FORM 2THE PATENT ACT 1970 (39 of 1970) &The Patents Rules, 2003 PROVISIONAL SPECIFICATION(See section 10 and rulel3)
1. TITLE OF THE INVENTION: "PHARMACEUTICAL COMBINATION"
2. APPLICANT(a) NAME: CIPLA LTD.(b)NATIONALITY: Indian Company incorporated under the IndianCompanies ACT, 1956 (c) ADDRESS: 289, Bellasis Road, Mumbai Central, Mumbai - 400 008, Maharashtra, India
3.PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention.

Field of invention
The present invention relates to a pharmaceutical composition for the treatment of human immunodeficiency virus (HIV). It further provides methods of preparing the same and to their use in therapy.
Background of Invention
Human Immunodeficiency virus (HIV) infection and related diseases are a major and global problem in today's world. A human immunodeficiency virus (HIV) is the etiological agent of the complex disease that includes progressive suppression or destruction of the immune system known as Acquired Immune Deficiency Syndrome or AIDS and degeneration of the central and peripheral nervous system. This HIV also predisposes subjects to fatal opportunistic infections.
HIV is called retrovirus because it is capable of copying RNA into DNA. HIV's genetic material is stored in the form of RNA. To allow incorporation of this genetic material into the host cell DNA, this viral RNA needs to be transformed into viral DNA. Reverse transcriptase is a necessary enzyme for this reaction. To build its viral DNA, HIV uses nucleotides from the cell cytoplasm (nucleosides are composed of a sugar and a base, nucleotides are composed of a sugar, a base, and a phosphate).
Nucleoside reverse transcriptase inhibitors (NRTI) are in fact nucleoside analogues that lack a 3' hydroxyl group. After these nucleoside analogues have been phosporilated (to the corresponding nucleotide), they can be incorporated into the growing DNA chain. Because of the missing 3' hydroxyl group in these analogues, the newly made DNA strand is terminated early and polymerization by the reverse transcriptase is stopped. The activity of these NRTIs is not limited to HIV reverse transcriptase only, but can be used against other retroviruses also.
A common feature of retrovirus replication is the extensive post-translation processing of precursor polyproteins by a virally encoded protease to generate mature viral proteins
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required for virus assembly and function. Inhibition of this processing prevents the production of normally infectious virus. Literature reports that genetic inactivation of the HIV encoded protease resulted in the production of immature, non-infectious virus particles. These results indicate that inhibition of the HIV protease represents a viable method for the treatment of AIDS and the prevention or treatment of infection by HIV.
One substantial and persistent problem in the treatment of AIDS has been the ability of the HIV virus to develop resistance to the individual therapeutic agents employed to treat the disease.
Now-a-days various combinations have been made available for this purpose and several attempts have been made to formulate combination regimens like Limivudine 150 mg and Zidovudine 300 mg which is commercially available as Combivir of Glaxosmithkline. Other such combination is Abacavir and Lamivudine which has been disclosed in Galxo's patent WO0310I467.
Combinations of lamivudine with other reverse transcriptase inhibitors, in particular
zidovudine, are described in WO 92/20344, WO 9818477, and W09955372 etc.
US patent application no. 2004/0224917 discloses the combination of Tenofovir and
Emtricitabine.
In spite of such combinations, there still remains a need to develop combination for acute therapy and for resistant HIV viruses.
The present invention, thus, provides an effective combination which attempts to provide a solution to the above mentioned problem.
Combination therapy reduces the daily dosages to be taken by patients and simplifies dosing schedule thereby increases patient compliance. Combination therapy also increases the drug efficacy. Use of combination therapy can yield an equivalent antiviral effect with reduced toxicity.
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Object of the invention
The present invention provides a pharmaceutical composition, which, inter alia, will assist in treating the human immunodeficiency virus (HIV).
Another object of present invention is to provide combination of nucleoside reverse transcriptase inhibitors or pharmaceutically acceptable salt, solvate, ester, prodrug or derivative, polymorph thereof and nucleotide reverse transcriptase inhibitors or pharmaceutically acceptable salt solvate, ester, prodrug or derivative, polymorph thereof.
It is another object of present invention to provide for a formulation, which provides the advantages of potent and selective therapeutic activity by employing combination of nucleoside reverse transcriptase inhibitors or pharmaceutically acceptable salt, solvate, ester, prodrug or derivative, polymorph thereof and nucleotide reverse transcriptase inhibitors or pharmaceutically acceptable salt, solvate, ester, prodrug or derivative, polymorph thereof.
It is yet another object of the present invention to provide for a formulation, which comprises a combination of nucleoside reverse transcriptase inhibitors or pharmaceutically acceptable salt, solvate, ester, prodrug or derivative, polymorph thereof and nucleotide reverse transcriptase inhibitors or pharmaceutically acceptable salt, solvate, ester, prodrug or derivative, polymorph thereof.
It is yet another object of the present invention to provide for a formulation, which comprises a combination of nucleoside reverse transcriptase inhibitors or pharmaceutically acceptable salt, solvate, ester, prodrug or derivative, polymorph thereof and nucleotide reverse transcriptase inhibitors or pharmaceutically acceptable salt, solvate, ester, prodrug or derivative, polymorph thereof, thereby providing a more potent formulation and therefore avoiding the resistance associated with single drug therapy.
It is yet another object of the invention to provide for a method of preparation of the pharmaceutical composition of the invention.
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Another object of this invention is to provide for a pharmaceutical composition for treatment of HIV and related disorders resulting in AIDS.
Summary of the Invention
According to the present invention, there is provided a pharmaceutical composition for oral administration, which composition comprises a combination of nucleoside reverse transcriptase inhibitors or pharmaceutically acceptable salt, solvate, ester, prodrug or derivative, polymorph thereof and nucleotide reverse transcriptase inhibitors or pharmaceutically acceptable salt, solvate, ester, prodrug or derivative, polymorph thereof. There are also provided, by the present invention, methods for preparing pharmaceutical compositions according to the invention.
There is also provided, by the present invention, pharmaceutical compositions for the treatment or prevention of human immunodeficiency virus (HIV).
Detailed description of the Invention
The need for a an efficacious and long lasting therapy for AIDS which lowers HIV levels in patients to undetectable level and raises CD4 cell counts for prolonged periods without the development of resistance.
Thus, the present invention provides a combination of nucleoside reverse transcriptase inhibitors or pharmaceutically acceptable salt, solvate, ester, prodrug or derivative, polymorph thereof with nucleotide reverse transcriptase inhibitors or pharmaceutically acceptable salt, solvate, ester, prodrug or derivative, polymorph thereof This combination therapy is a step ahead in the method for enhancing the effectiveness in treating AIDS and to preclude the development of resistance to individual therapeutic agents.
The nucleoside reverse transcriptase inhibitors can be but not limited to Lamivudine, Abacavir, Emitricitabine, zidovudine, stavudine or pharmaceutically acceptable salt, solvate, ester, prodrug or derivative, polymorph thereof, preferably Lamivudine.
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The nucleotide reverse transcriptase inhibitors can be but not limited to Tenofovir, adefovir or pharmaceutical^ acceptable salt, solvate, ester, prodrug or derivative, polymorph thereof, preferably Tenofovir.
According to the invention, a pharmaceutical composition may include combination of Lamivudine, Abacavir, Emitricitabine, zidovudine, stavudine or pharmaceutically acceptable salt, solvate, ester, prodrug or derivative, polymorph thereof and Tenofovir, Adefovir or pharmaceutically acceptable salt, solvate, ester, prodrug or derivative, polymorph thereof.
The composition may be provided as an oral dosage form.
The combination includes nucleoside reverse transcriptase inhibitors with nucleotide reverse transcriptase inhibitors or pharmaceutically acceptable salt, solvate, ester, prodrug or derivative, polymorph thereof.
The nucleoside reverse transcriptase inhibitors includes, but not limited to, compounds such as Lamivudine, Abacavir, Emitricitabine, zidovudine, stavudine etc. The nucleotide reverse transcriptase inhibitors include, but not limited to, compounds such as Tenofovir, Adefovir etc.
Further according to the invention, a method for treating, reducing or inhibiting retroviral infections, in particular HIV infections in a mammal, which includes administering to a human a safe and effective amount of the pharmaceutical composition as set out herein. Yet further according to the invention, use of a composition for the treatment of viral infections, particularly retroviral infections, which may include human immunodeficiency virus (HIV) infections.
Reference to word "treatment" as used herein extends to both the prophylaxis and the treatment of an established malady, infection or its symptoms.
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HIV causes a variety of clinical conditions including acquired immunodeficiency syndrome (AIDS) and chronic neurological disorders. Multiple drug regimes dramatically improve the treatment of HIV infected patients.
Single drug treatment regimens typically require long term treatment increasing the evidence of unwanted side effects. Moreover, single drug therapies are particularly vulnerable to mutation in the HIV runs, leading to drug resistant variants of HIV. Particularly, Tenofovir is given in combination with other anti-viral drugs. Studies have shown that Tenofovir in combination with other anti-viral drugs gives better results.
The use of multiple drug therapies may reduce the development of drug resistant strains of HIV because one drug will usually cancel out mutations against other drugs. Multiple drug therapies even inhibit replication of HIV viruses for a period of time sufficient to eliminate HIV from the body.
The effective multiple drug treatments for HIV often require strict compliance with a complex treatment regimen that can require the administration of many different drugs per day, administered at a precisely timed intervals with careful attention to diet. Patient non-compliance is a well-known problem accompanying such complex treatment regimens in the treatment of HIV because such non-compliance may lead to the emergence of multiple drug resistant strains of HIV and also abandonment of treatment in the middle of the therapy.
The combination therapy in accordance with the invention thus provides a method to enhance the effectiveness in treating AIDS and to prevent the development of resistance to the individual therapeutic agents.
Lamivudine (also known as 3TC) is a synthetic nucleoside analogue, chemically known
as (2R,cis)-4-amino-l-(2-hydroxymethyl-l:3-oxathiolan-5-yl)-(lH)-pyrimidin-2-one
(Epivir - RTM., Lamivudine) and its use in the treatment and prophylaxis of viral
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infections has been disclosed in US 5,047,407. Lamivudine has proven antiviral activity against HIV and other viruses such as HBV.
Intracellularly, lamivudine is phosphorylated to its active 5'-triphosphate metabolite, lamivudine triphosphate (L-TP). The principal mode of action of L-TP is the inhibition of HIV-1 reverse transcriptase (RT) via DNA chain termination after incorporation of the nucleoside analogue into viral DNA. L-TP is a weak inhibitor of mammalian DNA polymerases (alpha) and (beta), and mitochondrial DNA polymerase (gamma).
Lamivudine has also been referred to as (-)-l-[(2R, 5S) 2-(Hydroxymethyl)-l,3-oxathiolan-5-yl] cystosine, (Hydroxymethyl)-l,3-oxathiolan-5-yl] cystosine and it has proven antiviral activity against human immunodeficiency virus (HIV) and other viruses such as hepatitis B. Lamivudine is commercially available from Glaxo Wellcome Inc under trade name EPIVIR. Lamivudine and its use against HIV are described in WO 91/17159 and EP 0382526. Crystalline forms of lamivudine are described in WO 92/21676.
Tenofovir also known as PMPA. Tenofovir disoproxil fumarate (a prodrug of tenofovir) which is a fumaric acid salt of bis-isopropoxycarbonyloxymethyl ester derivative of tenofovir. Tenofovir disoproxil fumarate is 9-[(R)-2-[[bis[[(isopropoxycarbonyl) oxy] methoxy] phosphinyl] methoxy] propyl] adenine fumarate (1:1). Tenofovir disoproxil fumarate requires initial diester hydrolysis for conversion to tenofovir and subsequent phosphorylations by cellular enzymes to form tenofovir diphosphate. Tenofovir diphosphate inhibits the activity of HIV reverse transcriptase by competing with the natural substrate deoxyadenosine 5'-triphosphate and, after incorporation into DNA, by DNA chain termination. Tenofovir diphosphate is a weak inhibitor of mammalian DNA polymerases alpha & beta and of mitochondrial DNA polymerase.
Tenofovir disoproxil fumarate is an analog of adefovir and is classified as a nucleotide reverse transcriptase inhibitor (NtRTI). Tenofovir is a competitive inhibitor of other naturally occurring nucleotides, and its ultimate biological activity is viral DNA chain
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termination. Tenofovir is a novel nucleotide analog with antiviral activity against both HIV and HBV. The mechanism of tenofovir is similar to that of nucleoside analogs, which interferes with Reverse Transcriptase and prevents translation of viral genetic material into viral DNA. Unlike the nucleoside analogs, the nucleotide reverse transcriptase inhibitors are chemically pre-activated with the presence of phosphate group. Since the phosphorylation step is not necessary, nucleotide analogs can incorporate into viral DNA chain more rapidly than nucleoside analogs. More importantly, this will bypass a viral mechanism of nucleoside resistance.
Tenofovir is commercially available from Gilead Science Inc. under the trade name VIREAD. Tenofovir disoproxil fumarate has been disclosed in US patent no. 5,935,946, 5,922,695, 5,977,089, 6,043,230 & 6,069,249 while PMPA or Tenofovir has been disclosed in US patent nos. 4,808,716, 5,733,788 & 6,057,305.
Lamivudine and Tenofovir when intimately mixed to form a single layered tablet; it showed undesirable properties on stability. The appearance of tablets changed to brown colour at Controlled Room Temperature and even at Accelerated temperature. However, such change in appearance was not found in case of a bilayered tablet.

Product : Lamivudine & Tenofovir Disoproxil Fumarate Tablets
CONDITION PARAMETERS INITIAL 25°C / 60% RH 40°C/75%RH
1 M 1 M
Appearance Single layered tablets Brown colouration Brown colouration with gas formation.
Appearance Bilayered tablets No change in the colour No change in the colour
By means of the pharmaceutical composition in accordance with the invention, treatment regimens for HIV and other viruses can be simplified with the goal of enhancing patient compliance by providing a simplified dosage therapy containing a combination of pharmaceutically acceptable amounts of Lamivudine and Tenofovir or pharmaceutically acceptable derivative thereof
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The skilled person may also include appropriate binders in the tablet. Most preferably, the binder is starch, Maltodextrins, HPMC, HPC, povidone etc. added at a level of about l%to 50%.
The skilled person may also include appropriate lubricants in the tablet. Most preferably, the lubricants used are magnesium stearate, zinc stearate, calcium stearate, Sodium stearyl fumarate and more preferably magnesium stearate, added at a level of about 0.25% to 3%.
The person skilled in the art may include wetting agent such as Polysorbate 80, SLS, Sucrose esters, Polyethylene glycols, Lutrols, Cremophor etc. added at a level of about 0.1% to 5%.
In another preferred embodiment the composition may substantially include the following ingredients in %.
The first layer of tablet containing nucleoside reverse transcriptase inhibitors or pharmaceutical^ acceptable derivative thereof in the formulation according to the present invention may comprise about 5 to 55% of Lamivudine or its salt or derivative, about 1 to 50 % diluents, about 1 to 50 % binders, about 1 to 30% disintegrant and about 0.25 to 3.0 % of a lubricant.
Preferably, the first layer of tablet containing Lamivudine or pharmaceutically acceptable derivative thereof in the formulation according to the present invention comprises about 5 to 55% of Lamivudine, about 10 to 50 % Microcrystalline cellulose, about 2 to 30% Sodium starch glycolate, about 1 to 10% of Starch and about 0.25 to 2.5 % of a Magnesium stearate.
The second layer of tablet containing nucleotide reverse transcriptase inhibitors or its salts or derivatives in the formulation according to the present invention may comprise
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about 10 to 85% of Tenofovir or its salts or derivatives, about 1 to 50 % diluents, about 1 to 50 % binders, about 0.5 to30 % disintegrant and about 0.25 to 3 % of a lubricant.
Preferably, the second layer of tablet containing nucleotide reverse transcriptase inhibitors or its salts or derivatives in the formulation according to the present invention comprises about 35 to 85 % of Tenofovir, about 5 to 50 % Lactose or microcrystalline cellulose, about 1 to 10 % starch and about 0.2 to 2 % of Magnesium stearate.
Although different techniques known in the art may be employed to formulate granules, preferably, according to the present invention, a method of preparing a pharmaceutical composition for layer-I preferably includes the steps of blending a diluent and disintegrant with Lamivudine; granulating it further with water and suitable binder into granules; drying the resulting granules and sizing; lubricating the granules.
Preferably, according to the present invention, a method of preparing a pharmaceutical
composition for layer-II preferably includes the steps of blending a diluent with
Tenofovir; granulating it further with water and suitable binder into granules; drying the
resulting granules and sizing: again blending with suitable colour and disintegrant;
lubricating the granules.
Lubricated granules of both the layers then compressed together using suitable
compression machine.
Both the layers may optionally contain colouring agents.
Dry powder, oral suspension, syrup, can be formulated using suitable excipients known in the art e.g. suspending agents, diluents, disintegrants, preservatives, sweetners, flavouring agents, colorants, buffer and / or lubricants and other suitable excipients in order to formulation above mentioned dosage forms.
The present invention may be formulated as a multi-layered tablet formulation, preferably bi-layered which can typically be administered to patients and permits or achieves delivery of pharmaceutical!}' active agents effective for the prevention or treatment of
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infection by HIV and in prevention or treatment of the resulting acquired immunodeficiency syndrome (AIDS).
The each part of the tablet of the present invention can be prepared by wet granulation or direct compression or dry granulation. Further, both the parts i.e. blends may be formulated together as a tablet or capsule or dry syrup or any other suitable dosage form. Present invention may also be formulated as a trilayered tablets wherein placebo layer is used as an intermediate layer between Lamivudine & Tenofovir layer.
In a preferred embodiment of the present invention, a pharmaceutical formulation comprises a bilayered system including the pharmaceutically active agents effective for the treatment of HIV. More particularly, a bilayered formulation according to the present invention comprises a first layer comprising at least one nucleoside reverse transcriptase inhibitors or pharmaceutically acceptable derivatives thereof and further comprising other pharmaceutical excipients and a second layer comprising at least nucleotide reverse transcriptase inhibitors or pharmaceutically acceptable derivatives thereof and further comprising other pharmaceutical excipients: forming a pharmaceutically stable preparation together.
The following examples further describe but are not limited to the pharmaceutical composition in accordance with the present invention.
Example I

INGREDIENTS QTY (mg /tab)
Layer I
Lamivudine 150.00
Microcrystalline cellulose 99.50
Sodium starch glycollate 40.00
Magnesium stearate 4.00
Color 5.00
Layer II
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Tenofovir 300.00
Lactose 170.00
Microcrystalline cellulose 95.00
Starch 30.00
Polysorbate 80 6.50
Croscarmellose sodium 25.00
Magnesium stearate 6.50
Colour 0.50
As per the present invention, a method of preparing a pharmaceutical composition for tablet-I includes the steps of blending a diluent, disintegrant and optionally suitable colour with Lamivudine and then lubricating the blend.
As per the present invention, a method of preparing a pharmaceutical composition for layer-II includes the steps of blending a diluent with Tenofovir; granulating it further with water and suitable binder into granules; drying the resulting granules and sizing; again blending with suitable colour and disintegrant; and lubricating the granules. Lubricated granules of both the layers then compressed together using suitable compression machine.
Example -II
Pharmaceutical composition containing Lamivudine and Tenofovir

INGREDIENTS QTY (mg /tab)
Layer I
Lamivudine 300.00
Microcrystalline cellulose 250.00
Sodium starch glycollate 22.50
Colloidal silicon dioxide 2.25
Magnesium stearate 5.63
Film coat with colour 5.00
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Layer II
Tenofovir disoproxil fumarate to Tonofovir equivalent 300.00
Dibasic Calcium Phosphate 103.50
Starch 20.00
Polysorbate 80 6.5
Water q.s.
Magnesium stearate 4.00
Croscarmellose sodium 6.00
Process of manufacture for part-I includes the steps of- blending the suitable excipients with Lamivudine to form lubricated granules; and compressing the lubricated granules into tablets. The method includes the step of suitably coating the tablets.
Process of manufacture for part-II includes the steps of - blending suitable excipients with Tenofovir; granulating with suitable binder or water; drying the resulting granules and sizing; blending the granules with disintegrant; and lubricating the granules.
This Lamivudine tablet is then dry coated with blend of Tenofovir using suitable
technique.
Example -III

INGREDIENTS QTY (mg /tab)
Layer I
Lamivudine 300.00
Mannitol 195.00
Sodium starch glycollate 85.00
Starch 15.00
Water q.s.
Magnesium stearate 7.00
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Example - VI

INGREDIENTS QTY(mg/tab)
Layer I
Lamivudine 150.00
Microcrystalline cellulose 99.50
Croscarmellose sodium 25.00
Starch (binder) 7.50
Water q.s.
Croscarmellose sodium 15.00
Magnesium stearate 4.00
Colour 5.00
Placebo layer
Lactose monohydrate 104.00
Microcrystalline cellulose 90.00
Crosscarmellose sodium 4.00
Magnesium stearate 1.00
Colloidal silicon dioxide 1.00
Layer II
Tenofovir disoproxil fumarate equivalent to Tonofovir 300.00
Lactose 170.00
DCP 100.00
Starch 28.00
Polysorbate 80 6.00
Water q.s.
Magnesium stearate 6.50
L-HPC 23.00
Process of manufacture for part-I includes the steps of- blending diluent/diluents with Lamivudine; further granulating with suitable binder or water into granules; drying the
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resulting granules and sizing; blending the granules with disintegrant, colour; and lubricating the granules.
Placebo layer is prepared by blending all the suitable excipients.
Process of manufacture for part-II includes the steps of-blending suitable excipients with Tenofovir; granulating with suitable binder or water into granules; drying the resulting granules and sizing; blending the granules with disintegrant and colour; and lubricating the granules.
Both the blends are then compressed together along with Placebo layer using suitable compression machine.
Example - VII
INGREDIENTS QTY (mg /sachet)

Lamivudine 300.0
Hydroxy Propyl Methyl Cellulose 10.00
Tenofovir Disoproxil Fumarate 300.0
Mannitol 900.0
Mannitol (Pearlitol SD 200) 1828.0
Colloidal silicon dioxide 60.0
Sodium saccharin 40.0
Banana Flavour 80.0

Lamivudine coated with Hydroxy Propyl Methyl Cellulose using a process known in the art. All the excipients along with Tenofovir are then mixed together along with coated lamivuine in geometric proportion and filled in sachets.
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Example - VIII
INGREDIENTS QTY (mg /bottle)

Lamivudine 300.0
Hydroxy Propyl Methyl Cellulose 10.00
Tenofovir Disoproxil Fumarate 300.0
Methyl paraben NF 44.0
Propyl paraben NF 4.4
Flavour Banana 150.0
Sodium saccharin USP 7.5
Lake Quinoline Yellow WS 0.75
Titanitum dioxide 30.0
Saccharin Sodium 10.0
Sugar 21.15 g

Lamivudine coated with Hydroxy Propyl Methyl Cellulose using a process known in the art. All the excipients along with Tenofovir are then mixed together along with coated lamivudine in geometric proportion and filled in bottles.
Dated this 14th day of December 2005
Dr. Gopakumar G. Nair Agent for the Applicant
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