PT99362B - PROCESS FOR THE PREPARATION OF ADDITIVES FOR HYGIENE PRODUCTS INTIMA FEMININA CONTAINING MONOESTERS AND DIESTERS OF POLYHYDRIC ALPHATIC ALCOHOLS - Google Patents

Abstract

The invention relates to a process for the preparation of a liquid composition for use in intimate feminine hygiene, characterized by incorporating, in a proportion from 0.1% to 10% by weight, a compound chosen from the group consisting of: a) monoesters of an aliphatic polyhydric alcohol and a fatty acid containing eight to eighteen carbon atoms and in which said monoester has at least one hydroxyl group associated with the aliphatic alcohol residue thereof, b) diesters of an aliphatic polyhydric alcohol and a fatty acid containing eight to eighteen carbon atoms and in which said diester has at least one hydroxyl group associated with the aliphatic alcohol residue thereof, and c) mixtures of said monoesters and diesters, said compound being present in a proportion which is effective for inhibiting the reduction of the toxic shock syndrome of toxin-1 by the bacteria Staphylococcus aureus when said product is exposed to said bacteria, in combination with a pharmaceutically acceptable vehicle.

Description

DESCRIPTION

GIVES

INVENTION PATENT

No. 99 362 <.. ··

APPLICANT: McNeil-PPC, Inc., North American, based at Van Liew Ave., Millttown, NJ 08850, United States of America

EPIGRAPH: PROCESS FOR THE PREPARATION OF ADDITIVES FOR PRODUCTS OF INTIMATE FEMALE HYGIENE CONTAINING MONOES TERES AND DIETHERS OF POLYHYDRATE ALIFATIC ALCOHOLS

INVENTORS: Susan K. Brown-Skrobot and Mary M. Irving, residing in the United States of America

Claim of the right of priority under Article 4 of the Paris Convention of 20 March 1883.

United States of America on October 30, 1990 and May 3, 1991, under serial numbers 605,899 and 695,471, respectively.

INPI. MOD. 113 RF 16Z32

Description relating to McNeil-PPC, Inc., North American, industrial and commercial patent, established at Van Liew Ave., Millttown, NJ 08850, United States of America, (inventors: Susan K. Brown-Skrobot and Mary M. Irving, residing in the United States of America), for “PROCESS FOR THE PREPARATION OF ADDITIVES FOR PRODUCTS OF INTIMATE FEMALE HYGIENE CONTAINING MONOESTH

POLYHYDRIC ALYPHYTIC ALIFYL ALCOHOL RES AND DIETHERS ”.

DESCRIPTION

1. Scope of the Invention

The present invention relates to the preparation of non-absorbent products used in intimate feminine hygiene such as irrigations, suppositories, gels, washes, and also contraceptives. More particularly, the invention relates to an active component which, when incorporated into feminine hygiene products, reduces the amount of certain toxins produced by bacteria.

2. Background of the Invention

Many hygiene and cleaning products in

Women's suits are used predominantly by ladies in the form of liquids. More specifically, many women use liquid vaginal irrigations to irrigate and clean the vagina and prevent vaginal infections, for contraception and sterility and to promote abortion (Feminine Hygiene Products:

Why Your Advice Is Needed, U.S. Pharmacist, May, 1986, p. 20-27, Thomas A. Gossel). Compositions for vaginal irrigations may consist of several compositions. Vinegar is the substance most commonly used for irrigations for vaginal hygiene purposes. Vinegar consists of approximately 4 to 6% acetic acid. However, there are insufficient data to definitively prove that vinegar is effective in changing the vaginal pH value for a sufficient period of time to promote the growth of. normal vaginal flora, and thus avoid infections.

British Patent Specification NQ. 1,374,105, published on November 13, 1974 and entitled Effervescent Compositions describes compositions for vaginal irrigations containing a silica gel. The compositions can be compressed and used with teeth cleaning, antacids, analgesic laxatives and vaginal irrigations. The compositions des. substances contain carbon dioxide and / or oxygen producing substances, for example, persulphate / perborate mixtures, and suitable pharmaceutical products, diluents, for example, sodium chloride, chelating agents such as AEDT (ethylene-diamino-tetraacetic acid) , surfactants, lubricants, flavorings and odorizers.

North American Patent No. 3,584,119 assigned on June 8, 1971 to Daniel B. Langley, describes compositions for vaginal irrigations that contain 3 to 8 ppb in weight of water-soluble agent, sodium dodecylbenzene sulfonate, potassium or sodium lauryl sulfate or 2-5 ppb by weight of alkaline metal nopersulfate or 5-10 ppb by weight of alkali metal borate, used as aqueous solutions of 5-60 g per liter.

United States Patent No. 3,219,525 assigned on November 23, 1965 to Samuel G. Berkow et al., Describes a pressurized container that contains a solution with

1.5 to 2.5 mg of an active cationic quaternary ammonium germicide, 30 to 60 mg of an antiseptic wetting agent. The resulting composition is an aerosol foam.

It has been reported that when acidic or alkaline lotions are used daily for irrigation, there are no overall changes in the pH value of the vagina or the vaginal mucosa. It was also demonstrated, in turn, that during the irrigation period, the vaginal pH value assumes the value of the irrigation solution. Thirty minutes after irrigation with an acidic solution, the pH value becomes effectively alkaline.

Strobino et al. reported that some irrigations are toxic to sperm, and are therefore used with a contraceptive. On the contrary, irrigations with sodium bicarbonate are another type of irrigation, used to increase the survival of sperm and thus increase fertility (Sodium Bicarbonate Douching For Improvement Of The Postcoital Test, Fertility and Sterility, Vol. 33, NQ. 6 , June 1980, pages 608-612, Ansari, Gould and Ansari).

One type of disease that occurs during menstruation is toxic shock syndrome (TSS), it is a serious and sometimes fatal multi-system disease that is associated with infection or colonization by Staphylococcus aureus bacteria (£ 3. aureus). This disease has been linked to the use of tampons during menstruation. The disease is thought to be caused by toxin-1 toxic shock syndrome (TSST-1), which is the toxin produced by most staphylococcal strains isolated from patients with menstrual TSS disease.

After the publication of reports associating the toxic shock syndrome with the use of tampons, several investigators have carried out studies to evaluate the effect of tampons on the growth of S. aureus bacteria as well as the effect of tampons on the production of TSST-1 by these bacteria . Initial efforts to understand the role of tampons in TSS disease have revealed mixed data. Slievert et al. (Obstet. Gynecol. Vol. 64, pages 666-670, November 1984) studied the effect of buffers on S. aureus to assess whether or not the components of the buffers increase the growth of S. aureus and the production of toxi in -1 of the toxic shock syndrome. They concluded that,

test conditions in your study, the buffer components do not provide nutrients for the growth of toxic shock syndrome S. aureus nor factors that induce toxin-1 production of toxic shock syndrome above control levels. After six hours of incubation, some commercial buffers that were tested were inhibitors of bacterial growth and suppressed toxin production. Others suppressed toxin production but did not inhibit cell growth. A buffer inhibited cell growth but increased the amount of toxin produced. On the other hand, Tierno and Hanna (Contraception, Vol. 31, p. 185-194, 1985) reported that in their experiments, buffers stimulated S. aureus bacteria to produce TSST -1.

Reiser et al., (J. Clin. Microbiol.,

Vol. 25, No. 8, p. 1450-1452, August 1987) then referred to the results of the tests they conducted to determine the effect of four types of buffers on the production of toxin-1 of the toxic shock syndrome. The amount of air available for the buffers that were tested was limited to that contained in the bags (made of cellulose with a cut molecular weight less than 10,000) into which the buffers were introduced during the test. This process was considered advantageous given the limited amount of available air that could more adequately simulate the in vivo conditions in the vagina during menstruation with a tampon in place, and in which one of the tampons that were tested was not changed before the test. The results of the tests conducted by Reiser et al. indicated that the buffers provided an increased surface area for bacteria! 3. aureus grow and adequate oxygen for the production of toxins. There was no significant inhibition of the growth of staph bacteria or TSST-1 production by any of the buffers used.

Robbins et al., Published in J. Clinicai Microbiol., Vol. 25, NS. 8, p. 1446-1449, August 1987 at the same time as Raiser et al., Reported the effect of 17 commercial buffers on the production of TSST-1 toxins using a disk-membrane agar (DMA) process, with an incubation at 37 ° C for 19 hours with 5% CO ₂ in air. The filter membranes

that overlapped the agar medium (with or without blood) in small petri dishes were inoculated with a £ 3 strain. aureus producing TSST-1. Robbin et al. Concluded that the main role of buffers in TSS may be to provide a fibrous surface for high and or sufficient colonization for the production of TSST-1. In addition, they verified the evidence of TSST-1 production by additives such as the deodorant / surfactant used in a commercial deodorant buffer and a decrease in TSST-1 production by inhibiting the growth of S. aureus as observed in case of a different commercial buffer. It is thought that both inhibition of TSST-1 production and inhibition of growth of £ 3. aureus may prove to be important in reducing the risk of TSS.

S. Notermans et al., (Journal of Food Safety, Vol. 3 (1981), pages 83-88) reported that glyceryl monolaurate, when used at a rate of 5 g per kg of meat suspension (pH 6, 0-6.2) inhibited toxin production by Clostridium botulium, type A, type B and type E. This article does not mention Staphylococcus aureus or any toxins produced from it, nor does it mention the feminine hygiene compositions that use monolaurate of glyceryl or the toxic shock syndrome.

In toxic shock syndrome (TSS), whether or not associated with menstruation, symptoms include fever, hypotension, redness, and scaling of the skin. TSST-1 is very much associated with menstrual conditions but is less frequently isolated from Staphylococcus aureus in non-menstrual conditions of the disease. Since TSST-1 can induce many with clinical behaviors of TSS in rabbits and other species, it is generally thought that it is the toxin in question in TSS (Schlievert, Staphylococcal Enterotoxin B and Toxic Shock Syndrome Toxin-1 Are Significantly Associated With Non-menstrual TSS, The Lancet, Vol. 1 (8490), May 17, 1986). However, Garbe (Garbe, Arko, Reingold et al.,

Staphylococcus aureus isolates from patients with non-menstrual toxic shock syndrome: Evidence for additional toxins, JAMA, 1985, Vol. 253; p. 2538-42) noted that many TSS isolated from non-menstrual situations did not express TSST-1 although fos5

without the cause of TSS-like symptoms in a rabbit model. Of the toxins formed by non-menstrual aureus isolates, TSST-1 was produced by 40% of those reported by Schlievert, 1986.

The production of TSST-1 for £ 5. aureus has been predominantly associated with menstrual TSS related to the use of tampons. Experiments were initiated to determine whether TSST-1 production could be minimized or stopped with fibrous absorbent materials. A group of compounds has unexpectedly been identified which is described in the co-pending North American patent applications No. 343,965, April 27, 1989 and 316,742 on April 27, 1990, which are incorporated herein by reference.

However, there is a need to find an irrigation composition that is able to combat the production of TSST-1 by Staphylococcus aureus within the vaginal cavity.

Summary of the Invention

The invention relates to non-absorbent compositions for irrigating the vagina containing a compound chosen from the group consisting of:

a) a monoester of a polyhydric aliphatic alcohol and a fatty acid containing eight to eighteen carbon atoms and wherein said monoester has at least one hydroxyl group associated with its aliphatic alcohol residue,

b) diesters of a polyhydric aliphatic alcohol and a fatty acid containing from eight to eighteen carbon atoms and wherein said diester has at least one hydroxyl group associated with its aliphatic alcohol residue, and

c) mixtures of said monoesters and diesters.

The fatty acid portion of the aforementioned monoesters and diesters can be derived from caprylic, capric, lauric, myristic, palmitic and stearic acids, which are saturated fatty acids whose chain lengths are respectively C _g , C _1Q , C ₁₂ , C ₁₄ , C _lg and C _lg . The fatty acid portion of the aforementioned monoesters and diesters can also be derived from unsaturated fatty acids with

carbon chain compounds which also vary from Cg to C ^g, an example of such unsaturated fatty acids being oleic acid. The preferred fatty acid for use in the practice of the present invention is wooluric acid, which is a saturated fatty acid whose chemical formula is C ^H ^COOH.

As used in these specifications and in the appended claims, the term aliphatic has the meaning usually attributed in organic chemistry, that is, factual refers to organic compounds characterized by having a linear or branched composition of the chain of carbon atoms.

As used in this specification and in the appended claims, the term polyhydric refers to the presence in a chemical compound of at least two hydroxy groups (OH). Thus, an aliphatic polyhydric alcohol is one that has at least two hydroxyl groups and that the carbon skeleton is linear or branched.

Are polyhydric alcohols suitable for forming monoesters and / or diesters for use in the practice of the present invention 1,2-ethanediol? 1,2,3-propanotriol (glycerol); 1,3-propanediol; 1,4-butanediol; 1,2,4-butanotriol and similar products. The preferred polyhydric aliphatic alcohol for use in the practice of the present invention is 1,2,3-propanotriol (commonly known as glycerol) whose chemical formula is HOOE ^ CH (OH) C ^ OH.

Esters that are useful in the practice of the present invention have at least one hydroxyl group associated with its aliphatic alcohol residue. Thus, it should be understood that the 1,2-ethanediol monoester and one of the fatty acids mentioned above can be used in the practice of the present invention since said ester, whose general formula is

CH _n . ₁ —C — 0 — CH _O —CH _n OH n 2n + l 2 2 have at least one hydroxyl group (ie the hydroxyl group at the right end of the structural formula presented above) in the ester portion derived from the aliphatic alcohol which is the 1,2-ethanediol. On the other hand, it should be noted that the diester of

1,2-ethanediol and one of the aforementioned fatty acids cannot be used in the practice of the present invention since I have mentioned it, of the ester, whose general formula is ^C n ^H 2n + 1- ^C - ° - ^CH 2 ^CH 2 ° - ^C - ^C n ^H 2n + 1 does not have at least one hydroxyl group in the ester part derived from 1,2-ethanediol.

glycerol monoester and one of the designated fatty acids can be used in the practice of the present invention since the ester will have two hydroxyl groups associated with it which are derived from glycerol. The glycerol diester and one of said organic acids can also be used since that ester will have a hydroxyl group associated with it and that it is derived from aliphatic alcohol and glycerol. In fact / as will be seen below, it has been found that mixtures of glycerol monolaurate and glycerol dilaurate are useful in the practice of the present invention. Finally, it should be noted that the glycerol triester and one of the defined fatty acids cannot be used in the practice of the present invention since that ester does not have at least one hydroxyl group in its part which is derived from aliphatic alcohol, i.e. is glycerol.

Preferred esters for use in the practice of the present invention are glyceryl monolaurate, glyceryl dilaurate and mixtures thereof.

Glyceryl monolaurate sold under the Monomuls 90 L-12 trademark by Henkel Corporation is particularly preferred. This compound contains about 96% by weight of glyceryl monolaurate. Glyceryl dilaurate is not detected in the samples of this compound used in the Examples presented below. Glycerol monolaurate is a compound referred to by GRAS by the FDA (Food and Drug Administratin) for use as an emulsifier for food. This substance is non-toxic to humans and has antimicrobial properties.

Other preferred esters for use according to this invention include C-3 alkanol monolaurate derivatives such as 2-hydroxy-1-pro8 laurate

pylus and 3-hydroxy-1-propyl laurate. C-3 alkanol dilaurate derivatives such as glycerol 1,3-dilaurate, glycerol 1,2-dilaurate should also be active in reducing the amount of enterotoxins, A, B, C and TSST-1 with the produced enterotoxin . Ethylene glycol derivatives such as ethylene glycol monolaurate as well as polyethylene glycol laurates, for example, diethylene glycol monolaurate and triethylene glycol monolaurate, are also believed to be active. It is also expected that some polymers will have an activation. toxin reduction capacity, for example, polyethylene glycol monolaurate (200 PM), polyethylene glycol monolaurate (400 PM), polyethylene glycol monolaurate (1 000 PM), and polypropylene glycol laura such as for example polypropylene glycol monolaurate.

Other compounds believed to be active against the TSST-1 toxin in the compositions of this invention are: glyceryl monocaprylate, glyceryl caprate, a mixture of glyceryl monocaprylate with glyceryl caprate, glyceryl monopalatate, glyceryl monostearate and glyceryl monoleate glyceryl.

According to the invention, the non-absorbent compositions of this invention contain an amount of the above-described ester that is effective to inhibit Toxic Shock Syndrome (TSST-1) toxin when said product is exposed to aureus. the effective amounts were found to be from about 0.1% and above, and preferably at least 0.5% by weight of the monoester or diester compound (or mixture thereof), based on the weight of the prepared solution.

The glyceryl monolaurate / glyceryl dilaurate mixtures of this invention preferably contain at least 90% glyceryl monolaurate; more preferably they contain at least 95% glyceryl monolaurate. More preferably, the mixture of the compounds should preferably be made up entirely of glyceryl monolaurate.

The active component of the des.

This invention can be formulated by various formulations such as those used in standard commercial formulations for irrigation, or for irrigations of lower viscosity. For example

Therefore, the active component of the compositions of this invention can be formulated with surfactants, preferably non-ionic surfactants, such as Cremophos RH60, Tween 20 or similar products. The compositions of this invention can also contain preservatives such as methyl paraben or propyl paraben or similar products. Compounds that can impart higher viscosity, such as propylene glycol, can also be added to the compositions of this invention. Generally, compositions with higher viscosity are preferred in order to create formulations that tend to remain in the vagina for a relatively long time after administration. A typical formulation is as follows: 0.30% by weight of glyceryl monolaurate, 0.50% by weight of Cremophos RH60, 2.00% by weight of Tween 20, 0.30% by weight of methyl paraben, 0, 10% by weight of propyl paraben, 1.0% by weight of propylene glycol, 0.04% by weight of Blue FD&C (American pharmacopoeia) = (= (= 1 / one dye and 95.76% by weight of deionized water) Another typical formulation contains 0.50% by weight of glyceryl mouse monolau, 1.50% by weight of Cremophos RH60, 1.00% by weight of Tween 20, 0.30% by weight of methyl paraben, 0, 10% by weight of propyl paraben, 0.04% by weight of blue FD&C = (= (= 1, a dye and 96.56% by weight of deionized water).

Glyceryl monolaurate has been described as a useful active ingredient in combating toxin-1 toxic shock syndrome in US patent applications under Serial Numbers 343,965, filed on April 27, 1989 and 316,742 filed on April 27, 1990 , which are incorporated herein by reference. These applications describe glyceryl monolaurate and its analogs as a material which, when exposed to S. aureus in absorbent products, can reduce the formation of the TSST-1 toxin. It is also believed that the active compounds in the compositions of this invention are effective in combating the production of other types of Staphylococcus toxins, in particular Staphylococcus A, B, Ce and TSST-1 with A. Enterotoxins with this efficiency. to the above-mentioned enterotoxins when the active compound is placed in an absorbent material. The efficiency of the compounds of the invention against the formation of Staphylococcus A, B, C and TSST-1 enterotoxins with A is described in the North American patent applications co-pending with the Serial number 07/605910 (accession number PPC 369 ) presented on October 30, 1990. Glyceryl monolaurate was also found to be effective in inhibiting the production of pyrogenic extrexocins from Streptococci (SPE) A, B and C, as well as hemolysin produced by Groups A, B streptococci , F and G. Glyceryl monolaurate analogs are also thought to be effective in inhibiting the production of these toxins. The efficiency of glyceryl monolaurate in inhibiting the production of toxins by streptococci when used in solution and in absorbent products is illustrated in the US patent application co-pending with the NQ. Serial _, (PPC 389 access no.) presented on May 3, 1991.

The following examples are illustrative of the effects of the compositions of this invention on the production of TSST-1. These examples obviously only illustrate the products of the invention without limiting the scope of the invention.

EXAMPLE 1

Glyceryl monolaurate was added to commercially available irrigations and these mixtures were heated to dissolve the glyceryl monolaurate. The irrigation compositions were prepared to contain glyceryl monolaurate in concentrations of 0.1, 1.0 and 10.0% by weight of liquid irrigation. Specifically, the irrigation of Massengil Vinegar and Water Douche containing only vinegar and water with glyceryl monolaurate was combined in this same Example. The solutions in this Example include Mas irrigation. Vinegar and Water Douche which is a commercial product manufactured by Beecham Products, a division of Beecham Inc., Pittsburgh, Pennsylvania, in isolation and the three solutions containing Vinegar Massengil and Water Douche with 0.1% by weight of monolau rat glyceryl, Massengil of Vinegar and Water Douche with 1.0% by weight of glyceryl monolaurate and irrigation of Massengil of Vinegar and Water Douche with 10% by weight of gli monolaurate. ceryl.

Glyceryl monolaurate activity was determined using the Buffer Bag Process referenced

laughed by Reiser et al. in Journal of Clinicai Microbiology, Volume 25, August 1987, p. 1450-1452. The process was adjusted to allow the use of this procedure without tampons or absorbent materials as described below.

Staphylococcus aureus strain FRI-1169, obtained in lyophilized form from Dr. Merlin Bergdoll, Food Research Institute, University of Wisconsin, Madison, Wisconsin, United States of America, was used in the assays. The £ [. aureus was prepared by mixing one (1) ml well. ligram (mg) of strain £ 3. aureus lyophilized with one (1) milliliter (ml) of Brain and Heart Infusion (BHI) (obtained from Difco Laboratories, Detroit, Michigan, United States of America), transferring the mixture to a test tube containing five (5 ) ml of BHI broth, mixing again very well, and incubating for twenty-four (24) hours at 37 ° C before use.

microorganism with the medium was subsequently added to sterilized centrifuge tubes, centrifuged at 2,000 rpm for twenty minutes in a refrigerated evaporator, the supernatant was separated by decantation, the obtained pellet was resuspended in phosphate saline, pH 4.0 . This process was repeated for five washes in a sequence of 10 minutes each. The pH value of the solution of 4.0 allowed inoculation without excess nutrients and prevented the bacteria from being shocked by exposure to solutions of pH 4.0. This procedure was performed in the manner described for Massengil and Summer's Eve Douche irrigation products.

One hundred ml of BHI agar (also obtained from Difco Laboratories) was placed in each of ten 3.8 cm x 20 cm culture tubes. Cellulose dialysis bags were prepared and sterilized in the manner referred to by Reiser et al. The sterile cellulose dialysis bags (8,000 PM restrictive) were inoculated with the above mentioned suspension of S. aureus in an amount sufficient to provide a concentration of 5.20 x 10 cfu / ml of S. aureus bacteria at the start of the test. .

The test solutions were prepared

by adding solid glyceryl monolaurate to commercial irrigation solutions (Massengil and Summer's Eve), heating to 60 ° C in concentrations that result in a glyceryl monolaurate content of 0.1, 1.0 and 10% by weight in the solutions heated irrigation system. These solutions were cooled with agitation and then added to the inoculated dialysis bags containing aureus in quantities of 1.0 g by weight.

One gram of sterile distilled water was added to dialysis bags inoculated with S. aureus only without other additives and this mixture is referred to as inoculation control. Each dialysis bag or sachet was then inserted into culture tubes containing BHI agar and allowed to harden. Two inoculation controls were used, each in duplicate. Thus, ten culture tubes were used in this test, four of them containing the controls (two with Massengill Vinegar and Water Douche and two without), and six culture tubes containing the glyceryl monolaurate added to conventional showers in concentrations in order to result in Massengill Douche with 0.1% by weight of glyceryl monolaurate, Massengill Douche with 1.0% by weight of glyceryl mololaurate, and Massengill Douche with 10% by weight of glyceryl monolaurate, in duplicate. After a 24 hour incubation period, the culture tubes were measured to determine the cell concentration and TSST-1 concentration. The results obtained in this example are shown in Table 1.

The data presented in Table 1 demonstrate that for a concentration of 0.1% by weight of glyceryl monolaurate, there is a 66% reduction in TSST-1 whereas for 1.0% by weight of glyceryl monolaurate, it was observed a 95% reduction. There was a 99% reduction in the presence of TSST-1 formation to a concentration of 10% by weight of glyceryl monolaurate. The viable cell count allowed to verify that the cells increased when exposed to Massengill Douche containing 0.1% glyceryl monolaurate from the logarithmic concentration of 7.21 in the commercial isolated control formulation for an increase of 8.35. On the contrary, Massen gill Douche with 1% by weight of glyceryl monolaurate resulted in a logarithmic concentration of 6.85 (4.9% decrease) again resulting in 10% by weight glyceryl monolaurate with Massengill Douche in an increase in logarithmic concentration of

7.21 observed in the control to 7.35. The methodology used did not prevent the migration of the active ingredient through dialysis bags. However, even taking into account this migration, an effect was still observed and registered.

EXAMPLE 2

TSST-1 growth and production for £> was also assessed. aureus in the presence of Summer’s Eve Douche with and without glyceryl monolaurate. Summer's Eve Douche is manufactured by C.B. Fleet Co. Inc. in Lynchburg, Virginia and can be purchased in the United States of America. Although there are several irrigation products sold under the Summer’s Eve trademark, the product tested in this Example contained purified water, vinegar and benzoic acid. Solutions containing Summer's Eve Douche and various concentrations of glyceryl monolaurate were heated in order to dissolve the active ingredient as described in Example 1. Solutions containing concentrations of glyceryl monolaurate in amounts of 0.1, 1.0 and 10% by weight was included in this Example. The solutions were prepared and tested in 1.0 gram quantities as described in Example 1. The results obtained are shown in Table 2.

EFFECT OF GML ADDED TO MESSENGIL DOUCHE ON FORMATION

OF TSST-1 AND GROWTH OF STAPHYLOCOCCUS AUREUS

TABLE 1

SAMPLE FINAL CONCENTRATION OF S. _n AUREUS (x lo ^b CFU / ML) FINAL CONCENTRATION OF S. AUREUS (Log _ig CFU / ml) FINAL QUANTITY TSST-l ^b ' ^c - (ng) Only the Inoculate 91.20 9.96 6,309.80 Massengill Douche 0.16 7.21 6,512.52 Massengill Douche (0.1% GML) 2.25 8.35 2,161.60 Massengill Douche (1.0% GML) 0.71 6.85 315.80 Massengill Douche (10% GML) 0.22 7.35 35.78

Number of viable ÊL aureus cells expressed as logarithm relative to base 10.

As determined by the ELISA method reported by Reiser et al. in Applied and Environmental Microbiology, December 1982, p. 1349-55.

Average determination of duplicate samples.

TABLE 2

EFFECT OF GML ADDED TO SUMMER’S EVE DOUCHE'S TRAINING

TSST-1 AND GROWTH OF STAPHYLOCOCCUS AUREUS SAMPLE FINAL CONCENTRATION OF S. _O AUREUS (x 10 CFU / ML) FINAL CONCENTRATION OF S. AUREUS (LOg ₁₀ CFU / ml) FINAL QUANTITY TSST-l ^b ' ^c (ng) Only Inoculated 91.20 9.96 6,309.80 Summer ¹ s Eve Douche (Control) 0.021 6.34 670.94 Summer's Eve Douche (0.1% GML) 0.005 5.74 27.87 Summer's Eve Douche (1.0% GML) 0.006 5.83 4.60 Summer’s Eve 0.12 7.08 4.00

Douche (10% GML)

Number of viable aureus cells expressed as logarithm in relation to base 10.

'As determined by the ELISA method reported by Reiser et al. in Applied and Environmental Microbiology, December 1982, p. 1349-55.

I

Average determination of duplicate samples.

The results reported in Table 2 reveal a significant reduction in the viable cell count from a logarithmic concentration from 9.96 to 6.34 (decrease of 36%) in the presence of commercial irrigations, together with a significant reduction in the formation of TSST-1 , from 6 309.80 to 670.94 (decrease of 89%). The reduction in the concentration of viable cells is more likely due to the presence of benzoic acid, which is a well-known bactericide, in addition to acetic acid in the commercial formulation. The data presented in Table 2 resulted in a 96% reduction in TSST-1 formation with a 0.1% concentration of glyceryl monolaurate and

99% reductions in the presence of 1.0 and 10% by weight of glyceryl monolaurate when compared to those obtained with the control system containing only Summer's Eve Douche.

The presence of 0.1% by weight of glyceryl monolaurate resulted in a logarithmic concentration of 5.74 (9% decrease) of viable cells when compared to the control. In the presence of 1.0% by weight of glycerol monolaurate, the log concentration was 5.83 (8% decrease). On the contrary, a 10% by weight solution of glyceryl monolaurate in Summer's Eve Douche resulted in a lithium concentration of 7.08 (11% decrease).

EXAMPLE 3

In this Example, growth and production of TSST-1 by S. aureus in the presence of a suspension of glyceryl monolaurate in distilled water in concentrations of 0.1, 1.0 and 10% by weight was evaluated. The solutions were evaluated in 1.0 gram quantities as described in Example 1, with the exception that no commercial product was used in the test. The results obtained are shown in Table 3.

TABLE 3

EFFECT OF GML SUSPENSION IN WATER ON TSST-1 FORMATION AND STAPHYLOCOCCUS AUREUS GROWTH

SAMPLE FINAL CONCENTRATION OF S. AUREUS (X 10 ~ CFU / ML) FINAL CONCENTRATION OF S. AUREUS (Log ₁₀ CFU / ML) AMOUNT FINAL TSST-lbfc (ng) Inoculated Water (control) 91.20 9.96 6,309.80 GML (0.1%) IN WATER 0.35 7.55 3.45 GML (1.0%) in water 0.002 4.31 2.32 GML ~ (10%) in water 0.03 6.53 4.02

Number of viable S. aureus cells expressed as logarithm in relation to base 10.

As determined by the ELISA method reported by Reiser et al. in Applied and Environmental Microbiology, December 1982, pgs. 1349-1355.

C Λ »X»

Average determination of duplicate samples.

The data presented in Table 3 reveal a significant reduction in the formation of TSST-1 (99.9%) in concentrations of glyceryl monolaurate suspended in distilled water in concentrations of 0.1% by weight of glyceryl monolaurate and higher. In addition, the thousand-fold reduction in toxin formation also had a corresponding 99.6% reduction in the number of viable S. aureus cells that were noted at a concentration of 0.1% by weight of glyceryl monolaurate. For increasing concentrations of glyceryl monolaurate, the antimicrobial effect was more prevalent for the value than 1.0% by weight. An antimicrobial effect reduced to 10% by weight of glyceryl monolaurate, which could be attributed to the insolubility of the compound tested. Reductions in the formation of TSST-1 did not change significantly from 0.1% to 10% glyceryl monolaurate, while changes resulted in 'U5' »a» t

significant number of viable cells.

EXAMPLE 4

This Example was conducted in order to determine the growth and production of TSST-1 by S. aureus in the presence of a non-absorbent contraceptive product, Today Sponge, with and without glyceryl monolaurate. Today Sponge is manufactured by Whitehall Laboratories Inc., New York, United States of America. Today Sponge is described by the manufacturer as containing Nonoxynol-9, benzoic acid, citric acid, sodium dihydrogen citrate, sodium metabisulfite, sorbic acid and water in an expanded polyurethane sponge. The sponge has an average weight of 7.0 grams.

Glyceryl monolaurate solutions in water were heated until the glyceryl monolaurate had dissolved and then were placed using a pipette in quantities of 1.0 grams of Today Sponge in order to obtain the glyceryl monolaurate concentrations of 0 , 1, 1.0 and 10% by weight based on the weight of Today Sponge. The 1.0 gram samples were inserted into inoculated dialysis sachets containing 8 TSure-1 producing aureus at a concentration of 5.20 x 10 10 Cfu / ml at the initial height. The aforementioned samples were evaluated as described in Example 1. The results obtained in the assay are reported in Table 4.

TABLE 4

EFFECT OF GML SUSPENSION IN WATER ON TSST-1 FORMATION AND STAPHYLOCOCCUS AUREUS GROWTH

SAMPLE FINAL CONCENTRATION OF S. & UREUS (x 10 CFU / ml) FINAL CONCENTRATION OF S. AUREUS (Log ₁₀ CFU / ml) FINAL QUANTITY TSST-l ^b ' ^c (ng) Today Sponge (control) 5.66 8.75 12,635.21 Today Sponge (0.1% GML) 4.60 8.66 2,185.88 Today Sponge (1.0% GML) 4.46 8.65 799.99 Today Sponge (10% GML) 0.13 7.13 3.29 ^L Number of » S. aureus cells viable expressed as logarithm regarding to base 10.

As determined by the ELISA method reported by Reiser et al. in Applied and Environmental Microbiology, December 1982, p. 1349-1355.

Average determination of duplicate samples.

The results reported in Table 4 show a reduction in the formation of TSST-1 to a value of 82% m and toxins with 0.1% by weight of glyceryl monolaurate, and reductions of 93% and 99.9%, respectively, in the presence of 1.0 and 10% by weight of glyceryl monolaurate.

Regarding the viability of the cells, the presence of 0.1% by weight of glyceryl monolaurate resulted in a log concentration of 8.66 (reduction of 1.0%) when compared to the control. In the presence of 1.0% by weight of glyceryl monolaurate, a logarithmic concentration of

8.65 (1.1% reduction), with 10% by weight of glyceryl monolaurate, there was a superior microbial effect, resulting in a di. 18% decrease in viable cells.

EXAMPLE 7

Since commercial irrigations are often used in excess as contraceptives, experimentation with glyceryl monolaurate was carried out to assess spermicidal activity. Glyceryl monolaurate was tested to determine its spermicidal activity using the Sander-Cramer Assay, which uses human semen. The compound was prepared in saline at a concentration of 100 mg / ml. One (1) ml of this solution was rapidly mixed with 0.2 ml of human semen. A drop droplet of this mixture was prepared and it was examined microscopically to determine the mobility of the sperm within 20 seconds after the start of the mixture. For this higher concentration, sperm mobility lasted for more than three minutes, indicating the absence of an effect on sperm mobility.

These examples demonstrate that the glyceryl mouse monolau is an active toxin inhibitor for the TSST-1 toxin produced by the £ 3 bacteria. aureus. In addition, the compound does not significantly inhibit cell growth or viability at the concentrations used in commercial compositions of the present invention. In addition, glyceryl monolaurate does not inhibit sperm mobility.

EXAMPLE 8

In this experiment, conducted by Dr. Patrick Schlievert of the University of Minnesota, glyceryl monolaurate in various concentrations was added to 50 ml of Brain and Heart Infusion Broth. These solutions were in g

then inoculated with 1.0 x 10 CFU / ml of group A streptococcus strain C203, or, S. aureus MN8, which is a known producer of TSST-1. Samples containing group A C203 streptococci were incubated at 37 ° C for 12 hours without shaking in order to reduce the exposure of the organism to oxygen, in the presence of 7% C0 ₂ · Samples containing MN8 were incubated comparatively with the exception of shaking (at a rate of 200 rpm) and in a conventional standard incubator. The results obtained in this experiment are summarized in Table 5

TABLE 5

EFFECT OF GLYCERYL MONOLAURATE ON TOXIN PRODUCTION AND CELL VIABILITY IN GROUP A AND STREPTOCOCKS

S. AUREUS MN8

C203

MN 8

Sample

Type SPE

GML mg / 100 CFU THE B 0 3.1 x 10 ⁸ 6.0 3.0 0.05 3.5 x 10 ⁸ 6.0 3.0 0.1 3.2 x 10 ⁸ 1.5 0.75 0.25 3.3 x 10 ⁸ N.D. N.D. 0.5 3.2 x 10 ⁸ N.D. N.D. 0.75 2.0 x 10 ⁸ N.D. N.D. 1.0 4.0 x 10 ⁷ N.D. N.D. 1.25 3.5 x 10 ⁶ N.D. N.D. 1.50 0 N.D. N.D. 1.75 0 N.D. N.D. 2.0 0 N.D. N.D. 5.0 0 N.D. N.D. 10.0 0 N.D. N.D. 0 8.4 x 10 ⁹ 0.05 9.0 x 10 ⁹ 0.1 9.4 x 10 ⁹ 0.25 6.2 x 10 ⁹ 0.5 10 1.8 x 10 ^U .75 9.7 x 10 ⁹ 1.0 2.1 x 10 ^1θ 1.25 7.0 x 10 ⁹ 10 1.5 1.4 x 10 1.75 ϊ ί in ¹⁰ 1.1 x 10 2.0 4.0 x 10 ⁵ 2.25 2.0 x 10 ⁵ 2.5 2.3 x 10 ⁴ 5.0 2.1 x 10 ⁴ 10.0 2.9 x 10 ⁴

TSST-1

N.D.

N.D.

N.D.

N.D.

N.D.

N.D. N.D. N.D. N.D. N.D. N.D.

EXAMPLE 9

In this Example, conducted by Dr. Patrick Schlievert, strains of group A streptococci that individually express SPEA, SPEB or SPEC and strains of groups B, F and G of streptococci were evaluated to determine the effect of glyceryl monolaurate on exotoxin production. Using the process referred to in Example 8, the microorganisms were exposed to various concentrations of glyceryl monolaurate in a Brain and Heart Infusion broth. Strain 594, which produces SPEA, Strain 86-858, which produces SPEB and Strain T18P, which produces SPEC toxins, respectively, were used. The production of toxins was measured by Western immunorevolution for periods of up to 96 hours. The results obtained in this experiment to determine the effect of glyceryl monolaurate on the production of SPEA, SPEB and SPEC toxins are shown in Table

6.

The £ 3 strain. aureus Mn8 was also exposed to glyceryl monolaurate. The amount of TSST-1 production by S. aureus strain Mn8 was measured. The results obtained in this test are shown in Table 7.

Streptolysins 0 and S, also produced by strains 594, 86-858 and T18P, were measured, as well as Group B streptococci hemolysin, Group F streptococcus hemolysin and Group G streptococci hemolysin. mixture of 0.1% of sheep's erythrocytes and 0.014% of 2-mercaptoethanol as reducing agent carried out in 0.75% of agarose in a phosphate buffered solution (PBS), 4.5 ml per plate 0 PBS consisted of phosphate of sodium 0.005 MOlar, with 0.15 Molar NaCl at pH 7.0. Hemolysis induced by a 20 µl cell-free culture added to plate holes after 24 hours was used as a measure of lysine production.

Lipase was measured in the same way as hemolysin, with the exception that 0.1% tributyrin was used as the standard.

The results of reduced streptolysin 0 and S are also shown in Table 6. The results obtained in the experiments demonstrate the effect of

glyceryl in the production of toxins by Groups B, F and G streptococci in Tables 8, 9 and 10, respectively. The data reveal a notable reduction in the amounts of toxins and / or hemolysin produced by Groups A, B, F and G streptococci in the presence of glyceryl monolaurate.

TABLE 6

EFFECT OF GLYCERYL MONOLAURATE ON GROUP A STREPTOCOCKS

Bacterium ^a GML (ug / ml) Log CFU / ml SPE (ug / ml) Hemoline ^ ⁵ Reduced 594 (SPEA) 0 8.6 3.2 7.0 2.5 8.5 0.3 4.0 10.0 8.3 0.3 0.0 20.0 6.0 0.0 0.0 86-858 (SPEB) 0 8.0 0.8 4.0 2.5 7.7 0.0 2.0 10.0 7.7 0.0 0.0 20.0 5.8 0.0 0.0 T18P (SPEC) 0 7.9 0.4 8.0 2.5 7.9 0.0 8.0 10.0 6.1 0.0 0.0

s E fi - Size of inoculate between IO ³ and 10 ° CFU / ml - Includes streptolysin O and S measured in mm of diameter in the lysis

- 24 4

EFFECT OF GML ON Staphylococcus aureus— MN8

Eh 40 O A 00 • 3 * THE frog THE THE tf M THE THE 00 CN THE THE tf THE THE in LT) THE THE Ch THE THE THE THE 0 THE THE THE THE tnH • • • • 0 g O O O O THE THE THE THE THE O O 00 O Eh THE M · frog The The τί < O THE tf M THE THE X! THE tf oo rH The THE THE ιφ, Α n THE THE ϊ — 1 THE THE O O tnH • • • • 0 g O O O Γ ' THE I — 1 THE THE O THE O O EH THE tf frog THE THE O O Λ tf K THE THE 0 ι-1 ft M * tf g A rH The THE frog Φ i (D, A THE THE EH Ί 0 dn CN O THE σι t 0 6 • • • • THE O O 00 THE 0 THE THE t 0 Ή Eh CN σ O O tf Φ ft frog tf K σ » O O O tf THE THE tf THE O THE THE THE 00 'Φ.Α THE Ί tnH O THE THE frog 0 g • • • • THE σι r ~ THE THE tl EH O O O O raol tf M O O O O THE THE Λ tf, AQl »0 A CN CN CN THE 0 tfl ι — 1 00 00 00 THE 0 g • • • THE THE THE THE THE , _, THE d s The \ O O O O the u> CN O O THE THE

r- |

U THE m O Pí i — 1 0 X The Γ — 1 O Φ • 0 rH O 0 g t g tf THE Φ tf frog > —S O • rl iH 0 THE g tf THE tf &> g tf tf 0 g • rl t frog Φ • rl THE 0 frog I — 1 1 rtf tf 0 EH tf Praça g ω tf • rl Φ ω g tfl ω Eh tf % EH THE w Eh tf | ΛΙ Pl t |

TABLE 8 EFFECT OF GML ON GROUP B STREPTOCOCUS GML Period of 8 hr Time 24 hr (pg / ml) Log cells / ml Hemolysin Log cells / ml Hemolysin 0 8.7 2 8.5 2 2.5 8.1 0 8.4 0 10.0 4.0 0 3.0 0

Inoculate size 2.0 x 10 ³ / ml

TABLE 9

EFFECT OF GML ON GROUP F STREPTOCOCUS

Period of time

GML _8 hr_24 hr_ (pg / ml) Log cells / ml Hemolysin Log cells / ml Hemolysin

0 8.3 7 8.3 7 2.5 8.5 0 8.3 0 10.0 4 10 0 10 ³ 0 Size inoculated 2. 0 x 10 ⁵ / ml TABLE 10 GML EFFECT IN STREPTOCOCUS GROUP G Period of time GML 8 hr 24 hr (pg / ml) Log cells / ml Hemolysin Log cells / ml Hemolysin O 8.9 8 10.0 8 2.5 8.1 5 10.0 6 10.0 10 ⁴ 0 10 ³ 0 Size inoculated 8 x 10 ^ / ml

EXAMPLE 10

In this experiment, conducted by Dr. Pa trick Schlievert, attempts were made to induce the growth of strep Coc3 strain and staphylococcal strain MN8 on plates containing glyceryl monolaurate. The minimum inhibitory concentration of glyceryl monolaurate for strain C203 was 1 mg / 100 ml on agar plates when 5 x 10 CFU were plated. The 2 mg / 100 ml plate showed no growth. The minimum inhibitory concentration of glyceryl monolaurate for strain MN8 was 5 mg / 100 ml when plate 8 out of 7 x 10 CFU. The 7.5 mg / 100 ml plate showed no growth. This experiment was conducted an average of twice a week for a period of six months. The data indicate that there were no mutants likely to grow in the presence of inhibitory levels of glyceryl monolaurate.

Claims (4)

- wool Process for the preparation of a liquid composition for use in women's intimate hygiene characterized by incorporating in a proportion of 0.1% to 10% by weight, a compound chosen from the group consisting of: a) monoesters of a polyhydric aliphatic alcohol and a fatty acid containing eight to eighteen carbon atoms and wherein said monoester has at least one hydroxyl group associated with its aliphatic alcohol residue, b) diesters of a polyhydric aliphatic alcohol and a fatty acid containing eight to eighteen carbon atoms and wherein said diester has at least one hydroxyl group associated with its aliphatic alcohol residue, and c) mixtures of said monoesters and diesters, said compound being present in a proportion that is effective to inhibit the production of toxin-1 toxic shock syndrome by Staphilococus aureus bacteria when said product is exposed to said bacteria, in combination with a pharmaceutically acceptable vehicle. - 22 Process according to claim 1, characterized in that said fatty acid is lauric acid - 3§ Process according to claim 1, characterized in that said polyhydric alcohol is glycerol. - 4th Process according to claim 1, characterized in that said compound is glyceryl monolaurate. - 5th Process according to claim 4, characterized in that said glyceryl monolaurate is present in a proportion of at least 0.1% by weight based on the weight of said composition. Process according to claim 4, characterized in that said glyceryl monolaurate is present in a proportion of at least 0.5% by weight based on the weight of said composition. Process according to claim 1, characterized in that said compound comprises a mixture of glyceryl monolaurate and glyceryl dilaurate. - 8th Process according to claim 7, characterized in that said mixture is present in a proportion of at least about 0.1% based on the weight of said composition. - 9th Process according to claim 8, characterized in that said mixture comprises at least 90% by weight of glyceryl monolaurate. Process according to claim - 10§ 29 7, characterized in that said mixture is present in a proportion of at least about 0.1% based on the weight of said composition. 11. A process according to claim 1, characterized in that said compound comprises glyceryl monocaprylate. 12. A process according to claim 1, characterized in that said compound comprises glyceryl caprate. 133. The method of claim 1, wherein said compound comprises a mixture of glyceryl monocaprylate and glyceryl caprate. 143. The method of claim 1, wherein said compound comprises glyceryl monomyristate. - 15§ 30 155. The method of claim 1, wherein said compound comprises glyceryl monopalmitate. 16. A process according to claim 1 characterized in that said compound comprises glyceryl monostearate. 17. A process according to claim 1, characterized in that said compound comprises glyceryl monooleate. Process for the preparation of a liquid composition for use in feminine intimate hygiene characterized by incorporating in a proportion of 0.1 to 10% by weight a compound chosen from the group consisting of: a) monoesters of a polyhydric aliphatic alcohol and a fatty acid containing eight to eighteen carbon atoms and in which said monoester has at least one hydroxyl group associated with its aliphatic alcohol residue, b) diesters of a polyhydric aliphatic alcohol and a fatty acid containing eight to eighteen carbon atoms and wherein said diester has at least one hydroxyl group associated with its aliphatic alcohol residue, and c) mixtures of said monoesters and diesters, said compound being present in a proportion that is effective to inhibit the reduction of the toxic shock syndrome of Enterotoxin A, Enterotoxin B or Enterotoxin C by the Staphi lococus aureus bacterium when said composition is exposed to said bacteria in combination with a pharmaceutically acceptable carrier. 19. A process according to claim 18, characterized in that said fatty acid is wooluric acid. 20. A process according to claim 18, characterized in that said polyhydric alcohol is the glycerol. 21. The method of claim 18, wherein said compound is glyceryl monolaurate. 22. The method of claim 21, wherein said glyceryl monolaurate is present in a proportion of at least 0.1% based on the weight of said composition. 23. The method of claim 21, wherein said glyceryl monolaurate is present in a proportion of at least 1.0% based on the weight of said composition. 24. The method of claim 18, wherein said compound comprises a mixture of glyceryl monolaurate and glyceryl dilaurate. - 25§ Process according to claim 24, characterized in that said mixture is present in a proportion of at least about 0.1% based on the weight of said composition. - 26§ Process according to claim 25, characterized in that said mixture comprises at least 90% by weight of glyceryl monolaurate. 27th Process according to claim 25, characterized in that said mixture comprises at least 95% by weight of glyceryl monolaurate. 28. The method of claim 25, wherein said mixture is present in a proportion of at least about 0.1% based on the weight of said composition. - 29§ Process according to claim 28, characterized in that said mixture comprises at least 90% by weight of glyceryl monolaurate. - 30é Process according to claim 29, characterized in that said mixture comprises at least 95% by weight of glyceryl monolaurate. - 31§ Process according to claim 18, characterized in that said compound comprises glyceryl monocaprylate. 32. The method of claim 18, wherein said compound comprises glyceryl caprate. 33. The method of claim 18, wherein said compound comprises a mixture of glyceryl monocaprylate and glyceryl caprate. 34. The method of claim 18, wherein said compound comprises glyceryl monomyrstate. 35. The process of claim 18, wherein said compound comprises glyceryl monopalmitate. - 36th 35 - 36â Process according to claim 18, characterized in that said compound comprises glyceryl mouse monostea. - 37th Process according to claim 18, characterized in that said compound comprises glyceryl monooleate. - 38a Process for the preparation of a liquid composition for use in feminine intimate hygiene, characterized by incorporating a pharmaceutically acceptable vehicle and a compound chosen from the group consisting of: a) monoesters of a polyhydric aliphatic alcohol and a fatty acid containing eight to eighteen carbon atoms and wherein said monoester has at least one hydroxyl group associated with its aliphatic alcohol residue, b) diesters of a polyhydric aliphatic alcohol and a fatty acid containing from eight to eighteen carbon atoms and wherein said diester has at least one hydroxyl group associated with its aliphatic alcohol residue, and c) mixtures of said monoesters and diesters, said compound being present in a proportion that is effective to inhibit the production of pyrogenic exotoxins A, B and C of staphylococci, and hemolysins produced by Groups A, B, F and G streptococci when said composition is exposed to said bacteria. The applicant further claims the priorities of US patent applications filed on October 30, 1990 and May 3, 1991, under serial numbers 605,899 and 695,471, respectively.