WO2021170782A2 - Epi-x4 based peptides and derivatives thereof - Google Patents

Epi-x4 based peptides and derivatives thereof Download PDF

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Publication number
WO2021170782A2
WO2021170782A2 PCT/EP2021/054794 EP2021054794W WO2021170782A2 WO 2021170782 A2 WO2021170782 A2 WO 2021170782A2 EP 2021054794 W EP2021054794 W EP 2021054794W WO 2021170782 A2 WO2021170782 A2 WO 2021170782A2
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WIPO (PCT)
Prior art keywords
seq
pal
ilrwsrk
glu
peptide
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PCT/EP2021/054794
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English (en)
French (fr)
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WO2021170782A3 (en
Inventor
Jan MÜNCH
Frank Kirchhoff
Mirja HARMS
Ashraf H. Abadi
Monika Maged Wadie HABIB
Alexander Zelikin
Sheiliza CARMALI
Elsa SANCHEZ GARCIA
Pandian SOKKAR
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Universitaet Ulm
Universitaet Duisburg Essen
Baden Wuerttemberg Stiftung gGmbH
Aarhus Universitet
GERMAN UNIVERSITY IN CAIRO
Original Assignee
Universitaet Ulm
Universitaet Duisburg Essen
Baden Wuerttemberg Stiftung gGmbH
Aarhus Universitet
GERMAN UNIVERSITY IN CAIRO
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Priority to EP21707688.4A priority Critical patent/EP4110790A2/en
Priority to JP2022551673A priority patent/JP7733664B2/ja
Priority to US17/801,684 priority patent/US20230131339A1/en
Publication of WO2021170782A2 publication Critical patent/WO2021170782A2/en
Publication of WO2021170782A3 publication Critical patent/WO2021170782A3/en
Anticipated expiration legal-status Critical
Priority to JP2025138725A priority patent/JP2025186263A/ja
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/547—Chelates, e.g. Gd-DOTA or Zinc-amino acid chelates; Chelate-forming compounds, e.g. DOTA or ethylenediamine being covalently linked or complexed to the pharmacologically- or therapeutically-active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • IVRWSK(Pal)KVP-NH2 (SEQ ID NO.: 63, JM#166)
  • ILRWSRK(Pal)LP-NH2 (SEQ ID NO.: 68, JM#171)
  • d-ILRWSRKLP-NH2 SEQ ID NO.: 71, JM#174
  • ILRWSRK(Glu-Lau)LPCVS (SEQ ID NO.: 104, JM#213)
  • ILRWSRK(Glu-Ole)LPCVS (SEQ ID NO.: 108, JM#217)
  • ILRWSRK(C18diacid)LPCVS (SEQ ID NO.: 119,
  • ILRWSRK(Glu-C16diacid)-NH2 (SEQ ID NO.: 141 , JM#246) ILRWSRK(Glu-C18diacid)-NH2 (SEQ ID NO.: 142, JM#247) d-LLRWSRK(C16diacid)-NH2(SEQ ID NO.: 143, JM#248) d-LLRWSRK(C18diacid)-NH2(SEQ ID NO.: 144, JM#249) d-LLRWSRK(Glu-C16diacid)-NH2 (SEQ ID NO.: 145,
  • ILRWSRK(Glu-Ara)LPCVS (SEQ ID NO.: 148, JM#253)
  • ILRWSRK(Glu-Ste)-NH2 (SEQ ID NO.: 150, JM#255) d-LLRWSRK(Ste)-NH2 (SEQ ID NO.: 151 , JM#256) d-LLRWSRK(Glu-Ste)-NH2 (SEQ ID NO.: 152, JM#257) d-LLRWSRK(Glu-Myr)-NH2 (SEQ ID NO.: 157, JM#260)
  • ILRWSRK(Myr)-NH2 SEQ ID NO.: 159, JM#262
  • Group 1 consists of
  • Group 2 consists of ILRWSRK(Pal)L-NH2 (SEQ ID NO.: 69, JM#172) d-LMRWSRK(Pal)MP-NH2 (SEQ ID NO.: 75, JM#178) d-LMRWSRK(Pal)-NH2 (SEQ ID NO.: 77, JM#180)
  • ILRWSRK(Glu-Lau)LPCVS SEQ ID NO.: 104, JM#213) d-LLRWSRK(Glu-Pal)-NH2 (SEQ ID NO.: 130, JM#235) d-ILRWSRK(Glu-Pal)-NH2 (SEQ ID NO.: 133, JM#238) d-LLRWSRK(Glu-Ste)-NH2 (SEQ ID NO.: 152, JM#257)
  • Group 3 consists of d-LLRWSRK(Pal)MPCVS (SEQ ID NO.: 53, JM#141 )
  • IVRWSK(Pal)KVP-NH2 (SEQ ID NO.: 63, JM#166)
  • IVRWSKK(Pal)VP-NH2 (SEQ ID NO.: 64, JM#167)
  • ILRWSRK(Pal)LP-NH2 (SEQ ID NO.: 68, JM#171 )
  • ILRWSRK(Myr)LPCVS SEQ ID NO.: 107, JM#216
  • ILRWSRK(Ste)-NH2 (SEQ ID NO.: 149, JM#254)
  • d-LLRWSRK(Glu-Myr)-NH2 (SEQ ID NO.: 157, JM#260)
  • MLRWSRKLPCVS (SEQ ID NO.: 41 , JM#39)
  • ILRWSRKLPSVS (SEQ ID NO.: 51 , JM#122) d-LLRWSRK(Glu-Pal)MPCVS (SEQ ID NO.: 52, JM#140) ILRWSRK(Pal)MPCLS (SEQ ID NO.: 59, JM#149) d-LMRWSRKK(Glu-Pal)-NH2 (SEQ ID NO.: 92, JM#195) ILRWSRK-AcLPCVS (SEQ ID NO.: 97, JM#200)
  • ILRWSRK(Lau)LPCVS (SEQ ID NO.: 105, JM#214)
  • d-LLRWSRK(Ste)-NH2 SEQ ID NO.: 151 , JM#256
  • ILRWSRK(Myr)-NH2 SEQ ID NO.: 159, JM#262
  • ILRWSKKVPCVS SEQ ID NO.: 3, JM#1
  • IVRWSRKVPCVS SEQ ID NO.: 5, JM#3
  • IVRWSHKVPCVS SEQ ID NO.: 6, JM#4
  • IVRWSKKLPCVS (SEQ ID NO.: 7, JM#5)
  • ILRWSHKVPCVS SEQ ID NO.: 11 , JM#9
  • IIRWSRKMPCVS SEQ ID NO.: 18, JM#16
  • GLRWSRKMPCVS (SEQ ID NO.: 33, JM#31 )
  • Id-LRWSRKLPCVS (SEQ ID NO.: 43, JM#41 )
  • IVRWSKKVP-NH2 (SEQ ID NO.: 47, JM#106)
  • ILRWSRKLP-NH2 SEQ ID NO.: 49, JM#114
  • ILRWSRK-NH2 (SEQ ID NO.: 50, JM#118)
  • IVRWSKKVPSVS (SEQ ID NO.: 60, JM#151 )
  • IVRWSK(Pal)K-NH2 (SEQ ID NO.: 61 , JM#164)
  • IVRWSKK(Pal)-NH2 (SEQ ID NO.: 62, JM#165)
  • IVRWSK(Pal)KVPCVS SEQ ID NO.: 65, JM#168
  • d-ILRWSRK-NH2 SEQ ID NO.: 70, JM#173
  • d-ILRWSRKLP-NH2 SEQ ID NO.: 71 , JM#174
  • d-ILRWSRK(Pal)LP-NH2 SEQ ID NO.: 72, JM#175)
  • d-LMRWSRK(Pal)MPCVS SEQ ID NO.: 73, JM#176
  • ILRWSRK(Chl)LPCVS (SEQ ID NO.: 81 , JM#184)
  • VLRWSRKLPCVS (SEQ ID NO.: 86, JM#189)
  • d-VLRWSRKLPCVS SEQ ID NO.: 87, JM#190
  • d-MLRWSRK(Glu-Pal)-NH2 SEQ ID NO.: 93,
  • ILRWSRK(Glu-Ste)LPCVS SEQ ID NO.: 95, JM#198) ILRWSRK(Glu-Myr)LPCVS (SEQ ID NO.: 99, JM#204) ILRWSRK(Dec)LPCVS (SEQ ID NO.: 106, JM#215) d-LLRWSRK(Pal)-NH2 (SEQ ID NO.: 131 , JM#236) d-ILRWSRK(Pal)-NH2 (SEQ ID NO.: 132, JM#237) d-LLRWSRK(Myr)-NH2 (SEQ ID NO.: 158, JM#261 ) ILRWSRK(Pal-Glu)LPSVS (SEQ ID NO.: 163) ILRWSRK(Glu-Ste)LPSVS (SEQ ID NO.: 164)
  • Group 6 consists of d-LMRWSRKK(Glu-Pal)-NH2 (SEQ ID NO.: 92,
  • ILRWSRK(Glu-Ste)LPCVS SEQ ID NO.: 95, JM#198
  • ILRWSRK(Glu-Ste)LPSVS SEQ ID NO.: 164)
  • ILRWSRK(Dec)LPCVS (SEQ ID NO.: 106, JM#215) d-LLRWSRK(Myr)-NH2 (SEQ ID NO.: 158, JM#261 )
  • a ninth aspect of the invention is related to the inventive peptide or the inventive pharmaceutical composition for use in the prophylaxis and/or treatment of cancer, viral diseases, metabolic disorders, neurologic disorders, diseases of the immune system, or disorders of the blood clotting cascade and hematopoiesis in a mammal, wherein the mammal preferably is a human.
  • a tenth aspect of the invention is related to a method for manufacturing the inventive peptide by solid phase synthesis.
  • a further aspect of the invention is related to a conjugate in which the peptide according to the invention is coupled to cholesterol.
  • a further aspect of the invention is related to a conjugate in which the peptide according to the invention is coupled to a drug.
  • a further aspect of the invention is related to a conjugate in which the peptide according to the invention is coupled to human serum albumin.
  • Figure 1 shows two diagrams of X4-HIV-1 assays, wherein infection rates of cells are depending on concentrations of different peptide derivatives.
  • Figure 2 shows two diagrams of antibody competition assays, wherein the percentage of bound antibody is dependent on concentrations of truncated palmitoylated variants of peptide JM#21 (SEQ ID NO.: 23) (A) and of truncated palmitoylated variants of peptide JM#21 (SEQ ID NO.: 23) which are terminally modified (B).
  • Figure 3 shows a diagram of inhibition of CXCL12-induced Akt and Erk signaling, wherein the percentage of phosphorylated Akt and Erk is depending on concentrations of different peptide derivatives.
  • Figure 5 shows a diagram of antibody competition assays, wherein the percentage of bound antibody is depending on concentrations of DOTA- coupled peptides.
  • Figure 6 shows two diagrams of X4-FIIV-1 assays, wherein infection rates of cells are depending on concentrations of different peptides which are coupled to polyethylene glycol (A) or poly(vinyl alcohol) and poly(vinyl pyrrolidone) (B).
  • Figure 7 shows two diagrams of antibody competition assays, wherein the percentage of bound antibody is dependent on concentrations of truncated palmitoylated variants of peptides which are coupled to polyethylene glycol (A) or poly(vinyl alcohol) and poly(vinyl pyrrolidone) (B).
  • Figure 8 shows two diagrams of assays testing the activity of DSPE-coupled peptides, wherein (A) shows a diagram of X4-HIV-1 assays, and (B) shows a diagram of antibody competition assays.
  • Figure 11 shows illustrations of the computational models developed for the design of novel peptide derivatives: A) peptide - protein model in explicit water and membrane environment, B) investigation of tentative binding sites,
  • Figure 12 shows diagrams of stability of different peptides in human S9 liver fractions.
  • Figure 13 shows a diagram of in vivo stability of different peptides.
  • Figure 15 shows a diagram of cellular uptake and distribution of a 177 Lu/ 68 Ga- labeled DOTA-conjugated peptide and 177 Lu/ 68 Ga-labeled Pentixather in GHOST-CXCR4+ cells.
  • Figure 16 shows a diagram of cellular uptake of a 177 Lu-labeled DOTA- conjugated peptide and 177 Lu-labeled Pentixather in Jurkat cells.
  • IVRWSKK(Pal)VP-NH2 (SEQ ID NO.: 64, JM#167)
  • ILRWSRK(Pal)LP-NH2 (SEQ ID NO.: 68, JM#171 )
  • d-ILRWSRKLP-NH2 SEQ ID NO.: 71 , JM#174
  • ILRWSRK(Glu-Lau)LPCVS (SEQ ID NO.: 104, JM#213)
  • ILRWSRK(Glu-Ole)LPCVS (SEQ ID NO.: 108, JM#217)
  • ILRWSRK(Glu-C16diacid)-NH2 (SEQ ID NO.: 141 , JM#246) ILRWSRK(Glu-C18diacid)-NH2 (SEQ ID NO.: 142, JM#247) d-LLRWSRK(C16diacid)-NH2(SEQ ID NO.: 143, JM#248) d-LLRWSRK(C18diacid)-NH2(SEQ ID NO.: 144, JM#249) d-LLRWSRK(Glu-C16diacid)-NH2 (SEQ ID NO.: 145,
  • ILRWSRK(Glu-Ara)LPCVS (SEQ ID NO.: 148, JM#253)
  • ILRWSRK(Glu-Ste)-NH2 (SEQ ID NO.: 150, JM#255) d-LLRWSRK(Ste)-NH2 (SEQ ID NO.: 151 , JM#256) d-LLRWSRK(Glu-Ste)-NH2 (SEQ ID NO.: 152, JM#257) d-LLRWSRK(Glu-Myr)-NH2 (SEQ ID NO.: 157, JM#260)
  • Group 1 consists of
  • ILRWSRK(Glu-Pal)-NH2 (SEQ ID NO.: 89, JM#192) d-LMRWSRK(Glu-Pal)-NH2 (SEQ ID NO.: 91 , JM#194) ILRWSRK(Glu-Ste)-NH2 (SEQ ID NO.: 150, JM#255)
  • Group 2 consists of ILRWSRK(Pal)L-NH2 (SEQ ID NO.: 69, JM#172) d-LMRWSRK(Pal)MP-NH2 (SEQ ID NO.: 75, JM#178) d-LMRWSRK(Pal)-NH2 (SEQ ID NO.: 77, JM#180)
  • ILRWSRK(Glu-Lau)LPCVS SEQ ID NO.: 104, JM#213) d-LLRWSRK(Glu-Pal)-NH2 (SEQ ID NO.: 130, JM#235) d-ILRWSRK(Glu-Pal)-NH2 (SEQ ID NO.: 133, JM#238) d-LLRWSRK(Glu-Ste)-NH2 (SEQ ID NO.: 152, JM#257)
  • ILRWSRK(Pal)LP-NH2 (SEQ ID NO.: 68, JM#171 )
  • ILRWSRK(Myr)-NH2 SEQ ID NO.: 159, JM#262
  • IVRWSHKVPCVS SEQ ID NO.: 6, JM#4
  • IVRWSKKLPCVS (SEQ ID NO.: 7, JM#5)
  • ILRWSHKVPCVS SEQ ID NO.: 11 , JM#9
  • IVRWSKKMPCVS (SEQ ID NO.: 14, JM#12)
  • IIRWSRKMPCVS SEQ ID NO.: 18, JM#16
  • GLRWSRKMPCVS (SEQ ID NO.: 33, JM#31 )
  • Id-LRWSRKLPCVS (SEQ ID NO.: 43, JM#41 )
  • IVRWSKKVP-NH2 (SEQ ID NO.: 47, JM#106)
  • ILRWSRKLP-NH2 SEQ ID NO.: 49, JM#114
  • ILRWSRK-NH2 (SEQ ID NO.: 50, JM#118)
  • IYRWSRKMPCLS (SEQ ID NO.: 57, JM#146)
  • IVRWSK(Pal)K-NH2 (SEQ ID NO.: 61 , JM#164)
  • IVRWSKK(Pal)-NH2 (SEQ ID NO.: 62, JM#165)
  • ILRWSRK(Chl)LPCVS (SEQ ID NO.: 81 , JM#184)
  • ILRWSRK(Glu-Ste)LPCVS SEQ ID NO.: 95, JM#198
  • ILRWSRK(Glu-Ste)LPSVS SEQ ID NO.: 164)
  • Group 8 consists of d-LLRWSRKMPCVS (SEQ ID NO.: 31 , JM#29)
  • ILRWSRK(Dec)LPCVS (SEQ ID NO.: 106, JM#215) d-LLRWSRK(Myr)-NH2 (SEQ ID NO.: 158, JM#261 )
  • IVRWSK(Pal)KVP-NH2 (SEQ ID NO.: 63, JM#166)
  • IVRWSKK(Pal)VP-NH2 (SEQ ID NO.: 64, JM#167)
  • ILRWSRK(Pal)LP-NH2 (SEQ ID NO.: 68, JM#171 )
  • ILRWSRK(Glu-Ste)-NH2 (SEQ ID NO.: 150, JM#255)
  • d-LLRWSRK(Glu-Ste)-NH2 (SEQ ID NO.: 152, JM#257)
  • d-LLRWSRK(Glu-Myr)-NH2 (SEQ ID NO.: 157, JM#260)
  • IPRW(d-C)RKC-NH2 wherein the d-cysteine at position 5 is bound to the cysteine at position 8 via a disulfide bond to form a cyclic peptide (SEQ ID NO.: 113, JM#220)
  • ILRWSKKLPCVS wherein the lysine at position 6 is bound to the carboxyl-terminus via a peptide bond to form a cyclic peptide (SEQ ID NO.: 116, JM#222)
  • IPRW(d-C)RKCP-NH2 wherein the d-cysteine at position 5 is bound to the cysteine at position 8 via a disulfide bond to form a cyclic peptide (SEQ ID NO.: 155, JM#259) wherein d- in front of an amino acid indicates a D-amino acid, Pal indicates a palmitic acid on the preceding amino acid, Glu-Pal indicates a palmitic acid on the preceding amino acid with a glutamate linker, Dec indicates decanoic acid on the preceding amino acid, Glu-Dec indicates a decanoic acid on the preceding amino acid with a glutamate linker, Myr indicates myristic acid on the preceding amino acid, Glu-Myr indicates a myristic acid on the preceding amino acid with a glutamate linker, Ole indicates oleic acid on the preceding amino acid, Ste indicates stearic acid on the preceding amino acid, Glu-Ste indicates a ste
  • the inventive peptide derivatives are about 100 times more effective in binding to CXCR4 than previously known peptides. Furthermore, the invention provides peptides with a significantly higher plasma stability than peptides of the state of the art. Furthermore, the invention provides peptides with a significantly higher in vivo circulation half-life than peptides of the state of the art. Consequently, the invention provides peptides with a high therapeutic potential compared to drugs of the state of the art, because smaller doses will be sufficient in order to provide the desired effect.
  • the term derivative means all length fragments of the peptide EPI-X4 (SEQ ID NO.: 1) including truncations at the N and C terminus, the peptide of the invention containing amino acid residue substitutions including D-amino acid residues and modified amino acid residues as well as peptides containing disulfide bonds and extension at the N and C terminus.
  • the terms peptides, peptide derivatives and derivatives are used synonymously.
  • the term peptides does also extent to cyclized peptides, if the inventive peptides can be provided in cyclized form.
  • EPI-X4 derivatives dose dependently and specifically inhibited infection of reporter cells by X4-HIV-1.
  • Peptides EPI-X4 (SEQ ID NO.: 1) and WSC02 (SEQ ID NO.: 2) (EP 3 007 717 B1) were used as reference peptides.
  • Fig. 1 results of two assays are shown by way of diagrams, wherein the percentage of infected cells depends on the log concentration of different tested peptides ( Figures 1A and 1B).
  • JM#21 SEQ ID NO.: 23
  • Peptide derivatives coupled to fatty acids e.g.
  • Fig. 2 the activity was tested by an antibody competition assay (Fig. 2). Values determined by the competition assay represent the potency of a compound to compete with an antibody that specifically binds to the binding pocket (ECL2) of CXCR4. Those values most probable correlate with CXCR4 binding affinity of the compounds to CXCR4.
  • Fig. 2 results of two assays are shown by way of diagrams, wherein the percentage of bound antibody depends on the concentration of different peptides. To the right of the diagrams, an assignment of the curves to the tested peptides is explained.
  • JM#21 SEQ ID NO.: 23
  • WSC02 SEQ ID NO.: 2
  • JM#21 SEQ ID NO.: 23
  • AMD3100 Reduction of AKT phosphorylation at 10 pM by about 60
  • EPI-X4 SEQ ID NO.: 1
  • JM#18 SEQ ID NO.: 20
  • JM#18 (SEQ ID NO.: 20) blocked CXCL12-induced AKT and ERK signaling by about 40 % at a concentration of 0.1 pM and by almost 70 % at a concentration of 1 pM (not shown).
  • palmitic acid coupling led to an increased antagonistic effect.
  • At a concentration of 10 pM all tested palmitic acid coupled derivatives blocked CXCL12 induced AKT and ERK signaling by 100 %.
  • JM#143 (SEQ ID NO.: 54), a fatty acid coupled derivative of JM#21 (SEQ ID NO.: 23), blocked AKT and ERK phosphorylation at a concentration of 1 pM by almost 70 % and even at a concentration of 0.1 pM signaling was reduced by 20 - 25 %.
  • Strikingly, palmitic acid coupled WSC02 (JM#169, SEQ ID NO.: 66) had a very strong antagonistic activity and reduced AKT signaling by 85 % and ERK signaling by over 70% at a concentration of already 0.1 mM, at a concentration of 1 mM AKT signaling was completely and ERK signaling almost completely blocked.
  • Two or three independent experiments were carried out in triplicates for each peptide in each assay. The error bars refer to the standard deviation.
  • JM#173 SEQ ID NO.: 70
  • JM#174 SEQ ID NO.: 71
  • JM#114 SEQ ID NO.: 49
  • JM#118 SEQ ID NO.: 50
  • JM#173 SEQ ID NO.: 70
  • JM#174 SEQ ID NO.: 71
  • EPI-X4 (SEQ ID NO.: 1) derivatives that are coupled to fatty acids (e.g. palmitic acid).
  • Most of the fatty acid coupled derivatives had a strongly increased plasma stability as shown e.g. for fatty acid coupled JM#21 (JM#143 (SEQ ID NO.: 54) - JM#145 (SEQ ID NO.: 56)) that did not loose activity at all after 8 hours of plasma incubation. Strikingly, the same is also true for most truncated and fatty acid coupled versions of JM#21 (SEQ ID NO.: 23), WSC02 (SEQ ID NO.: 2) or similar (e.g.
  • JM#170 (SEQ ID NO.: 67), Fig. 4D - JM#172 (SEQ ID NO.: 69), Fig. 4E and JM#191 (SEQ ID NO.: 88) - JM#193 (SEQ ID NO.: 90) (Fig. 4F for JM#192)).
  • JM#194 (SEQ ID NO.: 91) - JM#197 (SEQ ID NO.: 94)
  • Fig. 4 D - F are based on one representative experiment. Fifth, it was shown that the peptide derivatives inhibit calcium-signaling. CXCL12 stimulation of CXCR4 expressing B-cells leads to a strong calcium- release. In the presence of CXCR4 antagonists this response is reduced. The inventors saw a reduction of cytokine induced calcium-signaling for 1 mM of JM#21 (SEQ ID NO.: 23) that was much stronger than the reduction seen for WSC02 (SEQ ID NO.: 2) at the same concentration.
  • Variants JM#192 (SEQ ID NO.: 89) and JM#255 (SEQ ID NO.: 150) have an unmodified N-terminus and were more rapidly degraded compared to their N-terminally modified variants (JM#194 (SEQ ID NO.: 91), JM#235 (SEQ ID NO.: 130) and JM#257 (SEQ ID NO.: 152), respectively) (Fig. 12B).
  • JM#194 SEQ ID NO.: 91
  • JM#235 SEQ ID NO.: 130
  • JM#257 SEQ ID NO.: 152
  • peptides were injected into the tail vein of mice. 4 hours post injection, mice were sacrificed and blood taken by heart punctation. Plasma was obtained by centrifugation and tested for remaining activity in antibody competition assay. As control, peptide was spiked into native plasma (ex vivo). ICso values were determined by non-linear regression assuming 1.8 ml in vivo blood volume. Remaining activity was determined by ICso (ex vivo)/ ICso ⁇ in vivo) x 100.
  • the peptides of group 1 are characterized by an inhibitory activity characterized by a half maximal inhibitory concentration (ICso) of below 5 nM, as measured in an X4-HIV-1 inhibition assay (in order to estimate the capability of blocking X4-HIV-1 infection).
  • ICso half maximal inhibitory concentration
  • the X4-HIV inhibition assay is designed to measure the activity of the inventive peptides by their efficiency of blocking the infection of tissue culture cells by CXCR4-tropic HIV-1 variants.
  • the peptides of group 5 are characterized by an inhibitory activity characterized by an ICso of above 150 nM, as measured in an HIV inhibition assay.
  • the peptides of group 8 are characterized by a relative activity of 100% after 2 hours of plasma incubation, but less (75 - 99%) after 8 hours of plasma incubation.
  • ILRWCRKPC-NH2 (SEQ ID NO.: 110, JM#218 linear)
  • ILRW(d-C)RKPC-NH2 (SEQ ID NO.: 112, JM#219 linear)
  • IPRW(d-S)RKP (SEQ ID NO.: 127, JM#233 linear)
  • IMRWCRKPC-NH2 (SEQ ID NO.: 154, JM#258 linear)
  • IPRW(d-C)RKCP-NH2 (SEQ ID NO.: 156, JM#259 linear)
  • the linear equivalents are used as control peptides in assays for characterizing the cyclized peptides such as activity and stability assays.
  • fatty acids i.e. of palmitic acid, decanoic acid, myristic acid, oleic acid, and stearic acid
  • the coupling of fatty acids could prolong the circulation of the peptides at a certain level of concentration in vivo.
  • other fatty acids such as lauric acid, saturated C16 fatty diacid, saturated C18 fatty diacid, and saturated C20 fatty acid.
  • a second aspect of the invention is directed to a peptide consisting of one of the following amino acid sequences, selected from any of the groups 1 to 11 , wherein
  • - Group 7 consists of d-ILRWSRK-NH2 (SEQ ID NO.: 70, JM#173)
  • IVRWSK(Pal)KVP-NH2 (SEQ ID NO.: 63, JM#166)
  • IVRWSKK(Pal)VP-NH2 (SEQ ID NO.: 64, JM#167)
  • ILRWSRK(Pal)LP-NH2 (SEQ ID NO.: 68, JM#171 )
  • d-ILRWSRKLP-NH2 SEQ ID NO.: 71 , JM#174
  • ILRWSRK(Glu-Ole)LPCVS (SEQ ID NO.: 108, JM#217)
  • ILRWSRK(Glu-Pal)-NH2 (SEQ ID NO.: 89, JM#192) d-LMRWSRK(Glu-Pal)-NH2 (SEQ ID NO.: 91 , JM#194) ILRWSRK(Glu-Ste)-NH2 (SEQ ID NO.: 150, JM#255)
  • ILRWSRK(Pal)L-NH2 (SEQ ID NO.: 69, JM#172) d-LMRWSRK(Pal)MP-NH2 (SEQ ID NO.: 75, JM#178) d-LMRWSRK(Pal)-NH2 (SEQ ID NO.: 77, JM#180) ILRWSRK(Ole)LPCVS (SEQ ID NO.: 80, JM#183)
  • Group 3 consists of d-LLRWSRK(Pal)MPCVS (SEQ ID NO.: 53, JM#141 )
  • IVRWSKK(Pal)VP-NH2 (SEQ ID NO.: 64, JM#167)
  • ILRWSRKLP-NH2 SEQ ID NO.: 49, JM#114
  • ILRWSRK(Glu-Ste)LPCVS SEQ ID NO.: 95, JM#198
  • ILRWSRK(Glu-Ste)LPSVS SEQ ID NO.: 164)
  • IVRWSKK(Pal)VP-NH2 (SEQ ID NO.: 64, JM#167)
  • ILRWSRK(Pal)LP-NH2 (SEQ ID NO.: 68, JM#171 )
  • ILRW(d-C)RKPC-NH2 wherein the d-cysteine at position 5 is bound to the cysteine at position 9 via a disulfide bond to form a cyclic peptide (SEQ ID NO.: 111 , JM#219)
  • IPRW(d-C)RKC-NH2 wherein the d-cysteine at position 5 is bound to the cysteine at position 8 via a disulfide bond to form a cyclic peptide (SEQ ID NO.: 113, JM#220)
  • ILRWSKKLPCVS wherein the lysine at position 6 is bound to the carboxyl-terminus via a peptide bond to form a cyclic peptide (SEQ ID NO.: 116, JM#222)
  • Deferoxamine may alternatively be conjugated to the peptide via a lysine residue.
  • the above statements regarding the conjugation of deferoxamine via a cysteine residue likewise apply to the conjugation of deferoxamine via a lysine residue.
  • the complexing agent is the chelator DOTA.
  • the peptide is conjugated to the chelator DOTA, wherein the peptide preferably is C-terminally conjugated to DOTA.
  • DOTA also known as tetraxetan
  • the molecule consists of a central 12-membered tetraaza (i.e., containing four nitrogen atoms) ring.
  • DOTA for dodecane tetraacetic acid
  • DOTA for dodecane tetraacetic acid
  • DOTA-conjugated peptides are suitable for labelling with radioactive nuclides, e.g. 68 Ga and 177 Lu. Consequently, these peptides are useful in applications in diagnostic and therapeutic approaches.
  • the inventive EPI-X4 (SEQ ID NO.: 1) derivatives, which are specific for the CXCR4, can be used for blending diagnostic and therapeutic with the same molecule (radiotheranostics). Radiotheranostics based on these peptides is offering new imaging tests and therapeutic options to patients suffering from CXCR4-expressing malignancies.
  • Fig. 5 shows a diagram of an antibody competition assay (based on one representative experiment per peptide), wherein the percentage of bound antibody is depending on the molar concentration of the shown peptides.
  • the DOTA-conjugated peptides were radioactively labelled with 177 Lu or with 68 Ga (see Example further below, Fig. 14-16).
  • the inventors confirmed the suitability of radiolabeled deferoxamine- conjugated peptides as tumor imaging probes and as probes for analyzing the distribution of the peptides in e.g. mouse models.
  • the inventors synthesized the following deferoxamine-conjugated peptides: C- deferoxamine linked JM#122 (SEQ ID NO.: 51), C-deferoxamine linked JM#194 (SEQ ID NO.: 91), C-deferoxamine linked peptide of SEQ ID NO.: 163 and C-deferoxamine linked peptide of SEQ ID NO.: 164, wherein C indicates the additional cysteine that was coupled to the C-terminal amino acid of the peptides and deferoxamine was conjugated to the peptides via this additional cysteine as (succinimido-propionyl-desferrioxamine) acetate.
  • the deferoxamine-conjugated peptides were
  • a third aspect of the invention is directed to a peptide consisting of one of the following amino acid sequences, selected from any of the groups 1 to 11 , wherein
  • Group 7 consists of d-ILRWSRK-NH2 (SEQ ID NO.: 70, JM#173)
  • IVRWSK(Pal)KVP-NH2 (SEQ ID NO.: 63, JM#166)
  • IVRWSKK(Pal)VP-NH2 (SEQ ID NO.: 64, JM#167)
  • ILRWSRK(Pal)LP-NH2 (SEQ ID NO.: 68, JM#171 )
  • d-ILRWSRKLP-NH2 SEQ ID NO.: 71 , JM#174
  • ILRWSRK(Glu-Lau)LPCVS (SEQ ID NO.: 104, JM#213)
  • ILRWSRK(Glu-Ole)LPCVS (SEQ ID NO.: 108, JM#217)
  • ILRWSRK(C16diacid)LPCVS (SEQ ID NO.: 117, JM#226) ILRWSRK(Glu-C16diacid)LPCVS (SEQ ID NO.: 118,
  • ILRWSRK(C18diacid)LPCVS (SEQ ID NO.: 119,
  • ILRWSRK(C16diacid)-NH2 (SEQ ID NO.: 139, JM#244) ILRWSRK(C18diacid)-NH2 (SEQ ID NO.: 140, JM#245) ILRWSRK(Glu-C16diacid)-NH2 (SEQ ID NO.: 141 , JM#246) ILRWSRK(Glu-C18diacid)-NH2 (SEQ ID NO.: 142, JM#247) d-LLRWSRK(C16diacid)-NH2(SEQ ID NO.: 143, JM#248) d-LLRWSRK(C18diacid)-NH2(SEQ ID NO.: 144, JM#249) d-LLRWSRK(Glu-C16diacid)-NH2 (SEQ ID NO.: 145,
  • ILRWSRK(Glu-Ara)LPCVS (SEQ ID NO.: 148, JM#253)
  • ILRWSRK(Glu-Ste)-NH2 (SEQ ID NO.: 150, JM#255) d-LLRWSRK(Ste)-NH2 (SEQ ID NO.: 151 , JM#256) d-LLRWSRK(Glu-Ste)-NH2 (SEQ ID NO.: 152, JM#257) d-LLRWSRK(Glu-Myr)-NH2 (SEQ ID NO.: 157, JM#260)
  • ILRWSRK(Myr)-NH2 SEQ ID NO.: 159, JM#262
  • Group 1 consists of
  • ILRWSRK(Glu-Pal)-NH2 (SEQ ID NO.: 89, JM#192) d-LMRWSRK(Glu-Pal)-NH2 (SEQ ID NO.: 91 , JM#194) ILRWSRK(Glu-Ste)-NH2 (SEQ ID NO.: 150, JM#255)
  • ILRWSRK(Glu-Lau)LPCVS SEQ ID NO.: 104, JM#213) d-LLRWSRK(Glu-Pal)-NH2 (SEQ ID NO.: 130, JM#235) d-ILRWSRK(Glu-Pal)-NH2 (SEQ ID NO.: 133, JM#238) d-LLRWSRK(Glu-Ste)-NH2 (SEQ ID NO.: 152, JM#257)
  • Group 3 consists of d-LLRWSRK(Pal)MPCVS (SEQ ID NO.: 53, JM#141 )
  • IVRWSK(Pal)KVP-NH2 (SEQ ID NO.: 63, JM#166)
  • IVRWSKK(Pal)VP-NH2 (SEQ ID NO.: 64, JM#167)
  • ILRWSRK(Pal)LP-NH2 (SEQ ID NO.: 68, JM#171 )
  • ILRWSRK(Myr)LPCVS SEQ ID NO.: 107, JM#216
  • ILRWSRK(Ste)-NH2 (SEQ ID NO.: 149, JM#254)
  • d-LLRWSRK(Glu-Myr)-NH2 (SEQ ID NO.: 157, JM#260)
  • MLRWSRKLPCVS (SEQ ID NO.: 41 , JM#39)
  • ILRWSRKLPSVS (SEQ ID NO.: 51 , JM#122) d-LLRWSRK(Glu-Pal)MPCVS (SEQ ID NO.: 52, JM#140) ILRWSRK(Pal)MPCLS (SEQ ID NO.: 59, JM#149) d-LMRWSRKK(Glu-Pal)-NH2 (SEQ ID NO.: 92, JM#195) ILRWSRK-AcLPCVS (SEQ ID NO.: 97, JM#200)
  • ILRWSRK(Lau)LPCVS (SEQ ID NO.: 105, JM#214)
  • d-LLRWSRK(Ste)-NH2 SEQ ID NO.: 151 , JM#256
  • ILRWSRK(Myr)-NH2 (SEQ ID NO.: 159, JM#262)
  • Group 5 consists of
  • ILRWSKKVPCVS SEQ ID NO. 3, JM#1
  • IVRWSRKVPCVS SEQ ID NO. 5, JM#3
  • IVRWSHKVPCVS SEQ ID NO. 6, JM#4
  • IVRWSKKLPCVS SEQ ID NO. 7, JM#5
  • ILRWSHKVPCVS SEQ ID NO. 11 , JM#9
  • IFRWSHKVPCVS SEQ ID NO. 13, JM#11
  • IIRWSRKMPCVS SEQ ID NO. 18, JM#16
  • GLRWSRKMPCVS (SEQ ID NO. 33, JM#31 )
  • Id-LRWSRKLPCVS (SEQ ID NO. 43, JM#41 )
  • IVRWSKKVP-NH2 (SEQ ID NO. 47, JM#106)
  • IVRWSKK-NH2 (SEQ ID NO.: 48, JM#110)
  • ILRWSRKLP-NH2 SEQ ID NO.: 49, JM#114
  • ILRWSRK-NH2 (SEQ ID NO.: 50, JM#118)
  • IYRWSRKMPCLS (SEQ ID NO.: 57, JM#146)
  • IVRWSKKVPSVS (SEQ ID NO.: 60, JM#151 )
  • IVRWSK(Pal)K-NH2 (SEQ ID NO.: 61 , JM#164)
  • IVRWSKK(Pal)-NH2 (SEQ ID NO.: 62, JM#165)
  • IVRWSK(Pal)KVPCVS SEQ ID NO.: 65, JM#168
  • d-ILRWSRK-NH2 SEQ ID NO.: 70, JM#173
  • d-ILRWSRKLP-NH2 SEQ ID NO.: 71 , JM#174
  • d-ILRWSRK(Pal)LP-NH2 SEQ ID NO.: 72, JM#175)
  • d-LMRWSRK(Pal)MPCVS SEQ ID NO.: 73, JM#176
  • ILRWSRK(Chl)LPCVS (SEQ ID NO.: 81 , JM#184)
  • VLRWSRKLPCVS (SEQ ID NO.: 86, JM#189)
  • d-VLRWSRKLPCVS SEQ ID NO.: 87, JM#190
  • d-MLRWSRK(Glu-Pal)-NH2 SEQ ID NO.: 93,
  • ILRWSRK(Glu-Ste)LPCVS SEQ ID NO.: 95, JM#198) ILRWSRK(Glu-Myr)LPCVS (SEQ ID NO.: 99, JM#204) ILRWSRK(Dec)LPCVS (SEQ ID NO.: 106, JM#215) d-LLRWSRK(Pal)-NH2 (SEQ ID NO.: 131 , JM#236) d-ILRWSRK(Pal)-NH2 (SEQ ID NO.: 132, JM#237) d-LLRWSRK(Myr)-NH2 (SEQ ID NO.: 158, JM#261 ) ILRWSRK(Pal-Glu)LPSVS (SEQ ID NO.: 163) ILRWSRK(Glu-Ste)LPSVS (SEQ ID NO.: 164)
  • Group 6 consists of d-LMRWSRKK(Glu-Pal)-NH2 (SEQ ID NO.: 92,
  • ILRWSRK(Glu-Ste)LPCVS SEQ ID NO.: 95, JM#198
  • ILRWSRK(Glu-Ste)LPSVS SEQ ID NO.: 164)
  • Group 8 consists of d-LLRWSRKMPCVS (SEQ ID NO.: 31 , JM#29)
  • JM#140 d-LMRWSRK(Pal)MP-NH2 (SEQ ID NO.: 75, JM#178) ILRWSRK(Dec)LPCVS (SEQ ID NO.: 106, JM#215) d-LLRWSRK(Myr)-NH2 (SEQ ID NO.: 158, JM#261 ) ILRWSRK(Glu-Myr)-NH2 (SEQ ID NO.: 160, JM#263)
  • Group 10 consists of ILRWSRK(Pal)LPCVS (SEQ ID NO.: 55, JM#144)
  • IVRWSK(Pal)KVP-NH2 (SEQ ID NO.: 63, JM#166)
  • ILRWSRKLK(Glu-Pal)-NH2 (SEQ ID NO.: 90, JM#193) d-LMRWSRK(Glu-Pal)-NH2 (SEQ ID NO.: 91 ,
  • ILRWSRK(Glu-Lau)LPCVS SEQ ID NO.: 104, JM#213) d-LLRWSRK(Glu-Pal)-NH2 (SEQ ID NO.: 130, JM#235) d-ILRWSRK(Glu-Pal)-NH2 (SEQ ID NO.: 133, JM#238)
  • a sixth aspect of the invention is related to the inventive peptide or the inventive pharmaceutical composition for use in medicine.
  • a seventh aspect of the invention is related to the use of the inventive peptide or the inventive pharmaceutical composition for the preparation of a formulation for oral administration, inhalation, intravenous administration, topical administration, intranasal administration, intraperitoneal administration, subcutaneous administration and/or any other injectable form.
  • the pharmaceutical composition may be administered, for example, in the form of liquid formulations including solutions, suspensions and emulsions, and in the form of pills, tablets, film tablets, coated tablets, capsules, liposomal formulations, micro- and nano-formulations, and powders.
  • JM#21 (SEQ ID NO.: 23) blocked the CXCR4 12G5 epitope of WM cells in presence or absence of different CXCR4 mutations in a dose dependent manner, impaired migration of WM cells with or without S338X mutation along a CXCL12 gradient, and reduced CXCL12-induced ERK phosphorylation of CXCR4 mutant WM cells in a dose-dependent manner.
  • a tenth aspect of the invention is related to a method for manufacturing the inventive peptide by solid phase synthesis. If this is not possible, e.g. for peptides coupled to polymers, other methods are selected for manufacture of those derivates.
  • monomeric peptides are provided and coupled under oxidative reaction conditions which are capable to oxidize SH bonds to yield -S-S-bonds.
  • the peptide that is conjugated to the maleimide linker for example JM#173 (SEQ ID NO.: 70), is supposed to react with albumin in vivo (binding of the peptide to Cys34 on albumin via the maleimide linker) and therefore is highly stable in human plasma, but not lipophilic (as with the fatty acid linked peptide versions).
  • a method of prophylaxis and/or treatment of cancer, viral diseases, metabolic disorders, neurologic disorders, diseases of the immune system, or disorders of the blood clotting cascade and hematopoiesis in a mammal comprising administering the inventive peptide or the inventive pharmaceutical composition to the mammal, wherein the mammal preferably is a human.
  • the cancer preferably is a CXCR4- expressing cancer.
  • the CXCR4-expressing cancer preferably is a CXCR4- expressing liver, pancreas, prostate, or breast cancer or another CXCR4- expressing solid tumor.
  • 12G5-APC antibody competition was then performed as described before.
  • samples were thawed and centrifuged at 14,000 rpm to remove cells and debris. The supernatant was then diluted in PBS and 12G5-antibody competition assay was performed. After the 2 hours incubation, the cells were washed and 50 pi of 1 -step-Fix/Lyse solution (Thermo Fisher #00-5333- 54) was added for 15 minutes at room temperature. Afterwards cells were washed again and analyzed for bound antibody.
  • 1 -step-Fix/Lyse solution Thermo Fisher #00-5333- 54
  • ERK/AKT signaling assay CXCL12 induced ERK and AKT phosphorylation was determined in SupT1 cells. For this, 100,000 cells were seeded per well in a 96-V well plate in 100 mI medium supplemented with 1% FCS. Cells were incubated for 2 hours at 37°C before 5 mI of compounds were added. After 15 min incubation at 37°C cells were stimulated by adding 5 mI CXCL12 diluted in PBS to reach a final concentration of 100 ng/ml. Cells were further incubated for 2 min before the reaction was stopped by adding 20 pi of 10% PFA.
  • Migration assay were performed using 96-well transwell assay plates (Corning Incorporated, Kennebunk, ME, USA) with 5 pm polycarbonate filters. First, the lower chambers were filled with 235 pi assay buffer (RPMI supplemented with 0.1 % BSA) with or without 100 ng/ml CXCL12 and serial dilutions of the CXCR4-inhibiting compounds (in assay buffer). Next, 75 pL (0.5x105 cells) of Jurkat cells (in assay buffer), together with/without the compounds, were added into the upper chambers.
  • Fig. 11 A For each binding site, the inventors built CXCR4 - EPI-X4 models in explicit solvent and lipid membrane (an example is shown in Fig. 11 A). The models consisted of 257 POPC lipids, approximately 40000 TIP3P water molecules, 50 mM KOI and the CXCR4 - EPI-X4 complex. d. The inventors performed atomistic molecular dynamics simulations (MD) of each model to analyze factors like ligand flexibility, interaction interface area, solvent accessible surface and hydrogen bonding interactions in the different binding modes. The analysis of all these parameters indicated that D is the preferred binding motif. In D, the N-terminus of EPI-X4 is inserted in CXCR4 while the C-terminus of the peptide is solvent-exposed (Fig. 11 B).
  • MD atomistic molecular dynamics simulations
  • the inventors performed an energetic analysis of the electrostatic and van der Waals contributions to the interaction energy in each binding motif as well as the contribution to the interaction energy of individual residues of EPI-X4 (Fig. 11 C).
  • the inventors also performed extensive coarse-grained (CG) MD simulations to investigate the self-assembly of the CXCR4 - EPI-X4 complex from the unbound state, using non-equilibrium dynamics. With these CG simulations, we further established D as the most favored mode, as predicted by the atomistic MDs (Fig. 11 D).
  • EPI-X4 SEQ ID NO.: 1
  • SEQ ID NO.: 1 the inventors designed shortened peptide derivatives with neutral C-terminus that the inventors predict to be more efficient than EPI-X4 (SEQ ID NO.: 1). A set of peptides was thus identified and their experimental activity assessed.
  • SDS PAGE SDS-PAGE analysis was conducted with a NuPAGE® bis-tris 4-12 % precast gel (Invitrogen) in an electrophoresis tank. Samples were prepared with NuPAGE® LDS sample buffer (4*). Samples volumes loaded were typically 10 pL. The running buffer was NuPAGE® MOPS SDS running buffer (20x). The voltage applied for electrophoresis was 150 V and the run time was 1 hour. The SDS PAGE gel was first stained with Coomassie blue stain for 30 minutes, then washed with distilled water for 1 hour.
  • maleimide PVA 20 kDa Amine-terminated polyvinylalcohol (PVA-NH2, 19 800 g/mol, 100 mg, 1 equiv, 5.05 pmol) was dissolved in anhydrous DMSO (2 ml_) and heated to 60 °C. After complete dissolution, maleimide-PEG2-succinimidyl ester (8.59 mg, 4 equiv, .20.2 pmol) was added and the mixture was stirred at room temperature for 48 h. After this time, the product was precipitated in heptane and isolated by centrifugation.
  • PVA-NH2 Amine-terminated polyvinylalcohol
  • di-maleimide PEG 20 kDa 81.4 mg, 0.5 equiv., 3.57 pmol
  • the mixture was incubated for 4 h at room temperature.
  • the bioconjugate was isolated from the native peptide by LH20 gel filtration with ACN / MQ water as the eluent.
  • the collected fractions were analysed by UV-Vis spectroscopy at 280 nm to determine the presence of peptide and combined respectively. After freeze drying, the peptide conjugate was obtained as a solid.
  • DOTA-conjugated peptides were used in this Example: DOTA-K-JM#21 (SEQ ID NO.: 101 , JM#206) (JM#21 (SEQ ID NO.: 23) with DOTA conjugated to the peptide via an additional lysine that is coupled to the C-terminal amino acid of the peptide),
  • DOTA-K-JM#122 (SEQ ID NO.: 102, JM#207) (JM#122 (SEQ ID NO.: 51 ) with DOTA conjugated to the peptide via an additional lysine that is coupled to the C-terminal amino acid of the peptide),
  • DOTA-K-JM#29 (SEQ ID NO.: 165) (JM#29 (SEQ ID NO.: 31) with DOTA conjugated to the peptide via an additional lysine that is coupled to the C- terminal amino acid of the peptide),
  • DOTA-JM#1 18 (SEQ ID NO.: 166) (JM#118 (SEQ ID NO.: 50) with DOTA conjugated to the peptide via the C-terminal lysine of the peptide
  • DOTA-K-JM#173 (SEQ ID NO.: 169) (JM#173 (SEQ ID NO.: 70) with DOTA conjugated to the peptide via an additional lysine that is coupled to the C- terminal amino acid of the peptide)
  • DOTA-JM#1 18 (SEQ ID NO.: 166) (JM#118 (SEQ ID NO.: 50) with DOTA conjugated to the peptide via the C-terminal lysine of the peptide)
  • DOTA-K-JM#173 (SEQ ID NO.: 169)
  • JM#173 (SEQ ID NO.: 70) with DOTA conjugated to the peptide via an additional lysine that is coupled to the C- terminal amino acid of the peptide
  • DOTA-K-JM#235 (SEQ ID NO.: 171 ) (JM#235 (SEQ ID NO.: 130) with DOTA conjugated to the peptide via an additional lysine that is coupled to the C- terminal amino acid of the peptide).
  • 177 Lu-labeled versions of DOTA-conjugated peptides were prepared in ammonium acetate buffer (0.4 M, pH 5.2) after incubation of 3 nmol of the peptide with different activities of [ 177 Lu]LuCl3 (150-450 MBq). Ten % ethanol (except Pentixather) was added to the reaction mixture to prevent radiolysis at 75°C for 30 min for cysteine-containing peptides and at 95°C for 30 min for cysteine-free peptides. In addition, DTT (10 mM) was added to prevent the dimer formation for cysteine-containing peptides.
  • 68 Ga-labeled versions of DOTA-conjugated peptides were prepared in sodium acetate buffer (0.2 M, pH 4-4.5) after incubating 3 nmol of the peptide with different activities of [ 68 Ga]GaCh (10-200 MBq) at 95°C for 15 mins.
  • For quality control 5 pi of this solution was added to 50 mI of Ca-DTPA solution and analysed via RP-HPLC. After determination of the radiochemical purity (> 95%) the reaction mixture was diluted with human serum albumin (HSA) 1 % to the desired activity concentration and used as such for evaluation.
  • HSA human serum albumin
  • the stability of 177 Lu/ 68 Ga-labeled DOTA-conjugated peptides in ammonium acetate buffer (0.4 M, pH 5.2) and sodium acetate buffer (0.2 M, pH 4-4.5) was assessed by determining the radiochemical purity (RCP) of each radiolabeled conjugate at different time points (0, 1 , 2, 4, and 24 h for 177 Lu- complexes and at 0, 1 and 2 h for 68 Ga-complexes) at room temperature. For this, an aliquot of labeling solution was stored at room temperature. RP- HPLC injections were consecutively performed at the desired time points.
  • RCP radiochemical purity
  • Radiolysis-induced instability of [ 177 Lu]Lu-labeled DOTA-conjugated peptides over time was tracked by determining the radiochemical purity (Table 3). Results are means ⁇ standard deviation from a minimum of two separate experiments. At room temperature, the most stable were [ 177 Lu]Lu-DOTA- JM#118 with 80 ⁇ 2 %, [ 177 Lu]Lu-DOTA-K-JM#235 with 80 ⁇ 10 % and [ 177 Lu]Lu-DOTA-K-JM#207 with 78 ⁇ 1 % of remaining radiolabeled conjugate after 24 h.
  • Table 3 Radiochemical purity of radiolabeled conjugates in NhU-acetate buffer pH 5.2 and radiolysis scavenger ethanol at different time points
  • the hydrophilic/lipophilic character of the 177 Lu/ 68 Ga-labeled conjugates was determined by the “shake-flask”method. To a pre-saturated solution containing 500 pL of n-octanol and 500 pl_ of phosphate-buffered saline (PBS) at pH 7.4, 10 pL of 1 picomol 177 Lu/ 68 Ga-labeled conjugates was added. The solutions were vortexed for 1 h to reach equilibrium and then centrifuged (3000 rpm) for 10 min. Hundred mI of the sample was removed from each phase and measured in a g-counter.
  • PBS phosphate-buffered saline
  • Lipophilicity is an important physicochemical property of a potential radiotracer, playing a role in distribution in the body, excretion, pharmacokinetics and in plasma protein binding.
  • [ 177 Lu]Lu-Pentixather shows the lowest log DO/PBS P H7.4 value -3.23 ⁇ 0.23
  • [ 177 Lu]Lu-DOTA-K-JM#235 is the most lipophilic compound (log DO/PBS P H7.40.29 ⁇ 0.10).
  • the receptor binding and internalization rates of 177 Lu/ 68 Ga-labeled conjugates were studied in GHOST-CXCR4+ cells seeded in 24-well plates (1 x 10 5 cells/well).
  • the radiolabeled conjugate (1 nM) was added and the cells were incubated at 37°C for different time points (15, 30 and 60 min). Incubation was interrupted by the removal of the medium and washing the cells twice with ice-cold PBS.
  • Membrane-bound radiolabeled conjugate was obtained by washing the cells twice with ice-cold glycine buffer pH 2.8, followed by collection of the internalized fraction with 1M NaOH. The activity in each fraction was measured in a g-counter.
  • Non-specific binding was determined in the presence of 100’000-fold excess of AMD3100 (blocking agent). The results are expressed as a percentage of the applied radioactivity and are demonstrated in Fig. 14 and Fig. 15, both showing the results for cellular uptake at 60 min.
  • [ 177 Lu]Lu-DOTA-K-JM#173 shows the highest overall cellular uptake as compared to all other 177 Lu-labeled conjugates (Fig. 14). More specifically, [ 177 Lu]Lu-DOTA-K-JM#173 is predominantly cell-membrane bound, while the more lipophilic [ 177 Lu]Lu-DOTA-K-JM#235 gets mainly internalized (Fig. 14). [ 177 Lu]Lu-DOTA-K-JM#173 displayed superiority over the reference molecule [ 177 Lu]Lu-Pentixather in this assay.
  • SPECT/CT The total body distribution of [ 177 Lu]Lu-Pentixather was compared to [ 177 Lu]Lu-DOTA-K-JM#173 and [ 177 Lu]Lu-DOTA-K-JM#235. Healthy Balb/c mice were injected into the tail vein with 15-20 MBq (100 pmol) of 177 Lu-labeled complexes and SPECT/CT images was acquired 4 h post injection (p.i.). For acquiring the images, mice were euthanized by CO2 inhalation after 4 hours, measured in a suitable dose calibrator and imaged supine, head first, using a SPECT/CT system dedicated to imaging small animals (NanoSPECT/CTTM Bioscan Inc.). The images were reconstructed using proprietary HiSPECT iterative reconstruction and fused with CT images using proprietary InVivoScope (Bioscan) software.
  • the radiolabeled DOTA-conjugated peptides have been evaluated in terms of lipophilicity, stability, and cellular uptake in GHOST-CXCR4+ cells.
  • [ 177 Lu]Lu-DOTA-K-JM#173 along with its diagnostic counterpart [ 68 Ga]Ga-DOTA-K-JM#173 is the most promising radiolabeled DOTA-conjugated peptide, as it exhibited the highest cellular uptake on GHOST-CXCR4+ cells compared to the other conjugates and the reference [ 177 Lu]Lu-Pentixather.
  • [ 177 Lu]Lu-DOTA-K-JM#173 and [ 68 Ga]Ga-DOTA-K-JM#173 showed no specific uptake in any organ in vivo. However, no uptake in other organs can also be attributed to the fact that these compounds are specific to human CXCR4.
  • its renal accumulation is attributed to urinary excretion. Renal accumulation is preferable instead of hepatic accumulation as for [ 177 Lu]Lu-Pentixather. Renal uptake of radioactivity can be reduced by using nephroprotective agents, therefore lowering off-target radiotoxicity. Hepatic uptake instead, cannot be lowered and remains a major drawback for both, imaging and therapy.
  • [ 177 Lu]Lu-DOTA-K- JM#173 seems to be a suitable radiopharmaceutical.
  • a peptide consisting of one of the following amino acid sequences, selected from any of the groups 1 to 10, wherein
  • Group 1 consists of
  • IMRWSRKMPCVS (SEQ ID NO. : 19, JM#17)
  • ILRWSRKMPCMS (SEQ ID NO. : 22, JM#20)
  • ILRWSRKLPCVS (SEQ ID NO. : 23, JM#21 )
  • ILRWSRKMPCFS (SEQ ID NO.: 21 , JM#19)
  • Group 3 consists of d-LLRWSRK(Pal)MPCVS (SEQ ID NO.: 53, JM#141 )
  • IVRWSK(Pal)KVP-NH2 (SEQ ID NO.: 63, JM#166)
  • IVRWSKK(Pal)VP-NH2 (SEQ ID NO.: 64, JM#167)
  • ILRWSRK(Pal)LP-NH2 (SEQ ID NO.: 68, JM#171 )
  • ILRWSRKK(Glu-Ste)LPCVS SEQ ID NO.: 96, JM#199
  • ILRWSRK(Glu-Dec)LPCVS SEQ ID NO.: 98, JM#203
  • ILRWSRK(Glu-Ste)LPCVS SEQ ID NO.: 100, JM#205
  • MLRWSRKLPCVS (SEQ ID NO.: 41 , JM#39)
  • ILRWSRKLPSVS (SEQ ID NO.: 51 , JM#122) d-LLRWSRK(Glu-Pal)MPCVS (SEQ ID NO.: 52, JM#140) ILRWSRK(Pal)MPCLS (SEQ ID NO.: 59, JM#149) d-LMRWSRKK(Glu-Pal)-NH2 (SEQ ID NO.: 92, JM#195) ILRWSRK-AcLPCVS (SEQ ID NO.: 97, JM#200)
  • ILRWSKKVPCVS SEQ ID NO.: 3, JM#1
  • IVRWSRKVPCVS SEQ ID NO.: 5, JM#3
  • IVRWSHKVPCVS SEQ ID NO.: 6, JM#4
  • IVRWSKKLPCVS (SEQ ID NO.: 7, JM#5)
  • ILRWSHKVPCVS SEQ ID NO.: 11 , JM#9
  • IFRWSHKVPCVS SEQ ID NO.: 13, JM#11
  • IVRWSKKMPCVS (SEQ ID NO.: 14, JM#12)
  • IIRWSRKMPCVS SEQ ID NO.: 18, JM#16
  • GLRWSRKMPCVS (SEQ ID NO.: 33, JM#31 )
  • Id-LRWSRKLPCVS (SEQ ID NO.: 43, JM#41 )
  • IVRWSKKVP-NH2 (SEQ ID NO.: 47, JM#106)
  • ILRWSRKLP-NH2 SEQ ID NO.: 49, JM#114
  • ILRWSRK-NH2 (SEQ ID NO.: 50, JM#118)
  • IYRWSRKMPCLS (SEQ ID NO.: 57, JM#146)
  • IVRWSKKVPSVS (SEQ ID NO.: 60, JM#151 )
  • IVRWSK(Pal)K-NH2 (SEQ ID NO.: 61 , JM#164)
  • IVRWSKK(Pal)-NH2 (SEQ ID NO.: 62, JM#165)
  • IVRWSK(Pal)KVPCVS SEQ ID NO.: 65, JM#168
  • d-ILRWSRK-NH2 SEQ ID NO.: 70, JM#173
  • d-ILRWSRKLP-NH2 SEQ ID NO.: 71 , JM#174
  • d-ILRWSRK(Pal)LP-NH2 SEQ ID NO.: 72, JM#175)
  • d-LMRWSRK(Pal)MPCVS SEQ ID NO.: 73, JM#176
  • ILRWSRK(Ste)LPCVS (SEQ ID NO.: 79, JM#182)
  • ILRWSRK(Chl)LPCVS SEQ ID NO.: 81 , JM#184)
  • VLRWSRKLPCVS (SEQ ID NO.: 86, JM#189)
  • d-VLRWSRKLPCVS SEQ ID NO.: 87, JM#190
  • d-MLRWSRK(Glu-Pal)-NH2 SEQ ID NO.: 93,
  • ILRWSRK(Glu-Ste)LPCVS (SEQ ID NO.: 95, JM#198)
  • ILRWSRK(Glu-Myr)LPCVS SEQ ID NO.: 99, JM#204
  • Group 6 consists of d-LMRWSRKK(Glu-Pal)-NH2 (SEQ ID NO.: 92,
  • IVRWSK(Pal)KVP-NH2 (SEQ ID NO.: 63, JM#166)
  • IVRWSKK(Pal)VP-NH2 (SEQ ID NO.: 64, JM#167)
  • ILRWSRK(Pal)LP-NH2 (SEQ ID NO.: 68, JM#171 ) d-ILRWSRK-NH2 (SEQ ID NO.: 70, JM#173) d-ILRWSRKLP-NH2 (SEQ ID NO.: 71 , JM#174)
  • Group 8 consists of d-LLRWSRKMPCVS (SEQ ID NO.: 31 , JM#29) ILRWSRK(Pal)L-NH2 (SEQ ID NO.: 69, JM#172) ILRWSRK(Glu-Pal)-NH2 (SEQ ID NO.: 89, JM#192)
  • IVRWSK(Pal)KVP-NH2 (SEQ ID NO.: 63, JM#166)
  • IVRWSKK(Pal)VP-NH2 (SEQ ID NO.: 64, JM#167)
  • ILRWSRK(Pal)LP-NH2 (SEQ ID NO.: 68, JM#171 )
  • ILRWSRKLK(Glu-Pal)-NH2 (SEQ ID NO.: 90, JM#193) d-LMRWSRK(Glu-Pal)-NH2 (SEQ ID NO.: 91 ,
  • d- in front of an amino acid indicates a D-Amino acid
  • Pal indicates a palmitic acid on the preceding amino acid
  • Glu-Pal indicates a palmitic acid on the preceding amino acid with a glutamate linker
  • Dec indicates decanoic acid on the preceding amino acid
  • Glu-Dec indicates a decanoic acid on the preceding amino acid with a glutamate linker
  • Myr indicates myristic acid on the preceding amino acid
  • Glu-Myr indicates a myristic acid on the preceding amino acid with a glutamate linker
  • Ole indicates oleic acid on the preceding amino acid
  • Ste indicates stearic acid on the preceding amino acid
  • Glu-Ste indicates a stearic acid on the preceding amino acid with a glutamate linker
  • Chi indicates Cholesterol
  • Ac indicates a substitution of an amino group by an acetyl group.
  • a peptide consisting of one of the following amino acid sequences,
  • Group 1 consists of
  • IMRWSRKMPCVS (SEQ ID NO. : 19, JM#17)
  • ILRWSRKMPCMS (SEQ ID NO. : 22, JM#20)
  • ILRWSRKLPCVS (SEQ ID NO. : 23, JM#21 )
  • ILRWSRK(Pal)L-NH2 (SEQ ID NO.: 69, JM#172) d-LMRWSRK(Pal)MP-NH2 (SEQ ID NO.: 75, JM#178) d-LMRWSRK(Pal)-NH2 (SEQ ID NO.: 77, JM#180) ILRWSRK(Ole)LPCVS (SEQ ID NO.: 80, JM#183)
  • Group 3 consists of d-LLRWSRK(Pal)MPCVS (SEQ ID NO.: 53, JM#141 )
  • IVRWSK(Pal)KVP-NH2 (SEQ ID NO.: 63, JM#166)
  • IVRWSKK(Pal)VP-NH2 (SEQ ID NO.: 64, JM#167)
  • ILRWSRK(Pal)LP-NH2 (SEQ ID NO.: 68, JM#171 )
  • MLRWSRKLPCVS (SEQ ID NO.: 41 , JM#39)
  • ILRWSRKLPSVS (SEQ ID NO.: 51 , JM#122) d-LLRWSRK(Glu-Pal)MPCVS (SEQ ID NO.: 52, JM#140) ILRWSRK(Pal)MPCLS (SEQ ID NO.: 59, JM#149) d-LMRWSRKK(Glu-Pal)-NH2 (SEQ ID NO.: 92, JM#195) ILRWSRK-AcLPCVS (SEQ ID NO.: 97, JM#200)
  • ILRWSKKVPCVS SEQ ID NO.: 3, JM#1
  • IVRWSRKVPCVS SEQ ID NO.: 5, JM#3
  • IVRWSHKVPCVS SEQ ID NO.: 6, JM#4
  • IVRWSKKLPCVS (SEQ ID NO.: 7, JM#5)
  • IVRWSKKIPCVS SEQ ID NO.: 8, JM#6
  • IVRWSKKFPCVS SEQ ID NO.: 9, JM#7
  • ILRWSHKVPCVS SEQ ID NO.: 11 , JM#9
  • IFRWSHKVPCVS SEQ ID NO.: 13, JM#11
  • IVRWSKKMPCVS (SEQ ID NO.: 14, JM#12)
  • IIRWSRKMPCVS SEQ ID NO.: 18, JM#16
  • GLRWSRKMPCVS (SEQ ID NO.: 33, JM#31 )
  • Id-LRWSRKLPCVS (SEQ ID NO.: 43, JM#41 )
  • IVRWSKKVP-NH2 (SEQ ID NO.: 47, JM#106)
  • ILRWSRKLP-NH2 SEQ ID NO.: 49, JM#114

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WO2025114560A1 (en) * 2023-12-01 2025-06-05 Pharis Biotec Gmbh Alb408-423 peptides for use in the treatment of cancer in combination with radiotherapy

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EP4483892A1 (en) * 2023-06-29 2025-01-01 Pharis Biotec GmbH Composition comprising lekti polypeptides for the treatment of disorders

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EP3007717A1 (en) 2013-06-12 2016-04-20 Pharis Biotec GmbH Peptides with antagonistic activities against natural cxcr4
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024100391A3 (en) * 2022-11-07 2024-08-02 University Of Liverpool New antibacterial products
WO2025114560A1 (en) * 2023-12-01 2025-06-05 Pharis Biotec Gmbh Alb408-423 peptides for use in the treatment of cancer in combination with radiotherapy

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