WO1996025163A1 - Treatment of herpes simplex viruses - Google Patents

Abstract

Both herpes simplex viruses (HSV-1 and HSV-2) produce a variety of infections involving mucocutaneous surfaces, the central nervous system, and occasionally visceral organs. HSV is a neurotrophic virus: following initial infection, the HSV may remain dormant for long periods of time within the cell bodies of neurons of the trigeminal ganglion. Periodically the virus reactivates, travelling down the branches of the trigeminal nerve to the ends, where it causes painful and unsightly skin lesions, or into the central nervous system or viscera, where it may produce debilitating or life-threatening tissue damage; administration of β-adrenergic antagonists, or α-adrenergic agonists, blocks reactivation of HSV, and thus can prevent recurrence of HSV infection.

Description

TREATMENT OF HERPES SIMPLEX VIRUSES

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to a method for treating Herpes Simplex Virus (HSV) by administering to an infected mammal a therapeutically effective amount of an adrenergic receptor ligand. More specifically, this invention relates to the use of or-adrenergic receptor agonists or _/3-adrenergic receptor antagonists, or salts thereof, in suppressing and inhibiting the reactivation of HSV in humans.

2. Description of the Related Art

HSV infections are pandemic: over 90 percent of adults have antibodies to HSV-1 by the age of 50; in lower socioeconomic populations, most persons will acquire HSV-1 infection before the age of 30. HSV-2 primarily infects the genitalia and can be acquired by newborns through contact with the birth canal; in adults, HSV-2 is most commonly acquired during sexual activity. Transmission of HSV results from contact with ulcerative lesions or asymptomatically excreting patients; asymptomatic salivary excretion of HSV-1 has been reported in 2-9% of adults and 5-8% of children; patients infected with HSV-2 also excrete virus. Exposure to HSV at mucosal surfaces or abraded skin permits entry of virus and initiation of replication in cells of the epidermis and der is. Whether or not clinically apparent lesions develop, sufficient viral replication to permit infection of either sensory or autonomic nerve endings may occur. Virus—or, more likely, nucleocapsid-is then thought to be transported intraaxonally to the nerve cell bodies in ganglia, particularly the trigeminal ganglia. During the initial phase of infection, viral replication occurs in ganglia and contiguous neural tissue. Virus then spreads to other mucosal skin surfaces through centrifugal migration of infectious virions via peripheral sensory nerves.

Following resolution of primary disease, infectious virus can no longer be recovered in the ganglia. However, this does not appear to be due to the death of HSV-infected neurons. Instead, viral genomes are maintained by the cell in a repressed state, compatible with survival and normal activities of the cell, a process called latency. Subsequently, activation of the viral genome may occur, resulting in viral replication and, in some cases, the redevelopment of herpetic lesions, a process called reactivation. The mechanisms by which various stimuli cause reactivation of HSV infection are not known. Ultraviolet light, immunosuppression, and trauma to the skin or ganglia are associated with reactivation.

The clinical manifestations of HSV most frequently include gingivostomatitis and pharyngitis during the first episode of the infection. Recurrent herpes labialis is the most frequent clinical manifestation of reactivation HSV infection. Reactivation of HSV from the trigeminal ganglia may also be associated with asymptomatic excretion in the saliva, development of intraoral mucosal ulcerations, or herpetic ulcerations on the vermilion border of the lip or external facial skin. However, HSV also commonly infects the eye and central nervous system. Genital HSV infection causes vaginal and penile lesions and also asymptomatic cervical lesions and urogenital disease.

HSV infection of the eye is the most frequent cause of corneal blindness in the United States. HSV keratitis presents with acute onset of pain, blurring of vision, chemosis, conjunctivitis, and characteristic dendritic lesions of the cornea. The use of topical corticosteroids may exacerbate symptoms and lead to involvement of deep structures of the eye. Debridement, topical antiviral treatment, and/or interferon therapy hastens healing. However, recurrences are common, and immunopathologic injury of the deeper structures of the eye may occur. Chorioretinitis, usually as a manifestation of disseminated HSV infection, may occur in neonates or in patients with HIV infection. Acute necrotizing retinitis due to HSV is an uncommon but severe manifestation of HSV infection. HSV encephalitis is the most common identified cause of acute, sporadic viral encephalitis in the United States, comprising 10-20% of all cases. Most adults with HSV encephalitis have clinical or serologic evidence of mucocutaneous HSV-1 infection prior to the onset of the CNS symptoms. Two theories have been proposed to explain the development of actively replicating HSV in localized areas of the CNS. Reactivation of latent trigeminal or autonomic nerve root HSV-1 infection may be associated with extension of the virus into the CNS via nerves innervating the middle cranial fossa. HSV DNA has been demonstrated by DNA hybridization in human autopsy brain tissue. Reactivation of long-standing CNS infection may be another potential mechanism for the development of HSV encephalitis. Antiviral chemotherapy reduces the mortality of HSV encephalitis. Even with therapy, however, neurologic sequelae are frequent, especially in those over 35 years of age.

HSV-2 is latent in the sacral ganglia; on reactivation the virus causes genital recurrences and cutaneous disease of the urogenitals and lower limbs. Neonates ( < 6 weeks of age) have the highest frequency of visceral and/or CNS infections of any HSV-infected patient population; neonatal herpes infection is commonly caused by HSV-2. Untreated, over 70 percent of neonatal herpes cases will disseminate or develop into CNS infection. Without therapy, the overall mortality of neonatal herpes is 65 percent, and less than 10 percent of neonates with CNS infection experience normal development. While skin lesions are the most commonly recognized features of the disease, many infants do not develop lesions until well into the course of the disease. Neonatal HSV infections may be acquired from genital HSV-2 infection in the mother, through postnatal contact with immediate family members with symptomatic or asymptomatic oral-labial HSV-1 infection, or from nosocomial transmission within the hospital. Antiviral chemotherapy has reduced the mortality of neonatal herpes to 25% ; however, morbidity remains high. Many aspects of mucocutaneous and visceral HSV infections are amenable to antiviral chemotherapy. For mucocutaneous infections, acyclovir has been the mainstay of therapy. Several antivirals are available for topical use in HSV eye infections: idoxuridine, trifluorothymidine, and topical vidarabine. For HSV encephalitis, intravenous acyclovir is the treatment of choice. For neonatal HSV infections, high-dose intravenous vidarabine and acyclovir are effective.

Treatment of HSV and human cytomegalovirus (HCMV) with calcium- channel blockers has also been suggested (Albrecht et al U.S. Patents 4,663,317, 4,782,065, 4,800,081, and 4,849,412). Calcium channel blockers, when administered in combination with cyclic nucleotide modulators, are reported to inhibit the expression of HCMV and HSV.

Acyclovir has been shown to be effective in shortening the duration of symptoms and lesions of mucocutaneous HSV infections in immune-compromised patients and first-episode genital herpes in immunocompetent patients. Intravenous and oral acyclovir also will prevent reactivation of HSV in seropositive immunocompromised patients during induction chemotherapy for acute leukemia or in the period immediately following bone marrow transplantation.

Oral acyclovir also has been shown to speed the healing and resolution of symptoms in first and recurrent episodes of genital HSV-1 infection. The benefit of treating acute episodes of recurrent genital disease with oral acyclovir is modest, and thus routine use for recurrent episodes of disease, especially for mild episodes, is not recommended. Chronic daily suppressive therapy reduces the frequency of reactivation disease among patients with frequent genital herpes. Chronic suppressive oral acyclovir does not eliminate sacral ganglionic latency, and reactivation of genital herpes occurs after discontinuation of therapy. Oral acyclovir is of benefit in primary gingivostomatitis but has limited benefit in the treatment of recurrent oral-labial lesions; lesions are not aborted and total healing time is not reduced. If started early, the duration of the ulcerative stage of the lip lesion may be reduced. The major side effect associated with intravenous acyclovir is transient renal insufficiency, usually due to crystallization of the compound in renal parenchyma. This can be avoided if the medication is given slowly over 1 hour and the patient is well-hydrated. Acyclovir-resistant strains are being identified with increasing frequency, especially in HIV-infected persons. Almost all clinically significant acyclovir resistance has been seen in immunocompromised patients who have received multiple intermittent courses of therapy. The frequent reactivation of virus and high virus titers in the lesions of immunocompromised patients in combination with the use of the medication selects out these resistant variants. Most acyclovir-resistant strains of HSV have altered substrate specificity for phosphorylating acyclovir. Thus intracellular levels of acyclovir triphosphate are low. In some patients, higher doses of acyclovir will be associated with clearing of lesions. In others, clinical disease will progress despite high-dose therapy. Isolation of HSV from persisting lesions despite adequate dosages and blood levels of acyclovir should raise the suspicion of acyclovir resistance. Therapy with the antiviral drug foscarnet may be effective against acyclovir-resistant HSV; however, because of its toxicity and cost, this drug is usually reserved for patients with extensive mucocutaneous infections. Effective HSV vaccines are not currently available. Heterologous vaccines such as smallpox, bacillus Calmett-Guerin, and influenza, which have been used as therapies for genital HSV infection, have been ineffective.

Therefore, in view of the aforementioned deficiencies attendant with prior art methods of treating HSV, it should be apparent that there still exists a need in the art for a safe, effective treatment for HSV. SUMMARY OF THE INVENTION

Accordingly, a major object of the present invention is to provide a method of preventing the reactivation of dormant HSV, in particular to prevent the reactivation of HSV-1 and HSV-2.

In general, this invention relates to a method of inhibiting the reactivation of dormant HSV, in particular dormant HSV-1 and HSV-2, by administering an adrenergic receptor ligand, for example, an α-adrenergic receptor agonist or β- adrenergic receptor antagonist, to a previously infected mammal. A further aspect of this invention is an anti-HSV composition comprising at least one adrenergic receptor ligand selected from an -adrenergic receptor agonist and a /-.-adrenergic receptor antagonist; and a pharmaceutically-acceptable carrier therefor.

With the foregoing and other objects, advantages and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the preferred embodiments of the invention and to the appended claims.

BRIEF DESCRDTTION OF THE DRAWINGS

Fig. 1. Slot blot comparison of viral DNA in the ganglia of propranolol- treated and control mice. Lane A: Viral DNA in the ganglia of a propranolol- treated mouse. Lane B: Viral DNA in the ganglia of a control mouse.

Fig. 2. Recurrences of ocular herpetic lesions in rabbit eyes over the 30- day observation period. DETAD ED DESCRIPTION OF THE PREFERRED EMBODIMENTS

OF THE INVENTION

This invention arose from a desire of the inventors to improve on previously available methods of treating HSV infection. More specifically, the inventors sought to provide a safe, effective treatment for HSV, in particular a treatment which would prevent or inhibit the reactivation of HSV in an infected mammal.

It is generally known in the art that a patient, in particular a mammal, including a human, that has been infected by HSV carries this virus in a latent state in cells in the nervous system. A variety of stimuli, including stress, will initiate viral reactivation in the nervous system. This reactivation can result in recurrent infection in the skin, eyes, and other areas of the body. The mechanisms whereby physical and chemical stimuli initiate viral reactivation, however, are not well understood by those skilled in the art.

The inventors have found that administration of adrenergic receptor ligands, in particular α-adrenergic receptor agonists or 0-adrenergic receptor antagonists, effectively blocks reactivation of dormant HSV. The phrases "receptor ligands", "receptor agonists" , and "receptor antagonists" used herein are understood to refer to pharmacologically active compounds, and to salts thereof. Preferred α-agonists include agmatine, /7-aminoclonidine, clonidine, iodoclonidine, guanabenz, lopidine, methoxamine, norepinephrine, octopamine, oxymetazoline, phenylephrine, xylazine, UK 14,304, and pharmaceutically acceptable salts thereof. Preferred non-selective -.-antagonists include alpenolol, pindolol, or propranolol, and pharmaceutically acceptable salts thereof.

Preferred selective /_?, -antagonists include atenolol, timolol, CGP20712A (i.e. , 4- [3-(l , l-dimethylethyl)amino]-2-hydroxypropoxy]-l,3-dihydro-2H-benzimidazol-2- one), and pharmaceutically acceptable salts thereof. Preferred selective β₂- antagonists include ICI-118,551 (i.e., (±)-l-[2,3-(dihydro-7-methyl-lH-inden-4- yl)oxyl-3-[(l-methylethyl)amino]2-butanol), and pharmaceutically acceptable salts thereof. Particularly preferred adrenergic receptor ligands include propranolol and timolol. These compounds have been found by the present inventors to inhibit reactivation of HSV.

As used herein, the α-adrenergic receptor agonists or 3-adrenergic receptor antagonists contemplated by the present invention include derivatives of those known in the art, in particular, the above-identified compounds, having any substitutions which do not eliminate or significantly reduce their ability to bind to α-adrenergic receptors or 3-adrenergic receptors. For example, the compounds of the present invention are optionally substituted with a functional group. Any art- recognized functional group which does not eliminate or significantly reduce the compound's ability to bind to α-adrenergic receptors or /3-adrenergic receptors are contemplated, including, but not limited to, ester, amide, acid, amine, alcohol, ether, thioether, etc. Solvates, e.g., hydrates of the compounds useful in the methods of the present invention, are also included within the scope of the present invention. Methods of solvation to produce such solvates are generally known in the art.

Pharmaceutical salts of the α-adrenergic receptor agonists and β- adrenergic receptor antagonists suitable for administration by a variety of routes are known in the art and need not be described herein in detail. Examples of pharmaceutically acceptable salts of the compounds and derivatives thereof according to the invention, include base salts, e.g., derived from an appropriate base, such as alkali metal (e.g. , sodium), alkaline earth metal (e.g. , magnesium), ammonium, and NW_nH_m bases and salts wherein each of n and m are 0 to 4 and n + m is 4, and wherein W is a (C,-C₁₈)alkyl. Pharmaceutically acceptable salts of an acid group or an amino group include, but are not limited to, salts of organic carboxylic acids such as acetic, lactic, tartaric, malic, isothionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and -tolylsulfonic acids, and inorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamic acids. Pharmaceutically- acceptable salts of a compound with a hydroxy group include, but are not limited to, the anion of the compound in combination with a suitable cation such as Na⁺, and NW_nH_m, wherein W is a (C,-C,_g)alkyl group, and n and m are 0 to 4, and n+m is 4.

A still further part of this invention is a pharmaceutical composition of matter for treating or preventing reactivation of HSV that comprises at least one of the α-adrenergic receptor agonists or jS-adrenergic receptor antagonists described above, mixtures thereof, and/or pharmaceutical salts thereof, and a pharmaceutically-acceptable carrier therefor. Such compositions are prepared in accordance with accepted pharmaceutical procedures, for example, as described in Remington's Pharmaceutical Sciences, seventeenth edition, ed. Alfonso R. Gennaro, Mack Publishing Company, Easton, PA (1985).

For therapeutic use in a method of inhibiting reactivation of HSV, an α- adrenergic agonist or 3-adrenergic antagonist, or its salt, can be conveniently administered in the form of a pharmaceutical composition containing an α- adrenergic agonist or /3-adrenergic antagonist, or its salt, and a pharmaceutically acceptable carrier therefor. Suitable carriers are well known in the art and vary with the desired form and mode of administration of the pharmaceutical composition. For example, they may include diluents or excipients such as fillers, binders, wetting agents, disintegrators, surface-active agents, lubricants, and the like. Typically, the carrier may be a solid, liquid, or vaporizable carrier, or combinations thereof. In one preferred embodiment, the composition is a therapeutic composition and the carrier is a pharmaceutically acceptable carrier.

The compound of the invention or its salt may be formulated together with the carrier into any desired unit dosage form. Typical unit dosage forms include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories; injectable solutions and suspensions are particularly preferred.

Each carrier must be "acceptable" in the sense of being compatible with the other ingredients in the formulation and not injurious to the patient. The carrier must be biologically acceptable and inert, i.e., it must permit the cell to conduct its metabolic reactions so that the compound of this invention may effect its inhibitory activity.

Formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and transdermal) administration, with topical ointment formulations, and formulations appropriate for oral administration, being preferred.

For example, to prepare formulations suitable for injection, solutions and suspensions are sterilized and are preferably isotonic to blood. In making injectable preparations, carriers which are commonly used in this field can also be used, for example, water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitate esters. In these instances, adequate amounts of isotonicity adjusters such as sodium chloride, glucose or glycerin can be added to make the preparations isotonic. The aqueous sterile injection solutions may further contain anti-oxidants, buffers, bacteriostats, and like additions acceptable for parenteral formulations.

The formulations may conveniently be presented in unit dosage form and may be prepared by any method known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which may encompass one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. Various unit dose and multidose containers, e.g., sealed ampules and vials, may be used, as is well known in the art.

In addition to the ingredients particularly mentioned above, the formulations of this invention may also include other agents conventional in the art for this type of pharmaceutical formulation. The compound of the invention may be present in the composition in an broad proportion to the carrier. For instance, the compound may be present in the amount of 0.01 to 99.9 wt% , and more preferably in about 0.1 to 99 wt%. Still more preferably, the compound may be present in an amount of about 1 to 70 wt% of the composition.

Also part of this invention is a method of treating HSV, or inhibiting the reactivation of HSV, in an infected mammal by treating infected neurons in that mammal with an effective amount or an anti-reactivation effective amount of an α-adrenergic receptor agonist or 3-adrenergic receptor antagonist. In this application, "treating" will encompass any means by which the compound of this invention contacts the neural circuitry and neurochemical mediators of viral infection or viral reactivation. Also, in this application "patient" will encompass any mammal in need of antiviral treatment, particularly a mammal infected with a HSV, more particularly a mammal infected with HSV-1 or HSV-2. An HSV-infected neuron is preferably treated with an α-adrenergic receptor agonist or /-.-adrenergic receptor antagonist at a preferred concentration of about 0.1 μM to 10 mM, more preferably about 0.1 to 1 mM, and still more preferably about 10 to 500 μM.

The invention also provides a method of inhibiting the reactivation of HSV in an infected patient comprising administering to the patient an effective amount of an α-adrenergic receptor agonist or β-adrenergic receptor antagonist, pharmaceutically acceptable salts thereof, or mixtures thereof.

The dosage of the α-adrenergic receptor agonists or β-adrenergic receptor antagonists, pharmaceutically acceptable salts thereof, or mixtures thereof, in the compositions of the invention administered to a patient will vary depending on several factors, including, but not limited to, the age, weight, and species of the patient, the general health of the patient, the severity of the symptoms, whether the composition is being administered alone or in combination with other antiviral agents, the incidence of side effects and the like. In general, a dose suitable for application in the treatment of HSV infection is about 0.001 to 100 g/kg body weight/dose, preferably about 0.01 to 60 mg/kg body weight/dose, and still more preferably about 0.1 to 40 mg/kg body weight/dose per day. The desired dose may be administered as 1 to 6 or more subdoses administered at appropriate intervals throughout the day. The compounds may be administered repeatedly over a period of months or years, or it may be slowly and constantly infused to the patient. Higher and lower doses may also be administered.

The daily dose may be adjusted taking into account, for example, the above-identified variety of parameters. Typically, the present compositions may be administered in an amount of about 0.001 to 100 mg/kg body weight/day. However, other amounts may also be administered.

To achieve good plasma concentrations, the active compounds may be administered, for instance, by intravenous injection of an approximate 0.1 to 1 % solution of the active ingredient, optionally in saline, or orally administered as a bolus.

The active ingredient may be administered for therapy by any suitable routes, including topical, oral, rectal, nasal, vaginal and parenteral (including intraperitoneal, subcutaneous, intramuscular, intravenous, intradermal, and transdermal) routes. It will be appreciated that the preferred route will vary with the condition and age of the patient, the nature of the disorder and the chosen active ingredient including other therapeutic agents. Preferred is the oral route. Also preferred is the topical route. However, other routes may also be utilized depending on the conditions of the patient and how long-lasting the treatment is. While it is possible for the active ingredient to be administered alone, it is preferably present as a pharmaceutical formulation. The formulations of the present invention comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof and optionally other therapeutic agents. The above method may be practiced by administration of the compounds by themselves or in a combination with other active ingredients, including antiviral compounds and/or therapeutic agents in a pharmaceutical composition. Other therapeutic agents suitable for use herein are any compatible drugs that are effective by the same or other mechanisms for the intended purpose, or drugs that are complementary to those of the present agents. These include agents that are effective for the treatment of viral infections and/or associated conditions in humans. Examples are acyclovir, vidarabine, idoxuridine, trifluorothymidine, and foscarnet, among others. The compounds utilized in combination therapy may be administered simultaneously, in either separate or combined formulations, or at different times than the present compounds, e.g., sequentially, such that a combined effect is achieved. The amounts and regime of administration will be adjusted by the practitioner, by preferably initially lowering their standard doses and then titrating the results obtained. The therapeutic method of the invention may be used in conjunction with other therapies as determined by the practitioner.

Having now generally described this invention, the same will be better understood by reference to certain specific examples, which are included herein for purposes of illustration only and are not intended to be limiting of the invention or any embodiment thereof, unless so specified.

EXAMPLE 1: Propranolol Suppresses Reactivation of Herpesvirus

Several lines of investigation suggest that sympathomimetic amines may modulate viral reactivation in the nervous system (Kwon et al. Invest.

Ophthalmol. Vis. Sci. 21 :442-449, 1981; Laibson et al. Arch Ophthalmol. 75:254-260, 1966; Nesburn et al. Proc. Soc. Exp. Biol. Med. 172:316-323, 1983). Therefore, we conducted experiments to determine if a 3-adrenergic receptor blocker, propranolol, could affect the incidence of viral reactivation following heat stress induction in mice. MΛTER1ALS AND METHODS Animals.

Female BALB/c strain mice, 5 to 6 weeks of age were obtained from the Jackson Laboratories, Bar Harbor, ME. Throughout these experiments the animals were handled in accordance with the NIH guidelines on the care and use of animals in research.

Virus.

The McKrae strain of HSV- 1 was propagated in Vero cells and the plaque forming units/ml (PFU) were determined using CV-1 cells. At the time of inoculation, virus was diluted to a concentration of 3 x 10⁵ PFU/ml in tissue culture medium and used immediately to infect the animals.

Experimental design. Mice were infected with the McKrae strain of HSV-1 by topical ocular application. The corneas of the mice were gently scratched in a crosshatched pattern with a 25-gauge needle and each eye received a 25-μl volume of the virus suspension at a concentration of 3 x 10⁵ PFU/ml. Following inoculation, the eyelids were gently held closed for 10 seconds. For the controls, the corneas were scratched and a 25-μl drop of diluent not containing virus was applied using the same procedure.

Viral infection was documented by visual examination 3, 5, and 7 days after infection. The characteristic clinical signs of photophobia, blinking, and periocular exudate were seen in all infected animals. (Deaths due to encephalitis resulted in a loss of 10% or less of the infected animals). Uninfected animals did not exhibit these clinical signs. Thirty-five days after primary infection, the animals were randomly divided into groups for use in experiments.

Infected and uninfected animals were given intraperitoneal injections of 0.1 ml of propranolol at a concentration of 1 mg/kg of body weight or saline (controls) on two consecutive days. On the following day, one group of infected and one group of uninfected animals were subjected to hypothermia (immersion in 43° temperature water for 10 minutes) (Sawtell et al., J. Virol. 66:2150-2156, 1992), after which the propranolol-treated and saline-treated (control) animals were given one additional intraperitoneal injection. Twenty-four hours later all animals were anesthetized by intraperitoneal injection of a xylazine-ketamine mixture, the ocular surface of each eye was swabbed, and the swabs placed into culture. The animals were sacrificed and tissues removed for analysis. The corneas and trigeminal ganglia of each animal were processed separately. The experiment was repeated three times.

Ocular swab assay for infectious virus.

CV-1 cells were subcultured into 24-well tissue culture plates. The cultures were incubated until confluence was achieved and then the plates were used to assay for infectious virus. Each ocular surface swab was gently swirled in the culture medium in one well of a 24-well plate. The plates were incubated and observed for 21 days and the occurrence of cytopathic effect (CPE) was recorded by an unbiased observer.

Assay qfcorneal and trigeminal ganglionic tissue for infectious virus. Tissues were homogenized in 0.2 ml of complete tissue culture medium consisting of RPMI-1640 containing 10% fetal bovine serum supplemented with antibiotics. Following homogenization, the homogenate was diluted to 1 ml, centrifuged at 12,000 xg for 5 min, and the supernatant plated onto CV-1 indicator cells. All cultures were observed daily for 21 days for the appearance of cytopathic effect (CPE).

Assay of ganglionic tissue for viral DNA.

The pellets obtained from the ganglion homogenates were immediately resuspended in DNA extraction buffer containing proteinase K and detergent and immediately processed for DNA extraction, slot-blotting, and Southern hybridization (Javier et al., J. Virol. 61: 1978-1986, 1987; Southern, J. Mol. Biol. 98: 503-517, 1975; Thompson et al., J. Virol. 55:504-508, 1985). Pairs of trigeminal ganglia from propranolol-treated and saline-treated control mice were separately homogenized in DNA extraction buffer and the DNA collected for quantitation by spectrophotometry at 260 nm, slot-blotting, and hybridization with a biotinylated probe complementary to the viral DNA polymerase gene. The hybridized probe was detected using a streptavidin alkaline phosphatase conjugate and the adamantyl-disodium 3-(4-methoxyspiro[l,2-dioxetane-3,2- tricyclo[3.3.1.1]decan]-4-yl)phenyl phosphate (CSPD, Tropix, Bedford, MA) substrate.

RESULTS

Effect of propranolol treatment on viral reactivation.

In all three experiments, fewer of the propranolol-treated animals had infectious virus in the precorneal tear film 24 hours after induction of reactivation by hyperthermia, compared with the saline-treated control animals (Table 1).

No difference in the time of appearance of CPE was noted in cultures derived from the propranolol-treated and saline-treated controls. Neither infected animals that were not stressed nor uninfected animals had virus in the precorneal tear film. Similarly, fewer corneal and trigeminal ganglionic cultures from propranolol-treated animals contained infectious virus, compared with the tissues from saline-treated control animals (Table 1). Again, infectious virus was not found in the corneal and ganglionic homogenates of infected stressed animals or uninfected animals. Table 1

Numbers of cultures from the ocular surface, corneal tissue, and trigeminal ganglionic tissue that were positive for herpes simplex virus.

Infectious Virus (No. Positive/Total)

Treatment Groups Experiment 1 Experiment 2 Experiment 3

Ocular Surface

+ propranolol 4/14 (29%) 5/11* (45%) 3/12 (25%)

- propranolol (control) 4/8 (50%) 8/1 T (73%) 8/12 (67%) Cornea

+ propranolol 5/14 (36%) 6/l l^a (55%) 4/12 (33%)

- propranolol (control) 5/8 (63%) 9/l l^a (82%) 9/12 (75%) Trigeminal ganglia

+ propranolol 6/14 (43%) 7/12 (64%) 5/12 (42%)

- propranolol (control) 6/8 (75%) 10/12 (83%) 10/12 (83%)

All of these swabs of the ocular surface and corneal and trigeminal ganglionic tissues were obtained from HSV-1-infected, hyperthermia-stressed mice. Chi-square analysis of the results in each experiment indicated that there was a statistically significant difference between the numbers of virus-positive cultures from the propranolol-treated animals compared with cultures from the saline-treated control animals (p < 0.05). No virus was found in any of the cultures from infected, unstressed mice or from uninfected mice, The denominator represents the number of swabs/tissue samples followed for 21 days.

* One culture from this group became infected with bacteria or fungus during the 21 -day observation period and was eliminated from the analysis

Viral DNA in the ganglia of propranolol-treated and control animals.

In a comparative analysis of viral DNA in the trigeminal ganglia of propranolol-treated and saline-treated control mice following heat stress-induced reactivation, Southern hybridization of ganglionic DNA extracts revealed a more intense hybridization signal in the extracts of ganglionic DNA from the control mice, compared with the propranolol-treated animals (Fig. 1). Densitometric readings were taken over the areas of the strongest hybridization signals for the DNA from the propranolol-treated and untreated ganglia shown in Fig. 1. The signal density was at least four times higher for the DNA from the untreated ganglion, compared with the DNA from the propranolol-treated ganglion. Analysis of ganglionic DNA from two additional experiments gave similar results; there was a two- to four- fold stronger signal in the untreated ganglion DNA sampled, compared with the propranolol-treated ganglion DNA.

EXAMPLE 2: Propranolol Suppression of Ocular Herpetic Recurrences in the Rabbit

In Example 1, above, we observed that injections of the /---antagonist propranolol in mice significantly reduced the likelihood of recurrences of herpes after the mice were placed in warm water. The purpose of this example is to demonstrate that propranolol will reduce spontaneous recurrences of ocular herpes in rabbits.

MATERIALS AND METHODS

Animals

Mixed male and female New Zealand white rabbits were handled in accordance with the NIH guidelines on the care and use of animals in research, the Institutional Animal Care and Use Committee of the LSU Medical Center in New Orleans, and the Association for Research in Vision and Ophthalmology

Statement for the Use of Animals in Ophthalmic and Vision Research. Virus

The McKrae strain of HSV-1 was propagated in primary rabbit kidney cells and had a titer of 1 x 108 PFU/ml. Twenty five microliters of undiluted virus suspension was used to infect the rabbit corneas.

Viral Cultures

All viral cultures were taken prior to the placement of fluorescein into the eye. A sterile Dacron-tipped swab was placed into the lower conjunctival cul-de- sac with care being taken to avoid touching the cornea. The swab was placed directly into a tube of primary rabbit kidney cells containing Earle's Minimal Essential Medium and allowed to remain there for 24 hours before being removed. The cells were incubated at 370 C until cytopathic effect(CPE) was noted. Blind passage was done of all cultures which did not show herpetic CPE.

Experimental Design

The corneas of 50 New Zealand white rabbits were anesthetized with topical proparacaine hydrochloride (Alcaine, Alcon, Humacao, PR) and lightly scratched with a 27 gauge needle. Twenty five microliters of the virus suspension was placed on each cornea, and the eyelids gently rubbed over the cornea for 10 seconds. Three days after infection, fluorescein (Fluor-1-Strip, Wyeth-Ayerst, Philadelphia, PA) was instilled into the eye, and, using the slit lamp, the presence of dendritic keratitis verified in all corneas. Starting at 19 days after infection, the corneas were examined for the presence of herpetic keratitis. The corneal examinations were performed 18 weekdays from the 19th to the 49th day after infection. On the 21st day after infection, 31 animals were randomized into two coded groups. One group was given intramuscular injections of sterile water twice a day at 7:30 am and at 5:00 pm; the second group was given intramuscular injections twice a day of 1 mg/kg propranolol in water for 30 days. Week day cultures of the conjunctival cul-de-sac were also started on day 19. All corneal evaluations and cultures were performed by persons without the knowledge of the treatments.

Statistical Analyses The outcome variable was recurrence days, or the count of days during the period of the experiment during which a specific herpetic lesion (dendrite, punctate lesion, stromal edema with keratic precipitate) was evident. Corneas with scars and persistent ulcers were excluded from the analysis. All data were entered into computer files and analyzed using Statistical Analysis System programs and procedures (SAS Institute, Cary, NC). A nested design in the analysis of variance was applied to analyze the number of recurrence days for the placebo-treated animals compared to the propranolol-treated animals. Rabbits within-treatment was the nested term, and treatment was the main effect. All comparisons were conducted at an alpha level of 0.05, and at a power of 80%.

RESULTS

The rabbits were examined two times prior to the initiation of treatment and 16 times during the treatment period and cultured 16 times during the treatment period. Only four positive viral cultures were obtained during the entire treatment period, which was not considered significant enough to warrant statistical analysis. This low level of culturable virus was probably due to the very gentle technique used to preclude corneal damage.

Thirty one rabbits were included at the start of treatment, and 24 were present at the completion. One rabbit developed dense stromal scars and heavy neovascularization in both corneas and was euthanized during the treatment period; the other animals died from either encephalitis or upper respiratory herpetic infections. There were 39 recurrence days in the propranolol-treated animals and 57 recurrence days in the water-treated animals. (Table 2, Figure 2). TABLE 2. Statistical Analysis of Recurrences

Treatment N Mean ± S.E.M. P Value*

Control 434 0.142 ± 0.0168 0.0079

Propranolol 349 0.094 ± 0.0156 *Resιult of a 1 degree of freedom F test on the two levels of treatment (control vs. propranolol) in the ANOVA described in the Statistical Methods section. Variable: Mean number of eyes with lesions over all experimental days. N represents total eyes over all days of study.

COMMENT In this model, propranolol significantly reduced the number of days the eyes showed signs of herpetic infection. We think that it is likely that in susceptible animals in which a continued supply of virus from the ganglion prolongs surface infection, that propranolol reduced viral multiplication in the ganglion. In Example 1, above, the quantitative PCR method to measure viral DNA in the trigeminal ganglia of mice subjected to thermal stress clearly showed that propranolol reduced the appearance of virus in the ganglion as well as the appearance of virus that can be cultured either from the tears or from the ocular tissue.

EXAMPLE 3: Stress-related Recurrences of Herpes are Adrenergically

Mediated and Blocking This Mediator Reduces Recurrences

We have found that propranolol, a non-selective /3-receptor blocker, can reduce HSV-1 recurrences that occur after thermal stress in mice (Example 1 , above) and reduces spontaneous recurrences in rabbits (Example 2, above). We will further define the receptor-mediated mechanisms of this phenomenon by determining: (1) whether other /-.-blockers prevent stress-induced herpes to be certain that this is not a property peculiar to propranolol; (2) whether selective 0-1 , β-2, and α-receptor blockers, such as the less selective epinephrine, the β-stimulator norepinephrine, and the longer acting ephedrine, prevent recurrences and which are most important, and (3) whether the α- agonist, lopidine, can initiate recurrences. It is possible that in these receptors, as in glaucoma, α-agonists might have an effect similar to the /-.-blocking effect they have in glaucoma.

OBJECTIVE

Examples 1 and 2, above, elucidate an efficient new model for studying HSV-1 reactivation in experimental animals. We have capitalized on a system for stimulating viral reactivation and the initiation of viral DNA synthesis in mice, and have shown that the 0-adrenergic blocker, propranolol, has an inhibitory effect on viral reactivation in this model. Based on our findings with propranolol, the following studies using 0-1 and β-2 receptor selective antagonists in a variety of treatment regimens and protocols in order to further dissect the role of adrenergic blockade in viral reactivation.

DESIGN

The basic design of these experiments is similar to Examples 1 and 2, above. Groups of mice, usually 40 animals per group, are infected with the McKrae strain of HSV-1. Forty days after primary infection, when latency has been established, the animals are used in experiments. In a typical experiment, the latent animals are divided into two groups of animals. One group of animals is treated with the /---adrenergic receptor blocker, propranolol, by intraperitoneal (IP) injection beginning 3 days before exposure to hyperthermia. The control animals receive saline injections IP. Three days after the initiation of the propranolol treatment, animals in the control, saline group, and the propranolol- treated group are further subdivided into two groups each. Half of the propranolol-treated animals are subjected to the hyperthermic stress paradigm of Sawtell and Thompson. Animals are immersed in 43°C water up to their necks for 10 minutes. After this time, the animals are rapidly dried and returned to their cages. In the typical experiment, a group of propranolol-treated and a group of control, untreated animals are exposed to the hyperthermic stress paradigm. In addition, a group of propranolol-treated animals and a control untreated group of animals are not subjected to the hyperthermic stress. Twenty- four hours after application of the stress, the animals are sacrificed and their ocular surfaces swabbed for viral culture, and their eyes and trigeminal ganglia collected for the analysis of infectious virus and viral DNA.

A flowchart of the experimental design is as follows: Day 0 . . . Infect mice with HSV-1 McKrae strain (40 mice per gram). Day 5 . . . Document corneal infection by slit lamp examination and ocular swab culture. Day 40 . . . Subdivide latent mice into two groups:

Group 1 - treat with propranolol IP Group 2 - treat with saline IP Day 41 . . . Repeat treatments of groups 1 and 2. Subdivide groups into:

Group 1A - treat with hyperthermic stress Group IB - no stress

Group 2A - treat with hyperthermic stress Group 2B - no stress Day 43 . . . Sacrifice animals In all groups, perform ocular surface swabs, analyze corneas, trigeminal ganglia for infections virus, viral DNA.

ANTICIPATED RESULTS AND INTERPRETATION

Groups of animals which have been treated with propranolol, the nonselective 3-receptor antagonist, and animals treated with β-l and β-2 selective antagonists will be sacrificed and their ocular surfaces tested for the presence of infectious virus 24 hours after exposure to hyperthermic stress. Infectious virus will be assayed by a viral co-culture on CV-1 indicator cells. The cell cultures will be observed for a period of 21 days for the appearance of cytopathic effect (CPE). The time of first appearance of the CPE and the number of cultures exhibiting this effect will be recorded and compared for the drug-treated and the control animals exposed to the hyperthermic stress and those animals that are not stressed.

At the time of sacrifice, the eyes and trigeminal ganglia of the animals in each group will be collected, and the corneas and trigeminal ganglia analyzed for both infectious virus and the amount of viral DNA in each tissue quantitated using a quantitative competitive-polymerase chain reaction. In this way, it will be possible to compare both the frequency of viral reactivation in terms of infectious virus and in terms of viral replication as reflected by viral DNA quantities in the tissues.

Throughout these studies, control animals will be employed to ensure that viral reactivation occurs with an acceptable frequency, and to ensure that there is a reproducible effect of the nonselective 0-receptor antagonists. Control animals include those not treated with the /-.-receptor antagonist and animals which were either treated with the antagonist or not, but were not subjected to the stress paradigm. Otherwise, throughout the experimental protocol these animals will be treated the same way as the experimental animals and will be sacrificed at the same time. Statistical analysis of the reactivation frequencies, time-course of reactivation and culture, the time-course of appearance of the CPE in culture, and the amount of viral DNA in the tissues will be performed.

RABBIT MODEL OF RECURRENT KERATITIS Objective

To determine whether specific blockers of adrenergic receptors change the frequency of recurrent herpetic keratitis.

Design

Recurrences of herpetic keratitis are compared in rabbits that have been infected by the ocular route with the McKrae strain of HSV-1. Rabbits are randomized into groups of 10 rabbits per group, one group of which is a placebo treated, concurrent control group.

Rationale New Zealand white rabbits infected by the corneal route experience spontaneous recurrences of herpetic keratitis starting approximately 21 days after infection. A minimum of 10 animals per treatment are necessary to obtain statistical significance, and a simultaneous control is necessary.

Methods

Both corneas of New Zealand white rabbits are anesthetized with proparacaine HCI, and the superficial corneal epithelium superficially traumatized with a 27 gauge needle using a # pattern in the center of the cornea. The corneas are examined three days after infection to verify the presence of herpetic keratitis by staining the corneas with fluorescein and examining the corneas with a slit lamp. Nineteen days after infection, daily examination for recurrent keratitis begins by an observer who is not aware of the treatment codes, and continues through thirty days of treatment. Twenty one days after infection, the animals are randomized into treatment groups.

Day 1 . . infect corneas with McKrae HSV-1

Day 3 verify presence of herpetic keratitis

Day 19 . . start daily examinations and continue until day 32

Day 21 . . randomize animals into coded treatment groups, start treatment D Daayy 3322 .. .. . conclude treatment and break treatment code PR1MATE STUDIES Objective

To determine whether viral inhibitors have an effect on the recurrences of herpetic keratitis in squirrel monkeys that are subjected to cold stress.

Rationale

Squirrel monkeys infected with the Rodanus strain of HSV-1 exhibit spontaneous recurrences of herpetic keratitis. When these primates are exposed over night to temperatures that are minimally colder than their ideal temperature, there is a statistically increased incidence of keratitis the next day. Utilizing this cold stress increases the number of ocular recurrences, provides a model analogous to humans with no tissue damage to induce recurrences and allows for more economical use of possibly expensive and hard to obtain test compounds.

Design Day 1 . . . infect corneas with Rodanus HSV-1

Day 3 . verify presence of herpetic keratitis

Day 14 . . . start daily examinations to determine spontaneous recurrences, continue to day 38

Day 21 . . . start treatment and continue on day 22

Day 22 . . . lower room temperature to 62-65 ' F in the evening

Day 23 . . . give last treatment in the morning, examine corneas and raise temperature to normal 75 "F

Day 28 . . . start treatment as on day 21 and continue on day 29

Day 29 . . . lower temperature as on day 22

Day 30 . . . give last treatment in the morning, examine corneas and raise room temperature to normal

Day 34 . . . start treatment as on day 21 , continue on day 35

Day 35 . . . lower temperature as on day 22 Day 36 . . . give last treatment, examine corneas, raise room temperature to normal

QUANTITATIVE POLYMERASE CHAIN REACTION (Q-PCR) The Q-PCR method is based on competition between the experimental, template DNA and a competitor template DNA which is added to the reaction tubes in known quantities. In these studies, DNA from the corneas and trigeminal ganglia of animals will be extracted, quantitated by spectrophotometry at 260 nm, and 1 ng quantities of the cellular extract amplified in PCR. Tissues are homogenized suspended in a DNA extraction buffer containing 0.01 %

Triton-X detergent and 0.25% proteinase K in Tris EDTA buffer, pH 6.5. The tissue homogenates are suspended in this medium and incubated at 70 "C for 10 minutes, after which time the samples are diluted in a mixture of Tris EDTA and isopropyl alcohol. The extracts are transferred to filter cartridges and centrifuged for one minute at 8,000 X g. The DNA is trapped on the DNA- binding membrane in the filter cartridge and washed three times with isopropyl alcohol. The DNA is eluted from the DNA-binding membrane with 0.1 ml of water at 70^* C. Following quantitation by spectrophotometry, the DNA samples are amplified in a PCR system according to the vendor's instructions (Perkin- Elmer/ Applied Biosystems, Foster City, CA). In order to quantitate the amount of viral DNA in each tissue extract, triplicate 0.5 ml tubes each containing IOX PCR buffer, dNTPs, Taq polymerase, and a pair of oligonucleotide primers complimentary to a 476 base pair sequence of the viral DNA polymerase chain are prepared. Known amounts of a competitor viral DNA sequence, identical to the DNA polymerase gene sequence except that the competitor product yields a 530 base pair amplified signal are added to each tube. In a typical amplification, triplicate tubes each containing 104, 103, 102, 101, 100, 10-1, and 10-2 molecules of competitor DNA are prepared. To each of these tubes 1 ng of the cellular extract DNA is added along with the reagents described above. All tubes are subjected to an amplification profile consisting of 95 ^*C for one minute, 72 ^*C for two minutes, and 60^* C for one minute. These cycles are repeated 35 times. Subsequently, 20 μl quantities of the amplified samples are resolved on 2% agarose gels in Tris borate EDTA buffer, pH 8.0. The DNA bands in the agarose gel can be visualized following ethidium bromide staining. Using a videodensitometry system (Eagle Eye 11, Stratagene Corp., La Jolla, CA), it is possible to accurately determine the point at which the competitor viral DNA and the amplified viral DNA in the tissue sample are present in equal amounts. The videodensitometry system permits us to obtain a quantitative measure of the amount of viral DNA in each tissue sample.

STATISTICAL METHODS General.

In animal experiments on HSV recurrence the slit lamp score, or more simply the number of eyes which are positive for herpetic lesions is a commonly used outcome variable. Other potential outcome variables include numbers of positive swabs, numbers of ganglia positive for viral DNA. All of these outcome variables are expressed as frequencies of one outcome or the other, rather than as continuous variables such as drug or metabolite concentrations. The analysis of simple experiments where the outcome is a frequency of some event, will be accomplished by use of the Chi square technique, where the frequencies at some endpoint in the experiment are compared in two different treatment groups (typically drug and control).

In other experiments in this proposal animals are observed over time following a treatment (or multiple recurrence inducing stresses), and thus measures from one animal may show a correlation over time. In such cases, more complex models may be needed for the analysis of recurrence frequency, models which take into account repeated measures from animals over time, as well as several levels of treatments (drug or stress episodes). For these cases, methods for categorical models are applied such as described in Grizzle et al. (Biometrics 25:429-504, 1969) or Koch et al. (Biometrics 33: 133-158, 1977). Specific methods of procedure for data analysis.

Data is logged in research notebooks and entered from these into ASCII data sets for processing by programs and procedures in the Statistical Analysis System (SAS, SAS Institute Inc, Cary, NC). Where required the treatments are encoded and neither the person entering the data nor the statistician knows the identity of the treatments until all data are entered and analyzed.

SAS procedures for categorical analysis are used, procedure (proc) FREQ for simple Chi-square analysis, and proc CATMOD for more complex models in categorical analysis. SAS output from these procedures for each experiment analyzed are written up as reports. Graphical representation where required for presentation or publications are produced from the same data sets using SAS graphical procedures.

While the invention has been described and illustrated herein by references to various specific material, procedures and examples, it is understood that the invention is not restricted to the particular material, combinations of material, and procedures selected for that purpose. Numerous variations of such details can be implied and will be appreciated by those skilled in the art.

Claims

WHAT IS CLAIMED IS:

1. A method for the treatment of Herpes Simplex virus infection by prevention or inhibition of the reactivation of herpes simplex virus, said method comprising administering to a mammal in need of such treatment an adrenergic receptor ligand in an amount which is effective to prevent or inhibit the reactivation of a Herpes Simplex virus.

2. The method of Claim 1 wherein the Herpes Simplex virus is HSV-1 or HSV- 2.

3. The method of claim 1 , wherein said adrenergic receptor ligand is an α- adrenergic receptor agonist or a /3-adrenergic receptor antagonist.

4. The method of claim 1, wherein said adrenergic receptor ligand is an α- adrenergic receptor agonist.

5. The method of claim 1 , wherein said adrenergic receptor ligand is a β - adrenergic receptor antagonist.

6. The method of claim 1 , wherein said adrenergic receptor ligand is a specific (3,-adrenergic receptor antagonist.

7. The method of claim 1 , wherein said adrenergic receptor ligand is a specific β₂-adrenergic receptor antagonist.

8. The method of claim .1 , wherein the composition is administered in an amount of about 0.001 to 100 mg/kg body weight/dose.

9. The method of claim 1, wherein the composition is administered orally, intravenously, subcutaneously, topically, transdermally, intramuscularly, or intraperitoneally.

10. The method of claim 1 , wherein the composition is administered orally.

11. The method of claim 1, wherein the composition is administered topically.

12. The method of claim 1 , wherein the adrenergic receptor ligand is administered in the form of a pharmaceutical composition of matter which further comprises a pharmaceutically-acceptable carrier.

13. The method of claim 4, wherein the α-adrenergic receptor agonist is an ephedrine derivative.

14. The method of claim 4, wherein the α-adrenergic receptor agonist is selected from the group consisting of agmatine, /-•-aminoclonidine, clonidine, epinephrine, iodoclonidine, guanabenz, lopidine, methoxamine, norepinephrine, octopamine, oxymetazoline, phenylephrine, xylazine, UK 14,304, and pharmaceutically acceptable salts thereof.

15. The method of claim 5, wherein the 3-antagonist is an isoproterenol or phenyloxypropanolamine derivative.

16. The method of claim 5, wherein the /3-antagonist is selected from the group consisting of alprenolol , isoproterenol, dichloroisoproterenol, metalol, nifenalol, pindolol, propranolol, sotalol, and pharmaceutically acceptable salts thereof.

17. The method of claim 6, wherein the selective /3,-a*ntagonists is selected from the group consisting of atenolol, metoprolol, oxprenolol, practolol, timolol, 4-[3-(l , l-dimethylethyl)amino]-2-hydroxypropoxy]- 1 ,3-dihydro- 2H-benzimidazol-2-one, and pharmaceutically acceptable salts thereof.

18. The method of claim 7, wherein the selective /3₂-antagonists is selected from the group consisting of butoxamine, l-[2,3-(dihydro-7-methyl-lH- inden-4-yl)oxyl-3-[( 1 -methylethyl)amino]2-butanol , and pharmaceutically acceptable salts thereof.