CONTRACEPTIVE VAGINAL SUPPOSITORY CONTAINING NONOXYNOL-9 AND ZINC ACETATE SALT IN A CLINICAL TRIAL

Spermicidal agents are defined as drugs that have the ability to immobilize or kill the sperm upon contact. An ideal spermicide should immediately and irreversibly produce immobilization of the sperm, nonirritating to the vaginal and penile mucosa, not have adverse effects on the developing fetus, free from long term topical and systemic toxicity and should not be systemically absorbed. Spermicides as a contraceptive methods have advantages as that they do not depend on high skilled personnel for their prescription and use, they don’t interact systemically or interfere with absorption of other drugs, they are used on demand only not on exact time that may be not remembered also it isn’t hormones that may disturb woman body by effecting on ovulation, lactation or others. Spermicides disadvantages appear as higher rate of failure than hormonal methods, vaginal irritation and vaginal secretions which appear mainly on frequent use. N-9 spermicides have a failure rate of 18% per year on perfect use, and 29% under typical use and irritation rate was recorded 12% of participants sharing in clinical trial at 2000. on comparing advantages to disadvantages we found spermicide can be better contraceptive method than other hormonal method on treating their problems. N-9 which is the most popular spermicide had low acceptability (16.9%) and it offered to women looking for a short term, usercontrolled contraceptive.


INTRODUCTION
Spermicidal agents are defined as drugs that have the ability to immobilize or kill the sperm upon contact. An ideal spermicide should immediately and irreversibly produce immobilization of the sperm, nonirritating to the vaginal and penile mucosa, not have adverse effects on the developing fetus, free from long term topical and systemic toxicity and should not be systemically absorbed 1,2 . Spermicides as a contraceptive methods have advantages as that they do not depend on high skilled personnel for their prescription and use, they don't interact systemically or interfere with absorption of other drugs, they are used on demand only not on exact time that may be not remembered also it isn't hormones that may disturb woman body by effecting on ovulation, lactation or others 3 . Spermicides disadvantages appear as higher rate of failure than hormonal methods, vaginal irritation and vaginal secretions which appear mainly on frequent use 4 . N-9 spermicides have a failure rate of 18% per year on perfect use, and 29% under typical use 5 and irritation rate was recorded 12% of participants sharing in clinical trial at 2000 6 . on comparing advantages to disadvantages we found spermicide can be better contraceptive method than other hormonal method on treating their problems. N-9 which is the most popular spermicide had low acceptability (16.9%) and it offered to women looking for a short term, usercontrolled contraceptive 7 .
Zinc acetate is another approved spermicide which has a spermicidal effectiveness in a 1% concentration it appears this is due to the acetate in zinc acetate which can decrease oxygen utilization by sperm 8 . Zinc acetate came over N-9 as it doesn't cause irritation but it can reduce irritation of the mucosal tissue if found due to zinc ion that is effective in preventing or reducing irritation in a concentration of 0.5% which avoid zinc toxicity 9 . Effective concentrations of Nonoxynol-9, benzalkonium chloride, zinc acetate, cupric chloride, cysteamine, tannic acid and propranolol ranged at least from 0.15 to 1% 10 . N-9 spermicidal activity can withstand to 6 hours 11 . While, zinc acetate spermicidal activity can withstand for only one hour 12 . Addition of Zn (OAC) 2 to N-9 was a trial to produce a new product has advantages of both spermicides together.
In this study we suggest that the prepared spermicide vaginal suppository contain N-9 and Zn (OAC) 2 in concentration 10:1 respectively possess more advantages over presently marketed formulations which contains N-9 alone for the causes that mentioned before. New preparation contains N-9 plus Zn (OAC) 2 salt was tested invivo and in-vitro in compare with market suppositories containing N-9 alone.

Suppository formulation
Suppository formulations were prepared from water soluble bases (PEG 400 and PEG 6000) by melting method 13 . The molten base was poured into a mold of torpedo shape then refrigerated and packaged. Each 2 gm. vaginal suppository contains 100 mg N-9 and 10 mg Zn (OAC) 2 .

Drug release measurement of the prepared suppository
Dissolution was conducted in the USP dissolution apparatus 2 operating at 50 rpm using 500 ml of distilled water at 37ºC. 5 ml samples were taken at different time intervals and replaced with 5 ml of fresh dissolution medium maintained at the same temperature. Samples were taken with filter-tipped pipette and analyzed spectrophotometrically at 276 nm for N-9 13 , while zinc acetate was analyzed at 550 nm 14 . Results were plotted against time in the representative curve. Release of N-9 from the combination in the prepared suppository was compared with N-9 release lonely as reported by Parrott 12 and determined at the same time intervals so we can study changes made on addition of zinc acetate.

In-vitro study
Human males participating in this study were fertile semen donors selected after appropriate screening. Three specimens from each of three males were used in this study. Specimens were collected by masturbation following three days abstinence. Following collection, specimens were incubated at 37ºC for 15-30 minutes to allow for liquefaction. Semen volume, sperm aggregation and motility percentage were assessed using a light microscope. Sperm motility and aggregation recorded as percent and best spermicide produce 100% sperm immobility and 100% aggregation. Motility was calculated according to the formula: Motility = Motile sperms ∕ Motile + non motile sperms × 100 Sperm motility and sperm aggregation were measured before and after addition of spermicide on the semen samples after liquefaction and after one hour from liquefaction for testing different spermicides concentrations on it. Three dilutions were prepared from each suppository (with and without zinc acetate) A, B and C as seen in table 1 from each type of suppository and they tested on semen samples for comparing market (No Gravida®) and prepared suppository in different concentrations.

In-vivo study
The study recruited 78 participants referred for using spermicide suppository formulated of N-9 in PEG base (market or compounded) for conception purpose at family planning unite at General Abo-Korkase hospital and General Minia Hospital during the period from July 2010 to August 2011. Study was made to achieve two purposes from using the new spermicide which were decrease failure and irritation rates. This study is randomized prospective clinical trial. Randomization was computerized and blind.
We choose participants which have special criteria and accept the experiment and study 17,18 . Participants should know as much as possible about the clinical trial.
Participants in this study were informed and randomized to receive either N-9 suppository preparation or N-9 plus zinc acetate preparation as computer divides them. All participants would be instructed on the use of the test products 18 . Method of application is the most important point in clinical work-up as can change results at all; explain method for each volunteer. Verbal and written informed consent was obtained from all volunteers after giving information about the aim of the study and the procedure involved in it 18 .
Participants would be followed through 12 menstrual cycles (approximately 13 months) at least and would have 8 study visits and two studies phone call.
All volunteers had comprehensive evaluation full history of taking other contraceptive methods and after using spermicides (market or prepared). Special notes were made of the age, cycle length, literati, parity, previous method of contraception and lactation. Notes were taken about pregnancy if occur, vaginal irritation, coital problems, cycle irregularities, secretions and other compliances after use 19 .

RESULTS AND DISCUSSIN
Prepared suppository with zinc acetate mainly appeared to have similar physical quality properties to the market suppository as weight variations, melting point, hardness and others.
The dissolution studying of the prepared suppository with zinc acetate has high dissolution rate and high drug release profile as it start release with high concentration that reach >95% at first 18 min.
The concentrations of N-9 was measured spectrophotometrically at 276 nm and it was increasing manner until it started to make plateau level after 20 min. that was compared to that made for N-9 by Parrott 13 . Zinc acetate was measured spectrophotometrically at 550 nm and it was increasing manner at first 20 min and reaches to ≥95%. Table (2) and figure (1) illustrate the high dissolution rates of both N-9 and zinc acetate in the prepared suppository. Release start from first 3 min in increasing manner and the maximum amount released were at 18 min.   Table (3) and figure (2) show no significant difference in release of N-9 from market suppository as made by Parrote 13 and that of prepared suppository and so Zn (OAC) 2 don't change N-9 release profile.  High dissolution rate return to the formula which contains N-9 as it is Non-ionic surfactant 20 in non-ionic polymer of PEGs base. Zn (OAC) 2 do not interact with it or impair N-9 activity as the low concentration of Zn (OAC) 2 salt (in compare to N-9 concentration) prevents it from interference with N-9 add to that both active ingredients N-9 and Zn (OAC) 2 are highly stable components and need high temperature degrees to melt or interact.
The in-vitro test made on human semen samples (out human body) for testing changes on sperm motility and sperm aggregation on addition of different concentration of two group of spermicides (with and without Zn (OAC) 2 ) on it.
Prepared suppository (N-9 plus Zn (OAC) 2 ) show significant increase in efficacy of the formula in reduction of sperm motility and increasing sperm aggregation than market one in all dilutions especially before first hour passing.
As illustrated in table (4).   In-vitro test prove the role of zinc acetate addition to N-9 that can increase spermicidal efficacy of the N-9 not diminish it. These results come right with 16 and against 9 .
Different mechanisms of two spermicides (N-9 and Zn (OAC) 2 ) give the new formula of the combination high strength in reduction of sperm motility. Zinc acetate contains acetate ion which decreases oxygen utilization by sperm that the cause in decrease motility and increase in aggregation. 8 . Nonoxynol-9 vaginal spermicides interact with the lipoproteins of the cell membrane to permanently disrupt the cell membranes of spermatozoa, resulting in severe damage to the acrosome (head), neck, midpiece, and tail of the sperm and rapid, irreversible loss of function and motility within the vagina and viability 11 . Different mechanisms give the synergistic effect.
In the clinical studying, When discuss the results related to reduction of irritations we found that after application of drug on group Ӏ (market supp.) and group ӀӀ (prepared supp.) as seen in table (5) The results were observed in the positive side as vaginal irritation decrease with a high significant difference (P=0.02) when zinc acetate salt added to N-9. Irritation in this context may be evidenced by redness or other changes in coloration, inflammation or swelling, hypersensitivity, the occurrence of burning, itching or other painful stimuli. So, zinc ion that is effective in preventing or reducing irritation in a concentration of 0.5% which avoid zinc toxicity 9 .
The most common satisfied mechanism of zinc salt use as anti-irritant product was that zinc salts may prevent irritation by zinc ions which can bind to negatively-charged regions exposed on the surface of proteins and alter the charge configuration of the protein and prevent subsequent protein-protein interactions between irritants and exposed mucous membranes., thereby preventing its subsequent binding to the underlying tissue and so prevent irritation 16 .
Failure which means pregnancy occur show the following results in the table (6). The results show increase in the spermicidal efficacy of N-9, Zn acetate combination than N-9 alone so that Zn acetate spermicidal effect can increase the efficacy of N-9 not reduce it. Failure rate of N-9 also decreased significantly (P value equal 0.03). Zinc acetate (and zinc gluconate on adjusting pH to 7.0) was proven as strong spermicide between zinc salts and useful as a vaginal contraceptive 8 . And we assure on Combining two birth control methods, can increase their effectiveness to 95% or more for less effective methods 21 .

CONCLUSION
Nonoxynol-9 is the active ingredient in all of the over-thecounter (OTC) spermicidal products available in the markets and has been used for pregnancy prevention since the 1950s. but it start to be withdrawn from markets after compliance from it's high failure rate and low safety due to high irritation which lead to wounds and lesions that increase rate of sexual transmitted diseases. After screening in family planning clinics we found that local methods of contraception have high acceptability and high fair in the same time so, we tried to introduce a new formula which safe and effective. The new formula contains 0.5% concentration zinc acetate and 5% N-9 concentration which could produce a new spermicidal product with the best quality to the market as two components are approved spermicides at the mentioned concentrations. Different zinc salts as zinc lactate, zinc gluconate, zinc acetate, and other water-soluble organic zinc salts can reduce irritation caused by surfactants (nonoxynol or octoxynol) and other microbicides in topical genital formulations with different degrees but zinc acetate is the only spermicidal without any changes in its structure. Zinc-containing additives additionally can stabilize and protect cellular membranes, thereby helping protect genital surfaces against damage caused by repeated exposure to agents that attack the lipid membranes that surround mammalian cells. Suitable zinc Salts which have been tested and shown to be non-irritating during sexual intercourse include zinc acetate, zinc propionate, and zinc gluconate. Other zinc salts have also been identified which are soluble in water and have low pK values, which indicates a high rate of zinc ion release 22 .
Our results reached to that addition of Zn (OAC) 2 to N-9 produced an effective spermicide product plus the demulcent effect of zinc ion which appears well at 0.5% concentration that came with 16,12 .
So we recommended addition of zinc acetate salt to N-9 spermicidal formula to reach the best properties of spermicidal contraceptive.

ACKNOLEDGMENT
This work was developed in dialogue with the members of the Statistics, pathology, and doctor stuff of family planning unit in general Abo-korkase hospital for Clinical and Behavioral Studies; we gratefully acknowledge their contribution. We would like to thank Dr. Magdy Hassan for providing the data and assisting in their interpretation. Also, we thank several reviewers as well as the editor for their helpful suggestions.