Patent Description:
Human immunodeficiency virus (HIV) is the causative agent of Acquired immunodeficiency syndrome (AIDS). Introduction of combination antiretroviral therapy (cART) has radically improved the management of HIV-<NUM> infection and decreased both morbidity and mortality. However, despite initial hopes to cure HIV, treatments were unable to fully eliminate the virus due to latent persistence of HIV in cells such as the central memory CD4+ T-cells, hematopoietic stem cells, dendritic cells, and cells from the monocyte-macrophages lineage in the form of provirus. The large number of protease (PR), reverse transcriptase (RT), and integrase (IN) amino acid variants has implications for antiretroviral (ARV) therapy and presents a challenge to laboratories performing genotypic resistance testing.

Folic acid analogues are known to inhibit Dihydrofolate Reductase (DHFR) by competitively binding to the active site of the enzyme which ultimately inhibit DNA synthesis pathway in the cells. Trimethoprim, a folic acid analog may be used in combination sulfamethoxazole or dapsone for pneumocystis pneumonia in people with HIV/AIDS but this combination may cause some unwanted side effects. Therefore, pharmacokinetic drug interactions can significantly affect the efficacy and toxicity of chemotherapy.

Few prior arts disclose certain compounds that may be used against HIV infection. For instance, <CIT> discloses analogues of folic acid in combination with para-aminobenzoic acid acting synergistically as anti-fungal agents. However, WO'<NUM> fails to disclose that the combination may be used to inhibit DNA synthesis and act as an antiviral agent.

<CIT> pertains to use of folate mimetics and folate-receptor binding conjugates as the selective targeting of tumours expressing elevated levels of high-affinity folate receptors.

However, US'<NUM> fails to disclose that such compounds may inhibit DNA synthesis and may be used against severe viral infections.

The above cited prior art disclose folic acid analogues that may be used in treating fungal infections or tumor but not in viral infections. Selecting the right treatment strategies and combinations remain challenging in viral infections due to compatibilities among individual drugs and adherence, side-effects from long-term use of drugs and existence and emergence of drug-resistance profiles. Hence, an update or modification in the current approach is important to completely eradicate the viral infections. <CIT> discloses a combination comprising antiviral compounds with methotrexate for treating HIV infection. The invention is limited to specific methotrexates.

An object of the invention is to provide compounds and pharmaceutical composition having anti-HIV activity.

The present invention relates to compounds and pharmaceutical composition having antiviral effects. In particular the present invention pertains to compounds having anti-HIV activity. More specifically, the present invention pertains Methotrexate, combinations thereof and compounds antiviral other with combination their analogues of methotrexate and pharmaceutical composition having anti-HIV activity. The present invention may further relate to pharmaceutical composition comprising compounds in combination with active drugs having anti-HIV activity. The invention is limited by the appended claims.

The present invention is drawn to compounds that have anti-HIV activity. The compounds of the present invention are hitherto first reported for their anti-viral activity.

The present invention discloses the antiviral testing of around <NUM> compounds, in precise <NUM> compounds.

The present invention further discloses that <NUM> molecules are reported for the first time to have significant anti-viral activity. The molecules have antiviral activity at a statistically significant level and comprising an embodiment of the present invention is reported herein at Table <NUM> below.

In an embodiment, the present invention is directed towards compounds having antiviral effects.

In particular, the compounds of the present invention discloses Methotrexate or analogue of Methotrexate (MTX) or Methotrexate and its analogues in combinations with other antivirals compounds for their use as antiviral agents.

The methotrexate of the present invention is selected from methotrexate-hydrate, Methotrexate dihydrate and Methotrexate tetrahydrate.

In total, <NUM> molecules were further tested in T-cell line, CEM-GFP by determining their IC<NUM> value. The MTX-HYD of present invention was selected based on the lowest IC<NUM> value out of <NUM> molecules tested (<FIG>).

MTX-HYD has ~<NUM> fold better anti-HIV potential than MTX (<FIG>).

MTX-HYD shows similar cytotoxicity profile in all the cell types tested with CC<NUM> value ranging from <NUM> to <NUM> (<FIG>). Further, the anti-HIV activity of MTX-HYD is cell-type independent phenomena and it possess significant Therapeutic Index in T-cell lines (CEM-GFP and Jurkat J6), Monocytic cell line (U937) and primary cells (hPBMCs) (<FIG>).

MTX-HYD may possess highly conserved anti-HIV activity and may inhibit HIV-<NUM> replication in virus isolate and viral load independent manner (<FIG>).

DHFR, the well-established target of MTX-HYD may not play a vital role in HIV-<NUM> life cycle (<FIG>).

MTX-HYD may not inhibit the viral enzymes Reverse transcriptase, Integrase and Protease (<FIG>) and may inhibit HIV-<NUM>NL4. <NUM> replication at early stages of virus life cycle (<FIG>.

Mechanistically, MTX-HYD may inhibit IkB phosphorylation which in turn may inhibit NF-kB p65 activation and nuclear localization (<FIG>).

The Methotrexate analogue may be selected from group comprising MTX-methyl-d3-Dimethyl Ester, MTX Dimethyl Ester, MTX-methyl-d3, MTX heptaglutamate and MTX-d3 heptaglutamate, preferably, MTX-methyl-d3-Dimethyl Ester, MTX-Dimethyl Ester and MTX-methyl-d3 (<FIG>).

The analogues-mediated inhibition of virus may be performed in vitro on various virus isolates such as HIV-<NUM>NL4. <NUM>, HIV-<NUM>IndieC, HIV-<NUM>IIIB, HIV-<NUM> ADA, HIV-<NUM><NUM>. Preferably, the analogues may inhibit the replication of all HIV-<NUM> isolates tested with high efficiency.

The antiviral activity of the analogues in terms of efficacy may be tested in dose dependent manner by determining the Therapeutic Index (TI), performing MTT cell proliferation assay, HIV-<NUM> infection and anti-HIV activity assay, ELISA for calculating the percentage inhibition of virus production, RNA dependent DNA polymerase activity assay of HIV-<NUM> RT (RDDP Assay), Cell free assay for HIV-<NUM> Integrase activity and cell free assay for HIV-<NUM> Protease activity, anti-HIV activity on various cell lines such as Jurkat J6 (CD4 +T cell line), TZM-bl cell line, CEM-GFP, U937 cell-lines and primary cells (hPBMCs) to monitor HIV infection and effect of the compounds on inhibition of virus production.

The in vitro efficacy of the MTX-analogues may be based upon the CC<NUM> values (concentrations of drug required to reduce cell viability by <NUM>%) and IC<NUM> values (concentration of the drug required to inhibit <NUM> % viral replication). The relative effectiveness of the analogues in inhibiting viral replication compared to inducing cell death may be indicated by the ratio of CC<NUM> values to IC<NUM> values as the therapeutic index of the MTX analogues. The activity of the methotrexate analogues of the present invention are provided herein below at Table <NUM>.

The above Table <NUM> represents the Therapeutic Index (TI) of MTX analogues which may inhibit HIV-<NUM>NL4. <NUM> replication ><NUM>% at <NUM> concentration in CEM-GFP cells. The analogues such as MTX-methyl-d3-dimethyl ester, MTX dimethyl ester and MTX methyl-d3 may have higher therapeutic index than MTX-HYD (<FIG>).

The analogues such as MTX-methyl-d3-dimethyl ester, MTX dimethyl ester and MTX methyl-d3 inhibits replication of HIV-<NUM>NL4. <NUM> (<FIG>).

MTX-methyl-d3-dimethyl ester may inhibit HIV-<NUM>NL4. <NUM> replication with high efficiency in primary cells (hPBMCs) (<FIG>) and may also inhibit other virus isolates such as HIV-<NUM>IndieC, HIV-<NUM>IIIB, HIV-<NUM> ADA, HIV-<NUM><NUM> with higher Therapeutic Index (TI) than Methotrexate Hydrate (MTX-HYD) both in CEM-GFP and hPBMCs (<FIG>).

In another embodiment, the pharmaceutical composition of the present invention may comprise analogues of methotrexate and at least one active drugs having anti-HIV activity. In other words, the Methotrexate-Hydrate (<FIG>) and its analogues (<FIG>) may be used in combination with other antiviral compounds as synergistic combinations for anti-viral therapy.

The other anti-viral compounds that may be administered in addition to MTX-HYD or MTX analogues may be selected from the group comprising Tenofovir (TDF), Indinavir (IND), Raltegravir (RLT) and Maraviroc (MRV), Lamivudine (LMD), Nevirapine (NVP), Elvitegravir (EVG) and Ritonavir (RTN).

The present invention discloses Methotrexate and its analogue where the Methotrexate and its analogues are used in combination with other antiviral compounds as antiviral agents.

The present invention discloses Anti-viral compounds. The antiviral compounds may be selected from group comprising Tenofovir (TDF), Indinavir (IND), Raltegravir (RLT) and Maraviroc (MRV), Lamivudine (LMD), Nevirapine (NVP), Elvitegravir (EVG) and Ritonavir (RTN).

The present invention discloses Methotrexate and its analogue where the antiviral activity is performed in vitro on virus isolates such as HIV-INL4. <NUM>, HIV-<NUM>IndieC, HIV-<NUM>IIIB, HIV-<NUM> ADA, HIV-<NUM><NUM> with high efficiency.

The present invention discloses Methotrexate and its analogue where the dose to obtain the antiviral activity may be in the range of <NUM>/kg body weight to <NUM>/kg body weight, preferably <NUM>/kg body weight to <NUM>/kg body weight, and most preferably <NUM>/kg body weight to <NUM>/kg body weight.

The present invention discloses a pharmaceutical composition comprising Methotrexate or its analogue in combination with other antivirals as claimed in claim <NUM>, along with pharmaceutically acceptable excipients.

The present invention discloses pharmaceutical composition where the pharmaceutical composition is administered as oral, intradermal, transdermal, parenteral, intramuscular, intrathecal and suppository.

The following examples are given by the way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

HEK-293T (Human Embryonic Kidney Fibroblast) and Jurkat J6 (CD4 +T cell line) cell-lines were obtained from NCCS Cell Repository, NCCS, Pune, India. The HeLa based reporter cell line TZM-bl was initially generated from parental JC. <NUM> cells which contains two separate copies of Luciferase and β-Galactosidase downstream to HIV-<NUM><NUM>' Long Terminal Repeat (LTR) promoter. CEM-GFP, a reporter CD4+ T cell line contains GFP gene downstream to <NUM>' LTR promoter. Both TZM-bl and CEM-GFP were obtained from NIH AIDS Reagent Program, Division of AIDS, NIH. CEM-GFP, Jurkat J6 and U937 cell-lines were grown in RPMI-<NUM> medium (Invitrogen, USA) and HEK-293T and TZM-bl cells were grown in DMEM medium both containing <NUM>% fetal bovine serum (FBS) in presence of <NUM> U/ml of penicillin (Invitrogen, USA) and 100µg/ml streptomycin (Invitrogen, USA) to avoid bacterial contamination. <NUM>µg/ml G418 (Invitrogen, USA) was added to the media containing CEM-GFP cells as a selective antibiotic.

Blood of seronegative donors was obtained from Indian Serological Institute (ISI) Blood bank, Navi Peth, Pune. hPBMCs were isolated from the blood using Histopaque <NUM> by gradient centrifugation as described previously. Briefly, blood was diluted using serum free RPMI-<NUM>. Diluted blood was then carefully layered on top of Histopaque <NUM> without disturbing the gradient. PBMCs were separated by centrifugation at <NUM> rpm for <NUM> minutes. PBMCs were then collected and washed twice with serum free RPMI and seeded at the density of <NUM><NUM> cells/mL of media and activated with PHA and used for further experiments (<FIG>).

Cytotoxicity of MTX-HYD and MTX and their analogues was determined by MTT cell proliferation assay kit, (Roche, Germany) as described earlier. Briefly, cells were seeded at the density of <NUM>,<NUM> cells/well in <NUM> well plate containing <NUM>µL of RPMI. Molecules at different concentration was added to the media and incubated in humidified CO<NUM> incubator. Untreated cells were taken as negative control and DMSO treated cells were taken as vehicle control. <NUM>-hours post incubation, <NUM>µL of MTT (<NUM>/mL) was added in each well in dark and incubated at <NUM> for the formation of formazan crystals. <NUM>-hours post incubation, the crystals were dissolved using <NUM>µL isopropanol and the absorbance was read at <NUM>.

Molecular clones for HIV-<NUM>NL4. <NUM>, HIV-<NUM>IIIB, HIV-<NUM> ADA, HIV-<NUM><NUM> virus isolates were obtained from NIH, AIDS Reagent Program, NIH, USA. HIV-<NUM>IndieC, which is a full length infectious molecular clone of Subtype C HIV-<NUM> prevalent in India was a kind gift of Dr. Tatsumi, Japan. Virus stock was prepared by transfecting these molecular clones in HEK-293T cells using CalPhos Mammalian Transfection Kit (Clontech, USA). Briefly, <NUM> x <NUM><NUM> cells were seeded in <NUM> petri-dish and incubated at <NUM> for adherence. <NUM>-hours post seeding, cells were transfected with various molecular clones independently according to manufacturer's protocol. <NUM>-hours post transfection, supernatant was collected and virus was pelleted at <NUM>,<NUM> rpm at <NUM> for two and a half hours using ultracentrifuge in SW28 rotor. The pellet was suspended in serum free RPMI and used for infection.

For infectivity of the prepared virus stocks, β-galactosidase staining assay was performed. For this, TZM-bl cells were infected with different concentrations of the virus prepared and incubated at <NUM> in humidified CO<NUM> incubator. <NUM>-hours post incubation, cells were washed with 1X PBS and fixed with <NUM>% Glutaraldehyde. Staining solution was added post fixation and blue stained cells were counted within <NUM> hours of staining.

CEM-GFP, Jurkat J6, U937 and hPBMCs were infected with different isolates of HIV-<NUM> as described previously. Briefly, cells were incubated with different isolates of HIV-<NUM> in presence of polybrene (<NUM>µg/mL) at <NUM> in humidified CO<NUM> incubator. <NUM>-hours post incubation cells were washed with serum free RPMI and plated in complete RPMI. Cells were then treated with molecules at different concentrations. Untreated cells were taken as negative control and DMSO treated cells were taken as vehicle control. <NUM>-hours post infection, supernatant was collected and virus production was determined by p24 antigen capture ELISA.

HIV-<NUM> virus production was determined using p24 antigen capture ELISA kit (Advanced Bioscience Laboratories, USA) according to manufacturer's protocol. Briefly, <NUM>µL of viral particle containing supernatant was loaded in to the p24 primary antibody coated wells in presence of lysis buffer and incubated at <NUM>. <NUM>-hour post incubation, wells were washed using ELISA washer (Biorad, USA) and HRP-conjugate solution was added to the well and incubated at <NUM> for <NUM>-hour. After incubation, wells were again washed and substrate solution was added and incubated at room temperature for <NUM> minutes to allow to blue colour to develop. The reaction was stopped using stop solution and absorbance was read at <NUM> using Molecular Devices M5 microplate reader. Percentage Inhibition of virus production was calculated using the following formula.

Effect of MTX-HYD and MTX on RNA dependent DNA polymerase activity of HIV-<NUM> RT enzyme was tested with HIV-<NUM> reverse transcriptase colorimetric assay kit (Roche, Germany) according to manufacturer's protocol. Briefly, <NUM> ng of recombinant HIV-<NUM> reverse transcriptase enzyme in reaction buffer in presence or absence of MTX-HYD were incubated at room temperature for <NUM>-<NUM>. Nevirapine (<NUM>) treated wells were taken as positive control whereas untreated wells were taken as negative control and DMSO treated wells were taken as vehicle control. The reaction mixture containing Poly rA/OligodT (template/primers) and biotin or digoxigenin labelled dUTP nucleotide mixture was added to initiate the reverse transcription at <NUM> for <NUM>-<NUM> hours. After RT reaction, the mixture was loaded in to streptavidin-coated plates to allow binding of streptavidin with biotin in biotinylated nucleotides that were incorporated in newly synthesized DNA. Non-adhered nucleotides were removed by washing with wash buffer and anti-DIG-POD was added to allow the binding to digoxygenin nucleotides. ABTS substrate was then added to observe the colour reaction, which was read at <NUM>.

MTX-HYD was tested for its effect, if any, on HIV-<NUM> Integrase enzyme using HIV-<NUM> integrase assay kit (XpressBio, USA) according to manufacturer's protocol. Briefly, biotin coated <NUM>' Donor Strand (DS) was loaded on to streptavidin coated wells and incubated at <NUM>. <NUM>-minutes post incubation, wells were washed and unattached DS was removed. After washing, HIV-<NUM> integrase enzyme and Transfer Strand (TS) were added to the wells in presence of MTX-HYD and incubated at <NUM>. Raltegravir treated wells were taken as positive controls whereas untreated wells were taken as negative control and DMSO treated wells were taken as vehicle control. <NUM>-minutes post incubation, wells were washed and <NUM>µL/well TMB substrate was added and incubated for <NUM>-<NUM> at room temperature. <NUM>µL/well TMB stop solution was added and absorbance was measured at <NUM>.

Effect of MTX-HYD on HIV-<NUM> Protease enzyme was determined using HIV-<NUM> Protease Activity Assay Kit, Fluorometric (Biovision, USA) according to manufacturer's protocol. Briefly, in a single step reaction, HIV-<NUM> protease enzyme was diluted in reaction buffer and <NUM> nmol/well was loaded in <NUM> well plate in presence or absence of MTX-HYD (<NUM>). Indinavir (IND) (<NUM>) treated wells were taken as positive control and untreated wells were considered as negative control. <NUM>-minutes post addition, Fluorescence was measured using Molecular Devices M5 at Ex/Em = <NUM>/<NUM> (<FIG>).

TZM-bl cells were seeded at the density of <NUM>,<NUM>-<NUM>,<NUM> cells/well of <NUM> well plate with <NUM>µL DMEM (with <NUM>% FBS) in each well. <NUM>-hours post seeding, the media was replaced with fresh DMEM containing HIV-<NUM>NL4. <NUM> at <NUM> MOI and cells were treated with different molecules independently at <NUM> working concentration. Cells were incubated in humidified CO<NUM> incubator for <NUM>-hours. After incubation, the media was replaced and cells were washed twice with incomplete DMEM and <NUM>µL of complete DMEM was added in each wells along with the molecules of interest and incubated at <NUM> in humidified CO<NUM> incubator. <NUM>-hours post incubation, the media was removed and cells were washed twice with <NUM>µL of 1X PBS. After washing, cells were lysed using cell culture lysis reagent and Steady-Glo substrate was added at <NUM>:<NUM> ration in each well. Luminescence was measured using Molecular Devices M5 (Table <NUM>). % Inhibition was calculated using following formula and plotted. <MAT> (RLU = Relative Luminescence Unit).

We further validated the anti-HIV activity of selected candidates in reporter T-cell line, CEM-GFP by determining their IC<NUM> (Inhibitory Concentration <NUM>) values. These results indicate that LOPAC candidates inhibit HIV-<NUM> replication with different efficacy at non-cytotoxic concentrations with IC<NUM> value ranging from <NUM> to <NUM> (Figure - <NUM>).

Claim 1:
Methotrexate or analogue of methotrexate (MTX), or methotrexate and its analogues in combination with other anti-HIV compounds for use in the treatment of Human immunodeficiency virus (HIV) infections, wherein the methotrexate is selected from methotrexate-hydrate, Methotrexate dihydrate and Methotrexate tetrahydrate and wherein the analogues are selected from the group comprising MTX-methyl-d3-Dimethyl Ester, MTX Dimethyl Ester, MTX-methyl-d3, MTX heptaglutamate and MTX-d3 heptaglutamate.