WO1996001050A1

WO1996001050A1 - Prevention and treatment of human herpesvirus-6 infection with quaternary ammonium compounds and/or ganglionic blocking agents

Info

Publication number: WO1996001050A1
Application number: PCT/US1995/008484
Authority: WO
Inventors: Joseph A. Baldone
Original assignee: Baltech, Inc.
Priority date: 1994-07-05
Filing date: 1995-07-05
Publication date: 1996-01-18
Also published as: AU2963195A

Abstract

This invention provides a superior method of treating or preventing diseases caused by or related to human herpesvirus-6 (HHV-6) infection, comprising administering a therapeutically effective amount of a quaternary ammonium compound and/or a ganglionic blocking agent.

Description

PREVENTION AND TREATMENT OF HUMAN HERPESVIRUS-6 INFECTION WITH QUATERNARY AMMONIUM COMPOUNDS AND/OR GANGLIONIC BLOCKING AGENTS BACKGROUND OF THE INVENTION FIELD OF INVENTION The present invention relates to a method of preventing and treating infection and related disorders caused by human herpesvirus-6 (HHV-6) . More specifically, the invention relates to administration of a quaternary ammonium compound and/or a ganglionic blocking agent before or after HHV-6 infection of the host.

BACKGROUND Human herpesvirus-6 (HHV-6) is classified as a "herpes" family virus because it has an icosahedral core structure consisting of 162 capsomers, a characteristic feature of herpes viruses. It was originally named "human B- lymphotropic virus (HBLV) , " but was later renamed "human herpesvirus type 6 (HHV-6) ." Josephs et al. , "Molecular Studies of HHV-6," J^". of Virological Methods, 21 , 179-190 (1988) .

HHV-6 was first identified and described in 1986 by Salahuddin et al. , "Isolation of a New Virus, HBLV, in Patients with Lymphoproliferative Disorders, " Science, 234 596-601 (1986) . The virus was originally isolated from human peripheral blood leukocytes from patients with associated lymphoproliferative disorders.

HHV-6 is distinguishable from other known members of the human herpesvirus family, such as Epstein-Barr virus (EBV) , human cytomegalovirus (CMV) , herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) , and varicella zoster virus (VZV) , serologically, morphologically, and by nucleic acid analysis. Josephs et al. , in "Molecular Studies of HHV-6," Journal of Virological Methods, 21 , 179-190 (1988) . Serological comparisons conducted by Salahuddin et al. demonstrated a lack of immunological cross-reactivity between HHV-6 and other known herpes viruses. It has also been reported that HHV-6 is distinguishable from known human and nonhuman primate herpesviruses by host range, in vi tro biological effects, and antigenic features. Salahuddin et al. , cited above. Moreover, hybridization experiments revealed the unique genome of HHV-6. A 9000 base pair cloned probe of HHV-6 detected specific DNA and RNA sequences of herpesvirus infected cells, but it did not hybridize to genomic DNA of EBV, CMV, HSV-1, HSV-2, or VZV. Josephs et al . , cited above.

The natural host of HHV-6 is man and the primary mode of transmission is by close personal contact. The latency sites of HHV-6 in the human body are salivary glands and lymphocytes. In particular, HHV-6 productively infects primary CD2 T lymphocytes (CD4, CD7, and to some extent CD8) , and a minor population of B-lymphocytes in culture.

HHV-6 is now believed to be a cofactor in the pathogenesis of acquired immunodeficiency syndrome (AIDS) . It is most likely that HHV-6 is not exclusively an AIDS- associated agent because it has been isolated from human immunodeficiency virus (HIV) seronegative patients. It is more likely that HHV-6 plays a role in some lympho¬ proliferative and immune abnormalities. Salahuddin et al. , cited above.

Although infection by HIV Type 1 (HIV-1) and Type 2 (HIV-2) has been implicated as the primary cause of AIDS and related disorders, cofactorial mechanisms may be involved in the pathogenesis of the disease. HHV-6 has been suggested as a possible cofactor because it has been identified in most patients with AIDS by virus isolation, DNA amplification techniques, and serological analysis. The likelihood that HHV-6 is a cofactor of AIDS is also indicated by the fact that the virus is predominantly tropic and cytopathic in vi tro for CD4⁺ T lymphocytes. Thus, HHV-6 may be a cofactor in some cases of AIDS by contributing to the depletion of CD4 T lymphocytes.

This conclusion is also supported by research findings indicating that HHV-6 and HIV-1 can productively co-infect individual human CD4 T lymphocytes resulting in accelerated HIV expression and cellular death. Lusso et al. , "Productive Dual Infection of Human CD4⁺ T lymphocytes by HIV-1 and HHV- 6," Nature, 337, 370-373 (1989) . This reference also reported that HHV-6 may transactivate the HIV long terminal repeat (LTR) .

The cofactorial role of HHV-6 in AIDS is also explored by Ensoli et al. , "Human Herpes Virus-6 Increases HIV-1 Expression In Co-infected T cells via Nuclear Factors Binding to the HIV-1 Enhancer," The EMBO J. , 8, 3019-3027 (1989) . This reference reported that HHV-6 infection may play an important role in the progression of HIV-1 infection to AIDS by increasing HIV-1 gene expression and viral replication, consequently accelerating the cytopathic effect in co-infected CD4⁺ T cells.

Furthermore, it has also been reported that HHV-6 can activate the HIV promoter. By increasing HIV promoter activity, HHV-6 may increase HIV replication, leading to an increase in cytopathic effect on coinfected human T cells. Horvat et al. , "Transactivation of the Human Immunodeficiency Virus Promoter by Human Herpesvirus 6 (HHV-6) Strains GS and Z-29 and Primary Human T-lymphocytes and Identification of Transactivating HHV-6 (GS) Gene Fragments, " J^". of Virology, 65, 2895-2902 (1991) .

HHV-6 infection is currently treated with ganciclovir sodium (GCV) or foscarnet (FCN) . Ganciclovir sodium is sold under the trade name Cytovene® by Syntex Laboratories, Inc., and foscarnet sodium is sold under the trade name Foscavir® by Astra Pharmaceutical Products, Inc. Physicians ' Desk Reference, 47th Edition, pp. 643-646 and 2397-2402 (1993) . Effective alternative treatments are desirable because both GCV and FCN, which are nucleoside analogs, are highly toxic to humans and are likely to produce drug-resistant mutants, a great concern to virologists in the treatment of herpesvirus infections.

Because of the lack of effective treatments for HHV-6 infection, and because HHV-6 infection may play a significant role in related disorders, such as AIDS, there is a need in the art for an effective treatment and prevention of HHV-6 infection.

The present invention is directed to the use of a quaternary ammonium compound and/or a ganglionic blocking agent to treat or prevent HHV-6 infection.

Preferred quaternary ammonium compounds are tetraethylammonium (TEA) ion, hexamethonium ion, pentolinium ion, chlorisondamine ion, trimethidinium ion, trimethaphan ion, and homologs thereof. In particular, TEA ion is employed as the quaternary ammonium compound.

A preferred ganglionic blocking agent is mecamylamine. In addition to having properties of quaternary ammonium compounds, TEA, hexamethonium ion, pentolinium ion, chlorisondamine ion, trimethaphan ion, and trimethidinium ion have ganglionic blocking activity.

TEA chloride is, or has been in the past, commercially available from City Chemical Corp., 132 W. 22nd Street, New York, W 10011. American Druggist ' s Blue Book (1974) Am. ed. The TEA ion has the following structure:

A process for the manufacture of TEA is described in U.S. Patent No. 2,653,156, issued September 22, 1953, to Deutsch et al. Quaternary ammonium compounds have most _.often been. used as antiseptic or disinfectant agents. For example, the use of quaternary ammonium compounds in a low foaming medium for external use as a general disinfecting agent was described in U.S. Patent No. 4,165,375, issued August 21, 1979, to Berger et al. U.S. Patent No. 2,689,814, issued September 21, 1953, to Nichols et al . , also refers to the germicidal effectiveness of TEA as well as the general fungicidal and anesthetic properties of quaternary ammonium compounds. Disinfectant properties of quaternary ammonium compounds are also referred to in U.S. Patent No. 2,653,156, issued September 22, 1953, to Deutsch et al. The topical application of quaternary ammonium compounds for the control of infections is described in U.S. Patent No. 2,886,487, issued May 12, 1959, to Kupferberg et al .

Ganglionic blocking drugs function to block transmission in autonomic ganglia without producing any preceding or concomitant changes in the membrane potentials of the ganglion cells. Goodman and Gilman ' s The Pharmacological Basis of Therapeutics, Seventh Edition, page 219 (Macmillan Publishing Co., New York, 1985) . Blockade of sympathetic ganglia interrupts adrenergic control of arterioles and results in vasodilatation, improved peripheral blood flow in some vascular beds, and a fall in blood pressure. Ganglionic blockers do not modify the conduction of impulses in the preganglionic or postganglionic neurons, and they do not prevent the release of the neurotransmitter acetylcholine (ACh) by preganglionic impulses. Ganglionic blockers occupy receptor sites on the ganglion cells, thereby stabilizing the postsynaptic membranes against the actions of ACh liberated from the presynaptic nerve endings.

Various secondary side-effects are associated with ganglionic blocking activity. These effects include the lowering of blood pressure to pronounced vasodilator action, mydriasis (pupil dilation) , cycloplegia, which may cause temporarily blurred vision, ptosis, and similar impairment of physical responses generally associated with nerve impulse blocking action.

Historically, the major therapeutic use of ganglionic blocking agents was in the management of hypertensive cardiovascular disease and in the production of controlled hypotension, in which a reduction in blood pressure during surgery may be sought deliberately to minimize hemorrhage in the operative field. However, these drugs have largely been supplanted by superior agents. Pentolinium is still used to a limited extent in Europe. Goodman and Gilman 's The Pharmacological Basis of Therapeutics at 220 (1985) .

The bromide and chloride salts of TEA are short acting quaternary ammonium ganglion-blocking agents used in the treatment of hypertension, peripheral vascular diseases, and other disorders of the peripheral circulation. The ganglion blocking activity reversibly blocks both sympathetic and parasympathetic motor impulses. TEA also lowers the blood pressure due to pronounced vasodilator action, mydriasis (pupil dilation) , cycloplegia (which may cause temporarily blurred vision) , ptosis (eyelid drooping) , and similar impairment of physical responses associated with the nerve impulse blocking action. Dorland 's Illustrated Medical Dictionary, Twenty-sixth Edition, page 1351 (W.B. Saunders Co., Philadelphia, 1974) . This reference also notes that TEA compounds are seldom used, having been replaced by more effective drugs. The 1993 Physicians ' Desk Reference, 47th Edition (Medical Economics Data, Montvale, NJ) , does not have a reference for TEA.

TEA blocks transmission of nerve impulses; it does not modify the conduction of nerve impulses. This blocking action is fully reversible and effective treatments of excessive doses are known. For example, a neurotransmitter, such as acetylcholine (ACh) , can be administered. TEA does not impair cellular metabolic processes, and changes in cell morphology due to TEA administration are reversible. In 1952, the Sixth Edition of Physicians ' Desk Reference to Pharmaceutical Special ties and Biologicals, J.M. Jones ed. (Medical Economics, Inc., Rutherford, NJ) , reported that "Etamon™ Chloride" contained 0.1 g. tetraethylammonium chloride per cc, and that the compound is used in the treatment of herpes zoster (HZ) . The Eighth Edition of Physicians ' Desk Reference (1954) omitted the description of the use or efficacy of "Etamon® Chloride" against HZ. In 1960, the Fourteenth Edition of Physicians ' Desk Reference, no longer listed the product "Etamon® Chloride, " and the manufacture of "Etamon® Chloride" was subsequently discontinued.

It is questionable whether the tetraethylammonium chloride present in "Etamon™ Chloride" was the effective agent in the treatment of HZ because "Etamon® Chloride" also contained benzethonium chloride as a preservative. Benzethonium chloride was disclosed to be an effective agent in the treatment of HZ in U.S. Patent No. 4,262,007, issued April 14, 1981, to Sherrill.

The use of TEA as a pain reliever in various types of causalgia and related post traumatic states, HZ, and chest pain caused by embolism, neoplasm, pleuritis, etc. was referred to by Goodman and Gil an ' s The Pharmacological Basis of Therapeutics, Second Edition, page 633 (The MacMillan Co., New York, 1955) . The mechanism of the relief of thoracic pain and HZ by TEA was unknown, but it was suggested that the mechanism was related to the observation that TEA prevented the substernal burning induced in man by lobeline, presumably caused by stimulation of pleural chemoreceptors. Lobeline is a compound having the same action as nicotine, but with less potency.

The use of quaternary ammonium compounds, including TEA, as an effective treatment for HSV-1, HSV-2, and HZ infections is described in U.S. Patent No. 4,760,079, issued July 26, 1988, to Baldone; U.S. Patent No. 4,902,720, issued February 20, 1990, to Baldone; U.S. Patent No. 4,935,448, issued June 19, 1990, to Baldone; and U.S. Patent No. 5,158,980, issued October 27, 1992, to Baldone, the disclosures of which are incorporated by reference.

Because of the differing structures between HSV-1, HSV-2, HZ, and HHV-6, and because several drugs which are effective against HSV-1, HSV-2, and/or HZ infections are ineffective against HHV-6 infection, the successful results of treating HHV-6 infection with a quaternary ammonium compound, such as TEA, are surprising. For example, the antiviral agent acyclovir is effective against HSV-1, HSV-2, and varicella zoster, but is ineffective against HHV-6. The antiviral agent trifluridine is effective against HSV-1, HSV-2, and vaccinia virus, but is ineffective against HHV-6.

It is also noted that, though the inventor is not bound by theory, the efficacy of quaternary ammonium compounds against HSV-6 may be due to a different mechanism than the compound's mechanism of action against HSV-1, HSV-2, and HZ.

Hexamethonium ion is a quaternary ammonium ganglion- blocking agent used in the treatment of hypertension, usually in combination with other hypotensive drugs, but has been largely replaced by more effective drugs. It is most often used as the chloride, bromide, or tartrate. Dorland' s Illustrated Medical Dictionary at 607; and Stedman ' s Medical Dictionary, 25th Edition, page 714 (Williams & Wilkins, Baltimore, MD, 1990) .

Pentolinium ion, a quaternary ammonium compound with potent ganglionic blocking action of longer duration than hexamethonium, is used in the management of severe and malignant hypertension, peripheral vasospastic diseases, and to produce controlled hypotension in anesthesia. It is most often used as the tartrate. Dorland ' s Illustrated Medical Dictionary at 985; Stedman ' s Medical Dictionary at 1160; and Goodman and Gilman ' s The Pharmacological Basis of Therapeutics at 218-221 (1985) .

Chlorisondamine ion, a quaternary ammonium compound with ganglionic blocking action similar to, but more potent than, hexamethonium and pentolinium, is used in the management of severe hypertension. It is most often used as the chloride. Dorland ' s Illustrated Medical Dictionary at 256; Stedman ' s Medical Dictionary at 291; and Goodman and Gilman ' s The Pharmacological Basis of Therapeutics at 218-221 (1985) .

Trimethidium ion, a quaternary ammonium compound that blocks ganglionic transmission at sympathetic and parasympathetic ganglia, is used in the treatment of moderate to severe hypertension. It is most often used as the methosulfate. Dorland' s Illustrated Medical Dictionary at 1397; and Stedman ' s Medical Dictionary at 1637.

Trimethaphan ion is a quaternary ammonium compound having short-acting ganglionic blocking activity with direct vasodilator action. It is used in surgery, particularly neurosurgery, to produce a relatively bloodless operative field (controlled hypotension) . The camsylate form is used most often. Dorland ' s Illustrated Medical Dictionary at 985; and Stedman ' s Medical Dictionary at 1160.

There are no references to hexamethonium ion, pentolinium ion, chlorisondamine ion, or trimethidinium ion in the 47th Edition of Physicians ' Desk Reference (Medical Economics Data, Montvale, NJ, 1993) . Trimethaphan camsylate, sold under the trade name Arfonad® by Roche Laboratories, is described in the 47th Edition Physicians ' Desk Reference (1993) as useful for producing controlled hypotension during surgery, for the short term (acute) control of blood pressure in hypertensive emergencies, and in the emergency treatment of pulmonary edema in patients with pulmonary hypertension associated with systemic hypertension.

Finally, mecamylamine ion is a ganglionic-blocking agent used as a hypotensive, usually in the treatment of moderately severe hypertension. The compound is similar to, but more effective than, hexamethonium, although mecamylamine is not a quaternary ammonium compound. Dorland' s Illustrated Medical Dictionary at 784; and Stedman ' s Medical Dictionary at Mecamylamine HC1 is sold under the trade name Inversine® by Merck & Co. Physicians ' Desk Reference, 47th Edition (1993) .

Unlike quaternary ganglionic blocking agents, which do not readily reach the central nervous system (CNS) , large doses of mecamylamine can produce prominent central effects. Goodman and Gilman 's The Pharmacological Basis of Therapeutics , Seventh Edition, page 220 (1985) .

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantages associated with current strategies for treating HHV-6 infection, such as the toxic effects of GCV and FCN, and it reduces the likelihood of creating drug-resistant mutants of HHV-6. The invention provides a new therapy for the treating and preventing HHV-6 infection of animals comprising the parenteral, topical, and/or oral administration of an effective amount of a quaternary ammonium compound and/or a ganglionic blocking agent.

The preferred animal is a human.

More specifically, a halide salt of a quaternary ammonium compound is employed. Halide salts of TEA ion are preferred because TEA ion is readily available in deliverable form as a halide salt in single and multiple-dose forms, and because the systemic side effects of TEA halide salts in humans are known.

A preferred ganglionic blocking agent is mecamylamine, and homologs thereof. Other preferred ganglionic blocking agents are TEA ion, hexamethonium ion, pentolinium ion, chlorisondamine ion, trimethaphan ion, mecamylamine, and homologs thereof.

It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the claimed invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

This invention describes inhibition of HHV-6 replication and infection of cells with administration of a composition comprising a quaternary ammonium compound and/or a ganglionic blocking agent. It was completely unexpected that the administration of a quaternary ammonium compound and/or a ganglionic blocking agent would have any effect on the replication or infectivity of HHV-6. Although the compounds are known and characterized, HHV-6 was only recently discovered in 1986. The virus is morphologically and serologically distinct from known retroviruses.

The composition can be administered orally, parenterally, or topically, or a combination of oral, parenteral, and topical administration can be used.

The quaternary ammonium compound can be any compound having a central nitrogen bound to four organic groups, and which is capable of treating or preventing an infection or disease in a mammal caused by HHV-6. The organic groups on the central nitrogen can be alkyls, aryls, aliphatics, alicyclics, heterocyclics, aromatic cyclics, alcohols, ethers, esters, aldehydes, ketones, sulfonamides, carboxylic acids, or carbohydrates. The central nitrogen may also form a ring with one or more of the organic groups. The organic groups may be the same or different depending upon the particular compound.

The quaternary ammonium compound can be a simple monoquaternary ammonium compound, such as a tetraethyl¬ ammonium chloride (TEAC) ; a complex monoquaternary ammonium compound, such as phenacyl homotropinium; a bis-quaternary ammonium compound, such as pentamethonium chloride, hexamethonium chloride, and pentolinium tartrate; or an asymmetrical diquaternary ammonium compound, such as trimethidinium methasulfate and chlorisondamine chloride.

The aliphatic groups on the central nitrogen can be alkanes, olefins, or alkynes. The cyclic groups can be alicyclics, aromatics, and heterocyclics. For example, the organic groups on the central nitrogen can be alkyls having 1 to 20 carbons, hydroxalkyls, aminoalkyls, alkenyls, or substituted derivatives thereof. Preferably, the organic groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, allyl, or methallyl groups. The alicycles and heterocyclic groups can be cyclohexyl or cyclopentyl groups, attached either directly or through side chains to the central nitrogen. The organic groups on the central nitrogen can also be hydrophobic aliphatics, unsaturated aliphatics, or aliphaticaryl radicals. The hydrophobic aliphatics may be long-chain alkyl, long-chain alkoxyaryl, halogen substituted long-chain alkylaryl, long-chain alkylphenoxyalkyl, or arylalkyl.

The organic group can also be a low molecular weight group, such as fufuryl, tertahydrofufuryl, alkyl, carbethoxyalkyl, hydroxalkyl, aminoalkyl, and alkenyl groups, or it can be a high molecular weight aliphatic hydrocarbon group, either saturated or unsaturated, and either branched or straight chain. Samples of these groups include myristyl, cetyl, stearyl, and oleyl. These groups may also contain substituents, such as hydroxyl, halogen, carbethoxy and amino.

The organic groups can be joined together to form a heterocyclic ring when both ends of an organic group are attached to the central nitrogen. Examples of such rings include pyridine, piperidine, picoline, pipicoline, pyrrol, pyrrolidine, morpholine, quinoline, quinaldine, azole, thiazole, and substituted derivatives thereof.

Examples of the various quaternary ammonium compounds useful in the present invention include alkyl ammonium halides, such as cetyl trimethyl ammonium bromide and dioctadecyl dimethyl ammonium chloride; alkyl aryl ammonium halides, such as octadecyl dimethyl benzyl ammonium chloride; N-alkyl-pyridinium halides, such as N-laurylpyridinium chloride; quaternary ammonium salts containing ether linkages in the molecule, such as paratertiary octylphenoxyethoxy- ethyl dimethyl benzyl ammonium chloride; quaternary ammonium compounds having oxygen in the form of amide or ester linkages, such as N- (laurylcolaminoformylmethyl) pyridinium chloride; quaternary ammonium compounds containing a sulfonamide group, such as 2-phenyl-3-p-sulfamido-phenyl-5- undecyltetrazolium chloride; ammonium compounds having a substituted aromatic nucleus, such as lauryloxyphenyl trimethyl ammonium chloride, dodecylbenzyl trimethyl ammonium chloride, chlorinated dodecylphenyl trimethyl ammonium chloride, and dodecylbenzyl trimethyl ammonium methyosulfate; and complex monosulfonium compounds, such as trimethaphan camphorsulfonate; and the like.

The anion of the quaternary ammonium compound can be any anion that is compatible with the central nitrogen and the four organic groups of the compound. Typical anions include - Cl, -Br, -I, -N0₃, =S0₄, -C0₂CH₃, -C₆H₅S0₃, -OH, =P0₄, -C0₂C₆H₅, =C0₃, -HO-C₆H₄C0₂, fatty acids, and dicarboxylic acids and tricarboxylic acids, such as tartaric and citric acids.

The preferred quaternary ammonium compounds are TEA, hexamethonium (C6) , pentolinium, chlorisondamine, trimethidinium, and trimethaphan. As discussed above, other quaternary ammonium compounds may also be used in the present invention.

The preferred ganglionic blocking agent is mecamylamine, although other ganglionic blocking agents can be used, as discussed above.

With administration of the appropriate quaternary ammonium compound and/or a ganglionic blocking agent, HHV-6 infection of the animal will be prevented, or an existing infection, and conditions associated with the infection, will be alleviated.

In one embodiment, the quaternary ammonium compound and/or ganglionic blocking agent is administered to the animal in a dosage effective to produce an antiviral effect against HHV-6. In another embodiment, the quaternary ammonium compound and/or ganglionic blocking agent is administered to the animal in a dosage effective to inhibit the viral function of the virus. The administration of a quaternary ammonium compound and/or a ganglionic blocking agent can simultaneously achieve many of these effects.

The quaternary ammonium compounds and/or ganglionic blocking agents can be administered to the animal in a number of pharmaceutically acceptable methods, such as intravenously, intramuscularly, intraperitoneally, topically, orally, and/or vaginally. The particular quaternary ammonium compound and/or ganglionic blocking agent selected depends on a number of factors, including the host and the method of administration.

For treating and preventing HHV-6 infection, it is preferred that the quaternary ammonium compound is administered from about 5 to about 7 mg/kg body weight by slow intravenous drip once or twice daily. Alternatively, the quaternary ammonium compound is administered at up to about 20 mg/kg body weight intramuscularly once or twice daily. With the use of a composition comprising mecamylamine, it is preferred that the composition is administered from about 2.5 to about 5.0 mg/day/orally in two divided doses, up to about 25 mg/day/orally in three divided doses.

Pharmaceutically acceptable carriers for the quaternary ammonium compound and/or ganglionic blocking agent can also be employed, such as a normal sterile saline solution for systemic use or a hydrophilic ointment base for topical or vaginal administration. Pharmaceutically acceptable carriers can be sterile liquids, such as water, alcohol, dimethyl- sulphoxide (DMSO) , oils, including petroleum oil, animal oil, vegetable oil, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Also useful are carriers such as starch, sugar, lactose, magnesium stearate, stearic acid, cellulose, gelatin, talc, titanium dioxide, silica gel, tartaric acid, zinc stearate, povidone, glycerin, benzoic acid, iron oxide, silicone, and the like. Saline solutions, aqueous dextrose, and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in Remington 's Pharmaceutical Sciences, 18th Edi tion (A. Gennaro, ed. , Mack Pub., Easton, Pa., 1990) , incorporated by reference.

The concentration of the quaternary ammonium compound and/or ganglionic blocking agent is limited only by the amount that may be carried or dissolved in the carrier or diluent, but preferably is from about 1 to about 1000 mg/ml total volume, more preferably from about 50 to about 250 mg/ml. In an ointment, the concentration of the quaternary ammonium compound and/or ganglionic blocking agent can be from about 0.01% to about 99% of volume. The quaternary ammonium compound and/or ganglionic blocking agent can also be encapsulated or placed in pill form for oral administration to the host. The dosages of the quaternary ammonium compound and/or ganglionic blocking agent can be readily determined by the skilled artisan.

The above-referenced patents to Baldone, incorporated by reference, teach that quaternary ammonium compounds are not deactivated in vivo, and are able to reduce the infectivity of HSV-1, HSV-2, and HZ. Thus, the skilled artisan would be able to determine effective dosage of a quaternary ammonium compound for use against HHV-6 infections in animals.

In a preferred embodiment, the quaternary ammonium compound has ganglionic blocking activity. Preferred quaternary ammonium compounds having ganglionic blocking activity include TEA ion, hexamethonium ion, pentolinium ion, chlorisondamine ion, trimethidinium ion, trimethaphan ion, and homologs thereof. An appropriate anion, such as chloride or bromide, is present with the ganglionic blocking agent in pharmaceutical preparation.

Having generally described the invention, a more complete understanding can be obtained by reference to specific examples, which are provided herein for purposes of illustration only and are not intended to be limiting. Example 1

The objective of this experiment was to evaluate the potential toxicity of TEAC on Molt-3 cells.

The HHV-6 strain used was Z-29, deposited with the A.T.C.C., and the cell type was Molt-3, also deposited with the A.T.C.C.

TEAC was dissolved in water (100-200 mg/ml) and filter sterilized. Stock drug solution was serially diluted in RPMI media with 10% fetal bovine serum to obtain the desired final drug concentrations. The pH of the drug-containing media was between 7.9 and 8.0.

Molt-3 cells were incubated in six well Costar dishes

5

(Fisher Scientific) at 3 x 10 cells/well, in duplicate, in media with the appropriate drug concentrations at 37°C in a humidified C0₂ incubator. The results are shown in Table 1. The percentages reported are an average of duplicate values.

Drug concentrations of 1 mg/ml to 40 mg/ml were toxic to Molt-3 cells at day 3. Drug concentrations of 0.5 mg/ml or lower had no retarding effect on the growth of Molt-3 cells. A concentration of 0.5 mg/ml of TEAC was selected to maintain uninfected and HHV-6 infected Molt-3 cells.

TABLE 1

Effect of TEAC on the Viability of Molt-3 Cells In Vitro

Example 2

The objectives of this experiment were to determine the effect of TEAC on the replication of HHV-6 in vitro.

The same materials used in Example 1 were used in Example 2.

Molt-3 cells at 3 x 10 cells/well were incubated (in duplicate) with 0.4 ml of untreated virus (2 x 10 PFU/0.4 ml) for 3 hours. The cultures were rehydrated with 4 ml of growth media containing the appropriate drug concentration, and incubated at 37° in a humidified C0₂ incubator. The results are shown in Table 2. The percentages reported are an average of duplicate values.

At 0.5 mg/ml, TEAC did not have any effect on the replication of HHV-6. Drug concentrations of 1 to 2 mg/ml showed a 50% reduction in the replication of HHV-6 at day 3.

TABLE 2

Effect of TEAC on the Replication of HHV-6 (Antiviral Activity)

Example 3

The objective of this experiment was to determine the effect of TEAC on the infectivity of HHV-6 in vi tro.

The same materials used in Example 1 were used in Example 3.

To determine the effect of TEAC on the infectivity of HHV-6, 1.2 ml of HHV-6 (2 x 10⁵ PFU/0.4 ml) was incubated with the appropriate drug concentration at 37°C overnight. The virus was collected by centrifugation, washed with PBS IX, and re-suspended in 1.2 ml of RPMI media containing 10% fetal bovine serum. An aliquot of drug treated virus (0.4 ml) was mixed with Molt-3 cells at 3 x 10 cells/well (in duplicate) and incubated for 3 hours. The cultures were rehydrated with 4 ml of growth media containing 0.5 mg/ml TEAC. Incubation was continued at 37°C in a humidified C0₂ incubator.

At days 3 and 6, small aliquots from each well were removed and the cells were collected by centrifugation, plated on clean coverslips, and fixed in acetone. The fixed cells were subjected to immunofluorescence and polyclonal antisera to HHV-6. One hundred cells were counted on each coverslip and antigen positive cells were recorded (as percent infected cells) . The percentage of infected cells in each culture was also estimated microscopically.

The results are shown in Table 3. The percentages reported are an average of duplicate values. At 0.5 mg/ml, TEAC had no virucidal effect. However, at 5-10 mg/ml of TEAC, HHV-6 growth was reduced 20-50%. Similar experiments using ganciclovir (28.5 μM) showed 90-98% reduction in virus growth. These results demonstrate that TEAC has virucidal activity against HHV-6.

TABLE 3

Effect of TEAC on the Infectivity of HHV-6

(Virucidal)

*Virus incubated with 28.5 μM of GCV showed less than 25% infectivity at day 8 Example 4

The objective of this experiment was to determine the effective concentrations of TEAC required to reduce 50-90% of HHV-6 replication or infectivity in vi tro ("IC50" and "IC90") .

The same materials used in Example 1 were used in Example .

HHV-6 was incubated in wells with appropriate drug concentrations of TEAC overnight at 37°C. The virus was washed off the wells and re-suspended in drug-free media. Drug-treated and untreated (0 mg/ml TEAC) HHV-6 were adsorped to Molt-3 cells in the presence of 0.5 mg/ml TEAC for four hours at 37°C (in duplicate) . The treated virus was then rehydrated with growth media containing 0.5 mg/ml TEAC and incubated at 37°C. The untreated control virus was adsorped and rehydrated in drug-free media.

For the determination of percentages of infected cells, all cultures were monitored microscopically to assess cytopathic effect (CPE) . The percentage of infected cells in each culture was also estimated microscopically.

At day 4 and 8, aliquots from each well were removed and cells were collected by centrifugation, plated on clean coverslips, and fixed in acetone. The fixed cells were subjected to immunofluorescence staining using polyclonal antisera to HHV-6. One hundred cells were counted on each coverslip and antigen positive cells were recorded (as percent infected cells) .

The results are shown in Table 4. The percentages reported are an average of duplicate values. At day 4, cells were not fully infected. It is customary to evaluate the efficacy of drugs when the cells are fully infected (i.e., 100%) . The results are more meaningful when the efficacy of drugs are evaluated at days when all or 100% cells are infected.

At 0.5 mg/ml to 9 mg/ml, TEAC showed no antiviral efficacy during HHV-6 infection in vitro. However, 10 mg/ml of TEAC showed 40% virucidal activity. The estimated IC50 and IC90 for TEAC are 10 mg/ml and 20 mg/ml, respectively.

TABLE 4

Effect of TEAC on the Infectivity of HHV-6

(Virucidal)

Cone, of TEAC pre- % infected cells* during viral incubation (mσ/ml) day 4 day 8

0 85 100 5 75 100 6 75 100 7 75 100 8 65 100 9 65 85-90 10 60 60

*Virus incubated with 28.5 μM of GCV showed less than 25% infectivity at day 8

Example 5

The objective of this experiment was to determine whether treatment of cells with TEAC prior to HHV-6 infection enhances the virucidal or antiviral efficacy of the drug (the effect on viral receptors on the cell surface) .

The same materials used in Example 1 were used in Example 5.

Molt-3 cells were incubated in wells in the presence of 0.5 mg/ml TEAC or GCV (28.5 μM) . A control group of Molt- 3 cells was incubated in drug-free media. HHV-6 was incubated separately with 0 mg/ml as a control, 0.5 mg/ml, 10 mg/ml, and 20 mg/ml of drug. One day later, the drug was washed off the virus and the virus was re-suspended in drug-free media. Drug-treated and untreated cells were infected with drug- treated and untreated virus in parallel. The cells were then adsorped at 37°C for 4 hours, followed by rehydration in media containing 0.5 mg/ml TEAC at 37°C. At day 18, aliquots were plated and stained as in Example 4.

The results are shown in Table 5. The percentages reported are an average of duplicate values. A 50% reduction of HHV-6 infectivity was obtained with 10 mg/ml, and a 90% reduction of HHV-6 infectivity was obtained with 20 mg/ml TEAC. Prior treatment of cells overnight with TEAC (0.5 mg/ ml) further reduced the infectivity of treated virus at 10 mg/ ml to 10%. However, virus treated with 20 mg/ml showed an increase in efficacy, accomplished by cell toxicity. The cell toxicity could be due to an incomplete removal of the drug from the virus after an overnight incubation.

TABLE 5

Effect of TEAC on Viral Receptors and Cell Surfaces

* Observations from day 18 PI. Cultures containing cells and viruses treated with GCV (28.5 μM) showed less than 2% infectivity, and cultures with untreated cells and treated virus showed 10% infectivity.

** Cell toxicity observed.

The results of Examples 4 and 5 indicate that under these experimental conditions, the antiviral efficacy of TEAC does not differ significantly from that of the more toxic GCV. Moreover, the use of TEAC is preferable over the use of GCV to treat HHV-6 infections because unlike GCV, TEAC is a non- nucleoside analog. TEAC may therefore be less likely to produce drug resistant mutants. The data also suggests that TEAC disrupts cell receptors as well as interferes with viral replication. Example 6

The objective of this experiment was to evaluate the potential toxicity of mecamylamine on Molt-3 cells.

The same materials used in Example 1 were used in Example 6. For preparation of the stock drug solution, mecamylamine was dissolved in water (2 mg/ml) and filter sterilized. For preparation of growth media, the stock drug solution was serially diluted in RPMI media with 10% fetal bovine serum to obtain the desired final drug concentrations.

The pH of the drug media was between 7.9 and 8.0.

5 Molt-3 cells at 3 x 10 cells/well (in duplicate) were incubated in media with the appropriate drug concentration

(20, 30, 60, 125, 250, and 500 μg/ml) at 37°C in a humidified

C0₂ incubator.

When infected Molt-3 cells were grown in mecamylamine (20, 30, 60, 125, 250, and 500 μg/ml) , no toxic effect was visible on day 3. At day 6, drug concentrations of 30 and 60 μg/ml showed minimal toxicity, while all other drug concentrations were highly toxic to the cells. Example 7

The objective of this experiment was to determine the effect of mecamylamine on the replication of HHV-6 in vi tro .

The same materials used in Example 1 were used in

Example 7.

Untreated HHV-6 virus (4 ml at 2 x 10⁵ PFU/0.4 ml)

5 were added to Molt-3 cells (3 x 10 cells/well, in duplicate) and incubated for 3 hours. The cultures were rehydrated with

4 ml growth media containing the appropriate drug concentration. Incubation was continued at 37°C in a humidified C0₂ incubator.

All cultures were monitored microscopically to assess cytopathic effect (CPE) . The percentage of infected cells in each culture was also estimated microscopically.

At day 3 and 6, small aliquots from each well were removed and the cells were collected by centrifugation, plated on clean coverslips, and fixed in acetone. The fixed cells were subjected to immunofluorescence staining and polyclonal antisera to HHV-6. One hundred cells were counted on each coverslip and antigen positive cells were recorded (as percent infected cells) . Mecamylamine had no effect on the replication of HHV-6 at 30-60 μg/ml. When cells were treated with mecamylamine at 0.5 mg/ml for three days, washed and infected with HHV-6, a 80-90% inhibition of viral replication was observed. Similar experiments conducted with GCV (28.6 μM) and TEAC (0.5 mg/ml) produced only 5 and 25% reduction in replication, respectively. Example 8

The objective of this experiment was to determine the effect of mecamylamine on the infectivity of HHV-6 in vi tro.

The same materials used in Example 1 were used in Example 8.

HHV-6 (1.2 ml at 2 x 10⁵ PFU/0.4 ml) was incubated with the appropriate drug concentration at 37°C overnight. The virus was collected by centrifugation, washed with PBS IX, and re-suspended in 1.2 ml of RPMI media containing 10% fetal bovine serum. An aliquot of drug treated virus (0.4 ml) was

5 added to Molt-3 cells (3 x 10 cells/well, in duplicate) and incubated for 3 hours. The cultures were rehydrated with 4 ml of drug-free growth media. Incubation was continued at 37°C in a humidified C0₂ incubator.

The percent infected cells was determined as in Example 7. Mecamylamine had no effect on the infectivity of HHV-6 at 30-120 μg/ml.

The results of Examples 6-8 indicate that mecamylamine prevents the entry of HHV-6 into cells, most likely by disrupting cell receptors for the virus.

* * * * *

Having now fully described the invention, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention. The appended claims are not intended to be limiting.

Claims

I CLAIM :

1. A method for treating human herpesvirus-6 infection in an animal, comprising administering a therapeutically effective amount of a composition comprising a quaternary ammonium compound and/or a ganglionic blocking agent.

2. The method of claim 1, wherein said quaternary ammonium compound is a ganglionic blocking agent.

3. The method according to claim 1, wherein the animal is a human.

4. The method according to claim 1, wherein the composition is administered orally, parenterally, topically, or by a combination thereof.

5. The method of claim 4, wherein said composition further comprises a pharmaceutically acceptable carrier.

6. The method of claim 5, wherein a liquid diluent is employed as the pharmaceutically acceptable carrier, and wherein the quaternary ammonium compound and/or ganglionic blocking agent is present at about 1 to about 1000 mg/ml total volume.

7. The method of claim 6, wherein the quaternary ammonium compound and/or ganglionic blocking agent is present at about 50 to about 250 mg/ml total volume.

8. The method of claim 5, wherein said pharmaceutically acceptable carrier is a hydrophilic ointment base.

9. The method of claim 8, wherein the quaternary ammonium compound and/or ganglionic blocking agent is present at about 0.01% to about 99% of volume.

10. The method according to claim 1, wherein the quaternary ammonium compound and/or ganglionic blocking agent is present in an amount effective in diminishing the replication or infectivity of HHV-6.

11. The method of claim 10, wherein said quaternary ammonium compound is selected from the group consisting of tetraethylammonium ion, hexamethonium ion, pentolinium ion, chlorisondamine ion, trimethidinium ion, trimethaphan ion, and homologs thereof.

12. The method according to claim 11, wherein the quaternary ammonium compound is administered at from about 5 to about 7 mg/kg body weight.

13. The method according to claim 11, wherein the quaternary ammonium compound is administered at up to about 20 mg/kg body weight.

14. A method for treating human herpesvirus-6 infection in an animal, comprising administering a therapeutically effective amount of a composition comprising a tetraethylammonium halide.

15. The method according to claim 14, wherein the tetraethylammonium halide is present in an amount effective in diminishing the replication or infectivity of HHV-6.

16. The method according to claim 14 or 15, wherein the tetraethylammonium halide is administered at from about 5 to about 7 mg/kg body weight.

17. The method according to claim 14 or 15, wherein the quaternary ammonium compound is administered at up to about 20 mg/kg body weight.

18. A method for treating human herpesvirus-6 infection in an animal, comprising administering a therapeutically effective amount of a composition comprising mecamylamine.

19. The method according to claim 18, wherein mecamylamine is present in an amount effective in diminishing the replication or infectivity of HHV-6.

20. The method according to claim 18 or 19, wherein mecamylamine is administered at from about 2.5 to about 5.0 mg/day.

21. The method according to claim 18 or 19, wherein mecamylamine is administered at up to about 25 mg/day.

22. A method for preventing the infection of an animal by human herpesvirus-6 (HHV-6) , comprising administering a therapeutically effective amount of a composition comprising a quaternary ammonium compound and/or a ganglionic blocking agent.

23. The method of claim 22, wherein said quaternary ammonium compound is a ganglionic blocking agent.

24. The method of claim 22, wherein the animal is a human.

25. The method of claim 22, wherein the composition is administered orally, parenterally, topically, or by a combination thereof.

26. The method of claim 25, wherein said composition further comprises a pharmaceutically acceptable carrier.

27. The method of claim 26, wherein a liquid diluent is employed as the pharmaceutically acceptable carrier, and wherein the quaternary ammonium compound and/or ganglionic blocking agent is present at about 1 to about 1000 mg/ml total volume.

28. The method of claim 27, wherein the quaternary ammonium compound and/or ganglionic blocking agent is present at about 50 to about 250 mg/ml total volume.

29. The method of claim 26, wherein said pharmaceutically acceptable carrier is a hydrophilic ointment base.

30. The method of claim 29, wherein the quaternary ammonium compound and/or ganglionic blocking agent is present at about 0.01% to about 99% of volume.

31. The method of claim 22, wherein the quaternary ammonium compound and/or ganglionic blocking agent is present in an amount effective in diminishing the replication or infectivity of HHV-6.

32. The method of claim 31, wherein said quaternary ammonium compound is selected from the group consisting of tetraethylammonium ion, hexamethonium ion, pentolinium ion, chlorisondamine ion, trimethidinium ion, trimethaphan ion, and homologs thereof.

33. The method of claim 32, wherein said quaternary ammonium compound comprises a tetraethylammonium halide.

34. The method of claim 31, wherein said ganglionic blocking agent is mecamylamine.

35. The method of claim 32 or 33, wherein the quaternary ammonium compound is administered at from about 5 to about 7 mg/kg body weight.

36. The method of claim 32 or 33, wherein the quaternary ammonium compound is administered at up to about 20 mg/kg body weight.

37. The method of claim 34, wherein mecamylamine is administered at from about 2.5 to about 5.0 mg/day.

38. The method of claim 34, wherein mecamylamine is administered at up to about 25 mg/day/orally.