Title of Invention

A PHARMACEUTICAL COMPOSITION USEFUL AS CONTRACEPTIVE

Abstract The present invention provides for the use of oil of clove which causes instant immobilization of human spermatozoa both in presence of seminal fluid as well as in presence of female reproductive tract fluids, including uterine components and cervical mucus and also be useful for treating and/or preventing the common vaginal infections.
Full Text Novel Composition having antibacterial and antifungal
properties
The present application claims priority from the US Provisional application serial no 60/316,263 filed on September 4, 2001.
FIELD OF THE INVENTION
The present invention relates to the use of oil of clove which causes instant immobilization of human spermatozoa both in presence of seminal fluid as well as in presence of female reproductive tract fluids, including uterine components and cervical mucus. The invention may also be useful for treating and/or preventing the common vaginal infections.
BACKGROUND AND PRIOR ART REFERENCES
Conseivatives estimates predict a nearly 50% increase in the current world population of approximately six billion people by the year 2050 (1998 United Nations. 1998 Revision of the official United Nations population estimates and projections. World population nearing 6 billion projected close to 9 billion by 2050, Population Division of the Department of Economics and Social Affairs. United Nations, New York, 1998). This potential over-population, particularly in developing countries, is further complicated by the non-availability of proper easy-to-use, simple, cheap, least toxic and non-mvasive method of contraception. Several contraceptive devices have been introduced till date and all of them suffer from certain constraints as enumerated below:
Though hormonal methods (oral contraceptives) are highly effective in preventing pregnancies, various complications and side effects on prolonged use can not be ruled out.
On the other hand, vasectomy and ligation of Fallopian tube never became popular because of their various complications, irreversibility and problems of recannulation.
The intrauterine devices (lUDs) also cause several complications, including uterine perforations, occasional bleeding etc.

Novel Composition having antibacterial and antifungal
properties
The present application claims priority from the US Provisional application serial no 60/316,263 filed on September 4, 2001.
FIELD OF THE INVENTION
The present invention relates to the use of oil of clove which causes instant immobilization of human spermatozoa both in presence of seminal fluid as well as in presence of female reproductive tract fluids, including uterine components and cervical mucus. The invention may also be useful for treating and/or preventing the common vaginal infections.
BACKGROUND AND PRIOR ART REFERENCES
Conservatives estimates predict a nearly 50% increase in the current world population of approximately six billion people by the year 2050 (1998 United Nations. 1998 Revision of the official United Nations population estimates and projections. World population nearing 6 billion projected close to 9 billion by 2050, Population Division of the Department of Economics and Social Affairs. United Nations, New York, 1998). This potential over-population, particularly in developing countries, is further complicated by the non-availability of proper easy-to-use, simple, cheap, least toxic and non-invasive method of contraception. Several contraceptive devices have been introduced till date and all of them suffer from certain constraints as enumerated below:
Though hormonal methods (oral contraceptives) are highly effective in preventing pregnancies, various complications and side effects on prolonged use can not be ruled out.
On the other hand, vasectomy and ligation of Fallopian tube never became popular because of their various complications, irreversibility and problems of recannuiation.
The intrauterine devices (lUDs) also cause several complications, including uterine perforations, occasional bleeding etc.

The vaginal preparations are however relatively popular all over the world amongst the motivated couples, and because of their local and non-systemic application. However, these never give 100% contraceptive assurance.
The vaginal contraceptive compositions marketed in the USA primarily utilize nomonic detergents (surfactants) as their active ingredient. Of these, the most commonly used is nonoxynol-9 (N-9). Although N-9 is an extremely potent sperm immobilizing agent in vitro, but the marketed preparations are encountered with failures due to several reasons including the possibility of escape of first fraction of ejaculated sperms from its interaction with the active agents. In addition, N-9 can interfere with the natural vaginal environment and can cause irritation at a concentration that is recommended for use. N-9 and other spermicidal agents that have surfactant and cytotoxic properties have been proposed to prevent conception as a dispersing agent in spermicidal preparations (namely, Delfen, Gentersal and Ortho-Creme). However, relatively high failure rate of these surfactants in preventing pregnancies as well as evidence of adverse side effects have been documented (Stafford et al., 1998 - Stafford, MK, Ward, H, Flanagan, A, Rosenstein, U, Taylor-Robinson, D, Smith, JR, Weber, J and Kitchen, VS. Safety study of nonoxynol-9 as a vaginal microbicide : evidence of adverse effects. Journal of Acquired Immune Deficiency Syndromes, 17 : 327-331, 1998., Roddy et al., 1998 -Roddy, RE, Zekeng, L, Ryan, KA, Tamoufe, U, Weir, SS and Wong, EL. A controlled trial of nonoxynol-9 film to reduce male-to-female transmission of sexually transmitted diseases -New England Journal of Medicine, 339: 504-510, 1998 and Homn. RE, Foldesy, RG and Hahn, DW. ORF 13904, a new long-acting vaginal contraceptive. Contraception, 32: 267-274,1985).
For the last few decades, clove oil is being used in dental cream, mouthwash and various medicinal preparations at concentrations much higher than that cause sperm immobilization.
For these reasons, it is imperative that a new type of highly active ingredient be identified and developed for vaginal contraceptive use, as it will be noninvasive and is expected to have least or no side effects.

OBJECTS OF THE INVENTION
The main object of the present invention is to provide a highly specific, safe and stable contraceptive composition for women to protect themselves from unexpected pregnancies and vaginal infections.
Another object of the invention is to provide an anti-fertility composition comprising clove oil with a dilunet and optionally with one or more pharmaceutically acceptable additives.
Still another object of the invention is to provide a composition for spontaneous immobilization of sperms of human, hamster and rats.
Yet, another object of the invention is to provide a composition, which causes irreversible inhibition of hyaluronidase and acrosin, the essential enzymes, for fertilization.
Yet another object of the invention, is to provide a composition causes irreversible damage to the sperm ceils with complete loss of motility even in presence of seminal fluid of men, uterine washings and cervical mucus of women.

contraceptive, which results in instantaneous immobilization with such a low dose 10° to
Still another object of the invention is to provide an innovative noninvasive ;eptive, which resul ICT1 of active component.
Still another object of the invention is to provide a method of immobilizing mammalian spermatozoa.
SUMMARY OF THE INVENTION
Accordingly, the present invention relates to the use of oil of clove which causes instant immobilization of human spermatozoa both in presence of seminal fluid as well as in presence of female reproductive tract fluids, including uterine components and cervical mucus.
The present invention also relates a contraceptive composition, that can be used vaginally by women to protect themselves frcr t,! unexpected pregnancies and vaginal infections; a highly specific, safe, stable, and comparatively cheap active ingredient has been discovered.

The efficacy has been further proven by hamster and rat spermatozoa, which were immobilized instantaneously with very low concentration of (1:1000) oil. The present invention composes m vitro as well as in vivo methods for complete prevention of human sperm motility, thereby making sperm entry impossible into upper genital tract (i.e. uterus. Fallopian tube etc.) of women. The invention may also be useful for treating and/or preventing the common vaginal infections.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the present invention provides a composition useful as an and fertility agent and anti microbial agent in mammals including human beings, said composition comprising effective amount of clove oil, diluent and optionally one or more pharmaceutically acceptable additives.
In accordance to the objectives, the present invention provides Anti-fertility or a contraceptive composition comprising:
i) clove oil as an active component;
ii) aqueous alcohol;
lii) BWW medium as an diluent; and
iv) optionally one or more pharmaceutically acceptable additive.
In an embodiment, the aqueous alcohol used contains 30% alcohol
Another embodiment, the said composition comprising clove oil: 30% aq.alcohol:BWW medium in the ratio of 0.5:0.5:1000 to 0.5:0.5:10000000, and preferably consists of clove oil: 30% aq.alcohol:BWW medium in the ratio ranging between 0.5:0.5:1000 and 0.5:0.5:50,000.
Still another embodiment, the pharmaceutically acceptable ingredients used is selected from talc, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste and/or carriers, excipients, diluents or solvents.

Still another embodiment, BWW medium consists of 94 mM NaCl, 4.7mm KC1, 1.7 rnM CaCl2.1.2 mM KH2P04, 1.2 mM MgS047H20, 25 mM NaHC03.o.s mM Sodium pyruvate, 19 mM Sodium acetate, 5 mM glucose, 0.4 % BSA, 0.1 % penicillin / streptomycin, said medium having a pH 7.2.
Yet another embodiment, the said composition immobilize human spermatozoa spontaneously within 10 seconds by a 1:1 aqueous alcoholic clove oil solution diluted 1,000 times with BWW medium.
In still another embodiment, the said composition provides complete inhibition of motility in about a minute by 1:1 aqueous alcoholic clove oil solution diluted 10,000 times with BWW medium.
Yet another embodiment, the composition provides for an innovative noninvasive contraceptive, which results in instantaneous immobilization having the active component concentration, is in the range of 10"3 to 10"4.
Yet another embodiment, the composition causes irreversible inhibition of hyaluronidase and acrosin, the essential enzymes for fertilization
Yet another embodiment, the composition totally disrupts the plasma membrane of sperms and destroys the sperm acrosomal enzyme.
Yet another embodiment, hyaluronidase (EC 3.2.1.35), and acrosin (EC 3.2.21.10) are greatly inhibited to the extent of 90 %, by a 1:1 aqueous alcoholic clove oil solution diluted 1,000 times with BWW medium, resulting in irreversible inhibition of
enzymes.
Still another embodiment, 5'-nucleotidase, that is associated with the sperm plasma membrane, is totally dispersed and dissociated in the medium at 1,000 times dilution of oil indicating the destabilization of the sperm membrane system.
Yet another embodiment, the composition causes irreversible damage to the sperm cells with complete loss of motility even in the presence of seminal fluid, uterine washings and cervical mucus.

Still another embodiment, the said composition may be used as capsule, sponge, cream, vaginal film and any other preparations that can be controlled by self administration.
Yet another embodiment, the said composition immobilizes sperms from humans, hamster and rats.
Still another embodiment, the said composition further provides anti-microbial characteristics against vaginal infections.
Yet another embodiment, the said composition inhibits the microbial growth of fungi selected from a group consisting of cocci, Candida and trichomonas.
Yet another embodiment, wherein the composition inhibits the microbial growth of fungi by 1:1 aqueous alcoholic clove oil solution diluted 100 times with BWW medium.
Still another embodiment, the composition may be administered by in-vitro as well as in vivo methods for complete prevention of human sperm motility.
One more embodiment of the invention provides a method of immobilizing mammalian spermatozoa, said method comprising treating / contacting the spermatozoa with a composition comprising:
i) clove oil as an active component;
ii) aqueous alcohol;
iii) BWW medium as an diluent; and
iv) optionally one or more pharmaceutically acceptable additive
Another embodiment, 5'-nucleotidase, that is associated with the sperm plasma membrane, is totally dispersed and dissociated in the medium at 1,000 times dilution of oil indicating the destabilization of the sperm membrane system, which is essential for the maintenance of moiility.

In an embodiment of the invention, the clove oil and its ingredients have been found highly effective in immobilizing human spermatozoa. Spontaneous immobilization occurs within 10 seconds at more than 1,000 times dilution of the oil. Complete inhibition of motility has been observed in about a minute even at 10,000 dilution in all types of sperm samples obtained from proven fertile men (vide Tables 1 and 2).

contraceptive which results in instantaneous immobilization with such a low dose 10"3 to
Yet another embodiment of the invention provides for an innovative noninvasive
rac 10"4 of active component.
Further, yet another embodiment of the invention reveals the damage- and perforation of plasma membrane system, which surrounds the mammalian sperm cells, that causes the total immobilization with occasional agglutination of human sperms, along with loss membrane components that are necessary for the fertilization to occur.
Yet another embodiment of the invention relates to the use of the composition for total immobilization with occasional agglutination of human sperms, along with loss membrane components that are necessary for the fertilization occurs.
Yet another embodiment of the invention relates to irreversible inhibition of hyaluronidase and acrosin, the essential enzymes for fertilization.
Yet another embodiments of the invention provide total disruption of the plasma membrane of human sperms and destroy the sperm acrosomal enzyme.
Yet another embodiment relates to the irreversible damage to the sperm cells with complete loss of rnotility even in presence of seminal fluid of men, and uterine washings and cervical mucus of women.
Yet another embodiment of the invention relates to ami bacterial activity of the clove oil dilution. The clove oil inhibits the microbial growth of fungi including cocci, Candida and trichomonas.
Yet another embodiment invention re,,,',es to anti-bacterial and and fungalv activities of clove oil, that inhibits the microbial growth of fungi including cocci, Candida and trichomonas at 100 times dilution.
Yet another embodiment of the invention relates to activity of clove oil active principles, that are also effective in immobilizing rat and hamster spermatozoa collected from epididymis.
Yet another embodiment of the invention relates to wherein, sperms are totally immobilized almost instantaneously with more than 1000 times dilution of clove oil. By showing total inhibitory activity towards human sperms even with low concentration of clove oil.
Yet another embodiment of the invention, the ratio of clove oil to the diluent is in the range between 1:1000 to 1:50,000.
Yet another embodiment of the invention, the composition may be administered by in-vitro as well as in vivo methods for complete prevention of human sperm motility, thereby making sperm entry impossible into upper genital tract namely uterus, Fallopian tube etc. of women.
Yet another embodiment of the invention, the said composition may be used as capsule, sponge, cream, vaginal film and any other preparations that can be controlled by self administration.
Another embodiment relates to the synergic activity of antifertility and and microbial is due to common mechanism of action that exists for prevention of fertilization by sperm immobilization and inhibition of vaginal infection.
Another embodiment of the present invention relates to potential contraceptive agent that acts as an inhibitor of hyaluronidase and acrosin enzymes, essential for fertilization.
In another embodiment hyaluronidase (EC 3.2.1.35), and acrosin (EC 3.2.21.10) are greatly inhibited to the extent of 90 %, at a very low concentration, which is achieved on incubation of the sperm cells with 1,000 times diluted clove oil, resulting in irreversible inhibition of enzymes.
Another embodiment of the invention relates to 5'-nucIeotidase, that is associated with the sperm plasma membrane, is totally dispersed and dissociated in the medium at 1,000 times dilution of oil indicating the destabilization of the sperm membrane system -the organization and the intactness of which is essential for the maintenance of motility.
Another embodiment relates to clove oil acting as highly potent agents to disrupt the plasma membrane of human sperms and destroys the sperm acrosomal enzyme, which is an irreversible damage to the sperm cells with complete loss of motility even in presence of seminal fluid of men, and uterine washings and cervical mucus of women.
Another embodiment of the invention, consistent with its action on human sperm, clove oil active principles are also effective in immobilizing rat and hamster spermatozoa collected from epididymis. All sperms are totally immobilized almost instantaneously with more than 1000 times dilution of clove oil.
Another embodiment relates to the composition showing total inhibitory activity towards human sperms, at a very low concentration to exhibit the desired effect when inserted as capsule or otherwise in the vagina prior to copulation.
Brief description of the accompanying drawings
Fig. 1 shows the results of this experiment where different dilution of clove oil was examined on human, hamster and rat sperm motility. Clove oil addition in different dilution immobilized the sperm in a dose dependent manner. 10° dilution effected 100% immobilization of human, hamster and rat sperm in 15 sec. There was practically very little difference between the doses and the source of sperm sample.
The invention is further described in the form of the following examples, which shall not be construed as a limit to the scope of the invention.
Sperm Immobilizing Assays:
Inhibition and immobilization of human sperm motility were evaluated by mixing 100 U.L of test solution with 100 jaL of freshly ejaculated semen. Immediately after mixing (within 10 seconds), the percentage of motile spermatozoa was determined by microscopic examination (400x and lOOx). At higher dilution incubation time was 60 seconds. The dose-response data have been presented in
Other Techniques:
Hyaiuronidase (EC 3,2.1.35) activity was quantified with hyaluronic acid as a substrate. This was measured by determining the concentration of N-acetylglucosamine reactive material, resulting from enzyme action Table 4(a).
Human acrosin (EC 3.4.21.10) activity was measured by following the kinetics of change in optical density at 253nm; the hydrolysis of N-«=-benzoyl-L-arginine ethyl ester (BAEE) was quantified Table 4(b).
Nucleoddases were assayed by using 5'-AMP and (3-glycerophosphate as substrate, and the enzyme activities are expressed in terms of free phosphates that are released Table 4(c).
Caudal spermatozoa from rats and hamsters and ejaculated sperm from six normospermic human donor (aged between 30-37) were used to observe clove oil effect in the motility of sperm. Clove oil was diluted to 1000 times and added to the sperm sample. Human sperm after collection were allowed to liquefy at 37°C for 30 minutes. The liquefied samples were diluted with equal volume of Ham's F-10 medium and centnfuged at 5000 g for 10 minutes. Sperm pellets were then layered with 1.0 ml of Ham's F-10 medium and were incubated at 37°C which permitted the progressive swimming up of the motile cells and counted in a Makler's chamber. Only the cells with high motility were used in the experiments. Hamster and rat sperm were diluted in HWW medium (94 mM NaCl, 4.7 mM KCI, 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSCu, 7H2O, 25 mM NaHCCb, 0.5 mM sodium pyruvate, 19 mM sodium acetate, 5 mM glucose, 0.4% BSA, 0.1% penicillin/streptomycin, pH 7.2), pre equilibrated in a C02 incubator for 30 minutes to swim them up and only highly motile sperm were selected and centnfuged at 1000 g for 2 minutes. The pellet containing highly motile sperm were diluted in BWW medium and counted in a Makler's chamber. In all the cases, i.e. human, hamster and rat, 25-35 million sperm were taken for clove oil experiment.
her this immobilization was due to the death of the sperm by clove oil or not was tested by using cosin-nigrosin vital dye uptake and this clearly showed mortality of the sperm. The same concentration of clove oil did not show any adverse effect on vaginal epithelial cells; therefore appears to be safe for its probable use as vaginal contraceptive.
How clove oil at a dilution of 10"J can completely immobilize, human, hamster and rat sperm by causing death to them is not yet clear. Since there is a possibility to immobilize through the destabilization of sperm membrane architecture, we selected two sperm membrane bound enzyme, acrosin and 5'-nucleotidase as markers to study this aspect. The outer envelope of most of the mammalian spermatozoa is rich in polar phospholipid and any alteration to this phospholipid affects sperm motility or survival. Acrosome matrix is a part of this complex where acrosin remains in the bound form. Any disturbance in membrane architecture induces acrosome reaction, which in turn results acrosin release. Similarly, 5'-nucleotidase is an enzyme bound to the membrane of sperm. This enzyme anchors glycophosphatidylinositol network in the sperm membrane and therefore when it gets free from its anchorage, an alteration in membrane structure is expected. Tables 6 and 7 demonstrate the result of clove oil addition (at a dilution of 10~J) to the sperm of six human donors. Both acrosin and 5'-nucleotidase activities could be located in the supernatant fraction on clove oil treatment. In the control sperm, both the marker enzyme activities was obtained in the sperm pellet where supernatant showed very little activity. On treating the human sperm with clove oil, the marker enzyme activities were found to be located in the supernatant which clearly shows the detachment of the enzymes from the membrane architecture. These results strongly suggest clove oil target to be the sperm membrane and by acting through the membrane, it destabilizes the sperm structure causing immobilization and death. All these are novel findings with clove oil that supports its use as an effective contraceptive.
Composition of BWW medium: 94 mM NaCl,, 4.7 mM KC1, 1.7 mM CaCl2.1.2 mM KH:PO4, 1.2 mM MgS04, 7H2O, 25 mM NaHCC ,0.5 mM sodium pyruvate, 19 mM sodium acetate, 5 mM glucose, 0.4% BSA, and 0.1% antibiotic (penicillin/streptomycin) solution
ECTS OF CLOVE OIL ON HUMAN SPERM IMMOBILIZATION
(Table Removed) Oligoospermic, Count 19 million/ml Hyperspermic, Count 120 million/ml
All categories of sperm cells were incubated for 60 seconds, and checked under microscope at 100 x and 400 x.
IMMOBILIZATION OF RAT AND HAMSTER EPIDIDYMAL
SPERMS
(Table Removed)incubation time was 10 seconds: ** Incubation time was 60 seconds Inhibition of human sperm by hyaluronidase and acrosin by c13
Inhibition of cocci, Candida and trichomonas by clove oil


Claims
1. Anti-fertility or a contraceptive composition comprising:
i) clove oil as an active component;
ii) aqueous alcohol;
iii) BWW medium as an diluent; and
iv) optionally one or more pharmaceutically acceptable additive.
2. The composition as claimed in claim 1, wherein the aqueous alcohol used
contains 30% alcohol
3. The composition as claimed in claim 1, wherein ratio of clove oil: ^0%
aq.alcohol:BWW medium is in the range of 0.5:0.5:1000 to 0.5:0.5:10000000.
4. The composition as claimned in claim 1, preferably consists of clove oil: 30%
aq.alcohol.-BWW medium in the ratio ranging between 0.5:0.5:1000 and
0.5:0.5:50,000.
5. A composition as claimed in claim 1, wherein pharmaceutically acceptable
ingredients used is selected from talc, magnesium stearate, cellulose, calcium
carbonate, starch-gelatin paste and/or carriers, excipients, diluents or solvents.
6. A composition as claimed in claim 1, wherein BWW medium consists of 94 mM
NaCl, 4.7mrn KC1, 1.7 mM CaCl2. 1.2 mM KH2PO4, 1.2 mM MgSO4.7H2O, 25
mM NaHCCh. Q.S mM Sodium pyruvate, 19 mM Sodium acetate, 5 mM glucose,
0.4 % BSA, 0.1 % penicillin / streptomycin, said composition having a pH 7.2.
7. A composition as claimed in claim 1, wherein spontaneous immobilization of
human spermatozoa occurs within 10 seconds by a 1:1 aqueous alcoholic clove oil
solution diluted 1.000 times with BWW medium.
8. A composition as claimed in claim 1, /s'herein the said composition provides,
complete inhibition of motility in about a minute by 1:1 aqueous alcoholic clove
oil solution diluted 10,000 times with BWW medium.

9. A composition as claimed in claim 1, wherein the composition provides for an
innovative noninvasive contraceptive, which results in instantaneous
immobilization having the active component concentration, is in the range of 10°
to 10'a.
10. A composition as claimed in claim 1, wherein the composition causes irreversible
inhibition of hyaluronidase and acrosin, the essential enzymes for fertilization
11. A composition as claimed in claim 1, wherein the composition totally disrupts the
plasma membrane of sperms and destroys the sperm acrosomal enzyme.
12. A composition as claimed in claim 1, wherein hyaluronidase (EC 3.2.1.35), and
acrosin (EC 3.2.21.10) are greatly inhibited to the extent of 90 %, by a 1:1
aqueous alcoholic clove oil solution diluted 1,000 times with BWW medium,
resulting in irreversible inhibition of enzymes.
13. A composition as claimed in claim 1, wherein 5'-nucleotidase, that is associated
with the sperm plasma membrane, is totally dispersed and dissociated in the
medium at 1,000 times dilution of oil indicating the destabilization of the sperm
membrane system.
14. A composition as claimed in claim 1, wherein the composition causes irreversible
damage to the sperm cells with complete loss of motility even in the presence of
seminal fluid, uterine washings and cervical mucus.
15. A composition as claimed in claim 1, wherein, the said composition may be used
as capsule, sponge, cream, vaginal film and any other preparations that can be
controlled by self administration.
16. A composition as claimed in claim 1, wherein the said composition immobilizes
sperms from humans, hamster and rats.
17. A composition as claimed in claim 1 further provides anti-microbial
characteristics against vaginal infections.

IS. A composition as claimed in claim 1, wherein the said composition inhibits the microbial growth of fungi selected from u group consisting of cocci, Candida and trichomonas.
19. A composition as claimed in claim 1, wherein the composition inhibits the
microbial growth of fungi by 1:1 aqueous alcoholic clove oil solution diluted 100
times with BWW medium.
20. A composition as claimed in claim I, wherein, the composition may be
administered by in-vitro as well as in vivo methods for complete prevention of
human sperm motility.
2L A method of immobilizing mammalian spermatozoa, said method comprising treating / contacting the spermatozoa with a composition as claimed in claim 1.
22. A method as claimed in claim 21, wherein spermatozoa are selected from humans,
hamster and rats.
23. A method as claimed in claim 21, wherein pharmaceutically acceptable
ingredients used is selected from talc, magnesium stearate, cellulose, calcium
carbonate, starch-gelatin paste and/or carriers, excipients, diluents or solvents.
24. A method as claimed in claim 21, wherein BWW medium consists of 94 mM
NaCl, 4.7mm KC1. 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4.7H2O, 25
mM NaHCOs. o.s noM Sodium pyruvate, 19 mM Sodium acetate, 5 mM glucose,
0.4 % BSA, 0.1 % penicillin / streptomycin, said composition having a pH 7.2.
25. A method as claimed in claim 21, wherein spontaneous immobilization of human
spermatozoa occurs within 10 seconds by a 1:1 aqueous alcoholic clove oil
solution diluted 1,000 times with BWW medium
26. A method as claimed in claim 21, wherein the said composition provides
complete inhibition of motility in about a minute by 1:1 aqueous alcoholic clove
oil solution diluted 10,000 times with BWW medium.

27. A method as claimed in claim 21, wherein the composition provides for an
innovative noninvasive contraceptive, which results in instantaneous
immobilization having the active component concentration, is in the range of 10"J
to ID'4.
28. A method as claimed in claim 21, wherein the composition causes irreversible
inhibition of rivaluronidase and acrosin, the essential enzymes for fertilization
29. A method as claimed in claim 21, wherein the composition totally disrupts the
plasma membrane of human sperms and destroys the sperm acrosomal enzyme.
30. A method as claimed in claim 21, wherein, 5'-nucleotidase, that is associated with
the sperm plasma membrane, is totally dispersed and dissociated in the medium at
1,000 times dilution of oil indicating the destabilization of the sperm membrane
system, which is essential for the maintenance of motility.
31. A method as claimed in claim 21, wherein the composition causes irreversible
damage to the sperm cells with complete loss of motility even in presence of
seminal fluid, uterine washings and cervical mucus.
32. A method as claimed in claim 21, wherein, the said composition may be used as
capsule, sponge, cream, vaginal film and any other preparations that can be
controlled by self administration.
33. A method as claimed in claim 21, wherein, the composition showing total
inhibitory activity towards human sperms, at a very low concentration to exhibit
the desired effect when inserted as capsule or otherwise in the vagina prior to
copulation.
34. A method as claimed in claim 21, wherein the said composition immobilize
sperms selected from humans, hamster and rats.
35. A method as claimed in claim 21 further provides anti-microbial characteristics
against vaginal infections.

36. A method as claimed in claim 21, wherein the said composition inhibits the
microbial growth of fungi selected from a group consisting of cocci, Candida and
trichomonas.
37. A method as claimed in claim 21, wherein the composition inhibits the microbial
growth of fungi by 1:1 aqueous alcoholic clove oil solution diluted 100 times with
BWW medium.
38. A method as claimed in claim 21, wherein, the composition may be administered
by in-vitro as well as in vivo methods for complete prevention of human sperm
motility.







Documents:

146-DEL-2003-Abstract-(17-03-2008).pdf

146-del-2003-abstract.pdf

146-DEL-2003-Claims-(17-03-2008).pdf

146-del-2003-claims.pdf

146-del-2003-correspondence-(others).pdf

146-DEL-2003-Correspondence-Others-(17-03-2008).pdf

146-del-2003-correspondence-po.pdf

146-DEL-2003-Description (Complete)-(17-03-2008).pdf

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

146-DEL-2003-Drawings-(17-03-2008).pdf

146-del-2003-drawings.pdf

146-DEL-2003-Form-1-(17-03-2008).pdf

146-del-2003-form-1.pdf

146-del-2003-form-18.pdf

146-DEL-2003-Form-2-(17-03-2008).pdf

146-del-2003-form-2.pdf

146-DEL-2003-Form-3-(17-03-2008).pdf

146-DEL-2003-Petition-137-(17-03-2008).pdf


Patent Number 221050
Indian Patent Application Number 146/DEL/2003
PG Journal Number 31/2008
Publication Date 01-Aug-2008
Grant Date 13-Jun-2008
Date of Filing 19-Feb-2003
Name of Patentee COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110001, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 ALOK PAL IICB CALCUTTA, INDIA.
2 ASHOK KUMAR BHATTACHARYA IICB CALCUTTA, INDIA.
3 SAMIR BHATTACHARYA IICB CALCUTTA, INDIA.
PCT International Classification Number A01N 65/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA