FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
PROVISIONAL/COMPLETE SPECIFICATION (See section 10 and rule 13)
Title: INTERFERON CONJUGATES
Intas Biopharmaceuticals Limited
An Indian company having its registered office at
Plot No: 423/P/A/GIDC
Sarkhej-Bavla Highway
Moraiya, Tal.: Sanand
Ahmedabad-382 210
Gujarat, India
The following specification describes the invention.

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INTERFERON CONJUGATES
INTRODUCTION TO THE INVENTION
The present invention relates to interferon conjugates comprising conjugates of interferon or mixture(s) of conjugates of interferon.
The present invention also relates to process(s) for preparing pharmaceutical compositions comprising conjugates of interferon or mixture(s) of conjugates of interferon and their method of use and treatment.
Interferon belongs to a group of cytokines which are classified in accordance with their cellular origin and the type of receptors to which they bind. There are three classes of interferons, namely alpha, beta and gamma. With respect to interferon alpha, there are two subtypes, namely interferon alpha 2a and interferon alpha 2b which differ only in one amino acid residue. In interferon alpha 2a, residue number 23 is lysine, whereas the same residue in interferon alpha 2b is an arginine.
Two commercially available products exist namely interferon alpha 2a marketed by Hoffmann La Roche under the tradename Roferon-A®, and interferon alpha 2b marketed by Schering Plough Corporation under the tradename Intron A®. Interferon alpha is used for a number of indications, including hepatitis B and C and various other cancer indications. Interferon alpha therapy has been limited by a number of protein characteristics, such as antigenicity, solubility, stability in the blood stream and half-life of the protein.
Pegylation technology has emerged as a means to improve the pharmacokinetic and pharmcodynamic properties of biopharmaceuticals without altering the parent molecule. Some of the benefits of pegylation includes improved clinical properties, enhanced solubility, sustained absorption and

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release, reduced immunogenicity and proteolysis, reduced clearance from circulation by the kidneys, increased dosing intervals due to higher in-vivo half-lives owing to increased circulation time and the like.
The longer circulation of interferon results in beneficial therapeutic effects such as prolongation of it's presence in the human body, effective therapeutic treatment of disease and conditions thereof.
Pegylated interferon alpha 2a (PEGASYS) is an interferon alpha 2a that has been pegylated using a branched PEG moiety to a number of lysine residues of recombinant human interferon alpha 2a. The branched PEG moiety has a molecular weight of 40 kDa consisting of two monomethoxy polyethyleneglycol (mPEG) chains each having an average molecular weight of 20 kDa, linked to a lysine molecule via urethane bonds, one at the alfa -amino group and the other at the epsilon amino group of lysine. The carboxyl group of lysine is derivatised to an N-hydroxy-succinimide ester. N-succinimidyl hydroxyl is the active group of PEG reagent PEG2-NHS. PEG2-NHS is also sometimes referred to as Y-PEG-LYS-NHS-40kDa.
Pegylated interferon alpha 2 b "PEG-lntron" is interferon alpha 2b derivatised with a linear monomethoxy polyethyleneglycol of a molecular weight of 12 kDa. The resulting protein has an overall molecular weight of 31.2 kDa.
EP 0809996 B1 [Indian Patent No. 198952 (1032/MAS/97)] describes a physiological active pegylated Interferon a conjugate having the Formula I, wherein R and R' are independently C1-C6-alkyl; X is NH or O; n and n'are integers having a sum of from 600 to 1500; and the average molecular weight of the polyethylene glycol units in said conjugate is from 26,000 daltons to 66,000 daltons. In formula 1 X is NH or O (X is at least one of the functional groups in the IFNa molecule selected from NH2 or OH). The conjugate is produced by covalent linkage of IFNa to PEG where the hydroxyl is replaced by a linking

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group forming a reagent which is an N-hydroxy succinimide ester derivative of PEG (monomethoxy PEG). The linkage is via a free lysine amino group of IFNa
2a forming an amide or ester bond. The composition described in EP 0809996 is 40 kDa branched mono PEG-IFNa 2a which is currently marketed as Pegasys for treatment of Hepatitis C.

EP 0788515 B1 describes a branched substantially non-antigenic polymer comprising the formula (R) nL-A, wherein: (R) is a water soluble, substantially non-antigenic polymer containing a terminal C1-C4 group; (n) = 2 or 3; (L) is an aliphatic linking moiety selected from the group consisting of substituted alkyl diamines and triamines, lysine esters and malonic ester derivatives covalently linked to each (R); and (A) is a functional group capable of bonding with a biologically active nucleophile or a moiety capable of being functionalized to react with said nucleophile.
Globally, drug regulatory authorities require therapies to be therapeutically beneficial coupled with better patient compliance and effective patient adherence to the treatment regimen. Earlier introduction of such therapies is increasingly being recommended.

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Further, the conjugates of interferon alfa2b with polyethylene glycol as described in context of the present invention results in the longer in-vivo half-life thus improving patient compliance due to the ease of administration, reduced frequency of dosing and reduction in the doses of the active used, hence resulting in reduction of side effects.
These and other needs are addressed by this invention.
SUMMARY OF THE INVENTION
The present invention relates to interferon conjugates comprising conjugates of interferon or mixture(s) of conjugates of interferon.
The present invention also relates to process(s) for preparing pharmaceutical compositions comprising conjugates of interferon or mixture(s) of conjugates of interferon and their method of use and treatment.
The present invention also relates to interferon conjugate comprising interferon, branched polyethylene glycol with aspartic acid core as a linker in the conjugate
DETAILED DESCRIPTION OF DRAWINGS
Figure 1 describes Peg Interferon alfa 2b produced by recombinant human (rHu) Interferon alfa 2b. PEG Interferon alfa 2b was formed as a result of the reaction between nitro phenyl carbonate (NPC) derivative of a 40K-branched PEG molecule and rHu IFN alfa 2b.
Figure 2 describes the reaction mechanism for 40K-branched PEG molecule and rHu IFN alfa 2b.

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DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to interferon conjugates comprising conjugates of interferon or mixture(s) of conjugates of interferon, process(s) for preparing pharmaceutical compositions comprising conjugates of interferon or mixture(s) of conjugates of interferon, their method of use and treatment.
Further, the present invention relates to interferon conjugate comprising
pegylated interferon alpha-2b (PEG-IFNa-2b) with aspartic acid core as a linker in the conjugate attached to the branched polyethyleneglycol (PEG) moiety having from about 20KD to about 60 KD molecular weight.
In one embodiment of the present invention, it was surprisingly found by our formulation scientists that pegylated interferon alpha-2b (PEG-IFNa-2b) with
aspartic acid core as a linker was easy to prepare and formulate under
manufacturing and processing conditions. Also the interferon conjugates of the present invention were found to be economically viable and feasible.
In an embodiment of the present invention, pegylated interferon alfa- 2b is manufactured by attaching a 40kD branched PEG moiety through an aspartic acid core to recombinant human interferon alpha 2b (rHu IFN alfa 2b) which is a non-glycosylated protein manufactured in Escherichia coli cells.
In another embodiment of the present invention, pegylated interferon alpha as shown in Formula I comprises of R and R' which are methyl, X is NH, each n is from about 420 to about 510 and the average molecular weight of the polyethylene glycol unit is about 40 KD.
Further in an embodiment, PEG derivative to be used in PEG-IFN may have linker molecule such as aspartic acid, glutamic acid and the like.

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In an embodiment, PEG derivative to be used in PEG-IFN has an aspartic acid linker molecule.
In context of the present invention, the functional group of derivative may be but not limited to nitrophenyl carbonate (NPC), succinimidyl succinate, succinimidyl glutarate, isocyanate, aldehydes and the like.
The interferon conjugates of the present invention can be administered by the routes known to the one skilled in the art such as but not limited to parenteral such as oral; intravenous, intra-peritoneal, intramuscular, subcutaneous and the like; intranasal or by inhalation, transmucosal such as vaginal, rectal and the like; intra-ocular, and the like.
The interferon conjugates of the present invention can be manufactured by following the techniques known to the one skilled in the art. For parenteral administration, sterile aqueous solutions or dispersions may be used. The vehicle for the same could be either oily such as sesame oil, peanut oil and the like or aqueous such as propylene glycol thereof. Such sterile aqueous solutions are preferably and suitably buffered and made isotonic using saline (0.9% sodium chloride) or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. The oily solutions are suitable for intramuscular and subcutaneous injection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art. The selection of a parenteral formulation for administration of interferon conjugates of the present invention should be stable, compatible with the inactive ingredients (s) used to avoid denaturation and loss of biological activity of the therapeutic protein. Furthermore, the compositions can
be in the form of sterile powders prepared by lyophillisation and other techniques known in the art In all cases, the final injectable form must be sterile.

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Interferon conjugates of the present invention may also be formulated using vesicular systems by the routes of administration known to the one skilled in the art such as liposomes, niosomes, microspheres and the like.
In context of the present invention, one or more pharmaceutically acceptable excipients may be used optionally to prepare the formulations comprising PEG-IFNa-2b. These pharmaceutically acceptable excipients may include but are not limited to solvents and co-solvents, solubilizing, wetting, suspending, thickening and emulsifying agents, chelating agents, buffers and pH adjusters, antioxidants and reducing agents, antimicrobial preservatives, bulking agents, protectants and tonicity adjusters and the like.
Solubilizing, wetting, suspending agents that is surfactants may include anionic surfactants such as chenodeoxycholic. acid, 1-octanesulfonic acid sodium salt, sodium deoxycholate, glycodeoxycholic acid sodium salt, N-lauroylsarcosine sodium salt, lithium dodecyl sulfate, sodium cholate hydrate, sodium lauryl sulfate (SLS) and sodium dodecyl sulfate (SDS); cationic surfactants such as cetylpyridinium chloride monohydrate and hexadecyltrimethylammonium bromide; nonionic surfactants such as N-decanoyl-N-methylglucamine, octyl a-D-glucopyranoside, n-Dodecyl b-D-maltoside (DDM), polyoxyethylene sorbitan esters like polysorbates and the like.
Further, thickening and emulsifying agents that may be used include such as glucose, lactose, mannitol and the like; chelating agents such as ethylenedinitrilotetraacetic acid and its salts and the like; buffers and pH adjusters such as acetic acid, hydrochloric acid, sodium hydroxide, sodium acetate trihydrate and the like; antioxidants and reducing agents such as butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), a-tocopherol and the like; tonicity adjusters such as sodium chloride and the like; antimicrobial preservatives that may be used include such as benzalkonium chloride, benzoic acid powder, benzyl alcohol, benzyl benzoate and the like.

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Formulations of the interferon conjugates comprising PEG-IFNa-2b as disclosed in context of the present invention may be used in the treatment of viral infections such as hepatitis B, hepatitis C and the like and various cancer indications.
Further in an embodiment, compositions comprising PEG-IFNa-2b as disclosed in context of the present invention can be administered either alone or as fixed dose combination optionally with pharmaceuticaily acceptable excipients, with atleast one of the drug form the class of anti-viral drugs. Anti-viral drugs include but are not limited to anti-herpes virus drugs such as Aciclovir, Cidofovir, Docosanol, Famciclovir, Fomivirsen, Foscarnet, Ganciclovir, Idoxuridine, Penciclovir, Trifluridine, Tromantadine, Valaciclovir, Valganciclovir, Vidarabine and the like; anti-influenza drugs such as Amantadine, Oseltamivir, Peramivir, Rimantadine, Zanamivir and the like; antiretroviral drugs such as Abacavir, Didanosine, Emtricitabine, Lamivudine, Stavudine, Zalcitabine, Zidovudine and the like; other antiviral drugs such as Adefovir, Fomivirsen, Imiquimod, Inosine.lnterferon, Podophyllotoxin, Ribavirin, Viramidine and the like.
The amount of dose to be administered depends upon a variety of factors including the age, body weight, general health, sex, diet, time of administration,
route of administration, rate of excretion, drug combination and the severity of the particular pathophysiological condition.
The following examples will further illustrate certain aspects and embodiments of the invention in greater detail and are not intended to limit the scope of the invention.
Examples

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Example 1
Composition of pegylated interferon alpha-2b (PEG-IFNa-2b)

S.No. Ingredients Quantity/ml
1 PEG-IFNa-2b 180ng
2 Sodium acetate trihydrate 2.6mg
3 Acetic acid 0.05mg
4 Polysorbate 80 0.05mg
5 Benzyl alcohol 10mg
6 Sodium chloride 0.02mg
8 Water for injection 1 ml
Manufacturing process:
Peg Interferon alfa 2b was produced by recombinant human (rHu) Interferon alfa 2b. PEG Interferon alfa 2b was formed as a result of the reaction between a Nitro phenyl carbonate derivative of a 40K-branched PEG molecule (as shown in Figure 1) and the interferon rHu IFN alfa 2b. The reaction mechanism is also enclosed. (Figure 2)
Under the experimental conditions, pegylation reaction mixture contained from about 25%-45% mono-PEG, from about 5%-10% di-PEG and from about 45%-70% unmodified interferon rHu IFN alfa 2b, as. determined by size exclusion chromatography HPLC (SEC-HPLC), reverse phase HPLC(RP-HPLC) and ion-exchange chromatography HPLC (IEC-HPLC). The monopegylated IFN was purified from the mixture.
After pegylation, Interferon conjugate was purified by cation exchanger chromatography and eluted by using salt gradient. This step yielded the pure protein. The pure protein solution was concentrated using tangential flow filtration

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technique using 30 KD molecular weight cut of membrane in flat-sheet cassette filter followed by its buffer exchange into the formulation buffer.
The purity of Interferon conjugate was about 98%-99%, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for both protein and PEG staining and SEC-HPLC analysis. The Interferon conjugate was characterized by using various analytical techniques like MALDI-TOF, CD spectroscopy SEC-HPLC, RP-HPLC and SDS-PAGE.
The conjugate of interferon was also characterized using In-vitro & In-vivo bioactivity. In-vitro bioactivity of Interferon conjugate was done by cell culture based assay which measured a dose dependent reduction in Cytopathic Effect (CPE) by PEG-IFN.
The Pharmacokinetics and In-vivo bioactivity of the product was further determined in mice.
Dated: 18th day of January 2007 Signed
Gautam Bakshi Manager
Intellectual Property Rights (IPR) Intas Biopharmaceuticals Limited Plot No: 423/P/A/GIDC
Sarkhej-Bavla Highway
Moraiya, Tal.: Sanand Ahmedabad - 382 210
Gujarat, India.