+

WO2004087105A1 - Formulations associant du platine et des fluoropyrimidines - Google Patents

Formulations associant du platine et des fluoropyrimidines Download PDF

Info

Publication number
WO2004087105A1
WO2004087105A1 PCT/CA2004/000508 CA2004000508W WO2004087105A1 WO 2004087105 A1 WO2004087105 A1 WO 2004087105A1 CA 2004000508 W CA2004000508 W CA 2004000508W WO 2004087105 A1 WO2004087105 A1 WO 2004087105A1
Authority
WO
WIPO (PCT)
Prior art keywords
composition
carboplatin
liposomes
fudr
fluoropyrimidine
Prior art date
Application number
PCT/CA2004/000508
Other languages
English (en)
Other versions
WO2004087105A8 (fr
Inventor
Lawrence Mayer
Marcel Bally
Murray Webb
Paul Tardi
Clifford Shew
Original Assignee
Celator Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Celator Pharmaceuticals, Inc. filed Critical Celator Pharmaceuticals, Inc.
Publication of WO2004087105A1 publication Critical patent/WO2004087105A1/fr
Publication of WO2004087105A8 publication Critical patent/WO2004087105A8/fr

Links

Classifications

    • 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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers

Definitions

  • the invention relates to compositions and methods for improved delivery of combinations of therapeutic agents. More particularly, the invention concerns delivery systems which provide combinations of pyrimidine and platinum agents and analogs or derivatives thereof.
  • Platinum-based drugs such as cisplatin, carboplatin and oxaliplatin, are primarily employed because the platinum atom allows them to form DNA adducts which inhibit DNA synthesis and often induce programmed cell death (apoptosis). They exhibit activity against a wide range of tumors and are commonly used in combination therapies for solid tumors such as ovarian, lung, testicular, bladder, colorectal, gastric, head and neck and melanoma cancers. Specific platinum-based drugs are known to bind DNA in unique arrangements which may explain the tumor-specific activity demonstrated with different platinum agents. This has proven beneficial for second-line treatments against cancers that have exhibited resistance against a first-line treatment with a different platinum agent.
  • Pyrimidine analogs such as 5-FU and cytarabine (cytosine arabinoside or araC) are antimetabolites that resemble pyrimidine nucleotides. Most antimetabolites have different modes of action.
  • 5-FU acts as a suicide inactivator of thymidylate synthase, covalently modifying the enzyme's active site.
  • Thymidylate synthase which is the limiting irreversible step in de novo synthesis of DNA, catalyzes the conversion of dUMP to dTMP. Temporary blockage of this step results in cell death.
  • cytarabine is bioactivated to araCMP by cellular enzymes which allows it to compete with CTP as an alternate substrate for DNA polymerase.
  • the araCMP incorporates into the DNA therefore inhibiting further synthesis of the growing DNA strand.
  • liposomes have the ability to provide this 'shielding' effect and they are thus able to, extend the half-life of therapeutic agents. Encapsulation into well-designed delivery vehicles can also result in coordinated pharmacokinetics of encapsulated drugs. However, formulation of specific drugs or more than one drug into delivery vehicles has proven to be difficult because the lipid composition of the vehicle often differentially affects the pharmacokinetics of individual drugs. Thus a composition that is suitable for retention and release of one drug may not be suitable for the retention and release of a second drug.
  • European patent application EPO 715854 by Oguru and Ohnishi describes a combination of certain platinum agents with carcinostatic drugs (such as 5-FU) to promote the anticancer activity of the carcinostatic agent; however, no formulations designed to control delivery or half-lives of the drugs were suggested.
  • carcinostatic drugs such as 5-FU
  • the invention relates to compositions and methods for administering effective amounts of fluoropyrimidine/platinum drug combinations using liposomal delivery vehicles that are stably associated therewith at least one fluoropyrimidine and one platinum-based drug.
  • These compositions allows the two or more agents to be delivered to the disease site in a coordinated fashion, thereby assuring that the agents will be present at the disease site at a desired ratio. This result will be achieved whether the agents are co-encapsulated in lipid-based delivery vehicles, or are separately encapsulated in a single lipid-based delivery vehicle administered such that desired ratios are maintained at the disease site.
  • the pharmacokinetics (PK) of the composition are controlled by the lipid-based delivery vehicles themselves such that coordinated delivery is achieved (provided that the PK of the delivery systems are comparable).
  • the invention provides a liposome composition for parenteral administration comprising at least one fluoropyrimidine and one platinum agent associated with the liposomes at therapeutically effective ratios especially those that are non-antagonistic.
  • the therapeutically effective 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, as well as tumor cell lysates from individual patient biopsies, over a range of concentrations.
  • Preferred combinations are carboplatin and fluorouracil (5-FU), carboplatin and fluorodeoxyuridine (FUDR), oxaliplatin and 5-FU or oxaliplatin and FUDR. Any method which results in determination of a ratio of agents which maintains a desired therapeutic effect may be used.
  • the composition comprises at least one fluoropyrimidine and one platinum agent in a mole ratio of the fluoropyrimidine to the platinum agent which exhibits a desired biologic effect to relevant cells in culture, cell-free systems or tumor cell lysates.
  • the ratio is that at which the agents are non-antagonistic.
  • relevant cells applicants refer to at least one cell culture or cell line which is appropriate for testing the desired biological effect.
  • “relevant” cells are those of cell lines identified by the Developmental Therapeutics Program (DTP) of the National Cancer Institute (NCI)/National Institutes of Health (NIH) as useful in their anticancer drug discovery program.
  • DTP Developmental Therapeutics Program
  • NCI National Cancer Institute
  • NH National Institutes of Health
  • tumor cell lysate refers to cells generated from the homogenization of patient biopsies or tumors. Extraction of whole tumors or tumor biopsies can be achieved through standard medical techniques by a qualified physician and homogenization of the tissue into single cells can be carried out in the laboratory using a number of methods well-known in the art.
  • the invention is directed to a method to deliver a therapeutically effective amount of a fluoropyrimidine/platinum agent combination to a desired target by administering the compositions of the invention.
  • the invention is also directed to a method to deliver a therapeutically effective amount of a fluoropyrimidine/platinum agent combination by administering a fluoropyrimidine stably associated with a first delivery vehicle and a platinum agent stably associated with a second delivery vehicle.
  • the first and second delivery vehicles may be contained in separate vials, the contents of the vials being administered to a patient simultaneously or sequentially.
  • the ratio of the fluoropyrimidine and the platinum agent is non-antagonistic.
  • leucovorin a compound related to the vitamin, folic acid
  • compositions of the invention in order to stabilize the fluoropyrimidines in vivo.
  • the invention is directed to a method to prepare a therapeutic composition comprising liposomes containing a ratio of at least one fluoropyrimidine and one platinum agent which provides a desired therapeutic effect
  • a method to prepare a therapeutic composition comprising liposomes containing a ratio of at least one fluoropyrimidine and one platinum agent which provides a desired therapeutic effect
  • method comprises providing a panel of at least one fluoropyrimidine and one platinum agent wherein the panel comprises at least one, but preferably a multiplicity of ratios of said drugs, testing the ability of the members of the panel to exert a biological effect on a relevant cell culture, cell-free system or tumor cell lysate over a range of concentrations, selecting a member of the panel wherein the ratio provides a desired therapeutic effect on said cell culture, cell-free system or tumor cell lysate over a suitable range of concentrations; and stably associating the ratio of drugs represented by the successful member of the panel into lipid-based drug delivery vehicles.
  • the non-antagonistic ratios are selected as those that have a combination index (CI) of ⁇ 1.1.
  • suitable liposomal formulations are designed such that they stably incorporate an effective amount of a fluoropyrimidine/platinum agent combination and allow for the sustained release of both drugs in vivo.
  • Preferred formulations contain at least one negatively charged lipid, such as phosphatidylglycerol.
  • Figure 1 A is a graph showing the combination index (CI) plotted as a function of the fraction of HCT-116 human colorectal carcinoma cells affected by combinations of FUDR: carboplatin at various mole ratios: 10:1 (circles); 5:1 (squares); 1:1 (upward triangles); 1 :5 (inverted triangles); and 1:10 (diamonds).
  • Figure IB is a graph showing the combination index (CI) plotted as a function of the fraction of L1210 murine lymphocytic leukemia cells affected by combinations of FUDR:carboplatin at various mole ratios: 10:1 (circles); 5:1 (squares); 1:1 (upward triangles); 1:5 (inverted triangles); and 1:10 (diamonds).
  • Figure 1C is a graph showing the combination index (CI) plotted as a function of the fraction of NCI-H322 human bronchoalveolar carcinoma cells affected by combinations of FUDR: carboplatin at various mole ratios: 10:1 (circles); 5:1 (squares); 1 :1 (upward triangles); 1:5 (inverted triangles); and 1:10 (diamonds).
  • Figure ID is a graph showing the compiled data sets from various tumor types plotted as a function of their relative synergy values from FUDRxarboplatin at a mole ratio of 1:5.
  • Figure 2A is a graph comparing carboplatin retention (% carboplatin to lipid) over time in DSPC/DSPG liposomes containing 150 mM sucrose, 10 mM HEPES, pH 7.4 with passively entrapped FUDR and carboplatin at a 1 :5 FUDR/carboplatin mole ratio.
  • Figure 2B is a graph comparing FUDR retention (% FUDR to lipid) over time in DSPC/DSPG liposomes containing 150 mM sucrose, 10 mM HEPES, pH 7.4 with passively entrapped FUDR and carboplatin at a 1 :5 FUDR/carboplatin mole ratio.
  • Figure 2C is a graph comparing carboplatin retention (% carboplatin to lipid) over time in DSPC/DSPG (60:40 and 55:45 mole ratios), DSPC/DSPG/Chol (55:40:5 mole ratio) and DSPC/DSPG/SM (60:30:10 mole ratio) liposomes containing 150 mM sucrose, 10 mM Hepes, pH 7.4 with passively entrapped FUDR and carboplatin at a 1 :5 FUDR/carboplatin mole ratio.
  • Figure 2D is a graph comparing FUDR retention (% FUDR to lipid) over time in DSPC/DSPG (60:40 and 55:45 mole ratios), DSPC/DSPG/Chol (55:40:5 mole ratio) and DSPC/DSPG/SM (60:30:10 mole ratio) liposomes containing 150 mM sucrose, 10 mM Hepes, pH 7.4 with passively entrapped FUDR and carboplatin at a 1:5 FUDR/carboplatin mole ratio.
  • Figure 2E is a graph comparing carboplatin retention (% carboplatin to lipid) over time in DSPC/DSPG (90:10 and 80:20 mole ratios) liposomes containing 150 mM sucrose, 10 mM Hepes, pH 7.4 with passively entrapped FUDR and carboplatin at a 1 :1 FUDR/carboplatin mole ratio.
  • Figure 2F is a graph comparing carboplatin retention (% carboplatin to lipid) over time in DSPC/DSPG (70:30, 60:40 and 50:50 mole ratios) liposomes containing 150 mM sucrose, 10 mM Hepes, pH 7.4 with passively entrapped FUDR and carboplatin at a 1 :5 FUDR/carboplatin mole ratio.
  • Figure 2G is a graph comparing FUDR retention (% FUDR to lipid) over time in DSPC/DSPG (90:10 and 80:20 mole ratios) liposomes containing 150 mM sucrose, 10 mM Hepes, pH 7.4 with passively entrapped FUDR and carboplatin at a 1 :1 FUDR/carboplatin mole ratio.
  • Figure 2H is a graph comparing FUDR retention (% FUDR to lipid) over time in DSPC/DSPG (70:30, 60:40 and 50:50 mole ratios) liposomes containing 150 mM sucrose, 10 mM Hepes, pH 7.4 with passively entrapped FUDR and carboplatin at a 1 :5 FUDR/carboplatin mole ratio.
  • Figure 3 A is a graph comparing carboplatin retention in plasma over time in DAPC/DSPG/Chol (60:30:10 mole ratio) and DSPC/DSPG/Chol (60:30:10 mole ratio) liposomes containing 150 mM sucrose, 10 mM HEPES, pH 7.4 with passively entrapped FUDR and carboplatin at a 1 :2 FUDR/carboplatin mole ratio.
  • Figure 3B is a graph comparing FUDR retention in plasma over time in DAPC/DSPG/Chol (60:30:10 mole ratio) and DSPC/DSPG/Chol (60:30:10 mole ratio) liposomes containing 150 mM sucrose, 10 mM HEPES, pH 7.4 with passively entrapped FUDR and carboplatin at a 1 :2 FUDR/carboplatin mole ratio.
  • Figure 3C is a graph comparing carboplatin retention (% initial carboplatin to lipid) over time in DAPC/DSPG/Chol (60:30:10 mole ratio) and DSPC/DSPG/Chol (60:30:10 mole ratio) liposomes containing 150 mM sucrose, 10 mM HEPES, pH 7.4 with passively entrapped FUDR and carboplatin at a 1 :2 FUDR/carboplatin mole ratio.
  • Figure 3D is a graph comparing FUDR retention (% initial FUDR to lipid) over time in DAPC/DSPG/Chol (60:30:10 mole ratio) and DSPC/DSPG/Chol (60:30:10 mole ratio) liposomes containing 150 mM sucrose, 10 mM HEPES, pH 7.4 with passively entrapped FUDR and carboplatin at a 1 :2 FUDR/carboplatin mole ratio.
  • Figure 4 is a graph of the carboplatin:FUDR ratio (mole ratio) over time in the plasma as a function of time after intravenous administration of DAPC/DSPG/Chol (60:30:10 mole ratio) and DSPC/DSPG/Chol (60:30:10 mole ratio) liposomes containing 150 mM sucrose, 10 mM HEPES, pH 7.4 with passively entrapped FUDR and carboplatin at a 1 :2 FUDR/carboplatin mole ratio.
  • Figure 5 A is a graph of the carboplatin/FUDR ratio (mol/mol) in the plasma as a function of time after intravenous administration of carboplatin/FUDR (1 :1) dual-loaded liposomes with varying DSPG content.
  • Figure 5B is a graph of the carboplatin/FUDR ratio (mol/mol) in the plasma as a function of time after intravenous administration of carboplatin/FUDR (1 :5) dual-loaded liposomes with varying DSPG content.
  • Figure 6 is a graph of the carboplatin/FUDR ratio (mol/mol) in the plasma as a function of time after intravenous administration of carboplatin/FUDR (1:5) in DSPC/DSPG (60:40 and 55:45 mole ratios), DSPC/DSPG/Chol (55:40:5 mole ratio) and DSPC/DSPG/SM (60:30:10 mole ratio) liposomes.
  • Figure 7 is a graph of FUDR: Carboplatin (1 :2 molar ratio) in vivo efficacy when administered intravenously into CD-I nude mice inoculated with Colon-26 murine colorectal solid tumors.
  • Figure 8A is a graph comparing carboplatin retention (% carboplatin to lipid) over time in DSPC/DSPG (60:40 mole ratio) liposomes containing 150 mM sucrose, 10 mM Hepes, pH 7.4 with passively entrapped FUDR and carboplatin at 1:1, 1 :2 and 1 :5 FUDR/carboplatin mole ratios.
  • Figure 8B is a graph comparing FUDR retention (% FUDR to lipid) over time in DSPC/DSPG (60:40 mole ratio) liposomes containing 150 mM sucrose, 10 mM Hepes, pH 7.4 with passively entrapped FUDR and carboplatin at 1:1, 1 :2 and 1 :5 FUDR/carboplatin mole ratios.
  • Figure 9A is a graph comparing carboplatin retention (% carboplatin to lipid) over time in DSPC/DSPG/SM (60:30:10 mole ratio) liposomes containing 150 mM sucrose, 10 mM Hepes, pH 7.4 with passively entrapped FUDR and carboplatin at 1:1, 1 :2 and 1 :5 FUDR/carboplatin mole ratios.
  • Figure 9B is a graph comparing FUDR retention (% FUDR to lipid) over time in DSPC/DSPG/SM (60:30:10 mole ratio) liposomes containing 150 mM sucrose, 10 mM Hepes, pH 7.4 with passively entrapped FUDR and carboplatin at 1 :1, 1 :2 and 1 :5 FUDR/carboplatin mole ratios.
  • compositions comprising liposomes stably associated therewith at least one fluoropyrimidine and one platinum agent, wherein the fluoropyrimidine and platinum agent are present at fluoropyrimidine/platinum mole ratios that exhibit a desired cytotoxic, cytostatic or biologic effect to relevant cells or tumor cell lysates.
  • liposomal compositions provided herein will include liposomes stably associated therewith at least one fluoropyrimidine and one platinum agent in a mole ratio of the fluoropyrimidine/platinum agent which exhibits a non-antagonistic effect to relevant cells or tumor cell lysates.
  • liposomal compositions of the invention will include liposomes stably associated therewith either 5-FU or FUDR and carboplatin. More preferably, 5-FU or FUDR and carboplatin will be present in compositions of the invention at a 5-FU (or FUDR): carboplatin mole ratio of between 100:1 and 1 :100, even more preferably the mole ratio of 5-FU or FUDR to carboplatin will be in the range of 1 : 1 and 1:10.
  • the above described lipid-based delivery vehicles comprise a third or fourth agent. Any therapeutic, diagnostic or cosmetic agent may be included.
  • DSPC distearoylphosphatidylcholine
  • PG phosphatidylglycerol
  • DSPG distearoylphosphatidylglycerol
  • PI phosphatidylinositol
  • SM sphingomyelin
  • Choi or CH cholesterol
  • CHE cholesteryl hexadecyl ether
  • SUV small unilamellar vesicle
  • LUV large unilamellar vesicle
  • MLV multilamellar vesicle
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide
  • EDTA ethyl enediaminetetraacetic acid
  • HEPES hydrogen peroxide
  • liposomes which comprise sphingomyelin are provided.
  • liposomes which comprise a sterol are provided.
  • the sterol is cholesterol.
  • the lipid-based delivery vehicles of the present invention may be used not only in parenteral administration but also in topical, nasal, subcutaneous, intraperitoneal, intramuscular, aerosol or oral delivery or by the application of the delivery vehicle onto or into a natural or synthetic implantable device at or near the target site for therapeutic purposes or medical imaging and the like.
  • the lipid-based delivery vehicles of the invention are used in parenteral administration, most preferably, intravenous administration.
  • leucovorin is administered with compositions of the invention in order to stabilize the fluoropyrimidines in vivo.
  • Fluoropyrimidine analogs of uracil, cytosine, or thymine, and the corresponding nucleosides are well known anticancer agents. Many such pyrimidine analogs or derivatives act as antimetabolites in that they closely resemble an essential metabolite and therefore interfere with physiological reactions involving it.
  • a common mechanism of action for pyrimidine analogs is to inhibit the enzyme, thymidylate synthase. This inhibition prevents the methylation of dUMP (deoxyuridine monophosphate) and subsequent generation of dihydrofolate and thymidylate, which is an essential precursor in DNA synthesis. The result of this interference is inhibition of DNA biosynthesis.
  • fluorinated pyrimidine analogs such as fluorouracil (5-FU) and fluorodeoxyuridine (floxuridine or FUDR), which have been shown to have significant antitumor activity in humans.
  • Fluoropyrimidines refers to pyrimidine analogs that have been derivatized with a fluorine atom. Fluoropyrimidines of the present invention are recognized in the art as being equivalents of 5-FU or FUDR, including but not limited to, UFT (uracil-tegafur), Capecitabine, Futraful (FT-207) and prodrugs, precursors, metabolic products of 5-FU or FUDR such as FdUMP (5 fluoro-deoxyuridine monophosphate) and FUTP (fluoro-uridine triphosphate) and the like.
  • UFT uracil-tegafur
  • Capecitabine Capecitabine
  • Futraful Futraful
  • prodrugs precursors, metabolic products of 5-FU or FUDR such as FdUMP (5 fluoro-deoxyuridine monophosphate) and FUTP (fluoro-uridine triphosphate) and the like.
  • UFT uracil and tegafur
  • Capecitabine is a prodrug that is selectively tumour-activated to its cytotoxic moiety, fluorouracil, by thymidine phosphorylase. Fluorouracil is further metabolized to two active metabolites, 5-fluoro-2-deoxyuridine monophosphate (FdUMP) and 5-fluorouridine triphosphate (FUTP), within normal and tumour cells.
  • FdUMP causes a reduction in normal thymidine production which inhibits DNA synthesis, while FUTP inhibits RNA and protein synthesis by competing with uridine triphosphate.
  • fluoropyrimidines for use in the invention are 5-FU, FUDR or tegafur/uracil. More preferably, the fluoropyrimidine is FUDR or 5-FU. Most preferably, the fluoropyrimidine is FUDR.
  • Leucovorin may also be administered in conjunction with compositions of the invention. This compound has no antineoplastic activity; however, it is a standard practice of care with the FDA when treating patients with 5-FU as it results in a significant increase in the life span of 5-FU. Leucovorin acts by stabilizing the binding of 5-FU (and FUDR) to its target enzyme, Thymidylate synthase, therefore protecting it from mechanisms that would otherwise lead to its clearance from the blood. The reduced clearance of the fluoropyrimidine allows it to exhibit a higher cytotoxic effect.
  • Platinum Agents [0054] In recent years metal-based therapeutic agents have played a relevant role in chemotherapy treatments. In particular, platinum-based compounds, are proving to be some of the most effective anticancer drugs used in clinical practice. Platinum agents, as incorporated herein, are used primarily because they form DNA adducts that block DNA and RNA synthesis and apoptosis. The nature of the platinum/DNA adducts has been widely investigated by hydrolysis of DNA into nucleotides. Studies have shown that the adduct is typically a cross link involving the N-7 of DNA purine (adenine (A) and guanine (G)) bases.
  • A DNA purine
  • G guanine
  • the preferential complex for platinum compounds such as cisplatin is an intrastrand crosslink at the dinucleotides GG (62% occurrence) and AG (22% occurrence).
  • intrastrand cross linking has been shown to correlate with the clinical response to . cisplatin therapy.
  • a platinum agent must have two relatively labile leaving groups to react with the DNA bases.
  • platinum agents have a central platinum atom bonded to four ligands, two of which are reactive. In the case of cisplatin, the platinum atom is linked to two amino groups and two chloride (leaving) groups.
  • Platinum agents therefore refers to therapeutic drugs that contain a reactive platinum atom, including derivatized and underivatized forms of cisplatin and its related compounds which have the essential features of containing a reactive platinum atom.
  • Approximately 3,000 platinum analogs have been synthesized over the past 30 years; however, only 6 are presently in clinical development, including cisplatin, carboplatin and oxaliplatin.
  • Cisplatin and carboplatin dominate the world platinum/cancer market and have become critical elements in the standard practice of care for numerous solid tumors including, ovarian, lung, testicular, bladder, gastric, melanoma and head and neck cancers.
  • Oxaliplatin (a newer platinum) has a different mechanism of action than either cisplatin or carboplatin which has proven to be especially important in cisplatin-resistant models and cell lines expressing resistance genes.
  • Cisplatin cw-diamminedichoroplatinum (II)
  • II cw-diamminedichoroplatinum
  • cisplatin is its toxicity. Common side effects include kidney damage, nerve damage, high-end hearing loss, and prolonged nausea and vomiting. Because of this, development of structurally related cisplatin analogs (of the cis isomer) or second-generation drugs, which are incorporated herein, has focused on those platinum-based complexes which may act through different mechanisms of action than cisplatin. Ideally these agents would also demonstrate reduced toxicity, enhanced efficacy, and/or activity against cisplatin-resistant cancers. [0059] Carboplatin (cis-diammine- 1 , 1 -cyclobutanedicarboxylateplatinum) is one of the best-characterized cisplatin analogs.
  • Oxaliplatin trans- 1 -diaminocyclohexane oxalatoplatinum
  • DACH diaminocyclohexane platinum family
  • Oxaliplatin is a new platinum salt that belongs to the DACH (diaminocyclohexane) platinum family, and is the only such cisplatin analog that has entered clinical development and achieved approval for marketing. It demonstrates good clinical tolerance with the absence of renal or auditory toxicity. The exact mechanism of action of oxaliplatin is unclear. It is known to form reactive platinum complexes which are believed to inhibit DNA synthesis by forming interstrand and intrastrand cross-linking of DNA molecules; however, it binds in a different location on DNA than cisplatin or carboplatin.
  • oxaliplatin It is also a larger and bulkier compound than either cisplatin or carboplatin, which makes it harder to separate from the DNA once bound.
  • Another difference between oxaliplatin and the other platinum compounds is its spectrum of cytotoxicity, or ability to induce cell death. In particular, it has shown activity against six colorectal cell lines that both cisplatin and carboplatin have limited cytotoxicity, demonstrating the selective nature of these platinum-based compounds.
  • the "platinum agent” is selected based on its activity against a particular cell type or tumor.
  • the platinum agent is cisplatin, carboplatin or oxaliplatin. Most preferably, the platinum agent is carboplatin or oxaliplatin.
  • platinum agents and fluoropyrimidines will be encapsulated into liposomes at synergistic or additive (i.e. non-antagonistic) ratios. Determination of ratios of agents that display synergistic or additive combination effects may be carried out using various algorithms, based on the types of experimental data described below. These methods include isobologram methods (Loewe, et al, Arzneim-Forsch (1953) 3:285-290; Steel, et «/., Int. J. Radiol. Oncol. Biol Phys. (1979) 5:27-55), the fractional product method (Webb, Enzyme and Metabolic Inhibitors (1963) Vol. 1, pp.
  • D D m [f a /(l-f a )] 1/m in which D is the dose of the drug used, f a is the fraction of cells affected by that dose, D m is the dose for median effect signifying the potency and m is a coefficient representing the shape of the dose-effect curve (m is 1 for first order reactions).
  • This equation can be further manipulated to calculate a combination index (CI) on the basis of the multiple drug effect equation as described by Chou and Talalay, Adv. Enzyme Reg. (1984) 22:27-55; and by Chou, et al, in: Synergism and Antagonism in Chemotherapy. Chou and Rideout, eds., Academic Press: New York 1991:223-244.
  • CI combination index
  • the combination index equation is based on the multiple drug-effect equation of Chou-Talalay derived from enzyme kinetic models.
  • An equation determines only the additive effect rather than synergism and antagonism.
  • synergism is defined as a more than expected additive effect
  • antagonism as a less than expected additive effect.
  • Equation 1 or equation 2 dictates that drug 1, (D) l5 and drug 2, (D) 2 , (in the numerators) in combination inhibit x % in the actual experiment. Thus, the experimentally observed x % inhibition may not be a round number but most frequently has a decimal fraction.
  • (D x )i and (D x ) 2 (in the denominators) of equations 1 and 2 are the doses of drug 1 and drug 2 alone, respectively, inhibiting x %.
  • a two-drug combination may be further used as a single pharmaceutical unit to determine synergistic or additive interactions with a third agent.
  • a three-agent combination may be used as a unit to determine non-antagonistic interactions with a fourth agent, and so on.
  • the underlying experimental data are generally determined in vitro using cells in culture or cell-free systems.
  • the combination index (CI) is plotted as a function of the fraction of cells affected (f a ) as shown in Figures 1A to 1C which, as explained above, is a surrogate parameter for concentration range.
  • Preferred combinations of agents are those that display synergy or additivity over a substantial range of f a values. Combinations of agents are selected that display synergy over at least 5% of the concentration range wherein greater than 1% of the cells are affected, i.e., an f a range greater than 0.01.
  • a larger portion of overall concentration exhibits a favorable CI; for example, 5% of an f a range of 0.2-1.0. More preferably 10% of this range exhibits a • favorable CI. Even more preferably, 20 % of the f a range, preferably over 50 % and most preferably over at least 70 % of the f a range of 0.2 to 1.0 are utilized in the compositions. Combinations that display synergy over a substantial range of f a values may be re-evaluated at a variety of agent ratios to define the optimal ratio to enhance the strength of the non-antagonistic interaction and increase the f a range over which synergy is observed.
  • the optimal combination ratio may be further used as a single pharmaceutical unit to determine synergistic or additive interactions with a third agent.
  • a three-agent combination may be used as a unit to determine non-antagonistic interactions with a fourth agent, and so on.
  • the combination of agents is intended for anticancer therapy.
  • Appropriate choices will then be made of the cells to be tested and the nature of the test.
  • tumor cell lines are suitable subjects and measurement of cell death or cell stasis is an appropriate end point.
  • other target cells and criteria other than cytotoxicity or cell stasis could be employed.
  • cell lines may be obtained from standard cell line repositories (NCI or ATCC for example), from academic institutions or other organizations including commercial sources.
  • Preferred cell lines would include one or more selected from cell lines identified by the Developmental Therapeutics Program of the NCI/NIH.
  • the tumor cell line screen used by this program currently identifies 60 different tumor cell lines representing leukemia, melanoma, and cancers of the lung, colon, brain, ovary, breast, prostate and kidney.
  • the required non-antagonistic effect over a desired concentration range need be shown only on a single cell type; however, it is preferred that at least two cell lines exhibit this effect, more preferably three cell lines, more preferably five cell lines, and more preferably 10 cell lines.
  • the cell lines maybe established tumor cell lines or primary cultures obtained from patient samples.
  • the cell lines may be from any species but the preferred source will be mammalian and in particular human.
  • the cell lines may be genetically altered by selection under various laboratory conditions, and/or by the addition or deletion of exogenous genetic material.
  • Cell lines may be transfected by any gene-transfer technique, including but not limited to, viral or plasmid-based transfection methods. The modifications may include the transfer of cDNA encoding the expression of a specific protein or peptide, a regulatory element such as a promoter or enhancer sequence or antisense DNA or RNA.
  • tissue culture cell lines may include lines with and without tumor suppressor genes, that is, genes such as p53, pTEN and pl6; and lines created through the use of dominant negative methods, gene insertion methods and other selection methods.
  • Preferred tissue culture cell lines that maybe used to quantify cell viability, e.g., to test antitumor agents include, but are not limited to, H460, MCF-7, SF-268, HT29, HCT-116, LSI 80, B16-F10, A549, Capan pancreatic, CAOV-3, IGROV1, PC-3, ' MX-1 and MDA-MB-231.
  • the given effect (f a ) refers to cell death or cell stasis after application of a cytotoxic agent to a cell culture.
  • Cell death or viability may be measured, for example, using the following methods:
  • BCECF Bis-carboxyethyl-carboxyfluorescein
  • SRB Sulforhodamine B
  • the "MTT assay” is preferred.
  • Non-antagonistic ratios of two or more agents can be determined for disease indications other than cancer and this information can be used to prepare therapeutic formulations of two or more drugs for the treatment of these diseases.
  • many measurable endpoints can be selected from which to define drug synergy, provided those endpoints are therapeutically relevant for the specific disease.
  • tumor cell lysates generated from homogenization of the tumor sample(s) into single cells.
  • the given effect (f a ) refers to cell death or cell stasis after application of a cytotoxic agent to a "relevant" cell culture or “tumor cell lysate” (see Example 1).
  • Cell death or viability may be measured using a number of methods known in the art.
  • the “MTT” assay Mosmann, J. Immunol Methods (1983) 65(l-2):55-63) detailed in Example 1 is preferred .
  • lipid carriers for use in this invention are liposomes.
  • Liposomes can be prepared as described in Liposomes: Rational Design (A.S. Janoff, ed., Marcel Dekker, Inc., New York, NY), or by additional techniques known to those knowledgeable in the art.
  • Suitable liposomes for use in this invention include large unilamellar vesicles (LUVs), multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs) and interdigitating fusion liposomes.
  • Liposomes for use in this invention may be prepared to contain a sphingolipid, such as sphingomyelin. Liposomes of the invention may also contain a sterol, such as cholesterol. Liposomes may also contain therapeutic lipids, which examples include ether lipids, phosphatidic acid, phosphonates, ceramide and ceramide analogs, sphingosine and sphingosine analogs and serine-containing lipids.
  • Liposomes may also be prepared with surface stabilizing hydrophilic polymer-lipid conjugates such as polyethylene glycol-DSPE, to enhance circulation longevity.
  • hydrophilic polymer-lipid conjugates such as polyethylene glycol-DSPE
  • PG phosphatidylglycerol
  • PI phosphatidylinositol
  • Preferred embodiments of this invention may make use of liposomes containing phosphatidylglycerol (PG) or phosphatidylinositol (PI) to prevent aggregation thereby increasing the blood residence time of the carrier.
  • liposome compositions in accordance with this invention are preferably used to treat cancer. Delivery of encapsulated drugs to a tumor site is achieved by administration of liposomes of the invention. Preferably liposomes have a diameter of less than 300 nm. Most preferably liposomes have a diameter of less than 200 nm. Tumor vasculature is generally leakier than normal vasculature due to fenestrations or gaps in the endothelia. This allows the delivery vehicles of 200 nm or less in diameter to penetrate the discontinuous endothelial cell layer and underlying basement membrane surrounding the vessels supplying blood to a tumor. Selective accumulation of the delivery vehicles into tumor sites following extravasation leads to enhanced anticancer drug delivery and therapeutic effectiveness.
  • Encapsulation includes covalent or non-covalent association of an agent with the lipid-based delivery vehicle. For example, this can be by interaction of the agent with the outer layer or layers of the liposome or entrapment of an agent within the liposome, equilibrium being achieved between different portions of the liposome.
  • encapsulation of an agent can be by association of the agent by interaction with the bilayer of the liposomes through covalent or non-covalent interaction with the lipid components or entrapment in the aqueous interior of the liposome, or in equilibrium between the internal aqueous phase and the bilayer.
  • Loading refers to the act of encapsulating one or more agents into a delivery vehicle.
  • Encapsulation of the desired combination can be achieved either through encapsulation in separate delivery vehicles or within the same delivery vehicle. Where encapsulation into separate liposomes is desired, the lipid composition of each liposome may be quite different to allow for coordinated pharmacokinetics.
  • release rates of encapsulated drugs can be matched to allow desired ratios of the drugs to be delivered to the tumor site. Means of altering release rates include increasing the acyl-chain length of vesicle forming lipids to improve drug retention, controlling the exchange of surface grafted hydrophilic polymers such as PEG out of the liposome membrane and incorporating membrane-rigidifying agents such as sterols or sphingomyelin into the membrane.
  • a first and second drag are desired to be administered at a specific drag ratio and if the second drag is retained poorly within the liposome composition of the first drag (e.g., DMPC/Chol), that improved pharmacokinetics may be achieved by encapsulating the second drag in a liposome composition with lipids of increased acyl chain length (e.g., DSPC/Chol).
  • a liposome composition with lipids of increased acyl chain length e.g., DSPC/Chol.
  • ratios of platinum agents-to-fluoropyrimi dines that have been determined on a patient-specific basis to provide optimal therapeutic activity will be generated for individual patients by combining the appropriate amounts of each liposome-encapsulated drag prior to administration.
  • two or more agents may be encapsulated within the same liposome.
  • Techniques for encapsulation are dependent on the nature of the delivery vehicles.
  • therapeutic agents may be loaded into liposomes using both passive and active loading methods.
  • Passive methods of encapsulating active agents in liposomes involve encapsulating the agent during the preparation of the liposomes. This includes a passive entrapment method described by Bangham, et al. (J. Mol Biol (1965) 12:238). This technique results in the formation of multilamellar vesicles (MLVs) that can be converted to large unilamellar vesicles (LUVs) or small unilamellar vesicles (SUVs) upon extrusion.
  • MLVs multilamellar vesicles
  • LUVs large unilamellar vesicles
  • SUVs small unilamellar vesicles
  • Active methods of encapsulation include the pH gradient loading technique described in U.S. patent Nos, 5,616,341, 5,736,155 and 5,785,987 and active metal-loading.
  • a preferred method of pH gradient loading is the citrate-base loading method utilizing citrate as the internal buffer at a pH of 4.0 and a neutral exterior buffer.
  • Other methods employed to establish and maintain a pH gradient across a liposome involve the use of an ionophore that can insert into the liposome membrane and transport ions across membranes in exchange for protons (see U.S. patent No. 5,837,282).
  • a recent technique utilizing transition metals to drive the uptake of drags into liposomes via complexation in the absence of an ionophore may also be used. This technique relies on the formation of a drag-metal complex rather than the establishment of a pH gradient to drive uptake of drag.
  • Passive and active methods of entrapment may also be coupled in order to prepare a liposome formulation containing more than one encapsulated agent.
  • the delivery vehicle compositions of the present invention may be administered to warm-blooded animals, including humans as well as to domestic avian species.
  • a qualified physician will determine how the compositions of the present invention should be utilized with respect to dose, schedule and route of administration using established protocols.
  • Such applications may also utilize dose escalation should agents encapsulated in delivery vehicle compositions of the present invention exhibit reduced toxicity to healthy tissues of the subject.
  • the pharmaceutical compositions of the present invention are administered parenterally, i.e., intraarterially, intravenously, intraperitoneally, subcutaneously, or intramuscularly. More preferably, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection.
  • parenterally i.e., intraarterially, intravenously, intraperitoneally, subcutaneously, or intramuscularly.
  • the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection.
  • a bolus injection for example, see Rahman, et al, U.S. patent No. 3,993,754; Sears, U.S. patent No. 4,145,410; Papahadjopoulos, et al, U.S. patent No. 4,235,871; Schneider, U.S. patent No. 4,224,179; Lenk, et al, U.S. patent No.
  • the pharmaceutical or cosmetic preparations of the present invention can be contacted with the target tissue by direct application of the preparation to the tissue.
  • the application may be made by topical, "open” or “closed” procedures.
  • topical it is meant the .direct application of the multi-drag preparation to a tissue exposed to the environment, such as the skin, oropharynx, external auditory canal, and the like.
  • Open procedures are those procedures that include incising the skin of a patient and directly visualizing the underlying tissue to which the pharmaceutical preparations are applied.
  • a surgical procedure such as a thoracotomy to access the lungs, abdominal laparotomy to access abdominal viscera, or other direct surgical approach to the target tissue.
  • "Closed" procedures are invasive procedures in which the internal target tissues are not directly visualized, but accessed via inserting instruments through small wounds in the skin.
  • the preparations may be administered to the peritoneum by needle lavage.
  • the preparations may be administered through endoscopic devices.
  • compositions comprising delivery vehicles of the invention are prepared according to standard techniques and may comprise water, buffered water, 0.9% saline, 0.3% glycine, 5% dextrose and' the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, and the like. These compositions may be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and the like.
  • the delivery vehicle suspension may include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage. Lipophilic free-radical quenchers, such as alpha-tocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.
  • Leucovorin may also be administered with compositions of the invention through standard techniques to enhance the life span of administered fluoropyrimidines.
  • the concentration of delivery vehicles in the pharmaceutical formulations can vary widely, such as from less than about 0.05%, usually at or at least about 2-5% to as much as 10 to 30% by weight and will be selected primarily by fluid volumes, viscosities, and the like, in accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment. Alternatively, delivery vehicles composed of irritating lipids may be diluted to low concentrations to lessen inflammation at the site of administration. For diagnosis, the amount of delivery vehicles administered will depend upon the particular label used, the disease state being diagnosed and the judgment of the clinician.
  • the pharmaceutical compositions of the present invention are administered intravenously. Dosage for the delivery vehicle formulations will depend on the ratio of drag to lipid and the administrating physician's opinion based on age, weight, and condition of the patient.
  • suitable formulations for veterinary use may be prepared and administered in a manner suitable to the subject.
  • Preferred veterinary subjects include mammalian species, for example, non-human primates, dogs, cats, cattle, horses, sheep, and domesticated fowl.
  • Subjects may also include laboratory animals, for example, in particular, rats, rabbits, mice, and guinea pigs.
  • kits which include, in separate containers, a first composition comprising delivery vehicles stably associated with at least a first therapeutic agent and, in a second container, a second composition comprising delivery vehicles stably associated with at least one second therapeutic agent. The containers can then be packaged into the kit.
  • the kit will also include instructions as to the mode of administration of the compositions to a subject, at least including a description of the ratio of amounts of each composition to be administered.
  • the kit is constructed so that the amounts of compositions in each container is pre-measured so that the contents of one container in combination with the contents of the other represent the correct ratio.
  • the containers may be marked with a measuring scale permitting dispensation of appropriate amounts according to the scales visible.
  • the containers may themselves be useable in administration; for example, the kit might contain the appropriate amounts of each composition in separate syringes. Formulations which comprise the pre-formulated correct ratio of therapeutic agents may also be packaged in this way so that the formulation is administered directly from a syringe prepackaged in the kit.
  • lipids were dissolved in a chloroform/methanol/water solution, combined together, dried under a stream of nitrogen gas and placed in a vacuum pump to remove residual solvent. Trace levels of radioactive lipid lA C-CHE were added to quantify lipid. The resulting lipid film was placed under high vacuum for a minimum of 2 hours. The lipid film was hydrated in the solution indicated to form multilamellar vesicles (MLVs). The resulting preparation was extruded 10 times through stacked polycarbonate filters with an extrusion apparatus (Lipex Biomembranes, Vancouver, BC) to achieve a mean liposome size between 80 and 150 nm.
  • an extrusion apparatus Lipex Biomembranes, Vancouver, BC
  • Measuring additive, synergistic or antagonistic effects was performed using FUDR/carboplatin at 10:1, 5:1, 1:1, 1:5 and 1:10 mole ratios in HCT-116 human colon adenocarcinoma, L1210 murine lymphocytic leukemia, and NCI-H322 human bronchoalveolar carcinoma cells.
  • the standard tetrazolium-based colorimetric MTT viability assay protocol (Mosmarm, et al, J. Immunol Methods (1983) 65(l-2):55-63) was utilized to determine the readout for the fraction of cells affected.
  • viable cells reduce the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazplium bromide (MTT) to a blue formazan which can be read spectrophotometrically.
  • Cells such as the HCT-116 colon cell line used here, are grown in 25 cm flasks and passaged (passage number ⁇ 20), resuspended in fresh cell culture medium and added into 96-well cell culture plates at a concentration of 1000 cells per well in 100 ⁇ L per well. The cells are then allowed to incubate for 24 hours at 37°C, 5% C0 2 . The following day, serial drug dilutions are prepared in 12-well cell culture plates.
  • agents previously prepared in various solutions, are diluted in fresh cell culture media. Agents are administered to the appropriate wells for single agents (20 ⁇ L) and at specific fixed ratio dual agent combinations (increments of 20 ⁇ L) using a Latin square design or "checkerboard" dilution method. The total well volumes are made up to 200 ⁇ L with fresh media. The drag exposure is for a duration of 72 hours.
  • MTT reagent (1 mg/niL in RPMI) is added to each well at a volume of 50 ⁇ L per well and incubated for 3-4 hours.
  • the well contents are then aspirated and 150 ⁇ L of dimethylsulfoxide (DMSO) is added to each well to disrupt the cells and to solubilize the formazan precipitate within the cells.
  • DMSO dimethylsulfoxide
  • the 96-well plates are shaken on a plate shaker, and read on a microplate spectrophotometer set at a wavelength of 570 nm.
  • the optical density (OD) readings are recorded and the OD values of the blank wells (containing media alone) are subtracted from all the wells containing cells.
  • the cell survival following exposure to agents is based as a percentage of the control wells (cells not exposed to drug). All wells are performed in triplicate and mean values are calculated.
  • a combination index was then determined for each FUDR/carboplatin dose using CalcuSyn which is, based on Chou and Talalay's theory of dose-effect analysis, in which a "median-effect equation" has been used to calculate a number of biochemical equations that are extensively used in the art. Derivations of this equation have given rise to higher order equations such as those used to calculate Combination Index (CI).
  • CI can be used to determine if combinations of more than one drug and various ratios of each combination are antagonistic (CI > 1.1), additive (0.9 ⁇ CI > 1.1) or synergistic (CI ⁇ 0.9).
  • CI plots are typically illustrated with CI representing the y-axis versus the proportion of cells affected, or fraction affected (f a ), on the x-axis.
  • FUDR/carboplatin at ratios of 1 : 10 or 1 : 5 in HCT- 116 cells synergy is observed over the entire range of fraction affected values (0.2 to 1.0) ( Figure 1 A). This demonstrates that a 1:10 or 1:5 ratio is synergistic independent of the concentration of drug used.
  • a 5:1 or 1 :1 ratio is non-antagonistic at f a values up to 0.75 while synergistic at f a values less than 0.6.
  • a 10:1 ratio of FUDR/carboplatin is additive (slightly antagonistic) over almost all f a values.
  • Figure IB presents results of the combinations on L 1210 cells and
  • Figure 1C presents results of the combinations on NCI-H322 cells.
  • FIG. 1 A further representation used to identify an optimal drug ratio is to prepare a relative synergy plot, Figure ID. This method of analysis is useful for identifying a common synergistic drag:drag ratio from many cell types.
  • Figure ID consists of compiled data sets at a 1 :5 molar ratio from various cell types, as indicated on the ordinate axis.
  • the relative synergy values shown on the abscissa are computed CI values obtained from CalcuSyn that have been normalized to zero by subtracting 1 from the original CI value, i.e., CI values of 0, 1, and 2 are equivalent to relative synergy values of -1, 0, and 1, respectively.
  • a mole ratio of 1 :5 or 1 :1 FUDR arboplatin was selected for pharmacokinetic studies described in the following examples as these ratios demonstrate synergistic effects over a broad concentration range and over a variety of cell types.
  • liposomes of various compositions were used to passively entrap both drags. Variations of DSPC/DSPG liposomes were investigated.
  • lipids are commonly used in the art to decrease aggregation and thus enhance the circulation lifetime of lipid-based carriers.
  • a negatively charged lipid e.g., DSPG
  • DSPC/DSPG liposomes were prepared using varying amounts of DSPG.
  • a fluoropyrimidine and platinum agent were both passively encapsulated during liposome preparation and the in vivo drag release from each liposomal composition was determined in the plasma of male SCID-Rag2M mice.
  • Lipid films were prepared by dissolving lipids together to 50 mg/ml, in chloroform/methanol/water (1/2/0.0625 volume ratio). Following solvent removal, the resulting lipid films were hydrated at 70°C with a solution consisting of 150 mM sucrose, 10 mM HEPES, pH 7.4 plus 5 mg of FUDR (with trace amounts of 3 H-FUDR)/50 mg lipid and 40 mg of carboplatin/50 mg of lipid. This corresponds to a 1 :5 mole ratio of FUDR to carboplatin. The resulting MLVs were extruded at 70°C to generate LUVs.
  • the mean diameter of these liposomes was then determined by QELS (quasi-elastic light scattering). Subsequently, the liposomes were buffer exchanged into 150 mM sucrose, 10 mM HEPES, pH 7.4, using a hand-held tangential flow column to remove any unencapsulated FUDR and carboplatin.
  • DSPC/DSPG liposomes were prepared as described above at molar lipid ratios of 90:10, 80:20, 70:30, 60:40 and 50:50 DSPC to DSPG.
  • FUDR and carboplatin were passively entrapped into liposomes during preparation at a 1:5 mole ratio, identified as being synergistic.
  • Doses of the liposomal formulations administered were 2.65 mg/kg of FUDR, 20 mg/kg of carboplatin (a 1 :5 molar ratio of FUDR/carboplatin) and 425.8 mg/kg of lipid.
  • Figure 2A shows the percent of initial carboplatin encapsulated, as measured in the plasma, at various time points after intravenous administration to mice. Data points represent the mean concentration (percent initial) of drug determined in plasma (+/- standard deviation) at the specified time points. The trend in the graph shows that as the amount of DSPG increases, there is a corresponding increase in the release of carboplatin, with the most extensive leakage seen in DSPC/DSPG (50:50) liposomes.
  • Figure 2B shows the percent of FUDR encapsulated in the same liposomal preparations used in Figure 2A. Similar to the release of carboplatin, as the amount of DSPG is increased, there is a corresponding increase in the release of FUDR. All formulations show a gradual release of drag with the exception of a 50:50 molar lipid ratio where there is an initial burst of FUDli released followed by a leveling off where minimal FUDR is released.
  • DSPC/DSPG liposomes were prepared as described above at molar lipid ratios of DSPC/DSPG 60:40, DSPC/DSPG 55:45, DSPC/DSPG/cholesterol 55:40:5, and DSPC/DSPG/sphingomyelin 60:30:10.
  • FUDR and carboplatin were passively entrapped into liposomes during preparation at a 1:5 mole ratio.
  • Doses of liposomal formulations administered were 10.8 mg/kg of FUDR, 53.9 mg/kg of carboplatin (a 1:5 molar ratio of FUDR/carboplatin) and 539 mg/kg of lipid.
  • Figure 2C shows the percent of initial carboplatin encapsulated, as measured in the plasma, at various time points after intravenous administration to mice. Data points represent the mean concentration (percent initial) of drag determined in plasma (+/- standard deviation) at the specified time points. In each of the four liposomal compositions a slow, steady release of carboplatin is seen, with a slightly faster release in DSPC/DSPG (55:45) liposomes.
  • Figure 2D shows the percent of FUDR encapsulated, as determined in the plasma of SCID-Rag2M, after intravenous administration. As seen in the graph in Figure 2D, similar release kinetics are observed for each of the four liposomal compositions. A desired, gradual release of both drugs over time is observed for all formulations.
  • DSPC/DSPG liposomes were prepared as described above at molar lipid ratios of 90:10, 80:20, 70:30, 60:40 and 50:50 DSPC to DSPG.
  • FUDR and carboplatin were passively entrapped into liposomes during preparation at either a 1 :1 or 1 :5 mole ratio.
  • Doses of the liposomal formulations administered to Rag2M mice were 20.0 mg/kg FUDR, 20.0 mg/kg carboplatin and 200 mg/kg lipid for a 1 : 1 FUDR/carboplatin mole ratio ( Figures 2E and 2G); and 10.8 mg/kg FUDR, 53.9 mg/kg carboplatin and 539 mg/kg lipid for a 1 :5 FUDR/carboplatin mole ratio ( Figures 2F and 2H).
  • Figure 2E shows the percent of initial carboplatin encapsulated, as determined in the plasma, in DSPC/DSPG liposomes (co-loaded with FUDR and carboplatin) formulated at a 90:10 and 80:20 DSPC:DSPG molar lipid ratio.
  • Figure 2F shows the amount of carboplatin encapsulated in 70:30, 60:40 and 50:50 DSPC/DSPG liposomes co-formulated with FUDR and carboplatin. The trend in both graphs shows that as the amount of DSPG increases, there is a corresponding increase in the release of liposomal carboplatin, with the most extensive leakage seen in DSPC/DSPG (50:50) liposomes.
  • FIGS. 2G and 2H respectively show the percent of FUDR encapsulated in the same liposomal preparations used in Figures 2E and 2F. Similar to the release of carboplatin, as the amount of DSPG is increased, there is a corresponding increase in the release of FUDR. All formulations in Figures 2E-2H show a gradual release of drag with the exception of a 50:50 molar lipid ratio where there is an initial burst of FUDR released followed by a leveling off where minimal FUDR is released.
  • DAPC/DSPG/Chol (6:3:1 molar ratio)
  • DSPC/DSPG/Chol (6:3:1 molar ratio) liposomes were prepared.
  • DAPC lipid consists of a 20 carbon chain structure
  • DSPC lipid consists of an 18 carbon chain structure.
  • a fluoropyrimidine and platinum agent were both passively encapsulated during liposome preparation and the in vivo drug release from each liposomal composition was determined in the plasma of female CD-I mice.
  • DAPC or DSPC/DSPG/Chol liposomes were prepared as described in Example 2. Cholesterol was added as a component because it is well known to enhance drag retention, reduce lipid aggregation and/or extend the circulation lifetime of the vehicles in vivo FUDR and carboplatin were passively entrapped into liposomes during preparation at a 1 :2 mole ratio.
  • Figure 3A and 3B shows the carboplatin and FUDR concentration in the plasma (expressed in ⁇ moles per ml of plasma) for the DAPC:DSPG:Chol and the DSPC:DSPG:Chol liposomal formulations.
  • Figure 3C and 3D shows the carboplatin and FUDR concentrations in the plasma (expressed as % initial drag:lipid molar ratio). The trend in the graphs shows that DAPC containing liposomes have better retention of the drags, mainly carboplatin, than DSPC containing liposomes.
  • DAPC/DSPG/Chol (6:3:1 molar ratio) and DSPC/DSPG/Chol (6:3:1 molar ratio) liposomes were prepared as described in Example 2.
  • FUDR and carboplatin were passively entrapped into liposomes during preparation at a 1 :2 mole ratio.
  • FIG 4 shows plasma levels of FUDR and carboplatin were effectively maintained at a synergistic mole ratio, as determine in Example 1.
  • Plasma levels of FUDR were roughly equal to that of carboplatin at various time points after intravenous administration to CD-I mice when they were delivered in DAPC/DSPG/Chol (6:3:1 molar ratio) and DSPC/DSPG/Chol (6:3:1 molar ratio).
  • DSPC/DSPG liposomes were prepared as described in Example 2 and at molar lipid ratios of 90:10, 80:20, 70:30, 60:40 and 50:50 DSPC to DSPG.
  • FUDR and carboplatin were passively entrapped into liposomes during preparation at either a 1 :1 or 1 :5 mole ratio, identified in Example 1 as being synergistic.
  • Figure 5 A shows plasma levels of FUDR and carboplatin were effectively maintained at a 1:1 mole ratio as plasma levels of FUDR were roughly equal to that of carboplatin at various time points after intravenous administration to SCID-Rag2M mice when they were delivered in DSPC/DSPG liposomes at 80:20 and 70:30 molar lipid ratios.
  • DSPC/DSPG 90:10 liposomes resulted in a rapid and uncontrolled change of the drag/drag ratio from an optimum ratio required (1 :1) for synergistic killing of cancer cells.
  • Figure 5B clearly demonstrates that a 60:40 DSPC/DSPG liposome formulation is optimal for maintaining a 1 :5 FUDR to carboplatin ratio in the plasma as the amount of carboplatin was sustained at roughly five times the amount of FUDR over time.
  • DSPC/DSPG co-loaded liposomes which are formulated with a DSPC to DSPG ratio between about 70:30 to 60:40 are effective for maintaining a synergistic ratio of FUDR/carboplatin after in vivo administration.
  • DSPC/DSPG/SM 60:30:10 liposomes are as effective as DSPC/DSPG (60:40) liposomes in maintaining FUDR and carboplatin at a synergistic ratio after in vivo administration.
  • Maintaining a ratio after administration is important as it will allow for controlled delivery of a synergistic drag combination to the delivery site (i.e., tumor) which will likely result in enhanced efficacy compared to administration of a synergistic drag combination in which there are no parameters designed to maintain the drag ratio after in vivo administration. In the latter case, based on literature in the art, it is expected that an uncontrolled change of the drug/drug ratio from the optimum ratio will occur rapidly after administration of the free drag cocktail.
  • DSPC/DSPG/Chol (60:30:10 molar ratio) liposomes were identified as being the optimal formulation for allowing for coordinated release of a 1 :2 molar ratio of FUDR and carboplatin.
  • the formulation was administered intravenously into CD-I nude mice inoculated with Colon-26 murine colorectal solid tumors.
  • DSPC/DSPG/Chol (60:30: 10 molar ratio) liposomes were prepared as previously described with FUDR and carboplatin passively entrapped during liposome preparation at a 1 :2 mole ratio.
  • Liposomal FUDR and Liposomal Carboplatin were also prepared as a comparison.
  • CD-I nude mice were inoculated subcutaneously with 1 x 10 6 Colon-26 tumor cells. Tumors were allowed to grow, then injections of the formulation were given on day 12, 15 and 18 post-inoculation (Q3Dx3). Tumor weight was monitored during treatment.
  • Liposomal preparations were injected intravenously via the tail vein into female CD-I mice. Administered doses were 15 mg/kg carboplatin and 301 mg/kg lipid for all formulations and 5 mg/kg FUDR.
  • Figure 7 shows that the DSPC/DSPG/Chol (60:30:10 molar ratio) formulation of FUDR and carboplatin co-loaded liposomes increases the efficacy against the tumor growth as compared to the saline control, liposomal FUDR and liposomal carboplatin formulations.
  • Example 4 DSPC/DSPG (60:40) and DSPC/DSPG/SM (60:30:10) liposomes were identified as being effective formulations for allowing for coordinated release of a 1 :5, synergistic ratio of FUDR and carboplatin.
  • the investigators measured the release of FUDR and carboplatin in the abovementioned liposome formulations at 1 :5, 1 :2 and 1 :1 FUDR/carboplatin mole ratios.
  • DSPC/DSPG 60:40
  • DSPC/DSPG/SM 60:30:10 liposomes were prepared as previously described with FUDR and carboplatin passively entrapped during liposome preparation at either a 1:5, 1:2 or 1:1 FUDR:carboplatin mole ratio.
  • Liposomal preparations were injected intravenously via the tail vein into male CD-I mice. Administered doses were 53.9 mg/kg carboplatin and 539 mg/kg lipid for all formulations and 10.8 mg/kg FUDR, 27 mg/kg FUDR and 53.9 mg/kg FUDR for 1 :5, 1 :2 and 1:1 molar FUDR/carboplatin ratios, respectively.
  • blood was collected by cardiac puncture (3 mice per time point) and placed into EDTA coated microcontainers. The samples were centrifuged to separate plasma and plasma was then transferred to another tube.
  • Liquid scintillation counting was used to quantitate radiolabeled lipid and FUDR in the plasma.
  • Plasma carboplatin levels were quantified by atomic absorption (AA).
  • Figures 8A and 8B show that carboplatin and FUDR, respectively, are released from co-loaded DSPC/DSPG (60:40) liposomes at a gradual rate, independent of the FUDR/carboplatin ratio.
  • Figures 9A and 9B respectively demonstrate that release of carboplatin and FUDR from co-loaded DSPC/DSPG/SM (60:30:10) liposomes occurs at a similar rate when FUDR and carboplatin are loaded at either a 1 :5, 1 :2 or 1 : 1 mole ratio.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Inorganic Chemistry (AREA)
  • Dispersion Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)

Abstract

L'invention concerne des compositions qui comprennent des liposomes auxquels sont associés, de manière stable, du platine et un antimétabolite. Ces compositions permettent d'obtenir des effets thérapeutiques améliorés lorsque les combinaisons de ces médicaments sont administrées.
PCT/CA2004/000508 2003-04-02 2004-04-02 Formulations associant du platine et des fluoropyrimidines WO2004087105A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US46025103P 2003-04-02 2003-04-02
US60/460,251 2003-04-02

Publications (2)

Publication Number Publication Date
WO2004087105A1 true WO2004087105A1 (fr) 2004-10-14
WO2004087105A8 WO2004087105A8 (fr) 2005-03-10

Family

ID=33131918

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA2004/000508 WO2004087105A1 (fr) 2003-04-02 2004-04-02 Formulations associant du platine et des fluoropyrimidines

Country Status (1)

Country Link
WO (1) WO2004087105A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006032136A1 (fr) * 2004-09-20 2006-03-30 British Columbia Cancer Agency Branch Gemcitabine libre ou encapsulee dans des liposomes seule ou en association avec de l'idarubicine libre ou encapsulee dans des liposomes
WO2006055903A1 (fr) * 2004-11-18 2006-05-26 Celator Pharmaceuticals, Inc. Procede pour charger des agents multiples dans des vehicules d'administration
WO2007042647A1 (fr) * 2005-10-06 2007-04-19 Erytech Pharma Erythrocytes contenant du 5-fluorouracile (5-fu) et/ou de l’oxaliplatine
WO2009097011A1 (fr) 2007-08-17 2009-08-06 Celator Pharmaceuticals, Inc. Préparations améliorées de médicaments au platine
EP2123258A1 (fr) * 2008-05-23 2009-11-25 Liplasome Pharma A/S Liposomes pour l'administration de médicaments
EP1959940A4 (fr) * 2005-11-30 2013-04-03 Celator Pharmaceuticals Inc Administration localisee de combinaisons de medicaments
US10905775B2 (en) 2004-07-19 2021-02-02 Celator Pharmaceuticals, Inc. Particulate constructs for release of active agents
CN114746124A (zh) * 2019-10-10 2022-07-12 北卡罗来纳-查佩尔山大学 包含活性剂沉淀物的递送系统复合物和使用方法
US11980636B2 (en) 2020-11-18 2024-05-14 Jazz Pharmaceuticals Ireland Limited Treatment of hematological disorders

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0715854A2 (fr) * 1994-11-11 1996-06-12 Tanaka Kikinzoku Kogyo K.K. Compositions carcinostatiques contenant du cis-oxalata et un ou plusieurs autres carcinogènes
WO1998055103A1 (fr) * 1997-06-06 1998-12-10 Max-Delbrück-Centrum für Molekulare Medizin Agent pour therapie antitumorale
WO2001034130A1 (fr) * 1999-11-05 2001-05-17 Teni Boulikas Therapie pour cancers humains utilisant la cisplatine et d'autres medicaments ou genes encapsules dans des liposomes
WO2001082892A2 (fr) * 2000-05-02 2001-11-08 Pharmacept Gmbh Liposomes a base de principes actifs
WO2001087313A1 (fr) * 2000-05-18 2001-11-22 Anormed Inc Chimiotherapie combinee
US20020173482A1 (en) * 2001-03-06 2002-11-21 Ajani Jafferhusen Abdulhusen Method and dosage form for treating tumors by the administration of tegafur, uracil, folinic acid, paclitaxel and carboplatin
WO2003028696A2 (fr) * 2001-10-03 2003-04-10 Celator Technologies Inc. Compositions pour l'administration de combinaisons medicinales

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0715854A2 (fr) * 1994-11-11 1996-06-12 Tanaka Kikinzoku Kogyo K.K. Compositions carcinostatiques contenant du cis-oxalata et un ou plusieurs autres carcinogènes
WO1998055103A1 (fr) * 1997-06-06 1998-12-10 Max-Delbrück-Centrum für Molekulare Medizin Agent pour therapie antitumorale
WO2001034130A1 (fr) * 1999-11-05 2001-05-17 Teni Boulikas Therapie pour cancers humains utilisant la cisplatine et d'autres medicaments ou genes encapsules dans des liposomes
WO2001082892A2 (fr) * 2000-05-02 2001-11-08 Pharmacept Gmbh Liposomes a base de principes actifs
WO2001087313A1 (fr) * 2000-05-18 2001-11-22 Anormed Inc Chimiotherapie combinee
US20020173482A1 (en) * 2001-03-06 2002-11-21 Ajani Jafferhusen Abdulhusen Method and dosage form for treating tumors by the administration of tegafur, uracil, folinic acid, paclitaxel and carboplatin
WO2003028696A2 (fr) * 2001-10-03 2003-04-10 Celator Technologies Inc. Compositions pour l'administration de combinaisons medicinales

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FICHTNER I ET AL: "PHARMACOLOGICAL EFFECTS OF CARBOPLATIN-LIPOSOMES -CPL0 IN MICE: A REVIEV OF PRESENT KNOWLEDGE", JOURNAL OF LIPOSOME RESEARCH, MARCEL DEKKER, NEW YORK, US, vol. 5, no. 1, 1 February 1995 (1995-02-01), pages 75 - 89, XP000491778, ISSN: 0898-2104 *
LEVI F ET AL: "A CHRONOPHARMACOLOGIC PHASE II CLINICAL TRIAL WITH 5 FLUOROURACIL FOLINIC ACID AND OXALIPLATIN USING AN AMBULATORY MULTICHANNEL PROGRAMMABLE PUMP HIGH ANTITUMOR EFFECTIVENESS AGAINST METASTATIC COLORECTAL CANCER", CANCER, AMERICAN CANCER SOCIETY, PHILADELPHIA, PA, US, vol. 69, no. 4, 1992, pages 893 - 900, XP009018850, ISSN: 0008-543X *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10905775B2 (en) 2004-07-19 2021-02-02 Celator Pharmaceuticals, Inc. Particulate constructs for release of active agents
WO2006032136A1 (fr) * 2004-09-20 2006-03-30 British Columbia Cancer Agency Branch Gemcitabine libre ou encapsulee dans des liposomes seule ou en association avec de l'idarubicine libre ou encapsulee dans des liposomes
WO2006055903A1 (fr) * 2004-11-18 2006-05-26 Celator Pharmaceuticals, Inc. Procede pour charger des agents multiples dans des vehicules d'administration
WO2007042647A1 (fr) * 2005-10-06 2007-04-19 Erytech Pharma Erythrocytes contenant du 5-fluorouracile (5-fu) et/ou de l’oxaliplatine
EP1959940A4 (fr) * 2005-11-30 2013-04-03 Celator Pharmaceuticals Inc Administration localisee de combinaisons de medicaments
WO2009097011A1 (fr) 2007-08-17 2009-08-06 Celator Pharmaceuticals, Inc. Préparations améliorées de médicaments au platine
EP2123258A1 (fr) * 2008-05-23 2009-11-25 Liplasome Pharma A/S Liposomes pour l'administration de médicaments
WO2009141450A3 (fr) * 2008-05-23 2010-07-08 Bio-Bedst Liposomes pour l’administration de medicaments et leurs procédés de préparation
CN114746124A (zh) * 2019-10-10 2022-07-12 北卡罗来纳-查佩尔山大学 包含活性剂沉淀物的递送系统复合物和使用方法
US11980636B2 (en) 2020-11-18 2024-05-14 Jazz Pharmaceuticals Ireland Limited Treatment of hematological disorders

Also Published As

Publication number Publication date
WO2004087105A8 (fr) 2005-03-10

Similar Documents

Publication Publication Date Title
AU2004226889B2 (en) Combination compositions of camptothecins and fluoropyrimidines
US10722464B2 (en) Compositions for delivery of drug combinations
EP2187869B1 (fr) Préparations améliorées de médicaments au platine
US8431806B2 (en) Liposomal formulations of anthracycline agents and cytidine analogs
US7850990B2 (en) Compositions for delivery of drug combinations
EP1432402B1 (fr) Compositions pour l'administration de combinaisons medicinales
US20090074848A1 (en) Combination formulations of cytidine analogs and platinum agents
AU2002331480A1 (en) Compositions for delivery of drug combinations
US20080075762A1 (en) Compositions for delivery of drug combinations
WO2004087105A1 (fr) Formulations associant du platine et des fluoropyrimidines
AU2007237323B2 (en) Compositions for delivery of drug combinations

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
CFP Corrected version of a pamphlet front page

Free format text: UNDER (54) PUBLISHED TITLE REPLACED BY CORRECT TITLE

DPEN Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed from 20040101)
122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载