Title of Invention	"METHOD OF PREPARING AN INEXPENSIVE, NON-MESSY, SAFE AND FAST ACTING AQUEOUS SYNERGISTIC SPERMICIDAL COMPOSITION"
Abstract	The present invention relates to a method of preparing an inexpensive, non-messy, safe and fast acting aqueous synergistic spermicidal composition of 100% efficacy useful as a vaginal contraceptive free of side effects in animals in human beings, said method comprising steps of preparing sodium chloride solution of concentration ranging between 0.4 to 3% in water, mixing hydrogen peroxide to said sodium chloride solution to obtain a mixture having hydrogen peroxide in the concentration ranging between 1 to 3%, and adjusting pH of the mixture ranging between 4 to 4.5, and obtaining said composition.

Title of Invention

"METHOD OF PREPARING AN INEXPENSIVE, NON-MESSY, SAFE AND FAST ACTING AQUEOUS SYNERGISTIC SPERMICIDAL COMPOSITION"

Abstract

The present invention relates to a method of preparing an inexpensive, non-messy, safe and fast acting aqueous synergistic spermicidal composition of 100% efficacy useful as a vaginal contraceptive free of side effects in animals in human beings, said method comprising steps of preparing sodium chloride solution of concentration ranging between 0.4 to 3% in water, mixing hydrogen peroxide to said sodium chloride solution to obtain a mixture having hydrogen peroxide in the concentration ranging between 1 to 3%, and adjusting pH of the mixture ranging between 4 to 4.5, and obtaining said composition.

Full Text	METHOD OF PREPARING AN INEXPENSIVE, NON-MESSY, SAFE AND FAST ACTING AQUEOUS SYNERGISTIC SPERMICIDAL COMPOSITION" Field of the present invention The present invention relates to a method of preparing an inexpensive, non-messy, safe and fast acting aqueous spermicidal composition of 100% efficacy useful as a vaginal contraceptive free of side effects, said method comprising steps of preparing sodium chloride solution of concentration ranging between 0.4 to 3% in water, mixing hydrogen peroxide to said sodium chloride solution to obtain a mixture having hydrogen peroxide in the concentration ranging between 1 to 3%, adjusting pH of the mixture ranging between 4 to 4.5, and obtaining said composition, and also, the composition thereof. The said composition is capable of maintaining vaginal pH and has no adverse effect on the beneficial vaginal flora. Background and prior art references Maintenance of functional integrity of the spermatozoa is vital for successful fertilization with the oocyte. Impairment with any of the functional properties of the sperm might lead to affect the ultimate process of fertilization. Development of a contraceptive composition that affects majority of sperm functions is always considered more efficacious. Sperm motility is one of the important functions of the sperms, which enables the sperms to reach the oocytes for fertilization. Spermatozoa without forward progressive motility will not be able to perform this ultimate task, a key aspect while making a contraceptive composition. Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male fertility (Armstrong JS, Rajasekharan M, Chamulitrat W, Gatti P, Helistrom WJ and Sikka SC (1998) Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism. Free Radical Biology Medicine 26: 869-880) There are two sources of oxidative stress that affect human spermatozoa. The first source is the reactive oxygen species generated by the defective spermatozoa due to some defect in the spermatogenesis. The second source is from seminal leukocytes, infiltration of which is a common feature of the human ejaculate (Gavella M, Lipovac V, Vicic M and Rocic B (1996) Superoxide anion scavanging capacity of human seminal plasma. Intl J of Andrology 19: 82-90). The deleterious effects of ROS on sperm functions have been reviewed by Aitkin et al, 1994a. Aitkin RJ (1994) A free radical theory of male infertility. Reproduction Fertility and Development 6: 19-24. Hydrogen peroxide is the most common and toxic ROS to spermatozoa till recognized. Hydrogen peroxide induced cell death was reported in cerebellar granule cells, which was characterized by chromatin condensation and DNA fragmentation. During the process, the fluidity of the cell membrane decreased markedly and the confirmation of the membrane proteins altered significantly (Wei T, Ni Y, Hou J, Chen C, Zhao B and Xin W (2000) Hydrogen peroxide-induced oxidative damage and apopotosis in cerebellar granule cells: Protection by Ginkgo Biloba extract. Pharmacological Research 41: 427-433.). Use of hydrogen peroxide has also been reported (patent no. 778 886 by Shihata et al, Shihata (1998) vaginal contraception compositions combining a spermicidal agent and a peroxygen compound. United States Patent No 778 886.) along with the known spermicidal Nanoxynol -9 to restore the alterations to pH caused by the spermicidal application affecting the vaginal flora itself. A letter to the editor {Strohmer et al in Human reproduction 1997 Jul; 12(7):1599}cites a case of a 37 year old patient who was using 3 % H2O2 soaked in cotton and inserting deep into vagina to the cervical os to avoid pregnancy. Applicants work is different from the work of Strohmer et al in many aspects and a few of such aspects are given below: 1. The contraceptive composition in instant application has normal saline along with H2O2 concentration ranging between 1 to 3%. Therefore the beneficial effects of the saline could neutralize the cyto-toxic effects of H2O2 on the vaginal epithelium as observed in said letter to editor. 2. The efficacy of the method as stated by Strohmer et al. (1997) is questionable since putting a soaked cotton deep into the vagina could also block the opening of the cervical os preventing the sperms to pass through. Therefore, the resultant effect can be due to mechanical blockade and need not necessarily be due to H2O2 only. 3. No validation of the effect was submitted in the report by Strohmer (1997). 4. The contraceptive composition of instant application has synergistic spermicidal effect, with toxicity towards the sperms but having no irritation or any other adverse effects on vaginal epithelium and is safe for application on animal including humans. Concentrations of hydrogen peroxide 100 to 200^1 are reported Daru et al (effects of hydrogen peroxide on NDA and plasma membrane integrity of human spermatozoa; Fertility and sterility 74 (6): 1200-1206) to increase the oxidative stress on human sperms affecting their progressive motility and plasma membrane integrity and inducting DNA fragmentation. Also, there is a need for development of new and low cost contraceptives with greater efficacy, safety, and acceptability. Available vaginal contraceptives are oily preparations, either gel or foam based. These become messy during and following the coitus and as a result, the sexual pleasure is also claimed to be affected. This may be the reason for which the acceptability of such contraceptives is on decline. Therefore, developing a new, water-based contraceptive with good efficacy would go a long way in increasing the acceptability of the vaginal contraceptive use in the country. Objects of the present invention The main object of the present invention is to develop an effective vaginal contraceptive. Another object of the present invention is to develop a safe vaginal contraceptive. Yet another object of the present invention is to develop a vaginal contraceptive with an ability to bring down the sperm motility. Still another object of the present invention is to develop a vaginal contraceptive with an ability to maintain vaginal pH. Still another object of the present invention is to develop a vaginal contraceptive with an ability to bring down sperm viability. Still another object of the present invention is to develop an easy to use vaginal contraceptive. Still another object of the present invention is to develop an economical vaginal contraceptive. Still another object of the present invention is to develop a non-messy vaginal contraceptive. Still another object of the present invention is to provide a shelf-stable composition. Further object of the present invention is to provide a method to develop a safe aqueous vaginal contraceptive with spermicidal activity. Another main object of the present invention is to develop a simple method of preparing an efficient vaginal contraceptive with spermicidal activity. In addition, another main object of the present invention is to develop a method of treating sperms with the synergistic spermicidal composition. Further, another main object of the present invention is to use the synergistic spermicidal composition of hydrogen peroxide and sodium chloride as vaginal contraceptive. Summary of the present invention The present invention relates to a method of preparing an inexpensive, non-messy, safe and fast acting aqueous spermicidal composition of 100% efficacy useful as a vaginal contraceptive free of side effects, said method comprising steps of preparing sodium chloride solution of concentration ranging between 0.4 to 3% in water, mixing hydrogen peroxide to said sodium chloride solution to obtain a mixture having hydrogen peroxide in the concentration ranging between 1 to 3%, adjusting pH of the mixture ranging between 4 to 4.5, and obtaining said composition, also, the composition thereof. The said composition is capable of maintaining vaginal pH and has no adverse effect on the beneficial vaginal flora. Detailed description of the present invention Accordingly, the present invention relates to a method of preparing an inexpensive, non-messy, safe and fast acting aqueous synergistic spermicidal composition of 100% efficacy useful as a vaginal contraceptive free of side effects, said method comprising steps of preparing sodium chloride solution of concentration ranging between 0.4 to 3% in water, mixing hydrogen peroxide to said sodium chloride solution to obtain a mixture having hydrogen peroxide in the concentration ranging between 1 to 3%, adjusting pH of the mixture ranging between 4 to 4.5, and obtaining said composition, also, the composition thereof. The said composition is capable of maintaining vaginal pH and has no adverse effect on the beneficial vaginal flora. embodiment of the present invention, wherein a water based composition useful as contraceptive, said composition comprising hydrogen peroxide of concentration ranging between 1 to 3%, and sodium chloride of concentration ranging between 0.4 to 3.0%, with pH ranging between 4 to 4.5 and showing synergistic contraceptive effect, and said composition is an inexpensive, non-messy, fast acting vaginal contraceptive, with 100% efficacy and without any side effects. In another embodiment of the present invention, wherein the composition has preferred concentration of hydrogen peroxide of about 2%. In yet another embodiment of the present invention, wherein the composition has preferred pH of about 4.15. In still another embodiment of the present invention, wherein the composition is economical than the commercials available vaginal contraceptives. In still another embodiment of the present invention, wherein the composition has no adverse effect on the vaginal tissue. In still another embodiment of the present invention, wherein the composition has no adverse effect on the vaginal flora. In still another embodiment of the present invention, wherein the composition has no adverse effect on the vaginal pH. In still another embodiment of the present invention, wherein the composition has shelf life of about 30 days. In still another embodiment of the present invention, wherein the composition is safe for application in animals including humans. In still another embodiment of the present invention, wherein the composition start functioning within 20 seconds of application. In still another embodiment of the present invention, wherein applying volume of the said composition varies between 10µ1 to 5000µ1. In still another embodiment of the present invention, wherein the composition is most effective in the semen volume in the ratio ranging between 1:1 to 1:5. In still another embodiment of the present invention, wherein the composition show decrease in sperm viability to about 10%. In still another embodiment of the present invention, wherein the composition show 100% non-motility in sperms. In still another embodiment of the present invention, wherein the composition show decrease in sperm hypo-osmotic swelling to about 8%. In still another embodiment of the present invention, wherein the composition show decrease in nuclear chromatin de-condensation to ranging between 25 to 30%. In still another embodiment of the present invention, wherein the composition show 100% contraceptive efficacy in first two hours of application. In still another embodiment of the present invention, wherein the composition show 77% contraceptive efficacy in first 24 hours of application. In still another embodiment of the present invention, wherein the composition show reversibility of contraceptive effect immediately after stopping the same. In an embodiment of the present invention, wherein a method of preparing a water based composition useful as vaginal contraceptive, said composition comprising hydrogen peroxide of concentration ranging between 1 to 3%, and sodium chloride of concentration ranging between 0.4 to 3.0%, with pH ranging between 4 to 4.5 and showing synergistic contraceptive effect, and said composition is an inexpensive, non-messy, fast acting vaginal contraceptive, with 100% efficacy and without any side effects. In another embodiment of the present invention, wherein adding sodium chloride to water. In yet another embodiment of the present invention, wherein obtaining sodium chloride solution of concentration ranging between 0.4 to 3%. In still another embodiment of the present invention, wherein mixing hydrogen peroxide to said sodium chloride solution. In still another embodiment of the present invention, wherein obtaining said mixture with hydrogen peroxide of concentration ranging between 1 to 3%. In still another embodiment of the present invention, wherein adjusting pH ranging between 4 to 4.5 In still another embodiment of the present invention, wherein obtaining said composition. In still another embodiment of the present invention, wherein the said solution has the capacity to increase the oxidative stress on the sperms limiting their progressive motility and affecting other sperm functions as well. In still another embodiment of the present invention, wherein isotonicity of the said composition would also be able to minimize any undesired effect of the hydrogen peroxide on the existing beneficial vaginal flora. In still another embodiment of the present invention, wherein chemical composition of the said vaginal contraceptive (i) NaCl (0.4 - 3.0%) in deionized triple distilled water, (ii) (1.0-3.0 %) H2O2, said composition at pH ranging between 4.0 to 4.5 In still another embodiment of the present invention, wherein volume of ejaculate varies with each ejaculate, thus effect of the test solution to varied volumes of semen is checked systematically for the loss of sperm motility over time. In still another embodiment of the present invention, wherein as not all the non-motile sperms are necessarily non-viable, thus, viability testing provides information on the live and dead ratio of the sperm population, both motile and non-motile. In still another embodiment of the present invention, wherein testing spermicidal efficacy of any composition is thus important to find out the live and dead ratio of the sperms following an in vitro challenge. In still another embodiment of the present invention, wherein said composition is stored in dark bottles. In still another embodiment of the present invention, wherein one application of said composition is effective only during one intercourse. hi still another embodiment of the present invention, wherein use of an inexpensive, non-messy, safe and fast acting aqueous synergistic spermicidal composition of 100% efficacy as vaginal contraceptive in animals including humans, wherein the said composition is applied in the vaginal region. In still another embodiment of the present invention, wherein the composition starts functioning within 20 seconds of application. hi still another embodiment of the present invention, wherein the applying volume of the composition is ranging between l0µl to 5000µl In still another embodiment of the present invention, wherein the composition is to be applied every time before intercourse. fln still another embodiment of the present invention, wherein the composition shows 100% contraceptive efficacy in first two hours of application. In still another embodiment of the present invention, wherein a method of treating sperms with an inexpensive, non-messy, safe and fast acting aqueous synergistic spermicidal composition of claim 16 of 100% efficacy as vaginal contraceptive in animals including humans, said method comprising step of exposing sperms to the said composition. In still another embodiment of the present invention, wherein said method shows composition starting functioning within 20 seconds of application. In still another embodiment of the present invention, wherein applying volume of the said composition varies between l0µl to 5000µl. In still another embodiment of the present invention, wherein said method producing composition showing decrease in sperm viability to about 10%. In still another embodiment of the present invention, wherein said method shows producing composition showing 100% non-motility in sperms. In still another embodiment of the present invention, wherein said method shows producing composition showing decrease in sperm hypo-osmotic swelling to about 8%. In still another embodiment of the present invention, wherein said method shows producing composition showing decrease in nuclear chromatin de-condensation to ranging between 25 to 30%. In still another embodiment of the present invention, wherein said method shows producing composition showing 100% contraceptive efficacy in first two hours of application. In still another embodiment of the present invention, wherein said method shows producing composition showing 77% contraceptive efficacy in first 24 hours of application. In still another embodiment of the present invention, wherein said method shows producing composition showing reversibility of contraceptive effect immediately after stopping the same. In still another embodiment of the present invention, wherein use of an inexpensive, non-messy, safe and fast acting aqueous synergistic spermicidal composition of claim 16 of 100% efficacy as vaginal contraceptive inn animals including humans, wherein the said composition is applied in the vaginal region. In still another embodiment of the present invention, wherein a method of treating sperms with an inexpensive, non-messy, safe and fast acting aqueous synergistic spermicidal composition of claim 16 of 100% efficacy as vaginal contraceptive in animals including humans, said method comprising step of exposing sperms to the said composition. A method of preparing an aqueous spermicidal composition, substantially as herein described with respect to the accompanying drawings and below-stated examples. A water based composition, substantially as herein described with respect to the accompanying drawings and below-stated examples. A method of preparing an aqueous spermicidal composition, substantially as herein described with respect to the accompanying drawings and aforesaid examples. A water based composition, substantially as herein described with respect to the accompanying drawings and aforesaid examples. The said composition is extremely economical and superior to commercially available vaginal contraceptive and can be of great use for a country like India. The details are as follows: Cost of commercially available drugs: (a) Deflon — 50 gms - cream -Rs. 10.00 approx. (for one applications) (b) Orthognal - 85 gms - gelly - Rs. 4.00 approx. ( for one applications) (c) Today - 5 pessaries - tablet -Rs. 14.00 approx. (for one application) (d) Applicants soln. - water based - Rs. 3-47- approx. (for about 100 applications) Table 1 (Table Removed) The components of the said composition are not capable of producing desired results individually. The synergistic contraceptive activity is shown by the said composition. The NaCl alone at concentration about 0.9% show 8% contraceptive activity, and hydrogen peroxide alone at concentration about 2% show 42% contraceptive activity. Surprisingly, both the said components put together shows synergistic contraceptive activity of 100% Brief description of the accompanying drawings Fig 1 shows sperm nuclear chromatin decondensation with about 70 % in control groups. Fig 2 shows after immobilization with test solution, a significantly (p Fig 3 shows viable sperms in the semen smear as cells with bright and colorless head, as viable cells do not pick color strain. (Control) Tig 4 shows dead sperms in the semen smear, as only dead cells pick up the strain after adding test solution. Fig 5 shows Control vagina with convoluted vaginal epithelium, which appeared normal with smooth boundaries. No inflammation of any kind was seen in any of the glandular cellular layers. The outer most epithelium lining was found a shade darker than the immediate innermost layer and could be differentiated from the other glandular cells. Fig 6 shows vaginal tissue after administering NaCl, with convoluted epithelium with inflammation at the outermost layer. As a result, the height of the cellular lining was found markedly increased. Fig 7 shows vaginal tissue after administering H2O2 with the outermost epithelium depicted less inflammatory but more pyknotic-staining pattern. This differential intense staining and contrast is mostly considered as apoptotic, inducing cell death in the affected region. This region can be differentiated from other glandular cells because of this typical staining pattern. The cells were more compressed and the cell height decreased. Increased vascularization in the innermost layers was noticed. Fig 8 shows vaginal tissue after administering test solution with differential staining of the innermost layer demarcated as was seen in control vagina. However, inflammatory changes in the innermost layer were still persisting similar to what has been observed in the vagina flushed with NaCl only. The significant improvement over raw H2O2 treatment was that there was no vascularization of tissue adjacent to the epithelial lining. The epithelial lining depicted restoration in the cellular height indicating normal secretary activity. There was no intense or pyknotic staining of the cellular epithelial lining. The following examples are given by the way of illustration of the present invention and should not be construed to limit the scope of the present invention. Examples: Example 1. - Sperm motility iemen and test solution was mixed in 1:1 proportion and a drop of the mixture was examined at 400 x under a phase contrast microscope to record the motility of the sperm in the preparation (WHO, 1999). Example 2: In vitro testing of human sperm motility with different dilutions of composition in semen Method: Effect of the Test Soln.[2% H2O2 in normal saline (0.9 % NaCl)] on Human Sperm (Table Removed) Table 2 Motility following admixing with semen in different proportions Table 3 (Table Removed) Example 3: Sperm viability Method: Eosin-Nigrosin staining (WHO, 1999) was utilized for recording sperm viability in control and test incubations. Briefly, 50µ1 of semen was properly mixed with 50 µl of test solution. 150µl Eosin Y (1.0 %).was added and after 30 seconds, three drops of Nigrosin (10 %) was added and mixed well. A smear on a clean glass slide was made from the mixture and allowed to air dry. Pink stained (dead) ones were (Table Removed) Table 4 differentiated from unstained, bluish fluorescent (live) sperms through examination using a phase contrast microscope. (As shown in figures 3 and 4) Fig 3 shows viable sperms in the semen smear as cells with bright and colorless head, as viable cells do not pick color strain. (Control). Fig 4 shows dead sperms in the semen smear, as only dead cells pick up the strain after adding test solution. Example 4. Hypo-osmotic swelling test Method: A hypo-osmotic swelling solution was prepared with sodium citrate 0.735 % and fructose 1.351 % in distilled water. Semen and the test solution was mixed in 1:1 proportion. 100 ul of the sperm mixture was added in a pre-warmed 1-ml hypo-osmotic swelling solution and incubated at 37°C for 30 min. Swelling as indicated by various types of coiling of the sperm tail (in 100 random sperms) was examined using a phase contrast microscope (Jeyendran et al., 1984). Table 5 (Table Removed) Example 5: Nuclear chromatin de-condensation test Method: Spermatozoa pelleted from plasma or media by centrifugation at 400 g for 15 min. were resuspended in borate buffer (0.05 M) containing dithiotheritol (DTT, 2mM). Following incubation for 30 min at room temperature, 1 % lauryl sulfate (SDS) was added and mixed gently. The mixture was diluted with equal volume of borate buffer (0.05 M) containing 2.5 % glutaraldehyde. An aliquot of the mixture was stained with Rose Bengal (0.8 %) and was examined under a phase contrast microscope. The number of condensed and decondensed nuclear chromatin were recorded (Rodriguez et al., 1985). Sperm nuclear chromatin decondensation was 70 % in control groups (as shown in figure 1). Fig 1 shows sperm nuclear chromatin decondensation with about 70 % in control groups. However, after immobilization with H2O2, a significantly (p Example 6: pH of different test solution Method: pH was measured using a pH meter against a standard solution. pH level in different test solutions Table 6 (Table Removed) *pH in the vaginal cavity ranges from 4-4.5 Example 7. Tissue compatibility of the test solution in the vagina Cyto-toxicity to the epithelial lining is conventionally validated through the following observations: (a) Decrease in cell height confirms cyto-toxicity. (b) Shrinkage of the secretary glands confirms cyto-toxicity. (c) Increased vascularization confirms cyto-toxicity. (d) Pyknotic (thickened) epithelial cell margin confirms cyto-toxicity. After 24 hrs of administration vaginal tissue were processed for histology and analyzed under microscope. Fig 5 shows Control vagina with convoluted vaginal epithelium, which appeared normal with smooth boundaries. No inflammation of any kind was seen in any of the glandular cellular layers. The outer most epithelium lining was found a shade darker than the immediate innermost layer and could be differentiated from the other glandular cells. Fig 6 shows vaginal tissue after administering NaCl, with convoluted epithelium with inflammation at the outermost layer. As a result, the height of the cellular lining was found markedly increased. Fig 7 shows vaginal tissue after administering H2O2 with the outermost epithelium depicted less inflammatory but more pyknotic-staining pattern. This differential intense staining and contrast is mostly considered as apoptotic, inducing cell death in the affected region. This region can be differentiated from other glandular cells because of this typical staining pattern. The cells were more compressed and the cell height decreased. Increased vascularization in the innermost layers was noticed. Fig 8 shows vaginal tissue after administering test solution with differential staining of the innermost layer demarcated as was seen in control vagina. However, inflammatory changes in the innermost layer were still persisting similar to what has been observed in the vagina flushed with NaCl only. The significant improvement over raw H2O2 treatment was that there was no vascularization of tissue adjacent to the epithelial lining. The epithelial lining depicted restoration in the cellular height indicating normal secretary activity. There was no intense or pyknotic staining of the cellular epithelial lining. Table 7 showing the conditions of the vaginal epithelium following the administration of various solutions: (Table Removed) Example 8: In vivo contraceptive efficacy testing of the chemical composition/test solution Method: Groups of rats in the proestrus and estrus stage of the cycle were vaginally administrated the test solutions at a specific volume as indicated below and immediately put to mating with a proven fertile male. After completion of specified time duration of co-habitation, mating was confirmed over the presence of spermatozoa in the vaginal swab analysis under microscope. The males were separated and the females were continued observation for 21 days till parturition for the final confirmation on the contraceptive efficacy of the test solution. In vivo testing of the Test composition (100µl) for efficacy as a vaginal contraceptive Table 8 (Table Removed) Many a times in some rats the test solutions drop back following administration into the vaginal cavity once the rat is brought back to the normal dorsal position. This probably seems to be one of the problems in lower animals (rats) for testing a vaginal contraceptive. In contrast, potential space is available in the posterior fernix of the vagina in humans, which can hold back the contraceptive following administration. Example 9 Comparative contraceptive efficacy of test solution Method - Groups of rats in the proestrus and estrus stage of cycle were vaginally administered the test solutions at a specific volume as indicated below and immediately put to mating with a proven fertile male. After completion of specified time duration of co-habitation, mating was confirmed over the presence of spermatozoa in the vaginal swab analysis under microscope. The males were separated and the females were under continued observation for 21 days till parturition for the final confirmation on the contraceptive efficacy of the test solution. Table 9 (Table Removed) Example 10 Studies on reversibility of the contraceptive effect Method - groups of rates in the proestrus and estrus stage of the cycle were vaginally administered the test solutions at a specific volume for one time and for three consecutive cycles below and immediately put to mating with a proven fertile male (1:1); After continuation of the efficacy in the three consecutive cycles and after 21 days (gestation period in arts), the rats were not administered the contraceptive test solution but allowed to mate with the male. The females were again watched till parturition (21 days) for confirmation of pregnancy and the size of the litters. The litters do not show any abnormality after administering the test solution of 0.9% NaCl and about 2 % hydrogen peroxide. Result: females not administered the composition of the instant Application became pregnant thus confirming the reversibility of the composition. Table 10 (Table Removed) We Claim 1. A method of preparing an inexpensive, non-messy, safe and fast acting aqueous synergistic spermicidal composition of 100% efficacy useful as a vaginal contraceptive free of side effects in animals in human beings, said method comprising steps of: (i) Preparing sodium chloride solution of concentration ranging between 0.4 to 3% in water, (ii) mixing hydrogen peroxide to said sodium chloride solution to obtain a mixture having hydrogen peroxide in the concentration ranging between 1 to 3%, and (iii) adjusting pH of the mixture ranging between 4 to 4.5, and (iv) obtaining said composition. 2. A method as claimed in claim 1, wherein said method shows preferred concentration of hydrogen peroxide of 2%. 3. A method as claimed in claim 1, wherein said method shows preferred pH of 4.15. 4. A method of preparing an inexpensive, non-messy, safe and fast acting aqueous synergistic spermicidal composition of 100% efficacy useful as a vaginal contraceptive free of side effects substantially as herein described and illustrated therein.

Full Text

METHOD OF PREPARING AN INEXPENSIVE, NON-MESSY, SAFE AND FAST ACTING AQUEOUS SYNERGISTIC SPERMICIDAL COMPOSITION"
Field of the present invention
The present invention relates to a method of preparing an inexpensive, non-messy, safe and fast acting aqueous spermicidal composition of 100% efficacy useful as a vaginal contraceptive free of side effects, said method comprising steps of preparing sodium chloride solution of concentration ranging between 0.4 to 3% in water, mixing hydrogen peroxide to said sodium chloride solution to obtain a mixture having hydrogen peroxide in the concentration ranging between 1 to 3%, adjusting pH of the mixture ranging between 4 to 4.5, and obtaining said composition, and also, the composition thereof. The said composition is capable of maintaining vaginal pH and has no adverse effect on the beneficial vaginal flora. Background and prior art references
Maintenance of functional integrity of the spermatozoa is vital for successful fertilization with the oocyte. Impairment with any of the functional properties of the sperm might lead to affect the ultimate process of fertilization. Development of a contraceptive composition that affects majority of sperm functions is always considered more efficacious.
Sperm motility is one of the important functions of the sperms, which enables the sperms to reach the oocytes for fertilization. Spermatozoa without forward progressive motility will not be able to perform this ultimate task, a key aspect while making a contraceptive composition.
Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male fertility (Armstrong JS, Rajasekharan M, Chamulitrat W, Gatti P, Helistrom WJ and Sikka SC (1998) Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism. Free Radical Biology Medicine 26: 869-880)
There are two sources of oxidative stress that affect human spermatozoa. The first source is the reactive oxygen species generated by the defective spermatozoa due to some defect in the spermatogenesis. The second source is from seminal leukocytes, infiltration of which is a common feature of the human ejaculate (Gavella M, Lipovac V, Vicic M and

Rocic B (1996) Superoxide anion scavanging capacity of human seminal plasma. Intl J of Andrology 19: 82-90).
The deleterious effects of ROS on sperm functions have been reviewed by Aitkin et al, 1994a. Aitkin RJ (1994) A free radical theory of male infertility. Reproduction Fertility and Development 6: 19-24.
Hydrogen peroxide is the most common and toxic ROS to spermatozoa till recognized. Hydrogen peroxide induced cell death was reported in cerebellar granule cells, which was characterized by chromatin condensation and DNA fragmentation. During the process, the fluidity of the cell membrane decreased markedly and the confirmation of the membrane proteins altered significantly (Wei T, Ni Y, Hou J, Chen C, Zhao B and Xin W (2000) Hydrogen peroxide-induced oxidative damage and apopotosis in cerebellar granule cells: Protection by Ginkgo Biloba extract. Pharmacological Research 41: 427-433.).
Use of hydrogen peroxide has also been reported (patent no. 778 886 by Shihata et al, Shihata (1998) vaginal contraception compositions combining a spermicidal agent and a peroxygen compound. United States Patent No 778 886.) along with the known spermicidal Nanoxynol -9 to restore the alterations to pH caused by the spermicidal application affecting the vaginal flora itself.
A letter to the editor {Strohmer et al in Human reproduction 1997 Jul; 12(7):1599}cites a case of a 37 year old patient who was using 3 % H2O2 soaked in cotton and inserting deep into vagina to the cervical os to avoid pregnancy. Applicants work is different from the work of Strohmer et al in many aspects and a few of such aspects are given below:
1. The contraceptive composition in instant application has normal saline along with
H2O2 concentration ranging between 1 to 3%. Therefore the beneficial effects of the
saline could neutralize the cyto-toxic effects of H2O2 on the vaginal epithelium as
observed in said letter to editor.
2. The efficacy of the method as stated by Strohmer et al. (1997) is questionable since
putting a soaked cotton deep into the vagina could also block the opening of the
cervical os preventing the sperms to pass through. Therefore, the resultant effect can
be due to mechanical blockade and need not necessarily be due to H2O2 only.
3. No validation of the effect was submitted in the report by Strohmer (1997).

4. The contraceptive composition of instant application has synergistic spermicidal effect, with toxicity towards the sperms but having no irritation or any other adverse effects on vaginal epithelium and is safe for application on animal including humans.
Concentrations of hydrogen peroxide 100 to 200^1 are reported Daru et al (effects of
hydrogen peroxide on NDA and plasma membrane integrity of human spermatozoa;
Fertility and sterility 74 (6): 1200-1206) to increase the oxidative stress on human sperms
affecting their progressive motility and plasma membrane integrity and inducting DNA
fragmentation.
Also, there is a need for development of new and low cost contraceptives with greater
efficacy, safety, and acceptability. Available vaginal contraceptives are oily preparations,
either gel or foam based. These become messy during and following the coitus and as a
result, the sexual pleasure is also claimed to be affected. This may be the reason for
which the acceptability of such contraceptives is on decline. Therefore, developing a
new, water-based contraceptive with good efficacy would go a long way in increasing the
acceptability of the vaginal contraceptive use in the country.
Objects of the present invention
The main object of the present invention is to develop an effective vaginal contraceptive.
Another object of the present invention is to develop a safe vaginal contraceptive.
Yet another object of the present invention is to develop a vaginal contraceptive with an
ability to bring down the sperm motility.
Still another object of the present invention is to develop a vaginal contraceptive with an
ability to maintain vaginal pH.
Still another object of the present invention is to develop a vaginal contraceptive with an
ability to bring down sperm viability.
Still another object of the present invention is to develop an easy to use vaginal
contraceptive.
Still another object of the present invention is to develop an economical vaginal contraceptive.

Still another object of the present invention is to develop a non-messy vaginal contraceptive.
Still another object of the present invention is to provide a shelf-stable composition. Further object of the present invention is to provide a method to develop a safe aqueous vaginal contraceptive with spermicidal activity.
Another main object of the present invention is to develop a simple method of preparing an efficient vaginal contraceptive with spermicidal activity.
In addition, another main object of the present invention is to develop a method of treating sperms with the synergistic spermicidal composition.
Further, another main object of the present invention is to use the synergistic spermicidal composition of hydrogen peroxide and sodium chloride as vaginal contraceptive. Summary of the present invention
The present invention relates to a method of preparing an inexpensive, non-messy, safe and fast acting aqueous spermicidal composition of 100% efficacy useful as a vaginal contraceptive free of side effects, said method comprising steps of preparing sodium chloride solution of concentration ranging between 0.4 to 3% in water, mixing hydrogen peroxide to said sodium chloride solution to obtain a mixture having hydrogen peroxide in the concentration ranging between 1 to 3%, adjusting pH of the mixture ranging between 4 to 4.5, and obtaining said composition, also, the composition thereof. The said composition is capable of maintaining vaginal pH and has no adverse effect on the beneficial vaginal flora. Detailed description of the present invention
Accordingly, the present invention relates to a method of preparing an inexpensive, non-messy, safe and fast acting aqueous synergistic spermicidal composition of 100% efficacy useful as a vaginal contraceptive free of side effects, said method comprising steps of preparing sodium chloride solution of concentration ranging between 0.4 to 3% in water, mixing hydrogen peroxide to said sodium chloride solution to obtain a mixture having hydrogen peroxide in the concentration ranging between 1 to 3%, adjusting pH of the mixture ranging between 4 to 4.5, and obtaining said composition, also, the composition thereof. The said composition is capable of maintaining vaginal pH and has no adverse effect on the beneficial vaginal flora.

embodiment of the present invention, wherein a water based composition useful as contraceptive, said composition comprising hydrogen peroxide of concentration ranging between 1 to 3%, and sodium chloride of concentration ranging between 0.4 to 3.0%, with pH ranging between 4 to 4.5 and showing synergistic contraceptive effect, and said composition is an inexpensive, non-messy, fast acting vaginal contraceptive, with 100% efficacy and without any side effects.
In another embodiment of the present invention, wherein the composition has preferred concentration of hydrogen peroxide of about 2%.
In yet another embodiment of the present invention, wherein the composition has preferred pH of about 4.15.
In still another embodiment of the present invention, wherein the composition is economical than the commercials available vaginal contraceptives. In still another embodiment of the present invention, wherein the composition has no adverse effect on the vaginal tissue.
In still another embodiment of the present invention, wherein the composition has no adverse effect on the vaginal flora.
In still another embodiment of the present invention, wherein the composition has no adverse effect on the vaginal pH.
In still another embodiment of the present invention, wherein the composition has shelf life of about 30 days.
In still another embodiment of the present invention, wherein the composition is safe for application in animals including humans.
In still another embodiment of the present invention, wherein the composition start functioning within 20 seconds of application.
In still another embodiment of the present invention, wherein applying volume of the said composition varies between 10µ1 to 5000µ1.
In still another embodiment of the present invention, wherein the composition is most effective in the semen volume in the ratio ranging between 1:1 to 1:5. In still another embodiment of the present invention, wherein the composition show decrease in sperm viability to about 10%.

In still another embodiment of the present invention, wherein the composition show
100% non-motility in sperms.
In still another embodiment of the present invention, wherein the composition show
decrease in sperm hypo-osmotic swelling to about 8%.
In still another embodiment of the present invention, wherein the composition show
decrease in nuclear chromatin de-condensation to ranging between 25 to 30%.
In still another embodiment of the present invention, wherein the composition show
100% contraceptive efficacy in first two hours of application.
In still another embodiment of the present invention, wherein the composition show 77%
contraceptive efficacy in first 24 hours of application.
In still another embodiment of the present invention, wherein the composition show
reversibility of contraceptive effect immediately after stopping the same.
In an embodiment of the present invention, wherein a method of preparing a water based
composition useful as vaginal contraceptive, said composition comprising hydrogen
peroxide of concentration ranging between 1 to 3%, and sodium chloride of concentration
ranging between 0.4 to 3.0%, with pH ranging between 4 to 4.5 and showing synergistic
contraceptive effect, and said composition is an inexpensive, non-messy, fast acting
vaginal contraceptive, with 100% efficacy and without any side effects.
In another embodiment of the present invention, wherein adding sodium chloride to
water.
In yet another embodiment of the present invention, wherein obtaining sodium chloride
solution of concentration ranging between 0.4 to 3%.
In still another embodiment of the present invention, wherein mixing hydrogen peroxide
to said sodium chloride solution.
In still another embodiment of the present invention, wherein obtaining said mixture with
hydrogen peroxide of concentration ranging between 1 to 3%.
In still another embodiment of the present invention, wherein adjusting pH ranging
between 4 to 4.5
In still another embodiment of the present invention, wherein obtaining said composition.

In still another embodiment of the present invention, wherein the said solution has the capacity to increase the oxidative stress on the sperms limiting their progressive motility and affecting other sperm functions as well.
In still another embodiment of the present invention, wherein isotonicity of the said composition would also be able to minimize any undesired effect of the hydrogen peroxide on the existing beneficial vaginal flora.
In still another embodiment of the present invention, wherein chemical composition of the said vaginal contraceptive (i) NaCl (0.4 - 3.0%) in deionized triple distilled water, (ii) (1.0-3.0 %) H2O2, said composition at pH ranging between 4.0 to 4.5 In still another embodiment of the present invention, wherein volume of ejaculate varies with each ejaculate, thus effect of the test solution to varied volumes of semen is checked systematically for the loss of sperm motility over time.
In still another embodiment of the present invention, wherein as not all the non-motile sperms are necessarily non-viable, thus, viability testing provides information on the live and dead ratio of the sperm population, both motile and non-motile. In still another embodiment of the present invention, wherein testing spermicidal efficacy of any composition is thus important to find out the live and dead ratio of the sperms following an in vitro challenge.
In still another embodiment of the present invention, wherein said composition is stored in dark bottles.
In still another embodiment of the present invention, wherein one application of said composition is effective only during one intercourse.
hi still another embodiment of the present invention, wherein use of an inexpensive, non-messy, safe and fast acting aqueous synergistic spermicidal composition of 100% efficacy as vaginal contraceptive in animals including humans, wherein the said composition is applied in the vaginal region.
In still another embodiment of the present invention, wherein the composition starts functioning within 20 seconds of application.
hi still another embodiment of the present invention, wherein the applying volume of the composition is ranging between l0µl to 5000µl

In still another embodiment of the present invention, wherein the composition is to be
applied every time before intercourse.
fln still another embodiment of the present invention, wherein the composition shows
100% contraceptive efficacy in first two hours of application.
In still another embodiment of the present invention, wherein a method of treating sperms
with an inexpensive, non-messy, safe and fast acting aqueous synergistic spermicidal
composition of claim 16 of 100% efficacy as vaginal contraceptive in animals including
humans, said method comprising step of exposing sperms to the said composition.
In still another embodiment of the present invention, wherein said method shows
composition starting functioning within 20 seconds of application.
In still another embodiment of the present invention, wherein applying volume of the said
composition varies between l0µl to 5000µl.
In still another embodiment of the present invention, wherein said method producing
composition showing decrease in sperm viability to about 10%.
In still another embodiment of the present invention, wherein said method shows
producing composition showing 100% non-motility in sperms.
In still another embodiment of the present invention, wherein said method shows
producing composition showing decrease in sperm hypo-osmotic swelling to about 8%.
In still another embodiment of the present invention, wherein said method shows
producing composition showing decrease in nuclear chromatin de-condensation to
ranging between 25 to 30%.
In still another embodiment of the present invention, wherein said method shows
producing composition showing 100% contraceptive efficacy in first two hours of
application.
In still another embodiment of the present invention, wherein said method shows
producing composition showing 77% contraceptive efficacy in first 24 hours of
application.
In still another embodiment of the present invention, wherein said method shows
producing composition showing reversibility of contraceptive effect immediately after
stopping the same.

In still another embodiment of the present invention, wherein use of an inexpensive, non-messy, safe and fast acting aqueous synergistic spermicidal composition of claim 16 of 100% efficacy as vaginal contraceptive inn animals including humans, wherein the said composition is applied in the vaginal region.
In still another embodiment of the present invention, wherein a method of treating sperms with an inexpensive, non-messy, safe and fast acting aqueous synergistic spermicidal composition of claim 16 of 100% efficacy as vaginal contraceptive in animals including humans, said method comprising step of exposing sperms to the said composition. A method of preparing an aqueous spermicidal composition, substantially as herein described with respect to the accompanying drawings and below-stated examples. A water based composition, substantially as herein described with respect to the accompanying drawings and below-stated examples.
A method of preparing an aqueous spermicidal composition, substantially as herein described with respect to the accompanying drawings and aforesaid examples. A water based composition, substantially as herein described with respect to the accompanying drawings and aforesaid examples.
The said composition is extremely economical and superior to commercially available vaginal contraceptive and can be of great use for a country like India. The details are as follows: Cost of commercially available drugs:
(a) Deflon — 50 gms - cream -Rs. 10.00 approx. (for one applications)
(b) Orthognal - 85 gms - gelly - Rs. 4.00 approx. ( for one applications)
(c) Today - 5 pessaries - tablet -Rs. 14.00 approx. (for one application)
(d) Applicants soln. - water based - Rs. 3-47- approx. (for about 100 applications)
Table 1

(Table Removed)

The components of the said composition are not capable of producing desired results individually. The synergistic contraceptive activity is shown by the said composition. The NaCl alone at concentration about 0.9% show 8% contraceptive activity, and hydrogen peroxide alone at concentration about 2% show 42% contraceptive activity. Surprisingly, both the said components put together shows synergistic contraceptive activity of 100% Brief description of the accompanying drawings
Fig 1 shows sperm nuclear chromatin decondensation with about 70 % in control groups. Fig 2 shows after immobilization with test solution, a significantly (p Fig 3 shows viable sperms in the semen smear as cells with bright and colorless head, as
viable cells do not pick color strain. (Control)
Tig 4 shows dead sperms in the semen smear, as only dead cells pick up the strain after
adding test solution.
Fig 5 shows Control vagina with convoluted vaginal epithelium, which appeared normal
with smooth boundaries. No inflammation of any kind was seen in any of the glandular
cellular layers. The outer most epithelium lining was found a shade darker than the
immediate innermost layer and could be differentiated from the other glandular cells.
Fig 6 shows vaginal tissue after administering NaCl, with convoluted epithelium with
inflammation at the outermost layer. As a result, the height of the cellular lining was
found markedly increased.
Fig 7 shows vaginal tissue after administering H2O2 with the outermost epithelium
depicted less inflammatory but more pyknotic-staining pattern. This differential intense
staining and contrast is mostly considered as apoptotic, inducing cell death in the affected
region. This region can be differentiated from other glandular cells because of this typical
staining pattern. The cells were more compressed and the cell height decreased.
Increased vascularization in the innermost layers was noticed.
Fig 8 shows vaginal tissue after administering test solution with differential staining of
the innermost layer demarcated as was seen in control vagina. However, inflammatory
changes in the innermost layer were still persisting similar to what has been observed in
the vagina flushed with NaCl only. The significant improvement over raw H2O2
treatment was that there was no vascularization of tissue adjacent to the epithelial lining.
The epithelial lining depicted restoration in the cellular height indicating normal secretary
activity. There was no intense or pyknotic staining of the cellular epithelial lining.
The following examples are given by the way of illustration of the present invention and
should not be construed to limit the scope of the present invention.

Examples:
Example 1. - Sperm motility
iemen and test solution was mixed in 1:1 proportion and a drop of the mixture was
examined at 400 x under a phase contrast microscope to record the motility of the sperm
in the preparation (WHO, 1999).
Example 2: In vitro testing of human sperm motility with different dilutions of
composition in semen
Method:
Effect of the Test Soln.[2% H2O2 in normal saline (0.9 % NaCl)] on Human Sperm
(Table Removed)
Table 2
Motility following admixing with semen in different proportions
Table 3

(Table Removed)
Example 3: Sperm viability

Method: Eosin-Nigrosin staining (WHO, 1999) was utilized for recording sperm viability in control and test incubations. Briefly, 50µ1 of semen was properly mixed with 50 µl of test solution. 150µl Eosin Y (1.0 %).was added and after 30 seconds, three drops of Nigrosin (10 %) was added and mixed well. A smear on a clean glass slide was made from the mixture and allowed to air dry. Pink stained (dead) ones were
(Table Removed)

Table 4
differentiated from unstained, bluish fluorescent (live) sperms through examination using a phase contrast microscope. (As shown in figures 3 and 4) Fig 3 shows viable sperms in the semen smear as cells with bright and colorless head, as viable cells do not pick color strain. (Control). Fig 4 shows dead sperms in the semen smear, as only dead cells pick up the strain after adding test solution. Example 4. Hypo-osmotic swelling test
Method: A hypo-osmotic swelling solution was prepared with sodium citrate 0.735 % and fructose 1.351 % in distilled water. Semen and the test solution was mixed in 1:1 proportion. 100 ul of the sperm mixture was added in a pre-warmed 1-ml hypo-osmotic swelling solution and incubated at 37°C for 30 min. Swelling as indicated by various types of coiling of the sperm tail (in 100 random sperms) was examined using a phase contrast microscope (Jeyendran et al., 1984).

Table 5
(Table Removed)
Example 5: Nuclear chromatin de-condensation test
Method: Spermatozoa pelleted from plasma or media by centrifugation at 400 g for 15 min. were resuspended in borate buffer (0.05 M) containing dithiotheritol (DTT, 2mM). Following incubation for 30 min at room temperature, 1 % lauryl sulfate (SDS) was added and mixed gently. The mixture was diluted with equal volume of borate buffer (0.05 M) containing 2.5 % glutaraldehyde. An aliquot of the mixture was stained with Rose Bengal (0.8 %) and was examined under a phase contrast microscope. The number of condensed and decondensed nuclear chromatin were recorded (Rodriguez et al., 1985). Sperm nuclear chromatin decondensation was 70 % in control groups (as shown in figure 1). Fig 1 shows sperm nuclear chromatin decondensation with about 70 % in control groups.
However, after immobilization with H2O2, a significantly (p Example 6: pH of different test solution
Method: pH was measured using a pH meter against a standard solution. pH level in different test solutions
Table 6

(Table Removed)
*pH in the vaginal cavity ranges from 4-4.5
Example 7. Tissue compatibility of the test solution in the vagina
Cyto-toxicity to the epithelial lining is conventionally validated through the following observations:
(a) Decrease in cell height confirms cyto-toxicity.
(b) Shrinkage of the secretary glands confirms cyto-toxicity.
(c) Increased vascularization confirms cyto-toxicity.
(d) Pyknotic (thickened) epithelial cell margin confirms cyto-toxicity.
After 24 hrs of administration vaginal tissue were processed for histology and analyzed under microscope.
Fig 5 shows Control vagina with convoluted vaginal epithelium, which appeared normal with smooth boundaries. No inflammation of any kind was seen in any of the glandular cellular layers. The outer most epithelium lining was found a shade darker than the immediate innermost layer and could be differentiated from the other glandular cells. Fig 6 shows vaginal tissue after administering NaCl, with convoluted epithelium with inflammation at the outermost layer. As a result, the height of the cellular lining was found markedly increased.
Fig 7 shows vaginal tissue after administering H2O2 with the outermost epithelium depicted less inflammatory but more pyknotic-staining pattern. This differential intense staining and contrast is mostly considered as apoptotic, inducing cell death in the affected region. This region can be differentiated from other glandular cells because of this
typical staining pattern. The cells were more compressed and the cell height decreased. Increased vascularization in the innermost layers was noticed.
Fig 8 shows vaginal tissue after administering test solution with differential staining of the innermost layer demarcated as was seen in control vagina. However, inflammatory changes in the innermost layer were still persisting similar to what has been observed in the vagina flushed with NaCl only. The significant improvement over raw H2O2 treatment was that there was no vascularization of tissue adjacent to the epithelial lining. The epithelial lining depicted restoration in the cellular height indicating normal secretary activity. There was no intense or pyknotic staining of the cellular epithelial lining. Table 7 showing the conditions of the vaginal epithelium following the administration of various solutions:

(Table Removed)
Example 8: In vivo contraceptive efficacy testing of the chemical composition/test
solution
Method: Groups of rats in the proestrus and estrus stage of the cycle were vaginally
administrated the test solutions at a specific volume as indicated below and immediately
put to mating with a proven fertile male. After completion of specified time duration of
co-habitation, mating was confirmed over the presence of spermatozoa in the vaginal
swab analysis under microscope. The males were separated and the females were

continued observation for 21 days till parturition for the final confirmation on the contraceptive efficacy of the test solution.
In vivo testing of the Test composition (100µl) for efficacy as a vaginal contraceptive
Table 8

(Table Removed)
Many a times in some rats the test solutions drop back following administration into the vaginal cavity once the rat is brought back to the normal dorsal position. This probably seems to be one of the problems in lower animals (rats) for testing a vaginal contraceptive. In contrast, potential space is available in the posterior fernix of the vagina in humans, which can hold back the contraceptive following administration. Example 9
Comparative contraceptive efficacy of test solution
Method - Groups of rats in the proestrus and estrus stage of cycle were vaginally administered the test solutions at a specific volume as indicated below and immediately put to mating with a proven fertile male. After completion of specified time duration of co-habitation, mating was confirmed over the presence of spermatozoa in the vaginal swab analysis under microscope. The males were separated and the females were under continued observation for 21 days till parturition for the final confirmation on the contraceptive efficacy of the test solution.
Table 9

(Table Removed)
Example 10
Studies on reversibility of the contraceptive effect
Method - groups of rates in the proestrus and estrus stage of the cycle were vaginally
administered the test solutions at a specific volume for one time and for three consecutive
cycles below and immediately put to mating with a proven fertile male (1:1); After
continuation of the efficacy in the three consecutive cycles and after 21 days (gestation
period in arts), the rats were not administered the contraceptive test solution but allowed
to mate with the male. The females were again watched till parturition (21 days) for
confirmation of pregnancy and the size of the litters.
The litters do not show any abnormality after administering the test solution of 0.9%
NaCl and about 2 % hydrogen peroxide.
Result: females not administered the composition of the instant Application became
pregnant thus confirming the reversibility of the composition.
Table 10

(Table Removed)

We Claim
1. A method of preparing an inexpensive, non-messy, safe and fast acting aqueous
synergistic spermicidal composition of 100% efficacy useful as a vaginal
contraceptive free of side effects in animals in human beings, said method
comprising steps of:
(i) Preparing sodium chloride solution of concentration ranging between 0.4 to
3% in water, (ii) mixing hydrogen peroxide to said sodium chloride solution to obtain a
mixture having hydrogen peroxide in the concentration ranging between 1 to
3%, and
(iii) adjusting pH of the mixture ranging between 4 to 4.5, and (iv) obtaining said composition.
2. A method as claimed in claim 1, wherein said method shows preferred
concentration of hydrogen peroxide of 2%.
3. A method as claimed in claim 1, wherein said method shows preferred pH of
4.15.
4. A method of preparing an inexpensive, non-messy, safe and fast acting aqueous
synergistic spermicidal composition of 100% efficacy useful as a vaginal
contraceptive free of side effects substantially as herein described and illustrated
therein.

Documents:

8-del-2002-abstract.pdf

8-del-2002-claims.pdf

8-del-2002-correspondence-others.pdf

8-del-2002-correspondence-po.pdf

8-del-2002-description (complete).pdf

8-del-2002-drawings.pdf

8-del-2002-form-1.pdf

8-del-2002-form-13.pdf

8-del-2002-form-19.pdf

8-del-2002-form-2.pdf

8-del-2002-form-26.pdf

8-del-2002-form-3.pdf

8-del-2002-form-4.pdf

8-del-2002-form-5.pdf

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Patent Number

217810

Indian Patent Application Number

8/DEL/2002

PG Journal Number

17/2008

Publication Date

25-Apr-2008

Grant Date

28-Mar-2008

Date of Filing

03-Jan-2002

Name of Patentee

NATIONAL INSTITUTE OF HEALTH & FAMILY WELFARE,

Applicant Address

1860, NEW MEHRAULI ROAD, MUNIRKA, NEW DELHI-110067, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	MANMOHAN MISRO	DEPARTMENT OF REPRODUCTIVE BIOMEDICINE NATIONAL INSTITUTE OF HEALTH AND FAMILY WELFARE MUNIRKA, NEW DELHI-110067, INDIA.
2	SANKAR PRASAD CHAKI	DEPARTMENT OF REPRODUCTIVE BIOMEDICINE NATIONAL INSTITUTE OF HEALTH AND FAMILY WALFARE, MUNIRKA, NEW DELHI-110067

PCT International Classification Number

A61K 9/08

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA