TITLE:
A water soluble PEGylated pemetrexed & a process for preparing the same.
FIELD OF INVENTION:
This invention relates to a water soluble PEGylated pemetrexed and a process for
preparing the same.
BACKGROUND OF THE INVENTION:
Antimetabolites have been used for the treatment of malignant diseases for 50 years.
They exert their anticancer action either as folate antagonists or as nucleoside analogues.
In recent years, however, several new antimetabolites have emerged in cancer treatment
and thus have provided the basis for further research. Pemetrexed is a novel pyrrolo [2, 3-
d] pyrimidine-based folate analogue that has been widely effective in the treatment of
various tumors [1]. It is a multitargeted antimetabolite that acts on the enzymes
thymidylate synthase (TS), dihydrofolate reductase (DHFR), glycinamide ribonucleotide
formlytransferase (GARFT) and aminoimidazole carboxamide ribonucleotide
formyltransferase (AICARFT) thereby depleting nucleotide pools and blocking DNA
synthesis [2]. Pemetrexed can be considered a prodrug, because it shows its activity in
pentaglutamate form which is predominantly, an intracellular form. Unlike the
aforementioned antimetabolites, pemetrexed has a unique pattern of cellular uptake and
metabolism, which may render it insusceptible to the common mechanisms of resistance
to nucleoside analogues [3]. Pemetrexed exerts its action by getting transported into the
cell via the reduced folate carrier
and the folate binding proteins. Once inside the cell, pemetrexed is polyglutamated by the
enzyme folyl-polyglutamatyl synthase to its tri- and pentaglutamate derivatives for the
significant inhibitory activity on multiple folate-dependent enzyme systems that lead to
DNA synthesis [4].

Conversely, the physiochemical characteristics limit the pharmacokinetic behavior of
pemetrexed. In the form of free acid, pemetrexed is poorly water soluble, sensitive to
light, heat and moisture, and also has a strong tendency of degradation [5, 6]. In addition,
the administration of pemetrexed in the form of disodium salt fails to provide high
aqueous solubility and more stable storage [7]. Like most other anti cancerous agent, the
rapid renal elimination of pemetrexed, can be due to the lowest molecular weight of
471.37 gm/mol [8, 9]. Pemetrexed
is primarily eliminated in the urine, with 70% to 90% of the dose recovered unchanged
within the first 24 h following administration. Pharmacokinetic studies have shown the
total systemic clearance of pemetrexed is 91.8 ml/min and the elimination half-life of
pemetrexed is 3.5 h in patients with normal renal function (creatininine clearance of 90
ml/min) [10]. Thus, restraining to the administration of the drug in a high dose along with
a frequent dosage schedule, that results in systematic toxicity.
Due to the futile properties of pemetrexed, various strategies are in the upcoming
approach. Polymer-drug conjugation is one of the major strategies of polymer
therapeutics for drug modifications, valuable for overcoming undesirable pharmaceutical
properties of drugs [11]. The field is driven by the belief that polymer conjugation will
contribute significantly by increasing the efficacy of currently used drugs as well as
providing opportunities for the use of new agents currently precluded from the clinic due
to challenges including low solubility and systematic toxicity [12] .This desired increase
in activity could be derived from a number of effects including prolonged circulation
times, reduced toxicity of drugs, increased drug solubility, and perhaps most importantly,
selective delivery to tumors through either active targeting with appended ligands for
tumor-specific receptors or passive targeting that results from the enhanced permeability
and retention (EPR) effect [13]. Further, the conjugation of polymer to drug enables its
endocytic internalization by all types of tumor cell. The intracellular delivery of a drug
into the endosomal or lysosomal compartment is in many cases not only essential for
therapeutic activity, it also provides the opportunity to bypass mechanisms of drug
resistance that are reliant on membrane efflux pumps such as P-glycoprotein (P-gPS) and
multi drug resistance proteins (MDRs) [14]. Among the various polymers used in
polymer therapeutics, Poly ethylene glycol (PEG), approved by FDA for pharmaceutical
applications; is used most often since it is water soluble, bi-compatible and nontoxic. It
facilitates its application for conjugation with low molecular weight anticancer agents to
improve their water solubility, plasma clearance and biodistribution [15]. Commercially,
PEG is available with a variety of activated functional groups attached to it. Among them
the heterobifunctional PEG have the advantage of endowing two different functional
groups for conjugating more than one therapeutic agent and availing itself as a multi-
tasking agent [8, 16]. The effects of PEGylation on drug pharmacokinetics include the
avoidance of reticuloendothelial [RES] clearance, the mitigation of immunogenicity, the
avoidance of enzymatic degradation and reducing renal filtration, with potentially
beneficial changes to biodistribution [13,15].
Based on a central assumption that pemetrexed can be modified by PEGylation, we
developed PEGylation pemetrexed which are highly water soluble, stable during storage
conditions and have an enhanced bioavailability during their administration. These
effects dramatically increased the potential index of pemetrexed as evident from the
various in vitro cellular experiments.
OBJECTS OF THE INVENTION:
• An object of this invention is to prepare a water soluble PEGylated pemetrexed.
• Another object of this invention is a process of preparing water soluble PEGylated
pemetrexed.
• Further object of this invention is to propose a PEGylation pemetrexed which are
stable during storage conditions.

• Still another object of this invention is to propose PEGylation pemetrexed having
enhanced bioavailability during their administration.
• Still further object of this invention is to prepare a PEGylation pemetrexed which
has enhanced antiproliferative activity in various cancer cells.
BRIEF DESCRIPTION OF THE INVENTION:
According to this invention, a process is provided for preparing a water soluble
PEGylated pemetrexed comprising the amphiphilic polymer conjugated to the
carboxylate form of pemetrexed. In accordance with this invention, the process provided
for preparing PEGylated pemetrexed comprises the activation of carboxylate group of
pemetrexed and then adding the polymer (NH2-PEG-COOH) in the form of solution to
the activated solution of pemetrexed. Thus, subjecting the resultant solution to dilution
followed by dialysis. The dialyzed solution is subjected to freeze drying using a
lyophilizer to form the final product.
DETAILED DESCRIPTION OF THE INVENTION;-
Synthesis of PEGylated pemetrexed
The synthesis of PEGylated pemetrexed was done in a sequence involving the activation
of carboxylate group of pemetrexed followed by the conjugation the activated
pemetrexed with amine group of heterobifunctional PEG (NH2-PEG-COOH). Initially,
pemetrexed is activated at its carboxylate group by a reaction in presence of NHS and
DCC in DMSO solution. Briefly, pemetrexed is dissolved in 10 ml of DMSO and reacted
with NHS and DCC under nitrogen atmosphere at room temperature for 12 h
(Pemetrexed /NHS/DCC molar ratio = 1:2:2). To the activated drug solution, 0.04 mM of
NH2-PEG-COOH dissolved in 5 ml of DMSO was added and the reaction was
performed under nitrogen atmosphere at room temperature for 4 h. The resultant solution
was diluted with deionozed water. The diluted solution was later dialyzed extensively
against deionized water and freeze-dried at temperature of -48°C and 0.05 mbar using a
lyophilizer (LABCONCO Corporation, USA) to obtain the powdered form of the
PEGylated pemetrexed.
Characterization of PEGylated pemetrexed
The characterization of the PEGylated pemetrexed was done by UV spectroscopy, FT-IR
and 'H-NMR spectroscopy and reverse-phase high pressure liquid chromatography (RP-
HPLC).
UV spectroscopy
The amount of pemetrexed conjugated to the polymer was determined
spectrophotometrically. 1 mg of PEGylated pemetrexed conjugate was dissolved in 1 ml
of deionized water, and UV spectra was recorded at 226nm.
Fourier transforms infrared spectroscopy
The FTIR spectra for native pemetrexed, NH2-PEG-COOH and PEGylated pemetrexed
were obtained from SPECTRUM RX I (Perkin Elmer, FTIR Spectrometer, USA) for
characterizing the chemical integrity of the PEGylated pemetrexed. Briefly, the samples
were pressed into a potassium bromide pellet before obtaining their IR absorption
spectra. The spectra were detected in KBr disks over a range of 4400-400 cm"1

1H NMR Spetroscopy
NMR spectra were recorded for native pemetrexed, NH2-PEG-COOH and PEGylated
pemetrexed from FT NMR spectrometer (JOEL 400 MHZ, Japan) using DMSO as the
solvent.
Pharmacokinetic investigation
Pharmacokinetic studies were carried out to study the bioavailability of the native
pemetrexed and PEGylated pemetrexed. The experiment on animals was performed with
the permission of Institutional Animal Ethics Committee of Institution of Life Sciences,
Bhubaneswar, India. The in vivo pharmacokinetic study was performed on female Balb/C
(25-28 g) mice divided into two groups. Group 1 received native pemetrexed and group 2
received PEGylated pemetrexed. Each mouse was administered with either native
pemetrexed or PEGylated pemetrexed dissolved in aqueous solution via tail vein, under
light anesthesia at a drug dose of 10 mg/kg. The experiment was performed in triplicates
for each time period. Blood samples were withdrawn from the retro-orbital plexus at
various times (30 mins, 1 h, 6 h and 24 h). Plasma samples (0.1 ml) containing
pemetrexed or PEGylated pemetrexed were precipitated with ice-cold methanol (0.2 ml)
for deproteinisation. After vortex mixing for 15 seconds, samples were kept on ice for 15
minutes. This was followed by centrifugation at 14,000 rpm for 4 minutes at 4°C using
SIGMA 1-15K (Germany). The aqueous-methanol supernatant obtained was analyzed by
RP-HPLC using a C18 column (Nova-Pak® C18, 3.9 mm x 300 mm, Waters Associates)
operated at 25 °C with Waters 2489 UV/Visible Detector (Water Associates) at a
wavelength of 226 nm. The mobile phase was composed of water/acetonitrile (86:14)
with 0.1 % of phosphoric acid at a flow rate of 1 ml/min. The injection volume was 20 pi.

Cell Culture
Medium A549, MCF 7, HCT 116 and MIA PaCa 2 cells were cultured in DMEM
(DMEM, PAN-Biotech GmbH, Aidenbach, Germany) and K562 and Y79 cells were
cultures 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), MCF 7 (human breast carcinoma), HCT 116 (human
colon carcinoma), MIA PaCa 2 (human pancreatic carcinoma) and Y79 (human
retinoblastoma) cells were purchased from American Type Culture Collection (Rockville,
MD, USA). K562 (human chronic myeloid leukemia) cell line was kindly gifted by Dr.
Soumen Chakrabarty, Institute of Life Sciences, Bhubaneswar, India.
Cells Cytotoxicity Studies
The cytotoxic effect of native pemetrexed and PEGylated pemetrexed was assayed
colorimetrically by the MTT staining method. The adherent cells (A549, MCF 7, HCT
116 and MIA PaCa 2) were plated at a density of 2000 cells per well and the suspension
cells (K562 and Y79) were plated at a density of 3000 cells per well separately in 96-well
plates (Corning, USA).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 pemetrexed or PEGylated pemetrexed (0.0003 µg,
0.003 µg, 0.03 µg, 0.3 µg, 3 µg and 30 µg) were added and the cells were incubated for 5
days in all the cell lines. 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-diphenyltetrazolium
bromide (MTT) was added and incubated for 3 h. The extent of cell viability is indicated
by the conversion of MTT into purple formazon crystals by metabolically active cells.
The crystals of produced formazon were dissolved with 100 µl of DMSO and optical
density was measured at 540 run using the EL-ISA plate Reader (Synergy™ HT, 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.
Apoptosis studies
The measurement of apoptosis was conducted by the Annexin V-FITC/PI apostosis
detection that recognizes the changes in phosphatidylserine (PS) during apoptosis, in
K562, A549, MCF 7, HCT 116, MIA PaCa 2 and Y79 cell lines. Annexin V-FITC and PI
labeling was done using Annexin V apostosis kit (Sigma) according to the prescribed
protocol. Briefly, 1 X 106 A549, MCF 7, HCT 116 and MIA PaCa 2 cells were plated in
25-cm2 culture flasks (Corning, NY, USA) and allowed to attach for 24 h while K562 and
Y79 were collected at 37 °C. After drug treatment with native pemetrexed or PEGylated
pemetrexed (0.5 µg/ml) for 3 days, adherent cells (A549, MCF 7, HCT 116 and MIA
PaCa 2 cells) were harvested by trypsinization and collected by centrifugation (3500
rpm,5 min) while the non-adherent cells (K562 and Y79) were simply collected by
centrifugation (3500 rpm, 5 min). This was followed by washing of the cells twice with
PBS (0.01 M, pH 7.4). Then 500 µl of Annexin V binding buffer was added to the cells.
Additionally, 5 µl of Annexin V-FITC and 10 µl of PI were added and incubated at room
temperature in dark for 10 min. The stained cells were analyzed directly by flow
cytometry (FACSCalibur; Becton-Dickison, San Jose, CA) using the Cell Quest™
programme (Becton Dickison, San Jose, CA). Cells displaying PS on their surface

sitive Annexin-V fluorescence) were considered to be apoptotic, regardless of
viability (PI staining). Cells staining positive for PI uptake was considered dead,
regardless of Annexin-V staining. This procedure was adopted for the above mentioned
cell lines. All experiments were performed in triplicates.
Synthesis and Characterization of PEGylated penmetrexed
The PEGylated pemetrexed can be synthesized by a two step process, as shown in Fig. 6.
To synthesis the reaction intermediate, succinylated pemetrexed i.e., Pemetrexed-NHS as
a precursor for polymer conjugation, pemetrexed was succinylated with an excess of
succinic anhydride (NHS). The similar procedure was adopted for the synthesis of
PEGylated methotrexate [16, 17]. This is because complete succinylation is essential to
provide pemetrexed cross-linking during polymer conjugation. It should be noted that
pemetrexed being a folate analogue has a- and y-carboxylic acid; both of them could be
activated when using the DCC/NHS chemistry. However, we presume that the terminal y-
carboxylic acid group of pemetrexed was activated by DCC/NHS and conjugated to the
terminal amine groups of a heterobifunctional PEG derivate (COOH-PEG-NH2). It is
generally known that among the two a and y-carboxylic acids; y-carboxylic acid is more
selectively activated due to its higher activity [16]. For further confirmation of the
PEGylated pemetrexed, various analytical techniques were used.
UV analysis:
The PEGylated pemetrexed was assessed by UV spectroscopy for the evaluation of the
amount of pemetrexed conjugated to PEG. The amount of pemetrexed conjugated to PEG
in 1 mg of PEGylated pemetrexed was found to be 25.8 µg.
FT IR ANALYSIS:
FT IR analysis was used to investigate the conjugation efficiency of pemetrexed with the
polymer. The NH2-PEG-COOH exhibited the characteristic peaks on IR spectrum at
1114.3 cm-11467 cm -1,1507.91 cm-1, 1636.49 cm-1, 2887.1 cm and 3447 cm-1 as shown
in figure 1 a. The absorption band at 1114.3 cm -1 is attributed to C-O-C stretching
vibration of repeated -O-CH2 -CH2- units of PEG backbone. The broad band at 3447 cm'
is due to the stretching vibration of -OH group corresponding to enhanced hydrogen
bonding and 2887.1 cm-1 is due to -CH stretching vibrations due to symmetric and anti-
symmetric modes of methylene groups. The peak at 1467 cm -1 is due to the -CH2 and
bend of the polymer [18, 19]. Furthermore, characteristic bands appearing in between
2500 to 3000 cm-1 and 1636.49 cm -1 correspond to the -COOH groups, while the peak at
1507.91 cm -1 relates to -NH2 group of heterobifunctional PEG. Figure 1 b shows the FT
IR spectra of native pemetrexed exhibits its characteristic peak at 1636.69 cm-1 and along
with the peak at 2934.22 cm -1 for the presence of -CQOH groups [20]. After
conjugation, the PEGylated pemetrexed, as shown in Figure 1 c, displays the
characteristic peaks at 2887 cm -1, 1715.49 cm-1, 1653.98 cm-1 and 1466.44 cm -1. The
peak of PEGylated pemetrexed due to the stretching vibration of-OH group is shifted to
a lower frequency, appearing at 3432 cm -1 due to intermolecular hydrogen bonding. In
addition, the larger and sharper C-H stretching band appeared at 2887 cm-1 in PEGylated
pemetrexed was similar to the peak displayed in PEG. Also, the strong absorption bands
attributed to amide bond formation at 1715.5 cm-1 and 1654 cm-1 in PEGylated
pemetrexed signifies the amide linkage between Pemetrexed -NHS and NH2- PEG-
COOH.
1H NMR analysis:
The synthesized PEGylated pemetrexed conjugate was also corroborated by 1 H NMR
spectra using DMSO solvent as shown in Figure 2. The typical 1 H-NMR spectrum of
linear NH2-PEG-COOH is shown in Figure 2 a, gives signal at the range of d = 3.3-3.6
ppm for the protons of O-CH2-CH2- of PEG chain [6,18]. The pemetrexed gives
characteristics signals at 2.0-2.9 ppm and 3.3-3.8 ppm (-CH2-), 4.0-5.0 ppm and 6.2-6.3
ppm (-CH- of pyrrole), 7.2 and 7.7 ppm (Aryl-CH) and the signal at 10.5 ppm
corresponds to the pteridine moiety proton from the pemetrexed and the signal at 11.2
ppm corresponds to the protons of the carboxylic groups, as shown in Figure 2 b [21, 22].
On the other hand, Figure 2 c shows the NMR signals of PEGylated pemetrexed at the
range of d = 3.3-3.6 ppm corresponding to PEG backbone along with the signals 1.8 -2.8
ppm, 3.3-3.8 ppm, 4.0 -5.0 ppm, 6.2 ppm, 6.4 ppm, 7.2 -8.8 ppm of pemetrexed with a
slight shift. However, the presence of an amide signal (d =10.5 ppm) and the absence of
the signal of the carboxyl group (d = 11.2 ppm) in the PEGylated pemetrexed confirms
the covalent bond formation between pemetrexed and NH2 - PEG -COOH.
RP-HPLC analysis:
The PEGylated pemetrexed was further assessed by RP-HPLC for the evaluation of the
amount of pemetrexed conjugate to PEG. The amount of pemetrexed conjugated to PEG
in 1 mg of PEGylated pemetrexed was found to be 30 µg.
Pharmacokinetic studies:
The plasma concentration level of pemetrexed was evaluated after intravenous injection
of pemetrexed, either in native form PEGylated at the dose of 10 mg/kg in female Balb/c
mice, as shown in Figure 3 for the period of 24 h. The native pemetrexed diminished
from the circulation with time due to its short half-life. In contrast, the PEGylated
pemetrexed showed a much longer circulation time. Both formulations reached the
highest level by 30 min (0.5 h) after the injection, which corresponds to 53 ug/ml for the
native pemetrexed and 50 ug/ml for the PEGylated pemetrexed. The drug concentration
of the PEGylated pemetrexed achieved further higher level in the plasma than the native
pemetrexed thereafter, all the time in the experiment. During 6 h when the concentration
of native pemetrexed was about 35 µg/ml in plasma circulation, the concentration of the
drug in PEGylated product was 47 µg/ml and the concentration of pemetrexed in
PEGylated form was twice that of native pemetrexed after 24 h i.e., 37.6 µg/ml of
pemetrexed in PEGylated form and 19.7 µg/ml in native form. Thus, PEGylatation allows
a higher pemetrexed bioavailability of pemetrexed in the in vivo condition. The prolong
availability of pemetrexed in blood plasma is an important parameter because it will
overcome one of the most relevant pemetrexed shortcomings, i.e., the fast in vivo
clearance due to short half-life.
In vitro cytotoxity studies:
As a preliminary experiment, PEGylated pemetrexed was evaluated for in vitro antitumor
activity by using the MTT assay based for 5 days on six different cancer cell lines: K562,
A549, MCF 7 and HCT 116, MIA PaCa 2 and Y79, as shown in Figure 4. The incubation
in the presence of PEGylated pemetrexed induced an increase in cytotoxicity in all cancer
cell lines when compared to the free drug. In particular, the findings showed that the
PEGylated pemetrexed exhibited significantly higher cytotoxicity at low drug
concentration and comparable cytotoxicity at high drug concentration in comparison with
the native pemetrexed from the 5 days treatment. This demonstrates that the enhanced
cytotoxicity of the PEGylated pemetrexed comes from the conjugation strategy of
prolonged release. The IC50 value i.e., the drug concentration at which 50 % cells has
been killed in a given period, is listed in Table 1. It can be concluded from table, that the
PEGylated pemetrexed achieved much lower IC50 values than the free drug pemetrexed in
all cases for MCF 7 and K562 cells. It is then evidenced in vitro, that the PEGylation
greatly enhanced the therapeutic effects of pemetrexed for a longer period of time.
Apopstis Study:
The apoptosis study was carried for 3 days using Annexin V-FITC staining of the native
pemetrexed and PEGylated pemetrexed treated six cancer cell lines (K562, A549, MCF
7, HCT 116, MIA PaCa2 and Y79), as shown in Figure 5.
Interestingly, with the 3 days treatment, all the cancer cells demonstrated a significant
percentage of apoptosis by native as well as PEGylated pemetrexed. Nevertheless, in
comparison to the free form, PEGylated pemetrexed lead to a marked amount of
apoptosis in the various cancer cell lines. In particular, K.562 and MCF 7 cell lines
exhibited the highest amount of apoptosis after the 3 days treatment of PEGylated
pemetrexed. Therefore, the PEGylated pemetrexed was able to induce apoptosis guiding
to a substantially higher cytotoxicity in comparison to the native pemetrexed.
In conclusion, PEGylated pemetrexed seemed to be more potent than the native
pemetrexed towards the six tested cell lines ( K562, A549, MCF 7, HCT 116, MIA PaCa
2 and Y79) and is worthy of further investigation.
Table 1: IC50 values of native pemetrexed and PEGylated pemetrexed were calculated on 5 day
in different cancer cell lines (A549, HCT116, K562, MCF 7, MIA PaCa 2 and Y79).
Table 1
We Claim:
1. A water soluble PEGylated pemetrexed formulation comprising the amphiphilic
polymer conjugated to the carboxylate form of pemetrexed.
2. The formulation according to claim 1, wherein the said amphiphilic polymer is
linear and heterobifunctional NH2 -PEG-COOH, 5 kDa.
3. The formulation according to claim 1, wherein the said pemetrexed carboxylate is
sodium salt of pemetrexed drug.
4. The formulation as claimed in claim 1 to 3, wherein the amine group of the
polymer reacts with carboxylate group of pemetrexed.
5. A process for preparing the PEGylated pemetrexed according to claims 1 to 4,
comprises the step of activation of carboxylate group of pemetrexed followed by
conjugation upon addition of the polymer (NH2-PEG-COOH) in the form of
solution to the activated solution of pemetrexed.
6. The process as claimed in claim 5, wherein the step of activation is performed by
dissolving pemetrexed in DMSO and reacted with NHS & DCC in presence of
nitrogen atmosphere at room temperature for 12hours.
7. The process as claimed in claim 6, when the molar ratio of pemetrexed: NHS:
DCC is 1:2:2.
8. The method as claimed in claim 5, wherein the polymer (NH2 -PEG-COOH) was
dissolved in DMSO to form a solution.
9. The method as claimed in claim 5,wherein the polymer solution is added to the
pemetrexed solution in presence of nitrogen atmosphere at room temperature and
reacted for 4 hrs.
10. The method as claimed in claim 5, wherein the final solution is diluted and
dialyzed followed by freeze dried at temperate of -48° C & 0.05 mbar using a
lyophilizer.
11. The pharmaceutical composition according to claims 1 to 10 exhibit enhanced in
vivo bioavailability along with improved in vitro Antiproliferative activity against
various cancer cells.
12. The formulation of claim 1 to 11, wherein the -COOH group of PEG can be
further functionalization by DCC/NHS method to conjugate any therapeutic
peptide, antibody or another low molecular weight anti cancer drug.

According to this invention, a process is provided for preparing a water soluble
PEGylated pemetrexed comprising the amphiphilic polymer conjugated to the
carboxylate form of pemetrexed.
In accordance with this invention, the process provided for preparing PEGylated
pemetrexed comprising of two steps. Initially, the carboxylate group of pemetrexed is
subjected to the step of activation by reacting with NHS and DCC. This is followed by
adding the polymer (NH2 - PEG-COOH) in the form of solution to the activated solution
of pemetrexed. Then the resultant solution is made to the step of dilution dialyzing the
diluted solution subjecting the dialyzed solution to the step of freeze drying using a
lyophilizer to form the final product.