Field of the invention
The present invention relates to stable bilayered tablets of combination of antiretroviral agents. More particularly, the present invention relates to stable dosage forms comprising lamivudine, tenofovir disoproxil fumarate and efavirenz prepared by wet granulation.
Background of the invention
The human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). This disease is characterized by the destruction of the immune system, particularly of the CD4 and T-cell making the host susceptible to opportunistic infections. HIV is also associated with a precursor AIDS-related complex (ARC), a syndrome characterized by symptoms such as persistent generalized lymphadenopathy, fever and weight loss.
Anti-retroviral drugs, such as reverse transcriptase inhibitors and viral protease inhibitors, have been used to treat HIV infection. These treatments can effectively suppress viral production when used in combinations known as HAART (Highly Active Anti-Retroviral Therapy).
Tenofovir disoproxil fumarate (a prodrug of tenofovir) is a fumaric acid salt of bis-isopropoxycarbonyloxymethyl ester derivative of tenofovir. Chemically, tenofovir disoproxil fumarate (tenofovir DF) is 9-[(R)-2-[[bis[[(isopropoxycarbonyl)oxy]methoxy]phosphinyl]methoxy]propyl]adenine fumarate and is commercially available as tablets under the trade name Viread®. Tenofovir disoproxil fumarate was first disclosed in US patent No. 5,922,695.
Chemically, lamivudine is (2R,cis)-4-amino-l-(2-hydroxymethyl-l,3-oxathiolan-5-yl)-(lH)-pyrimidin-2-one and is commercially available as tablets and oral solution under the trade name Epivir®. Lamivudine and method of treating HIV using lamivudine was first disclosed in US 5,047,407.
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. Thus, a need remains for an efficacious and long lasting therapy for AIDS which lowers HIV viral levels of patients to undetectable levels and raises CD4 cell counts for prolonged periods of time without the development of resistance. Hence, there is a need to develop combination of drugs to treat AIDS called as fixed dose combinations (FDC).
Chemically, efavirenz is (S)-6-chloro-4-(cyclopropylethynyl)-l,4-dihydro-4-(trifluoromethyl)-2H-3,l-benzoxazin- 2-one and is commercially available as tablets and capsules under the trade name Sustiva®. Efavirenz and method of treating HIV using efavirenz was first disclosed in US 5,519,021.
The various advantages of FDCs when compared to the separate ARV regimens are ease of use, better adherences to the dosage schedules, reduced risk of drug resistance and increased affordability. Combination therapy reduces the daily dosages to be taken by patients and simplifies dosing schedule thereby increases patient compliance.
Following are few patents/publications, which disclose combinations of antiretrovirals.
US 5,627,186 disclose combination of lamivudine and zidovudine for treating HIV infections.
US 6,417,191 discloses combination of abacavir and lamivudine; abacavir, lamivudine and zidovudine for treating HIV infections.
US 2004/0224917 discloses therapeutic combinations of emtricitabine and tenofovir DF useful for treatment of HIV infections.
US 2007/0077295 discloses composition of tenofovir DF and emtricitabine prepared by dry granulation for treating HIV infections.
US 2007/099902 discloses a multi component unitary dosage form comprising tenofovir DF and emtricitabine in first component and efavirenz in second component useful for treatment of HIV infections.
ZA 2001/10500 discloses pharmaceutical composition of lamivudine, zidovudine and nevirapine and process, which comprises wet granulating lamivudine, zidovudine, nevirapine and diluent with water; drying, sizing and

blending the granules with disintegrant; lubricating the granules; and compressing the lubricated granules into tablets.
WO 2004/089382 discloses combination of lamivudine, zidovudine and efavirenz for treating HIV infections.
WO 2004/089383 discloses combination of lamivudine, stavudine and efavirenz for treating HIV infections.
WO 2006/001029 discloses a process for preparing a composition of lamivudine, zidovudine and nevirapine and process which comprises granulating lamivudine, zidovudine, nevirapine, microcrystalline cellulose, starch, croscarmellose sodium with a solution of polyvinylpyrrolidone k-30 and drying the granules, blending the dried granules with magnesium stearate, croscarmellose sodium, colloidal anhydrous silica, crospovidone and compressing the blend into tablets.
WO 2006/086865 discloses a composition comprising lamivudine, zidovudine and nevirapine in a single coated tablet prepared by dry granulation.
WO 2006/114709 discloses a composition comprising lamivudine, zidovudine and nevirapine prepared by a granulation process comprising the steps of preparing granules of lamivudine plus zidovudine and granules of nevirapine separately, blending the obtained granules with excipients and finally compressing the granules into tablets.
WO 2007/026156 discloses a composition comprising lamivudine, stavudine and nevirapine for pediatric treatment of viral infections.
WO 2007/068934 discloses a bilayered formulation comprising lamivudine and tenofovir. It is disclosed in this publication that lamivudine and tenofovir DF when intimately mixed to form a single layered tablet showed undesirable properties in stability testing. The appearance of tablets changed to brown colour at controlled room temperature (25°C) and even at accelerated temperature (40°C). Inorder to avoid the discoloration, lamivudine and tenofovir DF are formulated as a bilayered tablet. This patent publication further discloses a kit comprising bilayered tablet of lamivudine and tenofovir as first formulation and efavirenz tablet as second formulation.

WO 2008/043829 discloses combination of emtricitabine, tenofovir and nevirapine for once a day administration.
WO 2008/096369 discloses a formulation comprising lamivudine and tenofovir DF prepared by granulating tenofovir DF seperately by dry granulation and blending the granules with extragranular lamivudine and finally compressing into tablets. This publication further discloses monolithic tablets comprising emtricitabine/lamivudine, tenofovir DF and efavirenz.
The above prior art disclose compositions of various combinations of antiretroviral agents prepared as single and/or double layered tablets. However, still there is a need to develop stable dosage form comprising lamivudine, tenofovir and efavirenz. The inventors of the present invention have surprisingly found that when lamivudine, tenofovir DF and efavirenz are granulated separately and compressed into a bilayered tablet results in a stable dosage form.
Objective of the invention
Accordingly, the main objective of the present invention is to provide a stable dosage form comprising lamivudine, tenofovir disoproxil fumarate and efavirenz and process for preparing the dosage form.
Yet another objective of the present invention is to provide stable dosage form of lamivudine, tenofovir disoproxil fumarate and efavirenz in such a way that it will comply with the reference products of each of these approved individual drugs in terms of in vitro parameters like dissolution, disintegration etc. and in vivo parameters like bioequivalence.
Summary of the invention
Accordingly, the present invention provides stable bilayered tablets comprising lamivudine, tenofovir and efavirenz prepared by wet granulation process.
Detailed description of the invention
In an embodiment, the stable dosage form further comprises one or more excipients selected from diluents, binders, disintegrants, surfactants, glidants and lubricants.

Efavirenz practically insoluble in water and is freely soluble in methanol. Hence, in order to improve the solubility and thereby bioavailability, the commercially available tablets of efavirenz i.e available in the US under the trade name Sustiva® tablets and capsules contain sodium lauryl sulfate as a solubilizer. However, the inventors of the present invention found that when tablets containing lamivudine, tenofovir and efavirenz are prepared using single step process, tenofovir degrades on storage.
Initial efforts to simply combine lamivudine, tenofovir and efavirenz into a unitary composition failed to produce a chemically stable tablet. The tenofovir in this combination tablet was highly unstable and rapidly degraded in stability studies. The efavirenz formulation containing sodium lauryl sulfate was unexpectedly incompatible with tenofovir.
Suitable diluents of the present invention are selected from mannitol, lactose, microcrystalline cellulose, maltitol, sorbitol, maltodextrin, maltose, starch, calcium carbonate, calcium phosphate dibasic, calcium sulfate or a combination thereof. The diluent may be used in the range of 3-50% by weight of the tablet.
Suitable binders of the present invention are selected from hydroxy propyl cellulose, hydroxypropyl methylcellulose, gelatin, hydroxy ethyl cellulose, povidone, starch and methylcellulose or a combination thereof. The binder may be used in the range of 0.5-10% by weight of the tablet.
Suitable disintegrants of the present invention are selected from sodium starch glycolate, croscarmellose sodium, crospovidone, starch, hydroxypropyl cellulose, magnesium aluminum silicate, pregelatinized starch or a combination thereof. The disintegrant may be used in the range of 1-10% by weight of the tablet.
Suitable surfactants of the present invention are selected from sodium lauryl sulphate, polysorbate, sorbitan monolaurate, polyoxyethylene-polyoxypropylene block copolymer (poloxamer), polyethylene glycol derivatives, cetyl alcohol or a combination thereof. The surfactant may be used in the range of 0.01-1% by weight of the tablet.

Suitable glidants of the present invention are selected from magnesium trisilicate, talc, tribasic calcium phosphate, glyceryl monostearate, glyceryl stearate and silica dioxide or a combination thereof. The glidant may be used in the range of 0.1-2% by weight of the tablet.
Suitable lubricants of the present invention are selected from calcium stearate, magnesium stearate, hydrogenated vegetable oil, stearic acid, magnesium aluminum silicate, sodium stearyl fumarate, glyceryl behenate or a combination thereof. The lubricant may be used in the range of 0.1-5% by weight of the tablet.
Tenofovir as used herein refers to tenofovir disoproxil fumarate. The stable bilayered tablets comprise 50-500 mg of lamivudine, 50-500 mg of tenofovir and 100-600mg of efavirenz.
In a preferred embodiment, the stable bilayered tablets comprise i) compressed granules of lamivudine, tenofovir, 2-20% w/w of diluent selected from microcrystalline cellulose, lactose and mannitol; 1-5% w/w of disintegrant selected from croscarmellose sodium, crospovidone and sodium starch glycolate prepared by wet granulation in first layer and ii) compressed granules of efavirenz, 5-20% w/w of diluent selected from microcrystalline cellulose, lactose and mannitol, 1-5% w/w of disintegrant selected from croscarmellose sodium, crospovidone and sodium starch glycolate; 0.5 to 5% w/w of binder selected form hydroxy propyl cellulose, hydroxypropyl methylcellulose, povidone; sodium lauryl sulfate in 0.1 to 2% w/w of sodium lauryl sulfate prepared by wet granulation in the second layer.
In another preferred embodiment of the invention, the stable bilayered tablets comprise
i) compressed granules of lamivudine, efavirenz, 2-20% w/w of diluent selected from microcrystalline cellulose, lactose and mannitol; 1-5% w/w of disintegrant selected from croscarmellose sodium, crospovidone and sodium starch glycolate; .5 to 5% w/w of binder selected form hydroxy propyl cellulose, hydroxypropyl methylcellulose, povidone; sodium lauryl sulfate in

0.1 to 2% w/w of sodium lauryl sulfate prepared by wet granulation in the first layer and
ii) compressed granules of tenofovir, 5-20% w/w of diluent selected from microcrystalline cellulose, lactose and mannitol, 1-5% w/w of disintegrant selected from croscarmellose sodium, crospovidone and sodium starch glycolate prepared by wet granulation in the second layer.
In another embodiment of the present invention, stable bilayered tablets comprising lamivudine, tenofovir and efavirenz are prepared by wet granulation process using solvents, which comprises the steps of:
a) preparing granules comprising lamivudine, tenofovir and one or more pharmaceutically acceptable excipients,
b) preparing granules of efavirenz and one or more pharmaceutically acceptable excipients,
c) separately blending and lubricating the granules of step (a) and (b), with extragranular excipients and
d) finally compressing the granules into bi-layer tablets.
In yet another embodiment of the present invention, stable bilayered tablets comprising lamivudine, tenofovir and efavirenz are prepared by wet granulation process using solvents, which comprises the steps of:
a) preparing granules comprising lamivudine, efavirenz and one or more pharmaceutically acceptable excipients,
b) blending and lubricating the granules of step (a) with extragranular excipients,
c) preparing granules of tenofovir and one or more pharmaceutically acceptable excipients,
d) blending and lubricating the granules of step (c) with extragranular excipients,
e) compressing the lubricated granules of step (b) and step (d) into bi-layer tablets.

In another embodiment of the present invention, stable bilayered tablets comprising lamivudine, tenofovir and efavirenz are prepared by wet granulation process using solvents, which comprises the steps of:
a) preparing granules comprising lamivudine and one or more pharmaceutically acceptable excipients,
b) preparing granules comprising tenofovir and one or more pharmaceutically acceptable excipients,
c) preparing granules of efavirenz and one or more pharmaceutically acceptable excipients,
d) blending the granules of step (a) and (b) or step (a) and (c), with extragranular excipients and
e) processing the blend of (a) and (b) or (a) and (c) with the blend of (c) or (b),
e) finally compressing the granules into bi-layer tablets.
In another embodiment, the solvents used for granulation process may be selected from water, isopropyl alcohol, acetone, ethanol, methylene chloride or combination thereof.
In another embodiment of the present invention, the tablets of the present invention may optionally be coated to prevent the degradation of lamivudine from light with coating polymers.
Suitable film forming polymers used according to the present invention are selected from hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxy ethyl cellulose, xanthan gum and the like or a combination there of.
In another embodiment, the stable dosage form of the present invention is used for the treatment or prevention of symptoms of HIV infection in an infected individual.
The following examples further exemplify the invention and are not intended to limit the scope of the invention. It is obvious to those skilled in the art to find out the composition for other dosage forms and substitute the equivalent excipients as described in this specification or with the one known to the industry.

The tenofovir present in the single layer tablets of lamivudine, tenofovir and efavirenz tablets prepared to the above examples were found to be unstable. The stability data obtained after 2 months at 40°C/75% RH is shown in Table 1.

Example 1
Bilayered tablets of lamivudine, tenofovir and efavirenz


The processing steps involved in manufacturing stable bilayered tablets of lamivudine, tenofovir and efavirenz are given below:
a) preparation of granules of lamivudine plus tenofovir DF:
i) sifted and blended lamivudine, tenofovir DF, microcrystalline cellulose and
croscarmellose sodium,
ii) granulated the blend using water,
iii) dried and sized the wet mass to obtain granules,
b) preparation of granules of efavirenz:
i) sifted and blended efavirenz, microcrystalline cellulose, croscarmellose sodium, sodium lauryl sulfate,
ii) granulated the blend using binder solution of hydroxypropyl cellulose, iii) dried and sized the wet mass to obtain granules,
c) blending and lubrication of granules:
i) the granules of lamivudine and tenofovir obtained in stage (a) were
lubricated with magnesium stearate,
iii) the granules of efavirenz were lubricated with magnesium stearate,
iv) compressed the blends of step (i) and (ii) on bilayered compression
machine, and
v) coated the tablets with Opadry white.
Example 2
Bilayered tablets of lamivudine, tenofovir and efavirenz



The processing steps involved in manufacturing stable bilayered tablets of lamivudine, tenofovir and efavirenz are given below:
a) preparation of granules of lamivudine plus efavirenz:
i) sifted and blended lamivudine, efavirenz, microcrystalline cellulose,
croscarmellose sodium and sodium lauryl sulphate,
ii) granulated the blend using binder solution of hydroxypropyl cellulose,
iii) dried and sized the wet mass and lubricated the dried granules with
magnesium stearate,
b) preparation of granules of tenofovir disoproxil fumarate:
i) sifted and blended tenofovir disoproxil fumarate, microcrystalline cellulose
and croscarmellose sodium,
ii) granulated the blend of step (i) using water,
iii) dried and sized the wet mass and lubricated the dried granules with
magnesium stearate,
c) preparation of tablets:
i) compressed the lubricated granules of step (a) and (b) on bilayered compression machine with lamivudine and efavirenz granules into one layer and tenofovir disoproxil fumarate granules into second layer, and ii) coated the tablets with coating agent.
Example 3
Bilayered tablets of lamivudine, tenofovir and efavirenz


4 Hydroxypropyl methylcellulose 5,00
Efavirenz part
5 Efavirenz 600.00
6 Microcrystalline Cellulose 37.50
7 Croscarmellose Sodium 25.00
8 Hydroxypropylcellulose 7.50
9 Sodium Lauryl Sulfate 30.00
10 Purified Water q.s.
Tenofovir DF part
11 Tenofovir Disoproxil Fumarate 300.00
12 Microcrystalline Cellulose 42.00
13 Croscarmellose Sodium 40.00
14 Hydroxypropyl methylcellulose 8.00
15 Purified Water q.s.
Extra-granular for Tenofovir part
16 Magnesium Stearate 15.00
Extra-granular for Lamivudine par
17 Croscarmellose Sodium 15.00
18 Magnesium Stearate 5.00
Extra-granular for Efavirenz part
19 Croscarmellose Sodium 15.00
20 Magnesium Stearate 5.00
Core Tablet weight
21 Opadry II white 45.30
22 Purified Water q.s.
The processing steps involved in manufacturing stable bilayered tablets of lamivudine, tenofovir and efavirenz are given below:
a) preparation of granules of lamivudine:
i) sifted and blended lamivudine, microcrystalline cellulose, croscarmellose
sodium,
ii) granulated the blend using binder solution of hydroxypropyl
methylcellulose,
iii) dried and sized the wet mass to obtain granules,
b) preparation of granules of efavirenz:
i) sifted and blended efavirenz, microcrystalline cellulose and croscarmellose sodium,
14

ii) granulated the blend using binder solution of hydroxypropyl cellulose and
sodium lauryl sulphate,
iii) dried and sized the wet mass to obtain granules,
c) preparation of granules of tenofovir disoproxil fumarate:
i) sifted and blended tenofovir disoproxil fumarate, microcrystalline cellulose
and croscarmellose sodium,
ii) granulated the blend using binder solution of hydroxypropyl
methylcellulose,
iii) dried and sized the mass to obtain granules,
d) preparation of tablets;
i) lubricated the granules of tenofovir disoproxil fumarate with magnesium
stearate,
ii) blended the lubricated tenofovir disoproxil fumarate granules with
lamivudine granules along with croscarmellose sodium and again lubricated
with magnesium stearate,
iii) blended the granules of efavirenz with croscarmellose sodium and
lubricated with magnesium stearate,
iv) compressed the lubricated granules of step (ii) and (iii) on bilayered
compression machine with lamivudine and tenofovir granules into one layer
and efavirenz granules into second layer, and
vi) coated the tablets with coating agent.
Dissolution profile of bilayered tablets of lamivudine, tenofovir and efavirenz prepared according to example 3 was carried out in water with 2% SLS as dissolution medium using USP Apparatus II with 1000 ml at 75 rpm speed. The release profile (% drug released in min) is given in Table 1.



The processing steps involved in manufacturing stable bilayered tablets of lamivudine, tenofovir and efavirenz are given below: a) preparation of granules of lamivudine:

i) sifted and blended lamivudine, microcrystalline cellulose, croscarmellose
sodium,
ii) granulated the blend using binder solution of hydroxypropyl
methylcellulose,
iii) dried and sized the wet mass to obtain granules,
b) preparation of granules of efavirenz;
i) sifted and blended efavirenz, microcrystalline cellulose and croscarmellose
sodium,
ii) granulated the blend using binder solution of hydroxypropyl cellulose and
sodium lauryl sulphate,
iii) dried and sized the wet mass to obtain granules,
c) preparation of granules of tenofovir disoproxil fumarate:
i) sifted and blended tenofovir disoproxil fumarate, microcrystalline cellulose
and croscarmellose sodium,
ii) granulated the blend using binder solution of hydroxypropyl
methylcellulose,
iii) dried and sized the wet mass,
d^ preparation of tablets:
i) lubricated the granules of tenofovir disoproxil fumarate with magnesium
stearate,
ii) blended the granules of lamivudine with croscarmellose sodium and
lubricated with magnesium stearate,
iii) blended the granules of efavirenz with croscarmellose sodium and
lubricated with magnesium stearate,
iv) mixed the lubricated granules of lamivudine and efavirenz,
v) compressed the lubricated granules of step (i) and (iv) on bilayered
compression machine with lamivudine and efavirenz granules into one layer
and tenofovir disoproxil fumarate granules into second layer, and
vi) coated the tablets with coating agent.

Dissolution profile of bilayered tablets of lamivudine, tenofovir and efavirenz prepared according to example 4 was carried out in water with 2% SLS as dissolution medium using USP Apparatus II with 1000 ml at 75 rpm speed. The release profile (% drug released in min) is given in Table 2.

The bilayered tablets of lamivudine, tenofovir and efavirenz tablets according to the present invention were found to be stable. The stability data obtained after 1 month at 45°C/75RH is shown in Table 3.

1. A stable bilayered tablet comprising lamivudine, tenofovir and efavirenz prepared by wet granulation process.
2. The bilayered tablet as claimed in claim 1, further comprises one or more excipients selected from diluents, binders, disintegrants, surfactants, glidants and lubricants.
3. The dosage form as claimed in claim 2, wherein the diluent is selected from mannitol, lactose, microcrystalline cellulose, maltitol, sorbitol, maltodextrin, maltose, starch, calcium carbonate, calcium phosphate dibasic, calcium sulfate or a combination thereof.
4. The dosage form as claimed in claim 2, wherein the binder is selected from hydroxypropyl cellulose, hydroxypropyl methylcellulose, gelatin, hydroxy ethyl cellulose, povidone, starch and methylcellulose or a combination thereof.
5. The dosage form as claimed in claim 2, wherein the disintegrant is selected from sodium starch glycolate, croscarmellose sodium, crospovidone, starch, hydroxypropyl cellulose, magnesium aluminum silicate, pregelatinized starch or a combination thereof.
6. The dosage form as claimed in claim 2, wherein the surfactant is selected from sodium lauryl sulphate, polysorbate, sorbitan monolaurate, polyoxyethylene-polyoxypropylene block copolymer (poloxamer), polyethylene glycol derivatives, cetyl alcohol or a combination thereof.
7. The dosage form as claimed in claim 2, wherein the lubricant is selected from calcium stearate, magnesium stearate, hydrogenated vegetable oil, stearic acid, magnesium aluminum silicate, sodium stearyl fumarate, glyceryl behenate or a combination thereof.
8. A stable bilayered tablet comprising
i) compressed granules of lamivudine, tenofovir, 2-20% w/w of diluent selected from microcrystalline cellulose, lactose and mannitol; 1-5% w/w of disintegrant selected from croscarmellose sodium, crospovidone and sodium starch glycolate prepared by wet granulation in first layer and

ii) compressed granules of efavirenz, 5-20% w/w of diluent selected from microcrystalline cellulose, lactose and mannitol, 1-5% w/w of disintegrant selected from croscarmellose sodium, crospovidone and sodium starch glycolate; 0.5 to 5% w/w of binder selected form hydroxy propyl cellulose, hydroxypropyl methylcellulose, povidone; sodium lauryl sulfate in 0.1 to 2% w/w of sodium lauryl sulfate prepared by wet granulation in the second layer. 9. A stable bilayered tablet comprising
i) compressed granules of lamivudine, efavirenz, 2-20% w/w of diluent selected from microcrystalline cellulose, lactose and mannitol; 1-5% w/w of disintegrant selected from croscarmellose sodium, crospovidone and sodium starch glycolate; .5 to 5% w/w of binder selected form hydroxy propyl cellulose, hydroxypropyl methylcellulose, povidone; sodium lauryl sulfate in 0.1 to 2% w/w of sodium lauryl sulfate prepared by wet granulation in the first layer and
ii) compressed granules of tenofovir, 5-20% w/w of diluent selected from microcrystalline cellulose, lactose and mannitol, 1-5% w/w of disintegrant selected from croscarmellose sodium, crospovidone and sodium starch glycolate prepared by wet granulation in the second layer.