US20110166063A1 - Polymer conjugates of therapeutic peptides - Google Patents

Polymer conjugates of therapeutic peptides Download PDF

Info

Publication number
US20110166063A1
US20110166063A1 US13/119,297 US200913119297A US2011166063A1 US 20110166063 A1 US20110166063 A1 US 20110166063A1 US 200913119297 A US200913119297 A US 200913119297A US 2011166063 A1 US2011166063 A1 US 2011166063A1
Authority
US
United States
Prior art keywords
conjugate
peptide
insulin
pharmaprojects
vaccine
Prior art date
Legal status (The legal status 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 status listed.)
Abandoned
Application number
US13/119,297
Other languages
English (en)
Inventor
Mary J. Bossard
Steven O. Roczniak
Harold Zappe
Yujun Wang
Ping Zhang
Dawei Sheng
C. Simone Jude-Fishburn
Elizabeth Louise Minamitani
Xiaofeng Liu
Haim Moskowitz
Dennis G. Fry
Cherie F. Ali
Christine Taylor Brew
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nektar Therapeutics
Original Assignee
Nektar Therapeutics
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 Nektar Therapeutics filed Critical Nektar Therapeutics
Priority to US13/119,297 priority Critical patent/US20110166063A1/en
Assigned to NEKTAR THERAPEUTICS reassignment NEKTAR THERAPEUTICS ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BOSSARD, MARY J., WANG, YUJUN, MINAMITANI, ELIZABETH L., ZAPPE, HAROLD, FRY, DENNIS G., SHENG, DAWEI, ZHANG, PING, ROCZNIAK, STEVEN O., ALI, CHERIE F., BREW, CHRISTINE TAYLOR, JUDE-FISHBURN, C. SIMONE, LIU, XIAOFENG, MOSKOWITZ, HAIM
Publication of US20110166063A1 publication Critical patent/US20110166063A1/en
Assigned to NEKTAR THERAPEUTICS reassignment NEKTAR THERAPEUTICS ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WANG, YUJUN, MINAMITANI, ELIZABETH LOUISE, ZAPPE, HAROLD, ZHANG, PING, BOSSARD, MARY J., FRY, DENNIS G., SHENG, DAWEI, ALI, CHERIE F., MOSKOWITZ, HAIM, BREW, CHRISTINE TAYLOR, LIU, XIAOFENG, JUDE-FISHBURN, C. SIMONE, ROCZNIAK, STEVEN O.
Assigned to WELLS FARGO BANK, NATIONAL ASSOCIATION, AS COLLATERAL AGENT reassignment WELLS FARGO BANK, NATIONAL ASSOCIATION, AS COLLATERAL AGENT GRANT OF SECURITY INTEREST Assignors: NEKTAR THERAPEUTICS
Assigned to NEKTAR THERAPEUTICS reassignment NEKTAR THERAPEUTICS RELEASE OF SECURITY INTEREST RECORDED AT REEL 28571, FRAME 0141 Assignors: WELLS FARGO BANK, NATIONAL ASSOCIATION, AS COLLATERAL AGENT
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • 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
    • A61K47/51—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
    • A61K47/61—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 the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention relates to conjugates comprising a therapeutic peptide moiety covalently attached to one or more water-soluble polymers.
  • peptides are naturally occurring molecules made up of amino acid building blocks, and are involved in countless physiological processes. With 20 naturally occurring amino acids, and any number of non-naturally occurring amino acids, a nearly endless variety of peptides may be generated. Additionally, peptides display a high degree of selectivity and potency, and may not suffer from potential adverse drug-drug interactions or other negative side effects. Moreover, recent advances in peptide synthesis techniques have made the synthesis of peptides practical and economically viable. Thus peptides hold great promise as a highly diverse, highly potent, and highly selective class of therapeutic molecules with low toxicity.
  • the present invention provides conjugates comprising a therapeutic peptide moiety covalently attached to one or more water-soluble polymers.
  • the water-soluble polymer may be stably bound to the therapeutic peptide moiety, or it may be releasably attached to the therapeutic peptide moiety.
  • the invention further provides methods of synthesizing such therapeutic peptide polymer conjugates and compositions comprising such conjugates.
  • the invention further provides methods of treating, preventing, or ameliorating a disease, disorder or condition in a mammal comprising administering a therapeutically effective amount of a therapeutic peptide polymer conjugate of the invention.
  • FIG. KISS 1 . 1 Cation exchange purification of the PEGylation reaction mixture.
  • FIG. KISS 1 . 2 RP-HPLC analysis of purified [mono]-[mPEG-ButyrALD-30K]-[Kisspeptin-13].
  • FIG. KISS 1 3 MALDI-TOF spectrum of purified [mono]-[mPEG-ButyrALD-30K]-[Kisspeptin-13].
  • FIG. KISS 2 . 1 Typical reversed phase purification profile of [mono]-[mPEG-ButyAldehyde-10K]-[Kisspeptin-10].
  • FIG. KISS 2 2 Purity analysis of mono-[ButyrAldehyde-10K]-[Kisspeptin-10] by Reversed Phase HPLC.
  • FIG. KISS 2 . 3 MALDI-TOF spectrum of purified mono-[mPEG-butyraldehyde-10k]-[Kisspeptin-10].
  • FIG. KISS 3 . 1 Typical reversed phase purification profile of [mono]-[mPEG-ButyAldehyde-30K]-[Kisspeptin-10].
  • FIG. KISS 3 . 2 Purity analysis of mono-[ButyrAldehyde-30K]-[Kisspeptin-1] by Reversed Phase HPLC.
  • FIG. KISS 3 . 3 MALDI-TOF spectrum of purified mono-[mPEG-Butyraldehyde-30K]-[Kisspeptin-10].
  • FIG. KISS 4 . 1 Typical reversed phase purification profile of mono-[mPEG2-CAC-FMOC-40K]-[Kisspeptin-10].
  • FIG. KISS 4 . 2 Purity analysis of [mono]-[CAC-PEG2-FOMC-40K]-[Kisspeptin-10] by Reversed Phase HPLC.
  • FIG. KISS 4 . 3 MALDI-TOF spectrum of purified mono-[CAC-PEG2-FMOC-40K]-[Kisspeptin-10].
  • FIG. KISS 5 . 1 Typical reversed phase purification profile of mono-[mPEG-SBC-30K]-[Kisspeptin-10].
  • FIG. KISS 5 . 2 SDS-PAGE, with Coomassie blue staining) of purified mono-[mPEG-SBC-30K]-[Kisspeptin-10].
  • FIG. KISS 5 . 3 Purity analysis of mono-[mPEG-SBC-30K]-[Kisspeptin-10] by Reversed Phase HPLC.
  • FIG. KISS 5 . 4 MALDI-TOF spectrum of purified mono-[mPEG-SBC-30k]-[Kisspeptin-10].
  • FIG. KISS 6 1 Typical cation exchange purification profile of mono-[mPEG2-ButyrAldehyde-40K]-[Kisspeptin-54].
  • FIG. KISS 6 . 2 Purity analysis of [mono]-[mPEG2-ButyrAldehyde-40K]-[Kisspeptin-54] conjugate by Reversed Phase HPLC.
  • FIG. KISS 6 . 3 SDS-PAGE with Coomassie staining of purified [mono]-[mPEG2-ButyrAldehyde-40K]-[Kisspeptin-54].
  • FIG. KISS 6 . 4 MALDI-TOF spectrum of purified [mono]-[mPEG2-ButyrAldehyde-40K]-[Kisspeptin-54].
  • FIG. KISS 8 . 1 Agonist activity at GPR54 for stable PEG conjugates of Kisspeptin 10, Kisspeptin 13, and Kisspeptin 54.
  • FIG. KISS 8 . 2 Agonist activity at GPR54 for releasable PEG conjugate of Kisspeptin 10.
  • FIG. KISS 8 . 3 Agonist activity at GPR54 for releasable PEG conjugate of Kisspeptin 10.
  • FIG. ZIC 2 . 1 Cation exchange purification of mono-mPEG-C2-FMOC-20K-ziconotide from the PEGylation reaction mixture.
  • FIG. ZIC 2 . 2 RP-HPLC analysis of purified mono-mPEG-C2-FMOC-20K-ziconotide.
  • FIG. ZIC 2 . 3 MALDI-TOF analysis of purified mono-mPEG-C2-FMOC-20K-ziconotide.
  • FIG. ZIC 3 . 1 Cation exchange purification of mono-mPEG-CAC-FMOC-40K-ziconotide from the PEGylation reaction mixture.
  • FIG. ZIC 3 . 2 RP-HPLC analysis of purified mono-mPEG-CAC-FMOC-40K-ziconotide.
  • FIG. ZIC 3 . 3 MALDI-TOF analysis of purified mono-mPEG-CAC-FMOC-40K-ziconotide.
  • FIG. ZIC 4 . 1 Cation exchange purification of mono-mPEG-SBA-30K-ziconotide from the PEGylation reaction mixture.
  • FIG. ZIC 4 . 2 RP-HPLC analysis of purified mono-mPEG-SBA-30K-ziconotide.
  • FIG. ZIC 4 . 3 MALDI-TOF analysis of purified mono-mPEG-SBA-30K-ziconotide.
  • FIG. ZIC 5 . 1 Cation exchange FPLC chromatography of the PEGylation reaction mixture between ziconotide and mPEG-SBC-30K-NHS.
  • FIG. ZIC 6 . 1 Mean ( ⁇ SEM) percent specific binding of ziconotide conjugates to calcium channel, N-type, in rat cortical membranes.
  • FIG. BIP 2 . 1 (SPA-2K)2-biphalin purification with CG-71S resin.
  • FIG. BIP 2 . 2 RP-HPLC analysis of reconstituted (SPA-2K)2-biphalin.
  • FIG. BIP 2 . 3 MALDI TOF MS analysis of reconstituted (SPA-2K)2-biphalin.
  • FIG. BIP 3 . 1 (C2-20K) 2 -biphalin purification with CG-71 S resin.
  • FIG. BIP 3 . 2 RP-HPLC analysis of reconstituted (C2-20K) 2 -biphalin.
  • FIG. BIP 3 3 MALDI-TOF analysis of reconstituted (C2-20K) 2 -biphalin.
  • FIG. BIP 4 . 1 (CAC-20K) 2 -biphalin purification with CG-71S resin.
  • FIG. BIP 4 . 2 (CAC-20K) 2 -biphalin re-purification with CG-71S resin.
  • FIG. BIP 4 . 3 RP-HPLC analysis of reconstituted (CAC-20K) 2 -biphalin.
  • BIP 4 . 4 MALDI-TOF analysis of reconstituted (CAC-20K) 2 -biphalin.
  • FIG. BIP 5 . 1 RP-HPLC analysis of SBC-30K and biphalin conjugation reaction mixture.
  • FIG. BIP 5 . 2 The purification of (SBC-30K) 2 -biphalin from the reaction mixture.
  • FIG. BIP 6 . 1 Competition binding assay of biphalin and di-CAC-20K-biphalin conjugate at human (A) ⁇ opioid and (B) ⁇ opioid receptors.
  • FIG. BIP 6 . 2 Competition binding assay of biphalin and di-C2-20K-biphalin, di-SBC-30K-biphalin, and di-SPA-2K-biphalin conjugate at human (A) ⁇ opioid and (B) ⁇ opioid receptors.
  • FIG. BNP 2 . 1 PEGylation rate of BNP-32 with mPEG2-40 kDa Butyr-ALD.
  • FIG. BNP 2 . 2 Typical purification profile for the 40 kDa mPEG2-Butyr-ALD mono-PEG conjugate of BNP-32.
  • FIG. BNP 2 . 3 HPLC analysis of the 40 kDa mPEG2-Butyr-ALD mono-PEG conjugate of BNP-32.
  • FIG. BNP 2 . 4 MALDI-TOF analysis of the 40 kDa mPEG2-Butyr-ALD mono-PEG conjugate of BNP-32.
  • FIG. BNP 2 . 5 SDS-PAGE (4-12% Bis-Tris-Nu-PAGE, Invitrogen) analysis of BNP-32 and purified [mono]-[mPEG2-Butyr-ALD-40K]-[BNP-32] conjugate.
  • FIG. BNP 4 . 1 Typical cation-exchange purification profile of [mono]-[mPEG-Butyr-ALD-10K]-[BNP-32].
  • FIG. BNP 4 . 2 SDS-PAGE analysis of BNP-32 and the purified [mono]-[mPEG2-Butyr-ALD-40K]-[BNP-32] conjugate.
  • FIG. BNP 4 . 3 RP-HPLC analysis of the purified [mono]-[mPEG-Butyr-ALD-10K]-[BNP-32] conjugate.
  • FIG. BNP 4 . 4 MALDI-TOF analysis of the purified [mono]-[mPEG-Butyr-ALD-10K]-[BNP-32] conjugate.
  • FIG. BNP 5 . 1 Typical first cation-exchange purification profile for [mono]-[mPEG-SBC-30K]-[BNP-32].
  • FIG. BNP 5 . 2 SDS-PAGE analysis of the purified [mono]-[mPEG-SBC-30K]-[BNP-32] conjugate.
  • FIG. BNP 5 . 3 RP-HPLC analysis of the purified [mono]-[mPEG-SBC-30K]-[BNP-32] conjugate.
  • FIG. BNP 5 . 4 MALDI-TOF analysis of the purified [mono]-[mPEG-SBC-30K]-[BNP-32] conjugate.
  • FIG. BNP 6 . 1 Typical first cation-exchange purification profile of [mPEG2-C2-fmoc-NHS-40K].
  • FIG. BNP 6 . 2 SDS-PAGE analysis of the purified [mPEG2-C2-fmoc-NHS-40K]-[BNP-32] conjugate.
  • FIG. BNP 6 . 3 RP-HPLC analysis of the purified [mPEG2-C2-fmoc-NHS-40K]-[BNP-32] conjugate.
  • FIG. BNP 6 . 4 MALDI-TOF analysis of the purified [mPEG2-C2-fmoc-NHS-40K]-[BNP-32] conjugate.
  • FIG. BNP 7 . 1 shows the mean plasma concentration-time profiles of for C2-FMOC-PEG2-40K-BNP, its corresponding metabolite and released BNP.
  • FIG. BNP 7 . 2 shows the non-released PEG-BNP levels after the administration of the two non-releasable PEG constructs (ButyrALD-40K-BNP, ButyrALD-10K-BNP).
  • FIG. PRO 2 . 1 Typical cation exchange purification profile of mono-[mPEG2-CAC-FMOC-40K]-[PG-1].
  • FIG. PRO 2 . 2 SDS-PAGE of purified [mono]-[CAC-PEG2-FOMC-NHS-40K]-[Protegrin-1].
  • FIG. PRO 2 . 3 Purity analysis of [mono]-[CAC-PEG2-FOMC-40K]-[Protegrin-1] by RP-HPLC.
  • FIG. PRO 2 . 4 MALDI-TOF spectrum of purified mono-[CAC-PEG2-FMOC-40K]-[Protegrin-1].
  • FIG. PRO 3 1 Typical cation exchange purification profile of mono-[mPEG-SBC-30K]-[PG-1].
  • FIG. PRO 3 . 2 SDS-PAGE of purified [mono]-[mPEG-SBC-30K-]-[Protegrin-1].
  • FIG. PRO 3 . 3 Purity analysis of [mono]-[mPEG-SBC-30K-]-[Protegrin-1] by RP-HPLC.
  • FIG. PRO 3 . 4 MALDI-TOF spectrum of purified [mono]-[mPEG-SBC-30K-]-[Protegrin-1].
  • FIG. PRO 4 1 Typical reversed phase purification profile of [Protegrin-1]-[PEG-di-ButyrAldehyde-5K]-[Protegrin-1].
  • FIG. PRO 4 . 2 SDS-PAGE of purified [Protegrin-1]-[PEG-di-butyraldehyde-5K]-[Protegrin-1].
  • FIG. PRO 4 . 3 Purity analysis of [Protegrin-1]-[PEG-di-butyraldehyde-5K]-[Protegrin-1] by reversed phase HPLC.
  • FIG. PRO 4 . 4 MALDI-TOF spectrum of [Protegrin-1]-[PEG-di-butyraldehyde-5K]-[Protegrin-1].
  • FIG. PRO 5 . 1 Typical cation-exchange chromatography profile of dextran-butryaldehyde-40K-protegrin-1.
  • FIG. PRO 5 . 2 SDS-PAGE analysis (4-12% gel) of purified dextran-butryraldehyde-40K-protegrin-1.
  • FIG. PRO 6 . 1 PG-1 and (ALD) 2 2K conjugates purification with CM Sepharose FF resin.
  • FIG. PRO 6 . 2 RP-HPLC analysis of (PG-1)-(ALD) 2 2K-(PG-1).
  • FIG. PRO 6 . 3 MALDI analysis of (PG-1)-(ALD) 2 2K-(PG-1).
  • FIG. PRO 7 . 1 . 1 and 7 . 1 . 2 ALD40K-PG-1 purification with SP Sepharose HP resin.
  • FIG. PRO 7 . 2 SDS-PAGE of the purified and concentrated ALD40K-PG-1.
  • FIG. PRO 7 . 3 RP-HPLC analysis of ALD40K-PG-1 (lot #YW-pgALD40K-01).
  • FIG. PRO 7 . 4 MALDI analysis of ALD40K-PG-1 (lot #YW-pgALD40K-01).
  • FIG. PRO 8 . 1 CG40K-PG-1 purification with SP Sepharose HP resin.
  • FIG. PRO 8 . 2 RP-HPLC analysis of purified CG40K-PG-1.
  • FIG. PRO 8 . 3 MALDI-TOF analysis of purified CG40K-PG-1.
  • FIG. PRO 9 . 1 Hemolysis relative to the 100% hemolysis produced by 0.25% Triton X-100.
  • FIG. PRO 9 . 1 Hemolysis by PEG reagent controls.
  • FIG. PRO 9 . 3 Hemolysis at the maximum concentration.
  • FIG. PRO 9 . 4 Hemolytic activities of PG-1
  • FIG. PRO 10 . 1 and PRO 10 . 2 show the mean plasma concentration-time profiles for CG-PEG 2 -FMOC-40K-PG-1 and CAC-PEG 2 -FMOC-40K-PG-1, their corresponding PEG-metabolite and released Protegrin-1.
  • FIG. PRO 10 . 3 shows the released Protegrin-1 levels after the administration of the two releasable PEG constructs versus the level of Protegrin-1 given as native protein at the same dose (mg/kg).
  • FIG. PRO 10 . 4 shows the mean plasma concentration-time profiles for mPEG 2 -PG-1, PG-1[PEG 2k -PG-1, PG-1-PEG 5k -PG-1.
  • FIG. V 2 . 1 Typical cation-exchange purification profile of [mPEG2-NHS-20K]-[V681(V13AD)].
  • FIG. V 2 . 2 SDS-PAGE analysis of V681(V13AD) PEGylation.
  • FIG. V 2 . 3 Purity analysis of [mono]-[mPEG2-NHS 20K]-[V681(V13AD)] conjugate by reverse phase HPLC.
  • FIG. V 2 . 4 MALDI-TOF spectra for [mono]-[mPEG2-NHS 20K]-[V681(V13AD)].
  • FIG. V 3 . 1 Typical cation-exchange purification profile of [mPEG-SMB-301(]-[V681(V13AD)].
  • FIG. V 3 . 2 SDS-PAGE analysis of V681(V13AD) PEGylation and purification on the SP ion-exchange column.
  • FIG. V 3 . 3 Purity analysis of [mono]-[mPEG-SMB-30K]-[V681(V13AD)] conjugate by reverse phase HPLC.
  • FIG. V 3 . 4 MALDI-TOF spectra for [mono]-[mPEG-SMB 30K]-[V681(V13AD)].
  • FIG. V 4 . 1 shows the mean plasma concentration-time profiles for V681 (V13AD), SMB-30K-V681 (V13AD), and NHS-20K-V681 (V13AD).
  • FIG. V 5 . 1 Hemolysis relative to the 100% hemolysis produced by 0.25% Triton X-100.
  • FIG. C-PEP 2 . 1 Typical anion-exchange chromatography profile of [[mono]-[mPEG-ru-MAL-30K]-[C-peptide(S20C)].
  • FIG. C-PEP 2 . 2 Purity analysis of [[mono]-[mPEG-ru-MAL-30K]C-peptide(S20C)] by reversed phase HPLC.
  • FIG. C-PEP 2 . 3 MALDI-TOF spectrum for [mono]-[mPEG-ru-MAL-30K]-[C-peptide(S20C)].
  • FIG. C-PEP 3 . 1 Typical anion-exchange chromatography profile of [[mono]-[mPEG-Butyraldehyde-30K]-[C-peptide(S20C)].
  • FIG. C-PEP 3 . 2 Purity analysis of [mono]-[mPEG-Butyraldehyde-30K]-[C-peptide(S20C)] by reversed phase HPLC.
  • FIG. C-PEP 3 . 3 MALDI-TOF spectrum for [mono]-[mPEG-Butyraldehyde-30K]-[C-peptide(S20C)].
  • FIG. C-PEP 4 . 1 Typical anion-exchange chromatography profile of [mono]-[C2-PEG2-FMOC-40K]-[C-peptide(S20C)].
  • FIG. C-PEP 4 . 2 Purity analysis of [[mono]-[C2-PEG2-FMOC-40K]C-peptide(S20C)] by reversed phase HPLC.
  • FIG. C-PEP 4 . 3 MALDI-TOF spectrum for [mono]-[C2-PEG2-FMOC-40K]-[C-peptide(S20C)].
  • FIG. C-PEP 5 . 1 Typical anion-exchange purification profile of [[mono]-[CAC-PEG2-FMOC-40K]-[C-peptide(S20C)].
  • FIG. C-PEP 5 . 2 Purity analysis of [mono]-[CAC-PEG2-FMOC-40K]-[C-peptide(S20C)] by reversed phase HPLC.
  • FIG. C-PEP 6 1 Typical anion-exchange chromatography profile of dextran-butryaldehyde-40K-C-peptide(S20C).
  • FIG. C-PEP 6 . 2 Concentration of fraction II from the anion-exchange chromatogram shown in FIG. c-pep 6 . 1 by a second anion-exchange chromatography run.
  • FIG. C-PEP 6 . 3 Purity analysis of [[mono]-[Dextran-40K]-[C-peptide(S20C)] by reversed phase HPLC.
  • FIG. C-PEP 6 . 4 MALDI-TOF spectrum for [mono]-[Dextran-40K]-[C-peptide(S20C)].
  • FIG. OGF 2 . 1 Typical CG71 S reversed phase purification profile of mono-[mPEG2-CAC-FMOC-40K]-[OGF].
  • FIG. OGF 2 . 2 Purity analysis of [mono]-[CAC-PEG2-FOMC-40K]-[OGF] by reversed phase HPLC.
  • FIG. OGF 2 . 3 MALDI-TOF spectrum of purified mono-[mPEG2-FMOC-CAC-40K]-[OGF].
  • FIG. OGF 3 . 1 Typical CG71S reverse phase purification profile of mono-[mPEG2-C2-FMOC-40K]-[OGF].
  • FIG. OGF 3 . 2 Purity analysis of mono-[mPEG2-FMOC-C2-40K]-[OGF] by reversed phase HPLC.
  • FIG. OGF 3 . 3 MALDI-TOF spectrum of purified mono-[mPEG2-FMOC-C2-40K]-[OGF].
  • FIG. OGF 4 . 1 Typical CG71S reversed phase purification profile of mono-[mPEG-Butyraldehyde-30K]-[OGF].
  • FIG. OGF 4 . 2 Purity analysis of mono-[mPEG-ButyrAldehyde-30K]-[OGF] by reversed phase HPLC.
  • FIG. OGF 5 . 1 Typical CG71S reversed phase purification profile of mono-[mPEG-epoxide-5K]-[OGF].
  • FIG. OGF 5 . 2 Purity analysis of mono-[mPEG-epoxide-5K]-[OGF] by reversed phase HPLC.
  • FIG. OGF 6 . 1 Typical CG71 S reversed phase purification profile of mono-[mPEG-Butyraldehyde-10K]-[OGF].
  • FIG. OGF 6 . 2 Purity analysis of mono-[mPEG-ButyrAldehyde-10K]-[OGF] by reversed phase HPLC.
  • FIG. OGF 7 . 1 Competition binding assay of OGF at human (A) ⁇ opioid and (B) ⁇ opioid receptors: effects of incubation treatment conditions.
  • FIG. OGF 7 . 2 Competition binding assay of OGF and PEG-OGF conjugates (released and unreleased) at human (A) ⁇ opioid and (B) ⁇ opioid receptors.
  • FIG. OGF 7 . 3 Competition binding assay of OGF and free PEGs at human (A) ⁇ opioid and (B) ⁇ opioid receptors.
  • FIG. INS 1 . 1 Typical anion-exchange chromatography profile of the conjugation reaction mixture with partially acetylated insulin.
  • FIG. INS 1 . 2 SDS-PAGE analysis of fractions containing dextran-butyrALD-40K-insulin collected from anion-exchange chromatography.
  • FIG. INS 1 . 3 Concentration of purified dextran-butyrALD-40K-insulin by anion-exchange chromatography.
  • FIG. INS 1 . 4 SDS-PAGE analysis of purified dextran-butyrALD-40K-insulin.
  • FIG. INS 1 . 5 Typical anion-exchange chromatography profile of the conjugation reaction mixture with non-acetylated insulin.
  • FIG. INS 3 . 1 Glucose levels after compound administration (0-8 hr).
  • a polymer includes a single polymer as well as two or more of the same or different polymers
  • reference to “an optional excipient” or to “a pharmaceutically acceptable excipient” refers to a single optional excipient as well as two or more of the same or different optional excipients, and the like.
  • therapeutic peptide and “therapeutic peptides” mean one or more peptides having demonstrated or potential use in treating, preventing, or ameliorating one or more diseases, disorders, or conditions in a subject in need thereof, as well as related peptides. These terms may be used to refer to therapeutic peptides prior to conjugation to a water-soluble polymer as well as following the conjugation.
  • Therapeutic peptides include, but are not limited to, those disclosed herein, including in Table 1.
  • Therapeutic peptides include peptides found to have use in treating, preventing, or ameliorating one or more diseases, disorders, or conditions after the time of filing of this application.
  • peptides include fragments of therapeutic peptides, therapeutic peptide variants, and therapeutic peptide derivatives that retain some or all of the therapeutic activities of the therapeutic peptide.
  • modifications may be made to peptides that do not alter, or only partially abrogate, the properties and activities of those peptides. In some instances, modifications may be made that result in an increase in therapeutic activities.
  • therapeutic peptide or “therapeutic peptides” are meant to encompass modifications to the therapeutic peptides defined and/or disclosed herein that do not alter, only partially abrogate, or increase the therapeutic activities of the parent peptide.
  • therapeutic activity refers to a demonstrated or potential biological activity whose effect is consistent with a desirable therapeutic outcome in humans, or to desired effects in non-human mammals or in other species or organisms.
  • a given therapeutic peptide may have one or more therapeutic activities, however the term “therapeutic activities” as used herein may refer to a single therapeutic activity or multiple therapeutic activites.
  • “Therapeutic activity” includes the ability to induce a response in vitro, and may be measured in vivo or in vitro. For example, a desirable effect may be assayed in cell culture, or by clinical evaluation, EC 50 assays, IC 50 assays, or dose response curves.
  • Therapeutic activity includes treatment, which may be prophylactic or ameliorative, or prevention of a disease, disorder, or condition.
  • Treatment of a disease, disorder or condition can include improvement of a disease, disorder or condition by any amount, including elimination of a disease, disorder or condition.
  • peptide refers to polymers comprised of amino acid monomers linked by amide bonds.
  • Peptides may include the standard 20 ⁇ -amino acids that are used in protein synthesis by cells (i.e. natural amino acids), as well as non-natural amino acids (non-natural amino acids may be found in nature, but not used in protein synthesis by cells, e.g., ornithine, citrulline, and sarcosine, or may be chemically synthesized), amino acid analogs, and peptidomimetics.
  • the amino acids may be D- or L-optical isomers.
  • Peptides may be formed by a condensation or coupling reaction between the ⁇ -carbon carboxyl group of one amino acid and the amino group of another amino acid.
  • the terminal amino acid at one end of the chain (amino terminal) therefore has a free amino group, while the terminal amino acid at the other end of the chain (carboxy terminal) has a free carboxyl group.
  • the peptides may be non-linear, branched peptides or cyclic peptides.
  • the peptides may optionally be modified or protected with a variety of functional groups or protecting groups, including on the amino and/or carboxy terminus.
  • Amino acid residues in peptides are abbreviated as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; and Glycine is Gly or G.
  • therapeutic peptide fragment refers to a polypeptide that comprises a truncation at the amino-terminus and/or a truncation at the carboxyl-terminus of a therapeutic peptide as defined herein.
  • therapeutic peptide fragment or “fragments of therapeutic peptides” also encompasses amino-terminal and/or carboxyl-terminal truncations of therapeutic peptide variants and therapeutic peptide derivatives.
  • Therapeutic peptide fragments may be produced by synthetic techniques known in the art or may arise from in vivo protease activity on longer peptide sequences. It will be understood that therapeutic peptide fragments retain some or all of the therapeutic activities of the therapeutic peptides.
  • therapeutic peptide variants or “variants of therapeutic peptides” refer to therapeutic peptides having one or more amino acid substitutions, including conservative substitutions and non-conservative substitutions, amino acid deletions (either internal deletions and/or C- and/or N-terminal truncations), amino acid additions (either internal additions and/or C- and/or N-terminal additions, e.g., fusion peptides), or any combination thereof.
  • Variants may be naturally occurring (e.g. homologs or orthologs), or non-natural in origin.
  • therapeutic peptide variants may also be used to refer to therapeutic peptides incorporating one or more non-natural amino acids, amino acid analogs, and peptidomimetics. It will be understood that, in accordance with the invention, therapeutic peptide fragments retain some or all of the therapeutic activities of the therapeutic peptides.
  • therapeutic peptide derivatives or “derivatives of therapeutic peptides” as used herein refer to therapeutic peptides, therapeutic peptide fragments, and therapeutic peptide variants that have been chemically altered other than through covalent attachment of a water-soluble polymer. It will be understood that, in accordance with the invention, therapeutic peptide derivatives retain some or all of the therapeutic activities of the therapeutic peptides.
  • amino terminus protecting group or “N-terminal protecting group,” “carboxy terminus protecting group” or “C-terminal protecting group;” or “side chain protecting group” refer to any chemical moiety capable of addition to and optionally removal from a functional group on a peptide (e.g., the N-terminus, the C-terminus, or a functional group associated with the side chain of an amino acid located within the peptide) to allow for chemical manipulation of the peptide.
  • PEG polyethylene glycol
  • poly(ethylene glycol) are interchangeable and encompass any nonpeptidic water-soluble poly(ethylene oxide).
  • PEGs for use in accordance with the invention comprise the following structure “—(OCH 2 CH 2 ) n —” where (n) is 2 to 4000.
  • PEG also includes “—CH 2 CH 2 —O(CH 2 CH 2 O) n —CH 2 CH 2 —” and “—(OCH 2 CH 2 ) n O—,” depending upon whether or not the terminal oxygens have been displaced.
  • PEG includes structures having various terminal or “end capping” groups and so forth.
  • PEG also means a polymer that contains a majority, that is to say, greater than 50%, of —OCH 2 CH 2 — repeating subunits.
  • the PEG can take any number of a variety of molecular weights, as well as structures or geometries such as “branched,” “linear,” “forked,” “multifunctional,” and the like, to be described in greater detail below.
  • end-capped and “terminally capped” are interchangeably used herein to refer to a terminal or endpoint of a polymer having an end-capping moiety.
  • the end-capping moiety comprises a hydroxy or C 1-20 alkoxy group, more preferably a C 1-10 alkoxy group, and still more preferably a C 1-5 alkoxy group.
  • examples of end-capping moieties include alkoxy (e.g., methoxy, ethoxy and benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and the like.
  • the end-capping moiety may include one or more atoms of the terminal monomer in the polymer [e.g., the end-capping moiety “methoxy” in CH 3 —O—(CH 2 CH 2 O) n — and CH 3 (OCH 2 CH 2 ) n —].
  • the end-capping group can also be a silane.
  • the end-capping group can also advantageously comprise a detectable label.
  • the amount or location of the polymer and/or the moiety (e.g., active agent) to which the polymer is coupled can be determined by using a suitable detector.
  • suitable detectors include photometers, films, spectrometers, and the like.
  • the end-capping group can also advantageously comprise a phospholipid.
  • phospholipids include, without limitation, those selected from the class of phospholipids called phosphatidylcholines.
  • Specific phospholipids include, without limitation, those selected from the group consisting of dilauroylphosphatidylcholine, dioleylphosphatidylcholine, dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine, behenoylphosphatidylcholine, arachidoylphosphatidylcholine, and lecithin.
  • targeting moiety is used herein to refer to a molecular structure that helps the conjugates of the invention to localize to a targeting area, e.g., help enter a cell, or bind a receptor.
  • the targeting moiety comprises of vitamin, antibody, antigen, receptor, DNA, RNA, sialyl Lewis X antigen, hyaluronic acid, sugars, cell specific lectins, steroid or steroid derivative, RGD peptide, ligand for a cell surface receptor, serum component, or combinatorial molecule directed against various intra- or extracellular receptors.
  • the targeting moiety may also comprise a lipid or a phospholipid.
  • Exemplary phospholipids include, without limitation, phosphatidylcholines, phospatidylserine, phospatidylinositol, phospatidylglycerol, and phospatidylethanolamine. These lipids may be in the form of micelles or liposomes and the like.
  • the targeting moiety may further comprise a detectable label or alternately a detectable label may serve as a targeting moiety.
  • the conjugate has a targeting group comprising a detectable label
  • the amount and/or distribution/location of the polymer and/or the moiety (e.g., active agent) to which the polymer is coupled can be determined by using a suitable detector.
  • Such labels include, without limitation, fluorescers, chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g., dyes), metal ions, radioactive moieties, gold particles, quantum dots, and the like.
  • Non-naturally occurring with respect to a polymer as described herein, means a polymer that in its entirety is not found in nature.
  • a non-naturally occurring polymer of the invention may, however, contain one or more monomers or segments of monomers that are naturally occurring, so long as the overall polymer structure is not found in nature.
  • water soluble as in a “water-soluble polymer” is any polymer that is soluble in water at room temperature. Typically, a water-soluble polymer will transmit at least about 75%, more preferably at least about 95%, of light transmitted by the same solution after filtering. On a weight basis, a water-soluble polymer will preferably be at least about 35% (by weight) soluble in water, more preferably at least about 50% (by weight) soluble in water, still more preferably about 70% (by weight) soluble in water, and still more preferably about 85% (by weight) soluble in water. It is most preferred, however, that the water-soluble polymer is about 95% (by weight) soluble in water or completely soluble in water.
  • Hydrophilic e.g, in reference to a “hydrophilic polymer,” refers to a polymer that is characterized by its solubility in and compatibility with water. In non-cross linked form, a hydrophilic polymer is able to dissolve in, or be dispersed in water.
  • a hydrophilic polymer possesses a polymer backbone composed of carbon and hydrogen, and generally possesses a high percentage of oxygen in either the main polymer backbone or in pendent groups substituted along the polymer backbone, thereby leading to its “water-loving” nature.
  • the water-soluble polymers of the present invention are typically hydrophilic, e.g., non-naturally occurring hydrophilic.
  • Molecular weight in the context of a water-soluble polymer can be expressed as either a number average molecular weight or a weight average molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to the weight average molecular weight. Both molecular weight determinations, number average and weight average, can be measured using gel permeation chromatography or other liquid chromatography techniques. Other methods for measuring molecular weight values can also be used, such as the use of end-group analysis or the measurement of colligative properties (e.g., freezing-point depression, boiling-point elevation, and osmotic pressure) to determine number average molecular weight, or the use of light scattering techniques, ultracentrifugation or viscometry to determine weight average molecular weight.
  • colligative properties e.g., freezing-point depression, boiling-point elevation, and osmotic pressure
  • the polymers of the invention are typically polydisperse (i.e., number average molecular weight and weight average molecular weight of the polymers are not equal), possessing low polydispersity values of preferably less than about 1.2, more preferably less than about 1.15, still more preferably less than about 1.10, yet still more preferably less than about 1.05, and most preferably less than about 1.03.
  • active when used in conjunction with a particular functional group refers to a reactive functional group that reacts readily with an electrophile or a nucleophile on another molecule. This is in contrast to those groups that require strong catalysts or highly impractical reaction conditions in order to react (i.e., a “non-reactive” or “inert” group).
  • spacer moiety refers to an atom or a collection of atoms optionally used to link interconnecting moieties such as a terminus of a polymer segment and a therapeutic peptide or an electrophile or nucleophile of a therapeutic peptide.
  • the spacer moiety may be hydrolytically stable or may include a physiologically hydrolyzable or enzymatically degradable linkage. Unless the context clearly dictates otherwise, a spacer moiety optionally exists between any two elements of a compound (e.g., the provided conjugates comprising a residue of a therapeutic peptide and a water-soluble polymer that can be attached directly or indirectly through a spacer moiety).
  • Alkyl refers to a hydrocarbon, typically ranging from about 1 to 15 atoms in length. Such hydrocarbons are preferably but not necessarily saturated and may be branched or straight chain, although typically straight chain is preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 2-methylbutyl, 2-ethylpropyl, 3-methylpentyl, and the like. As used herein, “alkyl” includes cycloalkyl as well as cycloalkylene-containing alkyl.
  • “Lower alkyl” refers to an alkyl group containing from 1 to 6 carbon atoms, and may be straight chain or branched, as exemplified by methyl, ethyl, n-butyl, i-butyl, and t-butyl.
  • Cycloalkyl refers to a saturated or unsaturated cyclic hydrocarbon chain, including bridged, fused, or spiro cyclic compounds, preferably made up of 3 to about 12 carbon atoms, more preferably 3 to about 8 carbon atoms.
  • Cycloalkylene refers to a cycloalkyl group that is inserted into an alkyl chain by bonding of the chain at any two carbons in the cyclic ring system.
  • Alkoxy refers to an —O—R group, wherein R is alkyl or substituted alkyl, preferably C 1-6 alkyl (e.g., methoxy, ethoxy, propyloxy, and so forth).
  • substituted refers to a moiety (e.g., an alkyl group) substituted with one or more noninterfering substituents, such as, but not limited to: alkyl; C 3-8 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g., fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl; substituted phenyl; and the like.
  • “Substituted awl” is aryl having one or more noninterfering groups as a substituent. For substitutions on a phenyl ring, the substituents may be in any orientation (i.e., ortho, meta, or para).
  • Noninterfering substituents are those groups that, when present in a molecule, are typically nonreactive with other functional groups contained within the molecule.
  • Aryl means one or more aromatic rings, each of 5 or 6 core carbon atoms.
  • Aryl includes multiple aryl rings that may be fused, as in naphthyl or unfused, as in biphenyl.
  • Aryl rings may also be fused or unfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclic rings.
  • aryl includes heteroaryl.
  • Heteroaryl is an aryl group containing from one to four heteroatoms, preferably sulfur, oxygen, or nitrogen, or a combination thereof. Heteroaryl rings may also be fused with one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl rings.
  • Heterocycle or “heterocyclic” means one or more rings of 5-12 atoms, preferably 5-7 atoms, with or without unsaturation or aromatic character and having at least one ring atom that is not a carbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen.
  • “Substituted heteroaryl” is heteroaryl having one or more noninterfering groups as substituents.
  • Substituted heterocycle is a heterocycle having one or more side chains formed from noninterfering substituents.
  • An “organic radical” as used herein shall include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl.
  • Electrode and “electrophilic group” refer to an ion or atom or collection of atoms, that may be ionic, having an electrophilic center, i.e., a center that is electron seeking, capable of reacting with a nucleophile.
  • Nucleophile and nucleophilic group refers to an ion or atom or collection of atoms that may be ionic having a nucleophilic center, i.e., a center that is seeking an electrophilic center or with an electrophile.
  • a “physiologically cleavable” or “hydrolyzable” or “degradable” bond is a bond that reacts with water (i.e., is hydrolyzed) under physiological conditions.
  • the tendency of a bond to hydrolyze in water will depend not only on the general type of linkage connecting two central atoms but also on the substituents attached to these central atoms.
  • Appropriate hydrolytically unstable or weak linkages include but are not limited to carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides.
  • the therapeutic peptide thus released will typically correspond to the unmodified parent or native therapeutic peptide, or may be slightly altered, e.g., possessing a short organic tag.
  • the unmodified parent therapeutic peptide is released.
  • An “enzymatically degradable linkage” means a linkage that is subject to degradation by one or more enzymes.
  • a “hydrolytically stable” linkage or bond refers to a chemical bond, typically a covalent bond, that is substantially stable in water, that is to say, does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time.
  • hydrolytically stable linkages include, but are not limited to, the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, urethanes, and the like.
  • a hydrolytically stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks.
  • linkages can be hydrolytically stable or hydrolyzable, depending upon (for example) adjacent and neighboring atoms and ambient conditions.
  • One of ordinary skill in the art can determine whether a given linkage or bond is hydrolytically stable or hydrolyzable in a given context by, for example, placing a linkage-containing molecule of interest under conditions of interest and testing for evidence of hydrolysis (e.g., the presence and amount of two molecules resulting from the cleavage of a single molecule).
  • Other approaches known to those of ordinary skill in the art for determining whether a given linkage or bond is hydrolytically stable or hydrolyzable can also be used.
  • pharmaceutically acceptable excipient and “pharmaceutically acceptable carrier” refer to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.
  • “Pharmacologically effective amount,” “physiologically effective amount,” and “therapeutically effective amount” are used interchangeably herein to mean the amount of a polymer-(therapeutic peptide) conjugate that is needed to provide a desired level of the conjugate (or corresponding unconjugated therapeutic peptide) in the bloodstream or in the target tissue.
  • the precise amount will depend upon numerous factors, e.g., the particular therapeutic peptide, the components and physical characteristics of the therapeutic composition, intended patient population, individual patient considerations, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein.
  • Multi-functional means a polymer having three or more functional groups contained therein, where the functional groups may be the same or different.
  • Multi-functional polymeric reagents of the invention will typically contain from about 3-100 functional groups, or from 3-50 functional groups, or from 3-25 functional groups, or from 3-15 functional groups, or from 3 to 10 functional groups, or will contain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups within the polymer backbone.
  • a “difunctional” polymer means a polymer having two functional groups contained therein, either the same (i.e., homodifunctional) or different (i.e., heterodifunctional).
  • subject refers to a vertebrate, preferably a mammal.
  • Mammals include, but are not limited to, murines, rodents, simians, humans, farm animals, sport animals, and pets.
  • substantially means nearly totally or completely, for instance, satisfying one or more of the following: greater than 50%, 51% or greater, 75% or greater, 80% or greater, 90% or greater, and 95% or greater of the condition.
  • conjugates comprising a therapeutic peptide covalently attached (either directly or through a spacer moiety or linker) to a water-soluble polymer.
  • the conjugates generally have the following formula:
  • PEP is a therapeutic peptide as defined herein
  • X is a covalent bond or is a spacer moiety or linker
  • POLY is a water soluble polymer
  • k in an integer ranging from 1-10, preferably 1-5, and more preferably 1-3.
  • the conjugates of the invention comprise a therapeutic peptide as disclosed and/or defined herein.
  • Therapeutic peptides include those currently known to have demonstrated or potential use in treating, preventing, or ameliorating one or more diseases, disorders, or conditions in a subject in need thereof as well as those discovered after the filing of this application.
  • Therapeutic peptides also include related peptides.
  • PEP is a therapeutic peptide selected from the group consisting of carperitide; alpha-neoendorphin; 348U87; A-3847; A-4114; A-68552; A-75998; A-84861; AN-1792; AAMP-1; exenatide; AC-625; ACE-inhibitors, Aventis; ACE-inhibitors, SRI; ACTH, Amgen; ruprintrivir; AI-102; AI-202; NeuroVax; AI-402; AI-502; AIDS therapeutic vaccine, Repl; AIDS therapy, Inst Pasteur; AIDS vaccine, J&J; AIDS vaccine, Liposome Co; AIDS vaccine, Arana; AIDS vaccine, Peptimmune; AIDS vaccine, Sanofi Past-3; AIDS vaccine, Protherics; AIDS vaccine, SSVI; AIDS vaccine, SWFBR; AIDS vaccine, United-1; AIDS vaccine, United-2; AIDS vaccine-2, Yokohama;
  • PEP is a therapeutic peptide selected from the therapeutic peptides listed in Table 1.
  • SEQ ID NOs. 1-301 describe sequences that are required to be provided with the Sequence Listing and are therefore appended with the instant Specification. In some instances, these peptides contain features that are either inconsistent with or not amenable to inclusion in the Sequence Listing. For example, a sequence with less than four-amino acids; a sequence with a D-amino acid; or certain modification that cannot be described in the Sequence Listing presently, and therefore are not provided in the Sequence Listing. However, for the ease of use and description, a SEQ ID NO. has been provided to these peptides (i.e., SEQ ID NOs: 302-469).
  • Pat. No. 5,470,831 Anti- inflammatory Thr-Thr-Ser-Gln-Val-Arg-Pro-Arg inflammatory peptide Val-Lys-Thr-Thr-Ser-Gln-Val-Arg-Pro-Arg. Immuno- Ser-Gln-Val-Arg-Pro-Arg suppressant Val-Arg-Pro-Arg Multiple Thr-Thr-Ser-Gln-Val-Arg-Pro-Arg-His-Ile-Thr.
  • Patent No. EP0078228 GSHK; ASHK; A D SHK; Antithrombotic 183-185 LSHK; TSHK; YSHK; GSHKCH 3 COOH•H 2 O; SAR-SHK; and 178 PSHK; (PYR)ESHK; WSHK; GSHK•2TosOH 411 Pharmaprojects N-methyl-D-Phe-Pro-Arg-H Antithrombotic No. 2363 186 Pharmaprojects N-3-(4-hydroxyphenyl)propionyl-Pro-Hyp- Antiarrhythmic No.
  • 3415 4-hydroxy-N-[1′-isopropyl-1′-(4- HIV isopropylcarbonylimidazol-2-yl)]methyl- 6-phenyl-2-phenylmethyl-hexanamide 415 Pharmaprojects Piv-1-Ser-Leu-GABA, and Piv-Ser-Leu-GABA Neurological No. 4004 416 Pharmaprojects (1R,4aR,8aR)-1,2,3,4,5,6,7,8- Antithrombotic No.
  • YM-216391 The unusual polyoxazole- thiazole-based cyclopeptide 1, designated YM-216391, was recently isolated from Streptomyces nobilis .1 It shares both a structural and biological homology with the potent telomerase inhibitor telomestatin 2 which is showing promise in cancer chemotherapy.2
  • the structure of YM-216391 comprises a continuum of five azoles which have their origins in serine, cysteine and phenylalanine, linked via a glycine-valine- isoleucine tripeptide tether. The complete stereochemical assignment of YM-216391 has not been established.
  • the therapeutic peptides are selected from the group consisting of peptide G, OTS-102, Angiocol (antiangiogenic peptide group), ABT-510 (antiangiogenic peptide group), A6 (antiangiogenic peptide group), islet neogenesis gene associated protein (INGAP), tendamistat, recombinant human carperitide (alpha-atrial natriuretic peptide) (natriuretic peptide group), urodilatin (natriuretic peptide group), desirudin, Obestatin, ITF-1697, oxyntomodulin, cholecystokinin, bactericidal permeability increasing (BPI) protein, C-peptide, Prosaptide TX14(A), sermorelin acetate (GHRFA group), pralmorelin (GHRFA group), growth hormone releasing factor (GHRFA group), examorelin (GHRFA group), gonadorelin (LH-
  • the therapeutic peptides of the invention may comprise any of the 20 natural amino acids, and/or non-natural amino acids, amino acid analogs, and peptidomimetics, in any combination.
  • the peptides may be composed of D-amino acids or L-amino acids, or a combination of both in any proportion.
  • the therapeutic peptides may contain, or may be modified to include, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more non-natural amino acids.
  • non-natural amino acids and amino acid analogs that can be use with the invention include, but are not limited to, 2-aminobutyric acid, 2-aminoisobutyric acid, 3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine, 3-methylhistidine, 3-pyridylalanine, 4-chlorophenylalanine, 4-fluorophenylalanine, 4-hydroxyproline, 5-hydroxylysine, alloisoleucine, citrulline, dehydroalanine, homoarginine, homocysteine, homoserine, hydroxyproline, N-acetylserine, N-formylmethionine, N-methylglycine, N-methylisoleucine, norleucine, N- ⁇ -methylarginine, O-phosphoserine, ornithine, phenylglycine, pipecolinic acid, piperazic acid, pyroglutamine, sarcosine,
  • the therapeutic peptides may be, or may be modified to be, linear, branched, or cyclic, with our without branching.
  • the therapeutic peptides may optionally be modified or protected with a variety of functional groups or protecting groups, including amino terminus protecting groups and/or carboxy terminus protecting groups.
  • Protecting groups, and the manner in which they are introduced and removed are described, for example, in “Protective Groups in Organic Chemistry,” Plenum Press, London, N.Y. 1973; and Greene et al., “P ROTECTIVE G ROUPS IN O RGANIC S YNTHESIS ” 3 rd Edition, John Wiley and Sons, Inc., New York, 1999. Numerous protecting groups are known in the art.
  • protecting groups includes methyl, formyl, ethyl, acetyl, t-butyl, anisyl, benzyl, trifluoroacetyl, N-hydroxysuccinimide, t-butoxycarbonyl, benzoyl, 4-methylbenzyl, thioanizyl, thiocresyl, benzyloxymethyl, 4-nitrophenyl, benzyloxycarbonyl, 2-nitrobenzoyl, 2-nitrophenylsulphenyl, 4-toluenesulphonyl, pentafluorophenyl, diphenylmethyl, 2-chlorobenzyloxycarbonyl, 2,4,5-trichlorophenyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, triphenylmethyl, and 2,2,5,7,8-pentamethyl-chroman-6-sulphonyl.
  • the therapeutic peptides contain, or may be modified to contain, functional groups to which a water-soluble polymer may be attached, either directly or through a spacer moiety or linker.
  • Functional groups include, but are not limited to, the N-terminus of the therapeutic peptide, the C-terminus of the therapeutic peptide, and any functional groups on the side chain of an amino acid, e.g. lysine, cysteine, histidine, aspartic acid, glutamic acid, tyrosine, arginine, serine, methionine, and threonine, present in the therapeutic peptide.
  • the therapeutic peptides can be prepared by any means known in the art, including non-recombinant and recombinant methods, or they may, in some instances, be commercially available. Chemical or non-recombinant methods include, but are not limited to, solid phase peptide synthesis (SPPS), solution phase peptide synthesis, native chemical ligation, intein-mediated protein ligation, and chemical ligation, or a combination thereof.
  • SPPS solid phase peptide synthesis
  • solution phase peptide synthesis native chemical ligation
  • intein-mediated protein ligation and chemical ligation, or a combination thereof.
  • the therapeutic peptides are synthesized using standard SPPS, either manually or by using commercially available automated SPPS synthesizers.
  • the subsequent amino acid to be added to the peptide chain is protected on its amino terminus with Boc, Fmoc, or other suitable protecting group, and its carboxy terminus is activated with a standard coupling reagent.
  • the free amino terminus of the support-bound amino acid is allowed to react with the carboxy-terminus of the subsequent amino acid, coupling the two amino acids.
  • the amino terminus of the growing peptide chain is deprotected, and the process is repeated until the desired polypeptide is completed.
  • Side chain protecting groups may be utilized as needed.
  • the therapeutic peptides may be prepared recombinantly.
  • Exemplary recombinant methods used to prepare therapeutic peptides include the following, among others, as will be apparent to one skilled in the art.
  • a therapeutic peptide as defined and/or described herein is prepared by constructing the nucleic acid encoding the desired peptide or fragment, cloning the nucleic acid into an expression vector, transforming a host cell (e.g., plant, bacteria such as Escherichia coli , yeast such as Saccharomyces cerevisiae , or mammalian cell such as Chinese hamster ovary cell or baby hamster kidney cell), and expressing the nucleic acid to produce the desired peptide or fragment.
  • a host cell e.g., plant, bacteria such as Escherichia coli , yeast such as Saccharomyces cerevisiae , or mammalian cell such as Chinese hamster ovary cell or baby hamster kidney cell
  • the expression can occur via exogenous expression or via endogenous expression (when the host cell naturally contains the desired genetic coding).
  • Methods for producing and expressing recombinant polypeptides in vitro and in prokaryotic and eukaryotic host cells are known to those of ordinary skill in the art. See, for example, U.S. Pat. No. 4,868,122, and Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989).
  • nucleic acid sequences that encode an epitope tag or other affinity binding sequence can be inserted or added in-frame with the coding sequence, thereby producing a fusion peptide comprised of the desired therapeutic peptide and a peptide suited for binding.
  • Fusion peptides can be identified and purified by first running a mixture containing the fusion peptide through an affinity column bearing binding moieties (e.g., antibodies) directed against the epitope tag or other binding sequence in the fusion peptide, thereby binding the fusion peptide within the column. Thereafter, the fusion peptide can be recovered by washing the column with the appropriate solution (e.g., acid) to release the bound fusion peptide.
  • binding moieties e.g., antibodies
  • the tag may subsequently be removed by techniques known in the art.
  • the recombinant peptide can also be identified and purified by lysing the host cells, separating the peptide, e.g., by size exclusion chromatography, and collecting the peptide. These and other methods for identifying and purifying recombinant peptides are known to those of ordinary skill in the art.
  • therapeutic peptides are used herein in a manner to include not only the therapeutic peptides defined and/or disclosed herein, but also related peptides, i.e. peptides that contain one or more modifications relative to the therapeutic peptides defined and/or disclosed herein, wherein the modification(s) do not alter, only partially abrogate, or increase the therapeutic activities as compared to the parent peptide.
  • Related peptides include, but are not limited to, fragments of therapeutic peptides, therapeutic peptide variants, and therapeutic peptide derivatives. Related peptides also include any and all combinations of these modifications.
  • a related peptide may be a fragment of a therapeutic peptide as disclosed herein having one or more amino acid substitutions.
  • any reference to a particular type of related peptide is not limited to a therapeutic peptide having only that particular modification, but rather encompasses a therapeutic peptide having that particular modification and optionally any other modification.
  • Related peptides may be prepared by action on a parent peptide or a parent protein (e.g. proteolytic digestion to generate fragments) or through de novo preparation (e.g. solid phase synthesis of a peptide having a conservative amino acid substitution relative to the parent peptide).
  • Related peptides may arise by natural processes (e.g. processing and other post-translational modifications) or may be made by chemical modification techniques. Such modifications are well-known to those of skill in the art.
  • a related peptide may have a single alteration or multiple alterations relative to the parent peptide. Where multiple alterations are present, the alterations may be of the same type or a given related peptide may contain different types of modifications. Furthermore, modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the N- or C-termini.
  • related peptides include fragments of the therapeutic peptides defined and/or disclosed herein, wherein the fragment retains some of or all of at least one therapeutic activity of the parent peptide.
  • the fragment may also exhibit an increase in at least one therapeutic activity of the parent peptide.
  • therapeutic peptides include related peptides having at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 contiguous amino acid residues, or more than 125 contiguous amino acid residues, of any of the therapeutic peptides disclosed, herein, including in Table 1.
  • therapeutic peptides include related peptides having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues deleted from the N-terminus and/or having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues deleted from the C-terminus of any of the therapeutic peptides disclosed herein, including in Table 1.
  • Related peptides also include variants of the therapeutic peptides defined and/or disclosed herein, wherein the variant retains some of or all of at least one therapeutic activity of the parent peptide.
  • the variant may also exhibit an increase in at least one therapeutic activity of the parent peptide.
  • therapeutic peptides include variants having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 conservative and/or non-conservative amino acid substitutions relative to the therapeutic peptides disclosed herein, including in Table 1. Desired amino acid substitutions, whether conservative or non-conservative, can be determined by those skilled in the art.
  • therapeutic peptides include variants having conservative amino substitutions; these substitutions will produce a therapeutic peptide having functional and chemical characteristics similar to those of the parent peptide.
  • therapeutic peptides include variants having non-conservative amino substitutions; these substitutions will produce a therapeutic peptide having functional and chemical characteristics that may differ substantially from those of the parent peptide.
  • therapeutic peptide variants have both conservative and non-conservative amino acid substitutions. In other embodiments, each amino acid residue may be substituted with alanine.
  • Natural amino acids may be divided into classes based on common side chain properties: nonpolar (Gly, Ala, Val, Leu, Ile, Met); polar neutral (Cys, Ser, Thr, Pro, Asn, Gln); acidic (Asp, Glu); basic (His, Lys, Arg); and aromatic (Tip, Tyr, Phe).
  • nonpolar Gly, Ala, Val, Leu, Ile, Met
  • polar neutral Cys, Ser, Thr, Pro, Asn, Gln
  • acidic Asp, Glu
  • basic His, Lys, Arg
  • aromatic Teip, Tyr, Phe
  • amino acid substitutions are conservative.
  • Conservative amino acid substitutions may involve the substitution of an amino acid of one class for that of the same class.
  • Conservative amino acid substitutions may also encompass non-natural amino acid residues, including peptidomimetics and other atypical forms of amino acid moieties, and may be incorporated through chemical peptide synthesis,
  • Amino acid substitutions may be made with consideration to the hydropathic index of amino acids.
  • the importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte et al., 1982 , J. Mol. Biol. 157:105-31). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics.
  • the hydropathic indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine ( ⁇ 0.4); threonine ( ⁇ 0.7); serine ( ⁇ 0.8); tryptophan ( ⁇ 0.9); tyrosine ( ⁇ 1.3); proline ( ⁇ 1.6); histidine ( ⁇ 3.2); glutamate ( ⁇ 3.5); glutamine ( ⁇ 3.5); aspartate ( ⁇ 3.5); asparagine ( ⁇ 3.5); lysine ( ⁇ 3.9); and arginine ( ⁇ 4.5).
  • amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity.
  • substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine ( ⁇ 0.4); proline ( ⁇ 0.5 ⁇ 1); alanine ( ⁇ 0.5); histidine ( ⁇ 0.5); cysteine ( ⁇ 1.0); methionine ( ⁇ 1.3); valine ( ⁇ 1.5); leucine ( ⁇ 1.8); isoleucine ( ⁇ 1.8); tyrosine ( ⁇ 2.3); phenylalanine ( ⁇ 2.5); and tryptophan ( ⁇ 3.4).
  • the substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • therapeutic peptides include variants having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid deletions relative to the therapeutic peptides disclosed herein, including in Table 1.
  • the deleted amino acid(s) may be at the N- or C-terminus of the peptide, at both termini, at an internal location or locations within the peptide, or both internally and at one or both termini.
  • the deletions may be of contiguous amino acids or of amino acids at different locations within the primary amino acid sequence of the parent peptide.
  • therapeutic peptides include variants having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid additions relative to the therapeutic peptides disclosed herein, including in Table 1.
  • the added amino acid(s) may be at the N- or C-terminus of the peptide, at both termini, at an internal location or locations within the peptide, or both internally and at one or both termini.
  • the amino acids may be added contiguously, or the amino acids may be added at different locations within the primary amino acid sequence of the parent peptide.
  • Addition variants also include fusion peptides. Fusions can be made either at the N-terminus or at the C-terminus of the therapeutic peptides disclosed herein, including in Table 1. In certain embodiments, the fusion peptides have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid additions relative to the therapeutic peptides disclosed herein, including in Table 1. Fusions may be attached directly to the therapeutic peptide with no connector molecule or may be through a connector molecule. As used in this context, a connector molecule may be an atom or a collection of atoms optionally used to link a therapeutic peptide to another peptide. Alternatively, the connector may be an amino acid sequence designed for cleavage by a protease to allow for the separation of the fused peptides.
  • the therapeutic peptides of the invention may be fused to peptides designed to improve certain qualities of the therapeutic peptide, such as therapeutic activity, circulation time, or reduced aggregation.
  • Therapeutic peptides may be fused to an immunologically active domain, e.g. an antibody epitope, to facilitate purification of the peptide, or to increase the in vivo half life of the peptide.
  • therapeutic peptides may be fused to known functional domains, cellular localization sequences, or peptide permeant motifs known to improve membrane transfer properties.
  • therapeutic peptides also include variants incorporating one or more non-natural amino acids, amino acid analogs, and peptidomimetics.
  • the present invention encompasses compounds structurally similar to the therapeutic peptides defined and/or disclosed herein, which are formulated to mimic the key portions of the therapeutic peptides of the present invention. Such compounds may be used in the same manner as the therapeutic peptides of the invention. Certain mimetics that mimic elements of protein secondary and tertiary structure have been previously described. Johnson et al., Biotechnology and Pharmacy, Pezzuto et al. (Eds.), Chapman and Hall, NY, 1993.
  • peptide mimetics The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions. A peptide mimetic is thus designed to permit molecular interactions similar to the parent peptide. Mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains. Methods for generating specific structures have been disclosed in the art. For example, U.S. Pat. Nos.
  • related peptides comprise or consist of a peptide sequence that is at least 70% identical to any of the therapeutic peptides disclosed herein, including in Table 1.
  • related peptides are at least 75% identical, at least 80% identical, at least 85% identical, 90% identical, at least 91% identical, at least 92% identical, 93% identical, at least 94% identical, at least 95% identical, 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any of the therapeutic peptides disclosed herein, including in Table 1.
  • Sequence identity (also known as % homology) of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to those described in Computational Molecular Biology (A. M. Lesk, ed., Oxford University Press 1988); Biocomputing: Informatics and Genome Projects (D. W. Smith, ed., Academic Press 1993); Computer Analysis of Sequence Data (Part 1, A. M. Griffin and H. G. Griffin, eds., Humana Press 1994); G. von Heinle, Sequence Analysis in Molecular Biology (Academic Press 1987); Sequence Analysis Primer (M. Gribskov and J. Devereux, eds., M. Stockton Press 1991); and Carillo et al., 1988 , SIAM J. Applied Math., 48:1073.
  • Preferred methods to determine sequence identity and/or similarity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are described in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al., 1984 , Nucleic Acids Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., 1990 , J. Mol. Biol. 215:403-10).
  • GCG program package including GAP (Devereux et al., 1984 , Nucleic Acids Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., 1990 , J. Mol. Biol. 215:403-10).
  • the BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (Altschul et al., BLAST Manual (NCB NLM NIH, Bethesda, Md.); Altschul et al., 1990, supra).
  • NCBI National Center for Biotechnology Information
  • the well-known Smith Waterman algorithm may also be used to determine identity.
  • GAP Genetics Computer Group, University of Wisconsin, Madison, Wis.
  • two polypeptides for which the percent sequence identity is to be determined are aligned for optimal matching of their respective amino acids (the “matched span,” as determined by the algorithm).
  • a gap opening penalty (which is calculated as 3 ⁇ the average diagonal; the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix)
  • a gap extension penalty which is usually 0.1 ⁇ the gap opening penalty
  • a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm.
  • Related peptides also include derivatives of the therapeutic peptides defined and/or disclosed herein, wherein the variant retains some of or all of at least one therapeutic activity of the parent peptide.
  • the derivative may also exhibit an increase in at least one therapeutic activity of the parent peptide.
  • Chemical alterations of therapeutic peptide derivatives include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, biotinylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginy
  • Therapeutic peptide derivatives also include molecules formed by the deletion of one or more chemical groups from the parent peptide. Methods for preparing chemically modified derivatives of the therapeutic peptides defined and/or disclosed herein are known to one of skill in the art.
  • the therapeutic peptides may be modified with one or more methyl or other lower alkyl groups at one or more positions of the therapeutic peptide sequence.
  • groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, etc.
  • arginine, lysine, and histidine residues of the therapeutic peptides are modified with methyl or other lower alkyl groups.
  • the therapeutic peptides may be modified with one or more glycoside moieties relative to the parent peptide.
  • any glycoside can be used, in certain preferred embodiments the therapeutic peptide is modified by introduction of a monosaccharide, a disaccharide, or a trisaccharide or it may contain a glycosylation sequence found in natural peptides or proteins in any mammal.
  • the saccharide may be introduced at any position, and more than one glycoside may be introduced. Glycosylation may occur on a naturally occurring amino acid residue in the therapeutic peptide, or alternatively, an amino acid may be substituted with another for modification with the saccharide.
  • Glycosylated therapeutic peptides may be prepared using conventional Fmoc chemistry and solid phase peptide synthesis techniques, e.g., on resin, where the desired protected glycoamino acids are prepared prior to peptide synthesis and then introduced into the peptide chain at the desired position during peptide synthesis.
  • the therapeutic peptide polymer conjugates may be conjugated in vitro. The glycosylation may occur before deprotection. Preparation of aminoacid glycosides is described in U.S. Pat. No. 5,767,254, WO 2005/097158, and Doores, K., et al., Chem. Commun., 1401-1403, 2006, which are incorporated herein by reference in their entireties.
  • alpha and beta selective glycosylations of serine and threonine residues are carried out using the Koenigs-Knorr reaction and Lemieux's in situ anomerization methodology with Schiff base intermediates. Deprotection of the Schiff base glycoside is then carried out using mildly acidic conditions or hydrogenolysis.
  • a composition comprising a glycosylated therapeutic peptide conjugate made by stepwise solid phase peptide synthesis involving contacting a growing peptide chain with protected amino acids in a stepwise manner, wherein at least one of the protected amino acids is glycosylated, followed by water-soluble polymer conjugation, may have a purity of at least 95%, such as at least 97%, or at least 98%, of a single species of the glycosylated and conjugated therapeutic peptide.
  • Monosaccharides that may by used for introduction at one or more amino acid residues of the therapeutic peptides defined and/or disclosed herein include glucose (dextrose), fructose, galactose, and ribose. Additional monosaccharides suitable for use include glyceraldehydes, dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose, xylose, ribulose, xylulose, allose, altrose, mannose, N-Acetylneuraminic acid, fucose, N-Acetylgalactosamine, and N-Acetylglucosamine, as well as others.
  • Glycosides such as mono-, di-, and trisaccharides for use in modifying a therapeutic peptide, may be naturally occurring or may be synthetic.
  • Disaccharides that may by used for introduction at one or more amino acid residues of the therapeutic peptides defined and/or disclosed herein include sucrose, lactose, maltose, trehalose, melibiose, and cellobiose, among others.
  • Trisaccharides include acarbose, raffinose, and melezitose.
  • the therapeutic peptides defined and/or disclosed herein may be chemically coupled to biotin.
  • the biotin/therapeutic peptide molecules can then to bind to avidin.
  • modifications may be made to the therapeutic peptides defined and/or disclosed herein that do not alter, or only partially abrogate, the properties and activities of these therapeutic peptides. In some instances, modifications may be made that result in an increase in therapeutic activity.
  • modifications to the therapeutic peptides disclosed herein including in Table 1, that retain at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and any range derivable therein, such as, for example, at least 70% to at least 80%, and more preferably at least 81% to at least 90%; or even more preferably, between at least 91% and at least 99% of the therapeutic activity relative to the unmodified therapeutic peptide.
  • modification to the therapeutic peptides disclosed herein including in Table 1, that have greater than 100%, greater than 110%, greater than 125%, greater than 150%, greater than 200%, or greater than 300%, or greater than 10-fold or greater than 100-fold, and any range derivable therein, of the therapeutic activity relative to the unmodified therapeutic peptide.
  • the level of therapeutic activity of a given therapeutic peptide, or a modified therapeutic peptide may be determined by any suitable in vivo or in vitro assay.
  • therapeutic activity may be assayed in cell culture, or by clinical evaluation, EC 50 assays, IC 50 assays, or dose response curves.
  • In vitro or cell culture assays are commonly available and known to one of skill in the art for many therapeutic peptides as disclosed herein, including in Table 1. It will be understood by one of skill in the art that the percent activity of a modified therapeutic peptide relative to its unmodified parent can be readily ascertained through a comparison of the activity of each as determined through the assays disclosed herein or as known to one of skill in the art.
  • One of skill in the art will be able to determine appropriate modifications to the therapeutic peptides defined and/or disclosed herein, including those disclosed herein, including in Table 1.
  • suitable areas of the therapeutic peptides that may be changed without abrogating their therapeutic activities, one of skill in the art may target areas not believed to be essential for activity.
  • one of skill in the art may compare those amino acid sequences to identify residues that are conserved among similar peptides. It will be understood that changes in areas of a therapeutic peptide that are not conserved relative to similar peptides would be less likely to adversely affect the thereapeutic activity.
  • one of skill in the art can review structure-function studies identifying residues in similar peptides that are important for activity or structure. In view of such a comparison, one can predict the importance of an amino acid residue in a therapeutic peptide that corresponds to an amino acid residue that is important for activity or structure in similar peptides. One of skill in the art may opt for amino acid substitutions within the same class of amino acids for such predicted important amino acid residues of the therapeutic peptides.
  • one of skill in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar peptides. In view of such information, one of skill in the art may predict the alignment of amino acid residues of a therapeutic peptide with respect to its three dimensional structure. One of skill in the art may choose not to make significant changes to amino acid residues predicted to be on the surface of the peptide, since such residues may be involved in important interactions with other molecules. Moreover, one of skill in the art may generate variants containing a single amino acid substitution at each amino acid residue for test purposes. The variants could be screened using therapeutic activity assays known to those with skill in the art. Such variants could be used to gather information about suitable modifications.
  • Additional methods of predicting secondary structure include “threading” (Jones, 1997 , Curr. Opin. Struct. Biol. 7:377-87; Sippl et al., 1996 , Structure 4:15-19), “profile analysis” (Bowie et al., 1991 , Science, 253:164-70; Gribskov et al., 1990 , Methods Enzymol. 183:146-59; Gribskov et al., 1987 , Proc. Nat. Acad. Sci. U.S.A. 84:4355-58), and “evolutionary linkage” (See Holm et al., supra, and Brenner et al., supra).
  • a conjugate of the invention comprises a water-soluble polymer covalently attached (either directly or through a spacer moiety or linker) to a therapeutic peptide.
  • a water-soluble polymer covalently attached (either directly or through a spacer moiety or linker) to a therapeutic peptide.
  • there will be about one to five water-soluble polymers covalently attached to a therapeutic peptide wherein for each water-soluble polymer, the water-soluble polymer can be attached either directly to the therapeutic peptide or through a spacer moiety).
  • a therapeutic peptide conjugate of the invention typically has about 1, 2, 3, or 4 water-soluble polymers individually attached to a therapeutic peptide. That is to say, in certain embodiments, a conjugate of the invention will possess about 4 water-soluble polymers individually attached to a therapeutic peptide, or about 3 water-soluble polymers individually attached to a therapeutic peptide, or about 2 water-soluble polymers individually attached to a therapeutic peptide, or about 1 water-soluble polymer attached to a therapeutic peptide.
  • the structure of each of the water-soluble polymers attached to the therapeutic peptide may be the same or different.
  • One therapeutic peptide conjugate in accordance with the invention is one having a water-soluble polymer releasably attached to the therapeutic peptide, particularly at the N-terminus of the therapeutic peptide.
  • Another therapeutic peptide conjugate in accordance with the invention is one having a water-soluble polymer stably attached to the therapeutic peptide, particularly at the N-terminus of the therapeutic peptide.
  • Another therapeutic peptide conjugate is one having a water-soluble polymer releasably attached to the therapeutic peptide, particularly at the C-terminus of the therapeutic peptide.
  • Another therapeutic peptide conjugate in accordance with the invention is one having a water-soluble polymer stably attached to the therapeutic peptide, particularly at the C-terminus of the therapeutic peptide.
  • therapeutic peptide conjugates in accordance with the invention are those having a water-soluble polymer releasably or stably attached to an amino acid within the therapeutic peptide. Additional water-soluble polymers may be releasably or stably attached to other sites on the therapeutic peptide, e.g., such as one or more additional sites.
  • a therapeutic peptide conjugate having a water-soluble polymer releasably attached to the N-terminus may additionally possess a water-soluble polymer stably attached to a lysine residue.
  • one or more amino acids may be inserted, at the N- or C-terminus, or within the peptide to releasably or stably attach a water soluble polymer.
  • a mono-therapeutic peptide polymer conjugate i.e., a therapeutic peptide having one water-soluble polymer covalently attached thereto.
  • the water-soluble polymer is one that is attached to the therapeutic peptide at its N-terminus.
  • a therapeutic peptide polymer conjugate of the invention is absent a metal ion, i.e., the therapeutic peptide is not chelated to a metal ion.
  • the therapeutic peptide may optionally possess one or more N-methyl substituents.
  • the therapeutic peptide may be glycosylated, e.g., having a mono- or disaccharide, or naturally-occurring amino acid glycosylation covalently attached to one or more sites thereof.
  • the compounds of the present invention may be made by various methods and techniques known and available to those skilled in the art.
  • a conjugate of the invention comprises a therapeutic peptide attached, stably or releasably, to a water-soluble polymer.
  • the water-soluble polymer is typically hydrophilic, nonpeptidic, and biocompatible.
  • a substance is considered biocompatible if the beneficial effects associated with use of the substance alone or with another substance (e.g., an active agent such a therapeutic peptide) in connection with living tissues (e.g., administration to a patient) outweighs any deleterious effects as evaluated by a clinician, e.g., a physician.
  • a substance is considered nonimmunogenic if the intended use of the substance in vivo does not produce an undesired immune response (e.g., the formation of antibodies) or, if an immune response is produced, that such a response is not deemed clinically significant or important as evaluated by a clinician.
  • the water-soluble polymer is hydrophilic, biocompatible and nonimmunogenic.
  • water-soluble polymer is typically characterized as having from 2 to about 300 termini, preferably from 2 to 100 termini, and more preferably from about 2 to 50 termini.
  • poly(alkylene glycols) such as polyethylene glycol (PEG), poly(propylene glycol) (“PPG”), copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly( ⁇ -hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), and combinations of any of the foregoing, including copolymers and terpolymers thereof.
  • PEG polyethylene glycol
  • PPG poly(propylene glycol)
  • the water-soluble polymer is not limited to a particular structure and may possess a linear architecture (e.g., alkoxy PEG or bifunctional PEG), or a non-linear architecture, such as branched, forked, multi-armed (e.g., PEGs attached to a polyol core), or dendritic (i.e. having a densely branched structure with numerous end groups).
  • the polymer subunits can be organized in any number of different patterns and can be selected, e.g., from homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, and block tripolymer.
  • a PEG used to prepare a therapeutic peptide polymer conjugate of the invention is “activated” or reactive. That is to say, the activated PEG (and other activated water-soluble polymers collectively referred to herein as “polymeric reagents”) used to form a therapeutic peptide conjugate comprises an activated functional group suitable for coupling to a desired site or sites on the therapeutic peptide.
  • a polymeric reagent for use in preparing a therapeutic peptide conjugate includes a functional group for reaction with the therapeutic peptide.
  • polymeric reagents and methods for conjugating such polymers to an active moiety are known in the art, and are, e.g., described in Harris, J. M. and Zalipsky, S., eds, Poly ( ethylene glycol ), Chemistry and Biological Applications , ACS, Washington, 1997; Veronese, F., and J. M Harris, eds., Peptide and Protein PEGylation , Advanced Drug Delivery Reviews, 54(4); 453-609 (2002); Zalipsky, S., et al., “ Use of Functionalized Poly ( Ethylene Glycols ) for Modification of Polypeptides ” in Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications , J. M. Harris, ed., Plenus Press, New York (1992); Zalipsky (1995) Advanced Drug Reviews 16:157-182, and in Roberts, et al., Adv. Drug Delivery Reviews, 54, 459-476 (2002).
  • PEG reagents suitable for use in forming a conjugate of the invention and methods of conjugation are described in the Pasut. G., et al., Expert Opin. Ther. Patents ( 2004), 14(5).
  • PEG reagents suitable for use in the present invention also include those available from NOF Corporation, as described generally on the NOF website (http://nofamerica.net/store/). Products listed therein and their chemical structures are expressly incorporated herein by reference.
  • Additional PEGs for use in forming a therapeutic peptide conjugate of the invention include those available from Polypure (Norway) and from QuantaBioDesign LTD (Ohio), where the contents of their online catalogs (2006) with respect to available PEG reagents are expressly incorporated herein by reference.
  • water soluble polymer reagents useful for preparing peptide conjugates of the invention can be prepared synthetically. Descriptions of the water soluble polymer reagent synthesis can be found in, for example, U.S. Pat. Nos. 5,252,714, 5,650,234, 5,739,208, 5,932,462, 5,629,384, 5,672,662, 5,990,237, 6,448,369, 6,362,254, 6,495,659, 6,413,507, 6,376,604, 6,348,558, 6,602,498, and 7,026,440.
  • the weight-average molecular weight of the water-soluble polymer in the conjugate is from about 100 Daltons to about 150,000 Daltons. Exemplary ranges include weight-average molecular weights in the range of from about 250 Daltons to about 80,000 Daltons, from 500 Daltons to about 80,000 Daltons, from about 500 Daltons to about 65,000 Daltons, from about 500 Daltons to about 40,000 Daltons, from about 750 Daltons to about 40,000 Daltons, from about 1000 Daltons to about 30,000 Daltons. In a preferred embodiment, the weight average molecular weight of the water-soluble polymer in the conjugate ranges from about 1000 Daltons to about 10,000 Daltons.
  • the range is from about 1000 Daltons to about 5000 Daltons, from about 5000 Daltons to about 10,000 Daltons, from about 2500 Daltons to about 7500 Daltons, from about 1000 Daltons to about 3000 Daltons, from about 3000 Daltons to about 7000 Daltons, or from about 7000 Daltons to about 10,000 Daltons.
  • the weight average molecular weight of the water-soluble polymer in the conjugate ranges from about 20,000 Daltons to about 40,000 Daltons.
  • the range is from about 20,000 Daltons to about 30,000 Daltons, from about 30,000 Daltons to about 40,000 Daltons, from about 25,000 Daltons to about 35,000 Daltons, from about 20,000 Daltons to about 26,000 Daltons, from about 26,000 Daltons to about 34,000 Daltons, or from about 34,000 Daltons to about 40,000 Daltons.
  • a molecular weight in one or more of these ranges is typical.
  • a therapeutic peptide conjugate in accordance with the invention when intended for subcutaneous or intravenous administration, will comprise a PEG or other suitable water-soluble polymer having a weight average molecular weight of about 20,000 Daltons or greater, while a therapeutic peptide conjugate intended for pulmonary administration will generally, although not necessarily, comprise a PEG polymer having a weight average molecular weight of about 20,000 Daltons or less.
  • Exemplary weight-average molecular weights for the water-soluble polymer include about 100 Daltons, about 200 Daltons, about 300 Daltons, about 400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1,000 Daltons, about 1,500 Daltons, about 2,000 Daltons, about 2,200 Daltons, about 2,500 Daltons, about 3,000 Daltons, about 4,000 Daltons, about 4,400 Daltons, about 4,500 Daltons, about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 55,000 Daltons,
  • Branched versions of the water-soluble polymer e.g., a branched 40,000 Dalton water-soluble polymer comprised of two 20,000 Dalton polymers or the like
  • the conjugate is one that does not have one or more attached PEG moieties having a weight-average molecular weight of less than about 6,000 Daltons.
  • the PEG will typically comprise a number of (OCH 2 CH 2 ) monomers.
  • the number of repeat units is typically identified by the subscript “n” in, for example, “(OCH 2 CH 2 ) n .”
  • the value of (n) typically falls within one or more of the following ranges: from 2 to about 3400, from about 100 to about 2300, from about 100 to about 2270, from about 136 to about 2050, from about 225 to about 1930, from about 450 to about 1930, from about 1200 to about 1930, from about 568 to about 2727, from about 660 to about 2730, from about 795 to about 2730, from about 795 to about 2730, from about 909 to about 2730, and from about 1,200 to about 1,900.
  • n examples include from about 10 to about 700, and from about 10 to about 1800.
  • n the number of repeating units
  • the conjugate comprises a therapeutic peptide covalently attached to a water-soluble polymer having a molecular weight greater than about 2,000 Daltons.
  • a polymer for use in the invention may be end-capped, that is, a polymer having at least one terminus capped with a relatively inert group, such as a lower alkoxy group (i.e., a C 1-6 alkoxy group) or a hydroxyl group.
  • a relatively inert group such as a lower alkoxy group (i.e., a C 1-6 alkoxy group) or a hydroxyl group.
  • mPEG methoxy-PEG
  • mPEG methoxy-PEG
  • the -PEG- symbol used in the foregoing generally represents the following structural unit: —CH 2 CH 2 O—(CH 2 CH 2 O) n —CH 2 CH 2 —, where (n) generally ranges from about zero to about 4,000.
  • Multi-armed or branched PEG molecules such as those described in U.S. Pat. No. 5,932,462, are also suitable for use in the present invention.
  • the PEG may be described generally according to the structure:
  • poly a and poly b are PEG backbones (either the same or different), such as methoxy poly(ethylene glycol); R′′ is a non-reactive moiety, such as H, methyl or a PEG backbone; and P and Q are non-reactive linkages.
  • the branched PEG molecule is one that includes a lysine residue, such as the following reactive PEG suitable for use in forming a therapeutic peptide conjugate.
  • the branched PEG below is shown with a reactive succinimidyl group, this represents only one of a myriad of reactive functional groups suitable for reacting with a therapeutic peptide.
  • the polymeric reagent (as well as the corresponding conjugate prepared from the polymeric reagent) may lack a lysine residue in which the polymeric portions are connected to amine groups of the lysine via a “—OCH 2 CONHCH 2 CO—” group.
  • the polymeric reagent (as well as the corresponding conjugate prepared from the polymeric reagent) may lack a branched water-soluble polymer that includes a lysine residue (wherein the lysine residue is used to effect branching).
  • Additional branched-PEGs for use in forming a therapeutic peptide conjugate of the present invention include those described in co-owned U.S. Patent Application Publication No. 2005/0009988.
  • Representative branched polymers described therein include those having the following generalized structure:
  • POLY 1 is a water-soluble polymer
  • POLY 2 is a water-soluble polymer
  • (a) is 0, 1, 2 or 3
  • (b) is 0, 1, 2 or 3
  • (e) is 0, 1, 2 or 3
  • (f′) is 0, 1, 2 or 3
  • (g′) is 0, 1, 2 or 3
  • (h) is 0, 1, 2 or 3
  • (j) is 0 to 20
  • each R 1 is independently H or an organic radical selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl
  • X 1 when present, is a spacer moiety
  • X 2 when present, is a spacer moiety
  • X 5 when present, is a spacer moiety
  • X 6 when present, is a spacer moiety
  • X 7 when present, is a spacer moiety
  • X 8 when present, is a spacer moiety
  • a preferred branched polymer falling into the above classification suitable for use in the present invention is:
  • Branched polymers suitable for preparing a conjugate of the invention also include those represented more generally by the formula R(POLY) y , where R is a central or core molecule from which extends 2 or more POLY arms such as PEG.
  • the variable y represents the number of POLY arms, where each of the polymer arms can independently be end-capped or alternatively, possess a reactive functional group at its terminus.
  • a more explicit structure in accordance with this embodiment of the invention possesses the structure, R(POLY-Z) y , where each Z is independently an end-capping group or a reactive group, e.g., suitable for reaction with a therapeutic peptide.
  • the resulting linkage can be hydrolytically stable, or alternatively, may be degradable, i.e., hydrolyzable.
  • at least one polymer arm possesses a terminal functional group suitable for reaction with, e.g., a therapeutic peptide.
  • Branched PEGs such as those represented generally by the formula, R(PEG) y above possess 2 polymer arms to about 300 polymer arms (i.e., n ranges from 2 to about 300).
  • such branched PEGs typically possess from 2 to about 25 polymer arms, such as from 2 to about 20 polymer arms, from 2 to about 15 polymer arms, or from 3 to about 15 polymer arms.
  • Multi-armed polymers include those having 3, 4, 5, 6, 7 or 8 arms.
  • Core molecules in branched PEGs as described above include polyols, which are then further functionalized.
  • Such polyols include aliphatic polyols having from 1 to 10 carbon atoms and from 1 to 10 hydroxyl groups, including ethylene glycol, alkane diols, alkyl glycols, alkylidene alkyl diols, alkyl cycloalkane diols, 1,5-decalindiol, 4,8-bis(hydroxymethyl)tricyclodecane, cycloalkylidene diols, dihydroxyalkanes, trihydroxyalkanes, and the like.
  • Cycloaliphatic polyols may also be employed, including straight chained or closed-ring sugars and sugar alcohols, such as mannitol, sorbitol, inositol, xylitol, quebrachitol, threitol, arabitol, erythritol, adonitol, ducitol, facose, ribose, arabinose, xylose, lyxose, rhamnose, galactose, glucose, fructose, sorbose, mannose, pyranose, altrose, talose, tagitose, pyranosides, sucrose, lactose, maltose, and the like.
  • sugar alcohols such as mannitol, sorbitol, inositol, xylitol, quebrachitol, threitol, arabitol, erythritol,
  • Additional aliphatic polyols include derivatives of glyceraldehyde, glucose, ribose, mannose, galactose, and related stereoisomers.
  • Other core polyols that may be used include crown ether, cyclodextrins, dextrins and other carbohydrates such as starches and amylose.
  • Typical polyols include glycerol, pentaerythritol, sorbitol, and trimethylolpropane.
  • linkage is degradable, designated herein as L D , that is to say, contains at least one bond or moiety that hydrolyzes under physiological conditions, e.g., an ester, hydrolyzable carbamate, carbonate, or other such group.
  • L D degradable, designated herein as L D , that is to say, contains at least one bond or moiety that hydrolyzes under physiological conditions, e.g., an ester, hydrolyzable carbamate, carbonate, or other such group.
  • the linkage is hydrolytically stable.
  • Multi-armed activated polymers for use in the method of the invention include those corresponding to the following structure, where E represents a reactive group suitable for reaction with a reactive group on the therapeutic peptide.
  • E represents a reactive group suitable for reaction with a reactive group on the therapeutic peptide.
  • E is an —OH (for reaction with a therapeutic peptide carboxy group or equivalent), a carboxylic acid or equivalent (such as an active ester), a carbonic acid (for reaction with therapeutic peptide —OH groups), or an amino group.
  • PEG is —(CH 2 CH 2 O) n CH 2 CH 2 —, and m is selected from 3, 4, 5, 6, 7, and 8.
  • typical linkages are ester, carboxyl and hydrolyzable carbamate, such that the polymer-portion of the conjugate is hydrolyzed in vivo to release the therapeutic peptide from the intact polymer conjugate.
  • the linker L is designated as L D .
  • the polymer may possess an overall forked structure as described in U.S. Pat. No. 6,362,254. This type of polymer segment is useful for reaction with two therapeutic peptide moieties, where the two therapeutic peptide moieties are positioned a precise or predetermined distance apart.
  • one or more degradable linkages may additionally be contained in the polymer segment, POLY, to allow generation in vivo of a conjugate having a smaller PEG chain than in the initially administered conjugate.
  • Appropriate physiologically cleavable (i.e., releasable) linkages include but are not limited to ester, carbonate ester, carbamate, sulfate, phosphate, acyloxyalkyl ether, acetal, and ketal. Such linkages when contained in a given polymer segment will often be stable upon storage and upon initial administration.
  • the PEG polymer used to prepare a therapeutic peptide polymer conjugate may comprise a pendant PEG molecule having reactive groups, such as carboxyl or amino, covalently attached along the length of the PEG rather than at the end of the PEG chain(s).
  • the pendant reactive groups can be attached to the PEG directly or through a spacer moiety, such as an alkylene group.
  • a therapeutic peptide polymer conjugate according to one aspect of the invention is one comprising a therapeutic peptide releasably attached, preferably at its N-terminus, to a water-soluble polymer.
  • Hydrolytically degradable linkages useful not only as a degradable linkage within a polymer backbone, but also, in the case of certain embodiments of the invention, for covalently attaching a water-soluble polymer to a therapeutic peptide, include: carbonate; imine resulting, for example, from reaction of an amine and an aldehyde (see, e.g., Ouchi et al.
  • phosphate ester formed, for example, by reacting an alcohol with a phosphate group
  • hydrazone e.g., formed by reaction of a hydrazide and an aldehyde
  • acetal e.g., formed by reaction of an aldehyde and an alcohol
  • orthoester formed, for example, by reaction between a formate and an alcohol
  • esters and certain urethane (carbamate) linkages.
  • Additional PEG reagents for use in the invention include hydrolyzable and/or releasable PEGs and linkers such as those described in U.S. Patent Application Publication No. 2006-0293499.
  • the therapeutic peptide and the polymer are each covalently attached to different positions of the aromatic scaffold, e.g., Fmoc or FMS, structure, and are releasable under physiological conditions.
  • the aromatic scaffold e.g., Fmoc or FMS
  • one such polymeric reagent comprises the following structure:
  • POLY 1 is a first water-soluble polymer
  • POLY 2 is a second water-soluble polymer
  • X 1 is a first spacer moiety
  • X 2 is a second spacer moiety
  • the polymeric reagent can include one, two, three, four or more electron altering groups attached to the aromatic-containing moiety.
  • Preferred aromatic-containing moieties are bicyclic and tricyclic aromatic hydrocarbons.
  • Fused bicyclic and tricyclic aromatics include pentalene, indene, naphthalene, azulene, heptalene, biphenylene, as-indacene, s-indacene, acenaphthylene, fluorene, phenalene, phenanthrene, anthracene, and fluoranthene.
  • a preferred polymer reagent possesses the following structure,
  • mPEG corresponds to CH 3 O—(CH 2 CH 2 O) n CH 2 CH 2 —
  • X 1 and X 2 are each independently a spacer moiety having an atom length of from about 1 to about 18 atoms, n ranges from 10 to 1800, p is an integer ranging from 1 to 8, R 1 is H or lower alkyl, R 2 is H or lower alkyl, and Ar is an aromatic hydrodrocarbon, preferably a bicyclic or tricyclic aromatic hydrocarbon.
  • FG is as defined above.
  • FG corresponds to an activated carbonate ester suitable for reaction with an amino group on therapeutic peptide.
  • Preferred spacer moieties, X 1 and X 2 include —NH—C(O)—CH 2 —O—, —NH—C(O)—(CH 2 ) q —O—, —NH—C(O)—(CH 2 ) q —C(O)—NH—, —NH—C(O)—(CH 2 ) q —, and —C(O)—NH—, where q is selected from 2, 3, 4, and 5.
  • the nitrogen in the preceding spacers is proximal to the PEG rather than to the aromatic moiety.
  • Another such branched (2-armed) polymeric reagent comprised of two electron altering groups comprises the following structure:
  • R e1 is a first electron altering group
  • R e2 is a second electron altering group.
  • An electron altering group is a group that is either electron donating (and therefore referred to as an “electron donating group”), or electron withdrawing (and therefore referred to as an “electron withdrawing group”).
  • an electron donating group is a group having the ability to position electrons away from itself and closer to or within the aromatic-containing moiety.
  • an electron withdrawing group is a group having the ability to position electrons toward itself and away from the aromatic-containing moiety.
  • Hydrogen is used as the standard for comparison in the determination of whether a given group positions electrons away or toward itself.
  • Preferred electron altering groups include, but are not limited to, —CF 3 , —CH 2 CF 3 , —CH 2 C 6 F 5 , —CN, —NO 2 , —S(O)R, —S(O)Aryl, —S(O 2 )R, —S(O 2 )Aryl, —S(O 2 )OR, —S(O 2 )OAryl, —S(O 2 )NHR, —S(O 2 )NHAryl, —C(O)R, —C(O)Aryl, —C(O)OR, —C(O)NHR, and the like, wherein R is H or an organic radical.
  • An additional branched polymeric reagent suitable for use in the present invention comprises the following structure:
  • POLY 1 is a first water-soluble polymer
  • POLY 2 is a second water-soluble polymer
  • X 1 is a first spacer moiety
  • X 2 is a second spacer moiety
  • Ar 1 is a first aromatic moiety
  • Ar 2 is a second aromatic moiety
  • H ⁇ is an ionizable hydrogen atom
  • R 1 is H or an organic radical
  • R 2 is H or an organic radical
  • (FG) is a functional group capable of reacting with an amino group of therapeutic peptide to form a releasable linkage, such as carbamate linkage.
  • Another exemplary polymeric reagent comprises the following structure:
  • each of POLY 1 , POLY 2 , X 1 , X 2 , Ar 1 , Ar 2 , H ⁇ , R 1 , R 2 , and (FG) is as previously defined, and R e1 is a first electron altering group. While stereochemistry is not specifically shown in any structure provided herein, the provided structures contemplate both enantiomers, as well as compositions comprising mixtures of each enantiomer in equal amounts (i.e., a racemic mixture) and unequal amounts.
  • each of POLY 1 , POLY 2 , X 1 , X 2 , Ar 1 , Ar 2 , H ⁇ , R 1 , R 2 , and (FG) is as previously defined, and R e1 is a first electron altering group; and R e2 is a second electron altering group.
  • a preferred polymeric reagent comprises the following structure:
  • each of POLY 1 , POLY 2 , X 1 , X 2 , R 1 , R 2 , H ⁇ and (FG) is as previously defined, and, as can be seen from the structure above, the aromatic moiety is a fluorene.
  • the POLY arms substituted on the fluorene can be in any position in each of their respective phenyl rings, i.e., POLY 1 -X 1 — can be positioned at any one of carbons 1, 2, 3, and 4, and POLY 2 -X 2 — can be in any one of positions 5, 6, 7, and 8.
  • Yet another preferred fluorene-based polymeric reagent comprises the following structure:
  • each of POLY 1 , POLY 2 , X 1 , X 2 , R 1 , R 2 , H ⁇ and (FG) is as previously defined, and R e1 is a first electron altering group; and R e2 is a second electron altering group as described above.
  • Yet another exemplary polymeric reagent for conjugating to a therapeutic peptide comprises the following fluorene-based structure:
  • each of POLY 1 , POLY 2 , X 1 , X 2 , R 1 , R 2 , H ⁇ and (FG) is as previously defined, and R e1 is a first electron altering group; and R e2 is a second electron altering group.
  • fluorene-based polymeric reagents for forming a releasable therapeutic peptide polymer conjugate in accordance with the invention include the following:
  • Still another exemplary polymeric reagent comprises the following structure:
  • each of POLY 1 , POLY 2 , X 1 , X 2 , R 1 , R 2 , H ⁇ and (FG) is as previously defined, and R e1 is a first electron altering group; and R e2 is a second electron altering group.
  • Branched reagents suitable for preparing a releasable therapeutic peptide conjugate include N- ⁇ di(mPEG(20,000)oxymethylcarbonylamino)fluoren-9-ylmethoxycarbonyloxy ⁇ succinimide, N-[2,7di(4mPEG(10,000)aminocarbonylbutyrylamino)fluoren-9 ylmethoxycarbonyloxy]-succinimide (“G2PEG2Fmoc 20k -NHS”), and PEG2-CAC-Fmoc 4k -BTC.
  • PEGs of any molecular weight as set forth herein may be employed in the above structures, and the particular activating groups described above are not meant to be limiting in any respect, and may be substituted by any other suitable activating group suitable for reaction with a reactive group present on the therapeutic peptide.
  • polymeric reagent generally refers to an entire molecule, which can comprise a water-soluble polymer segment, as well as additional spacers and functional groups.
  • the particular linkage between the therapeutic peptide and the water-soluble polymer depends on a number of factors. Such factors include, for example, the particular linkage chemistry employed, the particular spacer moieties utilized, if any, the particular therapeutic peptide, the available functional groups within the therapeutic peptide (either for attachment to a polymer or conversion to a suitable attachment site), and the possible presence of additional reactive functional groups or absence of functional groups within the therapeutic peptide due to modifications made to the peptide such as methylation and/or glycosylation, and the like.
  • the linkage between the therapeutic peptide and the water-soluble polymer is a releasable linkage. That is, the water-soluble polymer is cleaved (either through hydrolysis, an enzymatic processes, or otherwise), thereby resulting in an unconjugated therapeutic peptide.
  • the releasable linkage is a hydrolytically degradable linkage, where upon hydrolysis, the therapeutic peptide, or a slightly modified version thereof, is released.
  • the releasable linkage may result in the water-soluble polymer (and any spacer moiety) detaching from the therapeutic peptide in vivo (and in vitro) without leaving any fragment of the water-soluble polymer (and/or any spacer moiety or linker) attached to the therapeutic peptide.
  • exemplary releasable linkages include carbonate, carboxylate ester, phosphate ester, thiolester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, carbamates, and orthoesters. Such linkages can be readily formed by reaction of the therapeutic peptide and/or the polymeric reagent using coupling methods commonly employed in the art.
  • Hydrolyzable linkages are often readily formed by reaction of a suitably activated polymer with a non-modified functional group contained within the therapeutic peptide.
  • Preferred positions for covalent attachment of a water-soluble polymer induce the N-terminal, the C-terminal, as well as the internal lysines.
  • Preferred releasable linkages include carbamate and ester.
  • a preferred therapeutic peptide conjugate of the invention will possess the following generalized structure:
  • POLY is a water-soluble polymer such as any of the illustrative polymeric reagents provided in Tables 2-4 herein
  • X is a linker, and in some embodiments a hydrolyzable linkage (L D ), and k is an integer selected from 1, 2, and 3, and in some instances 4, 5, 6, 7, 8, 9 and 10.
  • L D refers to the hydrolyzable linkage per se (e.g., a carbamate or an ester linkage)
  • POLY is meant to include the polymer repeat units, e.g., CH 3 (OCH 2 CH 2 ) n ,—.
  • At least one of the water-soluble polymer molecules is covalently attached to the N-terminus of therapeutic peptide.
  • k equals 1 and X is —O—C(O)—NH—, where the —NH— is part of the therapeutic peptide residue and represents an amino group thereof.
  • the linkage between the therapeutic peptide and the water-soluble polymer (or the linker moiety that is attached to the polymer) may be a hydrolytically stable linkage, such as an amide, a urethane (also known as carbamate), amine, thioether (also known as sulfide), or urea (also known as carbamide).
  • a hydrolytically stable linkage such as an amide, a urethane (also known as carbamate), amine, thioether (also known as sulfide), or urea (also known as carbamide).
  • One such embodiment of the invention comprises a therapeutic peptide having a water-soluble polymer such as PEG covalently attached at the N-terminus of therapeutic peptide. In such instances, alkylation of the N-terminal residue permits retention of the charge on the N-terminal nitrogen.
  • a conjugate in one or more embodiments of the invention, comprises a therapeutic peptide covalently attached at an amino acid residue, either directly or through a linker comprised of one or more atoms, to a water-soluble polymer.
  • the conjugates may or may not possess a measurable degree of therapeutic peptide activity. That is to say, a conjugate in accordance with the invention will typically possess anywhere from about 0% to about 100% or more of the therapeutic activity of the unmodified parent therapeutic peptide.
  • compounds possessing little or no therapeutic activity contain a releasable linkage connecting the polymer to the therapeutic peptide, so that regardless of the lack of therapeutic activity in the conjugate, the active parent molecule (or a derivative thereof having therapeutic activity) is released by cleavage of the linkage (e.g., hydrolysis upon aqueous-induced cleavage of the linkage).
  • Such activity may be determined using a suitable in vivo or in vitro model, depending upon the known activity of the particular moiety having therapeutic peptide activity employed.
  • cleavage of a linkage is facilitated through the use of hydrolytically cleavable and/or enzymatically cleavable linkages such as urethane, amide, certain carbamate, carbonate or ester-containing linkages.
  • hydrolytically cleavable and/or enzymatically cleavable linkages such as urethane, amide, certain carbamate, carbonate or ester-containing linkages.
  • clearance of the conjugate via cleavage of individual water-soluble polymer(s) can be modulated by selecting the polymer molecular size and the type of functional group for providing the desired clearance properties.
  • a mixture of polymer conjugates is employed where the polymers possess structural or other differences effective to alter the release (e.g., hydrolysis rate) of the therapeutic peptide, such that one can achieve a desired sustained delivery profile.
  • One of ordinary skill in the art can determine the proper molecular size of the polymer as well as the cleavable functional group, depending upon several factors including the mode of administration. For example, one of ordinary skill in the art, using routine experimentation, can determine a proper molecular size and cleavable functional group by first preparing a variety of polymer-(therapeutic peptide) conjugates with different weight-average molecular weights, degradable functional groups, and chemical structures, and then obtaining the clearance profile for each conjugate by administering the conjugate to a patient and taking periodic blood and/or urine samples. Once a series of clearance profiles has been obtained for each tested conjugate, a conjugate or mixture of conjugates having the desired clearance profile(s) can be determined.
  • conjugates possessing a hydrolytically stable linkage that couples the therapeutic peptide to the water-soluble polymer will typically possess a measurable degree of therapeutic activity.
  • such conjugates are typically characterized as having a therapeutic activity satisfying one or more of the following percentages relative to that of the unconjugated therapeutic peptide: at least 2%, at least 5%, at least 10%, at least 15%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 100%, more than 105%, more than 10-fold, or more than 100-fold (when measured in a suitable model, such as those presented here and/or known in the art).
  • conjugates having a hydrolytically stable linkage e.g., an amide linkage
  • a therapeutic peptide provides a point of attachment between the therapeutic peptide and the water-soluble polymer.
  • a therapeutic peptide may comprise one or more lysine residues, each lysine residue containing an s-amino group that may be available for conjugation, as well as one amino terminus.
  • the amino group extending from the therapeutic peptide designation “ ⁇ NH -PEP” represents the residue of the therapeutic peptide itself in which the ⁇ NH— is an amino group of the therapeutic peptide.
  • One preferred site of attachment for the polymeric reagents shown below is the N-terminus.
  • conjugates in Tables 2-4 herein illustrate a single water-soluble polymer covalently attached to a therapeutic peptide
  • the conjugate structures on the right are meant to also encompass conjugates having more than one of such water-soluble polymer molecules covalently attached to therapeutic peptide, e.g., 2, 3, or 4 water-soluble polymer molecules.
  • Conjugation of a polymeric reagent to an amine group of a therapeutic peptide can be accomplished by a variety of techniques.
  • a therapeutic peptide is conjugated to a polymeric reagent functionalized with an active ester such as a succinimidyl derivative (e.g., an N-hydroxysuccinimide ester).
  • an active ester such as a succinimidyl derivative (e.g., an N-hydroxysuccinimide ester).
  • the polymeric reagent bearing the reactive ester is reacted with the therapeutic peptide in aqueous media under appropriate pH conditions, e.g., from pHs ranging from about 3 to about 8, about 3 to about 7, or about 4 to about 6.5.
  • Most polymer active esters can couple to a target peptide such as therapeutic peptide at physiological pH, e.g., at 7.0.
  • activated PEGs can be attached to a peptide such as therapeutic peptide at pHs from about 7.0 to about 10.0 for covalent attachment to an internal lysine.
  • lower pHs are used, e.g., 4 to about 5.75, for preferential covalent attachment to the N-terminus.
  • different reaction conditions e.g., different pHs or different temperatures
  • PEG polymer
  • Coupling reactions can often be carried out at room temperature, although lower temperatures may be required for particularly labile therapeutic peptide moieties.
  • Reaction times are typically on the order of minutes, e.g., 30 minutes, to hours, e.g., from about 1 to about 36 hours), depending upon the pH and temperature of the reaction.
  • N-terminal PEGylation e.g., with a PEG reagent bearing an aldehyde group, is typically conducted under mild conditions, pHs from about 5-10, for about 6 to 36 hours.
  • Varying ratios of polymeric reagent to therapeutic peptide may be employed, e.g., from an equimolar ratio up to a 10-fold molar excess of polymer reagent. Typically, up to a 5-fold molar excess of polymer reagent will suffice.
  • the PEG reagent may be incorporated at a desired position of the therapeutic peptide during peptide synthesis. In this way, site-selective introduction of one or more PEGs can be achieved. See, e.g., International Patent Publication No. WO 95/00162, which describes the site selective synthesis of conjugated peptides.
  • Exemplary conjugates that can be prepared using, for example, polymeric reagents containing a reactive ester for coupling to an amino group of therapeutic peptide, comprise the following alpha-branched structure:
  • POLY is a water-soluble polymer, (a) is either zero or one; X 1 , when present, is a spacer moiety comprised of one or more atoms; R 1 is hydrogen an organic radical; and “ ⁇ NH -PEP” represents a residue of a therapeutic peptide, where the underlined amino group represents an amino group of the therapeutic peptide.
  • any of the water-soluble polymers provided herein can be defined as POLY, any of the spacer moieties provided herein can be defined as X 1 (when present), any of the organic radicals provided herein can be defined as R 1 (in instances where R 1 is not hydrogen), and any of the therapeutic peptides provided herein can be employed.
  • POLY is a poly(ethylene glycol) such as H 3 CO(CH 2 CH 2 O) n —, wherein (n) is an integer having a value of from 3 to 4000, more preferably from 10 to about 1800; (a) is one; X 1 is a C 1-6 alkylene, such as one selected from methylene (i.e., —CH 2 —), ethylene (i.e., —CH 2 —CH 2 —) and propylene (i.e., —CH 2 —CH 2 —CH 2 —); R 1 is H or lower alkyl such as methyl or ethyl; and PEP corresponds to any therapeutic peptide disclosed herein, including in Table 1.
  • X 1 is a C 1-6 alkylene, such as one selected from methylene (i.e., —CH 2 —), ethylene (i.e., —CH 2 —CH 2 —) and propylene (i.e., —CH 2 —CH 2 —
  • Typical of another approach for conjugating a therapeutic peptide to a polymeric reagent is reductive amination.
  • reductive amination is employed to conjugate a primary amine of a therapeutic peptide with a polymeric reagent functionalized with a ketone, aldehyde or a hydrated form thereof (e.g., ketone hydrate and aldehyde hydrate).
  • the primary amine from the therapeutic peptide e.g., the N-terminus
  • the Schiff base in turn, is then reductively converted to a stable conjugate through use of a reducing agent such as sodium borohydride or any other suitable reducing agent.
  • a reducing agent such as sodium borohydride or any other suitable reducing agent.
  • Selective reactions are possible, particularly with a polymer functionalized with a ketone or an alpha-methyl branched aldehyde and/or under specific reaction conditions (e.g., reduced pH).
  • Exemplary conjugates that can be prepared using, for example, polymeric reagents containing an aldehyde (or aldehyde hydrate) or ketone or (ketone hydrate) possess the following structure:
  • POLY is a water-soluble polymer; (d) is either zero or one; X 2 , when present, is a spacer moiety comprised of one or more atoms; (b) is an integer having a value of one through ten; (c) is an integer having a value of one through ten; R 2 , in each occurrence, is independently H or an organic radical; R 3 , in each occurrence, is independently H or an organic radical; and “ ⁇ NH -PEP” represents a residue of a therapeutic peptide, where the underlined amino group represents an amino group of the therapeutic peptide.
  • k ranges from 1 to 3
  • n ranges from 10 to about 1800.
  • any of the water-soluble polymers provided herein can be defined as POLY
  • any of the spacer moieties provided herein can be defined as X 2 (when present)
  • any of the organic radicals provided herein can be independently defined as R 2 and R 3 (in instances where R 2 and R 3 are independently not hydrogen)
  • any of the PEP moieties provided herein can be defined as a therapeutic peptide.
  • POLY is a poly(ethylene glycol) such as H 3 CO(CH 2 CH 2 O) n —, wherein (n) is an integer having a value of from 3 to 4000, more preferably from 10 to about 1800; (d) is one; X 1 is amide [e.g., —C(O)NH—]; (b) is 2 through 6, such as 4; (c) is 2 through 6, such as 4; each of R 2 and R 3 are independently H or lower alkyl, such as methyl when lower alkyl; and PEP is therapeutic peptide.
  • Another example of a therapeutic peptide conjugate in accordance with the invention has the following structure:
  • each (n) is independently an integer having a value of from 3 to 4000, preferably from 10 to 1800;
  • X 2 is as previously defined;
  • (b) is 2 through 6;
  • (c) is 2 through 6;
  • R 2 in each occurrence, is independently H or lower alkyl; and
  • ⁇ NH -PEP represents a residue of a therapeutic peptide, where the underlined amino group represents an amino group of the therapeutic peptide.
  • Additional therapeutic peptide polymer conjugates resulting from reaction of a water-soluble polymer with an amino group of therapeutic peptide are provided below.
  • the following conjugate structures are releasable.
  • One such structure corresponds to:
  • mPEG is CH 3 O—(CH 2 CH 2 O) n CH 2 CH 2 —, n ranges from 10 to 1800, p is an integer ranging from 1 to 8, R 1 is H or lower alkyl, R 2 is H or lower alkyl, Ar is an aromatic hydrocarbon, such as a fused bicyclic or tricyclic aromatic hydrocarbon, X 1 and X 2 are each independently a spacer moiety having an atom length of from about 1 to about 18 atoms, ⁇ NH-PEP is as previously described, and k is an integer selected from 1, 2, and 3. The value of k indicates the number of water-soluble polymer molecules attached to different sites on the therapeutic peptide. In a preferred embodiment, R 1 and R 2 are both H.
  • the spacer moieties, X 1 and X 2 preferably each contain one amide bond.
  • X 1 and X 2 are the same.
  • Preferred spacers, i.e., X 1 and X 2 include —NH—C(O)—CH 2 —O—, —NH—C(O)—(CH 2 ) q —O—, —NH—C(O)—(CH 2 ) q —C(O)—NH—, —NH—C(O)—(CH 2 ) q —, and —C(O)—NH—, where q is selected from 2, 3, 4, and 5.
  • the spacers can be in either orientation, preferably, the nitrogen is proximal to the PEG rather than to the aromatic moiety.
  • aromatic moieties include pentalene, indene, naphthalene, indacene, acenaphthylene, and fluorene.
  • Additional therapeutic peptide conjugates resulting from covalent attachment to amino groups of therapeutic peptide that are also releasable include the following:
  • n ranges from about 10 to about 1800.
  • Additional releasable conjugates in accordance with the invention are prepared using water-soluble polymer reagents such as those described in U.S. Pat. No. 6,214,966.
  • water-soluble polymers result in a releasable linkage following conjugation, and possess at least one releasable ester linkage close to the covalent attachment to the active agent.
  • the polymers generally possess the following structure, PEG-W—CO 2 —NHS or an equivalent activated ester, where
  • releasable conjugates of this type include: mPEG-O—(CH 2 ) b —COOCH 2 C(O)—NH-therapeutic peptide, and mPEG-O—(CH 2 ) b —COO—CH(CH 3 )—CH 2 —C(O)—NH-therapeutic peptide, where the number of water-soluble polymers attached to therapeutic peptide can be anywhere from 1 to 4, or more preferably, from 1 to 3.
  • Carboxyl groups represent another functional group that can serve as a point of attachment to the therapeutic peptide.
  • the conjugate will have the following structure:
  • the C(O)—X linkage results from the reaction between a polymeric derivative bearing a terminal functional group and a carboxyl-containing therapeutic peptide.
  • the specific linkage will depend on the type of functional group utilized. If the polymer is end-functionalized or “activated” with a hydroxyl group, the resulting linkage will be a carboxylic acid ester and X will be O. If the polymer backbone is functionalized with a thiol group, the resulting linkage will be a thioester and X will be S.
  • the C(O)X moiety, and in particular the X moiety may be relatively more complex and may include a longer linker structure.
  • Polymeric reagents containing a hydrazide moiety are also suitable for conjugation at a carbonyl.
  • a carbonyl moiety can be introduced by reducing any carboxylic acid functionality (e.g., the C-terminal carboxylic acid).
  • carboxylic acid functionality e.g., the C-terminal carboxylic acid.
  • any polymeric reagent comprising an activated ester e.g., a succinimidyl group
  • an activated ester e.g., a succinimidyl group
  • a hydrazide moiety by reacting the polymer activated ester with hydrazine (NH 2 —NH 2 ) or tert-butyl carbamate [NH 2 NHCO 2 C(CH 3 ) 3 ].
  • the variable (n) represents the number of repeating monomeric units and “ ⁇ C -(PEP)” represents a residue of a therapeutic peptide following conjugation to the polymeric reagent were the underlined C is part of the therapeutic peptide.
  • the hydrazone linkage can be reduced using a suitable reducing agent.
  • Thiol groups contained within the therapeutic peptide can serve as effective sites of attachment for the water-soluble polymer.
  • the thiol groups contained in cysteine residues of the therapeutic peptide can be reacted with an activated PEG that is specific for reaction with thiol groups, e.g., an N-maleimidyl polymer or other derivative, as described in, for example, U.S. Pat. No. 5,739,208, WO 01/62827, and in Table 4 below.
  • cysteine residues may be introduced in the therapeutic peptide and may be used to attach a water-soluble polymer.
  • reagents themselves, along with the corresponding conjugates, are provided in Table 4 below.
  • the variable (n) represents the number of repeating monomeric units and “— S -(PEP)” represents a residue of a therapeutic peptide following conjugation to the water-soluble polymer, where the S represents the residue of a therapeutic peptide thiol group. While each polymeric portion [e.g., (OCH 2 CH 2 ) n or (CH 2 CH 2 O) n ] presented in Table 4 terminates in a “CH 3 ” group, other end-capping groups (such as H and benzyl) or reactive groups may be used as well.
  • end-capping groups such as H and benzyl
  • the corresponding maleamic acid form(s) of the water-soluble polymer can also react with the therapeutic peptide.
  • the maleimide ring will “open” to form the corresponding maleamic acid.
  • the maleamic acid in turn, can react with an amine or thiol group of a therapeutic peptide.
  • Exemplary maleamic acid-based reactions are schematically shown below.
  • POLY represents the water-soluble polymer
  • ⁇ S-PEP represents a residue of a therapeutic peptide, where the S is derived from a thiol group of the therapeutic peptide.
  • Thiol PEGylation is specific for free thiol groups on the therapeutic peptide.
  • a polymer maleimide is conjugated to a sulfhydryl-containing therapeutic peptide at pHs ranging from about 6-9 (e.g., at 6, 6.5, 7, 7.5, 8, 8.5, or 9), more preferably at pHs from about 7-9, and even more preferably at pHs from about 7 to 8.
  • a slight molar excess of polymer maleimide is employed, for example, a 1.5 to 15-fold molar excess, preferably a 2-fold to 10 fold molar excess.
  • Reaction times generally range from about 15 minutes to several hours, e.g., 8 or more hours, at room temperature.
  • Thiol-selective conjugation is preferably conducted at pHs around 7. Temperatures for conjugation reactions are typically, although not necessarily, in the range of from about 0° C. to about 40° C.; conjugation is often carried out at room temperature or less. Conjugation reactions are often carried out in a buffer such as a phosphate or acetate buffer or similar system.
  • an excess of the polymeric reagent is typically combined with the therapeutic peptide.
  • the conjugation reaction is allowed to proceed until substantially no further conjugation occurs, which can generally be determined by monitoring the progress of the reaction over time.
  • the reaction can be monitored by withdrawing aliquots from the reaction mixture at various time points and analyzing the reaction mixture by SDS-PAGE or MALDI-TOF mass spectrometry or any other suitable analytical method. Once a plateau is reached with respect to the amount of conjugate formed or the amount of unconjugated polymer remaining, the reaction is assumed to be complete. Typically, the conjugation reaction takes anywhere from minutes to several hours (e.g., from 5 minutes to 24 hours or more).
  • the resulting product mixture is preferably, but not necessarily purified, to separate out excess reagents, unconjugated reactants (e.g., therapeutic peptide) undesired multi-conjugated species, and free or unreacted polymer.
  • the resulting conjugates can then be further characterized using analytical methods such as MALDI, capillary electrophoresis, gel electrophoresis, and/or chromatography.
  • An illustrative therapeutic peptide conjugate formed by reaction with one or more therapeutic peptide thiol groups may possess the following structure:
  • POLY is a water-soluble polymer
  • X is an optional linker
  • Z is a heteroatom selected from the group consisting of O, NH, and S
  • Y is selected from the group consisting of C 2-10 alkyl, C 2-10 substituted alkyl, aryl, and substituted aryl
  • —S-PEP is a residue of a therapeutic peptide, where the S represents the residue of a therapeutic peptide thiol group.
  • polymeric reagents suitable for reacting with a therapeutic peptide thiol group those described here and elsewhere can be obtained from commercial sources.
  • methods for preparing polymeric reagents are described in the literature.
  • the attachment between the therapeutic peptide and water-soluble polymer can be direct, wherein no intervening atoms are located between the therapeutic peptide and the polymer, or indirect, wherein one or more atoms are located between the therapeutic peptide and polymer.
  • a “spacer moiety or linker” serves as a link between the therapeutic peptide and the water-soluble polymer.
  • the one or more atoms making up the spacer moiety can include one or more of carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinations thereof.
  • the spacer moiety can comprise an amide, secondary amine, carbamate, thioether, and/or disulfide group.
  • specific spacer moieties include those selected from the group consisting of —O—, —S—, —S—S—, —C(O)—, —C(O)O—, —OC(O)—, —CH 2 —C(O)O—, —CH 2 —OC(O)—, —C(O)O—CH 2 —, —OC(O)—CH 2 —, —C(O)—NH—, —NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—, —CH 2 —, —CH 2 —CH 2 —, —CH 2 —CH 2 —, —CH 2 —CH 2 —CH 2 —CH 2 —CH 2 —CH 2 —CH 2 —CH 2 —CH 2 —CH
  • spacer moieties have the following structures: —C(O)—NH—(CH 2 ) 1-6 —NH—C(O)—, —NH—C(O)—NH—(CH 2 ) 1-6 —NH—C(O)—, and —O—C(O)—NH—(CH 2 ) 1-6 —NH—C(O)—, wherein the subscript values following each methylene indicate the number of methylenes contained in the structure, e.g., (CH 2 ) 1-6 means that the structure can contain 1, 2, 3, 4, 5 or 6 methylenes.
  • any of the above spacer moieties may further include an ethylene oxide oligomer chain comprising 1 to 20 ethylene oxide monomer units [i.e., —(CH 2 CH 2 O) 1-20 ]. That is, the ethylene oxide oligomer chain can occur before or after the spacer moiety, and optionally in between any two atoms of a spacer moiety comprised of two or more atoms. Also, the oligomer chain would not be considered part of the spacer moiety if the oligomer is adjacent to a polymer segment and merely represent an extension of the polymer segment.
  • the water-soluble polymer-(PEP) conjugate will include a non-linear water-soluble polymer.
  • a non-linear water-soluble polymer encompasses a branched water-soluble polymer (although other non linear water-soluble polymers are also contemplated).
  • the conjugate comprises a therapeutic peptide covalently attached, either directly or through a spacer moiety comprised of one or more atoms, to a branched water-soluble polymer, at in a non-limiting example, an internal or N-terminal amine.
  • an internal amine is an amine that is not part of the N-terminal amino acid (meaning not only the N-terminal amine, but any amine on the side chain of the N-terminal amino acid).
  • conjugates include a branched water-soluble polymer attached (either directly or through a spacer moiety) to a therapeutic peptide at an internal amino acid of the therapeutic peptide
  • additional branched water-soluble polymers can also be attached to the same therapeutic peptide at other locations as well.
  • a conjugate including a branched water-soluble polymer attached (either directly or through a spacer moiety) to a therapeutic peptide at an internal amino acid of the therapeutic peptide can further include an additional branched water-soluble polymer covalently attached, either directly or through a spacer moiety comprised of one or more atoms, to the N-terminal amino acid residue, such as at the N-terminal amine.
  • One preferred branched water-soluble polymer comprises the following structure:
  • each (n) is independently an integer having a value of from 3 to 4000, or more preferably, from about 10 to 1800.
  • multi-armed polymer conjugates comprising a polymer scaffold having 3 or more polymer arms each suitable for capable of covalent attachment of a therapeutic peptide.
  • R is a core molecule as previously described
  • POLY is a water-soluble polymer
  • X is a cleavable, e.g., hydrolyzable linkage
  • y ranges from about 3 to 15.
  • conjugate may comprise the structure:
  • m is selected from 3, 4, 5, 6, 7, and 8.
  • the therapeutic peptide conjugate may correspond to the structure:
  • R is a core molecule as previously described
  • X is —NH—P—Z—C(O)P is a spacer
  • Z is —O—, —NH—, or —CH 2 —
  • —O-PEP is a hydroxyl residue of a therapeutic peptide
  • y is 3 to 15.
  • X is a residue of an amino acid.
  • the therapeutic peptide polymer conjugates described herein can be purified to obtain/isolate different conjugate species. Specifically, a product mixture can be purified to obtain an average of anywhere from one, two, or three or even more PEGs per therapeutic peptide. In one embodiment of the invention, preferred therapeutic peptide conjugates are mono-conjugates.
  • the strategy for purification of the final conjugate reaction mixture will depend upon a number of factors, including, for example, the molecular weight of the polymeric reagent employed, the therapeutic peptide, and the desired characteristics of the product—e.g., monomer, dimer, particular positional isomers, etc.
  • conjugates having different molecular weights can be isolated using gel filtration chromatography and/or ion exchange chromatography.
  • Gel filtration chromatography may be used to fractionate different therapeutic peptide conjugates (e.g., 1-mer, 2-mer, 3-mer, and so forth, wherein “1-mer” indicates one polymer molecule per therapeutic peptide, “2-mer” indicates two polymers attached to therapeutic peptide, and so on) on the basis of their differing molecular weights (where the difference corresponds essentially to the average molecular weight of the water-soluble polymer).
  • Gel filtration columns suitable for carrying out this type of separation include SuperdexTM and SephadexTM columns available from Amersham Biosciences (Piscataway, N.J.). Selection of a particular column will depend upon the desired fractionation range desired. Elution is generally carried out using a suitable buffer, such as phosphate, acetate, or the like.
  • the collected fractions may be analyzed by a number of different methods, for example, (i) optical density (OD) at 280 nm for protein content, (ii) bovine serum albumin (BSA) protein analysis, (iii) iodine testing for PEG content (Sims et al. (1980) Anal. Biochem, 107:60-63), and (iv) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), followed by staining with barium iodide.
  • OD optical density
  • BSA bovine serum albumin
  • SDS PAGE sodium dodecyl sul
  • Separation of positional isomers is typically carried out by reverse phase chromatography using a reverse phase-high performance liquid chromatography (RP-HPLC) C18 column (Amersham Biosciences or Vydac) or by ion exchange chromatography using an ion exchange column, e.g., a DEAE- or CM-SepharoseTM ion exchange column available from Amersham Biosciences. Either approach can be used to separate polymer-therapeutic peptide isomers having the same molecular weight (positional isomers).
  • RP-HPLC reverse phase-high performance liquid chromatography
  • ion exchange column e.g., a DEAE- or CM-SepharoseTM ion exchange column available from Amersham Biosciences.
  • Either approach can be used to separate polymer-therapeutic peptide isomers having the same molecular weight (positional isomers).
  • compositions are preferably substantially free of the non-conjugated therapeutic peptide.
  • compositions preferably are substantially free of all other non-covalently attached water-soluble polymers.
  • compositions comprising any one or more of the therapeutic peptide polymer conjugates described herein.
  • the composition will comprise a plurality of therapeutic peptide polymer conjugates.
  • such a composition may comprise a mixture of therapeutic peptide polymer conjugates having one, two, three and/or even four water-soluble polymer molecules covalently attached to sites on the therapeutic peptide.
  • a composition of the invention may comprise a mixture of monomer, dimer, and possibly even trimer or 4-mer.
  • the composition may possess only mono-conjugates, or only di-conjugates, etc.
  • a mono-conjugate therapeutic peptide composition will typically comprise therapeutic peptide moieties having only a single polymer covalently attached thereto, e.g., preferably releasably attached.
  • a mono-conjugate composition may comprise only a single positional isomer, or may comprise a mixture of different positional isomers having polymer covalently attached to different sites within the therapeutic peptide.
  • a therapeutic peptide conjugate may possess multiple therapeutic peptides covalently attached to a single multi-armed polymer having 3 or more polymer arms.
  • the therapeutic peptide moieties are each attached at the same therapeutic peptide amino acid site, e.g., the N-terminus.
  • the composition will typically satisfy one or more of the following characteristics: at least about 85% of the conjugates in the composition will have from one to four polymers attached to the therapeutic peptide; at least about 85% of the conjugates in the composition will have from one to three polymers attached to the therapeutic peptide; at least about 85% of the conjugates in the composition will have from one to two polymers attached to the therapeutic peptide; or at least about 85% of the conjugates in the composition will have one polymer attached to the therapeutic peptide (e.g., be monoPEGylated); at least about 95% of the conjugates in the composition will have from one to four polymers attached to the therapeutic peptide; at least about 95% of the conjugates in the composition will have from one to three polymers attached to the therapeutic peptide; at least about 95% of the conjugates in the composition will have from one to two polymers attached to the therapeutic peptide; at least about 95% of the conjugates in the composition will have one polymers attached to the therapeutic peptide;
  • the conjugate-containing composition is free or substantially free of albumin.
  • a pharmaceutical composition comprising a conjugate comprising a therapeutic peptide covalently attached, e.g., releasably, to a water-soluble polymer, wherein the water-soluble polymer has a weight-average molecular weight of greater than about 2,000 Daltons; and a pharmaceutically acceptable excipient.
  • Control of the desired number of polymers for covalent attachment to therapeutic peptide is achieved by selecting the proper polymeric reagent, the ratio of polymeric reagent to the Therapeutic peptide, temperature, pH conditions, and other aspects of the conjugation reaction.
  • reduction or elimination of the undesired conjugates can be achieved through purification mean as previously described.
  • the water-soluble polymer-(therapeutic peptide) conjugates can be purified to obtain/isolate different conjugated species.
  • the product mixture can be purified to obtain an average of anywhere from one, two, three, or four PEGs per therapeutic peptide, typically one, two or three PEGs per therapeutic peptide.
  • the product comprises one PEG per therapeutic peptide, where PEG is releasably (via hydrolysis) attached to PEG polymer, e.g., a branched or straight chain PEG polymer.
  • a therapeutic peptide conjugate composition of the invention will comprise, in addition to the therapeutic peptide conjugate, a pharmaceutically acceptable excipient. More specifically, the composition may further comprise excipients, solvents, stabilizers, membrane penetration enhancers, etc., depending upon the particular mode of administration and dosage form.
  • compositions of the invention encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted as well as liquids, as well as for inhalation.
  • suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic endotoxin-free water for injection, dextrose 5% in water, phosphate-buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof.
  • solutions and suspensions are envisioned.
  • Exemplary pharmaceutically acceptable excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.
  • Representative carbohydrates for use in the compositions of the present invention include sugars, derivatized sugars such as alditols, aldonic acids, esterified sugars, and sugar polymers.
  • Exemplary carbohydrate excipients suitable for use in the present invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), pyranosyl sorbitol, myoinositol and the like
  • non-reducing sugars are non-reducing sugars, sugars that can form a substantially dry amorphous or glassy phase when combined with the composition of the present invention, and sugars possessing relatively high glass transition temperatures, or Tgs (e.g., Tgs greater than 40° C., or greater than 50° C., or greater than 60° C., or greater than 70° C., or having Tgs of 80° C. and above).
  • Tgs glass transition temperatures
  • Such excipients may be considered glass-forming excipients.
  • Additional excipients include amino acids, peptides and particularly oligomers comprising 2-9 amino acids, or 2-5 mers, and polypeptides, all of which may be homo or hetero species.
  • Exemplary protein excipients include albumins such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, hemoglobin, and the like.
  • the compositions may also include a buffer or a pH-adjusting agent, typically but not necessarily a salt prepared from an organic acid or base.
  • Representative buffers include organic acid salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid.
  • Other suitable buffers include Tris, tromethamine hydrochloride, borate, glycerol phosphate, and phosphate. Amino acids such as glycine are also suitable.
  • compositions of the present invention may also include one or more additional polymeric excipients/additives, e.g., polyvinylpyrrolidones, derivatized celluloses such as hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose, FICOLLs (a polymeric sugar), hydroxyethylstarch (HES), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl- ⁇ -cyclodextrin and sulfobutylether- ⁇ -cyclodextrin), polyethylene glycols, and pectin.
  • additional polymeric excipients/additives e.g., polyvinylpyrrolidones, derivatized celluloses such as hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose, FICOLLs (a polymeric sugar), hydroxyethylstarch (HES), dextrates (e.g.,
  • compositions may further include flavoring agents, taste-masking agents, inorganic salts (e.g., sodium chloride), antimicrobial agents (e.g., benzalkonium chloride), sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80,” and pluronics such as F68 and F88, available from BASF), sorbitan esters, lipids (e.g., phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, although preferably not in liposomal form), fatty acids and fatty esters, steroids (e.g., cholesterol), and chelating agents (e.g., zinc and other such suitable cations).
  • inorganic salts e.g., sodium chloride
  • antimicrobial agents e.g., benzalkonium chloride
  • sweeteners e.g., benzalkonium chloride
  • compositions according to the present invention are listed in “Remington: The Science & Practice of Pharmacy,” 21 st ed., Williams & Williams, (2005), and in the “Physician's Desk Reference,” 60th ed., Medical Economics, Montvale, N.J. (2006).
  • the amount of the therapeutic peptide conjugate (i.e., the conjugate formed between the active agent and the polymeric reagent) in the composition will vary depending on a number of factors, but will optimally be a therapeutically effective amount when the composition is stored in a unit dose container (e.g., a vial).
  • a pharmaceutical preparation if in solution form, can be housed in a syringe.
  • a therapeutically effective amount can be determined experimentally by repeated administration of increasing amounts of the conjugate in order to determine which amount produces a clinically desired endpoint.
  • the amount of any individual excipient in the composition will vary depending on the activity of the excipient and particular needs of the composition.
  • the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects.
  • the excipient or excipients will be present in the composition in an amount of about 1% to about 99% by weight, from about 5% to about 98% by weight, from about 15 to about 95% by weight of the excipient, or with concentrations less than 30% by weight. In general, a high concentration of the therapeutic peptide is desired in the final pharmaceutical formulation.
  • a composition of the invention may also comprise a mixture of water-soluble polymer-(therapeutic peptide) conjugates and unconjugated therapeutic peptide, to thereby provide a mixture of fast-acting and long-acting therapeutic peptide.
  • compositions in accordance with the invention include those comprising, in addition to an extended-action therapeutic peptide water-soluble polymer conjugate as described herein, a rapid acting therapeutic peptide polymer conjugate where the water-soluble polymer is releasably attached to a suitable location on the therapeutic peptide.
  • the therapeutic peptide conjugates of the invention can be administered by any of a number of routes including without limitation, oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal), intrathecal, and pulmonary.
  • routes including without limitation, oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal), intrathecal, and pulmonary.
  • Preferred forms of administration include parenteral and pulmonary.
  • Suitable formulation types for parenteral administration include ready-for-injection solutions, dry powders for combination with a solvent prior to use, suspensions ready for injection, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration, among others.
  • compositions comprising the peptide-polymer conjugates may further be incorporated into a suitable delivery vehicle.
  • delivery vehicles may provide controlled and/or continuous release of the conjugates and may also serve as a targeting moiety.
  • Non-limiting examples of delivery vehicles include, adjuvants, synthetic adjuvants, microcapsules, microparticles, liposomes, and yeast cell wall particles.
  • Yeast cells walls may be variously processed to selectively remove protein component, glucan, or mannan layers, and are referred to as whole glucan particles (WGP), yeast beta-glucan mannan particles (YGMP), yeast glucan particles (YGP), ⁇ Rhodotorula yeast cell particles (YCP). Yeast cells such as S.
  • yeast cells exhibit different properties in terms of hydrodynamic volume and also differ in the target organ where they may release their contents.
  • the methods of manufacture and characterization of these particles are described in U.S. Pat. Nos. 5,741,495; 4,810,646; 4,992,540; 5,028,703; 5,607,677, and US Patent Applications Nos. 2005/0281781, and 2008/0044438.
  • a method comprising delivering a conjugate to a patient, the method comprising the step of administering to the patient a pharmaceutical composition comprising a therapeutic peptide polymer conjugate as provided herein.
  • Administration can be effected by any of the routes herein described.
  • the method may be used to treat a patient suffering from a condition that is responsive to treatment with therapeutic peptide by administering a therapeutically effective amount of the pharmaceutical composition.
  • the method of delivering a therapeutic peptide polymer conjugate as provided herein may be used to treat a patient having a condition that can be remedied or prevented by administration of therapeutic peptide.
  • conjugates of the invention include those effective to release the therapeutic peptide, e.g., by hydrolysis, over a period of several hours or even days (e.g., 2-7 days, 2-6 days, 3-6 days, 3-4 days) when evaluated in a suitable in-vivo model.
  • days e.g., 2-7 days, 2-6 days, 3-6 days, 3-4 days
  • a conjugate of the invention will be delivered such that plasma levels of a therapeutic peptide are within a range of about 0.5 picomoles/liter to about 500 picomoles/liter.
  • the conjugate of the invention will be delivered such that plasma leves of a therapeutic peptide are within a range of about 1 picomoles/liter to about 400 picomoles/liter, a range of about 2.5 picomoles/liter to about 250 picomoles/liter, a range of about 5 picomoles/liter to about 200 picomoles/liter, or a range of about 10 picomoles/liter to about 100 picomoles/liter.
  • a therapeutically effective dosage amount of a therapeutic peptide conjugate as described herein will range from about 0.01 mg per day to about 1000 mg per day for an adult.
  • dosages may range from about 0.1 mg per day to about 100 mg per day, or from about 1.0 mg per day to about 10 mg/day.
  • corresponding doses based on international units of activity can be calculated by one of ordinary skill in the art.
  • the unit dosage of any given conjugate (again, such as provided as part of a pharmaceutical composition) can be administered in a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth.
  • the specific dosing schedule will be known by those of ordinary skill in the art or can be determined experimentally using routine methods.
  • Exemplary dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Once the clinical endpoint has been achieved, dosing of the composition is halted.
  • a water-soluble polymer reagent is used in the preparation of peptide conjugates of the invention.
  • a water-soluble polymer reagent is a water-soluble polymer-containing compound having at least one functional group that can react with a functional group on a peptide (e.g., the N-terminus, the C-terminus, a functional group associated with the side chain of an amino acid located within the peptide) to create a covalent bond.
  • polymeric reagents and methods for conjugating such polymers to an active moiety are known in the art, and are, e.g., described in Harris, J. M. and Zalipsky, S., eds, Poly ( ethylene glycol ), Chemistry and Biological Applications , ACS, Washington, 1997; Veronese, F., and J. M Harris, eds., Peptide and Protein PEGylation , Advanced Drug Delivery Reviews, 54(4); 453-609 (2002); Zalipsky, S., et al., “ Use of Functionalized Poly ( Ethylene Glycols ) for Modification of Polypeptides ” in Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications , J. M. Harris, ed., Plenus Press, New York (1992); Zalipsky (1995) Advanced Drug Reviews 16:157-182, and in Roberts, et al., Adv. Drug Delivery Reviews, 54, 459-476 (2002).
  • PEG reagents suitable for use in forming a conjugate of the invention are described in Shearwater Corporation, Catalog 2001; Shearwater Polymers, Inc., Catalogs, 2000 and 1997-1998, and in Pasut. G., et al., Expert Opin. Ther. Patents (2004), 14(5).
  • PEG reagents suitable for use in the present invention also include those available from NOF Corporation (Tokyo, Japan), as described generally on the NOF website (2006) under Products, High Purity PEGs and Activated PEGs. Products listed therein and their chemical structures are expressly incorporated herein by reference.
  • Additional PEGs for use in forming a GLP-1 conjugate of the invention include those available from Polypure (Norway) and from QuantaBioDesign LTD (Powell, Ohio), where the contents of their online catalogs (2006) with respect to available PEG reagents are expressly incorporated herein by reference.
  • water-soluble polymer reagents useful for preparing peptide conjugates of the invention is prepared synthetically. Descriptions of the water-soluble polymer reagent synthesis can be found in, for example, U.S. Pat. Nos. 5,252,714, 5,650,234, 5,739,208, 5,932,462, 5,629,384, 5,672,662, 5,990,237, 6,448,369, 6,362,254, 6,495,659, 6,413,507, 6,376,604, 6,348,558, 6,602,498, and 7,026,440.
  • Peptide G is an amino acid synthetic peptide containing residues 161-189 of the 40 kDa laminin binding domain of 67LR, which has been found to inhibit laminin-coated melanoma cells from attaching to endothelial cells that express the 67 kDa laminin receptor (Gastronovo et al., J. Biol. Chem. 1991, 266, 20440-6.
  • the 20 amino acid sequence is Ile-Pro-Cys-Asn-Asn-Lys-Gly-Ala-His-Ser-Val-Gly-Leu-Met-Trp-Trp-Met-Leu-Ala-Arg, has been proposed as potential new antimetastatic agent. (Gastronovo et al., Cancer Res. 1991, 51, 5672-8).
  • Peptide G is prepared and purified according to standard automated peptide synthesis or recombinant techniques known to those skilled in the art.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content. The mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Peptide G prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The reaction is optionally quenched to terminate the reaction. The pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1 M HCl, if necessary, to bring the pH of the final solution to about 5.5. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -Peptide G conjugate formation.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the C-terminus of Peptide G, to provide a C ter -conjugate form of the peptide.
  • a protected Peptide G e.g, Fmoc-Ile-Pro-Cys(tBu)-Asn-Asn-Lys(Fmoc)-Gly-Ala-His-Ser(Dmab)-Val-Gly-Leu-Met-Trp-Trp-Met-Leu-Ala-Arg(Tos)
  • a protected Peptide G e.g, Fmoc-Ile-Pro-Cys(tBu)-Asn-Asn-Lys(Fmoc)-Gly-Ala-His-Ser(Dmab)-Val-Gly-Leu-Met-Trp-Trp-Met-Leu-Ala-Arg(To
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content. The mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot-Peptide G is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-Peptide G-C ter -mPEG conjugate formation. The remaining protecting groups are removed under standard deprotection conditions to yield the Peptide G-C ter -mPEG conjugate.
  • mPEG-Maleimide having a molecular weight of 5 kDa and having the basic structure shown below:
  • Peptide G which has a thiol-containing cysteine residue, is dissolved in buffer. To this peptide solution is added a 3-5 fold molar excess of mPEG-MAL, 5 kDa. The mixture is stirred at room temperature under an inert atmosphere for several hours. Analysis of the reaction mixture reveals successful conjugation of this peptide.
  • mPEG-N-Hydroxysuccinimide having a molecular weight of 5 kDa and having the basic structure shown below:
  • mPEG-Succinimidyl ⁇ -Methylbutanoate Derivative, 5 kDa (“mPEG-SMB”)
  • mPEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock Peptide G solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • OTS-102 is an angiogenesis inhibitor for cancer treatment consisting of KDR169, the nine amino acid sequence starting at residue 169 of VEGFR2.
  • KDR169 activates CD8-positive CTL's in an HLA-A2402 dependent manner. Augmented CTL exerts cytotoxicity to tumor-associated neovascular endothelial cells expressing KDR (VEGF receptor), and shows anti-tumor activity (see, U.S. Patent Application No. 2006/216301 A1 and OncoTherapy Sciences, Inc web site, http://www.oncotherapy.co.jp/eng/rd/page3.html).
  • KDR169 has the sequence, Arg-Phe-Val-Pro-Asp-Gly-Asn-Arg-Ile (RFVPDGNRI) (see, Seq. No. 8, in US2006/216301A1).
  • the 9-aa KDR169 peptide is prepared and purified according to standard automated peptide synthesis or recombinant techniques known to those skilled in the art.
  • An illustrative polymeric reagent, mPEG-SPC reagent, is covalently attached to the N-terminus of KDR169, to provide a N ter -conjugate form of the peptide.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content.
  • the mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of KDR169 prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved.
  • the stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the reaction is optionally quenched to terminate the reaction.
  • the pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1M HCl, if necessary, to bring the pH of the final solution to about 5.5.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -OTS102 conjugate formation.
  • mPEG-NH 2 reagent is covalently attached to the C-terminus of KDR169, to provide a C ter -conjugate form of the peptide.
  • a protected KDR169 (Prot-KDR169, e.g., Fmoc-Arg(Tos)-Phe-Val-Pro-Asp(OBz)-Gly-Asn-Arg(Tos)-Ile-OH) is prepared and purified according to standard automated peptide synthesis techniques known to those skilled in the art.
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • the reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content.
  • the mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar. A solution of Prot-KDR169 is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-KDR169-C ter -mPEG conjugate formation.
  • the remaining protecting groups are removed under standard deprotection conditions to yield the OTS102-C ter -mPEG conjugate.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the Asp residue of KDR169, to provide a Asp-conjugate form of the peptide.
  • a protected KDR169 (Prot2-KDR169, e.g., Fmoc-Arg(Tos)-Phe-Val-Pro-Asp(OBz)-Gly-Asn-Arg(Tos)-Ile-O(tBu)
  • a protected KDR169 (Prot2-KDR169, e.g., Fmoc-Arg(Tos)-Phe-Val-Pro-Asp(OBz)-Gly-Asn-Arg(Tos)-Ile-O(tBu)
  • Fmoc-Arg(Tos)-Phe-Val-Pro-Asp(OBz)-Gly-Asn-Arg(Tos)-Ile-O(tBu) is prepared and purified according to standard automated peptide
  • a 5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content.
  • the mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot3-KDR169 is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot3-KDR169-(Asp-O-mPEG) conjugate formation.
  • the remaining protecting groups are removed under standard deprotection conditions to yield the OTS102-Asp(O-mPEG) conjugate.
  • mPEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock OTS102 solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • AngiocolTM is a recombinant protein derived from the non-collagenous domain (alpha-2) of type IV collagen, which has been shown in preclinical studies to inhibit macrovascular endothelial cell proliferation (new blood vessel growth), as well as tumour growth, in in vitro and in vivo models by targeting the assembly and organization of the vascular basal lamina.
  • AngiocolTM has been proposed for the treatment of retinal neovascularization (Coleman et al., Microcirculation 2004, 11, 530).
  • AngiocolTM is prepared and purified according to standard automated peptide synthesis or recombinant techniques known to those skilled in the art.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content. The mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • AngiocolTM prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The reaction is optionally quenched to terminate the reaction. The pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1 M HCl, if necessary, to bring the pH of the final solution to about 5.5. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -Angiocol conjugate formation.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the C-terminus of AngiocolTM, to provide a C ter -conjugate form of the peptide.
  • a protected AngiocolTM Prot-AngiocolTM
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature.
  • mPEG-NH 2 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content.
  • the mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot-AngiocolTM is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-Angiocol-C ter -mPEG conjugate formation.
  • the remaining protecting groups are removed under standard deprotection conditions to yield the Angiocol-C ter -mPEG conjugate.
  • mPEG-N-Hydroxysuccinimide having a molecular weight of 5 kDa and having the basic structure shown below:
  • mPEG-Succinimidyl ⁇ -Methylbutanoate Derivative, 5 kDa (“mPEG-SMB”)
  • mPEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock AngiocolTM solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • ABT-510 is nonapeptide analogue that mimics the anti-angiogenic activity of the endogenous protein thrombospondin-1 (TSP-1) which is in development for treatment of advanced malignancies.
  • TSP-1 endogenous protein thrombospondin-1
  • ABT-510 blocks the actions of multiple pro-angiogenic growth factors known to play a role in cancer related blood vessel growth, such as VEGF, bFGF, HGF, and IL-8 (Haviv et al., J. Med. Chem. 2005, 48, 2838; Baker et al., J. Clin. Oncol. 2005, 23, 9013).
  • VEGF endogenous protein thrombospondin-1
  • bFGF thrombospondin-1
  • HGF thrombospondin-1
  • IL-8 pro-angiogenic growth factors known to play a role in cancer related blood vessel growth, such as VEGF, bFGF, HGF, and IL-8 (Haviv et al.,
  • ABT-510 is prepared and purified according to standard automated peptide synthesis or recombinant techniques known to those skilled in the art, without the N-terminal acetyl group (NH 2 -ABT-510).
  • An illustrative polymeric reagent, mPEG-SPC reagent, is covalently attached to the N-terminus of NH 2 -ABT-510, to provide a N ter -conjugate form of the peptide.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature.
  • mPEG-SPC 20 kDa reagent About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content.
  • the mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of NH 2 -ABT-510 prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved.
  • the stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the reaction is optionally quenched to terminate the reaction.
  • the pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1 M HCl, if necessary, to bring the pH of the final solution to about 5.5.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -ABT-510 conjugate formation.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the C-terminus of ABT-510, to provide a C ter -conjugate form of the peptide.
  • a protected ABT-510, lacking the C-terminal ethyl amide (Prot-ABT-510, e.g., NAc-Sar(tBu)-Gly-Val-(d-allo-Ile)-Thr(tBu)-Nva-Ile-Arg(Tos)-Pro-OH) is prepared and purified according to standard automated peptide synthesis techniques known to those skilled in the art.
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content. The mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot-ABT-510 is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-ABT-510-C ter -mPEG conjugate formation. The remaining protecting groups are removed under standard deprotection conditions to yield the ABT-510-C ter -mPEG conjugate.
  • mPEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock NH 2 -ABT-510 (as in Example 4a) solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • A6 is a urokinase-derived eight amino-acid peptide, NAc-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-NH 2 , with anti-angiogenic properties which has been shown to suppres metastases and prolong the life span of prostate tumor-bearing mice (Boyd et al., Am. J. Pathology 2003, 162. 619).
  • A6 is prepared and purified according to standard automated peptide synthesis or recombinant techniques known to those skilled in the art, without the N-terminal acetyl group (NH 2 -A6).
  • An illustrative polymeric reagent, mPEG-SPC reagent is covalently attached to the N-terminus of NH 2 -A6, to provide a N ter -conjugate form of the peptide.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content.
  • the mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of NH 2 -A6 prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved.
  • the stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the reaction is optionally quenched to terminate the reaction.
  • the pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1M HCl, if necessary, to bring the pH of the final solution to about 5.5.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -A6 conjugate formation.
  • mPEG-NH 2 reagent is covalently attached to the C-terminus of A6, to provide a C ter -conjugate form of the peptide.
  • a protected A6, lacking the C-terminal amide (Prot-A6, e.g., NAc-Lys(Fmoc)-Pro-Ser(tBu)-Ser(tBu)-Pro-Pro-Glu(tBu)-Glu(tBu)-OH) is prepared and purified according to standard automated peptide synthesis techniques known to those skilled in the art.
  • the stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-A6-C ter -mPEG conjugate formation.
  • the remaining protecting groups are removed under standard deprotection conditions to yield the ABT-510-C ter -mPEG conjugate.
  • mPEG-N-Hydroxysuccinimide having a molecular weight of 5 kDa and having the basic structure shown below: mPEG-SMB, 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature. A five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution. The 10% reagent solution is quickly added to the aliquot of a stock NH 2 -A6 (as in Example 4a) solution and mixed well. After the addition of the mPEG-SMB, the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the Glu residue of A6, to provide a Glu-conjugate form of the peptide.
  • a protected A6 e.g., NAc-Lys(Fmoc)-Pro-Ser(tBu)-Ser(tBu)-Pro-Pro-Glu(OBz)-Glu(tBu)-O(tBu)
  • a protected A6 (Prot2-A6, e.g., NAc-Lys(Fmoc)-Pro-Ser(tBu)-Ser(tBu)-Pro-Pro-Glu(OBz)-Glu(tBu)-O(tBu)
  • NAc-Lys(Fmoc)-Pro-Ser(tBu)-Ser(tBu)-Pro-Pro-Glu(OBz)-Glu(tBu)-O(tBu) is prepared and purified according to standard automated peptide synthesis techniques known to those
  • a 5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content.
  • the mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot3-A6 is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot3-A6-(Glu-O-mPEG) conjugate formation.
  • the remaining protecting groups are removed under standard deprotection conditions to yield the A6-Glu(O-mPEG) conjugate.
  • Islet Neogenesis-Associated Protein is a member of the Reg family of proteins implicated in various settings of endogenous pancreatic regeneration. The expression of INGAP and other RegIII proteins has also been linked with the induction of islet neogenesis in animal models of disease and regeneration. Administration of a peptide fragment of INGAP (INGAP peptide) has been demonstrated to reverse chemically induced diabetes as well as improve glycemic control and survival in an animal model of type 1 diabetes. (Lipsett et al., Cell Biochem. Biophys. 2007, 48, 127).
  • INGAP peptide is a 15 amino acid sequence contained within the 175 amino acid INGAP (see, amino acids 103-117 of SEQ ID. NO: 2 of U.S. Pat. No. 5,834,590): Ile-Gly-Leu-His-Asp-Pro-Ser-His-Gly-Thr-Leu-Pro-Asn-Gly-Ser.
  • INGAPP is prepared and purified according to standard automated peptide synthesis or recombinant techniques known to those skilled in the art.
  • An illustrative polymeric reagent, mPEG-SPC reagent, is covalently attached to the N-terminus of INGAPP, to provide a N ter -conjugate form of the peptide.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content.
  • the mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of INGAPP prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved.
  • the stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the reaction is optionally quenched to terminate the reaction.
  • the pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1M HCl, if necessary, to bring the pH of the final solution to about 5.5.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -INGAPP conjugate formation.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the C-terminus of INGAPP, to provide a C r -conjugate form of the peptide.
  • a protected INGAPP Prot-INGAPP, e.g Fmoc-Ile-Gly-Leu-His-Asp(tBu)-Pro-Ser(tBu)-His-Gly-Thr(tBu)-Leu-Pro-Asn-Gly-Ser(tBu)-OH
  • a protected INGAPP Prot-INGAPP, e.g Fmoc-Ile-Gly-Leu-His-Asp(tBu)-Pro-Ser(tBu)-His-Gly-Thr(tBu)-Leu-Pro-Asn-Gly-Ser(tBu)-OH
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content. The mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot-INGAPP is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-INGAPP-C ter -mPEG conjugate formation. The remaining protecting groups are removed under standard deprotection conditions to yield the INGAPP-C ter -mPEG conjugate.
  • mPEG-N-Hydroxysuccinimide having a molecular weight of 5,000 Daltons and having the basic structure shown below: mPEG-SMB, 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature. A five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution. The 10% reagent solution is quickly added to the aliquot of a stock INGAPP solution and mixed well. After the addition of the mPEG-SMB, the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the Asp residue of INGAPP, to provide a Asp-conjugate form of the peptide.
  • a protected INGAPP Prot2-INGAPP, e.g., Fmoc-Ile-Gly-Leu-His-Asp(OBz)-Pro-Ser(tBu)-His-Gly-Thr(tBu)-Leu-Pro-Asn-Gly-Ser(tBu)-O(tBu)
  • a protected INGAPP Prot2-INGAPP, e.g., Fmoc-Ile-Gly-Leu-His-Asp(OBz)-Pro-Ser(tBu)-His-Gly-Thr(tBu)-Leu-Pro-Asn-Gly-Ser(tBu)-O(tBu)
  • a 5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content.
  • the mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot3-INGAPP is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot3-INGAPP-(Asp-O-mPEG) conjugate formation.
  • the remaining protecting groups are removed under standard deprotection conditions to yield the INGAPP-Asp(O-mPEG) conjugate.
  • Tendamistat (HOE 467) is 74 residue alpha-amylase inactivator which effectively attenuates starch digestion (Meyer et al., S. Afr. Med. J. 1984, 66, 222), having the sequence, Asp-Thr-Thr-Val-Ser-Glu-Pro-Ala-Pro-Ser-Cys-Val-Thr-Leu-Tyr-Gln-Ser-Tip-Arg-Tyr-Ser-Gln-Ala-Asp-Asp-Gly-Cys-Ala-Glu-Thr-Val-Thr-Val-Lys-Val-Val-Tyr-Glu-Asp-Asp-Thr-Glu-Gly-Leu-Cys-Tyr-Ala-Val-Ala-Pro-Gly-Gln-Ile-Thr-Thr-Val-Gly-Asp-Gly-Tyr-Ile-Gly-Ser-His-Gly-His
  • Tendamistat is prepared and purified according to standard automated peptide synthesis or recombinant techniques known to those skilled in the art.
  • An illustrative polymeric reagent, mPEG-SPC reagent is covalently attached to the N-terminus of Tendamistat, to provide a N tre -conjugate form of the peptide.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content.
  • the mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Tendamistat prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved.
  • the stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the reaction is optionally quenched to terminate the reaction.
  • the pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1M HCl, if necessary, to bring the pH of the final solution to about 5.5.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -Tendamistat conjugate formation.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the C-terminus of Tendamistat, to provide a C ter -conjugate form of the peptide.
  • a protected Tendamistat Prot-Tendamistat, e.g., Fmoc-Asp(tBu)-Thr(tBu)-Thr(tBu)-Val-Ser(tBu)-Glu(tBu)-Pro-Ala-Pro-Ser(tBu)-Cys(tBu)-Val-Thr(tBu)-Leu-Tyr(tBu)-Gln-Ser(tBu)-Trp-Arg(Tos)-Tyr-Ser(tBu)-Gln-Ala-Asp(tBu)-Asp(tBu)-Gly-Cys(tBu)-Ala-Glu(tBu)-Thr(tBu)-Val-Thr
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content. The mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot-Tendamistat is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-Tendamistat-C ter -mPEG conjugate formation. The remaining protecting groups are removed under standard deprotection conditions to yield the Tendamistat-C ter -mPEG conjugate.
  • Tendamistat which has a thiol-containing cysteine residue, is dissolved in buffer. To this peptide solution is added a 3-5 fold molar excess of mPEG-MAL, 5 kDa. The mixture is stirred at room temperature under an inert atmosphere for several hours. Analysis of the reaction mixture reveals successful conjugation of this peptide.
  • mPEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock Tendamistat solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the Glu residue of Tendamistat, to provide a Glu-conjugate form of the peptide.
  • a protected Tendamistat Prot2-Tendamistat, e.g, Fmoc-Asp(tBu)-Thr(tBu)-Thr(tBu)-Val-Ser(tBu)-Glu(OBz)-Pro-Ala-Pro-Ser(tBu)-Cys(tBu)-Val-Thr(tBu)-Leu-Tyr(tBu)-Gln-Ser(tBu)-Trp-Arg(Tos)-Tyr-Ser(tBu)-Gln-Ala-Asp(tBu)-Asp(tBu)-Gly-Cys(tBu)-Ala-Glu(tBu)-Thr(tBu)-Val-Thr(tBu)-
  • the stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot3-Tendamistat-(Glu-O-mPEG) conjugate formation.
  • the remaining protecting groups are removed under standard deprotection conditions to yield the Tendamistat-Glu(O-mPEG) conjugate.
  • Carperitide ( ⁇ -atriopeptin) is secreted by the heart, is a member of the natriuretic peptide family which is comprised of peptides secreted by various organs. Carperitide is has been proposed for the treatment of acute heart failure and shown therapeutic potential to treat peripheral arterial diseases refractory to conventional therapies (Park et al., Endocrinology 2008, 149, 483).
  • Carperitide has the amino acid sequence Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr (SLRRSSCFGGRMDRIGAQSGLGCNSFRY).
  • Carperitide can be prepared and purified according to standard automated peptide synthesis or recombinant techniques known to those skilled in the art.
  • An illustrative polymeric reagent, mPEG-SPC reagent is covalently attached to the N-terminus of Carperitide, to provide a N ter -conjugate form of the peptide.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used, based upon absolute peptide content.
  • the mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Carperitide prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is rapidly stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved.
  • the stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the reaction is optionally quenched to terminate the reaction.
  • the pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1M HCl, if necessary, to bring the pH of the final solution to about 5.5.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -Carperitide conjugate formation.
  • conjugates can be prepared using mPEG derivatives having other weight-average molecular weights that also bear an N-hydroxysuccinimide moiety.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the C-terminus of Carperitide, to provide a C ter -conjugate form of the peptide.
  • a protected Carperitide (Prot-Carperitide, e.g., Fmoc-Ser(tBu)-Leu-Arg(Tos)-Arg(Tos)-Ser(tBu)-Ser(tBu)-Cys(tBu)-Phe-Gly-Gly-Arg(Tos)-Met-Asp(tBu)-Arg(Tos)-Ile-Gly-Ala-Gln-Ser(tBu)-Gly-Leu-Gly-Cys(tBu)-Asn-Ser(tBu)-Phe-Arg(Tos)-Tyr(tBu)-OH) can be prepared and purified according to standard automated peptide synthesis or re
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content. The mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot-Carperitide is prepared in N,N-dimethylformamide is added and the mixture is rapidly stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-Carperitide-C ter -mPEG conjugate formation. The remaining protecting groups are removed under standard deprotection conditions to yield the Carperitide-C ter -mPEG conjugate.
  • conjugates can be prepared using mPEG derivatives having other weight-average molecular weights that also bear an amino moiety.
  • Carperitide which has a thiol-containing cysteine residue, is dissolved in buffer. To this peptide solution is added a 3-5 fold molar excess of mPEG-MAL, 5 kDa. The mixture is stirred at room temperature under an inert atmosphere for several hours. Analysis of the reaction mixture reveals successful conjugation of this peptide.
  • mPEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock Carperitide solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • conjugates can be prepared using mPEG derivatives having other weight-average molecular weights that also bear an N-hydroxysuccinimide moiety.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the Asp residue of Carperitide, to provide a Asp-conjugate form of the peptide.
  • a protected Carperitide (Prot2-Carperitide, e.g., Fmoc-Ser(tBu)-Leu-Arg(Tos)-Arg(Tos)-Ser(tBu)-Ser(tBu)-Cys(tBu)-Phe-Gly-Gly-Arg(Tos)-Met-Asp(OBz)-Arg(Tos)-Ile-Gly-Ala-Gln-Ser(tBu)-Gly-Leu-Gly-Cys(tBu)-Asn-Ser(tBu)-Phe-Arg(Tos)-Tyr(tBu)-O(tBu)) is prepared and purified according to standard automated peptide synthesis techniques known to those
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. A 5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content. The mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot3-Carperitide is prepared in N,N-dimethylformamide is added and the mixture is rapidly stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot3-Carperitide-(Asp-O-mPEG) conjugate formation. The remaining protecting groups are removed under standard deprotection conditions to yield the Carperitide-Asp(O-mPEG) conjugate.
  • Urodilatin is a member of the natriuretic peptide family which is comprised of peptides secreted by various organs, has been studied for use in treating various conditions, including renal failure or congestive heart failure (see, e.g., U.S. Pat. Nos. 5,571,789 and 6,831,064; Kentsch et al., Eur. J. Clin. Invest. 1992, 22, 662; Kentsch et al., Eur. J. Clin. Invest. 1995, 25, 281; Elsner et al., Am. Heart J. 1995, 129, 766; Forssmann et al., Clinical Pharmacology and Therapeutics 1998, 64, 322; and US Patent Application Publication No.
  • Urodilatin has the amino acid sequence set forth in GenBank Accession No. 1506430A; Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr (TAPRSLRRSS CFGGRMDRIG AQSGLGCNSF RY). Urodilatin is also the 95-126 fragment [ANP(95-126)] of atrial natriuretic peptide (ANP).
  • Urodilatin is prepared and purified according to standard automated peptide synthesis or recombinant techniques known to those skilled in the art.
  • An illustrative polymeric reagent, mPEG-SPC reagent is covalently attached to the N-terminus of Urodilatin, to provide a N ter -conjugate form of the peptide.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content.
  • the mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Urodilatin prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved.
  • the stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the reaction is optionally quenched to terminate the reaction.
  • the pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1M HCl, if necessary, to bring the pH of the final solution to about 5.5.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -Urodilatin conjugate formation.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the C-terminus of Urodilatin, to provide a C ter -conjugate form of the peptide.
  • a protected Urodilatin e.g., Fmoc-Thr(tBu)-Ala-Pro-Arg(Tos)-Ser(tBu)-Leu-Arg(Tos)-Arg(Tos)-Ser(tBu)-Ser(tBu)-Cys(tBu)-Phe-Gly-Gly-Arg(Tos)-Met-Asp(tBu)-Arg(Tos)-Ile-Gly-Ala-Gln-Ser(tBu)-Gly-Leu-Gly-Cys(tBu)-Asn-Ser(tBu)-Phe-Arg(Tos)-Tyr(tBu)-OH) is
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content. The mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot-Urodilatin is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-Urodilatin-C ter -mPEG conjugate formation. The remaining protecting groups are removed under standard deprotection conditions to yield the Urodilatin-C ter -mPEG conjugate.
  • mPEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock Urodilatin solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the Asp residue of Urodilatin, to provide a Asp-conjugate form of the peptide.
  • a protected Urodilatin (Prot2-Urodilatin, e.g., Fmoc-Thr(tBu)-Ala-Pro-Arg(Tos)-Ser(tBu)-Leu-Arg(Tos)-Arg(Tos)-Ser(tBu)-Ser(tBu)-Cys(tBu)-Phe-Gly-Gly-Arg(Tos)-Met-Asp(OBz)-Arg(Tos)-Ile-Gly-Ala-Gln-Ser(tBu)-Gly-Leu-Gly-Cys(tBu)-Asn-Ser(tBu)-Phe-Arg(Tos)-Tyr(tBu)-NH 2 ) is prepared and
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. A 5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content. The mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot3-Urodilatin is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot3-Urodilatin-(Asp-O-mPEG) conjugate formation. The remaining protecting groups are removed under standard deprotection conditions to yield the Urodilatin-Asp(O-mPEG) conjugate.
  • Desirudin a recombinant hirudin, is a member of a class of anticoagulants that act by directly inhibiting thrombin. Desirudin acts via a bivalent binding arrangement with both the active site and fibrinogen-binding site (exosite 1) of thrombin, and has been shown to be useful in the prevention and management of thromboembolic disease, reducing the incidence of deep vein thrombosis (DVT) in patients undergoing elective hip replacement, preventing restenosis after coronary angioplasty for unstable angina, and in the treatment of acute coronary syndromes for patients in whom heparin therapy is not a viable option (Matheson and Goa, Drugs 2000, 60, 679).
  • DVDTT deep vein thrombosis
  • Desirudin has the primary sequence Val-Val-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu-Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu-Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val-Thr-Gly-Glu-Gly-Thr-Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Glu-Tyr-Leu-Gln.
  • Desirudin is prepared and purified according to standard automated peptide synthesis or recombinant techniques known to those skilled in the art.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content. The mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Desirudin prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The reaction is optionally quenched to terminate the reaction. The pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1 M HCl, if necessary, to bring the pH of the final solution to about 5.5. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -Desirudin conjugate formation.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the C-terminus of Desirudin, to provide a C ter -conjugate form of the peptide.
  • a protected Desirudin Prot-Val-Val-Tyr(tBu)-Thr(tBu)-Asp(tBu)-Cys(tBu)-Thr(tBu)-Glu(tBu)-Ser(tBu)-Gly-Gln-Asn-Leu-Cys(tBu)-Leu-Cys-Glu(tBu)-Gly-Ser(tBu)-Asn-Val-Cys(tBu)-Gly-Gln-Gly-Asn-Lys(Fmoc)-Cys(tBu)-Ile-Leu-Gly-Ser(tBu)-Asp(tBu)-Gly-Glu(tBu)-L
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content. The mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot-Desirudin is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-Desirudin-C ter -mPEG conjugate formation. The remaining protecting groups are removed under standard deprotection conditions to yield the Desirudin-C ter -mPEG conjugate.
  • Desirudin which has a thiol-containing cysteine residue, is dissolved in buffer. To this peptide solution is added a 3-5 fold molar excess of mPEG-MAL, 5 kDa. The mixture is stirred at room temperature under an inert atmosphere for several hours. Analysis of the reaction mixture reveals successful conjugation of this peptide.
  • mPEG-N-Hydroxysuccinimide having a molecular weight of 5 kDa and having the basic structure shown below:
  • mPEG-Succinimidyl ⁇ -Methylbutanoate Derivative, 5 kDa (“mPEG-SMB”)
  • mPEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock Desirudin solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the Asp residue of Desirudin, to provide a Asp-conjugate form of the peptide.
  • a protected Desirudin Prot2-Desirudin, e.g.
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. A 5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content. The mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot3-Desirudin is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot3-Desirudin-(Asp-O-mPEG) conjugate formation. The remaining protecting groups are removed under standard deprotection conditions to yield the Desirudin-Asp(O-mPEG) conjugate.
  • Obestatin is 28-amino acid, acylated, orexigenic peptide that is a ligand for growth hormone secretagogue receptors and is encoded by the same gene that also encodes ghrelin, a peptide hormone that increases appetite.
  • Treatment of rats with obestatin suppressed food intake, inhibited jejunal contraction, and decreased body-weight gain (Zhang et al., Science 2005, 310, 996).
  • Synthetic human obestatin is available from California Peptide Research, Inc (Napa, Calif.), having the sequence, Phe-Asn-Ala-Pro-Phe-Asp-Val-Gly-Ile-Lys-Leu-Ser-Gly-Val-Gln-Tyr-Gln-Gln-His-Ser-Gln-Ala-Leu-NH 2 (PubChem Substance ID: 47205412).
  • mPEG-SPC reagent An illustrative polymeric reagent, mPEG-SPC reagent, is covalently attached to the N-terminus of Obestatin, to provide a N ter -conjugate form of the peptide.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content.
  • the mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Obestatin prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The reaction is optionally quenched to terminate the reaction. The pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1M HCl, if necessary, to bring the pH of the final solution to about 5.5. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-Mer-Obestatin conjugate formation.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the C-terminus of Obestatin, to provide a C ter -conjugate form of the peptide.
  • a protected Obestatin lacking the C-terminus amide (Prot-Obestatin, e.g., Fmoc-Phe-Asn-Ala-Pro-Phe-Asp(tBu)-Val-Gly-Ile-Lys(Fmoc)-Leu-Ser(tBu)-Gly-Val-Gln-Tyr(tBu)-Gln-Gln-His-Ser(tBu)-Gln-Ala-Leu-OH) is prepared and purified according to standard automated peptide synthesis techniques known to those skilled in the art.
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content. The mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot-Obestatin is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-Obestatin-C ter -mPEG conjugate formation. The remaining protecting groups are removed under standard deprotection conditions to yield the Obestatin-C ter -mPEG conjugate.
  • Obestatin which has a thiol-containing cysteine residue, is dissolved in buffer. To this peptide solution is added a 3-5 fold molar excess of mPEG-MAL, 5 kDa. The mixture is stirred at room temperature under an inert atmosphere for several hours. Analysis of the reaction mixture reveals successful conjugation of this peptide.
  • mPEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock Obestatin solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • PEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature. A five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock protected Obestatin (e.g., Fmoc-Phe-Asn-Ala-Pro-Phe-Asp(tBu)-Val-Gly-Ile-Lys-Leu-Ser(tBu)-Gly-Val-Gln-Tyr(tBu)-Gln-Gln-His-Ser(tBu)-Gln-Ala-Leu-NH 2 ) solution and mixed well.
  • a stock protected Obestatin e.g., Fmoc-Phe-Asn-Ala-Pro-Phe-Asp(tBu)-Val-Gly-Ile-Lys-Leu-Ser(tBu)-Gly-Val-Gln-Tyr(tBu)-Gln-Gln-His-Ser(tBu)-Gln-Ala-Leu-NH 2
  • the reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • the remaining protecting groups are removed under standard deprotection conditions to yield the Obestatin-Lys(O-mPEG) conjugate.
  • ITF-1697 is a tetrapeptide, Gly-(N-Et)Lys-Pro-Arg (PubChem Compound ID: 216295), which reduces mortality and tissue damage in lipopolysaccharide (LPS)-induced systemic endotoxemia and coronary ischemia and ischemia/reperfusion (see, International Patent Application Publication WO 1995/10531.).
  • LPS lipopolysaccharide
  • ITF-1697 is prepared and purified according to standard automated peptide synthesis or recombinant techniques known to those skilled in the art.
  • An illustrative polymeric reagent, mPEG-SPC reagent, is covalently attached to the N-terminus of ITF-1697, to provide a N ter -conjugate form of the peptide.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content.
  • the mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of ITF-1697 prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved.
  • the stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the reaction is optionally quenched to terminate the reaction.
  • the pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1M HCl, if necessary, to bring the pH of the final solution to about 5.5.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -ITF-1697 conjugate formation.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the C-terminus of ITF-1697, to provide a C ter -conjugate form of the peptide.
  • a protected ITF-1697 (Prot-ITF-1697, e.g., Fmoc-Gly-(N-Et)Lys(Fmoc)-Pro-Arg(Tos)-OH) is prepared and purified according to standard automated peptide synthesis techniques known to those skilled in the art.
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature.
  • mPEG-NH 2 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content.
  • the mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot-ITF-1697 is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-ITF-1697-C ter -mPEG conjugate formation.
  • the remaining protecting groups are removed under standard deprotection conditions to yield the ITF-1697-C ter -mPEG conjugate.
  • mPEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock ITF-1697 solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • PEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock protected ITF-1697 (e.g., Fmoc-Gly-(N-Et)Lys-Pro-Arg(Tos)-O(tBu)) solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • the reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • the remaining protecting groups are removed under standard deprotection conditions to yield the ITF-1697-Lys(O-mPEG) conjugate.
  • Oxyntomodulin (Amylin) is a 37-amino acid peptide derived from proglucagon found in the colon, produced by the oxyntic (fundic) cells of the oxyntic mucosa and is known to bind both the Glucagon-like peptide-1 (GLP-1) and the glucagon receptors.
  • GLP-1 Glucagon-like peptide-1
  • Oxyntomodulin suppresses appetite and food intake (Cohen et al., J. Clin. Endocrin. Met. 2003, 88, 4696).
  • Oxyntomodulin is commercially available from GenScript Corporation (Piscataway, N.J.; Cat. No.
  • mPEG-SPC reagent An illustrative polymeric reagent, mPEG-SPC reagent, is covalently attached to the N-terminus of Oxyntomodulin, to provide a N ter -conjugate form of the peptide.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content.
  • the mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Oxyntomodulin prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The reaction is optionally quenched to terminate the reaction. The pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1M HCl, if necessary, to bring the pH of the final solution to about 5.5. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -Oxyntomodulin conjugate formation.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the C-terminus of Oxyntomodulin, to provide a C ter -conjugate form of the peptide.
  • a protected Oxyntomodulin lacking the C-terminus amide (Prot-Oxyntomodulin, e.g., Fmoc-Lys(Fmoc)-Cys(tBu)-Asn-Thr(tBu)-Ala-Thr(tBu)-Cys(tBu)-Ala-Thr(tBu)-Gln-Arg(Tos)-Leu-Ala-Asn-Phe-Leu-Val-His-Ser(tBu)-Ser(tBu)-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser(tBu)-Ser(tBu)-Thr(tBu)-Asn
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content. The mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot-Oxyntomodulin is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-Oxyntomodulin-C ter -mPEG conjugate formation. The remaining protecting groups are removed under standard deprotection conditions to yield the Oxyntomodulin-C ter -mPEG conjugate.
  • Oxyntomodulin which has a thiol-containing cysteine residue, is dissolved in buffer. To this peptide solution is added a 3-5 fold molar excess of mPEG-MAL, 5 kDa. The mixture is stirred at room temperature under an inert atmosphere for several hours. Analysis of the reaction mixture reveals successful conjugation of this peptide.
  • mPEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock Oxyntomodulin solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • PEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature. A five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • Oxyntomodulin e.g., Fmoc-Lys-Cys(tBu)-Asn-Thr(tBu)-Ala-Thr(tBu)-Cys(tBu)-Ala-Thr(tBu)-Gln-Arg(Tos)-Leu-Ala-Asn-Phe-Leu-Val-His-Ser(tBu)-Ser(tBu)-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser(tBu)-Ser(tBu)-Thr(tBu)-Asn-Val-Gly-Ser(tBu)-Asn-Thr(tBu)-Tyr(tBu)-O(tBu)) solution and mixed well.
  • Oxyntomodulin e.g., Fmoc-Lys-Cys(tBu)-Asn-Thr(tBu)-Ala-Thr(tBu)-Cys(tBu
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • the reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • the remaining protecting groups are removed under standard deprotection conditions to yield the Oxyntomodulin-Lys(O-mPEG) conjugate.
  • Cholecystokinin is a peptide hormone secreted by the upper intestinal mucosa which increases gallbladder contraction, release of pancreatic exocrine (or digestive) enzymes, and is responsible for stimulating the digestion of fat and proteins. Cholecystokinin has also been shown to be a physiologic regulator of gastric emptying in humans (Liddle et al., J. Clin. Invest. 1986, 77, 992).
  • Cholecystokinin has the sequence, Met-Asn-Ser-Gly-Val-Cys-Leu-Cys-Val-Leu-Met-Ala-Val-Leu-Ala-Ala-Gly-Ala-Leu-Thr-Gln-Pro-Val-Pro-Pro-Ala-Asp-Pro-Ala-Gly-Ser-Gly-Leu-Gln-Arg-Ala-Glu-Glu-Ala-Pro-Arg-Arg-Gln-Leu-Arg-Val-Ser-Gln-Arg-Thr-Asp-Gly-Glu-Ser-Arg-Ala-His-Leu-Gly-Ala-Leu-Leu-Ala-Arg-Tyr-Ile-Gln-Gln-Ala-Arg-Lys-Ala-Pro-Ser-Gly-Arg-Met-Ser-Ile-Val-Lys-As-As-As-Leu-Gl
  • Cholecystokinin is prepared and purified according to standard automated peptide synthesis or recombinant techniques known to those skilled in the art.
  • An illustrative polymeric reagent, mPEG-SPC reagent, is covalently attached to the N-terminus of Cholecystokinin, to provide a N ter -conjugate form of the peptide.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content.
  • the mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Cholecystokinin prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved.
  • the stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the reaction is optionally quenched to terminate the reaction.
  • the pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1M HCl, if necessary, to bring the pH of the final solution to about 5.5.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -Cholecystokinin conjugate formation.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the C-terminus of Cholecystokinin, to provide a C ter -conjugate form of the peptide.
  • a protected Cholecystokinin Prot-Cholecystokinin, e.g., Fmoc-Met-Asn-Ser(tBu)-Gly-Val-Cys(tBu)-Leu-Cys(tBu)-Val-Leu-Met-Ala-Val-Leu-Ala-Ala-Gly-Ala-Leu-Thr(tBu)-Gln-Pro-Val-Pro-Pro-Ala-Asp(tBu)-Pro-Ala-Gly-Ser(tBu)-Gly-Leu-Gln-Arg(Tos)-Ala-Glu(tBu)-Glu(tBu)-Ala-Pro-Arg
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content. The mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot-Cholecystokinin is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-Cholecystokinin-C ter -mPEG conjugate formation. The remaining protecting groups are removed under standard deprotection conditions to yield the Cholecystokinin-C ter -mPEG conjugate.
  • Cholecystokinin which has a thiol-containing cysteine residue, is dissolved in buffer. To this peptide solution is added a 3-5 fold molar excess of mPEG-MAL, 5 kDa. The mixture is stirred at room temperature under an inert atmosphere for several hours. Analysis of the reaction mixture reveals successful conjugation of this peptide.
  • mPEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock Cholecystokinin solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the Glu residue of Cholecystokinin, to provide a Glu-conjugate form of the peptide.
  • a protected Cholecystokinin Prot2-Cholecystokinin, e.g., Fmoc-Met-Asn-Ser(tBu)-Gly-Val-Cys(tBu)-Leu-Cys(tBu)-Val-Leu-Met-Ala-Val-Leu-Ala-Ala-Gly-Ala-Leu-Thr(tBu)-Gln-Pro-Val-Pro-Pro-Ala-Asp(tBu)-Pro-Ala-Gly-Ser(tBu)-Gly-Leu-Gln-Arg(Tos)-Ala-Glu(OBz)-Glu(tBu)-Ala-Pro-Arg(Tos)-Ala-Glu(OB
  • the stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot3-Cholecystokinin-(Glu-O-mPEG) conjugate formation.
  • the remaining protecting groups are removed under standard deprotection conditions to yield the Cholecystokinin-Glu(O-mPEG) conjugate.
  • Bactericidal permeability increasing protein is a 487 residue ( ⁇ 50 kDa) protein which is part of the innate immune system and which displays selective cytotoxicity toward gram-negative bacteria through binding to lipopolysaccharides produced by the bacteria.
  • BPI has the sequence, MRENMARGPC NAPRWVSLMV LVAIGTAVTA AVNPGVVVRI SQKGLDYASQ QGTAALQKEL KRIKIPDYSD SFKIKHLGKG HYSFYSMDIR EFQLPSSQIS MVPNVGLKFS ISNANIKISG KWKAQKRFLK MSGNFDLSIE GMSISADLKL GSNPTSGKPT ITCSSCSSHI NSVHVHISKS KVGWLIQLFH KKIESALRNK MNSQVCEKVT NSVSSKLQPY FQTLPVMTKI DSVAGINYGL VAPPATTAET LDVQMKGEFY SENHHNPPPF APPVMEFPAA HDRMVYLGLS DYFFNTAGLV YQEAGVLKMT LRDDMIPKES KFRLTTKFFG TFLPEVAKKF PNMKIQIHVS ASTPPHLSVQ PTGLTFYPAV DVQAFAVLPN SSLASLFLIG MHTT
  • BPI is prepared and purified according to standard recombinant techniques known to those skilled in the art.
  • An illustrative polymeric reagent, mPEG-SPC reagent is covalently attached to the N-terminus of BPI, to provide a M′′-conjugate form of the peptide.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content.
  • the mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of BPI prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The reaction is optionally quenched to terminate the reaction. The pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1M HCl, if necessary, to bring the pH of the final solution to about 5.5. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -BPI conjugate formation.
  • mPEG-NH 2 reagent is covalently attached to the C-terminus of BPI, to provide a C ter -conjugate form of the peptide.
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content.
  • the mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of BPI is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved.
  • the stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of BPI-C ter -mPEG conjugate formation.
  • the C ter conjugate is isolated and purified according the general procedure outlined above.
  • BPI which has a thiol-containing cysteine residue
  • buffer To this peptide solution is added a 3-5 fold molar excess of mPEG-MAL, 5 kDa. The mixture is stirred at room temperature under an inert atmosphere for several hours. Analysis of the reaction mixture reveals successful conjugation of this peptide.
  • mPEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock BPI solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • the reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • the N ter conjugate is isolated and according the general procedure outlined above.
  • PEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock BPI solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • the reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • the Lys conjugate is isolated and purified according the general procedure outlined above to yield the BPI-Lys-mPEG conjugate.
  • C-peptide is a product of the cleavage of proinsulin, consisting of the B and A chains of insulin linked together via a connecting C-peptide, produced when proinsulin is released into the blood stream in response to a rise in serum glucose.
  • C-peptide has the sequence, Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln (U.S. Pat. No. 6,610,649).
  • C-peptide alone has been proposed for the treatment of diabetes (EP 132 769); insulin in combination with C-peptide can be administered for the prevention of diabetic complications (SE 460334).
  • C-peptide is prepared and purified according to standard automated peptide synthesis or recombinant techniques known to those skilled in the art.
  • An illustrative polymeric reagent, mPEG-SPC reagent is covalently attached to the N-terminus of C-peptide, to provide a N ter -conjugate form of the peptide.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content.
  • the mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of C-peptide prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved.
  • the stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the reaction is optionally quenched to terminate the reaction.
  • the pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1M HCl, if necessary, to bring the pH of the final solution to about 5.5.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -C-peptide conjugate formation.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the C-terminus of C-peptide, to provide a C ter -conjugate form of the peptide.
  • a protected C-peptide (Prot-C-peptide, e.g., Fmoc-Glu(tBu)-Ala-Glu(tBu)-Asp(tBu)-Leu-Gln-Val-Gly-Gln-Val-Glu(tBu)-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser(tBu)-Leu-Gln-Pro-Leu-Ala-Leu-Glu(tBu)-Gly-Ser(tBu)-Leu-Gln) is prepared and purified according to standard automated peptide synthesis techniques known to those skilled in the art.
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content. The mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot-C-peptide is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-C-peptide-C ter -mPEG conjugate formation. The remaining protecting groups are removed under standard deprotection conditions to yield the C-peptide-C ter -mPEG conjugate.
  • mPEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock C-peptide solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the Glu residue of C-peptide, to provide a Glu-conjugate form of the peptide.
  • a protected C-peptide (Prot2 C-peptide, e.g., Fmoc-Glu(tBu)-Ala-Glu(tBu)-Asp(tBu)-Leu-Gln-Val-Gly-Gln-Val-Glu(OBz)-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser(tBu)-Leu-Gln-Pro-Leu-Ala-Leu-Glu(tBu)-Gly-Ser(tBu)-Leu-Gln)-O(tBu)) is prepared and purified according to standard automated peptide synthesis techniques known to those skilled in the art.
  • the stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot3-C-peptide-(Glu-O-mPEG) conjugate formation.
  • the remaining protecting groups are removed under standard deprotection conditions to yield the C-peptide-Glu(O-mPEG) conjugate.
  • Prosaptide TX14(A) is a 14-mer amino acid sequence derived from the active neurotrophic region in the amino-terminal portion of the saposin C domain. Prosaptides are active on a variety of neuronal cells, stimulating sulfatide synthesis and increasing sulfatide concentration in Schwann cells and oligodendrocytes. This indicates that prosaposin and prosaptides are trophic factors for myelin formation. Prosaptide TX14(A) may have potential for therapeutic use in neuropathic pain syndromes in humans (Otero et al. Neurosci. Lett. 1999, 270, 29).
  • Prosaptide TX14(A) is commercially available from AnaSpec (San Jose, Calif.) with the sequence, Thr-(D-Ala)-Leu-Ile-Asp-Asn-Asn-Ala-Thr-Glu-Glu-Ile-Leu-Tyr.
  • mPEG-SPC reagent An illustrative polymeric reagent, mPEG-SPC reagent, is covalently attached to the N-terminus of Prosaptide TX14(A), to provide a N ter -conjugate form of the peptide.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content.
  • the mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prosaptide TX14(A) prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The reaction is optionally quenched to terminate the reaction. The pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1M HCl, if necessary, to bring the pH of the final solution to about 5.5. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -Prosaptide TX14(A) conjugate formation.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the C-terminus of Prosaptide TX14(A), to provide a C ter -conjugate form of the peptide.
  • a protected Prosaptide TX14(A) (Prot-Prosaptide TX14(A), e.g., Fmoc-Thr(tBu)-(D-Ala)-Leu-Ile-Asp(tBu)-Asn-Asn-Ala-Thr(tBu)-Glu(tBu)-Glu(tBu)-Ile-Leu-Tyr(tBu) is prepared and purified according to standard automated peptide synthesis techniques known to those skilled in the art.
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content. The mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot-Prosaptide TX14(A) is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-Prosaptide TX14(A)-C ter -mPEG conjugate formation. The remaining protecting groups are removed under standard deprotection conditions to yield the Prosaptide TX14(A)-C ter -mPEG conjugate.
  • mPEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock Prosaptide TX14(A) solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the Glu residue of Prosaptide TX14(A), to provide a Glu-conjugate form of the peptide.
  • a protected Prosaptide TX14(A) (Prot2-Prosaptide TX14(A), e.g., Fmoc-Thr(tBu)-(D-Ala)-Leu-Ile-Asp(tBu)-Asn-Asn-Ala-Thr(tBu)-Glu(OBz)-Glu(tBu)-Ile-Leu-Tyr(tBu)-O(tBu)) is prepared and purified according to standard automated peptide synthesis techniques known to those skilled in the art.
  • a 5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content.
  • the mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot3-Prosaptide TX14(A) is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot3-Prosaptide TX14(A-(Glu-O-mPEG) conjugate formation.
  • the remaining protecting groups are removed under standard deprotection conditions to yield the Prosaptide TX14(A)-Glu(O-mPEG) conjugate.
  • Sermorelin is the biologically active fragment of human growth hormone-releasing factor, consisting of GHRH (1-29)-amide, which can be used as a provocative test of growth hormone deficiency (Prakash and Goa, Biodrugs 1999, 12, 139). Sermoline may also increase IGF-1 levels and improve body composition (increased lean mass and reduced truncal and visceral fat) in patients with HIV (Koutkia et al, JAMA 2004, 292, 210).
  • Synthetic sermorelin acetate is commercially available from Gelacs Innovation (Hangzhou, China) with the sequence, Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH 2
  • mPEG-SPC reagent An illustrative polymeric reagent, mPEG-SPC reagent, is covalently attached to the N-terminus of Sermorelin, to provide a N ter -conjugate form of the peptide.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content.
  • the mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Sermorelin prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The reaction is optionally quenched to terminate the reaction. The pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1M HCl, if necessary, to bring the pH of the final solution to about 5.5. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -Sermorelin conjugate formation.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the C-terminus of Sermorelin, to provide a C a -conjugate form of the peptide.
  • a protected Sermorelin lacking the C-terminus amide (Prot-Sermorelin, e.g., Fmoc-Tyr-Ala-Asp(tBu)-Ala-Ile-Phe-Thr-Asn-Ser(tBu)-Tyr(tBu)-Arg(Tos)-Lys-Val-Leu-Gly-Gln-Leu-Ser(tBu)-Ala-Arg(Tos)-Lys(Fmoc)-Leu-Leu-Gln-Asp(tBu)-Ile-Met-Ser(tBu)-Arg(Tos)-OH) is prepared and purified according to standard automated peptide synthesis techniques known to
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content. The mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot-Sermorelin is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-Sermorelin-C ter -mPEG conjugate formation. The remaining protecting groups are removed under standard deprotection conditions to yield the Sermorelin-C ter -mPEG conjugate.
  • mPEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature.
  • a five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock Sermorelin solution and mixed well.
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • PEG-SMB 5 kDa, stored at ⁇ 20° C. under argon, is warmed to ambient temperature. A five-fold excess (relative to the amount of the peptide) of the warmed mPEG-SMB is dissolved in buffer to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of a stock protected Sermorelin (e.g., Fmoc-Tyr-Ala-Asp(tBu)-Ala-Ile-Phe-Thr-Asn-Ser(tBu)-Tyr(tBu)-Arg(Tos)-Lys-Val-Leu-Gly-Gln-Leu-Ser(tBu)-Ala-Arg(Tos)-Lys-Leu-Leu-Gln-Asp(tBu)-Ile-Met-Ser(tBu)-Arg(Tos)-NH 2 ) solution and mixed well.
  • Sermorelin e.g., Fmoc-Tyr-Ala-Asp(tBu)-Ala-Ile-Phe-Thr-Asn-Ser(tBu)-Tyr(tBu)-Arg(Tos)-Lys-Val-Leu-Gly-Gln-Le
  • the pH of the reaction mixture is determined and adjusted to 6.7 to 6.8 using conventional techniques.
  • the reaction solution is stirred for several hours (e.g., 5 hours) at room temperature in the dark or stirred overnight at 3-8° C. in a cold room, thereby resulting in a conjugate solution.
  • the reaction is quenched with a 20-fold molar excess (with respect to the peptide) of Tris buffer.
  • the remaining protecting groups are removed under standard deprotection conditions to yield the Sermorelin-Lys(O-mPEG) conjugate.
  • Pralmorelin is a growth-hormone releasing peptide having the composition, D-Ala-([3-(naphthalen-2-yl)]-D-Ala)-Ala-Trp-(D-Phe)-Lys-NH 2 .
  • Pralmorelin has been proposed for the diagnosis of serious growth hormone deficiency and for treatment of short stature (Furata et al. Arz .- Forsch. 2004, 54, 868), and fro treating acute heart failure, chronic heart failure at a phase of acute exacerbation, and heart failure at a phase of transition to chronic heart failure (U.S. Pat. No. 6,878,689).
  • Pralmorelin is prepared and purified according to standard automated peptide synthesis or recombinant techniques known to those skilled in the art.
  • An illustrative polymeric reagent, mPEG-SPC reagent is covalently attached to the N-terminus of Pralmorelin, to provide a N ter -conjugate form of the peptide.
  • mPEG-SPC 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-SPC 20 kDa reagent is used based upon absolute peptide content.
  • the mPEG-SPC reagent is weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Pralmorelin prepared in phosphate buffered saline, PBS, pH 7.4 is added and the mixture is stirred using a magnetic stirrer until the mPEG-SPC is fully dissolved.
  • the stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product.
  • the reaction is optionally quenched to terminate the reaction.
  • the pH of the conjugate solution at the end of the reaction is measured and further acidified by addition of 0.1M HCl, if necessary, to bring the pH of the final solution to about 5.5.
  • the conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of mPEG-N ter -Pralmoreling conjugate formation.
  • An illustrative polymeric reagent, mPEG-NH 2 reagent is covalently attached to the C-terminus of Pralmorelin, to provide a C a -conjugate form of the peptide.
  • a protected Pralmorelin lacking the C-terminus amide (Prot-Pralmorelin, e.g., Fmoc-D-Ala-([3-(naphthalen-2-yl)]-D-Ala)-Ala-Trp-(D-Phe)-Lys(Fmoc)-OH) is prepared and purified according to standard automated peptide synthesis techniques known to those skilled in the art.
  • mPEG-NH 2 20 kDa stored at ⁇ 20° C. under argon, is warmed to ambient temperature. The reaction is performed at room temperature. About 3-5-fold molar excess of mPEG-NH 2 , PyBOP (benzotriazol-1-yloxy)tripyrrolidinonophosphonium hexafluorophosphate), and 1-hydroxybenzotriazole (HOBt) are used, based upon absolute peptide content. The mPEG-NH 2 , PyBOP, HOBt are weighed into a glass vial containing a magnetic stirrer bar.
  • a solution of Prot-Pralmorelin is prepared in N,N-dimethylformamide is added and the mixture is stirred using a magnetic stirrer until the mPEG-NH 2 is fully dissolved. The stirring speed is reduced and the reaction is allowed to proceed to formation of conjugate product. The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) to determine the extent of Prot-Pralmorelin-C ter -mPEG conjugate formation. The remaining protecting groups are removed under standard deprotection conditions to yield the Pralmorelin-C ter -mPEG conjugate.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Diabetes (AREA)
  • Endocrinology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Emergency Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Obesity (AREA)
  • Medicinal Preparation (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
US13/119,297 2008-09-19 2009-09-17 Polymer conjugates of therapeutic peptides Abandoned US20110166063A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US13/119,297 US20110166063A1 (en) 2008-09-19 2009-09-17 Polymer conjugates of therapeutic peptides

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US19267208P 2008-09-19 2008-09-19
US20808909P 2009-02-18 2009-02-18
US15396609P 2009-02-19 2009-02-19
US13/119,297 US20110166063A1 (en) 2008-09-19 2009-09-17 Polymer conjugates of therapeutic peptides
PCT/US2009/005192 WO2010033207A1 (en) 2008-09-19 2009-09-17 Polymer conjugates of therapeutic peptides

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2009/005192 A-371-Of-International WO2010033207A1 (en) 2008-09-19 2009-09-17 Polymer conjugates of therapeutic peptides

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14/664,582 Continuation US9682153B2 (en) 2008-09-19 2015-03-20 Polymer conjugates of therapeutic peptides

Publications (1)

Publication Number Publication Date
US20110166063A1 true US20110166063A1 (en) 2011-07-07

Family

ID=41226622

Family Applications (5)

Application Number Title Priority Date Filing Date
US13/119,297 Abandoned US20110166063A1 (en) 2008-09-19 2009-09-17 Polymer conjugates of therapeutic peptides
US13/119,276 Active 2030-04-27 US8680263B2 (en) 2008-09-19 2009-09-17 Carbohydrate-based drug delivery polymers and conjugates thereof
US14/167,271 Active US9173951B2 (en) 2008-09-19 2014-01-29 Carbohydrate-based drug delivery polymers and conjugates thereof
US14/664,582 Expired - Fee Related US9682153B2 (en) 2008-09-19 2015-03-20 Polymer conjugates of therapeutic peptides
US15/581,348 Abandoned US20190070251A1 (en) 2008-09-19 2017-04-28 Polymer conjugates of therapeutic peptides

Family Applications After (4)

Application Number Title Priority Date Filing Date
US13/119,276 Active 2030-04-27 US8680263B2 (en) 2008-09-19 2009-09-17 Carbohydrate-based drug delivery polymers and conjugates thereof
US14/167,271 Active US9173951B2 (en) 2008-09-19 2014-01-29 Carbohydrate-based drug delivery polymers and conjugates thereof
US14/664,582 Expired - Fee Related US9682153B2 (en) 2008-09-19 2015-03-20 Polymer conjugates of therapeutic peptides
US15/581,348 Abandoned US20190070251A1 (en) 2008-09-19 2017-04-28 Polymer conjugates of therapeutic peptides

Country Status (7)

Country Link
US (5) US20110166063A1 (enrdf_load_stackoverflow)
EP (3) EP2341942A1 (enrdf_load_stackoverflow)
JP (2) JP5765813B2 (enrdf_load_stackoverflow)
AU (1) AU2009292643B2 (enrdf_load_stackoverflow)
CA (1) CA2737040C (enrdf_load_stackoverflow)
MX (1) MX2011003117A (enrdf_load_stackoverflow)
WO (2) WO2010033207A1 (enrdf_load_stackoverflow)

Cited By (60)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110165113A1 (en) * 2008-09-19 2011-07-07 Nektar Therapeutics Polymer conjugates of v681-like peptides
US20110171161A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of protegrin peptides
US20110171164A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of glp-2-like peptides
US20110171166A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of osteocalcin peptides
US20110171163A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of ziconotide peptides
US20110171165A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of opioid growth factor peptides
US20110171160A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of kiss1 peptides
US20110171162A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of thymosin alpha 1 peptides
US20110171312A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Modified therapeutic peptides, methods of their preparation and use
US20110237524A1 (en) * 2008-09-19 2011-09-29 Nektar Therapeutics Polymer conjugates of aod-like peptides
WO2013059525A1 (en) * 2011-10-18 2013-04-25 Aileron Therapeutics, Inc. Peptidomimetic macrocyles
WO2013078222A1 (en) * 2011-11-23 2013-05-30 Quest Diagnostics Investments Incorporated Kisspeptin-54 detection by tandem mass spectrometry
US20140161884A1 (en) * 2012-12-10 2014-06-12 The Massachusetts General Hospital Multistage Nanoparticle Drug Delivery System for the Treatment of Solid Tumors
US20150050213A1 (en) * 2013-08-14 2015-02-19 University Of Virginia Patent Foundation Compositions and methods for imaging inflammation of traumatic brain injury
US20150299257A1 (en) * 2012-05-21 2015-10-22 Allotech Co. Ltd. Bioactive peptide complexes
WO2015048072A3 (en) * 2013-09-24 2015-10-29 Board Of Regents, The University Of Texas System Mucin-associated surface protein as vaccine against chagas disease
US9173950B2 (en) 2012-05-17 2015-11-03 Extend Biosciences, Inc. Vitamin D-ghrelin conjugates
US9249407B2 (en) 2006-02-03 2016-02-02 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
WO2016019340A1 (en) * 2014-07-31 2016-02-04 Serina Therapeutics, Inc. Polyoxazoline antibody drug conjugates
CN105658234A (zh) * 2013-06-14 2016-06-08 里约热内卢联邦大学 拟胰岛淀粉样多肽化合物与聚乙二醇的非-凝聚生物缀合物
CN105777892A (zh) * 2014-12-17 2016-07-20 深圳翰宇药业股份有限公司 普兰林肽聚乙二醇衍生物、其制备方法及其应用
US9458444B2 (en) 2006-02-03 2016-10-04 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US9522945B2 (en) 2012-04-19 2016-12-20 Opko Biologics Ltd. Long-acting oxyntomodulin variants and methods of producing same
EP3135690A1 (en) 2012-06-26 2017-03-01 Sutro Biopharma, Inc. Modified fc proteins comprising site-specific non-natural amino acid residues, conjugates of the same, methods of their preparation and methods of their use
US9585934B2 (en) 2014-10-22 2017-03-07 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
US9616109B2 (en) 2014-10-22 2017-04-11 Extend Biosciences, Inc. Insulin vitamin D conjugates
US9663778B2 (en) 2009-07-09 2017-05-30 OPKO Biologies Ltd. Long-acting coagulation factors and methods of producing same
US9789197B2 (en) 2014-10-22 2017-10-17 Extend Biosciences, Inc. RNAi vitamin D conjugates
US9808534B2 (en) 2012-11-20 2017-11-07 Opko Biologics Ltd. Method of increasing the hydrodynamic volume of polypeptides by attaching to gonadotrophin carboxy terminal peptides
US9828417B2 (en) 2006-02-03 2017-11-28 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US9845287B2 (en) 2012-11-01 2017-12-19 Aileron Therapeutics, Inc. Disubstituted amino acids and methods of preparation and use thereof
US9884901B2 (en) 2006-02-03 2018-02-06 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US9896494B2 (en) 2006-02-03 2018-02-20 Opko Biologics Ltd. Long-acting polypeptides and methods of producing same
US9908924B2 (en) 2006-02-03 2018-03-06 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US9957299B2 (en) 2010-08-13 2018-05-01 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10213477B2 (en) 2012-02-15 2019-02-26 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10221228B2 (en) 2006-02-03 2019-03-05 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US10227380B2 (en) 2012-02-15 2019-03-12 Aileron Therapeutics, Inc. Triazole-crosslinked and thioether-crosslinked peptidomimetic macrocycles
US10253067B2 (en) 2015-03-20 2019-04-09 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
US10279023B2 (en) 2013-09-24 2019-05-07 Board Of Regents, The University Of Texas System Mucin-associated surface protein as a vaccine against chagas disease
US10301351B2 (en) 2007-03-28 2019-05-28 President And Fellows Of Harvard College Stitched polypeptides
US10351615B2 (en) 2006-02-03 2019-07-16 Opko Biologics Ltd. Methods of treatment with long-acting growth hormone
US10471120B2 (en) 2014-09-24 2019-11-12 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
US10537646B2 (en) * 2012-06-04 2020-01-21 Opko Biologics Ltd. Pegylated OXM variants
CN111494318A (zh) * 2020-04-28 2020-08-07 华中科技大学同济医学院附属协和医院 一种肿瘤靶向和还原敏感型复合胶束及其制备方法和应用
WO2021030326A1 (en) * 2019-08-12 2021-02-18 Northwestern University Brush polymers for therapeutic applications
US10960058B2 (en) 2015-06-19 2021-03-30 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US10961289B2 (en) 2015-10-02 2021-03-30 The University Of Copenhagen Small molecules blocking histone reader domains
WO2021072349A1 (en) * 2019-10-11 2021-04-15 Tobebio Novel Drug Co., Ltd. Synergistic combination of chemotherapy and peptide for treating cancer
CN113134075A (zh) * 2021-04-29 2021-07-20 江苏普莱医药生物技术有限公司 一种抗菌肽乳膏及其制备方法
US11077198B2 (en) 2016-05-24 2021-08-03 Amgen Inc. Pegylated carfilzomib compounds
US11197915B2 (en) 2013-10-21 2021-12-14 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
WO2023036878A1 (en) 2021-09-08 2023-03-16 Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Use of diazirine linker for drug conjugates
CN116036234A (zh) * 2023-01-17 2023-05-02 青岛市中心医院 一种用于治疗骨质疏松的药剂
US11938218B2 (en) 2014-08-08 2024-03-26 The Regents Of The University Of California High density peptide polymers
US11976106B2 (en) 2016-07-11 2024-05-07 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
EP4072592A4 (en) * 2019-12-03 2024-06-12 Indian Institute of Technology, Delhi METHOD FOR PRODUCING PEGYLATED THERAPEUTIC PROTEINS
CN118440155A (zh) * 2024-07-11 2024-08-06 中国药科大学 一种双激动多肽化合物及其医药用途
US12203113B2 (en) 2009-07-09 2025-01-21 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US12233115B2 (en) 2022-09-30 2025-02-25 Extend Biosciences, Inc. Long-acting parathyroid hormone

Families Citing this family (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060182692A1 (en) 2003-12-16 2006-08-17 Fishburn C S Chemically modified small molecules
ES2444695T3 (es) 2006-06-23 2014-02-26 Alethia Biotherapeutics Inc. Polinucleótidos y polipéptidos implicados en el cáncer
US20110206633A1 (en) 2008-09-19 2011-08-25 Nektar Therapectics Polymer conjugates of cd-np peptides
WO2010033207A1 (en) 2008-09-19 2010-03-25 Nektar Therapeutics Polymer conjugates of therapeutic peptides
EP2362908A4 (en) 2008-11-03 2013-09-18 Alethia Biotherapeutics Inc ANTIBODIES THAT SPECIFICALLY BLOCK THE BIOLOGICAL ACTIVITY OF A TUMOR ANTIGEN
US8916693B2 (en) 2009-09-17 2014-12-23 Nektar Therapeutics Monoconjugated chitosans as delivery agents for small interfering nucleic acids
EP2553019A1 (en) 2010-03-26 2013-02-06 Mersana Therapeutics, Inc. Modified polymers for delivery of polynucleotides, method of manufacture, and methods of use thereof
GB201007356D0 (en) 2010-04-30 2010-06-16 Leverton Licence Holdings Ltd Conjugated factor VIIa
NZ603399A (en) * 2010-05-17 2014-09-26 Cebix Inc Pegylated c-peptide
WO2012054822A1 (en) 2010-10-22 2012-04-26 Nektar Therapeutics Pharmacologically active polymer-glp-1 conjugates
US9023986B2 (en) 2010-10-25 2015-05-05 Hoffmann-La Roche Inc. Glucose-dependent insulinotropic peptide analogs
CN102008732B (zh) * 2010-11-08 2012-10-24 武汉华耀生物医药有限公司 一种叶酸偶联抗体药物及其制备方法与应用
KR20200070407A (ko) 2010-11-12 2020-06-17 넥타르 테라퓨틱스 Ｉｌ-2 부분 및 중합체의 접합체
CA2824471C (en) 2011-01-04 2020-07-21 Bender Analytical Holding B.V. Cross-linked polymers and implants derived from electrophilically activated polyoxazoline
CA2893376A1 (en) 2011-03-31 2012-10-04 Alethia Biotherapeutics Inc. Antibodies against kidney associated antigen 1 and antigen binding fragemnts thereof
ES2981891T3 (es) * 2011-06-02 2024-10-11 Opko Biologics Ltd Agonistas del receptor de GLP-1/glucagón de acción prolongada
US10166295B2 (en) 2011-06-02 2019-01-01 Opko Biologics Ltd. Pegylated OXM variants
PL2718318T3 (pl) 2011-06-10 2019-02-28 Hanmi Science Co., Ltd. Nowe pochodne oksyntomoduliny i zawierająca je kompozycja farmaceutyczna do leczenia otyłości
KR101577734B1 (ko) 2011-06-17 2015-12-29 한미사이언스 주식회사 옥신토모듈린과 면역글로불린 단편을 포함하는 결합체 및 그의 용도
EP2755690B1 (en) * 2011-08-04 2020-04-01 Merck Sharp & Dohme B.V. Kisspeptide-pentasaccharide conjugates
WO2013020914A1 (en) * 2011-08-10 2013-02-14 Celares Gmbh Peg-conjugated peptides
KR101997939B1 (ko) 2011-08-12 2019-07-08 아센디스 파마 에이에스 담체-연결된 트레프로스티닐 전구약물
US9180202B2 (en) 2012-01-02 2015-11-10 Universiteit Gent Polyoxazoline polymers and methods for their preparation, conjugates of these polymers and medical uses thereof
ES2731665T3 (es) 2012-01-09 2019-11-18 Adc Therapeutics Sa Agentes para tratar cáncer de mama triple negativo
ES2425448B8 (es) * 2012-03-12 2015-03-17 Universidade De Santiago De Compostela Uso de la obestatina para la regeneración muscular
WO2013137736A1 (en) 2012-03-16 2013-09-19 Bender Analytical Holding B.V. Cross-linked polymers and medical products derived from nucleophilically activated polyoxazoline
KR101968344B1 (ko) 2012-07-25 2019-04-12 한미약품 주식회사 옥신토모듈린 유도체를 포함하는 고지혈증 치료용 조성물
KR101993393B1 (ko) 2012-11-06 2019-10-01 한미약품 주식회사 옥신토모듈린 유도체를 포함하는 당뇨병 또는 비만성 당뇨병 치료용 조성물
PT2916819T (pt) 2012-11-06 2019-10-28 Hanmi Pharm Ind Co Ltd Formulação líquida de conjugado proteico que compreende a oxintomodulina e um fragmento de imunoglobulina.
SI24318A (sl) * 2013-03-13 2014-09-30 Diagen D.O.O. Nove stabilne soli pentadekapeptida, postopek za njihovo pripravo, njihova uporaba za izdelavo farmacevtskih pripravkov in njihova uporaba v terapiji
WO2014139182A1 (en) * 2013-03-15 2014-09-18 Shenzhen Hightide Biopharmaceutical, Ltd. Compositions and methods of using islet neogenesis peptides and analogs thereof
HK1220626A1 (zh) 2013-03-15 2017-05-12 The Centre For Drug Research And Development 具细胞毒性和抗有丝分裂的化合物以及其使用方法
WO2015095953A1 (en) 2013-12-27 2015-07-02 The Centre For Drug Research And Development Sulfonamide-containing linkage systems for drug conjugates
CA2953371C (en) * 2014-06-30 2021-08-24 Tarveda Therapeutics, Inc. Targeted conjugates and particles and formulations thereof
AU2015308987B2 (en) * 2014-08-26 2021-02-18 Kaass Discovery As Methods, agents and compositions for treatment of inflammatory conditions
TWI802396B (zh) 2014-09-16 2023-05-11 南韓商韓美藥品股份有限公司 長效glp-1/高血糖素受體雙促效劑治療非酒精性脂肝疾病之用途
SG11201702143PA (en) 2014-09-17 2017-04-27 Zymeworks Inc Cytotoxic and anti-mitotic compounds, and methods of using the same
KR102514142B1 (ko) 2014-10-06 2023-03-27 가트 테크놀로지스 비.브이. 조직-접착 다공성 지혈 제품
KR102418477B1 (ko) 2014-12-30 2022-07-08 한미약품 주식회사 글루카곤 유도체
AU2016335860B2 (en) 2015-10-08 2023-08-10 Nektar Therapeutics Combination of an IL-2Rbeta-selective agonist and a long-acting IL-15 agonist
KR101642962B1 (ko) 2015-11-05 2016-07-26 (주)제이월드 봉합사 및 그 제조 방법
RU2605385C2 (ru) * 2015-11-23 2016-12-20 Федеральное государственное бюджетное учреждение науки институт биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова Российской академии наук (ИБХ РАН) Способ получения моноконъюгата полисиаловой кислоты с тимозином бета 4 и ковалентный моноконъюгат полисиаловой кислоты с тимозином бета 4, устойчивый к деградации в токе крови
RU2607527C2 (ru) * 2015-11-23 2017-01-10 Федеральное государственное бюджетное учреждение науки институт биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова Российской академии наук (ИБХ РАН) Ковалентный моноконъюгат полиэтиленгликоля с тимозином бета 4, устойчивый к деградации в токе крови, и способ его получения
US10039801B2 (en) 2016-02-16 2018-08-07 Strongbridge Ireland Limited Pharmaceutical compositions of water soluble peptides with poor solubility in isotonic conditions and methods for their use
JP7030720B2 (ja) * 2016-02-16 2022-03-07 ストロングブリッジ ダブリン リミテッド 先端巨大症、先端巨大症がん、ｓｓｔ－ｒ５発現腫瘍、２型糖尿病、高血糖症、及びホルモン関連腫瘍の治療において使用するための、生理学的条件において難溶性であるベルドレオチド
US20180078342A1 (en) * 2016-09-21 2018-03-22 William Graham Gardner Dental Appliance For Use as an Aligner and as a Tool to Reduce Temporomandibular Dysfunction
CN107987170B (zh) * 2016-10-27 2018-12-18 浙江道尔生物科技有限公司 一种用于治疗肠道疾病的融合蛋白
CN111093688A (zh) 2017-05-15 2020-05-01 尼克塔治疗公司 长效白细胞介素-15受体激动剂以及相关免疫治疗组合物和方法
EP4545553A2 (en) * 2017-07-05 2025-04-30 ShangPharma Innovation Inc. Peptide compound and application thereof, and composition containing peptide compound
JP7137625B2 (ja) * 2017-12-29 2022-09-14 エフ．ホフマン－ラ ロシュ アーゲー Ｐｅｇ化タンパク質組成物を提供するための方法
IL293852A (en) * 2019-12-13 2022-08-01 Folium Labs Inc Complexes comprising a carbohydrate polymer and an active ingredient and processes for their preparation
WO2022035947A1 (en) * 2020-08-12 2022-02-17 Wisconsin Alumni Research Foundation Conjugation of proteins to polysaccharides using a phosphate bridge
KR20220076375A (ko) * 2020-11-30 2022-06-08 세종대학교산학협력단 신규 퇴행성 신경질환 치료용 약학적 조성물
WO2022189463A1 (en) * 2021-03-08 2022-09-15 Akershus Universitetssykehus Hf Clearance assay
CN114395597B (zh) * 2021-12-17 2024-04-09 佛山汉腾生物科技有限公司 Glp-1或其变体的羟基化检测方法
WO2024249889A1 (en) * 2023-05-31 2024-12-05 University Of Connecticut Genetic sequence/small molecule-carbohydrate conjugates for enhanced lung-specific targeting
CN118666949B (zh) * 2024-06-27 2025-07-29 首都医科大学 熊果酰-Arg-Gly-Asp-Ser的四聚体,其合成,活性和应用

Citations (94)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4010148A (en) * 1975-05-12 1977-03-01 Board Of Regents Of The University Of Texas System Purified thymosin and process
US4013075A (en) * 1974-07-15 1977-03-22 I.S.F. S.P.A. Inhalers and insufflators having a cutting means
US4148788A (en) * 1978-01-23 1979-04-10 Hoffmann-La Roche Inc. Synthesis of thymosin α1
US4247066A (en) * 1978-02-21 1981-01-27 General Dynamics Corporation Airfoil variable cambering device and method
US4321260A (en) * 1980-05-22 1982-03-23 Ayerst, Mckenna & Harrison Inc. Method of using gonadorelin for the treatment of benign prostatic hyperplasia
US4461724A (en) * 1981-10-28 1984-07-24 Nippon Zoki Pharmaceutical Co., Ltd. Peptide compounds, a process for manufacturing them, pharmaceutical compositions containing them, and methods for treating ulcer and thrombus with them
US4585754A (en) * 1984-01-09 1986-04-29 Valcor Scientific, Ltd. Stabilization of proteins and peptides by chemical binding with chondroitin
US4652548A (en) * 1981-08-27 1987-03-24 Eli Lilly And Company Pharmaceutical formulations comprising human insulin, human C-peptide, and human proinsulin
US4667668A (en) * 1981-07-08 1987-05-26 Aktiebolaget Draco Dosage inhalator
US4668281A (en) * 1985-05-20 1987-05-26 E. I. Du Pont De Nemours And Company Thiophenesulfonamides
US4801612A (en) * 1986-07-03 1989-01-31 Regents Of The University Of California Method of inhibiting inflammatory response
US4805811A (en) * 1985-03-29 1989-02-21 Aktiebolaget Draco Dosage device
US4810646A (en) * 1984-11-28 1989-03-07 Massachusetts Institute Of Technology Glucan compositions and process for preparation thereof
US4846876A (en) * 1986-10-14 1989-07-11 Bayer Aktiengesellschaft Herbicidal imidazo-pyrrolo-pyridine derivatives
US4992540A (en) * 1984-11-28 1991-02-12 Massachusetts Institute Of Technology Glucan composition and process for preparation thereof
US5028703A (en) * 1988-03-11 1991-07-02 Massachusetts Institute Of Technology Glucan composition and process for preparation thereof
US5118494A (en) * 1990-03-23 1992-06-02 Minnesota Mining And Manufacturing Company Use of soluble fluorosurfactants for the preparation of metered-dose aerosol formulations
US5221736A (en) * 1988-12-21 1993-06-22 Bionebraska, Inc. Sequential peptide and oligonucleotide syntheses using immunoaffinity techniques
US5288708A (en) * 1989-09-12 1994-02-22 Predrag Sikiric Pharmacologically active substance BPC, the process for its preparation and its use in the therapy
US5320094A (en) * 1992-01-10 1994-06-14 The Johns Hopkins University Method of administering insulin
US5388572A (en) * 1993-10-26 1995-02-14 Tenax Corporation (A Connecticut Corp.) Dry powder medicament inhalator having an inhalation-activated piston to aerosolize dose and deliver same
US5492688A (en) * 1993-04-28 1996-02-20 The Center For Innovative Technology Metered dose inhaler fomulations which include the ozone-friendly propellant HFC 134a and a pharmaceutically acceptable suspending, solubilizing, wetting, emulsifying or lubricating agent
US5521184A (en) * 1992-04-03 1996-05-28 Ciba-Geigy Corporation Pyrimidine derivatives and processes for the preparation thereof
US5522385A (en) * 1994-09-27 1996-06-04 Aradigm Corporation Dynamic particle size control for aerosolized drug delivery
US5605674A (en) * 1988-12-06 1997-02-25 Riker Laboratories, Inc. Medicinal aerosol formulations
US5607677A (en) * 1989-06-15 1997-03-04 Alpha-Beta Technology, Inc. Glucan drug delivery system and adjuvant
US5610136A (en) * 1996-01-30 1997-03-11 Milkhaus Laboratory, Inc. Method for treatment of benign prostatic hypertrophy
US5612460A (en) * 1989-04-19 1997-03-18 Enzon, Inc. Active carbonates of polyalkylene oxides for modification of polypeptides
US5618914A (en) * 1991-02-07 1997-04-08 Molecumetics, Ltd. Conformationally restricted mimetics of beta turns and beta bulges and peptides containing the same
US5629384A (en) * 1994-05-17 1997-05-13 Consiglio Nazionale Delle Ricerche Polymers of N-acryloylmorpholine activated at one end and conjugates with bioactive materials and surfaces
US5650234A (en) * 1994-09-09 1997-07-22 Surface Engineering Technologies, Division Of Innerdyne, Inc. Electrophilic polyethylene oxides for the modification of polysaccharides, polypeptides (proteins) and surfaces
US5710245A (en) * 1993-06-18 1998-01-20 Molecumetics, Ltd. Alpha-helix mimetics and methods relating thereto
US5739208A (en) * 1993-11-12 1998-04-14 Shearwater Polymers, Inc. Isolatable, water soluble, and hydrolytically stable active sulfones of poly(ethylene glycol) and related polymers for modification of surfaces and molecules
US5740794A (en) * 1994-09-21 1998-04-21 Inhale Therapeutic Systems Apparatus and methods for dispersing dry powder medicaments
US5758637A (en) * 1995-08-31 1998-06-02 Aerogen, Inc. Liquid dispensing apparatus and methods
US5767254A (en) * 1992-12-15 1998-06-16 The Arizona Board Of Regents On Behalf Of The University Of Arizona Method for making amino acid glycosides and glycopeptides
US5859184A (en) * 1995-10-27 1999-01-12 Molecumetics Ltd. Alpha-helix mimetics and methods relating thereto
US5880131A (en) * 1993-10-20 1999-03-09 Enzon, Inc. High molecular weight polymer-based prodrugs
US5888762A (en) * 1990-06-05 1999-03-30 Centre National De La Recherche Scientifique (Cnrs) Neurotropic growth factors comprising a homeobox peptide
US5922675A (en) * 1994-11-17 1999-07-13 Eli Lilly And Company Acylated Insulin Analogs
US5929237A (en) * 1995-10-27 1999-07-27 Molecumetics Ltd. Reverse-turn mimetics and methods relating thereto
US6014970A (en) * 1998-06-11 2000-01-18 Aerogen, Inc. Methods and apparatus for storing chemical compounds in a portable inhaler
US6080762A (en) * 1998-04-08 2000-06-27 Eli Lilly And Company Pulmonary and nasal delivery of raloxifene
US6083909A (en) * 1996-07-08 2000-07-04 Fresenius Ag Haemoglobin-hydroxyethyl starch conjugates as oxygen carriers
US6085740A (en) * 1996-02-21 2000-07-11 Aerogen, Inc. Liquid dispensing apparatus and methods
US6205999B1 (en) * 1995-04-05 2001-03-27 Aerogen, Inc. Methods and apparatus for storing chemical compounds in a portable inhaler
US6214966B1 (en) * 1996-09-26 2001-04-10 Shearwater Corporation Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution
US6257233B1 (en) * 1998-06-04 2001-07-10 Inhale Therapeutic Systems Dry powder dispersing apparatus and methods for their use
US20020013266A1 (en) * 1999-10-04 2002-01-31 Shearwater Corporation Polymer stabilized neuropeptides
US6348558B1 (en) * 1999-12-10 2002-02-19 Shearwater Corporation Hydrolytically degradable polymers and hydrogels made therefrom
US6347936B1 (en) * 1995-05-10 2002-02-19 Allports Llc International Liquid vaporization and pressurization apparatus and methods
US20020028189A1 (en) * 2000-04-14 2002-03-07 Seongbong Jo Biocompatible macromers
US6357490B1 (en) * 2000-08-22 2002-03-19 Advanced Inhalation Research, Inc. System, method and apparatus for filling containers
US6362254B2 (en) * 1998-03-12 2002-03-26 Shearwater Corporation Poly(ethylene glycol) derivatives with proximal reactive groups
US6376470B1 (en) * 1999-09-23 2002-04-23 Enzon, Inc. Polymer conjugates of ara-C and ara-C derivatives
US6376604B2 (en) * 1999-12-22 2002-04-23 Shearwater Corporation Method for the preparation of 1-benzotriazolylcarbonate esters of poly(ethylene glycol)
US6379670B1 (en) * 1994-11-18 2002-04-30 Neurocrine Biosciences, Inc. Methods for treatment of multiple sclerosis using peptide analogs of human myelin basic protein
US20020065397A1 (en) * 2000-10-12 2002-05-30 Joseph Roberts Protecting therapeutic compositions from host-mediated inactivation
US6413507B1 (en) * 1999-12-23 2002-07-02 Shearwater Corporation Hydrolytically degradable carbamate derivatives of poly (ethylene glycol)
US6504005B1 (en) * 1996-08-07 2003-01-07 Yeda Research And Development Co. Ltd. Long-acting drugs and pharmaceutical compositions comprising them
US6518239B1 (en) * 1999-10-29 2003-02-11 Inhale Therapeutic Systems, Inc. Dry powder compositions having improved dispersivity
US6524591B1 (en) * 1998-06-29 2003-02-25 Cbr Monocyte locomotion inhibitory factor
US20030069170A1 (en) * 2001-09-07 2003-04-10 Richard Soltero Pharmaceutical compositions of drug-oligomer conjugates and methods of treating diseases therewith
US6579968B1 (en) * 1999-06-29 2003-06-17 Palatin Technologies, Inc. Compositions and methods for treatment of sexual dysfunction
US6583408B2 (en) * 2001-05-18 2003-06-24 Battelle Memorial Institute Ionization source utilizing a jet disturber in combination with an ion funnel and method of operation
US6585509B2 (en) * 1995-05-10 2003-07-01 Allports Llc International Vaporization and pressurization of liquid in a porous material
US20030143596A1 (en) * 2001-11-07 2003-07-31 Shearwater Corporation Branched polymers and their conjugates
US6701922B2 (en) * 2001-12-20 2004-03-09 Chrysalis Technologies Incorporated Mouthpiece entrainment airflow control for aerosol generators
US6706289B2 (en) * 2000-10-31 2004-03-16 Pr Pharmaceuticals, Inc. Methods and compositions for enhanced delivery of bioactive molecules
US20040096402A1 (en) * 2001-06-05 2004-05-20 Alexza Molecular Delivery Corporation Delivery of aerosols containing small particles through an inhalation route
US20040116649A1 (en) * 2002-09-09 2004-06-17 Antoni Kozlowski Water-soluble polymer alkanals
US20040203081A1 (en) * 2002-11-26 2004-10-14 James Kenneth D. Natriuretic compounds, conjugates, and uses thereof
US6838259B2 (en) * 1999-12-17 2005-01-04 Takeda Chemical Industries, Ltd. Process for producing kiss-1 peptide
US20050009988A1 (en) * 2003-05-23 2005-01-13 Harris J. Milton Polymer derivatives having particular atom arrangements
US20050014903A1 (en) * 2003-01-06 2005-01-20 Antoni Kozlowski Thiol selective water soluble polymer derivatives
US6858580B2 (en) * 2001-06-04 2005-02-22 Nobex Corporation Mixtures of drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US20050059600A1 (en) * 1999-12-17 2005-03-17 Mayo Foundation For Medical Education And Research Chimeric natriuretic peptides
US20050074425A1 (en) * 2003-07-02 2005-04-07 Polycord, Inc. Method for delivering polymerized therapeutic agent compositions and compositions thereof
US6878689B2 (en) * 2000-06-23 2005-04-12 Kaken Pharmaceutical Co., Ltd. Preventives or remedies for heart failure
US6890518B2 (en) * 2001-05-21 2005-05-10 Nektar Therapeutics Compositions of chemically modified insulin
US20060074009A1 (en) * 2004-05-26 2006-04-06 James Kenneth D Natriuretic compounds, conjugates, and uses thereof
US7030084B2 (en) * 1999-06-19 2006-04-18 Nobex Corporation Drug-oligomer conjugates with polyethylene glycol components
US20060105948A1 (en) * 2003-03-24 2006-05-18 Novo Nordisk A/S GLP-2 derivatives
US20060121062A1 (en) * 2002-09-11 2006-06-08 Wolfram Eichner Conjugated hydroxyalkyl starch allergen compounds
US7060675B2 (en) * 2001-02-15 2006-06-13 Nobex Corporation Methods of treating diabetes mellitus
WO2007034498A1 (en) * 2005-09-26 2007-03-29 Compugen Ltd. Atrial natriuretic peptide (anp) splice variants and methods of using same
US20070071764A1 (en) * 2005-04-22 2007-03-29 Sullivan John K Toxin peptide therapeutic agents
US7314754B2 (en) * 1998-10-27 2008-01-01 Takeda Pharmaceutical Company Limited Polynucleotide encoding the hOT7T175 G protein coupled receptor
US20080044438A1 (en) * 2006-03-17 2008-02-21 Ostroff Gary R Yeast Cell Particles As Oral Delivery Vehicles For Antigens
US20080085862A1 (en) * 2003-11-13 2008-04-10 Hanmi Pharm. Ind. Co., Ltd. Natriuretic peptide conjugate using carrier substance
US20080090758A1 (en) * 2006-09-06 2008-04-17 Aeterna Zentaris Gmbh Conjugates of disorazoles and their derivatives with cell-binding molecules, novel disorazole derivatives, processes of manufacturing and uses thereof
US20080139459A1 (en) * 2005-02-01 2008-06-12 N.V. Organon Conjugates A Polypeptide And A Pentasaccharide
US20080152668A1 (en) * 2001-11-01 2008-06-26 Sciclone Pharmaceuticals, Inc. Thymosin Alpha 1 Peptide/Polymer Conjugates
US20110165111A1 (en) * 2008-09-19 2011-07-07 Nektar Therapeutics Polymer conjugates of nesiritide peptides

Family Cites Families (140)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2224175B1 (enrdf_load_stackoverflow) 1973-04-04 1978-04-14 Isf Spa
US4554101A (en) 1981-01-09 1985-11-19 New York Blood Center, Inc. Identification and preparation of epitopes on antigens and allergens on the basis of hydrophilicity
IL66611A0 (en) 1981-08-27 1982-12-31 Lilly Co Eli Pharmaceutical formulations comprising human insulin and human c-peptide
DE3274798D1 (en) 1981-10-05 1987-02-05 Nicholas P Plotnikoff Process for using endorphins as antitumour agents
GB8314308D0 (en) 1983-05-24 1983-06-29 Matburn Holdings Ltd Medical administration devices
DE3326473A1 (de) 1983-07-22 1985-01-31 Hoechst Ag, 6230 Frankfurt Pharmazeutisches mittel zur behandlung des diabetes mellitus
US5256549A (en) 1986-03-28 1993-10-26 Chiron Corporation Purification of synthetic oligomers
AT387234B (de) 1987-03-05 1988-12-27 Vogelbusch Gmbh Vorrichtung zur zuechtung von dauerformen insbesondere filamentoeser mikroorganismen
US4966753A (en) 1987-08-18 1990-10-30 Molecular Rx, Inc. Immunotherapeutic methods and compositions employing antigens characteristic of malignant neoplasms
US5468481A (en) 1988-06-23 1995-11-21 Amergen, Inc. MHC class II-peptide conjugates useful in ameliorating autoimmunity
GB8909891D0 (en) 1989-04-28 1989-06-14 Riker Laboratories Inc Device
ATE136315T1 (de) * 1989-05-27 1996-04-15 Sumitomo Pharma Verfahren für die herstellung von polyethylenglykolderivate und modifizierte proteine.
US5137871A (en) 1989-07-28 1992-08-11 Regents Of The University Of California Treatment to reduce edema for brain and musculature injuries
GB8921222D0 (en) 1989-09-20 1989-11-08 Riker Laboratories Inc Medicinal aerosol formulations
IE67185B1 (en) 1990-02-02 1996-03-06 Fisons Plc Propellant compositions
SG45171A1 (en) 1990-03-21 1998-01-16 Boehringer Ingelheim Int Atomising devices and methods
US5545719A (en) 1990-05-01 1996-08-13 Neuromedica, Inc. Nerve growth peptides
EP0536250B1 (en) 1990-06-28 2000-09-06 Glaxo Wellcome Inc. Aerosol drug formulations
IT1243344B (it) 1990-07-16 1994-06-10 Promo Pack Sa Inalatore plurimonodose per medicamenti in polvere
US5252714A (en) 1990-11-28 1993-10-12 The University Of Alabama In Huntsville Preparation and use of polyethylene glycol propionaldehyde
ES2117045T3 (es) 1990-12-21 1998-08-01 Curative Tech Inc Peptidos angiogenicos.
US5475085A (en) 1991-02-07 1995-12-12 Molecumetics, Ltd. Conformationally restricted mimetics of beta turns and beta bulges and peptides containing the same
HU222249B1 (hu) 1991-03-08 2003-05-28 Amylin Pharmaceuticals Inc. Eljárás amilin agonista peptidszármazékok és ezeket tartalmazó gyógyszerkészítmények előállítására
US5938117A (en) 1991-04-24 1999-08-17 Aerogen, Inc. Methods and apparatus for dispensing liquids as an atomized spray
WO1993000951A1 (en) 1991-07-02 1993-01-21 Inhale, Inc. Method and device for delivering aerosolized medicaments
DE4216133A1 (de) 1992-05-15 1993-11-18 Bissendorf Peptide Gmbh Anwendung von Urodilatin bei Lungen- und Bronchialerkrankungen
US6582728B1 (en) 1992-07-08 2003-06-24 Inhale Therapeutic Systems, Inc. Spray drying of macromolecules to produce inhaleable dry powders
US5817628A (en) 1992-12-02 1998-10-06 The Rockefeller University Dynorphin a suppression of natural killer cell activity
US5672581A (en) 1993-01-29 1997-09-30 Aradigm Corporation Method of administration of insulin
US6191105B1 (en) 1993-05-10 2001-02-20 Protein Delivery, Inc. Hydrophilic and lipophilic balanced microemulsion formulations of free-form and/or conjugation-stabilized therapeutic agents such as insulin
US5359030A (en) 1993-05-10 1994-10-25 Protein Delivery, Inc. Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same
WO1995000162A1 (en) 1993-06-21 1995-01-05 Enzon, Inc. Site specific synthesis of conjugated peptides
US6653442B1 (en) 1993-07-20 2003-11-25 Intrabiotics Pharmaceuticals, Inc. Protegrins
US5804558A (en) 1993-07-20 1998-09-08 University Of California Protegrins
IS1736B (is) 1993-10-01 1999-12-30 Astra Ab Aðferð og tæki sem stuðla að aukinni samloðun agna
IT1271486B (it) 1993-10-12 1997-05-28 Italfarmaco Spa Oligopeptidi immunomodulatori derivati di frammenti della proteina c- reattiva
US5840900A (en) 1993-10-20 1998-11-24 Enzon, Inc. High molecular weight polymer-based prodrugs
US5457044A (en) 1993-10-29 1995-10-10 Genentech, Inc. Method for collection of aerosolized proteins by inert filtration
NZ276637A (en) 1993-12-20 1997-07-27 Minnesota Mining & Mfg Aerosol containing flunisolide, ethanol, and tetrafluoroethane and/or heptafluoropropane propellant
US6051256A (en) 1994-03-07 2000-04-18 Inhale Therapeutic Systems Dispersible macromolecule compositions and methods for their preparation and use
WO1995024183A1 (en) 1994-03-07 1995-09-14 Inhale Therapeutic Systems Methods and compositions for pulmonary delivery of insulin
US5932462A (en) * 1995-01-10 1999-08-03 Shearwater Polymers, Inc. Multiarmed, monofunctional, polymer for coupling to molecules and surfaces
US5834590A (en) 1995-02-22 1998-11-10 Eastern Virginia Medical School Of The Medical College Of Hampton Roads Ingap protein involved in pancreatic islet neogenesis
ES2237767T3 (es) 1995-04-14 2005-08-01 Nektar Therapeutics Composiciones farmaceuticas en polvo que tienen una dispersabilidad mejorada.
US6309671B1 (en) 1995-04-14 2001-10-30 Inhale Therapeutic Systems Stable glassy state powder formulations
US5692095A (en) 1995-05-10 1997-11-25 Allports, Inc. Capillary feed boiler
US5654007A (en) 1995-06-07 1997-08-05 Inhale Therapeutic Systems Methods and system for processing dispersible fine powders
NZ310804A (en) 1995-06-07 1998-10-28 Ortho Pharma Corp Compounds and peptides that bind to the erythropoietin receptor
US5795864A (en) 1995-06-27 1998-08-18 Neurex Corporation Stable omega conopetide formulations
US5672662A (en) 1995-07-07 1997-09-30 Shearwater Polymers, Inc. Poly(ethylene glycol) and related polymers monosubstituted with propionic or butanoic acids and functional derivatives thereof for biotechnical applications
DE19536902A1 (de) 1995-10-04 1997-04-10 Boehringer Ingelheim Int Vorrichtung zur Hochdruckerzeugung in einem Fluid in Miniaturausführung
US5826633A (en) 1996-04-26 1998-10-27 Inhale Therapeutic Systems Powder filling systems, apparatus and methods
SE520392C2 (sv) 1996-09-27 2003-07-01 Creative Peptides Sweden Ab C Specifika peptider för behandling av diabetes mellitus
EP0951300A4 (en) 1996-12-31 2006-07-26 Nektar Therapeutics PROCESS FOR SPRAY DRYING OF SOLUTIONS OF HYDROPHILIC DRUGS WITH HYDROPHILIC BINDERS AND COMPOSITIONS MADE BY THIS METHOD
US5990237A (en) 1997-05-21 1999-11-23 Shearwater Polymers, Inc. Poly(ethylene glycol) aldehyde hydrates and related polymers and applications in modifying amines
ATE377026T1 (de) 1997-09-08 2007-11-15 Metabolic Pharm Ltd Behandlung von fettleibigkeit
HRP20000175B1 (en) 1997-09-29 2004-06-30 Nektar Therapeutics Perforated microparticles and methods of use
US6448369B1 (en) 1997-11-06 2002-09-10 Shearwater Corporation Heterobifunctional poly(ethylene glycol) derivatives and methods for their preparation
US5932548A (en) 1998-06-03 1999-08-03 Deghenghi; Romano Lysine containing peptides for treatment of heart disease
GB9814527D0 (en) 1998-07-03 1998-09-02 Cyclacel Ltd Delivery system
DE19835346A1 (de) 1998-08-05 2000-02-10 Boehringer Ingelheim Pharma Zweiteilige Kapsel zur Aufnahme von pharmazeutischen Zubereitungen für Pulverinhalatoren
US6703381B1 (en) 1998-08-14 2004-03-09 Nobex Corporation Methods for delivery therapeutic compounds across the blood-brain barrier
GB2346616B (en) 1998-11-13 2004-04-21 Cyclacel Ltd Transport vectors
AU777167B2 (en) 1999-04-23 2004-10-07 Battelle Memorial Institute Directionally controlled EHD aerosol sprayer
SE9902627D0 (sv) 1999-07-08 1999-07-08 Siemens Elema Ab Medical nebulizer
US6323311B1 (en) 1999-09-22 2001-11-27 University Of Utah Research Foundation Synthesis of insulin derivatives
DE19951471A1 (de) 1999-10-26 2001-05-03 Forssmann Wolf Georg Verwendung von Urodilatin zur Behandlung chronischer Niereninsuffizienz mit Nierenrestfunktionen
AUPQ387599A0 (en) 1999-11-05 1999-12-02 Metabolic Pharmaceuticals Limited Product and method for control of obesity
ATE317868T1 (de) 1999-12-22 2006-03-15 Nektar Therapeutics Al Corp Sterisch gehinderte derivate von wasserlöslichen polymeren
WO2001062827A2 (en) 2000-02-22 2001-08-30 Shearwater Corporation N-maleimidyl polymer derivatives
WO2001075104A1 (fr) 2000-03-30 2001-10-11 Takeda Chemical Industries, Ltd. Nouvelle proteine, adn codant pour celle-ci, et son procede de production
US7971588B2 (en) 2000-05-05 2011-07-05 Novartis Ag Methods and systems for operating an aerosol generator
EP1970072A1 (en) 2000-09-18 2008-09-17 Sanos Bioscience A/S Use of GLP-2 peptides for the treatment of hyperparathyroidism
WO2002085399A1 (en) 2001-04-20 2002-10-31 Takeda Chemical Industries, Ltd. Peptide-containing preparations
US6803565B2 (en) 2001-05-18 2004-10-12 Battelle Memorial Institute Ionization source utilizing a multi-capillary inlet and method of operation
US6835802B2 (en) 2001-06-04 2004-12-28 Nobex Corporation Methods of synthesizing substantially monodispersed mixtures of polymers having polyethylene glycol moieties
US6828297B2 (en) 2001-06-04 2004-12-07 Nobex Corporation Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US7312192B2 (en) 2001-09-07 2007-12-25 Biocon Limited Insulin polypeptide-oligomer conjugates, proinsulin polypeptide-oligomer conjugates and methods of synthesizing same
EP1428835B1 (en) 2001-09-19 2007-04-18 Takeda Pharmaceutical Company Limited Antibody against metastin and use of the same in the diagnosis of pregnancy
EP1442294A4 (en) 2001-11-16 2005-01-19 Waters Investments Ltd CONCENTRATION, DESALINATION AND DEPOT IN PARALLEL ON MALDI TARGETS
US7458373B2 (en) 2002-01-15 2008-12-02 Philip Morris Usa Inc. Aerosol generator for drug formulation
AU2003213152A1 (en) 2002-02-15 2003-09-09 Nektar Therapeutics Al, Corporation Hydrolytically degradable alkylene oxide based polymers
US6634864B1 (en) 2002-02-19 2003-10-21 Vapore, Inc. High fluid flow and pressure in a capillary pump for vaporization of liquid
PL205811B1 (pl) 2002-08-13 2010-05-31 Iwona Bonney Zastosowanie peptydów o działaniu analgetycznym jako substancji czynnej w urządzeniach do podawania leku bezpośrednio do miejsca ich spodziewanego działania przeciwbólowego
RU2329274C2 (ru) * 2002-09-11 2008-07-20 Фрезениус Каби Дойчланд Гмбх Способ получения производных гидроксиалкилкрахмала
EP2014679B1 (en) 2002-09-12 2010-05-05 Oncotherapy Science, Inc. KDR peptides and vaccines comprising the same
KR101238517B1 (ko) * 2002-12-26 2013-02-28 마운틴 뷰 파마슈티컬즈, 인크. 생물학적 효능이 향상된 인터페론-베타의 중합체 접합체
GEP20084487B (en) 2002-12-26 2008-09-25 Mountain View Pharmaceuticals Polymer conjugates of cytokines, chemokines, growth factors, polypeptide hormones and antagonists thereof
US6800611B2 (en) 2003-01-06 2004-10-05 Takeda Chemical Industries, Ltd. Metastin derivatives and their use
CA2521784C (en) 2003-04-08 2012-03-27 Yeda Research And Development Co. Ltd. Reversible pegylated drugs
EP2599502B1 (en) 2003-04-11 2017-03-01 Antriabio, Inc. Method for preparation of site-specific protein conjugates
BRPI0409711A (pt) 2003-04-23 2006-05-02 Sciclone Pharmaceuticals Inc tratamento ou prevenção de infecções respiratórias virais com peptìdeos da alfa timosina
US20080206182A1 (en) * 2003-08-08 2008-08-28 Fresenius Kabi Deutschland Gmbh Conjugates of a Polymer and a Protein Linked by an Oxime Group
ES2294535T3 (es) 2003-08-12 2008-04-01 Lipoxen Technologies Limited Derivados del acido polisialico.
US7744861B2 (en) 2003-09-17 2010-06-29 Nektar Therapeutics Multi-arm polymer prodrugs
WO2005032556A1 (en) 2003-10-02 2005-04-14 Elan Pharmaceuticals, Inc. Method for reducing pain
CA2540831A1 (en) 2003-10-21 2005-06-02 Vapore, Inc. Improved capillary pumps for vaporization of liquids
DK1732607T3 (da) 2004-03-23 2019-07-22 Ascendis Pharma Gmbh Polymer-prodrug med en selvofrende linker
JP2007530697A (ja) 2004-03-29 2007-11-01 ジ・アリゾナ・ボード・オブ・リージェンツ・オン・ビハーフ・オブ・ザ・ユニバーシティー・オブ・アリゾナ 両親媒性グリコペプチド
US7946291B2 (en) 2004-04-20 2011-05-24 Novartis Ag Ventilation systems and methods employing aerosol generators
US7290541B2 (en) 2004-04-20 2007-11-06 Aerogen, Inc. Aerosol delivery apparatus and method for pressure-assisted breathing systems
AU2005249373A1 (en) 2004-04-23 2005-12-15 Laboratoires Serono Sa Use of GPCR54 ligands for the treatment of infertility
CN100355786C (zh) 2004-04-28 2007-12-19 江苏豪森药业股份有限公司 聚乙二醇修饰的胸腺肽1衍生物
US7740861B2 (en) 2004-06-16 2010-06-22 University Of Massachusetts Drug delivery product and methods
AU2005275108B2 (en) * 2004-07-16 2011-05-12 Nektar Therapeutics Conjugates of a GM-CSF moiety and a polymer
CN101111256B (zh) 2004-12-15 2012-04-25 科罗拉多大学 抗菌肽及其使用方法
WO2006076471A2 (en) 2005-01-12 2006-07-20 Nobex Corporation Bnp conjugates and methods of use
GB0501008D0 (en) 2005-01-18 2005-02-23 Amura Therapeutics Ltd Method of producing conjugate vaccines
US7393833B2 (en) 2005-03-09 2008-07-01 The Board Of Regents Of The University Of Oklahoma Chimeric proteins with phosphatidylserine binding domains
EP1863840A1 (en) 2005-03-18 2007-12-12 Novo Nordisk A/S Pegylated single-chain insulin
PT2510942E (pt) 2005-04-07 2015-12-28 Cardiorentis Ag Utilização de péptido natriurético para o tratamento de insuficiência cardíaca
EP1885403B1 (en) * 2005-04-12 2013-05-08 Nektar Therapeutics Poly(ethyleneglycol) conjugates of Lysostaphin
US9108211B2 (en) 2005-05-25 2015-08-18 Nektar Therapeutics Vibration systems and methods
ES2536450T3 (es) 2005-06-16 2015-05-25 Nektar Therapeutics Conjugados que tienen una unión degradable y reactivos poliméricos útiles en la preparación de tales conjugados
EP1937716A2 (en) 2005-09-08 2008-07-02 Uutech Limited Analogs of gastric inhibitory polypeptide as a treatment for age related decreased pancreatic beta cell function
CN100577685C (zh) 2005-11-10 2010-01-06 中国人民解放军军事医学科学院毒物药物研究所 长效胸腺素α1的聚乙二醇化修饰物
EP1797901A1 (en) 2005-12-16 2007-06-20 Diatos Cell penetrating peptide conjugates for delivering nucleic acids into cells
JP5096363B2 (ja) * 2005-12-16 2012-12-12 ネクター セラピューティックス Ｇｌｐ−１のポリマ複合体
EP2001500A4 (en) 2006-03-10 2010-07-28 Dyax Corp FORMULATIONS FOR ECALLANTIDE
CA2653061A1 (en) 2006-06-21 2007-12-27 Enzon Pharmaceuticals, Inc. Stabilized proteins
US8268948B2 (en) * 2006-07-21 2012-09-18 Nektar Therapeutics Polymeric reagents comprising a terminal vinylic group and conjugates formed therefrom
NZ575804A (en) 2006-09-13 2012-01-12 Univ Columbia Undercarboxylated/uncarboxylated osteocalcin increases beta-cell proliferation, insulin secretion, insulin sensitivity, glucose tolerance and decreases fat mass
JP2008156283A (ja) * 2006-12-25 2008-07-10 Kyowa Hakko Kogyo Co Ltd 水溶性ポリマー修飾ポリペプチドの製造方法
AU2007340382B2 (en) * 2006-12-27 2013-06-27 Nektar Therapeutics Von Willebrand Factor- and Factor VIII-polymer conjugates having a releasable linkage
RU2007137048A (ru) 2007-10-05 2009-04-10 Общество С Ограниченной Ответственностью "Концерн О3" (Ru) Лекарственное средство для лечения и предупреждения осложнений при сахарном диабете
DK2209494T3 (en) 2007-10-09 2016-10-03 Polytherics Ltd New conjugated proteins and peptides
EP2070950A1 (en) 2007-12-14 2009-06-17 Fresenius Kabi Deutschland GmbH Hydroxyalkyl starch derivatives and process for their preparation
US20120195847A1 (en) 2008-09-19 2012-08-02 Nektar Therapeutics Polymer conjugates of biphalin peptides
WO2010033223A1 (en) 2008-09-19 2010-03-25 Nektar Therapeutics Polymer conjugates of opioid growth factor peptides
WO2010033215A2 (en) 2008-09-19 2010-03-25 Nektar Therapeutics Polymer conjugates of aod-like peptides
US20110171160A1 (en) 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of kiss1 peptides
EP2334338A2 (en) 2008-09-19 2011-06-22 Nektar Therapeutics Polymer conjugates of c-peptides
WO2010033207A1 (en) 2008-09-19 2010-03-25 Nektar Therapeutics Polymer conjugates of therapeutic peptides
US20110171166A1 (en) 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of osteocalcin peptides
EP2344199A1 (en) 2008-09-19 2011-07-20 Nektar Therapeutics Polymer conjugates of thymosin alpha 1 peptides
WO2010033221A1 (en) 2008-09-19 2010-03-25 Nektar Therapeutics Polymer conjugates of protegrin peptides
WO2010033222A2 (en) 2008-09-19 2010-03-25 Netkar Therapeutics Polymer conjugates of ziconotide peptides
EP2334333A1 (en) 2008-09-19 2011-06-22 Nektar Therapeutics Polymer conjugates of v681-like peptides
US20110171164A1 (en) 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of glp-2-like peptides
WO2010033220A2 (en) 2008-09-19 2010-03-25 Nektar Therapeutics Modified therapeutics peptides, methods of their preparation and use

Patent Citations (101)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4013075A (en) * 1974-07-15 1977-03-22 I.S.F. S.P.A. Inhalers and insufflators having a cutting means
US4010148A (en) * 1975-05-12 1977-03-01 Board Of Regents Of The University Of Texas System Purified thymosin and process
US4148788A (en) * 1978-01-23 1979-04-10 Hoffmann-La Roche Inc. Synthesis of thymosin α1
US4247066A (en) * 1978-02-21 1981-01-27 General Dynamics Corporation Airfoil variable cambering device and method
US4321260A (en) * 1980-05-22 1982-03-23 Ayerst, Mckenna & Harrison Inc. Method of using gonadorelin for the treatment of benign prostatic hyperplasia
US4667668A (en) * 1981-07-08 1987-05-26 Aktiebolaget Draco Dosage inhalator
US4652548A (en) * 1981-08-27 1987-03-24 Eli Lilly And Company Pharmaceutical formulations comprising human insulin, human C-peptide, and human proinsulin
US4461724A (en) * 1981-10-28 1984-07-24 Nippon Zoki Pharmaceutical Co., Ltd. Peptide compounds, a process for manufacturing them, pharmaceutical compositions containing them, and methods for treating ulcer and thrombus with them
US4585754A (en) * 1984-01-09 1986-04-29 Valcor Scientific, Ltd. Stabilization of proteins and peptides by chemical binding with chondroitin
US4810646A (en) * 1984-11-28 1989-03-07 Massachusetts Institute Of Technology Glucan compositions and process for preparation thereof
US4992540A (en) * 1984-11-28 1991-02-12 Massachusetts Institute Of Technology Glucan composition and process for preparation thereof
US4805811A (en) * 1985-03-29 1989-02-21 Aktiebolaget Draco Dosage device
US4668281A (en) * 1985-05-20 1987-05-26 E. I. Du Pont De Nemours And Company Thiophenesulfonamides
US4801612A (en) * 1986-07-03 1989-01-31 Regents Of The University Of California Method of inhibiting inflammatory response
US4846876A (en) * 1986-10-14 1989-07-11 Bayer Aktiengesellschaft Herbicidal imidazo-pyrrolo-pyridine derivatives
US5028703A (en) * 1988-03-11 1991-07-02 Massachusetts Institute Of Technology Glucan composition and process for preparation thereof
US5605674A (en) * 1988-12-06 1997-02-25 Riker Laboratories, Inc. Medicinal aerosol formulations
US5221736A (en) * 1988-12-21 1993-06-22 Bionebraska, Inc. Sequential peptide and oligonucleotide syntheses using immunoaffinity techniques
US5612460A (en) * 1989-04-19 1997-03-18 Enzon, Inc. Active carbonates of polyalkylene oxides for modification of polypeptides
US5741495A (en) * 1989-06-15 1998-04-21 Alpha-Beta Technology, Inc. Glucan drug delivery system and adjuvant
US5607677A (en) * 1989-06-15 1997-03-04 Alpha-Beta Technology, Inc. Glucan drug delivery system and adjuvant
US5288708A (en) * 1989-09-12 1994-02-22 Predrag Sikiric Pharmacologically active substance BPC, the process for its preparation and its use in the therapy
US5118494A (en) * 1990-03-23 1992-06-02 Minnesota Mining And Manufacturing Company Use of soluble fluorosurfactants for the preparation of metered-dose aerosol formulations
US5888762A (en) * 1990-06-05 1999-03-30 Centre National De La Recherche Scientifique (Cnrs) Neurotropic growth factors comprising a homeobox peptide
US5618914A (en) * 1991-02-07 1997-04-08 Molecumetics, Ltd. Conformationally restricted mimetics of beta turns and beta bulges and peptides containing the same
US5320094A (en) * 1992-01-10 1994-06-14 The Johns Hopkins University Method of administering insulin
US5521184A (en) * 1992-04-03 1996-05-28 Ciba-Geigy Corporation Pyrimidine derivatives and processes for the preparation thereof
US5767254A (en) * 1992-12-15 1998-06-16 The Arizona Board Of Regents On Behalf Of The University Of Arizona Method for making amino acid glycosides and glycopeptides
US5492688A (en) * 1993-04-28 1996-02-20 The Center For Innovative Technology Metered dose inhaler fomulations which include the ozone-friendly propellant HFC 134a and a pharmaceutically acceptable suspending, solubilizing, wetting, emulsifying or lubricating agent
US5710245A (en) * 1993-06-18 1998-01-20 Molecumetics, Ltd. Alpha-helix mimetics and methods relating thereto
US5880131A (en) * 1993-10-20 1999-03-09 Enzon, Inc. High molecular weight polymer-based prodrugs
US5388572A (en) * 1993-10-26 1995-02-14 Tenax Corporation (A Connecticut Corp.) Dry powder medicament inhalator having an inhalation-activated piston to aerosolize dose and deliver same
US5739208A (en) * 1993-11-12 1998-04-14 Shearwater Polymers, Inc. Isolatable, water soluble, and hydrolytically stable active sulfones of poly(ethylene glycol) and related polymers for modification of surfaces and molecules
US5629384A (en) * 1994-05-17 1997-05-13 Consiglio Nazionale Delle Ricerche Polymers of N-acryloylmorpholine activated at one end and conjugates with bioactive materials and surfaces
US5650234A (en) * 1994-09-09 1997-07-22 Surface Engineering Technologies, Division Of Innerdyne, Inc. Electrophilic polyethylene oxides for the modification of polysaccharides, polypeptides (proteins) and surfaces
US5785049A (en) * 1994-09-21 1998-07-28 Inhale Therapeutic Systems Method and apparatus for dispersion of dry powder medicaments
US5740794A (en) * 1994-09-21 1998-04-21 Inhale Therapeutic Systems Apparatus and methods for dispersing dry powder medicaments
US5522385A (en) * 1994-09-27 1996-06-04 Aradigm Corporation Dynamic particle size control for aerosolized drug delivery
US5922675A (en) * 1994-11-17 1999-07-13 Eli Lilly And Company Acylated Insulin Analogs
US6379670B1 (en) * 1994-11-18 2002-04-30 Neurocrine Biosciences, Inc. Methods for treatment of multiple sclerosis using peptide analogs of human myelin basic protein
US6205999B1 (en) * 1995-04-05 2001-03-27 Aerogen, Inc. Methods and apparatus for storing chemical compounds in a portable inhaler
US6585509B2 (en) * 1995-05-10 2003-07-01 Allports Llc International Vaporization and pressurization of liquid in a porous material
US6347936B1 (en) * 1995-05-10 2002-02-19 Allports Llc International Liquid vaporization and pressurization apparatus and methods
US5758637A (en) * 1995-08-31 1998-06-02 Aerogen, Inc. Liquid dispensing apparatus and methods
US5929237A (en) * 1995-10-27 1999-07-27 Molecumetics Ltd. Reverse-turn mimetics and methods relating thereto
US5859184A (en) * 1995-10-27 1999-01-12 Molecumetics Ltd. Alpha-helix mimetics and methods relating thereto
US5610136A (en) * 1996-01-30 1997-03-11 Milkhaus Laboratory, Inc. Method for treatment of benign prostatic hypertrophy
US6085740A (en) * 1996-02-21 2000-07-11 Aerogen, Inc. Liquid dispensing apparatus and methods
US6083909A (en) * 1996-07-08 2000-07-04 Fresenius Ag Haemoglobin-hydroxyethyl starch conjugates as oxygen carriers
US6504005B1 (en) * 1996-08-07 2003-01-07 Yeda Research And Development Co. Ltd. Long-acting drugs and pharmaceutical compositions comprising them
US6214966B1 (en) * 1996-09-26 2001-04-10 Shearwater Corporation Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution
US6864350B2 (en) * 1996-09-26 2005-03-08 Nektar Therapeutics Al, Corporation Soluble, degradable poly (ethylene glycol) derivatives for controllable release of bound molecules into solution
US6515100B2 (en) * 1996-09-26 2003-02-04 Shearwater Corporation Soluble, degradable poly (ethylene glycol) derivatives for controllable release of bound molecules into solution
US6362254B2 (en) * 1998-03-12 2002-03-26 Shearwater Corporation Poly(ethylene glycol) derivatives with proximal reactive groups
US6080762A (en) * 1998-04-08 2000-06-27 Eli Lilly And Company Pulmonary and nasal delivery of raloxifene
US6257233B1 (en) * 1998-06-04 2001-07-10 Inhale Therapeutic Systems Dry powder dispersing apparatus and methods for their use
US6014970A (en) * 1998-06-11 2000-01-18 Aerogen, Inc. Methods and apparatus for storing chemical compounds in a portable inhaler
US6524591B1 (en) * 1998-06-29 2003-02-25 Cbr Monocyte locomotion inhibitory factor
US7314754B2 (en) * 1998-10-27 2008-01-01 Takeda Pharmaceutical Company Limited Polynucleotide encoding the hOT7T175 G protein coupled receptor
US7030084B2 (en) * 1999-06-19 2006-04-18 Nobex Corporation Drug-oligomer conjugates with polyethylene glycol components
US6579968B1 (en) * 1999-06-29 2003-06-17 Palatin Technologies, Inc. Compositions and methods for treatment of sexual dysfunction
US6376470B1 (en) * 1999-09-23 2002-04-23 Enzon, Inc. Polymer conjugates of ara-C and ara-C derivatives
US20020013266A1 (en) * 1999-10-04 2002-01-31 Shearwater Corporation Polymer stabilized neuropeptides
US20040038899A1 (en) * 1999-10-04 2004-02-26 Shearwater Corporation Polymer stabilized neuropeptides
US6518239B1 (en) * 1999-10-29 2003-02-11 Inhale Therapeutic Systems, Inc. Dry powder compositions having improved dispersivity
US6348558B1 (en) * 1999-12-10 2002-02-19 Shearwater Corporation Hydrolytically degradable polymers and hydrogels made therefrom
US6838259B2 (en) * 1999-12-17 2005-01-04 Takeda Chemical Industries, Ltd. Process for producing kiss-1 peptide
US20050059600A1 (en) * 1999-12-17 2005-03-17 Mayo Foundation For Medical Education And Research Chimeric natriuretic peptides
US6376604B2 (en) * 1999-12-22 2002-04-23 Shearwater Corporation Method for the preparation of 1-benzotriazolylcarbonate esters of poly(ethylene glycol)
US6413507B1 (en) * 1999-12-23 2002-07-02 Shearwater Corporation Hydrolytically degradable carbamate derivatives of poly (ethylene glycol)
US6899867B2 (en) * 1999-12-23 2005-05-31 Nektar Therapeutics Al, Corporation Hydrolytically degradable carbamate derivatives of poly(ethylene glycol)
US20020028189A1 (en) * 2000-04-14 2002-03-07 Seongbong Jo Biocompatible macromers
US6878689B2 (en) * 2000-06-23 2005-04-12 Kaken Pharmaceutical Co., Ltd. Preventives or remedies for heart failure
US6357490B1 (en) * 2000-08-22 2002-03-19 Advanced Inhalation Research, Inc. System, method and apparatus for filling containers
US20020065397A1 (en) * 2000-10-12 2002-05-30 Joseph Roberts Protecting therapeutic compositions from host-mediated inactivation
US6706289B2 (en) * 2000-10-31 2004-03-16 Pr Pharmaceuticals, Inc. Methods and compositions for enhanced delivery of bioactive molecules
US7060675B2 (en) * 2001-02-15 2006-06-13 Nobex Corporation Methods of treating diabetes mellitus
US6583408B2 (en) * 2001-05-18 2003-06-24 Battelle Memorial Institute Ionization source utilizing a jet disturber in combination with an ion funnel and method of operation
US6890518B2 (en) * 2001-05-21 2005-05-10 Nektar Therapeutics Compositions of chemically modified insulin
US6858580B2 (en) * 2001-06-04 2005-02-22 Nobex Corporation Mixtures of drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US20040096402A1 (en) * 2001-06-05 2004-05-20 Alexza Molecular Delivery Corporation Delivery of aerosols containing small particles through an inhalation route
US20030069170A1 (en) * 2001-09-07 2003-04-10 Richard Soltero Pharmaceutical compositions of drug-oligomer conjugates and methods of treating diseases therewith
US20080152668A1 (en) * 2001-11-01 2008-06-26 Sciclone Pharmaceuticals, Inc. Thymosin Alpha 1 Peptide/Polymer Conjugates
US20030143596A1 (en) * 2001-11-07 2003-07-31 Shearwater Corporation Branched polymers and their conjugates
US7026440B2 (en) * 2001-11-07 2006-04-11 Nektar Therapeutics Al, Corporation Branched polymers and their conjugates
US6701922B2 (en) * 2001-12-20 2004-03-09 Chrysalis Technologies Incorporated Mouthpiece entrainment airflow control for aerosol generators
US20040116649A1 (en) * 2002-09-09 2004-06-17 Antoni Kozlowski Water-soluble polymer alkanals
US20060121062A1 (en) * 2002-09-11 2006-06-08 Wolfram Eichner Conjugated hydroxyalkyl starch allergen compounds
US20040203081A1 (en) * 2002-11-26 2004-10-14 James Kenneth D. Natriuretic compounds, conjugates, and uses thereof
US20050014903A1 (en) * 2003-01-06 2005-01-20 Antoni Kozlowski Thiol selective water soluble polymer derivatives
US20060105948A1 (en) * 2003-03-24 2006-05-18 Novo Nordisk A/S GLP-2 derivatives
US20050009988A1 (en) * 2003-05-23 2005-01-13 Harris J. Milton Polymer derivatives having particular atom arrangements
US20050074425A1 (en) * 2003-07-02 2005-04-07 Polycord, Inc. Method for delivering polymerized therapeutic agent compositions and compositions thereof
US20080085862A1 (en) * 2003-11-13 2008-04-10 Hanmi Pharm. Ind. Co., Ltd. Natriuretic peptide conjugate using carrier substance
US20060074009A1 (en) * 2004-05-26 2006-04-06 James Kenneth D Natriuretic compounds, conjugates, and uses thereof
US20080139459A1 (en) * 2005-02-01 2008-06-12 N.V. Organon Conjugates A Polypeptide And A Pentasaccharide
US20070071764A1 (en) * 2005-04-22 2007-03-29 Sullivan John K Toxin peptide therapeutic agents
WO2007034498A1 (en) * 2005-09-26 2007-03-29 Compugen Ltd. Atrial natriuretic peptide (anp) splice variants and methods of using same
US20080044438A1 (en) * 2006-03-17 2008-02-21 Ostroff Gary R Yeast Cell Particles As Oral Delivery Vehicles For Antigens
US20080090758A1 (en) * 2006-09-06 2008-04-17 Aeterna Zentaris Gmbh Conjugates of disorazoles and their derivatives with cell-binding molecules, novel disorazole derivatives, processes of manufacturing and uses thereof
US20110165111A1 (en) * 2008-09-19 2011-07-07 Nektar Therapeutics Polymer conjugates of nesiritide peptides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Roberts et al, Chemistry for peptide and protein PEGylation, Advanced Drug Delivery Reviews 54 (2002) 459-476 *

Cited By (94)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9458444B2 (en) 2006-02-03 2016-10-04 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US10221228B2 (en) 2006-02-03 2019-03-05 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US9884901B2 (en) 2006-02-03 2018-02-06 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US9249407B2 (en) 2006-02-03 2016-02-02 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US9896494B2 (en) 2006-02-03 2018-02-20 Opko Biologics Ltd. Long-acting polypeptides and methods of producing same
US9908924B2 (en) 2006-02-03 2018-03-06 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US10119132B2 (en) 2006-02-03 2018-11-06 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US10351615B2 (en) 2006-02-03 2019-07-16 Opko Biologics Ltd. Methods of treatment with long-acting growth hormone
US9828417B2 (en) 2006-02-03 2017-11-28 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US10144921B2 (en) 2006-02-03 2018-12-04 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US11066658B2 (en) 2006-02-03 2021-07-20 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US10640758B2 (en) 2006-02-03 2020-05-05 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US10030060B2 (en) 2006-02-03 2018-07-24 Opko Biologics Ltd. Long-acting polypeptides and methods of producing same
US10301351B2 (en) 2007-03-28 2019-05-28 President And Fellows Of Harvard College Stitched polypeptides
US20110171160A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of kiss1 peptides
US20110165113A1 (en) * 2008-09-19 2011-07-07 Nektar Therapeutics Polymer conjugates of v681-like peptides
US20110237524A1 (en) * 2008-09-19 2011-09-29 Nektar Therapeutics Polymer conjugates of aod-like peptides
US20110171312A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Modified therapeutic peptides, methods of their preparation and use
US20110171162A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of thymosin alpha 1 peptides
US20110171165A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of opioid growth factor peptides
US20110171163A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of ziconotide peptides
US20110171166A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of osteocalcin peptides
US20110171164A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of glp-2-like peptides
US20110171161A1 (en) * 2008-09-19 2011-07-14 Nektar Therapeutics Polymer conjugates of protegrin peptides
US9763995B2 (en) 2008-09-19 2017-09-19 Nektar Therapeutics Polymer conjugates of protegrin peptides
US10538755B2 (en) 2009-07-09 2020-01-21 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US12203113B2 (en) 2009-07-09 2025-01-21 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US9663778B2 (en) 2009-07-09 2017-05-30 OPKO Biologies Ltd. Long-acting coagulation factors and methods of producing same
US9957299B2 (en) 2010-08-13 2018-05-01 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
WO2013059525A1 (en) * 2011-10-18 2013-04-25 Aileron Therapeutics, Inc. Peptidomimetic macrocyles
US10308699B2 (en) 2011-10-18 2019-06-04 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
CN104039342A (zh) * 2011-10-18 2014-09-10 爱勒让治疗公司 拟肽大环化合物
US9522947B2 (en) 2011-10-18 2016-12-20 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US9096684B2 (en) 2011-10-18 2015-08-04 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US20130165627A1 (en) * 2011-11-23 2013-06-27 Cory E. Bystrom Kisspeptin-54 detection by tandem mass spectrometry
US9874569B2 (en) * 2011-11-23 2018-01-23 Quest Diagnostics Investments Incorporated Kisspeptin-54 detection by tandem mass spectrometry
US9583322B2 (en) 2011-11-23 2017-02-28 Quest Diagnostics Investments Incorporated Kisspeptin-54 detection by tandem mass spectrometry
US10712348B2 (en) 2011-11-23 2020-07-14 Quest Diagnostics Investments Incorporated Kisspeptin-54 detection by tandem mass spectrometry
US20170176451A1 (en) * 2011-11-23 2017-06-22 Quest Diagnostics Investments Incorporated Kisspeptin-54 detection by tandem mass spectrometry
WO2013078222A1 (en) * 2011-11-23 2013-05-30 Quest Diagnostics Investments Incorporated Kisspeptin-54 detection by tandem mass spectrometry
US8916680B2 (en) * 2011-11-23 2014-12-23 Quest Diagnostics Investments, Inc. Kisspeptin-54 detection by tandem mass spectrometry
US10203337B2 (en) 2011-11-23 2019-02-12 Quest Diagnostics Investments Incorporated Kisspeptin-54 detection by tandem mass spectrometry
US10227380B2 (en) 2012-02-15 2019-03-12 Aileron Therapeutics, Inc. Triazole-crosslinked and thioether-crosslinked peptidomimetic macrocycles
US10213477B2 (en) 2012-02-15 2019-02-26 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US9522945B2 (en) 2012-04-19 2016-12-20 Opko Biologics Ltd. Long-acting oxyntomodulin variants and methods of producing same
US9884124B2 (en) 2012-05-17 2018-02-06 Extend Biosciences, Inc. Carriers for improved drug delivery
US9173950B2 (en) 2012-05-17 2015-11-03 Extend Biosciences, Inc. Vitamin D-ghrelin conjugates
US9289507B2 (en) 2012-05-17 2016-03-22 Extend Biosciences, Inc. Carriers for improved drug delivery
US20150299257A1 (en) * 2012-05-21 2015-10-22 Allotech Co. Ltd. Bioactive peptide complexes
US9499585B2 (en) * 2012-05-21 2016-11-22 Alloferon Inc. Bioactive peptide complexes
US10537646B2 (en) * 2012-06-04 2020-01-21 Opko Biologics Ltd. Pegylated OXM variants
EP3135690A1 (en) 2012-06-26 2017-03-01 Sutro Biopharma, Inc. Modified fc proteins comprising site-specific non-natural amino acid residues, conjugates of the same, methods of their preparation and methods of their use
US10669230B2 (en) 2012-11-01 2020-06-02 Aileron Therapeutics, Inc. Disubstituted amino acids and methods of preparation and use thereof
US9845287B2 (en) 2012-11-01 2017-12-19 Aileron Therapeutics, Inc. Disubstituted amino acids and methods of preparation and use thereof
US9808534B2 (en) 2012-11-20 2017-11-07 Opko Biologics Ltd. Method of increasing the hydrodynamic volume of polypeptides by attaching to gonadotrophin carboxy terminal peptides
US9919059B2 (en) * 2012-12-10 2018-03-20 Massachusetts Institute Of Technology Multistage nanoparticle drug delivery system for the treatment of solid tumors
US20140161884A1 (en) * 2012-12-10 2014-06-12 The Massachusetts General Hospital Multistage Nanoparticle Drug Delivery System for the Treatment of Solid Tumors
AU2014280869B2 (en) * 2013-06-14 2017-11-02 Universidade Federal Do Rio De Janeiro Non-agglomerating bioconjugates of amylin-mimetic compounds and polyethyleneglycol
CN105658234A (zh) * 2013-06-14 2016-06-08 里约热内卢联邦大学 拟胰岛淀粉样多肽化合物与聚乙二醇的非-凝聚生物缀合物
US20160331811A1 (en) * 2013-06-14 2016-11-17 Universidade Federal Do Rio De Janeiro Non-agglomerating bioconjugates of amylin-mimetic compounds and polyethyleneglycol
US20150050213A1 (en) * 2013-08-14 2015-02-19 University Of Virginia Patent Foundation Compositions and methods for imaging inflammation of traumatic brain injury
US10279023B2 (en) 2013-09-24 2019-05-07 Board Of Regents, The University Of Texas System Mucin-associated surface protein as a vaccine against chagas disease
US9566320B2 (en) 2013-09-24 2017-02-14 The Board Of Regents Of The University Of Texas System Mucin-associated surface protein as vaccine against Chagas disease
WO2015048072A3 (en) * 2013-09-24 2015-10-29 Board Of Regents, The University Of Texas System Mucin-associated surface protein as vaccine against chagas disease
US12029780B2 (en) 2013-10-21 2024-07-09 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US11197915B2 (en) 2013-10-21 2021-12-14 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US11065340B2 (en) 2014-07-31 2021-07-20 Serina Therapeutics, Inc. Polyoxazoline antibody drug conjugates
US10071168B2 (en) 2014-07-31 2018-09-11 Serina Therapeutics, Inc. Polyoxazoline antibody drug conjugates
WO2016019340A1 (en) * 2014-07-31 2016-02-04 Serina Therapeutics, Inc. Polyoxazoline antibody drug conjugates
US11938218B2 (en) 2014-08-08 2024-03-26 The Regents Of The University Of California High density peptide polymers
US10471120B2 (en) 2014-09-24 2019-11-12 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
US9789197B2 (en) 2014-10-22 2017-10-17 Extend Biosciences, Inc. RNAi vitamin D conjugates
US10420819B2 (en) 2014-10-22 2019-09-24 Extend Biosciences, Inc. Insulin vitamin D conjugates
US12076366B2 (en) 2014-10-22 2024-09-03 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
US10406202B2 (en) 2014-10-22 2019-09-10 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
US9585934B2 (en) 2014-10-22 2017-03-07 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
US9616109B2 (en) 2014-10-22 2017-04-11 Extend Biosciences, Inc. Insulin vitamin D conjugates
US10702574B2 (en) 2014-10-22 2020-07-07 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
US11116816B2 (en) 2014-10-22 2021-09-14 Extend Biosciences, Inc. Therapeutic vitamin d conjugates
CN105777892A (zh) * 2014-12-17 2016-07-20 深圳翰宇药业股份有限公司 普兰林肽聚乙二醇衍生物、其制备方法及其应用
US10253067B2 (en) 2015-03-20 2019-04-09 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
US10960058B2 (en) 2015-06-19 2021-03-30 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US10961289B2 (en) 2015-10-02 2021-03-30 The University Of Copenhagen Small molecules blocking histone reader domains
US11077198B2 (en) 2016-05-24 2021-08-03 Amgen Inc. Pegylated carfilzomib compounds
US11976106B2 (en) 2016-07-11 2024-05-07 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
WO2021030326A1 (en) * 2019-08-12 2021-02-18 Northwestern University Brush polymers for therapeutic applications
WO2021072349A1 (en) * 2019-10-11 2021-04-15 Tobebio Novel Drug Co., Ltd. Synergistic combination of chemotherapy and peptide for treating cancer
EP4072592A4 (en) * 2019-12-03 2024-06-12 Indian Institute of Technology, Delhi METHOD FOR PRODUCING PEGYLATED THERAPEUTIC PROTEINS
CN111494318A (zh) * 2020-04-28 2020-08-07 华中科技大学同济医学院附属协和医院 一种肿瘤靶向和还原敏感型复合胶束及其制备方法和应用
CN113134075A (zh) * 2021-04-29 2021-07-20 江苏普莱医药生物技术有限公司 一种抗菌肽乳膏及其制备方法
WO2023036878A1 (en) 2021-09-08 2023-03-16 Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Use of diazirine linker for drug conjugates
US12233115B2 (en) 2022-09-30 2025-02-25 Extend Biosciences, Inc. Long-acting parathyroid hormone
CN116036234A (zh) * 2023-01-17 2023-05-02 青岛市中心医院 一种用于治疗骨质疏松的药剂
CN118440155A (zh) * 2024-07-11 2024-08-06 中国药科大学 一种双激动多肽化合物及其医药用途

Also Published As

Publication number Publication date
CA2737040C (en) 2017-05-02
JP2012502990A (ja) 2012-02-02
US20150051371A1 (en) 2015-02-19
US20160022828A1 (en) 2016-01-28
AU2009292643A1 (en) 2010-03-25
JP2015134832A (ja) 2015-07-27
WO2010033240A3 (en) 2010-06-17
WO2010033207A1 (en) 2010-03-25
WO2010033240A2 (en) 2010-03-25
AU2009292643B2 (en) 2016-02-18
MX2011003117A (es) 2011-04-21
US8680263B2 (en) 2014-03-25
US20190070251A1 (en) 2019-03-07
EP2341942A1 (en) 2011-07-13
EP2898900A1 (en) 2015-07-29
EP2350118B1 (en) 2016-03-30
US20110171716A1 (en) 2011-07-14
CA2737040A1 (en) 2010-03-25
JP5765813B2 (ja) 2015-08-19
EP2350118A2 (en) 2011-08-03
US9682153B2 (en) 2017-06-20
EP2898900B1 (en) 2017-11-15
US9173951B2 (en) 2015-11-03

Similar Documents

Publication Publication Date Title
US9682153B2 (en) Polymer conjugates of therapeutic peptides
US20110171312A1 (en) Modified therapeutic peptides, methods of their preparation and use
US8449872B2 (en) Polymer conjugates of nesiritide peptides
JP5431874B2 (ja) ポリエチレングリコール−ｇｒｆ結合体の部位特異的調製方法
AU2006306236B2 (en) Selective VPAC2 receptor peptide agonists
CN101870728A (zh) 新型Exendin变体及其缀合物
US20110171164A1 (en) Polymer conjugates of glp-2-like peptides
CN113710692A (zh) 长效glp-2类似物
US20120195847A1 (en) Polymer conjugates of biphalin peptides
US20110171160A1 (en) Polymer conjugates of kiss1 peptides
US20110171165A1 (en) Polymer conjugates of opioid growth factor peptides
US20110165112A1 (en) Polymer conjugates of c-peptides
US20060105948A1 (en) GLP-2 derivatives
US9763995B2 (en) Polymer conjugates of protegrin peptides
US20110165113A1 (en) Polymer conjugates of v681-like peptides
US20110237524A1 (en) Polymer conjugates of aod-like peptides
US20110171162A1 (en) Polymer conjugates of thymosin alpha 1 peptides

Legal Events

Date Code Title Description
AS Assignment

Owner name: NEKTAR THERAPEUTICS, CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BOSSARD, MARY J.;ROCZNIAK, STEVEN O.;ZAPPE, HAROLD;AND OTHERS;SIGNING DATES FROM 20091013 TO 20091026;REEL/FRAME:023461/0201

AS Assignment

Owner name: NEKTAR THERAPEUTICS, CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BOSSARD, MARY J.;ROCZNIAK, STEVEN O.;ZAPPE, HAROLD;AND OTHERS;SIGNING DATES FROM 20110727 TO 20110829;REEL/FRAME:026878/0588

AS Assignment

Owner name: WELLS FARGO BANK, NATIONAL ASSOCIATION, AS COLLATE

Free format text: GRANT OF SECURITY INTEREST;ASSIGNOR:NEKTAR THERAPEUTICS;REEL/FRAME:028571/0141

Effective date: 20120711

Owner name: WELLS FARGO BANK, NATIONAL ASSOCIATION, AS COLLATERAL AGENT, NEW YORK

Free format text: GRANT OF SECURITY INTEREST;ASSIGNOR:NEKTAR THERAPEUTICS;REEL/FRAME:028571/0141

Effective date: 20120711

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION

AS Assignment

Owner name: NEKTAR THERAPEUTICS, CALIFORNIA

Free format text: RELEASE OF SECURITY INTEREST RECORDED AT REEL 28571, FRAME 0141;ASSIGNOR:WELLS FARGO BANK, NATIONAL ASSOCIATION, AS COLLATERAL AGENT;REEL/FRAME:036866/0700

Effective date: 20151005