WO2014102755A1

WO2014102755A1 - Bortezomib formulations

Info

Publication number: WO2014102755A1
Application number: PCT/IB2013/061430
Authority: WO
Inventors: Pradeep SHIVAKUMAR; Shivamurthy RAMAKRISHNAIAH; Badrinath ALAMPALLI; Akshaykant CHATURVEDI
Original assignee: Shilpa Medicare Limited
Priority date: 2012-12-31
Filing date: 2013-12-31
Publication date: 2014-07-03
Also published as: WO2014102755A4

Abstract

The present invention relates to Bortezomib pharmaceutical compositions and processes for preparing the same.

Description

BORTEZOMIB FORMULATIONS FIELD OF THE INVENTION

BACK GROUND OF THE INVENTION

Boronic acid and ester compounds display a variety of pharmaceutically useful biological activities. Shenvi et al., U.S. Pat. No. 4,499,082 (1985), discloses that peptide boronic acids are inhibitors of certain proteolytic enzymes. Kettner and Shenvi, U.S. Pat. No. 5,187,157 (1993); U.S. Pat. No. 5,242,904 (1993); and U.S. Pat. No. 5,250,720 (1993), describe a class of peptide boronic acids that inhibit trypsin-like proteases. Kleeman et al. in U.S. Pat. No. 5,169,841 (1992) discloses N-terminally modified peptide boronic acids that inhibit the action of renin leading to effect in regulation of blood pressure. Kinder et al. in U.S. Pat. No. 5,106,948 (1992) discloses that certain tripeptide boronic acid compounds inhibit the growth of cancer cells.

Unfortunately, alkyl boronic acids are relatively difficult to obtain in analytically pure form. Snyderet al. in J. Am. Chern. Soc, 3611(1958) demonstrates that alkyl boronic acid compounds readily form boroxines (anhydrides) under dehydrating conditions. Also, alkyl boronic acids and their boroxines are often air-sensitive. Korcek et al., J. Chern. Soc, Perkin Trans. 2242 (1972), teaches that butyl boronic acid is readily oxidized by air to generate I-butanol and boric acid. These difficulties limit the pharmaceutical utility of boronic acid compounds, complicating the characterization of pharmaceutical agents comprising boronic acid compounds and limiting their shelf life.

In normal course of cellular functioning, it requires processing of proteins regulating Cell cycle, Growth, and Apoptosis. The ubiquitin-proteasome pathway (UBP) modulates intracellular protein degradation. Specifically, the 26S proteasome is a multi-enzyme protease that degrades misfolded or redundant proteins; conversely, blockade of the proteasomal degradation pathways results in accumulation of unwanted proteins and cell death. Cancer cells are more highly proliferative than normal cells and their rate of protein translation and degradation is also faster. This typical behavior led to the development of various proteasome inhibitors as useful therapeutics in cancer. The FDA approved the first proteasome inhibitor bortezomib (Velcade) for the treatment of newly diagnosed and relapsed/refractory multiple myeloma. Other improved and II generation proteasome inhibitor approved recently includes Carfilzomib. Further on-going studies are examining other novel proteasome inhibitors, in addition to bortezomib, for the treatment of multiple myeloma and other cancers. Well known Proteasome inhibitors known today are Bortezomib (A), Carfilzomib (B), Marizomib (C)

Bortezomib chemically known as ((N-(2-pyrazine) carbonyl-L-phenylalanine-L-leucine boronic acid) is a 26S proteasome inhibitor that is approved for use in treatment of relapsed multiple myeloma and mantle cell lymphoma. It is believed that the boron atom in bortezomib binds to the catalytic site of the proteasome, ultimately leading to proteasome inhibition and reduced degradation of pro-apoptotic factors, which in turn triggers apoptosis in treated cells.

Stability of aminoalkyl boronic acids (including bortezomib) has remained a concern as they often undergo a spontaneous 1,3 -rearrangement to give the homologous amines, owing to the instability of free a-amino groups. These compounds yield boric acids and alcohols by degradation and undergo oxidative reactions that easily destroy the C-B bond which is longer and weaker than the corresponding C-C bond (see e.g., Adele Bolognese, Anna Esposito, MicheleManfra, Lucio Catalano, Fara Petruzziello, Maria CarmenMartorelli, Raffaella Pagliuca, VittoriaMazzarelli, Maria Ottiero, Melania Scalfaro, and Bruno Rotoli. Advances in Hematology, 2009 (2009) 1-5). Such instability is borne out in stress testing and accelerated stability studies of bortezomib that has established that bortezomib in aqueous solution for injection is intrinsically unstable. For example, in an ethanol: normal saline solution (2:98, pH 2.8), Bortezomib (0.5 mg/mL) degraded 20% at 25°C in 1 month, and in propylene glycol: ethanol: water (50: 10:40), the stability of the compound improved, but still degraded 20% in 8 months when stored at 25° C. Among other factors, it was speculated that the degradation of Bortezomib observed in PEG300: EtOH: H20 (40: 10:50) solvent might be due to the presence of peroxides, as PEG300 is known to undergo auto -oxidation with concomitant peroxide generation. (Journal of Pharmaceutical Sciences, 89, 2000 758-765).

In other studies, bortezomib was reported to be susceptible to oxidative degradation under a number of experimental conditions, and that the oxidation of alkyl boranes (which yields the ester of boric acid) can also be due to reaction with alkyl peracids, alkyl peroxides, or oxygen radical species. (Brown H C. 1972. Boranes in organic chemistry. Ithaca, N.Y.: Cornell University Press.). The initial oxidation can be attributed to peroxides or molecular oxygen and its radicals and as light, metal ions, and alkaline conditions normally facilitate oxidation. These conditions are therefore not considered favorable to the stability of bortezomib or any other alkyl boronic acid derivative. (Hussain M A, Knabb R, Aungust B J, Kettner C.1991. Anticoagulant activity of a peptide boronic acid thrombin inhibitor by various routes of administration in rats have been demonstrated in Peptides 12: 1153-1154.

Formation of boronic esters from diol and polyols was reported by Kuivila et al. reporting the preparation of several esters of phenyl boronic acid by reaction with sugars like mannitol and sorbitol, and 1, 2-diols like catechol and pinacal. (J. Org. Chem. 1954, 8, 780-783), and reversible formation of boronic ester by the interaction of boronic acids and polyols in water was first noted by Lorand and Edwards. (J. Org. Chem. 1959, 24, 769-774).

Adams et al in US5780454 discloses Bortezomib, its pharmaceutically acceptable salts, pharmaceutical composition and use in inhibiting the proteosome function in a mammal. Further, it discloses a process for the preparation of Bortezomib and its analogues.

Gupta et al in US6713446 discloses lyophilized formulation of Bortezomib esters. This patent mentions that Bortezomib prepared by the process as described in US5780454 is white amorphous powder.

Attempts to form the ester of boronic acid with alpha-hydroxy and beta-carboxylic acids like citric acid and with buffers were disclosed in WO 2009/154737. Namdeo et al in WO2010089768 discloses parenteral pharmaceutical composition comprising therapeutically effective amounts of N-(2-pyrazine) carbonyl-L-phenylalanine-L-leucine boronic acid or its salts or its derivatives and tromethamine wherein the composition is stable.

Kocherlakota et al in WO2010039762 disclose pharmaceutical compositions comprising bortezomib for oral or parenteral administration. Specific aspects relate to stable, sugar free pharmaceutical compositions of bortezomib, including its pharmaceutically acceptable salts or solvates, in the form of ready-to-use solutions, lyophilized forms, or physical admixtures, and the preparation thereof.

Bricout et al in WO2010114982 disclose a lyophilized cake comprising bortezomib or a pharmaceutically acceptable salt or ester thereof, a cyclodextrin and atleast one member selected from the group of bulking agents and surfactants.

Soppimath et al in US8263578 disclose a storage- stable liquid pharmaceutical composition that includes bortezomib in a therapeutically effective amount, the composition comprising: a single-phase liquid formulation comprising a substantially non-aqueous solvent system suitable for injection, an aqueous acetate buffer, and bortezomib, wherein the bortezomib is present in the formulation at a therapeutically effective concentration; wherein the solvent system comprises as a predominant component propylene glycol, and wherein the buffer has a pH of 3; and wherein the solvent system, the buffer, and the pH are selected such as to be effective to suppress formation of at least one of an amide degradation product, a first carbinolamide degradation product, and a second carbinolamide degradation product when the liquid formulation is stored under storage conditions. Soppimath et al describes degradation of bortezomib in solution as a well-known phenomenon and an exemplary degradation scheme is depicted in Scheme I below. Here, compound II is a first carbinolamide degradation product, compound III is a second carbinolamide degradation product (which is a stereo isomer of II). Hydrolysis of II or III will lead to the formation of the corresponding amide IV, which can be further hydrolyzed to the carboxylic acid product V. ScJiei&e I

in

MW :>S6 MW 354

5W :?7 MW 271

Usayapant et al in WO2012047845 disclose a bortezomib composition includes bortezomib and boric acid in a mass ratio of boric acid to bortezomib is from 1: 1 to 10: 1.

Anderson et al in WO2012148799 disclose a non-aqueous, homogeneous solution comprising a solubilized lipophilic pharmaceutical agent and an amphiphilic liquid polymeric solvent, the formulation being essentially free of non-polymeric organic solvents, water and non- solubilized particles, wherein the solubilized lipophilic pharmaceutical agent has a concentration of at least about 0.5 mg/niL, and further wherein the solution remains stable and essentially free of non-solubilized particles for at least 40 days when stored at room temperature.

Navin et al in WO2013128419 disclose a pharmaceutical composition comprising Bortezomib or pharmaceutically acceptable salt or solvate thereof, tromethamine and an organic carboxylic acid, wherein the pH of the composition is from about 3.0 to 6.0.

Further known disclosures for Bortezomib include- WO2008075376, WO2011099018, WO2011107912 and WO2012131707.

Even though there are several lyophilized formulations for bortezomib known in the art, however, most of them often suffer from drawbacks of either limited stability when bortezomib is in solution as well as over extended periods, and/or delayed reconstitution time of lyophilized cakes. Consequently, currently used products fail to provide flexibility of dosing suitability. Therefore, there remains a need of pharmaceutical composition comprising bortezomib or pharmaceutically acceptable salt or solvate thereof, with properties which improve its utility as a pharmaceutical agent. Ideally, such compositions would be conveniently prepared, would exhibit enhanced stability and longer shelf life and would readily liberate the bioactive boronic acid compound when administered to a subject in need of boronic acid therapy.

In their endeavors, inventors of the present application have developed compositions comprising bortezomib Form-SB, devoid of any pharmaceutically acceptable excipient; or with components particularly polyvinylpyrrolidone, lactic acid and an antioxidant with improved properties. The pharmaceutical compositions of the present invention are obtained by lyophilization of a mixture comprising neat Bortezomib monohydrate- Form-SB, or bortezomib or pharmaceutically acceptable salt or solvate thereof, with components particularly, polyvinylpyrrolidone, lactic acid and an antioxidant. Preferably, the composition comprises either neat Bortezomib or with additional components particularly- polyvinylpyrrolidone, lactic acid and sodium metabisulphite. The pre-lyophilized solutions of the present invention remains much convenient and acceptable, having well controlled levels of specified impurities during the product manufacturing stage, and the lyophilized compositions also have controlled levels of specified impurities and good reconstitution time. SUMMARY OF THE INVENTION

Aspects of the present invention relate to a pharmaceutical composition comprising Bortezomib (I) as monohydrate Form-SB

(I),

devoid of any pharmaceutically acceptable excipient, wherein said composition is having moisture content in the range of 3.5%-6.5 %w/w.

Aspects of the present invention also relate to a pharmaceutical composition comprising Bortezomib or pharmaceutically acceptable salt or solvate, with components particularly polyvinylpyrrolidone, lactic acid, and an antioxidant.

Aspects of the present invention further relate to a pharmaceutical composition comprising Bortezomib or pharmaceutically acceptable salt or solvate , polyvinylpyrrolidone, lactic acid, and an antioxidant; wherein the antioxidant is selected from sodium metabisulphite, butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, DL-tocopherol, tocopherol acetate, tocopherol polyethylene glycol Succinate, L-cysteine, or mixtures thereof.

In a particular aspect of the present invention-it relates to a pharmaceutical composition comprising Bortezomib, polyvinylpyrrolidone, lactic acid and sodium metabisulphite.

Aspects of the present invention also relate to a process of preparing pharmaceutical composition comprising Bortezomib, polyvinylpyrrolidone, lactic acid and sodium metabisulphite.

Aspects of the present invention relate to a pharmaceutical composition comprising Bortezomib, polyvinylpyrrolidone, lactic acid and sodium metabisulphite, wherein impurity 2 is not more than 0.5% and the said compositions are useful in the treatment of cancer. BRIEF DESCRIPTION OF THE DRAWING

Fig.l is Illustration of X-ray powder diffraction (XRPD) pattern of Bortezomib (I) as monohydrate Form-SB.

Fig.2 is Illustration of DSC thermogram of Bortezomib (I) as monohydrate Form-SB. Fig.3 is Illustration of IR spectra of Bortezomib (I) as monohydrate Form-SB. DETAILED DESCRIPTION OF THE INVENTION

In an embodiment of the present invention, provides a pharmaceutical composition comprising Bortezomib (I) as monohydrate Form-SB

devoid of any pharmaceutically acceptable excipient, wherein said composition is having moisture content in the range of 4%-5%w/w. In an embodiment of the present invention, provides a process of preparing a pharmaceutical composition comprising Bortezomib (I) as monohydrate Form-SB

devoid of any pharmaceutically acceptable excipient, wherein the said process comprising: i) Subjecting Water for injection to nitrogen purging till the dissolved oxygen content less than 2 ppm.

ii) Dissolving Bortezomib (I) monohydrate Form-SB in tertiary butanol to form a uniform white suspension;

iii) Mix the suspension of step (ii) with (i) under stirring.

iv) Filtering the solution obtained in step (iii) to obtain a filtered solution;

v) Lyophilizing the filtered solution of step (iv), wherein the lyophilized composition is having moisture content of 3.5%-6.5 %w/w.

In an embodiment of the present invention, provides a pharmaceutical composition comprising Bortezomib or pharmaceutically acceptable salt or solvate, optionally comprising polyvinylpyrrolidone, lactic acid, and an antioxidant.

In an embodiment of the present invention, provides a pharmaceutical composition comprising Bortezomib or pharmaceutically acceptable salt or solvate, polyvinylpyrrolidone, lactic acid, and an antioxidant selected from sodium metabisulphite, butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, DL-tocopherol, tocopherol acetate, tocopherol polyethylene glycol Succinate, L-cysteine, or mixtures thereof.

In an embodiment of the present invention, provides a pharmaceutical composition comprising Bortezomib, polyvinylpyrrolidone, lactic acid and sodium metabisulphite.

In a preferred embodiment of the present invention, provides a lyophilized composition comprising Bortezomib Form-SB, polyvinylpyrrolidone, lactic acid and sodium metabisulphite. In an embodiment of the present invention, provides a process of preparing a pharmaceutical composition comprising:

a) Dissolving polyvinylpyrrolidone in water for injection, and adding sodium metabisulfite and lactic acid, and mixed to get clear solution.

b) Dispersing Bortezomib in Tertiary Butanol and added to the step (a), and mixed to get clear solution.

c) Step (b) solution is made upto final volume with water for injection.

d) Lyophilizing the solution of step (c).

In a preferred embodiment of the present invention, provides a process of preparing a pharmaceutical composition comprising:

b) Dispersing Bortezomib Form-SB in Tertiary Butanol and added to the step (a), and mixed to get clear solution.

c) Step (b) solution is made upto final volume with water for injection.

d) Lyophilizing the solution of step (c).

In an embodiment of the present invention, provides a pharmaceutical composition comprising Bortezomib, polyvinylpyrrolidone, lactic acid and sodium metabisulphite; wherein the Bortezomib is in the range of 1 to 3.5mg.

In an embodiment of the present invention, provides a pre-lyophilized solution comprising Bortezomib Form-SB, polyvinylpyrrolidone, lactic acid and sodium metabisulphite, where in the pH of pre-lyophilized solution is between 3.5 to 5.5.

In a preferred embodiment of the present invention, provides a pre-lyophilized solution comprising Bortezomib Form-SB, polyvinylpyrrolidone, lactic acid and sodium metabisulphite, where in the pH of pre-lyophilized solution is between 4 to 5.

In an embodiment of the present invention, provides a pharmaceutical composition comprising Bortezomib, polyvinylpyrrolidone, lactic acid and sodium metabisulphite; wherein the Bortezomib is in the range of 1 to 3.5mg. In an embodiment of the present invention, provides a pharmaceutical composition comprising Bortezomib, polyvinylpyrrolidone, lactic acid and sodium metabisulphite; wherein the polyvinylpyrrolidone is in the range of 20 to 200 mg, for 3.5mg of bortezomib.

In an embodiment of the present invention, provides a pharmaceutical composition comprising Bortezomib, polyvinylpyrrolidone, lactic acid and sodium metabisulphite; wherein the lactic acid is in the range of 0.1 to 0.8 mg, for 3.5mg of bortezomib.

In an embodiment of the present invention, provides a pharmaceutical composition comprising Bortezomib, polyvinylpyrrolidone, lactic acid and sodium metabisulphite; wherein, sodium metabisulfite is in the range of 3 to 6 mg, for 3.5mg of bortezomib.

In an embodiment of the present invention, provides a pharmaceutical composition comprising Bortezomib, polyvinylpyrrolidone, lactic acid and sodium metabisulphite; wherein the impurity 2 in the composition is not more than 0.5%.

In a preferred embodiment of the present invention, provides a pre-lyophilized solution comprising Bortezomib, polyvinylpyrrolidone, lactic acid and sodium metabisulphite; wherein the impurity 2 in the solution is BLD.

In an embodiment of the present invention, provides bortezomib lyophilized composition comprising Bortezomib, polyvinylpyrrolidone, lactic acid and sodium metabisulphite; and the reconstitution time of the lyophilized composition is in the range of below 1 to 2 minutes.

In a preferred embodiment of the present invention, provides a lyophilized composition comprising Bortezomib Form-SB, polyvinylpyrrolidone, lactic acid and sodium metabisulphite; and the reconstitution time of the lyophilized composition is below 1 minute.

In a preferred embodiment of the present invention, provides a lyophilized composition comprising Bortezomib Form-SB, polyvinylpyrrolidone, lactic acid and sodium metabisulphite, wherein the pH of the reconstituted solution is between 6-8.

The term "pharmaceutical composition" as used herein shall mean a composition that is made under conditions such that it is suitable for administration to humans, e.g., it is made under GMP conditions and optionally contains pharmaceutically acceptable excipients, e.g., without limitation, stabilizers, bulking agents, antioxidants, buffers, carriers, diluents, vehicles, solubilizers, and binders. As used herein pharmaceutical composition includes but is not limited to a pre-lyophilization solution or dispersion, lyophilized composition, as well as a liquid form ready for injection, or a reconstituted solution of a lyophilized composition, meant for IV bolus and/or for subcutaneous administration.

According to the context of the discussion under the specification, "pharmaceutical composition" or "lyophilized composition" or "lyophilized formulation" are used interchangeably, and is meant for any composition having sufficient stability to have utility as a pharmaceutical agent. Preferably, the formulation has sufficient stability to allow storage at a convenient temperature, preferably between 0°C and 40°C, for a reasonable period of time, preferably longer than one month, more preferably longer than three months, even more preferably longer than six months, and most preferably longer than one year. Also, the term "lyophilized composition" as used herein means that the pharmaceutical composition when in the form of a lyophilized cake or powder that is the composition is not reconstituted, remains unaltered in terms of physical and chemical parameters for a prolonged period of time when packed in container which are either protected or unprotected against light, under various storage conditions. For instance, when the containers such as vials are not opened and are stored at controlled room temperature 25°C (77°F) with variation to a range of about 15 to 30°C (59 °F to 86°F) the pharmaceutical composition of the present invention remains stable for 6 months. The pharmaceutical composition when reconstituted with a suitable reconstitution medium such as water for injection or physiological saline, the reconstituted solution is said to be stable when there is no significant chemical degradation for at least 8 hours and there are no signs of precipitation or appearance of particles in the clear solution on storage at room temperature for the said time.

The term "pharmaceutically acceptable" refers to an ingredient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes those acceptable for veterinary use as well as human pharmaceutical use.

The term "Specified impurities" refer to any of impurity- 1, impurity-2 and impurity-3. Specified impurity- 1: (S)-3-Phenyl-2-(Pyrazine-2-Carboxamido) Propanoic acid.

Specified impurity-2: (N-(l-[((R)-l-Hydroxy-3-Methylbutyl) amino)- l-oxo-3-Phenylpropan- 2yl]Pyrazine-2-carboxamide).

Specified impurity-3: N-(l-amino-l-oxo-3-phenylpropan-2-yl) pyrazine-2-carboxamide.

The term "Total impurities" as referred in the specification relates to sum of all Specified impurities and unspecified impurities.

The term "BLD" as referred in the specification relates to below the limit of detection. The term "LOD" as referred in the specification relates to the limit of detection.

The term "LOQ" as referred in the specification relates to the limit of quantification.

The term "water for injections or WFI" as referred in the specification relates to distilled or sterile water for injection.

The term "PPM" as referred in the specification relates to Parts Per Million.

The term "NMT" as referred in the specification relates to Not more than.

The technique known as lyophilization is sometimes employed to process injectable pharmaceuticals that exhibit poor active ingredient stability in aqueous solutions. Lyophilization processing is suitable for injectables because it can be conducted under sterile conditions, which is a primary requirement for parenteral dosage forms. Lyophilization or freeze-drying is a process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from a solid to a vapor, without passing through a liquid phase. The process consists of three separate, unique, and interdependent processes; a freezing phase, a primary drying phase (sublimation), and a secondary drying phase (desorption). These processes may be optimized to enhance the product stability as well as decrease the manufacturing costs. Freezing Phase:

A primary function of the freezing phase is to ensure that the entire container having the complex solution is completely frozen, prior to proceeding to a subsequent phase. Additionally, it is usually desired that these containers freeze in a uniform manner. While there are different ways that this can be accomplished, one option is to chill the containers after they are loaded onto the lyophilizer shelves and holding for 30-60 minutes prior to initiation of the freezing cycle. It is generally not practical to equilibrate the shelves to a freezing temperature, because of frost accumulation during the filling and loading of the containers.

Primary Drying Phase:

Once the formulation is brought to the desired frozen state, primary drying via sublimation can proceed. The primary drying phase involves the removal of bulk water or solvent, at a product temperature below the ice transition temperature under a vacuum (pressures typically between 50-300 mTorr). This phase can be a critical one for stabilizing an active. The goal is to identify the glass transition temperature (Tg') for the formulation. The Tg' is the temperature at which there is a reversible change of state between a viscous liquid and a rigid, amorphous glassy state. One can measure the Tg' of candidate formulations using a differential scanning calorimeter (DSC), in particular with modulated DSC. Generally, the collapse temperature is observed to be about 2-5°C greater than the Tg'. Hence, the shelf temperature is set such that the target product temperature is maintained near or below the Tg' of the formulation throughout the removal of solvent during the primary dry phase.

As the solvent is progressively removed from the formulation containers, the product temperature will approach and reach the shelf temperature since it is no longer cooled by water sublimation. To optimize the duration of the primary dry phase, the removal of solvent vapor can be tracked using a moisture detector, or by monitoring the decrease in pressure difference between a capacitance manometer and a thermocouple pressure gauge or by a pressure drop measurement. The optimization of the primary dry cycle involves a removal of solvent as quickly as possible without causing cake collapse and subsequent product instability.

Secondary Drying Phase: The secondary drying phase is the final segment of the lyophilization cycle, where residual moisture is removed from a formulation's interstitial matrix by desorption with elevated temperatures and/or reduced pressures. The final moisture content of a lyophilized formulation, which can be measured by Karl Fischer or other methods, is important because if the solid cake contains too much residual moisture, the stability of the active can be compromised. Hence, it is imperative that one achieves a moisture level as low as possible.

To accomplish a low residual moisture, the shelf temperature is typically elevated to accelerate desorption of water molecules. The duration of the secondary drying phase is usually short. When microstructure collapse occurs, the residual moisture is generally significantly greater than desired. One alternative is to purge the sample chamber of the lyophilizer with alternating cycles of an inert gas such as nitrogen, to facilitate displacement of bound water. However, another solution is to properly formulate the drug product and run an optimal lyophilization cycle.

Example 1

Bortezomib Lyophilized composition for injection 3.5mg / Vial.

Brief method of preparation: i) Water for injection is subjected to nitrogen purging till the dissolved oxygen content is less than 2 ppm.

iii) Mix the suspension of step (ii) with (i) under stirring.

iv) Filtering the solution obtained in step (iii) to obtain a filtered solution; v) Lyophilizing the filtered solution of step (iv), wherein the lyophilized composition is having moisture content of 3.5%-6.5 %w/w; wherein the lyophilization cycle involves following steps: a. Precooling of shelf at -5°C.

b. Freezing at -45°C for 4 to 6 hours.

c. Primary drying at -45° C for 2 to 3 hours at 0.1 mbar.

d. Primary drying at -35° C for 16 to 20 hours at 0.2 mbar.

e. Primary drying at -15°C for 4 to 6 hours at 0.2 mbar.

f. Primary drying at 0°C for 4 to 6 hours at 0.2 mbar.

g. Primary drying at 25° C for 10 to 14 hours at 0.06 mbar.

h. Secondary drying at 40°C for 6 to 10 hours at 0.06 mbar.

vi) After the completion of lyophilization, completely stopper the vials under Nitrogen, unload and seal with flip off aluminium seals.

Analytical methods adopted for the determination of Assay and Related substances are discussed below:

Test method: Assay by HPLC -

Reagent preparation:

Preparation of Mobile phase: Prepare a filtered and degassed mixture of water, Acetonitrile, Formic acid in 715:285: 1 ratio.

Preparation of Diluent: Prepare a filtered and degassed mixture of 250 volumes of water and 750 volumes of Acetonitrile, mixed well and filtered.

Chromatographic conditions:

Column: X-Terra RP8, 150mm x 4.6 mm, 5μιη or Equivalent.

Wavelength: 270nm.

Flow rate: 1.5 ml / minute.

Injection volume: 10 μ

Run Time: 10 minutes.

Column Temperature: 40°C.

Sampler Temperature: 25°C.

Retention Time for Bortezomib: (about) 3.4 minutes

Standard Solution:

2.8 mg of Bortezomib working Standard into 20 ml volumetric flask. Add 5 ml of diluent dissolve and diluted to volume with diluent (or prepare the equivalent to 0.140 mg/ml).

Sample preparation: Take 5 vials and reconstitute each with 3.5 ml of diluent and pool the sample in 25 ml volumetric flask and dilute to volume with diluent. Further transfer 2 mL of this sample solution and transfer in to a 10 mL volumetric flask, add 5 mL of diluent. Sonicate and dilute to volume with diluent.

Content of uniformity:

Sample preparation: Take 1 vial and reconstitute with 5 ml of diluent. Further transfer 2 mL of this sample solution in to a 10 mL volumetric flask, add 5 mL of diluent and dilute to volume with diluent.

Procedure: Separately inject Blank (diluent) (one injection) and standard solution (five injections) into chromatograph and check the system suitability.

System Suitability Parameters:

1. The % relative standard deviation (RSD) for 5 injections of standard solution should be NMT 2.0.

2. The Tailing factor for Bortezomib peak in standard solution should be NMT 2.0.

3. Column Efficiency is Column Efficiency should be NLT 2000.

Bortezomib 3.5mg powder for solution for injection:

If passes the system suitability parameters, then inject sample preparations (duplicate injection) and record the chromato grams.

Acceptance criteria: Between 95% w/w and 105% w/w

Calculate the assay of Bortezomib in % by using following formula:

AT WS DT P 100

= X— x— x— χ

.45 ^" DS V ^' 100 LC

Where

AT: Average Area of Bortezomib peak from sample preparation chromatogram.

AS: Average area of Bortezomib peak from standard preparation chromatogram.

WS: Weight of Bortezomib Standard taken in mg.

DS: Dilution of Standard solution.

DT: Dilution of sample solution.

P: Potency of Bortezomib Standard in %w/w on as is basis.

V: Number of vials taken for sample preparation

LC: Label claim Bortezomib 3.5mg powder for solution for injection.

Test method: Related Substances by HPLC:

Reagent preparation: Preparation of Mobile phase: A

Prepare a filtered and degassed mixture of water, Acetonitrile, Formic acid in the ratio of 715:285: 1.

Preparation of Mobile phase: B

Transfer 200 ml of Water to a 1000 ml volumetric flask. Added 800ml of methanol to the volumetric flask and Sonicate and degassed.

Diluent 1: Weigh and transfer 9.0gm of NaCl into a 1000ml volumetric flask and dilute to volume with water.

Diluent 2: Prepare a filtered and degassed mixture of 250 ml of water and 750 ml of Acetonitrile.

Chromatographic conditions:

Column: Waters symmetry shield RP-18; 250 mm X 4.6mm, 5μιη

Wavelength: 270nm

Flow rate: 1.0 ml / minute

Injection volume: 20 μΙ_,

Diluent 1: 0.9% NaCl

Diluent 2: Water: ACN

Run Time: 60 minutes for Blank, System sensitivity solution Sample & Placebo; 20 minutes for Diluted standard.

Column Temperature: 30°C

Sampler Temperature: 5°C

Retention time of Bortezomib: 14.5 -15.5 minutes.

Gradient Programme: for Samples, Blank & Placebos

Time (Minutes) Mobile Phase A (%) Mobile Phase B (%)

0.0 100 0

20.0 100 0

35.0 0 100

50.0 0 100

52.0 100 0

60.0 100 0 Specified Specified Specified

S.no Bortezomib

impurity- 1 impurity-2 impurity-3

LOD (PPM) 0.112 0.071 0.084 0.071

LOQ (PPM) 0.370 0.200 0.265 0.213

LOD (%) 0.011 0.006 0.008 0.007

LOQ (%) 0.038 0.020 0.026 0.022

Example 1 bortezomib lyophilized cake is analyzed for Assay & Related substances:

The reconstitution time of lyophilized cake of example- 1 formulation in 0.9% sodium chloride solution is 2 minutes.

Example 2

Brief method of preparation (Note: Batch fabricated using WFI at Room temperature (RT):

1. About 110% of desired quantity of Water for injection was collected in cleaned Duran bottle and nitrogen purged for 30 minutes.

2. About 60% of the total batch quantity of WFI was collected from the above step in to the Duran bottle and weighed batch quantity of Povidone K12 was added and stirred for 2 minutes to get a clear solution.

3. Dispensed quantity of Tertiary Butanol was taken in a separate duran bottle and weighed quantity of Bortezomib Form-SB, was added, stirred for 15 minutes to get Milky dispersion.

4. The solution from the above step 3 was transferred to solution of step 2 and stirred, for 30 minutes to get clear solution.

5. The volume of the above solution was made up to 100% using WFI and stirred for 5 minutes. Assay and Related substances results of Example 2 pre-lyophilized solution are given under table below:

Example 3

Brief method of preparation (Note: Batch fabricated using WFI at 2-8 C).

5. The volume of the above solution was made up to 100% using WFI and stirred for 5 minutes.

Assay and Related substances results of Example 3 pre-lyophilized solution are given under table below:

Time Description Assay Related substances

Specified Specified Specified Unspecified Total impurity 1 impurity 2 impurity 3 impurity impurities

Initial clear solution 98.00 BLD 1.66 0.01 0.02 1.75 Example 4

Brief method of preparation (Note: Batch fabricated using WFI at Room temperature (RT) and also hold time study carried at RT):

2. About 60% of the total batch quantity of WFI was collected from the above step in to the Duran bottle and weighed batch quantity of Povidone K12 was added and stirred for 2 minutes to get a clear solution. Batch quantity of Lactic acid was then added to the clear solution and stirred for 2 minutes to get clear solution.

Hold time study of pre-lyophilized solutions were carried out at successive time intervals where samples were withdrawn from the Duran container, and submitted for the analysis for Assay and Related substances of Example 4 formulation and the results are discussed under table below:

Time Description Assay Related substances

Initial clear 98.63 BLD 0.95 BLD 0.07 1.19 solution

4^th clear

96.11 0.01 2.50 BLD 0.71 3.47 hour solution

Example 5

Brief method of preparation (Note: Batch fabricated using WFI at 2-8 C and also hold time study carried at 2-8°C):

Hold time study of pre-lyophilized solutions were carried out at successive time intervals where samples were withdrawn from the Duran container, and submitted for the analysis for Assay and Related substances of Example 5 formulation and the results are discussed under table below: The results of initial and storage vials under 2-8°C condition are discussed under table below:

Example 6:

2. About 60% of the total batch quantity of WFI was collected from the above step in to the Duran bottle and weighed batch quantity of Povidone K12 was added and stirred for 2 minutes to get a clear solution. To the clear solution batch quantity of Sodium meta bisulfite was added and stirred for 5 minutes to get clear solution. Batch quantity of Lactic acid was then added to the clear solution and stirred for 2 minutes to get clear solution.

5. The volume of the above solution was made up to 100% using WFI and stirred for 5 minutes. 6. The above bulk solution was filled in to 10mL/20mm flint vial with a fill volume of 2mL and half stoppered and loaded into lyophillizer and lyophilization carried out under the below recipe:

Hold time study of pre-lyophilized solutions were carried out at successive time intervals where samples were withdrawn from the Duran container, and submitted for the analysis for Related substances and the results are discussed under the table below:

Example 6 Bortezomib lyophilized cake is analyzed for Assay & Related substances:

Assay Related substances Specified Specified Specified Unspecified Total impurity 1 impurity 2 impurity 3 impurity impurities

98.0 0.01 0.41 0.01 0.12 0.89

The lyophilized cake of example-6 is characterized for the following:

The hold time study results of Pre-lyophilized solution of example 6 indicate the impurity 2 is restricted to BLD, and the impurity 2 content in the bortezomib lyophilized cake is also well controlled to less than 0.5%.

Example 7:

3. Dispensed quantity of Tertiary Butanol was taken in a separate duran bottle and weighed quantity of Bortezomib Form-SB, was added, stirred for 15 minutes to get Milky dispersion. 4. The solution from the above step 3 was transferred to solution of step 2 and stirred, for 30 minutes to get clear solution.

6. The above bulk solution was filled in to 10mL/20mm flint vial with a fill volume of 2mL and half stoppered and loaded into lyophillizer and lyophilization carried out under the below recipe:

Hold time study of pre-lyophilized solutions were carried out at successive time intervals where samples were withdrawn from the Duran container, and submitted for the analysis for Related substances of Example 6 formulation and the results are discussed under table below:

Related substances of Example 7 formulation, at initial and after storage of vials under RT condition; are discussed under table below:

Related substances

Time Description Specified Specified Specified Unspecified Total impurity 1 impurity 2 impurity 3 impurity impurity

Initial

clear solution BLD BLD BLD 0.01 0.05 (lmg/mL)

15 hours

clear solution 0.04 BLD BLD BLD 0.34 (lmg/mL)

24 hours clear solution 0.06 0.01 0.01 BLD 0.15

The lyophilized cake of example-7 is characterized for the following:

The following observations are drawn from the studies demonstrated under Formulation Examples 2, 3, 4, 5, 6 & 7: a) Examples 2&3 : The pre-lyophilized solutions containing Bortezomib and polyvinyl pyrrolidone do not have adequate chemical stability of bortezomib in solution form, as evident from high levels of impurity 2. Further, the temperature (25°C or 2-8°C) do not show any effect in improvement of the bortezomib chemical stability in solution state.

b) Examples 4&5: The pre-lyophilized solutions containing Bortezomib, polyvinyl pyrrolidone and lactic acid do not have adequate chemical stability of bortezomib in solution form, as evident from high levels of impurity 2. Further, the temperature (25°C or 2-8°C) do not show any effect in improvement of the bortezomib chemical stability in solution state. c) Examples 6&7: The pre-lyophilized solutions containing Bortezomib, polyvinyl pyrrolidone, lactic acid and sodium metabisulphite have superior chemical stability of bortezomib in solution form, as evident from BLD levels of impurity 2, at zero hours.

Further, it is observed that the hold-time study on bortezomib pre-lyophilized solutions prepared under 2-8°C, has total impurity level NMT 0.2%, at 28 hours, as compared to the hold-time study on bortezomib pre-lyophilized solutions prepared under 25°C, with total impurity level NMT 0.6%, at 28 hours. This suggests that for compositions comprising Bortezomib, polyvinyl pyrrolidone, lactic acid and sodium metabisulphite, the bortezomib solution stability at 2-8°C processing conditions are more suitable as compared to that of 25°C,. However, 25°C also appears to be acceptable to obtain adequate bortezomib solution stability throughout the manufacturing process steps, and therefore, the lyophilized cakes prepared from Pre-lyophilized solutions of Example 6&7, have good cake appearance and have rapid reconstitution time, less than one minute. Example 6&7 Bortezomib lyophilized cakes are subjected to accelerated stability at 40°/75%RH and real time stability at 25°C/60%RH, and at each appropriate stability time intervals, the product characteristics can be observed like the assay, related substances and reconstitution time.

Claims

Claims:

1. A pharmaceutical composition comprising Bortezomib (I) as monohydrate Form-SB

devoid of any pharmaceutically acceptable excipient, wherein the said composition is having moisture content in the range of 3.5%-6.5 %w/w.

2. A process for preparing a pharmaceutical composition according to claim- 1 , which process comprises the steps of:

i) Water for injection is subjected to nitrogen purging till the dissolved oxygen content is less than 2 ppm.

iii) Mix the suspension of step (ii) with (i) under stirring.

3. A pharmaceutical composition comprising Bortezomib according to claim- 1, optionally comprising polyvinyl pyrrolidone, lactic acid, and an antioxidant.

4. The pharmaceutical composition according to claim 3, wherein the antioxidant is selected from sodium metabisulphite, butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, DL-tocopherol, tocopherol acetate, tocopherol polyethylene glycol Succinate, L-cysteine, or mixtures thereof.

5. A pharmaceutical composition comprising Bortezomib, polyvinylpyrrolidone, lactic acid and sodium metabisulphite.

6. A process of preparing pharmaceutical composition of claim 5 comprising:

c) Step (b) solution is made upto final volume with water for injection.

d) Lyophilizing the solution of step (c).

7. The pharmaceutical composition according to claims 3-6, wherein the polyvinylpyrrolidone is in the range of 20 to 200 mg, for 3.5mg of bortezomib.

8. The pharmaceutical composition according to claims 3-7, wherein the lactic acid is in the range of 0.1 to 0.8 mg, for 3.5mg of bortezomib.

9. The pharmaceutical composition according to claims 3-8, wherein the sodium metabisulfite is in the range of 3 to 6 mg, for 3.5mg of bortezomib.

10. A pharmaceutical composition according to claims 3-9, wherein impurity 2 is not more than 0.5%.

11. The reconstitution time of bortezomib lyophilized composition according to claims 3-10, is below 1 minute.