US20090156638A1

US20090156638A1 - Uses of nordihydroguaiaretic acid (NDGA) and derivatives thereof in preventing transmission of sexually transmitted diseases

Info

Publication number: US20090156638A1
Application number: US12/152,037
Authority: US
Inventors: Niharika Khanna
Original assignee: Individual
Current assignee: Individual
Priority date: 2007-05-11
Filing date: 2008-05-12
Publication date: 2009-06-18
Also published as: US20170312115A1; EP2150218A4; NZ581837A; ES2588235T3; CA2687080A1; MX343303B; JP2010526595A; WO2008141180A1; AU2008251471A1; EP2150218B1; PL2150218T3; HUE029409T2; MX2009012223A; DK2150218T3; US20100241092A1; AU2008251471B2; US9707120B2; HRP20161017T1; CA2687080C; EP2150218A1

Abstract

Provided herein are methods for inhibiting proliferation of a microbe associated with a sexually transmitted disease (STD) or infection and for preventing transmission of one or more sexually transmitted diseases in a subject at-risk for acquiring the same, including an HIV positive subject at risk for acquiring another sexually transmitted disease. The methods comprise contacting a microbe or cell thereof associated with the STD or administering to the subject nordihydroguaiaretic acid or a derivative or analog thereof. In HIV positive at-risk subjects one or more anti-retroviral drugs are co-administered.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This nonprovisional application claims benefit of priority under 35 U.S.C. §119(e) to provisional U.S. Ser. No. 60/917,344, filed May 11, 2007, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention
The present invention relates to the fields of virology, bacteriology and public health.
Specifically, the present invention provides methods for treating or prophlactically treating sexually transmitted diseases or infections using a topical microbicide.
2. Description of the Related Art
HIV is a world wide epidemic with 14,000 new, preventable, sexually acquired infections per day. HIV remains a devastating illness with poor access to adequate therapy in most parts of the world, making it the most compelling area of focus. Despite the knowledge of successful HIV prevention strategies, i.e. condom use, reduction in the number of sexual partners, diagnosis and treatment of sexually transmitted infections, HIV continues to spread at an alarming rate, especially among women in developing countries. Evidence is mounting that the male and female genital tract represent a distinct replication compartment for HIV-1, and that such compartments may serve as viral reservoirs (1). Accordingly, current protocols for testing and treating women for sexually transmitted diseases, such as, for example, HSV and HPV and HIV-1, face new and emerging challenges.
Heterosexual transmission is the main mode of HIV-1 transmission worldwide and accounts for nearly 90% of all HIV infections in women. Further, about 45 million Americans over the age of 14 suffer from genital herpes (1 in 5), about 20 million from human papilloma virus (HPV) (1 in 10), and more than half a million each have sexually transmitted hepatitis B virus infection (HBV). Vaginal microbicides may improve prevention outcomes for high-risk populations, such as those women in urban areas (2).
With HIV vaccines in early stages of development, there is an urgent need to contain the spread of infection, thus suggesting the need for innovative modalities to prevent HIV transmission. The development and use of a safe, effective and acceptable microbicide will empower women and their sexual partners to protect themselves from HIV and other organisms that cause sexually transmitted infections, thus shifting sexual decision making from the male partner to the couple. A recent cost-benefit analysis conducted at the London School of Hygiene and Tropical Medicine indicates that the introduction of a microbicide in 73 lower-income countries that reduced the risk of infection by 40%, at 30% coverage, would avert approximately 6 million HIV infections over 3 years in men, women and children. In addition, this would reduce the health care costs (excluding the cost of antiretroviral therapy) by a staggering 3.2 billion US dollars. This implies that a microbicide with relatively low effectiveness could have a substantial impact against the global HIV epidemic if it were used by a significant number of women.
Thus, there is a recognized need in the art for improved prophylactic and therapeutic methods of treating any sexually-transmitted disease or infection using microbicides. More specifically, the prior art is deficient in prophylactic and therapeutic methods against sexually-transmitted diseases or infections using nordihydroguaiaretic acid or derivatives or analogs thereof as a topical microbicide. The present invention fulfills this long-standing need and desire in the art.

SUMMARY OF THE INVENTION

The present invention is directed to a method for inhibiting proliferation of a microbe associated with a sexually transmitted disease or infection. The method comprises contacting the microbe or a microbial cell thereof with an amount of one or more microbicidal compounds effective to inhibit an activity required for proliferation thereof. The compound has a structural formula
where R¹-R⁴are independently H, C₁-C₂alkyl, —C(═O)(C₁-C₂alkyl), —C(═O)CH₂NH₂, —C(═O)CH₂N(H)(C₁-C₂alkyl)₂, or —C₁-C₂alkyl-N-piperidinyl, or a pharmacologically effective salt or hydrate thereof.
The present invention also is directed to a method for preventing transmission of one or more sexually transmitted diseases in a subject at-risk for acquiring the same. The method comprises administering one or more of a microbicidal compound to the subject. The microbicidal compound(s) have the structural formula described supra. The present invention is directed to a related method comprising the further step of administering one or more anti-STD drugs to a subject that already has a sexually-transmitted disease.
The present invention is directed further to a method for preventing transmission of one or more sexually transmitted diseases in an HIV positive subject at-risk for acquiring the same.
The method comprises co-administering one or more of a microbicidal compound and one or more anti-retroviral drugs to the subject. The microbicidal compound(s) have the structural formula described supra.
The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described herein, which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that any conception and specific embodiment disclosed herein may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that any description, figure, example, etc. is provided for the purpose of illustration and description only and is by no means intended to define the limits the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict the chemical structures of tetra-O-methyl-nordihydroguaiaretic acid (FIG. 1A), tetra-O-dimethylaminoglycyl-nordihydroguaiaretic acid (FIG. 1B), 3′-O-methyl nordihydroguaiaretic acid (FIG. 1C), and tetra-O—(N-ethylpiperidinyl)-nordihydroguaiaretic acid (FIG. 1D).

FIG. 2 depicts the screening algorithm used to screen and develop the microbicidal compounds.

FIG. 3 illustrates the lack of viral cross-resistance between tetra-O-methyl-nordihydroguaiaretic acid (M₄N) and acyclovir (ACV). SM44: ACV and M₄N sensitive; M₄N-10: passages in M₄N; ACV-10: passages in ACV.

FIG. 4 illustrates the inhibition of acyclovir-resistant strains of HSV-1 by tetra-O-dimethylaminoglycyl-nordihydroguaiaretic acid (G₄N) and acyclovir

FIG. 5 illustrates the comparative potencies between M₄N and acyclovir at different HSV-passages of drug treatments.

FIGS. 6A-6D illustrate the efficacy of M₄N topical treatment of HSV-1 induced cutaneous lesions at 10 minutes (FIG. 6A), 24 hours (FIG. 6B), 48 hours (FIG. 6C), and 72 hours (FIG. 6D) post-infection over a six day period.

FIGS. 7A-7B illustrate the efficacy of G₄N topical treatment of HSV-1 induced cutaneous lesions. FIG. 7A shows the dose-dependence of topical G₄N treatment. FIG. 7B shows the varying severities of HSV-1 infection.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
As used herein, the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
As used herein, the term “sexually transmitted disease” is used interchangeably with “STD”, “sexually transmitted infection”, “STI” and/or the plural thereof. An STD or STI is an illness or pathophysiological condition that has a significant probability of transmission between humans by means of any form of sexual contact, including kissing for some STIs. Particularly, the term sexually transmitted infection (STI) encompasses a person who may be infected, and may potentially infect others, without showing signs of disease or infection.
As used herein, the terms “effective amount” or “therapeutically effective amount” are interchangeable and refer to an amount that results in an improvement or remediation of the symptoms of the sexually-transmitted or sexually-acquired disease or infection. Those of skill in the art understand that the effective amount may improve the patient's or subject's condition, but may not be a complete cure of the disease and/or condition.
As used herein, the term “treat” refers to act upon with a therapeutic agent to improve or alter an outcome. It is contemplated that the effect of such may be whole or partial with respect to the desired outcome. It is also contemplated that the term “treat” encompasses prophylaxis for or preventing transmission of a sexually transmitted disease or infection.
As used herein, the term “contacting” refers to any suitable method of bringing one or more of the microbicidal compounds described herein into contact with a sexually-transmitted or sexually-acquired microbe or microbial cell, as described herein. In vitro or ex vivo this is achieved by exposing the microbe or microbial cell to the microbicide in a suitable medium. For in vivo applications, topical methods of administration are suitable as described herein.
As used herein, the term “NDGA” refers to the plant derived compound meso-1,4-bis(3,4-dimethoxyphenyl)-(2R,3S)-dimethylbutane or nordihydroguaiaretic acid which is extracted from Larrea tridentata.
As used herein, the terms “M4N”, “G4N”, Mal.4”, and “P4N” refer to derivatives or analogs of NDGA that are tetra-O-methyl-nordihydroguaiaretic acid, tetra-O—(N,N-dimethyl)glycyl-nordihydroguaiaretic acid, 3′-O-methyl nordihydroguaiaretic acid, and tetra-O—(N-ethylpiperidinyl)-nordihydroguaiaretic acid or the tetrakis hydrochloride salt thereof, respectively.
As used herein, the terms “microbicide” or “microbicidal compound” refers to a topically administrable formulation of a nordihydroguaiaretic acid (NDGA) compound or its derivatives or analogs having a therapeutic or pharmacological effect against the viral, bacterial, or fungal agents or parasites, such as, crab lice or skin mites of sexually transmitted or sexually-acquired diseases or infections.
As used herein, “subject” refers to a recipient of the topical microbicide.

II. Present Invention

In one embodiment of the present invention there is provided a method for inhibiting proliferation of a microbe associated with a sexually transmitted disease or infection, comprising contacting the microbe or a microbial cell thereof with an amount of one or more microbicidal compounds effective to inhibit an activity required for proliferation thereof, where the compound has a structural formula:
where R¹-R⁴are independently H, C₁-C₂alkyl, —C(═O)(C₁-C₂alkyl), —C(═O)CH₂NH₂, —C(═O)CH₂N(H)(C₁-C₂alkyl)₂, or —C₁-C₂alkyl-N-piperidinyl, or a pharmacologically effective salt or hydrate thereof.
In this embodiment, R¹-R⁴independently may be H, CH₃, —C(═O)CH₂N(H)(CH₃)₂, —C(═O)CH₂N(H)(CH₃)₂.Cl, or —CH₂CH₂—N-piperidinyl. For example the microbicidal compound may be nordihydroguaiaretic acid (NDGA), tetra-O-methyl-nordihydroguaiaretic acid (M₄N), tetra-O—(N,N-dimethyl)glycyl-nordihydroguaiaretic acid (G₄N) or the hydrochloride salt thereof, 3′-O-methyl nordihydroguaiaretic acid (Mal.4), or tetra-O—(N-ethyl)piperidinyl-nordihydroguaiaretic acid (P₄N). Also, in this embodiment the microbe may be a human immunodeficiency virus (HIV), a herpes simplex virus (HSV), a human papilloma virus (HPV), a hepatitis virus, a Molluscipox virus, Haemophilus ducreyi, Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Sarcoptes scabiei or a crab louse.
In another embodiment of the present invention there is provided a method for preventing transmission of one or more sexually transmitted diseases in a subject at-risk for acquiring the same, comprising administering one or more of a microbicidal compound to the subject, where the compound(s) have a structural formula where the compound(s) have a structural formula as described supra, where R¹-R⁴are independently H, C₁-C₂alkyl, —C(═O)(C₁-C₂alkyl), —C(═O)CH₂NH₂, —C(═O)CH₂N(H)(C₁-C₂alkyl)₂, —C(═O)CH₂N(H)(CH₃)₂.Cl, or —C₁-C₂alkyl-N-piperidinyl, or a pharmacologically effective salt or hydrate thereof.
Further to this embodiment, the at-risk subject may already have a sexually transmitted disease and the method may comprise administering one or more other anti-STD drugs thereto. In this further embodiment the at-risk subject may have HIV and the other anti-STD(s) drug may comprises one or more anti-retroviral drugs.
In both embodiments the microbicidal compound(s) may be formulated as a topical pharmaceutical composition comprising the compounds and a pharmaceutically acceptable carrier suitable for topical administration. Also, the microbicidal compound(s) may be administered vaginally, rectally or bucally and may be administered before or after sexual intercourse. In addition, the sexually transmitted disease may be HIV, genital warts, herpes, chancroid, chlamydia, crab lice, gonorrhea, hepatitis, lympogranuloma venereum, molluscum contagiosum, nongonococcal urethritis, pelvic inflammatory disease, scabies, syphilis, or vaginitis. Furthermore, the R¹-R⁴substituents and the specific microbicidal compounds are as described supra.
In yet another embodiment of the present invention there is provided a method for preventing transmission of one or more sexually transmitted diseases in an HIV positive subject at-risk for acquiring the same, comprising co-administering one or more of a microbicidal compound to the subject, where the compound(s) have a structural formula as described supra, where R¹-R⁴are independently H, C₁-C₂alkyl, —C(═O)(C₁-C₂alkyl), —C(═O)CH₂NH₂, —C(═O)CH₂N(H)(C₁-C₂alkyl)₂, —C(═O)CH₂N(H)(CH₃)₂.Cl, or —C₁-C₂alkyl-N-piperidinyl, or a pharmacologically effective salt or hydrate thereof, and one or more anti-retroviral drugs. In this embodiment, the R¹-R⁴substituents, the specific microbicidal compounds, the topical pharmaceutical composition comprising the microbicidal compounds, the methods and timing of administration of the microbicidal compounds, and the types of sexually transmitted diseases are as described supra.
Provided herein are topical microbicides and therapeutic or prophylactice methods effective against sexually transmitted diseases for an at-risk subject. The microbicide comprises the therapeutic agent NGDA and/or a derivative or an analog thereof. The microbicide has broad spectrum anti-microbial effects, low toxicity, an excellent safety profile in clinical studies, mutation-insensitivity, and low cost manufacturing capabilities.
The therapeutic or prophylactic agent of the topical microbicide comprises nordihydroguaiaretic acid (NDGA) or derivatives or analogs thereof in which one or more of the four NDGA hydroxy oxygens are substituted with substituents such as, but not limited to, short C_1-2alkyl groups, or glycinyl or piperidinyl groups that are amino or N-substituted with C_1-2alkyl groups. Preferably, these microbicidal compounds are water soluble. Generally, the microbicidal compound has the chemical structural formula:
where R¹-R⁴independently may be H, C₁-C₂alkyl, —C(═O)(C₁-C₂alkyl), —C(═O)CH₂NH₂, C(═O)CH₂N(H)(C₁-C₂alkyl)₂, or C₁-C₂alkyl-N-piperidinyl. Alternatively, these microbicidal compounds may be in the form of a salt or hydrate. Preferably, but without being limiting, the microbicidal compound is tetra-O-methyl-nordihydroguaiaretic acid (M₄N), tetra-O—(N-dimethyl)glycyl-nordihydroguaiaretic acid (G₄N) or the hydrochloride salt thereof, 3′-O-methyl nordihydroguaiaretic acid (Mal.4), or tetra-O—(N-ethylpiperidinyl)-nordihydroguaiaretic acid (P₄N).
It is contemplated that the NDGA or its derivative employed in the compositions and methods of the present invention are synthetic, semi-synthetic, natural, or recombinant in origin. Also, it is contemplated that additional NDGA derivatives or analogs may be isolated or designed and synthesized using standard chemical synthetic techniques and their efficacy determined using the screening algorithm described herein.
The microbicide may comprise a pharmaceutical composition that comprises the therapeutic agent and a pharmaceutically acceptable carrier or diluent as are standard and known in the art. One example of a suitable pharmaceutically acceptable diluent is petrolatum, such as white petrolatum. The pharmaceutical composition may be in the form of a gel, a semi-solid, a cream, and/or a lotion. It is also contemplated that the topical microbicide comprises a drug delivery system such as a bioadhesive or mucoadhesive that adheres to human epithelials. For example, a polycarbophil-based vaginal gel may be used as a delivery vehicle.
Generally, the microbicide may be administered as a topical ointment applied to the lining of the vagina and/or cervix and/or rectum, which can be accomplished as a gel, cream, lotion, non-aqueous or aqueous solution used to flush the vaginal or rectal cavity, and/or a vaginal or rectal suppository. Also, the microbicide or microbicidal compound may be administered in a spray formulation. In addition the microbicidal compounds may be delivered using microbicide-impregnated diaphragms and female and male condoms
The microbicides are useful to inhibit proliferation of a microbial cell associated with a sexually transmitted disease or infection. Contacting the microbial cell with one or more of the microbicides interferes with, inhibits or prevents a function or activity of the cell necessary for cellular proliferation. Cell proliferation assays, as are known and standard in the art, determine the efficacy of the microbicide as an anti-proliferative agent.
The microbicides are useful in a method of treating a subject at risk for a sexually transmitted disease or preventing transmission of the same. An effective amount of the NDGA and/or its derivative is administered topically to the subject in need thereof before or after sexual intercourse. It is contemplated that the topical microbicides are effective against drug-resistant sexually transmitted diseases as well as emerging infectious diseases transmitted via body fluids, particularly semen, vaginal fluid, saliva, and rectal or anal mucus. The sexually transmitted disease or infection may be HIV, HPV (also called genital warts), HSV, chancroid (Haemophilus ducreyi), chlamydia (Chlamydia trachomatis), crab lice, gonorrhea (Neisseria gonorrhoeae), hepatitis, lympogranuloma venereum (LGV, Chlamydia trachomatis), molluscum contagiosum (poxvirus of the Molluscipox virus genus), nongonococcal urethritis (NGU), pelvic inflammatory disease (PID), scabies (the skin mite Sarcoptes scabiei), syphilis (Treponema pallidum), and/or vaginitis (trichomoniasis).
The subject at risk includes any individual that is sexually active. The subject may be female. In such cases, the microbicide is administered as a vaginal or rectal ointment, which is applied buccally, vaginally and/or rectally, such as, for example, as a suppository. An average vaginal dose of microbicidal compounds may be delivered in a 2% ointment with about 18 mg/mL of microbicide, delivering about 90 mg per 5 mL or a 1% ointment with about 9 mg/mL of microbicide delivering about 45 mg per 5 mL.
Alternatively, the subject is male. In such cases, the microbicide may be administered via the rectum as a topical ointment applied buccally and/or rectally, such as, for example, as a suppository, or to the penis as a cream, ointment, spray, or lubricant for use with or without a condom. Alternatively, the subject at risk has been diagnosed with a sexually transmitted disease or infection.
Methods of the present invention also encompass co-administering to a patient in need of the microbicide and already having a sexually transmitted disease, another drug effective against a sexually transmitted disease or infection (anti-STD drug). For example, and without being limiting, a person with HIV may have both one or more of the microbicides or microbicidal compounds co-administered with highly active antiretroviral therapies (HAART). These include, for example, Nucleoside Reverse Transcription Inhibitor (NRTIs) such as Zidovudine, Didanosine, Zalcitabine, Stavudine, Lamivudine, Abacavir, Tenofovir; Nonnucleoside Reverse Transcriptase Inhibitors (NNRTIs) such as Nevirapine, Delavirdine, Efavirenz; Protease Inhibitors such as Indinavir, Ritonavir, Nelfinavir, Saquinavir, Amprenavir, Lopinavir. One of ordinary skill in the art is well able to determine which drug therapy, including dose and dosing schedule, is suitable in combination with the microbicide based on a subject's medical history and the progression of the disease.
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.

EXAMPLE 1

Microbicidal Compounds

Nordihydroguaiaretic Acid (NDGA)
NDGA is a purified extract of Larrea tridentata (Erimos Technologies).
NDGA Derivatives and Analogs
Tetra-O-methyl-nordihydroguaiaretic acid (M₄N; FIG. 1A), tetra-O—(N,N-dimethyl)glycyl-nordihydroguaiaretic acid (G₄N; FIG. 1B), 3′-O-methyl nordihydroguaiaretic acid (Mal.4; FIG. 1C), or tetra-O—(N-ethyl)piperidinyl-nordihydroguaiaretic acid (P₄N) are derivatives of NDGA and are the active pharmaceutical ingredients of the microbicide. The microbicides may be hydrophobic, such as M₄N, or water soluble such as G₄N, Mal.4 or P4N.
Tetra-O-methyl-nordihydroguaiaretic acid (M₄N) is a semi-synthetic derivative of NDGA synthesized by a two stage chemical methodology (Sigma Aldrich Fine Chemicals). tetra-O—(N,N-dimethyl)glycyl-nordihydroguaiaretic acid (G₄N) is synthesized as described (3). 3′-O-methyl-nordihydroguaiaretic acid (Mal.4) also can be isolated from Larrea tridentata as disclosed in U.S. Pat. No. 6,291,524. Tetra-O—(N-ethyl)piperidinyl-nordihydroguaiaretic acid (P₄N) is synthesized as described (4).

EXAMPLE 2

Screening Algorithm

The screening algorithm depicted in FIG. 2 is used to develop the microbicide. In summary, an HIV R5 cell attachment assay is conducted with a potential microbicidal compound and followed with an R5 cell to cell transmission assay. If the compound is found to be active, the next stage of secondary screening occurs, in which an HIV X4 attachment and fusion assay is performed, as well as an HIV X4 cell to cell transmission assay. In addition, cervical explant studies and Lactobacillus toxicity testing are performed. HIV R5 cell to cell transmission assays in seminal plasma and assessment of effects on semen and sperm are also performed.

Antiviral Assays in Fresh Human Peripheral Blood Mononuclear Cells (PBMCs)

PBMCs are isolated following Ficoll-Hypaque density centrifugation, and re-suspended at a cell density of 1×10⁶in medium supplemented with 2 μg/mL PHA. Following incubation for 24 hrs, the cells are collected by centrifugation, washed and suspended in media supplemented with 20 U/mL recombinant IL-2. Mixed PBMC populations are suspended at 1×10⁶cells/mL and distributed to the inner 60 wells of a 96-well round-bottom microtiter plate. Serially diluted test materials are added to the plate in triplicate followed by the appropriate pre-titered strain of HIV-1. The culture is incubated for 6 days at 37° C./5% CO₂Following the incubation supernatants are collected for analysis of virus replication by supernatant RT activity and cells analyzed for viability by XTT dye reduction. AZT is used as an internal assay standard. Ghost X4/R5 CD4 dependent assay
GHOST X4/R5 cells are plated in a 96-well flat-bottomed plate at a density of 1×10⁴cells/well in assay media 24 hours prior to performing the assay. Following the incubation H9/SK1 cells treated with 200 μg/mL of mitomycin C are washed and re-suspended in assay media, and added to the plate at a density of 5×10³. Serially diluted test compound is to the plate in triplicate and the cultures are incubated for 4 hours at 37° C./5% CO2. Following the incubation the cell monolayers were washed 3 times and 200 μL of assay media is added to each well of the culture which is incubated for and additional 24 hours at 37° C./5% CO₂. Following the overnight incubation, the cell monolayers are washed an additional 3 times. One-hundred microliters p24 lysis buffer is added to the efficacy plates and the plate is frozen at −20° C. overnight. Samples are then evaluated for intracellular p24. A toxicity plate is set-up and evaluated in parallel using XTT staining.

Beta-Gal HL2/3 Cell: Cell Fusion Assay

HeLa-LTR-beta-Gal cells are plated in 96-well flat-bottomed microtiter plates at a density of 5×10³cells/well. Cells are added to duplicate plates in order to evaluate both efficacy and toxicity in parallel. Serially diluted test compounds are added in triplicate and the plates are incubated for 1 hour at 37° C./5% CO₂. Following the incubation, HL2/3 cells at a density of 5×10³/well are added to all wells of both the efficacy and toxicity plates except for the negative control wells. The plates are incubated for and additional 48 hours at 37° C./5% CO₂. Following the incubation, toxicity plates are evaluated by staining with XTT and the efficacy plates are evaluated using chemiluminescence detection of beta-galactosidase.

HIV-beta-Gal Attachment Assay

Twenty-four hours prior to the assay, HeLa-CD4-LTR-beta-Gal cells are seeded in all wells of a 96-well flat-bottom microtiter plate at a concentration of 1×10⁴cells/well and incubated at 37° C./5% CO₂. Following the incubation, media is removed and serially diluted compound is added in triplicate to the cells and incubated for 15 to 30 minutes at 37° C./5% CO₂. A pre-determined titer of virus is then added and the culture is incubated for an additional 2 hours. At the end of the incubation, residual virus and compound are removed by washing, fresh media is added and the culture is allowed to incubated for and additional 48 hours. beta-galactosidase expression is then determined by chemiluminescence for the efficacy plate. Compound toxicity is evaluated on a separate plate run in parallel using XTT dye reduction.

Lactobacillus Toxicity Assay

Evaluation of toxicity to normal flora of the vagina is performed in order to assure that any test compound that progresses to more detailed testing will not have an impact on the peroxide producing Lactobacilli, which play an important anti-infective role by maintaining the vaginal environment at a lower natural pH. MRS media is inoculated with a Lactobacillus crispatus, jensenii or acidophilus and is incubated for 24 hours at 37° C., anaerobically. Following the incubation, media is inoculated with bacteria to an OD₆₇₀of 0.06 and is added to a 96-well, round-bottom microtiter plate. Test compound is diluted serially and is added in triplicate to the plate. Plates are incubated anaerobically at 37° C. for 24 hours. Following the incubation plates are read on a Spectramax Plus 384 microtiter plate reader at 490 nm.

ME180 CD4 Independent Assay

ME180 cells are plated at a density of 2.5×10³/well in a 96-well flat-bottom plate. A duplicate toxicity plate for each infectious assay plate is evaluated in parallel. Plates are incubated at 37° C./5% CO₂overnight. On the day of assay, the compounds to be tested are diluted to the requested concentration and are added to ME180 cells. H9/SK1 cells treated with 200 μg/mL of mitomycin C for 1 hour are washed and then added to the plates at a concentration of 2×10⁴cells/well. The cultures are incubated at 37°/5% CO₂for 4 hours. Following the incubation, the monolayers are washed to remove residual compound and infected cells. Assay media is added to all the wells and the cultures are incubated at 37° C./5% CO₂. After 24 hours, the cultures are thoroughly washed and fresh medium is added. This step is repeated at 48 hours post infection. On day 6 post-infection, the toxicity plates are stained with XTT and p24 analysis is performed on the efficacy plates.

EXAMPLE 3

In Vitro Anti-Viral Activity

Sp1 Transcription Factor Binding

Tetra-O-dimethylaminoglycyl-nordihydroguaiaretic acid (G₄N) binds the major grove of the free DNA when it is uncomplexed with other proteins. The binding constant for the G₄N/Sp1 enhancer sequence was calculated by the ethidium displacement technique at various phosphate buffer concentrations and pH values using a spectrofluorometer with excitation at 517 nm and emission at 596 nm. An apparent equilibrium binding constant (K_app) of 3×10⁶M⁻¹was obtained by the equation K_app=K_EtBr[EtBr]/[G₄N] which represents the concentration of the G₄N causing a 50% reduction of the fluorescent intensity of DNA-ethidium solution. Tetra-O—(N-ethylpiperidinyl)-nordihydroguaiaretic acid (P₄N) has been found to disrupt Sp1 transcription factor binding to the major groove of Sp1 site either by competition for binding or by altering the overall DNA conformation such that the major groove becomes incompatible for Sp1 binding G₄N has been tested on the growth of Herpes Simplex Virus Type 1 and Type 2 (HSV-1 and HSV-2). The early (IE) ICP4 gene of HSV is essential for HSV replication. Its promoter region possesses eight Sp1 consensus binding sites, five of which are required for ICP4 gene expression, making the ICP4 gene a good candidate target for M₄N. M₄N was previously determined to effectively inhibit ICP₄gene transcription and thus, HSV-1 replication. M₄N dramatically reduces Sp1 regulated transcription of HPV E₆/E₇in luciferase assays and in HPV induced mouse cervical C₃tumor lines. This inhibition is HPV promoter specific.

Mutation Insensitivity

The novel anti-viral mode of action of the NDGA derivatives confers several clinically relevant advantages which may significantly enhance their therapeutic value with regard to drug resistance, the prevention of HSV-1 reactivation, and as topically applied agents for the treatment of herpes labialis. Because the inhibitory mechanism of this class of compounds is completely distinct from the antiviral mechanism of existing approved anti-HIV drugs, there is little possibility of cross-resistance between these classes of drugs. Chen et al., demonstrated that M₄N retains complete efficacy against acyclovir-resistant strains of HSV-1 (FIG. 3) and the studies also showed that mutant acyclovir-resistant strains of HSV-1 (HSV-1^R) have no cross-resistance against G₄N (FIG. 4).
The capacity of harmful viral and bacterial strains in general, and of HIV-1 and HSV-1 specifically, to mutate in response to conventional chemical agents poses a rapidly increasing problem in new drug design and the development of therapeutic strategies. Viral inhibitors such as nucleoside analogues which target viral factors have the advantages of high specificity and low cytotoxicity. However in the clinical setting, prolonged use of these drugs places selective pressure upon the viral population to generate harmful resistant strains containing mutant proteins against which the drug is ineffective.
Mutant acyclovir-resistant strains of HSV-1 have been commonly reported to contain altered TK and DNA polymerase enzymes, and repeated use of AZT and even the most effective reverse transcriptase (RT) and protease inhibitors leads to resistant HIV strains containing mutations found within HIV RT and protease enzymes. In contrast, host factors, unlike viral factors, are under no selective pressure to mutate and are in general structurally invariable. In comparative studies of mutation-insensitivity between M₄N and acyclovir, there was no increase in the IC₅₀of M₄N against HSV-1 following ten passages of virus, indicating HSV-1 was incapable of developing resistance to M₄N over the short-term (FIG. 5). In contrast, the IC₅₀of acyclovir increased from 7.54 μM in the first passage to 444 μM in the tenth passage of HSV-1 (FIG. 5). Table 1 compares the IC₅₀of M4N and acyclovir for ten passages.

	TABLE 1

	IC50 (μM)

HSV-1 Passage	M4N	Acyclovir

1	11.7	7.54
2	4.4	37.8
3	8.24	88.8
4	5.97	138.4
5	6.48	111
6	6.98	130.7
7	6.98	189.8
8	9.91	444
9	8.82	378.8
10	4.77	444

Spectrum of Activity of G₄N and M₄N

Table 2 provides in vitro data on the inhibitory concentrations of G₄N and M₄N against various microbes associated with sexually transmitted diseases or infections.

TABLE 2

Infected		Inhibitory
Cell	Virus	Concentration
Type	Strain	(IC₅₀or EC₅₀)	Drug

HIV-1		H9	HIV-1_RTMF	12	μM	G4N
antiviral		Cos	HIV-1 (WM,	11	μM	M4N
activity			MN, VS,
			JR-CSF,
			IIIB)
		Cos	HIV-1	36	μM	G4N
		Cos	HIV-1	25	μM	Mal.4
HIV-1	X
transmission
inhibition
HIV-2	X
SIV		174 × CEM	SIV_mac239	5.0	μM	G4N
SHIV	X
HPV		C33A	HPV 16	30	uM	M4N
GC	X
CT	X

HSV-1

Vero

KOS

3.6

G4N

	McKrae	4.5	μM	G4N
HSV-1	HSV-1^R	2.1	μM
	F59878
	HSV-1^R	4.3	μM
	T28387

HSV	Explanted	HSV-1^R	40 μM had 67%	G4N
	Mouse		decrease in HSV
	trigeminal		reactivation
	ganglia

EXAMPLE 4

Preliminary Rabbit Irritation Study of M₄N

Cottontail rabbit vaginal irritation toxicology studies used 20 mg of M₄N vaginal ointment, concentration of 200 mg/mL, applied to the rabbit vagina daily for 14 days. No evidence of vaginal irritation or systemic toxicity was noted. In toxicokinetic studies of M₄N intravenous administration in dogs, doses up to 90 mg/kg/ were administered with reversible CNS toxicity at the highest dose. In humans, doses up to 3300 mg (˜45 mg/kg) were administered intravenously with generally good tolerability (5).

Fourteen Day Rabbit Irritation Study of M₄N

Ten female New Zealand White rabbits were randomly assigned to 1 of 2 groups. Rabbits were dosed via intravaginal administration with 0 or 2% w/w M₄N once daily for 14 days at a dose volume of 1 mL per administration. Specimens were taken prior to the start of the study and prior to necropsy on Study Day (SD) 15. Animals were food fasted overnight, with water available, prior to blood sample collection. There were no early deaths; animals were terminated in accordance with the protocol.

Hematology

Prior to dosing, there were no statistically significant differences between the rabbits assigned to the treatment group when compared to the rabbits assigned to the control group. On SD 15, the Group 2 rabbits has statistically significant lower absolute basophil count (ABBAS) when compared to the controls due to individual animal variation.

Coagulation

Prior to dosing, the rabbits assigned to Group 2 had statistically significant lower prothrombin time (PT) when compared to the rabbits assigned to the control group due to individual animal variation. There was no apparent test article-related effect on measured clinical pathology parameters following repeated intravaginal administration of M₄N for 14 days in the New Zealand White rabbit. The changes observed were considered to be incidental findings with no biological effect.

Clinical Chemistry

Prior to dosing, the rabbits assigned to Group 2 had statistically significant higher sodium (NA), total protein (TPROT), and albumin (ALB), and statistically significant lower alanine aminotransferase (ALT) when compared to the rabbits assigned to the control group. The lower ALT was due to individual animal variation, and the higher values for NA, TPROT, and ALB were most likely due to slight hemoconcentration. On SD 15, the Group 2 rabbits had statistically significant lower ALT when compared to the control group due to individual animal variation.

Macroscopic and Microscopic Findings

Gross pathology observations and organ weights are summarized and presented individually in the main report. Group 2 rabbit 32092 had an enlarged liver and an irregularly shaped right femur. These were considered to be incidental findings and not test article related. It was undetermined if the inflammation observed in the urinary bladder of Group 2 rabbit 32092 and the infiltrates of heterophils transmigrating the mucosa of the vagina into the lumen of Group 2 rabbit 32095 were test article-related effects or related to dosing. The enlarged liver of rabbit 32092 correlated with diffuse hepatic congestion; however, there was no histologic correlation for the irregularly shaped femur. The other observed lesions were considered to be incidental findings.

Toxicity

Blood samples were taken from the female rabbits administered M₄N topically per vaginam pre dose, 4 hours post first dose, and pre and post dose on day 14. Some evidence of absorption was seen with levels after the first dose generally greater than the LOQ. However after day 14 dosing, these levels were not seen, with no resultant evidence of cumulation. Concentrations seen did not exceed 10 ng/mL.

Single Dose Rabbit Irritation Study

4 male and 2 female New Zealand White rabbits received single s.c. injections of saline (1 mL), DMSO (1 mL), 20 mg (0.1 mL) M₄N, and 200 mg (1 mL) M₄N at each of four sites. Mortality, clinical observations, body weights, dermal irritation, gross necropsy findings, and microscopic pathology were evaluated. Treatment sites were marked and examined approximately 24 hours post-dose using the standardized evaluations (grades 0-4) of the Draize Scoring System. Minimal to mild irritation was scored in 4 out of 6 animals at the 24-hour observation interval; therefore, the treatment sites for these animals were evaluated daily through Day 5. All injection sites were preserved in 10% neutral-buffered formalin (NBF).
No treatment-related effect was seen on body weight or mortality in any dose groups. Clinical observations, dermal irritation, and gross and microscopic pathology indicated that a single s.c. injection of 20 mg M₄N resulted in minimal erythema at the injection site of a single male rabbit and a single s.c. injection of 200 mg M₄N resulted in slight irritation at the injection site of all rabbits.
No other clinical signs of toxicity were noted during the conduct of the study. No edema was noted in any rabbit and no erythema was noted in any female rabbit at the DMSO injection site. The DMSO treatment site showed mild erythema in one male rabbit on Days 2 and 3. Treatment with 20 mg M₄N produced no reaction in any female rabbit but minimal erythema in one male rabbit at Days 2 through 5. No erythema or edema was observed at the treatment site in the remaining three male rabbits. Treatment with 200 mg M₄N produced mild-to-minimal erythema in one female and two male rabbits from Day 2 to Day 5. No edema was seen at the 200-mg M₄N site in any male or female rabbit. No gross lesions were observed in the rabbits without erythema, which were sacrificed on Day 2. A tan, semi-firm mass in the subcutis was observed in all four rabbits necropsied on Day 5.
Histopathology showed that, for rabbits necropsied on Day 2, microscopic lesions at the injection sites involved the subcutis and/or dermis. For the rabbits necropsied on Day 5, the injection site lesion involved the epidermis in three of the four rabbits. The latter injection site lesions showed an apparent increase in intensity of focal tissue necrosis accompanied by vascular exudative and/or thrombotic events and calcification. A slight increase in the intensity of reactions was evident in several M₄N injection sites compared to DMSO vehicle injection sites.
Based on clinical observations, dermal irritation, and gross and microscopic pathology, a single s.c. injection of 20 mg M₄N resulted in minimal erythema at the injection site of a single male rabbit, and a single s.c. injection of 200 mg M₄N resulted in slight irritation at the injection site of all rabbits. The dose of 200 mg represents 2 times the proposed starting clinical dose of 100 mg M₄N to be administered topically.

EXAMPLE 5

In Vivo Animal Safety. Tolerability and Pharmacokinetic Studies of Microbicides 14-Day Toxicity Study in Rats

The toxicokinetics study consisted of four dose-groups (0, 50, 100, 500 mg/kg) with 6 animals/sex/group. Approximately 0.7 mL of blood was collected from the jugular vein from 3 rats/sex/group rotated in ascending order for each dose group at 0.5, 1, 4, 8, and 12 hours post-dose on Days 1, 7, and 14. Plasma was isolated and analyzed for M₄N by HPLC assay.
M₄N plasma concentrations were below the limit of quantitation (LOQ) (0.4000 μg/mL) for all samples on Days 1 and 7. Groups 8 and 9 were below the LOQ for Day 14. Only Group 10 (500 mg/kg) on Day 14 had samples with concentrations higher than the LOQ with the mean being either near or below the LOQ. Maximum M₄N plasma concentrations (C_max) for males and females were reached at 0.5 and 4 hours (T_max), respectively. T_lastwas 8 hours for males and 12 for females.
C_maxvalues for males and females in Group 10 were near the LOQ on Day 14 with C_maxjust above the LOQ for females (0.5371 μg/mL) and males just below the LOQ (0.3329 μg/mL). AUC_lastwas nearly 4 times higher for females, but overall low concentrations preclude a definitive assessment of a sex difference exposure. Because of a lack of measurable plasma concentrations in Groups 8 and 9, the dose dependency of C_maxand AUC_lastcould not be evaluated.
It was determined that, at a dose level of 500 mg/kg/day, higher concentrations were present on Day 14 than on Days 1 and 7.

14-Day Toxicity Study in Dogs

Three beagle dogs/sex/group received doses of 0 (0.50 mL/kg DMSO), 10, 50, and 100 mg/kg (0.05, 0.25, 0.5 mL/kg) M₄N. Approximately 4 mL of blood was collected from the jugular vein at each sampling time point of 0.5, 1, 4, 8, and 12 hours after dosing on Days 1, 7, and 14. Three dogs/sex/group were rotated in ascending order for each dose group. Plasma was isolated and analyzed for M₄N by HPLC assay.
M₄N plasma concentrations were below the LOQ (0.4000 μg/mL) for all samples on Days 1 and 7. On Day 14, only one sample from Group 2 (10 mg/kg) had a concentration greater than the LOQ (0.4713 μg/mL). Many of the mean concentrations for Groups 3 (50 mg/kg) and 4 (100 mg/kg) were near or below the LOQ, the highest values being 1.004 μg/mL for Group 3 and 1.096 μg/mL for Group 4 animals.
Maximum M₄N plasma concentrations (C_max) for Groups 3 and 4 were reached at median times of 4 hours (T_max), except for Group 3 males, for which median T_maxwas 8 hours. The median T_lastwas 12 hours, except Group 3 females where T_lastwas 4 hours.
Mean C_maxwas slightly higher for males within each group, but all mean C_maxvalues were 1.0 μg/mL. There was no obvious increase in C_maxwith a dose increase from 50 mg/kg/day to 100 mg/kg/day for males or females. AUC_lastvalues for Group 3 on Day 14 were approximately 3 times higher for males and were nearly 2 times higher for males in Group 4. AUC_lastincreased with dose for males and females approximately proportionally with the dose increase form 50 mg/kg/day to 100 mg/kg/day.
Rat Rectal Irritation Study of Microbicides
Microbicides are administered rectally for 10 days to Sprague-Dawley rats at a volume of 0.25 ml/rat/day In-life evaluations include mortality checks twice daily; daily clinical signs, including evaluation of the rectum and weekly body weights. Samples of rectal tissue are obtained at necropsy on Day 11 and rectal irritation is visually graded for erythema and edema according to the method of Draize et al. (1944). Ay gross lesions identified during the necropsy will be fixed in phosphate-buffered 10% formalin. Microscopic assessment of the vaginal tissue is conducted according to a modification of the procedure of Eckstein et al. (1969), using the following criteria: characteristics of the epithelial lining (ulcerations or erosions), thickness of the submucosal layer (edema), leukocytic infiltration of the submucosal layer, and vascular injection (erythema). For each criterion, the tissue will be graded numerically from 0 (no effect) to 4 (severe effect). The mean irritation score (MIS) for grossly and microscopically observed responses will be calculated separately by adding the scores for each evaluation criterion for each treatment group and dividing by the number of animals in the group. A treatment group with a microscopic MIS of 0 to 8 will be considered acceptable, one with a score of 9 to 10 will be marginal, and one with an MIS>10 will be unacceptable for topical use.

EXAMPLE 6

In Vivo Human Safety. Tolerability and Pharmacokinetic Studies of Microbicides Test Subjects

Fourteen healthy women aged 18 and above with a body mass index between 18 and 35 kg/m2 with a) regular menstrual cycles, b) postmenopausal or c) taking hormonal methods of contraception with a current negative pregnancy test and who were willing to be abstinent during the study and to use a condom with one other form of contraception for 72 hrs after the study were recruited. The M₄N vaginal ointment or placebo was administered intravaginally in the morning daily for two periods of 7 days duration. A minimum of 7 days for washout was included between the two study periods and the study periods were scheduled around regular menstrual cycles to ensure continuity of 7-day application of medication or placebo.

Formulation and Dosing Scheme

M4N was formulated as a 2% wt/wt ointment in white petrolatum, packaged in 5 mL containers and remained stable at room temperature for 6 months. A total volume of approximately 5 mL was applied to the cervix by self-application using a disposable vaginal applicator. Placebo formulation comprised white petrolatum with no active ingredient packaged similarly using a 5 mL container with disposable applicator. Two dosing schemes were used.
Regimen A: Approximately 90 mg M4N (5 mL 2% wt/wt) ointment in white petrolatum administered intravaginally once per day for 7 days followed by a gap of 7 days before Regiment B. Regimen B: Placebo ointment with white petrolatum administered intravaginally once per day for 7 days.

Toxicity Grading

Systemic toxicity was graded according to the National Cancer Institute Common Toxicity Criteria (Version 2.0) found online. Local toxicity was graded by evaluating the treatment site. After M4N administration, each subject self-reported mucosal reactions including erythema, swelling bleeding/hemorrhage, necrosis, erosion, ulceration, and eschar formation. It was anticipated that subjects would not experience toxicities above grade 1. Subjects with a grade 2 reaction or higher would be discontinued as participants and the reaction treated appropriately.
The mucosal local toxicity grading scale is as follows. Grade 1: erythema, injection, mild pain not requiring analgesics or mild itching not requiring therapy. Grade 2 (mild): patchy mucositis, might develop inflammatory serosanguinous discharge, moderate pain requiring normarcotic analgesic. Grade 3 (moderate): confluent fibrinous mucositis, severe pain requiring narcotic. Grade 4 (severe): ulceration, hemorrhage or necrosis.
M4N 90 mg (2% wt/wt dosing strength) was well tolerated. The observed adverse events were sporadic, did not follow a consistent pattern and were not considered serious. With the exception of 1 reported application site irritation which might which might have been caused by the M4N ointment, the adverse effects were not directly attributable to administration of the M4N vaginal ointment. Headaches were reported in 3 subjects, upper respiratory tract infection in 2, application site irritation in 1, abdominal pain in 1, groin pain in 1, and tooth abscess in 1.

Pharmacokinetic Assessment

Assessments were performed on days 1 and 7 and blood samples were taken at 1, 2, 4, and 6 hours after dosing. The selection of time periods of observation was based on data from the rabbit vaginal irritation study where absorption into the blood was noted to start at 30 min and peak at 4 hour followed by a slow decline. Venous blood samples (5 mL) were withdrawn via an intravenous cannula or by venepuncture into nonheparinized blood/serum sampling tubes without serum/clot activators. The total amount of blood taken from each subject for the study was 114 mL. The samples were allowed to clot for at least 30 minutes and then centrifuged at approximately 1600 g for 10 min at 4° C. The serum fraction was split into 2 aliquots and frozen in labeled polypropylenne tubes (cryovials) at approximately −20° C. until the end of each study period and then at −70° C. or below until assayed. Pharmacokinetic studies were done using sensitive high performance liquid chromatographic assays with mass spectrometric detection. The lower limit of detection of terameprocol was 1 ng/mL in the serum with precision values <5% and accuracy of <15% relative error, respectively. There was no detection of M4N in the pharmacokinetic samples.

EXAMPLE 7

Effects Against HSV-1 Infection in Animal Models

M₄N and G₄N were tested or their abilities to prevent HSV-1 infection in two animal model systems. The dorsal cutaneous guinea pig model system is a well-established model for HSV-1 infection. Topical application of M₄N (FIGS. 6A-6D) and G₄N (FIG. 7) effectively inhibited HSV-1 induced cutaneous lesion formation and viral shedding, with no evidence of toxicity or dermal irritation even at the highest soluble concentrations (150 mM).
In FIGS. 6A-6D, for topical treatment with M₄N sections of shaved and depilated dorsal guinea pig skin were infected equally were infected with HSV-1. 8 sections each were treated initially at 10 min after infection with DMSO control and M₄N (170 μM; 60 mg/mL) and the lesions were scored on days 1-6 post-treatment (FIG. 6A). 4 sections each were treated initially at 24 h (FIG. 6A), 48 h (FIG. 6B) and 72 h (FIG. 6B) after infection with DMSO control and M₄N (170 μM; 60 mg/mL) and the lesions were scored on days 1-6 post-treatment. M₄N showed better efficacy when treatment commenced shortly, within 24, after infection.
In FIG. 7A, for topical treatment with G₄N, six (1-6) sections of shaved and depilated dorsal guinea pig skin were infected equally with high levels of HSV-1 (1×10⁸pfu/mL; McKrae strain). Regions 3 and 6 are controls and received only PBS, regions 2 and 5 were treated topically with 5 mM G₄N and regions 1 and 4 were treated topically with 10 mM G₄N. Treatments were administered twice daily and photogragphs were taken at day 6 post-infection. As shown in (FIG. 7A), without G₄N, HSV-1 infection was obvious. At 5 mM G₄N, the drug was semi-effective, at 10 mM G₄N it was more effective.
In FIG. 7B, regions 1-6 were treated topically with 150 mM G₄N or PBS twice a day. Regions 1 and 4 were infected with high levels of HSV-1 (1×10⁸pfu/mL), regions 2 and 5 were infected with moderate levels of HSV-1 (1×10⁶pfu/mL) and regions 3 and 6 were infected with low levels of HSV-1 (1×10⁴pfu/mL). Regions 4-6 were topically treated with G₄N and regions 1-3 received identical treatments with PBS. Photogragphs were taken at day 6 post-infection. 150 mM G₄N was extremely effective. At this concentration G₄N effectively inhibited HSV-1 induced cutaneous lesion formation and viral shedding, with no evidence of toxicity or dermal irritation even at the highest soluble concentrations (150 mM) (FIG. 7B).
The efficacies of G₄N (FIG. 8A) and M₄N (FIG. 8A) against HSV-2 in a mouse vaginal model system have also been examined. Progestin treated female CF-1 mice (10 per group) were infected with HSV-2 in the absence or presence of G₄N (in PBS), P₄N (in PB S), or M₄N in Vaseline (20 mg/ml). At the end of three days, the vaginal lavages were collected from the washings of the vagina regions. The status of the vaginal HSV-2 infection and replication were examined by their cytopathic effects on cultured human foreskin fibroblast cells. Monitoring and scoring were carried out by three persons independently. Data indicated that with no drug treatment, 9 out of 10 animals were infected with an average score of 3.5 (>75% infection) and with G₄N at 0.1 mM only 1 out of 10 was infected with an average score of 0.5 (app 12.5% infection). There were no infections detected with G₄N treatment at 10 mM or 100 mM. P₄N was also found to be quite effective. At 10 mM concentration only 2 of 10 mice were weakly infected. An average scale of 0.19 was obtained.
For M₄N treatment, because it was difficult to achieve total homogenous suspension of M₄N with Vaseline, data were less impressive. Out of 8 mice, 4 were fully infected with a score of 34. However, the other 4 showed very little infection at a M₄N concentration of 20 mg/ml. It is expected that better mixing of M₄N in Vaseline at a higher concentration than 20 mg/ml (55.8 mM) should improve the efficacy of the treatment. All these compounds showed little toxicity in animals. For example, topical application for six days of G₄N in guinea pigs at concentrations up to 50 mM showed no evidence of toxicity (34), and subdermal injection of M₄N also showed no toxicity in mice.

EXAMPLE 8

MN Efficacy Against Sexually Transmitted Infections

Trichomonias Vaginalis Susceptibility Testing

A cell suspension from a recently split culture exhibiting >90% cell viability evaluated by good motility is prepared. The suspension is adjusted to 5×10⁷cells/mL (1×10⁴cells/200 μL). Compound and the freshly prepard suspension are added to a 96-well flat-bottom plate and the plate is incubated at 35° C. for 24 hrs. Each well is then examined for motility. The MIC is scored as the minimum dilution where no motility is observed.
Bacteroides fragilis, Prevotella corporis and Mobiluncus curtisii Susceptibility Testing
Standard broth microdilution susceptibility testing is performed as specified in the CLSI M11-A6 standard “Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard-Sixth Edition”, Clinical and Laboratory Standards Institute, Wayne, Pa. The test media is Brucella Broth (BD-BBL, Sparks, Md.) supplemented with 5 μg/ml hemin, 1 μg/ml vitamin-K₁, and laked horse blood (5% v/v; Quad-Five, Ryegate, Mont.). Inoculum density is adjusted to ˜1×10⁷CFU/mL, and the final culture volume in 96-well plates is 5×10⁵CFU. Test compound is serially diluted and added to the plate in duplicate. Plates are incubated anaerobically at 37° C. for 48 hours. The MIC is defined as the lowest concentration of drug resulting in complete inhibition of growth as seen with the unaided eye.

BVDV (HCV Surrogate)

A CPE (virus-induced cytopathogenic effects)-inhibition assay procedure using an XTT endpoint is employed to evaluate compounds for antiviral activity against Madin-Darby bovine kidney (MDBK) cells. MDBK cells are resuspended at 5×10⁴cells per mL in assay media and are added to 96-well flat-bottom plates 24-hours prior to adding the compounds and virus. Compound is serially diluted and is added to the plate in triplicate. Each plate contains cell control wells (cells only), virus control wells (cells plus virus), drug toxicity control wells (cells plus drug only), drug colorimetric control wells (drug only) as well as experimental wells (drug plus cells plus virus). Virus is diluted to a pre-determined titer and added to the plate Cultures are evaluated by XTT-dye reduction on day 6 post-infection.
Gardnerella vaginalis and Candida albicans Susceptibility Testing Methodology
The susceptibility of the above microorganisms to the test microbicide compounds is evaluated by determining the MIC of each compound using micro-broth dilution analysis according to the methods recommended by the CLSI. For each organism, a standardized inoculum is prepared by direct suspension of freshly plated colonies in the appropriate broth indicated for each organism, to an optical density 625 nm (OD₆₂₅) of 0.1 (equivalent to a 0.5 McFarland standard). The suspended inoculum is diluted to a concentration of approximately 1×10⁶colony forming units per milliliter (CFU/mL) and added in triplicate to a 96-well plate containing serially diluted test compound. The plates are incubated for 24 hours at 37° C., and the microbial growth at each concentration of compound is determined by measuring the optical density at 625 nm on a Molecular Devices SpectraMax Plus-384 plate reader. The MIC for each compound is determined as the lowest compound dilution that completely inhibits microbial growth.

HBV Assay

HepG2.2.15 cells are treated with serial_-log dilutions of the test compound in triplicate wells of a 96-well flat-bottom tissue culture plate. As a control, HepG2.2.15 cells are cultured in replicate wells in the absence of test compound. Following six days of incubation at 37° C. 5% CO₂, 100 μL of cell culture supernatant from each well is transferred to individual wells of a round bottom 96-well tissue culture plate. Samples are stored at −20° C. until analyzed.
Following centrifugation, supernatant from each well is transferred to individual wells of a 96-well nucleic acid processing plate. Prior to DNA extraction, 10-fold serial dilutions of cell culture supernatant from triplicate wells of the untreated control are prepared in PBS and diluted material is added to individual wells of the 96-well plate. Protease K is added to each well and the plate is incubated at 37° C. for 90 minutes. Following incubation, DNA is precipitated, the plate is covered and incubated at room temperature for 45 minutes. Precipitated DNA is pelleted by centrifugation, washed with 70% ethanol and suspended in molecular grade water. The extracted DNA is stored at −20° until analyzed.
Quantitative real time PCR is performed using an Applied Biosystems 7900HT Sequence Detection System and the supporting SDS 2.1 software. Extracted DNA for each sample and the control dilutions are subjected to real time Q-PCR using Platinum Quantitative PCR SuperMix-UDG (Invitrogen) and specific DNA oligonucleotide primers and TaqMan probe. DNA extracted from the wells containing dilutions of the no compound control serve as a standard curve for relative quantification.

HSV-2 Cytoprotection Assay

Inhibition of virus-induced cytopathic effects (CPE) and cell viability following herpes simplex virus type 2 (strain MS) replication in Vero cells are measured by XTT dye reduction. Vero cells (10⁴cells per well) are grown in 96-well flat-bottom tissue culture plate. Six half-log dilutions of each compound are evaluated in triplicate against the challenge virus. Cell controls containing medium alone, virus-infected controls containing medium and virus, drug cytotoxicity controls containing medium and each drug concentration, reagent controls containing culture medium only (no cells), and drug colorimetric controls containing drug and medium (no cells) are evaluated in parallel with the test samples. The cultures are incubated at 37° C., 5% CO₂for 5 days, where maximum CPE is observed in the untreated virus control cultures. Inhibition of CPE (increased cell viability) is measured by reduction of the formazan dye XTT following a 4 hour incubation at 37° C. and is measured spectrophotometrically at 450/650 nm.

XTT Staining for Cell Viability

At assay termination, cell viability is determined by reduction of the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide). XTT is metabolized by mitochondrial enzymes of metabolically active cells to a soluble formazan product, allowing the rapid quantitative analysis cell viability and compound cytotoxicity. XTT solution is prepared at a concentration of 1.0 mg/mL in PBS. Phenazine methosulfate (PMS) solution is prepared at 0.15 mg/mL in PBS. XTT/PMS solution is added to each well of the plate and the plate is reincubated for 4 hours at 37° C. The plate is read spectrophotometrically at 450/650 nm with a Molecular Devices Vmax plate reader.
Susceptibility Testing for Chlamydia trachomatis
McCoy cells at a density of 2×10⁶/mL are seeded into 96-well flat-bottom tissue culture plates and incubated at 37° C. 5% CO ₂24 hours until they reach full confluency. Chlamydia trachomatis at a concentration determined to yield 1,000 to 5,000 inclusion forming units (IFU) per well (10,000 to 50,000 IFU per mL in growth media) is added to the plate and the plate is centrifuged at 1,200×g for 1 hour at room temperature to promote infection. Following centrifugation, the supernatants are removed and the compounds to be tested are added in triplicate in the presence of 1 mg/mL cyclohexamide. The plates are incubated for 48 hr at 37° C., 5% CO₂, the supernatants removed and the cells fixed with methanol. Inclusion bodies are detected by fluorescence using a genus specific FITC-labeled monoclonal antibody. The MIC is defined as the lowest concentration of compound that results in lack of inclusion body detection.
Susceptibility Testing of Neisseria gonorrhoeae
The test compound is diluted in two-fold serial increments for a total of 11 dilutions in chocolate agar that has been allowed to cool to 45° to 50° C. The agar is then poured into petri dishes and is allowed to solidify. Colonies are suspended directly until an OD625=0.1 is achieved (approximately 1×10⁸(CFU/mL). Cultures are adjusted to 1×10⁶CFU/mL and plated. The plates are incubated at 37° C./5% CO₂for 16 to 20 hours. The MIC is recorded as the lowest concentration of the antimicrobial agent that completely inhibits growth.

Evaluation of Virus Replication Inhibition

Chronically HIV infected cells are cultured with compound for various periods of time (1 minute through 1 hour) and measure the effect of M₄N on virus replication in the cells. AS a late acting microbicide, it will be important to understand the rate at which M₄N will be able to prevent the replication of virus from infected target cells in the semen. In addition we will also evaluate the time of pretreatment of uninfected target cells to evaluate the effects of preloading of target cells in the vaginal vault with M₄N prior to the introduction of virus into the environment. These mechanistic assays will demonstrate the ability of M₄N to act like a microbicide product and will be performed alone and in combination with surface active agents.

Vaginal pH Transition Assay

GHOST X4/R5 cells that have been adapted to grow at pH 4.5 are treated with antiviral compounds for 2-4 hrs at pH 4.5 and then infected with HIV-1_IIIBin the presence of compound at neutral pH for 1-2 hrs. Following the incubation the cells are washed to remove residual virus and compound and the culture is returned to low pH (4.5) and the cultures are incubated at 37° C./5% CO₂for 6 days at which time virus replication is evaluated by reverse transcriptase assay

Seminal/Vaginal Fluid Simulant Assays

To evaluate the effects of a compound on seminal and vaginal fluids these artificial additives are added to either the attachment inhibition or cell-free assays (described above). To make IL of vaginal stimulant combine 3.51 g of NaCl, 1.40 g of KOH, 0.22 g of Ca(OH)₂, 0.018 g bovine serum albumin, 2 g lactic acid, 1 g acetic acid, 0.16 glycerol, 0.4 g of urea and 5.0 g of glucose. Once combined the pH is adjusted to 4.2 using HCl. To make 100 mL of seminal stimulant combine 5.46 mL of 0.123 M sodium phosphate monobasic, monohydrate with 49.14 mL of 0.123 M sodium phosphate dibasic, anhydrate. Add 813 mg of sodium citrate dehydrate, 90.8 mg potassium chloride, 88.1 mg potassium hydroxide, 272 mg fructose, 102 mg glucose, anhydrous, 62 mg lactic acid, 45 mg urea and 5.04 mg bovine serum albumin. Separately mix 101 mg calcium chloride dehydrate in 15.13 mL of water, 92 mg of magnesium chloride hexahydrate in 15.13 mL of water and 34.4 mg of zinc chloride in 15.13 mL of water. Slowly add these solutions one at a time in that order. Raise the pH with sodium hydroxide to 7.7, sterile filter and freeze until ready for use.

Effects of Excipients

The effects of formulations used as excipients for microbicide candidates can be evaluated in any of our assays. The primary assays used to evaluate this are the PBMC assay and the attachment and fusion assays (described above).

Combination Therapy Assay

The IC₅₀value for both the test compound (compound A) and standard compound (compound B) are determined using the anti-HIV cytoprotection or PBMC assay. Five concentrations of compound A are evaluated alone and in combination with nine concentrations (2×−1/132×) of compound B. Fifty microliters of test or control compound are added to the test plate: The cells are re-suspended at 5×10⁴cells per mL and are added to the drug-containing microtiter plates in a volume of 50 μL. The selected strain of HIV-1 is diluted to a pre-determined titer and is added to the plate in a volume of 50 μL/well.
Six days post-infection endpoint quantification is performed by the XTT based viability assay. Effects of the drug combination are calculated based on the activity of the two compounds when tested alone. The expected additive antiviral protection (based on the dose response curves of the individual agents used alone) is subtracted from the experimentally determined antiviral activity at each combination concentration resulting in a positive value (synergy), a negative value (antagonism), or zero (additivity). Data are analyzed by the most stringent statistical means by assuming the compounds inhibited HIV replication by action at the same site (mutually exclusive). The results of the combination assays are expressed as a synergy volume (μM²%) calculated at the 95% confidence interval using the MacSynergy II template. For these studies, synergy is defined as drug combinations yielding synergy volumes greater than 50 μM²%. Slightly synergistic activity and highly synergistic activity are defined as yielding synergy volumes of 50-100 μM²% and >100 μM²%, respectively. Synergy volumes between −50 and 50 μM²% are considered additive and synergy volumes less than −50 μM²% are considered antagonistic.

Ex Vivo Toxicity Evaluation

Vaginal-ectocervical cells are purchased from MatTek Corporation. According to their product literature, the VEC-FF tissue model is infectable with HIV. The cells are prepared by seeding onto polycarbonate tissue culture treated microporous membrane cell culture inserts.
For compound toxicity test material is applied to the apical surface of the cultures for various time periods. After an allotted exposure time, the material is rinsed from the tissue using PBS and the cultures “loaded” with 1 mg/mL of MTT dye (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide) in culture medium. MTT is taken up by viable cells and reduced by mitochondrial dehydrogenases to form a purple formazan product. Since only viable cells with functioning mitochondria will perform this reaction, the amount of colored formazan produced has been shown to be proportional to the number of viable cells. This purple formazan dye is then extracted overnight using 2.0 mL of isopropyl alcohol and the extracted formazan is quantified by measuring optical density (OD) at 570 nm on a SpectraMax 384 Plus plate reader. The viability of the tissue is normalized as a percent of unexposed control tissues which are loaded with MTT and extracted in an identical manner. The % of viable cells remaining is determined using the equation: % viability=OD (treated tissue)/OD (control tissue). During initial studies to determine the optimal time of exposure and washing procedure, n=3 tissues will be utilized since in general the in vitro tissues are highly reproducible. Once an appropriate time of exposure and wash protocol is established, n=5 tissues will be used so that reproducibility between tissues can be established.

Transmission Assay

CEM-SS cells and HIV-1_IIIBare incubated for 1 hour at 37° C. Following the initial infection, the cells and virus suspension are transferred to a flask containing 5 mL of RPMI-1640 10% complete medium and a fixed concentration of experimental or control compound is added.
The concentrations range from an initial test concentration set at 10× the IC₅₀concentration, which is determined in the CPE inhibition assay, followed by 5 additional concentrations that are each 5-fold higher than the previous concentration. Three days after the initial infection the cultures are observed microscopically for virus induced cytopathic effect and the cell-free supernatant is evaluated for virus content by the RT assay. Twenty (20) % of the existing culture (1 mL of re-suspended cells and supernatant) is added to 4 mL of fresh cells at a cell density of 3×10⁵cells/mL and fresh test compound at the same fixed concentration. Each sub-culturing with new cells represents a new passage. Passaging is performed every 3^rdday for a total of 15 passages. At passage 10, the discarded cells are kept for extraction of DNA and further genetic sequence evaluation. Cells in passages 11 through 15 are cultured in the absence of test compound to observe virus outgrowth or confirm complete sterilization of the culture. Total DNA is extracted from cultured cells at passage 10 and 15. DNA is eluted in a total volume of 200 μL of elution buffer and the concentration of total DNA in each sample is determined by absorbance at 260 nm. PCR is performed on 500 ng of total DNA from each sample using TaqPro Complete containing 2.5 mM MgCl₂and HIV-1 oligonucleotides pNL4-3 2463F (5′-GCTATAGGTACAGTATTAGTAGGACCTACACC-3′; SEQ ID NO: 1) and pNL4-3 5353R (5′-GGTCTGCTAGGTCAGGG TCTACTTG-3′; SEQ ID NO: 2) in a total volume of 25 μL. Five microliters of the PCR products are resolved by electrophoresis on a 0.8% agarose TAE gel and the DNA bands are visualized by ethidium bromide staining.

Other Assays

It is contemplated that sperm motility and sperm inactivation can be evaluated in the presence of the microbicides. Also, compatibility of the microbicides with condoms can be evaluated.
The following references are cited herein:

1. Tirado et al., Virology, 2004, 324(2):577-586.
2. Weeks et al., Sex Transm. Dis., 2004, 31(11):682-690.
3. Huang et al., Antiviral Research, 2003, 58:57-64.
4. Dohm et al., J. Mol. Biol., 2005, 349:731-744.
5. Khanna et al. 2007, Gynecol Oncol, 107:554-562.

Any patents or publications mentioned in this specification are indicative of the level of those skilled in the art to which the invention pertains. Further, these patents and publications are incorporated by reference herein to the same extent as if each individual publication was specifically and individually incorporated by reference.
One skilled in the art would appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Claims

1. A method for inhibiting proliferation of a microbe associated with a sexually transmitted disease or infection, comprising:

contacting the microbe or a microbial cell thereof with an amount of one or more microbicidal compounds effective to inhibit an activity required for proliferation thereof, said compounds having a structural formula

wherein R¹-R⁴are independently H, C₁-C₂alkyl, C(═O)(C₁-C₂alkyl), —C(═O)CH₂NH₂, —C(═O)CH₂N(H)(C₁-C₂alkyl)₂, or —C₁-C₂alkyl-N-piperidinyl, or a pharmacologically effective salt or hydrate thereof.

2. The method of claim 1, wherein R¹-R⁴are independently H, CH₃, —C(═O)CH₂N(H)(CH₃)₂, —C(═O)CH₂N(H)(CH₃)₂.Cl, or —CH₂CH₂—N-piperidinyl.

3. The method of claim 2, wherein the microbicidal compound(s) is one or more of nordihydroguaiaretic acid (NDGA), tetra-O-methyl-nordihydroguaiaretic acid (M₄N), tetra-O—(N,N-dimethyl)glycyl-nordihydroguaiaretic acid (G₄N) or the hydrochloride salt thereof, 3′-O-methyl nordihydroguaiaretic acid (Mal.4), or tetra-O—(N-ethyl)piperidinyl-nordihydroguaiaretic acid (P₄N).

4. The method of claim 3, wherein the microbe is a human immunodeficiency virus (HIV), a herpes simplex virus (HSV), a human papilloma virus (HPV), a hepatitis virus, a Molluscipox virus, Haemophilus ducreyi, Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Sarcoptes scabiei or a crab louse.

5. A method for preventing transmission of one or more sexually transmitted diseases in a subject at-risk for acquiring the same, comprising:

administering one or more of a microbicidal compound to the subject, said compound(s) having a structural formula

6. The method of claim 5, wherein R¹-R⁴are independently H, CH₃, —C(═O)CH₂N(H)(CH₃)₂, —C(═O)CH₂N(H)(CH₃)₂.Cl, or —CH₂CH₂—N-piperidinyl.

7. The method of claim 6, wherein the microbicidal compound(s) is one or more of nordihydroguaiaretic acid (NDGA), tetra-O-methyl-nordihydroguaiaretic acid (M₄N), tetra-O—(N,N-dimethyl)glycyl-nordihydroguaiaretic acid (G₄N) or the hydrochloride salt thereof, 3′-O-methyl nordihydroguaiaretic acid (Mal.4), or tetra-O—(N-ethyl)piperidinyl-nordihydroguaiaretic acid (P₄N).

8. The method of claim 5, wherein the at-risk subject already has a sexually transmitted disease, the method further comprising:

administering one or more other anti-STD drugs thereto.

9. The method of claim 8, wherein the at-risk subject has HIV and the other anti-STD(s) drug comprises one or more anti-retroviral drugs.

10. The method of claim 5, wherein the microbicidal compound(s) is formulated as a topical pharmaceutical composition comprising the compounds and a pharmaceutically acceptable carrier suitable for topical administration.

11. The method of claim 5, wherein the microbicidal compound(s) is administered vaginally, rectally or bucally.

12. The method of claim 5, wherein the microbicidal compound(s) is administered before or after sexual intercourse.

13. The method of claim 5, wherein the sexually transmitted disease is HIV, genital warts, herpes, chancroid, chlamydia, crab lice, gonorrhea, hepatitis, lympogranuloma venereum, molluscum contagiosum, nongonococcal urethritis, pelvic inflammatory disease, scabies, syphilis, or vaginitis.

14. A method for preventing transmission of one or more sexually transmitted diseases in an HIV positive subject at-risk for acquiring the same, comprising:

co-administering one or more of a microbicidal compound to the subject, said compound(s) having a structural formula

wherein R¹-R⁴are independently H, C₁-C₂alkyl, C(═O)(C₁-C₂alkyl), —C(═O)CH₂NH₂, —C(═O)CH₂N(H)(C₁-C₂alkyl)₂, or —C₁-C₂alkyl-N-piperidinyl, or a pharmacologically effective salt or hydrate thereof and one or more anti-retroviral drugs.

15. The method of claim 14, wherein R¹-R⁴are independently H, CH₃, —C(═O)CH₂N(H)(CH₃)₂, —C(═O)CH₂N(H)(CH₃)₂.Cl, or —CH₂CH₂—N-piperidinyl.

16. The method of claim 15, wherein the microbicidal compound(s) is one or more of nordihydroguaiaretic acid (NDGA), tetra-O-methyl-nordihydroguaiaretic acid (M₄N), tetra-O—(N,N-dimethyl)glycyl-nordihydroguaiaretic acid (G₄N) or the hydrochloride salt thereof, 3′-O-methyl nordihydroguaiaretic acid (Mal.4), or tetra-O—(N-ethyl)piperidinyl-nordihydroguaiaretic acid (P₄N).

17. The method of claim 14, wherein the microbicidal compound(s) is formulated as a topical pharmaceutical composition comprising the compounds and a pharmaceutically acceptable carrier suitable for topical administration.

18. The method of claim 14, wherein the microbicidal compound(s) is administered vaginally, rectally or bucally.

19. The method of claim 14, wherein the microbicidal compound(s) is administered before or after sexual intercourse.

20. The method of claim 14, wherein the sexually transmitted disease is HIV, genital warts, herpes, chancroid, chlamydia, crab lice, gonorrhea, hepatitis, lympogranuloma venereum, molluscum contagiosum, nongonococcal urethritis, pelvic inflammatory disease, scabies, syphilis, or vaginitis.