Title of Invention

"A NOVEL COBALT COMPLEX USEFUL FOR REVERSAL OF DRUG RESISTANCE AND THE PREPARATION THEREOF"

Abstract The present invention relates to a cobalt N-(-2-hydroxyacetophenone) aminoacetate useful for reversal of drug resistance, said cobalt complex having general formula 1: Formula 1 wherein "Ar" is phenyl group; "R" is selected from methyl or ethyl group and the value of "n" is in the range of 1 to 3.
Full Text Field of invention:
The present work describes the synthesis of a novel cobalt complex with potassium salt of (N-2-hydroxy acetophenone) glycinate (PHAG). The structure of the complex, viz., cobalt (II) (N-2-hydroxy acetophenone) glycinate (CoNG) has been determined by spectroscopic studies. Antitumor property, toxicity and glutathione (GSH) depletion property in vivo of the complex has also been reported. CoNG enhances superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activity in various organs like spleen, heart, liver, kidney and lung. High and low doses of the complex deplete GSH; GSH depletion property of CoNG may be utilized to sensitize drug resistant cells to anticancer drugs where resistance is due to elevated level of GSH.
The novel metal complex cobalt (II) (N-2-hydroxyacetophenone) glycinate (CoNG) has been tested to reverse the drug resistance of doxorubicin in vivo in this tumor model. The reversal of drug resistance by CoNG in vivo was compared with the effect of EA (Ethacrynic acid) and BSO (L-buthionine-S, R-sulphoxamine).
Background and prior art
Coordination of organic compound with metal i.e., chelation causes drastic change in the biological properties of the ligand and also the metal moiety [1]. It has been reported that chelation is the cause and cure of many diseases including cancer [2]. Biocoordination chemistry that deals in the interactions of metal complexes with biological systems is receiving increasing interest. It has been reported by some authors that alkyne-cobalt carbonyl complexes inhibited the growth of human melanoma and lung carcinoma cell lines [3].
Cobalt compounds have antitumor activity in vivo [4-5]; cobalt complexes are also potential radiosensitizers [6,7]. Cobalt complexes would be of particular interest because of coordination capacity of the metal center and their ability to catalyze redox processes involving (di) oxygen and active oxygen species as well as biogenic substrates [7].
Coordination behavior of potassium salt of N-(2-hydroxy acetophenone) glycine [PHAG] towards organotin moiety has recently been reported [8]. Antitumor property of the ligand [PHAG] and some of its organotin complexes has been studied [9]. The nontoxic nature, water solubility and strong coordination behavior of PHAG deserve attention [10,11].
Multidrug resistance is one of the most significant problems in cancer treatment. For the last two decades tremendous effort was focused on understanding the cellular, molecular and physiological mechanisms underlying drug resistance. Several mechanisms have been described that mediate such resistance, e.g., a membrane glycoprotein of 170 kd (P-glycoprotein) [12], multidrug resistance-associated protein (MRP) [13], increased DNA repair mechanisms as a means to reverse cytoxicity [14] and altered expression of metabolic and detoxification processes that protect the cells against such damage [15]. The pathway of altered expression of metabolic and detoxification processes may be mediated by the glutathione/glutathione S-transferase (GSH/GST) detoxification system. Many reports have shown that resistance to alkylating agents anti cancer drug is associated with increased GSH levels and GST activity [15]. Other than the antioxidant GSH, Phase II detoxification system utilizes a number of antioxidant enzymes like GPx, catalase and SOD and protect the system from antioxidant damage [16]. Catalase arid GPx seek out hydrogen peroxide and convert it to water and diatomic oxygen. An increase in the production of SOD with concomitant increase of catalase and GPX has protective effect on the living system [17, 18]. Considering the structure and composition of CoNG and the elevation of SOD in all the organs by CoNG it seems that the present metal complex (CoNG) may have SOD mimetic property [19].
GSH and GSH related enzymes play a major role in the cellular detoxification of potentially harmful xenobiotics and oxygen related toxic species. The importance of GSH in altering cellular response to
certain chemotherapy drugs has been demonstrated by the agents that either inhibit or stimulate intracellular GSH synthesis. Depletion of intracellular GSH by L-buthionine-S,R-sulphoxamine (BSO) in a variety of cell types has been shown to markedly enhance the cytotoxicity of many chemotherapy drugs [20-22]. Conversely, elevating GSH levels prior to drug treatment by oxothiazolidine carboxylate can afford significant protection against chemotherapy drug-mediated cytotoxicity [23-25]. The report of reversal of drug resistance in a number of cell lines by BSO has prompted the clinical exploration of GSH modulation of tumor cells by a number of new chemicals [26]. Although ethacrynic acid (EA) and sulphosalazine deplete GSH and modulate drug resistance [27-30] both the chemicals have dose limiting toxicity [31].
Though numerous in vitro studies have described new resistance modifying agents (RMA), very few studies have shown beneficial effects of the use of such RMAs in vivo for MDR-tumor treatment in animal model. This is partly linked to the highly generalised toxicity of several of the RMAs.
The present study was conceived on the basis that a non toxic GSH depleting agent may sensitise multidrug resistant cells towards anti cancer drugs. To overcome the inherent difficulty in studying drug resistance due to heterogeneity of human cancer, or difficulty in obtaining several clinical specimens from the same tumor during the course of chemotherapeutic treatment, we developed an experimental drug resistant Ehrlich ascites carcinoma (EAC) model as reported earlier [32].
Novelty of the invention lies in identifying a GSH depleting agent, which is non-toxic and water soluble. Moreover, for the first time any metal complex having the properties of RMA has been disclosed.
Object of the present invention
The prime object of the present invention is to synthesize drug resistance modifying agent (RMA).
In another object of the present invention is to synthesize cobalt complex by the reaction of cobalt sulphate and PHAG having anti-tumor activity and reversal of drug resistance capability. In another object of the present invention is to develop non-toxic RMA.
In one more object of the present invention is to determine the structure of the cobalt complex.
Yet in another object of the present invention is to assess the anti-tumor property, toxicity, glutathione (GSH) depletion ability in vivo. Still in another object of the present invention is to assess role of cobalt complex on super-oxidase dismutase (SOD), catalase and glutathione peroxidase (GPx) activity in various organs like spleen, heart, liver, kidney and lung.
Another object of the present invention is to assess the reversal of drug resistance of doxorubicin in vivo.
Still in one more object of the present invention is to compare the cobalt complex with other RMA like ethacrynic acid (EA) and L-buthionine-S, R-sulphoxamine (BSO).
Further in another object of the present invention is to develop an anti-cancer drug resistance modifying agent which helps to penetrate higher amount of anti-cancer drug to the drug resistance cells. Statement of the invention A novel cobalt complex having structural formula 1
(Formula Removed)
Wherein Ar= Phenyl, substituted phenyl, napthyl, substituted napthyl, anthracene and substituted anthracene; R= CH3 and C2H5; n= 1, 2 and
useful for depleting glutathione (GSH) and sensitizing drug resistant cells to anticancer drugs where resistance is due to elevated level of GSH.
Detailed description of the invention
Accordingly, the present invention deals with a synthesis of non-toxic, water soluble, novel cobalt complex that increases super-oxidase dismutase (SOD), catalase and glutathione peroxidase (GPx) in all the organs like spleen, heart, liver, kidney and lung and depletes the elevated level of glutathione (GSH).
In another embodiment of the present invention the ligand, potassium, sodium or ammonium salt of N-(2-hydroxy substituted aryl ketone) aminoacetate (NHAG) is prepared using the following method.
a) mixing cold aqueous solution of NaOH, KOH or NH4OH of 0.1
Molar with cold aqueous solution of glycine, alanine or other
amino acid of 0.1 Molar at a temperature in the range of 15-20°C
in a ice bath with continuous stirring,
b) adding ethanolic solution of 2-hydroxy-substituted aryl ketone
0.1 Molar drop wise in the mixture of step (a),
c) stirring the mixture of step (b) for about 1 hr followed by 5 hrs
at room temperature to obtain a yellow colored mass,
d) evaporating the solvent from the mixture of step (c),
e) washing the yellow mass of step (d) with petroleum ether and
followed by precipitating with methanol-diethyl ether mixture,
and
f) recrystallizing the precipitated mass of step (e) to yield sodium,
potassium or ammonium salt of N-(2-hydroxy substituted aryl
ketone) aminoacetate (NHAG).
Yet in another embodiment of the present invention the aminoacetate is selected from a group comprising glycinate and alanate.
Still in another embodiment of the present invention the cobalt N-(2-hydroxy substituted aryl ketone) aminoacetate (CoNG) having following structural formula
(Formula Removed)
Wherein Ar= Phenyl, substituted phenyl, napthyl, substituted napthyl, anthracene and substituted anthracene; R= CH3 and C2H5; n= 1, 2 and 3.
and is synthesized used the following method.
a) dissolving sodium or potassium or ammonium salts of N-(-2-
hydroxy substituted aryl ketone) amino acetate (NHAG) and
cobalt salt separately in a suitable solvent to obtain a first
solution containing NHAG and a second solution containing the
cobalt salt,
b) cooling the first and second solutions,
c) drop wise adding the first solution containing NHAG to the
second solution containing cobalt salt solution, kept in ice bath,
d) stirring the mixture of step (c) to obtain crude CoNG in the form
of deep brown precipitate, and
e) separating the precipitate and recrystallizing the same in water
alcohol mixture to obtain CoNG.
f) MP >400°C, yield 40%. Anal. Calc. C10H11O4NCo: C, 44.77; H,
4.10; N, 5.2; Found: C, 45.44; H, 4.74; N, 4.98.
Still in another embodiment of the present invention the cobalt salt is selected from a group comprising cobalt (II) acetate, cobalt (II) nitrate and cobalt (II) chloride.
In another embodiment of the present invention Proton NMR peak of the metal complex (CoNG) in D2O appear at 6 7.1-7.8 (5H, S) and 6 6.3-
6.8 (3H, S) for aromatic protons. -CH2- protons appear at δ 3.53 (B).
CH3 protons appear at 5 2.53-2.84 (M, 4H).
Yet in another embodiment of the present invention the complex is
nontoxic as different doses of CoNG (50mg/kg, 100 mg/kg) causes no
toxic death up to 96h.
In one more embodiment of the present invention the long term (72h) and short term
(2h) effect of various doses CoNG on blood, spleen and bone marrow in male Swiss
mice show no toxic effect in vivo.
Yet in one more embodiment of the present invention the haemoglobin, RBC, WBC,
bone marrow cells remained unchanged after CoNG injection.
Still in another embodiment of the present invention CoNG at a dose
of 15, 20, 50 mg/kg depletes GSH markedly in all the organs like
heart, kidney, liver and lung in normal Swiss mice at 2h compared to
untreated control. Further in one more embodiment of the present
invention the level of GSH slightly restored at 4 hours and reached
almost to normal level at 24h of CoNG injection.
Yet in one more embodiment of the present invention in normal mice, CoNG does not deplete GST activity significantly in 2h, 4h and 24h. The level of GST activity remains almost unchanged with various doses of CoNG (15, 20, 50 mg/kg) when compared with untreated control.
Still in another embodiment of the present invention GPx activity increased at 2h with various doses of CoNG (15, 20, 50 mg/kg) significantly in all the organs compared to control (untreated) mice.
Yet in another embodiment of the present invention CoNG increases SOD and
catalase in all the organs like spleen, heart, kidney, liver and lung.
Still in one more embodiment of the present invention the amount of reduced
glutathione (GSH) has been found to be much less in drug treated cells than in
untreated cells (control). Enzymes involved in the metabolism of drugs and
xenobiotics are subdivided into two major categories viz., Phase I and Phase II. The
key component of the phase II detoxification parameter is GSH.
In one more embodiment of the present invention administering anticancer drug
doxorubicin (Dox) with CoNG (Dox + CoNG treated group) in drug resistant (EAC/Dox) cells, the concentration of Dox increased considerably. The concentration of Dox increased in CoNG treated cells by 276%, EA treated cells by 176% with respect to untreated drug resistant cells (control) that was considered to be 100%. Yet in another embodiment of the present invention the HPLC study shows no new compound or conjugate was detected after 4 hours of addition of CoNG and GSH. Retention time for GSH (0.95 mins.) and CoNG (2.303 mins) remained unchanged in the mixture of the two compounds where two peaks appeared at 0.938 minutes and 2.688 minutes were identified to be the peaks for GSH and CoNG respectively.
Further in one more embodiment of the present invention a non-toxic
anti-cancer drug resistance modifying agent comprising CoNG of
formula 1 in the range of lmg/ml to 50 mg/1 in a pharmaceutically
acceptable solvent/medium.
Still in one more embodiment of the present invention, wherein the
solvent selected from a group comprising water, saline and organic
solvent.
In one more embodiment of the present invention, the medium
selected from a group comprising milk, sugar, coffee and tea.
In one more embodiment of the present invention the dose of non toxic
anti-cancer drug resistance modifying agent is in the range of 1 to 500
mg/kg.


Statement of the Invention
An embodiment of the present invention relates to a cobalt N-(-2-hydroxyacetophenone) aminoacetate useful for reversal of drug resistance, said cobalt complex having general formula 1:
(Formula Removed)
Formula 1
wherein "Ar" is phenyl group; "R" is selected from methyl or ethyl group and the value of "n" is in the range of 1 to 3.
Brief description of Tables
Table 1. Long term (72h) and short term (2h) effect of various doses of cobalt (II)
(N-2-hydroxy acetophenone) glycinate (CoNG) on blood, spleen and
bone marrow in male Swiss albino mice.
Table 2. Effect of various doses of cobalt (N-2-hydroxy acetophenone) glycinate
(CoNG) on male Swiss albino mice bearing Ehrlich ascites carcinoma
(EAC) cells.
Table 3. Effect of cobalt (N-2-hydroxy acetophenone) glycinate (CoNG) on GSH,
GST, GPx in various organs of male Swiss albino mice.
Table 4. Effect of cobalt (N-2-hydroxy acetophenone) glycinate (CoNG) on
catalase and SOD in various organs of male Swiss mice.
Table 5. GSH and GST in drug treated and untreated
drug resistant (EAC/Dox) cells
Table 6. Effect of single dose of doxorubicin (Dox) on drug resistant
(EAC/Dox) and drug sensitive (EAC/S) cells up to maximum
tolerated dose (MTD).
Table 7. Effect of cobalt (II) (N-2-hydroxy acetophenone) glycinate (CoNG) as
resistance modifying agent (RMA) against doxorubicin (Dox) in male
Swiss albino mice bearing drug resistant (EAC/Dox) cells compared
to mice bearing sensitive (EAC/S) cells. Figure 1 Mass fragmentation of cobalt (N-2-hydroxyacetophenone)
glycinate (CoNG)
Figure 2 Interaction of CoNG and human serum albumin (HSA) Figure 3 Concentration of doxorubicin (Dox) in control and drug
treated cases.
Formula 1 Structure of Novel Cobalt complex Examples
The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the invention. Example 1
Synthesis of the CoNG
CoNG was synthesized by the reaction of PHAG with cobalt (II) sulphate; in brief, 230 mg of PHAG (0.01M) and 280 mg cobalt sulphate (0.01M) was dissolved in 5 ml double distilled water separately. Both the solutions were cooled to 8-10°C. The solution of PHAG was added drop wise to cobalt sulphate solution kept in ice bath. The mixture was rotated in a magnetic stirrer for 25-30 minutes, keeping the solution at 7-8°C. Deep brown precipitate deposited and was allowed to settle for 30 mins. in refrigerator. The precipitate was isolated by centrifugation and recrystallised in water-alcohol.
Example 2
Characterization of the CoNG
UV-VIS spectra were recorded in Shimadzu UV 160 A and in Varian Gary 100 Scan in the range of 800 -200 mµ.
I.R. spectra were recorded in Parkin-Elmer RX 1 FT spectrophotometer
in KBR discs in the range 4500-500 cm-1.
Proton NMR spectra was recorded in D2O on a Bruker ACF 300
spectrometer at 300.13 MHZ reference to Me4Si (0.0 ppm).
13C NMR spectrum was recorded in D2 O on a Bruker 200 MHZ
spectrometer.
Mass spectrum was recorded in an AEI MS-30 mass spectrometer.
C, H, N was measured by Parkin-Elmer 2400 Series II CHN analyzer.
EPR spectrum was recorded in Varian E 109C at room temperature at
field set: 3200G; scan range 4xlK; receiver gain 4.0xl03; scan time 4
mins.; modulation 1.25x10; time constant 0.250; frequency 9.1 GHz,
power 30 dB; temp. 295° K; state: microcrystalline.
Magnetic moment was measured in EG & G vibrating sample
magnetometer (Model 155).
UV spectrum for the ligand λmax (water): 211, 253, 325 nm.
UV-VIS spectrum for the complex λmax (water): 213, 245, 368, 665 nm.
The electronic absorption spectrum of the complex CoNG shows four
bands at 213 nm (ε18,600 M-1cm-1), 245 nm (ε17,000 M-1cm-1) 368 nm (ε
318 M-1cm-1), and at 665 nm (ε 56 M-1cm-1) in aqueous solution. The
three bands at 213, 245 and 365 nm in the complex are perhaps due to
n-n* or n- n* transition that are present in the ligand at 211, 253 and
325 nm. The band position of the ligand is shifted in the complex and
thus indicates coordination of the ligand to metal [33]. The 665 nm
band in the complex is assigned due to d-d transition. The d-d band
position suggests the complex to be predominantly four coordinated.
The band position of the d-d transition is an indication of a square
based geometry of the complex [34].
Important infrared (IR) bands for the ligand appear at: 3394, 1689,
1619, 1524, 1466, 1421, 1395, 1318, 1269, 1205, 1163, 969, 931, 752, 730
cm-1.
Important IR bands for the complex appear at: 3365-3162, 2340, 1625,
1598, 1541, 1438, 1372, 1303, 1328, 1225, 1160, 1138, 1027, 962, 865, 792,
752, 618, 547 cm-1.
The vCN characteristic stretching band in the ligand appears at 1619 cm-1 that shifts in the lower frequency region in the complex at 1598 cm-1 suggesting the coordination between nitrogen of the ligand and the Co-metal. In the ligand, one strong band appears at 1689 cm-1 due to asymmetric stretching vibration of vCOO and another strong band appears at 1395 cm-1 due to symmetric stretching vibration of vCOO group [35]. In the metal ligand system, 1689 cm-1 band is not observed and the band at 1395 cm-1 shifted to 1438 cm-1; So there is strong indication that the COO~ group coordinates through deprotonation. The vC-O ligand band (1269 cm-[) shifts towards high frequency region in the complex at 1303 cm '. This high frequency shift of vC-O phenolic band confirms the formation of covalent bond between oxygen atom of phenolic -OH and metal ion through deprotonation [36]. The -OH group participation in coordination is also indicated by the shift of 3394 cm-1 band (-OH group) towards 3162-3365 cm ' through deprotonation and formation of a metal-oxygen bond. Metal ligand vibrations are generally located in the region 600-250 cm-1. The skeletal vibrations of the ligand appearing in this region complicate the scope of interpretation. However, the comparison of the complex and ligand spectra allowed the assignment of metal sensitive bands. In the present case, we have assigned the band at 618 cnv1 to vM-N in the complex.
Proton NMR peak of the ligand in D2O appears at 7.38-7.51 (S, 5H) and 6.59-7.6 (S, 3H) for aromatic protons. CH2 protons appear at 4.09 (1H, M). CH3 protons appear at 2.26-2.29 (4H, M).
Proton NMR peak of the metal complex (CoNG) in D2O appear at 5 7.1-7.8 (5H, S) and 5 6.3-6.8 (3H, S) for aromatic protons. -CH2- protons appear at 5 3.53 (B). CH3 protons appear at δ 2.53-2.84 (M, 4H). The characteristic proton signals due to aryl group in the ligand (5 6.59-6.76 and 5 7.3-7.5) are almost unaffected in the complex and appear at 5 6.3-6.8 and 5 7.1 — 7.8. Complexation causes drastic changes of proton signals of -CH2 and -CH3 groups in the ligand. The signal in the ligand due to -CH2 group (5 4.09) shifts to higher field 6 3.53 in the complex; This is an indication of considerable drift of
electrons from two neighboring groups viz., -N=C- and -COOH to the
metal moiety [37]. The signal for CH3 shift to lower field in the
complex (in the complex at δ 2.53-2.84 and in the ligand at δ 2.26-2.29)
due to deshielding of protons and indicates participation of CH3-C = N-
group to coordination with cobalt atom [38].
In 13C NMR spectrum of CoNG in D2O, the peak at δ122.981 - 138.968
has been assigned for aromatic carbons; The aromatic -C-OH carbon
appears at 6166.570. The -COOH carbon appears at δ172.897, -CH=N
carbon appears at 6116.475, -CH2 carbon appears at 62.591 and --CH3
carbon appears at 5 20.626. The characteristic 13C peaks are in
agreement with the skeletal structure presented in Formula 1.
The electron paramagnetic resonance (EPR) spectra of CoNG have been
recorded as polycrystalline sample. EPR signal was not observed at
room temperature perhaps due to rapid spin lattice relaxation of Co
(II). Spin lattice relaxation of Co (II) broadens the lines at higher
temperature [39].
The room temperature magnetic moment of CoNG is found to be ueff =
3.48 BM. The value of ueff = 3.48 BM is a strong indication of the four
coordinated square planar geometry of the complex [40-41]. The high
magnetic moment value reveals that this complex is monomeric in
nature and there is no metal-metal interaction in the axial position
[39].
Mass spectral data is presented in Figure 1.
Example 3
Analytical method
Measurement of GSH
GSH was measured following the method of Sedlack and Lindsay [42].
Briefly, to 2xl05 cells in 0.2 ml PBS, 4.8 ml EDTA (0.2 M) was added and kept on ice bath for 10 minutes. Then 4 ml deionised water and 1ml of 5% trichloroacetic acid (TCA) were added. The mixture was again kept on ice for 10 tol5 minutes and then centrifuged at 3000 rpm for 15 minutes. 2 ml of supernatant was taken and 4 ml of 0.4 M tris buffer (pH 8.9) was added. O.lml of 5, 5'-dithio bis (2-nitrobenzoic acid) (DTNB) solution was added and vortexed thoroughly. Optical
density (O.D.) was read (within 2 to 3 minutes after addition of 0.1 ml 0.01M DTNB) at 412 nm against a reagent blank. Appropriate standards were taken and protein was measured according to Lowry [43]. The experiment was repeated for four times. Measurement of GPx activity
GPx activity was measured according to the reported methods [44, 45]. The homogenate was prepared with 0.15M KC1 and centrifuged at 10,000g for 20 min. supernatant was analyzed for GPx activity according to the method of Hofemann et. al. [32]. The enzyme assay tube containing GSH (0.2 mM), sodium azide (1 mM), sample, hydrogen peroxide (0.25 mM) were incubated at 37°C for 6 min. After addition of HPO3 ((1.67%) the mixture was centrifuged at 3000 rpm for 15 min. The supernatant was added to a mixture of Na2HPO4 (0.4 M) and 1 mM DTNB. After 10 min incubation at 37°C, the absorbance of the reaction mixture was measured at 412 nm. The unit of enzyme activity was measured by considering a decrease in the log [GSH] of 0.001 per minute after subtraction of the decrease in log [GSH] per min for the non enzymatic reaction [45]. Measurement of Catalase activity
Catalase was measured by the reported methods [44, 46]. In brief, tissue homogenate was prepared using 0.1 M phosphate buffer solution, pH 7 and centrifuged at 100,000 g for 1 h at 4°C. Tissue homogenate was transferred in 0.1 M phosphate buffer solution (PBS), pH 7 containing 0.45 M H2O2 Aliquots of the mixture (0.5 ml) were removed at 20 S intervals and added to 2.0 ml of a solutions containing 0.2 mg/ml O-dianisidine, 0.015 mg/ml peroxidase and 0.81mg/ml sodium azide. After alO-mins incubation at room temperature, 50% H2SO4 solution was added to stop the reaction. The absorbance of the reaction mixture was measured at 530 nm. One unit of enzyme activity (k1) was calculated as follows:
k' = 2.303/t log a/(a-x) [a, starting cone, of H2O2; a-x, H2O2Conc. after t time]. Measurement of SOD
SOD was measured by the reported method [45, 47]. In brief, tissue homogenate was prepared using 0.1 M PBS, pH 7 arid centrifuged at 100,000 g for 1 h. at 4°C. The
supernatant was dialyzed overnight against 0.1 M PBS, pH 7 and transferred to a reaction mixture containing 0.043 M NaaCO3 buffer (pH 10.2), O.lmM xanthine, O.lmM EDTA, 0.05 mg/ml bovine serum albumin, 0.025 mM nitro blue tetrazolium (NET) and the sample. After 10 min preincubation at 25°C, the reaction was started with 0.1 ml xanthine oxidase and incubation was performed for 20 min at 25°C. After addition of 0.2 mM CuCl2, the absorbance of the solution at 560 nm was measured. The activity of SOD required to inhibit the ratio of NBT reduction by 50% was defined as 1 unit of activity.
Measurement of glutathione S-transferase (GST) activity Glutathione S-transferase enzyme activity was assayed according to the method of Habig et al [48] with the use of l-chloro-2, 4-dinitro benzoic acid (CDNB) as substrate.
EAC/S and EAC/Dox cells were collected from mice after cervical dislocation and washed twice in PBS. To O.lml cell suspension (IxlO5 cells) in PBS, 500 µl sodium phosphate buffer (0.2M, PH 6.5) and GSH (20mM) were added. The final volume was made up to 1 ml. The reaction was monitored spectrophotometrically at 340 nm by increase in absorbance. A correction was made by measuring and subtracting the rate in the absence of enzyme. Example 4
Study of in vivo toxicity:
30 male Swiss albino mice of 6 weeks age were divided into six groups; different doses of CoNG viz., 15 mg/kg, 20 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg were injected IP to 5 group of male Swiss mice. Survivability of the animals was studied. The results shows the nontoxic nature of CoNG is indicated as all animals survived for 96 h.
Example 5
Effect of CoNG on blood:
10 mg CoNG was dissolved in 1 ml double distilled water. O.lml solution was
injected to male Swiss albino mice (i.e., 50 mg/kg).
Blood was obtained via closed cardiac puncture by means of a 22-guage hypodermic
needle and with a subxiphoid approach [49]. Blood from each group (CoNG treated
and untreated) was pooled into separate glass tubes and treated with anticoagulant
(heparin). Normal and differential blood count (WBC) was done for treated and
control mice. The experiment was repeated four times. Long term (72h) and short
term (2h) effect of various doses CoNG on blood, spleen and bone marrow in male
Swiss mice is presented in Table 1. Haemoglobin, RBC, WBC, bone marrow cells
remained unchanged after CoNG injection. Data indicate that the complex has no
long term or short term toxic effect in vivo.
Example 6
Effect of CoNG on spleen:
Aqueous solution of CoNG was injected (50 mg/kg) into male Swiss mice.
Preparation of spleen cell suspension: Normal and CoNG treated male Swiss mice
were anaesthetized and 70% alcohol was sprayed on abdominal region. Spleen was
removed aseptically and small amount of PBS was injected to it; spleen was rubbed
against the fine wire mesh of the tissue grinder. The cell suspension formed is
spinned at 1000-1500 rpm for 5-10 minutes. The supernatant was discarded and the
cells were washed by spinning in PBS twice at room temperature. Cell viability was
tested by trypan blue and cells were counted in a phase contrast microscope. The
experiment was repeated for four times.
The study of the effect of CoNG at varying doses and at different time intervals
indicated that CoNG had no splenic toxicity as no significant decrease in treated
spleen cells observed compared to untreated control (Table 1)
Example 7
Effect of CoNG on bone marrow:
50 mg/kg CoNG in ddw was injected i.p. to male Swiss mice.
Separation of bone marrow cells: Normal and CoNG treated mice were anaesthetized
and the femur bone was cut with the help of a vertebrate scissor. Bone marrow was
flushed with 0.56% KC1 solution and centrifuged at 3000 rpm for 15 mins at 37°C.
Cells were counted under microscope for treated and untreated animals. The
experiment was repeated for four times.
The study of the effect of CoNG at varying doses and at different time intervals
indicated that CoNG had no bone marrow toxicity as no significant decrease in
treated bone marrow cells observed compared to untreated control (Table 1)
Example 8
Study of anti-tumor property of CoNG in vivo:
55 male Swiss albino mice of 6 weeks age were divided into six groups; one control group with 5 mice and 5 drug treated groups with
10 mice in each group. IxlO6 EAC cells were injected IP to all mice on day one. On day two, various doses of CoNG dissolved in autoclaved doubled distilled water (ddw) were injected IP to mice of various groups. No drug was administered to the control group. Life monitoring was restricted to daily body weight measurement, recording the time of death. Animals were observed for a period of 60 days. Cell yield, ascites volume, packed cell volume, mean survival time (MST) and change in life span of the treated mice in comparison to control (T/C value) were recorded. The experiment was repeated for four times.
Effect of various doses of CoNG on male Swiss albino mice bearing Ehrlich ascites carcinoma (EAC) cells is presented in Table 2. The complex does not show any anti tumor effect. CoNG causes no change in cell count or packed cell volume; CoNG does not increase the life span (T/C %) ot the EAC bearing male Swiss mice (Table 2). Example 9
Effect of CoNG on survival of animals: Study of in vivo toxicity:
Various doses of CoNG dissolved in double distilled water (ddw) were injected i.p. to male Swiss mice. Animals were observed for a period of 72h. The average value of the animals living (percentage) of three independent experiments with respect to the doses of CoNG was studied.
CoNG at wide dose range (15-100 mg/kg) did not show antitumor property as life span of the CoNG treated cancerous mice was not increased as compared to untreated control (Table 2). Example 10
Effect of CoNG on GSH, GST, GPx in vivo:
Effect of various doses of CoNG on GSH, GPx, and GST in vivo is presented in Table 3.
CoNG at a dose of 15, 20, 50 mg/kg depletes GSH markedly in all the organs like heart, kidney, liver and lung in normal Swiss mice at 2h compared to untreated control. The level of GSH slightly restored at 4 hours and reached almost to normal level at 24h of CoNG injection. Various doses of CoNG were injected i.p. male Swiss albino mice. At different time intervals, the animals were killed by overdose of
anesthesia. Different organs were collected and GSH, GST, GPx were measured.
In normal mice, CoNG does not deplete GST activity significantly in 2h, 4h and 24h. The level of GST activity remains almost unchanged with various doses of CoNG (15, 20, 50 mg/kg) when compared with untreated control.
GPx increased at 2h with various doses of CoNG (15, 20, 50 mg/kg) significantly in all the organs [Table 3] compared to control (untreated) mice.
Example 11
Effect of CoNG on human serum albumin (HSA):
Aqueous solutions of HSA (10 5 M and 10-3 M) and CoNG (10-5 M and ID-3 M) were mixed in 1:1 molar ratio. The mixture was vortexed for 5 minutes and kept in 37°C incubator for 2 hours followed by UV-Vis spectral measurements. The effect of CoNG on HSA is presented in Fig.2.
In the UV-VIS spectra of CoNG + HSA the peak at 280 nm (protein peak of HSA) completely disappears and the sharp, high intensity peak for CoNG at 213 nm changes through intensity. A new peak at 450 nm (weak peak) appears in the spectrum of CoNG + HSA. The disappearance of protein peak at 280 nm, hypochromic shift of the peak at 213 nm and the formation of a new peak at 450 nm may be an indication of complexation between HSA and CoNG. Such complexation may help understanding the interaction of biologically relevant ligands (e.g., drugs) and proteins [50].
Example 12
Effect of CoNG on SOD and catalase activity in vivo:
Various doses of CoNG were injected i.p. to male Swiss mice. At different time intervals, the animals were killed by overdose of anesthesia. Different organs were collected for SOD and catalase measurement. The effect of CoNG on SOD and catalase is presented in Table 4
CoNG increases SOD and catalase activity in all the organs like spleen, heart, kidney, liver and lung. Enzymes involved in the metabolism of drugs and xenobiotics are subdivided into two major categories viz., Phase I and Phase II. Other than the antioxidant GSH, Phase II detoxification system utilizes a number of antioxidant enzymes like GPx, catalase and SOD and protect the system from antioxidant damage [51].
Catalase and GPx seek out hydrogen peroxide and convert it to water and diatomic oxygen. An increase in the production of SOD with concomitant increase of catalase and GPX has protective effect on the living system [52, 53]. Considering the structure and composition of CoNG and the elevation of SOD in all the organs by CoNG it seems that the present metal complex (CoNG) may have SOD mimetic property [54].
Example 13
Cell Line: EAC was maintained as an ascitic tumor in male Swiss albino mice weighing 18-20 g (6-8 weeks old), obtained from our own animal colony. A Dox resistant subline was developed following the reported methods [55, 56, 57] by sequential transfer of EAC cells to subsequent generation of host mice with continuous Dox treatment. The treatment regime consisted of 2.0 mg/kg /week Dox intraperitonaelly (i. p.). The daily treatment dose was 0.4 mg/kg for
five days. The drug was started 24 hours after inoculation of 1x10" ascites tumor cells i.p. to mice. The mean survival time ± standard error of the mean (MST + SEM) of untreated male Swiss mice bearing EAC cells was 22.4 ± 1.5 days (n=20). The MST ± SEM of the host mice bearing this tumor after 4 months treatment with Dox was 35.4 ± 3.1 days (n=20) whereas, MST of the 12th transfer generation of host mice was 19.8 ± 3.7 days (n=20). After this degree of resistance had been developed, the dose of Dox was increased to 4 mg /kg/week (daily treatment dose was 0.8 mg/kg for five days), which resulted in 25.5 ± 1.7 days MST (n=20). When this tumor subline was retreated with Dox after 17th transfer, MST was 21.1 ±1.4 days (n = 20). After 20th transfer, MST was noted to be 19.2 ± 2.9 days (n=20). The increased survival
(35.4 ± 1.5 days) of Dox treated (2 mg/kg/week for four months) mice was statistically significant (p Ascites fluid was withdrawn from the mice after 12 to 15 days of inoculation of tumor cells. No treatment was given during the last passage before an in vitro experiment. Example 14
Concentration and dose of the drug: Stock solution of CoNG (100 mg/ml), Dox (5 mg/ml), verapamil (10 mg/ml), EA (25 mg/ml) and BSO (25 mg/ml) were prepared in deionised water. When Dox was injected with CoNG (Dox + CoNG treated group) in drug resistant (EAC/Dox) cells, the concentration of Dox increased considerably. It is evident from Fig. 3 the concentration of Dox increased in CoNG treated cells by 276%, EA treated cells by 176% with respect to untreated drug resistant cells (control) that was considered to be 100%.
Example 15
Evaluation of chemotherapy assays: The MSTs were recorded following various drug treatment protocols [58]. In some cases, particularly when 1 or 2 mice in a group showed an abnormally short or long survival in comparison with the rest of the group and other comparable groups in independent experiments with the same protocol, median survival time were used to compare the groups. The statistical significance of the survival data of drug treated groups versus untreated groups or combined versus single therapy was evaluated by P values (Students' T test). Mouse survival times in the different groups were also compared as T/C ratios (percent) i.e., the ratio of the survival time (in days) for treated mice to untreated control mice. As in standard National Cancer Institute protocols for screening new anticancer drugs, it was considered that the increase in survival corresponding to T/C ratios around 120% to be "marginal", T/C ratios between 120% and 150% to be "clear" and T/C ratios equal or superior to 150% to be "marked".
For evaluation of chemotherapeutic assay, Standard International Protocol was followed (Table 7).
Example 16
Survival of animals in in vivo studies with tumor bearing mice
Drug resistant Ehrlich ascites carcinoma cells (EAC/Dox) and drug sensitive Ehrlich ascites carcinoma cells (EAC/S) were inoculated to
male Swiss mice at 1x106 cells/mouse. Twenty four hours later CoNG (15-100 mg/kg) was injected. Dox was injected 2 hours after CoNG injection. The control group received no drug but normal saline. Life monitoring was restricted to daily body weight measurement, examination of ascites development and recording of time of death. Animals were observed for a maximum period of 60 days and the mean survival time (MST) was calculated (Table 7). Example 17
Assays of combined therapy
The protocols of combined therapy were similar to those of showing the potentiating effects of verapamil and AHC-52 for vinca alkaloid-mediated increase of survival of mice [59]. The 6-8 week old male Swiss albino mice were pre inoculated i.p. at
day 0 with 1x10^ EAC/Dox cells and treated by combined therapy protocols or their single or placebo therapy control. The effect of CoNG, BSO, EA used in identical conditions were compared. Administration of Dox with resistance modifying agent (RMA): Although Dox alone at a dose of 1500 µg/kg could prolong the survival of mice bearing the drug sensitive tumor cells (EAC/S) from 22.4 days to 32 days, but could not prolong the survival of 6-8 week old Swiss albino male mice bearing drug resistant tumor cells (EAC/Dox). To study the effect of RMAs (Table 7), the mice were treated with the indicated amount of CoNG and Dox (i.p.). Dox was injected 2 hours after the treatment of CoNG. Dox alone could not prolong the survival of the EAC/Dox bearing mice, thus confirming in vivo the high degree of resistance found in vitro, reported earlier [17] (Table 2). The single dose of 3000 µg/kg Dox was so toxic that it killed the mice earlier than the usual time.
The CoNG at a dose of 70 mg/kg (double dose of 35mg/kg) with Dox (1500 µg/kg) increased the life span of EAC/Dox bearing mice
significantly (T/C=321 days) but with further increase of dose of
CoNG, the survival of animals decreased (Table 7).
EA at a dose of 25 mg/kg along with Dox (1.5mg/kg), i.e., (Dox + EA)
could prolong the survival of mice bearing drug resistant tumor cells
(EAC/Dox) markedly (T/C=147) but this value is much less in
comparison to the effect of CoNG, where T/C value is 321 days. BSO
at a dose of 25 mg/kg has toxic effect (T/C= 117) on the survival of
EAC/Dox bearing animals (Table 7),
Example 18
Measurement of cellular doxorubicin concentration
Drug resistant and drug sensitive EAC cells were inoculated to male
Swiss mice at 1x106 cells/mouse, as described above. Twenty four hours later, CoNG (15-100 mg/kg) was injected (Single dose). Dox was injected as a single dose (2-4 mg/kg). In combination therapy (CoNG+Dox), Dox was injected after 2 hours of CoNG injection. Tumor samples were obtained at fixed times, counted and homogenised. The extraction of Dox was performed [60] and the concentration of Dox was measured spectrofluorometrically with excitation and emission wave length at 470 and 585 nm respectively.
The CoNG increased Dox concentration inside resistant cells to 276% whereas ethacrynic acid (EA), a clinically used RMA increased Dox concentration inside the cells to 179%. CoNG has potential to be used as RMA (Fig.3).
Example 19
Glutathione inhibition study by ethacrynic acid, BSO, CoNG: EA
(1.5mg/kg), BSO (1.5mg/kg) and CoNG (15-100mg/kg) were injected in animals having EAC/S and EAC/Dox cell line. Ten days after injection of the chemicals, 103 cells were taken from the animals and GSH was measured according to the method described in detail [42]. The level of GSH had been reported to be elevated in a number of drug resistant cells [62] and hence by depleting GSH many drug resistant cells could be sensitized to anticancer drugs (ACD). CoNG depleted GSH better than clinically used RMAs like EA.
Example 20
Study of chemical reaction of GSH with RMAs (CoNG, BSO, EA)
2ml. aqueous solution of GSH (ImM) was mixed with 2ml aqueous solution of CoNG (ImM) and stirred for Ihour in magnetic stirrer at 37°C. The solution was kept in an incubator at the same temperature for 2hours. The mixture was taken out and stirred for one hour in magnetic stirrer at 37°C. The mixture was filtered under gravity in Whatman 40 filter paper. 100µl solution was taken and GSH was measured by the reported method [61]. The procedure was repeated thrice with GSH as control. Exactly the same procedure was followed for studying the reaction of GSH with BSO and EA. Chemical reaction of CoNG was studied with GSH. Inhibition of GSH was highest with BSO. The compound CoNG inhibits GSH much more than EA. Inhibition of GSH by various chemical compounds is in the following order: BSO> CoNG>EA. Example 21
Study of conjugate formation
The formation of conjugate between GSH and CoNG was studied by high performance liquid chromatography (HPLC) analysis in Novapack-C18 and a photodiode array detector (Waters model 996). The equimolecular amount (1:1) of GSH and CoNG was stirred for 1 hour in magnetic stirrer at 37 °C and kept in incubator at 37 °C for 2 hours. The mixture was taken out and stirred for one hour again at the same temperature. The retention time of the individual components i.e., GSH and CoNG were measured in analytical reverse phase column (150mm x 3.9 mm) eluting with methanol-water (50:50) at the flow rate of 1ml per minute and run time 8 minutes. To determine if any conjugate has been formed, the mixture of CoNG and GSH (1:1 mole) was injected in the column and eluted with the same solvents at the same flow rate and run time. In HPLC study no new compound or conjugate was detected after 4 hours of addition of CoNG and GSH. Retention time for GSH (0.95 mins.) and CoNG (2.303 mins.) remained unchanged in the mixture of the two compounds where two peaks
appeared at 0.938 minutes and 2.688 minutes were identified to be the peaks for GSH and CoNG respectively.
At room temperature CoNG did not form conjugate with GSH non enzymatically.
Example 22
Assays of combined therapy
Administration of Dox with resistance modifying agent (RMA) Although Dox alone at a dose of 1500 (j.g/kg could prolong the survival of mice bearing the drug sensitive tumor cells (EAC/S) from 22.4 days to 32 days, but could not prolong the survival of 6-8 week old Swiss albino male mice bearing drug resistant tumor cells (EAC/Dox). To study the effect of RMAs (Table 7), the mice were treated with the indicated amount of CoNG and Dox (i.p.). Dox was injected 2 hours after the treatment of CoNG. Dox alone could not prolong the survival of the EAC/Dox bearing mice, thus confirming in vivo the high degree of resistance found in vitro, reported earlier [62] (Table 6). The single dose of 3000 µg/kg Dox was so toxic that it killed the mice earlier than the usual time.
The CoNG at a dose of 70 mg/kg (double dose of 35mg/kg) with Dox (1500 µg/kg) increased the life span of EAC/Dox bearing mice significantly (T/C=321 days) but with further increase of dose of CoNG, the survival of animals decreased (Table 7).
EA at a dose of 25 mg/kg along with Dox (1.5mg/kg), i.e., (Dox + EA) could prolong the survival of mice bearing drug resistant tumor cells (EAC/Dox) markedly (T/C = 147) but this value is much less in comparison to the effect of CoNG, where T/C value is 321 days. BSO at a dose of 25 mg/kg has toxic effect (T/C= 117) on the survival of EAC/Dox bearing animals (Table 7).
Example 23
In vivo effects of CoNG on reversing drug resistance:
Search for an effective and maximum tolerated dose (MTT) of Dox:
Dox alone at incremental doses of 30 to 1500 µg/kg could prolong the survival of
mice bearing the drug sensitive (EAC/S) cells (T/C value increased from 100 to 142)
but could not prolong the survival of drug resistant (EAC/Dox) cells. At the doses of 30, 50, 100, 200 µg/kg in drug resistant cells, no effective increase in survival was noticed rather survival remains the same (T/C% value 100 to 110). Table 6 shows the effects of various doses of Dox on survival of animals that had been inoculated on day 0 with 1x106 cells. For drug sensitive cells (EAC/S) the maximum increase in survival is observed at a dose of 1500µg/kg. For drug resistant (EAC/Dox) cells no increase in life span (T/C=94) is observed at a dose of 1500µg/kg of Dox. Survival of mice with drug resistant and also drug sensitive cells gradually decreased when the dose of Dox was increased from 1500µg/kg to 10,000 µg/kg (Table 6).
Advantage of the present invention Glutathione depleting agent, which is non-toxic. The above said depleting agent is water soluble. For the first time any metal complex having the properties of RMA has been disclosed.
Table 1

(Table Removed)
N= neutrophil, L=lymphocyte.
The data are mean 1 SD of four independent experiments. CoNG has no short term
or long term effect on blood, bone marrow or spleen.
Table 2.

(Table Removed)
Survivability signifies life span after EAC injection.
The data are mean + SD of four independent experiments.
The animals were sacrificed after 12 days of cell injection. The cell count and cell
yield of the treated cases are not significant when compared to control (> 0.05).
Table 3.
Glutathione (GSH) jig/mg of protein

(Table Removed)
Glutathione peroxidase (GPx) uni0ng of protein/min

(Table Removed)
Glutathione S-transferase (GST) unit/ing of protein/min

(Table Removed)
Data are mean ±SD of four independent experiments.
CoNG (20 mg/kg) at 2h significantly:
i. Lowers GSH in all the organs (p ii. Enhances GPx in all the organs (p Table 4
Catalase K/mg of protein

(Table Removed)
Super oxide dismutase (SOD) U/mg of protein

(Table Removed)
Data are mean ±SD of four independent experiments.
The values of catalase and superoxide dismutase (SOD) are significantly higher in
CoNG treated
animals compared to untreated control (p Table 5.

(Table Removed)
Dox = doxorubicin, SHAG = Sodium (N-2- hydroxy acetophenone) glycinate.
The data are means ± SD of the four independent
experiments.
Depletion of GSH and GST activity by Dox and Dox + SHAG
are significant (p Table 6.
(Table Removed)
Table 7.

(Table Removed)
The data are mean + SD of four independent experiments.
n = number of animals; T/C % = life span of the treated animals compared to untreated control in percent; EA = ethacrynic acid; BSO = L-buthionine-S, R- sulphoxamine.
Combined with Dox, CoNG at double dose of 35 mg/kg (i.e., CoNG + Dox treated cases, applied on day 1 and day 8) shows highest increase of life span (T/C % = 321) in male Swiss mice bearing drug resistant (EAC/Dox) cells when compared with Dox + EA treated cases (T/C % = 168) and only-Dox treated cases (T/C % = 118). The effect of CoNG as RMA is significant (p References
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We Claim:
1. A cobalt N-(-2-hydroxyacetophenone) aminoacetate useful for reversal of
drug resistance, said cobalt complex having general formula 1:
(Formula Removed)
Formula 1
wherein "Ar" is phenyl group; "R" is selected from methyl or ethyl group and the value of "n" is in the range of 1 to 3.
2. A cobalt complex as claimed in claim 1, wherein, the cobalt complex is Cobalt (II) N-(-2-hydroxyacetophenone) glycinate [CoNG].
3. A process for the preparation of cobalt complex of claim 1, having structural formula 1.
(Formula Removed)
Formula 1
wherein " Ar" is selected from unsubstituted phenyl group; "R" is selected from methyl or ethyl group and the value of "n" is in the range of 1 to 3.
said process comprising the steps:
a) dissolving sodium or potassium or ammonium salts of N-(-2-hydroxy substituted aryl ketone) amino acetate (NHAG) and cobalt salt
separately in a suitable solvent to obtain a first solution containing NHAG and a second solution containing the cobalt salt,
b) cooling the first and second solutions,
c) drop wise adding the first solution containing NHAG to the second solution containing cobalt salt solution at a temperature in the range of 4 to 8°C and at a pH in the range of 7.5 to 8,
d) stirring the mixture of step (c) to obtain crude cobalt complex in the
form of deep brown precipitate, and
e) separating the precipitate and recrystallizing the same in water alcohol
mixture to obtain pure cobalt complex.
4. A process as claimed in claim 3, wherein in step (a) the cobalt salt is
selected from a group comprising cobalt (II) acetate, cobalt (II) nitrate and
cobalt (II) chloride.
5. A cobalt N-(-2-hydroxyacetophenone) aminoacetate and preparation
thereof substantially such as described herein with foregoing description,
tables and figures.

Documents:

1210-DEL-2004-Abstract (22-01-2010).pdf

1210-del-2004-abstract.pdf

1210-DEL-2004-Claims (22-01-2010).pdf

1210-del-2004-claims.pdf

1210-del-2004-Correspondence Others-(11-04-2012).pdf

1210-DEL-2004-Correspondence Others-(12-03-2012).pdf

1210-DEL-2004-Correspondence-Others (22-01-2010).pdf

1210-del-2004-correspondence-others.pdf

1210-DEL-2004-Description (Complete) (22-01-2010).pdf

1210-del-2004-description (complete).pdf

1210-del-2004-drawings.pdf

1210-DEL-2004-Form-1 (22-01-2010).pdf

1210-del-2004-form-1.pdf

1210-del-2004-form-18.pdf

1210-del-2004-form-2.pdf

1210-DEL-2004-Form-26 (22-01-2010).pdf

1210-del-2004-form-26.pdf

1210-del-2004-form-3.pdf

1210-del-2004-form-5.pdf

1210-del-2004-form-9.pdf

1210-del-2004-GPA-(11-04-2012).pdf

1210-DEL-2004-Petition-137 (22-01-2010).pdf

abstract.jpg


Patent Number 241119
Indian Patent Application Number 1210/DEL/2004
PG Journal Number 26/2010
Publication Date 25-Jun-2010
Grant Date 18-Jun-2010
Date of Filing 30-Jun-2004
Name of Patentee INDIAN COUNCIL OF MEDICAL RESEARCH
Applicant Address V. RAMALINGASWAMI BHAWAN, ANSARI NAGAR, POST BOX 4911, NEW DELHI, 110029,INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 SOUMITRA KUMAR CHOUDHURI DEPARTMENT OF ENVIRONMENTAL CARCINOGENESIS AND TOXICOLOGY, CHITTARANJAN NATIONAL CANCER RESEARCH INSTITUTE, 37, S.P. MUKHERJEE ROAD, CALCUTTA-700026, WEST BENGAL,INDIA
2 PRANABANANDA DUTTA DEPARTMENT OF ENVIRONMENTAL CARCINOGENESIS AND TOXICOLOGY, CHITTARANJAN NATIONAL CANCER RESEARCH INSTITUTE, 37, S.P. MUKHERJEE ROAD, CALCUTTA-700026, WEST BENGAL,INDIA
3 GOURISHANKAR PANDA DEPARTMENT OF ENVIRONMENTAL CARCINOGENESIS AND TOXICOLOGY, CHITTARANJAN NATIONAL CANCER RESEARCH INSTITUTE, 37, S.P. MUKHERJEE ROAD, CALCUTTA-700026, WEST BENGAL,INDIA
4 SURAJIT MAJUMDER DEPARTMENT OF ENVIRONMENTAL CARCINOGENESIS AND TOXICOLOGY, CHITTARANJAN NATIONAL CANCER RESEARCH INSTITUTE, 37, S.P. MUKHERJEE ROAD, CALCUTTA-700026, WEST BENGAL,INDIA
PCT International Classification Number A61K 31/28
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA