WO2022153211A1 - Composition liposomale d'un dérivé de camptothécine - Google Patents

Composition liposomale d'un dérivé de camptothécine Download PDF

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Publication number
WO2022153211A1
WO2022153211A1 PCT/IB2022/050264 IB2022050264W WO2022153211A1 WO 2022153211 A1 WO2022153211 A1 WO 2022153211A1 IB 2022050264 W IB2022050264 W IB 2022050264W WO 2022153211 A1 WO2022153211 A1 WO 2022153211A1
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liposomal composition
cancer
composition according
compound
cholesterol
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PCT/IB2022/050264
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English (en)
Inventor
Ajay Jaysingh Khopade
Raghavendra Chaluvayya MUNDARGI
Jayeshkumar Jasubhai HADIA
Ronak Jatinbhai VASHI
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Sun Pharma Advanced Research Company Limited
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Publication of WO2022153211A1 publication Critical patent/WO2022153211A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A61K9/1278—Post-loading, e.g. by ion or pH gradient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a liposomal composition of compound of formula (I): or a salt thereof.
  • the liposomal compositions of the present invention are useful as a targeted drug delivery system, wherein the compound of formula (I) or salt thereof: (i) is stably and efficiently encapsulated; (ii) remains in the bloodstream for a prolonged period of time; (iii) retains antitumor activity; and (iv) is efficiently delivered to the specific site of action (e.g., a tumor).
  • Topoisomerase inhibitors are effectively used for cancer therapy. These compounds inhibit the action of topoisomerase enzymes which play a role in the replication, repair, genetic recombination and transcription of DNA.
  • An example of a topoisomerase inhibitor is camptothecin, a natural compound that interferes with the activity of topoisomerase I, an enzyme involved in DNA replication and RNA transcription. Camptothecin and the camptothecin analogues topotecan and irinotecan are approved for clinical use.
  • camptothecin has its water insolubility, which hinders the delivery of the drug. Numerous analogues of camptothecin have been prepared to improve the compound's water solubility. Another problem with camptothecin and its analogues is that the compounds are susceptible in aqueous environments to hydrolysis at the a-hydroxy lactone ring. The lactone ring opens to the carboxylate form of the drug, a form that exhibits little activity against topoisomerase I.
  • RES reticuloendothelial system
  • Hong et al. discloses liposomal formulations of irinotecan comprising an uncharged lipid component and a neutral phospholipid.
  • the invention is based on the discovery that substituted ammonium and polyanion are useful for loading and retaining entities inside liposomes.
  • Hong et al specifically discloses liposomal composition comprising irinotecan and sucrose octasulfate, or irinotecan and sucrose octasulfate and a substituted ammonium compound.
  • SN-38 is topoisomerase I inhibitor and is an active metabolite of irinotecan. It is approximately 1000 times more potent than irinotecan.
  • SN-38 is limited by its poor aqueous solubility and also due to unwanted conversion of its pharmacologically active lactone ring into inactive carboxylate form at pH > 6 such as in plasma.
  • a metabolite of SN-38, that is SN-38-glucouronide causes severe diarrhoea.
  • SN-38 is unsuitable as an injectable form.
  • the carboxylate and also the lactone form are toxic and cause serious side effects like diarrhoea.
  • the compound of formula (I) is a potent SN-38 prodrug with potency similar to SN- 38 in various cancer cells.
  • a targeted drug delivery system which includes a compound of formula (I) or salt thereof, wherein the compound is efficiently encapsulated and can be effectively delivered to the specific site of action (e.g., a tumor).
  • the present invention provides a liposomal composition of a compound of formula (I) or a salt thereof comprising a counterion and a buffering agent.
  • One another object of the present invention is to provide a liposomal composition of a compound of formula (I) or a salt thereof for treating a tumor in a subject.
  • Fig. 1 shows liposomal structures of a compound of formula (I) prepared using different counterions.
  • Fig. 1(a) shows liposomal structures with ammonium citrate as the counterion showing closely packed dense strands.
  • Fig. 1(b) shows liposomal structures with ammonium sulfate as the counterion showing loosely packed thin strands.
  • Fig. 2 In-vitro release of a compound of formula (I) from liposomes of examples 2.2 and 2.3 in mice plasma.
  • Fig. 3 Tumor inhibition in SCLC NCI-H526 bearing xenograft mice.
  • Fig. 3(a) with a compound of formula (I) liposomes (2 mg/ml; citrate as gradient) and Comparator- Onivyde (Irinotecan liposomes) - Q7D*4.
  • Fig. 3(b) with a compound of formula (I) liposomes (2 mg/ml; citrate as gradient) and Comparator-Onivyde (Irinotecan liposomes) - Q14D*2.
  • Fig. 3(a) with a compound of formula (I) liposomes (2 mg/ml; citrate as gradient) and Comparator-Onivyde (Irinotecan liposomes) - Q14D*2.
  • Fig. 5 Tumor inhibition in MDA-MB 231 bearing xenograft mice model-TNBC.
  • Fig. 5(a) with a compound of formula (I) liposomes (2mg/ml; citrate as gradient) at 2.5mg/kg, 5mg/kg and lOmg/kg and Comparator - placebo of liposomes - Q7D*4.
  • Fig. 5(b) with a compound of formula (I) liposomes (2mg/ml; citrate as gradient) at 5mg/kg and Comparator - Onivyde (Irinotecan liposomes) and placebo of liposomes - Q7D*4.
  • targeted drug delivery system means compositions that acts as carriers and delivers the drug, either by active or passive means to the desired site of action, such as for example solid tumors.
  • loading or entrapment or encapsulation are used interchangeably and refers to the non-covalent association of compound of formula (I) with a liposome bilayer and/or the liposome's interior aqueous volume (also called the liposome's aqueous core).
  • terapéuticaally effective amount refers to an amount of the compound of formula (I), when administered as part of a desired dosage regimen (to a mammal, preferably a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated.
  • excipient typically refers to any pharmacologically inactive substance used for in the formulation or administration of the liposomal compositions of the present invention, for example, a pH adjusting agent (e.g., a buffer) a buffer, a carrier or vehicle, an osmotic agent and so on.
  • a pH adjusting agent e.g., a buffer
  • pH adjusting agent or buffering agents refers to any agent used to modify the pH of an aqueous solution. pH is adjusted by using acidifying (e.g., acids) and alkalizing agents (e.g., salts of acids or bases). Acidifying agents are used in a formulation to lower the pH and alkalizing agents are used to increase the pH.
  • the pH adjusting agents include buffering systems (e.g., combinations of acids and bases). Pharmaceutical compositions of the present invention can contain one or more of these agents to achieve a desirable pH either for preparation.
  • the “osmotic agent” used in present invention may be selected from monosaccharides or disaccharides, that is, consist of one or two monosaccharide units, each having from three to seven, preferably from three to six carbon atoms.
  • osmotic agents used in present invention are, without limitation, monosaccharide hexoses, such as glucose (dextrose), galactose, mannose, fructose; monosaccharide pentoses, such as xylose, ribose, arabinose and disaccharides, such as lactose, trehalose, sucrose, maltose and cellobiose.
  • tumor (which is used interchangeably with “cancer” in the present invention) specifically includes solid tumors such as esophageal cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, laryngeal cancer, lung cancer, prostate cancer, bladder cancer, breast cancer, skin cancer, intestinal cancer, brain cancer, uterine cancer, ovarian cancer, and Kaposi's sarcoma, and liquid tumors such as leukemia.
  • Sites where a tumor occurs are cells, tissues, organs or intestines and the inside thereof.
  • counterion includes a moiety capable of forming an insoluble salt with compound of formula I and does not reduce the activity of the therapeutic agent.
  • diammonium citrate or dibasic ammonium citrate refers to a salt of citric acid in which two of the three carboxy groups of citric acid are deprotonated and associated with ammonium ions.
  • compound loaded liposomes refers to the liposomes loaded or encapsulated with a compound of formula (I) or salt thereof.
  • polymerized lipid refers to a derivatized lipid, wherein the lipid is derivatized by a hydrophilic polymer.
  • the said hydrophilic polymers are selected from those which are suitable for derivatization of a lipid.
  • liposome refers to a spherical vesicle having at least one lipid bilayer and/or interior aqueous core, used as a drug delivery vehicle for administration of pharmaceutical drug.
  • liposomal composition of compound of formula (I) refers to a liposome intended as delivery vehicle for administration of compound of formula (I) and wherein compound of formula (I) is loaded or entrapped or encapsulated into the liposome bilayer and/or into the liposome's interior aqueous core.
  • the present invention provides a liposomal composition
  • a liposomal composition comprising a compound of formula (I) or a salt thereof having improved therapeutic efficacy:
  • the compound of formula (I) is a camptothecin derivative.
  • the targeted drug delivery system of the present invention has been found to effectively deliver the compound of formula (I) when administered to the target tissue through intravenous injection thereby resulting in a greater tumor deposition of SN-38. This reduces unintended effects or side effects of the compound of formula (I), minimizes its toxicity and enhances its therapeutic efficacy.
  • the targeted drug delivery system of the present invention was found to improve drug efficacy in solid tumors. The size of tumors measured in terms of mean tumour volume was found to reduce significantly, compared to known SN-38 prodrugs like irinotecan and camptothecin derivatives like topotecan.
  • the type of targeted drug delivery systems may vary depending on the drug delivery route selected.
  • One of the most preferred routes of delivery for drug targeting is the intravenous route of administration.
  • the targeted drug delivery systems of the present invention may be in the form of liposomes, niosomes, lipid micelles, polymeric micelles, nanoparticles, nanospheres or dendrimers wherein the compound of formula (I) or salt thereof is formulated in such a manner that it is not released until it reaches the target site, such as a solid tumor, upon intravenous administration.
  • one aspect the present invention provides a liposomal composition of compound of formula (I): or a salt thereof.
  • the liposomal composition of the present invention comprises at least one lipid component and at least one sterol component.
  • the lipid component is selected from the group consisting of phosphatidyl choline, phosphatidyl ethanolamine; phosphatidyl serine, phosphatidylglycerol, phosphatidylinositol, sphingomyelin, phosphatidic acid, lecithin or derivatives thereof.
  • the lipid component is phosphatidyl choline or a derivative thereof, wherein the phosphatidyl choline derivatives are selected from hydrogenated soy phosphatidyl choline, dipalmitoylphosphatidylcholine, egg phosphatidylcholine, hydrogenated egg phosphatidylcholine, partially hydrogenated egg phosphatidylcholine, distearylphosphatidyl choline, dipalmitoyl phosphatidyl choline, soy phosphatidyl choline or diarachidoyl phosphatidyl choline.
  • the lipid component is hydrogenated soy phosphatidyl choline.
  • the sterol component is selected from the group consisting of cholesterol, coprostanol, cholestanol, cholestane, cholic acid, cortisol, corticosterone, hydrocortisone or calciferol or derivatives thereof.
  • the sterol component is cholesterol or a derivative thereof, wherein the cholesterol derivatives are selected from cholesteryl sulfate, cholesteryl hemisuccinate, cholesterol phosphate, cholesteryl phosphocholine, hydroxycholesterol, amino cholesterol, cholesteryl succinate, cholesteryl oleate or polyethylene glycol derivative of cholesterol.
  • the sterol component is cholesterol.
  • the lipid component is hydrogenated soy phosphatidyl choline and the sterol component is cholesterol.
  • the present invention provides a liposomal composition wherein the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of lipid component and sterol is at least 0.0001 to 0.5. In a preferred embodiment, the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of lipid component and sterol is at least 0.001 to 0.4. In another preferred embodiment, the molar ratio of the compound of formula (I) to the combined molar ratio of lipid component and sterol is 0.020 to 0.3. In yet another embodiment, the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of lipid component and sterol is 0. 13 to 0.15.
  • the lipid component is preferably one having two hydrocarbon chains, typically acyl chains, and a head group, either a polar or nonpolar head group.
  • hydrocarbon chains typically acyl chains
  • head group either a polar or nonpolar head group.
  • lipid components are primarily selected from glycerophospholipids and sphingomyelins where the two hydrocarbon chains are typically between about 14-24 carbon atoms in length, and have varying degrees of unsaturation.
  • the glycerophospholipids may have a glycerol backbone wherein at least one, preferably two, of the hydroxyl groups is substituted by one or two of an acyl, alkyl or alkenyl chain, and the third hydroxyl group is substituted by a phosphate (phosphatidic acid) or a phospho-ester such as phosphocholine group (as exemplified in phosphatidylcholine), being the polar head group of the glycerophospholipid or combination of any of the above, and/or derivatives of same and may contain a chemically reactive group (such as an amine, acid, ester, aldehyde or alcohol).
  • a chemically reactive group such as an amine, acid, ester, aldehyde or alcohol
  • the sphingomyelins may comprise, as an example, N- palmitoyl sphingomyelin, N-stearoyl sphingomyelin and other ceramides (N-acyl sphingosines) varied in their acyl chains unit having a phosphocholine moiety attached to ceramide position 1 as the polar head group.
  • the amide of ceramides can be replaced by other types of bonds such as a C— C bond as is the case for ceramines.
  • the substituting chain e.g. the acyl, alkyl or alkenyl chain in the glycerophospholipids or sphingolipid, is between about 14 to about 24 carbon atoms in length, and has varying degrees of saturation, thus resulting in fully, partially or nonhydrogenated (liposome-forming) lipids.
  • the lipids may be of a natural source, semi-synthetic or a fully synthetic lipid, and may be neutral, negatively or positively charged.
  • the lipid component is a phospholipid and more specifically, a phosphatidylcholine (PC) based phospholipid (lipid having a phosphocholine headgroup), including, without being limited thereto, substituted PC, hydrogenated soy phosphatidylcholine (HSPC), Dipalmitoylphosphatidylcholine (DPPC), egg yolk phosphatidylcholine (EPC), 1 -palmitoyl -2 -oleoylphosphatidyl choline (POPC), distearoylphosphatidylcholine (DSPC), and dimyristoyl phosphatidylcholine (DMPC).
  • PC phosphatidylcholine
  • HSPC hydrogenated soy phosphatidylcholine
  • DPPC Dipalmitoylphosphatidylcholine
  • EPC egg yolk phosphatidylcholine
  • POPC 1 -palmitoyl -2 -oleoylphosphatidyl cho
  • the sphingolipid may include, without being limited thereto, sphingomyelin, N- palmitoyl sphingomyelin, N-stearyl sphingomyelin, and ceramide.
  • Lipid components having main phase transition temperatures from approximately 2° C-80° C are suitable for use as the primary liposome component of the present composition.
  • a lipid having a main phase transition temperature above about 37° C is used as the lipid component of the liposomes.
  • a lipid having a phase transition temperature between about 37-70° C is used as the lipid component of the liposomes.
  • the lipid distearoyl phosphatidylcholine (DSPC) has a main phase transition temperature of 55.1° C and the lipid hydrogenated soy phosphatidylcholine (HSPC) has a phase transition temperature of 58° C.
  • the sterol component that may be used in the liposomal composition according to the present invention includes, but is not limited to, cholesterol or its derivatives, such as cholesteryl sulfate (CS), cholesteryl hemisuccinate, cholesterol phosphate, cholesteryl phosphocholine and other hydroxycholesterol or amino cholesterol derivatives, cholesteryl succinate, cholesteryl oleate, polyethylene glycol derivatives of cholesterol (cholesterol- PEG); and coprostanol, cholestanol, cholestane, cholic acid, cortisol, corticosterone, hydrocortisone, calciferol and the like.
  • the sterol component is cholesterol.
  • the present invention provides a liposomal composition wherein the molar ratio of the lipid component and sterol component is at least 1 to 90. In a preferred embodiment, the molar ratio of the lipid component to sterol component is from about 1 to 30. In a more preferred embodiment, the molar ratio of the lipid component to the sterol component is 1 to 3. In another preferred embodiment, the molar ratio of lipid component to sterol component is 1 to 2.5.
  • the liposomal composition further comprises a polymerized lipid component, wherein the polymerized lipid component is a lipid derivatized by a hydrophilic polymer.
  • the hydrophilic polymers suitable for derivatization with a lipid include polyethyleneglycol, polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline , polymethacrylamide , poly dimethylacrylamide , polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, and polyaspartamide.
  • the hydrophilic polymers may be employed as homopolymers or as block or random copolymers.
  • a preferred hydrophilic polymer chain is polyethyleneglycol (PEG), preferably as a PEG chain having a molecular weight between 500-10,000 daltons, more preferably between 500-5,000 daltons, most preferably between 1,000-2,000 daltons.
  • PEG polyethyleneglycol
  • Methoxy or ethoxy-capped analogues of PEG are also preferred hydrophilic polymers, which are commercially available in a variety of polymer sizes, e.g., 120-20,000 daltons.
  • the polymerized lipid component is a phospholipid derivatized by polyethyleneglycol.
  • PEG-derivatized phospholipids include, polyethylene glycol conjugated phosphatidyl ethanolamine, (PEG- PE), methoxy polyethylene glycol conjugated hydrogenated soy phosphatidylcholine (mPEG-HSPC), and methoxy polyethylene glycol conjugated distearoylphosphatidyl ethanolamine (mPEG-DSPE).
  • the hydrophilic polymer derivatised lipid is methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine (mPEG-DSPE).
  • the molar ratio of the polymerized lipid to the combined molar ratio of lipid component and sterol ranges from about 1 to 90. In another embodiment, the molar ratio of the polymerized lipid to the combined molar ratio of lipid component and sterol ranges from about 10 to 50. In a more preferred embodiment, the molar ratio of the polymerized lipid to the combined molar ratio of lipid component and sterol ranges from about 90 to 10. In one embodiment, the molar ratio of the polymerized lipid to the combined molar ratio of lipid component and sterol ranges from about 0.0001 to 90. In a preferred embodiment, the molar ratio ranges from about 0.001 to 50.
  • the molar ratio ranges from about 0.01 to 1.
  • the hydrophilic polymer derivatized lipids incorporated in drug delivery system makes them long circulating in nature.
  • the term ‘long circulating’ as used herein means that the targeted drug delivery system such as liposomes escape the reticuloendothelial system (RES) which is responsible for removal of foreign particles from the bloodstream and thus circulates in bloodstream for prolonged period.
  • RES reticuloendothelial system
  • Long circulating targeted drug delivery systems entrap the drug and increase the mean residence time of the drug in the plasma, which helps the targeted drug delivery system to reach the desired site of action.
  • the liposomal composition further comprises a counterion and a buffering agent.
  • the counterion may include an acid, which comprises a sodium or an ammonium form of a monovalent anion, a divalent anion or trivalent anion.
  • the monovalent anion may be selected from chloride, acetate, lactobionate or formate.
  • the divalent anion may be selected from aspartate, succinate or sulfate.
  • the trivalent anion may be selected from citrate or phosphate.
  • the counterion is citrate. In another preferred embodiment, the counterion is sulfate.
  • the counterion is selected from ammonium sulphate, diammonium citrate, triammonium citrate, phytic acid, polyphosphoric acid and ammonium glycerine trisulfate. In a preferred embodiment, the counterion is diammonium citrate.
  • the counterion is present in the liposomal compositions in an amount ranging from about 0.01 mM to 2000 mM, preferably in an amount ranging from about 1 to 1000 mM, more preferably in an amount ranging from about 100 to 500 mM.
  • the buffering agent is selected from sucrose histidine buffer, sucrose phosphate buffer, HEPES buffer, sucrose phosphate EDTA buffer, acetate buffer, citrate buffer, sucrose HEPES buffer, HEPES EDTA buffer, HEPES saline, histidine and sucrose.
  • the buffering agent is sucrose histidine buffer.
  • the present invention provides a liposomal composition
  • a liposomal composition comprising: a. compound of formula (I): b. at least one lipid component; and c. at least one sterol component.
  • the lipid component is selected from the group consisting of phosphatidyl choline, phosphatidyl ethanolamine; phosphatidyl serine, phosphatidylglycerol, phosphatidylinositol, sphingomyelin, phosphatidic acid, lecithin or derivatives thereof.
  • the lipid component is phosphatidyl choline or a derivative thereof, wherein phosphatidyl choline derivatives are selected from hydrogenated soy phosphatidyl choline, dipalmitoylphosphatidylcholine, egg phosphatidylcholine, hydrogenated egg phosphatidylcholine, partially hydrogenated egg phosphatidylcholine, distearylphosphatidyl choline, dipalmitoyl phosphatidyl choline, soy phosphatidyl choline or diarachidoyl phosphatidyl choline.
  • the lipid component is hydrogenated soy phosphatidyl choline.
  • the sterol component is selected from the group consisting of cholesterol, coprostanol, cholestanol, cholestane, cholic acid, cortisol, corticosterone, hydrocortisone or calciferol or derivatives thereof.
  • the sterol component is cholesterol or a derivative thereof, wherein the cholesterol derivatives are selected from cholesteryl sulfate, cholesteryl hemisuccinate, cholesterol phosphate, cholesteryl phosphocholine, hydroxycholesterol, amino cholesterol, cholesteryl succinate, cholesteryl oleate or polyethylene glycol derivative of cholesterol.
  • the sterol component is cholesterol.
  • the lipid component is hydrogenated soy phosphatidyl choline and the sterol component is cholesterol.
  • the present invention provides a liposomal composition wherein the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of lipid component and sterol is at least 0.0001 to 0.5. In a preferred embodiment, the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of lipid component and sterol is at least 0.001 to 0.4. In another preferred embodiment, the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of lipid component and sterol is 0.020 to 0.3. In yet another embodiment, the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of lipid component and sterol is 0.13 to 0.15.
  • the present invention provides a liposomal composition wherein the molar ratio of the lipid component and sterol component is at least 1 to 90. In a preferred embodiment, the molar ratio of the lipid component to sterol component is from about 1 to 30. In a more preferred embodiment, the molar ratio of the lipid component to the sterol component is 1 to 3. In another preferred embodiment, the molar ratio of lipid component to sterol component is 1 to 2.5.
  • the liposomal composition further comprises a polymerized lipid component, wherein the polymerized lipid component is a lipid derivatized by a hydrophilic polymer.
  • the hydrophilic polymers suitable for derivatization with a lipid include polyethyleneglycol, polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, and polyaspartamide.
  • the hydrophilic polymers may be employed as homopolymers or as block or random copolymers.
  • a preferred hydrophilic polymer chain is polyethyleneglycol (PEG), preferably as a PEG chain having a molecular weight between 500-10,000 daltons, more preferably between 500-5,000 daltons, most preferably between 1,000-2,000 daltons.
  • PEG polyethyleneglycol
  • Methoxy or ethoxy-capped analogues of PEG are also preferred hydrophilic polymers, which are commercially available in a variety of polymer sizes, e.g., 120-20,000 daltons.
  • the polymerized lipid component is a phospholipid derivatized by polyethyleneglycol.
  • PEG-derivatized phospholipids include, polyethylene glycol conjugated phosphatidyl ethanolamine, (PEG- PE), methoxy polyethylene glycol conjugated hydrogenated soy phosphatidylcholine (mPEG-HSPC), and methoxy polyethylene glycol conjugated distearoylphosphatidyl ethanolamine (mPEG-DSPE).
  • the hydrophilic polymer derivatised lipid is methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine (mPEG-DSPE).
  • the molar ratio of the polymerized lipid to the combined molar ratio of lipid component and sterol ranges from about 1 to 90. In another embodiment, the molar ratio of the polymerized lipid to the combined molar ratio of lipid component and sterol ranges from about 10 to 50. In a more preferred embodiment, the molar ratio of the polymerized lipid to the combined molar ratio of lipid component and sterol ranges from about 90 to 10. In one embodiment, the molar ratio of the polymerized lipid to the combined molar ratio of lipid component and sterol ranges from about 0.0001 to 90. In a preferred embodiment, the molar ratio ranges from about 0.001 to 50. In more preferred embodiment, the molar ratio ranges from about 0.01 to 1.
  • the liposomal composition further comprise a counterion and a buffering agent.
  • the counterion may include an acid, which comprises a sodium or an ammonium form of a monovalent anion, a divalent anion or trivalent anion.
  • the monovalent anion may be selected from chloride, acetate, lactobionate or formate.
  • the divalent anion may be selected from aspartate, succinate or sulfate.
  • the trivalent anion may be selected from citrate or phosphate.
  • the counterion is citrate. In another preferred embodiment, the counterion is sulfate.
  • the counterion is selected from ammonium sulphate, diammonium citrate, triammonium citrate, phytic acid, polyphosphoric acid and ammonium glycerine trisulfate. In a preferred embodiment, the counterion is diammonium citrate.
  • the counterion is present in the liposomal compositions in an amount ranging from about 0.01 mM to 2000 mM, preferably in an amount ranging from about 1 to 1000 mM, more preferably in an amount ranging from about 100 to 500 mM.
  • the buffering agent is selected from sucrose histidine buffer, sucrose phosphate buffer, HEPES buffer, sucrose phosphate EDTA buffer, acetate buffer, citrate buffer, sucrose HEPES buffer, HEPES EDTA buffer, HEPES saline, histidine and sucrose.
  • the buffering agent is a sucrose histidine buffer.
  • the present invention provides a liposomal composition
  • the lipid component is selected from the group consisting of phosphatidyl choline, phosphatidyl ethanolamine; phosphatidyl serine, phosphatidylglycerol, phosphatidylionositol, sphingomyelin, phosphatidic acid, lecithin or derivatives thereof.
  • the lipid component is phosphatidyl choline or a derivative thereof, wherein phosphatidyl choline derivatives are selected from hydrogenated soy phosphatidyl choline, dipalmitoylphosphatidylcholine, egg phosphatidylcholine, hydrogenated egg phosphatidylcholine, partially hydrogenated egg phosphatidylcholine, distearylphosphatidyl choline, dipalmitoyl phosphatidyl choline, soy phosphatidyl choline or diarachidoyl phosphatidyl choline.
  • the lipid component is hydrogenated soy phosphatidyl choline.
  • the sterol component is selected from the group consisting of cholesterol, coprostanol, cholestanol, cholestane, cholic acid, cortisol, corticosterone, hydrocortisone or calciferol or derivatives thereof.
  • the sterol component is cholesterol or a derivative thereof, wherein the cholesterol derivatives are selected from cholesteryl sulfate, cholesteryl hemisuccinate, cholesterol phosphate, cholesteryl phosphocholine, hydroxycholesterol, amino cholesterol, cholesteryl succinate, cholesteryl oleate or polyethylene glycol derivative of cholesterol.
  • the sterol component is cholesterol.
  • the lipid component is hydrogenated soy phosphatidyl choline and the sterol component is cholesterol.
  • the present invention provides a liposomal composition wherein the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of lipid component and sterol is at least 0.0001 to 0.5. In a preferred embodiment, the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of lipid component and sterol is at least 0.001 to 0.4. In another preferred embodiment, the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of lipid component and sterol is 0.020 to 0.3. In yet another embodiment the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of lipid component and sterol is 0.13 to 0.15.
  • the present invention provides a liposomal composition wherein the molar ratio of the lipid component and sterol component is at least 1 to 90. In a preferred embodiment, the molar ratio of the lipid component to sterol component is from about 1 to 30. In a more preferred embodiment, the molar ratio of the lipid component to the sterol component is 1 to 3. In another preferred embodiment, the molar ratio of lipid component to sterol component is 1 to 2.5.
  • the polymerized lipid component is a lipid derivatized by a hydrophilic polymer
  • the hydrophilic polymers suitable for derivatization with a lipid include, but are not limited to, polyethyleneglycol, polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, and polyaspartamide.
  • the hydrophilic polymers may be employed as homopolymers or as block or random copolymers.
  • a preferred hydrophilic polymer chain is polyethyleneglycol (PEG), preferably as a PEG chain having a molecular weight between 500-10,000 daltons, more preferably between 500-5,000 daltons, most preferably between 1,000-2,000 daltons.
  • PEG polyethyleneglycol
  • Methoxy or ethoxy-capped analogues of PEG are also preferred hydrophilic polymers, which are commercially available in a variety of polymer sizes, e.g., 120-20,000 daltons.
  • the polymerized lipid component is a phospholipid derivatized by polyethyleneglycol.
  • PEG-derivatized phospholipids include, polyethylene glycol conjugated phosphatidyl ethanolamine, (PEG- PE), methoxy polyethylene glycol conjugated hydrogenated soy phosphatidylcholine (mPEG-HSPC), and methoxy polyethylene glycol conjugated distearoylphosphatidyl ethanolamine (mPEG-DSPE).
  • the hydrophilic polymer derivatized lipid is methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine (mPEG-DSPE).
  • the molar ratio of the polymerized lipid to the combined molar ratio of lipid component and sterol ranges from about 1 to 90. In another embodiment, the molar ratio of the polymerized lipid to the combined molar ratio of lipid component and sterol ranges from about 10 to 50. In a more preferred embodiment, the molar ratio of the polymerized lipid to the combined molar ratio of lipid component and sterol ranges from about 90 to 10. In one embodiment, the molar ratio of the polymerized lipid to the combined molar ratio of lipid component and sterol ranges from about 0.0001 to 90. In a preferred embodiment, the molar ratio ranges from about 0.001 to 50. In more preferred embodiment, the molar ratio ranges from about 0.01 to 1.
  • the counterion may include an acid, which comprises a sodium or an ammonium form of a monovalent anion, a divalent anion or trivalent anion.
  • the monovalent anion may be selected from chloride, acetate, lactobionate or formate.
  • the divalent anion may be selected from aspartate, succinate or sulfate.
  • the trivalent anion may be selected from citrate or phosphate.
  • the counterion is citrate. In another preferred embodiment, the counterion is sulfate.
  • the counterion is selected from ammonium sulphate, diammonium citrate, triammonium citrate, phytic acid, polyphosphoric acid and ammonium glycerine trisulfate. In a preferred embodiment, the counterion is diammonium citrate.
  • the counterion is present in the liposomal compositions in an amount ranging from about 0.01 mM to 2000 mM, preferably in an amount ranging from about 1 to 1000 mM, more preferably in an amount ranging from about 100 to 500 mM.
  • the buffering agent is selected from a sucrose histidine buffer, sucrose phosphate buffer, HEPES buffer, sucrose phosphate EDTA buffer, acetate buffer, citrate buffer, sucrose HEPES buffer, HEPES EDTA buffer, HEPES saline, histidine and sucrose.
  • the buffering agent is a sucrose histidine buffer.
  • the present invention provides a liposomal composition
  • HSPC hydrogenated soy phosphatidyl choline
  • cholesterol cholesterol
  • DSPE2000 e. diammonium citrate
  • sucrose and histidine e.g. sucrose and histidine
  • the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of hydrogenated soy phosphatidyl choline (HSPC) and cholesterol is at least 0.0001 to 0.5. In a preferred embodiment, the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of hydrogenated soy phosphatidyl choline (HSPC) and cholesterol is at least 0.001 to 0.4. In another preferred embodiment, the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of hydrogenated soy phosphatidyl choline (HSPC) and cholesterol is 0.020 to 0.3. In yet another embodiment, the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of hydrogenated soy phosphatidyl choline (HSPC) and cholesterol is 0.13 to 0.15.
  • the molar ratio of methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine (mPEG-DSPE2000) to the combined molar ratio of hydrogenated soy phosphatidyl choline (HSPC) and cholesterol ranges from about 1 to 90. In another embodiment, the molar ratio of methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine (mPEG-DSPE2000) to the combined molar ratio of hydrogenated soy phosphatidyl choline (HSPC) and cholesterol ranges from about 10 to 50.
  • the molar ratio of methoxy polyethylene glycol- distearoylphosphatidyl ethanolamine (mPEG-DSPE2000) to the combined molar ratio of hydrogenated soy phosphatidyl choline (HSPC) and cholesterol ranges from about 90 to 10. In one embodiment, the molar ratio ranges from about 0.01 to 1. In yet another embodiment, the molar ratio ranges from about 0. 1 to 0.3
  • the diammonium citrate is present in an amount ranging from about 0.01 mM to 2000 mM, preferably in an amount ranging from about 1 to 1000 mM, more preferably in an amount ranging from about 100 to 500 mM.
  • the compound of formula (I) may be present as free base or as a salt thereof.
  • the compound of formula (I) is present as a hydrochloride salt.
  • the liposomes may be prepared by a variety of techniques. Typically, the liposomes are multilame liar vesicles (MLVs), which can be formed by simple lipid-film hydration techniques. In this procedure, a mixture of liposome-forming lipids and including a vesicleforming lipid derivatized with a hydrophilic polymer are dissolved in a suitable organic solvent which is evaporated in a vessel to form a dried thin fdm.
  • MLVs multilame liar vesicles
  • the therapeutic agent of choice can be incorporated into liposomes by standard methods, including (i) passive entrapment of a water-soluble compound by hydrating a lipid film with an aqueous solution of the agent, (ii) passive entrapment of a lipophilic compound by hydrating a lipid film containing the agent, and (iii) loading an ionizable drug against an inside/outside liposome ion gradient, termed remote loading.
  • Other methods such as reverse evaporation phase liposome preparation, are also suitable.
  • the samples for TEM imaging were prepared by using a carbon coated grid with a carbon coated side as top and the grid was placed inside the glow discharge unit for plasma expose up to 1 minute.
  • the grid was used for liposome cryo sample preparation.
  • An ethane container assembly was cooled with liquid nitrogen and once assembly was cooled, and liquid ethane was prepared in a brass cup located in the center of the assembly using pressurized ethane gas.
  • the ethane container assembly was transferred onto a vitrobot. Blot time was set to 7.0 sec and the relative humidity was maintained above 80%. Further, the treated grid was mounted using tweezers to the vitrobot plunger.
  • the sample for cryo microscopy was prepared by applying 5 pL of a liposome sample on the carbon coated side of the grid.
  • the grid was transferred to the grid box located in the ethane container assembly using tweezers.
  • the grid box was maintained at liquid nitrogen temperature till the grid was transferred to cryo holder.
  • the sample grid was transferred to the cryo holder maintained below -165°C using liquid nitrogen. After transferring the grid, the holder was inserted into the TEM immediately and the temperature of the holder was checked using a smart set controller (-165°C). The sample was allowed to stabilize for about 10 minutes and the images were captured in TEM FEI Tecanai G2 Spirit BioTwin®.
  • the components of liposomal compositions are selected to control the rate of release of the entrapped compound of formula (I) or salt thereof in the liposome. In another embodiment, at least 50% of the compound or salt thereof was released in mice plasma for at least seven days.
  • the liposomal composition releases 90% of total compound of formula (I) or salt thereof after four days from administration. In a preferred embodiment, the liposomal composition releases 90% of total compound of formula (I) or salt thereof after seven days from administration.
  • the compound of formula (I) or salt thereof can be loaded or entrapped inside liposomes with high loading efficiency.
  • the compound of formula (I) or salt thereof can be loaded in the liposomal composition at a concentration of Img/ml or more.
  • the compound of formula (I) or salt thereof can be loaded in the liposomal composition at a concentration of 2mg/ml or more.
  • high entrapment efficiencies of more than 85%, typically more than 90%, are achieved with the liposomal compositions.
  • the present invention provides method of preparing liposome composition comprising a compound of formula (I) or a salt thereof, by active loading procedure comprising the steps of:
  • step (c) adding a solution of the compound of formula (I) or salt thereof to the liposomal suspension of step (b) to form compound loaded liposomes.
  • the lipid solution comprises a lipid component and a sterol component dissolved in an organic solvent.
  • the organic solvent may be selected from ethanol, methanol, chloroform, dimethylsulfoxide or mixture thereof.
  • the lipid component is selected from the group consisting of phosphatidyl choline; phosphatidyl ethanolamine; phosphatidyl serine, phosphatidylglycerol, phosphatidylionositol, sphingomyelin, phosphatidic acid, lecithin or derivatives thereof.
  • the lipid component is selected from phosphatidyl choline or its derivative selected from hydrogenated soy phosphatidyl choline, dipalmitoylphosphatidylcholine, egg phosphatidylcholine, hydrogenated egg phosphatidylcholine, partially hydrogenated egg phosphatidylcholine, distearylphosphatidyl choline, dipalmitoyl phosphatidyl choline, soy phosphatidyl choline or diarachidoyl phosphatidyl choline.
  • the sterol component includes, but is not limited to cholesterol or its derivatives, such as cholesteryl sulfate (CS), cholesteryl hemisuccinate, cholesterol phosphate, cholesteryl phosphocholine and other hydroxycholesterol or amino cholesterol derivatives, cholesteryl succinate, cholesteryl oleate, polyethylene glycol derivatives of cholesterol (cholesterol-PEG); and coprostanol, cholestanol, cholestane, cholic acid, cortisol, corticosterone, hydrocortisone, calciferol and the like.
  • the sterol component is cholesterol.
  • the present invention provides a liposomal composition wherein the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of lipid component and sterol is at least 0.0001 to 0.5. In a preferred embodiment, the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of lipid component and sterol is at least 0.001 to 0.4. In another preferred embodiment, the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of lipid component and sterol is 0.020 to 0.3. In yet another embodiment, the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of lipid component and sterol is 0.13 to 0.15.
  • the present invention provides a liposomal composition wherein the molar ratio of the lipid component to sterol component is at least 1 to 90. In a preferred embodiment, the molar ratio of the lipid component to sterol component is from about 1 to 30. In a more preferred embodiment, the molar ratio of the lipid component to the sterol component is 1 to 3. In another preferred embodiment, the molar ratio of lipid component to sterol component is 1 to 2.5.
  • the counterion solution is prepared by adding a counterion to the vehicle e.g. water for injection, and pH of the solution is maintained at 4.5-5.5.
  • the liposomal composition according to the present invention further comprises at least one counterion and a buffering agent.
  • the counterion may include an acid, which comprises a sodium or an ammonium form of a monovalent anion, a divalent anion or tri valent anion.
  • the monovalent anion may be selected from chloride, acetate, lactobionate or formate.
  • the divalent anion may be selected from aspartate, succinate or sulfate.
  • the trivalent anion may be selected from citrate or phosphate.
  • the counterion is citrate. In another preferred embodiment, the counterion is sulfate.
  • the counterion used for drug loading is diammonium citrate. In another preferred embodiment, the counterion used for drug loading is ammonium sulphate.
  • the counterion is present in the liposomal compositions in an amount ranging from about 0.01 mM to 2000 mM, preferably in an amount ranging from about 1 to 1000 mM, more preferably in an amount ranging from about 100 to 500 mM.
  • the buffering agents used to prepare the liposome composition according to the present invention may be selected from a sucrose histidine buffer, sucrose phosphate buffer, HEPES buffer, sucrose phosphate EDTA buffer, acetate buffer, citrate buffer, sucrose HEPES buffer, HEPES EDTA buffer, HEPES saline, histidine and sucrose.
  • the buffering agent is a sucrose histidine buffer.
  • the liposomal composition of compound of formula (I) or salt thereof according to the present invention selectively delivers higher concentrations of SN-38 in tumor cells compared to that of concentration in blood.
  • the concentration of actual SN-38 in tumor cells is at least two folds higher as compared to the concentration in blood when administered as liposomal compositions according to the present invention.
  • the concentration of combined SN-38 (actual SN-38 + uncleaved SN-38 from the pro-drug) in tumor cells is at least four folds higher as compared to the concentration in blood when administered as liposomal compositions according to the present invention.
  • the liposomal composition of the compound of formula (I) or salt thereof according to present invention provides higher exposure of SN-38 in tumor as compared to that in blood.
  • the exposure of actual SN-38 in tumor is at least two folds higher than in blood when administered as the liposomal composition of the compound of formula (I) or salt thereof according to the present invention.
  • the exposure of actual SN-38 and uncleaved SN-38 in tumor is at least four folds higher than in blood when administered as the liposomal composition of the compound of formula (I) or salt thereof according to the present invention.
  • the present invention relates to a process for the preparation of liposomal composition of a compound of formula (I) or a salt thereof, comprising the steps of: a. preparing a lipid solution by dissolving hydrogenated soy phosphatidyl choline (HSPC), cholesterol and methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine (mPEG-2000-DSPE) in ethanol; b. adding the lipid solution of step a into a solution comprising diammonium citrate and water for injection at about 65°C to form liposomal suspension; c. adding a solution comprising the compound of formula (I), sucrose and histidine buffer to the liposomal suspension of step b at about 55 ⁇ 3°C to form liposomal composition of the compound of formula (I).
  • HSPC hydrogenated soy phosphatidyl choline
  • mPEG-2000-DSPE methoxy polyethylene glycol-distearoylphosphatidyl
  • the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of hydrogenated soy phosphatidyl choline (HSPC) and cholesterol is at least 0.0001 to 0.5. In a preferred embodiment, the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of hydrogenated soy phosphatidyl choline (HSPC) and cholesterol is at least 0.001 to 0.4. In another preferred embodiment, the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of hydrogenated soy phosphatidyl choline (HSPC) and cholesterol is 0.020 to 0.3. In yet another embodiment, the molar ratio of the compound of formula (I) or salt thereof to the combined molar ratio of hydrogenated soy phosphatidyl choline (HSPC) and cholesterol is 0.13 to 0.15.
  • the molar ratio of methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine (mPEG-DSPE2000) to the combined molar ratio of hydrogenated soy phosphatidyl choline (HSPC) and cholesterol ranges from about 1 to 90. In another embodiment, the molar ratio of methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine (mPEG-DSPE2000) to the combined molar ratio of hydrogenated soy phosphatidyl choline (HSPC) and cholesterol ranges from about 10 to 50.
  • the molar ratio of methoxy polyethylene glycol- distearoylphosphatidyl ethanolamine (mPEG-DSPE2000) to the combined molar ratio of hydrogenated soy phosphatidyl choline (HSPC) and cholesterol ranges from about 90 to 10. In one embodiment, the molar ratio ranges from about 0.01 to 1. In a preferred embodiment, the molar ratio ranges from about 0. 1 to 0.3.
  • the diammonium citrate is present in an amount ranging from about 0.01 mM to 2000 mM, preferably in an amount ranging from about 1 to 1000 mM, more preferably in an amount ranging from about 100 to 500 mM.
  • the present invention relates to a liposomal composition of a compound of formula (I) or a salt thereof, which is obtained by the process comprising the steps of:
  • step (c) adding a solution of the compound of formula (I) or salt thereof to the liposomal suspension of step (b) to form compound loaded liposomes.
  • the present invention relates to a liposomal composition of a compound of formula (I) or a salt thereof, which is obtained by the processes comprising the steps of: a. preparing a lipid solution by dissolving hydrogenated soy phosphatidyl choline (HSPC), cholesterol and methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine (mPEG-2000-DSPE) in ethanol; b. adding the lipid solution of step a into a solution comprising diammonium citrate and water for injection at about 65°C to form liposomal suspension; c. adding a solution comprising the compound of formula (I) or salt thereof, sucrose and histidine buffer to the liposomal suspension of step b at about 55 ⁇ 3°C to form liposomal composition of the compound of formula (I) or salt thereof.
  • HSPC hydrogenated soy phosphatidyl choline
  • mPEG-2000-DSPE methoxy polyethylene glycol-diste
  • the liposomes of the present invention comprising the compound of formula (I) or salt thereof have a final size between 20 and 1000 nm, more preferably between 50 and 500 nm, even more preferably between 30 and 300 nm.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the liposomal composition of the compound of formula (I) or salt thereof according to any of the aspects of the present invention.
  • the liposomal composition of the compound of formula (I) or salt thereof comprises a lipid component and a sterol component. In yet another embodiment, the liposomal composition of the compound of formula (I) or salt thereof further comprises a counterion and a buffering agent.
  • the liposomal composition is an injection.
  • the present invention provides a method of inhibiting the growth of a tumor, said method comprises administering the targeted drug delivery system of the present invention having the compound of formula (I) or salt thereof to a patient.
  • the present invention provides a method of inhibiting the growth of a tumor, wherein said method comprises administering to a subject in need thereof the liposomal composition of the compound of formula (I) or a salt thereof.
  • the present invention provides a method of inhibiting the growth of a tumor, wherein said method comprises administering to a subject in need thereof the liposomal composition of the compound of formula (I) or a salt thereof, comprising at least one lipid component and at least one sterol component.
  • the present invention provides a method of inhibiting the growth of a tumor, wherein said method comprises administering to a subject in need thereof the liposomal composition of the compound of formula (I) or a salt thereof, comprising at least one lipid component, at least one sterol component, a polymerized lipid component, a counterion and a buffering agent.
  • the present invention provides a method of inhibiting the growth of a tumor, wherein said method comprises administering to a subject in need thereof the liposomal composition of the compound of formula (I) or a salt thereof, wherein the liposomal composition of the compound of formula (I) or salt thereof comprises hydrogenated soy phosphatidyl choline (HSPC), cholesterol, methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine (mPEG-DSPE2000), diammonium citrate, sucrose and histidine.
  • HSPC hydrogenated soy phosphatidyl choline
  • mPEG-DSPE2000 methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine
  • diammonium citrate sucrose and histidine.
  • the present invention provides a method of treating solid tumors, said method comprising administering a targeted drug delivery system having the compound of formula (I) or salt thereof in therapeutically effective amounts to the cancer patient.
  • the present invention provides a method of treating solid tumors, wherein said method comprises administering to a subject in need thereof the liposomal composition of the compound of formula (I) or a salt thereof.
  • the present invention provides a method of treating solid tumors, wherein said method comprises administering to a subject in need thereof the liposomal composition of the compound of formula (I) or a salt thereof, comprising at least one lipid component and at least one sterol component.
  • the present invention provides a method of treating solid tumors, wherein said method comprises administering to a subject in need thereof the liposomal composition of the compound of formula (I) or a salt thereof, comprising at least one lipid component, at least one sterol component, a polymerized lipid component, a counterion and a buffering agent.
  • the present invention provides a method of treating solid tumors, wherein said method comprises administering to a subject in need thereof the liposomal composition of the compound of formula (I) or a salt thereof, comprising hydrogenated soy phosphatidyl choline (HSPC), cholesterol, methoxy polyethylene glycol- distearoylphosphatidyl ethanolamine (mPEG-DSPE2000), diammonium citrate, sucrose and histidine.
  • HSPC hydrogenated soy phosphatidyl choline
  • mPEG-DSPE2000 methoxy polyethylene glycol- distearoylphosphatidyl ethanolamine
  • diammonium citrate sucrose and histidine.
  • the liposomal composition of the present invention is useful in the inhibiting the growth of a tumor.
  • the liposomal composition of the present invention is useful in the treatment of solid tumors.
  • Said tumor may arise from a cancer selected from the group consisting of breast cancer, pancreatic cancer, colorectal cancer, colon cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, skin cancer, ovarian cancer, cervix cancer, prostate cancer, gastric cancer, gastrointestinal cancer, stomach cancer, head and neck cancer, kidney cancer, liver cancer and etc.
  • a cancer selected from the group consisting of breast cancer, pancreatic cancer, colorectal cancer, colon cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, skin cancer, ovarian cancer, cervix cancer, prostate cancer, gastric cancer, gastrointestinal cancer, stomach cancer, head and neck cancer, kidney cancer, liver cancer and etc.
  • the liposomal composition of the compound of formula (I) or salt thereof are useful in the treatment of cancers such as breast cancer, pancreatic cancer, colorectal cancer, colon cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, skin cancer, ovarian cancer, cervix cancer, prostate cancer, gastric cancer, gastrointestinal cancer, stomach cancer, head and neck cancer, brain cancer, skin cancer, kidney cancer, intestinal cancer, liver cancer or metastatic lesions of these cancers.
  • cancers such as breast cancer, pancreatic cancer, colorectal cancer, colon cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, skin cancer, ovarian cancer, cervix cancer, prostate cancer, gastric cancer, gastrointestinal cancer, stomach cancer, head and neck cancer, brain cancer, skin cancer, kidney cancer, intestinal cancer, liver cancer or metastatic lesions of these cancers.
  • the liposomal composition of the compound of formula (I) or salt thereof may be administered in combination with other anticancer agents.
  • the liposomal composition of the compound of formula (I) or salt thereof is effective in reducing triple negative breast cancer, colon cancer and small cell lung cancer when tested in mice xenograft.
  • the liposomal composition of present invention significantly stasis tumor growth when tested in triple negative breast cancer orthotopic mice models.
  • the liposomal composition provides at least 60% therapeutic response at 2.5mg/kg, 5mg/kg and 10 mg/kg dose levels.
  • the liposomal formulation of the compound of formula (I) or salt thereof was effective in providing antitumor response for up to twenty days after cessation of treatment.
  • the liposomal composition of present invention are administered as injections by parenteral routes such as intramuscular, subcutaneous, intravenous, intraarterial, intraperitoneal, intraarticular, intraepidural, intrathecal, or others.
  • parenteral routes such as intramuscular, subcutaneous, intravenous, intraarterial, intraperitoneal, intraarticular, intraepidural, intrathecal, or others.
  • the liposomal compositions may be administered by means as depot injections, or erodible implants.
  • Table 1 Liposomal composition of the compound of Formula (I) having Dibasic ammonium citrate as counterion (350 mM)
  • Phospholipids hydrogenated soy phosphatidyl choline (HSPC), cholesterol and methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine (mPEG-2000-DSPE) were dissolved in ethanol (10 % v/v) by gently heating in a circulating water bath at 65°C.
  • a counterion solution comprising diammonium citrate (350 mM) was prepared in water for injection (WFI) and the pH of the solution was maintained at 4.5-5.5.
  • a sucrose (10%) solution was prepared in water for injection and filtered using cationic nylon filter.
  • the lipid solution (65 °C) was injected slowly into the preheated diammonium citrate solution at temperature of about 65°C under constant stirring at 500 rpm.
  • the resulting multi-lamellar vesicles were subjected to further size reduction through polycarbonate membranes in a thermo barrel extruder (Nozex-90, Noozle Fluid Technology Co. Ltd., Shanghai, China®) at a temperature of about 65°C and 250 mbar nitrogen pressure.
  • the polycarbonate membranes with pore size 200 nm followed by 80 nm pore size were utilized to achieve the desired particle size Z-average 80 nm to 100 nm.
  • the extruded liposomes size with Z-average 80 to 100 nm were further processed in a diafdtration unit at 2-8°C using the sucrose solution as feed to remove external citrate. Further, the resulting placebo liposomes were filtered through 0.2 pm poly ether sulfone (PES) filter and the filtered liposomal dispersion was stored at 2-8°C before initiating compound loading process.
  • PES poly ether sulfone
  • Drug solution was prepared by dissolving the compound of formula (I) in a buffer solution (sucrose 100 mg/ml + histidine 1.5 mg/ml) until a clear pale yellow solution was obtained.
  • the resulting solution was added slowly to the preformed placebo liposomal dispersion at 55 ⁇ 3°C under stirring at 500 rpm and stirred for 1 hr to complete the drug loading process.
  • the compound loaded liposomes were cooled to room temperature (24°C). Further, the volume of the compound loaded liposomes dispersion was made up to the actual batch size using water for injection to yield the final concentration of 2 mg/ml.
  • the compound loaded liposomal dispersion was sterile filtered through 0.2 pm polyether sulfone (PES) membrane filters and filled in sterile glass vials as finished product and was stored in refrigerator at 2-8°C.
  • PES polyether sulfone
  • the liposomes size were measured using a Malvern Zetasizer Nano ZS, ZEN3600, Malvern instrument, UK®. A dispersion media with sucrose (10%) and 1.5 mg/ml of L- Histidine was used during the sample preparation and measurement. The temperature was set at 25°C with equilibration time of 120 seconds, the measurement angle was set at 173° backscatter with 50 number of runs and duration of 5 seconds was set for all the measurements.
  • the liposomal samples were allowed to attain room temperature and 1 mL samples were transferred to a 5 mL clean volumetric flask to make volume up to mark with dispersion medium. Further, the diluted sample was transferred to a disposable cuvette and particle size measurements were recorded in triplicate by withdrawing three different aliquots from the volumetric flask. The histograms were recorded for size distribution by intensity and particle size results were reported as Z-average and D10, D50, D90.
  • Example 2 Preparation of liposome with different concentrations of dibasic ammonium citrate as counterion:
  • Liposomes were prepared in the same manner as given in Example 1 having compositions given in Table 2.
  • Example 2.1 300 mM dibasic ammonium citrate
  • Example 2.2 350 mM dibasic ammonium citrate
  • Example 2.3 400 mM dibasic ammonium citrate
  • Table 2.1 Liposomal composition of the compound of Formula (I) having dibasic ammonium citrate in different concentrations.
  • the compound of formula (I) loaded liposome samples were diluted to 2 ml with plasma in glass test tubes and mix well with horizontal hand shaking and the sample test tubes were incubated in a water bath at 37°C for liposome stability study up to 168 hours. Withdraw of 0. 1ml plasma sample from the test tube at specific time points (0, 24, 48, 72, 96, 120, 144, 168 and 240 hours) was done and the samples were transferred into centrifuge tubes, and lOpL of Topotecan intermediate standard solution (200 ppm) was added. The samples were mixed, and then 900 pL of dispersion media was added.
  • Free compound of formula (I) was eluted from the cartridge using (0.5ml x 2) of diluent and the eluent was collected in a clean and dry test tube and the estimation of the compound of formula (I) in eluent was performed using the below HPLC condition.
  • Mobile phase A Prepare a mixture of buffer solution and Acetonitrile in the ratio of 90: 10. Degas prior to use.
  • Diluent Prepare a mixture of DMSO and Methanol in the ratio of 50:50.
  • Orthophosphoric acid in water Dilute 0.5 ml Orthophosphoric acid to 100 ml with water and mix well.
  • Example 3 Preparation of liposome having ammonium sulfate as counterion Liposomes were prepared in the same as manner as given in Example 1.
  • Example 3.1 1 mg/ml SNP019 liposome
  • Example 3.2 2 mg/ml SNP019 liposome (prepared by concentrating 1 mg/ml SNP019 liposome in diafiltration process)
  • Table 3 Liposomal compositions of the compound of formula (I) having ammonium sulfate as counterion (250 mM)
  • Liposomes were prepared in the same as manner as given in Example 1.
  • the compound of formula (I) loaded liposome samples were transferred into a 20 ml volumetric flask, followed by adding dispersion medium to dilute the volume up to mark and mixed well. The final sample concentration obtained was 50 ppm. Further, the samples were processed in solid phase extraction to collect the samples for analysis. The samples were analysed using HPLC (Agilent system (1200 series) with following conditions in gradient programme:
  • Example 6 In-vitro Plasma Stability of Liposomal composition of the compound of formula (I) in mice and human plasma:
  • the compound of formula (I) when administered as liposomal compositions remains in plasma for a longer time compared to, when the compound of formula (I) was administered in non-encapsulated form i.e., solution form. Even after seven days the compound of formula (I) was stable without substantial cleavage in the liposomal compositions whereas when given as such in solution form compound of formula (I) gets cleaved almost completely in plasma within 24 hrs and only 5.7% of compound was available in plasma at 24 hrs after administration.
  • Table 6 In-vitro stability of compound of formula (I) in human plasma
  • the compound of formula (I) when administered as liposomal compositions remains in plasma for a longer time compared to, when compound of formula (I) was administered in non-encapsulated form i.e., solution form.
  • the compound of formula (I) was not released in plasma even after seven days when given as liposomal compositions, whereas when compound of formula (I) was given in solution form, it gets cleaved in plasma and only 11% of the compound of formula (I) was available in plasma at 24 hrs after administration.
  • Example 7 Plasma and tumor pharmacokinetics of liposomal compositions of the compound of formula (I) after i.v. bolus administration in triple negative breast cancer orthotopic mice models.
  • Table 7.2 Tumor pharmacokinetics of the compound of formula (I) loaded liposomes
  • the tumor exposure of SN-38 in liposomal compositions of compound of formula (I) was at least greater than two folds compared to that of blood with reference to actual SN-38 and four folds considering actual SN-38 + SN-38 uncleaved from prodrug in the tumor.
  • Liposomes were prepared in the same manner as given in Example 1 having compositions given in Table 8.
  • Table 8 Liposomal composition of compound of Formula (I) having Dibasic ammonium citrate as counterion (350 mM)
  • Example 9 Tumor inhibition in SCLC NCI-H526 bearing xenograft mice model
  • Example 9.1 Compound of formula (I) liposomes (2 mg/ml; citrate as gradient) and Comparator-Onivyde (Irinotecan liposomes) - Q7D*4
  • SCLC small cell lung cancer
  • Example 9.2 Compound of formula (I) liposomes (2 mg/ml; citrate as gradient) and Comparator-Onivyde (Irinotecan liposomes) Q14D*2
  • SCLC small cell lung cancer
  • Example 10 Tumor inhibition in NCI-H29 bearing xenograft mice model
  • the treatment with the compound of formula (I) resulted in >90% tumor growth inhibition starting from -3 weeks of dosing sustaining upto -7 weeks, i.e. 3 weeks after cessation of treatment.
  • Onivyde showed 69% tumor growth inhibition starting from -3 weeks of dosing sustaining up to -2 weeks after cessation of treatment.
  • the results are represented in Fig. 4.
  • Example 11 Tumor inhibition in MDA-MB 231 bearing xenograft mice model-TNBC
  • Example 11.1 Compound of formula (I) liposomes (2mg/ml; citrate as gradient) at 2.5mg/kg, 5mg/kg and lOmg/kg and Comparator - placebo of liposomes - Q7D*4
  • tumors were induced by inoculation of the breast cancer cell line MDA-MB-231 into the mammary fat pad of mice. Treatment was initiated when the initial average tumor volume reached -200 mm 3 .
  • the compound of formula (I) showed a dose-dependent inhibition of tumor growth. Significant stasis with 80% TGI was observed at 5 and 10 mg/kg dose. Tumor started reasserting after -1 week of cessation of dosing. The lower dose of 2.5 mg/kg showed 60% TGI with early reassertion of tumors. No mortality was noted at any dose level studied. The results are represented in Fig. 5(a).
  • Example 11.2 Compound of formula (I) liposomes (2mg/ml; citrate as gradient) at 5mg/kg and Comparator - Onivyde (Irinotecan liposomes) and placebo of liposomes at different dosing schedules
  • one group of mice received intravenous doses on day 0, 7, 28 and 35 and the other group received two doses 4 weeks apart.
  • Onivyde was administered at 15 mg/kg dose, on day 0, 7, 28 and 35.
  • the results presented in Fig. 5(b) suggest that there was no difference in efficacy of Onivyde and the compound of formula (I) given in a similar dose regimen.
  • Two doses of the compound of formula (I) administered 4 weeks apart showed tumor regression lasting 1 week after the second dose.

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Abstract

La présente invention concerne une composition liposomale d'un composé de formule I : ou d'un sel de celui-ci. Les compositions liposomales de la présente invention sont utiles en tant que système d'administration de médicament ciblé. Le composé de formule (I) : (I) est encapsulé de manière stable et efficace ; (ii) reste dans le système sanguin pendant un laps de temps prolongé ; (iii) conserve une activité antitumorale ; et (iv) est efficacement administré au site d'action spécifique (par exemple, une tumeur).
PCT/IB2022/050264 2021-01-13 2022-01-13 Composition liposomale d'un dérivé de camptothécine WO2022153211A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5552156A (en) 1992-10-23 1996-09-03 Ohio State University Liposomal and micellular stabilization of camptothecin drugs
US8147867B2 (en) 2004-05-03 2012-04-03 Hermes Biosciences, Inc. Liposomes useful for drug delivery
CN106109415B (zh) * 2016-07-26 2019-01-29 金华市人民医院 一种载喜树碱类抗肿瘤药物脂质体、制备方法及其应用
WO2021005583A1 (fr) * 2019-07-11 2021-01-14 Sun Pharma Advanced Research Company Ltd. Dérivés de camptothécine ayant une fraction disulfure et une fraction pipérazine

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5552156A (en) 1992-10-23 1996-09-03 Ohio State University Liposomal and micellular stabilization of camptothecin drugs
US8147867B2 (en) 2004-05-03 2012-04-03 Hermes Biosciences, Inc. Liposomes useful for drug delivery
CN106109415B (zh) * 2016-07-26 2019-01-29 金华市人民医院 一种载喜树碱类抗肿瘤药物脂质体、制备方法及其应用
WO2021005583A1 (fr) * 2019-07-11 2021-01-14 Sun Pharma Advanced Research Company Ltd. Dérivés de camptothécine ayant une fraction disulfure et une fraction pipérazine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SUBRAMANIANMULLER, ONCOLOGY RESEARCH, vol. 7, no. 9, 1995, pages 461 - 469

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