions) as the Al3+ entered the beads and formed three-dimensional network through ionic cross-linking. Figure 5(b) shows cross section of the drug free-CMXG/CS. The formation of thin PEC membrane is clear from these beads. Figure. 6: Swelling studies As accompanying in Figure 6, the swelling behavior of the CMXG beads and CMX/CS microcapsules in phosphate buffer solutions (pH6.8), swelling of the CMXG beads were more due to the ionization of the gel by basic ions(-OH, HPO42-.) of the buffer. The increase in the electrostatic repulsion between the ionized -COO- groups of the gel disrupts the cross-links and increases the mesh size encompassing a huge amount of solvent. While in case of CMXG /CS beads lower swelling can be attributed to mechanisms involved; the protonation of-COO- groups of CMXG and the potonation of -NH2 groups of the CS which enhanced the electrostatic interaction between the acidic form of-COOH groups and protonated form of-NH2 groups of CS Finally, it can be said that, the swelling of the CMXG /CS beads was markedly reduced due to the formation of a PEC membrane and membrane would remain more tightly packed and create a stronger barrier to the solvent molecules. Thus the ionization of the surface of the beads was restricted. Finally it can be concluded that chitosan formed a PEC membrane with CMXG. Figure 7: Drying studies As accompanying in Figure 7, A1CMX beads (•) have dried faster than the any chitosan coated beads. This indicates that chitosan has formed a PEC membrane. The formation of PEC was responsible for the slower drying of the A1CMX/CS microcapsules. Figure 8: Drug release studies As accompanying in Figure 8, A1CMX beads released the drug faster compared to any of the chitosan coated (CMX/CS) beads. The formation PEC on the A1CMX beads may be attributed to the slower drug release. The formation of PEC on the A1CMX beads is thus said to have produced the high strength gel-microbeads We claim :- 1) High strength drug-loaded gel- microcapsules comprising: a process for preparing drug-loaded gel- microcapsules formed by interaction of a modified natural polysaccharide, polyvalent metal-cations and a polycation; wherein modified natural polysaccharide is sodium salt of carboxymethyl xanthan gum; polyvalent metal-cation is aluminium (III); and polycation is chitosan; a therapeutically active agent is contained within the microcapsules. , 2) A process for preparing high strength gel- microcapsules as clamied in 1, including polyanions and polycation, the process comprising: i) dropping the aqueous solutions of modified natural polysaccharide into acidified aqueous solution containing a polyvalent metal- cations and a polycations, ii) curing gelled microcapsules, iii) harvesting and washing of gelled microcapsules, iv) drying of gelled microcapsules 3) high strength drug loaded microcapsules-as clamied in 1, wherein the drug is furosemide 4) high strength drug loaded microcapsules as clamed in 1, wherein the therapeutically active agent was at a level of 30%w/w of total solid content. 5) process as clamed in2, wherein aqueous solutions of modified natural polysaccharide was ata level of 2.25%w/v. 6) the process claimed in claim 1, wherein the polyvalent metal-cation, aluminium (III) was obtained from aluminium chloride. 7) process -as clamed in 2, wherein the aqueous solutions of polyvalent metal- cations obtained from aluminium chloride which was at l%w/v level. 8) process as clamed in 2, wherein the solutions of polycations were obtained from chitosan which was at a level of 0.1% to 0.3%w/v 9) process as clamed in 8, wherein the chitosan was dissolved in acidified aqueous solutions. 10) process -as clamed in 9, wherein acidified aqueous solution was obtained by dissolving glacial acetic acid in water. 11) process as clamed in 10, wherein the glacial acetic acid was present at level of 0.5% w/v 12) process as clamed in 2, wherein the gelled microcapsules are cured for 15 to 30 minutes. 13) process as clamed in 2, where in the gelled microcapsules harvested and washed 3 times in deionized water. 14) process as clamed in 2, where in the gelled microcapsules were dried for 12 hours in open air and then dried in vacuum oven for 4 hours at 60° 15) process as clamed in 2, wherein the aqueous solutions of modified natural polysaccharide were dropped into acidified aqueous solution containing polyvalent metal- cations and polymeric-cations using a syringe fitted with a needle (#16 bore) 16) high strength drug-loaded gel- microcapsules as clamed in 1, wherein the chitosan used was having a viscosityof 505.8 cps. Novel polymeric gel-microcapsules based on the sodium salt of carboxymehtyl xanthan gum, for drug delivery, is provided. Polyelectrolyte complex membrane was developed over the gel-microbeads of sodium salt of carboxymehtyl xanthan gum and the resulting high strength gel-microcapsules are provided as release controlling devices.

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00021-kol-2005 description(complete).pdf

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