WO1996008270A2

WO1996008270A2 - Method for inhibiting sexually transmitted diseases using magaining antimicrobials or squalamine compounds

Info

Publication number: WO1996008270A2
Application number: PCT/US1995/011675
Authority: WO
Inventors: Leonard Jacob; Michael Zasloff; Taffy Williams; Gurrinder Bedi
Original assignee: Magainin Pharmaceuticals Inc.
Priority date: 1994-09-13
Filing date: 1995-09-13
Publication date: 1996-03-21
Also published as: WO1996008270A3; AU3512595A

Abstract

Transmission of sexually transmitted disease is inhibited in humans by a method that comprises administering to a human one or more magainin antimicrobials or one or more squalamine compounds in an amount sufficient to inhibit transmission of sexually transmitted disease.

Description

METHOD FOR INHIBITING SEXUALLY TRANSMITTED DISEASES

USING MAGAININ ANTIMICROBIALS OR SQUALAMINE COMPOUNDS

BACKGROUND OF THE INVENTION

This invention relates to a method for inhibiting sexually transmitted diseases. More particularly, sexually transmitted diseases can be inhibited in humans by administering to a human a magainin antimicrobial, which is a polypeptide, or a

squalamine compound, which is an aminosterol.

A sexually transmitted disease (STD) is one that is transmitted through sexual contact. Sexually transmitted diseases (STDs) affect 12 million men and women in the United States each year. The highest risk of contracting STDs, comes from having sexual intercourse, vaginal, anal or oral, with an infected person.

STDs include a tremendous variety of diseases, including AIDS, Chlamydia, genital herpes, syphilis, genital warts, and Gonorrhea. These diseases exhibit a varied range of results: 1) Tubal pregnancies, sometimes fatal to the mother and always fatal to the unborn child; 2) death or severe damage to a baby born to an infected woman; 3) sterility; 4) cancer of the cervix; 5) damage to other parts of the body, including the heart, kidneys and brain; or 6) death to infected individuals.

The most commonly known organisms that cause sexually transmitted diseases are Chlamydia, Gonorrhoea, Candida, Herpes Simplex Virus, and HIV. The transmission of HIV is

substantially increased in individuals with other STDs.

Anyone can become infected through sexual intercourse with an infected person, regardless of socioeconomic status, race or geographic location, and many of those infected are teenagers or young adults. Sometimes, early in the infection, there may be no symptoms, or symptoms may be easily confused with other illnesses a problem particularly acute with teenagers and young adults.

Until now, other than abstinence, latex condom use has been the only method available to reduce the risk of STDs. Studies provide evidence that consistent condom use reduces the risk of HIV and other STDs, but condom use requires a cooperative male partner. Although the other partner may use spermicides,

researchers at the Centers for Disease Control and Prevention in Atlanta have reported that the effectiveness of spermicides against STDs, either alone or in combination with condoms, nas not been proven. Antibiotics are not a viable solution because many strains of organisms have developed resistance to certain

antibiotics. Thus, one way to prevent or at least retard the spread of STDs m epidemic proportions is development of a safe, effective, female-controlled chemical barrier that will block or at least inhibit transmission.

SUMMARY OF THE INVENTION

This invention aids in satisfying this need in the art by providing a method of inhibiting sexually transmitted diseases in humans. In one embodiment of the invention, the method comprises administering to a human one or more magainin antimicrobials in an amount sufficient to inhibit transmission of sexually transmitted disease. In another embodiment of the invention, the method comprises administering to a human one or more squalamine compounds in an amount sufficient to inhibit transmission of sexually transmitted disease. This invention makes it possible to prevent or at least retard the spread of sexually transmitted diseases in epidemic proportions.

DESCRIPTION OF THE PREFERRED EMBODIMENT

In one embodiment of this invention, the spread of sexually transmitted diseases is inhibited by administering to a human one or more magainin antimicrobials or squalamine compounds in an amount sufficient to inhibit transmission of sexually transmitted diseases. Magainin antimicrobials and squalamine compounds provide a safe, effective, female-controlled chemical barrier that can inhibit transmission of STDs. Inhibit means at least to reduce the risk of transmission of STDs in the subject treated. It is possible that the amount of inhibition will result in complete prevention of STDs in the subjects treated up to 6 hours before or up to 6 hours after sexual contact, and preferably 1 hour or less before or 1 hour or less after sexual contact.

The host to which the magainin antimicrobials or squalamine compounds are administered is a male or female human subject in need of treatment for the inhibition of a sexually transmitted disease. Because of the importance of female-controlled barriers to transmission, the subject will generally be a human female patient in need of such treatment.

This invention is useful for inhibiting the spread of

sexually transmitted diseases in general. The invention is particularly effective for inhibiting the spread of diseases caused by Chlamydia, Syphilis, Gonorrhoea, Candida Albacans, Herpes Simplex Virus (HSV), and Human Immunodeficiency Virus

(HIV). Preferably, human subjects are treated according to the invention to inhibit the spread of Chlamydia trachomatis, which is the most common sexually transmitted bacterial pathogen in the United States, as well as Herpes Simplex- 2, Gonorrhea and HIV which are also significant problems.

The magainin antimicrobials or squalamine compounds employed in this invention are administered to the human subject in an effective amount to inhibit transmission of sexually transmitted disease. Effective amounts of these compounds can be administered by any one of several methods, for example, orally as in capsules or tablets, parenterally in the form of sterile solutions or suspensions, and in some cases intravenously in the form of sterile solutions. In the preferred embodiment of this invention, these compounds are applied to the desired site by topical

application or by subcutaneous injection. In a particularly preferred embodiment of the invention, these compounds are

administered intravaginally to a female subject or applied to a condom. The magainin antimicrobials and squalamine compounds can be applied or administered to a location proximate the area of sexual contact in order to obtain the beneficial effects of the invention.

The magainin antimicrobials and squalamine compounds, which are effective themselves, can be formulated and administered in the form of their pharmaceutically acceptable addition salts for purposes of stability, convenience, increased solubility, and the like. Preferred pharmaceutically acceptable addition salts include salts of mineral acids, fcr example, hydrochloric acid, sulfuric acid, nitric acid, and the like; salts of monobasic carboxylic acids, for example, acetic acid, propionic acid, and the like; salts of dibasic carboxylic acids, for example, maleic acid, fumaric acid, and the like; and salts of tribasic carboxylic acids, such as carboxysuccinic acid, citric acid, and the like.

The magainin antimicrobials and squalamine compounds can be formulated into a form suitable for topical application by

incorporating these ingredients in a suitable vehicle. The vehicle can be a solid, semi-solid, or liquid vehicle that is pharmaceutically acceptable or physiologically acceptable, and , which enables the magainin antimicrobials and squalamine compounds to be conveyed to the locus of sexual contact at an appropriate dilution. The nature of the vehicle will depend upon the method chosen for topical administration. The vehicle can itself be inert or it can impart physiological or pharmaceutical benefits to the composition containing the other active ingredients.

The vehicle for topical application is a substance that acts as a diluent, dispersant, or solvent for the magainin

antimicrobials, squalamine compounds, and other reagents in the composition so that the composition can be applied to and

distributed substantially evenly over the site of sexual contact and at an appropriate concentration that inhibits the spread of sexually transmitted disease. The vehicle is preferably one that aids in retention of the active ingredients upon the site of sexual contact. Solutions or suspensions can also include the following components: a sterile diluent, such as water for injection, saline solution, oils, polyethylene glycols, glycerine, propylene glycol, or synthetic solvents, antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulphite; buffers, such as acetates, citrates, or

phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose.

The vehicle for topical application can be based on water or at least one pharmaceutically acceptable vehicle other than water It will be understood that non-aqueous pharmaceutically acceptable vehicles can also be combined with water to provide a composition suitable for topical application. Vehicles that can be employed in practicing this invention include solids or liquids, such as emollients, solvents, humectins, thickeners, and powders.

The magainin antimicrobials and squalamine compounds can be utilized in the methods of the present invention either alone or in combination with other known treatment agents. For example, these ingredients can be combined with other amino acids,

vitamins, male or female sex hormones, antibacterial agents, antiviral agents, anti-inflammatory agents, refrigerants, surfactants, perfumes, antioxidants, ultraviolet absorbers, and propeilants.

The magainin antimicrobials and squalamine compounds employed in this invention can be either directly applied or sprayed onto the area of sexual contact or applied by percutaneous injection. The dosage of these compounds employed in the method of this invention varies depending upon age, individual differences, symptoms, etc., but in the case of an adult, it is generally in a range of about 0.07 to about 7000 μg, and preferably in the range of about 1 to about 300 μg of the active ingredient per kg of body weight per day for topical administration.

Solutions that can be applied as coatings to condoms, sponges and other devices, suppositories, creams or gels, generally should contain about .02% to 5% of the active ingredient, and preferably in the range of 0.2% - 3.0%. The preferred vehicles for

administration allow the direct application of the active

ingredient to contact areas.

Effective quantities of the magainin antimicrobials and squalamine compounds can be administered orally, for example, with an inert diluent or with an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For purposes of oral therapeutic administration, they can be incorporated with an excipient and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums, and the like. These preparations should contain at least about 1% of active compound, but the amount can be varied depending upon the

particular form and can conveniently be between 1% to about 50% of the weight of the unit. The amount of active ingredient in such a composition is such that a suitable dosage will be obtained.

Preferred compositions and preparations according to the invention are prepared so that an oral dosage unit form contains between about 25 to about 400 mg of the magainin antimicrobial or the squalamine compound. For the purpose of parenteral therapeutic administration, the magainin antimicrobials and squalamine compounds can be

incorporated into a solution or suspension. These preparations should contain at least about 0.1% of active compound, but can be varied between about 0.1% and about 10% of the weight thereof.

The amount of magainin antimicrobials and squalamine compounds in such compositions is such that a suitable dosage will be obtained. Preferred compositions and preparations contain a parenteral dosage unit comprising between about 25 to about 400 mg of active ingredients.

The magainin antimicrobials and squalamine compounds can be administered to the human subject before, during, or after sexual contact. It is preferred that these active substances be

administered before and after sexual contact and in multiple dosages as needed. The active ingredients employed in the methods of this invention will now be described in greater detail.

As used herein, the term "magainin antimicrobials" means a polypeptide having a molecular weight of about 3500 or less, which is water soluble at a concentration of greater than 5 mg per ml at neutral pH or in an aqueous solution of physiologic ionic

strength, non-cytolytic to animal cells including red blood cells, amphiphilic, and which has a broad antibiotic spectrum at

physiologic ionic strength and pH. Magainin antimicrobials are reviewed in Bevins and Zasloff, 1990. The antimicrobial spectrum of magainin antimicrobials is described in U.S. Patent 4,810,777. More particularly, the magainins exhibit antimicrobial activity against at least several of the following organisms: Escheri cia coli, Acinetobacter caloaceticus, Shigella sonnei, Enterobacter somnes, Eschericia coli, Streptococcus pyogenes, Shigella

flexneri, Citrobacter freundii, Enterobacter aerogenes,

Klebisiella pneumonia, Staphyloccus epidermidis, Streptococcus faecalis, Pseudomonas aeruginosa, Salmonella typhimurium,

Staphyloccoccus aureus, Candida albicans, Proteus volgaris,

Sterratia marcescens.

The term magainin antimicrobials or magainins specifically includes peptides and peptidomimetics . Peptidomimetics are small organic molecules designed to mimic the function of the peptides.

The primary sequences of illustrative magainin

antimicrobials are:

Magainin I: (NH₂) GIGKFLHSAGKFGKAFVGEIMKS (OH)

Magainin II : (NH₂) GIGKFLHSAKKFGKAFVGEIMNS (OH)

Magainin IIIL: (NH₂) GIGKFLHSAKKFGKAFVGEIMN (OH).

It is noted, however, that these amino acid sequences show only essential portions required for antibiotic activity. In other words, the polypeptides are not limited to the sequences shown but must have, at least in part or in whole, the amino acid sequence shown. All analogs and derivatives that are equivalent in

structure and function to these magainins are encompassed within the scoυe of this invention. Some derivatives of Magainin I I are

Magainin II (a) : (NH₂) IGKFLHSAKKFGKAFVGEIMNS (OH)

Magainin II (b) : (NH₂) GKFLHSAKKFGKAFVGEIMNS (OH)

Magainin II (c) : (NH₂) KFLHSAKKFGKAFVGEIMNS (OH)

Magainin II (d) : (NH₂) FLHSAKKFGKAFVGEIMNS (OH)

Magainin II (e) : (NH₂) LHSAKKFGKAFVGEIMNS (OH)

Magainin II (f) : (NH₂) HSAKKFGKAFVGEIMNS (OH).

Other modified analogs and derivatives include those

disclosed in the following commonly assigned applications:

U.S.S.N. 07/908,455 to Maloy et al. for "Novel Peptide

Compositions and Uses Therefore"; U.S.S.N. 08/133,740 to Maloy for "Compositions of and Treatment with Biologically Active Peptides Having D-Amino Acid Residues"; U.S.S.N. 07/713,716 to Maloy and Kari for "Composition and Treatment with Biologically Active

Peptides Having C-terminal Substitutions"; U.S.S.N. 08/184,462 to Berkowitz and Jacob for "Biologically Active Peptides Having N- terminal Substitutions"; U.S.S.N. 08/199,553 to Kari for "Amino Acids and Peptides Having Modified C-terminals and N-terminals"; U.S.S.N. 07/944,370 to Maloy et al . for "Prophylaxis and Treatment of Adverse Oral Conditions with Biologically Active Peptides"; U.S.S.N. 08/226,108 to Baker et al . for "Treatment of

Gynecological Malignancies with Biologically Active Peptides"; all of which are specifically incorporated herein by reference.

In aqueous solution, a magainin antimicrobial exists in a random coiled conformation. Because of its overall cationic charge, the peptide binds electrostatically to membranes displaying accessible anionic phospholipid head groups. After binding to the membrane, magainin antimicrobials undergo a secondary structural transition into an amphipathic alpha helix (Guy and Ragunathan, 1988; Marion et al., 1988). The magainin antimicrobials appear to adopt an alpha-helical structure upon addition of 2.3 mol % of the organic solvent trifluoroethanol (Marion et al., 1988), and upon interaction with

phosphatidylserine vesicles (Matsuzaki et al., 1989) and other micelles (Williams et al., 1988a, b).

Magainins and PGLa (a frog antimicrobial Peptide, starting with Glycine, ending with Leucine, and in amide form) rapidly depolarize the bacterial cell membrane (Juretic et al., 1989;

Westerhoff et al., 1989a) by mechanisms consistent with ion-sized channels, which they can produce in synthetic bilayers (Cruciani et al., 1988). In an alpha-helical configuration, one face of the helix is populated by the side chains of hydrophobic amino acids and the other by hydrophilic residues including lysines. On interaction with the membrane, the peptide lies parallel to the plane of the membrane.

As yet, no evidence exists that magainin antimicrobials form classical transmembrane channels as suggested for certain

ionospheric peptides, such as gramicidin or alamethicin.

Regardless of the precise design of this higher order structure, these peptides disrupt membrane permeability. It may be that magainin antimicrobials and their analogs simply form a continuous spectrum of "holes" or imperfections of sizes ranging from those of ions to small molecules rather than a discrete series of precisely organized ion-sized pores (Westerhoff et al., 1989a, b) . Evidence for this mechanism include patch clamp analysis studies, which demonstrate for formation of channels in synthetic

phospholipid bilayers (Cruciani et al., 1988), and disruption of membrane-linked free-energy transduction (Westerhoff et al., 1989).

An important feature of magainin antimicrobials over other antimicrobial agents is, despite the magainins' activity on bacterial membranes, at antimicrobial concentrations they do not lyse erythrocytes, platelets and other formed circulating cells of vertebrates (Zasloff, 1987; Chen et al., 1988). This selectivity distinguishes the magainins from toxins, such as melittin, a bee venom constituent peptide.

Moreover , magainin efficacy has been demonstrated against antibiotic-resistant strains including, but not limited to, strains resistant to gentmicin, ciprofloxacin, cefotaxime, penicillin, oxacillin, methacillin, imipenem, piperacillin, carbenicillin, tobramycin, erythromycin, and mupirocin (Jacob, et al . , 1990) as set forth in Chart 1. This indicates a lack of cross-resistance between these drugs and magainin antimicrobials.

Since the discovery of Magainin-II, over 1300 analogs of other host defense compounds have been synthesized. The numerous analogs demonstrate a variety of in vi tro antibacterial

activities. Some molecules are selective for gram-positive or gram-negative bacteria while others are active against both gram-positive and gram-negative bacteria. Also a number of molecules are active against aerobes and anaerobes. Virtually all of the synthetic analogs have been designed based on in vi tro antibacterial activity structure-activity-relationship (SAR) studies.

Improving the amphipathic nature and stability of the

magainin alpha-helical structure is one means to enhance

antibacterial potency and spectrum. (See e.g., Chen et al ., 1988; Cuervo et al . , 1988) Based on this concept, analogs were made of Magainins I and II and PGLa. These peptides were tested for their activity against ATCC strains of Staphylococcus aureus,

Pseudomonas aeruginosa , and Escherichia coli , and demonstrated greatly improved potency and antibacterial spectrum. A few of the more potent analogs are shown in Table 1.

The in vi tro efficacy of magainin compounds in Table 1 as well as other tables and charts is determined by Minimum

Inhibitory Concentration (MIC) values expressed in μg/ml.

Bacteria which have been tested are clinical isolates along with the National Committee for Clinical Laboratory Standards

recommended reference strains of Staphylococcus aureus ATCC 29213 and ATCC 25923, Escheri chia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853. Efficacy results are traditionally given as an MIC expressed in μg/ml; however, if MIC values were expressed on a micromolar (μM) basis the variation in molecular weight (MW) among the compounds would not be a factor and MIC values for the compounds could be directly compared. For example, against P. aeruginosa ATCC 27853, the MICs in μg/ml for gentamicin (MW 464) and a magainin compound MSI-78 (MW 3078), characterized by the amino acid sequence: GIGKFLKKAKKFGKAFVKILKK-NH2-22, are 0.5 μg/ml and 4 μg/ml, respectively; however, the MICs on a micromolar basis are 1.1 μM and 1.3 μM.

As shown in Table 2, potent analogs were designed based on a 7 amino acid repeat of 2 or 3 amino acids separated by 2 or 3 hydrophobic or neutral hydrophilic ammo acids (i.e., BHHNBHH) where B is a basic ammo acid, H is a hydrophobic ammo acid and N is a neutral or basic hydrophilic ammo acid). The simplest analogs were similar to PGLa in which 3 seven amino acid units were present. These analogs are as potent as the magainm and PGLa analogs shown m Table 1, but allow a simpler and more cost- effective approach for large scale production.

Magainin antimicrobial peptide analogs with excellent antibacterial activity and spectrum, which also resist

proteolysis, have been designed through SAR. Substitutions were made in magainm analogs to remove sites of attack by proteolytic enzymes. For example, since the polymeric analog MIS-103 (Table 2) can be cleaved enzymatically, an analog was made where lysine 12 in MSI-103 was replaced by ornithine to enhance

resistance to lysis by serine proteases.

Other magainin antimicrobial peptide analogs were designed based on the premise that the magainin activity is only dependent on the ability to form an amphipathic alpha-helix, and therefore it does not matter if the helix is right-handed, formed with all L-amino acids, or left-handed, formed with all D-amino acids, sue: as those disclosed in the following commonly assigned application U.S.S.N. 07/908,455 to Maloy et al. for "Novel Peptide

Compositions and Uses Therefore", which is specifically

incorporated by reference. An MSI-103 analog was prepared using all D-amino acids (MSI-30) and was shown to have antibacterial activity (Table 3) similar to the MSI-103 of Table 2.

A class of peptidomimetics (a molecule that mimics the biological activities of the parent peptide) was developed with the following structure:

H₂N - C(= NH) NH-CH(R) -CONH (CH₂)_nCH₃ where R is side chain of an amino acid and n is the value varying from 6 to 17. In the case where R is -CH₂C₆H₅ and n = 11, the molecule exhibited good antibacterial properties.

A different class of closely related structures of the type

H₂N - C(= NH) NH-CH(R) -CONH (CH₂)_nNHCO CH(R)NH - C(=NH)NH₂ were synthesized and found to be equal or better than the previous structures.

On further investigation, a peptidomimetic of the structure

NH₂C(-NH) NH-CH(R) CO-N-{(CH₂)_n CH₃}₂ was designed which also exhibited good antimicrobial activities. As shown in Table 4, preliminary antibacterial activities against S. aureus, E. coli, P. aeruginosa and C. albicans varied from 0.25 μg/ml to 256 μg/ml.

In sum, magainin compounds exhibit potent microbial activity against many significant microbes as set forth in Table 5.

The potential for in vi vo on-therapy resistance development to magainin antimicrobials has been addressed in an in vi tro study in which bacteria were chronically exposed to a sub-inhibitory dose of compound MSI-78 for seven passages of growth (Table 6). Any significant change in MIC is used as a measure of resistance. For example, Table 6 contains the MIC data for the initial MIC and the MIC at the end of seven passages of the compound MSI-78 for two clinical strains of Staphylococcus aureus (one methicillin- sensitive and one methicillin-resistant) and two clinical strains of Pseudomonas aeruginosa (one gentamicin-sensitive and one gentamicm-resistant). There was no significant change in MIC indicating no induction of resistance or selection of resistant populations in the bacteria tested under the conditions of the assay.

Several magainin antimicrobials have been assayed against bacteria in this manner and have not demonstrated any potential to induce or select resistant organisms. These data suggest the development of resistance in patient populations will not occur.

The manufacture of magainin antimicrobials is well known in the art. For example, see U.S. Patent No. 4,810,777, which is incorporated by reference herein. The synthesis of magainin antimicrobials can be carried out by solid phase methodology

(Merrifield, 1963) either on an ABI-431A peptide synthesizer using t-Boc (tert-butyloxycarbonyl) chemistry or on the Milligen 9050 PepSynthesizer using Fmoc (9-fluorenylmethyloxycarbonyl)

chemistry. When t-Boc chemistry is used for the synthesis of the peptides appropriate benzyl-based side chain protection can be used and the appropriate resins for either C-terminal acids or amides. The final deblocking step can be carried out with liquid hydrogen fluoride (HF). With Fmoc chemistry, t-butyl-based side chain protection can be used with appropriate resins for

C-terminal acids or amide production. The final deblocking step can be carried out with a mixture of TFA (trifluoroacetic acid) and appropriate scavengers .

The cleaved free peptides can be desalted on reverse phase high performance liquid chromatography (RP-KPLC) and purified to >95% purity by preparative RP-HPLC using gradient elution. As needed, the peptides can also be purified by weak cation exchange HPLC, using a salt (NaCl) gradient. The fractions containing >95% of the desired peptide peak on analytical HPLC and capillary zone electrophoresis (CZE) can be pooled, converted to the acetate salt and lyophilized.

One or more magainin antimicrobials can be used to inhibit transmission of STDs. One or more means a single magainin

administered alone or a mixture comprising two or more magainins. Typically, an effective mixture contains 1 to 10 magainin

antimicrobials.

Magainin antimicrobials can be used in conjunction with other agents. For example, another agent that can be employed in conjunction with a magainin antimicrobial is a spermicidal agent, such as Nonoxynol-9. Spermicidal means an agent destructive to spermatozoa. In an alternative embodiment, the magainin

antimicrobials may also exhibit spermicidal activity.

Squalamine is another novel steroidal broad spectrum anti- infective agent, which has been the subject of analog synthesis and biological investigations. Squalamine, a novel aminosterol, is a host defense molecule isolated from the dog fish shark, Squalus acanthias . Squalamine has also been reported to be spermicidal at a dose of 0.123 mg/ml. (R. Blye, Personal Communication). Squalamine antiviral tests yielded data snowing a 70% plaque reduction in an HSV-1 absorption assay (Angelo Pinto, Personal Communication).

Squalamine inhibits the growth of gram-positive (S. aureus, E. faecalis), gram-negative (E. coli, P. aeruginosa), and fungal (C. albicans, C. tropicalis, C. parapsilosis, and C. kefyr) organisms with MIC values of between 0.5 and 16 μg/mL (Table 7, entry 1). Substantial progress has been made in understanding tre structural requirements for anti-infective activity against different types of organisms. Removal of the sulfate group, as in entry 2 (Table 7), decreases activity against four screening microbes. The structurally simple entries 3 and 4 contain

cholestane connected to butanediamine; both isomers at C3 retain reduced S. aureus activity, with the unnatural isomer (α) being more potent. Entries 5 and 6 contain the spermidme of

squalamine, but lack the 7-hydroxyl and C24 sulfate. Both isomers exhibited good potency in inhibiting the growth of S. aureus with the α-isomer again being more efficient The corresponding spermme analogs 7 and 8 were similar against S. aureus, however, they were also antifungal. This result is interesting in that only fungal organisms utilize spermme suggesting a possible polyamme mechanism in these organisms.

The importance of an acidic group at C24 and a hydroxyl group at C7 were then investigated. When the isopropyl group of 5 and 6 was replaced by a carboxylic acid group in entries 9 and 10, a dramatic decrease in potency against S. aureus was observed.

However, there was an improvement against the gram-negative organisms E. coli and F. aeruginosa . When the acidic group was combined with a 7-hydroxyl group as in 11 and 12, activity against all four screening organisms was obtained. The corresponding esters 13 and 14 were similarly efficacious suggesting that the acidic group is not contributing to activity. Although an agent as potent as squalamine has not been identified, compound 13 has a similar broad spectrum of activity. Perhaps more interestingly, selective analogs, such as entry 5, inhibit S . aureus ,

Heli cobacter pylori and Clos tridium difficile without affecting E. coli .

As used herein, the term "squalamine compound" means a compound of the formula:

Squalamine, as used herein, also includes analogs of

squalamine, such as those disclosed in the following commonly assigned application: U.S.S.N. 08/023,343 to Moriarety et al. entitled "Chemical Synthesis of Squalamine"; U.S.S.N. 08/029,018 to Frye and Zasloff entitled "Antibiotic Steroids"; PCT/US94/02397 to Frye and Zasloff entitled "Steroid Derivatives, Pharmaceutical Compositions Containing Them, and Their Use as Antibiotics or

Disinfectants" and PCT/US94/_______(to be assigned) filed

September 13, 1994 to Frye and Zasloff entitled "Steroid

Derivatives, Pharmaceutical Compositions Containing Them and Their Pharmaceutical Uses"; all of which are specifically incorporated by reference. Examples of squalamine analogs and their MIC values are shown in Chart 2 below.

As with magainin antimicrobials, mixtures of squalamine compounds can be employed in this invention. It will also be understood that magainin antimicrobials and squalamine compounds can be used in admixture.

Squalamine can be isolated from shark liver (sqrualus acanthias) by a previous published procedure (Moore, et al., 1993). A typical extraction of 40 kg of shark liver affords 300 mg of analytically pure squalamine after a sequence of C18 purification, ion exchange, salting-out, Folch extraction, and HPLC chromatography (C18). It has also been obtained by total synthesis (20 steps) in approximately 1% overall yield by Prof. Leah Frye at Rensselaer Polytechnic Institute.

This invention will now be described in greater detail in the following Examples.

Example 1

Studies on the In Vi tro Activity of Magainm

Antimicrobials Against C. trachomatis

The activity of magainin antimicrobials against

C. trachoma tis were carried out. Ten compounds were supplied for susceptibility testing of the type strain of C. trachoma tis serovar E. Serial twofold dilutions of each compound were

prepared in water and added to an equal volume containing 10 inclusion-forming units of C. trachomatis elementary bodies (EBs) in Bartels medium (Baxter Diagnostic, New Jersey). Final

antibiotic concentrations ranged from 0.5 μg/ml to 256 μg/ml.

Tubes were then incubated at 35° C for 60 minutes. A 0.1 ml inoculum of the suspension was applied to prepared McCoy cell cultures (10⁴ McCoy cells per tube) and the cultures were

centrifuged at 600 Xg for 1 hr at 35º C. The supernatant was then removed and cells were overlaid with 1 ml of antibiotic-free

Bartels medium containing 10% fetal bovine serum, 1% glucose, and

1 μg/ml of cycloheximide. Following 48 hrs of incubation at 35

C in an atmosphere of 3.5% CO₂-96.5% air, cell cultures were washed, fixed with methanol, and stained for inclusions using fluorescem-labelled monoclonal antibody (commer

McCoy cells were examined by microscopy for the presence of inclusions of C. trachomatis . Inclusions on the entire monolayer were counted. The anti-chlamydial activity of the magainin antimicrobials was determined as the percentage of inhibition

(% inhibition = 1.0 - number of inclusions in the presence of the agent/number of inclusions in control x 100%).

Referring to Table 8, three of the compounds tested (MSI -148, MSI-1262 and MSI-1296) gave 100% inhibition at 1 and 256 μg/ml. Two compounds (MSI-43 and MSI-63) gave 100% inhibition at

256 μg/ml, but reduced inhibition at 1 μg/ml. Two compounds

(MSI-1251 and MSI-1256D) gave slight to moderate inhibition at 256 μg/ml and two compounds (MSI-344 and MSI-1312) had no effect even at 256 μg/ml. Magainin antimicrobials themselves had no

cytopathic or toxic effects on McCoy cells in the uninfected controls, as determined by microscopic examination and trypan blue exclusion.

The amino acid sequence or formula for these magainins is provided below.

Example 2

Effects of Magainin Antimicrobials on

Infectivity of Cell-Free HIV-1 (IIIB)

The ability of selected magainin antimicrobials to inhibit

(reduce) the infectivity of cell-free HIV-1 (IIIB) was studied.

HIV-l was treated with various concentrations of magainin

antimicrobials for 1hr, then SupT1 cells, which will produce syncytia if live HIV-1 is present, were added. Several magainin antimicrobials reduced the infectivity of HIV-1 at concentrations that were nontoxic to the cells. The results are shown in

Table 9.

The amino acid sequences for these magainins, that have not already been provided herein, are shown below.

MSI 420 NH₂-KKLLKKLKKLLKKL-OH

MSI 591 OCT-LKKLLKKLKKL-NH2-11 Example 3

Studies on The Activity of 10 Selected

Magainin antimicrobials Against GC

Killing by magainin antimicrobials was measured at 37°C in

96-well plates in phosphate buffered saline or in gonococcal broth. A constant number of GC were incubated in 2-fold dilutions of antimicrobial, from 250 μg/ml to 1 μg/ml. All of the

antimicrobials showed increased killing over time, when viability was measured at 15, 30, and 60 min. However, most of the killing was complete by 15 min (see Table 10). For each of the

antimicrobials, there was a threshold concentration under which no bacteria were killed, regardless of incubation time (data not shown). This supports previous observations with magainin

antimicrobials against other bacteria, and suggests a

cooperativity, i.e., the antimicrobials seem to work in concert with each other, perhaps forming membrane homopolymeric pores or aggregates.

Example 4

Magainin Antimicrobials Show Antiviral

Activity Against HSV-1 and HSV-2

The ability of a variety of magainin antimicrobials to inhibit plaque formation in Vero cells infected with HSV-1 or HSV-2 was tested. To date, the magainin antimicrobials tested have shown little or no toxicity at concentrations as high as 200 μg/ml as assessed visually and by neutral red dye inclusion 48 and 72 hours after addition of peptide. Following toxicity screenings, various nontoxic concentrations of magainin

antimicrobials and virus were added together to monolayers of Vero cells and antiviral activity was determined by a plaque reduction assay (Pinto et al . , 1988).

The results clearly show that several Magainin antimicrobials have significant antiviral activity against both viruses. MSI-420 inhibited the infectivity of both HSV-1 and HSV-2 over a broad range of concentrations. Similarly, MSI-843 and MSI-1331 also showed excellent antiviral activity. MSI-843 inhibited plaque formation by 75-90% in Vero cells infected with virus and MSI-1331 completely inhibited HSV-1 and 2 at concentrations of 200 μg/ml.

A variety of other compounds have also been screened against HSV-1 in a plaque reduction assay. The results (Tables 11 and 12) clearly show that MSI-94, MSI-121, MSI-144, MSI-591 and MSI-1256C all have dose-dependent, good-to-excellent anti-HSV-1 activity. MSI-98, which is a helix disrupted peptide, and serves as a negative control, has failed to show significant antiviral

activity. These preliminary data clearly show that magainin antimicrobials have activity against both HSV-1 and -2. Furthermore, these results support our main hypothesis that magainin antimicrobials have potential as antiviral agents in the prevention of genital HSV infections.

The amino acid sequences for these magainins are provided below.

* * *

In summary, as set forth below in Chart 3, this invention provides methods for inhibiting sexually transmitted disease in female and male human subjects. The methods are particularly useful for providing a female-controlled chemical barrier to the spread of STD. The pharmaceutically active ingredients in compositions employed in the methods of the invention comprise magainin antimicrobials and squalamine compounds. These

ingredients can be administered proximate the locus of sexual contact where the pathogen responsible for the STD contacts the subject susceptible to infection. These active ingredients can inhibit STD by killing the pathogen responsible for the STD or by impeding the infection mechanism or replication cycle of the pathogen. The methods of the invention are especially

advantageous because they are easily and quickly implemented without special training.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art that various changes in form and detail can be made without departing from the true scope of the invention.

All publications mentioned hereinabove are hereby

incorporated in air entirety by reference.

LITERATURE

Bevins C.L. and Zasloff M., "Peptides From Frog Skin", Ann. Rev Biochem., 59:395-414 (1990).

Chen H.C., Brown J.H., Morell J.L., and Huang C.M., "Synthetic Magainm Analogs With Improved Antimicrobial Activity", FEBs.

Lett., 236:462-466 (1988).

Cruciani R.A., Stanley E.F., Zasloff M., Lewis D.L., and Barhem J.L., "Magainin 2, A Natural Antibiotic From Frog Skin, Forms Anicn-Selective Channels in Lipid Bilayer Membranes", Biophys. J., 53:9a (1988).

Cuervo J.H., Rodriguez B., and Houghten R.A., "The Magamms:

Sequence Factors Relevant to Increased Antimicrobial Activity and Decreased Hemolytic Activity", Peptide Res., 1:81-86 (1988).

Guy H.R. and Raghunathan G., "Structural Models for Membrane

Insertion and Channel Formation by Antiparallel Alpha Helical Membrane Peptides", Pullman A. (ed.), Transport Through Membranes, Carriers, Channels and Pumps, pp. 369-379 (1988).

Jacob L., MacDonald D., and Maloy L., "Magainm Peptides are

Active Against Organisms Resistant to Current Therapies", ICAAC 30th Meeting, Oct. 21-24, Atlanta, GA. (1990).

Juretic D., Chen H.C., Brown J.H., Morell J.L., Hendler R.W. and Westerhoff H.V., "Magainm 2 Amide and Analogs: Antimicrobial Activity, Membrane Depolarization and Susceptibility to

Proteolysis", FEBs. Lett., 249:219-223 (1989).

Marion D., Zasloff M., and Bax A., "A Two-Dimensional NMR Study of the Antimicrobial Peptide Magainin 2", FEBs. Lett., 227:21-26 (1988).

Matsuzaki K., Harada M., Handa T., Funakoshi S., Fujn N., Yajima H., and Miyajima K., "Magainin 1-Induced Leakage of Entrapped Calcium Out of Negatively-Charged Lipid Vesicles", Biochem.

Biophys. Acta., 981:130-134 (1989).

Merrifield R.B., "Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide", J. Am. Chem. Soc, 85:2149-2154 (1963)

Moore K.S., Wherli S., Roder H., Rogers M., Forest J.N. Jr.,

McCrimmon D., Zasloff M., "Squalamine: An Aminosterol Antibiotic From The Shark", Proc. Natl. Acad. Sci. USA, 90:1354-1358 (1993).

Pinto A. J., Morahan P.S., and Briton M.A., "Comparative Study of Various Immunodomodulators for Macrophage and Natural Killer Cell Activation and Antiviral Efficacy Against Exotic RNA Viruses", Int. J. Immunopharm., 10:197:209 (1988). Westerhoff H.V., Hendler R.W., Zasloff M. and Juretic D., "Interactions Between a New Class of Eukaryotic Antimicrobial Agents and Isolated Rat Liver Mitochondria", Biochem. Biophys. Acta., 975:361-369 (1989b).

Westerhoff H.V., Juretic D., Hendler R.W., and Zasloff M.,

"Magainins and the Disruption of Membrane-Linked Free-Energy

Transduction", "Squalamine: An Aminosterol Antibiotic From the Shark", Proc. Natl. Acad. Sci. USA, 86:6597-6601 (1989a).

Williams R.W., Covell D. and Chen H.C, J. Cell Biochem. Suppl., 13A:96, (1988a).

Williams R.W., Zasloff M. and Covell D., J. Cell Biochem. Suppl., 12B:64 (1988b).

Zasloff M., "Magainins, A Class of Antimicrobial Peptides From Xenopus Skin: Isolation, Characterization of Two Active Forms and Partial cDNA Sequence of a Precursor", Proc. Natl. Acad. Sci. USA, 84:5449-5453 (1987).

Claims

What is claimed is:

1. A method of inhibiting a sexually transmitted disease in a human, wherein the method comprises administering to said human at least one magainin in an amount sufficient to inhibit

transmission of the sexually transmitted disease.

2. The method of claim 1, wherein the magainin

antimicrobial is a peptide.

3. The method of claim 1, wherein the magainin

antimicrobial is a peptidomimetic.

4. The method of claim 1, wherein the sexually transmitted disease is caused by Chlamydia .

5. The method of claim 1, wherein the sexually transmitted disease is caused by human immunodeficiency virus.

6. The method of claim 1, wherein the sexually transmitted disease is caused by herpes simplex virus.

7. The method of claim 1, wherein the sexually transmitted disease is caused by Neisseria gonorrhoeae .

8. The method of claim 1, wherein the sexually transmitted disease is caused by Candida .

9. The method of claim 1, wherein the magainin

antimicrobial is MSI-420, MSI-591, MSI-751, MSI-753, or

squalamine.

10. The method of claim 1, wherein the magainin has

spermicidal activity.

11. A method of inhibiting a sexually transmitted disease in a human, wherein the method comprises topically administering to said human at a locus of sexual contact at least one magainin in an amount sufficient to inhibit transmission of the sexually transmitted disease, wherein the sexually transmitted disease is selected from the group consisting of Chlamydia, human

immunodeficiency virus, herpes simplex virus, Neisseria

gonorrhoeae, and Candida .

12. A method of inhibiting a sexually transmitted disease in a human, wherein the method comprises administering to said human at least one squalamine compound in an amount sufficient to inhibit transmission of the sexually transmitted disease.

13. The method of claim 12, wherein the sexually transmitted disease is caused by Chlamydia .

14. Th nethod of claim 12, wherein the sexually transmitted disease is caused by human immunodeficiency virus.

15. The method of claim 12, wherein the sexually transmitted disease is caused by herpes simplex virus.

16. The method of claim 12, wherein the sexually transmitted disease is caused by Neisseria gonorrhoeae .

17. The method of claim 12, wherein the sexually transmitted disease is caused by Candida .

18. A method of inhibiting a sexually transmitted disease in a human, wherein the method comprises topically administering to said human at a locus of sexual contact at least one squalamine compound in an amount sufficient to inhibit transmission of the sexually transmitted disease, wherein the sexually transmitted disease is caused by a microorganism selected from the group consisting of Chlamydia , human immunodeficiency virus, herpes simplex virus, Neisseria gonorrhoeae, and Candida .