| Title of Invention | PHARMACEUTICAL COMPOSITION USEFUL FOR THE TREATMENT OF DISEASES CAUSED BY VIRUSES AND A PROCESS FOR ITS PREPARATION |
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| Abstract | The invention disclosed in this application relates to a pharmaceutical composition in the form of capsules useful for the treatment of disease caused by viruses such as Hepatitis , AIDS and the like which comprises microspheres of Chitosan incorporating an appropriate antigen encapsulated in enteric coated gelatine capsules The invention also relates to a process for the preparation of the above said composition |
| Full Text | Field of invention This invention relates to a pharmaceutical composition useful for the treatment of diseases caused by viruses and a process for its preparation. This invention particularly relates to a pharmaceutical composition useful for the treatment of diseases of liver especially Hepatitis, more especially Hepatitis B and a process for its preparation. By using the composition of the present invention it is possible to administer the antigen orally. Therefore the system of the present invention is useful for the eradication of microbial infections and thereby addresses the difficulties and problems associated with the administration of such antigens It is well known that immunization against infectious diseases is recognized as the most cost-effective method for controlling and eradicating microbial infections (WHO, UNICEF document 1996). However, despite the introduction over the years of both human and animal vaccines against a variety of viral and bacterial pathogens, many diseases remain unconquered. This is due, in part, to problems associated with vaccine delivery, stability, and cost. Other major hurdles to be overcome include stimulating immunity at the most effective site, reducing the need for repeated injections to overcome short-lived immunological memory, and stimulating the necessary Cytotoxic T Lymphocyte (CTL) responses, and antigenic variation among the causative agents. The excellent approach of genetic engineering and the recombinant DNA technology has been responsible for the current increase in the production of proteins and peptides for therapeutic use and also for the development of a new generation of recombinant vaccines. These proteins and peptides have received much attention in recent years as drug candidates. Today, there are about 50 biotechnology products in the market, with more then 150 products in the pipeline, either at a human clinical test study level, or at FDA for review and approval. Biotechnology is now providing an impetus for cures for several life threatening diseases. It is to be noted that although several such products are being rapidly made available, their therapeutic utility is limited by the lack of convenient methods for their effective delivery. Unfortunately, despite the great potential of these new therapeutic and antigenic peptides and proteins, only a small number has received approval by the FDA and other regulatory agencies. This has been, in general a consequence of the lack of an appropriate route of administration, which would permit the therapeutic potential of these molecules to be exploited. In fact most of them still need to be administered repeatedly in an injection form. Therefore, the design of effective delivery systems and the search for new routes of administration for these new generations of drugs and vaccines are important challenges for the pharmaceutical scientists. It has been found that over the recent years, the effort of the World Health Organization (WHO) to highlight the need to use existing vaccines more effectively within childhood immunization programs, has drawn attention among other problems, to the drop out during a course of multiple vaccines delivery, especially for new vaccines, which are likely to involve higher production cost. In a country like India, where health care resources are limited, there is a significant fall-off between the number of individuals receiving the first vaccine dose and those receiving the full courses. Although there are a variety of reasons for this, the most significant reason include poor education and consequent lack of awareness of the need to return for booster doses, fear of injection and difficulties in reaching the healthcare centre. The objective of vaccination is to provide effective immunity by establishing adequate levels of antibody and CTL responses in many situations, and a primed population of cells which can rapidly expand on renewed contact with antigen. The first contact with an antigen should avoid the pathogenic effect of the organism yet provide an adequate stimulus to the immune system. A successful human vaccine is one that is able to induce rapid, long lasting protection, ideally after administration of a single dose. However, because the vaccine contains a non-replicating antigen, booster doses are required. Additional criteria for a successful vaccine include: the pathogen exists as a single subtype; there is no insect vector or reservoir in a second animal species; the epidemiology is known; the vaccine is effective early in life; the antigen, is stable and has a long shelf-life, even at relatively extreme temperatures; several antigens can be delivered simultaneously; the vaccine is easily administered, with minimal risk (orally, for example); an economic immunogen is available; the vaccine can be readily adapted to existing immunization programmes. Although the most recent human vaccines are often the result of the purification of the protein required to induce a protective response, or of recombinant DNA technology, subunit vaccines are poor immunogens and require the use of an adjuvant and often need to be given at monthly or longer intervals, in at least two or three doses. The only adjuvants suitable for human use are aluminium salts and gels. Over recent years, the World Health Organization (WHO), in highlighting the need to use existing vaccines more effectively within childhood immunization programmes, has drawn attention among 7 other problems, to the drop out during a course of three or four doses of immunization, which i results in poor coverage and to the high costs of multiple vaccine delivery, especially for new j vaccines, which are likely to involve higher production costs. ^ In countries where health care resources are limited, there is a significant fall-off between the number of individuals receiving the first vaccine dose and those receiving the full course. Although there are a variety of reasons for this, the most significant include poor education, and a consequent lack of awareness of the need to return for booster doses, difficulties in reaching health care centres, political instability, illness at the scheduled time for immunization, and fear of vaccine-related secondary effects. In addition to increasing vaccine efficacy, the cost of immunization would drop due to the reduced need for health care workers and logistics, which together can account for over 80% of the total cost of immunization. The majority of human and animal pathogens enter the host via a mucosal surface. In contrast, the majority of the currently available vaccines have been developed for systemic immunization. Although numerous studies have provided convincing evidence that protection can be obtained by oral (or intranasal) administration, the poor uptake of immunogens delivered by these routes has proved to be a major difficulty. It is a major goal of the WHO Global Programme for Vaccines and Immunization to promote and support the research and development of oral vaccines. WHO has encouraged research involving the use of microspheres to avoid problems in delivery to mucosal surfaces, such as proteolytic degradation, as well as possibly increase immunogenicity. The use of microspheres with a diameter of 10 µm or less are readily taken up by M cells lining the intestine, from where they are transported within macrophages to the regional lymph nodes and spleen. The use of Polylactide (PL)/Polyglycolide (PG) microspheres has been examined for the oral immunization of a number of immunogens. In addition to the use of model proteins such as ovalbumin, progress has been made with the oral delivery of a diverse range of proteins. The development of single-dose vaccines using inactivated biological products now appears to be an achievable goal. Rapid advances in the manufacture of microspheres using biodegradable polymers has been paralleled by studies designed to maximize the efficiency of entrapment of high molecular weight immunogens. The Controlled Release Tetanus Vaccine project, sponsored and directed by the WHO Global Programme for Vaccines and Immunization and the Children's Vaccine Initiative, is a unique example of international collaboration between scientists interested in controlled release drug delivery and vaccinologists wishing to develop new forms of antigen presentation systems. The major results of this project offer fresh insights for the development of other controlled release vaccines, particularly against diseases for which effective immunization is hampered by the need for multiple deliveries, poor immunogenicity, or both. Several aspects of the safety of microsphere-based vaccines remain to be carefully addressed, although there is every expectation that controlled release vaccines manufactured using the PL/PG or chitosan polymer system will prove both safe and effective. It is particularly important to keep in mind that using microspheres several antigens can be administered together, avoiding undesired interactions and facilitating thus the task of combination vaccines. In addition, as pointed out above, microspheres may represent the safest way to deliver DNA vaccines to mucosal surfaces. Thus , there is considerable excitement within the vaccine field, with the use of controlled release systems offering novel opportunities for the production of vaccines that could prove more effective against diseases for which vaccines already exist, as well as opening avenues to fight pathogens for which vaccines are not yet available. Despite the best efforts of WHO, UNICEF and others over recent years and the tremendous success of the Expanded program on Immunization (EPI), millions of children still die each year from vaccine preventable diseases. Disease prevention with EPI vaccines depends largely on the population at risk. New technological advances have brought many innovative drug delivery systems to the market and others to the brink of commercialization . A variety of approaches have been investigated for the controlled release of drugs and their targeting to selective sites; polymeric prodrugs, drug conjugates, liposomes, microspheres, monoclonal antibodies, and microcapsules, nanoparticles, films, and sponges are finding increasing use in drug delivery systems. They hold the promise of providing better drug efficacy, reducing toxicity, and improving patient compliance. Manufactures have also recognized the potential benefit of reformulating existing products in new delivery systems as an effective tactic to extend their proprietary position for drugs coming off patent. I. Immunization is the most important preventive action for protection against disease, disability, and death resulting from an infection. It is the act of artificially inducing an active or passive immune response, desired to ward off or even eradicate an infectious pathogen in the system. Passive or short-term immunity is conferred on an individual by exogeneously formed antibodies that prevent or ameliorate the advent of infection. Two situations in which passive immunization occurs are the transplacental transmission of antibodies to the fetus and the injection of immunoglobulins for a specific preventive purpose. The protection bestowed by passive immunity usually lasts for a short term and therefore may require repeated immunization each time the protection from infected wanes. Active immunization involved the induction of antibodies to develop defensive capability against the infection, and it is accomplished by exposing an individual to the immunogens. This concept serves as a cornerstone of immunoprophylactic or active vaccination (Spector, 1986). The three major approaches to active immunization employ the use of live-attenuated inactivated, killed, or detoxified infectious agents, the extracts of infectious agent and the use of live vectors capable of producing specific antigens to stimulate the immune response against the infectious agent. Live attenuated vaccines produce an immunological response most like that occurring from a natural infection and generally confer a lifelong protection with a single dose (Spector, 1986). By contrast, other forms of immunogens do not induce permanent immunity with one dose, making repeated vaccination and boosters necessary to develop and maintain sufficient levels of antibody. A. Types of Immunity Traditionally, vaccine development has been concerned mainly with the induction of systemic immunity by parenteral immunization. Because most infectious agents were detected parenterally following the course of infection, it appeared to be the most direct mode of warding off the infection. Consequently, a substantial number of vaccines developed employ the parenteral route of administration. Until the 1960s it was perceived that only systemic mechanisms existed for the production of immunity following an antigenic stimulus, and the detection of antibodies in external secretions was the result of an overflow of the conferred immunity (McGhee & Mestecky, 1990). However, the success of polio vaccine by oral administration and the poor induction of mucosal immunity by parenteral administration of cholera vaccine suggested the presence of an immunological system characteristic of certain external secretions. Considerable evidence has now been gathered that indicates the existence of an independent mucosal immune system (Tomasi et al., 1965) that can be anatomically and functionally divided into at least two distinct interconnected compartments. The Immunoglobulin A (IgA) derived from mucosal effector sites represents more than 75% of all antibody isotypes produced in humans (McGhee et al., 1992). These mucosal inductive sites include the gut-associated and the nasal-associated lymphoreticular tissue, strategically placed in the gastrointestinal tract and the nasopharyngeal tonsil area, respectively. Antigenic stimulation of these tissues cause dissemination of the T-helper cells and the IgA precursor B cells to the effector tissue and to the secretory glands for subsequent antigen-specific antibody response. The distribution of the effector tissue in the bronchus-associated lymphoreticular region may represent another site for immune response to antigenic stimuli in the respiratory tract (McGhee et al., 1992). B. Potential of Mucosal Immunization Therapy Because most infectious agents enter the body through mucosal surfaces,- immunization of these surfaces will represent a potent mechanism of warding off the pathogen at the site of entry. The gastrointestinal, nasopharyngeal, pulmonary, and genitourinary surfaces are bathed in mucus that contains immunoglobulins, almost exclusively of secretory IgA derived from the plasma cells underlying the mucosal membrane (Tomasi et al., 1965) .Manifestation of this IgA response has been achieved by a direct antigenic stimulus of the mucosally associated lymphoid tissue. However, the development of delivery systems targeted to these immunologically active sites remains a practical challenge. C. Routes of Mucosal Immunization The theoretical basis for oral immunization is ascribed to the existence of the compartmentalized common mucosal immune system. Following this concept, antigenic stimulus in the gut-associated lymphoid tissue or bronchus-associated lymphoid tissue will stimulate IgA precursor cells that migrate to distant surfaces where they express secretory IgA specific for that antigen. Oral immunization is both practical and relatively safer and, therefore, is a relatively preferred route compared with parenteral administration. Oral immunization thus should effectively stimulate antibody production in all associated secretory sites (McGhee & Kiyono, 1993; Cuff et al., 1992; Waldman et al., 1986). The feasibility of such oral immunization has been examined in a limited number of studies. Moderate immunogenic response achieved by this route has prompted the concurrent administration of adjuvants, such as cholera toxin, to amplify the immune response. D. Mucosal Immunization Delivery Systems The current strategy for the induction of a mucosal immune response involves the use of particulate-delivery systems. Formulation of antigens into particulate carrier systems offers the potential of optimizing delivery to immunoresponsive sites and also protection of the antigen against proteolytic degradation in the gastrointestinal tract. 1. Replicating Antigen-Delivery Systems The replicating antigen-delivery systems proliferate in the host tissues following immunization, resulting in a more prevalent and a effective presentation of the antigens (O' Hagan, 1992) and, therefore, are more likely to be effective at stimulating an immune response following oral delivery. Replicating systems may also be able to induce a cytotoxic T-lymphocyte response that can result in longer-lasting immunity. Typically, a replicating-delivery system involves the genetic alteration of a live virus to function as a vector. Multiple foreign genes can replace the nonessential regions of the viral genome, resulting in an immune response against multiple pathogens. This approach enables the recombinant virus to function as a vaccine for two or more infectious agents. The consequences of the host recombinant virus, however, need to be better defined before replicating systems can gain broad acceptability as a preferred antigen delivery system. 2. Nonreplicating Antigen-Delivery Systems Recent advances in DNA recombinant technology has led to some novel approaches that are focused on the production of particulate antigen-presentation systems (O'Hagan, 1992) (e.g., Ty-VLP; [Ty- virus-like particles], HBsAg [hepatitis B surface antigen] and others). The Ty-VLPs are produced by transposing the fusion proteins of the yeast on Ty-encoded particle- forming protein and the viral protein of interest. Each particle is approximately 50 nm in diameter and contains several hundred copies of the coupled antigen, expressed in a polyvalent form on the particulate surface. For HBsAg, the antigenic proteins self-assemble to form 22-nm particles that serves as the immunogen. Another interesting approach of antigen presentation involves construction of solid matrix antibody-antigen (SMAA) complexes by attachment of monoclonal antibodies (MAbs) to a suitable solid matrix. The desired antigens are complexed onto the monoclonal antibodies to produce SMAA particulates that are capable of presenting multiple antigens to the immune system and, thereby, evoking an enhanced multivalent immune response. The enhancement of the immune response is believed to be due to the particulate character of the system and the presence of the antibody-antigen complex, which is a potent stimulator of the humoral and cell mediated immunity (O' Hagan, 1990). A newer approach to the presentation of peptide antigens is by linking multiple radially branching peptide epitopes, 9-15 residues in length, to a core matrix of a trifunctional amino acid, such as lysine. These multiple antigen-peptide systems may also be sufficiently flexible to permit the inclusion of multiple antigenic peptides, such as the T- and B-cell epitopes to produce an enhanced antibody response to the proteins from which these peptides were derived. The use of biodegradable polymeric particulate carrier systems that has been pursued fairly vigorously during the past decade. The choice of the polymeric material has been primarily serum albumin, polylactide and polyglycolides, and poly(acrylic acid) systems (O'Hagan, 1990). Liposomal systems have also been investigated for the delivery of the antigenic load (Gilligan & Wan Po, 1991); however, the instability of these systems in the gastrointestinal tract has been the constraining factor in its successful implementation. ORAL IMMUNIZATION USING MICROSPHERES Table 1: Potential and Pitfalls of Microsphere Delivery System A. Biodegradable Polymers For more than two decades, use of polymeric materials to deliver bioactive agents have attracted the attention of investigators throughout the scientific community. When attempting to design a microparticulate antigen delivery system for targeting mucosal sites, it is important to choose a polymer that is biodegradable, biocompatible, and safe for use in humans. A biodegradable polymer is ideal for immunization purposes, for it can release antigen at the desired rate and does not necessitate an additional surgical step for retrieval of the depleted system. Release of entrapped molecules occurs primarily by bulk erosion of the polymer, although a combination of surface erosion, diffusion of the active agent through the polymer itself, or its release through the pores may also play a part. Biodegradable polymers are natural or synthetic polymers, which degrade to natural products such as small acids formed in metabolic pathways. These can be further metabolized or excreted via normal physiological pathways (Wise et al., 1987). Biodegradable polymers have properties of degrading in biological fluids with progressive release of dissolved or dispersed drug. The time scale over which the controlled release is envisaged can be weeks and frequently in months. Many natural and synthetic biodegradable polymers have been investigated as implants, microcapsules, microparticles, microspheres, and nanocapsules in order to achieve prolonged release and targeting of a variety of drugs (Deasy, 1984; Jalil, 1990; Deary et al., 1993; Venkatesan etal., 1995). B. Natural biodegradable polymers Natural polymers, particularly proteins, have been extensively investigated as drug carrier systems to achieve prolonged and site-specific targeted drug delivery. The use of natural polymers to deliver drugs continues to be an area of active research despite the advent of synthetic biodegradable polymers. Natural polymers remain attractive primarily because they are natural products of living organisms, readily available, relatively inexpensive, and capable of a multitude of chemical modifications. A majority of investigations of natural polymers as matrices in drug delivery systems have centered on proteins (collagen, gelatin, albumin, haemoglobin, casein etc.) and polysaccharides (chitosan, dextran, starch, hyaluronic acid etc.). The specific physical properties that contribute to the rate of degradation are summarized below: 1. The water permeability and water solubility, 2. The crystallinity of the polymer, 3. The glass transition temperature, and 4. The physical dimensions. C. CHITOSAN The use of natural polymers as drug carriers has received considerable attention in dosage form design, especially from the viewpoint of safety. The polycationic polysaccharide, chitosan ([1-4] 2-amino-2 deoxy-p -D-glucan) is , after cellulose , the most abundant polymer found in nature, Chitosan possesses valuable properties as a biopolymer. It has a molecular weight ranging from 1-3* 105 Da. It is the N-deacetylated product of chitin (1-4 - linked-2-acetamide-2~deoxy-D-glucan), which is one of the polysaccharides widely distributed in nature as the principle component of shells of crystaceaus and insects and of cell wall of bacteria and mushrooms. Chitosan's unique solubility, solution properties, polycationic character; physical attributes and chemical and biological activity make it an attractive biopolymer for many biomedical applications. Moreover, the biocompatibility and biodegradability of this novel polysaccharide have been well established. dye-binder for textiles, a strengthening additive in paper (Ashford et al., 1997) and a hypolipidis material in diets (Fukunda et al., 1991). It has been used extensively as a biomaterial (Shigemasa & Minami, 1995), owing to its immunostimulatory activities, anticoagulant properties, antibacterial and antifungal action and for its action as a promoter of wound healing in the field of surgery (Dutkiewicz & Kucharska, 1992). properties. Considering chitosan as a week base, a certain minimum amount of acid is required to transform the glucosamine units into the positively charged, water-soluble form. At neutral pH most chitosan molecules will lose their charge and precipitate from solution. Chitosan is a potential drug carrier, regulating drug release. High molecular mass chitosan is preferably used for sustained release preparations, partially hydrolyzed for the improvement of drug solubility. Table 4: Physicochemical properties of chitosan (Knapczyk et al 1989; Sanford 1990). ? to deliver DNA; ? considering chitosan as a cationic polymer for polycationic complex formation, it is obtained as a precipitate by mixing it with anionic polymers Thanoo B. C. et al., (1992) prepared chitosan microspheres with good spherical geometry and smooth surface by the glutaraldehyde cross-linking of an aqueous acetic acid dispersion of chitosan in paraffin oil using docusate as the stabilizing agent. Hassan E.E. et al., (1992) used as emulsion / polymer cross-linking /solvent evaporation method to prepare magnetic chitosan microspheres containing piroxantrone (oxantrazole) and the effects of formulation factors on various response variables were examined using a central composite experimental design. Knapczyk J. et al., (1992) evaluated the tests used to examine anti-mycotic effects of chitosan and concluded that the joint use of fungal growth and fungal adherence tests is useful for assessing the activity of anti-fungal preparations. Lehr CM. et al., (1992) screened various natural or partially modified polymers for mucoadhesive properties by measuring the force of detachment for swollen polymer from pig intestinal mucosa in a sodium chloride (saline) medium. Chitosan was found to be fairly mucoadhesive. S.R. Goskonda et al., (1993) prepared microbeads using a combination of carboxymethylcellulose sodium and cellulose microcrystalline (Avicel RC-591) and chitosan (sea cure) and the effects of chitosan viscosity on bead formulation and drug release were studied using acetaminophen as a model drug and concluded that spherical beads can be prepared using Avicel RC-501 and different viscosity grades of chitosan. Goskonda S. R. et al., (1993) prepared chitosan beads containing sulfadiazine by ionotropic gelation and studied to determine drug loading and release. Beads containing upto 90% drug could be prepared by this method. The efficiency of drug loading, bead size, opacity, and sphericity increased with drug loading. It was concluded that drug-loaded chitosan beads show potential for use as a dosage form. Genta L et al., (1995) loaded spray-dried microspheres of chitosan with a molecular weight of 70,000 or 750,000 with dexamethasone, and the microspheres were studied with respect to morphology, drug content, particle size distribution, thermal behaviour, powder x-ray diffraction properties, and drug releases profiles. The microspheres exhibited good morphological characteristics and a narrow size distribution. Dexamethasone was entrapped with good encapsulation efficiency. Drug entrapment in the chitosan polymeric network significantly improved the drug dissolution rate. Akbuga J. et al, (1994) prepared cross-linked chitosan microspheres containing furosemide from a w/o emulsion system, and determined the effect of several factors on microsphere properties. Discrete spherical furosemide microspheres having a 350-690 µm diameter range are produced. It was concluded that the properties of chitosan microspheres containing furosemide are affected by various preparation variables, including type and concentration of chitosan, drug concentration, stirring rate. Vural I. et al., (1994) prepared, sulfasalazine microspheres from human serum albumin that was treated with 0.5%-0.3% chitosan to develop a sustained- action sulfasalazine (sulphasalazine) formulation, and evaluated for release compared with microspheres prepared with either substance alone. Incorporation of chitosan into the microsphere retarded the rate of dissolution. In addition, the release rate was influenced by the chitosan concentration. To develop a sustained action diclofenac sodium formulation microspheres were prepared using chitosan and were characterized in vitro for particle size distribution, drug content, and release, and in rabbits for pharmacological activity and ulcerogenicity. A slow release of diclofenac was observed and a good fit to the Higuchi model was demonstrated. Although improvement in antiinflammatory activity was exhibited, no improvement in ulcerogenicity was observed (Acikgoz M. etal, 1995). D. Safety of Chitosan Chitosan is a collective term applied to deacetylated chitin in various stages of deacetylation and depolymerization. Almost all functional properties of chitosan depend on the chain length, change density and charge distribution. Numerous studies have demonstrated that the salt form (Lehr et al., 1992) molecular weight; degree of deacetylation (Sabnis et al., 1997) as well as the pH at which chitosan is used (Artursson et al., 1994) influence the properties of this polymer in drug delivery systems. Therefore, these factors must be considered carefully during formulation optimization of dosage forms. In addition, regulatory requirements concerning the use of chitosan in humans will be far more demanding (Weiner, 1992). It has been reported that the purity of chitosan influences its toxicological profile. Therefore, it would stand to reason that only the highest purity of chitosan would satisfy the standards set by regulatory agencies. E. Microspheres Many of the more biocompatible polymers can be used as small soluble molecular drug carriers or can be assembled as both soluble and particulate drug vehicles. These are free-flowing spherical microparticles, ranging in size from a few micrometers to about 200 µm. The term microcapsule usually refers to a reservoir-type system in which the active molecules are enclosed in the cavity surrounded by the polymeric membrane, whereas the term microsphere usually implies a monolithic system in which the active agent is uniformly distributed through the polymer matrix. Microcapsules are expected to provide diffusion-controlled zero-order release, whereas microspheres are expected to provide erosion-controlled first-order release of incorporated drug (Aguado & Lambert, 1992), although in practice, both mechanisms coexist in various proportions. Large amounts of drugs or agents can be incorporated through non-covalent forces into these assembled polymers. These particulate systems are best utilized as sustained release vehicles (Jayakrishnan & Latha, 1997). In the light of the studies reported so far, biodegradable polymeric microspheres appear to have potential applications in the controlled drug delivery. The most important characteristic of the microsphere is the microphase separation morphology, which endows it with controllable variability in degradation rate and drug release. The preparation of microspheres should satisfy certain criteria such as, 1. The ability to incorporate reasonably high concentration of the drug. 2. Stability of the preparations after formulation with a clinically acceptable shelf life. 3. Controllable particle size and dispersability in aqueous vehicles for injection. 4. Release of active agents with a good control over wide time scale. 5. Biocompatibility with a controllable biodegradability. 6. Susceptibility to chemical modification. F. Microencapsulation Several techniques are available for microencapsulation, and the choice of a method depends on the physical and chemical properties of the polymer and antigen to be encapsulated, and the function and desired size of microspheres. A high ratio of antigen to polymer is preferred to minimize the amount of mass that needs to be administered, without compromising the release kinetics. In addition, the microencapsulation technique must afford a pharmaceutically acceptable product relative to residual solvents and processing aids, batch-to-batch reproducibility, ease of scale-up, and high encapsulation efficiency and yields. For commercialization, cost-effectiveness is also an important requirement, especially for product isolation and drying and for solvent disposal. The microencapsulation techniques in use can be broadly classified as 1. Solvent extraction and solvent evaporation 2. Phase separation J. Spray drying Solvent Extraction and Solvent Evaporation The extraction-evaporation microencapsulation methods have been widely used because that can be easily set up in a laboratory and do not require any specialized equipment. A good review of procedures and modifications of solvent extraction and evaporation methods used for microsphere manufacture has been presented (Arshady, 1991). In both these processes, the polymer is first dissolved in a suitable volatile solvent, usually methylene chloride for solvent evaporation, or acetonitrile for solvent extraction. Either the active agent can be incorporated into the polymer solution as a aqueous solution, to form a primary emulsion, or as a solid matrix, which forms a dispersion. In such a system, droplet formation is a dynamic process in which droplets constantly form, collide, and coalesce or redivide. In the solvent extraction process, the solvent for polymer is dissolved away when the emulsion (or dispersion) is added to a suspension medium that is a nonsolvent for the polymer (e.g., heptane). This leads to the formation of solid microspheres in a short period, the microspheres can be recovered either by filtration or centrifugation. Solvent extraction has been used for encapsulation of various peptides and proteins (Reid et al., 1993). On the other hand, in the solvent evaporation process, droplet solidification occurs by evaporation of the volatile solvent at the continuous-phase-air-phase interface. Most commonly, the primary water-in-oil (W/O) emulsion is formed by the aqueous solution of the antigen in the polymer solution, which is later emulsified into a large volume of aqueous phase (typically, aqueous solution of a suitable emulsifier, such as polyvinyl alcohol) to form an water-in-oil-in-water (W/O/W) emulsion. In general, longer-processing times are required to obtain solid microspheres by solvent evaporation. The mixing rate and evaporation time needs to be carefully controlled for reproducibility. Solvent evaporation has been used to successfully encapsulate proteins, including bovine serum albumin, ovalbumin, tetanus toxoid, HBsAg, staphylococcal enterotoxin B toxoid, and peptides, such as leuprolide acetate (Cohen et al., 1991; Alonso et al., 1994; Eldridge et al., 1991; Ogawa et al., 1988). Subjecting the microspheres to vaccum drying can significantly reduce entrapped volatile solvent in the microspheres; however, trace amounts of organic solvents are often difficult to remove. To minimize the safety and regulatory concerns when dealing with organic solvents, liquid carbon dioxide under supercritical conditions has recently been used as a nonsolvent for the polymer. In general, porous, spherical particles, with a broad size distribution, that provide rapid release of incorporated active agent are obtained by the solvent extraction technique, whereas less porous microspheres are obtained by solvent evaporation. Various formulation factors affecting protein release kinetics and stability in biodegradable microspheres. Polymer • Molecular weight • Composition (hydrophilicity / hydrophobicity, amorphous / crystalline) • Uncapped or capped terminal end group • Residual amount of metallic catalyst Protein • Molecular weight • Isoelectric point • Aminoacid composition Formulation • Size and distribution of microspheres • Surface and internal morphology of microspheres • Protein loading amount within microspheres • Residual moisture content in microspheres • Addition of excipients Experimental factors for in vitro release • Buffer capacity of incubation medium • Volume of buffer solution for the incubation of microspheres • Amount of microspheres in the incubation medium • Method of sampling for released protein Protein stability problems • Covalent aggregation via thiol-disulphide exchange reaction • Non-covalent aggregation by hydrophobic interaction • Non-specific protein adsorption onto polymer surface • Ionic interaction between polymer and protein • Peptide hydrolysis • Deamidation • Oxidation Pharmaceutical and biopharmaceutical considerations in the development of a microsphere product (tomlinson & burger, 1984) • Core material • Route of preparation • Size • Type and amount of drug • Drug release (in vitro and in vivo) • Drug stability during preparation and storage • Microsphere stability (in vitro and in vivo) • Storage • Surface properties • Presentation (e.g. Free flowing, freeze dried powder) ACID RESISTANT POLYMERS The first attempt to prevent degradation of antigens by acid involve the administration of antacid solutions prior to vaccination. This method has been used in clinical trials on the development of an oral Cholera Vaccine (Clemens et al, 1986) and Typhoid Vaccine (Gilman et al., 1977). This procedure seemed to be efficient but may be difficult to execute on a large scale. The next stage was the use of polymers for enteric coating to supersede the use of buffer solutions. Enteric protective polymers have been used for the Ty 21 an oral typhoid vaccine (Levine et al., 1987) and have also been used in vaccines against Haemophilus influenza (Clancy et al., 1985) Hepatitis B (Lubeck et al., 1989) and Tuberculosis (Ishihara et al., 1986). Enteric coating is an efficient means of protecting antigens against inactivation by gastric acid. This type of coating may not be sufficient to protect against proteolytic degradation in the intestine. Therefore, the different oral additives like protease inhibitors and penetration enhancers which may be added internally or externally will also contribute to the overall effective response. ORAL ADJUVANTS An adjuvant is a substance administered concurrently with an antigen to potentiate the immune response. The only adjuvants suitable for human use are aluminium salts and gels. Aluminium adjuvanated vaccines have a number of limitations, for example, cell-mediated immunity is difficult to achieve and adverse stimulation of local IgE responses can occur. For these reasons, a number of alternative technologies have been investigated whereby the immunogenicity of such vaccines can be increased, while at the same time reducing the number of doses required. To promote retention in the gastrointestinal tract to aid absorption mucoadhesive microspheres can be used. Mucoadhesive microspheres, when used as antigen carrier for oral delivery, may achieve increased residence time within the gastrointestinal tract. Chitosan has been shown to have mucoadhesive properties. It is a polysaccharide prepared from crustacean shells, is biocompatible and naturally reabsorbed by the body and has been previously used for sustained drug release, bone induction material (Chandy et al., 1991; Klokkevold et al, 1992;). ABSORPTION ENHANCERS Oral route is with out question the most popular but yet the most complex route of drug delivery .The epithelial surface of the small and large intestine is different from that of the skin, buccal, and nasal epithelial. In order to promote oral absorption of antigens, co-administration of intestinal permeation enhancer is needed. Intestinal epithelial cells can be divided into several types: the goblet cells are mucus secreting cells; the enterocytes, the most numerous of the epithelium, are absorptive cells; and there are also some endocrine cells. Epithelial cells originate from the crypts of lieberkuhm and differentiate into various types of cells during migration to the top of the microvilli. The primary function of the intestine is absorption of nutrients and its permeability properties are quite different from those of other mucosal epitheliums. Because of the mucus covering of the epithelial surface of the intestine, the layer of water adjacent to the mucosal surface is essentially unstirred. This layer is often called the unstirred water layer (UWL) with a thickness up to 400 µm. Studies have shown that if the absorption of substances by the cell is fast, diffusion across this UWL layer becomes a rate limiting step (Csaky et al., 1984). Most small lipid soluble non-ionized drugs can be absorbed by diffusion across the epithelial membrane; small polar drugs are absorbed by a transporter-mediated absorption or via the paracellular route. Absorption enhancers, commonly called penetration enhancers, facilitate transport of coadministered substances across biological epithelial barriers. Human epithelial membranes exclude many substances from entry into the human body and limit others with low absorption rates based on their physical and chemical properties. As a result, many drug candidates with low permeability across human epithelial membranes have to be administered intravenously or intramuscularly. This greatly limits dosing frequency, can cause low patient compliance and, in many cases renders otherwise effective drugs useless. With the rapid development of biotechnology, more and more protein, peptide, and nucleotide drugs are becoming available, most of which have low membrane absorption characteristics including • A large size with high molecular weight • Domains of different hydrophobicity • Irregular shapes and • Delicate structures easily inactivated. These drugs are unable to cross membrane barriers in therapeutic amounts and thus research into penetration enhancers becomes even more important. Designing a Good Penetration Enhancer Many penetration enhancers are both drug and site specific. A good penetration enhancer for a certain drug at a certain site may not be a good enhancer for another drug at another site. Different drugs have different physico-chemical characteristics and compatibility with various penetration enhancers. Similarly, each administration site has its own distinct characteristics in terms of membrane thickness, membrane composition, lipid organization and enzyme activity. All these differences should be considered in the selection and design of an effective penetration enhancer. It is necessary to thoroughly understand the physical and chemical characteristics of the drug and application site before selecting or designing an effective penetration enhancer. Various relevant studies reported Bile salt can be used to enhance protein and peptide absorption by various routes of administration (Zhou and Po, 1991; Yamamoto et al., 1992). There are 3 major hypothesis to explain the mechanism of mucosal enhancement of protein absorption by Bile salt. 1. The reduction of protein degradation by the enzymes adsorbed within the mucosal layer (like aminopeptidase ) 2. The interaction of bile salts with cell membranes to form a reverse micelle, which acts as a channel to increase membrane permeation. 3. The dissociation of molecular aggregates through micellar solubilization (Li et al., 1992; Yamamoto et al., 1994). Protease Inhibitors. In order to promote oral absorption of peptides and proteins from the G.I. tract, many components of the enzymatic barrier must be controlled; in addition to pepsin from bile and tryspin and chymotrypsin from the pancreatic fluid, various digestive enzymes exist in the mucus, the brush border membrane, and the cytosol of the cell. These enzymes include peptidases, sucrose, maltase, lactase, and lipases. This is the primary metabolic region for digestion. For preventing the degradation of protein and peptides on that region a protease inhibitor is needed, for this study a serine protease inhibitor such as aprotinin is selected, which inhibit trypsin, chymotrypsin, plasmin, and kallikrein. Aprotinin. Aprotinin is a basic (pKa-10) polypeptide comprised of 58 amino acid residues with a molecular weight of 6,512D. Northop in 1936 (Northrop J. .EL, 1936) who defined it as a trypsin inhibitor in a preparation obtained from bovine pancreas. Aprotinin is a member of a family serpins (serine protease inhibitor),which are able to inhibit a range of proteases that have serine residues at their active sites. This inhibition is provided by inactivation of the active serine of the protease by the lysine residue at position 15 of the aprotinin molecule. The activity of aprotinin is expressed in various ways, kallikrein inactivator units (KIU) and trypsin inhibitor units (TIU) have been commonly used. It has its inhibitory effect on target serine protease by forming reversible stoichometric enzyme-inhibitor complexes (David toyston, 1992). In pure chemical systems (those without other plasma proteins) the concentration of aprotinin required to inhibit serine proteases that occur in nature such as trypsin, plasmin, or tissue and plasma kallikrein, varies for each of the enzymes with concentrations of approximately 50 KlU/ml required to inhibit plasmin and approximately 200 KlU/ml to inhibit plasma kallikrein (David Royston, 1992). IV. M CELL BIOLOGY Because mucosal surfaces serve as the portals of entry for a variety of bacterial, viral, and parasitic organisms, it seems logical to target these surfaces for development of immunity. But most current vaccination protocols call for parenteral immunizations, which do not effectively induce mucosal antibodies. The mucosal system differs from the systemic immune system in its main immunoglobulin, its method of stimulation, and also its function. In spite of the distinct advantages offered by oral immunization, only limited success has been achieved in the local immunization strategies that have been pursued. To design a successful antigenic delivery system for oral immunization, one needs to first understand the basics of the common mucosal immune system, with special emphasis on the gut-associated lymphoid tissues (GALT). The largest mass of lymphoid tissue found along the gastrointestinal tract (GIT) are the Peyer's patches (PP) which are the major sites of antigen uptake. Because Peyer's patches (PP) have been proved to be the primary sites of particle uptake, considerable effort has been directed toward specifically targeting antigens to these sites to facilitate antigenic stimulation, as well as to provide a means that will result in enhanced uptake by the M cells. In general, uptake of antigens in particulate form is superior to that in soluble form. Microspheres, because of their inherent particulate characteristics, serve as suitable vehicles for transport of antigens across the intestinal mucosa by way of M-cell uptake. However, the total particulate uptake remains quite insignificant and, therefore, quantitation of the extent of uptake of orally administered particles and evaluation of the factors affecting it have been of paramount interest. V. QUANTITATION OF MICROSPHERE UPTAKE Chess et al. suggested in 1950 that ingested, finely divided particles are taken up from the GIT and are responsible for production of chronic enteritis and systemic lesions. Although the phenomenon of particle uptake from the GIT was studied from time to time, it was not until the 1980s that definitive studies were conducted. Although the extent of uptake is still an area of disagreement, it is generally accepted that intact particle uptake does occur for particles smaller than 10 µm in diameter. It is also generally understood that the uptake is rather limited. This realization has led to several efforts directed toward quantitation of the extent of particle uptake and studies in improving particle characteristics to increase uptake. Because of poor bioavailability of orally delivered microspheres, there is a significant increase in modeling in vivo microsphere uptake using laboratory animals. Approaches to quantitate the uptake have been developed at the cellular and morphological (Pappo & Ermak, 1989; Howard et al., 1993) as well as whole-tissue levels (Ebel, 1990; Jani et al., 1990). Morphological studies have commonly been performed using the rabbit ligated-loop model. Typically, Peyer's patch sections are tied carefully in an anesthetized rabbit to maintain continuation of blood flow, but minimize movement of contents in the intestine into which a suspension of microspheres is introduced. The animal is kept alive for the duration of study (usually, not more than 2 h), after which the Peyer's patches are removed and processed for microscopic observation and counting of particles. Generally, fluorescent microspheres are used to aid in visualization. Howard et al. (1993) developed a unique approach for quantitation of particle uptake in which mesenteric vessels of ligated rabbit loop were monitored for fluorescent polystyrene microspheres. These morphological methods of determining uptake, although quantitatively definitive and anatomically specific, are tedious and require considerable skill. Additional pitfalls of the method are the artificiality of the ligated-loop approach and difficulty in providing longer-term observation of particle uptake. There is also a potential for recounting a microsphere split into consecutive tissue sections. To overcome some of these disadvantages, several researchers have taken a whole-tissue approach. Here, the animals are fed the microparticles orally, and the tissue in which particle uptake is to be quantitated is retrieved and then dissolved or handled otherwise to extract the microspheres, or their components. Jani et al. (1990) extracted freeze-dried ground tissue and quantitated uptake through analysis for polystyrene from the polystyrene microspheres. Particle-related factors that may affect the extent of uptake include the particle size, surface charge, hydrophobicity, and attachment of ligands. Extraneous factors affecting uptake include the formulation vehicle and its volume (Alpar et al., 1989; Lewis et al., 1992), as well as fasted versus fed state of the animal (Ebel, 1990). In general, smaller-sized particles have been reported to have a higher extent of up take. The effect of surface charge has been investigated using liposomes (Tomizawa et al., 1993), and it was observed that negatively charged liposomes were preferentially taken up by Peyer's patches. The effect of hydrophobicity on uptake was reported by Eldrich et al. (1990) from which it was concluded that hydrophobic polystyrene microspheres were preferentially taken up, poly(lactic acid) and poly(lactide-co-glycolideo microspheres were taken up to a lesser extent, and hydrophilic cellulose matrices were not taken up at all. Several ligands, with specific affinity to GALT, have been investigated. Generally the ligand is attached to the surface of the microspheres, either by adsorption or, by covalent linkage. VI. OVERVIEW OF HEPATITIS B VACCINE Hepatitis B- disease and significance Importance of Hepatitis B in persistent carriers: chronic liver disease, cirrhosis and hepatocarcinoma Prevalence: One-third of world population infected 350 million virus carriers 1-2 million deaths per year Risk to persistent infection: 90% in infants born to e antigen positive carrier mothers 30% in post-infant and pre-school children 5-10% in adults High risk factors for infection: Transfusion of contaminated blood Injection of contaminated blood - drug users Contaminated blood products Sexual promiscuity Hepatitis B virus is 100 times more infective than that of HTV Hepatitis Vaccines represent • World's first subunit vaccine • World's first licensed vaccine against human cancer • World's first recombinant expressed vaccine VII. DELIVERY SYSTEMS FOR MICROSPHERES For commercial use, microspheres should be placed in pharmaceutical acceptable oral delivery system. Potential oral delivery systems include tablets, capsules, and dry powder for reconstitution into a suspension. The former two are the most widely used oral delivery systems, but could pose significant technological challenges for delivery of microspheres. Tablets are prepared by compression forces may deform and fuse the microspheres, rendering them into large aggregates on disintegration in aqueous media. Also, compression forces and heat generated during compression may effect the stability of encapsulated antigen. A capsule formulation, although providing more favorable manufacturing conditions, could lead to chemical instability. Commercial capsules are made of gelatin or starch and may have up to 12% moisture to maintain flexibility. The moisture maintains a high relative humidity inside the capsule, promoting chances for hydrolytic cleavage of the biodegradable polymer. Hydrolysis of the polymer may increase antigen release rate and make the polymeric matrix acidic, resulting in potential stability problem for the antigen. Placing the microspheres in a moisture-resistant container for reconstitution with a suitable vehicle before administration presents a suitable alternative. This approach is commercially used with hydrolytically unstable antibiotics (such as erythromycin and cefprozil) and has been extended to clinical investigations of oral microsphere delivery (Tacket et al., 1994). VIII. DRUG RELEASE FROM MICROPARTICULATE SYSTEMS a. Disperse systems for drug delivery have found wide application in pharmacy; initially for external use as creams and ointments and later for oral administration and subcutaneous or intramuscular delivery. The release of a drug from a carrier system is actually a combination of a range of processes. The drug first has to move through the carrier, usually by diffusion through a solid or pores; it has to cross the interfacial layer, at which it may be bound if it is surface active; and finally it must diffuse into the continuous phase. If it has phase separated, the drug may have to dissolve in the carrier particle prior to diffusion, and the time varying hydration of the carrier particle may be important in many systems. The experimental release rate is the result of all these processes, although one is usually considered to be rate limiting and so dominates the kinetics. Some of these processes can be driven in reverse if the concentration of the solute in the external phase is sufficiently high; indeed, this may form a method for loading microspheres. Under these conditions, the reversible equilibrium nature of drug diffusion will be evident. The Fig 4 given below indicates the equilibrium of drug release between particle and continuous phase Consequently the experimental release rate is only indicative of the release process behavior at high dilutions; this is termed a perfect sink experiment, and it is generally desirable in order to simplify the interpretation of the experimental data. In concentrated systems, the experimental release kinetics is also influenced by the reverse reassociation reactions, which are represented here by kr (which may include other compartments, such as readsorption to a surface site on the particles). Obviously this model may be complicated by such factors as surface binding, carrier degradation, and diffusion in the carrier. More complex models involving ionization of the solute and surface binding have also been analyzed (Lostritto et al., 1987; Silvestri et al, 1992), but in general, it is simpler to understand the behavior of perfect sink systems, so experiments are normally performed under these conditions. b. Mechanisms of drug release It is rarely possible to make any interpretation of drug release data unless the experimenter has some preconceived model concerning the structure and composition of the system under study. Unfortunately, many workers give the impression that their systems consist wholly of the monosized spherical particles uniformly loaded with the drug; this is rarely the case, and it is useful to discuss some of the ways in which real formulations depart from this ideal. The particles can depart from a monosized distribution of spheres. They usually have a distribution of sizes, with the occasional large particle having a significant fraction of the total drug mass. Without giving the details of the release mechanisms, this will imply a range of release rates, with the smaller particles showing rapid release and the larger ones a slower component. In theory it is straightforward to integrate the release rate expression over the particle size distribution if this is known. The particles may also be nonspherical; many papers show electron micrographs of "spherical" particles which show minor or gross distortions; these can be induced by freeze drying, so that the dried formulation shows very different release profiles to the suspension formulation. Also the structure with the drug being nonuniformly distributed across the particle section, or multiple phases of drug and carrier. It is evident that if the particles are porous or have a fractal interior structure, an even greater range of uncertainty exists concerning drug distribution and release behavior. Finally, most polymer particles have varying degrees of crystallinity, which can be related to the drug release rate from them. c. Heterogenous particle mechanisms The majority of microparticle systems are heterogeneous, and so drug diffusion through them is complex. We could imagine, for example, polymeric microspheres, in which the crystallinity varies spatially with polymer blend; microspheres in which the drug and carrier are phase separated into distinct domains; and systems where drug diffusion is so slow that degradation of the microsphere was rate limiting. For example, Magenheim at al (1993) reported variations in the release of indomethacin from Polylactic co-glycolic acid (PLGA) and Polylactic acid (PLA) nanoparticles which could be attributed to variations in the solvent permeability of the polymer, (d) Dispersed and phase systems A potentially common system is that in which the drug has phase separated from the particle matrix into pure domains and is then released by diffusion through the polymer. Although systems of this type have been widely studied as macroscopic devices for controlled release many microscopic particles also display these characteristics. Baker and Lonsdale (1974) studied a system of this type, and they found that the release rate was given by: Where Cs is the drug solubility in the dissolution medium and A the drug loading per unit volume. The drug particles are assumed to be small compared with the particle radius. Release of drugs from heterogeneous systems has often been treated using the expression derived by Higuchi (1963) for the flux, Q, from a planar slab of polymer containing the drug: In this expression, cm is the solubility of the drug in the matrix, ct is the initial drug concentration, and cs is the drug solubility in the sink phase. Unfortunately, this expression refers to an infinitely deep matrix, which is not significantly depleted, and so it is rarely applicable if the majority of the particles drug load is released. However, the use of expressions in which release is proportional to the square root of time are so firmly ensconced in pharmaceuticals that little thought is normally given to their significance or applicability. As the loading of solid drug in the matrix is increased, ultimately a point will be reached at which the drug particles are in contact with each other; this normally occurs at loadings of around 10%-20%. In this case, as the drug diffuses out of the matrix, solvent-filled channels are left, which act as pathways through which remaining drug is preferentially released. The full expression for release from a spherical matrix of this type is rather complex (Higuchi, 1963) and involves terms for the porosity and tortuosity of the matrix. These terms can only be accessed with difficulty, and it would appear that such treatments are straining the limits of the pure analytical approach to drug-release modeling. Ultimately numerical simulation may prove useful for these systems, (e) Erodable and biodegradable systems There is an extensive literature on hydrating and eroding systems, which is largely applicable to macroscopic devices for sustained release applications. Eroding devices differ from those previously discussed, because drug release is by definition controlled by erosion of the particle rather than diffusion of drug within the matrix. This represents an increasingly important class of particles owing to the current interest in biodegradable particles for drug delivery and particularly for the delivery of macromolecules such as polypeptides and DNA fractions. The most commonly used materials for such applications are PLA, PLG and their copolymers PLGA; polyanhydrides are also receiving much attention (Tabata & Langer, 1993; Tabata et al., 1993). These polymers can be made to degrade in vivo on a timescale of hours to weeks, depending on the device size and polymer blend, and can easily be made into particles by solvent evaporation methods. The erosion rates are largely controlled by the crystallinity of the polymer, with amorphous polymers showing the most rapid degradation, and polymers degrading more rapidly above the glass transition temperature (Pitt et al., 1981; Izumikawa et al., 1991). The older literature on these systems has been reviewed by Heller (1984). More recent studies have provided a detailed description of the effects of polymer chain length and copolymer mix on release; for example, the work of Le Corre et al. (1994) demonstrated that release rates of bupivacaine from pure PLA microparticles varied with polymer chain length, with the shortest polymers showing the most rapid release rate, and an increased proportion of glycolide also causing an increase release rate. The drug release from a surface-eroding particle, in contrast to a bulk eroding particle is given by: Where ke is an erosion rate constant, r is the initial radius of the sphere, and Co is the concentration of drug in the sphere. There is little modeling work on the release from bulk eroding particles, although these appear to lose no mass until they have reached a critical chain length (m.w. 15,000), after which breakdown is rapid (Pitt et al., 1981). The other major candidates for the production of biodegradable microspheres are naturally occurring proteins and polysaccharides. Albumin microspheres have been studied for many years in this regard (Burger et al., 1985), as have a number of other proteins. Gelatin (Narayani & Rao, 1994) and Hyaluroinc acid (IIIum et al., 1994) are among a range of polysaccharides studied for this purpose. These systems are normally made by a water-in-oil emulsion technique, leading to microsphere sizes of a few micrometers down to hundreds of nanometers if high shear dispersion is used. There is some difficulty in the interpretation of release data from these systems, since they are normally added to the release medium in order to better simulate their degradation in vivo. It may thus appear that surface erosion models would be applicable; however the actual behaviour of the particles is more complex, since the microspheres normally swell prior to being digested, and so one must assume that some enzyme is transported into the core of the particle during this initial swelling phase. The behaviour was demonstrated by Magee et al (1993), who studied the size of albumin microspheres by laser diffraction as they were digested in a trypsin-containing medium; the microspheres swelled to several times their initial diameter over 20-30 mins prior to being degraded over a further 30-60 min by the enzyme. Control of release rates from protein or polysaccharides particles can be achieved by varying the particle size or the degree of cross linking. For example, Jayakrishnan et al. demonstrated that the release of methotrexate from casein microspheres was influenced by the degree of cross linking as a result of varying the amount of glutaraldehyde crosslinker used in the particle preparation; similar results were found by Rubino and by Dilova and Shishkova et al. (1993) for chemically and thermally denatured albumin microspheres, (f) Empirical models of drug release Many investigators have regarded the analytic modeling of drug release as unjustifiably complex and have used more empirical techniques for data analysis. These approaches vary from those, which provide useful data about the system dynamics to those which one is convinced have been adopted simply to minimize the size of error bars in a graph. Probably the most useful of the former group is the diffusional exponent approach described by Peppas and colleagues (1984, 1987), which goes some way to explaining behaviour of hydrating or eroding systems. In such systems, the diffusion coefficient is not constant, and the term anomalous diffusion is often used to indicate that a constant value of the diffusion coefficient does not satisfactorily fit the data. The terms Fickian and non-fickian are also used to indicate whether or not a material is diffusing with a temporally and spatially constant diffusion coefficient; these are somewhat misleading, since, at a particular instant and point in space, diffusion always occurs according to Fick's law. The diffusional exponent method proposes a power law relationship for drug release: The constant n is termed the diffusional exponent, and it should be equal 0.5 for diffusional (Fickian) release from a planar slab. Values greater than 0.5 indicate anomalous diffusion, and are generally indicative of a system, which swells in the solvent prior to diffusional release. Analysis of the model and comparison to the exact solutions demonstrates that n is equal to 0.5 for a flat slab and 0.432 for a sphere. Since simple diffusive release from spheres is often adequately fitted with n = 0.5, it is evident that this approach requires precise data to allow the extraction of a useful value for n. A corollary to this is that many of the experimental techniques in the literature require improvement before they can be used to discriminate between the various mechanisms of drug release. One of the more popular empirical relations is to describe release as a biexponential process: Where k1 and k2 are the rate constants of the two lifetime components into which the decay function is being decomposed. The exponentials usually consist of a rapid and a slow function, being assigned to "burst phase" and "sustained release" respectively. Ideally any new vaccine or antigen delivery system should be capable of being administered orally with all the added advantages. These include easy self-administration, without the need for trained personnel and reduced cost After the success of oral polio drops in mass immunization programs, there is an awakening for the development of oral vaccines for various viral and bacterial diseases, which are the major killer diseases in developed and developing countries. The oral route is by far , the most convenient and popular route for drug delivery. However , the enzymatic barrier remains the most important of the multitude of barriers limiting the absorption of natural protein and peptide drug from the Gl-tract. There is an inverse relationship between the amount of peptide transported across the intestine and its rate of hydrolysis in the intestine . Absorption is further compromised by the resistance exerted by the intestinal membrane to peptide and protein penetration through simple diffusion carrier mediated transport and endocytosis. In order to promote oral absorption of peptides and proteins from the Gl-tract, many of the enzymatic barrier must be controlled. This can be achieved by many ways such as , A. Restricting the release of peptide or protein drug in a region of the Gl-tract that favors its absorption. B. Co-administration of protease inhibitors. C. Co-administration of intestinal permeation enhancer. D. The peptide or protein drugs may be housed within a delivery system that is designed not only to protect the drug from contact with luminal protease but also to release the drug only upon reaching an area favourable to its absorption. Several relevant studies have been reported. The vaccines may be prepared by entrapping antigens in various carrier systems prepared from biodegradable polymers which can be administered orally. The carrier systems can be designed to release entrapped antigens at the appropriate site of the gut. The inhibition of protease activity with suitable inhibitor can protect the vaccines from degradation. The improvement of oral bioavailability can be achieved using suitable intestinal permeation enhancers. The primary candidates for the development of polymeric release vaccines are microparticles and microspheres. Since the microparticles are taken up from the intestine into lymphoid follicles (peyer's patches) following oral administration, they have considerable potential as oral vaccines. Several studies show that the novel vaccines can be formulated by entrapping the vaccines by various synthetic and natural polymers The carrier molecules like liposomes, niosomes, microspheres show sufficient entrapment and good adjuvant ability for the delivery of vaccines orally. Until the 1960's, it was perceived that only systematic mechanisms existed for the production of immunity following an antigen stimulus and detection of antibodies in external secretion was the result of an overflow of the conferred immunity. However the success of polio vaccine by oral administration and poor induction of mucosal immunity by parenteral administration of cholera independent mucosal immune system that can be anatomically and functionally divided into at least two distinct inter connected compartments. The IgA derived from mucosal effector represents more than 75% of all antibody isotype produced in humans. These mucosal inductive sites include the gut-associated and the nasal associated lymphopharyngeal tonsil area respectively. Antigenic stimulation of these tissues causes dissemination of T-helper cells and the IgA precursor β-cells to the effector tissue and to the secretary glands for the subsequent antigen a specific antibody response. Formulation of antigen into particulate carrier systems offer the potential of optimizing delivery to immune responsive sites and also protection of the antigen against proteolytic degradation in the Gl-tract. Therefore the present invention utilizes the induction of mucosal immune response involving the use of particulate delivery systems Therefore the main objective of the present invention is to provide a pharmaceutical composition for the treatment of diseases caused by viruses.. Another objective of the present invention is to provide a pharmaceutical composition for the treatment of diseases of liver, particularly Hepatitis ,more particularly Hepatitis B Still another objective of the present invention is to provide a pharmaceutical composition for the treatment of diseases of liver , particularly Hepatitis ,more particularly Hepatitis B which can be self administered orally in the form of capsules without the help of any trained personnel Still another objective of the present invention is to provide a a pharmaceutical composition for the treatment of diseases of liver , particularly Hepatitis ,more particularly Hepatitis B which is stable at room temperature. Another objective of the present invention is to provide a pharmaceutical composition for the treatment of diseases of liver , particularly Hepatitis ,more particularly Hepatitis B which is based on the use of chitosan microspheres Another objectives of the present invention is to provide a process for the preparation of a pharmaceutical composition for the treatment of diseases of liver , particularly Hepatitis ,more particularly Hepatitis B Over the past several decades, there has been an enormous increase in the development of strategies 7 for novel vaccines and improvement of existing vaccine. Till date composition for the treatment of \ hepatitis is available only in the form of injectables. Administration of such forms requires trained 1 The present invention has been developed to over come the above said problems .The invention is based on the exploitation of the phenomenon of microencapsulation by double emulsification techniques. We exploited the phenomenon of microencapsulation by double emulsification techniques. The antigen (Hepatitis B) is entrapped in microspheres using biodegradable polymers. The formulation includes some adjuvants to prevent the peptidic degradation of proteinaceous antigenic moiety and to enhance the absorption. The microspheres are released only in the intestine where they get absorbed through peyer's patches Accordingly , the present invention provides a pharmaceutical composition in the form of capsules i useful for the treatment of disease caused by viruses such as Hepatitis , AIDS and the like which ^ comprises microspheres of Chitosan incorporating an appropriate antigen encapsulated in enteric coated gelatine capsules The antigen especially Hepatitis B is incorporated into the microspheres of Chitosan , a biodegradable polymer. In order to increase the oral bioavailability of the antigen we have prepared the pharmaceutical composition in the form of gelatin capsules capable of oral administration , containing antigen particularly Hepatitis , more particularly ,Hepatitis B surface; antigen. Till date such a delivery system is not known . The composition may include some adjuvants to prevent the peptidic degradation of proteinaceous antigenic moiety and to enhance the absorption. The microspheres are released only in the intestine where they get absorbed through peyer's patches. The capsules has been developed by targeting the microspheres to intestinal Peyer's patches by encapsulating in enteric-coated gelatin capsule shell. This enteric coating protects the microspheres in contact with gastric medium and allows the microspheres get released in the intestine. In an embodiment of the invention there is also provided a process for the preparation of the pharmaceutical composition in the form of capsules useful for the treatment of disease caused by viruses such as Hepatitis , AIDS and the like as defined above which comprises (i) preparing a solution of Chitosan in a dilute acetic acid (ii) preparing an aqueous solution of the desired surface antigen (iii) mixing the solutions of the surface antigen and the solution of the Chitosan by stirring (iv) pouring the resulting homogenized mixture into liquid paraffin to form an emulsion (containing an emulsion stabilizer )and agitating the mixture (v) adding an organic solvent to the resulting mixture the solvent used being immiscible with the internal phase of the emulsion so as to get the microspheres (vi) centrifuging the microspheres and decanting to remove the oil phase (vii) washing the microspheres using the same solvent used in step(vi) (viii) repeating the washing followed with washing with a polar solvent (ix) encapsulating the microspheres in gelatine capsulesand (x) coating the resulting capsules with a enteric polymer The method followed for the preparation of chitosan microspheres used for preparing the solution was emulsion-solvent evaporation method as described by Thanoo et al., (1992). A solution of chitosan (molecular weight 3.15 X 105; degree of deacetylation 74%) was made using dilute mineral acid such as . 6% v/v aqueous acetic acid The aqueous solution of the Hepatitis B surface antigen was incorporated into this using homogenizer. A primary emulsion (w/o) was prepared using liquid paraffin as the external phase using a high-speed homogenizer. Span-80 was used as the preferred emulsion stabilizer at varying concentrations. After homogenizing, stable emulsion was formed, glutaraldehyde saturated toluene (GST) was added as cross-linking agent at varying concentrations at specified time intervals.The resulting reaction mixture was poured into bulk of liquid paraffin containing emulsion stabilizer. The mixture was agitated using a mechanical stirrer for 5 hours(. Appropriate volume of n-hexane was added and microspheres were collected by centrifugation at 1500 rpm for 30 min. The supernatant was decanted off and the pellets were resuspended in n-hexane for the purpose of washing off the oil phase. The washing /centrifugation steps were repeated 2-3 times followed by washing with methanol (once)and three times with ice cold distilled water. Finally, collected microspheres were vaccum dried and stored in an air tight, amber colored container under refrigeration. Emulsion stabilizers may be added may be in step(iv) for stabilization of the emulsion formed Such stabilizers may be selected from SPANS , like SPAN 20 , SPAN 40and the like .Their amount mat range from 0.5 to3 % w/v . The agitation in step (iii)may be done preferably for a period ranging from 10 tol5minutes and that for step(iv) may range from 5 to 6 hrs The organic solvent used in step (v ) may be selected from hexane , acetone , ethyl alcohol and the like The details of the invention are given in the examples given below which are provided only to illustrate the invention and therefore should not be construed to limit the scope of the invention We have prepared 11 formulations employing different concentrations of Chitosan antigen and stirring speed , solvent etc . The results of these experiments are given below From the information given in the Table shows that increasing the concentration of Chitosan resulted in the mean diameter of the microsphers . We have taken the composition identified as F VII in the above table , for evaluating the efficacy of the composition of the present invention Entrapment efficiency (antigen content) The efficacy of the microparticulate of the present invention will be affected by antigen loading and integrity. Thus accurate estimation of encapsulated antigen is an essential requirement for quality control of the proposed controlled release vaccines. Many methods have been reported for the estimation of the efficacy of antigen entrapment and of total core protein loading. One of those methods is measuring the total protein antigen loading of the known quantity of chitosan microspheres following complete digestion in solvents. The chitosan microspheres loaded with HBsAg were digested in an HC1 0.1 N / ethanol (1:1 v/v) mixture for 48 hours under mechanical agitation. The resultant solution was centrifuged and supernatant solution was collected and absorbance obtained by EOSA method was utilized for computing antigen content. The procedure was performed in triplicate for each of the batches. IN VITRO DRUG RELEASE STUDIES Dissolution of HBsAg from chitosan microspheres was carried out on all batches of HBsAg-loaded chitosan microspheres. Dissolution apparatus (USP II) with 50 rpm basket rotational speed was used in the studies. PBS (pH 7.4) was used as a dissolution medium. A known quantity of antigen loaded microspheres contained in a capsule were suspended in PBS in dissolution flasks maintained at 37±1° C at a speed setting of 50 rpm. The release study was carried out for a period of 30 hours. Aliquot of samples were withdrawn at regular predetermined intervals (for the first eighteen hours every three hours, next sample after six hours and last two samples after every three hours), filtered and analyzed for antigen content by ELISA method. To maintain a constant volume of release medium, a volume of fresh medium equivalent to the volume of sample withdrawn was added immediately after withdrawal of the sample. The in vitro studies were performed in triplicate for each of the batches. IN VIVO EVALUATION Animals The animals (Swiss Rabbits) were obtained from National Institute of Nutrition, Hyderabad. The experimental protocol for all the in vivo studies was approved by the Institutional Animal Ethical Committee (vide letter number - IAEC / KMC / 04 / 2002 dated 4 February 2002), which is an approved body under CPCSEA 1998. The animals were maintained under controlled conditions of temperature and humidity. They were fed balanced diet (obtained from Lipton India Ltd. Foe animal use) and water. Study Parameters 1. The appearance of antigen and its titre in serum for the composition (identified as F-VII in the above table) and modified (F-M) formulations. 2. Comparison of antigen titre of oral dosage form and intramuscular injection of HBsAg. Antigen Administration Four groups (Group A, B, C, & D) of animals, each having three rabbits were taken for the in vivo studies. The chitosan microsphere formulation ( F-VII shown in the table ) was taken for in vivo studies. The enteric coated capsules containing these antigen-loaded microspheres equivalent to 20 µg of antigen were administered to the over night fasting animal. The antigen administration modalities with other details are given in the following table 6. Sample collection After administering both HBsAg intramuscular injection and capsules to the respective groups, immediately 1 ml of blood was withdrawn from the marginal vein. Next samples were collected after every 24 hours intervals for fourteen days. The sera were separated by centrifugation and ELISA Technique detected antigen. *PA: Penetration enhancer (Sodium taurocholate) **PI: Protease inhibitor (Aprotinin) ELISA TECHNIQUE Hepanostika HBsAg Uni-Form II is an ELISA based on a one-step "sandwich principle. Antibody to HBsAg (anti-HBs) coupled to horseradish peroxidases (HRP) serves as the conjugate with tetramethylbenzidine (TMB) and peroxide as the substrate. Upon completion of the test, the development of color suggests the presence of HBsAg, and no or low color development suggests the absence of HBsAg. Specifically, microelisa wells are coated with anti-HBs (murine monoclonal). Each microelisa well contains an HRP-labeled anti-HBs (ovine) conjugate sphere. The test sample or appropriate control containing HBsAg is incubated in the microelisa wells for 60 or 90 min, at 37 °C .The conjugate sphere dissolves in the sample and a solid phase antibody / HBsAg enzyme-labeled antibody complex is formed. Following wash and incubation with TMB (tetramethylbenzidine) substrate a blue color is produced. The enzyme reaction is stopped by the addition of a sulfuric acid solution, which changes the color to yellow. When HBsAg is present in the sample, an intense color develops. However, if the sample is free of HBsAg, no or little color forms after the addition of substrate. Within limits, the amount of HBsAg in the sample is proportional to the degree of color development. Blank the reader on air and then microwell reader reads the absorbance of the solution in each well at 450 ± 5 nm. Reagents / Accessories REAGENT: 1 Sample diluent: Contains Tris buffer and detergent REAGENT: 2 Conjugate: Monoclonal anti HBsAg antibodies conjugated with peroxidase containing protein stabilizers. (Ready-to-use) REAGENT: 2A Conjugate Stabilizer: Tris buffer containing stabilizer, and preservatives. (Ready-to-use) REAGENT: 3 Washing Buffer (10X): Concentrated (10X) Tris buffer containing Tween-20 and Thiomersal (0.01%) as a preservative. Before use, dilute by adding one volume of concentrate to 9 volumes of Distilled or Reagent Grade water (Span product no. 2366A or equivalent). REAGENT: 4 Negative Control: HBsAg Negative serum containing preservative. (Ready-to-use) REAGENT: 5 Positive Control: HBsAg Positive serum containing preservative. (Ready-to-use) REAGENT: 6 Color Reagent A: Citrate Acetate buffer containing peroxide. (Ready-to-use) REAGENT: 7 Color Reagent B: 3, 3' 5, 5' Tetramethylbenzidine (TMB) solution. (Ready-to-use) REAGENT: 8 Stopping solution: Mineral Acid REAGENT: 9 Microwell Strips coated with anti-HBs antibodies Accessories Adhesive strips covers Setting up the Test 1. Washing buffer: Dilute the concentrated washing buffer 1:10 (1+9) with distilled or Reagent grade water. 500 ml of diluted buffer is enough to wash all the 96 wells. If all the strips are not used at a time, prepare the proportionate amount of Washing Buffer. All the other reagents are supplied ready to use. 2. General: Blank, Positive and Negative Controls must be included with each run. All liquid reagents must be gently mixed before use. Before addition, all reagents should be brought to room temperature. Assay Procedure 1. Add 100 µ of sample diluent (Reagent 1) to test and control wells of the microwell strips (Reagent 9). Add 100 µl of test serum or control (Reagent 4, 5) to the respective wells. Keep first well as reagent blank. 2. Cover with adhesive strips and incubate at room temperature (25°C to 40°C) for 1 hour. 3. Discard the adhesive strip cover and aspirate contents of the well and add 50 µl of conjugate stabilizer (Reagent 2A) first, followed by 100 µl of conjugate (Reagent 2) to each well. 4. Incubate at room temperature for 30 minutes. 5. Wash five times with 350 µ1 of washing buffer (Reagent 3), providing a soak time of 30 seconds in between each wash. 6. Add 50 µ1 of color Reagent A (reagent 6) and 50 |xl of color Reagent B (reagent 7). 7. Incubate at room temperature for 30 minutes (in dark). 8. Add 100 µl of Stopping solution (Reagent 8). 9. Read at 450 nm (using 630 nm as reference wavelength when bichromatic EOSA Reader is used). ELISA was chosen as a method of analysis for the product and preferred over the conventional Lowry's method for protein content determination because: 1. ELISA involves the determination of the exact Hepatitis B surface antigen (i.e. in ng quantities), whereas Lowryfs method does not give the information. 2. ELISA measures the HBsAg content with exact precision whereas there are always some chances of errors in the conventional methods. 3. ELISA requires very minute quantities of the sample to be analyzed whereas in the conventional Lowry's method the amount of the sample required for analysis is at least l ml. 4. Exact quantitative results can be obtained with positive and negative means in ELISA, which can then be correlated with the amount of antigen released from the microspheres, whereas in Lowry's method this is not possible. 5. Since ELISA reader is a fully automated machine employing an automated sample withdrawal probe, so chances of introduction of error due to volume of the sample introduced are very less. 6. ELISA measures and takes into account the specific wavelength criterion for measuring the Hepatitis B surface antigen and does not take into consideration the protein content obtained by other active peptide species as in Lowry's method. Interpretation of the Results The OD values obtained for each sample of the Group A (control i.e. no treatment group) were subtracted from the OD values for each sample of Group B, C & D (treatment groups) to get the exact OD value of the antigen. The antigen titre level was calculated by using regression equation of the calibration curve of HBsAg in rabbit serum. Form the above it can be observed that the system of the present invention can be self-administered and does not require the expertise of any trained personnel. It also does not produce side effects, such as serum sickness and immune complex deposition in chronic HBV carriers, seen with the present Hepatitis B vaccines administered intramuscularly. Further modification in the release rate by alteration of the polymer and cross linking agent concentration can lead to significant antibody titer levels. The system is stable at room temperature. The advantages of the invention are as follows: 1. The Delivery system overcomes the difficulties and problem associated with vaccine delivery and stability . The hurdles such as stimulating immunity at the most effective site, reducing the need for repeated injection to overcome short lived immunological memory are also solved. 2. The antigen delivery system can be administered orally. 3.The antigen delivery system is simple and economical. 4. The antigen delivery system can be self administered without the help of any trained personal 5. The antigen delivery system is stable at room temperature. We Claim . A pharmaceutical composition in the form of capsules useful for the treatment of disease I caused by viruses such as Hepatitis , AIDS and the like which comprises microspheres of Chitosan incorporating an appropriate antigen encapsulated in enteric coated gelatine capsules * —■—■—•—-— ■ ——— —.....—-—__ ■"* —..,—. 2. A pharmaceutical composition as claimed in claim 1 wherein the antigen used is Hepatitis B 3.A pharmaceutical composition as claimed in claims 1 & 2 wherein the composition contains enzyme inhibitors , stabilizers an absorption enhancers to prevent peptidic degradation of proteinaceous antigenic moiety and to enhance the absorption. 4.A pharmaceutical composition as claimed in claim 3 wherein the enzyme inhibitors are selected from Bacitracin , Arotinin , Chymostatin etc 5. A pharmaceutical composition as claimed in claims 3 and 4 wherein the amount of enzyme inhibitors used ranges from 1 to 2 % w/y 6. A pharmaceutical composition as claimed in claims 3 to 5 wherein the stabilizers are selected from SPAN such as SPAN 20, SPAN 60 and the like wide 7. A pharmaceutical composition as claimed in claim 6 wherein the amount of stabilizers used ranges from 0.5 to3 % w/v 8, j A pharmaceutical composition as claimed in claims 1 to7 wherein the absorption enhancers stabilizers are selected from EDTA, Sodium deoxycholate , oleic acid, Sodium taurocholate and the like wide 9, A pharmaceutical composition as claimed in claim 8 wherein the amount of absorption enhancers stabilizers used ranges froml to 2 % w/v 10. A pharmaceutical composition as claimed in claims 1 to 9 wherein the concentration of antigen in the composition ranges from 10 to20micro gams 11.A process for the preparation of the pharmaceutical composition in the form of capsules/useful for the treatment of disease caused by viruses such as Hepatitis , AIDS and the like as defined above which comprises (i) preparing a solution of Chitosan in a dilute acetic acid (ii) preparing an aqueous solution of the desired surface antigen (iii) mixing the solutions of the surface antigen and the solution of the Chitosan by stirring (iv) pouring the resulting homogenized mixture into liquid paraffin to form an emulsion and agitating the mixture (v) adding an organic solvent to the resulting mixture the solvent used being immiscible with the internal phase of the emulsion so as to get the microspheres (vi) centrifuging the microspheres and decanting to remove the oil phase (vii) washing the microspheres using the same solvent used in step(vi) (viii) repeating the washing followed with washing with a polar solvent (xi) encapsulating the microspheres in gelatine capsulesand (x) coating the resulting capsules with a enteric polymer 12. A process as claimed in claim 11 wherein the enzyme inhibitors are added to the mixture obtained in step (iii) 13. A process as claimed in claim 1 2 wherein the enzyme inhibitors are selected from Bacitracin Arotinin, Chymostatin etc 14. A pharmaceutical composition as claimed in claims 12 and 13 wherein the amount of enzyme inhibitors used ranges from 1 to 2 % w/v 15.A process as claimed in claim 11 to 14 wherein the stabilizers is added in step (iv) for stabilization of the emulsion formed 16.A process as claimed in claim 15 wherein the stabilizers used are selected from SPANS , like SPAN 20 , SPAN 40aitfl the like and their amount ranges from 0.5 to3 % w/v . 17.A process as claimed in claims 11 to 16 wherein the absorption enhancers are added to the mixture obtained in step(iii) 18. A process as claimed in claim 17 wherein the absorption enhancers used are selected from EDTA, Sodium deoxycholate , oleic acid, Sodium taurocholate and the like 19. A process as claimed in claims 17 & 18 wherein the amount of absorption enhancers used ranges froml to 2 % w/v 20. A process as claimed in claim 11 & 19wherein the agitation in step (iii) is effected for a period ranging from 10 tol5minutes and that for step (iv) ranges from 5 to 6 hrs 21. A process as claimed in claims 11 to 20 wherein the organic solvent used in step (v) is selected from hexane , acetone, ethyl alcohol and the like 22. A pharmaceutical composition in the form of capsules/useful for the treatment of disease caused by viruses such as Hepatitis , ADDS and the like substantially as herein described , 23. A process for the preparation of a pharmaceutical composition in the form of capsules (useful for the treatment of disease caused by viruses such as Hepatitis , AIDS and the like substantially as herein described |
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634-mas-2002-claims granted.pdf
634-mas-2002-correspondnece-others.pdf
634-mas-2002-correspondnece-po.pdf
634-mas-2002-description(complete) filed.pdf
634-mas-2002-description(complete) granted.pdf
634-mas-2002-description(provisional).pdf
| Patent Number | 222692 | |||||||||||||||
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| Indian Patent Application Number | 634/MAS/2002 | |||||||||||||||
| PG Journal Number | 47/2008 | |||||||||||||||
| Publication Date | 21-Nov-2008 | |||||||||||||||
| Grant Date | 20-Aug-2008 | |||||||||||||||
| Date of Filing | 29-Aug-2002 | |||||||||||||||
| Name of Patentee | THE COLLEGE OF PHARMACEUTICAL SCIENCES | |||||||||||||||
| Applicant Address | MANIPAL ACADEMY OF HIGHER EDUCATION (A DEEMED UNIVERSITY), MAHE, MANIPAL, MADHAV NAGAR, MANIPAL 576 119, | |||||||||||||||
Inventors:
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| PCT International Classification Number | A61K39/00 | |||||||||||||||
| PCT International Application Number | N/A | |||||||||||||||
| PCT International Filing date | ||||||||||||||||
PCT Conventions:
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