Title of Invention	"A CONTRACEPTIVE COMPOSITION"
Abstract	The present invention provides a novel composition comprising Nisin-Z useful as a contraceptive. The invention also provides a method for preparation of the composition.

Full Text

FIELD OF THE INVENTION The present invention provides a novel composition useful as a contraceptive. The invention also provides a method for preparation of the composition. BACKGROUND OF THE INVENTION Currently worldwide, there is a high incidence of reproductive tract infections (RTIs) taking place through heterosexual sex (Anderson, RA, Feathergill KA, Hui Diao X et al. Preclinical evaluation of sodium cellulose sulfate (4 sher cell) as a contraceptive antimicrobial agent. J. Androl, 2002; 23: 426-38). No cure exists for many of the RTIs of viral etiology, particularly infections caused by human immunodeficiency viruses (HIVs) and herpes simplex viruses (HSVs). Consequently, prevention is the strategy of choice for controlling the spread of infections (Zaneveld LJD, Waller DP, Anderson RA et al. Efficacy and safety of a new vaginal contraceptive antimicrobial formulation containing high molecular weight poly sodium 4-stryenesulfonate. Biol. Reprod. 2002; 66; 886-94). Today, unwanted pregnancies is another problem concerning sexually active persons and prevention of pregnancies is becoming as necessary as control of RTIs/HIV (Hardy E. Padma KA, Jimenez AL et al. Womens preferences for vaginal microbial contraceptive II, preferred characteristics according to women's age and socioeconomic status. Contraception 1998, 58: 239-44). The marketed vaginal contraceptive formulation contains detergent/Surfactant as an active ingredient, the most common one beiung nonoxynol-9 (N-9). Recently studies indicated that frequent use of clinically effective does of N-9 causes vaginal irritation, lesions on vaginal epithelium and inactivation normal vaginal flora (Piot D. Nonoxynol-9 not effective microbicide, trial shows. UNAIDS Press Release, 2000). Condoms, when used consistently and correctly, are the only method available that has been shown to be effective in preventing these infections. However, women are often unable to negotiate the use of condom with her partner, leaving them vulnerable. There is a real and urgent need to expand the range of preventive interventions for RTIs/HIV. It is not known when a safe and effective vaccine will be developed and become available. In the interim, non-vaccine prevention methods controlled by women are to be explored. Vaginal microbicides would likely be used repeatedly over decades, and hence selection of a compound with established safety record is crucial. Moreover, it should be inexpensive, and produced from commonly available resources. Towards this end, the invention provides a novel composition useful as a contraceptive. OBJECTS OF THE INVENTION The main object of the invention is to provide a novel contraceptive composition comprising Nisin-Z peptide as its active ingredient. Another object is to provide a process for the preparation of the composition. DESCRIPTION OF THE ACCOMPANYING DRAWINGS Figure 1 represents the structure of Nisin-Z peptide Figure 2 represents graphically the effect of Nisin-Z on sperm motility. STATEMENT OF THE INVENTION The present invention provides a composition useful as a contraceptive, comprising Nisin-Z peptide in a range of 0.1 to 1% along with pharmaceutically acceptable carriers and excipients, optionally with adjuvants, protease inhibitors or drugs. DETAILED DESCRIPTION OF THE INVENTION Accordingly, the invention provides a composition comprising Nisin-Z peptide of SEQ ID 1 in an amount sufficient to cause spermicide, together with a therapeutically acceptable carrier, additive or excipient. The amount of the peptide in the composition may be such as to cause spermicide or render sperms incapable of fertilizing a female subject, whether by immobilizing, altering or killing them. 'Spermicidal' as used herein means that the peptide of SEQ ID 1 employed herein inhibits, prevents or destroys the motility of the sperm. In general, the amount of Nisin-Z used in the composition may be between 0.1% to 1.0%. Preferably, the composition may contain 0.5% of Nisin-Z. The said active ingredient may be combined with a suitable non-toxic and therapeutically acceptable vehicle such as carrier, filler or additive. The amount of the vehicle is about 0.9%. The composition may be formulated as a tablet, paste, ointment, solution, lotion or otherwise employing conventional pharmacological techniques. In another aspect, the invention provides a method for destroying spermatozoa, which comprises the step of contacting spermatozoa with an effective amount of a composition comprising a biologically pure peptide Nisin-Z of SEQ ID No.l together with a therapeutics1 ly acceptable vehicle. While Nisin-Z is an anti-microbial peptide, the exact nature of its activity is yet unknown. Further, its action on sperms is not yet known or reported. Nisin's interaction with bacterial membrane is really with negatively charged phospholipid moieties. A sperm on the other hand is bound by a lipid bilayer plasma membrane and the action of Nisin-Z on this membrane is unpredictable. The applicants carried out various studies in this area and to their surprise, found that Nisin-Z successfully permeates this membrane and kills sperms. Thus, the said finding is surprising and unexpected. It is pertinent to note that Nisin-Z does not render the sperms inactive or immotile. It actually kills all the sperms. As shown in table 1, the percentage of sperms killed is 100% and that too within 20 seconds. In case of a bacteriocide, it would be sufficient if 80-90% killing of the bacteria is achieved, because the surviving bacteria may be killed by the body's own immune system. However, where the composition is intended to be used as a contraceptive, all sperms need to be killed, else, there is a possibility of fertilization. Further, the composition of the invention is safe since Nisin-Z has been used for the human being for other applications. Therefore, use of Nisin-Z as a spermicide is safe. Another aspect of the invention is the quantum of Nisin-Z used as a spermicide. It may be noted that for other applications of Nisin-Z, the amount of Nisin-Z used is a lot. However, in case of a spermicide, the amount used is very less and even such amount is effective. It is also found that if an amount higher that what has been described in this application is used, then adverse effects are observed. Therefore, the findings of the invention are surprising and unexpected. Yet another aspect of the invention is a method to control fertilization in a female subject comprising the step of administration of an effective amount of a composition containing the peptide Nisin-Z together with a therapeutically acceptable vehicle. The composition of the invention may be administered/used topically or systemically and in any suitable form such as a liquid, solid, semisolid, injectable solution, tablet, ointment, lotion, paste, capsule, or the like. The peptide composition may also be used in combination with adjuvants, protease inhibitors, or compatible drugs where such a combination is seen to be desirable or advantageous in controlling infection caused by harmful microorganisms including protozoa, viruses, and the like, as well as by parasites. A host defensive peptide Nisin-Z was selected for study. Nisin-Z is a 34 amino acid, cationic peptide having molecular mass of 3.5 Kda (Gross E, Morell JL. The structure of Nisin. J.Am. Chi. Soc. 1971; 93: 4634-35 and Morell, 1971). The said peptide is water soluble and has a +4 charge. It is amphiphatic in nature. The said peptide is a member of lantibiotic family and is produced by the bacterium, lactococcus lactis (Hansen JN, Nisin and related antimicrobial peptides. Strohl WR (ed), In: Biotechnology of antibiotics Marcell Dekker, Inc, New York, 1997; 437-70) in large quantities during lactic acid fermentation. Nisin-Z is also described in the sequence listing as SEQ ID 1. The characteristic features of Nisin-Z is the ring systems formed by thioether bonds and dehydrated amino acids. Nisin-Z possesses antibacterial activities (Breukink E, Van Kraaij C, Van Dalen A et al. The orientation of Nisin in membranes. Biochem. 1988; 37:8153-62) particularly against Gram positive organisms. Since 1953, Nisin-Z is being used as natural food preservative in USA and European countries. Nisin-Z was awarded the generally regarded as safe (GRAS) by FDA, WHO and EU in 1983. A detailed investigation conducted by the Applicant has revealed that Nisin-Z exhibits activity against sperms. The invention is described herebelow by the following examples which, however, are not intended to limit the scope of the invention. Example 1: Preparation of composition Nisin-Z was obtained from SIGMA, USA. Formulation (a) was prepared by mixing in a manner known to a skilled person, the following: i) peptide Nisin-Z: 1 mg ii) physiological saline: 9 mg Example 2: In-vitro activity Nisin-Z peptide is commercially obtained from SIGMA, USA. Milk solids and salts were removed by gel filtration chromatography on sephadex column. Different dilutions of Nisin-Z was prepared in physiological saline and tested under in vitro against spermatozoa of rat, rabbit, monkey and human (Sander FW, Cramer SD. A practical method for testing the spermicidal action of chemical contraceptives. Hum. Feril. 1941; 6: 134-37). The effect of Nisin-Z on sperm was found to be dose and time dependent (Figure 1). At 20 seconds time point - 300 |ug Nisin was found to be sufficient enough to immobilize the sperm present in 1 ml of semen (60-80 x 106). The effect was spermicidal not spermiostatic. Example 3: Evaluation in animals The composition of example 1 was administered intravaginally to a group of New Zealand white rabbits (n = 12) prior to mating. Thereafter, spermicidal activity was determined by examining semen sample immediately after mating at zero hour and half an hour after mating. Spermatozoa activity was determined by microscopic observation of sperm motility and of any detectable direction of movement of sperm. The results are as below: 14 days sub-acute toxicity studies in rabbits indicated that, vaginal application of high dose of Nisin-Z (40 mg/day) for 14 consecutive days was found to be safe as no treatment related abnormalities in vaginal epithelium, liver, kidney function and blood profiles. Table (Table Removed) claim: 1. A composition useful as a contraceptive, comprising a therapeutically effective amount of biologically active peptide Nisin-Z of SEQ ID 1 and a non-toxic vehicle. 2. A composition as claimed in claim 1 wherein the amount of the peptide is 0.1 to 1% or 4000µg. 3. A composition as claimed in claim 1, wherein the vehicle is saline. 4. A composition as claimed in claim 1, wherein the amount of the vehicle is about 0.9%. 5. A lotion comprising the composition of claim 1. 6. A solution comprising the composition of claim 1. 7. Use of Nisin-Z having seq ID No.l as a contraceptive and spermicidal agent. 8. Use of a composition comprising a therapeutically effective amount of Nisin-Z having seq ID No. 1 and a carrier as a contraceptive and spermicidal agent. 9. Use as claimed in claim 8 wherein the amount of Nisin-Z is 0.1 to 1%. 10. Use as claimed in claim 8 wherein the amount of the vehicle is about 0.9%. 11. A composition useful as a contraceptive substantially as hereindescribed and illustrated with reference to the examples and accompanying drawings. 12. Use of Nisin-Z as a contraceptive substantially as hereindescribed and illustrated with reference to the examples and accompanying drawings.

Documents:

2005-del-2004-abstract.pdf

2005-DEL-2004-Claims.pdf

2005-del-2004-complete specification (granded).pdf

2005-del-2004-correspondence-others.pdf

2005-del-2004-correspondence-po.pdf

2005-del-2004-description (complete).pdf

2005-del-2004-drawings.pdf

2005-del-2004-form-1.pdf

2005-del-2004-form-18.pdf

2005-del-2004-form-2.pdf

2005-del-2004-form-26.pdf

2005-del-2004-form-3.pdf

2005-del-2004-form-5.pdf