Description:FORM 2

THE PATENTS ACT 1970
(SECTION 39 OF 1970)
&
THE PATENT RULES, 2003

COMPLETE SPECIFICATION
(Section 10 and Rule 13)

LIPOSOMAL COMPOSITION OF DOXORUBICIN AND IRINOTECAN AND PROCESS FOR PREPARATION THEREOF

We, JODAS EXPOIM PVT. LTD.,
a company incorporated under the companies act, 1956 having address at 3rd Floor, NSL Centrum, Plot No S-1, Sy No: 1043 & 1048, KPHB Phase- III, Kukatpally, Hyderabad – 500072, Telangana, India.

The following specification particularly describes the invention and the manner in which it is to be performed.
FIELD OF INVENTION
The present invention relates to liposomal composition of Doxorubicin and Irinotecan.

The present invention also relates to liposomal composition of Doxorubicin or its pharmaceutically acceptable salt and Irinotecan or its pharmaceutically acceptable salt comprising one or more excipients, wherein the first drug Doxorubicin to second drug Irinotecan molar drug ratio is 1:1.

The present invention also relates to a process for the preparation of liposomal composition of Doxorubicin or its pharmaceutically acceptable salt and Irinotecan or its pharmaceutically acceptable salt.

The present invention also relates to liposomal composition of Doxorubicin or its pharmaceutically acceptable salt and Irinotecan or its pharmaceutically acceptable salt for cancer therapy.

BACKGROUND OF INVENTION
For overcoming life-threatening diseases such as cancer, AIDS and cardiovascular disorders, treatment with single drug agent may not be effective.

Camptothecin or topotecans compounds such as Irinotecan can prevent DNA replication by blocking the topoisomerase I (topo-I) by forming a topo-I-drug complex which leads to apoptosis. Irinotecan can be used to treat colorectal, lung, ovarian, breast, pancreatic and gynaecological malignancies/cancers.

Doxorubicin (DOX) is an anthracycline anticancer drug that sets inserted between adjacent base pairs of the double helical DNA strand and breaks them, they also further hinder nucleic acid synthesis .It also inhibit topoisomerase-II which induces apoptosis and interferes with DNA replication. DOX can be used to treat breast, ovarian, lung, bone marrow, brain and blood cancers.

Doxorubicin hydrochloride, (8S,10S)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl) oxy]-8-glycolyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacenedione hydrochloride. The molecular formula is C27H29NO11•HCl and the molecular weight is 579.99. The structural formula is:

Irinotecan hydrochloride, (S)-4, l l-diethyl-3,4, 12,14-tetrahydro-4-hydroxy-3,14-dioxo- lH-pyrano[3 4 6 7]-indolizino[l,2-b]quinolin-9yl[l,4'-bipiperidino]-r-carboxylate hydrochloride, having formula (1) is a camptothecin analog and a topoisomerase I inhibitor derived from camptothecin, a natural product extracted from a Chinese tree, camptotheca acumineta.

Recent clinical studies indicate synergistic activity of both drugs in which the Irinotecan at a dose of 50mg/m2 on day 1, 8, and 15 was injected as simple solution followed by doxorubicin simple injection at a dose of 40mg/m2 on day 3. However, this clinical study the dose limiting toxicity observed was neutropenia and diarrhea.

In another clinical trials of patients with previously treated solid tumors, the co-administration of topo I (Irinotecan at a dose of 100mg/m2) and topo-II (Pegylated liposomal Doxorubicin (PLD) at a dose of 20mg/m2) inhibitors was found to have adverse effects such as GIT toxicity, myelosuppression of irinotecan and cardio toxicity of doxorubicin which further limits its application as combination therapy. So there is a need for a drug delivery system such as liposome that co-encapsulates topo-I and topo-II inhibitors at their therapeutically synergistic ratio of approximately 1:1 (50mg/m2 of Doxorubicin hydrochloride and 70mg/m2 of Irinotecan hydrochloride trihydrate) to limit its dose limiting toxicity when individual drugs are administered.

The Doxorubicin liposome injection (DOXIL®) and Irinotecan liposome injection (ONIVYDE®) are in market where they are prepared by active drug loading with ammonium sulfate and triethylamine sucrose octasulfate gradient respectively. However, there is no combination product that is available in the market to reduce the dosing frequency and toxicities of this combination therapy.

The liposomal Irinotecan formulations (ONIVYDE®) have a drawback of formation of Lyso-PC due to hydrolysis of phospholipids. The Irinotecan and doxorubicin was co-encapsulated with the triethylamine sucrose octasulfate gradient showed higher tumor accumulation that can be attributed to high drug encapsulation and high D/PL ratio, which further improved drug delivery efficiency and relatively reduced the distribution of drugs to non-target sites.

KR 20160122310 A discloses a composition comprising 10 to 70 parts by weight of doxorubicin and 10 to 70 parts by weight of Irinotecan added to 100 parts by weight of a polyethylene glycol-block-polyaspartic acid copolymer. This document relates to polymeric drug delivery system whereas the present invention is liposomal delivery system.

US 8,147,867 B2 discloses a liposomal Irinotecan composition comprising liposomes in a an aqueous medium, the liposomes having an interior aqueous space separated from the aqueous medium by a membrane, said membrane comprising lipids, said lipids comprising an uncharged lipid component and a neutral phospholipid, with, entrapped inside the liposomes: 1) irinotecan and sucrose octasulfate, or 2) irinotecan and sucrose octasulfate and a substituted ammonium compound.

The inventors of the present invention have developed liposomal composition of Doxorubicin and Irinotecan. Further, liposomal composition of Doxorubicin or its pharmaceutically acceptable salt and Irinotecan or its pharmaceutically acceptable salt co-encapsulated in their synergistic molar ratio of 1:1 with ammonium dextran sulfate gradient for active drug loading. Dextran sulfate is a hydrophilic, biodegradable, biocompatible and negatively charged polysaccharide. It is a highly branched polyanionic polysaccharide with a sulfur content of about 17%, which is approximately equivalent to ~2.3 sulfate groups per glucose unit.

OBJECTIVE OF INVENTION
The objective of the present invention is to provide a liposomal composition of Doxorubicin and Irinotecan.

Another objective of the present invention is to provide a liposomal composition of Doxorubicin or its pharmaceutically acceptable salt and Irinotecan or its pharmaceutically acceptable salt comprising one or more excipients, wherein the first drug Doxorubicin to second drug Irinotecan molar drug ratio is 1:1.

Another objective of the present invention is to provide a process for the preparation of liposomal composition of Doxorubicin or its pharmaceutically acceptable salt and Irinotecan or its pharmaceutically acceptable salt.

Another objective of the present invention is to provide liposomal composition of Doxorubicin or its pharmaceutically acceptable salt and Irinotecan or its pharmaceutically acceptable salt for cancer therapy.

SUMMARY OF INVENTION
Accordingly, the present invention provides a liposomal composition of Doxorubicin and Irinotecan.

One embodiment of the present invention provides a liposomal composition of Doxorubicin or its pharmaceutically acceptable salt and Irinotecan or its pharmaceutically acceptable salt comprising one or more excipients, wherein the first drug Doxorubicin to second drug Irinotecan molar drug ratio is 1:1.

Another embodiment of the present invention provides a liposomal composition of Doxorubicin or its pharmaceutically acceptable salt and Irinotecan or its pharmaceutically acceptable salt for cancer therapy.

Another embodiment of the present invention provides a liposomal composition of Doxorubicin or its pharmaceutically acceptable salt and Irinotecan or its pharmaceutically acceptable salt comprising one or more excipients selected from phospholipid mixture, trapping agents, buffers and vehicles.

Another embodiment of the present invention provides a liposomal composition comprising:
a) Doxorubicin or its pharmaceutically acceptable salts,
b) Irinotecan or its pharmaceutically acceptable salts,
c) phospholipid mixture,
d) trapping agents,
e) buffers and
f) vehicles.

Another embodiment of the present invention provides a liposomal composition comprising:
a) Doxorubicin or its pharmaceutically acceptable salts,
b) Irinotecan or its pharmaceutically acceptable salts,
c) distearoylphosphatidylcholine (DSPC),
d) cholesterol,
e) methoxy-poly(ethylene glycol)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE),
f) ammonium dextran sulfate,
g) sucrose
h) histidine and
i) water for injection.

Another embodiment of the present invention provides a liposomal composition comprising:
a) Doxorubicin or its pharmaceutically acceptable salts,
b) Irinotecan or its pharmaceutically acceptable salts,
c) distearoylphosphatidylcholine (DSPC),
d) cholesterol,
e) methoxy-poly(ethylene glycol)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE),
f) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES),
g) sodium chloride,
h) ammonium dextran sulfate, and
i) water for injection.

Another embodiment of the present invention provides a liposomal composition comprising:
a) Doxorubicin or its pharmaceutically acceptable salts in the weight range of 1 mg/mL to 10 mg/mL,
b) Irinotecan or its pharmaceutically acceptable salts in the weight range of 1 mg/mL to 15 mg/mL,
c) distearoylphosphatidylcholine (DSPC) in the weight range of 1 mg/mL to 15 mg/mL,
d) cholesterol in the weight range of 1 mg/mL to 5 mg/mL,
e) methoxy-poly(ethylene glycol)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) in the weight range of 0.1 mg/mL to 1 mg/mL,
f) ammonium dextran sulfate in the weight range of 1 mM to 20 mM, and
g) water for injection.

Another embodiment of the present invention provides a process for the preparation of liposomal composition of Doxorubicin and Irinotecan.

Another embodiment of the present invention provides a process for the preparation of liposomal composition, wherein the process comprising:
a) preparing liposomes by dissolving the istearoylphosphatidylcholine (DSPC), methoxy-poly(ethylene glycol)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) and cholesterol in ethanol,
b) dissolving ammonium dextran sulfate in water for injection,
c) adding the ammonium dextran sulfate solution to ethanolic lipid solution,
d) extruding the coarse lipid dispersion under pressure through stacked polycarbonate membrane,
e) removing the unentrapped ammonium dextran sulphate by tangential flow filtration (TFF) against a 0.9 % (w/w) sodium chloride with HEPES buffer or 10% (w/w) sucrose solution, and
f) loading the Irinotecan hydrochloride trihydrate and Doxorubicin hydrochloride one after another or simultaneously in a suitable buffer.

BRIEF DESCRIPTION OF DRAWINGS
Fig.1: The In-vitro synergy effect of Doxorubicin: Irinotecan in different ratios.
Fig.2: Therapeutic activity of encapsulated Doxorubicin: Irinotecan at different ratios.
Fig.3: Mean survival time of individual products against combination of Doxorubicin: Irinotecan.

DETAILED DESCRIPTION OF THE INVENTION
The term "comprising", which is synonymous with "including", "containing", or "characterized by" here is defined as being inclusive or open-ended, and does not exclude additional, unrecited elements or method steps, unless the context clearly requires otherwise.

The present invention provides a liposomal composition of Doxorubicin or its pharmaceutically acceptable salt and Irinotecan or its pharmaceutically acceptable salt.

Doxorubicin salt is Doxorubicin hydrochloride and Irinotecan salt is Irinotecan hydrochloride.

One embodiment of the present invention provides a liposomal composition of Doxorubicin or its pharmaceutically acceptable salt and Irinotecan or its pharmaceutically acceptable salt comprising one or more excipients, wherein the first drug Doxorubicin to second drug Irinotecan molar drug ratio is 1:1.

Another embodiment of the present invention provides a liposomal composition of Doxorubicin or its pharmaceutically acceptable salt and Irinotecan or its pharmaceutically acceptable salt for cancer therapy.

Another embodiment of the present invention provides a liposomal composition of Doxorubicin or its pharmaceutically acceptable salt and Irinotecan or its pharmaceutically acceptable salt comprising one or more excipients selected from phospholipid mixture, trapping agents, buffers and vehicles.

Another embodiment of the present invention provides molar ratio of doxorubicin: Irinotecan is 1:1, preferably the concentration of doxorubicin hydrochloride is between 4.6mM to 18.4mM and Irinotecan hydrochloride trihydrate is between 5.165mM to 20.66mM.

The phospholipid mixture used in liposomes of present invention contains distearoylphosphatidylcholine (DSPC), cholesterol and methoxy-poly(ethylene glycol)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) at a molar ratio of 3:2:0.015 and the concentration of distearoylphosphatidylcholine (DSPC) is between 5.6mM to 11.6mM, Cholesterol is between 3.7mM to 7.7mM, MPEG 2000-DSPE is between 0.028mM to 0.058mM.

In one embodiment, the present invention relates to effective co-encapsulation of anticancer drugs such as topoisomerase-I and topoisomerase-II inhibitor or its pharmaceutically acceptable salt thereof in to liposome a mixture of at least one phospholipid and cholesterol, with 0.5mM to 15mM polyvalent counter ion donor or it’s pharmaceutically acceptable salt as trapping agent.

The trapping agent used in liposomes of present invention is the polyvalent counter ion donor that is ammonium salt of dextran sulfate which is prepared from sodium dextran sulfate to load both the drugs by active drug loading process. Ammonium dextran sulfate is used at concentration range of 0.5mM to 15Mm, preferably used at 6mM to 10mM was used.

The polyvalent counter ion donor such as dextran sulfate or its pharmaceutically acceptable salt more preferably its ammonium salt with molecular weight of 6,000 to 8,000 KD is used as entrapping agent at a concentration of 0.5mM to 15mM.

The buffers used in liposomes of present invention is Sucrose-histone buffer or HEPES-sodium chloride buffer at a suitable pH range of 6-7.5.

The resulting stable liposomal formulation, produces NMT 10mol% of Lyso-pc at refrigerated storage condition.

The vehicle used in the present invention is water for injection in quantity sufficient.

In another embodiment, having Lyso-PC generation of less than 10mmol% with respect to total phospholipids, at the refrigerated storage condition.

In another embodiment, the irinotecan and doxorubicin were co-encapsulated inside the liposome in precipitated or gelated state as dextran sulfate salt.

The encapsulation efficiency is not less than 90% for both drugs.

The liposomes were suspended in aqueous medium comprising
a) Hydroxyethylpiperazine-ethyl sulfonate (HEPES) at a concentration of 1mM to 30mM or histidine at a concentration of 1mM to 40 mM, as buffer component,
b) Sucrose or dextrose at a concentration 1mM to 60mM or NaCl at a concentration 100mM to 200mM, as tonicity modifiers.

The present invention is to provide liposomes have the mean diameter of less than 200 nm.

The following examples describe the nature of the invention and are given only for the purpose of illustrating the present invention in more detail and are not limitative.

EXAMPLES

Example 1: Preparation of Empty liposome

The liposomes were prepared by ethanol-injection method. Lipids including DSPC, MPEG-2000-DSPE and Cholesterol at a molar ratio of 3:2:0.015 were co-dissolved in ethanol at about 60°C. Ammonium dextran sulfate was dissolved in water for injection at a concentrations range of 0.5mM to15mM as per formulas provided in the examples. The prepared aqueous solution was added to ethanolic lipid solution. The bulk were homogenized at NLT 10,000 RPM and the temperature was maintained upto 80°C for 30mins to obtain the coarse liposomal dispersion. Then coarse lipid dispersion were extruded under pressure through two stacked polycarbonate membrane of size 200nm and100nm (0.2/0.1µ), extrusion was continued up to 8 to 10 cycles to obtain liposomes with size less than 200nm.The unentrapped ammonium dextran sulphate was removed by tangential flow filtration (TFF) against a 0.9 % (w/w) sodium chloride containing HEPES or 10% (w/w) sucrose solution. The prepared empty liposome can be stored in refrigerated for further drug loading.

Example 2: Preparation of liposome with co-encapsulated Doxorubicin and Irinotecan.
S.No Ingredients Quantity
Formula 1 Formula 2
1. Doxorubicin hydrochloride 5mg/mL 5mg/mL
2. Irinotecan hydrochloride trihydrate 7mg/mL 7mg/mL
3. DSPC 6.81mg/mL 6.81mg/mL
4. Cholesterol 2.22 mg/mL 2.22 mg/mL
5. MPEG-2000-DSPE 0.12mg/mL 0.12mg/mL
6. HEPES 17mM 17mM
7. NaCl 144mM 144mM
8. Ammonium Dextran Sulfate 6mM 8mM
9. Water for injection q.s q.s

Manufacturing process

Sodium dextran sulfate with a molecular weight of 6000 to 8000 Daltons is converted into ammonium dextran sulfate using ion exchange column. Two pharmaceutically acceptable composition of empty liposome with different concentration of ammonium dextran sulfate were prepared by the method in example 1 as per the formula 1 and 2. Remote drug loading is facilitated with gradient formed by removal of un-entrapped ammonium sulfate dextran with the TFF process with HEPES-sodium chloride buffer at pH 7.4. The concentration of ammonium dextran sulfate was 6mM and 8mM respectively. Then loading of drug with Irinotecan hydrochloride trihydrate and Doxorubicin hydrochloride can be done one after the other (or) simultaneously. The drug loading process continues till at least 90% to 100% of both drugs loaded into the preformed empty liposomes.

Example 3
S.No Ingredients Quantity
Formula 3 Quantity
Formula 4
1. Doxorubicin hydrochloride 7mg/mL 7mg/mL
2. Irinotecan hydrochloride trihydrate 10mg/mL 10mg/mL
3. DSPC 9.17mg/mL 9.17mg/mL
4. Cholesterol 2.99 mg/mL 2.99 mg/mL
5. MPEG-2000-DSPE 0.16mg/mL 0.16mg/mL
6. HEPES 17mM 17mM
7. NaCl 144mM 144mM
8. Ammonium Dextran Sulfate 10mM 12mM
9. Water for injection q.s q.s

Manufacturing process

Sodium dextran sulfate with a molecular weight of 6000 to 8000 Daltons is converted into ammonium dextran sulfate using ion exchange column. Two pharmaceutically acceptable composition of empty liposome with different concentration of ammonium dextran sulfate were prepared by the method in example 1 as per the formula 3 and 4. Remote drug loading is facilitated with gradient formed by removal of un-entrapped ammonium sulfate dextran with the TFF process with HEPES-sodium chloride buffer at pH 7.4. The concentration of ammonium dextran sulfate was 10mM and 12mM respectively. The loading of drug into bulk liposomes with irinotecan hydrochloride trihydrate and doxorubicin hydrochloride solution can be performed one after the other (or) simultaneously. The drug loading process continues till at least 90% to 100% of both drugs gets loaded into the preformed empty liposomes.

Example 4
S.No Ingredients Quantity
Formula 5
1. Doxorubicin hydrochloride 5mg/mL
2. Irinotecan hydrochloride trihydrate 7mg/mL
3. DSPC 6.81mg/mL
4. Cholesterol 2.22 mg/mL
5. MPEG-2000-DSPE 0.12mg/mL
6. Sucrose 30mM
7. Histidine 20mM
8. Ammonium Dextran Sulfate 8mM
9. Water for injection q.s

Manufacturing process

Sodium dextran sulfate with a molecular weight of 6000 to 8000 Daltons is converted into ammonium dextran sulfate using ion exchange column. A pharmaceutically acceptable composition of empty liposome with ammonium dextran sulfate were prepared by the method in example 1 as per the formula 5. Remote drug loading is facilitated with gradient formed by removal of un-entrapped ammonium sulfate dextran with the TFF process with 10% sucrose buffer. The concentration of ammonium dextran sulfate used was 8mM respectively. The loading of drugs into bulk liposomes were performed in sucrose-histidine buffer at pH - 7 either one after other (or) simultaneously. The drug loading process continues till at least 90% to 100% of both drugs gets loaded into the preformed empty liposomes.

In-vitro and in-vivo studies conducted are given below:

In-vitro study:
A panel of 08 tumor cell lines was utilized to examine drug ratio-dependent synergy for doxorubicin: Irinotecan combinations.
Cell line Cell Tissue
BxPc-3 Pancreatic
4T-1 Breast cancer cells
HT-29 Colon
Colon 26 Colon
HCT-116 Colon
A549 Non-small cell lung cancer cells
HT-29 Colon
IGROV-1 Ovarian

Adherent or non-adherent cell lines were collected in their log-arithmic growth phase using standard cell culture techniques. The harvested cells were assessed for viability with trypan blue and a hemacytometer. Cell concentrations were determined using a cell counter (Z2 Coulter, Beckman Coulter, Fullerton, CA, USA) and diluted in their respective media to the desired cell concentrations. Cells were then seeded into a 96-well plate (Falcon 353072, Becton Dickinson, Oakville, ON, Canada) across 9 columns and 6 rows, one column served as a cell-only control (no drug) while a tenth column served as a media-only control. Cell concentrations were optimized such that 72 h after cell plating an MTT assay performed on the untreated control cells would yield an A570 value of 1.5. Prior to drug addition, plates were incubated for 24 h at 37 °C and 5% CO2. The following day drug dilutions were prepared in 12-well plates (Falcon 353043, Becton Dickinson) using RPMI 1640 media (Stem Cell Technologies, Vancouver, Canada) containing 10% FBS and 2 mM l-glutamine as a diluent. Individual agents or combinations of agents at fixed doxorubicin: irinotecan molar ratios (1:1, 1:2, 2:1, and 2:2) were added to the cells in triplicate and returned to the incubator. After a 72 h incubation, cell viability was assessed by the MTT assay. Prior optimization experiments with the MTT assay indicated that the viability values in some cell lines was often dependent upon the time of drug exposure.

The absorbance of each well in a 96-well plate was measured at 570 nm using a multi-plate reader. Relative percent survival was determined by subtracting absorbance values obtained from media-only wells from drug-treated wells and then normalizing to the no-drug control wells (cell-only). The fraction of cells affected (fa) at each drug concentration was subsequently determined for each well. Three replicates were averaged and the repeated data sets (minimum of 3) were entered into CalcuSyn (Biosoft, Ferguson, MO, USA) for synergy analysis. The latter program employs the median-effect analysis algorithm which produces the Combination Index (CI) value as a quantitative indicator of the degree of synergy or antagonism. Using this analysis method, a CI = 0.9–1.1 reflects additive activity, CI > 1.1 signifies antagonism, and a CI < 0.9 indicates synergy. At 1:1 molar drug ratio of doxorubicin: irinotecan, maximum synergism was observed; i.e., 5.6 out of 8 (70%) cell lines showed synergism. The In-vitro synergy effect of doxorubicin and Irinotecan at different ratios is shown in Figure 1.

In-vivo study:
The in vivo antitumor efficacy of co-encapsulated doxorubicin and irinotecan liposomes was evaluated using A549 Non-small cell lung cancer cell line xenografts grown in mice. Mice were monitored twice weekly for signs of tumor growth .Once a palpable tumor was detected, measurements were taken twice a week with calipers. Tumor volume was calculated using the formula 0.5 × (longest measurement) × (shortest measurement) 2. When the mean tumor volume reached 700 mm3 the mice’s were divided into 6 groups of 5 mice in each group.1st group - Saline only, 2nd group- 1:2 ratio of Co-encapsulated doxorubicin and irinotecan liposome only, 3rd group- 2:1 ratio of Co-encapsulated doxorubicin and irinotecan liposome only, 4th group - 1:1 ratio of Co-encapsulated doxorubicin and irinotecan liposome only. All the above mentioned drugs are administered by intra-peritoneal injection on days 5, 6, and 7 then 12, 13 and 14 only. Three mice from each treatment group were sacrificed, the organs (heart, liver, spleen, lung, and kidney) and the tumors were collected, then washed in the saline, weighted and homogenized by a tissue homogenizer. Drug concentrations in tissues and tumors were analyzed by fluorescence quantification. Detection was completed at ?ex 380/?em 500 nm for IRN, and ?ex 500/?em 590 nm for DOX. 1:1 molar ratio co-encapsulated drugs has maximum tumor volume reduction of 26.47% whereas the other two ratios, i.e., 2:1 and 1:2 has tumor volume reduction of 57.35% and 53.67%, with respect to the saline control. Therapeutic activity encapsulated doxorubicin and irinotecan at different ratios are given in figure 2.

Clinical study:
The clinical study was a multi-center, open-label, active controlled and randomized trial with 12 patients in each group aged between 60-75, with newly diagnosed Non-small cell lung cancer and each group will get doxorubicin simple injection alone (ADRIAMYCIN®) (60mg/m2), Irinotecan simple injection (CAMPTOSAR®) alone (180mg/m2), Onivyde® Irinotecan liposome alone (70mg/m2) , Doxil® doxorubicin liposome (50mg/m2), Co-encapsulated doxorubicin and irinotecan liposome alone (1:1) (50mg/m2 of doxorubicin and 70mg/m2 of irinotecan).The overall survival rate for co-encapsulated doxorubicin and irinotecan liposome (1:1) ratio is found to be 14.43 months which is significantly higher than the survival times of Doxil® and Onivyde® individually.
SI.No Groups Mean survival time in days Mean survival time in months
1 ADRIAMYCIN® (Doxorubicin monoproduct) 158.583 5.29
2 CAMPTOSAR® (Irinotecan monoproduct) 194.167 6.47
3 Doxil® (Doxorubicin HCl liposome injection) 273.667 9.12
4 Onivyde® (Irinotecan liposome injection) 308.333 10.28
5 Co-encapsulated Doxorubicin and Irinotecan liposome of present application 432.917 14.43

The mean survival plot is shown in Figure 3. , Claims:WE CLAIM:
1. A liposomal composition of Doxorubicin and Irinotecan comprising one or more excipients.
2. The composition as claimed in claim 1, wherein the first drug Doxorubicin or its pharmaceutically acceptable salt to second drug Irinotecan or its pharmaceutically acceptable salt at a molar ratio of 1:1.
3. The composition as claimed in claim 1, wherein comprising excipients selected from phospholipid mixture, trapping agents, buffers and vehicles.
4. The composition as claimed in claim 3, wherein composition comprises phospholipid mixture in liposomes contains distearoylphosphatidylcholine, Cholesterol and methoxy-poly(ethylene glycol)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) at a molar ratio of 3:2:0.015 and the concentration of DSPC is between 5.6mM to 11.6mM, Cholesterol is between 3.7mM to 7.7mM, MPEG 2000-DSPE is between 0.028mM to 0.058mM.
5. The composition as claimed in claim 3, wherein trapping agent ammonium dextran sulfate is used at concentration range of 0.5mM to 15Mm.
6. The composition as claimed in claim 3, wherein buffers are selected from Sucrose-histone buffer, HEPES-sodium chloride buffer at a suitable pH range of 6-7.5.
7. The composition as claimed in claim 3, wherein vehicle is water for injection in quantity sufficient.
8. The process for the preparation of composition as claimed in claims 1, wherein the process comprising:
a) preparing liposomes by dissolving the istearoylphosphatidylcholine (DSPC), methoxy-poly(ethylene glycol)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) and cholesterol in ethanol,
b) dissolving ammonium dextran sulfate in water for injection,
c) adding the ammonium dextran sulfate solution to ethanolic lipid solution,
d) extruding the coarse lipid dispersion under pressure through stacked polycarbonate membrane,
e) removing the unentrapped ammonium dextran sulphate by tangential flow filtration (TFF) against a 0.9 % (w/w) sodium chloride containing HEPES or 10% (w/w) sucrose solution, and
f) loading the Irinotecan hydrochloride trihydrate and Doxorubicin hydrochloride one after another or simultaneously in a suitable buffer.
9. The composition as claimed in claim 3, where the mean particle diameter is less than 200 nm.
10. The composition as claimed in claim 3, wherein the encapsulation efficiency of both drugs is not less than 90%.

Date this Twenty Eighth (28th) day of December, 2023.

__________________________________
Dr. S. Padmaja
Agent for the Applicant
IN/PA/883