FIELD OF INVENTION:
This invention relates a water soluble polymeric drug formulation and to a process for preparing the same.
BACKGROUND OF THE INVENTION:
Gemcitabine (2', 2'- diflouro deoxycytidine), is currently a valuable cytotoxic drug for several solid tumors, e.g. pancreatic, lung and breast cancer. It is currently the first line drug available in the market, for locally advanced and metastatic pancreatic cancer. In addition, it is also effective in combination chemotherapy for the treatment of non-small cell lung cancer, bladder cancer and breast cancer.Gemcitabine's unique mechanism of action renders it an ideal candidate for chemotherapy. It accomplishes its anticancerous activity by incorporation into both DNA and RNA, resulting in masked DNA termination and inhibition of DNA synthase activities .Gemcitabine is said to be a cell cycle-dependent (S-phase-specific) deoxycitidine analogue of the antimetabolite class. It is a prodrug which requires cellular uptake and intracellular phosphorylation. Inside the cell, gemcitabine is first transported by human equilibrative nucleoside transporter -1 (h ENT1) via sodium-independent (equilibrative) mechanism and phosphorylated to monophosphate derivative (dFdCMP) by deoxycycitidine kinase (d CK), which is then converted to di- and triphospate derivatives (d FdCDP) and (dFdCTP, respectively). The emerging evidence demonstrates that incorporation of

gemcitabine derivatives into DNA is critical for gemcitabine to inhibit cell replication and induce apoptosis in cancer cells . dFdCDP inhibits ribonucleotide reductase (RR) liable for catalyzing the reaction that generates the deoxyribonucleotides required for synthesis and repair of DNA. dFdCTP incorporates into DNA as a false nucleoside, inhibiting DNA polymerase and thereby preventing the detection and repair of DNA repairing enzymes (masked chain termination).
Although the above described molecular events eventually contribute to the efficiency of gemcitabine for cancer treatment, the drug possesses certain drawbacks that are related to its unfavorable pharmacokinetic properties. Like most of the low molecular weight drugs, gemcitabine has very short plasma circulation time or bioavailability of 30-90 mins. It gets rapidly cleared from the body through renal excretion due to the enzymatic conversion in liver and kidney to the inactive and more soluble metabolite 2', 2'-difluorodeoxyuridine (d). Thus, a frequent administration scheduled at high doses is required, in turn leading to myelosuppression, high levels of hepatoxicity & renal toxicity along with toxicity towards other tissues or organs. The austerity of such treatment causes the patient to greatly weaken and the cancer, which may have seemed gone, often comes back with a vengeance. Therefore, new therapeutic strategies are needed aiming towards improved pharmacokinetics.
To circumvent the concerns of the high systematic toxicity, and improved body distribution together with prolonged blood circulation; the use of water soluble polymers as macromolecular carrier of low molecular weight conventional drugs is a very promising strategy in anticancer therapy. Poly (ethylene glycol) (PEG) is a water soluble amphiphilic polymer with high solubility and excellent biocompatibility. And as it is FDA approved, it is frequently used in numerous biomedical applications. PEG is commercially available in a variety of molecular weights with different functional groups and has been extensively used as ready-for-use forms by chemical. adtivation for covalent attachment to proteins. These activated PEGs are now being used for conjugation with small organic molecules acting as anticancer agents. This new low dose polymeric therapy referred to as "PEGylated drugs" offer the advantages of low-dose along with

long circulation displaying passive tumor targeting due to leakiness of angiogenic tumor blood vessels by Enhanced Permeability and Retention (EPR) effect; thereby facilitating superior therapeutic approach over the current chemotherapy regime for active or more aggressive cancer. Hence, use of a water soluble polymer like PEG act as a platform for drug targeting in non-immunogenic and non-toxic manner by increasing hydrodynamic volume after PEGylation and limiting its cellular uptake to the endocytic route leading to slower renal clearance and longer blood circulation time. Consequently,the pharmacokinetics of the drugs gets enhanced after conjugating with PEG. Recently, efforts have been made in the field of polymer conjugation to increase the therapeutic index of gemcitabine which include, conjugation of gemcitabine to hydrophilic synthetic polymers such as a, p-poly (N-2-hydroxyethyl) -DL-aspartamide (PHEA) and PEG using folic acid as targeting agent . Apparently, there has been very few published data available for the action of conjugated gemcitabine using bifunctional polymer PEG. The goal of the present study was to utilize the present efficiency of the polymer conjugation in the treatment of various cancers. We therefore designed PEGylated gemcitabine using homobifunctional PEG, HOOC-PEG-COOH for the conjugation with amine group of gemcitabine hydrochloride and studied its effect on pancreatic cancer cells. The, carboxylate group of PEG is initially reacted with N-hydroxysuccinimide (NHS) by l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) activation. NHS is chosen for amine coupling reactions in polymers due to its high reactivity at physiological pH in bioconjugation synthesis. Thus, in this paper we are reporting the novel method for the synthesis of the PEGylated gemcitabine by conjugating the amino group of PEG (HOOC-PEG-COOH) using triethylamine (TEA). The characterization of PEGylated gemcitabine is done by different analytical techniques like UV spectroscopy, lH NMR, FT IR spectroscopy and RP-HPLC". The cytotoxicity study of PEGylated gemcitabine in comparison to native gemcitabine is done in different cancer cell lines, MIA PaCa 2, A 549, K562 and Y79. Thus, this research aims to increase cytotoxic activity of gemcitabine through polymer conjugation. The intravenous administration of such polymeric conjugates can offers an excellent potential for the therapeutic approach in the treatment of cancer.

OBJECTS OF THE INVENTION:
An object of this invention is to propose a water soluble polymeric drug
formulation.
Another object of this invention is to propose as process for the preparation of
water soluble polymeric drug formulation.
Further object of this invention is to propose a water soluble polymeric drug
formulation which can be use as anticancer drug.
Still another object of this invention is to propose a polymeric drug formulation
with improved pharmacokinetics.
Still further object of this invention is to propose a polymeric drug formulation
with improved body distribution length with prolonged blood circulation.
Yet another object of this invention is to propose as polymeric drug formulation
with high solubility and excellent biocompatibility.
BRIEF DESCRIPTION OF THE INVENTION:
According to this invention, a water soluble polymeric drug formulation is provided comprising the amphiphilic polymer conjugated to the amino group of gemcitabine.
In accordance with this invention there is also provided a process for the preparation of water soluble polymeric drug formulation comprising the steps of:
(a) providing an N-hydroxysuccinimide ester of PEG by reaction of the carboxyl group of PEG with NHS in presence of EDC as a catalyst
(b) Conjugation of the said polymer with the amine group of the gemcitabine.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING:
SCHEME 1 - Schematic illustration of the reaction involved in the synthesis of the PEGylated gemcitabine.
FIGURE 1: UV spectra of PEGylated gemcitabine in comparison to gemcitabine in aqueous solution.
FIGURE 2: FT-IR spectra of (A) HOOC - PEG-COOH (B) PEGylated gemcitabine and (C) Gemcitabine.
FIGURE 3: 1H NMR spectra of (A) HOOC-PEG-COOH (B) Gemcitabine and (C) PEGylated gemcitabine.
FIGURE 4: Dose dependent cytotoxicity of native gemcitabine and PEGylated gemcitabine pemetrexed and Pegylated pemetrexed in A 549, MIA PaCa 2, K 562 and Y79 cells for 3 days and 5 days. Different concentrations of gemcitabine (-■-) and PEGylated gemcitabine (-•-) were added to the wells with medium as control. Medium was changed at 2 days and then every alternate day thereafter with no further dose of the drug added. The extent of growth inhibition was measured after stipulated time period by MTT assay. The inhibition was calculated with respect to respective controls. Data presented is mean ± standard deviation, n=6.

DETAILED DESCRIPTION OF THE INVENTION:
Synthesis of PEGvlated gemcitabine
The PEGylated gemcitabine was synthesized by conjugating gemcitabine to HOOC-PEG-COOH in dimethylsulfoxide (DMSO), in the presence of triethylamine (TEA). Briefly, HOOC-PEG-COOH (O.lmM) was dissolved in 2.5 ml of DMSO to which TEA (0.05 ml) was added. Further, NHS (100mM) and EDC (400 mM) were added to the above solution and the reaction mixture was stirred for 30 mins. Later, the PEG-(NHS)2 was coupled to gemcitabine. In brief, the gemcitabine (04 mM) was dissolved in 500ul and added drop wise to the PEG-(NHS) 2 solution, in presence of 2 mM of TEA (PEG -NHS/Gemcitabine/ TEA molar ratio = 1:4:20). The reaction mixture, was then kept on constant magnetic stirring for overnight at room temperature. The reaction mixture is later subjected to dialysis using a dialysis membrane (MWCO: molecular weight cut -off = 3.5 kDa) against distilled water to remove free and unreacted gemcitabine. Soon after, the dialyzed solution was freeze-dried using a lyophilizer (LABCONCO)—Corporation, USA) at a temperature of- 480 C and 0.05 mbar to obtain the powdered form of conjugate.
Characterization of PEGylated gemcitabine
The characterization of the PEGylated gemcitabine was done by UV
spectroscopy, FT-IR and H-MNR spectroscopy and reverse-phase
chromatography in HPLC.

UV Spectroscopy
The UV analysis for the PEGylated gemcitabine was determined spectrophotometrically. Briefly, 1 mg of PEGylated gemcitabine conjugates were dissolved in 1 ml of deionized water, and UV special scan was recorded with a wavelength ranging from 200 nm to 400nm using the ELISA plate Reader (Synergy ™ HT, Bio Tek Instruments Inc., USA).
FT IR spectroscopy
The FTIR spectra for native gemcitabine, HOOC-PEG-COOH and PEGylated gemcitabine were obtained from SPECTRUM RX 1 (Perkin Elmer, FTIR spectrometer, USA) for characterizing the chemical integrity of the PEGylated gemcitabine. Briefly, the samples were pressed into a potassium bromide pellet before obtaining their IP absorption spectra. The spectra were detected in KBr disks over a range of 4400-400 cm-1.
1H NMR Spectroscopy:
The native gemcitabine, HOOC -PEG -COOH and PEGylated gemcitabine were dissolved in DMSO -d6 (99.9 atom % deuterium-enriched, Sigma -Aldrich Inc., USA) with 0.1 % TMS serving as an internal reference. The NMR experiments were performed using a Bruker BioSpin (Fallanden, Switzerland) Avance -III 400 MHZ FT -NMR spectrometer (9.4 T, 54 nm vertical -bore magnet) equipped with a 5 mm BBFO - Plus multinuclear probehead with Z - Gradient, operating at a proton frequency of 400.13 MHz. The spectroscopic task was controlled by a HP xw-4600 workstation, and the spectral plotting was obtained using Bruker's TOPSPIN 2.1

RP - HPLC method for quantification of total gemcitabine
To determine the amount of gemcitabine conjugated to the PEGylated gemcitabine, alkaline hydrolysis of the PEGylated gemcitabine was performed to separate the native drug from the PEG moiety. The amount of gemcitabine obtained through this experiment was quantified by RP-HPCL. For this, 1 mg of PEGylated gemcitabine was subjected to.alkaline hydrolysis by dissolving in 1 ml of 1 N Na OH at 40 ° C for 1 h. The drug content in PEGylated gemcitabine was evaluated using RP - HPCL by injecting 20 ul of the above solution in the injection port. The amount of gemcitabine in the conjugate was measured with RP-HPCL system of Water ™ 600 (waters Co., Milford MA, USA) using a C 18 column (Nova-Pak® C-18, 3.9 MM X300MM. Water Associates) operated at 40° C with Waters 2489 UV/Visible Detector at a wavelength of 268 nm. As eluents , eluent A was 2.5 mM phosphate buffer, pH 7.0 and eluent B was composed by 50. % of A and 50 % of acetonitrile 95:5 v/v was used, at a flow rate of 1 ml/min . The total amount of gemcitabine in PEGYIated gemcitabine was determined from the peak area correlated with the standard curve. The standard curve of gemcitabine was prepared under identical conditions. All analysis was preformed in triplicates. Cell Culture:
Medium. A 549 and MIA Pa Ca 2 cells were cultured in DMEM(DMEM, PAN-Biotech GmbH, Aidenbach, Germany) and K562 and Y79 cells were cultured in RPMI (RPMI 1640, PAN- Biotech, GmbH, Aidenbach, Germany) with 1% L-Glutamine, 10% fetal bovine serum (GIBCO, USA) , 10,000 units/ml penicillin and streptomycin and maintained at 370 C in an incubator (Hera Cell, Thermo Scientific, Waltham, MA) in an atmosphere of 5 % carbon dioxide (CO2)

Cell Lines:
A549 (human lung carcinoma), MIA PaCa 2 (human pancreatic carcinoma) and Y79 (human retinoblastoma) cells were purchased form American Type Culture Collection (Rockville, MD). K 562 (human chronic myeloid leukemia) cell line was kindly gifted by DR. Soumen Chakraborty, Institute of Life Sciences, Bhubaneswar, India.)
Cells Cytotoxicity Studies
The cytotoxic effect of native gemcitabine and PEGylated gemcitabine was
assayed colorimetrically by the MTT staining method. The adherent cells (A 549
and MIA PaCa 2 ) were plated at a density of 2000 cells per wall and the
suspension cells (K562 and Y79) were plated at a density of 3000 cells per wall
separately in 96-well plates (Corning, YSA). The plated cells were then kept
overnight in appropriate growth medium with 10 % FBS and 10,000 units/ml
penicillin and streptomycin at 37 ° C. The next day, different concentrations of
gemcitabine or PEGylated gemcitabine (0.0lμ M, 0.1 M, 1 μM, 10 μM, 50 μM and
100 μM) were added in all the cell lines and the cells were incubated for 3 days
and 5 days. Cell culture medium with cells (without the drug treatment) served
as control in each experiment. Each test was performed in n= 6 wells. After
incubation, 100 μl of 5 mg/ml of 3-(4, 5 -dimethylthiazol -2-yl) - 2, 5-dipenyl-
tetrazolium bromide (MTT) was added and incubated for 3 h. The extent of cell
viability is indicated by conversion of MTT into purple formazon by
metabolically active cells. The crystals of produced formazon were dissolved with 100 μl of DMSO and optical density was measured at 540 nm using the ELISA plate Reader (Synergy ™' BioTek Instruments Inc., USA). The drug concentration which caused a 50 % inhibition of the control growth rate (IC50) was calculated by nonlinear regression analysis using the equation for a sigmoid plot.

RESULTS
Synthesis and characterization of PEGylated gemcitabine
The conjugation chemistry for low molecular weight drugs is less complex in comparison to the proteins because of the reduced number of functional groups present on a low molecular weight drug molecule, the absence of conformational constrains, and easier purification and characterization steps for the polymer drug conjugates. The macromolecular prodrug was synthesized by covalent linkage of gemcitabine hydrochloride (299 .68 gm/mol) to the HOOC-PEG-COOH (5 kDa) backbone. The selection of the functional group of polymer used for conjugation of amino drug is important. In this work, PEGylated gemcitabine was synthesized by the covalent of linkage of the carboxyl group of PEG (NHS) 2 with the amino group of gemcitabine at room temperature. The conjugation was preformed in two steps. Initially, the HOOC-PEG-COOH was activated to PEG -(NHS) 2 by using EDC as the catalyst. Further, the conjugation of gemcitabine was done by reacting with activated PEG:(NHS)2 in presence of TEA as a catalyst. The TEA helps in deprotonation of the gemcitabine primary ammonium salt, inducing its solubilization in organic solvent, here DMSO. The organic gemcitabine solution is then made to react with the N- hydroxysuccinimide moiety of PEG. The NHS ester of PEG reacts with the gemcitabine in presence of TEA dissolved in DMSO to yield the corresponding amide conjugate. The product was purified by dialysis of the reaction mixture against distilled water. UV spectra of conjugates showed disappearance of the typical UV peak of gemcitabine (268 nm) and formation of the new peaks of the PEGylated gemcitabine (246 and 298 nm), which are due to acylation of gemcitabine at N4 amino group due to amide bond formation with PEG (Figure 1). The spectra of the conjugates have shown the similar trends as those obtained from PEG-Ara-C.

FT IR analysis was used to investigate the conjugation efficiency of gemcitabine with the polymer. The HOOC-PEG-CCOH exhibited the characteristic peaks on IR spectrum at 2900.22 cm-1. 1743.89 cm-1, 1094,85 cm-1 and 3460.50 cm-1 as shown in figure 2 A. The absorption band at 1743.89 cm"1 is attributed to the carboxylic (-COOH) group, 1094. 85 cm-1 is attributed to C-O-C stretching vibration of repeated -O-CH2 - CH2 units of polyethylene glycol (PEG) backbone. The broad-band at 3460.50 cm-1 is due to the stretching vibration of -OH group corresponding to enhanced hydrogen bonding and 2900.22 cm-1 is due to -CH stretching vibrations due to symmetric and anti-symmetric modes of methylene groups. Figure 2 C shows the FT IP spectra of native gemcitabine with characteristic peaks of amide bands at 1681.51 cm-1 and 1724.64 cm-1 with 3256 cm-1 at 1681.51 cm-1 for stretching vibration of (-NH2). After conjugation, the PEGylated gemcitabine, as shown in figure 2 B, displays the characteristics peaks at 2887 cm-1, 1717 cm-1 , 1651 cm-1 , and 1578 cm-1. The peak of PEGylated gemcitabine due to the stretching vibration of -OH group is shifted to a higher frequency, appearing at 3431.55 cm-1 due to intermolecular hydrogen bonding. In addition, the larger and sharper C-H stretching band appeared at 2887 cm-1 in PEGylated gemcitabine that was similar to the peak displayed in PEG at 2882 cm-1. Also, the characteristic bands of carbonyl (C=O -NHR) at 1627.59 cm-1 in PEGylated gemcitabine signifies the amide bond formation between HOOC- -PEG - COOH and gemcitabine.
The synthesized PEGylated gemcitabine conjugate was also corroborated by 1H NMR spectra using DMSO solvent as shown in Figure 3. The typical H -NMR spectrum of. liner HOOC:PEG:CpOH is shown in figure 3A gives signal at the range of 5 =3.3-3.6 ppm for the protons of -O- CH2 CH2-(PEG chain )2. The gemcitabine gives the signal at 5 = 6.0-6.6 ppm for the protons of the 5' and 3'OH groups respectively, as shown in Figure 3B. In particular, gemcitabine gives the signals of 4' NH2 group at 5 =8.0 ppm. On the other hand figure 3C

shows the NMR signals of PEGyalated gemcitabine at the range of 6 = 3.7-4.3 ppm due to the pyranose ring of gemcitabine. In addition, the 1H-MMR spectrum showed a signal at 8.3 ppm for 0=CNH protons of amide carbonyl group in the PEGylated gemcitabine confirming the covalent bond formation between gemcitabine and PEG-NHS.
The PEGylated gemcitabine was further assessed by RP-HPCL for the evaluation of the amount of gemcitabine conjugated to PEG. The quantification of gemcitabine in the conjugate was determined by RP-HPCL analysis of the hydrolyzed product of the PEGylated gemcitabine. The hydrolysis was performed by the incubation of 1 ml of 1 N NaOH, which released gemcitabine from conjugate. The amount o f gemcitabine conjugated to PEG in 1 mg of PEGylated gemcitabine was found to be 70.0 μg.
Invitro cytotoxicity of PEGylated gemcitabine:
As a preliminary experiment) PERylated gencitabine was evaluated for in vitro antitumor activity by using the MTT assay based for 3 days and 5 days on four different cancer cell lines: MIA Pa Ca -2, A 549, K562 and Y79 as shown in Figure 4. The incubation in the presence of PEGylated gemcitabine induced an increase in cytotoxicity in all cancer lines when compared to the native drug. In particular the findings showed that the PEGylated gemcitabine. exhibited significantly higher cytotoxicity at low drug concentration and comparable cytotoxicity at high drug concentration in comparison with the native gemcitabine from both 3 days and 5 days treatment. This demonstrates that the enhanced cytotoxicity of the PEGylated gemcitabine comes form the conjugation strategy of prolonged release. The IC50 value, i.e., the drug concentration at which 50% cells have been killed at a given period, is listed is Table 1.It can be concluded from Table 1 that the PEGylated gemcitabine achieved much lower IC50 values

than the native gemcitabine in all the cases for the Mia Pa Ca 2 and Y79 cells. It is thus evidenced in vitro, that the PEGylation gemcitabine greatly enhanced that the therapeutic effects of gemcitabine for a longer period of time. The improved cytotoxicity of PEGylated gemcitabine exhibited in MTT assay can be attributed to its enhanced permeation and prolonged localization. Various studies have also shown that polymeric conjugation of the low molecular weight drugs provides a sustained release of the drug in the cells due to prolong retention, thus owing to higher cytotoxicity. The lower activity of native gemcitabine in inducing cellular cytotoxicity may be due to its less internalization and loss of stability by rapid degradation in the cancer cells. Thus, the in-vitro cytotoxicity study proves that the PEGylated gemcitabine more effectively in comparison to native gemcitabine for a prolonged period in all cancer cells lines.
Cell Culture:
Medium. A 549 and MIA Pa Ca 2 cells were cultured in DMEM(DMEM, PAN-Biotech GmbH, Aidenbach, Germany) and K562 and Y79 cells were cultured in RPMI (RPMI 1640, PAN- Biotech, GmbH, Aidenbach, Germany) with 1% L-Glutamine, 10% fetal bovine serum (GIBCO, USA) , 10,000 units/ml penicillin and streptomycin and maintained at 37 C in an incubator (Hera Cell, Thermo Scientific, Waltham, MA) in an atmosphere of 5 % carbon dioxide (CO2).
Cell Lines:
A549 (human lung carcinoma), MIA PaCa 2 (human pancreatic carcinoma) and Y79 (human retinoblastoma) cells were purchased form American Type Culture Collection (Rockville, MD). K 562 (human chronic myeloid leukemia) cell line was kindly gifted by DR. Soumen Chakraborty, Institute of Life Sciences, Bhubaneswar, India.)

WE CLAIM:
1) A water-soluble polymeric drug formulation comprising the amphiphilic polymer conjugated to the amino group of gemcitabine. 2.) A formulation according to claim 1, wherein the said amphiphilic polymeric
carrier is a linear and homobifunctional HOOC-PEG-COOH, 5 kDa. 3.) A formulation according to claim 1 to 2, wherein the said aminated
gemcitabine is the hydrochloride form of the gemcitabine drug. 4.) A formulation of any one of the claims 1 to 3, wherein the carboxylate
group of the polymer is reacted to the amino group of gemcitabine. 5.) A pharmaceutical composition according to claim 1 comprise the formulation
of PEGylated gemcitabine. 6.) A method for synthesizing the formulation of any of the claims 1 to 5,
comprising the steps of:
(a) providing an N-hydroxysuccinimide ester of PEG by reaction of the carboxyl group of PEG with NHS in presence of EDC as a catalyst.
(b) Conjugation of the said polymer with the amine group of the gemcitabine.
7.) The method of claim 5, wherein step (a) and (b) comprise forming the polymeric drug in room temperature by using DMSO as the solvent.

8.) The pharmaceutical composition according to claim 5 exhibit an enhanced in vitro cytotoxicity in various cancer cells.
9.) The use of pharmaceutical composition of claim 5 for producing a medication for the treatment and/or prevention of cancer characterized by enhanced angiogenic activity.
10) The formulation of claim 1 to 5, wherein the -COOH group of PEG is
functionalization by EDC and NHS method to conjugate any therapeutic peptide, antibody or another low molecular weight anti cancer drug.

A water soluble polymeric drug formulation and a process for preparing the same.
According to this invention, there is provided a water soluble polymeric drug formulation comprising a water-soluble polymeric drug formulation comprising the amphiphilic polymer conjugated to the amino group of gemcitabine. In accordance with this invention there is also provided a process for the preparation of water soluble polymeric drug formulation comprising the steps of:
(a) providing an N-hydroxysuccinimide ester of PEG by reaction of the carboxyl group of PEG with NHS in presence of EDC as a catalyst
(b) Conjugation of the said polymer with the amine group of the gemcitabine.