WO2011013128A2 - Vecteurs destinés à l'administration de neurothérapies au système nerveux central - Google Patents

Vecteurs destinés à l'administration de neurothérapies au système nerveux central Download PDF

Info

Publication number
WO2011013128A2
WO2011013128A2 PCT/IL2010/000612 IL2010000612W WO2011013128A2 WO 2011013128 A2 WO2011013128 A2 WO 2011013128A2 IL 2010000612 W IL2010000612 W IL 2010000612W WO 2011013128 A2 WO2011013128 A2 WO 2011013128A2
Authority
WO
WIPO (PCT)
Prior art keywords
bbb
residue
alkylene
compound according
radical
Prior art date
Application number
PCT/IL2010/000612
Other languages
English (en)
Other versions
WO2011013128A3 (fr
Inventor
Matityahu Fridkin
Yoram Shechter
Original Assignee
Yeda Research And Development Co. 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
Application filed by Yeda Research And Development Co. Ltd. filed Critical Yeda Research And Development Co. Ltd.
Publication of WO2011013128A2 publication Critical patent/WO2011013128A2/fr
Publication of WO2011013128A3 publication Critical patent/WO2011013128A3/fr

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
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
    • 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
    • A61K47/543—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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the present invention relates to vectors for the delivery of non-transportable drugs from the periphery to the central nervous system (CNS) through the blood-brain barrier (BBB), and their release in the CNS.
  • CNS central nervous system
  • BBB blood-brain barrier
  • BBB blood-brain barrier
  • CNS central nervous system
  • HSA human serum albumin
  • HPLC high-performance liquid chromatography
  • MIB-NHS m- maleimido benzoic acid N-hydroxy-succinimide ester
  • DTNB 5,5'-dithiobis (2- nitrobenzoic acid)
  • PBS phosphate buffer saline
  • EDC l-ethyl-3-(3- dimethylaminopropyl) carbodiimide
  • FMS 2-sulfo-9-fluorenyl-methoxycarbonyl
  • Fraoc 2-sulfo-9-fluorenyl-methoxycarbonyl
  • Cat-HSA-Fmoc-enkephalin a conjugate of cationized HSA linked to Leu-enkephalin via a MAL-Fmoc spacer
  • Cat-HSA-Benz-enkephalin a conjugate of cationized HSA, linked to Leu-enkephalin through a maleimido benzoate spacer
  • DTT dithiothreitol
  • the brain is one of the least accessible organs of the body, thus making the delivery of neurotherapeutics a challenge.
  • BBB blood brain barrier
  • CNS central nervous system
  • BBB blood brain barrier
  • BBB prevents effective treatment of many severe and life-threatening diseases and disorders such as brain cancer, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, neuro-AIDS disorders and schizophrenia.
  • a drug For treatment of brain disorders, a drug should possess several characteristic features following peripheral administration, to allow its arrival at the CNS and facilitate there a viable therapeutic effect.
  • the drug should resist degradation in the circulatory system; be sufficiently hydrophobic to transverse the BBB by passive diffusion; and preserve its active form, being capable of binding to brain receptor sites upon arrival at the brain.
  • the drug is a protein or a peptide it should also be immune to degradation by proteases and peptidases both in the circulatory system and in the brain.
  • Fmoc-OSu 9-fiuorenylmethoxycarbonyl-N- hydroxysuccinimide
  • the inventors synthesized an heterobifunctional agent, 9- hydroxymethyl-2-(amino-3-maleimidopropionate)-fluorene- ⁇ -hydroxy-succinimide (MAL-Fmoc-OSu), which allows the covalent linkage of SH-containing compounds to the amino-side chains of peptides through a hydrolizable chemical bond.
  • Peptides or proteins are released from such prodrug conjugates in their native form with ty 2 value in the range of 20-70 hrs (WO 2004/089280; Tsubery et al., 2004; Shechter et al., 2005a; Shechter et al., 2006; Shechter et al., 2005b).
  • Fmoc-type compounds when linked on the one side to a blood brain barrier (BBB)-non-transportable drug and on the other side to a residue capable of crossing the blood brain barrier, are capable of delivering BBB-non-transportable neurotherapeutics through the BBB and releasing them in the central nervous system (CNS) in a slow and continuous fashion, while maintaining their therapeutic effect.
  • BBB blood brain barrier
  • CNS central nervous system
  • the BBB transport vectors of the invention are capable of reversibly binding the BBB-nontransportable neuropeptide enkephalin; delivering it through the BBB; and releasing it in the brain while maintaining its analgesic effect.
  • the present invention thus relates in one aspect to a compound of the formula:
  • X is a radical of the formula I linked to Z through the S atom:
  • Ri is hydrogen, C 1 -C 6 alkyl, -SO 3 H, halogen, nitro, hydroxyl, sulfonamide, acyl, phosphoryl, or dialkylamine;
  • R 2 is C 2 -C 8 alkylene, optionally interrupted by an aryl ring, -(OCH 2 CH 2 )I 1 -, wherein n is 1 to 6, or a residue of a peptide containing from 2 to 4 hydrophobic amino acid residues;
  • Ra and R 4 are selected from hydrogen, C 1 -C 8 alkyl or C 6 - C 10 aryl;
  • R 5 is a leaving group
  • Z is a residue capable of crossing the BBB selected from:
  • a spacer linked to a second identical or different X radical through the S atom such as a C 2 -C 8 alkylene, optionally substituted by one or more hydroxy groups or interrupted by one or more oxygen atoms or by a phenylene radical, wherein R 5 of the second X radical is optionally replaced by a residue of a protein which is capable of crossing the BBB; or
  • Ri is -SO3H, halogen, nitro, hydroxyl, sulfonamide, acyl, phosphoryl, or dialkylamine
  • Z is not a C 8 -C 2O fatty acid residue.
  • the present invention relates to a conjugate of the formula:
  • X' is a radical of the formula Ia linked to Z through the S atom:
  • Ri is hydrogen, C 1 -C 6 alkyl, -SO3H, halogen, nitro, hydroxyl, sulfonamide, acyl, phosphoryl, or dialkylamine;
  • R 2 is C 2 -C 8 alkylene, optionally interrupted by an aryl ring * -(OCH 2 CHa) n , wherein n is 1 to 6, or a residue of a peptide containing 2 to 4 hydrophobic amino acid residues;
  • R 3 and R 4 are selected from hydrogen, Ci-C 8 alkyl or C 6 - Cio aryl; R's is a moiety of a BBB-non-transportable drug comprising at least one primary or secondary amino group, or a residue of a protein capable of crossing the BBB; and
  • Z is a residue capable of crossing the BBB selected from:
  • a spacer linked to a second identical or different X' radical through the S atom such as a C 2 -Cg alkylene, optionally substituted by one or more hydroxy groups or interrupted by one or more oxygen atoms or by a phenylene radical, wherein R' 5 of the second X radical is optionally a residue of a protein which is capable of crossing the BBB; or (iii) a residue of a protein which crosses the BBB;
  • Ri is -SO3H, halogen, nitro, hydroxyl, sulfonamide, acyl, phosphoryl, or dialkylamine
  • Z is not a Cg-C 20 fatty acid residue.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the conjugate of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the present invention relates to use of the conjugate of the invention, in delivery of a BBB-non-transportable drug from the periphery across the BBB to the CNS.
  • the present invention relates to a method for delivering a BBB- non-transportable drug from the periphery into the CNS of a subject through the BBB, comprising administering to said subject a therapeutically effective amount of the conjugate of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the present invention relates to a method for treatment of a disease or disorder in the CNS of a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a conjugate of the invention, wherein R' 5 is a moiety of a BBB-non-transportable drug capable of treating said disease or disorder.
  • the present invention relates to use of a conjugate of the invention, for the preparation of a pharmaceutical composition for the treatment of a disease or disorder in the CNS.
  • Fig. 1 shows HPLC-analysis of the purified synthetic product SuO-Fmoc-MAL-S- (CH 2 ) 10 -COOH.
  • Fig. 2 shows HPLC analysis of purified DTT-(MAL-Fmoc-OSu) 2
  • the mass-spectrum was determined using electrospray ionization technique.
  • Fig. 6 shows ultraviolet absorption spectrum of SuO-Fmoc-MAL-S-(CH 2 )io- COOH. The absorption spectrum was recorded for HPLC-purified SuO-Fmoc-MAL-S- (CH 2 ) 10 -COOH (70 nmoles/ml) in acetonitrile at 25 0 C.
  • Fig. 7 shows ultraviolet absorption spectrum of SuO-FmOC-MAL-S-(CH 2 ) I5 - COOH.
  • the absorption spectrum was recorded for HPLC-purified SuO-Fmoc-MAL-S- (CH 2 )i5-COOH (64 nmoles/ml) in acetonitrile at 25°C.
  • Fig. 8 shows ultraviolet absorption spectrum of DTT-(MAL-Fmoc-OSu) 2 .
  • the absorption spectrum was recorded for HPLC-purified DTT-(MAL-Fmoc-OSu) 2 (17 nmoles/ml) in acetonitrile at 25°C.
  • Fig. 9 shows the rate of enkephalin release from HSA-Fmoc-Leu-enkephalin and Fmoc-Met-enkephalin upon incubation at pH 8.5, 37°C.
  • Figs. 10A-10B show that IP administration of Fmoc-derived lipophilic Leu- enkephalin conjugates produces analgesia.
  • 1OA the indicated enkephalins and conjugates were administered IP, each at a concentration of 10 ⁇ moles/kg body weight, and analyzed for their analgesic effect in mice, 40 minutes after administration.
  • 1OB time course of the antinociceptive response following IP administration of HOOC-(CH 2 ) I o-FmOc-LeU- enkephalin (C10FME-1095). Each group consisted of 5 mice. Each point in the figure is the arithmetic mean ⁇ SEM from 5 male ICR mice.
  • Figs. 1 IA-I IB show the analgesic effect of intraperitoneally administered cationized HSA-Fmoc-enkephalin.
  • HA the indicated Leu-enkephalin conjugates covalently linked Leu-enkephalin were administered IP, each at a concentration of 2 ⁇ moles per 1 kg body-weight, and analyzed for their analgesic effect 60 minutes after administration.
  • HB time course of the antinociceptive response following IP administration of cationized HSA-Fmoc-Leu-enkephalin.
  • Each point in the Figure is the arithmetic mean ⁇ SEM from 5 male ICR mice.
  • the present invention provides blood brain barrier (BBB) transport vectors for the delivery of neurotherapeutics from the periphery across the BBB to the central nervous system (CNS).
  • BBB transport vectors of the invention are Fmoc-type compounds of the formula X-Z below, linked to a residue capable of crossing the BBB, which are capable of reversibly linking a BBB-non-transportable drug, delivering it through the BBB and releasing it in the CNS in a slow and continuous fashion, while maintaining its therapeutic effect.
  • the "residue capable of crossing the BBB” as defined herein can be either a lipophilic residue which increases the lipophilicity of the compound (e.g. a fatty acid residue or a spacer linked to a second X radical) and can passively cross the BBB or a protein which crosses the BBB by receptor-mediated transendocytosis.
  • Lipid solubility is the major factor that determines the rate by which a drug passively crosses the BBB.
  • elevating the lipophilicity of a given drug e.g. neuropeptide
  • This issue is further complicated with regard to peripherally administered drugs that should preserve some hydrophilicity in order to migrate from the intraperitoneal space into the bloodstream, before entering brain capillaries.
  • the BBB transport vectors of the invention when linked to either a fatty acid residue or to one another through a linker, are sufficiently lipophilic to passively cross the BBB on the one hand and sufficiently hydrophilic to be soluble in the blood on the other hand.
  • the same complexity is valid with regard to 'vector-mediated' or conjugated drugs. If the drugs are inactivated upon conjugation, a reasonable solution might be a reversible linkage between the BBB-transport-vector and the non-transportable drug. The drug must be released unmodified, in a slow, continuous fashion at physiological conditions. Indeed, the BBB-transport-vectors of the invention when linked to a protein which crosses the BBB, are capable of delivering drugs across the BBB and releasing them in the CNS in an active form.
  • the present invention thus relates in one aspect to a compound of the formula:
  • X is a radical of the formula I linked to Z through the S atom:
  • R 1 is hydrogen, C 1 -C 6 alkyl, -SO 3 H, halogen, nitro, hydroxyl, sulfonamide, acyl, phosphoryl, or dialkylamine;
  • R 2 is C 2 -C 8 alkylene, optionally interrupted by an aryl ring? -(OCH 2 CH 2 )I 1 -, wherein n is 1 to 6, or a residue of a peptide containing from 2 to 4 hydrophobic amino acid residues;
  • R 3 and R 4 are same or different, each is selected from hydrogen, C 1 -C 8 alkyl or C 6 - C 10 aryl;
  • R 5 is a leaving group
  • Z is a residue capable of crossing the BBB selected from:
  • a spacer linked to a second identical or different X radical through the S atom such as a C 2 -C 8 alkylene, optionally substituted by one or more hydroxy groups or interrupted by one or more oxygen atoms or by a phenylene radical, wherein R5 of the second X radical is optionally replaced by a residue of a protein which is capable of crossing the BBB; or
  • C 2 -C 8 alkylene refers to a straight or branched divalent hydrocarbon radical having 2-8 carbon atoms and includes, e.g. ethylene, n-propylene, isopropylene, n-butylene, sec-butylene, isobutylene, tert-butylene, n-pentylene, 2,2- dimethylpropylene, n-hexylene, n-heptylene, n-octylene, and the like.
  • alkyl alone or as part of a radical such as “aralkyl” or “alkylene” typically means a straight or branched saturated hydrocarbon radical having 1-8 or 1-6 carbon atoms and includes, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2,2- dimethylpropyl, n-hexyl, n-heptyl, n-octyl, and the like.
  • C 6 -C 10 aryl denotes a carbocyclic aromatic radical such as phenyl and naphthyl.
  • C 2 -C 8 alkylene interrupted by an aryl ring refers to any C 2 -C 8 alkylene interrupted by a C 6 -C 10 arylene-diyl, wherein said C 6 -C 10 arylene-diyl is optionally substituted by halogen, alkyl, alkoxy or nitro.
  • halogen refers to fluoro, chloro, bromo or iodo.
  • the BBB transport vector of the invention is a compound of the formula X-Z, wherein Z is a residue of a Cs-C 20 fatty acid, preferably a C 10 -C 16 fatty acid, more preferably undecanoic acid or hexadecanoic fatty acid.
  • Enkephalin did not show any analgesic effect (data not presented). It is therefore hypothesized that for BBB-transport vectors wherein Z is a residue of a C 8 -C 20 fatty acid and R 1 is -SO 3 H, halogen, nitro, hydroxyl, sulfonamide, acyl, phosphoryl, or dialkylamine, the vector might not be sufficiently lipophilic to cross the BBB.
  • R 1 is preferably either hydrogen or C 1 -C 6 alkyl.
  • the BBB transport vector is a compound of the formula X-Z, wherein Z is a spacer linked to a second identical or different X radical through the S atom, wherein said spacer is a C 2 -C 8 alkylene, preferably C 4 -C 6 alkylene, more preferably C 4 alkylene, optionally substituted by one or more hydroxy or interrupted by one or more oxygen atoms or by a phenylene radical.
  • the spacer is - (CH(OH)) 4 -.
  • a protein which is capable of crossing the BBB refers to any protein or peptide which crosses the BBB by receptor-mediated transendocytosis such as, but not limited to, leptin, leptin fragments, glucagon-like peptide- 1, transferrin, lactoferrin, rabies virus glycoprotein, ⁇ - MSH, IGF-I, LDL- receptor related proteins, nontoxic mutant of diphteria toxin (CRM- 197), ghrelin, or PYY 3- 36- Preferred proteins or peptides are those which are able to penetrate the BBB by receptor-mediated transendocytosis but do not activate brain receptors (which can produce side effects).
  • receptor-mediated transendocytosis such as, but not limited to, leptin, leptin fragments, glucagon-like peptide- 1, transferrin, lactoferrin, rabies virus glycoprotein, ⁇ - MSH, IGF-I, LDL- receptor related proteins, nontoxic
  • the protein which crosses the BBB by receptor-mediated transendocytosis is leptin, leptin fragments, rabies virus glycoprotein or nontoxic mutant of diphteria toxin.
  • leptin fragments are capable of penetrating the BBB without exerting the effect of leptin.
  • rabies virus glycoprotein and nontoxic mutant of diphteria toxin are not human the should not exert an effect in the CNS.
  • R 2 is C 2 -C 8 alkylene, preferably C 2 -C 4 alkylene, more preferably -(CH2) 2 -.
  • R 2 is C 2 -C 8 alkylene interrupted by an aryl, -(OCH2CH2)n-, wherein n is 1 to 6, or a residue of a peptide containing from 2 to 4 hydrophobic amino acid residues.
  • amino acid residue refers to both L- and D- stereoisomers of any natural or non-natural amino acid residue and derivatives and analogs thereof. While a natural amino acid is any one of the twenty amino acid residues typically occurring in proteins, the term non-natural amino acid refers to any amino acid, modified amino acid and/or an analog thereof, that is not one of the twenty natural amino acids. Examples of non-natural amino acids include but are not limited to norleucine, norvaline, and ⁇ -aminobutyric acid.
  • the amino acid derivatives include "chemical derivatives" that contain additional chemical moieties not normally a part of the peptide such as the esters and amides of carboxyl group or N-acyl derivatives.
  • hydrophobic amino acid residue refers to an amino acid residue having a hydrocarbyl side chain.
  • hydrophobic amino acids include the natural amino acids valine, leucine, isoleucine, methionine and phenylalanine, as well as the non-natural amino acids norvaline (Nva), norleucine (NIe), homovaline, homoleucine, bipyridyl alanine, p-carboxymethyl-L-phenylalanine and p-nitro-L-phenylalanine.
  • leaving group refers to any functional group or atom, which can be displaced by another functional group or atom in a substitution reaction, e.g., a nucleophilic substitution reaction.
  • Non-limiting examples of leaving groups include -O- (CH) 2 -CN, -Cl, 2,5-dioxopyrrolidin-l-olate also known as N-hydroxysuccinimide (herein designated -OSu), 4-nitrophenoxy, 2-nitro ⁇ henoxy, 2,3,4,5,6-pentachlorophenoxy, isoindoline-l,3-dione-2-oxy, benzenesulfanyl, and 2-nitrobenzenesulfanyl wherein -OSu is preferred.
  • the BBB transport vector is a compound of the formula X- Z as detailed above, wherein Ri is H or -SO 3 H, R 3 and R 4 are H, R 2 is C 2 -C 8 alkylene interrupted by an aryl ring, -(OCH2CH2) n -, wherein n is 1 to 6, or a residue of a peptide containing from 2 to 4 hydrophobic amino acid residues, and R5 is -OSu.
  • the BBB transport vector is a compound of the formula X-Z as detailed above, wherein Ri is H or -SO 3 H, R 3 and R 4 are H, R 2 is C 2 -C 8 alkylene, preferably C 2 -C 4 alkylene, more preferably -(CH 2 ) 2 -, and R 5 is -OSu.
  • the BBB transport vector is a compound of the formula X-Z as detailed above, wherein R 1 , R 3 and R 4 are H, R 2 is -(CH 2 ) 2 -, R 5 is -OSu, and Z is a C 8 -C 2 O fatty acid, preferably a C 1O -Ci 6 fatty acid, more preferably a residue of undecanoic acid or hexadecanoic acid.
  • the BBB transport vector is a compound of the formula X-Z as detailed above, wherein R 1 is H or -SO3H, R 3 and R 4 are H, R 2 is C 2 -C 8 alkylene, preferably C 2 -C 4 alkylene, more preferably -(CH 2 ) 2 - j R 5 is -OSu, and Z is a spacer linked to a second identical or different X radical through the S atom, wherein R 5 of the second X radical is optionally replaced by a residue of a protein which is capable of crossing the BBB.
  • this transport vector is not highly lipophilic, it is more effective in releasing the drug and can be used e.g. to bind a BBB-nontransportable drug on one side and a protein capable of crossing the BBB on the other side, thereby producing a protein-drug conjugate which is capable of penetrating the BBB and effectively releasing the drug.
  • the BBB transport vector is a compound of the formula X-Z as detailed above, wherein Ri is H or -SO 3 H, R 3 and R 4 are H, R 2 is C 2 -Cg alkylene, preferably C 2 -C 4 alkylene, more preferably -(CH 2 ) 2 - ; Rs is -OSu, and Z is a spacer linked to a second identical or different X radical through the S atom, wherein R 5 of the second X radical is replaced by a residue of a protein which is capable of crossing the BBB.
  • the BBB transport vector is a compound of the formula X-Z as detailed above, wherein Ri is H or -SO 3 H, R 3 and R 4 are H, R 2 is C 2 -C 8 alkylene, preferably C 2 -C 4 alkylene, more preferably -(CH 2 ⁇ -, R 5 is -OSu, and Z is a residue of a protein which is capable of crossing the BBB.
  • said protein is selected from leptin, glucagon-like peptide- 1, transferrin, lactoferrin, rabies virus glycoprotein, ⁇ -MSH, IGF-I, LDL-receptor related proteins, nontoxic mutant of diphteria toxin (CRM- 197), ghrelin, or PYY 3-36 , preferably leptin, leptin fragments, rabies virus glycoprotein or nontoxic mutant of diphteria toxin.
  • BBB transport vectors of the chemical structures depicted in Scheme I are described herein. They are herein represented as SuO-Fmoc-MAL-S-(CH 2 )i 0 -COOH; SuO-Fmoc-MAL-S-(CH 2 )i 5 - COOH and DTT-(MAL-Fmoc-OSu) 2 , and have the capability to covalently link to an amino group of a drug comprising at least one primary or secondary amino group thereby imparting lipophilicity to said drug.
  • the drug conjugated with the BBB transport vector becomes a hydrophobic species capable of permeating the BBB.
  • reversible lipophilization This process is reversible and is herein designated "reversible lipophilization".
  • the drug is released from the lipophilic tail of the BBB transport vector in its native and active form. The detachment takes place at a slow rate under physiological conditions by spontaneous chemical hydrolysis.
  • the choice of the BBB transport vector depends on the hydrophilicity of the drug to be transported to the brain. For a more hydrophilic drug, a more hydrophobic probe, e.g. SuO-Fmoc-MAL-S-(CH 2 )i5-COOH, is preferred.
  • the transport vector DTT-(MAL-Fmoc- OSu) 2 is unique in its capability to link either two drug moieties or a drug and a protein at the same time.
  • a BBB-nontransportable neurotherapeutic on one side and a protein which crosses the BBB on the other side, which protein has high-affinity receptor sites, located at the BBB, and can therefore enter the CNS by a mechanism defined as receptor-mediated transendocytosis.
  • Such selective substitution of the dimer transport vector e.g. DTT-(MAL-Fmoc- OSu) 2 or DTT-(MAL-FMS-OSU) 2
  • DTT-(MAL-FMS-OSU) 2 can be performed by reacting the dimer transport vector with the drug to be transported and a protein which crosses the BBB, and then purifying the desired conjugate from the mixture using HPLC.
  • An alternative option is to react one unit of the transport vector (e.g. MAL-Fmoc-OSu) with the drug to be transported and another unit of the transport vector with the protein transporter and then to react the two vectors with a linker (e.g. DTT) to obtain the desired conjugate.
  • the present invention relates to a conjugate of the BBB transport vector X-Z and a moiety of a BBB-non-transportable drug comprising at least one primary or secondary amino group, of the formula:
  • X' is a radical of the formula Ia linked to Z through the S atom:
  • R 1 is hydrogen, C 1 -C 6 alkyl, -SO 3 H, halogen, nitro, hydroxyl, sulfonamide, acyl, phosphoryl, or dialkylamine;
  • R 2 is C 2 -C 8 alkylene, optionally interrupted by an aryl ring, -(OCH 2 CH 2 ) n , wherein n is 1 to 6, or a residue of a peptide containing 2 to 4 hydrophobic amino acid residues;
  • R 3 and R 4 are selected from hydrogen, C 1 -C 8 alkyl or Ce- C 10 aryl;
  • R' 5 is a moiety of a BBB-non-transportable drug comprising at least one primary or secondary amino group, or a residue of a protein capable of crossing the BBB;
  • Z is a residue capable of crossing the BBB selected from:
  • a spacer linked to a second identical or different X' radical through the S atom such as a C 2 -Cs alkylene, optionally substituted by one or more hydroxy groups or interrupted by one or more oxygen atoms or by a phenylene radical, wherein R' 5 of the second X radical is optionally a residue of a protein which is capable of crossing the BBB; or
  • R 1 is -SO3H, halogen, nitro, hydroxyl, sulfonamide, acyl, phosphoryl, or dialkylamine, Z is not a C 8 -C 2 O fatty acid residue.
  • the BBB transport vector is reacted with a drug comprising at least one primary or secondary amino group whereby the leaving group R 5 is replaced by said drug of R' 5 in a substitution reaction, e.g., a nucleophilic substitution reaction, and the drug is linked to the BBB transport vector through said at least one amino group.
  • a substitution reaction e.g., a nucleophilic substitution reaction
  • drug refers to any compound biologically active in the brain, including any substance intended for use in the diagnosis, cure, alleviation, treatment or prevention of disease or disorder in the central nervous system (CNS), or in the enhancement of desirable physical or mental development.
  • primary or secondary amino group as use herein, relates to an amino group (-NH 2 ) comprising either two hydrogen atoms (primary) or one hydrogen atom is replaced by an organic moiety (secondary), wherein the secondary amino group is optionally a part of a cyclic ring. It should be noted that an amino side chain moiety (-CH 2 -NH 2 ) can be added to many drug molecules lacking such amino group, making them now capable of linking the
  • BBB-non-transportable drug or “BBB-non-transportable neurotherapeutic” refer to any compound biologically active in the brain, which is incapable of passing through the blood-brain barrier or shows very low blood-brain barrier permeability, and include but are not limited to any protein, peptide, amino-acid derivative, nucleotides and derivatives thereof, and hydrophilic drugs which do not inherently cross the BBB.
  • peptides active in the brain include, without being limited to, enkephalin (which, besides being analgesics, initiate epileptic activity in the brain at doses that are about ten-fold lower than for effecting analgesic activity), endorphin, NAP peptide (a peptide derived from activity-dependent neuroprotective protein (ADNF)), vasoactive intestinal peptide (VP), thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), laminin peptides, tachykinin, somatostatin, melanocortin stimulating hormone (MSH), angiotensins, substance-P, oxytocin, hemiasterlin, beta-sheat breaker peptides, vasopressin, adrenocorticotropic hormone (ACTH), cholecystokinin (CCK), bombesin, motilin, glicentin, glucagon, glucagon-
  • proteins active in the brain include, without being limited to, nerve nutrition factors of the neurotrophine family such as nerve growth factor (NGF), brain- derived neurotrophine factor (BDNF), neurotrophine 3 (NT-3), neurotrophine 4 (NT-4), neurotrophine 5 (NT-5) and ciliary neurotrophic factor (CNTF); growth factors active in the central nervous system such as basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF) and epithelial growth factor (EGF); cytokines which act directly or indirectly on the nervous system or are involved in gliacyte proliferation, differentiation and activation to exhibit nerve nutrition factor action, such as interferon-alpha (IFN- ⁇ ), interferon-beta (IFN- ⁇ ), interferon-gamma (IFN- ⁇ ), interleukin 1 (IL-I), interleukin 2 (IL- 2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6),
  • hydrophilic drugs active in the brain include, but are not limited to, antibiotics such as aminoglycosides (e.g. amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, and apramycin), antiviral agents (e.g.
  • antibiotics such as aminoglycosides (e.g. amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, and apramycin), antiviral agents (e.g.
  • acyclovir e.g. amphotericin B
  • anticancer drugs e.g. anticancer drugs
  • potassium channel openers e.g., fluorouracil, aminopterin, methotrexate actinomycins, doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin and mitomycin
  • potassium channel openers e.g.
  • diazoxide diazoxide, minoxidil, nicorandil, pinacidil, retigabine and flupirtine
  • histamine bradykinin
  • catecholamines epinephrine, noroepinephrin and others
  • serotonin choline esterases
  • GABA gamma-aminobutyric acid
  • dopamine a neurotransmitter and dopaminergic agent used, e.g., in the treatment of Parkinsonism or hyperprolactinemia
  • derivatives of amino-acids and nucleotides and derivatives thereof.
  • salts of the conjugates of the invention refers to both salts of carboxyl groups and to acid addition salts of amino groups of the drug, e.g. peptide or protein, molecule.
  • Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases such as those formed for example, with amines, such as triethanolamine, arginine, or lysine, piperidine, procaine, and the like.
  • Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids, such as, for example, acetic acid or oxalic acid.
  • mineral acids such as, for example, hydrochloric acid or sulfuric acid
  • organic acids such as, for example, acetic acid or oxalic acid.
  • Such chemical derivatives and salts are preferably used to modify the pharmaceutical properties of the peptide insofar as stability, solubility, etc., are concerned.
  • the invention is related to a conjugate of the formula X'-Z as detailed above, wherein R 1 , R 3 and R 4 are H, R 2 is C 2 -C 8 alkylene, preferably C 2 -C 4 alkylene, more preferably -(CH 2 ) 2 -, R'5 is a moiety of a BBB-non- transportable drug comprising at least one primary or secondary amino group, and Z is a C 8 -C 2 O fatty acid, preferably a C 1 O-C 16 fatty acid, more preferably a residue of undecanoic acid or hexadecanoic acid.
  • the invention is related to a conjugate of the formula X'-Z as detailed above, wherein R 1 is H or -SO 3 H, R 3 and R 4 are H, R 2 is C 2 -C 8 alkylene, preferably C 2 -C 4 alkylene, more preferably -(CH 2 ) 2 -, R' 5 is a moiety of a BBB- non-transportable drug comprising at least one primary or secondary amino group and Z is a spacer, preferably -(CH(OH)) 4 -, linked to a second identical or different X' radical through the S atom, wherein R' 5 of the second X radical is optionally a residue of a protein which is capable of crossing the BBB.
  • the invention is related to a conjugate of the formula X'-Z as detailed above, wherein Ri is H or -SO 3 H, R 3 and R 4 are H, R 2 is C 2 -C 8 alkylene, preferably C 2 -C 4 alkylene, more preferably -(CH 2 ) 2 -, R' 5 is a moiety of a BBB- non-transportable drug comprising at least one primary or secondary amino group and Z is a residue of a protein which is capable of crossing the BBB.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the conjugate of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • conjugates and pharmaceutical compositions of the present invention can be provided in a variety of formulations, e.g., in a pharmaceutically acceptable form and/or in a salt form, e.g., hydrates, as well as in a variety of dosages.
  • the pharmaceutical composition provided by the invention may be prepared by conventional techniques, e.g., as described in Remington: The Science and Practice of Pharmacy, 19th Ed., 1995.
  • the composition may further include pharmaceutically acceptable fillers, carriers or diluents, and other inert ingredients and excipients.
  • the pharmaceutical composition can be designed for a slow release of the binding compound.
  • the composition can be administered by any suitable route, which effectively transports the active compound, i.e., the conjugate of the invention, to the appropriate or desired site of action.
  • Suitable administration routes include, e.g., intravenous, intraarterial, intramuscular, subcutaneous, transdermal and topical administration; inhalation; and nasal, oral, sublingual, nasogastric, nasoenteric, orogastric, rectal and intraperitoneal administration.
  • the dosage will depend on the specific disease or disorder and the state of the patient, and will be determined as deemed appropriate by the practitioner.
  • compositions of the present invention may comprise the active agent, i.e., the conjugate of the invention, formulated for controlled release in microencapsulated dosage form, in which small droplets of the active agent are surrounded by a coating or a membrane to form particles in the range of a few micrometers to a few millimeters, or in controlled-release matrix.
  • the present invention relates to use of the conjugate of the invention, in delivery of a BBB-non-transportable drug from the periphery across the BBB to the CNS.
  • the present invention relates to a method for delivering a BBB- non-transportable drug from the periphery into the CNS of a subject through the BBB, comprising administering to said subject a therapeutically effective amount of the conjugate of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the present invention relates to a method for treatment of a disease or disorder in the CNS of a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a conjugate of the invention, wherein R' 5 is a moiety of a BBB-non-transportable drug capable of treating said disease or disorder.
  • the present invention relates to use of a conjugate of the invention, for the preparation of a pharmaceutical composition for the treatment of a disease or disorder in the CNS.
  • Diseases or disorders to be treated with the method of the present invention include but are not limited to: neurogenetic diseases (e.g. Huntington's disease and muscular dystrophy); developmental disorders (e.g. cerebral palsy); degenerative diseases of adult life (e.g. Parkinson's disease and Alzheimer's disease); metabolic diseases (e.g. Gaucher's disease); cerebrovascular diseases (e.g. stroke and vascular dementia); trauma (e.g. spinal cord and head injury); convulsive disorders (e.g. epilepsy); infectious diseases, (e.g. AIDS dementia); and brain tumors.
  • neurogenetic diseases e.g. Huntington's disease and muscular dystrophy
  • developmental disorders e.g. cerebral palsy
  • degenerative diseases of adult life e.g. Parkinson's disease and Alzheimer's disease
  • metabolic diseases e.g. Gaucher's disease
  • cerebrovascular diseases e.g. stroke and vascular dementia
  • trauma e.g. spinal cord and head injury
  • convulsive disorders e.
  • the applicability of the BBB transport vectors of the present invention was examined by assessing their ability to reversibly bind the BBB-nontransportable neuropeptide enkephalin; to deliver it through the BBB; and to release it in the brain while maintaining its analgesic effect.
  • Conjugates of enkephalin and representative BBB- transport-vectors of the invention were synthesized, namely: 3-thio [11-Undecanoic acid]- N-3-propiosuccinimido-4-amido-Fmoc-Leu-enkephalin, an example for a conjugate of the formula X'-Z, wherein Z is a fatty acid residue; S,S'-di [3-succinimido-N-3 propion-4- amido-Fmoc-Leu-enkephalin] dithiothreitol as an example for a conjugate of the formula X'-Z, wherein Z is a spacer linked to a second X radical through the S atom; and HSA- Fmoc-enkephalin and cationized-HSA-Fmoc-enkephalin as examples for conjugates of the formula X'-Z wherein.Z is a residue of a protein which crosses the BBB.
  • conjugates were examined for their analgesic effect using the tail-flick test, wherein, saline served as a negative control and morphine served as a positive control.
  • Our in-vivo experimental system was based on Leu-enkephalin or Met-enkephalin. This BBB-nontransportable neuropeptide facilitates analgesic effect only if centrally administered to rodents.
  • a variety of conjugates and derivatives, all linked to the single ⁇ - amino side-chain of enkephalin, were prepared and tested for their analgesic effect in mice following intraperitoneal administration.
  • Conjugates of Leu-enkephalin and BBB transport vectors of the invention such as
  • Fmoc-based peptide derivatization can turn a nontransportable drug into a lipophilic-prodrug derivative capable of reaching the brain, following peripheral administration.
  • the reversible linkage between such drugs and a protein capable of crossing the BBB through the Fmoc-type spacer can produce a prodrug that penetrates the BBB and confers therapeutic effect upon the release of the active peptide in the brain.
  • Scheme I depicts the structures and the nomenclature used herein of the BBB transport vectors synthesized, purified and characterized in examples 1-5 below: SuO- Fmoc-MAL-S-(CH 2 )io-COOH; SuO-FmOC-MAL-S-(CH 2 )! 5 -C00H, and DTT (MAL- Fmoc-OSu) 2 .
  • Scheme II depicts the structures and the nomenclature of the following conjugates prepared and examined in examples 6-16 below:
  • Fmoc-N ⁇ -Leu-enkephalin herein designated Fmoc-Enk-Leu or FLE-1077
  • FME-1091 Fmoc-N ⁇ -Met-enkephalin
  • Leu-Enk-Fmoc-Fmoc-Enk-Leu or LEFFLED-1031 cationized-HSA-Fmoc-Leu-enkephalin.
  • the dashed arrows indicate the sites of cleavage that take place upon incubation at physiological conditions.
  • HSA-Fmoc-enkephalin HSA-Fmoc-enkephalin
  • HSA-benz-enkephalin cationized HSA-benz-enkephalin
  • MAL-Fmoc-OSu and 16 mercaptohexadecanoic acid were dissolved in DMF at a concentration of 60 ⁇ moles/ml each. Pyridine 10 ⁇ l/ml was then added to obtain a concentration of 120 ⁇ moles/ml. The reaction mixture was stirred for 4 hrs. Ten volumes of cold 0.05M acetic acid were then added. The precipitate thus formed was collected by centrifugation, washed several times with cold H 2 O and desiccated.
  • the derivative SuO-Fmoc-MAL-S-(CH 2 )i 5 -COOH was purified by HPLC on Lichrospher RP- 18 column, under the purifying conditions applied for SuO-Fmoc-MAL-S-(CH 2 )io- COOH (see Example 5). The fractions corresponding to this derivative were collected and lyophilized.
  • DTT-(MAL-Fmoc-OSu) 2 was prepared by coupling two molecules of MAL-Fmoc- OSu to dithiothreitol [DTT, HS-(CHOH) 4 -SH; Sigma Chemical Co. St. Louis, MO, USA]. This was carried out by combining solution of DTT (60 mM, 0.5 ml) with that of MAL- Fmoc-OSu (120 mM, 0.5 ml) each dissolved in anhydrous DMF. Final concentration in the reaction mixture was 60 and 30 mM of MAL-Fmoc-OSu and DTT respectively. Pyridine was then added to obtain a final concentration of 12OmM.
  • reaction was then allowed to proceed for 1 hr at 25 0 C, and product formation was verified by the absence of free sulfhydryl group as determined by DTNB.
  • Ten volumes of cold 0.05 M acetic acid were then added. The precipitate thus formed was collected by centrifugation, washed several times with cold H 2 O and desiccated.
  • the derivative DTT-(MAL-Fmoc-OSu) 2 was purified by HPLC on Lichrospher RP- 18 column, under the purifying conditions applied for SuO-FmOC-MAL-S-(CH 2 ) I o-COOH and for SuO-Fmoc-MAL-S-(CH 2 )i5-COOH respectively (see Example 5). The fractions corresponding to this derivative were collected and lyophilized. Example 5. Chemical features of HPLC purified SuO-Fmoc-MAL-S-(CH 2 )io-COOH, SuO-Fmoc-MAL-S-(CH 2 )is-COOH and DTT-(MAL-Fmoc-OSu) 2 .
  • Biosystem 757 variable wavelength absorbance detector The column effluents were monitored by ultraviolent absorbance at 214nm.
  • Figs. 6-8 show the ultraviolet absorption spectra of all three synthetic compounds. All show identical absorption spectrum, with absorption maximum at 290.5 nm, and shoulder at 304 nm (Figs. 6-8).
  • Table 1 shows the chemical characteristic features of the HPLC-purified synthetic vectors.
  • Mass spactra were determined using electrospray ionization technique.
  • the Fmoc-peptides were prepared by the solid-phase methodology, using an APEX396 (Advanced ChemTech Louisville, Kentucky USA) automated synthesizer. The Fmoc strategy was employed throughout according to the company's commercial protocol. Following completion of chain assembly, the N-terminal Fmoc-moiety was not removed and the N-protected peptides were detached from the carrier by acidolysis (with TFA) and purified (>97%) by HPLC.
  • Example 7 Preparation of 3-thio (11-undecanoic acid) N-3 propion-succinimido-4- amido-Fmoc-Leu-enkephalin (10 FME-1095).
  • SuO-FmOC-MAL-S-(CH 2 ) I o-COOH was synthesized as described in Example 2 above.
  • a solution of SuO-Fmoc-MAL-S-(CH 2 ) 10 -COOH (1.06 mg/100 ml dimethylformamide (DMF)) was reacted with Leu-enkephalin (5.5 mg in 1.8 ml 0.1 M phosphate buffer, pH 8.5, 10 ⁇ moles). The reaction was carried out for 4 hours, at 25°C.
  • Cold 0.1 M HCl (20 ml) was added. The precipitate thus formed was collected by centrifugation, resuspended in cold 10 ml 0.1 M HCl and recentrifuged. This procedure was repeated three times.
  • DTT-(MAL-FmOc-OSu) 2 was synthesized as described in Example 4 above.
  • a solution of DTT-(MAL-FmOC-OSu) 2 (2.12 mg/100 ml DMF) was reacted with Leu- enkephalin (11 mg/in 1.8 ml of 0.1 M phosphate buffer, pH 8.5, 20 ⁇ moles).
  • the reaction was carried out for 7 hours at 25°C.
  • Cold 0.1M HCl (18 ml) was then added.
  • the precipitate thus formed was collected by centrifugation, resuspended in 10 ml 0.1 M HCl and recentrifuged. This procedure was repeated three times.
  • MAL-Fmoc-OSu 50 ⁇ l from a fresh solution of MAL- Fmoc-OSu in DMF 20 mg/ml was added. The reaction was carried out for 5 min at 25 0 C.
  • HPLC was conducted with a linear gradient from 0 to 100% of solution A (0.1% trifluoroacetic acid (TFA)) to solution B (acetonitrile - H 2 O, 75:25 in 0.1% TFA) over 10 min and then 4 min in solution B, using a ChromoliteTM RP 18 e (100 mm x 4 mm) column at a rate of 3 ml/min.
  • TFA trifluoroacetic acid
  • Dialysis removed any Leu-enkephalin and MAL-Fmoc- enkephalin that had not been covalently-linked to cationized HSA.
  • the amount of covalently-linked enkephalin was determined as described for HSA-Fmoc-enkephalin (Example 9 above).
  • the reagent (3 ⁇ l from a fresh solution of MIB-NHS, 20 mg/ml in DMF, 2 ⁇ moles) was reacted with Leu-enkephalin for 10 min at 25°C prior to the addition of cationized HAS.
  • All Fmoc-enkephalin conjugates prepared showed a decreased solubility in water, high solubility in organic solvents, such as dimethylsulfoxide (DMSO), and remained in solution upon 20 times dilution with 0.01 M NaHCO 3 .
  • concentration of each conjugate could be determined either by acid hydrolyzing an aliquot followed by amino acid analysis, or simply by the absorbance at 280 nm.
  • Analytic HPLC was performed using a prepacked ChromoliteTM R.P 18e column (4.0mm X 100mm Merck KGaA, Darmstadt Germany). The column was eluted with a binary gradient established between solution A (0.1% TFA in H 2 O) and solution B (acetonitrile :H 2 O; 75:25 in 0.1% TFA). A linear gradient from 0 to 100% of solution A to solution B was applied over a period of 10 min following 4 min in solution B at a rate of 3 ml/min.
  • Rt retention time
  • Table 2 summarizes the retention time (Rt) values, obtained by analytical HPLC analyses, for Met- and Leu-enkephalins and for the Fmoc-enkephalin conjugates prepared herein, using a ChromoliteTM RP-ISe column. This approach of 'hydrophobic chromatography' yielded an estimate of the relative lipophilic degree for each conjugate.
  • Rt-values in an increasing order, were 5.31, 5.69, 8.56, 8.77, 9.14 and 9.58 min for Met- enkephalin, Leu-enkephalin, Fmoc-Met-enkephalin, Fmoc-Leu-enkephalin, LEFFLED- 1031 and (ClOFJkffi-1095), respectively.
  • Fmoc hydrolysis was nearly identical to that obtained in normal human serum at 37 0 C (Gershonov et al., 1999; Gershonov et al., 2000).
  • Leu/Met-enkephalin is released from the conjugates in a slow and homogenous fashion, with half lives of 24 ⁇ 2 and 71 ⁇ 3 h for HSA-Fmoc-Leu-enkephalin, and Fmoc-Met-enkephalin, respectively.
  • the Tail Flick analgesia meter measures the movement of the animal's tail in response to a painful sensation. A focused beam of radiant heat was applied to the animal's tail, and then a timer begins. When the animal flicks its tail, the timer stops and the recorded time is a measure of the pain threshold.
  • Fmoc-Leu-enkephalin conjugates were synthesized, both of which are more lipophilic than Fmoc-Leu-enkephalin as judged by HPLC chromatography.
  • Rt value of Fmoc-Leu-enkephalin has shifted from 8.77 min to 9.14 min for S,S'-Di(3-succinimido-N- 3-propion-4-amido-Fmoc-Leu-enkephalin) di-thiothreitol (LEFFLED-1031) and to 9.58 min for 3-thio (11-undecanoic acid), N-3-propiosuccinimido-4-amido-Fmoc-Leu- enkephalin (ClOFME 1 - 1095) (Table 2).
  • Canonized HSA-Fmoc-enkephalin is analgesic following intraperitoneal administration.
  • cationized HSA-Fmoc-enkephalin has facilitated an antinociceptive effect following IP administration.
  • Analgesia was manifested at a low dose (2 ⁇ moles/kg body weight) and amounted to 55% of that obtained by IP administered morphine. Maximal effect was obtained 40 min after administration (Fig. HB).
  • Neither noncationized HSA-Fmoc-enkephalin nor the irreversibly linked cationized HSA-Benz- enkephalin were analgesic at this concentration (Fig. HA).
  • MAL-FMS-OSu i.e., 7-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl) propanamido)- 9-(((2,5-dioxopyrrolidin-l-yloxy)carbonyloxy) methyl)-9H-fluorene-2-sulfonic acid, is synthesized as described in Tsubery et al. (2004), starting from 9-hydroxymethyl-2- aminofluorene, and the final product was obtained in 65% yield following four steps of synthesis.
  • the dimer S,S' di (3-succinimido-N-3 propion-4-amido-FMS-Leu-enkephalin) dithiothreitol is produced from MAL-FMS-OSu and DTT, in the same manner that DTT- (MAL-FmOc-OSu) 2 was prepared from MAL-Fmoc-OSu and dithiothreitol (Example 4 above).
  • Met-enkephalin and FMRFa induces antinociception and attenuates development of tolerance to morphine antinociception.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne de nouveaux vecteurs de transport BBB qui sont capables d'administrer des neurothérapies non transportables BBB par le biais de BBB et de les libérer dans le cerveau d'une manière lente et continue, tout en maintenant leur effet thérapeutique.
PCT/IL2010/000612 2009-07-31 2010-07-29 Vecteurs destinés à l'administration de neurothérapies au système nerveux central WO2011013128A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US23016209P 2009-07-31 2009-07-31
US61/230,162 2009-07-31

Publications (2)

Publication Number Publication Date
WO2011013128A2 true WO2011013128A2 (fr) 2011-02-03
WO2011013128A3 WO2011013128A3 (fr) 2011-03-24

Family

ID=43382491

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IL2010/000612 WO2011013128A2 (fr) 2009-07-31 2010-07-29 Vecteurs destinés à l'administration de neurothérapies au système nerveux central

Country Status (1)

Country Link
WO (1) WO2011013128A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2437786B1 (fr) * 2009-06-01 2016-05-18 Yeda Research and Development Co. Ltd. Promédicaments contenant une sonde se liant à l'albumine

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4801575A (en) 1986-07-30 1989-01-31 The Regents Of The University Of California Chimeric peptides for neuropeptide delivery through the blood-brain barrier
US5442043A (en) 1992-11-27 1995-08-15 Takeda Chemical Industries, Ltd. Peptide conjugate
US5525727A (en) 1982-05-18 1996-06-11 University Of Florida Brain-specific drug delivery
WO1998005361A2 (fr) 1996-08-07 1998-02-12 Yeda Research And Development Co. Ltd. Medicaments a effet prolonge et compositions pharmaceutiques les renfermant
WO2002078597A2 (fr) 2001-04-01 2002-10-10 Yeda Research And Development Co. Ltd. Weizmann Institute Of Science Medicaments a absorption orale
EP1337270A2 (fr) 2000-11-01 2003-08-27 YEDA RESEARCH AND DEVELOPMENT Co. LTD. Derives de cytokine de longue action et compositions pharmaceutiques comprenant ces derives
WO2004089280A2 (fr) 2003-04-08 2004-10-21 Yeda Research And Development Co. Ltd. Medicaments pegyles reversibles

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4933324A (en) * 1988-02-26 1990-06-12 Shashoua Victor E Fatty acid-neuroactive drug conjugate as a prodrug
EP2437786B1 (fr) * 2009-06-01 2016-05-18 Yeda Research and Development Co. Ltd. Promédicaments contenant une sonde se liant à l'albumine

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5525727A (en) 1982-05-18 1996-06-11 University Of Florida Brain-specific drug delivery
US4801575A (en) 1986-07-30 1989-01-31 The Regents Of The University Of California Chimeric peptides for neuropeptide delivery through the blood-brain barrier
US5442043A (en) 1992-11-27 1995-08-15 Takeda Chemical Industries, Ltd. Peptide conjugate
WO1998005361A2 (fr) 1996-08-07 1998-02-12 Yeda Research And Development Co. Ltd. Medicaments a effet prolonge et compositions pharmaceutiques les renfermant
US6504005B1 (en) 1996-08-07 2003-01-07 Yeda Research And Development Co. Ltd. Long-acting drugs and pharmaceutical compositions comprising them
EP1337270A2 (fr) 2000-11-01 2003-08-27 YEDA RESEARCH AND DEVELOPMENT Co. LTD. Derives de cytokine de longue action et compositions pharmaceutiques comprenant ces derives
WO2002078597A2 (fr) 2001-04-01 2002-10-10 Yeda Research And Development Co. Ltd. Weizmann Institute Of Science Medicaments a absorption orale
WO2004089280A2 (fr) 2003-04-08 2004-10-21 Yeda Research And Development Co. Ltd. Medicaments pegyles reversibles

Non-Patent Citations (17)

* Cited by examiner, † Cited by third party
Title
ALBERICIO, F.; CRUZ, M.; DEBETHUNE, L.; ERITJA, R.; GIRALT, E.; GRANDAS, A.; MARCHAN, V.; PASTOR, J. J.; PEDROSO, E.; RABANAL, F., SYNTH. COMMUN., vol. 31, 2001, pages 225 - 232
D'AMOUR, F.E.; SMITH, D.L.: "A method for determining loss of pain sensation", J PHARMACOL. EXP. THER., vol. 72, 1941, pages 74 - 79
EGLETON, R.D.; MITCHELL, S.A.; HUBER, J.D.; TANDERS, J.; STRROPOVA, D.; POLT, R.; YAMAMURA, H.I.; HURBY V.J.; DAVIS, T.P.: "Improved bioavailability to the brain of glycosylated Met-enkephalin analogs", BRAIN RES., vol. 881, 2000, pages 37 - 46
GERSHONOV, E.; GOLDWASER, I.; FRIDKIN, M.; SCHECHTER, Y.: "A novel approach for a water-soluble long-acting insulin prodrug: Design preparation and analysis of [2-sulfo-9-fluorenylmethoxycarbnyl]3-insulin", J. MED. CHEM., vol. 43, 2000, pages 2530 - 2537
GERSHONOV, E.; SHECHTER, Y.; FRIDKIN, M.: "Spontaneous conversion of an inactive modified insulin to the active hormone in circulation: 9-fluorenylmethoxycarbonyl derivative of insulin", DIABETES, vol. 48, 1999, pages 1437 - 1442
GUPTA, S.; PASHA, S.; GUPTA, Y.K.; BARDWAY, D.K.: "Chimeric peptide of Met-enkephalin and FMRFa induces antinociception and attenuates development of tolerance to morphine antinociception", PEPTIDES, vol. 20, 1999, pages 471 - 478
GUPTA, S.; PASHA, S.; GUPTA, Y.K.; BARDWAY, D.K.: "Effects of intracerebro-ventricularly administered chimeric peptide of Met-enkephalin and FMRFa-[D-Ala2]YFa-on antinociception and its modulation in mice", BRAIN RES. BULL., vol. 55, 2001, pages 51 - 57
HRUBY, V.J.; KAZMIERSKI, W.; KAWASAKI, A.M.; MATSUNAGA, T.O.: "Synthetic chemistry and the design of peptide-based drugs. in peptide pharmaceuticals", 1991, OPEN UNIV. PRESS, pages: 135 - 184
KNAPP, R.J.; DAVIS, T.P.; BURKS, T.F.; YAMAMURA, H.I.: "pharmaceuticals", 1991, OPEN UNIV. PRESS, pages: 210 - 242
PARDRIDGE, W.M.: "Peptide Drug Delivery to the Brain", 1991, RAVEN
SHECHTER, Y.; GOLDWASER, I.; LAVON, 1.; GERSHONOV, E.; MESTER, B.; MIRONCHIK, M.; PATT, L.; FRIDKIN, M.: "A new approach for prolonging the half-life of peptide, proteins and low-molecular-weight drugs in vivo", DRUGS OF THE FUTURE, vol. 26, 2001, pages 669 - 676
SHECHTER, Y.; MIRONCHIK, M.; RUBINRAUT, S.; SAUL, A.; TSUBERY, H.; FRIDKIN, M.: "Albumin-insulin conjugate releasing insulin slowly under physiological conditions; A new concept for long-acting insulin", BIOCONJUGATE CHEM., vol. 16, 2005, pages 913 - 920
SHECHTER, Y.; MIRONCHIK, M.; SAUL, A.; GERSHONOV, E.; SASSON, K.; TSUBERY, H.; FRIDKIN, M.: "New technologies to prolong life-time of peptide and protein drugs in vivo (a review)", INT. J. PEPTIDE RES. THER., 2006
SHECHTER, Y.; PATT, P.; SCHREIBER, G.; FRIDKIN, M.: "Prolonging the half-life of human interferon-2 in circulation; design, preparation and analysis of (2-sulfo-9-fluorenylmethoxycarbonyl)7 interferon-2", PROC. NATL. ACAD. SCI. USA, vol. 98, 2001, pages 1212 - 1217
SHECHTER, Y.; TSUBERY, H.; FRIDKIN, M.: "N-[2-sulfo-9-fluorenylmethoxycarbonyl]3-gentamicin C1 is a long acting prodrug derivative", J. MED. CHEM., vol. 45, 2002, pages 4264 - 4270
SHECHTER, Y.; TSUBERY, H.; MIRONCHIK, M.; RUBINSTEIN, M.; FRIDKIN, M.: "Reversible pegylation of peptide PYY3-36 prolongs its inhibition of food intake in mice", FEBS. LETT., vol. 579, 2005, pages 2439 - 2444
TSUBERY, H.; MIRONCHIK, M.; FRIDKIN, M.; SHECHTER, Y.: "Prolonging the action of protein and peptide drugs by a novel approach of reversible polyethylene glycol modification", J. BIOL. CHEM., vol. 279, 2004, pages 38118 - 38124

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2437786B1 (fr) * 2009-06-01 2016-05-18 Yeda Research and Development Co. Ltd. Promédicaments contenant une sonde se liant à l'albumine

Also Published As

Publication number Publication date
WO2011013128A3 (fr) 2011-03-24

Similar Documents

Publication Publication Date Title
US10357572B2 (en) Prodrugs activated by caspase
CA2374964C (fr) Procede permettant de produire une preparation medicamenteuse injectable
JP3645283B2 (ja) リソゾーム酵素開裂性抗腫瘍剤結合体
CN109316605B (zh) 叶酸受体结合配体-药物偶联物
DK2566335T3 (en) Controlled release of active ingredients from macromolecular CONJUGATES
US8106007B2 (en) Conjugates of a polypeptide and a pentasaccharide
US6613879B1 (en) FAP-activated anti-tumour compounds
CN105934257B (zh) 用于含氮和羟基的药物的生物可逆引入基团
KR20040095228A (ko) 규제된 물질의 남용을 방지하기 위한 새로운 서방성 약학화합물
JP2023078367A (ja) 新規ｇｌｐ－１類似体
KR102436012B1 (ko) 항암제 프로드러그 컨쥬게이트의 새로운 용도
WO2011013128A2 (fr) Vecteurs destinés à l'administration de neurothérapies au système nerveux central
US7304037B2 (en) Cytostatic conjugates with integrin ligands
US7368431B2 (en) Polypeptide, the conjugate thereof with doxorubicine and a pharmaceutical composition based thereon
US20040038871A1 (en) Conjugates of aminodrugs
US8642555B2 (en) Prodrugs
JP2019515025A (ja) トポイソメラーゼ毒
KR101095396B1 (ko) 암 표적지향형 트랜스페린-폴리에틸렌 글리콜이 수식된 트레일, 이의 제조 및 용도
US10420845B2 (en) Taggable heteroaromatic drugs and conjugates thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10747681

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10747681

Country of ref document: EP

Kind code of ref document: A2