FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
PROVISIONAL SPECIFICATION / COMPLETE SPECIFICATION (See section 10 and rule 13)

1. TITLE OF THE INVENTION
2. APPLICANT(S)
(a) NAME
(b) NATIONALITY
(c) ADDRESS
PHARMACEUTICAL COMPOSITION OF ISONIAZID
CADILA PHARMACEUTICALS LIMITED
An INDIAN Company
"Cadila Corporate Campus", Sarkhej - Dholka Road, Bhat, Ahmedabad -382210, Gujarat, India

3. PREAMBLE TO THE DESCRITION
PROVISIONAL SPECIFICATION
The following specification describes the invention.
4. DESCRIPTION
(Description starts from next page)

FIELD OF THE INVENTION
The present invention relates to pharmaceutical composition comprising rifampicin, piperine and isoniazide, wherein the said pharmaceutical composition retains the bioenhancing property of piperine with respect to rifampicin.
The present invention further relates to process for preparing pharmaceutical composition comprising rifampicin, piperine and isoniazide.
BACKGROUND OF THE INVENTION
Tuberculosis (abbreviated as TB for tubercle bacillus or Tuberculosis) is a common and often deadly infectious disease caused by mycobacteria. Tuberculosis usually attacks the lungs (as pulmonary TB) but can also affect the central nervous system, the lymphatic system, the circulatory system, the genitourinary system, the gastrointestinal system, bones, joints, and even the skin. Other mycobacteria such as Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti, and Mycobacterium microti also cause tuberculosis, but these species are less common in humans.
The management of tuberculosis treatment is difficult due to development of drug resistance by the microbes. Fixed dose combination (FDC) tablets provide a simple approach to delivering the drugs at their correct dosage when the drugs are formulated in a single tablet.
Some marketed pharmaceutical composition of rifampicin and / or isoniazide involves the use of variety of excipients in the composition which are as under:
a) Rimactan® Capsules from Sandoz contains Lactose monohydrate, Soya-bean lecithin, Gelatin, Dimeticone, Shellac, Calcium stearate, Titanium dioxide, Iron oxide
b) Rifadin® Capsules from Ab Ferrosan contains Indigo carmine, Titanium dioxide, Starch, Magnesium stearate, Gelatin and Cochineal Red A
c) Rifampin Capsule from Actavis Totowa contains Lactose monohydrate, Magnesium stearate, Methylparaben, Propylparaben, Silicon dioxide, Sodium lauryl sulfate, Corn starch, Talc, and Titanium dioxide.
d) Rifampin Capsule from Lannett Company contains Pregelatinized starch, Colloidal silicon dioxide, Talc, Magnesium stearate, and Titanium dioxide.
e) Rifampin Capsule from VersaPharm Incorporated contains Lactose monohydrate, Magnesium stearate, Sodium lauryl sulfate, Talc, and Titanium dioxide.
f) Rimactazid® Tablets from Ciba contains Calcium stearate, Sodium lauryl sulphate, Maize starch, Sodium carboxymethylcellulose, Talc, Hydroxypropyl methylcellulose, Povidone, Titanium dioxide, Cellulose, Polyethylene glycol, Sugar (Sucrose), Red iron oxide, Yellow iron oxide
g) Tibinide® Tablets Recipharm Stockholm AB contains microcrystalline cellulose, Stearic acid, Talc and Colloidal silicondioxide.

US Patent No. 5,439,891 (US '891) discloses pharmaceutical composition for the treatment of tuberculosis and leprosy, said composition comprising piperine in combination with known antituberculosis drugs. A pharmaceutical composition of US '891 involve the use of piperine in the range of 0.4 to 0.9% by weight of antituberculosis (i.e. Rifampicin, Isoniazid, Ethambutol, Pyrazinamide) or antileprosy drugs (i.e. Rifampicin Dapsone). The patent is silent on process details of claimed pharmaceutical composition.
However, when we made the pharmaceutical composition comprising rifampicin and isoniazide with piperine, it was observed that the bioavailability of rifampicin was markedly reduced as compared to pharmaceutical composition containing rifampicin and piperine, suggesting loss of bioenhancement property of piperine with respect to rifampicin in presence of isoniazide.
Thus there is an unmet need to have a pharmaceutical composition comprising rifampicin, isoniazide and piperine wherein the bioavailability of rifampicin is not reduced.
SUMMARY OF THE INVENTION:
The object of present invention is to provide pharmaceutical composition comprising rifampicin, piperine and isoniazide wherein the said pharmaceutical composition retains the bioenhancing property of piperine with respect to rifampicin in presence of isoniazide.
Another object of the present invention is to provide process for manufacturing of pharmaceutical composition comprising rifampicin, piperine and isoniazide wherein the said pharmaceutical composition retains the bioenhancing property of piperine with respect to rifampicin in presence of isoniazide.
DETAILED DESCRIPTION OF THE INVENTION:
In accordance with present invention to provide pharmaceutical composition comprising rifampicin, piperine and isoniazide wherein the said pharmaceutical composition retains the bioenhancing property of piperine with respect to rifampicin in presence of isoniazide. According to present invention the quantity of piperine ranges from more than 1.0% by weight of antituberculosis or antileprosy drugs. The antituberculosis or antileprosy drugs may be selected from rifampicin, isoniazide and the like. The preferred range of piperine quantity is 1.0% to 8.0% by weight of antituberculosis or antileprosy drugs. The bioenhancement property of piperine with rifampicin containing formulation also depends on grade of rifampicin, selection of excipients and process of preparing pharmaceutical composition. The use of compacted rifampicin results in reduction of its bioavailability, whereas powdered rifampicin results to enhance its bioavailability, but further micronization doesn't improve bioavailability of rifampicin.
The pharmaceutically acceptable excipient include any of diluent, binder, disintegrant, lubricant, glidant, stabilizer, surfactant, organic solvent, water, film forming polymer, opacifier, plasticizer, modified release polymer and the like.

The pharmaceutical composition of the present invention may contain one or more diluents added to increase mass and, hence, make a dosage form easier for the patient and caregiver to handle. Common diluents are microcrystalline cellulose, directly compressible grade microcrystalline cellulose, lactose, starch, pregelatinized starch, calcium carbonate, calcium sulfate, sugar, dextrates, dextrin, dextrose, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, magnesium carbonate, magnesium oxide, maltodextrin, Mannitol and the like. The preferred diluent used in the instant invention is starch.
Binders also can be included in the pharmaceutical composition. Some typical binders are acacia, alginic acid, carbomer (e.g. carbopol), carboxymethylcellulose sodium, dextrin, ethyl cellulose, gelatin, guar gum, hydrogenated vegetable oil, hydroxyethyl cellulose, hydroxypropyl cellulose (e.g. Klucel®), Hydroxypropyl methyl cellulose (e.g. Methocel®), liquid glucose, magnesium aluminum silicate, maltodextrin, methylcellulose, polymethacrylates, povidone (e.g. Kollidon®, Plasdone®), pregelatinized starch, sodium alginate and starch.
The surfactant like polysorbate, anionic surfactant, and nonionic surfactant improves the effect of piperine on bioavailability of rifampicin. Examples of the above-mentioned nonionic surfactant are, for instance, polyoxyethylamine oxides, alkylamine oxides, polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene fatty acid esters, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, glycerine esters, polyoxyethylene alkylamine, the derivatives thereof, and the like, anionic surfactant, there can be used known ones, for example, higher fatty acid and its salt, alkyl sulfate, alkyl sulfonate, alkyl aryl sulfonate, alkyl phosphoric acid ester and the like. The preferred surfactant used in the instant invention is Sodium lauryl sulphate. The stabilizer like citric acid may also be useful in instant invention.
The lubricant like Magnesium stearate, Sodium stearyl fumarate, Stearic acid, Talc, Colloidal silicon dioxide and the like known to skilled person in the art.
The disintegrant can be used to accelerate disintegration of the tablet or granules in the patient's stomach. Disintegrants include alginic acid, carboxymethyl cellulose calcium, carboxymethylcellulose sodium, croscarmellose sodium, sodium starch glycolate, crospovidone, polacrilin potassium, powdered cellulose, pregelatinized starch, sodium alginate and starch. The preferred disintegrant used in the instant invention is croscarmellose sodium. The disintegrant is used in the instant invention in the range of 0.1% to 10% of total weight of dosage form. It was surprisingly found that use of croscarmellose sodium in piperine granules or tablets resulted in better bioavailability of rifampicin.
The organic solvents include but are not limited to alcohols, ketones, esters, ethers, halogenated solvents, hydrocarbons, nitriles, or mixtures thereof. Lower alkanols are useful and can be any alcohol such as for example one or more of the primary, secondary or tertiary alcohols having from one to about six carbon atoms. The lower alkanol can be for example one or more of methanol, ethanol, denatured spirits, n-propanol, isopropanol, n-

butanol, isobutanol, t-butanol and the like. The ketones can be any solvent from this class such as for example one or more of acetone, propanone, 2-butanone and the like. The halogenated solvent can be any solvent from this class such as for example one or more of chloroform, dichloromethane, 1, 2-dichloroethane, carbon tetrachloride and the like. The ester can be any solvent from this class such as for example one or more of ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate and the like. The ether can be any solvent from this class such as for example one or more of dimethyl ether, diethyl ether, methyl tertiary-butyl ether ethyl methyl ether, di isopropyl ether, tetrahydrofuran, dioxane and the like.
We have found that the pharmaceutical composition comprising rifampicin and isoniazide when mixed with piperine does not result the enhancement in bioavailability of rifampicin. The enhancement in bioavailability of rifampicin in presence of piperine is lost due to the presence of isoniazide in the pharmaceutical composition. In order to resolve this problem we need to improve the bioavailability of rifampicin with piperine in presence of isoniazide.
Accordingly, the invention further provides a solid oral pharmaceutical composition comprising rifampicin, piperine and isoniazide, wherein the said pharmaceutical composition retains the bioenhancing property of piperine wherein a substantial portion of isoniazide is separated from rifampicin and piperine.
The pharmaceutical composition comprising rifampicin, piperine and isoniazide wherein the said pharmaceutical composition retain the bioenhancing property of piperine with respect to rifampicin in presence of isoniazide wherein rifampicin and piperine are delivered in immediate release form and isoniazide is delivered in modified release form. The pharmaceutical composition may release rifampicin and piperine immediately in stomach and release isoniazide in intestine.
The modified release polymer may be natural, semi-synthetic and synthetic. Suitable materials include cellulose and cellulose derivatives like methyl cellulose, ethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, cellulose acetate phthalate, cellulose acetate, cellulose acetate butyrate, cellulose acetate propionate, cellulose acetate trimellitate, cellulose carboxymethyl ethers and their salts, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate and other modified release polymers like polymethacrylates.
We have observed that blending of Rifampicin powder, Starch and Magnesium stearate with piperine tablets or granules provided higher bioavailability of rifampicin as compared to other excipients. The piperine composition can be formulated with suitable excipients by wet granulation or compaction or direct blending. The rifampicin composition is formulated with suitable excipients through direct blending process. The preferable excipients for rifampicin are starch and magnesium stearate. The rifampicin and piperine composition can be filled in hard gelatin capsule or compressed into tablets. The isoniazide

composition to deliver as modified release dosage form can be prepared by processes like wet granulation, dry granulation, direct compression, extrusion spheronization or drug layering.
The term "immediate release" as used herein is NLT 70% of active ingredient is dissolved within 30 minutes in given dissolution conditions: Set I: The dissolution parameters for immediate release are as follows:

Dissolution Condition Rifampicin Piperine Isoniazide
Dissolution Medium 0.1 N Hydrochloric acid 0.01 N Hydrochloric acid + 2% SLS 0.1 N Hydrochloric acid + 2% SLS Basket
Apparatus Basket Basket

RPM 100 150 100
Volume 900 ml 900 ml 900 ml
The term "modified release" as used herein is NMT 10% of isoniazide is dissolved within 2 hours in 0.1 N HCI and NLT 70% of isoniazide is dissolved within 45 minutes in 6.8 pH phosphate buffer under given dissolution conditions: Set II: The dissolution parameters for modified release are as follows:

Dissolution Condition
Dissolution Medium

Isoniazide
0 - 2 hrs : 0.1 N Hydrochloric acid 2 - 3 hrs : 6.8 pH Phosphate Buffer

Apparatus

Basket

RPM

100

Volume

900 ml

The following examples will further illustrate certain aspects of the present invention in greater detail and are not intended to limit the scope of the invention.

Example I: The following example demonstrate the effect of isoniazide on the
bioenhancement property of piperine with respect to rifampicin Pharmaceutical composition containing rifampicin (Pharmaceutical Composition 1)
Ingredients Mg / Capsule
Rifampicin #200.00
Citric Acid 5.00
Lactose 20.00
Starch 10.00
Sodium Starch Glycolate 5.00
Magnesium Stearate 2.00
Sodium Lauryl Sulphate 4.00
Colloidal Silicon Dioxide 2.00
# powdered grade rifampicin
Process description:
1. Rifampicin was sifted through 16 # s.s screen.
2. Citric acid, Lactose, Starch, Sodium starch glycolate, Magnesium stearate, Sodium lauryl stearate and Colloidal silicon dioxide were sifted through 40 # s.s screen.
3. Rifampicin and blend from step 2 were mixed thoroughly.
4. The blend from step 3 was filled in hard gelatin capsule shell.
To the above Pharmaceutical Composition 1, addition of 10mg piperine per capsule (as Pharmaceutical Composition 2) resulted in significant enhancement of bioavailability of rifampicin as compared to Pharmaceutical Composition 1.
However, when isoniazide (300mg per capsule) was added to the formulation containing rifampicin (200 mg per capsule) and piperine (10mg per capsule) (as Pharmaceutical Composition 3), the bioavailability of rifampicin was found to be significantly reduced as compared to Pharmaceutical Composition 1.
The bioavailability of rifampicin in pharmaceutical composition containing rifampicin (200 mg per capsule) and isoniazide (300mg per capsule) (as Pharmaceutical Composition 4) was identical to Pharmaceutical Composition 1.

Pharmaceutical Composition 5 and 6

Sr. No. Ingredients Pharmaceutical Composition 5 (Mg / Capsule) Pharmaceutical Composition 6
(Mg / Capsule)
A. Rifampicin Blend
1. Rifampicin* 200.00 200.00
2. Citric Acid 5.00 5.00
3. Lactose 20.00 20.00
4. Starch 10.00 10.00
5. Sodium Starch Glycoiate 5.00 5.00
6. Magnesium Stearate 2.00 2.00
7. Sodium Lauryl Sulphate 4.00 4.00
8. Colloidal Silicondioxide 2.00 2.00
B. Piperine Tablets
1. Piperine 10.00 10.00
a. Base Granules
1. Lactose 41.83 41.83
2. Starch 21.27 21.27
3. Sodium Lauryl Sulphate 0.38 0.38
4. Starch (Paste) 2.57 2.57
5. Purified Water q.s q.s
b. Pre Lubrication Stage
1. Sodium Lauryl Sulphate 0.35 0.35
2. Croscarmellose Sodium 2.40 2.40
3. Colloidal Silicondioxide 0.30 0.30
4. Magnesium Stearate 0.30 0.30
c. Final Lubrication
1. Colloidal Silicondioxide 0.30 0.30
2. Magnesium Stearate 0.30 0.30
d Alsoniazide capsule - 300.00
Alsoniazide capsule containing 300mg isoniazide, administered separately Process description.
A. Rifampicin Blend
1. Rifampicin was sifted through 16 # s.s screen.
2. Citric acid, Lactose, Starch, Sodium starch glycoiate, Magnesium stearate, Sodium lauryl stearate and Colloidal silicon dioxide were sifted through 40 # s.s screen.
3. Rifampicin and blend from step 2 was mixed thoroughly.
B. Piperine Tablet
1. Piperine was sifted through 60 # s.s screen.

a. Base granules:
1. Lactose, Starch and Sodium lauryl sulphate were sifted through 40 # s.s screen.
2. Above blend was granulated with starch paste prepared in purified water.
3. The wet mass was dried in tray dryer.
4. Dried granules were sifted through 30 # s.s screen.
b. Pre Lubrication:
1. The base granules and piperine were mixed geometrically.
2. Sodium lauryl sulphate, Croscarmellose sodium, Colloidal silicondioxide and
Magnesium stearate were sifted through 40 # s.s screen.
3. Mix step 1 and step 2.
Slugging:
1. The above blend was slugged and was sifted through 30 # s.s screen. c. Final Lubrication:
1. Colloidal silicondioxide and Magnesium stearate were sifted through 40 # s.s screen. The slugged granules and lubricants were mixed and compressed into tablets
These batches were subjected to pharmacokinetic studies in human volunteers for evaluating bioavailability of Rifampicin. When the above formulation of Pharmaceutical Composition 5 was administered along with Isoniazide capsule (Isoniazide capsule containing 300mg isoniazide, administered separately) (Pharmaceutical Composition 6) the data of the same for rifampicin is summarized as follows:
The pharmacokinetic parameters Cmax (pg/ml), AUC(o-t) (hr X pg/ml) and AUC(0-) (hr X ug/ml) of pharmaceutical composition 6 were reduced significantly as compared to pharmaceutical composition 6. The pharmacokinetic parameters of pharmaceutical composition 6 verses pharmaceutical composition 5 fail according to bioequivalence criteria.
The above results demonstrate that bioenhancing property of piperine with respect to rifampicin is reduced in presence of immediate release isoniazide composition and isoniazide doesn't affect the bioavailability of rifampicin.

Example II: The following examples demonstrate enhanced bioavailability of
rifampicin:
Pharmaceutical Composition 7: This pharmaceutical composition demonstrate
bioenhancing property of piperine with respect to rifampicin

Sr. No. Ingredients Mg / Capsule

Pharmaceutical composition 7
A. Rifampicin Blend
1. Rifampicin 200.00
2. Starch 56.00
3. Magnesium Stearate 2.00
B. Piperine Tablets
1. Piperine 10.00
a. Base Granules
1. Lactose 41.83
2. Starch 21.27
3. Sodium Lauryl Sulphate 0.38
4. Starch (Paste) 2.57
5. Purified Water q.s
b. Pre Lubrication Stage
1. Sodium Lauryl Sulphate 0.35
2. Croscarmellose Sodium 2.40
3. Colloidal Silicondioxide 0.30
4. Magnesium Stearate 0.30
c. Final Lubrication
1. Colloidal Silicondioxide 0.30
2. Magnesium Stearate 0.30
Process description:
A. Rifampicin Blend
1. Rifampicin was sifted through 16 # s.s screen.
2. Starch and Magnesium stearate were sifted through 40# s.s screen.
3. Rifampicin and blend from step 2 was mixed thoroughly.
B. Piperine Tablet
1. Piperine was sifted through 60 # s.s screen. a. Base granules:
1. Lactose, Starch and Sodium lauryl sulphate were sifted through 4Q # s.s screen.
2. Above blend was granulated with starch paste prepared in purified water.
3. The wet mass was dried in tray dryer.
4. Dried granules were sifted through 30 # s.s screen.

b. Pre Lubrication:
1. The base granules and piperine were mixed geometrically.
2. Sodium lauryl sulphate, Croscarmellose sodium, Colloidal silicondioxide and Magnesium stearate were sifted through 40 # s.s screen.
3. Mix step 1 and step 2.
Slugging:
1. The above blend was slugged and was sifted through 30 # s.s screen.
c. Final Lubrication:
1. Colloidal Silicondioxide and Magnesium Stearate were sifted through 40 # s.s screen. The slugged granules and lubricants were mixed and compressed into tablets This batch was subjected to pharmacokinetic studies in human volunteers for evaluating bioavailability of Rifampicin. The data of the same for rifampicin is summarized as follows:
The results demonstrate pharmaceutical composition 7 having significantly enhanced bioavailability of rifampicin as compared to pharmaceutical composition 1.

Pharmaceutical composition 8: This pharmaceutical composition demonstrate bioenhancing property of piperine with respect to rifampicin in presence of isoniazide

Sr. No. Ingredients Mg / Capsule

Pharmaceutical Composition 8
A. Isoniazide Tablet
1 Core Tablet
1. Isoniazide 150.00
2. Sodium Starch Glycolate 5.00
3. Micro Crystalline Cellulose 8.00
4. Purified Water q.s.
5. Magnesium Stearate 2.00
II Seal Coating
1. Instacoat® Moist Shield (Cellulose based Coating) 1.65
2. Isopropyl Alcohol q.s.
3. Dichloromethane q.s.
Ill Coating-I
1. Instacoat® EN-HPMC-P (HPMC Phthalate based polymer) 16.62
2. Isopropyl Alcohol q.s.
3. Dichloromethane q.s.
B. Piperine Granules
1. Piperine 10.00
2. Starch 2.50
3. Croscarmellose Sodium 2.50
4. Sodium Lauryl Sulphate 0.25
5. Povidone K-30 0.10
6. Purified Water q.s
C. Rifampicin Blend

1. Rifampicin 200.00
2. Starch 56.00
3. Magnesium Stearate 2.50
Process description: A. Isoniazide Tablet I. Core Tablet
1. Isoniazide, Sodium starch glycolate, microcrystalline cellulose were sifted through 40# s.s screen and mixed thoroughly.
2. The above blend was granulated with Purified Water.
3. The wet mass was passed through 20 # s.s screen.
4. The wet granule s was dried.
5. The dried granules were sifted through 24# s.s screen.

6. Magnesium Stearate was sifted through 60 # s.s screen and mixed with step 5.
7. The above blend is compressed into Tablet
II. Seal Coating
1. The tablets were barrier coated with Instacoat Moist shield using Non-aqueous coating.
III. Coating-I
1. The tablets were coated with Instacoat® EN-HPMC-P using Non-aqueous coating.
B. Piperine Granules:
1. Piperine, Starch, Croscarmellose sodium, Starch, Sodium lauryl sulphate and Povidone K-30 were sifted through 30 # s.s screen and mixed thoroughly.
2. The blend from step B1 was granulated with purified water and dried.
3. The dried granules were sifted through 60 # s.s screen.
C. Rifampicin Blend:
1. Rifampicin, Starch and Magnesium stearate were sifted through 60 # s.s screen and
mixed well.
The two coated tablets of isoniazide from stage-A, granules of piperine from stage-B and blend of rifampicin from stage-C were filled in hard gelatin capsule.
This batch was subjected to pharmacokinetic studies in human volunteers for evaluating bioavailability of Rifampicin. The data of the same for rifampicin is summarized as follows:
The pharmacokinetic parameters CmaX (ug/ml), AUC(0-t) (hr X ug/ml) and AUC(0--) (hr X ug/ml) of pharmaceutical composition 8 are similar to that of pharmaceutical composition 7. There was no significant loss of bioavailability of rifampicin as compared to pharmaceutical composition 7. The bioenhancement property of piperine is retained in pharmaceutical composition 8.
The dissolution of pharmaceutical composition 8 was performed as dissolution conditions described above in Set I and Set II. The dissolution of piperine was more than 90% in twenty minutes in 0.01 N HCI, dissolution of rifampicin was more than 80% in 45 minutes in 0.1 N HCI and dissolution of isoniazide was more than 80% in 6.8 pH phosphate buffers.

For Cadila Pharmaceuticals Ltd.,
Dr. Bakulesh M. Khamar Executive Director - Research