Abstract:
A novel formulation having anti-viral and spermicidal properties has been developed. The formulation contains lipid vesicles having an outer bilayer formed of a non-ionic amphiphile, a surfactant such as having spermicidal and/or anti-viral activity, an oil and a sterol. The selection of the components making the vesicle is such that the formulation provides rapid spermicidal or anti-viral activity. The formulation and methods of the invention are particularly useful in the mucous membranes such as the vaginal tract and has been tested against viruses such as HIV and Vaccinia.

Description:
BACKGROUND OF THE INVENTION 
     The present invention relates to methods of constructing non-phospholipid-based liposomes (Novasome®) containing surfactants which are latex compatible and inactivate enveloped viruses and spermatozoa on contact. Formulations useful for coating on condoms or for vaginal insertion are also disclosed. NONOXYNOL-9 is the preferred surfactant for use in the formulation and methods. 
     The portal of entry of pathogenic viruses in humans are predominantly the mucus membranes and the respiratory tract. The first step in any infection is attachment or colonization with subsequent invasion and dissemination of the infectious pathogen. Inactivation of potential pathogens at the site of colonization could prevent many infectious diseases. The present inventions were designed to inactivate spermatozoa and enveloped viruses on contact. 
     Although there has been some experimentation using liposomes as a delivery system for a variety of materials, no successful use of liposomes or other lipid vesicles as a contact anti-viral agent or contact spermicide has been reported. The major problems are compatibility and stability, particularly since the environments for use are often perfect &#34;laboratories&#34; for growing bacteria which could attach and degrade the preparations. The formulations must be able to operate over a physiologically broad spectrum of pH and at physiological temperatures. The pH and temperature requirements have made use of many phospholipid formulations, or classic liposomes, inappropriate because of their instability. Further, many formulations using either phospholipids or non-phospholipids are not latex compatible, a requirement if the formulation is to be used in connection with condoms, or are irritating to the mucous membranes at the delivery site. Both of these properties, latex compatibility and lack of irritation, are important for a successful product. 
     In the last few years, several companies, primarily IGI, Inc., through its former subsidiaries Micro Vesicular Systems, Inc. and NovaVax, Inc., and L&#39;Oreal, have developed and popularized non-phospholipid lipid vesicles and their uses. U.S. Pat. No. 4,911,928, U.S. Pat. No. 4,895,452, U.S. Pat. No. 5,260,065, and United States Patent application Ser. No. 08/482,552, the disclosures of which are incorporated herein by reference, disclose various formulations and methods for making lipid vesicles which utilize a variety of non-phospholipid materials. These patents and this application, together with variations on the methods and materials disclosed therein, have been used to develop a variety of products ranging from vaccines to skin care products. However, to date, no formulations have been made which successfully meet all the requirements necessary for a spermicidal or anti-viral product which is latex compatible and stable. 
     Accordingly, an object of the present invention is to provide a virus-inactivating lipid vesicle which inactivates viruses on contact. 
     A further object of the invention to provide non-irritating, stable preparations that inactivate viruses, especially enveloped viruses, on skin or mucous membranes. 
     Another further object of the present invention is to provide a method of preventing a viral infection in an affected subject by administrating a virus-inactivating lipid vesicle to the subject. 
     A still further object of the present invention is to provide a method of inactivating spermatozoa by contacting the spermatozoa with a spermicidal lipid vesicle. 
     These and other objects and features of the invention will be apparent from the Description of the Preferred Embodiments and the Claims. 
     SUMMARY OF THE INVENTION 
     The present invention features methods of inactivating spermatozoa and enveloped viruses such as HIV and vaccinia viruses, on 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 be made having a sufficient portion of the anti-viral or spermaticidal surfactant carried in the lipid bilayers such that the formulation was active without vesicle breakdown. This formulation may 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 anti-enveloped viral or spermicidal activity. The preferred surfactant is a nonoxyphenol surfactant, most preferably NONOXYNOL-9. The non-ionic amphiphile, the oil and the surfactant are selected such that at least a portion of the surfactant is carried in the outer bilayers of the lipid vesicle. 
     Preferred non-ionic amphiphiles are selected from the group consisting of Polyoxyethylene (POE). ethers of fatty alcohols, POE esters of fatty acids, glycerol mono- and dilaurate, glycerol mono- and distearate, and POE sorbitan fatty acid ethers, with glycerol monostearate being most preferred. The oil should be one in which the surfactant has a low solubility, preferably an oil in which the surfactant is not dissolvable. If NONOXYNOL-9 is used as the surfactant, vegetable oils such as soybean oil are preferred, while mineral oils are not usable. It is theorized that if the surfactant is dissolvable in the oil, it is not in the lipid bilayers, but if it is not dissolvable, it must be carried at the bilayer surface. 
     While almost any sterol can be used, cholesterol or phytosterols such as soy sterol are preferred. The vesicles may also contain a charge producing agent, preferably a negative charge producing agent such as oleic acid. The vesicles may be formulated for delivery using a pharmaceutically acceptable carrier such as saline. The vesicles can be formulated into a cream, e.g., a cream suitable for vaginal insertion, or it may be adapted for use on or with a condom. 
     The methods of the invention use this formulation for its anti-enveloped viral activity or spermicidal activity. While some of the formulations of the invention have both anti-enveloped viral and spermicidal activity, e.g., formulations using NONOXYNOL-9 (poly(ethylene glycol) p-nonylphenyl ether; nonylphenoxypolyethoxyethanol, average number of ethylene oxide units per molecule is indicated by number following nonoxynol (e.g. nonoxynol-9 for n=9)) as the surfactant, it is also possible to use other surfactants which have only one of these properties. Some of the other surfactants which may be used include the TRITON-X (polyethylene glycol p-isooctylphenyl ether) family of surfactants which have been shown to have some spermicidal properties, or cetyl pyridinium chloride which has been shown to have some anti-viral properties. Briefly, the methods of the invention expose the spermatozoa or enveloped virus to the formulation and allow the formulation to act on the spermatozoa or virus. Since the formulations of the invention provide rapid activity, exposure can be very short, possibly on the order of seconds. For sexually transmitted viruses and spermatozoa, the formulations can be made into vaginal creams or condom lubricants. For other viruses, other forms, such as nasal sprays, could be used. 
     The following description and the claims will further clarify the scope of the invention. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present invention provides methods of inactivating enveloped viruses and spermatozoa, as well as a formulation useful in those methods. The invention uses lipid vesicles which include a spermicidal or anti-viral surfactant in the outer bilayers to provide rapid activity without the necessity of vesicle breakdown. 
     The basic vesicle of the invention is formed of a non-ionic amphiphile such as glycerol monostearate, a sterol such as soy phytosterol, a surfactant showing the anti-viral or spermicidal (or both) activities such as NONOXYNOL-9, and an oil such as soybean oil. The oil, non-ionic surfactant and surfactant are selected such that at least a portion of the surfactant is in the outer bilayers of the vesicle so it can provide the requisite activity without breakdown of the vesicles. This requires compatibility between the non-ionic amphiphile and the surfactant, and it further requires that the surfactant is not dissolvable in the oil. Selection of any surfactant requires the ability to be used in stable lipid vesicles as well as compatibility testing, all of which are standard in the art. For example, if the formulations are going to be used with latex; e.g., with latex condoms, they must be latex-compatible, which can be determined rapidly using standard latex-compatibility testing. One means of selecting the proper mix of materials is a screening assay using spermicidal activity; the lipid vesicles must be able to inactivate spermatozoa in less than 20 seconds. If none of the surfactant was in the outer bilayers, rapid spermicidal activity could not occur with immediate lysis of the lipid vesicles, which is not found. Tests for spermicidal activity and anti-viral activity are described in the Examples. 
     The invention is more clearly understood through the following, non-limiting Examples. 
     EXAMPLE 1 
     Preparation of GNP9SO Novasomes® Containing Nonoxynol-9 
     Table 1 contains the materials utilized to produce GNP9SO Novasomes® containing NONOXYNOL-9 as the surfactant, glycerol monostearate as the amphiphile, soybean oil, and oleic acid as the negative charge producing agent. 
     
                       TABLE 1______________________________________Chemical Component    Weight______________________________________Glycerol monostearate 13.7 gramsSoya sterol (or cholesterol)                  3.8 gramsNONOXYNOL-9            7.2 gramsSoybean oil             16 gramsOleic acid             250 microliters______________________________________ 
    
     The components shown in Table 1 form the lipid-oil phase which is heated for 1 hour at 86° C. to achieve uniformity. Water is heated to 65° C. The GNP9SO Novasomes® are then produced by injecting the water phase into the lipid phase on a volume/volume basis of 13 parts lipid-oil to 37 parts water. GNP9SO Novasomes® can be produced manually, with reciprocating syringe instrumentation or continuous flow instrumentation. 
     EXAMPLE 2 
     Characterization of NONOXYNOL-9 Novasome® Preparations 
     After production of Novasomes®, characterization studies are performed. All preparations are tested for sterility and pH. Preparations are also sized on a Coulter LS 230 Laser sizing instrument equipped with a circulating waterbath. Table 2 shows the physical characteristics of the vesicles. Tables 3 and 4 show tests for anti-viral activity in vitro utilizing cell lines which allow viral proliferation, Tables 5 and 6 show latex compatibility, Table 7 shows anti-spermicidal activity using the Sander-Cramer assay, and Table 8 shows oral toxicity data. 
     
                       TABLE 2______________________________________                        Mean                        Coulter                               Mean CoulterChemical Components        Charge   pH     Size in μm                               Range in μm______________________________________GNP9SO Novasomes ®        Negative 4.52   0.598  0.375-0.839______________________________________ 
    
     EXAMPLE 3 
     Anti-Viral Assays 
     A. HIV-1 MN Viricidal Assay 
     The Novasome® preparation of Example 1 was tested for anti-viral activity against HIV-1. 100 microliters of HIV-1 Mn 1000× pelleted virus Lot #50-013 (TC ID 50  /ml =10 -6 ) was mixed for 30 minutes with either 100 microliters of water for injection or 100 microliters of Novasome® preparations. 1.8 mL of tissue culture medium was then added to each preparation. Serial 10 fold dilutions were then performed from 10 -2  to 10 -6 . 0.4 mL of each dilution was then placed in tubes containing C8166 cells, a HTLV-1 transformed T cell line. These combined materials were then incubated at 37° C. for 2 hours, washed 3 times in phosphate buffered saline, resuspended in fresh medium and each dilution plated in four replicate wells. Cells were fed twice weekly. Cells were observed at 7 and 14 days for cytopathic effects and supernatants were collected for p24 assay determinations as confirmation of viral proliferation. Tissue Culture Infective Dose (TC ID 50 ) (the amount of virus required to infect 50% of a tissue culture) calculations were performed based on the results of the p24 determinations. 
     The results are illustrated in Table 3. The Novasome® preparation clearly has anti-HIV activity. 
     
                       TABLE 3______________________________________Test article  TC ID.sub.50 at 1 week                      TC ID.sub.50 at 2 weeks______________________________________Water         10.sup.-4.75 10.sup.-4.75GNP9SO Novasomes ®         0.sup.       0.sup.______________________________________ 
    
     B. Vaccinia WR Viricidal Assay 
     The Novasome® preparation of Example 1 was also tested for anti-viral activity against Vaccinia WR. 100 microliters of Vaccinia WR 1000× pelleted virus Lot #68-016 (TCID-50/ml=10 -9 ) was mixed for 30 minutes with either 100 microliters of water for injection or 100 microliters of the Novasome® preparation. 200 μL of tissue culture medium was then added to the preparations. Serial 10 fold dilutions were then performed on the virus and no virus controls from 10 -1  to 10 -6 . 40 μL of each dilution was then placed in 96 well tissue culture plates containing BSC-1 cells. There were five replicates at each dilution, and duplicate plates were prepared. These combined materials were then incubated at 37° C. for 2 hours, washed 1 time in phosphate buffered saline, resuspended in fresh medium. Cells were observed at 3 and 7 days for cytopathic effects. (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; also known as Owen&#39;s reagent) (MTS) was run on the first plate at 3 days; the second plate was refed at three days and the MTS was run on this plate at 7 days. 
     Table 4 shows the results of the Vaccinia WR assay. Again, it is clear that the Novasome® preparation has anti-viral activity. 
     
                       TABLE 4______________________________________Test article   TC ID.sub.50 at 3 days                      TC ID.sub.50 at 7 days______________________________________Water          10.sup.-7   10.sup.-7GNP9SO Novasomes ®          0.sup.      0.sup.______________________________________ 
    
     EXAMPLE 4 
     Latex Compatibility Testing 
     The Novasome® preparation of Example 1 was also tested for latex compatibility. Three types of condoms, Trojan-Enz Non-Lubricated LO316RP, Lifestyles 0602258800, and Aladan were tested in this assay. The Novasome® preparation was applied to 13 condoms of each brand in 5 seconds using a soft brush and placed on a cheesecloth. Condoms were then conditioned in a humidity chamber at 90% relative humidity (RH) @38° C. for 30 minutes. They were then washed, dried, and tested for elongation and tensile strength. Another 13 condoms of each brand were treated as above but after conditioning were mounted directly onto the Airburst test fixture and tested. 80 condoms of each brand without the Novasome® preparation were tested for these properties as controls. Table 5 shows the results of the latex compatibility testing without the Novasome® preparation while Table 6 shows the testing with the Novasome® preparation. It is clear that the Novasome® preparation does not effect the properties of the latex condoms in short time compatibility testing. 
     
                       TABLE 5______________________________________       Trojan EnzCondom Brand       Non-Lubricated                    Lifestyle                             Aladan______________________________________Elongation %       783          827      802Tensile Strength, MPa       25.9         29.8     29.0Pressure kPa       2.3          1.3      2.2Volume, Liters       39.4         40.7     36.5______________________________________ 
    
     
                       TABLE 6______________________________________       Trojan EnzCondom Brand       Non-Lubricated                    Lifestyle                             Aladan______________________________________Elongation %       777          841      848Tensile Strength, MPa       26.8         29.1     30.2Pressure kPa       2.1          1.2      1.9Volume, Liters       39.4         40.7     36.5______________________________________ 
    
     EXAMPLE 5 
     Sander-Cramer Spermicidal Assay 
     In this Example, the preparation of Example 1 was tested for spermicidal activity using the Sander-Cramer spermicidal assay. A series of dilutions of the test compound are prepared using 0.9% saline. 1 mL of test article is mixed with 0.2 mL of human semen in a 13×100 min. test tube by drawing the mixture into a pipet and forcibly expressing the mixture. This procedure is repeated 6 times. A hanging drop of the mixture is prepared and five fields are observed under low power magnification. The whole process from mixing to microscopic observation must be accomplished in less than 20 seconds. The end point is the highest dilution that immobilizes spermatozoa in less than 20 seconds. 
     Table 7 shows the results of the Sander-Cramer assay. The vesicles of the invention not only allowed a higher NP-9 concentration than standard DMSO preparations, it also killed the sperm at a higher dilution, showing greater activity. 
     
                       TABLE 7______________________________________        Highest        Spermicidal NP9Test article Dilution      Concentration______________________________________GNP9SO Novasomes ™        528           32.5 mg/mLNP9 Control   91             10 mg/mL in DMSO______________________________________ 
    
     EXAMPLE 6 
     Rat Oral Toxicity Study 
     In this Example, the preparation of Example 1 was tested for oral toxicity. One group of Sprague-Dawley rats consisting of five males and five females were fasted overnight. The following morning after weighing each rat received three milliliters by gavage of the GNP9SO Novasome® preparation, the maximum allowable oral dose for rats. Animals were observed daily for sickness and mortality and individual body weights were obtained on day 7 and 14. The results are reported in Table 8. 
     
                       TABLE 8______________________________________      Mean Total Weight GainRats       Day 0-14______________________________________Male       126.66 gFemale      24.13 g______________________________________ 
    
     Based on the results of the studies shown in the Examples, it is clear that the preparations of the invention show spermicidal and anti-viral activity while being latex compatible and safe orally. These preparations are particularly useful for spermicidal and viricidal vaginal creams. 
     The forgoing formulations and Examples are merely illustrative and the scope of the invention is governed by the following claims.