Title of Invention	"FORMULATION COMPRISING A PROTEIN ENRICHED FRACTION OBTAINED FROM A PLANT OF TINOSPORA SPECIES"
Abstract	The present invention provides a formulation comprising a protein enriched fraction obtained from a plant of Tinospora cordifolia together with additives selected from alum, lipids and immunomodulators.

Full Text

FIELD OF THE INVENTION The present invention relates to a method of enhancing the vaccine efficacy for prevention and treatment of infections and diseases using extract of Tinospora cordifolia, a composition including extract of Tinospora cord/folia used in the above method and a process for preparing such a formulation. BACKGROUND OF THE INVENTION: Vaccination represents an immunologicai strategy that sensitizes the immunocompetent cells of the host against specific antigens expressed on the disease causing agents for generation an immunological memory so that the host could elicit an appropriate and adequate immune response to subsequent exposures to the same disease causing agent. Based on this concept, the vaccines have primarily been used as a prophylactic measure, although they may also have therapeutic effects. Immune response is basically of two types: 1) humoral immunity involving an antibody mediated elimination of the disease causing agent and 2) a cell mediated immunity involving specific effector cells directed against infected or altered cells. Predominance of one of the immune responses, i.e., either antibody or cell mediated, has been linked to selective activation of Th2 or Th1 type of T eel! subsets, respectively. While an antibody mediated response may have protective effect against extracellular pathogens and their soluble products like toxins, cell meditaed immunity is more effective against intracellular parasites and tumors. The key factor involved in vaccines is an adjuvant which given along with arr antigen enhances or amplifies the antigen specific immune response. The adjuvant, basically an immunomodulator, provides a stimulus to the immunocompetant cells, especially the antigen presenting cells and/or T cell subsets, and modulates the immune response to generate either Th1 or Th2 response. Efficay of a vaccine therefore depends on the enhanced response of the desired type against a specific disease causing agent. In the present senario of AIDS, malaria and cancer, there appears to be great need for developing new immunoadjuvant formualations customized for induction of Th-1 specific responses that may provide protective immunity against such diseases. PRIOR ART: In the prior art six types of immunoadjuvants are known. These immunoadjuvants are 1) Alum, 2) Saponins, 3) non-ionic polymer surfactants, 4) monophosphoryl lipid A, 5) muramyl dipeptides and 6) cytokines (Audibert and Use, 1993). The essence of the above immunoadjuvants are given hereinbelow: 1) Alum: Alum has been widely used as an adjuvant for preventive vaccines. It has been shown to enhance predominently the humoral response. 2) Saponins: Quail-A, a saponin derived from Quillaia saponaria, mixed with cholesterol has been used as immunostimulating complexes (ISCOMS) alongwith a variety of antigens (Morein era/., 7987; Claasen and Osterhaus, 1992). It is known to enhance both humoral and cell mediated immune response. ISCOMS have also been shown to have adjuvant activity when given orally. 3) Non-ionic block polymer: Non-ionic block polymer (NBP) surfactants, consisting of hydrophobic polyoxypropylene and hydrophilic polyoxyethylene, have been used as adjuvant in oil-in-water emulsions against peptides, proteins and polysaccharides (Hunter et a/., 1991). NBP suractants bind proteins to the surface of oil drops and activate macrophages and enhance their ability to present antigens to T cells and shift response to Th-1 type. 4) Monophosphoryl lipid A: Monophosphoryl lipid A (MLA) is derivative of gram negative bacterial lipopolyscahharide (IPS). MLA promotes IFN-g production by T cells (Tomai and Johnson, 1989) and TNF-a by macrophages (Ulrich et a/., 1991). These cytokines in turn induce a Th-1 response (Takayama et at, 1991). 5) Muramyl dipeptides: Muramyl dipeptide (MDP) is a component of Mycobacterial cell wall with immunoadjuvant properties (Ellouz et a/., 1974). A number of MDP derivatives have been synthesized. MDP given as adjuvant in saline mainly enhances antibody response whereas given in water-in-oil emulsion increase both cell mediated and humoral immunity (Warren et a/., 1986). 6) Cytokines: Cytokines are mediators of immune response and have been shown to act as modultors of immune response. Recombinant cytokines like IL-1, IL-2, and IFN-y have been shown to have adjuvant activity (Heath and Playfair, 1992). More recntly IL-12 has been shown to induce protective immunity against Malaria. Tinospora cordifoiia: The new immunoadjuvant formulation described in this invention, uses a plant formulation prepared from Tinospora cordifoiia, belonging to family Menispermaceae. Tinospora species have been widely used in Indian traditional medicine for skin infection, arthritis, fever, dysentry, urinary tract infections, and diabetes (Gupta et al.,, 1967; Sharma and Sharma, 1981; Raghunathan and Sharma, 1969). Tinospora cordifoiia has also been reported to protect mice against E.coli induced abdomial sepsis (Thatte et al., 1987, Thatte and Dahanukar, 1989). Crude preparations of Tinospora cordifoiia has been used in preparing herbal formulation for treatment of skin diseases (Shah, US patent # 5,693,327, 1997), diabetes (Dhaliwal, US patent # 5,886,029, 1999), arthritis (Chavali et al, US patent # 5,683,698,1997). None of these reports/patents indicate the use of this plant material as described in this invention. SUMMARY OF THE INVENTION: The present invention provides a new method of including antigen specific cell mediated and Th-1 type antibody response for enhancing the efficacy of a vaccine against infections and tumors, using extract of Tinospora cordifoiia, and a formulation comprising extract of Tinospora cordifoiia for inducing antigen specific cell medicated and Th-1 type antibody response for enhancing the efficacy of a vaccine against infections and tumors. OBJECTS OF THE INVENTION The main object of the invention relates to a new method of including antigen specific cell mediated and Th-1 type antibody response for enhancing the efficacy of a vaccine against infections and tumors, using extract of Tinospora cordifolia. Another object of the present invention provides an immunoadjuvant formulation including extract of Tinsopora cordifolia for enhancing vaccine efficacy. Yet another object of the present invention relates to a process for preparing an immunoadjuvant formulation which includes extract of Tinospora cordifolia. Still another object of the present invention relates to a process for preparing the formulation comprising of a glycoprotein enriched fraction derived from Tinospora cordifolia DETAILED DESCRIPTION OF INVENTION The present invention describes a new formulation comprising of a plant product for inducing antigen specific cell mediated and Th-1 type antibody response for enhancing the efficacy of a vaccine against infections and tumors The present invention also provides a new formulation for enhancing immune response of vaccines. The immuno-enhancer formulation is comprised of a protein fraction prepared from Tinospora cordifolia, which induces antigen specific cell mediated immune response and Th-1 type antibody response. The immuno-enhancer formulation comprising of a glycoprotein enriched fraction from Tinospora cordifolia can be given as a physical mixture or chemically linking with the desired antigen. It can also be given separately during the time of conventional immunization. The formulation described here can be adsorbed on alum or complexed with lipids or combined with any other immunomodulator for synergistic action. It can also be given in the form of an emulsion/ suspension/ solution/ syrup/ capsule/ tablet or incorporated in liposomes, microspheres, cream, ointement, gel, spray, powder. The formulation can be administered through intra-muscular, subcutaneous, intra-dermal, intra-venous, intra-nasal, inhalation, oral, genital, rectal or topical application on skin or any mucosal openings. The formulation comprising of Tinospora protein fraction can be used with any vaccine against any infections or tumors. The present invention also relates to a method of prevention and treatment of infections and cancer in a patient, said method comprising administering a pharmaceutical effective amount of extract of Tinospora species preferably, Tinospora cordifolia to the patient. In the process of the present invention, any species of Tinospora can be used. The formulation of the present invention is a synergists composition having enhanced/surprising properties. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS: Figure 1 represents the effect of the glycoprotein fraction obtained from Tinospora cordifolia, on production of IL-12 by peritoneal macrophages, in vitro Figure 2 represents the effect of the glycoprotein fraction obtained from Tinospora cordifolia, on the antigen specific T cell recall response, in vitro Figure 3 represents the effect of the glycoprotein fraction obtained from Tinospora cordifolia, on the antigen induced production of IFN-g by T cells, in vitro Figure 4 represents the effect of in vivo treatment of mice with the glycoprotein fraction obtained from Tinospora cordifolia, on the antigen specific T cell recall, ex vivo. Figure 5 represents the effect of the glycoprotein fraction obtained from Tinospora cordifolia used as adjuvant on the production of antigen specific antibody level in the serum. Figure 6 represents the effect of the glycoprotein fraction obtained from Tinospora cordifolia used as adjuvant on the antigen specific isotypes of serum immunoglobulins. The method described herein is demonstrated by the following examples which should not be construed as limiting the scope of this application. 1. Method of preparation of Tinospora protein fraction for use as immuno-enhancer formulation: The Tinospora protein fraction for use as immuno-enhancer formulation is prepared by any of the following methods: Method 1: Fresh or dried parts of Tinospora cordifolia, preferably the stems, is grounded and soaked in water for .1 - 72 hours or longer, preferably 24 - 48 hours, at room temperature. The supernatant is filtered and is subjected to amonium sulphate (up to 90% saturation) precipitation and the precipitate thus obtained is resuspended in phosphate buffered saline (PBS) (0.1 M and pH 7.2 - 7.4). The solution thus obtained is dialysed against PBS to remove the salts for 24 - 48 hours at 4°C or at room temperature and this fraction comprising of total proteins (simple proteins, glycoproteins, proteoglycans, glycopeptides) can be used as such or concentrated or lypholized, or diluted with any diluent as per requirement. Method 2: Fresh or dried parts of Tinospora cordifolia, preferably the stems, is grounded and soaked with water for 1 - 72 hours or longer, preferably 24 - 48 hours, at room temperature. The supernatant is filtered and is subjected to amonium sulphate precipitation and the precipitate thus obtained is resuspended in phosphate buffered saline (PBS) (0.1 M and pH 7.2 - 7.4). The solution thus obtained is dialysed against PBS to remove the salts for 24 - 48 hours at 4°C or at room temperature. This preparation containing total proteins is then passed over the lectin column (Concanavalin A immobilized on 4% beaded Agarose). Glycoproteins absorbed on the column is then eluted using a Tris buffer (10mM pH 7.4) containing 0.2 M glucose and dialysed against PBS to remove free glucose and salts. The resulting preparation comprising of glycoproteins can be used as such or concentrated or lypholized or diluted with any diluent, as per requirement. Method 3: Fresh or dried parts of Tinospora cordifolia, preferably the stems, is grounded and soaked with water for 1 - 72 hours or longer, preferably 24 - 48 hours, at room temperature. The supernatant is filtered and directly passed over a lectin column (Concanavalin A - immobilized on 4% beaded Agarose). Glycoprotein thus absorbed is then eluted using a Tris buffer (10mM pH 7.4) containing 0.2 M glucose and dialysed against PBS to remove free glucose and salts. The resulting preparation comprising of glycoproteins can be used as such or concentrated or lypholized, or diluted with any diluent, as per requirement. Although the formulation comprising of a glycoprotein enriched fraction prepared from the stems of Tinospora cordifolia, as described above in Method 1-3, is stable even at room/ ambient temperature, preservatives and antibiotics can be added for long term preservation. This preparation can also be stored at 4°C or frozen below 0°C for long term storage. The use of the present invention is illustrated by way of the following experimental studies and such experiments should not be construed as limiting the scope of the invention. EXAMPLES: Example 1 Peritoneal macrophages from BALB/c mice were isolated and cultured in vitro with different concentrations (total protein content) of Tinospora glycoprotein fraction. Culture supernatants were collected after 48 hours and tested for IL-12 release. Results show that Tinospora glycoprotein formulation induces a dose dependent production of IL-12 by macrophages (Fig. 1). Example 2 BALB/c mice were sensitized to ovalbumin by immunization with this antigen through hind footpad and the draining lymph node cells were taken out on day 11 and challenged in vitro with the same antigen (ovalbumin) in the presence of different concentartions, ranging from 1 - 25 ug / ml of total protein content of Tinospora glycoprotein fraction. After 72 hours of incubation culture cells were evaluated for proliferative activity using 3H thymidine incorpoaration assay. Results showed that Tinospora glycoprotein fraction induces antigen specific T cell recall response at a low dose of 1 - 3 ug/ml (Fig. 2 ) Example 3 BALB/c mice were sensitized to ovalbumin by immunization with this antigen through hind footpad and the draining lymph node cells were taken out on day 11 and challenged in vitro with the same antigen (ovalbumin) in the presence of different concentartions, ranging from 1 - 100 ug /ml of total protein content of Tinospora glycoprotein fraction. After 48 hours of incubation culture supernatants were collected for cytokine analysis. Results showed that Tinospora glycoprotein fraction induces antigen specific T cell response of Th-1 type as indicated by prresence of IFN-y in the supernatant (Fig. 3); the production of IFN-y is dose dependent. Example 4 BALB/c mice were sensitized to ovalbumin by immunization with this antigen through hind footpad and animals were treated daily intra-peritoneally with different doses, ranging from 2.5, 5, 10 mg/kg of the total protein content of Tinospora glycoprotein fraction, for upto 10 days. On 11th day the draining lymph nodes were removed and challenged in vitro with the same antigen (ovalbumin) and incubated for 72 hours. Cell proliferation was evaluated for proliferative activity using 3H thymidine incorpoaration assay. The results showed that treatment of ovalbumin immunized animals with Tinospora glycoprotein fraction significantly enhances the antigen specific T cell recall response (Figure 4). Example 5 BALB/c mice were immunized intramuscularly with alum absorbed ovalbumin (100 ug) mixed with 10 mg/kg of the protein content of Tinospora glycoprotein fraction as an adjuvant. Booster was given on day 21. Blood samples were collected on every 10th day and analysed for the antigen specific antibody titre using ELISA. The results indicated that use of Tinospora glycoprotein fraction as an adjuvant significantly enhances the antigen specific antibody response (Fig. 5). Sub-class typing of these antibodies on day 30, revealed that Tinospora glycoprotein fraction induces TH-1 type of antibody response (Fig. 6) as indicated by predominance of lgG2a over other subclasses of immunoglobulin. We claim: 1. A formulation comprising of protein enriched fraction prepared from a plant of Tinospora species for enhancing vaccine efficacy by increasing the antigen specific immune response for prevention and treatment of infections and cancer. 2. A formulation as claimed in claim 1, wherein the plant is Tinospora cordifolia. 3. A formulation as claimed in claim 1, wherein the protein enriched fraction comprises of proteins conjugated to carbohydrates. 4. A formulation as claimed in claim 1, wherein the protein enriched fraction comprises of glycoproteins, glycopeptides or proteoglycans. 5. A formulation as claimed in claim 1 is used based on its total protein concentration per mililitre. 6. A formulation as claimed in claim 1, comprising other additives such as alum, lipids or other immunomodulators. 7. A formulation as claimed in any one of the preceding claims, said formulation can be in any form such as emulsion, suspension, solution, syrup, capsule, tablet or incorporated in liposomes, microspheres, cream, ointement, gel, spray, powder. 8. A formulation as claimed in claim 7, which can be administered through intra-muscular, sub-cutaneous, intra-dermal, intra venous, intra-nasal, inhalation, oral, genital, rectal or topical application on skin or any mucosal openings. 9. A method of prevention and treatment of infections and cancer in a patient, said method comprising administering a pharmaceutical effective amount of extract of Tinospora species preferably, Tinospora cordifolia to the patient. 10. A method as claimed in claim 9 wherein, said formulation is administered through intra-muscular, sub-cutaneous, intra- dermal, intra-venous, intra-nasal, inhalation, oral, genital, rectal or topical application on skin or any mucosal openings. 11. A formulation comprising of protein enriched fraction prepared from a plant of Tinospora species, substantially as herein described with reference to the examples and the accompanying drawings. 12. A method for the treatment of cancer, substantially as herein described and illustrated with reference to the examples and the accompanying drawings.

Documents:

55-del-2003-abstract.pdf

55-del-2003-claims.pdf

55-del-2003-correspondence-others.pdf

55-del-2003-correspondence-po.pdf

55-del-2003-description (complete).pdf

55-del-2003-drawings.pdf

55-del-2003-form-1.pdf

55-del-2003-form-19.pdf

55-del-2003-form-2.pdf

55-del-2003-form-26.pdf

55-del-2003-form-3.pdf

55-del-2003-form-4.pdf

55-del-2003-gpa.pdf