WO1996013263A2

WO1996013263A2 - Methods of treatment of viral disease with inhibitors of sphingolipid biosynthesis

Info

Publication number: WO1996013263A2
Application number: PCT/US1995/014070
Authority: WO
Inventors: Meir Lev; K. Soma Sundaram; Yaffa Mizrachi; Arye Rubinstein
Original assignee: Research Corp Technologies Inc
Current assignee: Research Corp Technologies Inc
Priority date: 1994-11-01
Filing date: 1995-10-30
Publication date: 1996-05-09
Anticipated expiration: 1997-05-01
Also published as: WO1996013263A3

Abstract

The present invention provides a novel method of inhibition of virus replication. In particular, the present invention is directed to the use of an inhibitor of sphingolipid biosynthesis for the treatment or prevention of infection caused by enveloped viruses, including HIV. L-cycloserine is the preferred inhibitor of sphingolipid biosynthesis. Pharmaceutical compositions comprising inhibitors of sphingolipid biosynthesis, including L-cycloserines, are also provided.

Description

96/13263 PCIYUS95/1407

METHODS OF TREATMENT OF VIRAL DISEASE WITH INHIBITORS OF SPHINGOLIPID BIOSYNTHESIS

The present invention relates to a novel method of inhibition of viral replication. Treatment of an infected host with an inhibitor of sphingolipid biosynthesis results in changes in the architecture of the host cell membrane- such that viruses that require the host cell membrane to form a viral envelope are unable to reproduce. The present invention thus provides a method of treatment of infection by enveloped viruses, including retroviruses such as HIV. Pharmaceutical compositions comprising inhibitors of sphingolipid biosynthesis are also provided.

Viruses are obligate intracellular parasites that subvert the genetic mechanisms of the host cell in order to replicate. The development of antiviral agents has thus been particularly difficult in that agents that inhibit a virus are also likely to be toxic to the host cell. Antiviral agents have been designed to target virus-specific enzymes, for example the retroviral reverse transcriptase. Reverse transcriptase is notoriously error-prone and permits a high degree of mispairing in the production of a new DNA strand. Moreover, reverse transcriptase cannot edit and thus does not repair mismatched bases as DNA synthesis proceeds. Various nucleoside analogs have thus been designed to inhibit viral DNA synthesis, without adversely affecting normal cellular DNA synthesis. 6/13263

However, side effects are frequently observed in patients treated with known nucleoside analogs as a result of incorporation of a nucleoside analog into cellular chromosomes or mitochondrial DNA. In practice, a nucleoside analog can also inhibit the conversion of normal nucleosides into the nucleoside triphosphate used to synthesize DNA. While inhibition of triphosphate formation can slow the rate of viral replication, inhibition of normal nucleoside triphosphate formation can also have detrimental effects upon the cell. While a number of nucleoside analogs are known to inhibit viral replication, these analogs cause side effects such as anemia, neutropenia, neuropathy, pancreatitis and other problems.

The first nucleoside analog reported to have activity against human immunodeficiency virus (HIV) in vitro was 3' -azido-3' -deoxythymidine (AZT) (Mitsuya et al.. 1985, Proc. Natl. Acad. Sci. USA 82:7096-7100). While AZT has proven efficacy against HIV, considerable toxicity problems have been encountered as a result of AZT therapy. The most serious of these has been suppression of bone marrow formation resulting in anemia and neutropenia (Richman e_t al. , 1987, N. Enq. J. Med. 317:192-197) . The effect of AZT on bone marrow is sufficiently severe to necessitate blood transfusion, AZT dose reduction or even cessation of AZT treatment. Moreover, AZT-resistant strains of HIV have developed in patients receiving AZT therapy (Larder et al., 1989, Science 243:1731-1734) . Clearly, improved non-AZT therapeutic agents are needed for treatment of HIV infection.

Several 2' ,3' -dideoxy (dd) nucleosides have been investigated for efficacy against HIV including, for example, dideoxycytidine (ddC) , dideoxyadenine (ddA) and dideoxyinosine (ddl) (Mitsuya et al. , 1986, Proc. Natl. Acad. Sci. USA 82:1911-1915) . m clinical trials ddC evidenced activity against HIV (Yarchoan et al. , 1988, Lancet i.:76-81; and Merigan e_t al. , 1989, Ann. Intern. Med. 110:189-194) . However, ddC causes peripheral neuropathy, possibly because ddC may inhibit mitochondrial DNA synthesis, and ddC is a potent inhibitor of mammalian nuclear DNA polymerase. The ddT analog has only weak activity against HIV and has not been further developed as an anti-retroviral agent. ddA and ddl are both converted to active ddATP species. Although ddATP is less potent than the AZT triphosphate (AZTTP) or ddCTP, the intracellular half- life of ddATP is 12 hr, at least 4-fold longer than AZTTP and ddCTP. However, both ddA and ddl are highly susceptible to solvolysis of the glycosidic linkage which liberates the free purine base. The free base of ddA, adenine, has been shown to cause renal damage (Lindbald et al. , 1973, Acta Pharmacol. Toxicol. 32:246- 256) . While ddl does not cause the severe anemia caused by AZT, ddl does have its own side effects: neuropathy and pancreatitis.

Accordingly, there is a long-standing need for effective, non-toxic agents to treat HIV infections and other viral infections. The present invention provides a novel method of inhibiting viral replication and treating and preventing conditions caused by viruses. By inhibiting sphingolipid biosynthesis in a host cell, the architecture of the host cell membrane is altered. The changes in the membrane architecture result in inhibition of replication by viruses that require the host membrane for infection or for formation of the virus envelope. Inhibition of the sphingolipid biosynthesis pathway thus presents a new method of chemotherapy of viral infection.

The present invention provides a method of inhibiting replication of an enveloped virus which comprises contacting a virally infected cell with a replication-inhibiting amount of at least one inhibitor of sphingolipid biosynthesis.

The present invention further provides a method of treatment or prevention of infection by an enveloped virus which comprises administering to a mammal in need of such treatment or prevention an antiviral effective amount of at least one inhibitor of sphingolipid biosynthesis.

The present invention still further provides a method of treatment or prevention of human immunodeficiency virus (HIV) infection which comprises administering to a patient in need of such treatment or prevention an anti-HIV effective amount of at least one inhibitor of sphingolipid biosynthesis. A pharmaceutical composition comprising at least one inhibitor of sphingolipid biosynthesis is also provided by the present invention.

The present invention also provides a compartmentalized kit for the treatment or prevention of infection by an enveloped virus which comprises a first compartment adapted to contain an inhibitor of sphingolipid biosynthesis.

Still further, the present invention provides an article of manufacture comprising a packaging material and a inhibitor of sphingolipid biosynthesis contained therein wherein the packaging material comprises a label that indicates that the inhibitor of sphingolipid biosynthesis can be used as an antiviral agent.

The present invention provides a method of inhibiting replication of an enveloped virus which comprises contacting a virally-infected cell or a cell susceptible to viral infection with a replication- inhibiting effective amount of at least one inhibitor of sphingolipid biosynthesis.

In many viruses, the protein capsid that surrounds the viral nucleic acid is enclosed by a protein-containing lipid bilayer membrane. In accordance with the present invention, such viruses are referred to as enveloped viruses. Enveloped viruses acquire the envelope in the process of budding from the host cell membrane. The lipid bilayer of the viral envelope is parasitized from the host cell membrane, whereas the proteins in the envelope are encoded by the viral genome. Enveloped viruses contemplated in accordance with the present invention include, for example, poxvirus, influenza virus, herpesvirus, hepadnavirus, togavirus, flavivirus, coronavirus, rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus, arenavirus and retroviruses, including human immunodeficiency viruses.

Viral envelope proteins bind to receptors on the host cell membrane to initiate the process of viral infection. Following receptor-mediated endocytosis, the virus exploits the host cell machinery to synthesize nucleic acids, capsid proteins, and envelope proteins. The envelope proteins are delivered to the host cell membrane and become embedded in host cell lipids. The assembled capsid containing viral nucleic acid interacts with envelope proteins at the host cell plasma membrane to form a bud that pinches off to release free virus. The envelope of the bud contains envelope proteins embedded in host cell lipids. Without the envelope, the virus cannot emerge from an infected cell or spread to and infect another cell.

Accordingly, enveloped viruses have extensive interactions with the host cell membrane, both at the stage of infection of a host cell and release of replicated free virus by budding from the host cell membrane.

It has been surprisingly discovered in accordance with the present invention that replication of enveloped viruses can be inhibited by altering the lipid composition of the host cell membrane. In 96/13263 PCIYUS95/14070

- 7-

particular, it has been discovered that inhibition of the biosynthesis of sphingolipids results in alteration of the host cell membrane such that replication of enveloped viruses is inhibited. Inhibition of viral replication may be due to loss of sphingolipids that act as receptors for viruses during infection, or by alteration of the membrane to prevent budding of the virus from the host cell.

Sphingolipids are membrane lipids in which the amino group of sphingosine is acylated by a fatty acid. Sphingolipids include cerebrosides and gangliosides. Inhibitors of sphingolipid biosynthesis are known. For example, L-cycloserine (L-4-amino-3-isoxazolidinone) inhibits the first enzyme of the sphingolipid pathway, palmitoylserine transferase (3-ketodihydro sphingosine synthase) so that the biosynthesis of major species of sphingolipids is inhibited, resulting in a cell membrane with an altered architecture. (Sundaram and Lev, 1985, J. Lipid Res. £6:473; 1984, J. Neurochem. 42:577.)

In accordance with the present invention, the contemplated inhibitors of sphingolipid synthesis are those which can inhibit replication of an enveloped virus at dosages that are not lethal to the host cell. In a preferred embodiment the inhibitor of sphingolipid synthesis is L-cycloserine. In another embodiment the inhibitor of sphingolipid synthesis is a derivative or analog of L-cycloserine wherein said derivative or analog is an inhibitor of sphingolipid biosynthesis and is capable of inhibiting replication of an enveloped virus. Dimeric and acetylated forms of L-cycloserine are specifically contemplated. L-cycloserine is commercially available. Derivatives and analogs of L- cycloserine can be chemically synthesized by methods known to one of ordinary skill in the art.

The ability of L-cycloserine, its derivatives and analogs, and other inhibitors of sphingolipid biosynthesis to inhibit replication of enveloped viruses can be determined, for example, by in vitro assays are known to one of ordinary skill in the art. For example, HIV replication is routinely measured in a CD4 positive cell line (CEM) . HIV replication is measured by assessing syncytia formation, HIV positive immunofluorescent cells, and p24 protein production three and seven days following infection of CEM cells with HIV. Accordingly, an inhibitor of sphingolipid biosynthesis can be identified as an inhibitor of HIV replication if, upon treatment of HIV infected CEM cells, there is a statistically significant reduction in syncytia formation, HIV positive immunofluorescent cells, and p24 protein production without lethality to the CEM cells. Corresponding assays for replication of other enveloped viruses are known to the ordinarily skilled artisan.

For example, the rate of viral reproduction can be determined by observing the number of viruses or the amount of viral antigen (e.g., p24 of HIV), or the amount of viral nucleic acid present over time. The detection of antibodies in animal body fluids (e.g., serum, urine and the like) which react with viral antigens is also diagnostic of viral infection and viral replication.

Procedures for detecting and quantitating viruses both in vitro and in vivo are available and known to the ordinarily skilled artisan. Agrawal et, al. (1988, Proc. Natl. Acad. Sci. USA 85:7079-7083): Balzarini et al. (1991, AIDS 5:21-28); Balzarini et al. (1988, Biochem. Pharmacol. 32=2847-2856) ; Goodchild et al. (1988, Proc. Natl. Acad. Sci. USA 85:5507-5511): Weislow et al. (1989, J. Natl. Cancer Inst. 81:577-586) : and Zamecnik et al. (1978, Proc. Natl. Acad. Sci. USA .25.280-284) provide useful procedures.

A replication-inhibiting effective amount of an inhibitor of sphingolipid biosynthesis is an amount that is sufficient to detectably reduce viral replication. Viral replication can be assayed as described hereinabove.

In accordance with the present invention it has been demonstrated that replication of an enveloped virus is inhibited by contacting a virally infected cell or a cell susceptible to viral infection with a replication-inhibiting amount of an inhibitor of sphingolipid biosynthesis, preferably L-cycloserine. It is known that chronic administration of L-cycloserine at dosages sufficient to inhibit sphingolipid biosynthesis is tolerated by mammals without significant side effects (Sundaram and Lev, 1989, Neurochemical Research .14.:245) . Accordingly, the present invention further provides a method of treatment or prevention of infection caused by an enveloped virus which comprises administering to a mammal in need of such treatment or prevention an antiviral effective amount of at least one inhibitor of sphingolipid biosynthesis. In a preferred embodiment the inhibitor of sphingolipid biosynthesis is L- cycloserine. In another preferred embodiment the mammal is human.

As used herein, treatment of viral infections means to slow the progress of the disease, to ameliorate symptoms of such infections which are already visible, and to preclude or diminish the onset of new symptoms. Prevention of viral infections refers to delaying or preventing the onset of initial symptoms of the infection. Amelioration of neurological symptoms of HIV is particularly contemplated, in that brain and nerve have high concentrations of sphingolipids.

Viral infections for which treatment or prevention is contemplated include infections caused by influenza virus, herpes virus, and retroviruses, including infections caused by a lentivirus, oncovirus C, oncovirus A, oncovirus B, cisternavirus, Spumavirus F and the like. Specifically contemplated infections are those caused by human immunodeficiency virus-1 (HIV-1) , human immunodeficiency virus-2 (HIV-2) , human intracisternal retrovirus, human T cell leukemia/lymphoma virus type I (HTLV-I) and human T cell leukemia/lymphoma virus type II (HTLV-II) .

In an especially preferred embodiment the method of the present invention is used in the prevention or treatment of a human immunodeficiency virus infection. An antiviral effective amount of an inhibitor of sphingolipid biosynthesis is an amount which detectably reduces the number of infective viruses, the viral infectivity, the symptoms or progression of a viral infection. Procedures for determining the number or infectivity of viruses are known and readily available to the skilled artisan as described hereinabove. Moreover the symptoms associated with retroviral disease are well documented and can be used to assess the progression of the disease (e.g., see Wilson et al., 1991, Harrison's Principles of Internal Medicine, twelfth edition, McGraw-Hill, Inc., New York; Centers for Disease Control, 1986, Morb. Mort. Week Rep. 3.5:334; Centers for Disease Control, 1987, Morb. Mort. Week Rep 3_ :1S; Centers for Disease Control, 1989, Morb. Mort. Week Rep. 3_8.:S-6) .

In a preferred embodiment the present invention provides a method of treatment or prevention of HIV infection which comprises administering to a patient in need of such treatment or prevention an anti- HIV effective amount of at least one inhibitor of sphingolipid biosynthesis. In a preferred embodiment the inhibitor of sphingolipid biosynthesis is L- cycloserine.

Αn anti-HIV effective amount is an amount of an inhibitor of sphingolipid biosynthesis sufficient to inhibit or reduce the replication of HIV RNA, the amount of HIV antigen, the number of HIV-induced syncytia, the number of infective HIV virions, the HIV infectivity or the progression of HIV infection. The amount of DNA replicated from HIV RNA can be measured in vivo or in vitro. Measurements of the amount of HIV DNA replicated include enzymatic assays, e.g., as described in Eriksson et al. (1989, .Antimicrobial Agents and Chemotherapy 33:663) and cell culture measurements, e.g., as described in Weislow et al. The amount of HIV antigen can be routinely detected by the skilled artisan in patient body fluids, e.g., blood (serum), urine and the like. Commonly available procedures for HIV antigen detection include enzyme-linked immunosorbant assays

(ELISA) , Western analyses, immunofluorescence assays and radioimmunoprecipitation assays. The number of infective HIV virions can be assayed e.g., as described in Balzarini et al.. (1991, AIDS 5_:21-28) and Balzarini et al. (1988, Biochem. Pharmacol. 32:2847-2856) . The progression of HIV infection in humans is well documented (e.g., Wilson e_t al. 1991; Centers for Disease Control, 1986, 1987 and 1989) .

In the methods of treatment or prevention, the inhibitors of sphingolipid biosynthesis are administered to mammals, including but not limited to humans. The route of administration can be determined by the ordinarily skilled artisan, and includes oral, topical, intradermal, intravenous, intramuscular, intraperitoneal and subcutaneous administration. In a preferred embodiment the route of administration is subcutaneous or oral.

A pharmaceutically effective amount of the present compounds is about .02 mg/kg/day to about 25 mg/kg/day as needed to attain beneficial therapeutic effects. In a preferred embodiment such a pharmaceutically effective amount of the present compounds is about 2 mg/kg/day to about 10 mg/kg/day. Specific dosage amounts can be readily determined by one or ordinary skill in the art taking into account factors which generally tend to modify drug action, e.g., age, weight, sex, diet, disease state, times and methods of administration, and the like. The dosage required for humans is generally less than that required for small warm-blooded animals such as mice.

A dosage unit can include a single inhibitor of the present invention or a mixture of the present inhibitors; a dosage unit can further include other therapeutic agents beneficial for the treatment of diseases caused by enveloped viruses. Such combinations of the present inhibitors with other therapeutic agents can be administered either sequentially or simultaneously.

The inhibitors of the present invention can be administered to an animal in a variety of forms adapted to the chosen route of administration, e.g., oral, topical, intradermal, intravenous, intramuscular, intraperitoneal or subcutaneous routes.

For oral administration the present inhibitors can be suitably protected, e.g., by enclosure in hard or soft shell gelatin capsules. For oral therapeutic administration, the active inhibitor may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, incorporated directly with the food of the diet and the like. The subject inhibitors can be incorporated into a cream, solution or suspension for topical administration. The active inhibitors may be incorporated into liposomes or liposomes modified with polyethylene glycol for parenteral administration. Incorporation of additional substances into the liposome, for example, antibodies reactive against membrane proteins found on specific target cells, can help target the present inhibitors to

10 specific cell types.

The percentage of such additives and stabilizers can be varied as needed, however the amount of active compound is at least 0.1%. More conveniently the active inhibitor can constitute about 2% to about ,,- 60% of the weight of the unit. The amount of active inhibitor in such therapeutically useful compositions is varied such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the following: a binder such as

20 gum tragacanth, acacia, corn starch or gelatin; an excipient such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid or the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, fructose, lactose or saccharin; or a flavoring agent such as peppermint, oil

25 of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it can also contain a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules

35 may be coated with shellac, sugar or both. A syrup or elixir can contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. In addition, the active inhibitor may be incorporated into sustained-release preparations and formulations. Any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.

10 The active inhibitor may also be administered parenterally or intraperitoneally. Such solutions can be mixed with a surfactant such as hydroxypropylcellulose or a dispersing agent such as glycerol, a liquid polyethylene glycol, an oil and a _,_ mixture thereof. Under ordinary conditions of storage and use these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or

20 dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and preserved against the contaminating action of microorganisms such as bacteria and fungi. Such pharmaceutical forms for injection must be fluid to the

25 extent that easy syringability exists. Preferably the pharmaceutical composition is stable under the conditions of manufacture and storage.

A pharmaceutical carrier can be a solvent or dispersion medium containing, for example, water,

30

35 ethanol, polyol (for example, glycerol, propylene glycol, polyethylene glycol and the like) , vegetable oil and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active inhibitor in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solutions thereof.

In another embodiment the present invention provides a pharmaceutical composition containing a pharmaceutically effective amount of at least one inhibitor of sphingolipid biosynthesis and a pharmaceutically acceptable carrier. In a preferred embodiment the inhibitor of sphingolipid biosynthesis is L-cycloserine or a derivative or analog thereof, for

10 example, dimeric or acetylated L-cycloserine. In a most preferred embodiment the inhibitor is L-cycloserine.

As used herein such a pharmaceutically effective amount is an anti-viral effective amount, a retrovirus replication-inhibiting amount, or a human ^,_ immunodeficiency virus-inhibiting amount.

As used herein, pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use

20 of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the

25 compositions.

The present invention further provides a compartmentalized kit for the treatment or prevention of infection by an enveloped virus which comprises a first compartment adapted to contain an inhibitor of

30

35 sphingolipid biosynthesis. In a preferred embodiment the inhibitor of sphingolipid biosynthesis is L- cycloserine or a derivative or analog thereof, for example dimeric or acetylated L-cycloserine. In a most preferred embodiment the inhibitor of sphingolipid biosynthesis is L-cycloserine.

In another embodiment the present invention provides an article of manufacture comprising a packaging material and an inhibitor of sphingolipid

10 biosynthesis contained therein wherein the packaging material comprises a label that indicates that the inhibitor of sphingolipid biosynthesis can be used as an antiviral agent. In a preferred embodiment the inhibitor of sphingolipid biosynthesis is L-cycloserine ,,- or a derivative thereof, for example dimeric or acetylated L-cycloserine. In a most preferred embodiment the inhibitor of sphingolipid synthesis is L- cycloserine and the label indicates use as an anti-HIV agent.

20 The following examples further illustrate the present invention.

25

30

35 EXAMPLE 1

L-Cycloserine Inhibits HIV Replication

Materials and Methods

CD4 positive (CEM) cells (1 x 10⁶/ml) were grown in RPMI medium supplemented with 5% FCS and 100 μg/ml penicillin and 200 μg/ml streptomycin. The cells were incubated with 0.625, 1.25, 2.5 and 5 μg/ml L- cycloserine for 24 hr and then subjected to HIV-1 infection, NIT isolate (Sakai et. al. , 1988, J. Virol. :4078) at TCID₅₀ of 1,000 for 1 hr. Excess virus was removed by 3x washing in RPMI medium and the cells incubated in 5 ml RPMI with 5% FCS + penicillin/ streptomycin and L-cycloserine at the stated concentrations. Fresh L-cycloserine was added at 3 day intervals. Cells were treated with L-cycloserine at similar concentrations used for viral inhibition and counted three days later. No toxic effects were observed as a result of the drug at the concentrations used in these experiments (from 0.5 μg/ml to 5 μg/ml L-

CS) . Experiments were repeated 3 times.

Results

1. Reduction of svncvtia formation by L- Cvcloserine. CEM cells were treated with L-cycloserine at 0.625, 1.25, 2.5 and 5 μg/ml for 24 hr. Cells were then subjected to HIV-1/NIT (Sakai et al. , 1988) infection at TCID₅₀ of 1,000 for 1 hr. The most striking observation was the complete inhibition of syncytia formation in CEM cultures treated with 5 μg/ml L- cycloserine (Table 1) . In cultures treated with lower L-cycloserine concentrations, syncytia were reduced compared to CEM cells infected with HIV-1 untreated with L-cycloserine (positive control) . In these positive control cultures syncytia appear 48 hr post infection and become most pronounced 72 hr following infection. As noted above, no syncytia were observed in cultures treated with 5 μg/ml L-cycloserine 72 hr post infection. A reduced number of syncytia could be observed in cultures treated with 2.5 μg/ml L-cycloserine. Continuous treatment of HIV infected CEM cells with 2.5 μg/ml L-cycloserine every three days up to seven days, resulted in a significant reduction in number of syncytia (c.60%).

2. Immunofluorescence studies. Slides were prepared three days following infection of the CEM cells. The number of HIV immunofluorescent cells were analyzed in cultures treated with 0, 1 and 5 μg/ml L- cycloserine. The cells were treated with anti HIV antibodies (pooled from 5 HIV-1 positive patients) for 30 min and then stained with rabbit and human IgG antibody tagged with fluorescein isothiocyanate (FITC) . At a level of 5 μg/ml L-cycloserine, there was a significant reduction in the number of immunofluorescent (IF positive) cells in the cultures (Table II) .

3. L-cvcloserine inhibited HIV-1 production. CEM cells were treated with L-cycloserine for 24 hr. prior to infection with HIV-1. L-cycloserine, at concentrations from 2.5 to 10 μg/ml, inhibited HIV production in CEM cells as measured by p24 production in the supernatant (Table III) . A significant effect on HIV replication, as detected by p24 protein production, was also observed in cultures treated with 1.25 μg/ml L- cycloserine.

L-cycloserine was non-toxic at the tested concentrations as evidenced by cell numbers in treated cells relative to controls (Table IV) .

In summary, L-cycloserine inhibited HIV replication in the CEM cells as determined by (a) reduction in syncytia formation, (b) the number of HIV-1 infected CEM cells in the cultures assayed as HIV positive immunofluorescent cells and (c) p24 protein production three and seven days following infection with

HIV.

TABLE I

Reduced Syncytia Formation Following HIV Infection by L-CS (# of syncytia per field)

L-CS μg/ml 24 hours 48 hours 72 hours

0 5 13 20

0.625 5 10 20

1.25 3 7 11

2.5 2 3 6

5 0 0 0

Infection was performed with HIV/NIT with 1,000 TCID 50 ' TABLE II

L-Cycloserine Induced Inhibition of HIV-1 Immunofluorescence

L-cycloserine IF* μg/ml %

0 70

0 50

1 70

1 50

5 10

5 5

TABLE III

Inhibition of HIV-1 Production by L-Cvcloserine (pq p24)

TABLE IV Cell Number with L-Cvcloserine (xlO⁶) 72 hr. Culture

Cell Number

Ctrl 1.12

Sodiumcarbonate 1.02

LCS 5μg/ml vl/10 1.24

LCS 5μg/ml vl/20 1.18

LCS 2.5μg/ml vl/10 0.94

LCS 2.5μg/ml vl/20 1.0

LCS 1.2μg/ml vl/10 1.0

LCS 1.2 μg/ml vl/20 1.04 virus 1/10 0.6

EXAMPLE 2

L-Cycloserine is non-toxic at levels used to inhibit cerebroside biosynthesis in vivo.

The present experiment was performed to determine whether there were any toxic effects of prolonged administration of L-cycloserine to mammals.

Materials and Methods

Male HSD (1CR) mice weighing 8-12 g, obtained from Harlan Sprague Dawley, Indianapolis, IN were used at 16 days of age (postweaned) for all experiments.

Food and water were given ad libitum. They were injected subcutaneously with L-cycloserine (25 mg/kg body wt.) dissolved in 0.05 ml of saline (prepared immediately before use) for alternate days for two months. After L-cycloserine treatment, the animals were decapitated and the brain, liver and spleen were removed and homogenized with 20 volumes (20 ml/g wet time) of chloroform-methanol 2:1 (v/v) . The lipids were extracted and partitioned by the procedure of Folch et al. (1957) J. Biol. Chem. 226:497. The upper phase was dialyzed, lyophilized and used for total ganglioside estimation by the resorcinol method (Svennerholm, 1957, Biochim. Biophvs. Acta.

2_4..*604) as modified employing free N-acetyl neuraminic 5 acid as the standard. The lower phase was concentrated and passed through a column containing 5g silicic acid in chloroform. The column was washed with 20 ml __Q chloroform and then eluted with 20 ml chloroform: methanol 1:1 (v/v) . From this, cerebrosides and sulfatides were separated by TLC (nonborate plates) using chloroform-methanol-water 66:25:4 (v/v/v) . The

15 bands co-chromatographing with standard cerebroside and sulfatide were visualized by iodine vapors, removed separately and used for galactose estimation. Glucocerebroside and galactocerebroside were estimated

20 together. In liver, these lipids were separated by the use of borate plates. Glucocerebrosides were separated by chloroform-methanol-water-15M NH₂0H (280:70:6:1). In these experiments 70% recovery of the lipids was

25 obtained following extraction from TLC plates. Sphingomyelin was separated by TLC using chloroform- methanol-acetic acid-water 25:15:4:2 (v/v/v/v) . The ryry phospholipid phosphorus was estimated without extraction

35 by direct gel digestion with 0.9 ml of 70% perchloric acid over a sand bath for 15 minutes. Inorganic phosphorous was estimated by the procedure of Bartlett

(1959) J. Biol. Chem. 234:466, as modified by Marinetti 5

(1962) J. Lipid Res. 3_:1.

Microsomes were prepared from the brains essentially according to the procedure of Morell and

-_j_0 Radin (1970) J. Biol. Chem. 245:342. with modification that suspending medium contained 0.25 M sucrose, 1 πiM pyridoxal phosphate, and 1 mM dithiothreitol in 50 mM phosphate buffer (pH 7.4) . 3KDS synthase activity was

15 assayed as described previously (Lev et al. , 1981, Arch. Biochem. Biophvs. 212:424) .

Results

20 The results of the subcutaneous injection of low levels of L-cycloserine to mice is shown in Table V. The body and brain weights of the animals were not changed. Sulfatide, ganglioside and sphingomyelin

25 levels showed no significant reduction. Of the lipids examined, the cerebrosides alone showed a significant reduction (approximately 39%) . The effect of a 2 month course of L-cycloserine treatment on liver and spleen

30

35 glucocerebroside and sulfatides is shown in Table VI.

In this experiment glucocerebroside was reduced by 26% in liver and by 47% in spleen whereas sulfatide levels were reduced to a smaller degree (17%) in liver. 5

These results indicate that the level of cerebrosides can be greatly reduced without impairing brain function. No neurological symptoms have been

-*_0 noted in these mice which appeared as coordinated and as lively as the control untreated mice. The examination, by electron microscopy, of the brains of the treated mice has shown no histological changes from those of

15 controls indicating that the reduction in cerebroside levels did not affect myelination or other aspects of brain morphology. Further experiments were performed to determine whether high doses of L-cycloserine

20 administered intraperitoneally could affect learning.

Chronic administration of L-cycloserine for 4 or 5 days resulted in no deficit in memory retention as compared to saline controls using the inhibitory avoidance task. 25

30

35 TABLE V

Inhibition of Brain Sphingolipids and 3KDS Synthase Activity Following Long-

Term Subcutaneous Administration of L-cycloserine

Percent- Control Experimental Reduction

Body Wt.(g) 38.2 ± 0.64 36.8 ± 0.67 4

Brain Wt. (g) 0.49 ± 0.002 0.49 ± 0.04 0

Sphingolipids

(mg/g wet tissue)

Cerebrosides 3.16 ± 0.18 1.94 ± 0.27* 39

Sulfatides 1.83 ± 0.07 1.72 ± 0.07 6

Gangliosides 0.38 ± 0.01 0.39 ± 0.00 0

Sphingomyelin 2.54 ± 0.25 2.50 ± 0.16 2

3 DS Svnthase Activity

(nmol 3KDS formed) 1.92 ± 0.09 1.65 ± 0.02** 15

Mice (10 per group) were injected with 25 mg/kg body wt. L-cycloserine on alternate days for 2 months. Controls received saline. All animals were killed after cessation of cycloserine treatment. Brain sphingolipids were estimated as described in materials and methods. Values are means ± SEM of ten animals per group.

* P < 0.001, ** P < 0.05.

TABLE VI

Inhibition of Liver and Spleen Glycolipids Followinε Lone-Term Administration of L-cvcloserine

Glycolipids Percent- mg/g wet wt.) Control Experimental Reduction

Liver (wet wt.) 2.02 ± 0.13 1.90 ± 0.1 1 6

Glucocerebrosides 0.56 ± 0.044 0.42 ± 0.015* 26

Sulfatides 0.49 ± 0.026 0.41 ± 0.041 17

Spleen (wet wt.) 0.10 0.08 20

G 1 ucocerebrosi des 2.51 1.33 47

Sulfatides 0.36 0.37 0

Five 16 day old mice per group were injected subcutaneously with L-cycloserine (25 mg/kg body wt) alternate days for 2 months. Controls received saline. The animals were killed, and glucocerebrosides were separated from liver and spleen lipids by borate TLC plates as described in Materials and Methods. Values are means ± SEM of five animals per group. Values for spleen were obtained from pooled samples.

* P < 0.005

Claims

WE CLAIM :

1. A method of inhibiting replication of an enveloped virus comprising contacting a virally infected cell or a cell susceptible to viral infection with a replication-inhibiting amount of at least one inhibitor of sphingolipid biosynthesis.

2. The method of Claim 1 wherein said inhibitor of sphingolipid biosynthesis is L-cycloserine.

3. The method of Claim 1 or 2 wherein said enveloped virus is a human immunodeficiency virus (HIV) .

4. A pharmaceutical composition comprising at least one inhibitor of sphingolipid biosynthesis and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition of Claim 4 wherein said inhibitor of sphingolipid biosynthesis is L-cycloserine.

6. A compartmentalized kit for the treatment or prevention of infection by an enveloped virus which comprises a first container adapted to contain an inhibitor of sphingolipid biosynthesis.

7. The compartmentalized kit of Claim 6 wherein said inhibitor of sphingolipid biosynthesis is L-cycloserine.

8. An article of manufacture comprising a packaging material and an inhibitor of sphingolipid biosynthesis contained therein wherein said packaging material comprises a label that indicates that said inhibitor of sphingolipid biosynthesis can be used as an antiviral agent.

9. The article of manufacture of Claim 8 wherein said inhibitor of sphingolipid biosynthesis is L-cycloserine.