MXPA00010328A - Formulations for the prevention or the treatment of diseases affecting mucosae or skin, or for pregnancy prevention, and an applicator for the delivery of topical formulations into mucosal cavities - Google Patents

Abstract

This invention relates to formulations for the prevention of infection and/or abnormal conditions of mucosae and/or skin caused by any pathogen and/or any disease, and more particularly for the prevention of sexually transmitted infections specially HIV and HSV. This invention also relates to formulations for the treatment of infection and/or abnormal conditions of skin and/or mucosae and more particularly for the treatment of herpetic lesions. The formulations could be used as a prophylactic agent to prevent accidental infection of health care workers. The formulations could be used for the healing and/or treatment of burn wounds and prevention of further infection. This invention also relates to the development of a unique vaginal/ano-rectal applicator for the uniform delivery of any topical formulations to treat and/or prevent any infection and/or abnormal conditions of mucosa cavity caused by any pathogen and/or disease.

Description

FORMULATIONS FOR THE PREVENTION OR TREATMENT OF DISEASES THAT AFFECT THE MUCOUS OR SKIN OR FOR THE PREVENTION OF PREGNANCY AND AN APPLICATOR FOR THE SUPPLY OF TOPICAL FORMULATIONS IN THE CAUCITIES OF THE MUCOSA FIELD OF THE INVENTION This invention relates to formulations comprising film forming formulations and any active ingredient, particularly to topical formulations. More particularly, this invention relates to topical formulations for preventing or treating diseases associated with or transmitted through the mucosa or skin, caused by any causative agent, particularly a pathogen. This invention also relates to an applicator for the uniform delivery of topical formulations to prevent or treat any disease associated with or transmitted through the mucosal cavity, or to prevent invasion by an external agent such as sperm or microbe.

BACKGROUND OF THE INVENTION The spread of sexually transmitted diseases (STDs) caused by the human immunodeficiency virus (HIV), herpes and other pathogens are attacking at a disconcerting pace. The global incidence, morbidity, and mortality of STDs is very significant. It is estimated worldwide that over 900 million individuals are infected with sexually transmitted pathogens. Each year more than 12 million people in the United States are newly infected with a pathogen responsible for STDs. Herpes Simplex virus type 1 (HSV-1) and type 2 (HSV-2) are the most common causes of genital ulceration in developed countries. The infection of genital herpes is lifelong and can result in painful and recurrent genital lesions, systemic complications, psychosocial morbidity and also several neonatal diseases after intrapartum transmission of HSV. The genital transmission of this pathogen is usually due to asymptomatic viral shedding by people who do not know they are infected. HSV-2 is now detected in 1 in 5 Americans 12 years of age and older. In addition, it is estimated that about a third of the world population has recurrent HSV infections and therefore has the capacity to transmit the virus through episodes of productive infection. Neisseria gonorrheae and Chlamydia trachomatis are recognized as two of the most prevalent sexually transmitted bacterial infections. Worldwide, there is an estimated annual incidence of 25 million cases of gonorrhea and 50 million cases of chlamydia. On the other hand, recent epidemiological data indicate that the number of individuals infected with HIV is growing dramatically worldwide. According to United Nations officials, estimates from epidemiological data suggest that as many as 16,000 individuals were infected with HIV each day during 1997. Recent statistics (date of late 1997) from the World Health Organization (WHO) ) indicate that there are approximately 31 million people infected with HIV worldwide and this number is projected to reach 40 million by the year 2000. Globally, heterosexual transmissions can account for 85-90% of HIV infection. As there is no vaccine against HIV, preventive measures are the only tools that can currently reduce the transmission of this retrovirus. The consistent and careful use of condoms represents an effective barrier against the sexual transmission of HIV and other sexually transmitted pathogens, although they should be used in all risky sexual relationships to significantly reduce the chances of acquiring the infection. In Africa, the most intensive prevention programs were only able to increase condom use in approximately 70% of all sexual relationships in prostituted women. Consequently, doubts arise about the possibilities of condom promotion to control the AIDS epidemic in high-risk groups. In situations where the heterosexual transmission of HIV is important, preventive measures where women could prevent their risk of contracting STDs could be an additional tool to restrict the epidemic. Such a protective tool would also be used in homosexual men's relationships since it could provide additional protection under the control of their receptive partner. Therefore, it is important to develop a barrier method that could be used as an alternative to condoms where the person could protect themselves against the infection without having to ask their sexual partners. Preventive measures aimed at blocking the initial transmission of pathogens that are the causative agents of AIDS, herpes and other STDs will, of course, lead to enormous benefits. The development of safe topical microbicides is currently a very high priority for the World Health Organization (WHO) and the National Institutes of Health (NIH) in the field of HIV prevention. A topical microbicide is often composed of an active ingredient and a vehicle. The active ingredients can act through a variety of mechanisms including: i) destabilizing the body's cell membrane, wrapping or encapsulating lipid or protein constituents (eg, spermicides / detergent-type microbicides such as nonoxynol-9), ii) blocking receptor-ligand interactions essential for infection (e.g., inhibitors of microbial adhesion such as sulfated compounds), iii) inhibiting intracellular or extracellular replication of the pathogen (e.g. antimicrobial drugs), iv) altering the vaginal environment and reducing susceptibility to infection (for example, regulating agents and products that keep the flora and environment of the vagina normal) or v) improving local immune responses (for example, modifiers of the immune response). The total efficiency of a topical microbicide against the sexual transmission of pathogens that cause STDs depends on the efficiency of the active ingredient to be delivered and its ability to cover the entire area of the vagina / cervix for maximum efficiency against the pathogens. The ability of these active agents to cover the entire vaginal cavity depends to a large extent on the type of vehicle used. Typical formulations of vehicles include gels, creams, foams, suppositories, sponges and films. The most commonly available vaginal formulations use the spermicide nonoxynol-9, a non-ionic surfactant, as a microbicide. In vitro, nonoxynol-9 inactivates coated viruses, such as HSV, HIV and other microorganisms that include Chlamydia trachomatis, Neisseria gonorrhoae. However, the potential efficacy of nonoxynol-9, against HIV is not yet clearly established and the results of the clinical trials are controversial. A recent controlled trial conducted among 1292 HIV-negative sexer women in Cameroon showed that the use of a vaginal film containing 70 mg of nonoxynol-9 did not reduce the proportion of new HIV infections, gonorrhea or chlamydia (Roody et al., 1998, N. Engl. J. Med., 339: 504-510). The failure of the nonoxynol-9 film to reduce the transmission of infectious agents can be attributed to the incomplete covering of the entire area of the vagina / cervix with the drug delivery system for nonoxynol-9 or the occurrence of mucosal toxicity that favors the infection of microorganisms. Due to the dramatic increase in the number of individuals worldwide who are infected with HIV, herpes or other sexually transmitted pathogens, there is an urgent need to develop active products and / or appropriate delivery systems that | can reduce the sexual transmission of these pathogens with minimal irritation of the mucosa and minimal effects on the flora of the vagina and pH. Sodium lauryl sulfate (SLS) is a sulphated surfactant that denatures the proteins of the membrane of the | pathogens. In this way it has a dual action as a detergent and as a chaotropic agent. With this concept, experiments have been carried out to evaluate the potential microbicidal effect of SLS on HSV and HIV. Preliminary studies clearly showed that SLS modifies infections in vitro | of both viruses. More recently, Howett et al., Have confirmed the findings that the SLS is also a potent activator of HSV-2, HIV-1 (Antimicrob Agents Chemother 43 (2): 314-321, 1999). In addition, it has been shown that SLS is effective against papillomavirus in rabbit, bovine and human | (uncoated virus) after brief treatment with low concentrations of this product. However, this reference does not teach the use of a vehicle to supply this potential microbicide. The selection of the vehicle is very important because it affects the concentration of available drugs, the duration of the availability of the drug and the degree of mucosal coverage by the formulation which are key factors to offer protection against invading pathogens. Another interesting category of candidate microbicides are microbial adhesion inhibitors, such as sulfated compounds, which block the interaction between the host cell receptor and the microbe. A known example of microbial adhesion inhibitors is dextran sulfate (DS), a polysulphated carbohydrate, which has been shown to inhibit in vitro HIV and herpesvirus infections. Recently, a gel formulation has been developed that can be applied to the vaginal, cervical or ano-rectal mucosa and that could be effective in preventing sexually transmitted pathogens. A superior feature of this gel formulation is its thermoreversible property. The transition from the liquid state at room temperature to the gel state at body temperature is of primary importance because when applied to rough biological surfaces such as vaginal or ano-rectal epithelium, the gel must penetrate into the smallest irregularities which form a good physical barrier against infectious agents. The gel formulation has the following key characteristics that both the FDA and NIH consider important: i) it is colorless, odorless and does not stain; i) it covers the entire vagina / cervix because when applied in a liquid state, iii) it is compatible with the latex male condom, iv) resists elusion by aqueous flow, v) has a pH to that of a healthy vagina (pH 4.0-4.5), vi) maintains the desired rheological properties under extreme heat conditions and cold and vii) does not affect, in vitro, the normal vaginal flora, especially Lactobacillus spp. The international publication (WO 97/42962) describes the use of formulations comprising components that form films capable of forming per se a physical barrier to pathogens. Thermoreversible gels such as poloxamers are particularly preferred for that use. The film forming formulations may additionally comprise microbicides, spermicides or any other drug, whose selection is guided by the pathogen, organism or the disease to be inactivated or treated. The formulations are therefore efficient as a physical barrier, and optionally as a chemical or pharmacological, as well as usable as a sustained drug delivery system at the site of administration. These formulations are intended for use in the prevention of sexually transmitted diseases, as well as in the treatment of infections, cancer, inflammation or any disease or condition that requires pharmacological treatment.

In addition, this publication teaches that the formulation decreases the toxicity of potent spermicides / microbicides such as nonoxynol-9. However, this publication does not specifically teach the use of SLS as a candidate chemical candidate incorporated within topical formulations. U.S. Patent Publication No. 5,275,805 discloses an oral composition comprising an antiplaque agent, a mixture of surfactants which may comprise SLS, a polyoxyethylene / polyoxypropylene block polymer and a taurate salt. It is said that the surfactant mixture is effective "to stabilize the oral composition for phase separation without compromising the antibacterial efficiency of the [antiplaque agent]." This purpose is assisted by using rather low concentrations of each component of the surfactant mixture; It is not believed that these low concentrations achieve antimicrobial activity and a physical barrier. European Patent Publication No. 386,960 discloses a vehicle for drug delivery comprising a thermoset gel component and a film forming component. The purpose of the film-forming component is to provide a firmer jelly. Spermicides can be included in that, and are part of a list of drugs intended for vaginal drug delivery. However, there is no evidence that a spermicide, a thermosetting gel component and a film-forming component could form a physicochemical barrier against pathogens, or that a sub-combination of these components would achieve the formation of a physicochemical barrier, or another, that any product that can disrupt the lipids of a cell membrane or the conformation of a protein (detergents in general, or chaotropic agents) would be effective against pathogens, alone or in combination with a thermosetting gel. For an efficient supply of semi-solid or liquid formulations within the body cavity, an applicator is a selection tool. The ideal applicator should be supplied immediately, during the operation, to the formulation to the mucosa and this, without expelling too much air before the formulation. US Patent No. 2,683,456 discloses a rectal applicator which comprises an elongated body pierced to be inserted into a body cavity, and a proximal enlarged portion, which remains outside the body cavity. A medication tube is located in close proximity to the junction between the enlarged and elongated body portions to discharge its contents directly into the elongated body in operation. The diffusion channel, through which the medication travels before being expelled into the body cavity, is simply formed by the elongated body duct, which allows the passage and expulsion of a fairly large volume of air. There is no description of a diffusion channel of a reduced volume, which would allow the medication to be expelled almost immediately without being preceded by such a large volume of air. The European Patent Publication No. 761246 discloses an applicator comprising a container, a pump having an inlet port in the container, an actuator button for driving the pump and a tube to be inserted into a body cavity, containing A distribution head on the tip of the tube, connecting the tube to an outlet port of the pump. The section of the tube defines a diffusion channel consisting simply of the conduit of the tube, through which a medication travels to the distribution head. The dispensing head is made of an enlarged section having a funnel portion and a plurality of perforations made through the entire thickness of the head. This document does not describe any applicator that could have a diffusion channel of a small volume which delivers a medication without supplying a substantially high volume of air. Patent publication number DE 3513645 discloses an applicator which comprises a non-perforated tip portion and a cylindrical body having a perforated outer wall. The defined duct in the body is simply outlined by the outer wall. This conduit constitutes a container in which a liquid or semi-solid formulation is placed. A plunger pushes the formulation into the container and, by compression against the non-perforated tip portion, the formulation is forced through the perforations. The conduit and the plunger may have a complementary conical shape to supply small volumes of the formulation. However, because the conduit has a relatively long volume, there may be a more or less high volume of air forced through the perforations before the formulation reaches them. HSV-1 and HSV-2 are neurotropic viruses that mainly infect neuroectodermal tissues including the skin, peripheral nerves and the central nervous system. The surfaces of the mucosa or skin are the usual sites of primary infection. Recurrent herpes labialis and genital herpes represent the most common clinical manifestations associated with HSV-1 and HSV-2 infections, respectively. Recurrences are spontaneous but are associated with physical or emotional tension, fever, exposure to ultraviolet light, tissue damage and immunosuppression. Although it is a moderate disease in immunocompetent individuals, HSV infections are annoying, especially for patients with frequent episodes. Patients compromised by either immunotherapy or underlying disease have an increased risk of developing HSV infections. The recipients of renal and cardiac transplant demonstrated an increase in the severity of the infection. In addition, the eruption of AIDS has reinforced the severity of clinical HSV disease in immunocompromised hosts. Currently available topical antiviral treatments have only limited efficacy particularly against symptomatic recurrent herpes. The limited efficiency of these topical formulations on the development of herpetic mucocutaneous lesions may be due to the poor ability of drugs to penetrate into the skin. The stratum corneum or corneal layer constitutes the barrier for the penetration of most substances into the skin. This layer consists of corneocytes embedded in a double layer lipid matrix composed of cholesterol, free fatty acids and ceramide. Consequently, the use of skin penetration enhancers may represent a convenient strategy for increasing the penetration of topical drug formulations into the skin. The SLS is a surfactant which has a skin penetration enhancing property by increasing the fluidity of the epidermal lipids. The skin penetration enhancing property of SLS combined with its ability to modify viral infection by its detergent and chaotropic properties could further increase the efficiency of topical drug formulations. In addition, due to its chaotropic properties, the SLS can have a broader spectrum of activity against sperm, bacteria, fungi and viruses than other simple detergents.

BRIEF DESCRIPTION OF THE INVENTION According to the present invention, it is a first object to provide formulations which comprise a film forming component, which is applied to the surface of the mucosa or skin, preferably in the form of gel, cream or ointment . The gel formulations are to be used to coat different types of mucosa such as vaginal, cervical, ano-rectal, eyes, mouth, nose or skin to prevent infection and / or abnormal conditions of the mucosa and / or skin. In addition, the gel formulations can be applied topically to the eye for the treatment and / or prevention of infection or ophthalmic conditions. Preferably, a thermoreversible gel is used, which is applied in a liquid form, spreads on the surface and forms a semi-solid coating after it reaches the temperature of this body surface. More preferably the thermoreversible gel is composed of poloxamer 407. Similar polymers such as poloxamines can also be used. The above formulation also comprises an agent capable of interfering with the cell membrane of the organism, enveloping or encapsulating lipid or protein constituents in a target cell, tissue or microbe. The above combination of the film forming component and the above agent can provide formulations with improved efficiency and reduced toxicity. In a specific embodiment, the agent is capable of interfering with the binding of an external microbial protein to a host receptor. In a more specific embodiment, the agent is a microbial adhesion inhibitor, or is a detergent or chaotropic agent capable of disrupting the integrity of the microbial outer protein. In yet a more specific embodiment, the microbial adhesion inhibitor is dextran sulfate; the detergent is selected from the group consisting of lauryl sulfate, benzalkonium chloride, lauroyl sarcosine, polyoxyethylene fatty acyl derivatives and fatty acyl ester derivatives polyoxyethylene sorbitan; and the chaotropic agent is sodium lauryl sulfate or guanidine. In the most specific modality the agent is SLS, the latter being a chemical candidate for selection due to its numerous properties as a detergent and a chaotropic agent and a putative microbial adhesion inhibitor. The SLS is only efficient against microbes. The SLS efficiency is further improved when incorporated into the present formulations. Therefore, it is contemplated that the SLS or any equivalent product may be used alone or in combination with the component forming the above film to prevent microbial infection. The SLS can be used alone or in combination with the above formulations in any suitable concentration, preferably at a concentration of about 0.1-25% (w / v), and more preferably at a concentration of about 1-15% (w / v) . The poloxamer concentration 407 can be used at any suitable concentration, preferably at a concentration of about 5-50% (w / v) and more preferably at a concentration of about 15-35% (w / v). The physical properties of the final formulation depend to a large extent on the drug to be incorporated in them, on the pH and solutes used in the manufacture of the formulations and on the viscosity sought for a given purpose. The above formulations may additionally comprise a drug that is effective in preventing infection and / or abnormal conditions of the mucosa or skin. Vaginal formulations constitute a physical and chemical barrier due to their components of microbial disorganization and film deformation. They agree, with an activity against infectious agents, these formulations can also be effective to prevent pregnancies. The SLS will advantageously replace nonoxynol-9 in the formulations. The SLS having a broader spectrum of activity against, inter alia, sperm, covered viruses and not covered, is a candidate of choice in the present formulation. The gel may contain a drug that is effective in preventing infection and / or abnormal conditions of the mucosa and / or skin. For the purpose of the invention, the term "drug" is intended to cover any antimicrobial, bactericidal, virucidal, chemotherapeutic, anti-inflammatory, antineoplastic, immunomodulatory or any other agent or combination thereof that is effective in preventing infection of the mucous and / or skin. The term "drug" also refers to cytokines or antigens that could stimulate an immune response that would protect against infection. The drugs could be incorporated into drug carriers such as gels, liposomes, nanoparticles or cyclodextrins, whose encapsulation results in improved prevention of infection. It is a further object of the present invention to provide a single applicator that can be used vaginally and / or ano-rectally to deliver topical formulations for the treatment and / or prevention of infection and / or abnormal conditions of the mucosa. The applicator can be designed in different ways to give the same required characteristics specified under a detailed description of the invention. Examples of some different concepts are also discussed under the detailed description which is intended to describe some of the general possibilities of design of the applicator, but in no way intended to limit the scope thereof. It is important to mention that the shape and final appearance of the applicator may differ from the examples given herein. In other preferred embodiments, the present formulations are used to treat viral diseases and additionally comprise as a drug an antiviral agent such as acyclovir or foscarnet or any other antimicrobial agents, used alone or in combination, at any suitable concentration. In a more preferred embodiment the formulation is composed of poloxamer 407 and contains foscarnet at a concentration ranging from 0.5 to 5% (weight / volume). In another more preferred embodiment, the formulation is comprised of poloxamer 407 and contains acyclovir at a concentration ranging from 0.5 to 5% (weight / volume). In yet another more preferred embodiment, the formulation is comprised of poloxamer 407 and contains SLS at a concentration ranging from 1 to 10% and foscarnet or acyclovir at the above concentrations. It is an object of the present invention to develop new topical formulations to prevent infection of the mucosa and / or skin, more particularly those sexually transmitted infections and even more particularly those caused by HIV and herpes. The microbicides or any other drug can be trapped within the gel formulations freely or encapsulated within drug carriers such as liposomes, nanoparticles or cyclodextrins. Such microbicidal gels could prolong the topical microbicidal activity, eliminate local irritation and reduce the systemic side effects of the incorporated active agents. It is also an object of the invention to develop, for vaginal applications, a single applicator which allows uniform distribution of contents to the entire vagina (supply to the sides) and cervix (supply to the front) for maximum protection against sexual transmission of pathogens. Therefore, a unique applicator has been designed which allows approximately a 360 degree distribution of its contents within the vagina and far into the cervix which is a great improvement over the existing conventional vaginal applicators which supply their content only in the front (only in the area of the cervix). It is another object of the present invention to develop topical formulations of drugs which could improve the efficiency of the agents chemically or pharmacologically active against mucocutaneous infections and more particularly those caused by HSV infections. The improved efficiency of the drugs when inorated into suitable matrices and / or drug carriers could reduce the dosage interval and consequently improve the quality of life of the patients. It is also an object of the present invention to develop topical formulations for the treatment and / or cure of burns as well as to prevent their potential infection. DESCRIPTION OF THE SPECIFIC MODALITIES OF THE PRESENT INVENTION This invention will be described later when referring to specific embodiments and appended figures, whose purpose is to illustrate the invention rather than limit its scope.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the infection of HSV-1 (strain F) in Vero cells after pretreatment of the virus with different concentrations of SLS (panel A) or DS (panel B) for 1 hour at 37 ° C ( •) or after the addition of SLS or DS to viruses without pretreatment (O). The plate-forming units (PFU) are expressed as a percentage of the control. The results are the mean ± SD of four independent experiments. Figure 1A: A: PFU (% control), B: Concentration of SLS (μM), C: LEGEND; D: INFECTION OF INCUBATED VIRUS WITH SLS (PANEL A) or DS (PANEL B) (NOT PRETRACTED); AND: Infectivity of the virus incubated with SLS (PANEL A) or DS (PANEL B) (1 h PRETATED). Figure 1B: A: PFU (% control); B: Concentration of dextran sulfate (nM); C: LEGEND; D: INFECTIVITY OF THE VIRUS INCUBATED WITH SLS (PANEL A) or DS (PANEL B) (NO PRETATED); E: Infectivity of the virus incubated with SLS (PANEL A) or DS (PANEL B) (1 h PRETATED) Figure 2 shows the efficiency of different concentrations of SLS (Panel A) or DS (Panel B) against HSV-1 (strain F) in Vero cells. Plaque forming units (PFU) are expressed as a percentage of control. The results are the mean ± SD of 4 independent experiments. Figure 2A: A: PFU (% control); B: Concentration of SLS (μM), Figure 2B: A: PFU (% control); B: Concentration of dextran sulfate (nM), Figure 3 illustrates the effect of pretreatment of HIV-1 (strain NL4-3) with 500 μ / M SLS for 1 hour at 37 ° C on its infection in 1G5 cells. The values represent the mean ± SD of 3 determinations. A: CELLS WITHOUT TREATING WITHOUT INFECTING; B: CELLS INFECTED WITH UNTREATED VIRUSES; C: CELLS INFECTED WITH PRETRACTED VIRUSES WITH 500 μm SLS; D: Luciferase activity (RLU); E: LEGEND. Figure 4 shows electron micrographs of Vero cells infected with HSV-1 (strain F) pretreated for 1 hour at 37 ° C with 50 μM (Panel B), 75 μM (Panel C), and 100 μM (Panel D) of SLS . Cells infected with HSV-1 (strain F) in the absence of SLS were used as control (Panel A). 70,000X amplification. Figure 5 shows the quantification of HSV-1 glycoprotein D (strain F) pretreated for 1 hour at 37 ° C with 12.5, 25, 50, 75 and 100 μM of SLS in Vero cells. Cells infected with HSV-1 (strain F) in EMEM + 2% FBS were used as control. The values are expressed as a percentage of the hybridization signal strength compared to the control. Figure 5B: A: SLS concentration (μM); B: Percentage of control.

Figure 6 shows the evolution of the survival time of intranasally infected mice with HSV-2 (strain 22) pretreated for 1 hour at 37 ° C with 6.25 (•), 25 (O) and 100 (A) μM of SLS. Mice infected with untreated virus were used as control (D). The results are expressed as the average of 8 animals per group. A: Survival (%); B: Days after infection; C: LEGEND; D: INFECTED MOUSE WITH UNTREATED VIRUSES; E: INFECTED MOUSE WITH PRETRACTED VIRUS WITH 6.25 μM SLS; F: INFECTED MOUSE WITH PRETRACTED VIRUS WITH 25 μM SLS; G: INFECTED MOUSE WITH PRETRACTED VIRUS WITH 100 μM SLS. Figure 7 shows the evolution of the time of the average result of lesions of mice infected cutaneously with HSV-1 (strain F) pretreated for 1 hour at 37 ° C with different concentrations (6.25 (•), 25 (O) and 100 (A) μM) of SLS (Panel A), or different concentrations (0.25 (•), 1 (O) and 10 (A) nM) of DS (Panel B). Mice infected with untreated virus were used as control (D). The results are expressed as the average of 6 animals per group. Figure 7A: A: Result of average injury, B: Days after infection C: LEGEND D: INFECTED MOUSE WITH UNDETRUPTED VIRUS E: INFECTED MOUSE WITH PRETRACTED VIRUS WITH 6.25 μM SLS (PANEL A) OR 0.25 Nm DS (PANEL B), F: MOUSE INFECTED WITH PRETRACTED VIRUS WITH 25 μM SLS (PANEL A) OR 1 Nm DS (PANEL B), G: INFECTED MOUSE WITH PRETRACTED VIRUS WITH 100 μM SLS (PANEL A) OR 10 Nm DS (PANEL B). Figure 7B: L: Average injury score, F: Days after infection G: LEGEND, H: INFECTED MOUSE WITH UNTREATED VIRUS I: INFECTED MOUSE WITH PRETRACTED VIRUS WITH 6.25 μM SLS (PANEL A) OR 0.25 Nm DS (PANEL B), J: INFECTED MOUSE WITH PRETRACTED VIRUS WITH 25 μM SLS (PANEL A) OR 1 Nm DS (PANEL B), K: MOUSE INFECTED WITH PRETRACTED VIRUS WITH 100 μM SLS (PANEL A) OR 10 Nm DS (PANEL B). Figure 8 shows the time course of the average result of the lesions of mice infected with HSV-1 (strain F) after pretreatment of the mice with the poloxamer formulation only 5 minutes (O) or 1 hour (?) before infection or with the poloxamer formulation containing 5% SLS also 5 minutes (•) or 1 hour (?) before infection. The infected untreated mice were used as control (D). The results are expressed as the average of 6 animals per group. A: Average injury result; B: Days after infection; C: LEGEND; D: UNATTENDED MICE INFECTED; E: PRETRACTED INFECTED MOUSE (5 min BEFORE THE INFECTION) WITH THE POLOXAMER ONLY; F: PRETRACTED INFECTED MOUSE (1 h BEFORE THE INFECTION) WITH THE POLOXAMER ONLY; G: PRETRACTED INFECTED MOUSE (5 min BEFORE THE INFECTION) WITH THE POLOXAMER + 5% SLS; H: PRETRACTED INFECTED MOUSE (1 h BEFORE THE INFECTION) WITH THE POLOXAMER + 5% SLS. Figure 9 shows the evolution of the time of the average result of the lesions (Panel A) and survival (Panel B) of mice infected intravaginally with HSV-2 (strain 333) pretreated with only the gel (M, A, •) 5 minutes before infection. The infected untreated mice were used as control (D,?, O). The results are the average of 8 animals per group. Figure 9A: A: Result of average injury, B: INFECTED INFRINGED CAT (REDNESS); C: INFECTED MOUSE PRETRACTED WITH THE GEL (REDNESS); D: NON-TREATED INFECTED MOUSE (FLAMMED); E: INFECTED MOUSE PRETRACTED WITH THE GEL; (SWOLLEN); F: Result of average injury. Figure 9B: A: Survival (%); B: INFECTED RATON WITHOUT TREAT; C: INFECTED MOUSE PRETRACTED WITH THE GEL; D: Days after infection; Figure 10 shows the evolution of survival time of mice infected intravaginally with HSV-2 (strain 333) pretreated with 2.5% SLS (*) or gel + 2.5% SLS (•) 5 minutes before infection. The infected untreated mice were used as control (D). The results are expressed as the average of 8 animals per group.

A: Survival (%); B: Days after infection; C: LEGEND; D: INFECTED RATON WITHOUT TREAT; E: INFECTED MOUSE PRETRACTED WITH GEL + 25% SLS; F: PRETRACTED INFECTED MOUSE WITH AN SLS SOLUTION AT 25%. Figure 11 shows the evolution of the survival time of mice infected intravaginally with HSV-2 (strain 333) pretreated with gel + 5% polyoxyethylene stearate 40 (•), gel + 5% guanidine (O), gel +2.5 % lauroyl sarcosine (A), gel + 2.5% benzalkonium chloride (?) or gel + 5% tween 80 (^) 5 minutes before infection. The infected untreated mice were used as control (D). The results are the average of 7 to 10 animals per group. A. Survival (%); B: Days after infection; C: LEGEND; D: INFECTED RATON WITHOUT TREAT; E: INFECTED MOUSE PRETRACTED WITH GEL + 5% POLIOXYETHYLENE ESTEARATE 40; F: INFECTED MOUSE PRETRACTED WITH GEL + 5% GUANIDINE; G: INFECTED MOUSE PRETRACTED WITH GEL + 2.5% LAUROIL SARCOSINE; H: INFECTED MOUSE PRETRACTED WITH GEL + 2.5% OF BENZALCONIO CHLORIDE; I: PRETRACTED INFECTED MOUSE WITH GEL + 5% TWEEN 80. Figure 12a is a perspective view illustrating a first embodiment of an applicator according to an aspect of the present invention.

Figure 12b is a side elevational view showing the inch dimensions of the applicator of Figure 12a. Figure 12c is an exploded view of the components of the applicator of Figure 12a. Figure 12d is a perspective view illustrating the details of the external surface of the proximal end of the inner wall of the applicator of Figure 12a. Figure 13a is a perspective view illustrating a second embodiment of an applicator according to an aspect of the present invention. Figure 13b is a side elevational view illustrating the inch dimensions of the applicator of Figure 13a in the insertion position and in the operating position. Figure 13c is an exploded view of the applicator of Figure 13a. Figure 14a is a perspective view of a third embodiment of an applicator according to an aspect of the present invention; the applicator being shown in an insertion position. Figure 14b is a perspective view of the applicator of Figure 14a shown in an operating position. Figure 14c is a side elevational view illustrating the internal details of the applicator of Figure 14a in the insertion position.

Figure 14d is a side elevational view illustrating the internal details of the applicator of Figure 14a in the operating position. Figure 15a is a perspective view of a fourth embodiment of the applicator according to an aspect of the present invention. Figure 15b is an exploded view of the applicator of Figure 15a. Figure 15c is a side elevation view of the external wall of the applicator of Figure 15a where the dimensions are given in inches. Figure 15d is a side elevational view of the piston / container of the applicator of Figure 15a where the dimensions are given in inches. Figure 15e is a sectional side elevation view of a portion of the applicator of Figure 15a illustrating details of the arrangement of the piston / container with respect to the internal and external walls of the applicator body. Figure 16 shows the evolution of the time of the average of the result of the lesions (Panel A) and survival (Panel B) of hairless mice infected cutaneously with HSV-1 and treated topically with the poloxamer alone (I), 0.5% foscarnet in aqueous solution (O) or poloxamer containing 0.5% foscarnet (•). The infected untreated mice were used as control (D). The treatment started 24 hours after the infection and was repeated 3 times daily for 4 days. The values are expressed as the average of 4 animals per group. Figure 16A: A. Result of average injury; B: Days after infection; A ': LEGEND; B ': UN INFECTED INFRINGED MOUSE; C: INFECTED MOUSE TREATED WITH POLOXAMER ONLY; D: INFECTED MOUSE TREATED WITH 0.5% OF FOSCARNET; E: INFECTED MOUSE TREATED WITH THE POLOXAMER + 5% OF FOSCARNET. Figure 16B: L. Survival (%); F: Days after infection; G: LEGEND; H: INFECTED RATON WITHOUT TREAT; I: INFECTED MOUSE TREATED WITH POLOXAMER ONLY; J: INFECTED MOUSE TREATED WITH ZOVIRAX OINTMENT; K: INFECTED MOUSE TREATED WITH POLAXAMER + 5% ACYCLOVIR. Figure 17 shows the evolution of the time of the average of the results of the lesions (Panel A) and survival (Panel B) of hairless mice infected cutaneously with HSV-1 (strain F) and treated 24 hours after the infection with a simple application of poloxamer containing 5% acyclovir (•) or Zovirax® (O) ointment. The infected untreated mice (D) were used as control. The values are expressed as the average of 7 to 10 animals per group. Figure 17A: A. Result of average injury; B: Days after infection; A ': LEGEND; B ': INFECTED MOUSE WITHOUT TREATING; C: INFECTED MOUSE TREATED WITH ZOVIRAX OINTMENT; D: INFECTED MOUSE TREATED WITH POLOXAMERO + 5% OF ACYCLOVIR. Figure 17B: J. Survival (%); E: Days after infection; F: LEGEND; G: INFECTED INFRINGED MOUSE; H: INFECTED MOUSE TREATED WITH ZOVIRAX OINTMENT; I: INFECTED MOUSE TREATED WITH THE POLAXAMER + 5% ACYCLOVIR. Figure 18 shows the evolution of time of the average of the results of the lesions (Panel A) and survival (Panel B) of hairless mice cutaneously infected with HSV-1 (strain F) and treated with poloxamer alone (I), poloxamer containing 5% acyclovir (•) or Zovirax® ointment (O). The infected untreated mice (D) were used as control. The treatment started 5 days after the infection and was repeated 3 times daily for 4 days. The values are expressed as the average of 7 to 10 animals per group. Figure 18A: A. Result of average injury; B: Days after infection; A ': LEGEND; B ': INFECTED MOUSE WITHOUT TREATING; C: INFECTED MOUSE TREATED WITH POLOXAMER ONLY; G: INFECTED MOUSE TREATED WITH ZOVIRAX OINTMENT; H: INFECTED MOUSE TREATED WITH THE POLAXAMERO + 5% ACYCLOVIR. Figure 18B: L. Survival (%); F: Days after infection; G: LEGEND; H: INFECTED RATON WITHOUT TREAT; I: INFECTED MOUSE TREATED WITH POLOXAMER ONLY; J: INFECTED MOUSE TREATED WITH 0.5% OF FOSCARNET; K: INFECTED MOUSE TREATED WITH THE POLOXAMER + 5% OF FOSCARNET. Figure 19 shows the distribution of foscarnet (?, A) and acyclovir (O, •) in skin tissue of uninfected (Panels A, C, E) and infected (Panels B, D, F) mice at 24 hours after of its topical application, either within the phosphate regulator (open symbols) or within poloxamer (full symbols). Panels A and B show the distribution of foscarnet and acyclovir in the stratum corneum strips. Panels C and D show the concentration of foscarnet and acyclovir in the epidermis while Panels E and F show the concentration foscarnet and acyclovir in the dermis. The values are expressed as the average of 4 to 6 animals per group. Figures 19A and 19B: A: Band tape number; B:% of applied dose. Figures 19C and 19D: A: acyclovir; B: foscarnet; C: dermis nmol / g. Figures 19E and 19F: A: acyclovir; B: foscarnet; C: dermis nmol / g. Figure 20 shows the concentration of acyclovir in infected and uninfected mice 24 hours after topical application, either in phosphate buffer (open bars) or in poloxamer (full bars) values are expressed as the mean of 4 to 6 animals per group.

A: Disinfected; B: Infected; C: LEGEND; D: CONCENTRATION OF ACICLOVIR PLASMA APPLIED IN SOLUTION; E: PLASMA CONCENTRATION OF ACICLOVIR APPLIED IN POLOXAMER; F: Plasma concentration (μM). Figure 21 shows the evolution of the time of the average of the results of the lesions (Panel A) and survival (Panel B) of hairless mice infected cutaneously with HSV-1 (strain F) treated with the poloxamer alone (I), poloxamer containing 3% foscarnet (O), poloxamer containing 5% SLS (•), or poloxamer containing 3% foscarnet + 5% SLS (?). The infected untreated mice (D) were used as control. The results are expressed as the average of 5 animals per group. Figure 21A: A. Mean injury result; B: Days after infection; A ': LEGEND; B ': INFECTED MOUSE WITHOUT TREATING; C: INFECTED MOUSE TREATED WITH POLOXAMER ONLY; D: INFECTED MOUSE TREATED WITH 3% OF FOSCARNET; E: INFECTED MOUSE TREATED WITH POLOXAMER SLS + 5%; F: INFECTED MOUSE TREATED WITH POLOXAMERO SLS + 3% OF FOSCARNET SLS + 5%. Figure 21B: G: Days after infection; H: LEGEND; I: INFECTED MOUSE WITHOUT TREAT; J: INFECTED MOUSE TREATED WITH POLOXAMER ONLY; K: INFECTED MOUSE TREATED WITH 3% OF FOSCARNET; L: INFECTED MOUSE TREATED WITH POLOXAMER SLS + 5%; M: INFECTED MOUSE TREATED WITH POLOXAMERO SLS + 3% OF FOSCARNET SLS + 5%; N: Survival (%). Figure 22 shows the susceptibility of HSV-1 (strain F) to combinations of different concentrations of foscarnet and SLS in Vero cells. The values are expressed as the mean ± SD of 3 determinations. Figure 22A: A: WITHOUT SLS ADDED; B: 375 μM SLS; C: 25 μm SLS; D: 125 μm SLS; E: Foscarnet concentration (μg / ml). Figure 22B: A: WITHOUT ADDED FOSCARNET; B: SLS + 33. 3 μm FORCARNET; C: SLS + 16.7 μm FORCARNET; D: SLS concentration (μM); E: PFU (% control).

GEL FORMULATIONS Poloxamer 407 is a block copolymer of polyoxyethylene and polyoxypropylene in a weight ratio of 7: 3 with an average molecular weight of 12500. An important feature of this block copolymer is its ability to form a thermoreversible gel. The transition from the liquid state at low temperature to the gel state at body temperature (the phase transition temperature being dependent, in part, on the concentration of the gel, the ionic strength and the incorporated solute) allows a number of medical applications interesting that include topical applications. Such a feature is of primary importance because when applied topically in its fluid state to the mucosa, the gel formulation should allow better penetration into skin and / or mucosal irregularities during application and a longer persistence once that the gel has reached body temperature. Due to the extremely low toxicity and irritation of the gel formulations, they represent an attractive proposition for topical drug delivery systems. The details for the preparation of the gel formulations are given below. This invention covers poloxamer 407 gel formulations of any suitable concentration, and more particularly those between about 10 and 35% w / w. This invention also covers any other component that forms film, gel, cream, ointment or thermoreversible substance that includes other poloxamers, poloxamines or chemicals.

Drugs Any antimicrobial, bactericidal, virucidal, chemotherapeutic, anti-inflammatory, antineoplastic, immunomodulatory or combination thereof that is effective in preventing or treating infection and / or abnormal conditions of the mucosa and / or skin caused by any pathogen and / or any disease is within the scope of this invention. Any detergent that may disorganize the membrane of pathogens, any skin penetration enhancer that increases the penetration of drugs and / or drug carriers within the mucosa and / or skin, any inhibitor of microbial adsorption which prevents entry of the pathogen within a target cell, any cytokine or antigen that could stimulate an immune response that would protect against infection of the pathogen is also within the scope of this invention. This invention also covers any combination of topical formulations and / or drugs.

Examples involving gel formulations for the prevention of infection The following examples are intended for the demonstration of the preparation of gel formulations that can be efficient to prevent infection and / or abnormal conditions of the mucosa and / or skin caused by any pathogen and / or any disease, but in no way intended to limit the scope of the present invention.

Preparation of gel formulations Gel formulations are prepared by adding an appropriate volume of distilled water, regulator or any other suitable aqueous solution to poloxamer 407 to obtain the desired concentration. An appropriate amount of drugs is then added to the powder or poloxamer solution to achieve the desired concentration. The pH of the gel formulation can be adjusted to meet the requirements of each target tissue to be coated with the present formulations. For example, if a formulation will be used to coat vaginal mucosa, an acid solution with a pH of about 4.0-4.5 will be used. Accordingly, the polymer percentage can be adjusted to obtain an adequate transition temperature from the liquid to the solid state. These adjustments are also within the knowledge and skill of the person skilled in the art. Even though the description of this invention is limited to specific cases, any film and / or drug forming component and / or liposomes (or other drug carriers) or any combination of the above are considered as potential candidates for the development of these topical presentations and are within the scope of this invention. The formulations also include any film and / or drug forming component and / or liposomes (or other drug carriers) or any combination of these products in any suitable concentration.

In vitro infection of herpes viruses pretreated with SLS or DS The effect of pretreating different strains of herpes virus with SLS or DS on their viral infections to susceptible cells has been evaluated. In brief, the cells were seeded in 24-well plates (Costar, Montreal, QC, Canada). Before infection, the viruses were suspended in a culture medium or phosphate-buffered saline (PBS), or incubated with different concentrations of SLS in PBS for 1 hour at 37 ° C. At confluence, the cells were incubated with viral suspensions by centrifuging the plates (750 x g for 45 minutes at 20 ° C) to allow adsorption of the virus. The virus was removed and the cell sheets were then covered with 0.5 ml of 0.6% Seaplaque agarose (Marine Colloids, Rockland, MA) prepared in an appropriate culture medium. The plates were incubated for 2 days at 37 ° C. The cells were then fixed with 10% formaldehyde in PBS for 20 minutes, washed with deionized water and stained with 0.05% methylene blue. The viral infection was evaluated by means of the determination of plaque forming units (PFU). Table 1 shows that pretreatment of various HSV-1 and HSV-2 strains with SLS for 1 hour at 37 ° C decreased, in a concentration-dependent manner, their infection on Vero cells. The infection of HSV-1 (strain F) was reduced to 21% when the viral particles were pretreated with 25 μM of SLS. Infections of all strains of HSV-2 were between 50 to 70% after preincubation with 25 μM of SLS. A complete loss of infection of all tested strains was obtained after pretreatment of the viruses with 50 μM of SLS. Pre-incubation of Vero cells for 1 hour at 37 ° C with SLS concentrations ranging from 6.25 to 100 μM before infection with HSV-1 (strain F) did not result in a loss of infection of the viruses (data not shown ). These results suggest that the SLS acts directly on the virus and not on the cells.

Table 1: Infection of various strains of HSV-1 and HSV-2 pretreated with different concentrations of SLS for 1 hour at 37 ° C. PFU (% control) for Concentration HSV-1 (F) to HSV-2 (333) to HSV-2 (22) to HSV-2 (6) b HSV-2 (15,589) c to SLS (μM) 6.25 101.1 ± 7.0 102.9 ± 23.5 128.0 ± 18.5 105.3 ± 12.4 108.7 ± 22.2 12.5 79.2 ± 36.4 115.4 ± 17.0 103.4 ± 14.9 82.1 ± 40.7 115.1 ± 17.5 25 21.2 ± 18.0 72.9 ± 9.1 63.8 ± 11.9 51.1 ± 30.1 59.0 ± 24.0 50 0 0 0 0 0 to wild type strain cceeppaa rreessiisstteennttee aa c ciclovir strain resistant to foscarnet Figure 1 shows the effect of pretreatment of HSV-1 (strain F) with different concentrations of SLS or DS on its infection in Vero cells. When the SLS was immediately added to the Vero cells after infection, the loss of viral infection was less dramatic compared to that obtained for the virus pretreated for 1 hour at 37 ° C with the same concentrations of SLS. After pretreatment, a 50% loss of viral infection was observed at a concentration of 20 μM compared to 75 μM when the virus was not pretreated. In addition, although complete inhibition of viral infection was obtained after preincubation with 50 μM of SLS, the inhibition was not complete yet at 100 μM without pretreatment. Similarly, the pretreatment of HSV-2 (strain 333) with SLS also influenced the infection of this strain (data not shown). On the other hand, DS reduces virus infection regardless of whether the virus was pretreated with DS. In this case, a 50% loss of viral infection was observed at a concentration of approximately 1 nM. The viability of Vero cells exposed for 1 hour at 37 ° C at SLS or DS concentrations similar to those used in Figure 1 and Table 1 was also tested using an MTS test. No signs of cytotoxicity could be demonstrated in the range of concentrations used (data not shown). Figure 2 shows the efficiency of different concentrations of SLS (Panel A) or DS (Panel B) against HSV-1 (strain F) in Vero cells. Soon, the cells were infected with the virus for 2 h at 37 ° C. Then, the supernatant was removed and the cells were covered with 0.5 ml of EMEM + 2% FBS containing 0.6% Seaplaque and SLS or DS agarose at the desired concentration. The plates were then incubated for 2 days at 37 ° C in a 5% CO2 atmosphere. The cells were fixed with 10% formaldehyde in PBS for 20 minutes, washed with deionized water and stained with 0.05% methylene blue. The viral infection was evaluated after the determination of PFU.

The results show that SLS and DS reduced in a concentration-dependent manner viral replication in a similar manner with full efficiency at 100 μM and 20 nM for SLS and DS, respectively. Without being tied to any mechanism, the above results suggest that the SLS may have an inhibitory effect on microbial adhesion.

In vitro infection of HIV-1 Pretreated with SLS The effect of pre-treating HIV-1 (strain NL4-3) with SLS on its infection in 1G5 cells, a Jurkat E6-1 derivative that protects two stably integrated constructs made of the luciferase gene under the control of HIV-1SF2LTR, has also been evaluated. In brief, before infection, the virus was incubated with a culture medium or 500 μM of SLS for 1 hour at 37 ° C. The cells (1x105 cells / well) were then incubated with HIV-1 strain NL4-3 (10 mg of p24) for 2 h at 37 ° C under an atmosphere of 5% CO2. The cells were then washed, resuspended in 200 μl of complete culture medium and transferred to a 96 well round bottom tissue culture plate (Microtest III, Falcon, Becton Dickinson, Lincoln Park, NJ). After an incubation period of 48 hours at 37 ° C, the cells were used, subjected to a freeze-thaw cycle and the luciferase activity was monitored using a microplate luminometer (MLX, Dynex Technologies, Chantilly, VA) . The results of this set of experiments clearly show that pretreatment of HIV-1 (strain NL4-3) with 500 μM of SLS for 1 hour at 37 ° C almost completely inhibited HIV-1 infection to 1G5 cells (Figure 3) .

Vero cell microscopy infected with HSV-1 (F strain) pretreated with SLS The appearance of HSV-1 (strain F) pretreated with various SLS concentrations (50, 75 and 100 μM) for 1 hour at 37 ° C has been evaluated in Vero cells using electron microscopy. Briefly, cells (80-90% confluence) were infected with the virus (approximately 70 PFU / ml in 14 ml) for 48 h at 37 ° C in a 5% CO2 atmosphere. The cells were scraped off the dishes and resuspended in a culture medium. The cells were centrifuged (515 x g for 10 minutes at 4 ° C) and the supernatant was decanted and the cells were resuspended in approximately 500 μl of medium. The cells were transferred in an eppendorf tube and centrifuged at (10,000 x g for 5 minutes at 4 ° C). The granule was resuspended in approximately 200 μl of 20% bovine serum albumin (BSA). A few 25% drops of glutaraldehyde were added to the mixture and the samples were immediately placed in an ice bath to allow BSA polymerization. The granule was then cut into 1 mm3 samples which were then fixed in 2% glutaraldehyde in PBS for 1 hour, 1% OsO in PBS for 1 hour and then with 0.1% tannic acid in PBS for 30 minutes. The samples were washed 3 times in PBS for 5 minutes between each stage. The samples were stained with 2% uranyl acetate in 10% ethanol for 30 minutes. The samples were dehydrated and imbibed in Epon following routine procedures. The sections (approximately 75 nm thick) were mounted on copper gratings (200 mesh). The specimens were stained with uranyl acetate, counterstained with lead citrate and observed with a JEOL 1010 electron microscope (JEOL Canada Inc., St-Hubert, QC, Canada). Figure 4 (Panel A) shows the normal appearance of the virus in the nuclei of Vero cells. Viral particles capsid, in hexagonal form and, containing a nucleus dense electron DNA. Complete viral particles formed by a nucleocapsid surrounded by a covering were also found in the cytoplasm of most cells. In Vero cells infected with virus pretreated with 50 (Panel B), 75 (Panel C) and 100 (Panel D) μM of SLS, the viral particles could recover in the nucleus but not in the cytoplasm of the cell. No mature nucleocapsid could be observed in the nucleus but the viral particles were constituted by capsids containing a discrete accumulation of dense electron material. The number of empty capsids found in the nucleus of cells infected with viruses pretreated with SLS decreased with the increased concentrations of drug used for pretreatment. In cells infected with virus pretreated with 100 μM of SLS, only a few cells with empty capsids in the nucleus could be detected. Taken together, these results may explain the loss of ineffectiveness of herpes viruses in the presence of SLS.

Quantification of the HSV glycoprotein D gene The quantification of the HSV-1 glycoprotein D gene (F strain) pretreated with SLS was also evaluated in Vero cells to determine the presence of viral DNA in the infected cells. In short, HSV-1 (strain F) was pretreated with various concentrations of SLS (12.5, 25, 50, 75 and 100 μM) in EMEM + 2% FBS for 1 hour at 37 ° C. Vero cells (80-90% confluence) were infected with the virus (100 PFU / ml in 20 ml) for 48 hours at 37 ° C in a 5% CO2 atmosphere. The culture medium was removed and the cell sheet was washed twice with 1X HBSS. The cells were scraped from the plates and resuspended in EMEM + 2% FBS. Total DNA was extracted using a standard phenol / chloroform procedure. Quantitation of total DNA was achieved using the Burton procedure. The probe used for this study corresponds to a part of HSV-2 glycoprotein D (strain 333), generated by PCR using the following primers: P1 (5'-GCCACCATGGGGCGTTTGACC-3 ') and P2 (5'-AAACTCAGTTATCTAGTCCTCGGGGTC-3' ) and [32P] was labeled by random priming. Hybridization was performed at 65 ° C in Na2HPO4 in 0.25 M (pH 6.8 with orthophosphoric acid) and 7% SDS. Washes were made in Na2HPO4 40 mm (pH 6.8 with orthophosphoric acid) and 1% SDS for 20 minutes at 65 ° C followed by 20 minutes at 25 ° C. Figure 5 (Panel A) shows the quantification of the HSV-1 glycoprotein D gene (strain F) pretreated with various concentrations of SLS in Vero cells. After an incubation of 48 hours, the cells were harvested and the DNA was extracted. Panel A samples aliquots of DNA fragmented in BglW (325 ng) applied to a 0.8% agarose gel, transferred to a nylon membrane, and hybridized with the glycoprotein D probe. Panel B shows the quantitative measurements of levels of HSV-1 DNA obtained by scanning densitometry of the autoradiogram using an Alphalmager. A major modification in the expression of the virus glycoprotein D gene in HSV-1 infected cells (strain F) pretreated with 12.5, 25 and 50 μM SLS compared to the control could not be observed. The quantitative measurements of the HSV-1 DNA levels obtained by scanning densitometry of the autoradiogram were similar (Panel B). However, when the virus was pretreated with higher concentrations of SLS (75 and 100 μM), a marked reduction in the expression of the glycoprotein D gene was observed with a reduction in DNA levels to 65.1% and 34.9% of the control values, respectively. These data suggest that SLS may interfere with the maturation of viral nucleocapsids either by slowing down their maturation speed or by interfering with the encapsulation of the DNA within the capsid shell.

In vivo infection of herpes virus pretreated with SLS (intranasal model) The effect of pretreating HSV-2 (strain 22) with SLS on viral infection has also been evaluated in a murine intranasal infection model. In short, female Balb / c mice (Charles River Breeding Laboratories Inc., St-Constant, QC, Canada) of 4 weeks of age were used throughout this study. Before infection, HSV-2 (strain 22) was incubated for 1 hour at 37 ° C with PBS or with different concentrations of SLS (6.25, 25 or 100 μM) to reach a final viral inoculum of 2,000 PFU / 20 μl. The mice were lightly anesthetized using Aerrane® (Isoflurane, USP; Janssen, North York, ON, Canada) and the viral suspension (20 μl total volume) was applied into the hole of the left external nose of the mice. The mice were then returned to their cages and survival was evaluated daily. Figure 6 shows that all mice infected with untreated virus died of encephalitis between day 9 and day 11. In contrast, 67% of mice infected with the viral inoculum pretreated with 6.25 and 25 μM of SLS survived the infection. Of primary interest, all mice infected with a viral suspension pretreated with 100 μM SLS survived the infection and showed no signs of disease.

In vivo infection of herpes virus pretreated with SLS or DS (cutaneous model) The effect of pretreating HSV-1 (strain F) with SLS on viral infection has also been evaluated in a model of murine skin infection. Female hairless mice (SKH1; Charles River Breeding Laboratories Inc., St-Constant, QC, Canada), 5-6 weeks of age were used throughout this study. Before infection, HSV-1 (strain F) was incubated for 1 hour at 37 ° C with PBS, with 6.25, 25 or 100 μM of SLS or with 0.25, 1 or 10 nM DS to obtain a viral inoculum of 3x105 PFU / 50 μl. Mice were anesthetized by intraperitoneal injection of a mixture containing 70 mg / kg ketamine hydrochloride (injection of Rogarsetic * USP; Rogar / STB Inc. Montreal, QC, Canada) and 11.5 mg / kg of xylazine (Rompun®; Canada Inc., Etobicoke; ON, Canada). The virus was inoculated on the lateral side of the body in the area of the left lumbar skin. The skin was scraped six times in a pattern of criss-crossed lines with a 27-gauge needle held vertically. The viral suspension (50 μl) was deposited on the scarified area and rubbed for 10 to 15 seconds with a cotton tip applicator saturated with EMEM + 2% FBS or SLS or DS solutions. The scarified area was protected with a corn pad which was kept on the body of the mice with surgical tape. The porous inner wall of the corn pad opening was waterproofed with adhesive cloth before being used to prevent absorption of the drug. The opening of the corn pad was also closed with surgical tape. The mice were then returned to their cages and observed twice daily. Figure 7 shows the time course of the mean lesion results of hairless mice infected cutaneously with HSV-1 (strain F) pretreated with different concentrations of SLS or DS for 1 hour at 37 ° C. The evaluation of the results of the lesion was performed according to the criteria presented in Table 2. In infected untreated mice, pathological signs of skin infection were not visible during the first four days after infection and only the scarified area remained. apparent. On day 5, herpetic skin lesions began to appear in some mice in the form of small vesicles distant from the site of inoculation. On day 6, almost all untreated mice developed herpetic skin lesions in the form of a 4-5 mm wide band extending from the spine to the ventral midline of the infected dtome similar to zoster-like infections . The maximum mean of the results of the lesion was observed on day 8. The mean of the results of the lesion decreased after day 11 until day 15 due to the spontaneous regression of the cutaneous lesions in some mice. Mice infected with the virus pretreated with 6.25 and 25 μM SLS did not demonstrate a significant reduction in the mean lesion results. However, mice infected with the virus pretreated with 100 μM of SLS did not show any signs of skin lesion. Of primary importance, all mice infected with the virus pretreated with 100 μM of SLS survived the infection (data not shown). On the other hand, the mice infected with the virus pretreated with 0.25 nM of DS showed a partial reduction of the average of the results of the lesion whereas the mice infected with the virus pretreated with either 1 or 10 nM of DS gave better protection against the development of herpetic lesions.

Table 2: Criterion used for the evaluation of herpetic skin lesions Result Appearance of the lesion 0 Infection not visible 1 Infection visible only at the site of inoculation, scarification area 2 I pfecture at the site of inoculation only, with swelling, scab and erythema 3 Infection at the site of inoculation with discrete lesions forming away from the site of inoculation 4 Ras visible around the middle of the body but not yet confluent 5 Ras confluent but not yet necrotic or ulcerated 6 Ras complete with necrosis or ulceration, paralysis of the hind limb, impanitis, death.

Prophylactic effect in vivo of poloxamer formulations containing or not SLS (cutaneous model) The efficiency of the poloxamer alone and of the poloxamer containing 5% SLS to prevent the development of skin lesions in mice has also been evaluated. Hairless female mice (5-6 weeks old) were used throughout this study. Briefly, the mice were anesthetized by intraperitoneal injection of a mixture containing 70 mg / kg of ketamine hydrochloride and 11.5 mg / kg of xylazine. The formulations were applied topically on the lateral side of the body in the area of the left lumbar skin. Five minutes and 1 hour after the application, a drop of the viral inoculum (3.15 x 108 PFU / ml) was deposited on the skin and a scarification was made with a 27G needle through a fall to mimic an accident that may occur to health care workers. In this model, the viral inoculum needs to be higher to obtain a complete zosteriform ras in almost all mice. However, the mortality associated with the infection was low and could not be used as a criterion to evaluate the efficiency of the treatments. The scarified area was protected with a corn pad which was kept on the body of the mouse with surgical tape. The opening of the corn pad was also closed with surgical tape. The mice were then returned to their cages and observed twice daily.

Figure 8 shows the time course of the mean of the lesion results of the untreated infected mice and of the mice pretreated with poloxamer alone or poloxamer containing 5% SLS 5 minutes or 1 hour before their cutaneous HSV infection -1 (strain F). The results show that mice pretreated with the gel only 5 minutes or 1 hour before infection gave only modest protection against the development of skin lesions. Of first interest, in mice pretreated 5 minutes or 1 hour with the poloxamer containing 5% SLS, complete protection against the development of skin lesions was observed. These results show the great potential of the formulations as a prophylactic proposal to prevent infection with pathogens. Such a tool could truly protect against the accidental infection of health care workers.

In vivo efficiency of gel formulations to protect against infection caused by herpes virus (intravaginal model) The efficiency of gel formulations to prevent genital transmission of HSV-2 has been evaluated in a murine intravaginal infection model . In short, female Balb / c mice 4 weeks of age were used for this study. To increase the susceptibility of the mouse to herpes, 2.5 mg of progesterone (Depo-Provera) was administered subcutaneously to each mouse 7 days before and one day after inoculation with HSV-2. The anesthetized mice were inoculated with 5 μl of 2.4 × 10 7 pfu / ml of HSV-2 (strain 333) after carving the vagina with a thin-tipped swab with calcium alginate. To determine the efficiency of the gel formulations to block herpes infection, 15 μl of the gel was delivered with a pipette tip into the vagina a few minutes before inoculation. The tip of the pipette moved in and out 4 times to simulate the shaking action of sexual intercourse while preventing itself from causing any bleeding. Figure 9 shows the average of the results of the lesion and the survival rate of the untreated infected mice and of the mice pretreated intravaginally with the gel alone before infection with HSV-2 (strain 333). Four days after infection, infected untreated animals showed perineal edema and redness and from 6 to 12 days, most of them died of encephalitis. Of primary importance, all mice pretreated with the gel only survived the infection and showed no signs of disease until 16 days after infection. The presence of the gel alone could thus abolish HSV-2 infection. Figure 10 shows the survival rate of infected untreated mice and mice pretreated intravaginally with 2.5% SLS or gel containing 2.5% SLS before infection with HSV-2 (strain 333). Four days after infection, infected untreated animals showed perineal edema and redness and from 6 to 12 days, most of them died of encephalitis. Of primary importance, all mice pretreated with either 2.5% SLS alone or gel containing 2.5% SLS survived the infection and showed no signs of disease until 16 days after infection. Taken together, these results clearly indicate that the use of the gel preparation may represent an innovative preventive measure to reduce the sexual transmission of herpes, HIV and other pathogens that cause STDs. Figure 11 shows the survival rate of the untreated infected mice and of the mice pre-treated intravaginally with gel containing various compounds before infection with HSV-2 (strain 333). Those compounds were selected to represent other sulfated and non-sulfated compounds that have or do not have detergent properties. They also represent various ionic (anionic and cationic) and nonionic compounds. This selection proposal was directed to find other potential candidate microbicides. The results showed that the gel formulation containing 2.5% lauroyl sarcosine gave complete protection against infection (100% survival). On the other hand, gel formulations containing 2.5% benzalkonium chloride, 5% polyoxyethylene stearate 40 and 5% guanidine gave 60, 60 and 30% survival, respectively. Preliminary results show that lauroyl sarcosine has good potential as a candidate microbicide that we are currently exploring. However, other compounds such as benzalkonium chloride, polyoxyethylene 40 stearate and guanidine that show potential partial microbicide can also be explored by optimizing their concentration for better efficiency. Alternatively, combinations of these compounds can also provide optimum efficiency, if compatible. Without joining any theory, it is contemplated that the combination of a detergent with a chaotropic agent can provide an efficiency as good as or even better than the SLS. These are specific examples of potential microbicides, but they are not intended to limit the scope of them.

Applicator design for vaginal / ano-rectal supply of the formulations As mentioned above, it is an object of the present invention to provide formulations for preventing infection and / or abnormal conditions of the mucosa and / or skin caused by any pathogen and / or any disease. For vaginal applications, any topical formulation should be administered using an applicator which allows uniform distribution of contents to the entire vagina (supply to the sides) and cervix (supply to the front) for maximum efficiency. Therefore, a unique applicator has been designed which allows a distribution of about 360 ° of its contents inside the vagina and away from the cervix which is a great improvement over the existing conventional vaginal applicators which supply their content only to the front (area of the cervix). The different objectives to be achieved in the main characteristics that the single applicator must have to supply the topical formulations include: a) Uniform distribution of topical formulations such as liquids or gels to the vagina / cervix b) Efficient and rapid supply of its contents c ) Resistance to temperature variations (-40 to 60 ° C) d) Compatibility of the polymer of the applicator with the gel formulations e) Ease of sterilization f) No leakage g) Ease of handling and insertion h) Resistance to breakage, expansion of the content and vibrations due to transport i) Compatibility with agents and / or conditions present in the surrounding environment Technical Background and Strategy The effectiveness of a formulation to block the sexual transmission of pathogens that cause STDs depends on i) the nature of the formulation to be delivered and ii) its ability to cover the entire vaginal / cervix area. Unlike other products, there is a unique formulation with termoreversible property which is supplied in liquid form with a good penetration of the formulation within the smallest irregularities of the vaginal / cervical mucosa. For maximum protection, such formulation should cover the entire vagina / cervix. However, existing conventional vaginal applicators have a unique orifice at the tip so that the contents are delivered only to the area of the cervix excluding the vagina, thereby limiting its efficiency. The single vaginal applicator will have multiple holes and / or slits, (at the tip and on the sides) to supply the formulation or any other component that forms film, gene, cream, ointment and / or antimicrobial agent, bactericidal, virucidal, chemotherapeutic, anti-inflammatory, antineoplastic, or immunomodulatory, detergents, microbial absorption inhibitor, skin penetration enhancing agent, cytokine, antigen, vaccines or combination thereof to treat or prevent STDs, cancer or any other disease, to uniformly cover the vagina and the cervix for maximum protection. The bibliographic investigations revealed that there are no applicators or similar products on the market that have such a design which allows the supply of its contents to the vagina / complete cervix.

Characteristics of the applicator All existing vaginal applicators supply the formulations in a gel / cream form which has the disadvantage of not covering the entire area of the vagina / cervix. On the other hand, the formulation has an important thermoreversible property being liquid at room temperature and gelling at body temperature. When supplied as a liquid, the formulation covers the entire vagina / cervix and penetrates through the smallest irregularities of the vaginal and cervical mucosa. For the single formulation or any other component that forms film, gel, cream, ointment and / or antimicrobial, bactericidal, virucidal, chemotherapeutic, anti-inflammatory, antineoplastic agent, or immunomodulatory, detergents, microbial adsorption inhibitor, skin penetration enhancing agent, cytokine, antigen, vaccines, or combination thereof to treat or prevent STDs, cancer or any other disease, a single applicator is needed to supply from the tip as well as from the sides to cover the entire vagina / cervix which is the key factor to offer maximum protection against the pathogens that cause the STDs. The main characteristics of the applicator are discussed later (see also Table 3): a) Uniform distribution of topical formulations such as liquid or gel to the vagina / complete cervix The applicator should supply the formulation uniformly and should cover the entire area of the vagina / entire cervix when delivered through apical and lateral holes. In addition, the applicator must provide sufficient amount to cover the cervix and vagina. This will allow maximum protection of individuals against pathogens that cause STDs. b) Efficient and rapid supply of its content Most of the existing vaginal applicators supply only a fraction of their content, limiting the efficiency of the formulation. Therefore, the applicator can supply all the contents without leaving residual material in the container or supplying the amount required for sufficient coating of the entire target mucosa. This will be achieved through the design of the container and calculating the average force of the fingers pressing on it to release its contents. The delivery time will vary depending on whether the content is supplied as a liquid, semi-cup or gel. However, the delivery of the applicator content must be fast c) Resistance to temperature variations (-40 to 60 ° C) The applicator must withstand temperature variations because the storage and transport environments will vary greatly from one country to another. It should be designed so that the applicator and the formulation remain unchanged under temperature conditions ranging from -40 to 60 ° C. d) Compatibility of the polymer of the applicator with the gel The polymer used for the development of the vaginal applicator must not affect the properties of the gel formulation (stability, viscosity parameters, non-cytotoxicity, efficiency to block pathogens, etc.). e) Sterilization facility The design and material of the applicator must ensure that they can be sterilized using an appropriate method and must not result in changes in the characteristics of their or their contents. f) No leakage The applicator must be leak proof under storage and transport conditions. If the boxes are stacked one on top of the other, the applicator should not spill its contents. g) Ease of handling and insertion The applicator should be used amicably as easy to handle and easy to insert without causing any discomfort to its user. In addition, it must be attractive to users. h) Resistance to breakage, for expansion of content and vibrations due to transport The applicator must resist breaking if it falls from the hands of the user or when it is handled during transport. It must also resist the expansion of its content. In addition, the applicator must be stable and resist vibrations during transport. i) Compatibility with agents and / or conditions present in the surrounding environment The applicator must resist the agents and / or various conditions present in the surrounding environment. For example, it should not be affected by vaginal acid pH, vaginal discharge or other similar conditions.

Table 3: Desired functions and target values of the applicator No Function Description Target value 1 Distributes the formulation Once introduced, Quantity as liquid, semi-viscous, proceed to ejection and approximately 3.5 ml gel, cream, ointment or distribution of any component that forms a film formulation 2 Distributes the formulation Distributes the formulation Distributes on uniformly to cover the vagina / cervix approximately 360 ° full in the vagina and on The following are examples of some different concepts which are intended to describe some of the general design possibilities of the applicator, but in no way intended to limit the scope from the same. It is important to mention that the final shape of the applicator may differ from the examples given herein. It is considered that such designs can be modified to adapt the ano-rectal application. Figures 12-15 illustrate specific examples of applicators according to an aspect of the present invention. The following description describes 4 embodiments of applicators illustrated in these figures. Generally declared, the present applicator is designed to uniformly supply any formulation such as liquid, semi-viscose, gel, cream, ointment or any other film-forming component described hereinbefore within a mucosal cavity, with the smallest residual amount thereof left inside. of the applicator. The present applicator comprises a longitudinally extending body which has proximal and distal ends. The proximal end is located near the external site of the mucosa cavity accessible to the patient. The body has external perforations, made as a series of slits or holes, for the uniform distribution of any formulation as described above to be delivered to the patient's mucosal cavity. Upon the insertion of the applicator and the expulsion of the formulation into the mucosa cavity, the formulation that is contained in a container, must travel advantageously through a diffusion channel having a small volume, before being expelled through the perforations. Indeed, this allows the rapid expulsion of the formulation and the minimization of the amount of formulation left in the applicator after the expulsion. The diffusion channel is created by a free space between the two walls that define the body. The first wall is an external wall of the body and includes openings. The second, non-perforated, inner wall is provided within the first wall to create the diffusion channel. The inner wall is sized and configured so that it can be slidably inserted into the first wall. Alternatively, the inner wall, sized to be smaller than the first one, can be molded integrally with the external wall of the body. The inner wall has a proximal end which has an inlet end for the formulation within the diffusion channel. A directing element may also be provided to direct the formulation within the input end of the broadcast channel. The director element thus prevents the entry of the formulation into another compartment other than the broadcast channel. A container capable of receiving the formulation is also part of the applicator. The container can be located close to the body of the applicator or inside the body. The container is operatively connected to an ejection element. The ejection element connects itself to the proximal end of the body through a connector element. The expulsion element is operated by the patient. Upon the application of compression, pushing or pulling movements, the ejection element releases the contents of the container, which is brought into contact with the proximal inlet end of the diffusion channel. The formulation therefore travels within the diffusion channel to the mucosa cavity, expelling through the perforations. Turning now to Figures 12a-12d of the accompanying drawings, a first embodiment of an applicator according to an aspect of the present invention will be described. Figure 12b shows an exploded view of this first applicator. The external wall (1) of the body of the applicator shows perforations (2) (only one shown) made as a single slit extending from one side of the body through the opposite side without interruption at the distal end of the outer wall (1) . The longitudinal slit thus defines lateral and distal perforations. In this embodiment, the container and the ejection element are a simple element (3) made of a compressible material. The formulation is contained in the container which expels its contents by pressing it with your fingers. The container is terminated in a membrane of low compressive strength (4). The container being the ejection element, is connected to the proximal end of the body through a connector element (5) represented by a lanyard or snap closure connector element. In this particular modality, the inner wall (6) of the body is provided as a separate element sized to be smaller than the outer wall. The proximal end of the inner wall ends with a protruding collar, which sits on the connector element formed at the proximal end of the outer wall. The proximal part of the internal wall comprises a closing element (7) which closes the internal duct formed by the internal wall. The closure element can have the shape of a plate. Alternatively, the proximal end of the inner wall can be molded integrally with the latter to simply close. Concentric to this closing element, there is an open concentric element (8) located on the periphery of the closing element. These elements provide a generally called director element, which directs the formulation within the diffusion channel formed between the inner and outer walls and away from the inner surface of the inner wall (6). Figure 12c also shows a conical element (9) located in the center of the steering means, provided to break the membrane (4) when the proper pressure is applied. A second embodiment of the applicator is illustrated in Figure 13. The same peripheral and internal walls as in Figure 12 are used in this applicator. However, it provides a plurality of grids spaced regularly from one another on the outer wall. In this specific version, the ejection element and the container are also a simple element. However, the ejection element is not a compressible container. Rather it is a piston-like structure (10) which comprises the formulation provided in a bag (11). In this embodiment, the connector element (5) is telescopically inserted into the piston-like structure (10). The bag is made of a material with low resistance to compression. To break this membrane, a conical element is provided at the proximal end of the inner wall. Figure 13 shows this conical element (9) as a plate provided with a tip portion. The plate sits at the proximal end of the inner wall, the pointed portion facing the bag (11). In use, the piston-like structure (10) is pressed by the user, the membrane is thus traversed by the tip portion, and the formulation is forced through the diffusion channel, and expelled through the perforations. Figure 14 illustrates a third embodiment of the present applicator. While the two previous embodiments show a container located near the proximal end of the diffusion channel, this third embodiment shows a container (12) provided away from the proximal end of the diffusion channel. In this case a seat (13) located away from the container is provided. The seat is operatively connected to the piston (14) located proximally to the container (12). The user pushes the piston and therefore compresses the container, the contents of which are engaged within the proximal inlet end of the diffusion channel. The formulation is expelled through the perforations made in the external wall of the body of the applicator, shown in Figure 14 as a plurality of holes (2). The orifices are spaced such that the formulation is uniformly distributed within the mucosal cavity. The holes are located in the longitudinal section of the outer wall as well as the distal end thereof. Figure 14 further shows that the inner and outer walls of the body of the applicator can be formed integrally. Alternatively, the inner wall can also take the form shown in Figures 12 and 13, without the need for a conical element. The container may include a membrane of low compressive strength such that, when compressed by the pushing movement of the piston (14), the membrane ruptures and discharges its contents into the diffusion channel. In this modality of the applicator, the director element is formed by the proximal inlet end of the diffusion channel and a closure element located this time at the proximal end of the body (not shown). Figure 15 shows a fourth embodiment of the applicator according to an aspect of the present invention. In this embodiment, the container and the ejecting element are a simple element. A low resistance membrane (4) is located near the proximal end of the body (1). The external wall of the applicator comprises grids that are practiced as a plurality of slits. The inner wall (6) is integrally formed with the outer wall. The inner wall ends at its proximal end with a conical element (15). The container / piston (16) has a diameter which is slightly larger than the external diameter of the inner wall, but smaller than the internal diameter of the external wall of the body of the applicator. In use, the container slides coupled between the two walls, the membrane is perforated and its contents are forced into the diffusion channel and the perforations located at the sides and at the distal end of the external wall. It should be noted that in all the embodiments described above, the steering element can be formed integrally with the proximal end of the inner wall of the body or be provided as a closure element or disk to block the passage of the formulation within the internal conduit formed by the internal wall and to direct the flow of the formation within the diffusion channel. In addition, for ease of use, fastener elements may be provided in some embodiments to assist the user in keeping the applicator in place while operating the ejector element. More specifically, in the second embodiment, the grasping element is defined by the annular collar (17) formed on the outer periphery of the connector element (5) the annular collar has an external thickness such that the user has sufficient space to grasp the distal end of the collar between the fingers and push the piston with another finger. In the third embodiment, the gripping element is provided at the proximal end of the piston (see number 18). The outer wall of the body being of a section larger than the piston, the user can maintain the body of the applicator by its proximal end with one hand and push the piston with another. Finally, in the fourth embodiment, the gripping element is provided as an elliptical handle (19) located at the proximal end of the body of the applicator and surrounding the connector element. This handle can be held between two fingers, while the piston is pushed with another finger.

Examples involving our poloxamer formulations for the treatment of infections In order to test the efficiency of our gel formulations in a murine model of cutaneous HSV-1 infection, the solutions were prepared inside a phosphate buffer (0.2 M, pH 6) to be compatible with the pH of the skin.

Comparative efficiency of topical formulations of foscarnet ointment. Acyclovir and of Zovirac against skin lesions of HSV-1 in mice The efficiency of our different topical formulations has been evaluated in a murine model of cutaneous HSV-1 infection. In short, female hairless mice (SKH1: Charles River Breeding Laboratories Inc., St-Constant, QC Canada) from 5 to 7 weeks of age were anesthetized by intraperitoneal injection of a mixture containing 70 mg / kg ketamine hydrochloride and 11.5 mg / kg of xylazine. The virus was inoculated on the lateral side of the body in the area of the left lumbar skin. The skin was scraped six times with a 27 gauge needle held vertically in a crisscross pattern. Fifty μl of viral suspension (HSV-1 strain F, 1.5 × 10 6 plaque-forming units (PFU / ml) were rubbed for 10 to 15 seconds on the scarified skin area with a cotton-tipped applicator saturated with culture medium [ minimal essential medium (MEM) supplemented with 100U / ml penicillin / streptomycin, 2 mM L-glutamine and 2% fetal bovine serum (MEM-E + 2% FBS).] The scarified area was protected with a pad of corn was kept on the mouse body with surgical tape.The porous internal wall of the corn pad opening was waterproofed with adhesive fabric before being used to prevent drug absorption, the opening of the corn pad was closed with tape The mice were then returned to their boxes and were observed twice daily.There were different treatment regimens evaluated in this study.Briefly, the tape that closes the opening of the corn pad was removed and the area scarified da was cleaned with a cotton-tipped applicator saturated with cold water. Fifteen μl of the different formulations were applied on the scarified area. The opening of the corn pad was closed with surgical tape to prevent rapid elimination of the drug by the mouse. This procedure also prevents accidental systemic treatment that could occur due to the potential licking of the lesions treated. The efficiency of the different formulations was evaluated using injury and survival results. Figure 16 (Panel A) shows the time evolution of the average lesion result of the untreated infected mice or mice treated with foscarnet in solution or incorporated within the poloxamer. The treatment started 24 hours after infection and was repeated 3 times daily for 4 days. In the mice treated with the poloxamer alone, a pattern substantially similar to that seen with the untreated mice is observed except that the regression of the skin lesions seems to go faster in the latter group. In mice treated with this 0.5% foscarnet solution, a considerable reduction in the mean of the lesion results was observed, which was more pronounced when the drug was associated with the poloxamer formulation. Figure 16 (Panel B) shows the corresponding survival for untreated infected mice and mice treated with the drug formulations. Death by encephalitis occurred in 75% of the untreated mice infected between day 7 and day 8. Mortality was similar in the mice that received the poloxamer alone and occurred between day 8 and 10. Half of the mice treated with foscarnet in solution survived the infection. Of primary interest, 75% of the mice treated with the poloxamer formulation of foscarnet survived the infection (p <0.05). Figure 17 (Panel A) shows the evolution of the time of the average result of the lesions of untreated infected mice and of mice treated with a simple application at 24 hours after the poloxamer infection containing 5% acyclovir or the Zovirax® ointment. Of first interest, the poloxamer formulation containing 5% acyclovir showed good efficiency against the development of skin lesions in mice, whereas the Zovirax® ointment exerted only a modest effect. However, acyclovir incorporated within the poloxamer significantly reduced lethality (p <0.05), but not the Zovirax® ointment (Panel B). The superior efficiency of the poloxamer formulation of acyclovir over the commercial ointment Zovirax® strongly suggests that the poloxamer could be a better vehicle for the local delivery of this drug. Figure 18 (Panel A) shows the evolution of the time of the average result of the lesions of control mice and of mice treated 3 times daily for 4 days and initiated 5 days after infection with the poloxamer alone, containing poloxamer 5 % acyclovir or Zovirax® ointment. In the mice that received the poloxamer alone, a reduction in the mean of the lesion results was observed compared to the untreated infected mice. Treatment with Zovirax® ointment exerted only a modest effect. However, a marked reduction in the mean of the lesion results was observed for the mice treated with the poloxamer formulation containing 5% acyclovir when compared with the untreated infected animals. Of primary interest, all mice treated with the poloxamer containing 5% acyclovir survived the infection (p <0.001) (Figure 18, Panel B). Treatment with Zovirax® ointment increased the survival of the infected mice to a lesser extent (p <0.05).

Penetration of antivirals in the skin in vivo Figure 19 shows the distribution of foscarnet and acyclovir in skin tissues of uninfected (Panels A, C, E) and infected (Panels B, D, F) mice at 24 hours after its topical application, either in phosphate regulator or in the poloxamer matrix. The distribution of both formulations of foscarnet and of the regulating solution of acyclovir was similar in the tape strips in the stratum corneum of the uninfected and infected mice. In contrast, the incorporation of acyclovir within the poloxamer markedly increased the amount of drug recovered in the stratum corneum of the uninfected and infected mice; being the increase of the penetration of the drug more pronounced in the infected mice. Insignificant or no amounts of foscarnet were found in the dermis and underlying epidermis of the uninfected mice or the carrier used for the application of the drug. The concentration of foscarnet in the epidermis and dermis of the infected mice was significantly higher when the drug was incorporated into the poloxamer. The concentration of acyclovir was higher than that of the foscarnet in the epidermis and dermis of the uninfected and infected mice and respectively of the carrier used. The concentration of acyclovir incorporated within the poloxamer in the epidermis of the uninfected mice was 6.1 times greater than that of the drug in the buffer. The infection of the mice did not significantly increase the amount of acyclovir in the epidermis. The concentration of acyclovir in the dermis of the infected mice was 7.9 times higher than that of the uninfected mice when the drug was administered in the poloxamer matrix. Figure 20 shows the concentration of acyclovir in plasma of uninfected and infected mice 24 hours after its local application, either in phosphate buffer or in the poloxamer matrix. Similar concentrations of acyclovir were found in the plasma of the uninfected mice for both formulations. The infection of the mice markedly increased the concentration of acyclovir in the plasma, especially when the drug was incorporated into the poloxamer matrix for which an increase of 4 times the concentration was reached. The concentration of acyclovir in the plasma of infected mice was 2.1 times higher when the drug was incorporated into the poloxamer matrix.

Effect of SLS on the efficiency of poloxamer formulations containing foscarnet or aciclovir against skin lesions of HSV-1 in mice The influence of SLS on the efficiency of poloxamer formulations containing foscarnet against HSV-1 infection 1 was also evaluated in mice. Figure 21 (Panel A) shows the time evolution of the average of the lesion results of the untreated infected mice and of the infected mice treated with a simple application (given 24 hours after infection) of the poloxamer alone, containing poloxamer 3% foscarnet, poloxamer containing 5% SLS, or poloxamer containing 3% foscarnet + 5% SLS. The poloxamer alone did not give any protection against the infection. In addition, a modest decrease in mean lesion results was observed in mice treated with poloxamer containing either 5% SLS or 3% foscarnet compared to untreated infected mice. Of first interest, the mice treated with the poloxamer containing 3% of foscarnet and 5% of SLS, we observed a marked and significant reduction (p <0.05) in the average of the results of the lesions compared with that of the mice without treat infected. The corresponding survival values for the same treatment groups are given in Panel B, which supports the results of the average of the results of the injuries. The skin penetration enhancing property of the SLS combined with its ability to modify the viral infection could explain the improved efficiency of the foscarnet formulation.

Susceptibility of HSV-1 in vitro to the combination of foscarnet and SLS The effect of SLS on the efficiency of foscarnet against HSV-1 (strain F) was investigated in Vero cells. In brief, the cells were seeded in 24-well plates (Costar, Montreal, QC, Canada) and incubated with HSV-1 strain F (approximately 100 PFU / ml) for 2 hours at 37 ° C to allow virus uptake . The virus was then removed and the cells were covered with 0.5 ml of 0.6% Seaplaque agarose (Marine Colloids, Rockiand, MA) containing different concentrations of foscarnet, SLS or combinations of both compounds. The plates were incubated for 2 days at 37 ° C. The cells were then fixed with 10% formaldehyde PBS for 20 minutes, washed with deionized water and stained with 0.05% methylene blue. The susceptibility of the virus was evaluated by means of the determination of PFU. Figure 22 shows the susceptibility of HSV-1 strain F to the combination of different concentrations of foscarnet and SLS on Vero cells. The results show that the presence of SLS improved the efficiency of foscarnet against HSV-1 (strain F) in Vero cells.

Potential applications The following examples described hereinafter are potential applications specific to local formulations, but are in no way intended to limit the scope thereof. As demonstrated in the above results, the gel formulations can be used for the prevention of skin and / or mucosal infection and more particularly for the prevention of HSV and HIV. In addition, the results show that gel formulations can serve as a prophylactic agent to prevent accidental infection of health care workers. As also demonstrated in the above results, the gel formulations can be used for the treatment and prevention of infection of skin and / or mucosal conditions and more particularly for the treatment and prevention of herpetic lesions. In addition to the above applications, potential additional applications for using the gel formulations are i) for the healing and / or treatment of burns and prevention of further infection and ii) for the treatment and / or prevention of infection of ophthalmic conditions . In the above examples, the gel formulations may contain any antimicrobial, bactericidal, virucidal, chemotherapeutic, anti-inflammatory, antineoplastic, immunomodulatory or any other agent or combination thereof which are effective for the treatment and / or prevention of infection and / or abnormal conditions of the mucosa and / or skin caused by any pathogen and / or any disease. The following examples described herein below are specific potential uses of the single applicator, but in no way attempt to limit the scope thereof. As described above, the applicator can be used for the delivery of any local formulation used to cover the cervical / vaginal / ano-rectal mucosa for the treatment and / or prevention of infection and / or abnormal conditions of the mucosa. The applicator can also be used to supply i) any topical formulation that can prevent the sexual transmission of pathogens that cause STDs, ii) vaginal contraceptive formulations, iii) topical microbicidal formulations against specific diseases and iv) any antimicrobial agent, bactericide, chemotherapeutic, antiinflammatory, antineoplastic or immunomodulatory virucide, detergents, microbial adsorption inhibitor, skin penetration enhancing agent, cytokine, vaccines antigen, radioactive agents or combinations thereof.

Claims (43)

1. CLAIMS 1. The use of an agent capable of disrupting the conformation of the membrane or proteins in a target cell, tissue or microbe, in the manufacture of a topical formulation to treat or to prevent a disease affecting the patient's mucosa or skin, or to prevent invasion by an external agent such as sperm or microbe, with the proviso that the agent does not consist of nonoxynol-9, benzalkonium chloride or menfegol.
2. 2. The use of a gel component and an agent according to claim 1, in the manufacture of a composition for preventing the transmission of a pathogen through the skin or mucosa of the person and to disorganize the conformation of the membrane or proteins of the pathogen characterized in that, on contact, the gel component forms an effective semi-solid protective layer to provide a physical barrier against the passage of the pathogen through the skin or mucosa, as well as a chemical barrier against it through of the disorganization of the conformation of the membrane or proteins by the agent.
3. 3. The use according to claim 1 or 2, characterized in that the agent comprises chaotropic components to improve the disorganization of the protein conformation.
4. 4. The use according to any of claims 1 to 3, characterized in that it additionally comprises an effective drug against a disease that affects the skin or mucosa or is transmitted through the skin or mucosa.
5. 5. The use according to claim 4, characterized in that the drug is one or more drugs selected from those that are antimicrobial, spermicidal, bactericidal, virucidal, chemotherapeutic, antineoplastic anti-inflammatory and immunomodulatory.
6. 6. The use according to claim 5, characterized in that it comprises an antiviral agent.
7. The use according to claim 6, characterized in that the antiviral agent is acyclovir or foscarnet.
8. The use according to any of claims 1 to 7, characterized in that the agent comprises a detergent comprising polyoxyethylene fatty acid.
9. 9. The use according to any of claims 3 to 7, characterized in that the agent comprises guanidine or lauryl sulfate sodium.
10. 10. The use according to any of claims 1 to 7, characterized in that the compound is lauroyl sarcosine.
11. 11. The use according to claim 9, characterized in that the sodium lauryl sulfate is used in a concentration of about 1% up to 15% (w / w).
12. 12. The use according to any of claims 2 to 11, characterized in that the gel component is poloxamer 407.
13. 13. The use according to claim 12, characterized in that poloxamer 407 is used in a concentration of about 5% to 50 % (p / p).
14. 14. The use according to claim 12, characterized in that the poloxamer 407 is used in a concentration of about 15% up to 35% (w / w).
15. 15. A barrier forming composition consisting essentially of a poloxamer, a regulatory solution and an effective amount of an agent capable of disrupting the conformation of the membrane or proteins in a target cell, tissue or mucosa, whereby, when applied To the surface of the skin or mucosa of the person, the composition forms a semisolid protective layer on the skin or mucosa, effective to provide a physical barrier and a chemical barrier against a pathogen, with the proviso that the agent is not nonoxynol.9, benzalkonium chloride or menfegol.
16. 16. The barrier forming composition according to claim 15, characterized in that it additionally includes an effective amount of an antiviral drug.
17. 17. The barrier forming composition according to claim 15 or 16, characterized in that the agent includes a chaotropic component in an amount sufficient to improve the disorganization effect towards the pathogen.
18. 18. The barrier forming composition according to claim 16 or 17, characterized because the antiviral is foscarnet or acyclovir.
19. 19. The barrier forming composition according to any of claims 15 to 18, characterized in that the agent comprises lauroyl sarcosine or polyoxyethylene stearate 40.
20. The barrier forming composition according to any of claims 15 to 18, characterized because the agent comprises lauryl sulfate or guanidine sodium.
21. The barrier forming composition according to any of claims 15 to 20, characterized in that the poloxamer is poloxamer 407.
22. 22. The barrier forming composition according to claim 21, characterized in that poloxamer 407 is present in a concentration of approximately 5% -50% (P / P)
23. 23. The barrier forming composition according to claim 21, characterized in that the poloxamer 407 is present in a concentration of about 15% -35% (w / w).
24. 24. An applicator for delivering a liquid to semi-solid formulation within the mucosal cavity of the patient characterized in that it comprises: a body having proximal and distal ends, a longitudinally extending outer wall provided with perforations and an internal wall extending longitudinally configured and dimensioned to be spaced from the outer wall to define a diffusion channel therebetween; the diffusion channel has a proximal inlet end; - a container capable of receiving the formulation; - an ejection element operatively connected to the container and operable by the patient to expel the formulation from the channel of the container to the diffusion channel; whereby, when operated, the ejection element forces the formulation out of the container into the diffusion channel through the proximal inlet end thereof, and the formulation is delivered to the mucosa cavity through the perforations.
25. 25. The applicator according to claim 24, characterized in that it additionally comprises: - a connecting element for connecting the ejection element with the proximal end of the body; and - an address element located at the proximal end of the body to direct any formulation to the proximal inlet end of the diffusion channel.
26. 26. The applicator according to claim 24, characterized in that the perforations of the external wall include longitudinal and distal perforations.
27. 27. The applicator according to claim 24, characterized in that the ejection element and the container are a simple element.
28. 28. The applicator according to claim 27, characterized in that the ejection element is a flexible element which is operated by compression.
29. 29. The applicator in accordance with the claim 27, characterized in that the ejection element is a piston operable by the patient.
30. 30. The applicator according to claim 24, characterized in that the ejection element comprises a piston operable by the patient.
31. The applicator according to claim 30, characterized in that the ejection element also includes a seat located distally from the container within a conduit formed by the internal wall, the seat being operatively connected to a piston located near the container; whereby, when the piston is pulled by the patient, the seat compresses the container which ejects the formulation into the diffusion channel.
32. 32. The applicator in accordance with the claim 30, characterized in that the piston has an external wall of a diameter greater than the outer diameter of the inner wall and smaller than the internal diameter of the external wall of the body and the piston is operated by the patient by occupying sliding the external wall of the body. same between the internal and external walls of the body.
33. 33. The applicator according to any of claims 24 to 30 and 32, characterized in that the steering element comprises a closing element for closing an internal conduit formed by the internal wall of the body.
34. The applicator according to claim 33, characterized in that the steering element additionally comprises an open element concentric to the closing element, opening the opening element within the proximal entrance end of the diffusion channel, and through which it is forced the formulation to operate the expulsion element.
35. 35. The applicator according to claim 33 or 34, characterized in that the steering element is formed integrally with the proximal end of the internal wall of the body.
36. 36. The applicator in accordance with the claim 31, characterized in that the steering element is formed by the proximal inlet channel and a closing element provided at the proximal end of the body.
37. 37. The applicator according to any of claims 24 to 36, characterized in that it additionally comprises a low resistance membrane located between the container and the proximal end of the body.
38. 38. The applicator in accordance with the claim 37, characterized in that it additionally comprises a conical element for breaking the membrane, the conical element being located at the proximal end of the steering element.
39. 39. The applicator according to any of claims 24 to 38, characterized in that the perforations are formed by at least two slits longitudinally spaced by a substantially equal distance and a distal perforation.
40. 40. The applicator according to claim 39, characterized in that the slit and distal perforations are continuous.
41. 41. The applicator according to any of claims 24 to 38, characterized in that the perforations are formed by a plurality of holes, which are spaced such that the formulation is substantially and uniformly delivered to the mucosa cavity.
42. 42. The applicator according to any of claims 24 to 41, characterized in that it additionally comprises a gripping element located on the outer periphery of the connector element.
43. 43. The applicator according to any of claims 24 to 41, characterized in that it additionally comprises a gripper element formed integrally with the connector element.