Technical field of invention:

The present invention relates to freeze dried pharmaceutical formulation of Rifabutin along with a suitable Solubilizing agent and Bulking agent for parenteral administration. The pharmaceutical formulation provides sufficient solubilization and stabilization of Rifabutin thus improving the shelf life and reduces the likelihood of precipitation during storage. The invention further relates to a process of preparation of freeze dried pharmaceutical formulation of Rifabutin.

Background of the invention:

Rifabutin is second line anti tubercular drug used in treatment of tuberculosis. It is related to Rifampin in structure and mechanism of action but it is less active against Mycobacterium tuberculosis and more active against Mycobacterium avium complex (MAC). Majority of M.tuberculosis isolates resistant to R are cross resistant to Rifabutin. Thus it is not an option for treatment of Multidrug-resistant tuberculosis (MDR-TB). The only place of Rifabutin in the treatment of TB is as 's' substitute for 'R' to minimize drug interaction due to strong enzyme inducing property of R. This is especially needed in HIV co-infected patients of TB who receive a protease inhibitor and/or a non-nucleoside reverse transcriptase inhibitor whose metabolism is markedly induced by R-rendering them ineffective.

The primary indication of Rifabutin is for prophylaxis and treatment of MAC infection in HIV-AIDS patients. For prophylaxis of MAC, Rifabutin alone 300mg/day is an alternative to Azithromycin / Clarithromycin; while for treatment of MAC infection, it is combined with two or three other anti-MAC drugs. Gastrointestinal intolerance, rashes, granulocytopenia, myalgia, and uveitis have been reported with Rifabutin. Reactions similar to those caused by R can also occur. Oral bioavailability of Rifabutin is low (about 20%), but t½ is much longer (730hrs.). Rifabutin may be administered orally as once a day at a dose ranging from 150 mg to 300mg (5mg/kg). The bioavailability of Rifabutin after oral administration as well as intravenous administration is very less and at about 20%.

Therefore, there remains a need in the art to provide Rifabutin with improved bioavailability. In the light of the above, the inventors of the present invention have decided to develop Rifabutin formulation for I.V. route, where much less dose also can work to achieve good blood plasma concentration.

Since, Rifabutin is insoluble in water, so it was found necessary to study the physical and chemical properties of product.

The chemical name for Rifabutin is l',4-didehydro-l-deoxy-l,4-dihydro-5'-(2- methylpropyl)-l-oxorifamycin XIV (Chemical Abstracts Service, 9th Collective Index) or (9S, 12E, 14S, 15R, 16S, 17R, 18R, 19R,20S,21 S,22E, 24Z)-6, 16, 18,20-tetrahydroxy- 1 '- isobutyl- 14-methoxy-7,9, 15,17,19,21 ,25-heptamethyl-spiro [9,4-

(epoxypentadecafl , 11 , 13]trienimino)-2H-furo[2',3':7,8]naphth[ 1 ,2-d] imidazole-2,4'- piperidine]-5,10,26-(3H,9H)-trione-16-acetate. Rifabutin has a molecular formula of C4 ₆ H ₆₂ N ₄ 0i l, a molec :

Rifabutin is a red-violet powder soluble in chloroform and methanol, sparingly soluble in ethanol, and very slightly soluble in water (0.19 mg/mL). Its log P value (the base 10 logarithm of the partition coefficient between n-octanol and water) is 3.2 (n- octanol/water). Parenteral drug preparation of Rifabutin for therapy of human and animal infectious diseases is disclosed in RU Patent No. 2481109. The excipients mentioned in Patent No. RU2481 109 along with chelating agent and antioxidant, however, it is experienced that « product is not soluble to the concentration mentioned in the patent.

Further, Rifabutin is sensitive to chemical degradation which limits the shelf-life of solutions in water as liquid injection and being insoluble in water is extremely dangerous to administer in parenteral state.

Chinese Patent Publication No.CN1775214 (A) includes main medicine, medicinal organic co-solvent, alkaline antioxidant, surfactant and sodium hydroxide for pH adjustment. This mass composition may lead to degradation or less stable due to undesirable reaction.

In view of the poor solubility of Rifabutin and the other shortcomings in preparing the lyophilized product for parenteral administration as mentioned above, the present inventors felt a need to provide a more stable Rifabutin injectable formulation which enhances the shelf life and is patient compliant.

Summary of the invention:

In accordance with above, the present invention provide freeze dried pharmaceutical formulation of Rifabutin or pharmaceutically acceptable salt thereof along with a suitable solubilising and bulking agent for parenteral administration.

The solubility of Rifabutin is increased by adding solubilizing agents selected from Polysorbate derivatives such as Polysorbate 20, Polysorbate 40, Polysorbate 60, Polysorbate 80.

The solubilizing agent is present in the ratio of 1 :1 to 1 :20, preferably 1 : 18, more preferably 1 : 17 by weight of the active. The Structure of lyophilized Rifabutin cake is formed by adding bulking agents selected from Mannitol, Sucrose, Lactose, Trehalose and mono and di-saccharides derivatives, cyclodextrins and the like.

The Bulking agent is present in the ratio of 1 :1 to 1 :20, preferably 1 : 15, more preferably 1 :12.5 by weight of the active.

In another aspect, the present invention provides a method for increasing solubility of Rifabutin in an aqueous solution.

In yet another aspect, the present invention provides a process for preparation of freeze dried pharmaceutical formulation of Rifabutin.

Detailed description of the invention:

The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be fully understood and appreciated.

Rifabutin is red-violet powder with a pKa of 3.2 and is insoluble in water. The solubility of Rifabutin decreases further strongly when the pH is below 4. However, it is observed by the current inventors that it is possible to provide stable pharmaceutically acceptable composition/formulation for parenteral administration or intravenous infusion comprising Rifabutin as the active using a suitable solubilizer and maintaining appropriate pH of the formulation.

Accordingly, in a preferred embodiment, the present invention discloses a freeze dried pharmaceutical formulation for parenteral administration comprising;

i. Rifabutin or a pharmaceutically acceptable salt thereof,

ii. A pharmaceutically acceptable solubilizing agent and

iii. A pharmaceutically acceptable bulking agent;

wherein pH of said formulation is in the range of 5 to 8. Rifabutin or a pharmaceutically acceptable salt is present in the formulation in an amount from lmg to lOOmg/vial; more preferably 60mg/vial and 80mg/vial.

The solubility of Rifabutin is increased using a suitable solubilizing agent where Rifabutin can be maintained in a dissolved state in the aqueous solution thereby preventing crystallization or crystalline growth of Rifabutin. The solubilizing agent is selected from Polysorbate derivatives such as Polysorbate 20 (Polyoxyethylene (20) sorbitan monolaurate); Polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate); Polysorbate 60 (polyoxyethylene (20) sorbitan monostearate) and Polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).

The solubilizing agent is present in the ratio of 1 : 1 to 1 :20, preferably 1 : 18, more preferably 1 : 17 by weight of the active.

The ^' Structure of lyophilized Rifabutin is formed by addition of bulking agents selected from Mannitol, Sucrose, Lactose, Trehalose and mono and di-saccharides derivatives, cyclodextrins and the like.

The Bulking agent is present in the ratio of 1 :1 to 1 :20, preferably 1 :15, more preferably 1 : 12.5 by weight of the active.

The freeze dried pharmaceutical formulation of the present invention optionally comprises acidifying agent and buffering agent.

The acidifying agent is selected from glacial acetic acid, citric acid, Ortho-phosphoric acid, Succinic acid and the like. The acidifying agent is present in the ratio of 1 : 0.166 by weight of the active.

The buffering agent is selected from dibasic sodium phosphate, disodium hydrogen orthophosphate, Trisodium citrate, Sodium hydroxide and the like. The buffering agent is present in the ratio of 1 : 0.32 by weight of the active. Such formulation after freeze drying is chemically and physically stable over an extended period of time and is suitable for intended pharmaceutical use after reconstitution with water for injection, sodium chloride injection or 5% dextrose injection which is represented in the examples below.

The aqueous intravenous formulation of the present invention may also comprise a physiologically and pharmaceutically acceptable compound effective to render the aqueous intravenous formulation isotonic, i.e. to have an osmotic pressure corresponding to that of a 0.9% solution of sodium chloride. Typical examples of such compounds are chloride salts such as NaCl and saccharide such as sorbitol, mannitol and dextrose/glucose. The preparation of isotonic solutions is well known for one skilled in the art. The diluents suitable for the purpose of present invention include 5% Dextrose, 5% Glucose, Ringers solution, Lactated Ringers solution, saline solution and half normal saline.

In another embodiment, the present invention provides a method of increasing solubility of Rifabutin in an aqueous solution comprising the step of combining Rifabutin aqueous solution with a solubilising agent and an acidifying agent. Alternately, adding solublizing agent and an acidifying agent to the aqueous solution of Rifabutin. The solubilizing agent being the pharmaceutically acceptable solubilizing agent selected from Polysorbatederivatives such as Polysorbate 20, Polysorbate 40, Polysorbate 60, and Polysorbate 80.

In another embodiment, the present invention provides a method of increasing solubility of Rifabutin in an aqueous solution comprising, adding 10% solution of Polysorbate 80 as solubilizing agent, Mannitol as bulking agent, 5% solution of glacial acetic acid as acidifying agent and dibasic sodium phosphate as buffering agent to the aqueous solution of Rifabutin.

In yet another embodiment, the invention provides a process for preparation of pharmaceutical formulation, in particular for intravenous infusion, comprising Rifabutin or a pharmaceutically acceptable salt thereof as the active ingredient, along with pharmaceutically acceptable solubilizing agent and bulking agent. Accordingly, Rifabutin or pharmaceutically acceptable salt thereof initially is solubilized in an aqueous solution containing solubilizing agent, preferably in the ratio 1 :17 followed by addition of bulking agent such as Mannitol 300 mg to 800 mg, more preferably 750 mg to an acceptable level of rangejhetween 6.0- 8.0 followed by filtration from 0.22 micron filter paper. The filtrate.ii nen freeze dried to render it sterile and filling 10 ml quantity of filter solution in sterile glass container of 20 ml vial size.

The formulation of the current invention is meant for administration via the IV route, the selection of the above ingredients is done by keeping in mind their compatibilities and stability during the rigorous process for lyophilization and also in terms of safety for use in patients as an intravenous injectable which is isotonic with blood tonicity.

Moreover, for intravenous pharmaceutical aqueous solution to be administrable to a patient, the active ingredient of the composition needs to be dissolved sufficiently in the composition i.e. be free from visible particles. To achieve this, freeze dried/lyophilized powder is used to obtain clear colourless solution, free from visible particles.

Freeze drying process involves cooling of product at suitable temperature not less than - 50°C, raising temperature to 0°Cat suitable pressure of 200 mtorr to 100 mtorr in 38 hours, then at 75 mtorr, further raising temperature to +30°C in 25 hrs.

The freeze dried Rifabutin when reconstituted with 10 ml of suitable vehicle contain final drug concentrate of 6.0 mg/ml.

In another embodiment, the pharmaceutical formulation of the invention described herein is freeze dried formulation, which may also be prepared by dissolving Rifabutin first in aqueous vehicle containing solubilizing agent and then adjusting pH to desired range using acidifying agent.

Developing freeze drying process for such a low soluble active ingredient needs special care and it is an art in its own way because of low concentration of solute (about 0.5%) and maximum amount of aqueous vehicle e.g. water for injection. The freeze dried drug may be diluted with suitable diluents before administration as IV injection. The final concentration of solution may be reduced to further desired level using 5% Dextrose infusion prior to administration to a patient.

In a further embodiment, the invention provides a method of treating or preventing an infection caused by a fungus or a parasite in an animal in need thereof which comprises administering to said animal an effective amount of the pharmaceutical formulation of the instant invention.

In another embodiment, the present invention relates to the use of Rifabutin parenteral formulation for treating or preventing an infectious disease caused by a fungus or a parasite in an animal in need thereof.

The pharmaceutical formulation of the present invention is administered to a patient according to a dosing regimen. It should be understood that the specific dosing regimen for any particular patient will depend on a variety of factors, including age, body weight, general health, sex, diet, time of administration, specific disease being treated, and the severity of the condition among other factors and the judgment of the treating physician.

Industrial Advantages:

1. The pharmaceutical formulation comprising Rifabutin as active is in parenteral form.

2. The pharmaceutical formulation is stable for the entire period of the shelf life.

Other features and embodiments of the invention will become apparent by the following examples which are given for illustration of the invention rather than limiting its intended scope. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art.

Experimental:

Several different trials were conducted and tested before narrowing down to the present formulation. Some of these trials are discussed in brief below to emphasize the inventiveness of the current invention. Example: 1

Rifabutin (active) or pharmaceutically acceptable salt was added to an aqueous solution containing pre-decided quantity of Kollidon PF 12 and stirred for some time. Solution was further sonicated. The solution was divided in to 4 equal parts.

To part one drop by drop 5% citric acid solution was added but the resulting clear solution changed color and assay of active went down and out of specifications when measured by spectrophotometer.

Assay: 35.75%

To second part drop by drop 5% Tartaric acid solution was added but the resulting clear solution-changed-color-and-assay- of~active-went-down-and-out-of-speeifieations-when- measured by spectrophotometer.

Assay: 45.15%

To Third part drop by drop 5% Acetic acid solution was added but the resulting clear solution changed color and assay of active went down and out of specifications when measured by spectrophotometer.

Assay: 40.0%

To fourth part drop by drop 1% Hydrochloric acid solution was added but Rifabutin did not dissolve in it and remained as un-dissolved particles.

Since product showed un-dissolved particles, no further testing was done.

Example: 2

In another trial,Rifabutin (active) or pharmaceutically acceptable salt was added to an aqueous solution containing pre-decided quantity of Kollidon PF 12 and stirred for some time. Solution was further sonicated.To this solution 1% sodium hydroxide solution was added drop wise but the resulting clear solution changed color and assay of active went down and out of specifications when measured by spectrophotometer.

Assay: 15.75% Example: 3

To a 2% solution of This buffer hydrochloride salt in water, calculated quantity of Rifabutin was added, Suspension was sonicated.

It was divided into two parts.

To part one drop by drop O.lNHydrochloric acid solution was added to observe if Rifabutin dissolved. Only, minor quantity of Rifabutin remained as un-dissolved particles.

To Second part drop by drop IN sodium hydroxide solution was added which dissolved Rifabutin at pH 10 but it rendered solution colored and assay of active went down to 10% measured on spectrophotometer.

Example: 4

In Rifabutin plus water, 1-50 times (weight of Rifabutin) powder of Disodium phosphate was added but the active remained partially insoluble and rendered the solution coloured. So no assay was done.

Example: 5

In Rifabutin plus water, 1 -20% This buffer was added drop- wise but the active remained insoluble.

So no assay was done. Example: 6

In Rifabutin plus water, 1% tri-sodium citrate solution was added drop wise but the active remained insoluble.

So no assay was done.

Example: 7

In Rifabutin plus water, 1-2% Kollidon PF 12 solution was added drop wise but the active remained insoluble.

So no assay was done. Example: 8

Like-wise other pre-formulation trials were checked with organic alkaline agent like either mono ethanolamine or triethanolamine and it was observed that the products were either getting solubilized partly or forming colored solution or assay was getting reduced to 10 to 15%.

Example 9:

Suitable trial was done using micelle forming surfactant like Polysorbate 80 to dissolve active product Rifabutin in aqueous base along with suitable acidifying agent like citric acid, buffering agent like Tri-sodium citrate and bulking agent like Mannitol, to check and compare solubility and stability of liquid preparation. Accordingly following process was followed:

Dissolved Polysorbate 80, Mannitol, citric acid and Trisodium citrate in water for injection. Added Rifabutin and stirred to dissolve. Filtered, filled 10ml in each vial and lyophilized it. The results are described in Table 1 herein below:

Tablel:

Period Assay pH Single Total Storage Colour

(%) impurity impurity temp.

(%) (%)

Initial Bulk 96.69 6.90 0.28 1.43 25°C Dark red solution coloursolution Lyophilized 96.17 6.80 0.30 1.54 25°C Red

coloredlyophilized cake

Example 10:

Suitable trial was done using micelle forming surfactant like Polysorbate 80 to dissolve active product Rifabutin in aqueous base along with suitable acidifying agent like Ortho- phosphoric acid, buffering agent like disodium hydrogen orthophosphate and bulking agent like Mannitol, to check and compare solubility and stability of liquid preparation. Accordingly following process was followed:

Dissolved Polysorbate 80, Mannitol, Disodium hydrogen orthophosphate and orthophosphoric acid in water for injection. Added Rifabutin and stirred to dissolve. Filtered, filled 10 ml in each vial and lyophilized it. The results are described in Table 2 herein below: Table 2:

Example 11:

Suitable trial was done using micelle forming surfactant like Polysorbate 80 to dissolve active product Rifabutin in aqueous base along with suitable acidifying agent like Succinic acid,buffering agent like Sodium hydroxide and bulking agent like Mannitol, to ^; check and compare solubility and stability of liquid preparation. Accordingly following process was followed:

Dissolved Polysorbate 80, Mannitol, and Sodium hydroxide and Succinic acid in water for injection. Added Rifabutin and stirred to dissolve. Filtered, filled 10 ml in each vial and lyophilized it. The results are described in Table 3 herein below: Table 3:

Example 12:

Suitable trial was done using micelle forming surfactant like Polysorbate 80 to dissolve active product Rifabutin in aqueous base along with suitable Bulking agent like Mannitol, to check and compare solubility and stability of liquid preparation. Accordingly following process was followed:

Dissolved Polysorbate 80 and Mannitol in water for injection. Added Rifabutin and stirred to dissolve. Filtered, filled 10 ml in each vial and lyophilized it. The results are described in Table 4 herein below: Table 4:

Conclusion: The pharmaceutical formulation was stable after 6 months at 25°C (Accelerated).

Example 12 is without addition of acidifying agent and buffering agent, which show fewer amounts of Total impurities at initial analysis as compared to Example 9 to 11.

The freeze-dried pharmaceutical formulation so obtained dissolved easily in 10 ml water for injection, to form clear red coloured solution.

In summary, the present invention provides water soluble Rifabutin as freeze dried stable formulation, stable for 6 months, when stored at 25°C.