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WO2025088367A1 - Composition liposomale d'idarubicine et de cytarabine et son procédé de préparation - Google Patents

Composition liposomale d'idarubicine et de cytarabine et son procédé de préparation Download PDF

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Publication number
WO2025088367A1
WO2025088367A1 PCT/IB2023/062448 IB2023062448W WO2025088367A1 WO 2025088367 A1 WO2025088367 A1 WO 2025088367A1 IB 2023062448 W IB2023062448 W IB 2023062448W WO 2025088367 A1 WO2025088367 A1 WO 2025088367A1
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Prior art keywords
cytarabine
liposomes
idarubicin
drug
composition
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PCT/IB2023/062448
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English (en)
Inventor
Munnavar SHAIK
Sivakumar Ramachandran
Tathagata Dutta
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Jodas Expoim Pvt. Ltd
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Publication of WO2025088367A1 publication Critical patent/WO2025088367A1/fr

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  • the present invention relates to liposomal composition of Idarubicin and Cytarabine.
  • the present invention also relates to liposomal composition of Idarubicin and Cytarabine comprising one or more excipients, wherein the first drug Idarubicin to second drug Cytarabine (Ara-C) molar drug ratio is 1:17.
  • the present invention also relates to a process for the preparation of liposomal composition of Idarubicin and Cytarabine.
  • the present invention also relates to liposomal composition of Idarubicin and Cytarabine which shows synergistic action against acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • Vyxeos® shows the better cure rate in the treatment of cancers particularly in Acute myeloid leukemia (AML) than the traditional “7+3 Regimen” of individual drugs.
  • Liposomal co-encapsulation of Daunorubicin and Cytarabine (Ara-C) in a 1:5 molar ratio for treating AML is demonstrated in marketed product of Vyxeos®.
  • AML is most common type of leukemia found in adults which is characterized by a heterogeneous group of hematological neoplastic disorders, caused by generation and aggregation of immature hematopoietic cells in the bone marrow and blood due to exposure to ionizing radiations (such as atomic bomb detonation in Hiroshima and Nagasaki during world war II), chemicals such as petroleum products, benzene, herbicides and pesticides and secondary to exposure to genotoxic chemotherapeutic agents.
  • ionizing radiations such as atomic bomb detonation in Hiroshima and Nagasaki during world war II
  • chemicals such as petroleum products, benzene, herbicides and pesticides and secondary to exposure to genotoxic chemotherapeutic agents.
  • the aetiology of AML which includes the consecutive genomic changes in the multipotent hematopoietic stem cells that leads to abnormal clonal proliferations that are distinct from myeloid cells.
  • AML Alzheimer's disease
  • the prevalence of AML increases with age with median age of diagnosis of 65 years.
  • the incidence of AML rise rapidly after 50 years of age with 3.5, 15, 22 per hundred thousand individuals of age 50, 70 and 80 years respectively.
  • the incidence of secondary AML (therapy related AML) caused by exposure to genotoxic chemotherapeutic agents is increasing and accounts to 10-20% of all cases of AML.
  • the Down syndrome associated AML is associated with mutations in exon 2 of the transcription factor GAT Al which is unique to Down syndrome and it is detectable in virtually all cases.
  • the AML1 gene which is present on the chromosome 21q22 is also known as Runt related protein 1 (RUNX1) or core binding factor acute myeloid leukemia 1 (CBFA), is a highly conserved transcription factor i.e. critical for normal hematopoiesis.
  • the AML1 mutation is present in the germ line of affected individuals but they do not develop AML until later in life.
  • the standard induction therapy for the treatment of AML includes the continuous infusion of a combination of cytidine analogs i.e. cytarabine (Ara-C) and the anthracycline analogs i.e. Idarubicin or Daunorubicin.
  • Cytarabine is given at 100-200 mg/m 2 dose for 7 days followed by the Daunorubicin given at 60 mg/m dose or Idarubicin (the most potent analogue of anthracyclines) at 12 mg/m dosage regimen for 3 days. This is also known as the standard “7+3 regimen”.
  • US 7,850,990 B2 discloses a delivery vehicle composition for parenteral administration comprising two or more agents encapsulated in the vehicle composition at a ratio that is synergistic or additive over a desired concentration range.
  • the delivery vehicle composition is prepared by a process comprising encapsulating the agents in the delivery vehicle composition at these ratios.
  • the non-antagonistic ratio of the agents is determined by assessing the biological activity or effects of the agents on relevant cell culture or cell-free systems over a range of concentrations and, in one embodiment, applying an algorithm to determine a “combination index,” (CI).
  • US 8,022,279 B2 discloses a composition for parenteral administration to a subject which composition comprises liposomes having associated therewith Daunorubicin and Cytarabine in a mole ratio of Daunorubicin : Cytarabine of about 1:5 wherein the Daunorubicin and Cytarabine are associated with the liposomes so that said mole ratio is maintained in the blood for at least one hour after administration to the subject.
  • This patent also discloses Lipid foams were prepared by dissolving lipids (DSPC: DSPG:CHOL (7:2: 1 mol ratio)).
  • US 8,518,437 B2 discloses a composition comprising liposomes that contain at least one biologically active agent wherein the ordered bilayer(s) of said liposomes consist(s) essentially of (a) one or more vesicle forming lipids, which are phospholipids or sphingolipids such that; (b) at least 1 mol % of said bilayer(s) is phosphatidylglycerol (PG) and/or phosphatidylinositol (PI); (c) 5-20 mol % cholesterol; and wherein said liposomes have a mean diameter between 80-200 nm +/-25 nm; and have a transition temperature (Tc) of at least 38° C which composition is cryostable in the absence of cryoprotectant.
  • PG phosphatidylglycerol
  • PI phosphatidylinositol
  • Tc transition temperature
  • US 10,166,184 B2 discloses a lyophilized gel-phase liposomal composition, which composition comprises gel-phase liposomes that exhibit a melting phase temperature (Tc) of at least 37° C and wherein the liposome membrane of said liposomes comprises no more than 20 mol % cholesterol and at least 1 mol % of a phosphatidylglycerol (PG) or a phosphatidylinositol (PI) or both; and wherein at least two therapeutic and/or diagnostic agents are stably associated with said liposomes wherein at least one of said agents is amphipathic or hydrophilic; and; a cryoprotectant external to said liposomes; and wherein said liposomes contain less than 50 mM internal cryoprotectant, and wherein when said lyophilized gel-phase liposomal composition is reconstituted in a pharmaceutical carrier, the mean diameter of the liposomes is maintained as compared to said composition prior to lyophilization and said agents are substantially retained in
  • the inventors of the present invention have developed liposomal composition of Idarubicin and Cytarabine.
  • the present invention corresponds to the composition and process of the manufacture for enhanced drug delivery of co-encapsulated active therapeutic agents.
  • the invention consists of lipid based drug delivery system which shows synergistic action against Acute myeloid leukemia (AML), wherein the first drug is the most potent anthracyclines i.e. Idarubicin and the second drug is from cytidine analogues i.e. Cytarabine (Ara-C) at 1:17 molar drug ratio and lipid carrier DSPC, DSPG and cholesterol at the molar ratio of 6:3:1.
  • AML Acute myeloid leukemia
  • the present invention uses a liposomal delivery of Idarubicin (a more potent, efficacious anthracycline analogue which has a high cure rate in the long term than Daunorubicin) and Cytarabine at their clinically effective dosage regimen of 1:17 molar ratio.
  • Idarubicin a more potent, efficacious anthracycline analogue which has a high cure rate in the long term than Daunorubicin
  • Cytarabine at their clinically effective dosage regimen of 1:17 molar ratio.
  • the objective of the present invention is to provide a liposomal composition of Idarubicin and Cytarabine.
  • Another objective of the present invention is to provide a liposomal composition of Idarubicin and Cytarabine comprising one or more excipients, wherein the first drug Idarubicin to second drug Cytarabine (Ara-C) molar drug ratio is 1:17.
  • AML Acute myeloid leukemia
  • the present invention provides a liposomal composition of Idarubicin and Cytarabine.
  • One embodiment of the present invention provides a liposomal composition of Idarubicin and Cytarabine comprising one or more excipients, wherein the first drug Idarubicin to second drug Cytarabine (Ara-C) molar drug ratio is 1:17.
  • Another embodiment of the present invention provides a liposomal composition of Idarubicin and Cytarabine which shows synergistic action against Acute myeloid leukemia (AML).
  • AML Acute myeloid leukemia
  • Another embodiment of the present invention provides a liposomal composition of Idarubicin and Cytarabine comprising one or more excipients, wherein the first drug Idarubicin to second drug Cytarabine (Ara-C) molar drug ratio is 1: 17 and lipid carrier DSPC, DSPG and cholesterol at the molar ratio of 6:3:1.
  • Another embodiment of the present invention provides a liposomal composition
  • a liposomal composition comprising: a) Idarubicin, b) Cytarabine, c) Distearoylphosphatidylcholine (DSPC), d) Distearoylphosphatidylglycerol (DSPG), e) Cholesterol, f) Copper Gluconate, g) Triethanolamine, and h) Sucrose
  • a liposomal composition comprising: a) Idarubicin in the weight range of 0.1 to 2.4 mg/mL, b) Cytarabine in the weight range of 1 to 20 mg/mL, c) Distearoylphosphatidylcholine (DSPC) in the weight range of 4 to 80 mg/mL, d) Distearoylphosphatidylglycerol (DSPG) in the weight range of 1 to 40 mg/mL, e) Cholesterol in the weight range of 0.3 to 7 mg/mL, f) Copper Gluconate in the weight range of 1 to 10 mg/mL, g) Triethanolamine in the weight range of 0.1 to 1 mg/mL, and h) Sucrose in the weight range of 50 to 200 mg/mL.
  • DSPC Distearoylphosphatidylcholine
  • DSPG Distearoylphosphatidylglycerol
  • Cholesterol in the weight range of 0.3 to 7 mg/
  • Another embodiment of the present invention provides a process for the preparation of liposomal composition of Idarubicin and Cytarabine.
  • 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 lipids DSPC: DSPG:
  • 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 lipids DSPC: DSPG: Cholesterol (6:3:1 molar ratio) in a mixture of organic solvent, b) Evaporating the solvent by vacuum rotary evaporator for up to 12 hours, c) Dissolving the copper-gluconate in WFI and adding tri-ethanolamine , d) Hydrating the lipid film with aqueous solution of copper-gluconate and triethanolamine, e) Reducing the size of liposomes using high pressure homogenizer and/or extruder, f) Buffering the liposomes and remove the unencapsulated Cytarabine (Ara-C), TEA and copper gluconate, g) Adding Cytarabine (Ara-C) and Idarubucin for passive drug encapsulation into liposomes, and h) Filtering
  • Fig.l The In-vitro synergy effect of Cytarabine: Idarubicin.
  • Fig.2 Therapeutic activity of free drug cocktail and encapsulated drug at different ratios.
  • Fig.3 Mean survival time of individual products against combinations. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention is to provide a liposomal composition of Idarubicin and Cytarabine comprising one or more excipients, wherein the first drug Idarubicin to second drug Cytarabine (Ara-C) molar drug ratio is 1:17.
  • the present invention is to provide a liposomal composition of Idarubicin and Cytarabine which shows synergistic action against Acute myeloid leukemia (AML).
  • AML Acute myeloid leukemia
  • the present invention is to provide a liposomal composition of Idarubicin and Cytarabine comprising one or more excipients, wherein the first drug Idarubicin to second drug Cytarabine (Ara-C) molar drug ratio is 1:17 and lipid carrier DSPC, DSPG and cholesterol at the molar ratio of 6:3:1.
  • the present invention is to provide the said liposomes consists of mixture of lipid at molar ratio of 6:3:1, wherein Liposome composition includes 60% of Zwitterionic neutral phospholipids selected from the group of phosphatidylcholine like DSPC, Liposome composition includes 30% of negatively charged phospholipid selected from the group of phosphatidylglycerol like DSPG and Liposome composition includes 10% of sterols like cholesterol.
  • the present invention is to provide liposomes have the mean diameter of less than 200nm.
  • 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.
  • the liposomes contained lipids i.e. DSPC, DSPG and cholesterol at the molar ratio of 6:3:1.
  • the liposomes were prepared by dissolving the lipids i.e. DSPC: DSPG: Cholesterol (6:3:1 molar ratio) in a mixture of organic solvent. The solvent was evaporated by vacuum rotary evaporator for up to 12 hours.
  • the resulting lipid film is hydrated with aqueous solution of copper- gluconate (250 mM), tri-ethanolamine (TEA; 550 mM) and cytarabine (Ara-C); wherein the copper-gluconate dissolved in WFI turns blue in color with a pH of approximately 3-4; color slightly changes to dark blue color upon addition of tri-ethanolamine (TEA) with resultant pH of 7.4 and further addition of cytarabine (Ara-C), 50 mg/ml, for passive drug encapsulation into liposomes.
  • the resultant mixture of crude liposomes were size reduced using high pressure homogenizer and/or extruder with 10 passes in stacked 0.2/ 0.1 micron polycarbonate membranes at ⁇ 70°C to get the small unilamellar vesicles (SUVs).
  • the mean diameter of liposomes was determined by zeta sizer and found to be less than 200 nm.
  • the resulting liposomes with passively loaded cytarabine were subjected to diafiltration by using tangential flow filtration (TFF), wherein the Eiposomes were buffer exchanged into SHE buffer (Sucrose - 300mM, HEPES - 20mM and EDTA - ImM; pH 7.4), that removes the unencapsulated cytarabine (Ara-C), TEA and copper gluconate and creates a concentration gradient of copper gluconate necessary to load the second drug actively into the preformed liposomes. This is followed by second diafiltration with 10% sucrose to remove EDTA and HEPES from the liposomal bulk.
  • THF tangential flow filtration
  • idarubicin The active drug loading of idarubicin is carried out by adding idarubicin in 300mM Sucrose solution and incubating at ⁇ 50°C for 30-min. After loading, the final purple bulk was cooled down to room temperature. Drug loading efficiency was determined by elution through sephadex G-50 column. Idarubicin and cytarabine were loaded at 1:17 molar ratio into the liposomes.
  • the resulting bulk solution is sterile filtered and lyophilized in vials to get a finished product of 12 mg of idarubicin and 100 mg of cytarabine per vial.
  • the lyophilized formulation was stable for 36 months at refrigerated condition when stored upright and protected from light. The resulting formulation released the drugs slowly for up to 7 days.
  • the liposomes contained lipids i.e. DSPC, DSPG and cholesterol at the molar ratio of 6:3:1.
  • the liposomes were prepared by dissolving the lipids i.e. DSPC: DSPG: Cholesterol (6:3:1 molar ratio) in a mixture of organic solvents. The solvent was evaporated by vacuum rotary evaporator for up to 12 hours.
  • the resulting lipid film is hydrated with aqueous solution of copper- gluconate (250 mM), tri-ethanolamine (TEA; 550 mM); wherein the copper-gluconate dissolved in WFI turns blue in color with a pH of approximately 3-4; color slightly changes to dark blue color upon addition of tri-ethanolamine (TEA) with resultant pH of 7.4.
  • the resultant mixture of crude liposomes were size reduced using high pressure homogenizer and/or extruder 10 passes in stacked 0.2/ 0.1 micron polycarbonate membranes at ⁇ 70°C to get the small unilamellar vesicles (SUVs).
  • the mean diameter of liposomes was determined by zeta sizer and found to be less than 200 nm were subjected to diafiltration by using tangential flow filtration (TFF), wherein the liposomes were buffer exchanged into SHE buffer (Sucrose - 300mM, HEPES - 20mM and EDTA - ImM; pH 7.4), that removes the unencapsulated TEA and copper gluconate and creates a concentration gradient of copper gluconate necessary to load idarubicin actively into the preformed liposomes. This is followed by second diafiltration with 10% sucrose to remove EDTA and HEPES from the liposomal bulk.
  • THF tangential flow filtration
  • the liposomal bulk was preheated to 40- 50°C for couple of minutes.
  • the simultaneous active loading of idarubicin (at drug to lipid ratio of 0.03:1) and passive loading of cytarabine (200 pmol/mL) is carried out by adding idarubicin and cytarabine in 300mM sucrose solution and incubating at 40- 50°C for 60-min. After loading, the final purple bulk was cooled down to room temperature. At regular intervals, drug loading efficiency was determined by elution through sephadex G-50 column. Idarubicin and cytarabine were loaded at 1:17 molar ratio into the liposomes.
  • the resulting bulk solution is sterile filtered and lyophilized in vials to get a finished product of 12 mg of idarubicin and 100 mg of cytarabine per vial.
  • the lyophilized formulation was stable for 36 months at refrigerated condition when stored upright and protected from light.
  • the resulting formulation released the drugs slowly for up to 7 days. In-vitro and in-vivo studies conducted are given below:
  • a panel of 15 tumor cell lines was utilized to examine drug ratio-dependent synergy for Cytarabine: Idarubicin combinations.
  • 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.
  • a cell-only control no drug
  • 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.
  • CI Combination Index
  • CI > 1.1 signifies antagonism
  • a CI ⁇ 0.9 indicates synergy.
  • maximum synergism was observed; i.e., 9 out of 15 (60%) cell lines showed synergism.
  • the In-vitro synergy effect of Cytarabine: Idarubicin is shown in Figure 1.
  • Therapeutic activity of free drug cocktail and encapsulated drug was determined in the P388 murine leukemia tumor models following i.p. tumor cell inoculation.
  • BDF-1 mice (Charles River Laboratories, St. Constant, QC, Canada) were inoculated i.p. with 1 x 10 6 P388 cells. The day of tumor cell inoculation was used as day zero.
  • BDF-1 mice received intravenous treatment on days 1, 4 and 7 with saline or a dual-drug liposome of Cytarabine and Idarubicin at different molar drug ratios. Ascitic tumor progression, survival and body weight were monitored daily. Moribund mice were euthanized and the time of death was logged as the following day. The day 55 survival rate was highest with the 17:1 Cytarabine: Idarubicin molar drug ratio.
  • the clinical study was a multi-center, open-label, active controlled and 2 2 randomized trial of Cytarabine alone (lOOmg/m ), Idarubicin alone (12mg/m ), Cytarabine (lOOmg/m ) + Idarubicin (12mg/m ) and liposomal Cytarabine (100mg/m 2 ) + Idarubicin (12mg/m 2 ) with 12 patients in each group aged between 60- 75, with newly diagnosed t-AML or AML-MRC.
  • Cytarabine (100 mg/m ) Idarubicin (12 mg/m ) and Liposomal Cytarabine (100 mg/m )

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Abstract

La présente invention concerne une composition liposomale d'idarubicine et de cytarabine. La présente invention concerne également une composition liposomale d'idarubicine et de cytarabine comprenant un ou plusieurs excipients, le rapport de médicament molaire du premier médicament, l'idarubicine au second médicament, la cytarabine (Ara-C) étant de 1 : 17. La présente invention concerne également un procédé de préparation d'une composition liposomale d'idarubicine et de cytarabine. La présente invention concerne également une composition liposomale d'idarubicine et de cytarabine qui présente une action synergique contre la leucémie myéloïde aiguë (LMA).
PCT/IB2023/062448 2023-10-27 2023-12-11 Composition liposomale d'idarubicine et de cytarabine et son procédé de préparation WO2025088367A1 (fr)

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