WO2011077100A1

WO2011077100A1 - Antiretroviral composition

Info

Publication number: WO2011077100A1
Application number: PCT/GB2010/002331
Authority: WO
Inventors: Amar Lulla; Geena Malhotra
Original assignee: Cipla Limited; Curtis, Philip Anthony
Priority date: 2009-12-24
Filing date: 2010-12-23
Publication date: 2011-06-30

Abstract

A pharmaceutical composition comprising a combination of Withania somnifera extract with at least one anti-retroviral agent and one or more optional pharmaceutically acceptable excipients.

Description

Antiretroviral composition

Field of the Invention:

The present invention pertains to a novel antiretroviral composition of an immunomodulating agent and anti-retroviral agents, its manufacturing process and use of the said composition for the prevention, treatment or prophylaxis of diseases caused by retroviruses, especially acquired immune deficiency syndrome or an HIV infection, in a human.

Background and Prior art: The spread of HIV in India has been diverse, with much of India having a low rate of infection and the epidemic being most extreme in the southern half of the country and in the far north-east. India has the second highest number of people living with HIV/AIDS in the world after South Africa. India accounts for almost 10 per cent of the 40 million people living with HIV/AIDS globally and over 60% of the 7.4 million people living with HIV/AIDS (PLWHA) in the Asia and Pacific region.

Patients suffering from immuno-suppressive diseases as consequence of AIDS or conditions such as human immunodeficiency virus (HIV) infection are immuno-compromised and experience decreased levels of lymphocytes and correspondingly decreased levels of cytokines. For example, where individuals have been infected by HIV, lymphocytes having a cell surface antigen known as CD4 are present in noticeably low numbers. While in healthy individuals these CD4 lymphocyte cells are present in concentrations of about 800 cells per millilitre of serum, HIV-infected individuals exhibit as few as 200 CD4 cells per millilitre of serum when opportunistic infections develop. Similarly, in the case of CD8 lymphocyte, the concentration is found to be very low in case of HIV affected person.

Currently the majority of the AIDS patients in the world population rely on the allopathic medicines which only reduce the mean viral load. Such synthetic antiretroviral compounds are broadly classified as nucleoside and nucleotide reverse transcription inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (Pis) are available. One of the standard care options is a three-drug combination, of two nucleoside analogues and one protease inhibitor. Unfortunately, non-availability, high cost, toxicity concerns and emergence of drug resistance of present anti-viral drugs, pose difficulty in therapeutic management of many patients (K Vermani, S Garg, J Ethnopharmacol, 80 (1): 49-66, 2002; J. A. Wu, A S Attele, L Zhang, C S Yuan, Am J Chin Med, 29 (1): 69-81, 2001).

However, development of resistance has recently become a major concern in the treatment retroviral infections such as AIDS with such medicines. Resistance usually occurs when the drugs being used are not potent enough to completely stop virus replication. If the virus can reproduce at all in the presence of drugs, it has the opportunity to make changes in its structure, called mutations, until it finds one that allows it to reproduce in spite of the presence of the drugs. Once a mutation occurs, it then grows unchecked and soon becomes the dominant strain of the virus in the individual. The drug becomes progressively weaker against the new strain. There is also increasing concern about cross-resistance. Cross- resistance occurs when mutations causing resistance to one drug also cause resistance to another. Several studies have proven that combining two drugs delays the development of resistance to one or both drugs compared to when either drug is used alone. Other studies suggest that three-drug combinations extend this benefit even further. As a result, it is commonly believed that the best way of preventing, or at least delaying, resistance is to use multi-drug combination therapies. The risk of drug interactions and toxicity to the patient increases as the number of drugs increases.

Several modern medications have been developed from ancient healing traditions associated with specific plants. The medicinal properties of many plants have been identified with specific chemical compounds, which have been isolated, purified and, in many cases, synthetically reproduced.

Plant extracts have also been reported to have antiretroviral activity. For example, the plant extract of Buxus sempervirens has shown a beneficial effect in asymptomatic HIV patients and was found to delay the progression of HIV disease (Durant J et al., Phytomedicine, 5, 1- 10, 1998). However, in this investigation, a very high dose (330 mg 3 times per day) was given to the HIV patients. A polyherbal formulation consisting of extracts of Ocimum sanctum, Withania somnifera, Emblica officinalis and Tinospora cordifolia has been reported to protect non-specific host defence mechanisms (S Chatterjee and S N Das, Ani Sci Life, 16, 200-205, 1997; Ani Sci Life, 15 (4): 250-253, 1996). Chatterjee et al also have shown that in immunodeficient and immunocompromised hosts, this polyherbal preparation restored and improved the immune status.

U.S. Pat. No. 5,529,778 has described a composition consisting of eight different plant extracts which have beneficial effects against AIDS, flu, tuberculosis, hepatitis, cirrhosis and immunodeficiency conditions. This patent also describes the use of a very high dose (1 g twice a day) to the patients. Plant extracts are not standardized against their bioactive principles. Hence, consistency of results is expected to be compromised and questionable.

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

U. S. Patent No. 5,178,865 discloses herbal extracts from ten Chinese Herbal Medicines for example extract from Salvia miltiorrhiza, Scutellaria baicaleusis, Coptis chineusis etc demonstrating significant in vitro and ex vivo anti-HIV activity and their use for the diagnosis and treatment of HIV and HIV -related disease. U. S. Patent No. 5,294,443 discloses the use of Tripterygium Wilfordii Hook F extracts for immunosuppression and treatment of autoimmune disorders.

The international patent WO 02/32444 discloses herbal compositions for treating patients with liver disease and HIV comprising various parts of different plants such as Herba Hedyotidis diffusae, Rhizoma Bistortae, Rhizoma Polygoni Cuspidati, and Fructus Schisandrae and so on, and a method for treatment using the compositions mentioned therein.

U. S. Patent No. 7,250,181 discloses a polyherbal composition for the treatment of viral infections caused by HIV containing plant extracts of Withania somnifera, Mangifera indica, an extract of purified Shilajit obtained from rock exudates. The international patent WO 02/079748 discloses a composition obtained from a plant having immuno-stimulant activity or anti-tumor activity, and more particularly to Withania somnifera fractions. In a study by Lu et al, it has been concluded that AIDS is a reversible disease and using medicinal herbs to enhance the immune function will facilitate the appearance of seroconversion, which has not been reported before (Lu W B, Wen R X, Guan C F. A report on 8 seronegative converted HIV/AIDS patients with traditional Chinese medicine. Zhongguo Zhong Xi Yi Jie He Za Zhi May 1997; 17(5):271-3).

It will be noted from the discussion above that allopathic and herbal medicines have been taken separately. However, none of the prior art disclose the combination of allopathic and herbal drug therapy. Accordingly, there exists a need for an alternative efficacious therapy for HIV infection by reducing the mean viral load and alongside improving the immune system of the immunocompromised AIDS patient.

Object of the invention: The object of the present invention is to provide a formulation that effectively inactivates or reduces the human retrovirus load.

Another object of the present invention is to boost immune system of the patient.

Another object of the present invention is to increase the count of CD 4 cells and CD 8 cells.

Another object of the present invention is to provide a formulation which stimulates the cell mediated immunity in human.

Yet another object of the present invention is to provide method of prevention, treatment or prophylaxis of diseases caused by retroviruses, especially acquired immune deficiency syndrome or an HIV infection, in a human. Summary of the invention:

According to one aspect of the present invention there is provided a pharmaceutical composition comprising a combination of Withania somnifera extract with at least one anti- retroviral agent and one or more optional pharmaceutically acceptable excipients. According to a further aspect of the present invention there is provided a process for manufacturing a pharmaceutical composition comprising a combination of Withania somnifera extract with at least one anti-retroviral agent and one or more optional pharmaceutically acceptable excipients.

According to another aspect of the present invention there is provided a method of boosting the immune system, reducing the retroviral load and increasing the CD 4 cell and CD 8 cell count, comprising administering a pharmaceutical composition comprising a combination of Withania somnifera extract and at least one anti-retroviral agent and one or more optional pharmaceutically acceptable excipients to a patient in need thereof.

According to another aspect of the present invention there is provided a method of preventing, treating or prophylaxis of diseases caused by retroviruses, especially acquired immune deficiency syndrome or an HIV infection, in a human comprising administering a pharmaceutical composition comprising a combination of Withania somnifera extract and at least one anti-retroviral agent and one or more optional pharmaceutically acceptable excipients to a patient in need thereof. Detailed Description:

As discussed above, there is a need to develop an alternative efficacious therapy for HIV infection by reducing the mean viral load and alongside improving the immune system of the immuno-compromised AIDS patient.

The present inventors now surprisingly have found that all the aforesaid need can be satisfied when the combination of an herbal extract having immuno modulating activity is administered with antiretroviral agents.

In particular the present inventors have found that on administering a composition comprising a combination of Withania somnifera extract and an antiretroviral agent in fixed dose proportion, the mean retroviral load may reduce, simultaneously increasing the CD4 cell and CD 8 cell count and thus may boost the immunity of the patient.

Thus, the present invention provides a combination of Withania somnifera extract and one or more antiretroviral agents. In particular, the present invention provides a pharmaceutical composition comprising combination of Withania somnifera extract and at least one antiretroviral agent with one or more optional pharmaceutically acceptable excipients.

The terms "Withania somnifera or Ashwagandha extract" are used interchangeably throughout the description in broad sense to include not only an extract prepared using Withania somnifera plant leaves but any parts of the plant such as roots, stem, flower, fruit may be used. Any suitable commercially available Withania somnifera extract may also be used, provided that the said extract possesses immune stimulating activity.

For the purpose of the present invention, suitable extract of Withania somnifera extract can be prepared using various _^extraction methods known in the art such as maceration, remaceration, digestion, agitation, agitation maceration, filtration, vortex extraction, centrifugation, ultrasonic extraction, counter current extraction, percolation, repermolation, evacolation (extraction under reduced pressure), diacolation and solid liquid extraction under continuous reflux in a Soxhlet extractor.

Chemical constituents of Withania somnifera include alkaloids (withanine, withasomnin) and steroidal glycosides (sitoindosides and withanolides; D. Lavie, Plzytochemistry, 14: 189 (1975)). Sitoindosides and Withanolides are suspected of being responsible for particular biological activities of Withania Somnifera, such as antiarthritic and antirheumatic activity (K. Anabalgen et al, Indian Journal of Experimental Biology, 19: 245-249 (1981) ; N. P. Bactor et al., Journal of Research in Indian Medicine, 5 (2): 72 (1971)).

For the purpose of the present invention, any parts of the plant of Withania somnifera may be used to obtain the extract, preferably devoid of trace amount of toxic tropane-type alkaloid, scopolamine. Preferred extract is the standardized extract containing glycowithanolides, withanolide aglycone and oligosaccharides and devoid or have a only a trace amount of toxic tropane-type alkaloid. Antiretroviral agents for the purpose of the present invention may be selected from nucleoside and nucleotide reverse Transcription inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (Pis).

The composition may contain as antiretroviral agent at least one of the materials in the following classes (i) to (iv) along with Ashwagandha

(i) at least one nucleoside reverse transcriptase inhibitor

(ii) at least one nucleotide reverse transcriptase inhibitor

(iii) at least one non-nucleoside reverse transcriptase inhibitor

(iv) at least one protease inhibitor. In an embodiment, the composition may include at least one compound from two of the classes (i) to (iv), in particular (i) and (ii); (i) and (iii); (i) and (iv); (ii) and (iii); (ii) and (iv); and (iii) and (iv).

In an embodiment, the composition may include at least one compound from three of the classes (i) to (iv), in particular (i), (ii) and (iii); (i), (iii) and (iv); and (ii), (iii) and (iv). In an embodiment, the composition may include at least one compound from all four of the classes (i) to (iv).

The term "nucleoside and nucleotide reverse transcriptase inhibitors" (NRTIs) as used herein means nucleosides and nucleotides and analogues thereof that inhibit the activity of HIV-1 reverse transcriptase, the enzyme which catalyzes the conversion of viral genomic HIV-1 RNA into proviral HIV-1 DNA. Recent progress in development of RTI and PI inhibitors has been reviewed (F. M. Uckun and O. J. D'Cruz, Exp. Opin. Ther. Pat. 2006 16:265-293; L. Menendez-Arias, Eur. Pharmacother. 2006 94-96 and S. Rusconi and O. Vigano, Future Drugs 2006 3(l):79-88).

Suitable protease inhibitors (Pis) that may be employed in the pharmaceutical composition of the present invention comprise one or more of saquinavir; ritonavir; nelfmavir; amprenavir; lopinavir, indinavir; nelfmavir; lasinavir; palinavir; TMC114; DMP450, a cyclic urea under development by Triangle Pharmaceuticals; BMS-2322623, an azapeptide under development by Bristol-Myers Squibb as a 2nd-generation HIV-1 PI; BMS-232623; GS3333; K I-413; KNI-272; LG-71350; CGP-61755; PD 173606; PD 177298; PD 178390; PD 178392; U- 140690; ABT-378 under development by Abbott; and AG-1549 an imidazole carbamate under development by Agouron Pharmaceuticals, Inc. Additional Pis in preclinical development include N-cycloalkylglycines by BMS, a-hydroxyarylbutanamides by Enanta Pharmaceuticals; a-hydroxy-y-[[(carbocyclic- or heterocyclic- substituted)amino)carbonyl]alkanamide derivatives; y-hydroxy-2-

(fluoroalkylaminocarbonyl)-l-piperazinepentanamides by Merck; dihydropyrone derivatives and a- and β-amino acid hydroxyethylamino sulfonamides by Pfizer; and N-aminoacid substituted L-lysine derivatives by Procyon.

Suitable nucleoside reverse transcriptase inhibitors (NsRTIs) that may be employed in the pharmaceutical composition of the present invention comprise one or more of Zidovudine; didanosine; stavudine; lamivudine; abacavir; adefovir; lobucavir; entecavir; apricitabine; emtricitabine; zalcitabine; dexelvucitabine; alovudine; amdoxovir; elvucitabine; AVX754; BCH-189; phosphazid; racivir; SP 1093V; stampidine; a nucleoside reverse transcriptase inhibitor disclosed in EP-0358154 and EP-0736533; BCH-10652, a reverse transcriptase inhibitor (in the form of a racemic mixture of BCH-10618 and BCH-10619) under development by Biochem Pharma; P-L-FD4 (also called -L-D4C and named P-L-2',3'- dicleoxy-5-fluoro-cytidene) licensed Vion Pharmaceuticals; DAPD, the purine nucleoside, (- )-p-D-2,6-diamino-purine dioxolane disclosed in EP-0656778 and licensed to Triangle Pharmaceuticals; and lodenosine (FddA), 9-(2,3-dideoxy-2-fluoro-P-D-threo- pentofuranosyl)adenine, an acid stable purine-based reverse transcriptase inhibitor under development by U.S. Bioscience Inc.

Suitable nucleoside reverse transcriptase inhibitors (NtRTIs) that may be employed in the pharmaceutical composition of the present invention comprise tenofovir and/or adefovir.

Suitable non-nucleoside reverse transcriptase inhibitors (NNRTIs) that may be employed in the pharmaceutical composition of the present invention comprise one or more of nevirapine, delaviridine, efavirenz, etravirine. Other NNRTIs currently under investigation include PNU- 142721, a furopyridine-thiopyrimide under development by Pfizer; capravirine (S-1153 or AG-1 549); 5-(3,5-dichlorophenyl)-thio-4-isopropyl-l-(4-pyridyl)methyl-lH-imidazol-2- - ylmethyl carbonate) by Shionogi and Pfizer; emivirine [MKC-442]; (l-(ethoxy-methyl)-5-(l - methylethyl)-6-(phenylmethyl)-(2,4(lH,3H)-pyrimid- inedione)] by Mitsubishi Chemical Co. and Triangle Pharmaceuticals; (+)-calanolide A (NSC-67545 1) and B, coumarin derivatives disclosed in NIH U.S. Pat. No. 5,489,697, licensed to Sarawak/Advanced Life Sciences; DAPY (TMC120); 4-{4-[4-((E)-2-cyano-vinyl)-2,6-dimethyl-phenylamino]-pyrimidin-2- ylamino- }-benzonitrile) by Tibotec-Virco and Johnson & Johnson; BILR-355 BS (12-ethyl- 8- [2-( 1 -hydroxy-quinolin-4-yloxy)-ethyl] -5-methyl- 1 1,12-dihydro- -5H- 1 ,5, 10, 12-tetraaza- dibenzo[a,e]cycloocten-6-one by Boehringer-Ingelheim; PHI-236 (7-bromo-3-[2-(2,5- dimethoxy-phenyl)-ethyl]-3,4-dihydro-lH-pyrido[l,2-a][- l,3,5]triazine-2-thione) and PHI- 443 (TMC-278), l-(5-bromo-pyridin-2-yl)-3-(2-thiophen-2-yl-ethyl)-thiourea) by Paradigm Pharmaceuticals.

The antiretroviral agents of the present invention may be used in the form of salts or esters derived from inorganic or organic acids. These salts include but are not limited to the following: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-ethanesulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3 -pheny .propionate, pi crate, pivalate, propionate, succinate, tartrate, thiocyanate, p-toluenesulfonate and undecanoate. Also, the basic nitrogen-containing groups can be quaternized with such agents as loweralkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides, and others.

In one embodiment, the pharmaceutical composition according to the present invention comprises combination of Withania somnifera extract and at least one nucleoside reverse transcriptase inhibitor (NRTI) with one or more pharmaceutically acceptable excipient. Preferably the NRTI is emtricitabine. In another embodiment, the pharmaceutical composition according to the present invention comprises combination of Withania somnifera extract and at least one nucleotide reverse transcriptase inhibitor (NtRTI) with one or more pharmaceutically acceptable excipient. Preferably the NtRTI is tenofovir, more preferably tenofovir disoproxil fumarate.

In another embodiment, the pharmaceutical composition according to the present invention comprises combination of Withania somnifera extract and at least one non-nucleoside reverse transcriptase inhibitor (NNRTI) with one or more pharmaceutically acceptable excipient. Preferably the NNRTI is efavirenz.

In yet another embodiment, the pharmaceutical composition according to the present invention comprises combination of Withania somnifera extract and at least one non- nucleoside reverse transcriptase inhibitor (NNRTI) with one or more pharmaceutically acceptable excipient. Preferably the NNRTI is Rilpivirine.

In a further embodiment, the pharmaceutical composition according to the present invention comprises combination of Withania somnifera extract, at least one of nucleoside reverse transcriptase inhibitor, at least one of nucleotide reverse transcriptase inhibitor and at least one non-nucleoside reverse transcriptase inhibitor (NNRTI) with one or more pharmaceutically acceptable excipient. Preferably, the said combination comprises Withania somnifera extract, tenofovir, efavirenz, emtricitabine with one or more pharmaceutically acceptable excipient.

In another embodiment, the pharmaceutical composition according to the present invention comprises combination of Withania somnifera extract, at least one of nucleotide reverse transcriptase inhibitor and at least one non-nucleoside reverse transcriptase inhibitor (NNRTI) with one or more pharmaceutically acceptable excipient. Preferably, the said combination comprises Withania somnifera extract, tenofovir, rilpivirine with one or more pharmaceutically acceptable excipient.

In yet another embodiment, the pharmaceutical composition according to the present invention comprises combination of Withania somnifera extract, at least one of nucleotide reverse transcriptase inhibitor and at least one non-nucleoside reverse transcriptase inhibitor (NNRTI) with one or more pharmaceutically acceptable excipient. Preferably, the said combination comprises Withania somnifera extract, tenofovir, rilpivirine, emtricitabine with one or more pharmaceutically acceptable excipient. In a further embodiment, the pharmaceutical composition according to the present invention comprises combination of Withania somnifera extract and at least one anti-retroviral agent selected from nucleoside reverse transcriptase inhibitor, nucleotide reverse transcriptase inhibitor and non-nucleoside reverse transcriptase inhibitor (NNRTI) with one or more pharmaceutically acceptable excipient. Preferably, the said combination comprises Withania somnifera extract, Rilpivirine with one or more anti-retroviral agent and one or more pharmaceutically acceptable excipient.

The amount of Withania somnifera extract and antiretroviral agent that may be combined with suitable pharmaceutically acceptable excipient to produce a single dosage form individually or in combination may vary depending upon the host treated and the particular mode of administration and the drug category chosen.

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy.

Alternatively the pharmaceutical composition of the present invention is in the form of a kit comprising separate dosage forms for each of the Withania somnifera extract and antiretroviral agents which may be administered simultaneously, sequentially or separately. The pharmaceutical composition of the present invention may be administered orally, parenterally, sublingually, by inhalation spray, rectally, or topically in dosage unit formulations containing pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.

Solid dosage forms for oral administration may include capsules, tablets including uncoated and coated tablets, pills, powders, powder for suspension, and granules. Uncoated tablets may be chewable tablet, effervescent tablet, lozenge tablet, soluble tablet, and sublingual tablet; coated tablets may be enteric coated tablet, film coated tablet, implant, sugar coated tablet, and modified-release tablet. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose lactose or starch. Such dosage forms may also comprise, additional substances known in the art, other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings. Other processes of manufacturing solid dosage forms may be employed to develop the pharmaceutical composition of the present invention, such as wet granulation, dry granulation, direct compression, hot melt extrusion, hot melt granulation or other methods known in the art. When provided as a tablet, the tablet may be multilayer, for example bilayer. A different antiretroviral agent may be provided in each layer. The Withania somnifera extract may be provided just one layer, in some of the layers or in every layer. In one embodiment, the composition comprises a bilayer tablet with Tenofovir Disoproxil fumarate and Emtricitabine and Efavirenz in the other layer; the Withania somnifera extract may be provided in the same layer as the Tenofovir Disoproxil fumarate and Emtricitabine.

According to the present invention suitable pharmaceutically acceptable excipients may be used for formulating the solid dosage forms.

According to the present invention, pharmaceutically acceptable excipients include but not limited to carriers, diluents or fillers like lactose (for example, spray-dried lactose, a-lactose, β-lactose) lactose available under the trade mark Tablettose, various grades of lactose available under the trade mark Pharmatose or other commercially available forms of lactose, lactitol, saccharose, sorbitol, mannitol, dextrates, dextrins, dextrose, maltodextrin, croscarmellose sodium, microcrystalline cellulose (for example, microcrystalline cellulose available under the trade mark Avicel), hydroxypropylcellulose, L-hydroxypropylcellulose (low substituted), hydroxypropyl methylcellulose (HPMC), methylcellulose polymers (such as, for example, Methocel A, Methocel A4C, Methocel A15C, Methocel A4M), hydroxyethylcellulose, sodium carboxymethylcellulose, carboxymethylene, carboxymethyl hydroxyethylcellulose and other cellulose derivatives, starches or modified starches (including potato starch, corn starch, maize starch and rice starch) and mixtures thereof. According to the present invention, glidants, anti-adherents and lubricants may also be incorporated in the pharmaceutical composition of the present invention, which may comprise one or more, but not limited to stearic acid and pharmaceutically acceptable salts or esters thereof (for example, magnesium stearate, calcium stearate, sodium stearyl fumarate or other metallic stearate), talc, waxes (for example, microcrystalline waxes) and glycerides, light mineral oil, PEG, silica acid or a derivative or salt thereof (for example, silicates, silicon dioxide, colloidal silicon dioxide and polymers thereof, crospovidone, magnesium aluminosilicate and/ or magnesium alumino metasilicate), sucrose ester of fatty acids, hydrogenated vegetable oils (for example, hydrogenated castor oil) , or mixtures thereof.

According to the present invention, suitable binders may also present in the in the pharmaceutical composition of the present invention, which may comprise one or more, but not limited to polyvinyl pyrrolidone (also known as povidone), polyethylene glycol(s), acacia, alginic acid, agar, calcium carragenan, cellulose derivatives such as ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethylcellulose, dextrin, gelatin, gum arabic, guar gum, tragacanth, sodium alginate, or mixtures thereof or any other suitable binder.

According to the present invention, suitable disintegrants may also be present in the in the pharmaceutical composition of the present invention, which may comprise one or more, but not limited to hydroxylpropyl cellulose (HPC), low density HPC, carboxymethylcellulose (CMC), sodium CMC, calcium CMC, croscarmellose sodium; starches exemplified under examples of fillers and also carboxymethyl starch, hydroxylpropyl starch, modified starch; crystalline cellulose, sodium starch glycolate; alginic acid or a salt thereof, such as sodium alginate or their equivalents and mixtures thereof.

According to one embodiment the pharmaceutical composition of the present invention, may be prepared by a process which comprises (i) mixing one or more drug with pre-sifted diluent and disintegrant

(ii) Adding binder in organic solvent and then adding onto step (i) blend to form granules. (iii) Drying and lubricating the granules with suitable lubricants and then blending with disintegrant and Withania somnifera extract, (iv) compressing the granules to form a tablet, (v) The tablets obtained were seal coated and then film coated.

The pharmaceutical composition of the present invention may also be applied in the form of topical composition comprising pharmaceutically acceptable excipients as desired for topical administration. Solid dosage forms for topical administration may include, spot-on, gel; a spray; a foam; a cream; a wash; a pessary; an ovule; a lotion; an ointment; a film; a foaming tablet; a tampon; a vaginal spray; solution; a bath; a liniment; a patch; a pad; a bandage

According to the present invention suitable pharmaceutically acceptable excipients may be used for formulating topical dosage forms. According to one embodiment, the pharmaceutically acceptable excipients for topical composition may include, but are not limited to, one or more surfactant, emollient or humectant, pH adjusting agent, fatty alcohol, preservative, organic solvent, gelling agents, chelating agents, film forming polymers, antioxidants, propellants or combinations thereof.

The surfactants may be selected from, but not limited to, Polyoxyethylene alcohol, alkylphenol ethoxylate, polysorbate 80, polysorbate 60, polymethylsiloxane, alkylphenol ethoxylate, poloxomer 407, sorbitan monostearate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monooleate, polyethylene glycol (PEG) stearic acid esters (e.g. polyethylene glycol 100 stearate).

Suitable humectants and/or emollients provide smoothness and lubricity which, in turn, facilitate the loading and dispensing of the formulation. The emollients and/or humectants may be selected from, but not limited to, polyhydric alcohols such as glycols, and polysaccharides, such as ethylene glycol, propylene glycol, butylene glycol, diethylene glycol, dipropylene glycol, glycerin, diglycerin, sorbitol, malvitol, trehalose, raffinose, xylitol, mannitol, polyethylene glycol, propylene glycol, polyglycerin, cholesterol, squaline, fatty acids, octyldodecanol, myristyl alcohol, urea, lanolin, lactic acid, esters such as isopropyl stearate, isopropyl myristate, isopropyl palmitate and isopropyl laurate and the like.

The pH adjusting agents may be selected from, but not limited to, lactic acid, sodium hydroxide, acetic acid, citric acid, tartaric acid, propionic acid, sodium phosphate, ammonia solution, triethanolamine, sodium borate, sodium carbonate, potassium hydroxide and like. The preservatives may be selected from, but not limited to, benzyl alcohol, hydroxybenzoates (parabens), Benzoic Acid, Chlorphenesin, Sorbic Acid, Phenoxyethanol and like.

The gelling agents may be selected from, but not limited to, alginic acid, sodium alginate, potassium alginate, agar, carrageenan, pectin, gelatin, calcium alginate, carbomers, methyl cellulose, sodium carboxy methyl cellulose, hydroxy ethyl cellulose and other cellulose derivatives, carbopol, bentonite (preferably carbomers) may be used in combination with bioadhesives which includes, but not limited to, gelatin, carbopol 934, polycarbophil, cross- linked polymethacrylic acid, hydroxypropyl methyl cellulose, ethyl cellulose, preferably carbopol & methyl cellulose. The chelating agents may be selected from, but not limited to disodium edetate, sodium citrate, condensed sodium phosphate, diethylenetriamine penta-acetic acid and like.

According to one embodiment the pharmaceutical composition of the present invention, may be prepared by a process which comprises (i) dissolving preservatives to pre-heated humectant. (ii) adding gelling agent to the above solution to form an organic phase, (iii) adding chelating agent to the purified water and then adding drug to it to form a drug phase, (iv) adding ashwagandha extract to the purified water and filtering through suitable filter to form a herbal phase, (v) adding drug phase to organic phase and stirring to form a gel. (vi) finally adding herbal phase to step (v) to form a gel.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavouring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1, 3-propanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Suppositories for rectal administration of the drug can be prepared by mixing the drug with suitable nonirritating pharmaceutically acceptable excipients. Examples of suitable excipients include but not limited to cocoa butter and polyethylene glycols which are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug. According to the present invention there is provided a method of boosting the immune system, reducing the retroviral load and increasing the CD 4 cells and CD 8 cell count comprising administering the pharmaceutical composition comprising a combination of Withania somnifera extract and at least one anti-retroviral agent. In a further embodiment, there is provided a method of preventing or treating HIV infection or alleviating the symptoms associated with HIV infection comprising administering the pharmaceutical composition comprising a combination of Withania somnifera extract and at least one anti-retroviral agent. The following examples are for the purpose of illustration of the invention only and are not intended to limit the scope of the present invention.

Examples:

Formula 1: Tablets containing Efavirenz, Emtricitabine, Tenofovir disoproxil fumarate and Withania somnifera extract

1. Tenofovir diso roxil fumarate, Emtricitabine & Withania somnifera layer

4 Red oxide of iron 0.06

5 Microcrystalline cellulose 83.94

Binder preparation

6 Hypromellose 15.00

7 Isopropyl alcohol q. s.

8 Methylene chloride q. s.

Lubrication

9 Crosscarmellose sodium 40.00

10 Corn starch 30.00

11 Magnesium stearate 6.00

12 Withania somnifera extract 200.00

Total 900.00 r:

(mg)

1 Hypromellose 5.00

2 Isopropyl alcohol q. s.

3 Purified water q. s.

4. Film coating:

Process:

(1) Tenofovir disoproxil fumarate and emtricitabine was mixed with pre-sieved and pre-sifted amounts of croscarmellose sodium, red iron oxide and microcrystalline cellulose.

(2) The above blend was then granulated using binder solution prepared using hypromellose, isopropyl alcohol and methylene chloride.

(3) The granules were lubricated using magnesium stearate and then blended with croscarmellose sodium, corn starch and Withania somnifera extract.

(4) Efavirenz was mixed with pre-sieved and pre-sifted quantities of sodium lauryl sulphate, croscarmellose sodium, microcrystalline cellulose and hydroxypropyl cellulose.

(5) Granules of the blend obtained in step (4) were then obtained using purified water which were then blended and lubricated with magnesium stearate.

(6) Granules obtained in step (3) and step (5) then compressed to form a bilayer tablet.

(7) The bilayer tablet was then seal coated using hypromellose, isopropyl alcohol and purified water solution which then film coated using Opadry ready colour mix solutions.

Example 2: Capsules containing Emtricitabine and Withania somnifera extract

1 Emtricitabine 200.00

2 Microcrystalline cellulose 76.35

3 Crospovidone 10.00

4 Povidone 3.00

5 Purified water q. s.

II Blending

6 Withania somnifera extract 200.00

7 Magnesium stearate 0.65

Total 490.00

Process:

(1) Emtricitabine was mixed with pre-sieved and pre-sifted amounts of croscarmellose sodium, crospovidone and microcrystalline cellulose, povidone and granulated using purified water.

(2) The obtained granules then lubricated with magnesium stearate and blended with Withania somnifera extract.

(3) Finally the above blend was filled in hard gelatin capsules. Example 3: Tablets containing Efavirenz and Withania somnifera extract

8 Withania somnifera extract 200.00

9 Magnesium stearate 15.00

IV Film coating

1 Opadry 03B 52570 Yellow 15.00

3 Purified water q. s.

Total 1515.00

Process:

(1) Efavirenz was mixed with pre-sieved and pre-sifted amounts of lactose monohydrate and sodium starch glycolate.

(2) The above blend was then granulated using sodium starch glycolate, pregelatinised starch. (3) The granules were then lubricated using magnesium stearate and then blended with Withania somnifera extract.

(6) The blend obtained in step (3) was then compressed to form tablets which then film coated using Opadry ready colour mix solution.

Example 4: Tablets containing Tenofovir disoproxil fumarate and Withania somnifera extract.

9 Croscarmellose sodium 20.00

10 Magnesium stearate 9.50

9 Withania somnifera extract 200.00

IV Film coating

1 Opadry 03B 52570 Yellow 15.00

3 Purified water q. s.

Total 875.00

Process:

(1) Tenofovir disoproxil fumarate was mixed with pre-sieved and pre-sifted amounts of croscarmellose sodium, corn starch and lactose monohydrate.

(2) The above blend was then granulated using binder solution prepared using corn starch, polysorbate 80 and purified water.

(3) The granules were then lubricated using magnesium stearate and then blended with croscarmellose sodium, microcrystalline cellulose and Withania somnifera extract.

(4) The blend obtained in step (3) was then compressed to form tablets which then film coated using Opadry ready colour mix solution.

Example 5: Gel formulation comprising Tenofovir and Ashwagandha.

Sr No. Ingredients Qty (% / )

1 Tenofovir disoproxil fumarate 1.00

2 Ashwagandha extract 0.50

3 Disodium edetate 0.05

4 Citric acid 1.00

5 Methyl Paraben 0.18

6 Propyl Paraben 0.02

7 Glycerin 20.0

8 Hydroxy Ethyl Cellulose 2.00

9 Purified Water Up to 100%

10 Sodium Hydroxide Up to pH 4.5

11 Hydrochloric acid q.s to dissolve drug 1. Methyl paraben and Propyl paraben were added to the heated glycerin and stirred to dissolve.

2. To the above formed solution, hydroxy ethyl cellulose (HEC) was added and dispersed. This was the organic phase.

3. Disodium edetate and citric acid were added to purified water in another SS vessel.

The mixture was stirred to get a clear colourless solution.

4. Tenofovir was added and dispersed in the above solution under stirring. The pH of the solution was adjusted to 4.5. This was the drug phase.

5. Ashwagandha extract was added to small amount of water under continuous stirring.

6. Above solution was then filtered through suitable filter to get brown coloured solution. This was herbal phase.

7. The drug phase was added to the organic phase under continuous stirring to form

clear, transparent colorless gel.

8. Herbal phase was then added to the above formed gel to form clear, transparent,

brown colored gel.

It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the spirit of the invention. Thus, it should be understood that although the present invention has been specifically disclosed by the preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and such modifications and variations are considered to be falling within the scope of the invention.

It is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having" and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a polymer" includes a single polymer as well as two or more different polymers; reference to a "plasticizer" refers to a single plasticizer or to combinations of two or more plasticizer, and the like.

Claims

CLAIMS:

1. A pharmaceutical composition comprising a combination of Withania somnifera extract with at least one anti-retroviral agent and one or more optional pharmaceutically acceptable excipients.

2. A pharmaceutical composition according to any one of the preceding claims, wherein the antiretroviral agent comprises at least one of the following groups (i) to (iv): (i) at least one nucleoside reverse Transcription inhibitor; (ii) at least one nucleotide reverse Transcription inhibitors (iii) at least one non-nucleoside reverse transcriptase inhibitor; (iv) and at least one protease inhibitor (PI). 3. A pharmaceutical composition according to any one of the preceding claims, wherein the nucleoside reverse transcription inhibitor comprises one or more of Zidovudine; didanosine; stavudine; lamivudine; abacavir; adefovir; lobucavir; entecavir; apricitabine; emtricitabine; zalcitabine; dexelvucitabine; alovudine; amdoxovir; elvucitabine;

AVX754; BCH-189; phosphazid; racivir; SP 1093V; stampidine; BCH- 10652, the purine nucleoside, (-)- -D-2,6-diammo-purine dioxolane, lodenosine (FddA), 9-(2,

3-dideoxy-2- fluoro-P-D-threo-pentofuranosyl)adenine.

4. A pharmaceutical composition according to any one of the preceding claims, wherein the nucleotide reverse transcription inhibitor comprises one or more of tenofovir and/or adefovir. 5. A pharmaceutical composition according to any one of the preceding claims, wherein the non-nucleoside reverse transcriptase inhibitor comprises one or more of nevirapine, delaviridine, efavirenz, etravirine. PNU- 142721, a furopyridine-thiopyrimide; capravirine (S-1 153 or AG-1 549); 5-(3,5-dichlorophenyl)-thio-4-isopropyl-l-(4-pyridyl)methyl-lH- imidazol-2- -ylmethyl carbonate); emivirine [MKC-442]; (l-(ethoxy-methyl)-5-(l - methylethyl)-6-(phenylmethyl)-(2,4( 1 H,3H)-pyrimid- inedione)] ; (+)-calanolide A (NSC- 67545 1) and B, coumarin derivatives; DAPY (TMC120); 4-{4-[4-((E)-2-cyano-vinyl)- 2,6-dimemyl-phenylamino]-pyrimidin-2-ylamino-}-benzonitrile); BILR-355 BS (12- ethyl-8-[2-(l -hydroxy-quinolin-4-yloxy)-ethyl]-5-methyl- 11,12-dihydro-5H- 1,5,10,12- tetraaza-dibenzo[a,e]cycloocten-6-one; PHI-236 (7-bromo-3-[2-(2,5-dimethoxy-phenyl)- ethyl]-3,4-dihydro-lH-pyrido[l,2-a][- l,3,

5]triazine-2-thione) and PHI-443 (TMC-278), l-(5-bromo-pyridin-2-yl)-3-(2-thiophen-2-yl-ethyl)-thiourea.

6. A pharmaceutical composition according to any one of the preceding claims, wherein the protease inhibitors comprises one or more of saquinavir; ritonavir; nelfinavir; amprenavir; lopinavir, indinavir; nelfinavir; lasinavir; palinavir; TMC1 14; DMP450, BMS-2322623, BMS-232623; GS3333; KNI-413; KNI-272; LG-71350; CGP-61755; PD 173606; PD 177298; PD 178390; PD 178392; U-140690; ABT-378; and AG-1549 N- cycloalkylglycines, a-hydroxyarylbutanamides; a-hydroxy-Y-[(carbocyclic- or heterocyclic-substituted)amino)carbonyl]alkanamide derivatives; y-hydroxy-2- (fluoroalkylaminocarbonyl)-l-piperazinepentanamides; dihydropyrone derivatives and a- and β-amino acid hydroxyethylamino sulfonamides; and N-aminoacid substituted L- lysine derivatives

7. A pharmaceutical composition according to any one of the preceding claims, wherein the antiretroviral agent includes emtricitabine.

8. A pharmaceutical composition according to any one of the preceding claims, wherein the antiretroviral agent includes tenofovir, preferably tenofovir disoproxil fumarate.

9. A pharmaceutical composition according to any one of the preceding claims, wherein the antiretroviral agent includes efavirenz.

10. A pharmaceutical composition according to any one of the preceding claims, wherein the antiretroviral agent includes tenofovir, preferably tenofovir disoproxil fumarate; efavirenz; and emtricitabine.

1 1. A pharmaceutical composition according to any one of the preceding claims, wherein the antiretroviral agent includes Rilpivirine.

12. A pharmaceutical composition according to any one of the preceding claims, wherein the antiretroviral agent includes Rilpivirine and Tenofovir.

13. A pharmaceutical composition according to any one of the preceding claims, wherein the antiretroviral agent includes Rilpivirine, Emtricitabine and Tenofovir.

14. A pharmaceutical composition according to any one of the preceding claims comprises one or more of antiretroviral agent and Rilpivirine.

15. A pharmaceutical composition according to any preceding claim, which is formulated for oral, parenteral, sublingual, inhalation, rectal, topical administration, powder for suspension, oral granules and the like.

16. A pharmaceutical composition according to any one of the preceding claims, for use in boosting the immune system, reducing the retroviral load and increasing the CD 4 cells and CD 8 cell count.

17. A pharmaceutical composition according to any one of claims 1 to 15, for use in preventing or treating HIV infection or alleviating the symptoms associated with HIV infection.

18. A method of boosting the immune system, reducing the retroviral load and increasing the CD 4 cells and CD 8 cell count comprising administering a therapeutically effective amount of a pharmaceutical composition according to any one of claims 1 to 15 to a patient in need thereof.

19. A method of preventing or treating HIV infection or alleviating the symptoms associated with HIV infection comprising administering a therapeutically effective amount of a pharmaceutical composition according to any one of claims 1 to 15 to a patient in need thereof.