MXPA99000008A - Lipid vesicles containing nonoxinol with antiviral and espermatic activity - Google Patents

Abstract

The present invention relates to a novel formulation having anti-viral and spermicidal properties. The formulation contains lipid vesicles formed from a non-ionic amphiphilic agent, a surfactant having a spermicidal and / or anti-viral activity, an oil and a sterol. The selection of the components constituting the vesicle is such that the formulation provides a fast spermicidal or anti-viral activity. The formulation and methods of the invention are especially useful in mucous membranes such as, for example, the vaginal tract and have been tested against viruses such as HIV and Vaccin.

Description

IPIDOS VESICLES CONTAINING NONOXINOL ON CTIVITY ANTIVIRAL AND ESPERMATICIDE BACKGROUND OF THE INVENTION The present invention relates to methods for constructing liposamas based on phosphates (Navasame (MR)) containing surfactants that are compatible with late-1 and deactivate enveloped viruses and spermatozoa by contact- Useful formulations are also presented pars. Coating in condoms s well for vaginal insertion. Is the nano preferred? pal-9 as a surfactant in the formulation and methods.

The entry of pathogenic viruses in humans is. dax essentially through the mucous membranes / d the respiratory tract. The first step in any infection is fixation or colonization with subsequent invasion and spread of the infectious pathogen. The deactivation of potential pathogens at the site of colonization could prevent many infectious diseases. The present inventions were designed to deactivate spermatozoa by contact. Even though some experiments have been carried out using liposomes co or delivery system for various materials, no successful use of liposomes or other lipid vesicles has been reported as an aptiviral contact agent or as an active agent. Contact. The main problems are compatibility? the stabilized, especially since the environments for their use are often "laboratories" perfect for cultivating bacteria that fix and degrade the preparations. The formulations must be able to operate in a wide physiological range of pH and at physiological temperatures. The pH and temperature requirements make many formulations of phospholipids, as well as classical liposomes, inappropriate due to their lack of stability. In addition, many formulations employing YA either phospholipids or l-O non-phospholipids are not compatible with latex, a requirement if one wishes to use the formulation with condoms, or they are irritant to the mucous membranes in the administration system. Both properties, compatibility with latex and lack of irritation, are important to obtain a successful product. In the last few years several companies, mainly ITI, Inc. through subsidiary Micro Vesicular Systems, Inc. and Nova Va- < , Inc., and L'Oreal, have developed and popularized non-phospholipid lipid vesicles and their 0 uses. U.S. Patent No. 4,911,928, U.S. Patent No. 4,895,452, U.S. Patent No. 5, 260.0é5, and U.S. Patent Application Serial No. 08 / 432,552, the disclosures of which are incorporated herein by reference, present various formulations. and methods for making elaborate vesicles employing various materials no phospholides. These patents and this application, with variations in the methods and materials presented here, have been employed to develop various products that range from vaccines to skin care products. However, to date no formulation has been made that successfully satisfies all the necessary requirements for a stable and latex-compatible spermatic product or antiviral. Accordingly, an object of the present invention is to provide a virus deactivation lipid vesicle that deactivates viruses upon contact with them. A further object of the present invention is to provide stable preparations not before they deactivate viruses, especially enveloped viruses, in the skin or mucous membranes. Another additional object of the present invention is to provide a method for preventing viral infection in a patient's environment by administering a virus vesicle that inactivates viruses to the subject. A further object of the present invention is to provide a method for deactivating spermatozoa by contacting the spermatozoa with a vesicle of spermicidal lipids. These and other objects and features of the invention they will be apparent from the description of the preferred modalities and from the revindications. COMPENDIUM OF THE INVENTION The present invention presents methods for deactivating spermatozoa and enveloped viruses such as HIV, for contact as well as a formulation having these properties. The invention is based, at least in part, on the discovery that a lipid vesicle formulation could have a sufficient part of spermatic or antiviral surfactant in lipid bilayers in such a manner that the formulation was active without rupture of the vesicle. This formulation can also be less irritating than other formulations. The formulation useful in the present invention contains lipid vesicles consisting of a non-ionic amphiphile, an oil, a sterol, and a surfactant having a spermatic activity and against enveloped viruses. The preferred surfactant is a nono-tifenol surfactant, more preferably nono-amol-9. The ionic naphionic acid, the oil and the surfactant are selected in such a way that at least a part of the surfactant is found in the outer bilayers of the lipid vesicle. Preferred non-ionic amphiphiles are selected from the group consisting of POE ethers of fatty alcohols, POE fatty acid esters, monolaurate and dilaurate glycerol, mannostearate and glycerol distearate, as well as POE fatty acid ethers sorbitan, glycerol monostearate being preferred. The oil must be an oil in which the surfactant has a low solubility, preferably an oil in which it can not dissolve. If nonxinol-9 co or surfactant is used, vegetable oils such as soybean oil are preferred, while mineral oils can not be used- It is theoretically stated that if the surfactant can be dissolved in the oil, it is not found in the bilayers of the oil. lipids, but if it can not be dissolved it must be carried on the surface of the bilayers. While almost any steral can be employed, cholesterol or phytosterols are preferred, for example, soy sterol. The vesicles also contain a bulking agent, preferably an agent that produces a c.a.rQ-3, negative as e.g. oleic acid. The vesicles can be formulated for administration using a pharmaceutically acceptable carrier, for example a saline solution. The vesicles can be formulated in a cream such as a cream suitable for vaginal insertion, or they can be adapted for use in a condom or with a condom. The methods of the present invention employ this formulation for their activity against enveloped viruses or for their spermicidal activity. While some of the Formulations of the invention have both an activity against both enveloped virus and a spermicidal activity, as for example, ormulations employing nono-inol-9 as a surfactant, it is also possible to employ other surfactants having only one of these properties. Some of the other surfactants that can be used include the Tritan-X family of surfactants from which it is known to have certain spermicidal properties, or cetylpidipipipium chloride which is known to have certain antiviral properties. In summary, the methods of the present invention expose the spermatozoa or the enveloped virus to the formulation and allow the formulation to act on the spermatozoa or virus. Since the formulations of the invention offer rapid activity the exposure can be very short, possibly in the order of a few seconds. In the case of viruses and sexually transmitted spermatozoa, the formulations may take the form of vaginal creams or lubricants for condoms. Other forms may be used for other viruses, such as nasal sprays. The following description and claims will further explain the scope of the present invention. Description of the preferred modalities. The present invention offers methods for deactivating enveloped viruses and spermatozoa, as well as an oral formulation in these methods. The invention employs lipid vesicles that include a spermicide or antiviral surfactant in the outer bilayers to provide rapid activity without the need for gallbladder rupture. The basic vesicle of the present invention is formed of a nonionic amphiphil such as for example glycerol monostearate, a stellar bed for example soy phytosterol a surfactant showing an antiviral or spermatid activity (or both) bed for example n ño mol -, and an oil such as soybean oil. The oil, non-ionic surfactant and surfactant is selected such that at least a part of the surfactant is found in the blotches and vesicles of the vesicle in such a way that it can provide the required activity without rupture of the vesicles. This requires compatibility between the naphonic amphiphile and the surfactant, and further requires that the surfactant can not be dissolved in the oil. The selection of a surfactant requires that it can be employed in stable lipid vesicles as well as compatibility testing, all of which is standard in the art. For example, if the formulations are used with latex, for example, latex condoms should be compatible with latex, which can be determined quickly using standard compatibility test with latex. One way to select the right mix of materials is to test sifted using spermicidal activity; The lipid vesicles may slow down the spermatozoa in less than 20 seconds. If no part of the surfactant is found in the outer bilayers, rapid spermicidal activity can not be provided with immediate lysis of the lipid vesicles. In the examples, tests to determine spermicidal activity and aptiviral activity are described. The invention is more fully understood through the following non-limiting examples. Example 1 - Preparation of SNP9S0 Novaso is (MR) containing nonoxmol-9. Table i contains the materials used to produce GNF9S0 Novasomes (MR) containing nonx? Pol-9 as a surfactant, glycerol manstearate as amphiphile, soybean oil and oleic acid as a negatively charged producing agent. TABLE 1 Chemical component Weight. Glycerol monostearate 13.7 grams Soy Sterol (or cholesterol) 3.8 grams Nonaxinal-9 7.2 grams Soybean oil 16 grams Oleic acid 250 micralitres The components illustrated in table i form the phase Lid dacite, which is heated for 1 hour at a temperature of 86 ° C to achieve uniformity. The water is heated to a temperature of 65 * C. The TNP9S0 Novasames (MR) was then produced by injecting the water phase in the liquid phase on a volume / volume basis of 13 parts of 1-oil-per-unit by 37 parts of aaua. GNP9S0 Novasomes (MR) - can be produced manually, with instrumentation with reciprocating syringe or with continuous flow instrumentation. Example 2 - Characterization of preparations of nonoinol-9 Novasomes (MR). After the production of Novasomes (MR), characterization studies were carried out. All the preparations were tested to determine their sterility and pH. The preparations were also measured on a Coulter LS 230 Laser measuring instrument equipped with a circulating water bath. Table 2 shows the physical characteristics of the vesicles. Tables 3 and 4 show tests to determine the antiviral activity in the cell lines that allow viral proliferation. Tables 5 and 6 show the compatibility with latte, Table 7 shows the anti-spermicidal activity using the Sander-Cramer test, and Table S shows oral suitability data. TABLE 2. Size Pro range mean of - of Coultei- Coulter en- in μm. Chemical components Load pH μm. TNP9S0 Novasames (MR) Negative 4.52 0.598 0.375-0.839 Example 3 - Tests of antiviral activity. A. Vipcida HIV-1 MN assays. The preparation of Novasome example 1 was tested to determine its anti-viral activity against HIV-1. 100 microliters of HIV-1 MN 1000 x virus were mixed in pellets lot No.50-013 .., TCID-50 / ml = l / 1000,000) for 30 minutes and either with 100 microliters of water for injection or 100 microliters of Novasomes (MR) preparations. An amount of 1.8 ml of tissue culture medium was then added to each preparation. Ten-fold serial dilutions of 1/100 to 1/1000,000 were then carried out. 0.4 ml of c & amp; amp;;gives. dilution in tubes containing C8166 cells, a HTLV-1 pair of transformed T cell line. These combined materials were then incubated at a temperature of 36 ° C for 2 hours, washed 3 times in a phosphate buffered saline, resuspended in fresh medium and each dilution was placed in dishes in 4 replication wells. The cells were then fed twice a week. The cells were observed at 7 and 14 days to determine the cytopathic effects and the leftovers were collected for p24 assay determinations as confirmation of viral proliferation. TCID-50 calculations were carried out based on the results of the p24 determinations. The results are illustrated in Table 3. The preparation of Novasome (MR) clearly had an apti-HIV activity. Table 3. Test item TC ID50 A 1 week TC ID50 A 2 weeks Water 10 (-4.75) 10 (-4.75) BNP9S0 Novasomes (MR) 0 0 B. Viricidal test against vaccinia WR. The Novassome (MR) preparation of example i was also tested to determine its viral activity against vaccinia WR '. 100 microliters of vaccinia WR 1000 x virus were mixed in pellets lot No.68-016 (TCID-50 / ml = l / l .000,000,000) for 30 minutes with either 100 microliters of water for injection or 100 micraliters of the preparation of Novasome (MR). 200 microliters of a tissue culture medium were then added to the preparations. Serial dilutions 10 times were then performed on the virus and controls without virus of 1/10 to 1 / 1,000,000. Then 40 μl of Z? Da dilution was placed in 96-well tissue culture dishes containing BSC-i cells. There were 5 replicates in each dilution, and plates were prepared in duplicate. These Combined materials were then incubated at 37 ° C for 2 hours washed 1 time in a phosphate buffered saline solution, resuspended in fresh medium. Cells were observed at 3 and 7 days to determine their cytostatic effects. MTS was performed on the first dish at 3 days; the second dish was fed back at 3 days and MTS was performed on this plate at 7 days. Table 4 shows the results of the test against Vaccinia WR. Again it turns out, it is evident that the preparation of Navasome (MR) has an antiviral activity. Table 4 Item of pruba TCID50 After 3 days TCID50 At 7 days Water 1 / 10,000,000 1 / 10,000,000 GNF9S0 Novaso me 0 0 Example 4 - Compatibility test with Latex. The Novasome (MR) preparation of Example 1 was also tested to determine its compatibility with the latex. Three types of condoms Truap-Enz Not lubricated L0316RP, Lifestyles 0602258800, and Aladan were tested in this trial. The preparation Novasa e (MR) was applied to 13 condoms of each msrca in 5 seconds using a soft brush and placed in a cheesecloth. The condoms were then conditioned in a humidity chamber at a relative humidity of 90% at a temperature of 30 ° C for 30 minutes the condoms were then washed, dried and tested to determine their Elongation and resistance to attraction. Another 13 condoms from each brand were treated in the same way as above but after conditioning they were mounted directly on the Airbust test device and tested. 80 Condoms of each brand without the preparation Novasome (MR) to determine its properties as controls. Table 5 shows the results of the compatibility test with te ----, without the preparation Navasome (MR) while table 6 shows the tests with the preparation Novasome (MR). It is evident that the Novasame (MR) preparation does not affect the properties of latex condoms in the short-term compatibility test. Table 5. Trojapi Epz Mark of Condom not lufcipcado Llfest le Aladan.

Elongation, 7. 783 827 802 Tensile strength. MPa 25.9 29.8 29.0 Pressure l Pa 2.3 1.3 2.2 Vol in, Liters 39.4 40.7 36.5 Table 6. Troj p Enz Non-lubricated condom mark Lifest / le Aladan.

Elongation, * -. 777 841 848 Tensile strength MPa. 26.8 29.1 30.1 Pressure kPa 2.1 1.2 1.9 Volume, Liters 3-9.4 40.7 36.5 Example 3 - Sandet-Cra er. In this example, the preparation of Example 1 was tested to determine its spermicidal activity using the Sander-'Cramer spermicidal assay. Several diffusions of the test compound were prepared using a 0.9% saline solution. An i ml test article is mixed with 0.2 ml of human semen in a tube of test of 13x100 mm taking the mixture in a pipette and expressing forcefully the mixture. This procedure is repeated 6 times a drop of the mixture is prepared and 5 fields are refracted under low power amplification. The entire process from mixing to microscopic observation should be carried out in a period of less than 20 seconds. The final point is the highest dilution that immobilizes the spermatozoa in less than 20 seconds. Table 7 shows the results of the San er-Cra assay and the vesicles of the invention not only allowed a higher concentration than the standard DMSO preparations., if not that I also kill the sperm at a higher dilution, showing greater activity. Table 7 Spermatid Dilution Highest NP9 test item. Cane in traction BNP9S0 Novasomes (MR) 528 32.5 mg / mL Control NP9 91 10 mg / mL in DMSO. Example 6 - Oral toxicity study in rats. In this example, the preparation of example i was tested to determine its actual toxicity. A group of 5 male and 5 female Sprague-Dawley rats were fasted overnight. On the following morning, after weighing, each rat received three milliliters by forced feeding of the preparation SNP9S0 Novaso e (MR), the maximum oral dose allowed for the rats. The animals were observed daily to determine disease and mortality and the individual body weights 7 and 14 were obtained. The results appear in table 8. Table 8.

Average total weight increase Rats Day 0-14 Males 126.66 g Females 24.13 g Based on the results of the studies illustrated in the examples it is clear that the preparations of the present invention show a spermicidal activity and an antiviral activity while being compatible with latex and orally safe. These preparations are especially useful for vipcida vaginal creams and spermicides. The foregoing formulations and the examples are merely illustrative and the scope of the present invention follows from the following revisions.

Claims (47)

1. CLAIMS I 1. A method for deactivating spermatozoa, comprising the step of exposing said spermatozoa to a lipid vesicle formulation compatible with latef having a topical spermicidal activity, said lipid vesicle formulation containing lipid vesicles manufactured from a non-ionic apdipile, an oil, a sterol, and a spermatid surfactant; said nonionic amphiphile, said oil, and said spermicidal surfactant are selected such that said spermicidal surfactant can not be dissolved in said oil and that at least a portion of said aticidal surfactant is in the outer bilayer of said formed vesicle of lxids. sporadically of said non-ionic amphiphile.
2. 2. The method of claim 1, wherein said spermicidal surfactant comprises a nonoxyphenol surfactant.
3. 3. The method of claim 2, wherein said nopaxyphenol surfactant comprises nonoxmol-9.
4. 4. The method of claim 1, wherein said nonionic amphiphile is selected from the group consisting of ethers POE of fatty alcohols, fatty acid POE esters, glycerol monolaurate and dilaurate, glycerol onaterate and distearate, and sorbitan POE fatty acid ethers.
5. 5. The method of claim 5 wherein said amphiphile is not ionic comprises olicerol monstearate.
6. 6. The method of claim 1"wherein said ester 1 is selected from the group consisting of cholesterol and phytosterols-
7. 7. The method of claim 6, wherein said sterol contains soy sterol.
8. 8. The method of claim 1, wherein said oil comprises a vegetable oil.
9. The method of claim 8, wherein said vegetable oil comprises soybean oil.
10. 10. The method of claim 1, wherein said lipid vesicle further comprises a charge producing agent.
11. 11. The method of claim 10, wherein said c & It comprises a load producing agent, negative.
12. The method of claim 11, wherein said negative charge producing agent comprises oleic acid.
13. 13. The method of claim 1, wherein said formulation further comprises a pharmaceutically acceptable carrier.
14. The method of claim 13, wherein said formulation is in the form of a cream.
15. 15. The method of claim 14, wherein said formulation is used for vaginal insertion.
16. 16. The method of claim 1, wherein said formulation is applied over a condom.
17. 17. A method for deactivating a enveloped virus comprising the step of exposing said enveloped virus to a lipoid compatible lipid vesicle formulation, said lipid vesicle formulation contains lipid vesicles made from a non-amphiphilic virus. ionic, an oil, a stearyl, a manoyiphenol surfactant, said nonionic aphypryl, said oil and said nano-phenol surfactant are selected such that said nanovifenol surfactant can be dissolved in said oil and at least a part of said surfing nonoxyphenol is found in the outer bilayer of said lysis vesicle formed primarily of said nonionic amphiphile.
18. 18. The method of claim 17, wherein said nona / ifenol surfactant comprises nono unal-9. 1 .
19. The method of claim 17, wherein said non-ionic amphotole is selected from the group comprising POE ethers of fatty alcohols, PQE esters of fatty acids, glycerol analaurate and glycerol dilaurate, glycerol onostearate and glycerol distearate. , and fatty acid ethers of Sarbitan FOE.
20. 20. The method of claim 19, wherein said nonionic apfxphila comprises glycerol monostearate.
21. 21. The method of claim 17, wherein said sterol is selected from the group consisting of cholesterol and phytosterols.
22. 22. The method of claim 2-1, wherein said sterel comprises soy sterol.
23. 23. The method of claim 17, where said oil co-prefers a vegetable oil.
24. The method of claim 23, wherein said vegetable oil comprises soybean oil.
25. 25. The method of claim 17, wherein said lipid vesicle further comprises a charge producing agent.
26. 26. The method of claim 25 wherein said charge producing agent comprises a negative charge producing agent.
27. 27. The method of claim 26, wherein said negative charge producing agent comprises oleic acid.
28. The method of claim 17, wherein said formulation further comprises a pharmaceutically acceptable carrier.
29. 29. The method of claim 28, wherein said formulation is in the form of a cream.
30. 30. The method of claim 29, wherein said formulation is used for vaginal insertion.
31. 31. The method of claim 17, wherein said formulation is applied on a condom.
32. 32. A formulation for inactivating spermatozoa and enveloped viruses comprising a lipid vesicle formulation compatible with latex, said lipid vesicle formulation contains lipid vesicles manufactured from an ionic naphthalate, an oil, an e = terol, and an onoxyphenol surfactant, said non-ionic amphiphile, said oil and said nonsxyphenol surfactant are selected in such a way that said nonoxyphenol surfactant can not be dissolved in said oil and at least a part of said nopoxyphenol surfactant is found in the bilayer. externally of said lipid vesicle formed primarily of said non-ionic amphiphile.
33. The formulation of claim 32, wherein said nono? Ifenol surfactant comprises nonoxypol-9.
34. The formulation of claim 32, wherein said non-ionic amphiphile is selected from the group consisting of POE ethers of fatty alcohols, POE fatty acid esters, glycerol monolaurate - glycerol dilaurate, glycerol monostearate and glycerol distearate. , and fatty acid ethers of POE sorbitap.
35. 35. The formulation of claim 34, wherein said non-ionic amphiphile comprises glycerol monostearate ".
36. 36. The formulation of claim 32, wherein said sterol is selected from the group consisting of cholesteral and phytosterols.
37. 37. The formulation of claim 36, wherein said sterol comprises soy steara.
38. 38. The formulation of claim 32, wherein said oil comprises a vegetable oil.
39. 39. The formulation of claim 38, wherein said vegetable oil comprises soybean oil.
40. 40. The formulation of claim 32, wherein said lipid vesicles further comprise a charge producing agent.
41. 41. The formulation of claim 40, wherein said charge producing agent comprises a negatively charged production agent.
42. 42. The formulation of claim 41, wherein said negative charge producing agent comprises oleic acid.
43. 43. The formulation of claim 32, wherein said formulation further comprises a pharmaceutically acceptable carrier.
44. 44. The formulation of claim 43, wherein said formulation is in the form of a cream.
45. 45. The formulation of claim 44, wherein said formulation is specially adapted for vaginal insertion.
46. 46. The formulation of claim 32. where said formulation is specially adapted for coating on a condom.
47. 47. A condom having a coating with attentive sperm activity and virus enveloped inactivation, said condom has the formulation of claim 32 applied thereon.