WO2007033208A2 - Use of indirubin and its derivatives in the treatments of hiv infection and heart failure - Google Patents

Abstract

Indirubin and its derivatives are described for reduction of replication of human immunodeficiency virus. Such therapeutic agent and its derivatives are also described in application to reducing the effects of heart failure, by administration of indirubin or a functionally active derivative thereof to modify cardiac muscle cell hypertrophy. Indirubin and its functional derivatives may also be employed in antiviral combination therapy compositions containing therapeutically effective chimeric polypeptides containing a virus coat polypeptide sequence and a viral receptor polypeptide sequence wherein the virus coat polypeptide sequence and the viral receptor polypeptide sequence are linked and exhibit ligand/receptor binding affinity.

Description

USE OF INDIRUBIN AND ITS DERIVATIVES IN THE TREATMENTS OF fflV

INFECTION AND HEART FAILURE

CROSS-REFERENCE TO RELATED APPLICATIONS

The benefit of priority of U. S. Provisional Patent Application No. 60/716,097 filed September 12, 2005 in the names of Robert R. Redfield, Alonso Heredia and Charles E. Davis, Jr., is hereby claimed under the provisions of 35 USC §119(e). The disclosure of said U.S. provisional patent application is incorporated herein in its entirety, for all purposes.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to the use of indirubin and derivatives thereof for the reduction of replication of HIV virus

Background of the Related Art

Despite the potent anti-HTV-1 activity of HAART, ongoing low-level virus replication and sporadic rises in viremia (blips) are detected in the majority of treated patients. Current HAART regimens contain inhibitors of HIV-Fs reverse transcriptase and protease. Targeting of additional virus proteins may help augment the antiviral effects of HAART. In this regard, an inhibitor of

HIV-I Tat protein activity would be desirable because transcriptional activation of the HIV- 1

LTR promoter element by Tat is an essential step in the virus life cycle. Recent reports have demonstrated that the cellular transcription factor P-TEFb (Positive transcription elongation factor b) serves as a Tat cofactor and is essential for virus replication.

Indirubin is a bis-indole described more than 30 years ago as being clinically active in the treatment of human chronic myelocytic leukemia. Indirubin can be obtained from 4 natural sources: several of indigo-producing plants, mollusks from the Muricidae family, various bacteria, and human urine. Efforts aimed at improving its solubility and absorption resulted in the derivatives Indirubin-3'-monoxime, 5-halogeno-indirubin, N-ethyl-indirubin and N- methylisoindigo, which exhibited similar or better antitumor activity than indirubin in animal models, Indirubin and its derivatives are thought to exert antileukemia activity through inhibition ofCDK2.

Thus, in light of the shortcomings of other antiviral treatments, it would be beneficial to develop an HIV treatment that uses a compound that inhibits HIV protein activity to prevent, delay or allow fewer cells from activating the HTV- 1 LTR promoter element in an essential step in the virus life cycle.

SUMMARY OF THE INVENTION

The present invention demonstrates that Indirubin-3'-monoxime (EVI) is a potent inhibitor of P- TEFb and Tat transactivation functions. Importantly, IM inhibited HFV-I replication in PBMCs and MDMs at concentrations that did not affect cell viability. It was found herein that from the mechanism of action, BVI was effective against both R5 and X4 strains. In addition, the drug inhibited drug-resistant strains of HFV- 1.

In one aspect the present invention provides for a pharmaceutical composition for reducing effects of Human Immunodeficiency Virus (HIV) infection, the composition comprising a therapeutically effective amount of Indirubin or a functionally active derivative thereof.

This pharmaceutical composition may further comprising at least one antiviral agent selected from the group consisting of Zidovudine (ZDV, AZT), Lamivudine (3TC), Stavudine (d4T), Didanosine (ddl), Zalcitabine (ddC), Abacavir (ABC), Emirivine (FTC), Tenofovir (TDF), Delaviradine (DLV), Efavirenz (EFV), Nevirapine (NVP), Fuzeon (T-20), Saquinavir (SQV), Ritonavir (RTV), Indinavir (JDV), Nelfmavir (NFV), Amprenavir (APV), Lopinavir (LPV), Atazanavir, Combivir (ZDV/3TC), Kaletra (RTV/LPV), Trizivir (ZDV/3TC/ABC), SCH-C, SCH-D, PRO 140, TAK 779, TAK-220, RANTES analogs, AK602, UK-427, 857, monoclonal antibodies, NB-2, NB-64, T-649, T-1249, and functional analog thereof.

The pharmaceutical composition comprising the indirubin or derivative thereof may be administered orally, rectally, nasally, topically, vaginally or parenterally.

In another aspect the present invention provides for a method for reducing the effects of HTV infection, the method comprising: administering a composition comprising indirubin or a functionally active derivative thereof, in an effective amount to inhibit the Tat transactivation of HIV-I. Preferably the functionally active derivative is indirubin-3' monoxime, 5-halogeno-indirubin, N-ethyl-indirubin or N-methylisoindigo.

In yet another aspect, the present invention provides for a therapeutically effective method to reduce replication of HIV in a HIV infected subject, the method comprising: a) administering a therapeutically effective amount of Indirubin or a functionally active derivative thereof, for a first predetermined time period; and b) administering the Indirubin or a functionally active derivative thereof with at least one antiviral agent, for a second predetermined time period, wherein the first and second time periods are sequential in a cyclic schedule.

In a still further aspect, the present invention provides for a method for reducing Tat transactivation thereby reducing replication of HIV, the method comprising administering to a subject a sufficient amount of Indirubin or a functionally active derivative thereof to inhibit the kinase activities of CDK9 a component of P-TEFb.

In yet another aspect the present invention provides for a method of manipulating the cell cycle in an activated lymphocytes with indirubin acting as a compound that prolongs or arrests the Gl phase of a cell cycle thereby increasing production or available levels of P-chemokine by the activated lymphocytes and or down-regulation of CCR5 expression, which, in turn reduces the effects of HIV and related complications.

In another aspect the present invention provides for an antiviral combination therapy, the combination comprising;

a) a therapeutically effective amount of a chimeric polypeptide containing a virus coat polypeptide sequence and a viral receptor polypeptide sequence wherein the virus coat polypeptide sequence and the viral receptor polypeptide sequence are linked by a spacer and wherein the virus coat polypeptide and the viral receptor polypeptide sequences exhibit ligand/receptor binding affinity; and b) a therapeutically effective amount of indirubin or functional derivative thereof which has the function of a Gl cytostatic agent that suppresses and/or reduces expression of CCR5, wherein the Gl cytostatic agent potentiates the effectiveness of the chimeric polypeptide thereby reducing the therapeutic concentration of both the chimeric polypeptide and Gl phase arresting agent.

In various embodiments, the virus coat polypeptide sequence of a chimeric polypeptide is an envelope polypeptide sequence (e.g., full-length gpl20 or a fragment), a virus that binds a co- receptor polypeptide, an immunodeficiency virus, including HIV (e.g., HIV-I or HTV-2), SIV, FIV, FeLV, FPV, and a herpes virus. In various additional embodiments, the viral receptor polypeptide sequence is a CD4 polypeptide sequence, full-length or a fragment thereof, such as the Dl, D2 domains and mutations thereof. Introducing envelope genes derived from viruses that use alternative co-receptors could further expand the potential of these single chain molecules affording protection from viral infection of different cell types that express the different co- receptors.

Chimeric polypeptides having heterologous domains also are provided. Such heterologous domains impart a distinct functionality and include tags, adhesins and immunopotentiating agents. For example, heterologous domains can have an amino acid sequence, such as a c-myc polypeptide sequence or an immunoglobulin polypeptide sequence (e.g., a heavy chain polypeptide sequence). The contents of U.S. Application Nos: 09/934,060 and 09/684,026 are incorporated herein for all purposes.

Other features and advantages of the invention will be apparent from the following detailed description, drawings and claims.

BRIEF DESCRIPTION OF THE FIGURES

Fig 1. Effect of BVI on the kinase activities of CDK2, CDK7 and CDK9. In vitro kinase reactions were performed by using inununoprecipitates prepared from the U937 cell line. DVI was added to the reaction at different concentrations. CDK2 kinase activity was measured using Histone Hl as substrate, whereas CDK7 and CDK9 kinase activities were measured using a peptide carrying 5 heptad repeat sequences (CTD5). Reactions were performed in the presence, of 32P-ATP. Phosphorylated peptides were separated in a 16% Novex Tricine gel. Gels were dried and exposed on a Phosphorimager. Fig 2. IM inhibits Tat activation of CDK9 kinase activity in P-TEFb imtmmoprecipitates from Ul cells, (a) Exogenously added Tat activates the kinase activity of CDK9, and this activation is sensitive to DRB. Kinase reactions using CTD5 as substrate were carried out in the presence and absence of Tat and DRB as indicated on top of the gel. (b) Inhibition of Tat activation of CDK9 kinase activity by IM. Kinase reactions were performed in the presence of Tat and different concentrations of IM. Phosphorylated peptides were separated on a gel. The gel was dried and exposed to a Phosphorimager.

Fig 3. IM inhibits Tat-induced HIV-I RNA expression and virus production in Ul cells. Cells were cultured in the presence of Tat (1 ug/ml) and different concentrations of IM for 48 h. (a) Total cellular RNA was isolated, and 20 ug RNA were separated on a formaldehyde-agarose gel, transferred and hybridized using a whole-genome HIV-I DNA probe (top panel). Lower panel shows hybridization of the gel with a β-actin probe, (b) Virus production was assessed by measuring p24 levels in the culture supernatants. (c) Viability of cells was assessed by Trypan blue staining.

Fig 4. IM inhibits Tat-mediated induction of HIV-I LTR. HLtat cells were transiently transfected with pHIVlacZ. Transfected ceils were cultured in the presence of IM for 48h. β-Gal activity was measured in cell lysates. Cytotoxicity was determined by measuring amount of protein in the lysates.

Fig 5. IM inhibits HIV-I replication in PBMCs. (a) Effect of IM on PBMC viability. PHA- activated PBMCs were cultured in the presence of IL-2 and the indicated concentrations of BVI for 7 days. Cell viability was determined by MTT. (b) HIV-I inhibition. PHA-activated PBMCs were infected with HW-I ADA (R5 strain) and with HW-I mb (X4 strain). Infected cells were cultured in the presence of IL-2 and DVI for 7 days. Day 7 virus replication and cell viability were determined by the RT and Trypan blue assays, respectively.

Fig 6. DVI inhibits HW- 1 replication in monocyte-derived macrophages (MDMs). MDMs were infected with the SF 162 strain of HW-I. Infected cells were cultured in the presence of DVI for 28 days, (a) Virus production was assessed weekly by RT. (b) Cell viability was determined by MTT on day 28. Fig 7. IM inhibits drag-resistant strains of HIV-I. PHA-activated PBMCs were infected with the RT multidrug resistant HIV-I RTMDR and with the CCRS antagonist resistant HIV-I CClOl.19 strains. Infected cells were cultured in the presence of IM for 7 days. Virus production (a) and cell viability (b) were determined by p24 and MTT assays, respectively.

Fig. 8. shows that administering EVI to lymphocytes arrests cells in the Gl phase.

DETAILED DESCRIPTION OF THE INVENTION

A method of treating a viral infection is meant herein to include "prophylactic" treatment or "therapeutic" treatment. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or who exhibits early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

The term "therapeutic," as used herein, means a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.

The term "therapeutically effective amount," as used herein means an amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered. A beneficial effect means rendering a virus incompetent for replication, inhibition of viral replication, inhibition of infection of a further host cell, or increasing CD4 T-cell count, for example.

The term "a virally-targeted cell," as used herein, means a cell in which virus is present and is infective or potentially infective and includes epithelial cells, nervous system cells, T- lymphocytes (activated or resting), macrophage, monocytes, tissue dendritic cells or the like.

The term "functional equivalent," as used herein, means that the agent retains some or all of the biological activity of the corresponding compound.

The term "functional analog," as used herein means compounds derived from a particular parent compound by straightforward substitutions that do not result in a substantial (i.e. more than 100X) loss in the biological activity of the parent compound, where such substitutions are modifications well-known to those skilled in the art, e.g., esterification, replacement of hydrogen by halogen, replacement of alkoxy by alkyl, replacement of alkyl by alkoxy, etc.

The Invention

The present invention demonstrates that IM inhibits CDK9 more potently than CDK2, and that this effect likely is associated with inhibition of HIV-I at non-toxic concentrations of drug and shows the potential use of IM in the treatment of HIV- 1. Further it was discovered that IM has the ability to manipulate the cell cycle in activated lymphocytes by arresting the Gl phase of the cell cycle, thereby disrupting the response of a lymphocyte to IL-2 (through the IL-2R) which governs the transition from Gl to S phase, as well as the progression through S phase.

By arresting the cell cycle in the Gl phase, transcription of CCR5 is suppress to reduce expression of CCR5 surface receptors thereby causing an accumulation or increase of chemokines at the cellular level. This accumulation of chemokines may be due to increase time for production thereof or a reduced number of surface CCR5 receptors for chemokine/ligand uptake.

Transcription of eukaryotic genes by RNA Pol II requires phosphorylation of its carboxyl- terminal domain (CTD) region in the large subunit. The phosphorylation of CTD begins with CDK7 (which is part of the transcription factor TFIIH) and is completed by CDK9 (which is part of the Positive transcription elongation factor or P-TEFb). P-TEFb is necessary for the transition of RNA Pol π into productive elongation in both human and Drosophila systems.

In addition to its role in eukaryotic gene transcription, P-TEFb is crucial for activation of transcription by HIV-I Tat in vitro and essential for HIV-I replication (Mancebo, Flores). Tat enhances the function of P-TEFb by further increasing Pol II CTD phosphorylation. Interestingly, studies using P-TEFb inhibitors or an inactive CDK9 subunit of P-TEFb have shown that HTV-I replication is more sensitive to reduction of P-TEFb activity than other viral or cellular promoters. Thus, inhibition of P-TEFb provides a novel approach to inhibit HIV-I

Screening efforts by Merck, in which over 100,000 compounds were screened for inhibition of Tat transactivation of the HTV-I promoter, found that the chemicals benzymidazole (T172298) and isoxazole (T276239) inhibit P-TEFb and block HTV-I replication at concentrations about 15 times lower than concentrations causing cytotoxic effects (Mancebo, Flores). Similar anti-HTV results at non-toxic concentrations of drug have been reported with Flavopiridol, a drug currently being tested (at high concentrations) by Aventis in clinical trials against cancer. A recent report by Stevenson's group has demonstrated inhibition of Tat transactivation and HIV-I replication in the absence of cell toxicity through knockdown of P-TEFb expression by RNA interference (RX4i).

Here we demonstrate that Indirubin-3'-monoxime (IM) (a previously described inhibitor of CDKl, CDK2 and CDK5) is a potent inhibitor of CDK9 kinase activity, and an inhibitor of Tat transactivation of HIV-I in HLtat cells. In addition, IM inhibited Tat mediated activation of gene expression and virus production in the cell lines Ul and 8E5 at non-toxic concentrations of drug. Similarly, IM was shown to inhibit HIV- 1 replication in primary PBMCs and MDMs. Moreover, IM was found to be effective against drug-resistant HIV-I.

Pharmaceutical Compositions

The present invention provides compositions comprising at IM and optionally at least one antiviral agent, as well as methods of preventing, treating and/or reducing the effects of HTV. The methods comprise administering said compositions comprising the BVI and optionally antiviral agents, wherein the two compounds can be administered, separately, simultaneously, concurrently or sequentially.

Pharmaceutically Acceptable Derivatives and Salts

The teπn "pharmaceutically acceptable derivative" is used herein to denote any pharmaceutically or pharmacologically acceptable salt, ester or salt of such ester of a compound according to the invention, or any compound which, upon administration to the recipient, is capable of providing (directly or indirectly) one or more of the compounds according to the invention, or an antivirally active metabolite or residue thereof.

Preferred esters include carboxylic acid esters in which the non-carbonyl moiety of the ester grouping is selected from straight or branched chain alkyl. e.g. n-propyl, t-butyl, n-butyl, alkoxyalkyl (e.g. methoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl), aryl (e.g. phenyl optionally substituted by halogen, C^alkyl or C^alkoxy or amino); sulfonate esters such as alkyl- or aralkylsulfonyl (e.g. methanesulfonyl); amino add esters (e.g. L-valyl or L- isoleucyl); and mono-, di- or triphosphate esters. The phosphate esters may be further esterified by, for example, a Q.₂0 alcohol or reactive derivative thereof, or by a 2,3-di C₂-₄ acyl glycerol.

Pharmaceutically acceptable salts include, without limitation, salts of organic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic, p-aminobenzoic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p- toluenesulfonic acids and inorganic adds such as hydrochloric, sulfuric, phosphoric and sulfamic acids.

Anti-viral compounds

In one aspect, the compositions and methods of the present invention further comprise a therapeutically effective amount of at least one antiviral agent, including, but not limited to nucleoside RT inhibitors, CCR5 inhibitors/antagonists, viral entry inhibitors and functional analogs thereof.

Preferably, the antiviral agent comprises nucleoside RT inhibitors, such as Zidovudine (ZDV, AZT), Lamivudine (3TC), Stavudine (d4T), Didanosine (ddl), Zalcitabine (ddC), Abacavir (ABC), Emirivine (FTC), Tenofovir (TDF), Delaviradine (DLV), Efavirenz (EFV), Nevirapine (NVP), Saquinavir (SQV), Ritonavir (RTV), Indinavir (IDV), Nelfmavir (NFV), Amprenavir (APV), Lopinavir (LPV), Atazanavir, Combivir (ZDV/3TC), Kaletra (RTVfLPV), Trizivir (ZDV/3TC/ABC);

CCR5 inhibitors/antagonists, such as SCH-C, SCH-D, PRO 140, TAK 779, TAK-220, RANTES analogs, AK602, UK-427, 857, monoclonal antibodies;

viral entry inhibitors, such as Fuzeon (T-20), NB-2, NB-64, T-649, T-1249, SCH-C, SCH-D, PRO 140, TAK 779, TAK-220, RANTES analogs, AK602, UK-427, 857; and functional analogs thereof.

In various embodiments, the EVI of the present invention may be administered with a chimeric polypeptide comprising a virus coat polypeptide sequence which may include an envelope polypeptide sequence (e.g., full-length gpl20 or a fragment), a virus that binds a co-receptor polypeptide, an immunodeficiency virus, including HTV (e.g., HIV-I or HTV-2), SIV, FIV, FeLV, FPV, and a herpes virus. In various additional embodiments, the viral receptor polypeptide sequence is a CD4 polypeptide sequence, full-length or a fragment thereof, such as the Dl, D2 domains and mutations thereof. Further CD4 mimetic may be used in the present invention such as CD4M9. Introducing envelope genes derived from viruses that use alternative co-receptors could further expand the potential of these single chain molecules affording protection from viral infection of different cell types that express the different co-receptors.

Chimeric polypeptides having heterologous domains also are provided. Such heterologous domains impart a distinct functionality and include tags, adhesins and immunopotentiating agents. For example, heterologous domains can have an amino acid sequence, such as a c-myc polypeptide sequence or an immunoglobulin polypeptide sequence (e.g., a heavy chain polypeptide sequence).

Antiviral Therapy

Although current treatment with antiretroviral (ARV) therapy causes suppression of HIV replication and results in improvements of immune function, it is limited by high costs, toxicities and adherence difficulties. Moreover, the chance of achieving long- term control of HIV infection with antiretroviral therapy alone seems very unlikely. To date, current antiretroviral therapy has been shown to be insufficient to completely eradicate HIV from infected individuals and there is no real data that the amount of residual virus is decreasing with time on typical antiretroviral therapy. Further, after stopping antiretroviral therapy, the viral load can rebound to higher levels than pretreatment viral loads.

Antiretroviral therapy demands stringent adherence to complex dosing regimens. The rate of virological failure over a 6-month period of time has been demonstrated to be as high as 60% in patients that cannot achieve greater then 95% adherence. The combination of multiple adverse side effects associated with antiretroviral therapy and the availability of this treatment to only 1 in 20 of the estimated 33 million people infected world wide has prompted reconsideration of the current strategies for achieving the goals of HIV therapy.

Moreover, HIV therapy is now thought to be a life-long process. Therefore, it is crucial to develop effective treatments that can be successfully administered for long periods of time for the suppression of retroviruses, and in particular, the prevention and/or inhibition of HTV. Further, it is desirable to eliminate, or at least minimize, the cytotoxicity associated with the administration of antiviral agents otherwise determined to be effective. It is generally recognized that the toxicity of an antiviral agent may be avoided or at least minimized by administration of a reduced dose of the antiviral agent; however, it is also recognized that the effectiveness of an antiviral agent generally decreases as the dose is reduced.

Thus, one embodiment of the present invention provides for reducing the dose of antiviral agents while maintaining or reducing viral load by using cyclic therapy and introducing IM of the present invention to a dosing regime for an HIV infected subject. Specifically, the use of IM that is considered to be a Gl phase arresting compound in combination with antiviral agents has shown promise to maintain viral suppression in a cycle therapy dosing program. By using 50% less medication, side effects associated with antiretroviral use have been shown to be reduced and adherence has shown to be increased. The other obvious impact is on overall cost of medications, which will facilitate expanding these drugs throughout the developed world.

Further, the compositions and methods of the present invention can be used to treat HIV viral infections by reducing viral load and replication of the virus by reducing binding sites for gpl20 ligands.

Doses to be administered are variable according to IM, the antiviral agent, the treatment period, frequency of administration, the host, and the nature and severity of the infection. The dose can be determined by one of skilled in the art without an undue amount of experimentation.

The compositions of the invention are administered in substantially non-toxic dosage concentrations sufficient to ensure the release of a sufficient dosage unit of the present combination into the patient to provide the desired inhibition of the HTV virus. The actual dosage administered will be determined by physical and physiological factors such as age, body weight, severity of condition, and/or clinical history of the patient. The active antiviral components are ideally administered to achieve in vivo plasma concentrations of an antiviral agent of about 0.01 MM to about 100 wM, more preferably about 0.1 to 10 «M, and most preferably about 1-5 wM, and of a Gl phase arresting agent of about 1 MM-25MM, more preferably about 2-20 iiM, and most preferably about 5-10 wM.

For example, in the treatment of HIV-positive and ADDS patients, the methods of the present invention may use compositions to provide from about 0.005-500 mg/kg body weight/day of an antiviral agent, more preferably from about 0.1-200 mg/kg/day, and most preferably 1-50 mg/kg/day; and from about 0.01-1000 mg/kg body weight/day of IM, more preferably from about 0.001-1000 mg/kg/day, or most preferably from about 0.5-50 mg/kg/day. Particular unit dosages of IM and optionally an antiviral agent of the present invention include 50 mg, 100 mg, 200 mg, 500 mg, and 1000 mg amounts, for example, formulated separately, or together as discussed infra.

It will be understood, however, that dosage levels that deviate from the ranges provided may also be suitable in the treatment of a given viral infection.

Therapeutic efficacy of IM can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining The LD50 (The Dose Lethal To 50% Of The Population) and The ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50- Compounds, which exhibit large therapeutic indexes, are preferred. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

The desired dose is preferably presented as two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, containing 0.01 to 1000 mg, preferably 1 mg to 50 mg, depending on the number of sub-doses .

While it is possible for the IM acting as a Gl phase arresting compound and an antiviral agent to be administered individually, either sequentially or simultaneously, it is preferable to present them together, as combined in a pharmaceutical composition.

The compositions of the present invention may comprise both the above-discussed ingredients, together with one or more acceptable carriers thereof and optionally other therapeutic agents. Each carrier must be "pharmaceutically acceptable " in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.

The present invention provides a method for the treatment or prophylaxis of a viral infection such as retroviral infections which may be treated or prevented in accordance with the invention include human retroviral infections such as human immunodeficiency virus.

The therapeutic compositions according to the present invention may be employed in combination with other-therapeutic agents for the treatment of viral infections or conditions. Examples of such further therapeutic agents include agents that are effective for the treatment of viral infections or associated conditions such as immunomodulatory agents such as thymosin, ribonucleotide reductase inhibitors such as 2-acetylpyridine 5-[(2-chloroanilino) thiocarbonyl) thiocarbonohydrazone, interferons such as alpha -interferon, 1- beta -D-arabinofuranosyl-5-(l- propynyl)uracil, 3'-azido-3'-deoxythymidine, ribavirin and phosphonoformic acid.

Routes of Administration

The compositions according to the present invention, may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal). It will be appreciated that the preferred route will vary with the condition and age of the recipient, the nature of the infection and the chosen active ingredient.

Pharmaceutical formulations of the present invention include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.

The formulations may conveniently be presented in unit dosage form and may be prepared by methods known in the art of pharmacy. Such methods include the step of bringing into association IM and optionally an antiviral agent with the carrier. The carrier optionally comprises one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the separate ingredients with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Such patches suitably contain IM and optionally an antiviral agent such as a CCR5 antagonist: 1) in an optionally buffered, aqueous solution; or 2) dissolved and/or dispersed in an adhesive; or 3) dispersed in a polymer.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, caches or tablets, each containing a predetermined amount of the ingredients; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the Gl phase arresting compound EVI and antiviral agent in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservatives, disintegrant (e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of one or more of the ingredients therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

Formulations suitable for topical administration in the mouth include lozenges comprising IM and optionally an antiviral agent in a flavored basis, usually sucrose or acacia; pastilles comprising one or more of the ingredients in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the one or more of the ingredients in a suitable liquid carrier. Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing, in addition to the one or more of the compounds of the present invention, such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multidose sealed containers, for example, ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

For a perinatal subject, the drug combination of the present invention may be, for example, administered orally after 36 weeks of pregnancy and continued through delivery. Interventions around the time of late gestation and delivery (when the majority of transmissions are thought to occur) are most efficacious.

In addition to the compositions described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Other suitable delivery systems include microspheres that offer the possibility of local noninvasive delivery of drugs over an extended period of time. The administered therapeutic is slowly released from these microspheres and taken up by surrounding tissue cells (e.g. endothelial cells).

The compositions may, if desired, be presented in a pack or dispenser device, which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

The present invention is further illustrated by the following examples that should not be construed as limiting in any way.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2^nd Ed., ed. by Sarnbrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization(B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, VoIs. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

EXAMPLES

IM is an inhibitor of CDK9 kinase activity

It has been reported that IM is an inhibitor of CDKl, CDK2 and CDKS kinase activities. The effect of IM was evaluated on the kinase activities of CDK7 and CDK9, which phosphorylate the CTD region of RNA Pol II and are required for Tat transactivation. For comparison, the effect of IM on CDK2 was also evaluated. Results are shown in Fig 1. IM inhibited the kinase activity of CDR2 with an IC50 value of 0.1-0.5 uM, which is in agreement with the 0.25 uM value reported by Hoessel et al. As expected from previous studies, phosphorylation of the CTD5 peptide by immuαoprecipitates complexes containing CDK7 or CDK9 resulted in underphosphorylated (Ha) and hyperphosphorylated (Ho) forms with different migration properties upon fractionation on SDS gels. IM was relatively inefficient against CDK-7 (IC50 8-16uM), but it strongly inhibited CDK9 kinase activity (IC50 0.01-0.05 uM). These results demonstrate that IM is a potent inhibitor of CDK9 and a weak inhibitor of CRK7. In addition, these data show that inhibition of kinase activity by IM is greater on CDK9 than on CDK2.

IM inhibits Tat activation of CDK9 kinase activity

Binding of the HIV-I Tat protein to P-TEFb strongly activates its CTD kinase activity, resulting in increased phosphorylation of the RNA Pol II CTD and efficient elongation of viral transcription. In in vitro, this reaction results in the formation of a novel form of RNA Pol II referred to as RNA Pol Ho*, which is sensitive to low concentrations of 5,6-dichloro-l-beta-D- ribofuranosyl benzimidazole (DRB). The effect of BVI was tested on the Tat-mediated phosphorylation of P-TEFb using P-TEFb immunoprecipitates prepared from Ul cells, a HIV-I infected cell line that produces defective Tat. In preliminary experiments we tested the ability of Tat to increase the phosphorylation of CTD in kinase reactions containing Ul derived P-TEFb immunoprecipitates. In agreement with previous reports, it was found that Tat dramatically up regulated the kinase activity of P-TEFb as manifested by greater intensities of the IIa and Ho CTD forms as well as by the apparition of the Ho* CTD form(Fig 2a, lane 2).

Moreover, addition of DRB inhibited the kinase reaction and led to the disappearance of the CTD Ho* form (Fig 2a, lanes 3-5). The effect of including IM was evaluated in the kinase reaction, and observed an inhibitory effect which was made by the diminished intensities of the different CTD forms (Fig 2b). In these experiments, DVI inhibited Tat activation of P-TEFb kinase activity with an IC50 value ranging 0.05-0.1 uM. Thus, these data demonstrate that BVI interferes with the ability of HIV-I Tat to activate the kinase activity of P-TEFb.

IM inhibits Tat transactivation of HIV-I LTR and virus production

Activation of P-TEFb by Tat leads to efficient elongation of HIV-I transcripts by the RNA Pol π. The presence of a defective Tat in the Ul cell line results in an aberrant pattern of viral transcription characterized for the absence of full-length viral RNA. Exogenous addition of Tat to Ul cells enables the synthesis of full-length transcripts and virus production. The effect of BVI on the Tat-mediated synthesis of HIV- 1 transcripts in Ul cells was evaluated. To this end, total RNA prepared from Ul cells cultured in the presence of Tat and IM was examined for HIV -1 RNA species by Northern Blot analysis. IM was used at concentrations <16 uM concentrations which were not toxic to cells (data not shown). Results are shown in Fig 3a. In Tat- treated cells, all 3 species of viral RNA (unspliced, single and multiply spliced) were readily detectable. However, in the presence of IM reduced viral transcript signals were observed. Probing of the same RNA membrane with a radioactive probe specific for the β-Actin gene indicated similar content of RNA among the samples. These results indicate that IM inhibits the synthesis of viral transcripts in Tat activated Ul cells. In addition, IM treatment resulted in suppression of virus production as measured by p24 antigen levels in the culture supernatants. At a drug concentration of 8 uM, ~90 % of virus inhibition was achieved in the absence of cell toxicity (Fig 3b). These experiments suggest that IM suppresses the activation by Tat of virus production in the latently infected cell line Ul .

A further confirmation of BVI ability to inhibit Tat transactivation of virus transcription was obtained in transfection studies. HLtat cells, that contain stably integrated copies of the HIV-I LTR promoter linked to the tat gene, were transfected with pHIVlacZ (plasmid containing the virus LTR driving the E. coli lacZ gene). Transfected cells were cultured in the presence of different concentrations of EVI for 48 h. time at which cell extracts were analyzed for β-Gal activity and protein content. β-Gal activity was inhibited by IM in a dose dependent manner (Fig 4). At drug concentrations of 0.5 and 1 uM the β-Gal activity was inhibited by 50 and 90 %, respectively. An almost complete suppression on β-Gal was observed at higher concentrations of BVI. As shown by the total amount of protein content in the cell lysates, the concentrations of IM used were not toxic to the cells. Together, these experiments in Ul and HLTat cells demonstrate that EVI suppresses Tat's ability to activate virus replication by interfering with its LTR- transactivation activity.

IM inhibits HIV-I replication in PBMCs and MDM

The studies thus far described demonstrate that EVI inhibits HIV-I production in the Ul cell line. The antiviral activity of EVI in primary cells was next investigated. In order to ascertain the effect of EVI on cell viability, PHA activated PBMCs from healthy donors were cultured in the presence of IL-2, (100 U/ml) and increasing concentrations of EVI. On day 7 after addition of drug cell viability was examined using the MTT assay (Fig 5a), decreased MTT values were observed at IM concentrations > 4 uM. Antiviral assays on PBMCs were next carried out using the R5 strain HIV-I ADA and the X4 strain HW-IEDB. Concentrations of IM ranging 0.5-2 uM inhibited both viruses from ~50 % to > 90 % in the absence of cellular toxicity (Fig 5b). These data indicate that IM inhibits the replication of HIV- 1 in PBMCs in the absence of cell toxicity.

The antiviral activity of IM in MDM was next investigated. MDMs were infected with HIV-I SF- 162 and cultured in the presence of EvI for 28 days. Results are depicted on Fig 6. Peak of virus production occurred on day 21 after infection, and at that time point EvI concentrations ranging 0.25-4 uM suppressed virus production 40-94 %. An MTT assay perfoπned on day 28 revealed no cytotoxic effects by the drug, Similar antiviral results were obtained in MDMs infected with the HTV-I ADA strain (data not shown). Collectively, these results show that IM inhibits the replication of HIV-I in both PBMCs and MDMs at concentrations that do not affect cell viability. In addition, these data suggest that EvI exerts antiviral activity against both R5 and X4 strains of HIV- I.

IM inhibits drug-resistant strains of HIV-I

Since our experiments suggested that EVI exerts antiviral activity through inhibition of the cellular factor CDK-9, we reasoned that EvI should also be effective against drug-resistant strains of HIV- 1. This hypothesis was tested by evaluating the antiviral activity of EvI on two drug-resistant isolates. Antiviral assays were performed by using PBMCs infected with the drug resistant strains HIV-I RTMDR and CClOl.19. HlV-I RTMDR is an X4 virus containing the 74V, 41L, 106A, and 215Y mutations in the RT gene, which confer resistance to AZT, ddl, nevirapine and other non-nucleoside RT inhibitors. CC 101.19 is an R5 virus, with the 305R, 30SP, 316V and 32 IE mutations in V3, and is resistant to the CCR5 inhibitor SCH-C. The results of these experiments are shown in Fig 7, and they indicate that EvI inhibited both drug-resistant strains at drug concentrations that did not affect cell viability. Thus, these data suggest that EVI exerts antiviral activity against drug-resistant HTV- 1.

IM arrest human lymphocytes in Gl phase

PBMC from 2 normal donors were activated in absence and presence of 4 uM BM for 24 hours. Cells were then fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry. Percentages of cells in each phase of the cell cycle are shown. In the presence of EVI, the % of cells in the 1 increases and this cycle arrest leads to lower percentages of cells in S and G2 phases as compared to untreated control as shown in Figure 8.

Claims

1. A pharmaceutical composition for reducing effects of Human Immunodeficiency Virus (HIV) infection, the composition comprising a therapeutically effective amount of Indirubin or a functionally active derivative thereof.

2. The pharmaceutical composition of claim 1, further comprising at least one antiviral agent.

3. The pharmaceutical composition of claim 1, wherein the functionally active derivative is indirubin-3'monoxime, 5-halogeno-indirubin, N-ethyl-indirubin or N-methylisoindigo.

4. The pharmaceutical composition of claim 2, wherein the antiviral agent is an HIV antiviral agent.

5. The pharmaceutical composition of claim 4, wherein the HIV antiviral agent is a nucleoside RT inhibitor, CCR5 inhibitors/antagonist, viral entry inhibitor or functional equivalent thereof.

6. The pharmaceutical composition of claim 2, wherein the antiviral agent is at least one member selected from the group consisting of: Zidovudine (ZDV, AZT), Lamivudine (3TC), Stavudine (d4T), Didanosine (ddl), Zalcitabine (ddC), Abacavir (ABC), Emirivine (FTC), Tenofovir (TDF), Delaviradine (DLV), Efavirenz (EFV), Nevirapine (NVP), Fuzeon (T-20), Saquinavir (SQV), Ritonavir (RTV), Indinavir (IDV), Nelfmavir (NFV), Amprenavir (APV), Lopinavir (LPV), Atazanavir, Combivir (ZDV/3TC), Kaletra (RTV/LPV), Trizivir (ZDV/3TC/ABC), SCH-C, SCH-D, PRO 140, TAK 779, TAK-220, RANTES analogs, AK602, UK-427, 857, monoclonal antibodies, NB-2, NB-64, T-649, T-1249, and functional analog thereof.

7. The pharmaceutical composition of claim 4, wherein the compound is administered orally, rectally, nasally, topically, vaginally or parenterally.

8. The pharmaceutical composition of claim 4, wherein the antiviral agent comprises tenofovir.

9. The pharmaceutical composition of claim 4, wherein the antiviral agent comprises tenofovir, 3TC or Efavirenz.

10. The pharmaceutical compositions of claim 2, wherein the composition is administered alone and in combination with the antiviral agent in a cyclic therapy program.

11. A method for reducing the effects of HIV infection, the method comprising: administering a composition comprising indirubin or a functionally active derivative thereof, in an effective amount to inhibit P-TEFb thereby inhibiting the Tat transactivation of HIV-I.

12. The method according to claim 11, wherein the functionally active derivative is indirabin-3'monoxime, 5-halogeno-indirubin, N-ethyl-indirubin or N-methylisoindigo.

13. The method of claim 11, further comprising at least one antiviral agent.

14. The method of claim 13, wherein the antiviral agent is an HTV antiviral agent.

15. The method of claim 14, wherein the HIV antiviral agent is a nucleoside RT inhibitor, CCR5 inhibitors/antagonist, viral entry inhibitor or functional equivalent thereof.

16. The method of claim 13, wherein the at least one antiviral agent is a member selected from the group consisting of: Zidovudine (ZDV, AZT), Lamivudine (3TC), Stavudine (d4T), Didanosine (ddl), Zalcitabine (ddC), Abacavir (ABC), Emirivine (FTC), Tenofovir (TDF), Delaviradine (DLV), Efavirenz (EFV), Nevirapine (NVP), Fuzeon (T-20), Saquinavir (SQV), Ritonavir (RTV), Indinavir (IDV), Nelfinavir (NFV), Amprenavir (APV), Lopinavir (LPV), Atazanavir, Combivir (ZDV/3TC), Kaletra (RTV/LPV), Trizivir (ZDV/3TC/ABC), SCH-C₃ SCH-D, PRO 140, TAK 779, TAK-220, RANTES analogs, AK602, UK.-427, 857, monoclonal antibodies, NB-2, NB-64, T-649, T-1249, and functional analog thereof.

17. The method of claim 13, wherein the compound is administered orally, rectally, nasally, topically, vaginally or parenterally.

18. A therapeutically effective method to reduce replication of HIV in a HIV infected subject, the method comprising: a) administering a therapeutically effective amount of Indirubin or a functionally active derivative thereof, for a first predetermined time period; and b) administering the Indirubin or a functionally active derivative thereof with at least one antiviral agent, for a second predetermined time period, wherein the first and second time periods are sequential in a cyclic schedule.

19. The therapeutically effective method according to claim 18, wherein the Indirubin or a functionally active derivative thereof is indirubin-3'monoxime, 5-halogeno-indirubin, N-ethyl- indirubin or N-methylisoindigo.

20. The therapeutically effective method according to claim 18, wherein the antiviral agent is a nucleoside RT inhibitor, CCR5 inhibitors/antagonist, viral entry inhibitor or functional equivalent thereof.

21. The therapeutically effective method according to claim 18, wherein the antiviral agent is at least one member selected from the group consisting of: Zidovudine (ZDV, AZT), Lamivudine (3TC), Stavudine (d4T), Didanosine (ddl), Zalcitabine (ddC), Abacavir (ABC), Emirivine (FTC), Tenofovir (TDF), Delaviradine (DLV), Efavirenz (EFV), Nevirapine (NVP), Fuzeon (T-20), Saquinavir (SQV), Ritonavir (RTV), Indinavir (IDV), Nelfinavir (NFV), Amprenavir (APV), Lopinavir (LPV), Atazanavir, Combivir (ZDV/3TC), Kaletra (RTV/LPV), Trizivir (ZDV/3TC/ABC), SCH-C, SCH-D, PRO 140, TAK 779, TAK-220, RANTES analogs, AK602, UK-427, 857, monoclonal antibodies, NB-2, NB-64, T-649, T-1249, and functional analog thereof.

22. A method to reduce the effects of heart failure, the method comprising administering an effective dosage of indirubin or a functionally active derivative thereof to modify cardiac muscle cell hypertrophy.

23. A method for reducing Tat transactivation thereby reducing replication of HTV, the method comprising administering to a subject a sufficient amount of Indirubin or a functionally active derivative thereof to inhibit the kinase activities of CDK9 a component of P-TEFb.

24 A method for reducing the replication of drug-resistant strains of HIV-I, the method comprising administering to a subject a sufficient amount of Ltidirubin or a functionally active derivative thereof.

25. An antiviral combination therapy composition, comprising:

a) a therapeutically effective amount of a chimeric polypeptide containing a virus coat polypeptide sequence and a viral receptor polypeptide sequence wherein the virus coat polypeptide sequence and the viral receptor polypeptide sequence are linked by a spacer and wherein the virus coat polypeptide and the viral receptor polypeptide sequences exhibit ligand/receptor binding affinity; and b) a therapeutically effective amount of indirubin or functional derivative thereof which has the function of a Gl cytostatic agent that suppresses and/or reduces expression of CCR5, wherein the Gl cytostatic agent potentiates the effectiveness of the chimeric polypeptide thereby reducing the therapeutic concentration of both the chimeric polypeptide and Gl phase arresting agent.