A SYNERGISTIC ANTICANCER ACTIVITY EXHIBITING FORMULATION

Abstract

TITLE: A SYNERGISTIC ANTICANCER ACTIVITY EXHIBITING FORMULATION APPLICANT: SRI RAMACHANDRA INSTITUTE OF HIGHER EDUCATION AND RESEARCH ABSTRACT The present invention discloses a synergistic anticancer activity exhibiting formulation. The synergistic anticancer activity exhibiting formulation of the present invention comprises of characterized combination of Abiraterone Acetate and CDRI 08.

Specification

Description:Form 2


THE PATENT ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)




"A SYNERGISTIC ANTICANCER ACTIVITY EXHIBITING FORMULATION"





in the name of SRI RAMACHANDRA INSTITUTE OF HIGHER EDUCATION AND RESEARCH an Indian National having address at, SRI RAMACHANDRA INSTITUTE OF HIGHER EDUCATION AND RESEARCH (DEEMED TO BE UNIVERSITY), NO.1, RAMACHANDRA NAGAR, PORUR, CHENNAI, CHENNAI-600116, TAMIL NADU, INDIA.




The following specification particularly describes the invention and the manner in which it is to be performed.

SOURCE AND GEOGRAPHICAL ORIGIN OF THE BIOLOGICAL MATERIAL:

SL.
NO COMMON NAME SCIENTIFIC NAME PART OF BIOLOGICAL SOURCES SOURCE OF ACCESS DETAILS OF GEOGRAPHICAL LOCATION
1. Waterhyssop Bacopamonnieri Leaf Trader (i) Name of the trader:
Lumen Marketing Company.

(ii) Contact details:
Second Floor
New No.22 / Old No.2 First Corss Street Second Avenue Ashok Nagar, Brindavan Extension, Chennai-600083, Tamil Nadu,
India.

FIELD OF THE INVENTION:

The present invention generally relates to the field of medicine. Specifically, the present invention relates to an anticancer formulation. More particularly, the present invention relates to a synergistic anticancer activity exhibiting formulation.


BACKGROUND OF THE INVENTION:

Prostate cancer is one of the malignancies that affects men and significantly contributes to increased mortality rates in men globally. Prostate cancer affects middle-aged men between the ages of 45 and 60. Patients affected with prostate cancer present with either a localized or advanced disease. The risk factors related to prostate cancer include family risk, ethnicity, age, obesity, and other environmental factors.

Prostate cancer diagnoses include a digital rectal examination, prostate-specific antigen analysis, and prostate biopsies. Mutations in certain genes are linked to the onset, progression, and metastasis of the cancer. Treatment for localized prostate cancer encompasses active surveillance, ablative radiotherapy, radical prostatectomy and androgen deprivation therapy (ADT). Men who relapse or present metastatic prostate cancer receive androgen deprivation therapy (ADT), salvage radiotherapy, and chemotherapy.

There are reports available in the state of art relating to treatment of prostate cancer.

US10449191B2 discloses methods for treating prostate cancer, including advanced prostate cancer, in a subject in need thereof, include administering once-daily to the subject, at least 80 mg of N-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxy-3-pyridazinyl)-2,4-dioxo-1,2,3,4- tetrahydrothieno[2,3-d]pyrimidin-6-yl)phenyl)-N′-methoxyurea, or a corresponding amount of a pharmaceutically acceptable salt thereof. Another method includes: administering once-daily to the subject in need thereof, an oral load dose formulation having from 240 mg to 480 mg of N-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxy-3-pyridazinyl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl)phenyl)-N′-methoxyurea, or a corresponding amount of a pharmaceutically acceptable salt thereof; and thereafter administering once-daily to the subject, an oral maintenance dose formulation having 80 mg to 160 mg of N-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxy-3-pyridazinyl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl)phenyl)-N′-methoxyurea, or a corresponding amount of a pharmaceutically acceptable salt thereof.

EA043716B1 discloses application of K-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxy-3-pyridazinyl)2,4-dioxo-1,2,3 ,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl)phenyl)-N'-methoxyurea or a pharmaceutically acceptable salt thereof for the treatment of prostate cancer in a subject in need thereof, wherein a loading dose of an oral formulation containing 360 mg K-(4-(1-(2,6-difluorobenzyl)-5((dimethylamino)methyl)-3-(6-methoxy-3-pyridazinyl)-2,4-dioxo-1,2,3,4 α-tetrahydrothieno[2,3-d]pyrimidin6-yl)phenyl)-N'-methoxyurea, or an appropriate amount of a pharmaceutically acceptable salt thereof, is administered to the subject once on the first day of the treatment period; and a maintenance dose of an oral formulation containing 120 mg K-(4-(1-(2,6-difluorobenzyl)5-((dimetulamino)methyl)-3-(6-methoxy-3-pyridazinyl)-2,4- dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin6-yl)phenyl)-N'-methoxyurea, or an appropriate amount of a pharmaceutically acceptable salt thereof, is administered to the subject once daily beginning on the second day of the treatment period.

US10398791B2 discloses field of radiopharmaceuticals and their use in nuclear medicine as tracers, imaging agents and for the treatment of various disease states of prostate cancer. Thus, the present invention concerns compounds that are represented by the general Formulae (Ia) or (Ib).


Though, conventional treatment method exhibits advantages in the prior arts they do have disadvantages such as side effects, recurrence, drug resistance, poor patient compliance and expensive.

Thus there exists a need in the state of art for developing an alternative method for treatment of prostate cancer.

Hence an attempt has been made to develop a synergistic anticancer activity exhibiting formulationovercoming the above said drawbacks.

OBJECT OF THE INVENTION:

The main object of the present invention is to develop a synergistic anticancer activity exhibiting formulation.

Another object of the present invention is to prepare a synergistic anticancer activity exhibiting formulation employing Abiraterone Acetate and CDRI 08.

Yet another object of the present invention is to study the anticancer activity of the prepared synergistic anticancer activity exhibiting formulation.

Further object of the present invention is to utilize the prepared synergistic anticancer activity exhibiting formulation for treating prostate cancer.

BRIEF DESCRIPTION OF DRAWINGS:

Figure 1 depicts individual dose dependent cytotoxic effect of AA and CDRI 08 in PC3 cell line.
A. Cytotoxic effect of AA in PC3 cell line
B. Cytotoxic effect of CDRI 08 in PC3 cell line

Figure 2 depicts dose response matrix of combination effect of AA and CDRI 08 in PC3 cell line in the absence and presence of growth factors.
A. Combination effect of AA and CDRI 08 in PC3 cell line in the absence of growth factors
B. Combination effect of AA and CDRI 08 in PC3 cell linein the presence of growth factors
Drug A: Abiraterone acetate (AA), Drug B: CDRI-08

Figure 3 depicts apoptosis induced cell death in control, individual treated groups and combination treated groups with and without GF. (1) Control, (2) CDRI 08 IC50, (3) CDRI 08 IC25, (4) CDRI 08 IC10, (5) AA IC50, (6) AA IC25, (7) AA IC10. C1, C2, C4 & C5 represent the combination treated groups. C1- IC25A + IC25B, C2- IC25A + IC50B, C4- IC50A +IC25B, C5- IC10A + IC50B
A. Apoptosis induced cell deathwithout GF
B. Apoptosis induced cell deathwith GF, EGF & DHT

Figure 4 depicts changes in protein expression in control, individual and combination groups, in the presence of GF, EGF & DHT.

SUMMARY OF THE INVENTION:

The present invention disclosesa synergistic anticancer activity exhibiting formulation. The synergistic anticancer activity exhibiting formulation of the present invention comprises of characterized combination of Abiraterone Acetate and CDRI 08.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention disclosesa synergistic anticancer activity exhibiting formulation.
The synergistic anticancer activity exhibiting formulation of the present invention comprises of characterized combination of Abiraterone Acetate and CDRI 08.The synergistic anticancer activity exhibiting formulation in which the Abiraterone Acetate is present in an amount of 13.5 µM/ml and CDRI 08 is present in an amount of 17.0 µg/ml..

Table 1: Composition of synergistic anticancer activity exhibiting formulation of the present invention:

S. NO INGREDIENTS QUANTITY
1 Abiraterone Acetate 13.5 µM/ml
2 CDRI 08 17.0 µg/ml

The prepared synergistic anticancer activity exhibiting formulation of the present invention was then subjected to experimental studies to determine its anticancer activity.

Methodology
The androgen-independent human Prostate cancer cell line, PC3 cell lines were obtainedfrom National Centre for Cell Science (NCCS), Pune, India. The cells were cultured inDMEM media with 10% FBS, and 1X Antibiotic/Antimycotic.Stock solutions of AA, & CDRI 08 were prepared in DMSO and stored at -20°C. 10nM ofEGF and DHT were used in the study. Working concentrations of AA (10-30 µM) &CDRI 08 (50-250 µg/ml) were freshly prepared in DMEM medium with & without GF.Similarly, the different combination ratios of the AA and CDRI 08 were freshly prepared.

Data analysis & statistics:
All the experiments were performed in triplicates and as three individual experiments. The values were expressed as means ± standard deviation (SD). The Student's t-test and the one-way ANOVA test were used to compare differences in the mean values, and a p value < 0.05 was considered statistically significant.The data were processed in Graph Pad Prism 5 (Graph Pad Software), and Synergy finder tools.

In-vitro Cytotoxicity Studies
MTT Assay:The 70% confluent PC3 cells were seeded into a 96-well plate. After 24hrs,the cells were treated with different concentrations of AA & CDRI 08, individually and incombinations, in the presence and absence of the GF, EGF & DHT. After 48 h, the cellswere rinsed with PBS and incubated with 100 µL of MTT for 4 h. The MTT metaboliteswere dissolved in 100 µL DMSO and the absorbance was read at 540 nm.The dose-response effect of CDRI 08 & AA was studied and inhibitory concentrationwas evaluated.

Individual effect of AA & CDRI 08
Dose-dependent cytotoxic effect was observed in AA, standard drug, treated PC3 cell lines with a cell viability of 45% at 30 µM (p <0.05) and the IC50 value was found to be 27 µM as shown in Figure 1A.
The CDRI 08 has also shown dose dependent cytotoxic effect and the cell viability was reduced to 12% at 250 µg/ml (p <0.05) as shown in Figure 1B. The IC50 value was found to be 33.6 µg/ml and 34 µg/ml of CDRI 08 was used further in the combination study.

Table 1: Individual Inhibitory Effect of AA & CDRI 08 at Different Concentrations.

Compounds Inhibitory Concentrations % Inhibition
Without GF With GF
CDRI 08
(µg/ml) IC50 (34) 47.42 59.2
1C25 (17) 28.7 27.6
IC10 (7) No effect No effect
AA (µM) IC50 (27) 48.6 47.4
IC25 (13.5) 29.5 26.3
IC10 (6) No effect No effect

The results were represented as mean± standard deviation (SD) with statistical significance of p < 0.05

With the calculated IC50 values, the IC25 and IC10 values were also validated both in the presence and absence of GF, as shown in Table 1.
With the individual cytotoxic effect of AA and CDRI 08, the drug synergy was analysed with the Bliss Independent model using the software, Synergy Finder as shown in Figure 2A and 2B. Combination effect of AA and CDRI 08 at different ratios has shown a significant decrease in cell viability compared to the individual treated groups, indicating synergismin PC3 cells, even in the presence of GF. The effective synergism was found in C1 & C4 combination with & without GF. The cytotoxic effect of individual and combination treated groups is represented as dose response matrix.

Combination Index Evaluation
The synergism in the combination study was analysed with the combination index (CI), based on the Bliss Independence model. The assessment is based on the evaluation of the observed combination effect(EAB) and the expected combination effect by the formula for probabilisticindependence.
CI = EA + EB - EAEB
EAB
where EA & EB are the observed inhibition rates with drug A alone at dose a and drug Balone at dose b and EAB is observed combination effect(0 = EA/EB/EAB = 1).

Following this, the combination index (CI) was calculated as shown in Table 2, which was found to be less than 1, indicating synergism for all the combination groups, except C3. The CI forC3 alone was found to be more than 1, representing an antagonistic effect. The CI for C1 & C4 in the absence of GF was found to be the lowest, 0.61 & 0.72 (p <0.05) respectively,while the same in the presence of GF was found to be 0.78 & 0.81 (p <0.05). The othercombination treated groups have also shown an effective synergism with CI <1. The CIvalues confirm an enhanced synergism of AA in the presence of CDRI 08.
Table 2: Combination Index of AA & CDRI 08 in the presence & absence of GF.

S.No
Combinations WithoutGF WithGF
AA
(µM) CDRI 08
(µg/ml) CI CI
C1 IC25 IC25 0.619 0.78
C2 IC25 IC50 0.86 0.9
C3 IC50 IC10 0.93 1.77
C4 IC50 IC25 0.85 0.86
C5 IC10 IC50 0.72 0.81

The results were represented as mean± standard deviation (SD) with statistical significance of p < 0.05

2. Apoptosis Analysis
Apoptosis induced morphological changes was studied with Acridine Orange andEthidium Bromide (AO/EB) dual staining. Cells were cultured in 24 well platesandtreated with individual & combination groups. After 48 hrs, the cells were stained withAO/EB and apoptosis was characterized by chromatin condensation and yellow andorangefluorescence.


The apoptosis induced cell death in PC3 cell lines was studied in individual andcombination treated groups in the presence and absence of growth factors. C1, C2, C4 &C5 combination treated groups were studied for apoptosis induced cell death as synergismwas not seen in C3 treated group with GF.

Observation of AO/EB stained cells under Fluorescence microscope showed nuclearchromatin condensation, with greenish-yellow and orange-stained cells indicating earlyand late apoptosis respectively. While the live cells (control) showed green fluorescence as shown in Figure 3A and 3B.

The study has shown a concentration dependent apoptotic activity in individual treatedgroups. Effective apoptosis was found in the IC50 & IC25 treated group of AA and CDRI08. The IC10 treated cells were found to be live cells with green fluorescence.The apoptosis induced cell death was observed in all the combination treated groups. TheC1 and C2 treated groups, in the absence of GF as shown in Figure 3A, have shown a good apoptosisinduced cell death with orange fluorescence indicating late apoptosis. While, earlyapoptosis with yellow fluorescence was observed in C4 & C5 treated groups. In thepresence of GF, the C1 and C4 treated groups have shown orangefluorescence, while theC2 & C5 treated groups have shown yellowfluorescence as shown in Figure 3B.

3. Western blot analysis
PC3 cells were plated into 6 dishes and were treated with individual and combination for48 hours, in the presence of GF, EGF & DHT. Then, the cells were lysed on ice and the cell lysate obtained was centrifuged at10,000 rpm at 4°C for 15. The protein samples (30 µg) were separated using 12%SDS-PAGE gels and transferred to nitrocellulose membrane. The membrane wasincubated overnight with primary antibodies against GAPDH, ERK, p-ERK, Akt, p-Akt,Casp3 in 4°C. The membrane was then incubated with horseradish peroxidase-conjugatedsecondary anti-mouse IgG antibody for 1 h at room temperature and chemi-luminiscence was visualized.

The p-AKT and p-ERK and Casp3 and its active form Cleaved casp3 expression levelswere studied in the individual and combination treated groups with the western blot experiment as shown in Figure 4. The cleaved Casp3 expression level was increased in CDRI 08(IC50) compared to AA (IC50). C-Casp3 was found to be increased in all thecombination treated groups compared to the control. C-casp3 was found to be increasedin the C1 and C2 combination groups, compared to the CDRI-08 (IC50). Thus, the studyshows an increased apoptotic activity of CDRI 08, which was found to be wellmaintainedeven in the combination treated groups. The presence of CDRI 08 B increasedthe activity of AA and apoptosis induced cell death in combinations even in the presenceof GF.

The AKT and ERK proteins are shown to be highly involved in activation of AR in theCRPC cells, in the absence of androgen resulting in drug resistance. CDRI-08 (IC50) shown a decrease in p-AKT expression levels, indicating an inhibitory effect, which wasnot found in AA (IC50). A decrease in p-AKT expression levels was found to be higherin C1 & C2 groups compared to other combination groups. There are no changes in theexpression levels of p-ERK found in the individual as well as the combination treatedgroups. The inhibitory effect of CDRI 08 on p-AKT may be contributing to the increasedcytotoxicity observed in the combination groups at a low dose, in the presence of GF.


An example of invention use.

Example 1
Individual Cytotoxicity of Abiraterone acetate (AA) and CDRI 08
AA tablets (250 mg) were dissolved and diluted in DMSO and stock solution wasprepared for 30 mM. CDRI 08, in powder form, was dissolved in DMSO and stocksolution was prepared for 250 mg/ml.
Working concentrations of AA (10-30 µM) was prepared in DMEM media without FBS. Working concentrations of CDRI (25-250 µg/ml) was prepared in DMEM media withoutFBS. Working concentrations of AA and CDRI were also prepared in DMEM media with10 nM of EGF and DHT.
PC3 cells were plated in 96 well plates at a density of 5×10in 2 different plates. After 24,one plate was treated with AA, 10, 15, 20, 25 30 µM and other plate was treated withCDRI, 25, 50, 100, 200, 250 µg/ml.
After 48 hrs of treatment, MTT assay was performed and IC50 values were calculated.All the experiments were performed in triplicates for 3 times. The statistical analysis ofthe samples was performed.
With the calculated IC50 values, IC25 and IC10 were also calculated and validated withanother MTT assay. The same was repeated in the presence of EGF and DHT and theinhibitory effect was calculated.

Example 2
Combination effect of CDRI and AA
Based the calculated effect, different concentration rations of AA and CDRI 08 wereadded to DMEM media to prepare the combination groups as explained in Table 03. The combination groups were also prepared with EGF and DHT

Table 3: Combination groups of AA and CDRI 08

CombinationGroups Concentrations
AA
(µM) CDRI 08
(µg/ml)
C1 IC25 IC25
C2 IC25 IC50
C3 IC50 IC10
C4 IC50 IC25
C5 IC10 IC50

The cytotoxicity study was repeated with MTT assay. Now the PC3 cells plated in 96well plates were treated with (i) AA (IC50, IC25, IC10) and CDRI (IC50, IC25, IC10)and (ii) the combination groups of AA and CDRI 08. The same treatment of AA, CDRI08 and combination groups was repeated with the EGF and DHT.

The MTT assay was performed and cell viability was studied. The changes in cellviability in AA alone and CDRI 08 alone treatment was compared with the combinationgroups treatment.


Example 3

Calculation of Synergism - Bliss independent Model
The expected cytotoxicity of the combination treated groups was compared with theobserved cytotoxicity effect and Combination index was calculated. The CI value of lessthan1 indicated synergism.

Example 4
Apoptosis induced cell death and western blot analysis
The PC3 cells were seeded in 6 well plate and the cells were treated with AA alone, CDRI 08 alone and the combination groups. The cells were also treated with AA, CDRI08 and the combination groups in the presence of EGF and DHT.
After 48 hrs, apoptosis study was performed by staining the cells with Acridineorangeandethidium bromide stain.
Western blot analysis was performed by blotting with GAPDH, ERK-T, ERK-p, Akt-T,AKT-p and Casp3 primary antibodies.
Thus from the above it was found that the synergistic anticancer activity exhibiting formulation of the present invention be a suitable candidate for treating prostate cancer.

In one of the preferred embodiments, the present invention shall disclose asynergistic anticancer activity exhibiting formulation. The synergistic anticancer activity exhibiting formulation of the present invention comprises of characterized combination of Abiraterone Acetate and CDRI 08.

As per the invention, the synergistic anticancer activity exhibiting formulation in which the Abiraterone Acetate is present in an amount of 13.5 µM/ml

In accordance with the invention, the synergistic anticancer activity exhibiting formulation in which the CDRI 08is present in an amount of 17.0 µg/ml.

Working example:
A synergistic anticancer activity exhibiting formulation comprises of 13.5 µM/mlof Abiraterone Acetate and 17.0 µg/mlCDRI 08.

Although the invention has now been described in terms of certain preferred embodiments and exemplified with respect thereto, one skilled in art can readily appreciate that various modifications, changes, omissions and substitutions may be made without departing from the scope of the following claims.
, Claims:WE CLAIM:
1. A synergistic anticancer activity exhibiting formulation comprises of characterized combination of Abiraterone Acetate and CDRI 08.

2. The synergistic anticancer activity exhibiting formulation as claimed in claim 1, wherein the said Abiraterone Acetate is present in an amount of 13.5 µM/ml

3. The synergistic anticancer activity exhibiting formulation as claimed in claim 1, wherein the said CDRI 08is present in an amount of 17.0 µg/ml


Dated this 25th day of OCT 2024



For SRI RAMACHANDRA INSTITUTE OF
HIGHER EDUCATION AND RESEARCH
By its Patent Agent

Dr.B.Deepa
IN/PA 1477

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