WO2000064484A2 - Conjugate having a cleavable linkage for use in a liposome - Google Patents

Conjugate having a cleavable linkage for use in a liposome Download PDF

Info

Publication number
WO2000064484A2
WO2000064484A2 PCT/US2000/010922 US0010922W WO0064484A2 WO 2000064484 A2 WO2000064484 A2 WO 2000064484A2 US 0010922 W US0010922 W US 0010922W WO 0064484 A2 WO0064484 A2 WO 0064484A2
Authority
WO
WIPO (PCT)
Prior art keywords
conjugate
therapeutic drug
mitomycin
drug
dithiobenzyl
Prior art date
Application number
PCT/US2000/010922
Other languages
French (fr)
Other versions
WO2000064484A3 (en
Inventor
Samuel Zalipsky
Alberto A. Gabizon
Original Assignee
Alza Corporation
Hadasit Medical Research Services & Development Ltd.
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
Priority to IL14605500A priority Critical patent/IL146055A0/en
Priority to KR1020017013571A priority patent/KR100669053B1/en
Priority to CA2369595A priority patent/CA2369595C/en
Priority to DE60030965T priority patent/DE60030965T2/en
Application filed by Alza Corporation, Hadasit Medical Research Services & Development Ltd. filed Critical Alza Corporation
Priority to JP2000613474A priority patent/JP4558952B2/en
Priority to HU0201425A priority patent/HUP0201425A3/en
Priority to EP00928321A priority patent/EP1173222B1/en
Priority to DK00928321T priority patent/DK1173222T3/en
Priority to AU46577/00A priority patent/AU769425B2/en
Priority to MXPA01010750A priority patent/MXPA01010750A/en
Publication of WO2000064484A2 publication Critical patent/WO2000064484A2/en
Priority to IL146055A priority patent/IL146055A/en
Priority to NO20015144A priority patent/NO20015144L/en
Publication of WO2000064484A3 publication Critical patent/WO2000064484A3/en
Priority to HK02103601.3A priority patent/HK1041820B/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • 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
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A61K47/544—Phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/10—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C323/18—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton
    • C07C323/19—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton with singly-bound oxygen atoms bound to acyclic carbon atoms of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
    • C07D239/545—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals with other hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/553—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals with other hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms with halogen atoms or nitro radicals directly attached to ring carbon atoms, e.g. fluorouracil
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
    • C07D487/14—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/12—Triazine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
    • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]
    • Y10T428/2984—Microcapsule with fluid core [includes liposome]

Definitions

  • the present invention relates to a conjugate comprised of a hydrophobic moiety, a cleavable linkage, and a therapeutic agent. More particularly, the present invention relates to conjugates comprised of a lipid, a cleavable linkage and a drug inco ⁇ orated into a liposomal formulation. The conjugates are cleavable under mild thiolytic conditions in vivo for release of the drug in an unmodified state.
  • Liposomes are closed lipid vesicles used for a variety of therapeutic purposes, a d in particular, for carrying therapeutic agents to a target region or cell by systemic administration of liposomes.
  • Liposomes having a surface grafted with chains of water- soluble, biocompatible polymer, in particular polyethylene glycol, have become important drug carries. These liposomes offer an extended blood circulation lifetime over liposomes lacking the polymer coating. The grafted polymer chains shield or mask the liposome, thus minimizing nonspecific interaction by plasma proteins. This in turn slows the rate at which the liposomes are cleared or eliminated in vivo since the liposome circulate unrecognized by macrophages and other cells of the reticuloendothelial system. Furthermore, due to the so-called enhanced permeability and retention effect, the liposomes tend to accumulate in sites of damaged or expanded vasculature, e.g. , tumors, sites of inflammation.
  • An extended blood circulation time is often desired to allow systemically administered liposomes to reach a target region, cell or site.
  • a blood circulation lifetime of greater than about 12 hours is preferred for liposomal-therapy to a tumor region, as the liposomes must systemically distribute and then extravasate into the tumor region.
  • liposome-based therapy One problem associated with liposome-based therapy is retention of drug within the liposome for a time sufficient for systemic distribution. This problem is of particular concern when long-circulating liposomes, i.e., liposomes with grafted polymer chains, are administered. Relatively few drugs can be efficiently loaded and retained for a long duration and subsequently released.
  • lipid bilayer components that render the bilayer less permeable to entrapped drug.
  • the lipid bilayer should be sufficiently fluidic such that the drug is released, for example by transport across the lipid bilayer or by lipid vesicle breakdown, at the desired time, e.g. , after localization at a target site or sufficient biodistribution.
  • Another approach to improving drug retention is to covalently attach the drug to a lipid in the liposomal lipid bilayer (Waalkes, et al., Selective Cancer Therap., 6: 15-22 (1990); Asai, et al, Biol. Pharm. Bull , 21:766-771 (1998)). It would be desirable to formulate a liposome composition having a long blood circulation lifetime and capable of retaining an entrapped drug for a desired time, yet able to release the drug on demand.
  • a liposome from a non-vesicle-forming lipid, such as dioleoylphosphatidylethanolamine (DOPE), and a lipid bilayer stabilizing lipid, such as methoxy-polyethylene glycol-distearoyl phosphatidylethanolamine (mPEG-DSPE) (Kirpotin, D, et al, FEBS Lett. 388:115-118 (1996)).
  • DOPE dioleoylphosphatidylethanolamine
  • mPEG-DSPE methoxy-polyethylene glycol-distearoyl phosphatidylethanolamine
  • the mPEG is attached to the DSPE via a cleavable linkage. Cleavage of the linkage destabilizes the liposome for a quick release of the liposome contents.
  • Labile bonds for linking PEG polymer chains to liposomes has been described (U.S. Patent Nos. 5,013,556, 5,891,468; WO 98/16201).
  • the labile bond in these liposome compositions releases the PEG polymer chains from the liposomes, for example, to expose a surface attached targeting ligand or to trigger fusion of the liposome with a target cell.
  • Senter describes a drug-antibody prodrug, where the antibody is linked to a drug using a disulfide benzyl carbamate or carbonate linker, and reduction of the disulfide bond effects release of the drug.
  • Senter' s teaching is specific to cleavage of a drug-ligand prodrug molecule, under the action of reducing agents such as 1,4- dithiothreitol, glutathione, NADH and NADPH.
  • an extended blood circulation time is a desirable feature of PEG- coated liposomes, with blood circulation lifetimes of greater than about 12 hours being preferred for liposomal-therapy to a tumor region.
  • the disclosure of Senter provides no guidance as to the release kinetics of a conjugate incorporated into a liposome under endogenous reducing conditions, such as during blood circulation of the liposome.
  • the invention includes a conjugate for use in a liposomal drug- delivery vehicle, the conjugate having the general structural formula:
  • L is a hydrophobic moiety suitable for incorporation into a liposomal lipid bilayer
  • R 1 represents a therapeutic drug covalently attached to the dithiobenzyl moiety, and where orientation of the CH 2 R' group is selected from the ortho position and the para position.
  • the therapeutic drug is covalently attached by a linkage selected from the group consisting of urethane, amine, amide, carbonate, thio- carbonate, ether and ester.
  • L is selected from the group consisting of cholesterol, a diacylglycerol, a phospholipid and derivatives thereof.
  • L is a diacylglycerol derivative to yield a conjugate having the general structural formula:
  • R 2 and R 3 are hydrocarbons having between about 8 to about 24 carbon atoms, or in another embodiment, from about 12 to about 22 carbon atoms. In still another embodiment, R 2 and R 3 are hydrocarbon chains of the same length.
  • the drug is selected from the group consisting of mitomycin C, mitomycin A, bleomycin, doxorubicin, daunorubicin, fluorodeoxyuridine, iododeoxyuridine, etoposide, AZT, acyclovir, vidarabine, arabinosyl cytosine, pentostatin, quinidine, atropine, chlorambucil, methotrexate, mitoxantrone and 5-fluorouracil.
  • the therapeutic drug is covalently linked to the dithiobenzyl moiety to form a conjugate having the structure:
  • R 4 represents a residue of the therapeutic drug.
  • R 4 in one embodiment is a therapeutic drug residue containing a primary or a secondary amine moiety thereby forming a urethane linkage between the dithiobenzyl and the therapeutic drug.
  • the therapeutic drug can be, for example, mitomycin A, mitomycin C, bleomycin or a polypeptide.
  • R 4 is a residue of a carboxyl-containing therapeutic drug, which forms an ester linkage between the dithiobenzyl and the therapeutic drug.
  • Examplary drugs in this embodiment include chlorambucil or methotrexate.
  • R 4 is a therapeutic drug residue containing a hydroxyl moiety thereby to form a carbonate linkage between the dithiobenzyl and the therapeutic drug.
  • exemplary drugs in this embodiment include fluorodeoxyuridine, iododeoxyuridine, etoposide, AZT, acyclovir, vidarabine, arabinosyl cytosine, pentostatin, quinidine, mitoxantrone and atropine.
  • the invention includes a liposome composition, comprising liposomes composed of vesicle-forming lipids including from about 1 to about 30 mole percent of a conjugate having the general structural described above.
  • the therapeutic drug is released from the conjugate in vivo in response to a physiologic condition or an artificially induced condition.
  • the invention includes a method for retaining a drug in a liposome, comprising preparing liposomes comprised of a vesicle-forming lipid and of between about 1 to about 30 mole percent of a conjugate described above.
  • the liposomes effectively retain the drug in the liposomes until release from the conjugate in response to a physiologic condition or an artificially induced condition.
  • Fig. 1 shows a synthetic reaction scheme for preparation of /r ⁇ ra-diacyldiglycerol- dithiobenzylalcohol for further reaction with amine-, hydroxy- or carboxyl-containing drugs;
  • Fig. 2 A shows a general reaction scheme for attachment of an amino-containing drug to a reactive diacyldiglycerol-dithiobenzylcarbonate
  • Fig. 2B shows the products after thiolytic cleavage of the conjugate in Fig. 2A;
  • Fig. 3 A shows a synthetic reaction scheme for preparation of a diacyldiglycerol- dithiobenzyl-5-fluorouracil conjugate ;
  • Fig. 3B shows the products after thiolytic cleavage of the conjugate in Fig. 3 A;
  • Fig. 4 shows an alternative synthetic reaction scheme for preparation of a diacyldiglycerol-dithiobenzyl-5-fluorouracil conjugate and the products after thiolytic cleavage of the conjugate;
  • Fig. 5 shows a synthetic reaction scheme for preparation of a diacyldiglycerol- dithiobenzyl-chlorambucil conjugate and the products after thiolytic cleavage of the conjugate;
  • Fig. 6 A shows a synthetic reaction scheme for preparation of a diacyldiglycerol- difhiobenzy 1-mitomycin-C conjugate ;
  • Fig. 6B shows the products after thiolytic cleavage of the conjugate in Fig. 6A
  • Fig. 7 shows a synthetic reaction scheme for preparation of a cholesterol- dithiobenzyl-mitomycin-C conjugate ;
  • Fig. 8 shows another synthetic reaction scheme for preparation of a cholesterol- dithiobenzy 1-mitomycin-C conjugate
  • Figs. 9A-9C show the structures of three lipid-dithiobenzy 1-mitomycin-C conjugates, ⁇ ra-distearoyl-DTB-mitomycin-C (Fig. 9A), /? ⁇ r ⁇ -dipalmitoyl-DTB- mitomycin-C (Fig. 9B) and ⁇ rtb ⁇ -dipalmitoyl-DTB- mitomycin-C (Fig. 9C);
  • Figs. 10A-10B are HPLC chromatograms for liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C (Fig. 10A) and HSPC/cholesterol/mPEG- DSPE/lipid-DTB-mitomycin C (Fig. 10B), where each figure shows a series of chromatograms as a function of time of incubation of the liposomes in the presence of cysteine;
  • Fig. 11 is a plot showing the percent of mitomycin C released from liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C (closed diamonds) and HSPC/cholesterol/mPEG-DSPE/lipid-DTB-mitomycin C (closed circles) as a function of time of incubation in the presence of cysteine;
  • Figs. 12A-12B are plots showing the percent of mitomycin C released from liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C (Fig. 12 A) and HSPC/cholesterol/mPEG-DSPE/lipid-DTB-mitomycin C (Fig. 12B) as a function of time of incubation in the presence of cysteine at concentrations of 150 ⁇ M (closed symbols) and at 1.5 mM (open symbols);
  • Fig. 13 is a plot of growth rate of Ml 09 cells, expressed as a percentage based on growth of M 109 cells in the absence of drug and cysteine, as a function of mitomycin C amount, in nM, for free mitomycin c (open triangles), liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C (closed squares), and liposomes comprised of HSPC/cholesterol/mPEG-DSPE/lipid-DTB-mitomycin C (open circles); Fig.
  • 14A is a plot of growth rate of Ml 09 cells, expressed as a percentage based on growth of M109 cells in the absence of drug or cysteine, as a function of mitomycin C concentration in nM. Shown are cells treated mitomycin C in free form (open triangles) and with mitomycin C in free form plus 1000 ⁇ M cystein (closed triangles). Also shown are cells treated with the liposome formulation comprised of HSPC/PEG- DSPE/lipid-DTB-mitomycin C (open circles) and with the liposome formulation with additional cysteine added at concentrations of 150 ⁇ M (open diamonds), 500 ⁇ M (closed circles) and 1000 ⁇ M (open squares);
  • Fig. 14B is a plot of growth rate of Ml 09 cells, expressed as a percentage based on growth of M109 cells in the absence of drug or cysteine, as a function of mitomycin C concentration in nM. Shown are cells treated mitomycin C in free form (open triangles) and with mitomycin C in free form plus 1000 ⁇ M cystein (closed triangles). Also shown are cells treated with the liposome formulation comprised of HSPC/cholesterol/mPEG-DSPE/lipid-DTB-mitomycin C (open circles) and with the liposome formulation with additional cysteine added at concentrations of 150 ⁇ M (open diamonds), 500 ⁇ M (closed circles) and 1000 ⁇ M (open squares); Fig. 15 is a plot showing the percent increase in cytotoxicity (as determined by
  • Fig. 16A is a plot showing the concentration of mitomycin C in the blood of rats as a function of time in hours following intravenous injection of free mitomycin C (open squares), liposomes comprised of HSPC/cholesterol/mPEG-DSPE/lipid-DTB- mitomycin C (closed diamonds), and liposomes comprised of HSPC/mPEG-DSPE/lipid- DTB-mitomycin C (closed circles); and
  • Fig. 16B is a plot showing the percent of injected dose remaining in the blood of rats as a function of time in hours following intravenous injection of free mitomycin C (open squares), liposomes comprised of HSPC/cholesterol/mPEG-DSPE/lipid-DTB- mitomycin C (closed diamonds), and liposomes comprised of HSPC/mPEG-DSPE/lipid- DTB-mitomycin C (closed circles).
  • hydrophobic moiety suitable for incorporation into a liposomal lipid bilayer intends any material comprising a hydrophobic portion capable of being integrated with the hydrophobic bilayer region of a liposomal lipid bilayer.
  • Such hydrophobic moieties are typically lipids, including amphipathic lipids having a hydrophobic lipid tail and a hydrophilic polar head, such as phospholipids and diacylglycerols.
  • Triglycerides, sterols, derivatives of phospholipids, diacylglyerols, sterols and triglycerides and other lipids derived from a natural source or synthetically prepared are also contemplated.
  • the term “residue” as in "therapeutic drug residue” intends a drug molecule that has been reacted to form an linkage with another molecule where at least one atom of the drug molecule is replaced or has been sacrificed to from the linkage.
  • Polypeptide refers to a polymer of amino acids and does not refer to a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, and enzyme are included within the definition of polypeptide. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like.
  • PEG poly(ethylene glycol); mPEG, methoxy-PEG; DTB, dithiobenzyl; DSPE, distearoyl phosphatidylethanolamine; HSPC, hydrogenated soy phosphatidylcholine; MMC, mitomycin C.
  • the invention includes a conjugate of the form:
  • L is a hydrophobic moiety suitable for incorporation into a liposomal lipid bilayer
  • R 1 represents a therapeutic drug residue covalently attached to the dithiobenzyl moiety, and where orientation of the CH 2 R' group is selected from the ortho position and the para position.
  • the hydrophobic moiety, L is typically a lipid such as a diacylglycerol, a sterol, a phospholipid, derivatives of these lipids, other naturally-occurring lipids and their synthetic analogs.
  • a therapeutic drug is attached to the dithiobenzyl moiety by a covalent linkage, thereby forming a drug residue, represented by R 1 in the structure.
  • the linkage will vary according to the drug and the reaction chemistry, as will be appreciated by those of skill in the art.
  • the therapeutic drug is covalently attached to the diithiobenzyl moiety by a linkage selected from the group consisting of urethane, amine, amide, carbonate, thio-carbonate, ether and ester.
  • a drug containing a primary or secondary amine such as mitomycin C, mitomycin A, bleomycin and therapeutic polypeptides to name a few, is reacted to from a urethane linkage with the amine moiety in the drug.
  • Exemplary drugs having such a moiety for reaction with dithiobenzyl alcohol to form a carbonate linkage include fluorodeoxyuridine, iododeoxyuridine, etoposide, AZT, acyclovir, vidarabine, arabinosyl cytosine, pentostatin, quinidine, mitoxantrone and atropine.
  • the linkage derives from reaction with a carboxylic acid moiety in the therapeutic drug, and an example of a conjugate having an ester linkage between chlorambucil and dithiobenzyl is described below.
  • Methotrexate is another example of a drug capable of forming an ester linkage with the dithiobenzyl moiety of the conjugate.
  • Conjugates having a urethane, carbonate or ester linkage attaching the drug to the dithiobenzyl moiety can generally be represented by the following structure:
  • R 4 represents a residue of the therapeutic drug.
  • the conjugate includes an ether linkage, which takes the form of O-R 4 , where R 4 represents the therapeutic drug residue.
  • the linkage typically derives from reaction with an alcohol functionality on the drug.
  • a conjugate with the drug 5-fluorouracil where an amine linkage is formed will be described below.
  • An amide linkage can also be formed with a peptide as the therapeutic agent, where the free carboxyl of an amino acid residue, such as an aspartic acid or glutamic acid, is condensed with dithiobenzylamine.
  • Fig. 1 shows a synthetic reaction scheme for preparation of exemplary conjugates in accord with the invention.
  • synthesis of an intermediate compound, ⁇ r ⁇ -diacyldiglyceroldithiobenzalcohol (Compound IV) is prepared for further reaction with a selected therapeutic drug.
  • Compound IV is prepared, as described in Example 1 , by reacting 3-mercapto-l ,2-propanediol (Compound I) with hydrogen peroxide to form r ⁇ c-3,3'-dithiobis(l ,2-propanediol) (Compound II).
  • Rac- 3,3'-dithiobis(l ,2-propanediol) is acylated with a hydrophobic moiety R.
  • R can be a fatty acid having from about 8 to about 24 carbon atoms.
  • Example 1 details the reaction procedure where R is stearic acid.
  • R is a fatty acid having from about 12 to about 22 carbon atoms.
  • Acylation of Compound II yields Rac- 3,3'-dithiobis(l ,2-propanedistearoyl) (Compound III), which is reacted with sulfuryl chloride and 4-mercaptobenzalcohol to form the desired intermediate product, para- diacyldiglycerol-dithiobenzalcohol (Compound IV).
  • Compound IV is readily reacted with a drug containing a reactive carboxyl moiety (R'C0 2 H) to form a lipid- dithiobenzyl (DTB)-drug conjugate where the drug is joined to the DTB via an ester linkage (Compound V).
  • Compound IV is also readily reacted with a drug containing a reactive amine moiety (R'-NH 2 ) to yield a lipid-DTB-drug conjugate where the drug is joined to the DTB by a urethane linkage (Compound VI).
  • Compound IV is also readily reacted with a drug containing a reactive hydroxyl moiety (R'OH) to form a lipid-DTB- drug conjugate where the drug is joined to the DTB by a carbonate linkage (Compound VII).
  • drugs are contemplated for use in the conjugate of the invention.
  • the invention contemplates drugs having an amine (NH or NH 2 ), carboxyl, sulfhydryl or hydroxyl moiety suitable for reaction.
  • "suitable for reaction” implies that the drug has one of the recited moieties capable of reacting with the dithiobenzyl moiety, in the form of, for example, dithiobenzyl alcohol.
  • Exemplary drugs include 5-fluorouracil, which has an NH group suitable for reaction, chlorambucil, which has a reactive carboxyl and mitomycin C, which has a reactive amine (aziridine group).
  • exemplary drugs contemplated for use include mitomycin C, mitomycin A, bleomycin, doxorubicin, daunorubicin, fluorodeoxyuridine, iododeoxyuridine, etoposide, AZT, acyclovir, vidarabine, arabinosyl cytosine, pentostatin, quinidine, atropine, chlorambucil, methotrexate, mitoxantrone and 5-fluorouracil.
  • polypeptides, aminoglycosides, alkaloids are all also suitable for use in the invention.
  • Example 1 also details the reaction conditions for preparation of ortho- diacyldiglyceroldithiobenzalcohol, which can serve as a intermediary compound to form the conjugate.
  • Figs. 2A-2B show preparation of a lipid-DTB-drug conjugate (Fig. 2A), and thiolytic cleavage of the conjugate in the presence of a reducing agent (Fig. 2B).
  • Fig. 2A Compound VII of Fig.
  • hydrophobic moiety R is derived from a fatty acid R"(CO)OH, such as stearic acid (CH 3 (CH 2 ) 16 CO 2 H), is reacted with an amine-containing drug, H 2 N-drug, in the presence of phosgene (COCl 2 ).
  • This reaction yields the lipid-DTB-drug conjugate illustrated in Fig. 2A.
  • the conjugate upon exposure to reducing conditions, i.e., a reducing agent such as cysteine or glutathione, decomposes to yield the products shown in Fig. 2B.
  • thiolytic cleavage of the conjugate results in regeneration of the drug in an unmodified, natural state.
  • the drug in conjugate can be readily incorporated into liposomes for administration in vivo to a subject. Further, the drug in the form of the conjugate is not toxic, as will also be shown below. After administration and upon exposure to endogeneous reducing agents or exposure to an exogeneous reducing agent, the conjugate decomposes to yield the drug in its native state and with biological activity.
  • Fig. 3 A a synthetic reaction scheme for preparation of a conjugate of 5- fluorouracil is illustrated.
  • Compound IV /r ⁇ ra-diacyl-diglycerol-dithiobenzalalcohol
  • /r ⁇ ra-toluenesulfonyl chloride to form the intermediate compound IX.
  • Reaction with 5-fluorouracil anion or sodium salt (Compound X) yields the desired lipid-DTB-5-fluorouracil conjugate (Compound XI).
  • Decomposition of the lipid-DTB- 5-fluorouracil conjugate (Compound XI) upon exposure to a reducing agent, R'-SH is shown in Fig. 3B. Thiolytic cleavage of the conjugate results in regeneration of 5- fluorouracil in an unmodified form.
  • Fig. 4 shows an alternative synthetic reaction scheme for preparation of a diacyldiglycerol-DTB-5-fluorouracil conjugate.
  • 1-chloroethyl chloroformate (Compound XII) is reacted with /? ⁇ r ⁇ -diacyl-diglycerol-dithiobenzalalcohol (Compound IV) to form a reactive chloroethyl carbonate-DTB-diacyldiglycerol intermediate (Compound XII).
  • the intermediate is subsequently reacted with 5-fluorouracil in the presence of ttiemanolamine (TEA) to yield a lipid-DTB-5-fluorouracil conjugate (Compound XIV).
  • TAA ttiemanolamine
  • a drug containing a reactive carboxyl moiety chlorambucil (Compound XV)
  • Compound IV ⁇ ra-diacyl-diglycerol-dithiobenzalalcohol
  • DCC 1,3- dicyclohexycarbodiimide
  • DMAP dimethylaminopyridine
  • a lipid- DTB-chlorambucil conjugate Compound XVI
  • DCC 1,3- dicyclohexycarbodiimide
  • DMAP dimethylaminopyridine
  • the conjugate thiolytically decomposes to the products shown. Chlorambucil is subsequently regenerated in an unmodified state.
  • Fig. 6 A shows the synthesis of a conjugate in accord with another embodiment of the invention.
  • mitomycin C Compound XVII, Fig. 6B
  • a drug containing a reactive amine moiety is reacted with /r ⁇ ra-diacyl-diglycerol- dithiobenzalalcohol (Compound IV) in the presence of phosgene to form a diacyldiglycerol-dithiobenzy 1-mitomycin-C conjugate (Compound XVIII).
  • Compound IV /r ⁇ ra-diacyl-diglycerol- dithiobenzalalcohol
  • phosgene diacyldiglycerol-dithiobenzy 1-mitomycin-C conjugate
  • Fig. 6B shows the thiolytic decomposition of a diacyldiglycerol-DTB-mitomycin-
  • hydrophobic moiety in the conjugate can be selected from any number of hydrophobic moieties, e.g. , lipids.
  • lipids e.g. lipids
  • a diacyldiglycerol lipid was used to form conjugates having the structure:
  • R 2 and R 3 are hydrocarbons having between about 8 to about 24 carbon atoms.
  • Fig. 7 shows another embodiment where cholesterol is used as the hydrophobic moiety in the conjugate.
  • Cholesterol (Compound XIV) is reacted with methanesulfonyl chloride in dichloromethane in the presence of triemylamine (TEA). The resulting intermediate is then converted into the thiol derivative and ultimately into the principal dithiobenzyl alcohol, which is used to link mitomycin C in a similar fashion as described above for diacylglycerol.
  • the invention includes a liposome composition comprised of a vesicle-forming lipid and a conjugate as described above.
  • Liposomes are closed lipid vesicles used for a variety of therapeutic purposes, and in particular, for carrying therapeutic agents to a target region or cell by systemic administration of liposomes.
  • liposomes having a surface coating of hydrophilic polymer chains, such as polyethylene glycol (PEG), are desirable as drug carries as these liposomes offer an extended blood circulation lifetime over liposomes lacking the polymer coating.
  • the polymer acts as a barrier to blood proteins thereby preventing binding of the protein and recognition of the liposomes for uptake and removal by macrophages and other cells of the reticuloendothelial system.
  • Liposomes include a conjugate in combination with a lipid, which in one embodiment is a vesicle-forming lipid, and, optionally, other bilayer components.
  • a lipid which in one embodiment is a vesicle-forming lipid, and, optionally, other bilayer components.
  • "Vesicle-forming lipids” are lipids that spontaneously form bilayer vesicles in water.
  • the vesicle-forming lipids preferably have two hydrocarbon chains, typically acyl chains, and a polar head group.
  • Examples include the phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), and sphingomyelin (SM).
  • a preferred lipid for use in the present invention is hydrogenated soy phosphatidylcholine (HSPC).
  • Another preferred family of lipids are diacylglycerols. These lipids can be obtained commercially or prepared according to published methods.
  • the vesicle-forming lipid may be selected to achieve a degree of fluidity or rigidity, to control the stability of the liposome in serum, and to control the rate of release of an entrapped agent in the liposome.
  • Liposomes having a more rigid lipid bilayer, or a liquid crystalline bilayer can be prepared by incorporation of a relatively rigid lipid, e.g., a lipid having a relatively high phase transition temperature, e.g. , up to about 80°C.
  • Rigid lipids i.e. , saturated, contribute to greater membrane rigidity in the lipid bilayer.
  • Other lipid components, such as cholesterol are also known to contribute to membrane rigidity in lipid bilayer structures.
  • Lipid fluidity is achieved by incorporation of a relatively fluid lipid, typically one having a lipid phase with a relatively low liquid to liquid-crystalline phase transition temperature, e.g. , at or below room temperature (about 20-25°C).
  • the liposome can also include other components that can be inco ⁇ orated into lipid bilayers, such as sterols. These other components typically have a hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and a polar head group moiety oriented toward the exterior, polar surface of the membrane.
  • Another lipid component in the liposomes of the present invention is a vesicle- forming lipid derivatized with a hydrophilic polymer. In this lipid component, a derivatized lipid results in formation of a surface coating of hydrophilic polymer chains on both the inner and outer lipid bilayer surfaces. Typically, between about 1-20 mole percent of the derivatized lipid is included in the lipid composition.
  • Hydrophilic polymers suitable for derivatization with a vesicle-forming lipid include polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, poly hydroxypropylmethacry late, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, and polyaspartamide.
  • the 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 about 500 to about 10,000 Daltons, preferably between about 1,000 to about 5,000 Daltons.
  • PEG polyethyleneglycol
  • Methoxy or ethoxy-capped analogues of PEG are also preferred hydrophilic polymers. These polymers are commercially available in a variety of polymer sizes, e.g. , from about 12 to about 220,000 Daltons.
  • Liposomes of the present invention include typically between about 1 and about 30 mole percent of the lipid-DTB-drug conjugate, preferably between about 5 and about 30 mole percent, more preferably between about 5 and about 20 mole percent.
  • liposomes comprised of the vesicle- forming lipid hydrogenated soy phosphatidylcholine (HSPC), distearoyl phosphatidylethanolamine derivatized with methoxy-polyethylene glycol (mPEG-DSPE) and the conjugate shown in Fig. 9A, ⁇ ra-distearoyl-DTB-mitomycin C (Compound XVIII) were prepared as described in Examples 4A-4B.
  • HSPC vesicle- forming lipid hydrogenated soy phosphatidylcholine
  • mPEG-DSPE distearoyl phosphatidylethanolamine derivatized with methoxy-polyethylene glycol
  • Fig. 9A ⁇ ra-d
  • One of the liposome formulations included cholesterol (Example 4A), with the lipids HSCP/cholesterol/mPEG-DSPE//r ⁇ r ⁇ -distearoyl-DTB-mitomycin C (Compound XVIII) present at a molar ratio of 60/30/5/5.
  • the lipids HSCP/mPEG-DSPE/ ⁇ ra-distearoyl-DTB-mitomycin C (Compound XVII) were present at a molar ratio of 90/5/5.
  • the liposome formulations e.g., HSPC/cholesterol/mPEG-DSPE/conjugate Compound XVffl (hereinafter the "cholesterol-containing formulation”) and HSPC/mPEG- DSPE/conjugate Compound XVffl (hereinafter the "cholesterol-free liposome formulation”) were incubated at 37°C in the presence of 150 ⁇ M cysteine for 24 hours. Samples were withdrawn at selected time points and analyzed by high performance liquid chromatography (HPLC) to quantify the amount of conjugate and of free mitomycin C. The HPLC conditions are described in Example 5.
  • HPLC high performance liquid chromatography
  • Figs. 10A-10B show HPLC chromatograms for two liposome formulations.
  • Fig. 10A the results for the cholesterol-free liposome formulation are shown.
  • time zero there is no detectable free mitomycin C and all measurable drug is in the form of a lipid-DTB-drug conjugate that is liposome bound.
  • Fig. 10B shows the results for the liposome formulation containing cholesterol.
  • Fig. 11 is a plot showing the percent of mitomycin C released from the two liposome formulations, as determined from the chromatograms in Figs. 10A-10B.
  • the cholesterol-free liposomes (closed diamonds) had a higher rate of release than the liposomes containing cholesterol (closed circles). More than 50% of the mitomycin C was released from the liposome-bound conjugate after 2 hours for the cholesterol-free formulation. For both formulations, greater than 80% of the drug was released at the end of the 24 hour incubation period.
  • Figs. 12A-12B show the results of mitomycin C released from the lipid-DTB-drug conjugate inco ⁇ orated into the cholesterol-free liposomes (HSPC/PEG- DSPE/lipid-DTB-mitomycin C). The percent release during incubation with 150 ⁇ M are also shown (closed diamonds) for comparison. As seen, incubation at a higher concentration of reducing agent (1.5 mM, open diamonds) causes an increase in the rate of conjugate decomposition and rate of drug release.
  • Fig. 12B shows the results for the liposome formulation containing cholesterol. Liposomes incubated in 1.5 mM (open circles) have a significantly higher decomposition rate than the same liposomes incubated in 150 ⁇ M cysteine (closed circles).
  • Liposomes prepared as described in Examples 4A-4B with the molar ratios specified in Example 6A were tested. Cysteine at concentrations of 150 ⁇ M, 500 ⁇ M and 1000 ⁇ m was added to some of the test cells to effect thioytic decomposition of the conjugate and release of mitomycin C.
  • IC50 values were taken as the drug concentration which caused a 50% inhibition of the control growth rate (IC 50 ), as described in Example 6. The results are shown in Table 1.
  • the percent growth rate of M109 mouse carcinoma cells determined from the cytotoxicity studies is shown in Fig. 13.
  • the percent growth rate is expressed as a percentage based on growth rate of Ml 09 cells in the absence of mitomycin C and of cysteine and is shown as a function of mitomycin C concentration, in nM.
  • the growth rate of cells was determined as described in Example 6. As seen, the percent of cell growth rate decreases as the cysteine concentration is increased for both the liposomes containing cholesterol (open circles) and the cholesterol-free liposome formulation (closed squares). It can also be seen that cysteine has no effect on the activity of free mitomycin c and that mitomycin C is released from the conjugate to effectively inhibit cell growth.
  • Figs. 14A-14B the results for the liposome formulation containing no cholesterol are shown.
  • the growth rate of M109 cells is expressed as a percentage based on growth of M109 cells in the absence of drug and cysteine and is shown as a function of mitomycin C concentration in nM.
  • the cells treated with mitomycin C in free form (open triangles) and with mitomycin C in free form plus 1000 ⁇ M cysteine (closed triangles) exhibit a decrease in growth rate due tl e toxicity of the drug in free form.
  • Fig. 14B is a similar plot for the liposome formulation containing cholesterol.
  • Cytotoxicity of free mitomycin C (closed squares) is not effected by the presence of cysteine.
  • the cytotoxicity data shows that the cholesterol-free liposome formulation is more affected by cysteine.
  • the IC50 of the cholesterol-free liposome formulation at certain cysteine concentrations is only 2-fold lower than that of the free drug alone.
  • the liposome formulation containing cholesterol is less cytotoxic than the cholesterol-free liposome formulation.
  • the data also shows that cysteine has no cytotoxic effect of the tumor cells and no effect on the cytotoxicity of free mitomycin C. It is also apparent from the data that cysteine increases in a dose-dependent fashion the cytotoxcity of liposome-bound mitomycin C. Thus, the cytotoxic effects observed for the liposomal formulations are mostly accounted for by cysteine-mediated release of mitomycin C from the lipid-DTB-drug conjugate.
  • the in vivo pharmacokinetics of the liposomes containing cholesterol and the cholesterol-free liposome formulation was determined in rats. As described in Example 7, the animals were treated with a single bolus intravenous injection of approximately 0.1 mg/mL mitomycin C in free form or inco ⁇ orated into liposomes in the form of the lipid-DTB-mitomycin C conjugate in accord with the invention. After injection, blood samples were taken and analyzed for amount of mitomycin C. The results are shown in Figs. 16A-16B.
  • Fig. 16A shows the concentration ( ⁇ g/mL) of mitomycin C in the blood of rats as a function of time in hours following intravenous injection.
  • free mitomycin C (open squares) administered intravenously in free form is rapidly cleared from the blood.
  • Mitomycin C in the form of a liposome-bound lipid-DTB-drug conjugate remains in circulation for a substantially longer period of time.
  • Mitomycin C associated with liposomes containing cholesterol (closed diamonds) and with cholesterol-free liposomes (closed circles) was detected in the blood at greater than 10 ⁇ g/mL for 20-25 hours.
  • Fig. 16B shows the percent of injected dose remaining in the blood as a function of time in hours following intravenous injection of the test formulations.
  • the rac-3,3'-dithiobis(l,2-propanediol) product (Compound U) was acylated by adding the compound (980 mg, 4.6 mmol) to an oven-dried 100 mL round bottom flask and dissolving in dry methylene chloride (40 mL). To this, stearic acid (4.92 g, 17.1 mmol) and 4-dimethylamino)pyridinium 4-toluenesulfonate (1.38 g, 4.6 mmol) as the catalyst was and stirred at room temperature (25 °C) for 20 minutes.
  • the mitomycin C solution was added drop-wise the acyl chloride solution. After 1 hour, the toluene was evaporated off and the crude product was chromatographed (1: 1 hexane: ethyl acetate) on silica. The purified product was then taken up in t-BuOH (50 mL) and lyophilized. The product was a pu ⁇ le solid (183 mg, 53%).
  • Liposomes Containing Cholesterol 1. Liposome Preparation 59 mg HSPC, 14.4 mg cholesterol, 17.4 mg mPEG-DSPE, and 7.4 ⁇ ngpara- distearoyl-DTB-mitomycin C (molar ratio of 60/30/5/5) were added to 1 mL dehydrated ethanol at 60-65 °C and mixed until dissolved, approximately 10 minutes. A hydration medium composed of 10 mM histidine and 150 mM NaCl in distilled water was warmed to 70 °C.
  • the warm lipid solution was rapidly added to the warm (63-67 °C) hydration medium, with mixing, to form a suspension of liposomes having heterogeneous sizes.
  • the suspension was mixed for one hour at 63-67 °C.
  • the liposomes were sized to the desired mean particle diameter by controlled extrusion through polycarbonate filter cartridges housed in Teflon-lined stainless steel vessels.
  • the liposome suspension was maintained at 63-65 °C throughout the extrusion process, a period of 6-8 hours.
  • Ethanol was removed from the liposome suspension by diafiltration.
  • a histidine/sodium chloride solution was prepared by dissolving histidine (10 mM) and sodium chloride (150 mM) in sterile water. The pH of the solution was adjusted to approximately 7. The solution was filtered through a 0.22 ⁇ m Durapore filter. The liposome suspension was diluted in approximately a 1 : 1 (v/v) ratio with the histidine/sodium chloride solution and diafiltered through a polysulfone hollow-fiber ultrafilter. Eight volume exchanges were performed against the histidine/sodium chloride solution to remove the ethanol. The process fluid temperature was maintained at about 20-30 °C. Total diafiltration time was approximately 4.5 hours.
  • lipid concentration and conjugate/drug concentration were determined by HPLC.
  • Liposome particle size was measured by dynamic light scattering and the amount of "free" , unbound mitomycin C in the external suspension medium was measured by HPLC.
  • Liposomes were prepared as described above with a lipid composition of HSPC, mPEG-DSPE and ⁇ ra-distearoyl-DTB-mitomycin C in a molar ratio of 90/5/5. Specifically, 88.5 mg HPSC, 17.9 mg mPEG-DSPE (PEG MW 2000 Daltons) and 7.3 mg of the conjugate were dissolved in 1 mL ethanol. Liposome size, lipid and drug concentration and free mitomycin C concentration in the external suspension medium were determined after each processing step.
  • Liposomes prepared as described in Examples 4A-4B were diluted in 0.6 M octaylglucopyranoside. The liposomes were incubated in the presence of 150 mM cysteine at 37 °C. Samples with withdrawn at time zero, 30 minutes, 1 hour, 2 hours, 4 hours and 24 hours. A 20 ⁇ L volume was analyzed by HPLC using a Water Symmetry C 8 3.5 x 5 cm column. The flow rate was 1 mL/min and the mobile phase gradient as follows:
  • Liposomes prepared as described in Example 4A-4B, were composed of
  • HSPC/cholesterol/mPEG-DSPE/distearoyl-DTB-mitomycin C (90/45/5/5).
  • the liposome preparations were sterile filtered through 0.45 ⁇ m cellulose membranes and were not downsized via extrusion. After liposome formation, mitomycin C concentration was determined by absorbance at 360 nm in liposomes solubilized by 10- 20 fold dilution in isopropanol and the phospholipid concentration was determined by inorganic phosphate assay.
  • the liposomes containing cholesterol had an average diameter of 275 ⁇ 90 nm.
  • the cholesterol-free liposomes had an average diameter of 150 + 50 nm.
  • the phospholipid concentration in both liposome formulations was 10 ⁇ M/mL and the concentration of mitomycin C in both formulations was 120 ⁇ g/mL.
  • the cytotoxic effect of free mitomycin C or mitomycin C in the form of a distearoyl-DTB-mitomycin C conjugate inco ⁇ orated into liposomes was assayed colorimetrically by a methylene blue staining method described previously (Horowitz, A.T. et l , Biochim. Biophys. Acta, 1 109:203-209 (1992)) with slight modifications. Upon completion of the assay, the cells were fixed and evaluated using the methylene blue staining assay.
  • the plates were washed three times with deionized water, once with 0.1 M borate buffer (pH 8.5) and then stained for 60 minutes with 100 ⁇ l methylene blue (1 % in 0.1 M buffer borate, pH 8.5) at room temperature (20-25°C) .
  • the plates were rinsed in five baths of deionized water to remove non-cell bound dye and then dried.
  • the dye was extracted with 200 ⁇ l 0.1 N HC1 for 60 minutes at 37°C and the optical density was determined using a microplate spectrophotometer .
  • the cell number determined by counting cells with a hemocytometer correlated well with the spectrophotometric absorbance.
  • the percent growth inhibition or percent of control growth rate was obtained by dividing the growth rate of drug-treated cells by the growth rate of the untreated, control cells.
  • the drug concentration which caused a 50% inhibition of the control growth rate (IC 50 ) was calculated by inte ⁇ olation of the two closest values of the growth inhibition curve.
  • Mitomycin C was assayed in the range 10 "8 -10 "5 M.
  • the liposomal formulations with conjugate-bound were assayed in the range 10 "8 - 3 x 10 "5 M.
  • For interaction stodies cysteine (SIGMA, St. Louis, MO) was added together with the mitomycin C or liposome formulations to final concentration of 150, 500, or 1000 ⁇ M. The results are shown in Table 1 and in Figs. 13, 14 and 15A-15B.
  • Liposomes containing cholesterol and cholesterol-free liposomes were prepared as described in Example 5 A and 5B.
  • a solution of mitomycin C in free form was prepared by dissolving 11.9 mg of mitomycin C in 119 ⁇ L ethanol. After dissolution, approximately 11.8 ⁇ L of a solution of 10 mM histidine/ 150 mM saline was added. Prior to use, the mitomycn C solution was diluted to 100 ⁇ g/mL with the histidine/saline solution and filtered.
  • a single intravenous injection of the test formulation was administered as a bolus dose.
  • Blood samples were taken from each animal at the following times after injection: 30 seconds, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours and 96 hours.
  • the quantity of mitomycin C in the blood samples was determined by the HPLC procedure given below.
  • a 200 mM iodoacetamine solution was prepared by placing 199.3 mg of iodoacetamide in 5.1 mL of 7.5% EDTA. 15 ⁇ L of the 200 mM iodoacetamide solution was placed in each 1 ⁇ L of blood sample.
  • a mobile phase of methanol and the aqueous buffer were mixed via a gradient program using a Waters Alliance binary pump.
  • the concentration ranges were 0.05-5.0 ⁇ g/mL and 0.1-5 ⁇ g/mL for mitomycin C and mitomycin C conjugate, respectively.
  • the final volume was adjusted to 1 mL with methanol.
  • a similar procedure was followed to prepare quality control samples.
  • the concentrations of quality control samples was 0.1, 0.5 and 5 ⁇ g/mL for mitomycin C and 0.1 , 1 and 5 ⁇ g/mL for mitomycin C conjugate in rat plasma.
  • the samples were spun down at 3,000 ⁇ m for 10 minutes at room temperature. 300 ⁇ L of supernatant was transferred to HPLC vials containing 300 ⁇ L insert for injection. 3.
  • a Supelco ® C-8, 5 ⁇ , 4.6mm x 5 cm column was used.
  • the mobile phase A was 10 mM ammonium phosphate, pH 7.
  • Mobil phase B was methanol.
  • the flow rate was 1 mL/min and detection was by UV at 360 nm.
  • the injection volume was 40 ⁇ L and the typical run time was 15 minutes.
  • the gradient program was as follows:

Abstract

Conjugates of a hydrophobic moiety, such as a lipid, linked through a cleavable dithiobenzyl linkage to a therapeutic agent are described. The dithiobenzyl linkage is susceptible to cleavage by mild thiolysis, resulting in release of the therapeutic agent in its original form. The linkage is stable under nonreducing conditions. The conjugate can be incorporated into liposomes for administration in vivo and release of the therapeutic agent in response to endogeneous in vivo reducing conditions or in response to administration of an exogeneous reducing agent.

Description

CONJUGATE HAVING A CLEAVABLE LINKAGE FOR USE IN A LIPOSOME
Field of the Invention
The present invention relates to a conjugate comprised of a hydrophobic moiety, a cleavable linkage, and a therapeutic agent. More particularly, the present invention relates to conjugates comprised of a lipid, a cleavable linkage and a drug incoφorated into a liposomal formulation. The conjugates are cleavable under mild thiolytic conditions in vivo for release of the drug in an unmodified state.
Background of the Invention
Liposomes are closed lipid vesicles used for a variety of therapeutic purposes, a d in particular, for carrying therapeutic agents to a target region or cell by systemic administration of liposomes. Liposomes having a surface grafted with chains of water- soluble, biocompatible polymer, in particular polyethylene glycol, have become important drug carries. These liposomes offer an extended blood circulation lifetime over liposomes lacking the polymer coating. The grafted polymer chains shield or mask the liposome, thus minimizing nonspecific interaction by plasma proteins. This in turn slows the rate at which the liposomes are cleared or eliminated in vivo since the liposome circulate unrecognized by macrophages and other cells of the reticuloendothelial system. Furthermore, due to the so-called enhanced permeability and retention effect, the liposomes tend to accumulate in sites of damaged or expanded vasculature, e.g. , tumors, sites of inflammation.
An extended blood circulation time is often desired to allow systemically administered liposomes to reach a target region, cell or site. For example, a blood circulation lifetime of greater than about 12 hours is preferred for liposomal-therapy to a tumor region, as the liposomes must systemically distribute and then extravasate into the tumor region.
One problem associated with liposome-based therapy is retention of drug within the liposome for a time sufficient for systemic distribution. This problem is of particular concern when long-circulating liposomes, i.e., liposomes with grafted polymer chains, are administered. Relatively few drugs can be efficiently loaded and retained for a long duration and subsequently released.
One approach to improving drug retention is to select lipid bilayer components that render the bilayer less permeable to entrapped drug. However, the lipid bilayer should be sufficiently fluidic such that the drug is released, for example by transport across the lipid bilayer or by lipid vesicle breakdown, at the desired time, e.g. , after localization at a target site or sufficient biodistribution.
Another approach to improving drug retention is to covalently attach the drug to a lipid in the liposomal lipid bilayer (Waalkes, et al., Selective Cancer Therap., 6: 15-22 (1990); Asai, et al, Biol. Pharm. Bull , 21:766-771 (1998)). It would be desirable to formulate a liposome composition having a long blood circulation lifetime and capable of retaining an entrapped drug for a desired time, yet able to release the drug on demand. One approach described in the art for achieving these features has been to formulate a liposome from a non-vesicle-forming lipid, such as dioleoylphosphatidylethanolamine (DOPE), and a lipid bilayer stabilizing lipid, such as methoxy-polyethylene glycol-distearoyl phosphatidylethanolamine (mPEG-DSPE) (Kirpotin, D, et al, FEBS Lett. 388:115-118 (1996)). In this approach, the mPEG is attached to the DSPE via a cleavable linkage. Cleavage of the linkage destabilizes the liposome for a quick release of the liposome contents.
Labile bonds for linking PEG polymer chains to liposomes has been described (U.S. Patent Nos. 5,013,556, 5,891,468; WO 98/16201). The labile bond in these liposome compositions releases the PEG polymer chains from the liposomes, for example, to expose a surface attached targeting ligand or to trigger fusion of the liposome with a target cell.
To date, however, a means of releasing polymer chains from liposomes under conditions suitable for in vivo use has not been achieved. For example, some releasable linkages require a potent thiolytic agent, such as 1,4-dithiothreitol, to achieve release of the polymer chains. This reducing agent is unacceptable for in vivo use. Another problem with known releasable linkages joining PEG to a liposome lipid is that cleavage of the releasable bond generates an unnatural and undesirable modified lipid. Accordingly, there remains a need in the art for a cleavable linkage that is suitable for in vivo use and which, after cleavage, yields the drug or therapeutic agent in its natural, unmodified form. In EP 0317957, Senter describes a drug-antibody prodrug, where the antibody is linked to a drug using a disulfide benzyl carbamate or carbonate linker, and reduction of the disulfide bond effects release of the drug. Senter' s teaching is specific to cleavage of a drug-ligand prodrug molecule, under the action of reducing agents such as 1,4- dithiothreitol, glutathione, NADH and NADPH. The behavior of such a linker, when incorporated into a liposome that circulates through the bloodstream, cannot be predicted based on the Senter disclosure. For example, in liposomes, the linker between the polymer and the lipid would be buried or masked in the PEG coating. It is relatively easy to envision release of a single drug-ligand prodrug as in Senter; however, release of the linker when a part of a densely packed barrier as in a liposome surface coating of polymer chains is less predictable.
As noted above, an extended blood circulation time is a desirable feature of PEG- coated liposomes, with blood circulation lifetimes of greater than about 12 hours being preferred for liposomal-therapy to a tumor region. The disclosure of Senter provides no guidance as to the release kinetics of a conjugate incorporated into a liposome under endogenous reducing conditions, such as during blood circulation of the liposome.
Summary of the Invention
Accordingly, it is an object of the invention to provide a liposome composition wherein the drug is retained for a desired period of time for release from the liposome . It is another object of the invention to provide a conjugate for use in a liposome, wherein the conjugate is comprised of a hydrophobic moiety for anchoring into the lipid bilayer, a linkage cleavable in response to mild thiolytic conditions and a therapeutic agent. In one aspect, the invention includes a conjugate for use in a liposomal drug- delivery vehicle, the conjugate having the general structural formula:
Figure imgf000005_0001
wherein L is a hydrophobic moiety suitable for incorporation into a liposomal lipid bilayer, R1 represents a therapeutic drug covalently attached to the dithiobenzyl moiety, and where orientation of the CH2R' group is selected from the ortho position and the para position.
In one embodiment, the therapeutic drug is covalently attached by a linkage selected from the group consisting of urethane, amine, amide, carbonate, thio- carbonate, ether and ester.
In another embodiment, L is selected from the group consisting of cholesterol, a diacylglycerol, a phospholipid and derivatives thereof.
In yet another embodiment, L is a diacylglycerol derivative to yield a conjugate having the general structural formula:
Figure imgf000006_0001
wherein R2 and R3 are hydrocarbons having between about 8 to about 24 carbon atoms, or in another embodiment, from about 12 to about 22 carbon atoms. In still another embodiment, R2 and R3 are hydrocarbon chains of the same length.
In another embodiment, the drug is selected from the group consisting of mitomycin C, mitomycin A, bleomycin, doxorubicin, daunorubicin, fluorodeoxyuridine, iododeoxyuridine, etoposide, AZT, acyclovir, vidarabine, arabinosyl cytosine, pentostatin, quinidine, atropine, chlorambucil, methotrexate, mitoxantrone and 5-fluorouracil.
In still another embodiment, the therapeutic drug is covalently linked to the dithiobenzyl moiety to form a conjugate having the structure:
Figure imgf000006_0002
wherein R4 represents a residue of the therapeutic drug.
R4 in one embodiment is a therapeutic drug residue containing a primary or a secondary amine moiety thereby forming a urethane linkage between the dithiobenzyl and the therapeutic drug. In this embodiment, the therapeutic drug can be, for example, mitomycin A, mitomycin C, bleomycin or a polypeptide.
In another embodiment, R4 is a residue of a carboxyl-containing therapeutic drug, which forms an ester linkage between the dithiobenzyl and the therapeutic drug. Examplary drugs in this embodiment include chlorambucil or methotrexate.
In another embodiment, R4 is a therapeutic drug residue containing a hydroxyl moiety thereby to form a carbonate linkage between the dithiobenzyl and the therapeutic drug. Exemplary drugs in this embodiment include fluorodeoxyuridine, iododeoxyuridine, etoposide, AZT, acyclovir, vidarabine, arabinosyl cytosine, pentostatin, quinidine, mitoxantrone and atropine.
In another aspect, the invention includes a liposome composition, comprising liposomes composed of vesicle-forming lipids including from about 1 to about 30 mole percent of a conjugate having the general structural described above. The therapeutic drug is released from the conjugate in vivo in response to a physiologic condition or an artificially induced condition.
In yet another aspect, the invention includes a method for retaining a drug in a liposome, comprising preparing liposomes comprised of a vesicle-forming lipid and of between about 1 to about 30 mole percent of a conjugate described above. The liposomes effectively retain the drug in the liposomes until release from the conjugate in response to a physiologic condition or an artificially induced condition.
These and other objects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying drawings.
Brief Description of the Drawings
Fig. 1 shows a synthetic reaction scheme for preparation of /røra-diacyldiglycerol- dithiobenzylalcohol for further reaction with amine-, hydroxy- or carboxyl-containing drugs;
Fig. 2 A shows a general reaction scheme for attachment of an amino-containing drug to a reactive diacyldiglycerol-dithiobenzylcarbonate;
Fig. 2B shows the products after thiolytic cleavage of the conjugate in Fig. 2A;
Fig. 3 A shows a synthetic reaction scheme for preparation of a diacyldiglycerol- dithiobenzyl-5-fluorouracil conjugate ; Fig. 3B shows the products after thiolytic cleavage of the conjugate in Fig. 3 A;
Fig. 4 shows an alternative synthetic reaction scheme for preparation of a diacyldiglycerol-dithiobenzyl-5-fluorouracil conjugate and the products after thiolytic cleavage of the conjugate;
Fig. 5 shows a synthetic reaction scheme for preparation of a diacyldiglycerol- dithiobenzyl-chlorambucil conjugate and the products after thiolytic cleavage of the conjugate;
Fig. 6 A shows a synthetic reaction scheme for preparation of a diacyldiglycerol- difhiobenzy 1-mitomycin-C conjugate ;
Fig. 6B shows the products after thiolytic cleavage of the conjugate in Fig. 6A; Fig. 7 shows a synthetic reaction scheme for preparation of a cholesterol- dithiobenzyl-mitomycin-C conjugate ;
Fig. 8 shows another synthetic reaction scheme for preparation of a cholesterol- dithiobenzy 1-mitomycin-C conjugate;
Figs. 9A-9C show the structures of three lipid-dithiobenzy 1-mitomycin-C conjugates, αra-distearoyl-DTB-mitomycin-C (Fig. 9A), /?αrα-dipalmitoyl-DTB- mitomycin-C (Fig. 9B) and ørtbø-dipalmitoyl-DTB- mitomycin-C (Fig. 9C);
Figs. 10A-10B are HPLC chromatograms for liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C (Fig. 10A) and HSPC/cholesterol/mPEG- DSPE/lipid-DTB-mitomycin C (Fig. 10B), where each figure shows a series of chromatograms as a function of time of incubation of the liposomes in the presence of cysteine;
Fig. 11 is a plot showing the percent of mitomycin C released from liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C (closed diamonds) and HSPC/cholesterol/mPEG-DSPE/lipid-DTB-mitomycin C (closed circles) as a function of time of incubation in the presence of cysteine;
Figs. 12A-12B are plots showing the percent of mitomycin C released from liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C (Fig. 12 A) and HSPC/cholesterol/mPEG-DSPE/lipid-DTB-mitomycin C (Fig. 12B) as a function of time of incubation in the presence of cysteine at concentrations of 150 μM (closed symbols) and at 1.5 mM (open symbols);
Fig. 13 is a plot of growth rate of Ml 09 cells, expressed as a percentage based on growth of M 109 cells in the absence of drug and cysteine, as a function of mitomycin C amount, in nM, for free mitomycin c (open triangles), liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C (closed squares), and liposomes comprised of HSPC/cholesterol/mPEG-DSPE/lipid-DTB-mitomycin C (open circles); Fig. 14A is a plot of growth rate of Ml 09 cells, expressed as a percentage based on growth of M109 cells in the absence of drug or cysteine, as a function of mitomycin C concentration in nM. Shown are cells treated mitomycin C in free form (open triangles) and with mitomycin C in free form plus 1000 μM cystein (closed triangles). Also shown are cells treated with the liposome formulation comprised of HSPC/PEG- DSPE/lipid-DTB-mitomycin C (open circles) and with the liposome formulation with additional cysteine added at concentrations of 150 μM (open diamonds), 500 μM (closed circles) and 1000 μM (open squares);
Fig. 14B is a plot of growth rate of Ml 09 cells, expressed as a percentage based on growth of M109 cells in the absence of drug or cysteine, as a function of mitomycin C concentration in nM. Shown are cells treated mitomycin C in free form (open triangles) and with mitomycin C in free form plus 1000 μM cystein (closed triangles). Also shown are cells treated with the liposome formulation comprised of HSPC/cholesterol/mPEG-DSPE/lipid-DTB-mitomycin C (open circles) and with the liposome formulation with additional cysteine added at concentrations of 150 μM (open diamonds), 500 μM (closed circles) and 1000 μM (open squares); Fig. 15 is a plot showing the percent increase in cytotoxicity (as determined by
(IC50no cysteme/IC50cyst.,ne)xl00)) of free mitomycin C (closed squares), mitomycin C associated with liposomes comprised of HSPC/cholesterol/mPEG-DSPE/lipid-DTB- mitomycin C (closed circles), and liposomes comprised of HSPC/mPEG-DSPE/lipid- DTB-mitomycin C (open triangles) to M109 cells in vitro at various concentrations of cysteine;
Fig. 16A is a plot showing the concentration of mitomycin C in the blood of rats as a function of time in hours following intravenous injection of free mitomycin C (open squares), liposomes comprised of HSPC/cholesterol/mPEG-DSPE/lipid-DTB- mitomycin C (closed diamonds), and liposomes comprised of HSPC/mPEG-DSPE/lipid- DTB-mitomycin C (closed circles); and
Fig. 16B is a plot showing the percent of injected dose remaining in the blood of rats as a function of time in hours following intravenous injection of free mitomycin C (open squares), liposomes comprised of HSPC/cholesterol/mPEG-DSPE/lipid-DTB- mitomycin C (closed diamonds), and liposomes comprised of HSPC/mPEG-DSPE/lipid- DTB-mitomycin C (closed circles).
Detailed Description of the Invention
I. Definitions
The phrase "hydrophobic moiety suitable for incorporation into a liposomal lipid bilayer" intends any material comprising a hydrophobic portion capable of being integrated with the hydrophobic bilayer region of a liposomal lipid bilayer. Such hydrophobic moieties are typically lipids, including amphipathic lipids having a hydrophobic lipid tail and a hydrophilic polar head, such as phospholipids and diacylglycerols. Triglycerides, sterols, derivatives of phospholipids, diacylglyerols, sterols and triglycerides and other lipids derived from a natural source or synthetically prepared are also contemplated. The term "residue" as in "therapeutic drug residue" intends a drug molecule that has been reacted to form an linkage with another molecule where at least one atom of the drug molecule is replaced or has been sacrificed to from the linkage.
"Polypeptide" as used herein refers to a polymer of amino acids and does not refer to a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, and enzyme are included within the definition of polypeptide. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like.
The following abbreviations are used herein: PEG, poly(ethylene glycol); mPEG, methoxy-PEG; DTB, dithiobenzyl; DSPE, distearoyl phosphatidylethanolamine; HSPC, hydrogenated soy phosphatidylcholine; MMC, mitomycin C. II. Conjugate Composition and Method of Preparation
In one aspect, the invention includes a conjugate of the form:
Figure imgf000011_0001
wherein L is a hydrophobic moiety suitable for incorporation into a liposomal lipid bilayer, R1 represents a therapeutic drug residue covalently attached to the dithiobenzyl moiety, and where orientation of the CH2R' group is selected from the ortho position and the para position. The hydrophobic moiety, L, is typically a lipid such as a diacylglycerol, a sterol, a phospholipid, derivatives of these lipids, other naturally-occurring lipids and their synthetic analogs.
In the conjugate, a therapeutic drug is attached to the dithiobenzyl moiety by a covalent linkage, thereby forming a drug residue, represented by R1 in the structure. The linkage will vary according to the drug and the reaction chemistry, as will be appreciated by those of skill in the art. In preferred embodiments, the therapeutic drug is covalently attached to the diithiobenzyl moiety by a linkage selected from the group consisting of urethane, amine, amide, carbonate, thio-carbonate, ether and ester.
A urethane linkage takes the form of 0(C=0)NH-R4 or 0(C=0)N=R\ where R4 represents the therapeutic drug residue. For example, a drug containing a primary or secondary amine, such as mitomycin C, mitomycin A, bleomycin and therapeutic polypeptides to name a few, is reacted to from a urethane linkage with the amine moiety in the drug.
A carbonate linkage takes the form of 0(C=0)0-R4, where R4 represents the drug residue and the carbonate linkage derives from a phenol or alcohol or hydroxyl moiety in the drug. A thio-carbonate takes the form of 0(C=0)S-R4, where R4 represents the drug residue and the linkage derives from a moiety in the drug.
Exemplary drugs having such a moiety for reaction with dithiobenzyl alcohol to form a carbonate linkage include fluorodeoxyuridine, iododeoxyuridine, etoposide, AZT, acyclovir, vidarabine, arabinosyl cytosine, pentostatin, quinidine, mitoxantrone and atropine. An ester linkage takes the form of 0(C=0)-R4, where R4 represents the drug residue. The linkage derives from reaction with a carboxylic acid moiety in the therapeutic drug, and an example of a conjugate having an ester linkage between chlorambucil and dithiobenzyl is described below. Methotrexate is another example of a drug capable of forming an ester linkage with the dithiobenzyl moiety of the conjugate.
Conjugates having a urethane, carbonate or ester linkage attaching the drug to the dithiobenzyl moiety can generally be represented by the following structure:
Figure imgf000012_0001
wherein R4 represents a residue of the therapeutic drug.
In another embodiment, the conjugate includes an ether linkage, which takes the form of O-R4, where R4 represents the therapeutic drug residue. The linkage typically derives from reaction with an alcohol functionality on the drug.
An amine linkage is of the form N=R4, where R4 represents the drug residue and the linkage is a direct attachment with the CH2 moiety of the dithiobenzyl with a N in the drug. A conjugate with the drug 5-fluorouracil where an amine linkage is formed will be described below. An amide linkage can also be formed with a peptide as the therapeutic agent, where the free carboxyl of an amino acid residue, such as an aspartic acid or glutamic acid, is condensed with dithiobenzylamine. An amide linkage takes the form of NH(C =0)-R4, where R4 represents the drug residue.
Fig. 1 shows a synthetic reaction scheme for preparation of exemplary conjugates in accord with the invention. In this embodiment, synthesis of an intermediate compound, αrα-diacyldiglyceroldithiobenzalcohol (Compound IV), is prepared for further reaction with a selected therapeutic drug. Compound IV is prepared, as described in Example 1 , by reacting 3-mercapto-l ,2-propanediol (Compound I) with hydrogen peroxide to form rαc-3,3'-dithiobis(l ,2-propanediol) (Compound II). Rac- 3,3'-dithiobis(l ,2-propanediol) is acylated with a hydrophobic moiety R. For example, R can be a fatty acid having from about 8 to about 24 carbon atoms. Example 1 details the reaction procedure where R is stearic acid. In another embodiment, R is a fatty acid having from about 12 to about 22 carbon atoms. Acylation of Compound II yields Rac- 3,3'-dithiobis(l ,2-propanedistearoyl) (Compound III), which is reacted with sulfuryl chloride and 4-mercaptobenzalcohol to form the desired intermediate product, para- diacyldiglycerol-dithiobenzalcohol (Compound IV). Compound IV is readily reacted with a drug containing a reactive carboxyl moiety (R'C02H) to form a lipid- dithiobenzyl (DTB)-drug conjugate where the drug is joined to the DTB via an ester linkage (Compound V). Compound IV is also readily reacted with a drug containing a reactive amine moiety (R'-NH2) to yield a lipid-DTB-drug conjugate where the drug is joined to the DTB by a urethane linkage (Compound VI). Compound IV is also readily reacted with a drug containing a reactive hydroxyl moiety (R'OH) to form a lipid-DTB- drug conjugate where the drug is joined to the DTB by a carbonate linkage (Compound VII).
A variety of drugs are contemplated for use in the conjugate of the invention. In particular, the invention contemplates drugs having an amine (NH or NH2), carboxyl, sulfhydryl or hydroxyl moiety suitable for reaction. As used herein, "suitable for reaction" implies that the drug has one of the recited moieties capable of reacting with the dithiobenzyl moiety, in the form of, for example, dithiobenzyl alcohol. Exemplary drugs include 5-fluorouracil, which has an NH group suitable for reaction, chlorambucil, which has a reactive carboxyl and mitomycin C, which has a reactive amine (aziridine group). These drugs are used to illustrate synthesis of exemplary embodiments of the invention and are discussed with respect to Figs. 3-9. Other exemplary drugs contemplated for use include mitomycin C, mitomycin A, bleomycin, doxorubicin, daunorubicin, fluorodeoxyuridine, iododeoxyuridine, etoposide, AZT, acyclovir, vidarabine, arabinosyl cytosine, pentostatin, quinidine, atropine, chlorambucil, methotrexate, mitoxantrone and 5-fluorouracil. It will be appreciated that polypeptides, aminoglycosides, alkaloids are all also suitable for use in the invention.
Example 1 also details the reaction conditions for preparation of ortho- diacyldiglyceroldithiobenzalcohol, which can serve as a intermediary compound to form the conjugate. Figs. 2A-2B show preparation of a lipid-DTB-drug conjugate (Fig. 2A), and thiolytic cleavage of the conjugate in the presence of a reducing agent (Fig. 2B). As shown in Fig. 2A, Compound VII of Fig. 1 where the hydrophobic moiety R is derived from a fatty acid R"(CO)OH, such as stearic acid (CH3(CH2)16CO2H), is reacted with an amine-containing drug, H2N-drug, in the presence of phosgene (COCl2). This reaction yields the lipid-DTB-drug conjugate illustrated in Fig. 2A. The conjugate, upon exposure to reducing conditions, i.e., a reducing agent such as cysteine or glutathione, decomposes to yield the products shown in Fig. 2B. As shown, thiolytic cleavage of the conjugate results in regeneration of the drug in an unmodified, natural state. This is a desirable feature, since, as will be shown below, the drug in conjugate can be readily incorporated into liposomes for administration in vivo to a subject. Further, the drug in the form of the conjugate is not toxic, as will also be shown below. After administration and upon exposure to endogeneous reducing agents or exposure to an exogeneous reducing agent, the conjugate decomposes to yield the drug in its native state and with biological activity.
In Fig. 3 A, a synthetic reaction scheme for preparation of a conjugate of 5- fluorouracil is illustrated. Compound IV, /røra-diacyl-diglycerol-dithiobenzalalcohol, is reacted with /røra-toluenesulfonyl chloride to form the intermediate compound IX. Reaction with 5-fluorouracil anion or sodium salt (Compound X) yields the desired lipid-DTB-5-fluorouracil conjugate (Compound XI). Decomposition of the lipid-DTB- 5-fluorouracil conjugate (Compound XI) upon exposure to a reducing agent, R'-SH, is shown in Fig. 3B. Thiolytic cleavage of the conjugate results in regeneration of 5- fluorouracil in an unmodified form.
Fig. 4 shows an alternative synthetic reaction scheme for preparation of a diacyldiglycerol-DTB-5-fluorouracil conjugate. In Fig. 4, 1-chloroethyl chloroformate (Compound XII) is reacted with /?αrα-diacyl-diglycerol-dithiobenzalalcohol (Compound IV) to form a reactive chloroethyl carbonate-DTB-diacyldiglycerol intermediate (Compound XII). The intermediate is subsequently reacted with 5-fluorouracil in the presence of ttiemanolamine (TEA) to yield a lipid-DTB-5-fluorouracil conjugate (Compound XIV). Thiolytic cleavage of the conjugate and regeneration of 5-fluorouracil is also shown in Fig. 4.
Another embodiment of the conjugate is shown in Fig. 5. In this embodiment, a drug containing a reactive carboxyl moiety, chlorambucil (Compound XV), is reacted with αra-diacyl-diglycerol-dithiobenzalalcohol (Compound IV) in the presence of 1,3- dicyclohexycarbodiimide (DCC) and dimethylaminopyridine (DMAP) to form a lipid- DTB-chlorambucil conjugate (Compound XVI). Upon exposure to a reducing agent, the conjugate thiolytically decomposes to the products shown. Chlorambucil is subsequently regenerated in an unmodified state.
Fig. 6 A shows the synthesis of a conjugate in accord with another embodiment of the invention. In the reaction scheme shown, mitomycin C (Compound XVII, Fig. 6B), a drug containing a reactive amine moiety, is reacted with /røra-diacyl-diglycerol- dithiobenzalalcohol (Compound IV) in the presence of phosgene to form a diacyldiglycerol-dithiobenzy 1-mitomycin-C conjugate (Compound XVIII). Details of the synthesis are provided in Example 2. Fig. 6B shows the thiolytic decomposition of a diacyldiglycerol-DTB-mitomycin-
C conjugate. In the presence of a reducing agent, the conjugate decomposes to regenerate mitomycin C (Compound XVII) and the other products shown.
As noted above, the hydrophobic moiety in the conjugate can be selected from any number of hydrophobic moieties, e.g. , lipids. In the examples above, a diacyldiglycerol lipid was used to form conjugates having the structure:
Figure imgf000015_0001
wherein R2 and R3 are hydrocarbons having between about 8 to about 24 carbon atoms.
In addition to diacylglycerols as the hydrophobic moiety, other lipids are contemplated. Fig. 7 shows another embodiment where cholesterol is used as the hydrophobic moiety in the conjugate. Cholesterol (Compound XIV) is reacted with methanesulfonyl chloride in dichloromethane in the presence of triemylamine (TEA). The resulting intermediate is then converted into the thiol derivative and ultimately into the principal dithiobenzyl alcohol, which is used to link mitomycin C in a similar fashion as described above for diacylglycerol.
An alternative reaction scheme for preparation of a cholesterol-DTB-mitomycin-C conjugate is shown in Fig. 8. Methoxycarbonyldithioethyl amine is directly reacted with cholesterol chloroformate forming a urethane linkage. Then mercaptobenzylalcohol is used to obtain the DTB-cholesterol compound. Mitomycin C is linked as described above and in Example 2.
It will be appreciated by one of skill in the art that the various conjugates described above are merely exemplary. A wide variety of other hydrophobic moieties and other drugs, such as doxorubicin and daunorubicin, are contemplated and suitable for use in the invention. In additional studies performed in support of the invention, described below, the conjugate prepared as described in Fig. 6A, Compound XVII, αra-distearoyl-DTB-mitomycin C, was used. For ease of reference, this conjugate is shown in Fig. 9A. It is to be appreciated that other diacyl lipids, such as a dipalmitoyl lipid, can be used, and Fig. 9B shows a/?αr -dipalmitoyl-DTB-mitomycin C conjugate. It will also be appreciated that the conjugate can also have an isomeric linkage. This is evident by the ortbo-dipalmitoyl-DTB-mitomycin C conjugate as shown in Fig. 9C.
III. Preparation of Liposomes Comprising Conjugate
In another aspect, the invention includes a liposome composition comprised of a vesicle-forming lipid and a conjugate as described above. Liposomes are closed lipid vesicles used for a variety of therapeutic purposes, and in particular, for carrying therapeutic agents to a target region or cell by systemic administration of liposomes. In particular, liposomes having a surface coating of hydrophilic polymer chains, such as polyethylene glycol (PEG), are desirable as drug carries as these liposomes offer an extended blood circulation lifetime over liposomes lacking the polymer coating. The polymer acts as a barrier to blood proteins thereby preventing binding of the protein and recognition of the liposomes for uptake and removal by macrophages and other cells of the reticuloendothelial system.
Liposomes, according to the invention, include a conjugate in combination with a lipid, which in one embodiment is a vesicle-forming lipid, and, optionally, other bilayer components. "Vesicle-forming lipids" are lipids that spontaneously form bilayer vesicles in water. The vesicle-forming lipids preferably have two hydrocarbon chains, typically acyl chains, and a polar head group. There are a variety of synthetic vesicle- forming lipids and naturally-occurring vesicle-forming lipids known in the art where the two hydrocarbon chains are typically from about 12 to about 24 carbon atoms in length, and have varying degrees of unsaturation. Examples include the phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), and sphingomyelin (SM). A preferred lipid for use in the present invention is hydrogenated soy phosphatidylcholine (HSPC). Another preferred family of lipids are diacylglycerols. These lipids can be obtained commercially or prepared according to published methods.
The vesicle-forming lipid may be selected to achieve a degree of fluidity or rigidity, to control the stability of the liposome in serum, and to control the rate of release of an entrapped agent in the liposome. Liposomes having a more rigid lipid bilayer, or a liquid crystalline bilayer, can be prepared by incorporation of a relatively rigid lipid, e.g., a lipid having a relatively high phase transition temperature, e.g. , up to about 80°C. Rigid lipids, i.e. , saturated, contribute to greater membrane rigidity in the lipid bilayer. Other lipid components, such as cholesterol, are also known to contribute to membrane rigidity in lipid bilayer structures. Lipid fluidity is achieved by incorporation of a relatively fluid lipid, typically one having a lipid phase with a relatively low liquid to liquid-crystalline phase transition temperature, e.g. , at or below room temperature (about 20-25°C).
The liposome can also include other components that can be incoφorated into lipid bilayers, such as sterols. These other components typically have a hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and a polar head group moiety oriented toward the exterior, polar surface of the membrane. Another lipid component in the liposomes of the present invention, is a vesicle- forming lipid derivatized with a hydrophilic polymer. In this lipid component, a derivatized lipid results in formation of a surface coating of hydrophilic polymer chains on both the inner and outer lipid bilayer surfaces. Typically, between about 1-20 mole percent of the derivatized lipid is included in the lipid composition.
Hydrophilic polymers suitable for derivatization with a vesicle-forming lipid include polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, poly hydroxypropylmethacry late, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, and polyaspartamide. The 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 about 500 to about 10,000 Daltons, preferably between about 1,000 to about 5,000 Daltons. Methoxy or ethoxy-capped analogues of PEG are also preferred hydrophilic polymers. These polymers are commercially available in a variety of polymer sizes, e.g. , from about 12 to about 220,000 Daltons.
Liposomes of the present invention include typically between about 1 and about 30 mole percent of the lipid-DTB-drug conjugate, preferably between about 5 and about 30 mole percent, more preferably between about 5 and about 20 mole percent. In studies performed in support of the invention, liposomes comprised of the vesicle- forming lipid hydrogenated soy phosphatidylcholine (HSPC), distearoyl phosphatidylethanolamine derivatized with methoxy-polyethylene glycol (mPEG-DSPE) and the conjugate shown in Fig. 9A, αra-distearoyl-DTB-mitomycin C (Compound XVIII) were prepared as described in Examples 4A-4B. One of the liposome formulations included cholesterol (Example 4A), with the lipids HSCP/cholesterol/mPEG-DSPE//rørø-distearoyl-DTB-mitomycin C (Compound XVIII) present at a molar ratio of 60/30/5/5. A second formulation, which contained no cholesterol, was prepared and characterized (Example 4B). In this formulation, the lipids HSCP/mPEG-DSPE/^αra-distearoyl-DTB-mitomycin C (Compound XVII) were present at a molar ratio of 90/5/5.
IV. In vitro Characterization of Liposomes Containing a Conjugate A. In vitro Drug Release Liposomes were prepared as described in Examples 4A-4B and were characterized in vitro to determine the rate of release of mitomycin C following exposure to reducing agent. For the in vitro studies, reducing conditions were induced by addition of cysteine, typically at a concentration of about 150 μM, to the test medium. It will be appreciated that in vivo, endogeneous reducing conditions may be sufficient to effect thiolytic decomposition of the lipid-DTB-drug conjugate for release of the drug. It is further contemplated that reducing conditions in vivo can be artificially induced by administration of a suitable reducing agent, such as cysteine or glutathione. The liposome formulations, e.g., HSPC/cholesterol/mPEG-DSPE/conjugate Compound XVffl (hereinafter the "cholesterol-containing formulation") and HSPC/mPEG- DSPE/conjugate Compound XVffl (hereinafter the "cholesterol-free liposome formulation") were incubated at 37°C in the presence of 150 μM cysteine for 24 hours. Samples were withdrawn at selected time points and analyzed by high performance liquid chromatography (HPLC) to quantify the amount of conjugate and of free mitomycin C. The HPLC conditions are described in Example 5.
Figs. 10A-10B show HPLC chromatograms for two liposome formulations. In Fig. 10A, the results for the cholesterol-free liposome formulation are shown. At time zero, there is no detectable free mitomycin C and all measurable drug is in the form of a lipid-DTB-drug conjugate that is liposome bound. As the incubation time increases, the amount of mitomycin C released from the liposomes and detectable in free form increases, with a corresponding decrease in the presence of conjugate-bound mitomycin C. Fig. 10B shows the results for the liposome formulation containing cholesterol.
In the first sample taken at time zero, there was no detectable free mitomycin C. After 1 hour of incubation in 150 μM cysteine, a small amount of free drug was detected, indicating decomposition of the liposome-bound lipid-DTB-mitomycin conjugate. In comparison with Fig. 10A, liposomes containing cholesterol yield a slower conjugate decomposition rate and accordingly slower release of the drug.
Fig. 11 is a plot showing the percent of mitomycin C released from the two liposome formulations, as determined from the chromatograms in Figs. 10A-10B. The cholesterol-free liposomes (closed diamonds) had a higher rate of release than the liposomes containing cholesterol (closed circles). More than 50% of the mitomycin C was released from the liposome-bound conjugate after 2 hours for the cholesterol-free formulation. For both formulations, greater than 80% of the drug was released at the end of the 24 hour incubation period.
In another study, the two liposome formulations were incubated in 1.5 mM cysteine. Analysis was done as described in Example 5 and the results are shown in Figs. 12A-12B. Fig. 12A shows the percent of mitomycin C released from the lipid-DTB-drug conjugate incoφorated into the cholesterol-free liposomes (HSPC/PEG- DSPE/lipid-DTB-mitomycin C). The percent release during incubation with 150 μM are also shown (closed diamonds) for comparison. As seen, incubation at a higher concentration of reducing agent (1.5 mM, open diamonds) causes an increase in the rate of conjugate decomposition and rate of drug release.
Fig. 12B shows the results for the liposome formulation containing cholesterol. Liposomes incubated in 1.5 mM (open circles) have a significantly higher decomposition rate than the same liposomes incubated in 150 μM cysteine (closed circles).
B. In vitro Cvtotoxicity The in vitro cytotoxicity of liposomes containing the lipid-DTB-mitomycin C conjugate (Compound XVIII) was evaluated using M-109 cells, a mouse lung carcinoma line. As described in Example 6, M109 cells were incubated in the presence of free mitomycin C or liposomes containing the distearoyl-DTB-mitomycin C conjugate.
Liposomes prepared as described in Examples 4A-4B with the molar ratios specified in Example 6A were tested. Cysteine at concentrations of 150 μM, 500 μM and 1000 μm was added to some of the test cells to effect thioytic decomposition of the conjugate and release of mitomycin C.
IC50 values were taken as the drug concentration which caused a 50% inhibition of the control growth rate (IC50), as described in Example 6. The results are shown in Table 1.
Table 1
IC50 Values for M109 tumor cells after 72 hour culture with continuous exposure to formulation
Figure imgf000021_0001
MMC = mιtomycm C HSPC/cholesterol/mPEG-DSPE/distearoyl-DTB-MMC (90/45/5/5) ΗSPC/mPEG-DSPE/distearoyl-DTB-MMC (90/5/5) "n.d. =not done
The percent growth rate of M109 mouse carcinoma cells determined from the cytotoxicity studies is shown in Fig. 13. The percent growth rate is expressed as a percentage based on growth rate of Ml 09 cells in the absence of mitomycin C and of cysteine and is shown as a function of mitomycin C concentration, in nM. The growth rate of cells was determined as described in Example 6. As seen, the percent of cell growth rate decreases as the cysteine concentration is increased for both the liposomes containing cholesterol (open circles) and the cholesterol-free liposome formulation (closed squares). It can also be seen that cysteine has no effect on the activity of free mitomycin c and that mitomycin C is released from the conjugate to effectively inhibit cell growth. The in vitro growth rate of Ml 09 mouse carcinoma cells treated with mitomycin
C in free form or with mitomycin C in the form a liposome-bound lipid-DTB-drug conjugate is shown in Figs. 14A-14B. In Fig. 14A the results for the liposome formulation containing no cholesterol are shown. In the plot, the growth rate of M109 cells is expressed as a percentage based on growth of M109 cells in the absence of drug and cysteine and is shown as a function of mitomycin C concentration in nM. The cells treated with mitomycin C in free form (open triangles) and with mitomycin C in free form plus 1000 μM cysteine (closed triangles) exhibit a decrease in growth rate due tl e toxicity of the drug in free form. Cells treated with the liposome formulation comprised of HSPC/PEG-DSPE/DSPE-DTB-mitomycin C (open circles) and with the liposome formulation with additional cysteine added at concentrations of 150 μM (open diamonds), 500 μM (closed circles) and 1000 μM (open squares) exhibited cell cytotoxicity in a cysteine-dose dependent fashion.
Fig. 14B is a similar plot for the liposome formulation containing cholesterol.
The same pattern was observed for cells treated with the liposome composition containing cholesterol plus additional cysteine at concentrations of 150 μM (open diamonds), 500 μM (closed circles) and 1000 μm (open squares). That is, as the concentration of cysteine increased, the cell growth rate decreased. This indicates a cysteine-induced release of mitomycin C in direct correlation with cysteine concentration. In contrast to the liposome formulations, the in vitro growth rate of cells treated with mitomycin C in free form (open triangles) was the same as the growth rate of cells treated with mitomycin C in free form plus 1000 μM cysteine (closed triangles). Fig. 15 shows the percent increase in cytotoxicity as a function of cysteine concentration, in μM, of free mitomycin C and of the liposome formulations. Increase in cytotoxicity was determined by the percent drop in IC50, e.g., IC50 in the presence of cysteine relative to IC50 in the absence of cysteine time 100 ((IC50no cysteιne y'IC50cy-te,ne)xl00)). As seen, the percent of cytotoxicity increases significantly as the cysteine concentration is increased for both the liposomes containing cholesterol (open triangles) and the cholesterol-free liposome formulation (closed circles).
Cytotoxicity of free mitomycin C (closed squares) is not effected by the presence of cysteine.
The cytotoxicity data shows that the cholesterol-free liposome formulation is more affected by cysteine. The IC50 of the cholesterol-free liposome formulation at certain cysteine concentrations is only 2-fold lower than that of the free drug alone. The liposome formulation containing cholesterol is less cytotoxic than the cholesterol-free liposome formulation. The data also shows that cysteine has no cytotoxic effect of the tumor cells and no effect on the cytotoxicity of free mitomycin C. It is also apparent from the data that cysteine increases in a dose-dependent fashion the cytotoxcity of liposome-bound mitomycin C. Thus, the cytotoxic effects observed for the liposomal formulations are mostly accounted for by cysteine-mediated release of mitomycin C from the lipid-DTB-drug conjugate. C. In vivo Pharmacokinetics
The in vivo pharmacokinetics of the liposomes containing cholesterol and the cholesterol-free liposome formulation was determined in rats. As described in Example 7, the animals were treated with a single bolus intravenous injection of approximately 0.1 mg/mL mitomycin C in free form or incoφorated into liposomes in the form of the lipid-DTB-mitomycin C conjugate in accord with the invention. After injection, blood samples were taken and analyzed for amount of mitomycin C. The results are shown in Figs. 16A-16B.
Fig. 16A shows the concentration (μg/mL) of mitomycin C in the blood of rats as a function of time in hours following intravenous injection. As seen, free mitomycin C (open squares) administered intravenously in free form is rapidly cleared from the blood. Mitomycin C in the form of a liposome-bound lipid-DTB-drug conjugate remains in circulation for a substantially longer period of time. Mitomycin C associated with liposomes containing cholesterol (closed diamonds) and with cholesterol-free liposomes (closed circles) was detected in the blood at greater than 10 μg/mL for 20-25 hours.
Fig. 16B shows the percent of injected dose remaining in the blood as a function of time in hours following intravenous injection of the test formulations. Virtually none of the dose of free mitomycin C (open squares) remains in the blood at time points greater than about 5 minutes. However, at 20 hours after injection of the liposome formulations, about 15-18 percent of the dose of mitomycin C remains in circulation. This indicates the mitomycin C-DTB-lipid conjugate remains stable in the liposome while in circulation and that minimal thiolytic cleavage occurs in plasma. Therefore, this system appear to be compatible with long-circulating liposomes (Stealth* liposomes) which have an extended blood circulation lifetime and enhanced accumulation in tumors.
V. Examples
The following examples further illustrate the invention described herein and are in no way intended to limit the scope of the invention. Materials
All materials were obtained from commercially suitable vendors, such as Aldrich Coφoration.
EXAMPLE 1
Synthesis of /7αrα-diacyIdiglyceroIdithiobenzalcohol (Compound TV) and ortho- diacyldiglyceroldithiobenzalcohol
A. para-diacyldiglyceroldithiobenzalcohol This reaction is illustrated in Fig. 1. The procedure of Snyder, W.R. (Journal of
Lipid Research, 28:949 (1987) was followed to prepare Compounds II and HI.
A 100 ml round bottom flask containing 3-mercapto-l,2-propanediol (Compound I, 1 g, 9.26 mmol) in 5 ml of water was placed in an ice-bath. To this rapidly stirring flask, hydrogenperoxide (exactly 0.5 mole equivalent, 525 μl, 4.63 mmol) was dropwise added while maintaining the temperature between 30-40 °C. At the end of the exothermic process, the reaction was allowed to stir overnight at room temperature. Water was azeotroped with rotary evaporation by successive addition of acetonitrile in 20 ml aliquots. The process of acetonitrile addition was repeated 3-4 times or until all water was removed, yielding a clear oil. After scratching the flask with a metal spatula and cooling overnight at -20 °C, the oily product solidified (Compound H, rac-3,3'-dithiobis(l,2-propanediol)). The chalky solid was dried in vacuo over P205. Yield: 630 mg, 63%. 'HNMR (CD3OD, 360 MHz) δ 2.77, 2.95 (2xd, CH2OH, 2H), 3.59 (M, SCH2, 2H), 3.87 (m, CH, 1H) ppm.
The rac-3,3'-dithiobis(l,2-propanediol) product (Compound U) was acylated by adding the compound (980 mg, 4.6 mmol) to an oven-dried 100 mL round bottom flask and dissolving in dry methylene chloride (40 mL). To this, stearic acid (4.92 g, 17.1 mmol) and 4-dimethylamino)pyridinium 4-toluenesulfonate (1.38 g, 4.6 mmol) as the catalyst was and stirred at room temperature (25 °C) for 20 minutes. Then diisopropylcarbodiimide (3.1 mL, 20 mmmol) was pipetted and reacted overnight at room temperature. TLC silic on GF (10% ethylacetate in hexane) showed the complete reaction of the diol group. (rαc-3,3'-dithiobis(l,2-propanediol) Rf=0.60; rαc-3,3'-dithiobis(l,2- propanedistearoyl) Rf=0.35). Amberlyst* A-21 slightly basic ion-exchange resin ("3 g) and Amberlyst" 15 strongly acidic ion-exchange resin ("3 g) were added to the reaction mixture. After 30 minutes of shaking, the resins were filtered and the filtrate was taken to dryness. The residue was recrystallized from isopropanol three time (100 mL each). The solid product, rac-3,3'-dithiobis(l,2-propanedistearoyl) (Compound DT), was collected and dried over P205. Yield: 70%, 4.1 g. Melting Point 54-55 °C. 'HNMR (CDC13, 360 MHz) δ 0.86, (t, CH3, 6H), 1.22 (s, lipid, 56H), 1.48 (m, CH2CH2(CO)0, 4H), 2.26
(2xt, CH2(CO)0, 4H), 2.87 (d, CH2S, 2H), 4.03 & 4.22 (2xd, CH2CH of lipid, 2H), 4.97 (m, CHCH2 of lipid)ppm.
In the next step, a solution of rac-3,3'-dithiobis(l,2-propanedistearoyl) (Compound πi) (2.97 g, 2.33 mmol) was dissolved in toluene (30 mL) and placed in an ice bath. Sulfuryl chloride (1.9 mL, 23.2 mmol) was pipetted into the flask and the mixmre was stirred at the cold ice bath temperature for 30 minutes. The flask was then placed at room temperature and stirred for another 30 minutes. Excess of sulfuryl chloride was removed with a rotary evaporator. A fresh (20 mL) aliquot of toluene was added to the reaction flask and placed on an ice bath. To this, a solution of 4-mercaptobenzalcohol (780 mg, 5.6 mmol) in toluene was added with a slow rate. After 5 hours of reaction time, all solvents were evaporated with rotary evaporation to dryness. Warm ethyl acetate (10 mL) was added to the reaction flask to dissolve the solid and insoluble matter was filtered. To the ethyl acetate solution, 50 mL of ether was added to precipitate, and the solid product (/?αra-diacyl-diglycerol-dithiobenzalalcohol, Compound TV) was collected by filtration. This process was repeated twice. Yield: 75%.
To purify the product ( αrα-diacyl-diglycerol-dithiobenzal-alcohol, Compound TV), a silica gel column (20 x 2.5 cm) in chloroform was prepared. The sample was dissolved in minimum amount of chloroform and was chromatographed with addition of two different mobile phases. First, 100% CHC13 (100ml) was eluted. This fraction contained the impurity dithiobenzyl alcohol. The confirmation was made by 'HNMR. Then,
Changing the mobile phase to 15% methanol in chloroform, the pure product was collected by flash chromatography. By eluting 500 ml of CH30H:CHC13 (15:85) pure DGTBA (one spot by TLC) was collected. After evaporation of the solvents, the solid was lyophilized from t-BuOH and dried in vacuo over P205. The final purification dropped the yield to 40%, 1.4 g. 'HNMR: (CDC13, 360MHz) δ 0.86 (t, CH3, 6H), 1.22 (s, lipid, 56H), 1.48 (m, CH2CH2(CO)0, 4H), 2.26 (2xt, CH2(CO)0, 4Η), 2.87 (d, CH2S, 2H), 4.03 & 4.22 (2xd, CH2CH of lipid, 2H), 4.69 (s, CH2, bz, 2H), 4.97 (m, CHCH2 of lipid), 7.36 &7.56 (d, CH2, aromatic, 4H) ppm.
5 mg of sample was submitted to a laboratory for elemental analysis (Midwest Micro Lab).
Figure imgf000026_0001
B. ort zo-diglvceroldithiobenzalcohol
A solution of rαc-3,3'-dithiobis(l,2-propanedistearoyl) (Compound ffl) (200 mg, 0.156 mmol) was dissolved in toluene (30 mL) and placed in an ice bath. Sulfuryl chloride (39 μl, 0.47 mmol) was pipetted into the flask and the mixture was stirred at the cold ice bath temperature for 30 minutes. The flask was then placed at room temperature and stirred for another 30 minutes. Excess of sulfuryl chloride was removed with a rotary evaporator. A fresh (20 mL) aliquot of toluene was added to the reaction flask and placed on an ice bath. To this, a solution of 2-mercaptobenzalcohol (48 mg, 35 mmol) in toluene was added with a slow rate. After 5 hours of reaction time, all solvents were evaporated with rotary evaporation to dryness. Warm ethyl acetate (10 mL) was added to the reaction flask to dissolve the solid and insoluble matter was filtered. To the ethyl acetate solution, 50 mL of ether was added to precipitate, and the solid product (ørtbø-diacyl-diglycerol- dithiobenzalalcohol) was collected by filtration. This process was repeated twice. The solid was dried in vacuo over P205. Yield: 75%, 190mg. 'HNMR: (CDC13, 360 MHz) δ 0.86 (t, CH3, 6H), 1.25 (s, lipid, 56H), 1.58 (m, CH2CH2(CO)0, 4H), 2.28 (2xt, CH2(CO)0, 4Η), 2.91 (d, CH2S, 2H), 4.14 & 4.35 (2xd, CH2CH of lipid, 2H), 4.86 (s, CH2, bz, 2H), 5.26 (m, CHCH2 of lipid), 7.31 (m, aromatic, 2H), 7.48 & 7.75 (d, aromatic, 2H) ppm. EXAMPLE 2 Synthesis of »αrα-diacyldiglyceroldithiobenzal-mitomycin C (Compound XVHI)
This reaction is illustrated in Fig. 6A.
A 50 mL round bottom flask was charged with phosgene (3.1 mmol) and toluene (5 mL) and the solution was cooled to 0 °C. A solution of /?αrα-diacyl-diglycerol- dithiobenzal-alcohol, (Compound IV, prepared as described in Example 1, 0.31 mmol) in toluene (2.5 mL) was prepared. The alcohol solution was then added dropwise to the phosgene solution. The mixture was allowed to warm to room temperature overnight. After 18 hours, the solution was concentrated in vacuo to remove excess phosgene. The crude acyl chloride was redissolved in toluene (5 mL).
A solution of mitomycin C (0.31 mmol), dimethylaminopyridine (0.031 mmol) and DMF (1 mL) was prepared. The mitomycin C solution was added drop-wise the acyl chloride solution. After 1 hour, the toluene was evaporated off and the crude product was chromatographed (1: 1 hexane: ethyl acetate) on silica. The purified product was then taken up in t-BuOH (50 mL) and lyophilized. The product was a puφle solid (183 mg, 53%). Rf = 0.38 (50% hexane: ethyl acetate); Η NMR (360 MHz, CDC13) δ 0.88 (t, J = 6.8 Hz, 6H), 1.26 (s, 58 H), 1.58 - 1.63 (m, 4H), 1.76 (s, 3H), 2.29 (t, J = 7.6 Hz, 4H), 2.93 - 2.96(m, 2H), 3.19 (s, 3H), 3.29 (dd, / = 4.7 and 2.9 Hz, IH), 3.41 (dd, J = 5.0 and 2.2 Hz, IH), 3.48 (dd, J = 13.7 and 2.5 Hz, IH), 3.67 (dd, J = 11.5 and 4.7 Hz, IH), (ddd, J = 12.2 and 5.8 and 2.5 Hz, IH), 4.27-4.36
(m, 2H), 4.43 (d, J = 13.3Hz, IH), 4.61 (s, 2H), 4.90 (ddd, J = 10.4 and 5.0 and 2.2 Hz, IH), 5.00 - 5.12 (m, 3H), 5.26 - 5.30 (m, IH), 7.32 (d, J = 8.6 Hz, 2H), 7.50 (d, J = 7.9 Hz, 2H); MALDI MS calcd for C62H99N4OuS2Na: 1164, found m/z 1164 (M + Na).
EXAMPLE 4
Liposome Preparation
A. Liposomes Containing Cholesterol 1. Liposome Preparation 59 mg HSPC, 14.4 mg cholesterol, 17.4 mg mPEG-DSPE, and 7.4 τngpara- distearoyl-DTB-mitomycin C (molar ratio of 60/30/5/5) were added to 1 mL dehydrated ethanol at 60-65 °C and mixed until dissolved, approximately 10 minutes. A hydration medium composed of 10 mM histidine and 150 mM NaCl in distilled water was warmed to 70 °C.
The warm lipid solution was rapidly added to the warm (63-67 °C) hydration medium, with mixing, to form a suspension of liposomes having heterogeneous sizes. The suspension was mixed for one hour at 63-67 °C.
2. Extrusion
The liposomes were sized to the desired mean particle diameter by controlled extrusion through polycarbonate filter cartridges housed in Teflon-lined stainless steel vessels. The liposome suspension was maintained at 63-65 °C throughout the extrusion process, a period of 6-8 hours.
3. Diafiltration
Ethanol was removed from the liposome suspension by diafiltration. A histidine/sodium chloride solution was prepared by dissolving histidine (10 mM) and sodium chloride (150 mM) in sterile water. The pH of the solution was adjusted to approximately 7. The solution was filtered through a 0.22 μm Durapore filter. The liposome suspension was diluted in approximately a 1 : 1 (v/v) ratio with the histidine/sodium chloride solution and diafiltered through a polysulfone hollow-fiber ultrafilter. Eight volume exchanges were performed against the histidine/sodium chloride solution to remove the ethanol. The process fluid temperature was maintained at about 20-30 °C. Total diafiltration time was approximately 4.5 hours.
4. Sterile Filtration The liposome suspension was heated to 33-38°C and filtered through a 0.2 μm
Gelman Supor polyethersulfone filter. Total filtration time was approximately 10 minutes.
After each processing step (hydration, extrusion, dialysis and filtration) the lipid concentration and conjugate/drug concentration were determined by HPLC. Liposome particle size was measured by dynamic light scattering and the amount of "free" , unbound mitomycin C in the external suspension medium was measured by HPLC.
Figure imgf000029_0001
1 Conjugate = Compound XVIII, /?αra-dιstearoyl-DTB-mιtomycin C 2 2MMMCC==r mnιittoormnvycciinn C C.
B. Cholesterol-Free Liposome Formulation
Liposomes were prepared as described above with a lipid composition of HSPC, mPEG-DSPE and αra-distearoyl-DTB-mitomycin C in a molar ratio of 90/5/5. Specifically, 88.5 mg HPSC, 17.9 mg mPEG-DSPE (PEG MW 2000 Daltons) and 7.3 mg of the conjugate were dissolved in 1 mL ethanol. Liposome size, lipid and drug concentration and free mitomycin C concentration in the external suspension medium were determined after each processing step.
Figure imgf000029_0002
'Conjugate = Compound XVIII, αrα-distearoyl-DTB-mitomycin C 2 !MMMCC == m mιittoommvyccιmn C C.
EXAMPLE 5
HPLC Conditions for in vitro Characterization
Liposomes prepared as described in Examples 4A-4B were diluted in 0.6 M octaylglucopyranoside. The liposomes were incubated in the presence of 150 mM cysteine at 37 °C. Samples with withdrawn at time zero, 30 minutes, 1 hour, 2 hours, 4 hours and 24 hours. A 20 μL volume was analyzed by HPLC using a Water Symmetry C 8 3.5 x 5 cm column. The flow rate was 1 mL/min and the mobile phase gradient as follows:
Figure imgf000030_0001
EXAMPLE 6 Cytotoxicity Studies
A. Liposome Preparation
Liposomes, prepared as described in Example 4A-4B, were composed of
HSPC/mPEG-DSPE/distearoyl-DTB-mitomycin C (90/5/5) or
HSPC/cholesterol/mPEG-DSPE/distearoyl-DTB-mitomycin C (90/45/5/5). The liposome preparations were sterile filtered through 0.45 μm cellulose membranes and were not downsized via extrusion. After liposome formation, mitomycin C concentration was determined by absorbance at 360 nm in liposomes solubilized by 10- 20 fold dilution in isopropanol and the phospholipid concentration was determined by inorganic phosphate assay. The liposomes containing cholesterol had an average diameter of 275 ± 90 nm.
The cholesterol-free liposomes had an average diameter of 150 + 50 nm. The phospholipid concentration in both liposome formulations was 10 μM/mL and the concentration of mitomycin C in both formulations was 120 μg/mL.
B. Chemosensitivity Assay and Growth Rate Determination
The cytotoxic effect of free mitomycin C or mitomycin C in the form of a distearoyl-DTB-mitomycin C conjugate incoφorated into liposomes was assayed colorimetrically by a methylene blue staining method described previously (Horowitz, A.T. et l , Biochim. Biophys. Acta, 1 109:203-209 (1992)) with slight modifications. Upon completion of the assay, the cells were fixed and evaluated using the methylene blue staining assay.
In the assay, 1500 M109 mouse carcinoma cells from exponentially growing cultures in 200 μl aliquots (RPMI-1640 medium + 10% fetal bovine serum) were plated onto 96 well flat-bottom microtiter plates. Following 20 hours in culture, during which cells attached and resumed growth, 20 μl of the test formulations (free mitomycin C or liposome formulations) was added to each well. For each 10-fold increase in drug concentration, four drug concentration points were tested. Each test was performed in triplicate wells and in two parallel plates. The cells were treated continuously for 72 hours. After the 72 hour treatment period, the cultures were fixed by the addition of 50 μl 2.5 % glutaraldehyde to each well for 10 minutes. The plates were washed three times with deionized water, once with 0.1 M borate buffer (pH 8.5) and then stained for 60 minutes with 100 μl methylene blue (1 % in 0.1 M buffer borate, pH 8.5) at room temperature (20-25°C) . The plates were rinsed in five baths of deionized water to remove non-cell bound dye and then dried. The dye was extracted with 200 μl 0.1 N HC1 for 60 minutes at 37°C and the optical density was determined using a microplate spectrophotometer .
The cell number determined by counting cells with a hemocytometer correlated well with the spectrophotometric absorbance. The initial cell plating density was chosen to ensure a linear relationship between cell number and absorbance at the end of the study. In each study, six wells were fixed before drug was added to determine the initial average absorbance. This value was used to calculate growth rate (GR) and doubling times (DT) of control and drug-treated cells using the following equation: DT = In 2/ln[(ODt/ODc)/h]; where DT = doubling time in hours; ODt = optical density of test well at the end of the stody; ODc = optical density of control well at the start of the study; h = duration of incubation in hours.
The growth rate was calculated as GR = (In 2/DT). The percent growth inhibition or percent of control growth rate was obtained by dividing the growth rate of drug-treated cells by the growth rate of the untreated, control cells. The drug concentration which caused a 50% inhibition of the control growth rate (IC50) was calculated by inteφolation of the two closest values of the growth inhibition curve.
Mitomycin C was assayed in the range 10"8 -10"5 M. The liposomal formulations with conjugate-bound were assayed in the range 10"8 - 3 x 10"5 M. For interaction stodies cysteine (SIGMA, St. Louis, MO) was added together with the mitomycin C or liposome formulations to final concentration of 150, 500, or 1000 μM. The results are shown in Table 1 and in Figs. 13, 14 and 15A-15B.
EXAMPLE 7 In vivo Pharmacokinetic Study
A. Liposome Formulations
Liposomes containing cholesterol and cholesterol-free liposomes were prepared as described in Example 5 A and 5B.
A solution of mitomycin C in free form was prepared by dissolving 11.9 mg of mitomycin C in 119 μL ethanol. After dissolution, approximately 11.8 μL of a solution of 10 mM histidine/ 150 mM saline was added. Prior to use, the mitomycn C solution was diluted to 100 μg/mL with the histidine/saline solution and filtered.
B. Animals
Eight rats were randomized into treatment groups as follows:
Figure imgf000032_0001
A single intravenous injection of the test formulation was administered as a bolus dose. Blood samples were taken from each animal at the following times after injection: 30 seconds, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours and 96 hours. The quantity of mitomycin C in the blood samples was determined by the HPLC procedure given below. A 200 mM iodoacetamine solution was prepared by placing 199.3 mg of iodoacetamide in 5.1 mL of 7.5% EDTA. 15 μL of the 200 mM iodoacetamide solution was placed in each 1 μL of blood sample.
C. HPLC Method for Measuring Mitomycin C in Plasma
1. Solution Preparation
An aqueous buffer containing 10 mM ammonium phosphate, pH=7 was prepared by placing 1.321 g of ammonium phosphate into a 1 L volumetric flask filled with deionized water. The mixture was stirred and the pH was adjusted to 7.0 with o- phosphoric acid. The buffer was filtered through a 0.45 μm nylon filter before use.
A mobile phase of methanol and the aqueous buffer were mixed via a gradient program using a Waters Alliance binary pump.
2. Preparation of Standard Solution and Quality Control Samples Two separate weights of mitomycin C and mitomycin C conjugate were prepared as standards and quality control samples. One mg of mitomycin C and of mitomycin C conjugate were weighed and dissolved in 1 mL diluent (20% chloroform and 80% methanol mixture) separately. The concentration of the stock solution for both compounds was 1 mg/mL. Several dilutions were made in diluent to obtain concentrations from 5 μg/mL to 100 μg/mL for standard and quality control samples. An aliquot of 0.1 mL rat plasma was spiked with appropriate volumes (10 μL-50 μL) of mitomycin C and mitomycin C conjugate standard solutions. The concentration ranges were 0.05-5.0 μg/mL and 0.1-5 μg/mL for mitomycin C and mitomycin C conjugate, respectively. The final volume was adjusted to 1 mL with methanol. A similar procedure was followed to prepare quality control samples. The concentrations of quality control samples was 0.1, 0.5 and 5 μg/mL for mitomycin C and 0.1 , 1 and 5 μg/mL for mitomycin C conjugate in rat plasma. The samples were spun down at 3,000 φm for 10 minutes at room temperature. 300 μL of supernatant was transferred to HPLC vials containing 300 μL insert for injection. 3. Sample Preparation
100 μL of plasma sample was denatured with 900 μL of methanol followed by centrifugation for 10 minutes at 3,000 φm. An aliquot of 300 μL supernatant was transferred to an HPLC vial containing a 300 μL insert for injection.
4. Chromatographic Conditions
A Supelco® C-8, 5 μ, 4.6mm x 5 cm column was used. The mobile phase A was 10 mM ammonium phosphate, pH 7. Mobil phase B was methanol. The flow rate was 1 mL/min and detection was by UV at 360 nm. The injection volume was 40 μL and the typical run time was 15 minutes. The gradient program was as follows:
Figure imgf000034_0001
5. Assay and Calculations The prepared linearity standards (six concentration levels) from low to high concentration were injected. The quality control and plasma samples were then injected for analysis.
Peak area and retention times were determined by the PE-Nelson Turbochrom
(Version 4.1) system. Concentrations of mitomycin C and mitomycin C conjugate were calculated using a linear regression program. The linearity of the method was evaluated suing standard responses from six concentration levels. The data were fit to the linear regression dquation y = B*x + A with a weighting factor of 1/x2. The precision and accuracy of the method were evaluated from the back-calculated concentrations of the standards as well as from the quality control samples. The results are shown in Figs. 16A-16B.
Although the invention has been described with respect to particular embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the invention.

Claims

We claim:
1. A conjugate for use in a liposomal drug-delivery vehicle, the conjugate having the general structural formula:
Figure imgf000035_0001
wherein L is a hydrophobic moiety suitable for incoφoration into a liposomal lipid bilayer, R' represents a therapeutic drug covalently attached to the dithiobenzyl moiety, and where orientation of the CH2R' group is selected from the ortho position and the para position.
2. The conjugate of claim 1, wherein the therapeutic drug is covalently attached by a linkage selected from the group consisting of urethane, amine, amide, carbonate, thio-carbonate, ether and ester.
3. The conjugate of claim 1, wherein L is selected from the group consisting of cholesterol, a diacylglycerol, a phospholipid and derivatives thereof.
4. The conjugate of claim 1, wherein L is a diacylglycerol derivative to yield a conjugate having the general structural formula:
Figure imgf000035_0002
wherein R and R are hydrocarbons having between about 8 to about 24 carbon atoms.
5. The conjugate of claim 4, wherein R2 and R3 are hydrocarbons having from about 12 to about 22 carbon atoms.
6. The conjugate of claim 4, wherein R2 and R3 are hydrocarbon chains of the same length.
7. The conjugate of claim 1, wherein said drug is selected from the group consisting of mitomycin C, mitomycin A, bleomycin, doxorubicin, daunorubicin, fluorodeoxyuridine, iododeoxyuridine, etoposide, AZT, acyclovir, vidarabine, arabinosyl cytosine, pentostatin, quinidine, atropine, chlorambucil, methotrexate, mitoxantrone and 5-fluorouracil.
8. The conjugate of claim 1, wherein the therapeutic drug is covalently linked to the dithiobenzyl moiety to form a conjugate having the structure:
Figure imgf000036_0001
wherein R4 represents a residue of the therapeutic drug.
9. The conjugate of claim 8, wherein R4 is a therapeutic drug residue containing a primary or a secondary amine moiety thereby forming a urethane linkage between the dithiobenzyl and the therapeutic drug.
10. The conjugate of claim 9, wherein said therapeutic drug is selected from the group consisting of mitomycin A, mitomycin C, bleomycin and a polypeptide.
11. The conjugate of claim 8, wherein R4 is a residue of a carboxyl-containing therapeutic drug, thereby to form an ester linkage between the dithiobenzyl and the therapeutic drug. 35
12. The conjugate of claim 11, wherein said drug is chlorambucil or methotrexate.
s 13. The conjugate of claim 8, wherein R4 is a therapeutic drug residue containing a hydroxyl moiety thereby to form a carbonate linkage between the dithiobenzyl and the therapeutic drug.
14. The conjugate of claim 13, wherein the therapeutic drug is selected from the o group consisting of fluorodeoxyuridine, iododeoxyuridine, etoposide, AZT, acyclovir, vidarabine, arabinosyl cytosine, pentostatin, quinidine, mitoxantrone and atropine.
15. A liposome composition, comprising liposomes comprised of vesicle-forming lipids including from about 1 to about 30 s mole percent of a conjugate having the general structural formula:
Figure imgf000037_0001
wherein L is a hydrophobic moiety suitable for incoφoration into a liposomal lipid bilayer, R' represents a therapeutic drug covalently attached to the dithiobenzyl 0 moiety, and where orientation of the CH2R' group is selected from the ortho position and the para position, wherein said therapeutic drug is released from the conjugate in vivo in response to a physiologic condition or an artificially induced condition.
5 16. The conjugate of claim 15, wherein the therapeutic drug is covalently attached to the dithiobenzyl moiety by a linkage selected from the group consisting of urethane, amine, amide, carbonate, thio-carbonate, ether and ester.
17. The conjugate of claim 15, wherein L is selected from the group consisting 0 of cholesterol, a diacylglycerol, a phospholipid, and derivatives thereof.
18. The conjugate of claim 15, wherein L is a diacylglycerol to yield a conjugate having the structural formula:
Figure imgf000038_0001
wherein R and R are hydrocarbons having between about 8 to about 24 carbon atoms.
19. The conjugate of claim 18, wherein R2 and R3 are hydrocarbons having from about 12 to about 22 carbon atoms.
20. The conjugate of claim 18, wherein R2 and R3 are hydrocarbon chains of the same length.
21. The conjugate of claim 15, wherein said drug is selected from the group consisting of mitomycin C, mitomycin A, bleomycin, doxorubicin, daunorubicin, fluorodeoxyuridine, iododeoxyuridine, etoposide, AZT, acyclovir, vidarabine, arabinosyl cytosine, pentostatin, quinidine, atropine, chlorambucil, methotrexate, mitoxantrone and 5-fluorouracil.
22. The conjugate of claim 15, wherein the therapeutic drug is covalently linked to the dithiobenzyl moiety to form a conjugate having the structure:
Figure imgf000038_0002
wherein R4 represents a residue of the therapeutic drug.
23. The conjugate of claim 22, wherein R4 is a therapeutic drug residue containing a primary or a secondary amine moiety thereby forming a urethane linkage between the dithiobenzyl and the therapeutic drug.
24. The conjugate of claim 23, wherein said therapeutic drug is selected from the group consisting of mitomycin A, mitomycin C, bleomycin and a polypeptide.
25. The conjugate of claim 22, wherein R4 is a residue of a carboxyl-containing therapeutic drug, thereby to form an ester linkage between the dithiobenzyl and the therapeutic drug.
26. The conjugate of claim 25, wherein said drug is chlorambucil or methotrexate.
27. The conjugate of claim 22, wherein R4 is a therapeutic drug residue containing a hydroxyl moiety thereby to form a carbonate linkage between the dithiobenzyl and the therapeutic drug.
28. The conjugate of claim 27, wherein the therapeutic drug is selected from the group consisting of fluorodeoxyuridine, iododeoxyuridine, etoposide, AZT, acyclovir, vidarabine, arabinosyl cytosine, pentostatin, quinidine, mitoxantrone and atropine.
29. A method for retaining a drug in a liposome, comprising preparing liposomes comprised of a vesicle-forming lipid and of between about 1 to about 30 mole percent of a conjugate having the general form:
Figure imgf000039_0001
wherein L is a hydrophobic moiety suitable for incoφoration into a liposomal lipid bilayer, R' represents a therapeutic drug covalently attached to the dithiobenzyl moiety, and where orientation of the CH2R' group is selected from the ortho position and the para position, whereby said preparing is effective to retain the drug in the liposomes until release from the conjugate in response to a physiologic condition or an artificially induced condition.
30. The method of claim 29, wherein said preparing includes preparing a conjugate where the therapeutic drug is covalently attached by a linkage selected from the group consisting of urethane, amine, amide, carbonate, thio-carbonate, ether and ester.
31. The method of claim 29, wherein said preparing includes preparing a conjugate wherein L is selected from the group consisting of cholesterol, a diacylglycerol, a phospholipid, and derivatives thereof.
32. The method of claim 29, wherein said preparing includes preparing a conjugate wherein L is a diacylglycerol to yield a conjugate having the general structural formula:
Figure imgf000040_0001
wherein R and R are hydrocarbons having between about 8 to about 24 carbon atoms.
33. The method of claim 18, wherein R2 and R3 are hydrocarbons having from about 12 to about 22 carbon atoms.
34. The conjugate of claim 32, wherein R2 and R3 are hydrocarbon chains of the same length.
35. The method of claim 29, wherein said preparing includes preparing a conjugate comprising a drug selected from the group consisting of mitomycin C, mitomycin A, bleomycin, doxorubicin, daunorubicin, fluorodeoxyuridine, iododeoxyuridine, etoposide, AZT, acyclovir, vidarabine, arabinosyl cytosine, pentostatin, quinidine, atropine, chlorambucil, methotrexate, mitoxantrone and 5- fluorouracil.
36. The method of claim 29, wherein said preparing includes preparing a conjugate comprising a therapeutic drug covalently linked to the dithiobenzyl moiety to form a conjugate having the structure:
Figure imgf000041_0001
wherein R4 represents a residue of the therapeutic drug.
37. The method of claim 36, wherein R4 is a therapeutic drug residue containing a primary or a secondary amine moiety thereby forming a urethane linkage between the dithiobenzyl and the therapeutic drug.
38. The method of claim 37, wherein said therapeutic drug is selected from the group consisting of mitomycin A, mitomycin C, bleomycin and a polypeptide.
39. The method of claim 36, wherein R4 is a residue of a carboxyl-containing therapeutic drug, thereby to form an ester linkage between the dithiobenzyl and the therapeutic drug.
40. The method of claim 39, wherein said drug is chlorambucil or methotrexate. WO 00/64484 PCT USOO/l 0922
40
41. The method of claim 36, wherein R4 is a therapeutic drug residue containing a hydroxyl moiety thereby to form a carbonate linkage between the dithiobenzyl and the therapeutic drug.
i 42. The method of claim 41, wherein the therapeutic drug is selected from the group consisting of fluorodeoxyuridine, iododeoxyuridine, etoposide, AZT, acyclovir, vidarabine, arabinosyl cytosine, pentostatin, quinidine, mitoxantrone and atropine.
PCT/US2000/010922 1999-04-23 2000-04-21 Conjugate having a cleavable linkage for use in a liposome WO2000064484A2 (en)

Priority Applications (13)

Application Number Priority Date Filing Date Title
HU0201425A HUP0201425A3 (en) 1999-04-23 2000-04-21 Conjugate having a cleavable linkage for use in a liposome
CA2369595A CA2369595C (en) 1999-04-23 2000-04-21 Conjugate having a cleavable linkage for use in a liposome
DE60030965T DE60030965T2 (en) 1999-04-23 2000-04-21 CONJUGATE CONTAINS A SPLICABLE BINDING FOR USE IN A LIPOSOM
DK00928321T DK1173222T3 (en) 1999-04-23 2000-04-21 Conjugate with a cleavable bond for use in a liposome
JP2000613474A JP4558952B2 (en) 1999-04-23 2000-04-21 Complexes with cleavable bonds for use in liposomes
KR1020017013571A KR100669053B1 (en) 1999-04-23 2000-04-21 Conjugate having a cleavable linkage for use in a liposome
EP00928321A EP1173222B1 (en) 1999-04-23 2000-04-21 Conjugate having a cleavable linkage for use in a liposome
IL14605500A IL146055A0 (en) 1999-04-23 2000-04-21 Conjugate having a cleavable linkage for use in a liposome
AU46577/00A AU769425B2 (en) 1999-04-23 2000-04-21 Conjugate having a cleavable linkage for use in a liposome
MXPA01010750A MXPA01010750A (en) 1999-04-23 2000-04-21 Conjugate having a cleavable linkage for use in a liposome.
IL146055A IL146055A (en) 1999-04-23 2001-10-18 Conjugate having a cleavable linkage for use in a liposome
NO20015144A NO20015144L (en) 1999-04-23 2001-10-22 Conjugate with a cleavable bond for use in a liposome
HK02103601.3A HK1041820B (en) 1999-04-23 2002-05-13 Conjugate having a cleavable linkage for use in a liposome

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13089799P 1999-04-23 1999-04-23
US60/130,897 1999-04-23

Publications (2)

Publication Number Publication Date
WO2000064484A2 true WO2000064484A2 (en) 2000-11-02
WO2000064484A3 WO2000064484A3 (en) 2001-11-15

Family

ID=22446872

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/010922 WO2000064484A2 (en) 1999-04-23 2000-04-21 Conjugate having a cleavable linkage for use in a liposome

Country Status (18)

Country Link
US (8) US6365179B1 (en)
EP (3) EP1173222B1 (en)
JP (1) JP4558952B2 (en)
KR (2) KR100642955B1 (en)
CN (2) CN100512879C (en)
AT (1) ATE340592T1 (en)
AU (1) AU769425B2 (en)
CA (1) CA2369595C (en)
DE (1) DE60030965T2 (en)
DK (1) DK1173222T3 (en)
ES (1) ES2272280T3 (en)
HK (1) HK1041820B (en)
HU (2) HUP0200797A3 (en)
IL (2) IL146055A0 (en)
MX (2) MXPA01010750A (en)
NO (1) NO20015144L (en)
WO (1) WO2000064484A2 (en)
ZA (2) ZA200108726B (en)

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1385479A1 (en) * 2001-03-26 2004-02-04 Alza Corporation Liposome composition for improved intracellular delivery of a therapeutic agent
WO2004110497A2 (en) 2003-04-30 2004-12-23 Alza Corporation Mitomycin conjugates cleavable by thiols
WO2005053749A2 (en) * 2003-11-26 2005-06-16 Alza Corporation Thiol-cleavable linkage between polymer and ligand
US7211440B2 (en) 2002-03-08 2007-05-01 Wallac Oy Dissociative fluorescence enhancement assay
US7276248B2 (en) 1999-04-23 2007-10-02 Alza Corporation Conjugate having a cleavable linkage for use in a liposome
CN101284136A (en) * 2002-12-03 2008-10-15 布朗歇特洛克菲勒神经科学研究所 Artificial low-density lipoprotein carriers for transport of substances across the blood-brain barrier
US9181321B2 (en) 2013-03-14 2015-11-10 Shire Human Genetic Therapies, Inc. CFTR mRNA compositions and related methods and uses
US9308281B2 (en) 2011-06-08 2016-04-12 Shire Human Genetic Therapies, Inc. MRNA therapy for Fabry disease
US9522176B2 (en) 2013-10-22 2016-12-20 Shire Human Genetic Therapies, Inc. MRNA therapy for phenylketonuria
US9629804B2 (en) 2013-10-22 2017-04-25 Shire Human Genetic Therapies, Inc. Lipid formulations for delivery of messenger RNA
US9717690B2 (en) 2011-06-08 2017-08-01 Rana Therapeutics, Inc. Cleavable lipids
US9850269B2 (en) 2014-04-25 2017-12-26 Translate Bio, Inc. Methods for purification of messenger RNA
GB2552301A (en) * 2016-07-11 2018-01-24 Evox Therapeutics Ltd Metabolic drug loading of EVs
US9877919B2 (en) 2012-03-29 2018-01-30 Translate Bio, Inc. Lipid-derived neutral nanoparticles
US9957499B2 (en) 2013-03-14 2018-05-01 Translate Bio, Inc. Methods for purification of messenger RNA
US10022455B2 (en) 2014-05-30 2018-07-17 Translate Bio, Inc. Biodegradable lipids for delivery of nucleic acids
US10138213B2 (en) 2014-06-24 2018-11-27 Translate Bio, Inc. Stereochemically enriched compositions for delivery of nucleic acids
WO2019016208A1 (en) * 2017-07-17 2019-01-24 Technische Universiteit Eindhoven Applicable chemical composition comprising an agent conjugated to a hydrophobic moiety and a carrier
US10576166B2 (en) 2009-12-01 2020-03-03 Translate Bio, Inc. Liver specific delivery of messenger RNA
US11173190B2 (en) 2017-05-16 2021-11-16 Translate Bio, Inc. Treatment of cystic fibrosis by delivery of codon-optimized mRNA encoding CFTR
US11174500B2 (en) 2018-08-24 2021-11-16 Translate Bio, Inc. Methods for purification of messenger RNA
US11224642B2 (en) 2013-10-22 2022-01-18 Translate Bio, Inc. MRNA therapy for argininosuccinate synthetase deficiency
US11253605B2 (en) 2017-02-27 2022-02-22 Translate Bio, Inc. Codon-optimized CFTR MRNA
US11254936B2 (en) 2012-06-08 2022-02-22 Translate Bio, Inc. Nuclease resistant polynucleotides and uses thereof
US11951181B2 (en) 2023-04-03 2024-04-09 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery

Families Citing this family (69)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7422902B1 (en) * 1995-06-07 2008-09-09 The University Of British Columbia Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
US5981501A (en) * 1995-06-07 1999-11-09 Inex Pharmaceuticals Corp. Methods for encapsulating plasmids in lipid bilayers
US6287591B1 (en) 1997-05-14 2001-09-11 Inex Pharmaceuticals Corp. Charged therapeutic agents encapsulated in lipid particles containing four lipid components
US7112337B2 (en) * 1999-04-23 2006-09-26 Alza Corporation Liposome composition for delivery of nucleic acid
GB9915074D0 (en) * 1999-06-28 1999-08-25 Cortecs Plc Ligand-binding composition
US7094423B1 (en) * 1999-07-15 2006-08-22 Inex Pharmaceuticals Corp. Methods for preparation of lipid-encapsulated therapeutic agents
DE60231868D1 (en) * 2001-04-24 2009-05-20 Purdue Research Foundation FOLAT MIMETICS AND THEIR FOLAT RECEPTOR BINDING CONJUGATES
CA2472055A1 (en) * 2001-11-07 2003-05-15 Inex Pharmaceuticals Corporation Improved mucosal vaccines and methods for using the same
US7842498B2 (en) * 2001-11-08 2010-11-30 Bio-Rad Laboratories, Inc. Hydrophobic surface chip
US6737524B2 (en) * 2002-03-25 2004-05-18 Paul K. Smith Activated polyethylene glycol compounds
US20040009944A1 (en) * 2002-05-10 2004-01-15 Inex Pharmaceuticals Corporation Methylated immunostimulatory oligonucleotides and methods of using the same
AU2003243226A1 (en) * 2002-05-15 2003-12-02 Endocyte, Inc. Vitamin-mitomycin conjugates
US8496961B2 (en) 2002-05-15 2013-07-30 Sutter West Bay Hospital Delivery of nucleic acid-like compounds
EP2517730A3 (en) 2003-01-27 2013-01-02 Endocyte, Inc. Vitamin receptor binding drug delivery conjugates
WO2005034979A2 (en) * 2003-10-11 2005-04-21 Inex Pharmaceuticals Corporation Methods and compositions for enhancing innate immunity and antibody dependent cellular cytotoxicity
JP2007520481A (en) * 2004-01-15 2007-07-26 アルザ・コーポレーシヨン Liposome composition for delivering therapeutic agents
US7282590B2 (en) * 2004-02-12 2007-10-16 The Research Foundation Of State University Of New York Drug conjugates
US7931693B2 (en) * 2004-02-26 2011-04-26 Endosphere, Inc. Method and apparatus for reducing obesity
US9592277B2 (en) * 2004-04-14 2017-03-14 Avirid, Inc. Compositions with modified nucleases targeted to viral nucleic acids and methods of use for prevention and treatment of viral diseases
JP2007533750A (en) * 2004-04-21 2007-11-22 アルザ コーポレイション Polymer conjugates releasable under mild thiol degradation conditions
CN1997399A (en) * 2004-04-21 2007-07-11 阿尔扎公司 Polymer conjugate releasable under mild thiolytic conditions
JP5149620B2 (en) 2004-07-23 2013-02-20 エンドサイト，インコーポレイテッド Bivalent linker and conjugate thereof
AU2005282463A1 (en) * 2004-09-03 2006-03-16 Alza Corporation Endogenously-formed conjugate of albumin
TW200612993A (en) * 2004-10-08 2006-05-01 Alza Corp Lipopolymer conjugates
JP2008518951A (en) * 2004-10-28 2008-06-05 アルザ コーポレイション Lyophilized liposome formulations and methods
JP5289935B2 (en) * 2005-03-16 2013-09-11 エンドサイト，インコーポレイテッド Synthesis and purification of pteroic acid and its conjugates
JP2009504783A (en) * 2005-08-19 2009-02-05 エンドサイト，インコーポレイテッド Ligand conjugates of vinca alkaloids, analogues and derivatives
RU2470668C2 (en) * 2005-08-19 2012-12-27 Эндосайт, Инк. Conjugates of ligand and drugs
EP2046389A2 (en) * 2006-07-19 2009-04-15 The Board of Regents of the University of Texas System Preparations of phospholipids and pharmaceuticals containing 5-amino salicylic acid for the treatment of inflammatory bowel disease
WO2008091655A2 (en) * 2007-01-23 2008-07-31 The Regents Of The University Of California Methods, compositions and device for directed and controlled heating and release of agents
US20100104626A1 (en) 2007-02-16 2010-04-29 Endocyte, Inc. Methods and compositions for treating and diagnosing kidney disease
CN103948937A (en) * 2007-03-14 2014-07-30 恩多塞特公司 Binding ligand linked drug delivery conjugates of tubulysins
ITRM20070327A1 (en) * 2007-06-11 2008-12-12 Univ Palermo COLLOIDAL VECTORS WITH POLYAMINOACIDIC STRUCTURE FOR THE ORAL RELEASE OF PEPTIDES AND PROTEINS AND ITS RELATED PRODUCTION METHOD.
US9877965B2 (en) 2007-06-25 2018-01-30 Endocyte, Inc. Vitamin receptor drug delivery conjugates for treating inflammation
RU2523909C2 (en) 2007-06-25 2014-07-27 Эндосайт, Инк. Conjugates, containing hydrophilic spacers of linkers
US20100210575A1 (en) * 2007-06-29 2010-08-19 Wisconsin Alumni Research Foundation Structuring effect of cholesterol in peg-phospholipid micelles, drug delivery of amphotericin b, and combination antifungals
EP2180881A2 (en) * 2007-07-20 2010-05-05 Basf Se Vesicles comprising a transmembrane transport trigger system
CA2702945C (en) 2007-10-23 2016-08-23 Nektar Therapeutics Al, Corporation Hydroxyapatite-targeting multiarm polymers and conjugates made therefrom
US9187521B2 (en) 2007-10-25 2015-11-17 Endocyte, Inc. Tubulysins and processes for preparing
US9259398B1 (en) * 2007-11-26 2016-02-16 Abbott Cardiovascular Systems Inc. Bioactive agent-loaded targeting micelles
KR101919093B1 (en) * 2008-05-23 2018-11-16 더 유니버시티 오브 브리티쉬 콜롬비아 Modified drugs for use in liposomal nanoparticles
CN102388073B (en) 2009-02-04 2015-11-25 布里格姆及妇女医院股份有限公司 Nano level platinic compound and using method thereof
BRPI1012036A2 (en) 2009-05-27 2017-10-10 Selecta Biosciences Inc nanocarriers that have components with different release rates
IN2012DN02589A (en) * 2009-08-26 2015-08-28 Selecta Biosciences Inc
US20120219538A1 (en) * 2009-11-02 2012-08-30 Therapeomic Ag Stabilized protein formulations and use thereof
WO2011119995A2 (en) 2010-03-26 2011-09-29 Cerulean Pharma Inc. Formulations and methods of use
DE102010042338A1 (en) * 2010-10-12 2012-04-12 Bayer Technology Services Gmbh A composition for the treatment of controlled release cancer of the active ingredient
WO2012061717A1 (en) 2010-11-05 2012-05-10 Selecta Biosciences, Inc. Modified nicotinic compounds and related methods
CA2834571A1 (en) 2011-04-29 2012-11-01 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for inducing regulatory b cells
EP2606884A1 (en) 2011-12-21 2013-06-26 Ecole Polytechnique Fédérale de Lausanne (EPFL) Inhibitors of notch signaling pathway and use thereof in treatment of cancers
US10080805B2 (en) 2012-02-24 2018-09-25 Purdue Research Foundation Cholecystokinin B receptor targeting for imaging and therapy
US20140080175A1 (en) 2012-03-29 2014-03-20 Endocyte, Inc. Processes for preparing tubulysin derivatives and conjugates thereof
WO2014062697A2 (en) 2012-10-16 2014-04-24 Endocyte, Inc. Drug delivery conjugates containing unnatural amino acids and methods for using
US9827552B2 (en) 2013-07-17 2017-11-28 Clemson University Functionalized lipid modification of solid phase surfaces for use in chromatography
US9937261B2 (en) * 2014-06-09 2018-04-10 Lipomedix Pharmaceuticals Ltd. Combination therapy comprising a liposomal prodrug of mitomycin C and radiotherapy
AU2016233143B2 (en) 2015-03-17 2021-03-25 Alberto Gabizon Methods for the treatment of bladder cancer
CN106519221B (en) * 2015-09-10 2019-04-26 中国科学院高能物理研究所 A kind of polyethylene glycol/daiamid copolymer, preparation method and the amphipathic siRNA carrier comprising the copolymer
CN106519211B (en) * 2015-09-10 2018-10-09 中国科学院高能物理研究所 A kind of amphipathic nature polyalcohol and the magnetic micella nano-carrier formed by it and its purposes
CN105866311B (en) * 2016-05-25 2017-05-31 福建出入境检验检疫局检验检疫技术中心 Determine the UPLC MS/MS methods of antiviral drugs residual in chicken
WO2018089481A1 (en) 2016-11-08 2018-05-17 Mallinckrodt Llc Mitomycin c prodrug liposome formulations and uses thereof
US10618896B2 (en) 2017-08-22 2020-04-14 Dynavax Technologies Corporation Alkyl chain modified imidazoquinoline TLR7/8 agonist compounds and uses thereof
WO2019099412A1 (en) 2017-11-14 2019-05-23 Dynavax Technologies Corporation Cleavable conjugates of tlr7/8 agonist compounds, methods for preparation, and uses thereof
WO2020144657A1 (en) 2019-01-11 2020-07-16 Lipomedix Pharmaceuticals Ltd. Liposome composition comprising liposomal prodrug of mitomycin c and method of manufacture
CA3134791A1 (en) 2019-04-10 2020-10-15 Cellestia Biotech Ag Inhibitors of notch signalling pathway and use thereof in treatment of cancers
WO2022122667A1 (en) 2020-12-07 2022-06-16 Cellestia Biotech Ag Pharmaceutical combinations for treating cancer
EP4008324A1 (en) 2020-12-07 2022-06-08 Cellestia Biotech AG Combinations comprising an inhibitor of an anti-apoptotic protein, such as bcl-2, bcl-xl, bclw or mcl-1, and a notch signaling pathway inhibitor for treating cancer
WO2022253794A2 (en) 2021-06-02 2022-12-08 Cellestia Biotech Ag Method for treating an autoimmune and inflammatory disease
WO2023079132A1 (en) 2021-11-08 2023-05-11 Cellestia Biotech Ag Pharmaceutical combinations for treating cancer
EP4223292A1 (en) 2022-02-07 2023-08-09 Cellestia Biotech AG Pharmaceutical combinations for treating cancer

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2254336A1 (en) * 1973-12-17 1975-07-11 Kyowa Hakko Kogyo Kk
EP0317957A2 (en) * 1987-11-23 1989-05-31 Bristol-Myers Squibb Company Drug-monoclonal antibody conjugates
EP0317956A2 (en) * 1987-11-23 1989-05-31 Bristol-Myers Squibb Company Anti-tumor prodrugs
EP0510197A1 (en) * 1990-01-11 1992-10-28 Nippon Shinyaku Company, Limited Fat emulsion
US5169934A (en) * 1990-05-14 1992-12-08 Anergen, Inc. Intracellularly cleavable compounds
WO1997036904A1 (en) * 1996-04-02 1997-10-09 Sagami Chemical Research Center Mitomycin c derivative and non-receptor tyrosine kinase inhibitor
WO1999029302A1 (en) * 1997-12-05 1999-06-17 Katarina Edwards Drug delivery system with two-step targeting

Family Cites Families (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
GB8430252D0 (en) 1984-11-30 1985-01-09 Beecham Group Plc Compounds
US4766106A (en) 1985-06-26 1988-08-23 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
US5059421A (en) 1985-07-26 1991-10-22 The Liposome Company, Inc. Preparation of targeted liposome systems of a defined size distribution
US4917888A (en) 1985-06-26 1990-04-17 Cetus Corporation Solubilization of immunotoxins for pharmaceutical compositions using polymer conjugation
JPH0615532B2 (en) * 1986-02-03 1994-03-02 テルモ株式会社 5-Fluorouracil derivative and pharmaceutical preparation containing the same
US4766105A (en) * 1986-10-31 1988-08-23 Shell Oil Company Ethylene oxide catalyst and process for preparing the catalyst
JPH01113391A (en) * 1987-10-24 1989-05-02 Kyowa Hakko Kogyo Co Ltd Mitomycin derivative
US5103556A (en) 1988-05-05 1992-04-14 Circon Corporation Method of manufacturing an electrohydraulic probe
US4902502A (en) 1989-01-23 1990-02-20 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate
US5585112A (en) 1989-12-22 1996-12-17 Imarx Pharmaceutical Corp. Method of preparing gas and gaseous precursor-filled microspheres
WO1993018751A1 (en) 1992-03-23 1993-09-30 Georgetown University Liposome encapsulated taxol and a method of using the same
JP2813511B2 (en) * 1992-06-10 1998-10-22 大日本スクリーン製造株式会社 Method of applying coating liquid to substrate surface using roll coater
WO1994005259A1 (en) 1992-09-02 1994-03-17 Georgetown University Method of encapsulating anthracycline glycosides in liposomes
US5395619A (en) 1993-03-03 1995-03-07 Liposome Technology, Inc. Lipid-polymer conjugates and liposomes
US5820873A (en) * 1994-09-30 1998-10-13 The University Of British Columbia Polyethylene glycol modified ceramide lipids and liposome uses thereof
RU2166512C2 (en) * 1995-01-16 2001-05-10 Коммонвелт Сайентифик Энд Индастриал Рисерч Организейшн Conjugates of therapeutic compound with fatty acid
AU4857796A (en) 1995-01-20 1996-08-07 Florida State University Sex-specific dna probe for parrots, methods and kits
TW520297B (en) 1996-10-11 2003-02-11 Sequus Pharm Inc Fusogenic liposome composition and method
EP0932390A1 (en) * 1996-10-11 1999-08-04 Sequus Pharmaceuticals, Inc. Therapeutic liposome composition and method
JPH1160499A (en) 1997-08-22 1999-03-02 Hiroshi Maeda Antitumor agent
US6180095B1 (en) 1997-12-17 2001-01-30 Enzon, Inc. Polymeric prodrugs of amino- and hydroxyl-containing bioactive agents
NZ507456A (en) 1998-04-28 2003-10-31 Applied Research Systems Process and conjugated forms of PEGylated interferon- beta with polyethylene glycol (PEG) wherein the thiol reactive polyol agent is mono-methoxylated
US7112337B2 (en) 1999-04-23 2006-09-26 Alza Corporation Liposome composition for delivery of nucleic acid
US7303760B2 (en) 1999-04-23 2007-12-04 Alza Corporation Method for treating multi-drug resistant tumors
JP4558952B2 (en) 1999-04-23 2010-10-06 アルザ コーポレイション Complexes with cleavable bonds for use in liposomes
IL146047A0 (en) 1999-04-23 2002-07-25 Alza Corp Releasable linkage and compositions containing same
US7238368B2 (en) 1999-04-23 2007-07-03 Alza Corporation Releasable linkage and compositions containing same
AU7868400A (en) 1999-10-08 2001-04-23 Alza Corporation Neutral-cationic lipid for nucleic acid and drug delivery
ES2367891T3 (en) 2000-09-29 2011-11-10 Schering Corporation INTERLEUCINA-10 PEGILADA.
US7260330B2 (en) 2002-11-04 2007-08-21 The Boeing Company Optical communication system using correlation receiver

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2254336A1 (en) * 1973-12-17 1975-07-11 Kyowa Hakko Kogyo Kk
EP0317957A2 (en) * 1987-11-23 1989-05-31 Bristol-Myers Squibb Company Drug-monoclonal antibody conjugates
EP0317956A2 (en) * 1987-11-23 1989-05-31 Bristol-Myers Squibb Company Anti-tumor prodrugs
EP0510197A1 (en) * 1990-01-11 1992-10-28 Nippon Shinyaku Company, Limited Fat emulsion
US5169934A (en) * 1990-05-14 1992-12-08 Anergen, Inc. Intracellularly cleavable compounds
WO1997036904A1 (en) * 1996-04-02 1997-10-09 Sagami Chemical Research Center Mitomycin c derivative and non-receptor tyrosine kinase inhibitor
WO1999029302A1 (en) * 1997-12-05 1999-06-17 Katarina Edwards Drug delivery system with two-step targeting

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Section Ch, Week 198923 Derwent Publications Ltd., London, GB; Class B02, AN 1989-170530 XP002159819 & JP 01 113391 A (KYOWA HAKKO KOGYO KK), 2 May 1989 (1989-05-02) *
DIAZ C ET AL: "Synthesis of disulfide-containing phospholipid analogs for the preparation of head group-specific lipid antigens: generation of phosphatidylserine antibodies" BIOCONJUGATE CHEMISTRY,US,AMERICAN CHEMICAL SOCIETY, WASHINGTON, vol. 9, no. 2, March 1998 (1998-03), pages 250-254, XP002125967 ISSN: 1043-1802 *
VAAGE J ET AL: "THERAPY OF PRIMARY AND METASTATIC MOUSE MAMMARY CARCINOMAS WITH DOXORUBICIN ENCAPSULATED IN LONG CIRCULATING LIPOSOMES" INTERNATIONAL JOURNAL OF CANCER, vol. 51, no. 6, 1992, pages 942-948, XP000979548 ISSN: 0020-7136 *

Cited By (81)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7608687B2 (en) 1999-04-23 2009-10-27 Alza Corporation Releasable linkage and compositions containing same
US7285622B2 (en) 1999-04-23 2007-10-23 Alza Corporation Releasable linkage and compositions containing same
US7238368B2 (en) 1999-04-23 2007-07-03 Alza Corporation Releasable linkage and compositions containing same
US7303760B2 (en) 1999-04-23 2007-12-04 Alza Corporation Method for treating multi-drug resistant tumors
US7592307B2 (en) 1999-04-23 2009-09-22 Alza Corporation Releasable linkage and compositions containing same
US7276248B2 (en) 1999-04-23 2007-10-02 Alza Corporation Conjugate having a cleavable linkage for use in a liposome
US7108863B2 (en) 2001-03-26 2006-09-19 Alza Corporation Liposome composition for improved intracellular delivery of a therapeutic agent
EP1385479A4 (en) * 2001-03-26 2006-12-06 Alza Corp Liposome composition for improved intracellular delivery of a therapeutic agent
EP1385479A1 (en) * 2001-03-26 2004-02-04 Alza Corporation Liposome composition for improved intracellular delivery of a therapeutic agent
US7211440B2 (en) 2002-03-08 2007-05-01 Wallac Oy Dissociative fluorescence enhancement assay
US7682627B2 (en) * 2002-12-03 2010-03-23 Blanchette Rockefeller Neurosciences Institute Artificial low-density lipoprotein carriers for transport of substances across the blood-brain barrier
JP2012131799A (en) * 2002-12-03 2012-07-12 Blanchette Rockefeller Neurosciences Inst Artificial low-density lipoprotein carrier for transport of substance across blood-brain barrier
CN101284136A (en) * 2002-12-03 2008-10-15 布朗歇特洛克菲勒神经科学研究所 Artificial low-density lipoprotein carriers for transport of substances across the blood-brain barrier
US7576055B2 (en) * 2002-12-03 2009-08-18 Blanchette Rockefeller Neurosciences Institute Artificial low-density lipoprotein carriers for transport of substances across the blood-brain barrier
AU2004247004B2 (en) * 2003-04-30 2010-10-14 Alza Corporation Mitomycin conjugates cleavable by thiols
WO2004110497A2 (en) 2003-04-30 2004-12-23 Alza Corporation Mitomycin conjugates cleavable by thiols
WO2004110497A3 (en) * 2003-04-30 2005-03-24 Alza Corp Mitomycin conjugates cleavable by thiols
WO2005053749A3 (en) * 2003-11-26 2006-11-09 Alza Corp Thiol-cleavable linkage between polymer and ligand
WO2005053749A2 (en) * 2003-11-26 2005-06-16 Alza Corporation Thiol-cleavable linkage between polymer and ligand
US10576166B2 (en) 2009-12-01 2020-03-03 Translate Bio, Inc. Liver specific delivery of messenger RNA
US10507183B2 (en) 2011-06-08 2019-12-17 Translate Bio, Inc. Cleavable lipids
US10238754B2 (en) 2011-06-08 2019-03-26 Translate Bio, Inc. Lipid nanoparticle compositions and methods for MRNA delivery
US9597413B2 (en) 2011-06-08 2017-03-21 Shire Human Genetic Therapies, Inc. Pulmonary delivery of mRNA
US11234936B2 (en) 2011-06-08 2022-02-01 Translate Bio, Inc. Cleavable lipids
US11185595B2 (en) 2011-06-08 2021-11-30 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US9717690B2 (en) 2011-06-08 2017-08-01 Rana Therapeutics, Inc. Cleavable lipids
US11291734B2 (en) 2011-06-08 2022-04-05 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US11338044B2 (en) 2011-06-08 2022-05-24 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US11052159B2 (en) 2011-06-08 2021-07-06 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US10888626B2 (en) 2011-06-08 2021-01-12 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US10702478B2 (en) 2011-06-08 2020-07-07 Translate Bio, Inc. Cleavable lipids
US10507249B2 (en) 2011-06-08 2019-12-17 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US10413618B2 (en) 2011-06-08 2019-09-17 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US10350303B1 (en) 2011-06-08 2019-07-16 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US11547764B2 (en) 2011-06-08 2023-01-10 Translate Bio, Inc. Lipid nanoparticle compositions and methods for MRNA delivery
US9308281B2 (en) 2011-06-08 2016-04-12 Shire Human Genetic Therapies, Inc. MRNA therapy for Fabry disease
US11730825B2 (en) 2011-06-08 2023-08-22 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US10786455B2 (en) 2012-03-29 2020-09-29 Translate Bio, Inc. Lipid-derived neutral nanoparticles
US10137086B2 (en) 2012-03-29 2018-11-27 Translate Bio, Inc. Lipid-derived neutral nanoparticles
US9877919B2 (en) 2012-03-29 2018-01-30 Translate Bio, Inc. Lipid-derived neutral nanoparticles
US11497716B2 (en) 2012-03-29 2022-11-15 Translate Bio, Inc. Lipid-derived neutral nanoparticles
US10137087B2 (en) 2012-03-29 2018-11-27 Translate Bio, Inc. Lipid-derived neutral nanoparticles
US11254936B2 (en) 2012-06-08 2022-02-22 Translate Bio, Inc. Nuclease resistant polynucleotides and uses thereof
US10420791B2 (en) 2013-03-14 2019-09-24 Translate Bio, Inc. CFTR MRNA compositions and related methods and uses
US10876104B2 (en) 2013-03-14 2020-12-29 Translate Bio, Inc. Methods for purification of messenger RNA
US11510937B2 (en) 2013-03-14 2022-11-29 Translate Bio, Inc. CFTR MRNA compositions and related methods and uses
US9713626B2 (en) 2013-03-14 2017-07-25 Rana Therapeutics, Inc. CFTR mRNA compositions and related methods and uses
US11820977B2 (en) 2013-03-14 2023-11-21 Translate Bio, Inc. Methods for purification of messenger RNA
US11692189B2 (en) 2013-03-14 2023-07-04 Translate Bio, Inc. Methods for purification of messenger RNA
US9181321B2 (en) 2013-03-14 2015-11-10 Shire Human Genetic Therapies, Inc. CFTR mRNA compositions and related methods and uses
US9957499B2 (en) 2013-03-14 2018-05-01 Translate Bio, Inc. Methods for purification of messenger RNA
US11890377B2 (en) 2013-10-22 2024-02-06 Translate Bio, Inc. Lipid formulations for delivery of messenger RNA
US9522176B2 (en) 2013-10-22 2016-12-20 Shire Human Genetic Therapies, Inc. MRNA therapy for phenylketonuria
US10959953B2 (en) 2013-10-22 2021-03-30 Translate Bio, Inc. Lipid formulations for delivery of messenger RNA
US10052284B2 (en) 2013-10-22 2018-08-21 Translate Bio, Inc. Lipid formulations for delivery of messenger RNA
US11377642B2 (en) 2013-10-22 2022-07-05 Translate Bio, Inc. mRNA therapy for phenylketonuria
US10208295B2 (en) 2013-10-22 2019-02-19 Translate Bio, Inc. MRNA therapy for phenylketonuria
US10493031B2 (en) 2013-10-22 2019-12-03 Translate Bio, Inc. Lipid formulations for delivery of messenger RNA
US11224642B2 (en) 2013-10-22 2022-01-18 Translate Bio, Inc. MRNA therapy for argininosuccinate synthetase deficiency
US9629804B2 (en) 2013-10-22 2017-04-25 Shire Human Genetic Therapies, Inc. Lipid formulations for delivery of messenger RNA
US11884692B2 (en) 2014-04-25 2024-01-30 Translate Bio, Inc. Methods for purification of messenger RNA
US10155785B2 (en) 2014-04-25 2018-12-18 Translate Bio, Inc. Methods for purification of messenger RNA
US11059841B2 (en) 2014-04-25 2021-07-13 Translate Bio, Inc. Methods for purification of messenger RNA
US9850269B2 (en) 2014-04-25 2017-12-26 Translate Bio, Inc. Methods for purification of messenger RNA
US10286083B2 (en) 2014-05-30 2019-05-14 Translate Bio, Inc. Biodegradable lipids for delivery of nucleic acids
US10286082B2 (en) 2014-05-30 2019-05-14 Translate Bio, Inc. Biodegradable lipids for delivery of nucleic acids
US10022455B2 (en) 2014-05-30 2018-07-17 Translate Bio, Inc. Biodegradable lipids for delivery of nucleic acids
US10293057B2 (en) 2014-05-30 2019-05-21 Translate Bio, Inc. Biodegradable lipids for delivery of nucleic acids
US10493166B2 (en) 2014-05-30 2019-12-03 Translate Bio, Inc. Biodegradable lipids for delivery of nucleic acids
US11433144B2 (en) 2014-05-30 2022-09-06 Translate Bio, Inc. Biodegradable lipids for delivery of nucleic acids
US10912844B2 (en) 2014-05-30 2021-02-09 Translate Bio, Inc. Biodegradable lipids for delivery of nucleic acids
US11104652B2 (en) 2014-06-24 2021-08-31 Translate Bio, Inc. Stereochemically enriched compositions for delivery of nucleic acids
US10138213B2 (en) 2014-06-24 2018-11-27 Translate Bio, Inc. Stereochemically enriched compositions for delivery of nucleic acids
GB2552301A (en) * 2016-07-11 2018-01-24 Evox Therapeutics Ltd Metabolic drug loading of EVs
US11253605B2 (en) 2017-02-27 2022-02-22 Translate Bio, Inc. Codon-optimized CFTR MRNA
US11173190B2 (en) 2017-05-16 2021-11-16 Translate Bio, Inc. Treatment of cystic fibrosis by delivery of codon-optimized mRNA encoding CFTR
WO2019016208A1 (en) * 2017-07-17 2019-01-24 Technische Universiteit Eindhoven Applicable chemical composition comprising an agent conjugated to a hydrophobic moiety and a carrier
US11174500B2 (en) 2018-08-24 2021-11-16 Translate Bio, Inc. Methods for purification of messenger RNA
US11951181B2 (en) 2023-04-03 2024-04-09 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US11951180B2 (en) 2023-04-03 2024-04-09 Translate Bio, Inc. Lipid nanoparticle compositions and methods for MRNA delivery
US11951179B2 (en) 2023-04-03 2024-04-09 Translate Bio, Inc. Lipid nanoparticle compositions and methods for MRNA delivery

Also Published As

Publication number Publication date
US6849270B2 (en) 2005-02-01
ZA200108726B (en) 2003-03-05
KR20010114247A (en) 2001-12-31
HK1041820A1 (en) 2002-07-26
KR20020005006A (en) 2002-01-16
HUP0200797A2 (en) 2002-07-29
CN1355713A (en) 2002-06-26
HUP0200797A3 (en) 2010-01-28
IL146055A (en) 2006-12-31
HK1041820B (en) 2007-03-30
AU4657700A (en) 2000-11-10
EP1579874A2 (en) 2005-09-28
AU769425B2 (en) 2004-01-29
CN1399561A (en) 2003-02-26
US7285622B2 (en) 2007-10-23
WO2000064484A3 (en) 2001-11-15
ES2272280T3 (en) 2007-05-01
EP1880736A1 (en) 2008-01-23
MXPA01010751A (en) 2002-05-14
CA2369595A1 (en) 2000-11-02
EP1173222B1 (en) 2006-09-27
MXPA01010750A (en) 2003-08-20
EP1173222A2 (en) 2002-01-23
US7608687B2 (en) 2009-10-27
KR100642955B1 (en) 2006-11-10
DE60030965D1 (en) 2006-11-09
KR100669053B1 (en) 2007-01-15
US20030054028A1 (en) 2003-03-20
NO20015144D0 (en) 2001-10-22
EP1579874A3 (en) 2006-01-25
DE60030965T2 (en) 2007-05-24
HUP0201425A2 (en) 2002-12-28
ATE340592T1 (en) 2006-10-15
JP2002542302A (en) 2002-12-10
CN100512879C (en) 2009-07-15
DK1173222T3 (en) 2007-01-29
JP4558952B2 (en) 2010-10-06
ZA200108724B (en) 2002-10-23
US7276248B2 (en) 2007-10-02
US6605299B2 (en) 2003-08-12
US6984396B2 (en) 2006-01-10
NO20015144L (en) 2001-12-11
HUP0201425A3 (en) 2006-07-28
CA2369595C (en) 2010-10-05
US6342244B1 (en) 2002-01-29
US20050271715A1 (en) 2005-12-08
US6365179B1 (en) 2002-04-02
US20030211079A1 (en) 2003-11-13
US20080152702A1 (en) 2008-06-26
US20020128195A1 (en) 2002-09-12
IL146055A0 (en) 2002-07-25
CN1244376C (en) 2006-03-08
US20050123597A1 (en) 2005-06-09

Similar Documents

Publication Publication Date Title
US6365179B1 (en) Conjugate having a cleavable linkage for use in a liposome
US7238368B2 (en) Releasable linkage and compositions containing same
AU2002305094B2 (en) Liposome composition for improved intracellular delivery of a therapeutic agent
KR100617921B1 (en) Liposome composition and method for administering a quinolone
US20040022842A1 (en) Liposome preparations containing oxaliplatin
Karathanasis et al. Preparation of in vivo cleavable agglomerated liposomes suitable for modulated pulmonary drug delivery
EP1426044A1 (en) Use of esters of L-carnitine or alkanoyl L-carnitines as cationic lipids for the intracellular delivery of pharmacologically active compounds
AU2004247004B2 (en) Mitomycin conjugates cleavable by thiols
AU770390B2 (en) Releasable linkage and compositions containing same
JPH06228012A (en) Liposome preparation
WO2005069750A2 (en) Therapeutic composition with nanoscale activation agents

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 00807901.3

Country of ref document: CN

AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE 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 NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

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

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
ENP Entry into the national phase

Ref document number: 2369595

Country of ref document: CA

Ref document number: 2369595

Country of ref document: CA

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2000 613474

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2000928321

Country of ref document: EP

Ref document number: 2001/08726

Country of ref document: ZA

Ref document number: 514991

Country of ref document: NZ

Ref document number: PA/a/2001/010750

Country of ref document: MX

Ref document number: 46577/00

Country of ref document: AU

Ref document number: 1020017013571

Country of ref document: KR

Ref document number: 200108726

Country of ref document: ZA

AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE 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 NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

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

WWP Wipo information: published in national office

Ref document number: 1020017013571

Country of ref document: KR

WWP Wipo information: published in national office

Ref document number: 2000928321

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWG Wipo information: grant in national office

Ref document number: 46577/00

Country of ref document: AU

WWG Wipo information: grant in national office

Ref document number: 2000928321

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 1020017013571

Country of ref document: KR