Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
  • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
  • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals

Definitions

• This invention relates to pharmaceutical agents for the treatment of viral diseases, and more specifically, to pharmaceutical agents containing acyclovir and fusaric acid or derivatives thereof.
• Acyclovir 2-amino- 1 ,9-dihydro-9- [(2-hydroxyethoxy)metyl] -6H-purin-6- one, has been used for the inhibition of herpes simplex virus types 1 (HSV-1), 2 (HSV-2), and varicella-zoster virus (VZV).
• Acyclovir is a synthetic purine nucleoside analogue with in vitro and in vivo inhibitory activity against HSV-1, HSV-2 and VZV, which can be found in capsule, tablet, topical and suspension formulations.
• the following structural formula represents acyclovir:
• acyclovir The inhibitory activity of acyclovir is highly selective due to its affinity for the enzyme thymidine kinase encoded by HSV-1, HSV-2 and VZV.
• This viral enzyme converts acyclovir into acyclovir monophosphate, a nucleotide analogue. The monophosphate is further converted into diphosphate by cellular guanylate kinase and into triphosphate by a number of cellular enzymes.
• acyclovir triphosphate stops replication of herpes viral DNA. This is accomplished by: 1) competitive inhibition of viral DNA polymerase, 2) incorporation into and termination of the growing viral DNA chain and 3) inactivation of the viral DNA polymerase.
• acyclovir While the use of acyclovir is useful in the treatment of some herpes viruses, it has limitations related to tissue permeability and viral resistance that make its use less desirable. For example, acyclovir is not effective in topical formulation against herpes labialis and herpes genitalis. As such, acyclovir may not be used topically to effectively treat herpes labialis, herpes genitalis or other viral diseases, disorders, or conditions. Moreover, prodrugs of acyclovir have only minimal effects in herpes labialis. For example, the use of denavir to treat herpes labialis reduces the median duration of the illness by 6 hours. However, herpes labialis typically lasts in the range of about 6 to 7 days.
• compositions which may be used topically to successfully treat a wide range of viral diseases.
• a composition including at least one nucleoside analogue inhibitor, a pharmaceutically acceptable salt, solvate, or prodrug thereof and fusaric acid, a derivative, or pharmaceutically acceptable salt of fusaric acid is provided.
• a pharmaceutical composition comprising the above- described composition of the invention and a pharmaceutically acceptable carrier is provided.
• a method of treating a viral disease, disorder, or condition comprising the administration of a pharmaceutically effective dose of the above- described pharmaceutical composition to a patient is provided.
• a topical preparation that includes at least one above-described composition of the invention and a liquid vehicle.
• a systemic preparation that includes at least one above-described pharmaceutical composition.
• Figure 1 illustrates the antiviral effect of fusaric acid on HS V as determined by plaque reduction assay.
• Figure 2 illustrates the cytotoxicity profiles of fusaric acid as determined by neutral red uptake assay.
• the composition of the invention comprises fusaric acid, or any pharmacologically acceptable salt or derivative thereof and at least one nucleoside analogue inhibitor or a pharmaceutically acceptable salt, solvate or prodrug thereof.
• Fusaric acid is the 5-butyl derivative of picolinic acid.
• nucleoside analogue inhibitor includes any pharmaceutically acceptable salt, solvate, or prodrug of a nucleoside analogue inhibitor.
• the structure of fusaric acid and many of its derivatives is represented by the following structure (formula (I)):
• Ri, R 2 and R_ ⁇ are selected from the group consisting of a peptide of sixteen amino acids, carboxyl group, methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, secondary butyl group, tertiary butyl group, pentyl group, isopentyl group, neopentyl group, fluorine, chlorine, bromine, iodine, and hydrogen and R 3 is a butyl group.
• "fusaric acid” includes the above-derivatives thereof.
• “pharmaceutically acceptable” means it is, within the scope of sound medical judgment, suitable for use in contact with the cells of humans and lower animals without undue toxicity, irritation, allergic response and the like, and is commensurate with a reasonable benefit/risk ratio.
• “animal” includes humans.
• “Pharmaceutically acceptable salt” means a relatively non-toxic, inorganic and organic acid addition salt, and base addition salt of compounds of the invention. These salts can be prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed.
• Exemplary acid addition salts include, but are not limited to the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulphamates, malonates, salicylates, propionates, methylene-bis-b-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methane-sulphonates, ethanesulphonates, benzenesulphonates, p-toluenesulphonates, cyclohexylsulphamates and quinates
• Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed.
• Base addition salts include pharmaceutically acceptable metal and amine salts.
• Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium and aluminum salts.
• Suitable inorganic base addition salts are prepared from metal bases which include, but are not limited to sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide and zinc hydroxide.
• Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use.
• Suitable amine base addition salts include, but are not limited to, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N'- dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)- aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, tri
• Fusaric acid was recognized in the early 1960's to have activity as an antihypertensive agent in vivo. It also has been shown that fusaric acid is an inhibitor of cancerous cell growth, showing an effect on the growth and viability of normal and cancerous cells in tissue culture.
• FIGS. 1 and 2 illustrate the antiviral effect of fusaric acid on HSV-1 and HSV-2 as determined by plaque reduction assay, a technique described in detail below.
• concentration of fusaric acid was approximately 19 ⁇ g/ml for HSV-1 and 18 ⁇ g/ml
• Fusaric acid is active against HSV-1 and HSV-2 infections.
• the addition of small amounts of acyclovir or another nucleoside analogue inhibitor to fusaric acid, a derivative or pharmaceutically acceptable salt thereof results in enhanced antiviral effects as compared to the antiviral effects of either fusaric acid or acyclovir alone.
• administration of the compositions of the invention comprising both fusaric acid and acyclovir to susceptible cells show about a 50% reduction of HSV-1 viral activity and about a 40% reduction in HSV-2 viral activity.
• compositions of the invention are prepared by admixing each component.
• elevated temperature may be necessary to ensure uniform dispersement of the pharmacologically active components through the chosen medium.
• compositions of the invention which may vary depending on the specific composition employed, the nature and presence of other therapeutic compositions or carriers within the composition of the invention and the quantity being produced.
• the compounds of the invention may alternatively be prepared by other methods well known in the art.
• At least one nucleoside analogue inhibitor, pharmaceutically acceptable salt, solvate or prodrug thereof must be present in the compositions of the invention.
• Suitable nucleoside analogue inhibitors include, but are not limited to acyclovir, zidovudine, didanosine, zalcitabine, lamivudine, stavudine, abacavir, combivir, valacyclovir, famciclovir, ganciclovir, ribavirin and denavir.
• the compositions of the inventions are comprised of acyclovir, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.
• the nucleoside analogue inhibitor is an acyclic guanosine analogue.
• Suitable acyclic guanosine analogues include, but are not limited to acyclovir, valacyclovir, famciclovir, gangciclovir and danavir.
• danavir may be optionally referred to as penciclovir.
• prodrugs are those prodrugs of the compounds that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compositions of the invention.
• prodrug means compounds that are rapidly transformed in vivo to yield the parent compound of the above formula, for example by hydrolysis in blood. Functional groups which may be rapidly transformed, by metabolic cleavage, in vivo form a class of groups reactive with the carboxyl group of the compositions of this invention.
• alkanoyl such as acetyl, propionyl, butyryl, and the like
• unsubstituted and substituted aroyl such as benzoyl and substituted benzoyl
• alkoxycarbonyl such as ethoxycarbonyl
• trialkylsilyl such as trimethyl- and triethysilyl monoesters formed with dicarboxylic acids (such as succinyl) and the like.
• the compounds bearing the metabolically cleavable groups have the advantage that they may exhibit improved bioavailability as a result of enhanced solubility and/or rate of abso ⁇ tion conferred upon the parent compound by virtue of the presence of metabolically cleavable group.
• prodrugs are provided in Glycolamide esters as biolabile prodrugs of carboxylic acid agents: synthesis, stability, bioconversion, and physicochemical properties, Nielsen NM, Bundgaard H., J Pharm Sci. 1988 Apr.;77(4):285-98, which is inco ⁇ orated herein by reference.
• compositions of the invention are dependent upon the specific usage for the compositions of the invention, e.g. the nature of the disease and the relative age, condition and size of the patient, as well as the method of administration and/or carrier employed.
• the compositions of the invention are comprised of in the range of about 0.2% to about 50% fusaric acid, a pharmaceutically acceptable salt, or derivative thereof and in the range of about 0.2% to 50% at least one nucleoside analogue inhibitor.
• compositions of the invention are comprised of in the range of about 1% to about 20% fusaric acid, a pharmaceutically acceptable salt, or derivative thereof and in the range of about 1% to about 20% at least one nucleoside analogue inhibitor.
• the compositions of the invention are comprised of about 5% fusaric acid, a pharmaceutically acceptable salt, or derivative thereof and about 5% at least one nucleoside analogue inhibitor. It will be appreciated by those of skill in the art that compositions including a combination of nucleoside analogue inhibitors are within the scope of the present invention. It will also be appreciated by those of skill in the art that compositions having concentrations outside of the stated ranges are within the scope of the present invention.
• the composition of the invention comprises about 5% acyclovir and about 5% fusaric acid. This particular formulation of the invention has been shown to effectively treat primary HSV-1 infection in vivo. See Example 4, supra.
• compositions of the invention may also be mixed with other therapeutic compounds to form pharmaceutical compositions (with or without diluent or carrier) which, when administered, provide simultaneous administration of a combination of active ingredients resulting in the combination therapy of the invention.
• a pharmaceutical composition comprising the above-described compositions of the invention and at least one other therapeutic compound selected from a group consisting of pharmaceutically acceptable carriers, diluents, for example saline solutions, adjuvants, excipients, or vehicles, such as preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents, dispensing agents and any combination thereof.
• the therapeutic compound may also be a liquid vehicle, including but not limited to both an ointment and abso ⁇ tion base.
• the therapeutic compound employed depends on the nature of the disease, mode of administration, dosage forms employed, as well as the overall age, size and condition of the patient.
• suspending agents include, but are not limited to, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth and any combination or mixture of these substances.
• active ingredients necessary in combination therapy may be combined in a single pharmaceutical composition for simultaneous administration.
• the choice of vehicle and the content of active substance in the vehicle are also generally determined in accordance with the solubility and chemical properties of the active compound, the particular mode of administration and the provisions to be observed in pharmaceutical practice.
• excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate and disintegrating agents such as starch, alginic acids and certain complex silicates combined with lubricants such as magnesium stearate, sodium lauryl sulfate and talc may be used for preparing tablets.
• aqueous suspensions When aqueous suspensions are used they can contain emulsifying agents or agents which facilitate suspension.
• Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol and chloroform or mixtures thereof may also be used.
• Any pharmaceutically acceptable carrier suitable to permit drug administration may be used. Suitable carriers include, but are not limited to colorants, flavorants, and other inactive or inert ingredients, for example, gelatin, corn starch, lactose, magnesium stearate, sodium lauryl sulfate, parabens, ink, microcrystalline cellulose, povidone, methyl paraben, preservatives and sodium starch glycolate.
• compositions of the invention are administered to a patient by any acceptable means, which results in effective antiviral activity according to the method of the invention.
• Acceptable means of administration include, but are not limited to topical, oral, parenteral, subcutaneous, intravenous, vaginal, intramuscular, systemic and intraperitoneal administration.
• the pharmaceutical compositions of the invention may be administered to a patient, among other routes of administration, in capsule forms, topical preparations or intravaginal preparations.
• patient includes both humans and other animals. It will be appreciated that the preferred route may vary with, for example, the nature of the disease and the condition, age and size of the patient.
• Topical preparations are provided in accordance with the invention, which comprise the above-described compositions of the invention and a liquid vehicle.
• Formulations suitable for topical administration are formulations, which are in a form suitable to be administered topically to a patient.
• the formulation may be presented as a topical ointment, salves, powders, sprays and inhalants, gels (water or alcohol based), creams, as is generally known in the art, or inco ⁇ orated into a matrix base for application in a patch, which would allow a controlled release of compound through the transdermal barrier.
• the active ingredients may be employed with either a paraffinic or a water-miscible ointment base.
• the active ingredients may be formulated in a cream with an oil-in-water cream base.
• Formulations suitable for topical administration in the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient.
• Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
• the liquid vehicle may be any liquid vehicle suitable to facilitate the topical application of the composition of the invention.
• Suitable liquid vehicles include but are not limited to ointment and abso ⁇ tion bases.
• the relative concentrations of fusaric acid and nucleoside analogue inhibitor in the topical preparations of the invention are dependent upon the specific usage for the composition of the invention, e.g. the nature of the disease and the relative age, condition and size of the patient, as well as the method of administration and/or carrier employed.
• the topical preparations of the invention are comprised of in the range of about 0.2% to about 50% fusaric acid and in the range of about 0.2% to 50% at least one nucleoside analogue inhibitor.
• the topical and intravaginal preparations of the invention are comprised of in the range of about 1% to about 20% fusaric acid, a pharmaceutically acceptable salt, or derivative thereof and in the range of about 1% to about 20% at least one nucleoside analogue inhibitor.
• the topical and intravaginal preparations of the invention are comprised of about 5% fusaric acid, a pharmaceutically acceptable salt, or derivative thereof and about 5% at least one nucleoside analogue inhibitor. It will be appreciated by those of skill in the art that compositions including a combination of nucleoside analogue inhibitors are within the scope of the present invention. It will also be appreciated by those of skill in the art that compositions having concentrations outside of the stated ranges are within the scope of the present invention.
• the topical preparations of the invention comprise about 5% acyclovir and about 5% fusaric acid.
• This particular formulation of the invention has been shown to effectively treat HSV-1 in vivo. See Example 4, supra.
• the compounds of the invention can be microencapsulated in, or attached to, a slow release or targeted delivery systems such as a biocompatible, biodegradable polymer matrices, e.g. poly(d,l-lactide co-glycolide), liposomes, and microspheres and subcutaneously or intramuscularly injected by a technique called subcutaneous or intramuscular depot to provide continuous slow release of the compounds for a period of 2 weeks or longer.
• a slow release or targeted delivery systems such as a biocompatible, biodegradable polymer matrices, e.g. poly(d,l-lactide co-glycolide), liposomes, and microspheres and subcutaneously or intramuscularly injected by a technique called subcutaneous or intramuscular depot to provide continuous
• the compounds may be sterilized, for example, by filtration through a bacteria retaining filter, or by inco ⁇ orating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
• Formulations suitable for systemic administration are formulations, which are in a form suitable to be administered systemically to a patient.
• the systemic formulation of the invention include the above-described compositions of the invention and at least one therapeutic agent, preferably a liquid vehicle.
• the fo ⁇ nulation is preferably administered to a patient by injection, including transmuscular, intravenous, intraperitoneal, and subcutaneous.
• the compounds useful according to the invention are formulated in liquid solutions, e.g., saline solutions, and preferably, in physiologically compatible buffers such as Hank's solution or Ringer's solution.
• the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
• Systematic administration also can be by transmucosal or transdermal means, or the compounds can be administered orally.
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• penetrants are generally known in the art, and include, for example, bile salts and fusaric acid derivatives for transmucosal administration.
• detergents may be used to facilitate permeation.
• Transmucosal administration may be through use of nasal sprays, for example, or suppositories.
• the compounds are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
• a therapeutically effective dose of the compositions of the invention are administered to patients according to the methods of the invention.
• a therapeutically effective dose is the dosage necessary to provide the desired physiological change, i.e., to kill the subject virus.
• the compositions of the invention can be prepared in unit dosage form by any of the methods well known in the art of pharmacy.
• the specific dosage is dependent upon the means of administration, the general health and physical condition of the patient, as well as the patient's age and weight, and the specific viral disease, disorder, or condition suffered by the patient. The existence of other concurrent treatments may also effect the specific dosage administered to the patient.
• composition of the invention administered to a patient is also determined by the attending clinicians taking into consideration the etiology and severity of the disease, the patient's condition, size and age, the potency of each component and other factors.
• compositions of the invention may be presented in unit-dose or multi- dose containers, for example sealed ampoules and vials with elastomeric stoppers, and may be stored in freeze-dried condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
• sterile liquid carrier for example water for injections, immediately prior to use.
• Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
• compositions of the invention are prepared as described herein or by the application or adaptation of known methods, by which is meant methods used heretofore or described in the literature.
• the invention also provides kits or single packages combining two or more active ingredients useful in treating the disease.
• a kit may provide alone or in combination with a pharmaceutically acceptable diluent or carrier, the above- described formula (I) and the above-described nucleoside analogue inhibitor, pharmaceutically acceptable salt, solvate or prodrug thereof, alone, or in combination with a diluent or carrier.
• compositions of the invention optionally are supplied as salts.
• Those salts which are pharmaceutically acceptable are of particular interest since they are useful in administering the foregoing compounds for medical pu ⁇ oses.
• Salts which are not pharmaceutically acceptable are useful in manufacturing processes, for isolation and purification pu ⁇ oses, and in some instances, for use in separating stereoisomeric forms of the compounds of this invention. The latter is particularly true of amine salts prepared from optically active amines.
• Example 1 The following examples are illustrative of specific embodiments of the invention and do not limit the scope of the invention in any way.
• the plaque reduction technique and cytotoxicity testing procedures described with reference to Example 1 are used to determine the percent control of the samples.
• DMSO sulfoxide
• HSV-2 He ⁇ es simplex virus standard strains G (HSV-2) and F (HSV-1) were used in each Example.
• Virus input for a 6-well plate was 200 pfu/well.
• HSV susceptible cell line, Vero cells were used in the virus yield reduction assay.
• the culture medium for Vero cells is 5% Minimum Essential Medium GIBCO/BRL (MEM)
• fetal bovine serum 200 U/ml penicillin and 200 ⁇ g/ml
• HSV-2 (200 pfu well) for 1 hour at 37°C. The cells were tilled every 20 minutes.
• infected cells were washed with PBS and overlaid with 0.5% methylcellulose in culture medium (equal volume of 1% methylcellulose mixed with 2 times culture medium) containing different doses of
• HSV-2 HSV-2. Control was calculated as the mean plaque count in the drug treated wells divided by the mean plaque count in the control wells. The concentrations giving a
• Vero cells using neutral red dye uptake assay. Confluent Vero cells were cultured in a medium containing various doses of picolinic acid for two days. The testing
• concentrations were 0, 10, 100, 250, 500 and 1000 ⁇ g/ml for picolinic acid and 0, 1,
• cytotoxic concentration giving 50% of cell death (CC 50 ) was
• Example 1 To examine the efficacy of the combination of fusaric acid and acyclovir on HSV-1 and HSV-2, the plaque reduction assay technique described in Example 1 was employed. The susceptible cells were infected with HSV-1 then incubated with
• acyclovir reduces HSV-1 plaque counts by about 50% over that seen with 1.75 ⁇ M
• HSV-1 virus stock (0.035 ml) was introduced to the guinea pig through its skin at well-spaced sites of equal size on each side of the dorsal midline of a shaved area of the pig by ten activations of a six-pronged spring-loaded vaccination instrument. The day of inoculation was day 0.
• each sample compound was applied two times per day for days 1, 2 and 3. In some cases, each sample compound was applied up to four times per day. In the case of the sample that included both acyclovir and fusaric acid, the sample was applied two times per day because of skin irritation. To better see the lesions, a depilatory cream was used to remove regrown hair on the dorsum on day 4. Lesions were then counted and lesions sizes were measured using pictures.
• the animals were then sacrificed and the full-thickness skin from different infected treatment areas of the guinea pig were removed.
• the skin from each of the infected treatment areas was placed into 15 milliliters (mils) of tissue culture medium with 2% Fetal Bovine Serum in an ice bath.
• the samples were then homogenized in a Stomacher 80 Biomaster lab blender, commercially available from Seward Company, London, United Kingdom. Debris was pelleted by centrifugation
• Percent differences between mean lesion severity at drug-treated sites compared to the vehicle-treated sites are shown A positive value indicates a reduction in lesion seventy for the test compound
• Day 0 is the day of infection 5% ACV in NVL vehicle, 5% Fusa ⁇ c Acid, 5% ACV + 5% Fusa ⁇ c Acid, 50% Picolinic Acid and 5% ACV/PEG were used 2x/day on Days 1, 2 and 3 Efficacy measurements were done on the morning of Day 4
• Day 0 is the day of infection.
• 5% ACV in NVL vehicle, 5% Fusaric Acid, 5% ACV + 5% Fusaric Acid, 5 0% Picolinic Acid and 5% ACV/PEG were used 2x/day on Days 1 , 2 and 3. Efficacy measurements were done on the morning of Day 4.
• compositions comprising 5% acyclovir as the active agent reduce the number of lesions present by 6%, the total lesion area by 20% and the virus titer by 46%.
• Compositions comprised of 5% fusaric acid as the active agent significantly reduced the number of lesions present by 19% total lesion area by 41% and virus titer by 75%.
• Compositions comprised of 5% acyclovir and 5% fusaric acid as the active agents statistically significantly reduced lesion number by
• the Mann- Whitney rank sum test was used to compare the efficacy of the samples.
• the Mann- Whitney rank sum test is well known to those skilled in the art. The results showed that the compositions that included 5% acyclovir and 5% fusaric acid as the active agents were better than the compositions that comprised 5% acyclovir, 5% fusaric acid and 20% picolinic acid independently as the active agents in all three parameters tested. Further, only the combination of acyclovir and fusaric acid was able to affect all three parameters in a statistically significant manner.

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