WO2011011073A1 - Site-specific pegylated or site-specific lipidated igf-1 and analogues of igf-1 - Google Patents

Site-specific pegylated or site-specific lipidated igf-1 and analogues of igf-1 Download PDF

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Publication number
WO2011011073A1
WO2011011073A1 PCT/US2010/002063 US2010002063W WO2011011073A1 WO 2011011073 A1 WO2011011073 A1 WO 2011011073A1 US 2010002063 W US2010002063 W US 2010002063W WO 2011011073 A1 WO2011011073 A1 WO 2011011073A1
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higf
seq
ala
arg
giu
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PCT/US2010/002063
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French (fr)
Inventor
Zheng Xin Dong
Nicholas C. Prairie
John S. Eynon
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Ipsen Pharma S.A.S.
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Publication of WO2011011073A1 publication Critical patent/WO2011011073A1/en

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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
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • 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
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel site-specific pegylated or site-specific lipidated insulin-like growth factor- 1 (IGF-I) and analogues of IGF-I, and the use of said compounds for treatment of IGF-I -receptor mediated conditions, such as short stature, diabetes therapy, neurodegenerative disease treatment, and cartilage repair.
  • IGF-I insulin-like growth factor- 1
  • IGF-I is a 70-amino-acid polypeptide hormone having insulin-like and mitogenic growth biological activities. This hormone enhances growth of cells in a variety of tissues including musculoskeletal systems, liver, kidney, intestines, nervous system tissues, heart, and lung.
  • the wild-type IGF-I has the following amino acid sequence with three intrachain disulfide bridges wherein the side-chains of residue pairs A 6 and A 48 , A 47 and A 52 , and A 18 and A 61 , each form a disulfide bond (SEQ ID NO: 112):
  • IGFBP IGF binding protein
  • IGF-I circulating IGF-I
  • trimolecular complex containing IGFBP-3 and acid labile submit.
  • the IGF-I within such complexes is unable to bind to surface receptors, and is therefore biologically inactive.
  • IGF-I present within the trimolecular complex also has a substantially longer half-life than uncomplexed IGF-I .
  • IGF-I action may contribute to a number of physiological disorders including neurodegenerative disorders such as motor neuron disease (i.e., amyotrophic lateral sclerosis (ALS)), muscular dystrophy and multiple sclerosis, cartilage disorders such as osteoarthritis, bone diseases such as osteoporosis, inflammatory disorders such as rheumatoid arthritis, ischemic injuries to organs such as to the heart, brain, or liver, and so forth.
  • motor neuron disease i.e., amyotrophic lateral sclerosis (ALS)
  • muscular dystrophy and multiple sclerosis i.e., muscular dystrophy and multiple sclerosis
  • cartilage disorders such as osteoarthritis
  • bone diseases such as osteoporosis
  • inflammatory disorders such as rheumatoid arthritis
  • ischemic injuries to organs such as to the heart, brain, or liver, and so forth.
  • IGF-I insulin growth factor-I
  • a number of studies report on the use of IGF-I as a potential therapeutic agent for treatment of neurodegenerative conditions. See, e.g., Kanje et al, Brain Res., 486:396-398 (1989); Hantai et al, J. Neurol. ScL, 129:122-126 (1995); Contreras et al, Pharmac. Exp.
  • IGF-I therapy has been indicated in numerous neurological conditions, including ALS, stroke, epilepsy, Parkinson's disease, Alzheimer's disease, acute traumatic injury and other disorders associated with trauma, aging, disease, or injury. See, e.g., U.S. Pat. Nos. 5,093,137; 5,652,214; 5,703,045; International Publication Nos. WO 90/1483 and WO 93/02695.
  • IGF-I therapy for a variety of other conditions has been referred to in a number of publications. See, e.g., Schalch et al, "Modern Concepts of Insulin-Like Growth Factors," ed. Spencer (Elsevier, New York), pp. 705-714 (1991); Clemmons and Underwood, J. Clin. Endocrinol Metab., 79(l):4-6 (1994); Langford et al., Eur. J. Clin. Invest, 23(9):5O3- 516 (1993) (referring to, e.g., insulin-resistant states and diabetes); and O' Shea et al., Am. J.
  • WO 91/12018 referring to, e.g., intestinal disorders
  • WO 92/09301 and WO 92/14480 referring to, e.g., wound healing
  • WO 93/08828 referring to, e.g., neuronal damage associated with ischemia, hypoxia, or neurodegeneration
  • WO 94/16722 referring to, e.g., insulin resistance
  • WO 96/02565A1 referring to, e.g., IGF/IGFBP complex for promoting bone formation and for regulating bone remodeling
  • U.S. Patent Application Publication No. 2003/0100505 referring to, e.g., osteoporosis
  • the present invention is directed to an amino-terminal pegylated IGF-I conjugate of formula (I),
  • W is wherein X is O or NH;
  • B is the wild-type IGF-I or an analogue of IGF-I
  • linker is alkyl, aryl, alkyl-aryl, substituted alkyl, substituted aryl, or substituted alkyl-aryl, which may contain other organic functional group(s), and which is preferably wherein p is an integer from 1 to 10 inclusive, and preferably 1; and wherein said is
  • the present invention is directed to an amino-terminal lipidated IGF-I conjugate of formula (II),
  • Y is or wherein Z is alkyl, aryl, alkyl-aryl, substituted alkyl, substituted aryl, or substituted alkyl-aryl which may contain other organic functional group(s), and which is preferably CH 3 -(CH 2 ),,,-, wherein m is an integer from 1 to 30 inclusive; and
  • B is the wild-type IGF-I or an analogue of IGF-I
  • a 1 is GIy, Ala, Asn, Asp, GIn, GIu, or deleted;
  • a 2 is Pro, Ala, Arg, Asp, GIn, GIu, Lys, or deleted;
  • a 3 is GIu, Ala, Asp, GIn, or deleted;
  • a 4 is Thr, Ala, Asn, Asp, GIn, GIu, Ser;
  • a 5 is Leu, Ace, Ala, He, or VaI
  • a 6 is Cys, D-Cys, hCys, D-hCys, ⁇ -Me-Cys, D- ⁇ -Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
  • a 7 is GIy, Ala, Asn, Asp, GIn or GIu;
  • a 8 is Ala, Arg, Asn, Asp, GIn, GIu, or Lys
  • a 9 is GIu, Ala, Asp, or GIn
  • a 10 is Leu, Ace, Ala, He, or VaI;
  • A" is VaI, Ala, lie, or Leu;
  • a 12 is Asp, Ala, Arg, Asn, GIn, GIu, or Lys;
  • a 13 is Ala, Asn, Asp, GIn, GIu, lie, Leu, or VaI;
  • a 14 is Leu, Ace, Ala, He, or VaI;
  • a 15 is GIn, Ala, Asn, Asp, or GIu;
  • a 16 is Phe, Ala, Asn, Asp, GIn, GIu, Trp, or Tyr;
  • a 17 is VaI, Ala, lie or Leu;
  • a 18 is Cys, D-Cys, hCys, D-hCys, ⁇ -Me-Cys, D- ⁇ -Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
  • a 19 is GIy, Ala, Asn, Asp, GIn, or GIu;
  • a 20 is Asp, Ala, Asn, GIn, or GIu;
  • a 21 is Arg, Ala, Asn, Asp, GIn, GIu, or Lys;
  • a 22 is GIy, Ala, Asn, Asp, GIn, or GIu;
  • a 23 is Phe, Ala, Trp, or Tyr
  • a 24 is Tyr, Ala, Phe, or Trp;
  • a 25 is Phe, Ala, Trp, or Tyr
  • a 26 is Asn, Ala, Asp, GIn, GIu, Ser, or Thr;
  • a 27 is Lys, Ala, Arg, Asn, Asp, GIn, GIu, or Pro;
  • a 28 is Pro, Ala, Arg, or Lys
  • a 29 is Thr, Ala, Asn, Asp, GIn, GIu, or Ser;
  • a 30 is GIy, Ala, Asn, Asp, GIn, or GIu;
  • a 31 is Tyr, Ala, Phe, or Trp;
  • a 32 is GIy, Ala, Asn, Asp, GIn, or GIu;
  • a 33 is Ser, Ala, Thr, or VaI
  • a 34 is Ser, Ala, Asn, Asp, GIn, GIu, or Thr;
  • a 35 is Ser, Ala, Asn, Asp, GIn, GIu, or Thr;
  • a 36 is Arg, Ala, Asn, Asp, GIn, GIu, or Lys;
  • a 37 is Arg, Ala, Asn, Asp, GIn, GIu, or Lys;
  • a 38 is Ala, Asn, Asp, GIn, or GIu;
  • a 39 is Pro, Ala, Arg, or GIu;
  • a 40 is GIn, Ala, Asn, Asp, or GIu;
  • a 41 is Thr, Ala, Asn, Asp, GIn, GIu, or Ser;
  • a 42 is GIy, Ala, Arg, Asn, Asp, GIn, GIu, or Lys;
  • a 43 is He, Ala, Arg, Asn, Asp, GIn, GIu, or Lys;
  • a 44 is VaI, Ala, Arg, Asn, Asp, GIn, GIu, lie, Leu, or Lys;
  • a 45 is Asp, Ala, Arg, Asn, GIn, GIu, or Lys;
  • a 46 is GIu, Ala, Arg, Asn, Asp, GIn, or Lys;
  • a 47 is Cys, D-Cys, hCys, D-hCys, ⁇ -Me-Cys, D- ⁇ -Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
  • a 48 is Cys, D-Cys, hCys, D-hCys, ⁇ -Me-Cys, D- ⁇ -Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
  • a 49 is Phe, Ala, Arg, He, Leu, Lys, Ser, Thr, Trp, Tyr, or VaI;
  • a 50 is Arg, Ala, Lys, Ser, or Thr;
  • a 51 is Ser, Aib, Ala, Arg, Lys, or Thr;
  • a 52 is Cys, D-Cys, hCys, D-hCys, ⁇ -Me-Cys, D- ⁇ -Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
  • a 53 is Asp, Ala, Arg, Asn, GIn, GIu, Lys, Ser, or Thr;
  • a 54 is Leu, Ace, Ala, Arg, He, Phe, or VaI;
  • a 55 is Arg, Ala, lie, Leu, Lys, Phe, Trp, Tyr, or VaI;
  • a 56 is Arg, Ala, Asn, Asp, GIn, GIu, or Lys;
  • a 57 is Leu, Ace, Ala, Ee, Phe, or VaI;
  • a 58 is GIu, Ace, Ala, Arg, Asn, Asp, GIn, or Lys;
  • a 59 is Met, Ace, Ala, Arg, Asn, Asp, GIn, GIu, He, Leu, Lys, NIe, Ser, D-Ser, Thr, Trp, Tyr, or VaI;
  • a 60 is Tyr, Ala, Arg, Ee, Phe, or Trp;
  • a 61 is Cys, D-Cys, hCys, D-hCys, ⁇ -Me-Cys, D- ⁇ -Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
  • a 62 is Ala, Arg, Asn, Asp, GIn, GIu, He, Leu, or VaI;
  • a 63 is Pro, D-Pro, Ala, Ser, Thr, or deleted;
  • a 64 is Leu, D-Leu, des-Leu, Ala, Arg, lie, Phe, VaI, or deleted;
  • a 65 is Lys, D-Lys, des-Lys, Ala, Arg, He, Leu, VaI, or deleted;
  • a 66 is Pro, D-Pro, Ala, or deleted
  • a 67 is Ala, D-AIa, Aib, Arg, or deleted;
  • a 68 is Lys, D-Lys, Ala, Arg, He, Leu, VaI, or deleted;
  • a 69 is Ser, D-Ser, Aib, Ala, Arg, Thr, or deleted;
  • a 70 is Ala, D-AIa, Asn, Asp, GIn, GIu, or deleted;
  • a 71 is Asn, Ala, Asp, GIn, GIu, Lys, Ser, Thr, or deleted;
  • R 1 is OH or NH 2 ;
  • the analogue contains at least one additional amino acid substitution or addition as defined herein.
  • preferred amino acid substitutions and additions are defined as follows:
  • a 1 is GIy, GIu, or deleted
  • a 2 is Pro, GIu, Lys, or deleted
  • a 3 is GIu or deleted
  • a 4 is Thr or GIu
  • a 6 is Cys, hCys, ⁇ -Me-Cys, N-Me-Cys, or Pen;
  • a 7 is GIy or GIu
  • a 8 is Ala, Arg or GIu;
  • a 11 is VaI or Leu
  • a 13 is Ala or GIu
  • a 15 is GIn or GIu
  • a 16 is Phe or GIu
  • a 17 is VaI or Leu
  • a 18 is Cys, hCys, ⁇ -Me-Cys, N-Me-Cys, or Pen;
  • a 19 is GIy or GIu;
  • a 20 is Asp or GIu
  • a 21 is Arg or GIu
  • a 22 is GIy or GIu
  • a 26 is Asn, GIu, or Thr;
  • a 27 is Lys, Arg, GIu, or Pro
  • a 28 is Pro or Lys
  • a 29 is Thr or GIu
  • a 30 is GIy or GIu
  • a 32 is GIy or GIu;
  • a 34 is Ser or GIu
  • a 35 is Ser or GIu
  • a 36 is Arg or GIu
  • a 37 is Arg or GIu
  • a 38 is Ala or GIu
  • a 40 is GIn or GIu
  • a 41 is Thr or GIu
  • a 42 is GIy, Arg, or GIu;
  • a 43 is Ee, Arg, or GIu;
  • a 44 is VaI, Arg, or GIu;
  • a 45 is Asp, Arg, GIn, or GIu;
  • a 46 is GIu, Arg, or GIn;
  • a 47 is Cys, hCys, ⁇ -Me-Cys, N-Me-Cys, or Pen;
  • A is Cys, hCys, ⁇ -Me-Cys, N-Me-Cys, or Pen;
  • a 49 is Phe, Arg, Leu, or Thr;
  • a 5 ⁇ is Arg or Ser
  • a 51 is Ser, Aib, Arg, or Thr;
  • a 52 is Cys, hCys, ⁇ -Me-Cys, N-Me-Cys, or Pen;
  • a 53 is Asp, Arg, or Ser
  • a 54 is Leu, A6c, Arg, or Phe;
  • a 55 is Arg, Tyr, or VaI
  • a 56 is Arg or GIn
  • a 57 is Leu or Phe
  • a 58 is GIu, A6c, or Arg
  • a 59 is Met, A6c, Arg, Asn, Asp, GIn, GIu, He, Leu, NIe, Ser, D-Ser, Trp, or Tyr;
  • a 60 is Tyr, Arg, De, or Phe;
  • a 61 is Cys, hCys, ⁇ -Me-Cys, N-Me-Cys, or Pen;
  • A is Ala, Arg, or Asn
  • a bi is Pro, D-Pro, Thr, or deleted
  • a 64 is Leu, D-Leu, des-Leu, Arg, Phe, or deleted;
  • a 65 is Lys, D-Lys, des-Lys, Arg, VaI, or deleted;
  • a 66 is Pro, D-Pro, or deleted
  • a 67 is Ala, D-AIa, Aib, Arg, or deleted;
  • a 68 is Lys, D-Lys, Arg, VaI, or deleted;
  • a 69 is Ser, D-Ser, Aib, Arg, Thr, or deleted;
  • a 70 is Ala, D-AIa, Arg, GIu, or deleted.
  • a 71 is Ser, Asp, GIu, Lys, or deleted.
  • Preferred analogues of IGF-I of the formula (IA) or formula (ILA) are:
  • Example 1 (Asn 59 )hIGF-l(l -7O)-OH; (SEQ ID NO: 1)
  • Example 4 (Pro 27 , Lys 28 , Asn 59 )hIGF-l (1-7O)-OH; (SEQ ID NO:4)
  • Example 5 (Pro 27 , Lys 28 , Asn 59 )hIGF-l(l-62)-OH; (SEQ ID NO:5)
  • Example 7 (Leu 17 )hIGF-l(l-70)-OH; (SEQ LD NO:7)
  • Example 8 (Asn 59 , Thr 63 , des-Leu 64 , des-Lys 65 , Glu 70 )hIGF-l(l -7O)-OH; (SEQ LD NO: 8)
  • Example 9 (Tyr 55 , Asn 59 )hIGF-l(l-70)-OH; (SEQ LD NO:9)
  • Example 10 (Thr 49 , Asn 59 )hIGF-l(l -7O)-OH; (SEQ IDNO:10)
  • Example 12 (Asn 59 , Phe 60 )hIGF-l(l -7O)-OH; (SEQ IDNO:12)
  • Example 14 (GIn 56 , Asn 59 )hIGF-l(l -7O)-OH; (SEQ IDNO:14)
  • Example 15 (Asn 59 , D-Pro 63 )hIGF- 1(1 -7O)-OH;
  • Example 17 (Asn 59 , D-Lys 65 )hIGF-l(l -7O)-OH;
  • Example 18 (Asn 59 , D-Pro 66 )hIGF-l(l -7O)-OH;
  • Example 19 (Asn 59 , D-Ala 67 )hIGF- 1(1 -7O)-OH;
  • Example 20 (Asn 59 , D-Lys 68 )hIGF-l(l -7O)-OH;
  • Example 21 (Asn 59 , D-Ser 69 )hIGF-l(l-70)-OH;
  • Example 22 (Asn 59 , D-Ala 70 )hIGF- 1(1 -7O)-OH;
  • Example 25 (Leu 49 ' 59 )hIGF-l(l -7O)-OH; (SEQ ID NO:17)
  • Example 26 ( ⁇ -Me-Cys 52 , Leu 59 )hIGF-l(l -7O)-OH; (SEQ ID NO:18)
  • Example 27 ( ⁇ -Me-Cys 47 , Leu 59 )hIGF- 1 ( 1 -7O)-OH; (SEQ IDNO:19)
  • Example 28 (Leu 59 , Glu 71 )hIGF-l(l-71)-OH; (SEQ ID NO:20)
  • Example 30 (Leu 59 , Lys 71 )hIGF-l(l-71)-OH; (SEQ IDNO:22)
  • Example 31 (Leu 59 , Ser 71 )hIGF-l(l-71)-OH; (SEQ ID NO:23)
  • Example 32 (Leu 59 , Thr 69 )hIGF-l(l -7O)-OH; (SEQ IDNO:24)
  • Example 36 (N-Me-Cys 48 , Nle 59 )hIGF-l(l -7O)-OH; (SEQ IDNO:28)
  • Example 37 (NIe 59 , Aib 67 )hIGF-l(l -7O)-OH; (SEQ IDNO:29)
  • Example 38 (hCys 52 , Nle 59 )hIGF- 1 ( 1 -7O)-OH; (SEQ IDNO:30)
  • Example 40 (Pen 52 , Nle 59 )hIGF-l(l -7O)-OH; (SEQ IDNO:32)
  • Example 42 (A6c 54 , Nle 59 )hIGF-l(l -7O)-OH; (SEQ IDNO:34)
  • Example 46 (Arg 58 , Ee 59 )hIGF-l(l -7O)-OH; (SEQ DD NO:38)
  • Example 47 (A6c 59 )hIGF-l(l -7O)-OH; (SEQ ID NO:39)
  • Example 48 (Asp 59 )hIGF-l (1 -7O)-OH; (SEQ ID NO:40)
  • Example 50 (Ser 59 )hIGF-l(l-70)-OH; (SEQ ID NO:42)
  • Example 51 (Tyr 59 )hIGF- 1(1 -7O)-OH; (SEQ ID NO:43)
  • Example 60 (GIu 13 )hIGF-l(l -7O)-OH; (SEQ ID NO:52)
  • Example 70 (Glu 30 )hIGF- 1 ( 1 -7O)-OH; (SEQ ED NO:62)
  • Example 83 (Pro 27 , Lys 28 )hIGF-l(l -7O)-OH; (SEQ ED NO:75)
  • Example 84 (Lys 2 )hIGF-l(l-70)-OH; (SEQ ID NO:76)
  • Example 85 (Thr 26 )hIFG- 1 ( 1 -7O)-OH; (SEQ BD NO:77)
  • Example 88 (VaI 65 ' 6S )hIGF- 1 ( 1 -7O)-OH; (SEQ ID NO:80)
  • Example 96 (Gln 46 )hIGF-l(l-70)-OH; (SEQ ID NO:88)
  • Example 100 (Phe 54 )hIGF- 1 ( 1 -7O)-OH; (SEQ ID NO:92)
  • Example 102 (Phe 64 )hIGF- 1 ( 1 -7O)-OH; (SEQ ID NO:94)
  • Example 104 (Arg 60 )hIGF-l(l-70)-OH; (SEQ ID NO:96)
  • Example 108 (Arg 67 )hIGF-l(l-70)-OH; (SEQ ID NO: 100)
  • Example 110 (Arg 69 )hIGF- 1(1 -7O)-OH; and (SEQ ID NO: 102)
  • Example 111 (Arg ⁇ 7'0 ⁇ -)hIGF-l(l-70)-OH. (SEQ ID NO: 103)
  • Preferred amino-terminal pegylated IGF-I conjugates of formula (I) are:
  • Example 112 [2-(ethoxyimino(2-carbamate-10K linear PEG))acetyl-Gly', Asn 59 ]hIGF-l(l- 7O)-OH; (SEQ ID NO: 104)
  • Example 113 [2-(ethoxyimino(2-carbamate-20K linear PEG))acetyl-Gly', Asn 59 ]hIGF-l(l- 7O)-OH; (SEQ ID NO: 105)
  • Example 114 [2-(ethoxyimino(2-carbamate-20K branched PEG))acetyl-Gly ⁇ Asn 59 ]hIGF- 1 ( 1 -7O)-OH; (SEQ ID NO: 106)
  • Example 115 [2-(ethoxyimino(2-carbamate-30K linear PEG))acetyl-Gly ' , Asn 59 ]hIGF- 1(1- 7O)-OH; and (SEQ ID NO: 107)
  • Example 116 [2-(ethoxyimino(2-carbamate-40K branched PEG))acetyl-Gly ⁇ Asn 59 ]hIGF- 1(1-7O)-OH. (SEQ ID NO:108)
  • Preferred amino-terminal lipidated IGF-I conjugate of formula (I) is:
  • Example 117 [2-(n-octadecoxyimino)acetyl-Gly', Asn 59 ]hIGF-l(l -7O)-OH.
  • the site-specifically modified IGF-I and analogues of IGF-I according to the present invention have improved pharmacokinetic profile, improved biological activity, and/or decreased immunogenicity.
  • Ace includes:
  • A3c 1 -amino- 1 -cyclopropanecarboxylic acid
  • A4c 1 -amino- 1 -cyclobutanecarboxylic acid
  • A5c 1 -amino- 1 -cyclopentanecarboxylic acid
  • A6c 1 -amino- 1 -cyclohexanecarboxylic acid
  • cystine disulfide dimer of cysteine
  • ⁇ -Me-Cys beta-methyl-cysteine, i.e.,
  • N-Me-Cys N-methyl-cysteine
  • proline i.e.,
  • a peptide of this invention is also denoted herein by another format, e.g., (Asn 59 )hIGF- 1(1 -7O)-OH (SEQ ID NO: 1) or [2-(n-octadecoxyimino)acetyl-Gly 1 , Asn 59 ]hIGF- 1(1-7O)-OH (SEQ ID NO: 109), with the substituted amino acids from the natural sequence placed between the parentheses or brackets (i.e., Asn for Met at position 59 of the wild-type IGF-I).
  • the range found within the parentheses refers to those amino acids found in the analogue.
  • IGF-I (4-68)-OH indicates that the analogue is comprised of amino acids 4 through 68 which correspond to the peptide sequence for the wild-type IGF-I.
  • NH 2 " in “IGF-I (1-7O)-NH 2 " (SEQ ID NO: 111) indicates that the C- terminus of the peptide is amidated.
  • IGF-l(l-70) or “IGF-I (1-7O)-OH” indicates that the C-terminus is the free acid (SEQ ID NO:112).
  • BSA bovine serum albumin
  • DIPEA diisopropylethylamine
  • DMEM Dulbecco's Modified Eagle's Medium
  • DMF dimethylformamide
  • FCS fetal calf serum
  • MALDI-TOF MS matrix-assisted laser dissorption ionization time-of-flight mass spectroscopy
  • TIS triisopropylsilane
  • Trt trityl
  • UV spectroscopy ultraviolet spectroscopy
  • Alkyl refers to a hydrocarbon group containing one or more carbon atoms wherein multiple carbon atoms, if present, are joined by single bonds. Examples of which include, but are not limited to, methyl, ethyl, propyl, and butyl.
  • the alkyl hydrocarbon group may be straight-chain or contain one or more branches or cyclic groups, examples of which include, but are not limited to, isopropyl and tert-butyl.
  • Substituted alkyl refers to an alkyl wherein one or more hydrogen atoms of the hydrocarbon group are replaced with one or more substituents selected from the group consisting of halogen, OH, CN, SH, NH 2 , NHCH 3 , NO 2 , (C 1-2 ) alkyl substituted with 1 to 6 halogens, CF 3 , OCH 3 , OCF 3 , and (CH 2 )( M -COOH. In different embodiments, 1, 2, 3 or 4 substituents are present.
  • Aryl refers to an optionally substituted aromatic group with at least one ring having a conjugated pi-electron system, containing up to three conjugated or fused ring systems.
  • Aryl includes carbocyclic aryl, heterocyclic aryl and biaryl groups.
  • the aryl is a 5 or 6 membered ring.
  • Preferred atoms for a heterocyclic aryl are one or more sulfur, oxygen, and/or nitrogen.
  • Examples of aryl include phenyl, 1-naphthyl, 2-naphthyl, indole, quinoline, 2-imidazole, and 9-anthracene.
  • Aryl substituents are selected from the group consisting of - C 1-20 alkyl, -Ci -20 alkoxy, halogen, -OH, -CN, -SH, -NH 2 , -NO 2 , -C L20 alkyl substituted with halogens, -CF 3 , -OCF 3 , and -(CH 2 ) O-20 -COOH.
  • the aryl contains O, 1, 2, 3, or 4 substituents.
  • Alkyl-aryl refers to an “alkyl” joined to an “aryl”.
  • PEG poly(ethylene glycol), which has the structure of
  • n an integer between 1 and 2,000, and which may be methylated as defined herein
  • mPEG methylated poly(ethylene glycol), which has the structure of
  • n is an integer between 1 and 2,000
  • 1OK PEG poly(ethylene glycol), which encompasses mPEG as defined herein, with an average molecular weight of about 10 kDa, and which may be either linear or branched
  • 2OK PEG poly(ethylene glycol), which encompasses mPEG as defined herein, with an average molecular weight of about 20 kDa, and which may be either linear or branched
  • 3OK PEG poly(ethylene glycol), which encompasses mPEG as defined herein, with an average molecular weight of about 30 kDa, and which may be either linear or branched
  • 4OK PEG poly(ethylene glycol), which encompasses mPEG as defined herein, with an average molecular weight of about 40 kDa, and which may be either linear or branched
  • n is an integer between 1 and 2,000 such that the "10K linear PEG” moiety has the average molecular weight of about 1O kDa
  • n is, independently for each occurrence, an integer between 1 and 2,000 such that the "2OK branched PEG" moiety has the combined average molecular weight of about 20 kDa
  • n is, independently for each occurrence, an integer between 1 and 2,000 such that the "4OK branched PEG" moiety has the combined average molecular weight of about 40 kDa
  • the PEGs used in the present invention are not restricted to any particular form or molecular weight range.
  • the PEG molecular weight may be between about 500 and about 100,000 Daltons.
  • the term "about” indicates that in preparations of PEGs, some molecules will weigh more and some less than the stated molecular weight; the stated molecular weight refers to the average molecular weight. It is understood that there is some degree of polydispersity associated with polymers such as PEGs. It is preferable to use PEGs with low polydispersity. Normally, a PEG with molecular weight of about 500 to about 60,000 Daltons is used.
  • a specific PEG molecular weight range of the present invention is from about 500 to about 50,000 Daltons.
  • the PEG molecular weight is about 20,000 to about 40,000 Daltons. Other sizes may be used, depending on the desired therapeutic profile (e.g., duration of sustained release desired, the effects, if any, on biological activity, the degree or lack of antigenicity and other known effects of the polyethylene to a therapeutic protein).
  • a PEG according to the present invention may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 Daltons.
  • the exemplified analogues of IGF-I of the present invention were prepared by a first step of peptide fragment synthesis, a second step of ligation, a third step of folding, and a fourth step of site-specific pegylation or site-specific lipidation.
  • the following synthetic procedures illustrate how a skilled chemist would be enabled to prepare any one of the exemplified analogues of IGF-I of the present invention.
  • Fmoc-based solid-phase peptide synthesis was used to assemble the titled peptide fragment using microwave assistance on a Liberty Peptide Synthesizer (CEM; Matthews, NC, USA).
  • the first 14-residue fragment i.e., residues 57-70 of hIGF-1, or the C-terminal acid peptide, was synthesized on a 1.0-mmole scale using Fmoc-Ala-Wang resin (0.72 meq/g).
  • the resulting peptide fragment was then split into four 0.25-mmole batches for elongation and differentiation.
  • a 1.36 g resin sample was placed in a 50-mL conical tube together with 15 mL of a 1:1 solution of DMF and DCM which was loaded into position in the synthesizer. The resin was then transferred to the reaction vessel via the synthesizer's automated process.
  • the standard Liberty protocol for 1.0-mmole scale synthesis was used.
  • the protocol involved removal of the N-terminal Fmoc protecting group by treatment with 20 mL of 20% piperidine containing 0.1M HOBt in DMF.
  • the initial de-protection step of microwave power (45 watts, maximum temperature of 75 C) and nitrogen bubbling (3 seconds on, 7 seconds off) lasted for 30 seconds.
  • the reaction vessel was drained and the resin was washed thoroughly with DMF several times.
  • the coupling protocol for Fmoc-Cys(Trt)-OH was a slightly modified version of the standard protocol. For Cys residues, no microwave power was applied for the first 2 minutes. A 4-minute session of microwave power (20 watts, maximum temperature of 50 ° C) followed. All amino acids were introduced similarly, employing a double coupling strategy throughout the entire sequence.
  • the resin was transferred back to the 50-mL conical tube using DMF as a solvent.
  • the resin was manually split evenly into four samples which were put into four 50-mL conical tubes which were then put back into the synthesizer.
  • the remaining portion of the titled peptide was synthesized on a 0.25-mmole scale.
  • the protocol used was the same as that used for the larger scale synthesis, however, lesser amounts of reagents were used.
  • Removal of the N-terminal Fmoc protecting group consisted of treatment with a solution containing 10 mL of 20% piperidine and 0.1M HOBt in DMF.
  • the initial de-protection step of microwave power (45 watts, maximum temperature of 75 ° C) with nitrogen bubbling (3 seconds on, 7 seconds off) lasted for 30 seconds.
  • the reaction vessel was then drained and the resin was washed several times thoroughly with DMF.
  • the next amino acid (Cycle 14), prepared as a 0.2M stock solution in DMF, was then introduced (5.0 mL, 4 equivalents) to the growing peptide (Fmoc-Gln(tBu)-OH). 2.0 mL of a 0.45M solution (4 equivalents) of HBTU in DMF was then added followed by 1.0 mL of a 2M solution (8 equivalents) of DIPEA in NMP.
  • Cycles 14, 16 and 21 employed a capping procedure which immediately followed the coupling step, which involved adding 7 mL of 0.5M acetic anhydride containing 0.015M HOBt and 2 mL of 2M DIPEA both in NMP while utilizing a multi-step microwave protocol (50 watts for 30 seconds with a maximum temperature of 65 C, then no power for 30 seconds, 50 watts for 30 seconds with a maximum temperature of 65 ° C, then no power for 30 seconds).
  • a multi-step microwave protocol 50 watts for 30 seconds with a maximum temperature of 65 C, then no power for 30 seconds, 50 watts for 30 seconds with a maximum temperature of 65 ° C, then no power for 30 seconds).
  • the N-terminal Fmoc-protecting group was removed and the resin was washed again with DMF. The resin was then transferred back to the 50-mL conical tube using DMF as the transfer solvent.
  • the resin was transferred into a reaction vessel with a sintered glass frit.
  • the DMF was removed and the resin was washed extensively with DCM.
  • the peptide fragment was cleaved and de-protected by treatment with the following reagent: 5% TIS : 5% water : 90% TFA.
  • the reaction was allowed to proceed for 3 hours at room temperature with constant shaking.
  • the solution was then filtered into a 50-mL conical tube. TFA was reduced by evaporation with nitrogen gas flow.
  • the peptide fragment was precipitated by the addition of 40 mL of cold ethyl ether followed by centrifugation at 3000 rpm for 30 minutes at 4 ° C within a refrigerated centrifuge (Sorvall Legend RT; Thermo Fisher, San Jose, CA, USA).
  • the resulting pellet was dissolved in 0.1% TFA water before purification by preparative HPCL equipped with a C18 reverse phase column (Luna, 10 ⁇ m, 250 x 21.2 mm column) utilizing a gradient of 0-60% acetonitrile (0.1% TFA) over 50 minutes with a flow rate of 10 mL/min.
  • the purified peptide fragment was analyzed by HPLC (Luna C 18, 3 ⁇ m, 4.6 x 100 mm column) with a gradient of 5-80% acetonitrile (0.08% TFA) over 30 minutes with a flow rate of 1 mL/min) and by mass spectrometry (LCQ Advantage; Thermo Fisher, San Jose, CA, USA).
  • HPLC Lisco C 18, 3 ⁇ m, 4.6 x 100 mm column
  • LCQ Advantage Thermo Fisher, San Jose, CA, USA
  • the N-terminal peptide fragment i.e., residues 1-47 of hIGF-1, was assembled using Boc-chemistry based solid-phase peptide synthesis.
  • An ABI 433A peptide synthesizer (Applied Biosystems; Foster City, CA, USA) modified to run the standard FastBoc protocol was utilized for the 0.5-mmole scale synthesis.
  • the reaction vessel containing 0.645 mg of 0.77 meq/g of Tampal Resin was placed on the synthesizer. To swell the resin, DMF was introduced.
  • the ABI FastBoc 0.5 protocol was used to generate the fragment. Each cycle consisted of de-blocking the N-terminal Boc protecting group with neat TFA followed by extensive DMF washing.
  • Pre-packaged 2.0-mmole (4 equivalents) cartridges of each amino acid were then dissolved in 0.40M HBTU and DMF. After complete dissolution of each amino acid, the solution was automatically transferred to the activation vessel. A DIPEA solution (neat) was introduced to the activation vessel and was exposed to the resin for an extended period. The reaction vessel was emptied and the resin was washed with DMF. For Arg/Asn cartridges, an extended activation time was required to ensure solubility. In addition, any amino acid added immediately after the coupling of a GIn residue was washed with DCM both before and after the deblocking protocol. The coupling times were 30 minutes.
  • Boc-Arg(Tos)- OH Boc-Asp(cHex)-OH, Boc-Glu(cHex)-OH, Boc-Asn(Xan)-OH, Boc-Cys(4Me-Bzl)-OH, Boc-Lys(ClZ)-OH, Boc-Gln-OH, Boc-Ser(OBzl)-OH, Boc-Thr(OBzl)-OH, and Boc- Tyr(BrZ)-OH.
  • the resin was washed with DCM and dried.
  • the peptide fragment was de-protected and cleaved from the resin using a treatment with 10 mL of hydrogen fluoride and anisole. The reaction was allowed to proceed for 70 minutes at which point the hydrogen fluoride was blown off with a stream of nitrogen. The residue was washed with ether and then the peptide was dissolved in 10-15 ml of TFA.
  • the peptide fragment was precipitated by filtering the TFA into 40 mL of cold ethyl ether followed by centrifugation at 3000 rpm for 30 minutes at 4°C within a refrigerated centrifuge (Sorvall Legend RT; Thermo Fisher, San Jose, CA, USA).
  • the resulting pellet was dissolved in 0.1% TFA water and was purified by preparative HPLC equipped with a Cl 8 reverse phase column (Luna, 10 ⁇ m, 250 x 21.2 mm column) utilizing a gradient of 20-40% acetonitrile (0.1% TFA) over 120 minutes with a flow rate of 10 mL/min.
  • the purified peptide fragment was analyzed by HPLC (Luna C18, 3 ⁇ m, 4.6 x 100 mm column) with a gradient of 5-80% acetonitrile (0.08% TFA) for 30 minutes with a flow rate of 1 mL/min and by mass spectrometry (LCQ Advantage; Thermo Fisher, San Jose, CA, USA).
  • the peptide fragment was subsequently lyophilized and stored at -50 ° C for future use.
  • hIGF-1 analogues were constructed using the chemical ligation method that naturally occurs between an N-terminal thioester fragment, e.g., hIGF-l(l-47)-S- (CH 2 ) 2 C(O)-Leu-NH 2 (SEQ ID NO: 114), and a C-terminal fragment, e.g., (GIn 56 , Asn 59 )hIGF-l (48-7O)-OH (SEQ ID NO: 113), which contains a cysteine residue at its N- terminus.
  • N-terminal thioester fragment e.g., hIGF-l(l-47)-S- (CH 2 ) 2 C(O)-Leu-NH 2 (SEQ ID NO: 114
  • a C-terminal fragment e.g., (GIn 56 , Asn 59 )hIGF-l (48-7O)-OH (SEQ ID NO: 113
  • the ligation product was purified by SemiPrep-HPLC (Vydac 218TP101510, Cl 8, 10-15 ⁇ m, 10 x 250 mm) with a gradient of 5-80% acetonitrile (0.1% TFA) over 40 minutes with a flow rate of 5 mL/min. The product peak was lyophilized and stored at -50 ° C. The mass of the unfolded ligation product was determined by physical measurement.
  • the protein prepared by the ligation process of step C) as described above, was dissolved in ligation buffer (20OmM sodium phosphate, pH 8.5, 6M guanidine hydrochloride) to a concentration of 1 mg/mL.
  • ligation buffer (20OmM sodium phosphate, pH 8.5, 6M guanidine hydrochloride)
  • Folding buffer (10OmM Tris, pH 8.5, ImM oxidized glutathione, 1OmM reduced glutathione) was then added to bring the final protein concentration to 0.25 mg/mL. The folding process was allowed to occur over 3 hours. Afterwards, the reaction was quenched by the drop-wise addition of TFA until the reaction mixture reached pH ⁇ 3.
  • the product was then purified by SemiPrep-HPLC (Vydac 218TP101510, C 18, 10-15 ⁇ m, 10 x 250 mm column) with a gradient of 5-60% acetonitrile (0.1% TFA) over 40 minutes with a flow rate of 5mL/min.
  • the product was lyophilized.
  • the protein content was determined by re-dissolving the product in 0.1% TFA water then measuring the absorbance at 280 nm (NanoDrop NDlOOO Spectrophotometer). The protein was then analyzed for QC (HPLC and MS).
  • step C The protein, prepared by the ligation process of step C) as described above, was dissolved in folding buffer A (2OmM Tris, pH 7.8, 5M guanidine hydrochloride) to a concentration of 10 mg/mL.
  • Folding buffer B (2OmM Tris, pH 7.8, ImM cystine, 8mM cysteine) was then added to bring the final protein concentration to 1 mg/mL. The folding process was allowed to occur over 3 hours. Afterwards, the reaction was quenched by the drop-wise addition of TFA until the reaction mixture reached pH ⁇ 3.
  • the product was then purified by SemiPrep-HPLC (Vydac 218TP101510, C18, 10-15 ⁇ m, 10 x 250 mm column) with a gradient of 5-60% acetonitrile (0.1% TFA) over 40 minutes with a flow rate of 5 mL/min.
  • the product was lyophilized.
  • the protein content was determined by re-dissolving the product in 0.1% TFA water then measuring the absorbance at 280 nm (NanoDrop NDlOOO Spectrophotometer). The protein was then analyzed for QC (HPLC and MS).
  • the protein prepared by the ligation process of step C) as described above, was dissolved in folding buffer (2OmM glycine, pH 10.5, 2M Urea, IM sodium chloride, 20% ethanol) and transferred to a 15-mL conical tube.
  • folding buffer (2OmM glycine, pH 10.5, 2M Urea, IM sodium chloride, 20% ethanol) and transferred to a 15-mL conical tube.
  • a 250 ⁇ M copper (II) bromide solution was added until the final copper concentration of the reaction was 0.5 ⁇ M.
  • the reaction was shaken vigorously for at least 6 hours occasionally stopping to open the vessel in order to exchange the air within the headspace.
  • the reaction was quenched and diluted by the addition of 10% TFA water to bring the final volume to 10 mL.
  • the product was purified by SemiPrep-HPLC (Vydac 218TP101510, C 18, 10-15 ⁇ m, 10 x 250 mm column) with a gradient of 0-60% acetonitrile (0.1% TFA) over 40 minutes with a flow rate of 5 mL/min.
  • the product was lyophilized.
  • the protein content was determined by re-dissolving the product in 0.1% TFA water then measuring the absorbance at 280 nm (NanoDrop NDlOOO Spectrophotometer). The protein was then analyzed for QC (HPLC and MS).
  • the mass of the folded hIGF-1 analogue was determined by absorbance at 280 nm in 0.1% TFA water (NanoDrop NDlOOO Spectrophotometer).
  • the protein prepared by the folding process of any one of steps D)-F) as described above, was re-dissolved in 5OmM imidazole buffer (pH 7.0) to a final concentration of 2 mg/mL (2.66 x 10 "4 M).
  • Sodium periodinate (NaIO 4 ) (4 equivalents) dissolved in an imidazole buffer was added and the resulting solution was gently mixed. The reaction was allowed to proceed at room temperature without further agitation. After 5 minutes, the reaction was quenched with the addition of 10 equivalents of ethylene glycol.
  • the mixture was allowed to stand for 15 minutes at room temperature.
  • the mixture was diluted with 0.1% TFA water to a final volume of 10 mL.
  • the product was then purified by SemiPrep-HPLC (Vydac 218TP101510, C18, 10-15 ⁇ m, 10 x 250 mm column) with a gradient of 5-60% acetonitrile (0.1% TFA) over 40 minutes with a flow rate of 5 mL/min.
  • the product was then lyophilized and stored at - 50 ° C until needed.
  • the titled protein was assembled through native chemical ligation from hIGF(l-46)- thio-propionyl-Leu-NH 2 (SEQ ID NO: 117) and the C-terminal fragment, i.e., ( ⁇ -Me-Cys 47 , Leu 59 )hIGF- 1(47-70) (SEQ ID NO: 118).
  • the protein thioester (7.4 mg, 1.45 ⁇ moles) and the C-terminal fragment (3.8 mg, 1.38 ⁇ moles) were dissolved in ligation buffer (6M guanidine hydrochloride in 20OmM sodium phosphate, pH 8.5, 400 ⁇ L) and TCEP (80 ⁇ L, 40 mg/mL, pH 7).
  • An MPAA catalyst was added (80 ⁇ L, 20 mg/mL, pH 7).
  • the reaction progress was monitored on a LCQ Deca XP LC-MS (Thermo Finnigan) with a Luna C 18(2) column (5 ⁇ m, 4.6 x 100 mm) with a gradient of 5-80% acetonitrile (0.1% TFA) for 30 minutes.
  • the reaction was quenched to a dilution of 1 : 10 with dH 2 O, 0.1% TFA (v/v).
  • the crude mixture was centrifuged and passed through a 1.0- ⁇ m glass filter to remove any MPAA precipitate.
  • the full length protein was purified using a 5-60% B linear gradient for 40 minutes with a flow rate of 5 mL/min on a Vydac C18 (10 ⁇ m, 10 x 250 mm).
  • the protein was quantitated by UV spectroscopy (NanoDrop NDlOOO Spectrophotometer) and lyophilized for future use.
  • the stored protein (1.8 mg, 235 nmoles) was dissolved in a 20OmM H 2 PO 4 " , 6M guanidinium-HCl solution having pH 8.5 to a concentration of 1.0 mg/mL.
  • Folding buffer (10OmM Tris, 1OmM glutathione, ImM oxidized glutathione at pH 8.5) was added to the solution until a final protein concentration of 250 ⁇ g/mL was achieved.
  • the mixture was allowed to incubate at room temperature while being monitored by HPLC. Once equilibrium was reached (as visualized by a stable HPLC profile), the reaction was quenched by stirring in either acetic acid or TFA to bring the solution to pH 3.
  • the solution was then passed through a 1.0- ⁇ m glass filter and further purified on a semi-preparative column.
  • the folded protein was purified using a 5-60% B linear gradient for 40 minutes with a flow rate of 5 mL/min.
  • the protein was quantitated by UV (NanoDrop NDlOOO Spectrophotometer) and lyophilized. Approximately 92 ⁇ g of purified product was obtained, representing a yield of 5%.
  • the mass of the protein was verified on a Finnigan LCQ Advantage MAX MS.
  • the titled protein was assembled utilizing native chemical ligation using MGF-I(I- 47)-thio-propionyl-Leu-NH 2 (SEQ ID NO: 114) and the C-terminal fragment, i.e., (N-Me- Cys 48 , Nle 59 )hIGF-l(48-70) (SEQ ID NO: 119).
  • the protein thioester (4.3 mg, 824 nmoles) and the C-terminal fragment (2.1 mg, 790 nmoles) were dissolved in ligation buffer (400 ⁇ L, 6M guanidine hydrochloride in 20OmM sodium phosphate, pH 8.5) and TCEP (80 ⁇ L, 40 mg/mL, pH 7).
  • An MPAA catalyst was added (80 ⁇ L, 20 mg/mL, pH 7).
  • the reaction progress was monitored using a Finnigan LCQ Deca XP LC-MS with a Luna C 18(2) column (5 ⁇ m, 4.6 x 100 mm) having a gradient of 5-80% acetonitrile (0.1% TFA) for 30 minutes.
  • the reaction was quenched to a dilution of 1: 10 with dH 2 O, 0.1% TFA (v/v).
  • the crude mixture was centrifuged and passed through a 1.0 ⁇ m glass filter to remove any MPAA precipitate.
  • the full length protein was purified using a Vydac Cl 8 (lO ⁇ m, 10 x 250 mm) with a 5-60% B linear gradient for 40 minutes with a flow rate of 5 mL/min.
  • the protein was quantitated by UV (NanoDrop NDlOOO Spectrophotometer) and lyophilized for future use.
  • the stored protein was dissolved using a 20OmM H 2 PO 4 " , 6M guanidinium-HCl solution (pH 8.5) until a concentration of 1.0 mg/mL was achieved.
  • Folding buffer (10OmM Tris, 1OmM glutathione, ImM oxidized glutathione, pH 8.5) was added to the solution until a final protein concentration of 250 ⁇ g/mL was achieved.
  • the mixture was allowed to incubate at room temperature while being monitored by HPLC. Once equilibrium was reached (as visualized by a stable HPLC profile), the reaction was quenched with either acetic acid or TFA to pH 3.
  • the solution was purified using first a 1.0- ⁇ m glass filter and then a semi- preparative column.
  • the folded protein was purified using a 5-60% B linear gradient with a flow rate of 5 mL/min for 40 minutes.
  • the protein was quantitated by UV (NanoDrop NDlOOO Spectrophotometer) and lyophilized. Approximately 0.415 mg of purified product was obtained, representing a yield of 10.6%.
  • the mass of the protein was verified on a Finnigan LCQ Advantage MAX MS.
  • the reaction was diluted with 0.1% TFA water then purified by SemiPrep-HPLC (Vydac 218TP101510, C18, 10-15 ⁇ m, 10 x 250 mm) with a gradient of 5- 95% acetonitrile (0.1% TFA) over 40 minutes at a flow rate of 5 mL/min.
  • the product was lyophilized, weighed, and then analyzed for QC (HPLC, MALDI-TOF MS).
  • the final protein concentration (2 mg/mL) required for this procedure was equivalent to that used for the PEGylation procedure described above in step J).
  • Isopropyl alcohol (approximately 15%) was used as a co-solvent.
  • Approximately 2.2 equivalents of aminooxy lipid were pre-dissolved.
  • the protein was dissolved in a 10OmM sodium acetate 10OmM aniline buffer having pH 4.6.
  • the folded protein was purified using a SemiPrep-HPLC (Vydac 218TP101510, C18, 10-15 ⁇ m, 10 x 250 mm column) with a gradient of 0-60% acetonitrile (0.1% TFA) and a flow rate of 5 mL/min for a 40-minute period.
  • the product was then lyophilized yielding 4.1 mg of folded (Ser 1 , Asn 59 )hIGF-l (0-7O)-OH.
  • the folded IGF-I analogue was then dissolved in 2 mL of imidazole buffer. Approximately 37 ⁇ L of a 13 mg/mL solution of sodium periodinate was added. The reaction was allowed to proceed for 5 minutes, and was quenched thereafter by the addition of 85 ⁇ L of ethylene glycol. The reaction was allowed to rest for 15 minutes. Approximately 6 mL of 0.1% TFA water was used to dilute the solution.
  • a SemiPrep-HPLC (Vydac 218TP101510, C18, 10-15 ⁇ m, 10 x 250 mm column) with a gradient of 5-60% acetonitrile (0.1% TFA) and a flow rate of 5 mL/min was employed for 40 minutes to purify the product which was analyzed by HPLC and MS. The remaining peptide was lyophilized, re-dissolved in 4 mL of 0.1% TFA water, divided into four equal 1.5-mL eppendorf tubes, and lyophilized again.
  • the sample was purified using a SemiPrep-HPLC (Vydac 218TP101510, C18, 10-15 ⁇ m, 10 x 250 mm) with a gradient of 5-95% acetonitrile (0.1% TFA) and a flow rate of 5 mL/min for 40 minutes.
  • the product was lyophilized, weighed, and analyzed for QC (HPLC, MALDI- TOF MS).
  • the reaction was monitored by HPLC and quenched/diluted after 30 minutes with 0.1% TFA water to a volume of 10 mL.
  • the reaction was purified using a SemiPrep-HPLC (Vydac 218TP101510, C18, 10-15 ⁇ m, 10 x 250 mm) with a gradient of 5-80% acetonitrile (0.1% TFA) and with a flow rate of 5mL/min for 40 minutes.
  • the product was lyophilized.
  • the protein content was determined by re-dissolving the product in 0.1% TFA water then measuring the absorbance at 280 nm (NanoDrop NDlOOO Spectrophotometer). It was also analyzed for QC (HPLC, MS).
  • Membranes were prepared for radioligand binding studies by homogenization of human MCF-7 cells expressing the native IGF-I receptor in 20 ml of ice-cold 5OmM Tris- HCl with a Brinkman Polytron (Westbury, NY, USA) (setting 6, 15 sec). The homogenates were washed twice by centrifugation (39,000 g / 10 minutes) and the final pellets were resuspended in 5OmM Tris-HCl containing 2.5mM MgCl 2 and 0.1% BSA.
  • Mouse 3T3/R cells obtained from Dr. E. Rozengurt at UCLA in Los Angeles, CA, USA were cultured on a 24-well plate (DMEM + 10% FCS) and maintained for 2 days in culture.
  • the media was removed and washed once with serum-free DMEM. The serum was then starved for 24 hours. After starvation, [ 3 H]thymidine and IGF-I peptides were added. The cells were then incubated for 24 hours at 37 0 C.
  • the media was aspirated.
  • the cells were then washed with an ice-cold 0.9% NaCl solution.
  • An ice-cold 5% TCA solution was then added for a 30- minute incubation at 4 0 C.
  • the TCA was aspirated and the wells were incubated with 95% ethanol for 4 hours.
  • the media was then transferred to a liquid scintillation vial for radioactivity counting.
  • IGF-I bioactivity data i.e., EC 50 values
  • Table 2 In vitro IGF-I bioactivity data (i.e., EC 50 values) for the compounds exemplified herein are also given in Table 2.
  • U2OS cells (Catalog # 93-0466C3, DiscoveRX Corporation, Fremont, CA, USA) were plated at 6 x 10 5 cells/mL in a 96-well poly-D-lysine plate 16 hours prior to assay in serum-free assay media.
  • the wild-type insulin (Catalog # 10908, Sigma, St. Louis, MO, USA), the wild-type IGF-I (Increlex®, Tercica, Inc., Brisbane, CA, USA ), or a test IGF-I peptide disclosed in the instant application was added at a dose range of lO ⁇ M (micromolar) to 0.15nm (nanomolar), and incubated for 3 hours at 37 0 C with 5% CO 2 .
  • PathHunterTM reagent (Catalog # 93-001, DiscoveRX) was prepared according to manufacturer's instructions, and added to each well. Plates were incubated at room temperature for 1 hour. Luminescence was read on an Envision 2104 multi-label plate reader (PerkinElmer, Inc., Waltham, MA, USA). Activity of each test peptide was analyzed and reported as
  • the analogues of IGF-I of this invention can be provided in the form of pharmaceutically acceptable salts.
  • such salts include, but are not limited to, those formed with organic acids (e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, methanesulfonic, toluenesulfonic, or pamoic acid), inorganic acids (e.g., hydrochloric acid, sulfuric acid, or phosphoric acid), and polymeric acids (e.g., tannic acid, carboxymethyl cellulose, polylactic, polyglycolic, or copolymers of polylactic-glycolic acids).
  • organic acids e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, methanesulfonic, toluenesulfonic, or pamoic acid
  • inorganic acids e.g., hydrochloric acid, sulfur
  • a typical method of making a salt of a peptide of the present invention is well known in the art and can be accomplished by standard methods of salt exchange.
  • the TFA salt of a peptide of the present invention (the TFA salt results from the purification of the peptide by using preparative HPLC eluting with TFA containing buffer solutions) was converted into another salt, such as an acetate salt, by dissolving the peptide in a small amount of 0.25 N acetic acid aqueous solution.
  • the resulting solution is applied to a SemiPrep HPLC column (Zorbax, 300 SB, C-8).
  • the column is eluted with (1) 0.1N ammonium acetate aqueous solution for 0.5 hours, (2) 0.25N acetic acid aqueous solution for 0.5 hours, and (3) a linear gradient (20% to 100% of solution B over 30 min) at a flow rate of 4 ml/min (solution A is a 0.25N acetic acid aqueous solution, and solution B is a 0.25N acetic acid in acetonitrile/water, with a 80:20 ratio).
  • solution A is a 0.25N acetic acid aqueous solution
  • solution B is a 0.25N acetic acid in acetonitrile/water, with a 80:20 ratio
  • the fractions containing the peptide are collected and lyophilized to dryness.
  • the dosage of active ingredient in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained.
  • the selected dosage depends on the desired therapeutic effect, on the route of administration, and on the duration of the treatment. Dosing is easily determined by the skilled, competent medical practitioner.
  • the compounds of this invention can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual, or topical routes of administration, and can be formulated with pharmaceutically acceptable carriers to provide dosage forms appropriate for each route of administration.
  • parenteral e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant
  • nasal, vaginal, rectal, sublingual, or topical routes of administration and can be formulated with pharmaceutically acceptable carriers to provide dosage forms appropriate for each route of administration.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch.
  • Such dosage forms can also comprise, as is normal practice, additional substances other than such inert diluents, e.g., lubricating agents such as magnesium stearate.
  • the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
  • Liquid dosage forms for oral administration include, without limitation, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, and the like, containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents.
  • Preparations according to this invention for parenteral administration include, without limitation, sterile aqueous or non-aqueous solutions, suspensions, emulsions, and the like.
  • non-aqueous solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
  • Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents.
  • Preparations may be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, and/or by heating the compositions.
  • Pharmaceutical compositions containing the novel IGF-I analogues described herein can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water or some other sterile injectable medium immediately before use.
  • compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as coca butter or a suppository wax.
  • compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.
  • a compound of this invention can be administered in a sustained release composition such as those described in the following patents and patent applications.
  • U.S. Patent No. 5,672,659 teaches sustained release compositions comprising a bioactive agent and a polyester.
  • U.S. Patent No. 5,595,760 teaches sustained release compositions comprising a bioactive agent in a gelable form.
  • U.S. Patent No. 5,821,221 teaches polymeric sustained release compositions comprising a bioactive agent and chitosan.
  • U.S. Patent No.5,916,883 teaches sustained release compositions comprising a bioactive agent and cyclodextrin.
  • PCT publication WO99/38536 teaches absorbable sustained release compositions of a bioactive agent.
  • PCT publication WO00/04916 teaches a process for making microparticles comprising a therapeutic agent such as a peptide in an oil-in-water process.
  • PCT publication WO00/09166 teaches complexes comprising a therapeutic agent such as a peptide and a phosphorylated polymer.
  • PCT publication WO00/25826 teaches complexes comprising a therapeutic agent such as a peptide and a polymer bearing a non-polymerizable lactone.
  • the invention disclosed in U.S. Pat. No. 7,258,864 features a method for treating a subject having insulin-like growth factor- 1 deficiency (IGFD) comprising administering to a human pediatric subject an effective amount of the unmodified IGF-I wherein the subject is characterized as follows: a) at the time of treatment or prior to initial treatment with IGF-I, has or had a height at least about 2 standard deviations (SD) below a normal mean for a corresponding age and gender, and b) at the time of treatment or prior to initial treatment with IGF-I, has or had a blood level of IGF-I at least about -1 SD below normal mean levels wherein the subject does not have Laron syndrome or partial growth hormone insensitivity syndrome, and wherein said administering is effective to treat IGFD in the subject.
  • SD standard deviations
  • the invention disclosed in WO 2006/130769 features a method for treating a subject having idiopathic short stature (ISS) comprising administering to a human pediatric subject suffering from ISS characterized by partial endogenous growth hormone activity or signaling, an amount of IGF-I effective to promote growth in the subject, wherein the subject is further characterized as follows: a) at the time of treatment or prior to initial treatment with IGF-I, has or had a height at least about 2.0 standard deviations (SD) below the normal mean height for a subject of the same age and gender, and b) has blood levels of GH and IGF-I that are at least normal for a subject of the same age and gender.
  • SD standard deviations
  • novel analogues disclosed herein are able to be administered alone or in combination with another therapeutic agent as determined by a skilled medical practitioner.

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Abstract

The present invention relates to novel site-specific pegylated or site-specific lipidated IGF-1 and analogues of IGF-1, e.g., [2-(ethoxyimino(2-carbamate-20K branched PEG))acetyl-Gly1, Asn59]hIGF-1(1 -7O)-OH (SEQ ID NO: 106) and [2-(n- octadecoxyimino)acetyl-Gly1, Asn59]hIGF- 1 ( 1 -7O)-OH (SEQ ID NO: 109), and the use of said compounds for treatment of IGF-1 -receptor mediated conditions, such as short stature, diabetes therapy, neurodegenerative disease treatment, and cartilage repair. The site- specifically modified IGF-1 and analogues of IGF-1 according to the present invention have improved pharmacokinetic profile, improved biological activity, and/or decreased immunogenicity.

Description

SITE-SPECIFIC PEGYLATED OR SITE-SPECIFIC LIPIDATED IGF-I AND
ANALOGUES OF IGF-I
FIELD OF THE INVENTION
The present invention relates to novel site-specific pegylated or site-specific lipidated insulin-like growth factor- 1 (IGF-I) and analogues of IGF-I, and the use of said compounds for treatment of IGF-I -receptor mediated conditions, such as short stature, diabetes therapy, neurodegenerative disease treatment, and cartilage repair.
BACKGROUND ART
IGF-I is a 70-amino-acid polypeptide hormone having insulin-like and mitogenic growth biological activities. This hormone enhances growth of cells in a variety of tissues including musculoskeletal systems, liver, kidney, intestines, nervous system tissues, heart, and lung.
The wild-type IGF-I has the following amino acid sequence with three intrachain disulfide bridges wherein the side-chains of residue pairs A6 and A48, A47 and A52, and A18 and A61, each form a disulfide bond (SEQ ID NO: 112):
Gly-Pro-Glu-Thr-Leu-Cys-Gly-Ala-Glu-Leu-Val-Asp-Ala-Leu-Gln-Phe-Val-Cys-
1 5 10 15
Gly-Asp-Arg-Gly-Phe-Tyr-Phe-Asn-Lys-Pro-Thr-Gly-Tyr-Gly-Ser-Ser-Ser-Arg-
20 25 30 35
Arg-Ala-Pro-Gln-Thr-Gly-Ile-Val-Asp-Glu-Cys-Cys-Phe-Arg-Ser-Cys-Asp-Leu-
40 45 50
Arg-Arg-Leu-Glu-Met-Tyr-Cys-Ala-Pro-Leu-Lys-Pro-Ala-Lys-Ser-Ala
55 60 65 70
While IGF-I is present in a wide variety of body tissues, it is normally found in an inactive form in which it is bound to an IGF binding protein (IGFBP). Six related IGFBPs are known and have been designated IGFBPl - IGFBP6. See, e.g., Holly and Martin, "Insulin-like Growth Factor Binding Proteins: A Review of Methodological Aspects of Their Purification, Analysis and Regulation," Growth Regul, 4(Suppl l):20-30 (1994). IGFBPs play an important role in IGF-I regulation by exerting inhibitory and/or stimulatory effects on IGF-I action. For example, about 90% of circulating IGF-I is present in a trimolecular complex containing IGFBP-3 and acid labile submit. The IGF-I within such complexes is unable to bind to surface receptors, and is therefore biologically inactive. IGF-I present within the trimolecular complex also has a substantially longer half-life than uncomplexed IGF-I . Disruption of IGF-I action may contribute to a number of physiological disorders including neurodegenerative disorders such as motor neuron disease (i.e., amyotrophic lateral sclerosis (ALS)), muscular dystrophy and multiple sclerosis, cartilage disorders such as osteoarthritis, bone diseases such as osteoporosis, inflammatory disorders such as rheumatoid arthritis, ischemic injuries to organs such as to the heart, brain, or liver, and so forth.
As is well known to those skilled in the art, the known and potential uses of IGF-I are varied and multitudinous. For example, a number of studies report on the use of IGF-I as a potential therapeutic agent for treatment of neurodegenerative conditions. See, e.g., Kanje et al, Brain Res., 486:396-398 (1989); Hantai et al, J. Neurol. ScL, 129:122-126 (1995); Contreras et al, Pharmac. Exp. Therap., 274: 1443-1499 (1995); Di Giulio et al., Society for Neuroscience, 22:1960 (1996); Di Giulio et al., Society for Neuroscience, 23:894 (1997); Hsu et al, Biochem. MoI. Med., 60(2):142-148 (1997); Gorio et al, Neuroscience, 82:1029-1037 (1998). IGF-I therapy has been indicated in numerous neurological conditions, including ALS, stroke, epilepsy, Parkinson's disease, Alzheimer's disease, acute traumatic injury and other disorders associated with trauma, aging, disease, or injury. See, e.g., U.S. Pat. Nos. 5,093,137; 5,652,214; 5,703,045; International Publication Nos. WO 90/1483 and WO 93/02695.
Use of IGF-I therapy for a variety of other conditions has been referred to in a number of publications. See, e.g., Schalch et al, "Modern Concepts of Insulin-Like Growth Factors," ed. Spencer (Elsevier, New York), pp. 705-714 (1991); Clemmons and Underwood, J. Clin. Endocrinol Metab., 79(l):4-6 (1994); Langford et al., Eur. J. Clin. Invest, 23(9):5O3- 516 (1993) (referring to, e.g., insulin-resistant states and diabetes); and O' Shea et al., Am. J. Physiol, 264:F917-F922 (1993) (referring to, e.g., reduced renal function). Also see U.S. Pat. No. 7,258,864 (referring to short stature); U.S. Pat. Nos. 5,110,604 and 5,427,778 (referring to, e.g., wound healing); U.S. Pat. No. 5,126,324 (referring to, e.g., cardiac disorders and growth retardation); U.S. Pat. No. 5,368,858 (referring to, e.g., defects or lesions in cartilage); U.S. Pat. Nos. 5,543,441 and 5,550,188 (referring to, e.g., tissue augmentation); U.S. Pat. No. 5,686,425 (referring to, e.g., scar tissue, localized muscular dysfunction, and urinary incontinence); and U.S. Pat. No. 5,656,598 (referring to, e.g., bone growth). Also see International Patent Publication Nos. WO 91/12018 (referring to, e.g., intestinal disorders); WO 92/09301 and WO 92/14480 (referring to, e.g., wound healing); WO 93/08828 (referring to, e.g., neuronal damage associated with ischemia, hypoxia, or neurodegeneration); WO 94/16722 (referring to, e.g., insulin resistance); WO 96/02565A1 (referring to, e.g., IGF/IGFBP complex for promoting bone formation and for regulating bone remodeling); U.S. Patent Application Publication No. 2003/0100505 (referring to, e.g., osteoporosis); and U.S. Patent Application Publication No. 2005/0043240 (referring to obesity). Although IGF-I therapy has been used for a number of physiological indications, results have sometimes been unpredictable. Short-term beneficial effects sometimes do not persist (see, e.g., Miller et al., Kidney International, 46:201-207 (1994)) and undesirable side effects can result, particularly from administration of high doses and/or long-term administration (see, e.g., Jabri et al., Diabetes, 43:369-374 (1994); Wilton, Acta Paediatr., 393:137-141 (1992)). Also, high levels of IGF-I have been reported to increase risk for prostate cancer (Chan et al, Science, 278:563-566 (1998)).
Accordingly, there is a need in the art for better ways to treat conditions responsive to IGF-I and/or other proteins that bind to insulin-like growth factor binding proteins. The present invention fulfills these needs and further provides other related advantages.
SUMMARY OF THE INVENTION
In one aspect, the present invention is directed to an amino-terminal pegylated IGF-I conjugate of formula (I),
W-B
(I)
wherein,
W is
Figure imgf000004_0001
wherein X is O or NH; and
B is the wild-type IGF-I or an analogue of IGF-I;
wherein said linker is alkyl, aryl, alkyl-aryl, substituted alkyl, substituted aryl, or substituted alkyl-aryl, which may contain other organic functional group(s), and which is preferably
Figure imgf000004_0002
wherein p is an integer from 1 to 10 inclusive, and preferably 1; and wherein said
Figure imgf000004_0003
is
Figure imgf000004_0004
Figure imgf000005_0001
In another aspect, the present invention is directed to an amino-terminal lipidated IGF-I conjugate of formula (II),
Y-B
(H)
wherein,
Y is
Figure imgf000005_0002
or wherein Z is alkyl, aryl, alkyl-aryl, substituted alkyl, substituted aryl, or substituted alkyl-aryl which may contain other organic functional group(s), and which is preferably CH3-(CH2),,,-, wherein m is an integer from 1 to 30 inclusive; and
B is the wild-type IGF-I or an analogue of IGF-I;
According to either formula (IA) or formula (HA) respectively:
(W or Y)^1^2-^^4-^^6-^-^^9^10^11^12^13^14^15^16^17^18^19- A20-A21-A22-A23-A24-A25-A26-A27-A28-A29-A30-A31-A32-A33-A34-A35-A36-A37-A38-A39-A40-A41- A42-A43-A44-A45-A46-A47-A48-A49-A50-A51-A52-A53-A54-A55-A56-A57-A58-A59-A60-A61-A62-A63-
A64,A65.A66.A67_A68.A69.A70_A7,_Rl )
(IA) or (HA)
wherein:
A1 is GIy, Ala, Asn, Asp, GIn, GIu, or deleted;
A2 is Pro, Ala, Arg, Asp, GIn, GIu, Lys, or deleted;
A3 is GIu, Ala, Asp, GIn, or deleted;
A4 is Thr, Ala, Asn, Asp, GIn, GIu, Ser;
A5 is Leu, Ace, Ala, He, or VaI;
A6 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A7 is GIy, Ala, Asn, Asp, GIn or GIu;
A8 is Ala, Arg, Asn, Asp, GIn, GIu, or Lys; A9 is GIu, Ala, Asp, or GIn;
A10 is Leu, Ace, Ala, He, or VaI;
A" is VaI, Ala, lie, or Leu;
A12 is Asp, Ala, Arg, Asn, GIn, GIu, or Lys;
A13 is Ala, Asn, Asp, GIn, GIu, lie, Leu, or VaI;
A14 is Leu, Ace, Ala, He, or VaI;
A15 is GIn, Ala, Asn, Asp, or GIu;
A16 is Phe, Ala, Asn, Asp, GIn, GIu, Trp, or Tyr;
A17 is VaI, Ala, lie or Leu;
A18 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A19 is GIy, Ala, Asn, Asp, GIn, or GIu;
A20 is Asp, Ala, Asn, GIn, or GIu;
A21 is Arg, Ala, Asn, Asp, GIn, GIu, or Lys;
A22 is GIy, Ala, Asn, Asp, GIn, or GIu;
A23 is Phe, Ala, Trp, or Tyr;
A24 is Tyr, Ala, Phe, or Trp;
A25 is Phe, Ala, Trp, or Tyr;
A26 is Asn, Ala, Asp, GIn, GIu, Ser, or Thr;
A27 is Lys, Ala, Arg, Asn, Asp, GIn, GIu, or Pro;
A28 is Pro, Ala, Arg, or Lys;
A29 is Thr, Ala, Asn, Asp, GIn, GIu, or Ser;
A30 is GIy, Ala, Asn, Asp, GIn, or GIu;
A31 is Tyr, Ala, Phe, or Trp;
A32 is GIy, Ala, Asn, Asp, GIn, or GIu;
A33 is Ser, Ala, Thr, or VaI;
A34 is Ser, Ala, Asn, Asp, GIn, GIu, or Thr;
A35 is Ser, Ala, Asn, Asp, GIn, GIu, or Thr;
A36 is Arg, Ala, Asn, Asp, GIn, GIu, or Lys;
A37 is Arg, Ala, Asn, Asp, GIn, GIu, or Lys;
A38 is Ala, Asn, Asp, GIn, or GIu;
A39 is Pro, Ala, Arg, or GIu;
A40 is GIn, Ala, Asn, Asp, or GIu;
A41 is Thr, Ala, Asn, Asp, GIn, GIu, or Ser;
A42 is GIy, Ala, Arg, Asn, Asp, GIn, GIu, or Lys;
A43 is He, Ala, Arg, Asn, Asp, GIn, GIu, or Lys;
A44 is VaI, Ala, Arg, Asn, Asp, GIn, GIu, lie, Leu, or Lys; A45 is Asp, Ala, Arg, Asn, GIn, GIu, or Lys;
A46 is GIu, Ala, Arg, Asn, Asp, GIn, or Lys;
A47 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A48 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A49 is Phe, Ala, Arg, He, Leu, Lys, Ser, Thr, Trp, Tyr, or VaI;
A50 is Arg, Ala, Lys, Ser, or Thr;
A51 is Ser, Aib, Ala, Arg, Lys, or Thr;
A52 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A53 is Asp, Ala, Arg, Asn, GIn, GIu, Lys, Ser, or Thr;
A54 is Leu, Ace, Ala, Arg, He, Phe, or VaI;
A55 is Arg, Ala, lie, Leu, Lys, Phe, Trp, Tyr, or VaI;
A56 is Arg, Ala, Asn, Asp, GIn, GIu, or Lys;
A57 is Leu, Ace, Ala, Ee, Phe, or VaI;
A58 is GIu, Ace, Ala, Arg, Asn, Asp, GIn, or Lys;
A59 is Met, Ace, Ala, Arg, Asn, Asp, GIn, GIu, He, Leu, Lys, NIe, Ser, D-Ser, Thr, Trp, Tyr, or VaI;
A60 is Tyr, Ala, Arg, Ee, Phe, or Trp;
A61 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A62 is Ala, Arg, Asn, Asp, GIn, GIu, He, Leu, or VaI;
A63 is Pro, D-Pro, Ala, Ser, Thr, or deleted;
A64 is Leu, D-Leu, des-Leu, Ala, Arg, lie, Phe, VaI, or deleted;
A65 is Lys, D-Lys, des-Lys, Ala, Arg, He, Leu, VaI, or deleted;
A66 is Pro, D-Pro, Ala, or deleted;
A67 is Ala, D-AIa, Aib, Arg, or deleted;
A68 is Lys, D-Lys, Ala, Arg, He, Leu, VaI, or deleted;
A69 is Ser, D-Ser, Aib, Ala, Arg, Thr, or deleted;
A70 is Ala, D-AIa, Asn, Asp, GIn, GIu, or deleted;
A71 is Asn, Ala, Asp, GIn, GIu, Lys, Ser, Thr, or deleted; and
R1 is OH or NH2;
provided that the side-chains of residue pairs A6 and A48, A47 and A52, and A18 and A61, each form a disulfide bond; and
further provided that when A59 is either Leu, Ee, NIe, Thr, or VaI, then the analogue contains at least one additional amino acid substitution or addition as defined herein. In the formula (IA) or formula (HA), preferred amino acid substitutions and additions are defined as follows:
A1 is GIy, GIu, or deleted;
A2 is Pro, GIu, Lys, or deleted;
A3 is GIu or deleted;
A4 is Thr or GIu;
A6 is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A7 is GIy or GIu;
A8 is Ala, Arg or GIu;
A11 is VaI or Leu;
A13 is Ala or GIu;
A15 is GIn or GIu;
A16 is Phe or GIu;
A17 is VaI or Leu;
A18 is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A19 is GIy or GIu;
A20 is Asp or GIu;
A21 is Arg or GIu;
A22 is GIy or GIu;
A26 is Asn, GIu, or Thr;
A27 is Lys, Arg, GIu, or Pro;
A28 is Pro or Lys;
A29 is Thr or GIu;
A30 is GIy or GIu;
A32 is GIy or GIu;
A34 is Ser or GIu;
A35 is Ser or GIu;
A36 is Arg or GIu;
A37 is Arg or GIu;
A38 is Ala or GIu;
A40 is GIn or GIu;
A41 is Thr or GIu;
A42 is GIy, Arg, or GIu;
A43 is Ee, Arg, or GIu;
A44 is VaI, Arg, or GIu;
A45 is Asp, Arg, GIn, or GIu;
A46 is GIu, Arg, or GIn; A47 is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A49 is Phe, Arg, Leu, or Thr;
A5υ is Arg or Ser;
A51 is Ser, Aib, Arg, or Thr;
A52 is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A53 is Asp, Arg, or Ser;
A54 is Leu, A6c, Arg, or Phe;
A55 is Arg, Tyr, or VaI;
A56 is Arg or GIn;
A57 is Leu or Phe;
A58 is GIu, A6c, or Arg;
A59 is Met, A6c, Arg, Asn, Asp, GIn, GIu, He, Leu, NIe, Ser, D-Ser, Trp, or Tyr;
A60 is Tyr, Arg, De, or Phe;
A61 is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A is Ala, Arg, or Asn;
Abi is Pro, D-Pro, Thr, or deleted;
A64 is Leu, D-Leu, des-Leu, Arg, Phe, or deleted;
A65 is Lys, D-Lys, des-Lys, Arg, VaI, or deleted;
A66 is Pro, D-Pro, or deleted;
A67 is Ala, D-AIa, Aib, Arg, or deleted;
A68 is Lys, D-Lys, Arg, VaI, or deleted;
A69 is Ser, D-Ser, Aib, Arg, Thr, or deleted;
A70 is Ala, D-AIa, Arg, GIu, or deleted; and
A71 is Ser, Asp, GIu, Lys, or deleted.
Preferred analogues of IGF-I of the formula (IA) or formula (ILA) are:
Example 1 : (Asn59)hIGF-l(l -7O)-OH; (SEQ ID NO: 1)
Example 2: (Asn59)hIGF-l(l-62)-OH; (SEQ ID NO:2)
Example 3: (Asn59)hIGF-l (4-7O)-OH; (SEQ ID NO: 3)
Example 4: (Pro27, Lys28, Asn59)hIGF-l (1-7O)-OH; (SEQ ID NO:4)
Example 5: (Pro27, Lys28, Asn59)hIGF-l(l-62)-OH; (SEQ ID NO:5)
Example 6: (Ser53, Asn59)hIGF-l(l -7O)-OH; (SEQ LD NO:6)
Example 7: (Leu17)hIGF-l(l-70)-OH; (SEQ LD NO:7)
Example 8: (Asn59, Thr63, des-Leu64, des-Lys65, Glu70)hIGF-l(l -7O)-OH; (SEQ LD NO: 8)
Example 9: (Tyr55, Asn59)hIGF-l(l-70)-OH; (SEQ LD NO:9) Example 10: (Thr49, Asn59)hIGF-l(l -7O)-OH; (SEQ IDNO:10)
Example 11: (Asn59' 62)hIGF-l(l-70)-OH; (SEQ IDNO:11)
Example 12: (Asn59, Phe60)hIGF-l(l -7O)-OH; (SEQ IDNO:12)
Example 13: (Ser50, Asn59)hIGF-l(l -7O)-OH; (SEQ IDNO:13)
Example 14: (GIn56, Asn59)hIGF-l(l -7O)-OH; (SEQ IDNO:14)
Example 15: (Asn59, D-Pro63)hIGF- 1(1 -7O)-OH;
Example 16: (Asn59, D-Leu^hlGF-lO -7O)-OH;
Example 17: (Asn59, D-Lys65)hIGF-l(l -7O)-OH;
Example 18: (Asn59, D-Pro66)hIGF-l(l -7O)-OH;
Example 19: (Asn59, D-Ala67)hIGF- 1(1 -7O)-OH;
Example 20: (Asn59, D-Lys68)hIGF-l(l -7O)-OH;
Example 21 : (Asn59, D-Ser69)hIGF-l(l-70)-OH;
Example 22: (Asn59, D-Ala70)hIGF- 1(1 -7O)-OH;
Example 23: (Arg27' 65' 68, Leu59)hIGF-l(l -7O)-OH; (SEQ ID NO:15)
Example 24: (Leu59, Arg65' 68)hIGF- 1(1 -7O)-OH; (SEQ ID NO:16)
Example 25: (Leu49' 59)hIGF-l(l -7O)-OH; (SEQ ID NO:17)
Example 26: (β-Me-Cys52, Leu59)hIGF-l(l -7O)-OH; (SEQ ID NO:18)
Example 27 : (β-Me-Cys47, Leu59)hIGF- 1 ( 1 -7O)-OH; (SEQ IDNO:19)
Example 28: (Leu59, Glu71)hIGF-l(l-71)-OH; (SEQ ID NO:20)
Example 29: (Leu59, Asp71)hIGF-l(l-71)-OH; (SEQIDN0:21)
Example 30: (Leu59, Lys71)hIGF-l(l-71)-OH; (SEQ IDNO:22)
Example 31 : (Leu59, Ser71)hIGF-l(l-71)-OH; (SEQ ID NO:23)
Example 32: (Leu59, Thr69)hIGF-l(l -7O)-OH; (SEQ IDNO:24)
Example 33: (Thr51, Leu59)hIGF-l(l -7O)-OH; (SEQ IDNO:25)
Example 34: (N-Me-Cys47, Nle59)hIGF-l(l -7O)-OH; (SEQ IDNO:26)
Example 35: (NIe59, Aib69)hIGF-l(l -7O)-OH; (SEQ IDNO:27)
Example 36: (N-Me-Cys48, Nle59)hIGF-l(l -7O)-OH; (SEQ IDNO:28)
Example 37: (NIe59, Aib67)hIGF-l(l -7O)-OH; (SEQ IDNO:29)
Example 38 : (hCys52, Nle59)hIGF- 1 ( 1 -7O)-OH; (SEQ IDNO:30)
Example 39: (Aib51, Nle59)hIGF-l(l -7O)-OH; (SEQIDN0:31)
Example 40: (Pen52, Nle59)hIGF-l(l -7O)-OH; (SEQ IDNO:32)
Example 41: (NIe59, Pen61)hIGF-l(l-70)-OH; (SEQ IDNO:33)
Example 42: (A6c54, Nle59)hIGF-l(l -7O)-OH; (SEQ IDNO:34)
Example 43 : (Arg53, Ile59)hIGF- 1 ( 1 -7O)-OH; (SEQ ID NO:35)
Example 44: (Arg49, Ile59)hIGF- 1 ( 1 -7O)-OH; (SEQ ID NO:36)
Example 45: (Arg51, Ile59)hIGF-l(l -7O)-OH; (SEQ IDNO:37)
Example 46: (Arg58, Ee59)hIGF-l(l -7O)-OH; (SEQ DD NO:38) Example 47: (A6c59)hIGF-l(l -7O)-OH; (SEQ ID NO:39)
Example 48: (Asp59)hIGF-l (1 -7O)-OH; (SEQ ID NO:40)
Example 49: (Trp59)hIGF- 1(1 -7O)-OH; (SEQ ID NO:41)
Example 50: (Ser59)hIGF-l(l-70)-OH; (SEQ ID NO:42)
Example 51 : (Tyr59)hIGF- 1(1 -7O)-OH; (SEQ ID NO:43)
Example 52: (Glu59)hIGF-l(l -7O)-OH; (SEQ ED NO:44)
Example 53: (Gln59)hIGF-l(l -7O)-OH; (SEQ ID NO:45)
Example 54: (Arg59)hIGF-l(l -7O)-OH; (SEQ ID NO:46)
Example 55: (Glu^hlGF-lO^-OH; (SEQ ID NO:47)
Example 56: (Glu2)hIGF-l(l -7O)-OH; (SEQ ID NO:48)
Example 57: (Glu4)hIGF-l(l -7O)-OH; (SEQ ED NO:49)
Example 58: (Glu7)hIGF- 1(1 -7O)-OH; (SEQ ID NO:50)
Example 59: (Glu8)hIGF-l(l-70)-OH; (SEQ ID NO:51)
Example 60: (GIu13)hIGF-l(l -7O)-OH; (SEQ ID NO:52)
Example 61: (Glu15)hIGF-l(l-70)-OH; (SEQ ID NO:53)
Example 62: (Glu16)hIGF-l(l -7O)-OH; (SEQ ID NO:54)
Example 63: (Glu19)hIGF-l(l-70)-OH; (SEQ ED NO:55)
Example 64: (Glu20)hIGF- 1 ( 1 -7O)-OH; (SEQ ED NO:56)
Example 65: (Glu21)hIGF-l(l-70)-OH; (SEQ ED NO:57)
Example 66: (Glu22)hIGF- 1(1 -7O)-OH; (SEQ ED NO:58)
Example 67: (Glu26)hIGF-l(l -7O)-OH; (SEQ ED NO:59)
Example 68: (Glu27)hIGF- 1(1 -7O)-OH; (SEQ ED NO:60)
Example 69: (Glu29)hIGF- 1 ( 1 -7O)-OH; (SEQ ED N0:61)
Example 70: (Glu30)hIGF- 1 ( 1 -7O)-OH; (SEQ ED NO:62)
Example 71 : (Glu32)hIGF- 1 ( 1 -7O)-OH; (SEQ ED NO:63)
Example 72: (Glu34)hIGF- 1(1 -7O)-OH; (SEQ ED NO:64)
Example 73: (Glu35)hIGF-l(l-70)-OH; (SEQ ED NO:65)
Example 74: (Glu36)hIGF-l(l -7O)-OH; (SEQ ED NO:66)
Example 75: (Glu37)hIGF-l(l -7O)-OH; (SEQ ED NO:67)
Example 76: (Glu38)hIGF-l(l -7O)-OH; (SEQ ED NO:68)
Example 77: (Glu40)hIGF-l(l-70)-OH; (SEQ ED NO:69)
Example 78: (Glu41)hIGF-l(l-70)-OH; (SEQ ED NO:70)
Example 79: (Glu42)hIGF-l(l -7O)-OH; (SEQ ED N0:71)
Example 80: (Glu43)hIGF-l(l -7O)-OH; (SEQ ED NO:72)
Example 81 : (Glu^hlGF-Kl -7O)-OH; (SEQ ED NO:73)
Example 82: (Glu45)hIGF-l(l -7O)-OH; (SEQ ED NO:74)
Example 83: (Pro27, Lys28)hIGF-l(l -7O)-OH; (SEQ ED NO:75) Example 84: (Lys2)hIGF-l(l-70)-OH; (SEQ ID NO:76) Example 85: (Thr26)hIFG- 1 ( 1 -7O)-OH; (SEQ BD NO:77)
Example 86: (Val65)hIGF-l(l-70)-OH; (SEQ ID NO:78)
Example 87: (Val68)hIGF-l(l-70)-OH; (SEQ ID NO:79)
Example 88: (VaI65' 6S)hIGF- 1 ( 1 -7O)-OH; (SEQ ID NO:80)
Example 89: (Arg8)hIGF-l(l-70)-OH; (SEQ ID NO:81)
Example 90: (Arg42)hIGF-l(l-70)-OH; (SEQ ID NO:82)
Example 91 : (Arg44)hIGF-l(l-70)-OH; (SEQ ID NO:83)
Example 92: (Arg45)hIGF-l(l-70)-OH; (SEQ ID NO:84)
Example 93: (Arg46)hIGF- 1 ( 1 -7O)-OH; (SEQ ID NO:85)
Example 94: (Arg43)hIGF-l(l-70)-OH; (SEQ ID NO:86)
Example 95: (Gln45)hIGF-l(l-70)-OH; (SEQ ID NO:87)
Example 96: (Gln46)hIGF-l(l-70)-OH; (SEQ ID NO:88)
Example 97: (Leuu)hIGF- 1 ( 1 -7O)-OH; (SEQ ID NO:89)
Example 98: (Leu49)hIGF-l(l-70)-OH; (SEQ ID NO:90)
Example 99: (Ile60)hIGF-l(l-70)-OH; (SEQ ID NO:91)
Example 100: (Phe54)hIGF- 1 ( 1 -7O)-OH; (SEQ ID NO:92)
Example 101: (Phe57)hIGF-l(l-70)-OH; (SEQ ID NO:93)
Example 102: (Phe64)hIGF- 1 ( 1 -7O)-OH; (SEQ ID NO:94)
Example 103: (Arg54)hIGF-l(l-70)-OH; (SEQ ID NO:95)
Example 104: (Arg60)hIGF-l(l-70)-OH; (SEQ ID NO:96)
Example 105: (Arg62)hIGF-l(l -7O)-OH; (SEQ ID NO:97)
Example 106: (Arg64)hIGF-l(l-70)-OH; (SEQ ID NO:98)
Example 107: (Arg65)hIGF-l(l-70)-OH; (SEQ ID NO:99)
Example 108: (Arg67)hIGF-l(l-70)-OH; (SEQ ID NO: 100)
Example 109: (Arg68)hIGF-l(l -7O)-OH; (SEQ ID NO: 101)
Example 110: (Arg69)hIGF- 1(1 -7O)-OH; and (SEQ ID NO: 102)
Example 111 : (Arg ~7'0υ-)hIGF-l(l-70)-OH. (SEQ ID NO: 103)
Preferred amino-terminal pegylated IGF-I conjugates of formula (I) are:
Example 112: [2-(ethoxyimino(2-carbamate-10K linear PEG))acetyl-Gly', Asn59]hIGF-l(l- 7O)-OH; (SEQ ID NO: 104)
Example 113: [2-(ethoxyimino(2-carbamate-20K linear PEG))acetyl-Gly', Asn59]hIGF-l(l- 7O)-OH; (SEQ ID NO: 105)
Example 114: [2-(ethoxyimino(2-carbamate-20K branched PEG))acetyl-Gly\ Asn59]hIGF- 1 ( 1 -7O)-OH; (SEQ ID NO: 106) Example 115: [2-(ethoxyimino(2-carbamate-30K linear PEG))acetyl-Gly ' , Asn59]hIGF- 1(1- 7O)-OH; and (SEQ ID NO: 107)
Example 116: [2-(ethoxyimino(2-carbamate-40K branched PEG))acetyl-Gly\ Asn59]hIGF- 1(1-7O)-OH. (SEQ ID NO:108)
Preferred amino-terminal lipidated IGF-I conjugate of formula (I) is:
Example 117: [2-(n-octadecoxyimino)acetyl-Gly', Asn59]hIGF-l(l -7O)-OH.
(SEQ ID NO: 109)
The site-specifically modified IGF-I and analogues of IGF-I according to the present invention have improved pharmacokinetic profile, improved biological activity, and/or decreased immunogenicity.
DETAILED DESCRIPTION OF THE INVENTION
The application employs the following commonly understood abbreviations:
Ace: 1 -amino- l-cyclo(C3-C9)alkyl carboxylic acid
Ace includes:
A3c: 1 -amino- 1 -cyclopropanecarboxylic acid
A4c: 1 -amino- 1 -cyclobutanecarboxylic acid
A5c: 1 -amino- 1 -cyclopentanecarboxylic acid
A6c: 1 -amino- 1 -cyclohexanecarboxylic acid
Aib: α-aminoisobutyric acid
Ala or A: alanine
Arg or R: argmine
Asn or N: asparagine
Asp or D: aspartic acid
Cys or C: cysteine
cystine: disulfide dimer of cysteine
hCys: homocysteine
β-Me-Cys: beta-methyl-cysteine, i.e.,
(2S, 3S)-2-amino-3-mercaptobutyric acid
N-Me-Cys: N-methyl-cysteine
GIn or Q: glutamine
GIu or E: glutamic acid
GIy or G: glycine
lie or I: isoleucine
Leu or L: leucine
des-Leu: deleted Leu Lys or K: lysine
des-Lys: deleted Lys
Met or M: methionine
NIe: norleucine
Pen: penicillamine
Phe or F: phenylalanine
Pro or P: proline
Ser or S: serine
Thr or T: threonine
Tip or W: tryptophan
Tyr or Y: tyrosine
VaI or V: valine
All abbreviations (e.g., Ala) of amino acids in this disclosure stand for the structure of -NR-CR'(R")-CO-, wherein R1 and R" each is, independently, hydrogen or the side chain of an amino acid (e.g., R1 = H and R" = CH3 for alanine) and wherein R = H or CH3, except for
proline, i.e.,
Figure imgf000014_0001
A peptide of this invention is also denoted herein by another format, e.g., (Asn59)hIGF- 1(1 -7O)-OH (SEQ ID NO: 1) or [2-(n-octadecoxyimino)acetyl-Gly1, Asn59]hIGF- 1(1-7O)-OH (SEQ ID NO: 109), with the substituted amino acids from the natural sequence placed between the parentheses or brackets (i.e., Asn for Met at position 59 of the wild-type IGF-I). The range found within the parentheses refers to those amino acids found in the analogue. For example, "IGF-I (4-68)-OH" (SEQ ID NO: 110) indicates that the analogue is comprised of amino acids 4 through 68 which correspond to the peptide sequence for the wild-type IGF-I. "NH2" in "IGF-I (1-7O)-NH2" (SEQ ID NO: 111) indicates that the C- terminus of the peptide is amidated. "IGF-l(l-70)" or "IGF-I (1-7O)-OH" indicates that the C-terminus is the free acid (SEQ ID NO:112).
Certain other abbreviations used herein are defined as follows:
Act: acetonitrile
Boc: tert-butyloxycarbonyl
BSA: bovine serum albumin
DCM: dichloromethane
DIPEA: diisopropylethylamine
DMEM: Dulbecco's Modified Eagle's Medium DMF: dimethylformamide
DTT: dithiothrieitol
ESI: electrospray ionization
FCS: fetal calf serum
Fmoc: 9-fluorenylmethyloxycarbonyl
HBTU: 2-(lH-benzotriazole-l -yl)-l , 1 ,3,3-tetramethyluronium
hexafluorophosphate
HOBt: N-hydroxybenzotriazole
HPLC: high performance liquid chromatography
LC-MS: liquid chromatography mass spectrometry
MALDI-TOF MS: matrix-assisted laser dissorption ionization time-of-flight mass spectroscopy
MPAA: 4-mercaptophenylacetic acid
NMP: N-methylpyrrolidinone
OrBu: O-tert-butyl ester
Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
QC: quality control
*Bu: tert-bnty\
TCA: trichloroacetic acid
TCEP tris-2-carboxyethyl-phosphine
TIS: triisopropylsilane
TFA: trifluoroacetic acid
Tris: 2-amino-2-(hydroxymethyl)-l ,3-propanediol
Trt: trityl
UV spectroscopy: ultraviolet spectroscopy
"Alkyl" refers to a hydrocarbon group containing one or more carbon atoms wherein multiple carbon atoms, if present, are joined by single bonds. Examples of which include, but are not limited to, methyl, ethyl, propyl, and butyl. The alkyl hydrocarbon group may be straight-chain or contain one or more branches or cyclic groups, examples of which include, but are not limited to, isopropyl and tert-butyl.
"Substituted alkyl" refers to an alkyl wherein one or more hydrogen atoms of the hydrocarbon group are replaced with one or more substituents selected from the group consisting of halogen, OH, CN, SH, NH2, NHCH3, NO2, (C1-2) alkyl substituted with 1 to 6 halogens, CF3, OCH3, OCF3, and (CH2)(M-COOH. In different embodiments, 1, 2, 3 or 4 substituents are present. "Aryl" refers to an optionally substituted aromatic group with at least one ring having a conjugated pi-electron system, containing up to three conjugated or fused ring systems. Aryl includes carbocyclic aryl, heterocyclic aryl and biaryl groups. Preferably, the aryl is a 5 or 6 membered ring. Preferred atoms for a heterocyclic aryl are one or more sulfur, oxygen, and/or nitrogen. Examples of aryl include phenyl, 1-naphthyl, 2-naphthyl, indole, quinoline, 2-imidazole, and 9-anthracene. Aryl substituents are selected from the group consisting of - C1-20 alkyl, -Ci-20 alkoxy, halogen, -OH, -CN, -SH, -NH2, -NO2, -CL20 alkyl substituted with halogens, -CF3, -OCF3, and -(CH2)O-20-COOH. In different embodiments the aryl contains O, 1, 2, 3, or 4 substituents.
"Alkyl-aryl" refers to an "alkyl" joined to an "aryl".
PEG: poly(ethylene glycol), which has the structure of
wherein n an integer between 1 and 2,000, and which may be methylated as defined herein mPEG: methylated poly(ethylene glycol), which has the structure of
H,cα J n
wherein n is an integer between 1 and 2,000
1OK PEG: poly(ethylene glycol), which encompasses mPEG as defined herein, with an average molecular weight of about 10 kDa, and which may be either linear or branched
2OK PEG: poly(ethylene glycol), which encompasses mPEG as defined herein, with an average molecular weight of about 20 kDa, and which may be either linear or branched
3OK PEG: poly(ethylene glycol), which encompasses mPEG as defined herein, with an average molecular weight of about 30 kDa, and which may be either linear or branched
4OK PEG: poly(ethylene glycol), which encompasses mPEG as defined herein, with an average molecular weight of about 40 kDa, and which may be either linear or branched
"2-(ethoxyimino(2-carbamate-10K linear PEG))acetyl-Gly" has the structure of:
Figure imgf000017_0001
wherein n is an integer between 1 and 2,000 such that the "10K linear PEG" moiety has the average molecular weight of about 1O kDa
"2-(ethoxyimino(2-carbamate-20K linear PEG))acetyl-Gly" has the structure of:
Figure imgf000017_0002
wherein n is an integer between 1 and 2,000 such that the "2OK linear PEG" moiety has the average molecular weight of about 2O kDa
"2-(ethoxyimino(2-carbamate-20K branched PEG))acetyl-Gly" has the structure of:
Figure imgf000017_0003
wherein n is, independently for each occurrence, an integer between 1 and 2,000 such that the "2OK branched PEG" moiety has the combined average molecular weight of about 20 kDa
"2-(ethoxyimino(2-carbamate-30K linear PEG))acetyl-Gly" has the structure of:
Figure imgf000017_0004
wherein n is an integer between 1 and 2,000 such that the "30K linear PEG" moiety has the average molecular weight of about 30 kDa
"2-(ethoxyimino(2-carbamate-40K branched PEG))acetyl-Gly" has the structure of:
Figure imgf000018_0001
wherein n is, independently for each occurrence, an integer between 1 and 2,000 such that the "4OK branched PEG" moiety has the combined average molecular weight of about 40 kDa
"2-(n-octadecoxyimino)acetyl-Gly" has the structure of:
Figure imgf000018_0002
The PEGs used in the present invention are not restricted to any particular form or molecular weight range. The PEG molecular weight may be between about 500 and about 100,000 Daltons. The term "about" indicates that in preparations of PEGs, some molecules will weigh more and some less than the stated molecular weight; the stated molecular weight refers to the average molecular weight. It is understood that there is some degree of polydispersity associated with polymers such as PEGs. It is preferable to use PEGs with low polydispersity. Normally, a PEG with molecular weight of about 500 to about 60,000 Daltons is used. A specific PEG molecular weight range of the present invention is from about 500 to about 50,000 Daltons. In another specific embodiment, the PEG molecular weight is about 20,000 to about 40,000 Daltons. Other sizes may be used, depending on the desired therapeutic profile (e.g., duration of sustained release desired, the effects, if any, on biological activity, the degree or lack of antigenicity and other known effects of the polyethylene to a therapeutic protein). For example, a PEG according to the present invention may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 Daltons.
Synthetic Procedures
The exemplified analogues of IGF-I of the present invention were prepared by a first step of peptide fragment synthesis, a second step of ligation, a third step of folding, and a fourth step of site-specific pegylation or site-specific lipidation. The following synthetic procedures illustrate how a skilled chemist would be enabled to prepare any one of the exemplified analogues of IGF-I of the present invention.
A) Peptide fragment synthesis of (GIn56. Asn59MGF- 1(48-70 VOH. i.e.. Cvs-Phe- Arg-Ser-Cys- Asp-Leu- Arg-Gln-Leu-Glu-Asn-Tyr-Cys- Ala-Pro-Leu- Lvs-Pro- Ala-Lvs-Ser- AIa-OH (SEO ID NO: 113)
Fmoc-based solid-phase peptide synthesis was used to assemble the titled peptide fragment using microwave assistance on a Liberty Peptide Synthesizer (CEM; Matthews, NC, USA). The first 14-residue fragment, i.e., residues 57-70 of hIGF-1, or the C-terminal acid peptide, was synthesized on a 1.0-mmole scale using Fmoc-Ala-Wang resin (0.72 meq/g). The resulting peptide fragment was then split into four 0.25-mmole batches for elongation and differentiation. A 1.36 g resin sample was placed in a 50-mL conical tube together with 15 mL of a 1:1 solution of DMF and DCM which was loaded into position in the synthesizer. The resin was then transferred to the reaction vessel via the synthesizer's automated process. The standard Liberty protocol for 1.0-mmole scale synthesis was used. The protocol involved removal of the N-terminal Fmoc protecting group by treatment with 20 mL of 20% piperidine containing 0.1M HOBt in DMF. The initial de-protection step of microwave power (45 watts, maximum temperature of 75 C) and nitrogen bubbling (3 seconds on, 7 seconds off) lasted for 30 seconds. The reaction vessel was drained and the resin was washed thoroughly with DMF several times. The next amino acid (Cycle 1) to be added to the growing peptide, (Fmoc- Ser(d3u)-OH) prepared as a 0.2M stock solution in DMF, was then added (15 mL, 3 equivalents). 6.0 mL of 0.45M (3 equivalents) HBTU in DMF was added followed by 3.0 mL of 2M (6 equivalents) DIPEA in NMP. The coupling step was performed using microwave power (20 watts, maximum temperature of 75°C) with nitrogen bubbling at the same rate as in the de-protection step for a period of 5 minutes. The reaction vessel was then drained to waste and the coupling step was repeated.
The coupling protocol for Fmoc-Cys(Trt)-OH was a slightly modified version of the standard protocol. For Cys residues, no microwave power was applied for the first 2 minutes. A 4-minute session of microwave power (20 watts, maximum temperature of 50°C) followed. All amino acids were introduced similarly, employing a double coupling strategy throughout the entire sequence. The synthesis cycles for the titled peptide fragment following the first Ser were as follows: Cycle 2, Fmoc-Lys(Boc)-OH; Cycle 3, Fmoc-Ala-OH; Cycle 4, Fmoc- Pro-OH; Cycle 5, Fmoc-Lys(Boc)-OH; Cycle 6, Fmoc-Leu-OH; Cycle 7, Fmoc-Pro-OH; Cycle 8, Fmoc-Ala-OH; Cycle 9, Fmoc-Cys(Trt)-OH; Cycle 10, Fmoc-Tyr(/Bu)-OH; Cycle 11, Fmoc-Asn(Trt)-OH; Cycle 12, Fmoc-Glu(OfBu)-OH; and Cycle 13, Fmoc-Leu-OH. Once the initial peptide fragment was completed, the resin was transferred back to the 50-mL conical tube using DMF as a solvent. The resin was manually split evenly into four samples which were put into four 50-mL conical tubes which were then put back into the synthesizer. The remaining portion of the titled peptide was synthesized on a 0.25-mmole scale. The protocol used was the same as that used for the larger scale synthesis, however, lesser amounts of reagents were used. Removal of the N-terminal Fmoc protecting group consisted of treatment with a solution containing 10 mL of 20% piperidine and 0.1M HOBt in DMF. The initial de-protection step of microwave power (45 watts, maximum temperature of 75°C) with nitrogen bubbling (3 seconds on, 7 seconds off) lasted for 30 seconds. The reaction vessel was then drained and the resin was washed several times thoroughly with DMF. The next amino acid (Cycle 14), prepared as a 0.2M stock solution in DMF, was then introduced (5.0 mL, 4 equivalents) to the growing peptide (Fmoc-Gln(tBu)-OH). 2.0 mL of a 0.45M solution (4 equivalents) of HBTU in DMF was then added followed by 1.0 mL of a 2M solution (8 equivalents) of DIPEA in NMP.
The coupling protocols for Fmoc-Cys(Trt)-OH and Fmoc-Arg(Pbf)-OH were slightly modified versions of the standard protocol. For the coupling of Cys residues, the microwave power was initially off for the first 2 minutes then turned on for 4 minutes (20 watts, maximum temperature of 5O C). For the coupling of Arg residues, microwave power was not employed in the first coupling, however, a second standard coupling step was required. Cycles 14, 16 and 21 employed a capping procedure which immediately followed the coupling step, which involved adding 7 mL of 0.5M acetic anhydride containing 0.015M HOBt and 2 mL of 2M DIPEA both in NMP while utilizing a multi-step microwave protocol (50 watts for 30 seconds with a maximum temperature of 65 C, then no power for 30 seconds, 50 watts for 30 seconds with a maximum temperature of 65°C, then no power for 30 seconds). The synthesis cycles for the titled peptide fragment after GIn were as follows: Cycle 15, Fmoc-Arg(Pbf)-OH; Cycle 16, Leu-OH; Cycle 17, Fmoc-Asp(O<Bu)-OH; Cycle 18, Fmoc- Cys(Trt)-OH; Cycle 19, Fmoc-Ser(fBu)-OH; Cycle 20, Fmoc-Arg(Pbf)-OH; Cycle 21, Fmoc- Phe-OH; and Cycle 22, Fmoc-Cys(Trt)-OH.
Following completion of the peptide backbone, the N-terminal Fmoc-protecting group was removed and the resin was washed again with DMF. The resin was then transferred back to the 50-mL conical tube using DMF as the transfer solvent.
The resin was transferred into a reaction vessel with a sintered glass frit. The DMF was removed and the resin was washed extensively with DCM. The peptide fragment was cleaved and de-protected by treatment with the following reagent: 5% TIS : 5% water : 90% TFA. The reaction was allowed to proceed for 3 hours at room temperature with constant shaking. The solution was then filtered into a 50-mL conical tube. TFA was reduced by evaporation with nitrogen gas flow. The peptide fragment was precipitated by the addition of 40 mL of cold ethyl ether followed by centrifugation at 3000 rpm for 30 minutes at 4°C within a refrigerated centrifuge (Sorvall Legend RT; Thermo Fisher, San Jose, CA, USA). The resulting pellet was dissolved in 0.1% TFA water before purification by preparative HPCL equipped with a C18 reverse phase column (Luna, 10 μm, 250 x 21.2 mm column) utilizing a gradient of 0-60% acetonitrile (0.1% TFA) over 50 minutes with a flow rate of 10 mL/min. The purified peptide fragment was analyzed by HPLC (Luna C 18, 3 μm, 4.6 x 100 mm column) with a gradient of 5-80% acetonitrile (0.08% TFA) over 30 minutes with a flow rate of 1 mL/min) and by mass spectrometry (LCQ Advantage; Thermo Fisher, San Jose, CA, USA). The peptide fragment was subsequently lyophilized and stored at -50°C for future use.
B) Peptide fragment synthesis of hIGF-iri-47Vthioester. i.e.. Glv-Pro-Glu-Thr- Leu-Cvs-Glv-Ala-Glu-Leu-Val-Asp-Ala-Leu-Gln-Phe-Val-Cvs-Gly-Asp-Arg-Glv- Phe-Tyr-Phe-Asn-Lvs-Pro-Thr-Glv-Tyr-Glv-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr- Glv-πe-Val-Asp-Glu-Cvs-thioester-propionyl-Leu-NH, (SEO ID NO: 114)
The N-terminal peptide fragment, i.e., residues 1-47 of hIGF-1, was assembled using Boc-chemistry based solid-phase peptide synthesis. An ABI 433A peptide synthesizer (Applied Biosystems; Foster City, CA, USA) modified to run the standard FastBoc protocol was utilized for the 0.5-mmole scale synthesis. The reaction vessel containing 0.645 mg of 0.77 meq/g of Tampal Resin was placed on the synthesizer. To swell the resin, DMF was introduced. The ABI FastBoc 0.5 protocol was used to generate the fragment. Each cycle consisted of de-blocking the N-terminal Boc protecting group with neat TFA followed by extensive DMF washing. Pre-packaged 2.0-mmole (4 equivalents) cartridges of each amino acid were then dissolved in 0.40M HBTU and DMF. After complete dissolution of each amino acid, the solution was automatically transferred to the activation vessel. A DIPEA solution (neat) was introduced to the activation vessel and was exposed to the resin for an extended period. The reaction vessel was emptied and the resin was washed with DMF. For Arg/Asn cartridges, an extended activation time was required to ensure solubility. In addition, any amino acid added immediately after the coupling of a GIn residue was washed with DCM both before and after the deblocking protocol. The coupling times were 30 minutes. The following amino acids were used for the titled peptide fragment: Boc-Arg(Tos)- OH, Boc-Asp(cHex)-OH, Boc-Glu(cHex)-OH, Boc-Asn(Xan)-OH, Boc-Cys(4Me-Bzl)-OH, Boc-Lys(ClZ)-OH, Boc-Gln-OH, Boc-Ser(OBzl)-OH, Boc-Thr(OBzl)-OH, and Boc- Tyr(BrZ)-OH.
Following the last coupling cycle, the resin was washed with DCM and dried. The peptide fragment was de-protected and cleaved from the resin using a treatment with 10 mL of hydrogen fluoride and anisole. The reaction was allowed to proceed for 70 minutes at which point the hydrogen fluoride was blown off with a stream of nitrogen. The residue was washed with ether and then the peptide was dissolved in 10-15 ml of TFA. The peptide fragment was precipitated by filtering the TFA into 40 mL of cold ethyl ether followed by centrifugation at 3000 rpm for 30 minutes at 4°C within a refrigerated centrifuge (Sorvall Legend RT; Thermo Fisher, San Jose, CA, USA). The resulting pellet was dissolved in 0.1% TFA water and was purified by preparative HPLC equipped with a Cl 8 reverse phase column (Luna, 10 μm, 250 x 21.2 mm column) utilizing a gradient of 20-40% acetonitrile (0.1% TFA) over 120 minutes with a flow rate of 10 mL/min. The purified peptide fragment was analyzed by HPLC (Luna C18, 3 μm, 4.6 x 100 mm column) with a gradient of 5-80% acetonitrile (0.08% TFA) for 30 minutes with a flow rate of 1 mL/min and by mass spectrometry (LCQ Advantage; Thermo Fisher, San Jose, CA, USA). The peptide fragment was subsequently lyophilized and stored at -50°C for future use.
C) General ligation procedure
Full length hIGF-1 analogues were constructed using the chemical ligation method that naturally occurs between an N-terminal thioester fragment, e.g., hIGF-l(l-47)-S- (CH2)2C(O)-Leu-NH2 (SEQ ID NO: 114), and a C-terminal fragment, e.g., (GIn56, Asn59)hIGF-l (48-7O)-OH (SEQ ID NO: 113), which contains a cysteine residue at its N- terminus.
To commence the process for the titled peptide, 5.5 mg of the C-terminal hIGF-1 fragment was dissolved in 0.5 mL of ligation buffer (20OmM sodium phosphate, pH 8.5, 6M guanidine hydrochloride) in a 1.5-mL eppendorf tube. To this solution, 100 μL of a TCEP solution (40 mg/mL) was added and the mixture was vortexed. The mixture was transferred to a second eppendorf tube containing 6.5 mg of the N-terminal hIGF-1 thioester fragment. The reactants were mixed thoroughly. A small sample (5 μL) was removed and analyzed by LC-MS (LCQ Deca XP; Thermo Fisher, San Jose, CA, USA). To the reaction mixture, 100 μL of a MPAA solution (20 mg/mL) was added followed by mixing. Samples (5 μL) were periodically extracted in order to follow the progress of the reaction using LC-MS. After approximately 3.5 hours when the reaction was near completion, the mixture was quenched and diluted by the addition of 9.5 mL of 0.1% TFA water. The ligation product was purified by SemiPrep-HPLC (Vydac 218TP101510, Cl 8, 10-15 μm, 10 x 250 mm) with a gradient of 5-80% acetonitrile (0.1% TFA) over 40 minutes with a flow rate of 5 mL/min. The product peak was lyophilized and stored at -50°C. The mass of the unfolded ligation product was determined by physical measurement. D) Folding procedure 1 (glutathione redox pair) for Example 87. i.e.. (VaI68MGFCl-TOVQH (SEO ID NO^
The protein, prepared by the ligation process of step C) as described above, was dissolved in ligation buffer (20OmM sodium phosphate, pH 8.5, 6M guanidine hydrochloride) to a concentration of 1 mg/mL. Folding buffer (10OmM Tris, pH 8.5, ImM oxidized glutathione, 1OmM reduced glutathione) was then added to bring the final protein concentration to 0.25 mg/mL. The folding process was allowed to occur over 3 hours. Afterwards, the reaction was quenched by the drop-wise addition of TFA until the reaction mixture reached pH < 3. The product was then purified by SemiPrep-HPLC (Vydac 218TP101510, C 18, 10-15 μm, 10 x 250 mm column) with a gradient of 5-60% acetonitrile (0.1% TFA) over 40 minutes with a flow rate of 5mL/min. The product was lyophilized. The protein content was determined by re-dissolving the product in 0.1% TFA water then measuring the absorbance at 280 nm (NanoDrop NDlOOO Spectrophotometer). The protein was then analyzed for QC (HPLC and MS).
E) Folding procedure 2 (cysteine/cystine redox pair) for Example 55, i.e., (Glu'MGF(l-70VOH (SEO ID NO:47)
The protein, prepared by the ligation process of step C) as described above, was dissolved in folding buffer A (2OmM Tris, pH 7.8, 5M guanidine hydrochloride) to a concentration of 10 mg/mL. Folding buffer B (2OmM Tris, pH 7.8, ImM cystine, 8mM cysteine) was then added to bring the final protein concentration to 1 mg/mL. The folding process was allowed to occur over 3 hours. Afterwards, the reaction was quenched by the drop-wise addition of TFA until the reaction mixture reached pH < 3. The product was then purified by SemiPrep-HPLC (Vydac 218TP101510, C18, 10-15 μm, 10 x 250 mm column) with a gradient of 5-60% acetonitrile (0.1% TFA) over 40 minutes with a flow rate of 5 mL/min. The product was lyophilized. The protein content was determined by re-dissolving the product in 0.1% TFA water then measuring the absorbance at 280 nm (NanoDrop NDlOOO Spectrophotometer). The protein was then analyzed for QC (HPLC and MS).
F) Folding procedure 3 (copper/oxygen redox pair) for Example 56. i.e., (GIu2MGF(I -70VOH (SEO ID NO:48)
The protein, prepared by the ligation process of step C) as described above, was dissolved in folding buffer (2OmM glycine, pH 10.5, 2M Urea, IM sodium chloride, 20% ethanol) and transferred to a 15-mL conical tube. A 250μM copper (II) bromide solution was added until the final copper concentration of the reaction was 0.5μM. The reaction was shaken vigorously for at least 6 hours occasionally stopping to open the vessel in order to exchange the air within the headspace. The reaction was quenched and diluted by the addition of 10% TFA water to bring the final volume to 10 mL. The product was purified by SemiPrep-HPLC (Vydac 218TP101510, C 18, 10-15 μm, 10 x 250 mm column) with a gradient of 0-60% acetonitrile (0.1% TFA) over 40 minutes with a flow rate of 5 mL/min. The product was lyophilized. The protein content was determined by re-dissolving the product in 0.1% TFA water then measuring the absorbance at 280 nm (NanoDrop NDlOOO Spectrophotometer). The protein was then analyzed for QC (HPLC and MS).
G) Oxidation procedure for the formation of (glvoxylyl-Glv1. Asn59)hIGF-l(l-70)- OH (SEO ID NO: 1151 from (Ser-Glv1. Asn59)hIGF-l(l-70VOH (SEO ID NOil lό)
The mass of the folded hIGF-1 analogue was determined by absorbance at 280 nm in 0.1% TFA water (NanoDrop NDlOOO Spectrophotometer). The protein, prepared by the folding process of any one of steps D)-F) as described above, was re-dissolved in 5OmM imidazole buffer (pH 7.0) to a final concentration of 2 mg/mL (2.66 x 10"4M). Sodium periodinate (NaIO4) (4 equivalents) dissolved in an imidazole buffer was added and the resulting solution was gently mixed. The reaction was allowed to proceed at room temperature without further agitation. After 5 minutes, the reaction was quenched with the addition of 10 equivalents of ethylene glycol. The mixture was allowed to stand for 15 minutes at room temperature. The mixture was diluted with 0.1% TFA water to a final volume of 10 mL. The product was then purified by SemiPrep-HPLC (Vydac 218TP101510, C18, 10-15 μm, 10 x 250 mm column) with a gradient of 5-60% acetonitrile (0.1% TFA) over 40 minutes with a flow rate of 5 mL/min. The product was then lyophilized and stored at - 50°C until needed.
H) Synthetic procedure for Example 27. i.e.. (β-Me-Cvs47. LeU59MGF- 1(1-70VOH (SEO ID NO: 19)
The titled protein was assembled through native chemical ligation from hIGF(l-46)- thio-propionyl-Leu-NH2 (SEQ ID NO: 117) and the C-terminal fragment, i.e., (β-Me-Cys47, Leu59)hIGF- 1(47-70) (SEQ ID NO: 118). The protein thioester (7.4 mg, 1.45 μmoles) and the C-terminal fragment (3.8 mg, 1.38 μmoles) were dissolved in ligation buffer (6M guanidine hydrochloride in 20OmM sodium phosphate, pH 8.5, 400 μL) and TCEP (80 μL, 40 mg/mL, pH 7). An MPAA catalyst was added (80 μL, 20 mg/mL, pH 7). The reaction progress was monitored on a LCQ Deca XP LC-MS (Thermo Finnigan) with a Luna C 18(2) column (5 μm, 4.6 x 100 mm) with a gradient of 5-80% acetonitrile (0.1% TFA) for 30 minutes. The reaction was quenched to a dilution of 1 : 10 with dH2O, 0.1% TFA (v/v). The crude mixture was centrifuged and passed through a 1.0-μm glass filter to remove any MPAA precipitate. The full length protein was purified using a 5-60% B linear gradient for 40 minutes with a flow rate of 5 mL/min on a Vydac C18 (10 μm, 10 x 250 mm). The protein was quantitated by UV spectroscopy (NanoDrop NDlOOO Spectrophotometer) and lyophilized for future use.
The stored protein (1.8 mg, 235 nmoles) was dissolved in a 20OmM H2PO4 ", 6M guanidinium-HCl solution having pH 8.5 to a concentration of 1.0 mg/mL. Folding buffer (10OmM Tris, 1OmM glutathione, ImM oxidized glutathione at pH 8.5) was added to the solution until a final protein concentration of 250 μg/mL was achieved. The mixture was allowed to incubate at room temperature while being monitored by HPLC. Once equilibrium was reached (as visualized by a stable HPLC profile), the reaction was quenched by stirring in either acetic acid or TFA to bring the solution to pH 3. The solution was then passed through a 1.0-μm glass filter and further purified on a semi-preparative column.
The folded protein was purified using a 5-60% B linear gradient for 40 minutes with a flow rate of 5 mL/min. The protein was quantitated by UV (NanoDrop NDlOOO Spectrophotometer) and lyophilized. Approximately 92μg of purified product was obtained, representing a yield of 5%. The mass of the protein was verified on a Finnigan LCQ Advantage MAX MS.
I) Synthetic procedure for Example 36. i.e.. (N-Me-Cvs48. NIe59MGF- U 1-70 V OH (SEO ID NO:28)
The titled protein was assembled utilizing native chemical ligation using MGF-I(I- 47)-thio-propionyl-Leu-NH2 (SEQ ID NO: 114) and the C-terminal fragment, i.e., (N-Me- Cys48, Nle59)hIGF-l(48-70) (SEQ ID NO: 119). The protein thioester (4.3 mg, 824 nmoles) and the C-terminal fragment (2.1 mg, 790 nmoles) were dissolved in ligation buffer (400 μL, 6M guanidine hydrochloride in 20OmM sodium phosphate, pH 8.5) and TCEP (80 μL, 40 mg/mL, pH 7). An MPAA catalyst was added (80 μL, 20 mg/mL, pH 7). The reaction progress was monitored using a Finnigan LCQ Deca XP LC-MS with a Luna C 18(2) column (5 μm, 4.6 x 100 mm) having a gradient of 5-80% acetonitrile (0.1% TFA) for 30 minutes. The reaction was quenched to a dilution of 1: 10 with dH2O, 0.1% TFA (v/v). The crude mixture was centrifuged and passed through a 1.0 μm glass filter to remove any MPAA precipitate. The full length protein was purified using a Vydac Cl 8 (lOμm, 10 x 250 mm) with a 5-60% B linear gradient for 40 minutes with a flow rate of 5 mL/min. The protein was quantitated by UV (NanoDrop NDlOOO Spectrophotometer) and lyophilized for future use.
The stored protein was dissolved using a 20OmM H2PO4 ", 6M guanidinium-HCl solution (pH 8.5) until a concentration of 1.0 mg/mL was achieved. Folding buffer (10OmM Tris, 1OmM glutathione, ImM oxidized glutathione, pH 8.5) was added to the solution until a final protein concentration of 250 μg/mL was achieved. The mixture was allowed to incubate at room temperature while being monitored by HPLC. Once equilibrium was reached (as visualized by a stable HPLC profile), the reaction was quenched with either acetic acid or TFA to pH 3. The solution was purified using first a 1.0-μm glass filter and then a semi- preparative column.
The folded protein was purified using a 5-60% B linear gradient with a flow rate of 5 mL/min for 40 minutes. The protein was quantitated by UV (NanoDrop NDlOOO Spectrophotometer) and lyophilized. Approximately 0.415 mg of purified product was obtained, representing a yield of 10.6%. The mass of the protein was verified on a Finnigan LCQ Advantage MAX MS.
J) General PEGylation procedure
Four equivalents of aminooxy-PEG (NOF, Tokyo, Japan) were weighted into a 1.5 mL eppendorf tube which was dissolved in PEGylation buffer (10OmM sodium acetate, 10OmM aniline, 10% ethanol, pH 4.6) until a final protein concentration of 2 mg/mL was achieved. The PEG solution was then transferred to a tube containing a measured amount of (glyoxylyl-Gly1, Asn59)hIGF-l(l-70)-OH. The reaction was allowed to run overnight at ambient temperature. The reaction was diluted with 0.1% TFA water then purified by SemiPrep-HPLC (Vydac 218TP101510, C18, 10-15 μm, 10 x 250 mm) with a gradient of 5- 95% acetonitrile (0.1% TFA) over 40 minutes at a flow rate of 5 mL/min. The product was lyophilized, weighed, and then analyzed for QC (HPLC, MALDI-TOF MS).
K) General lipidation procedure
The final protein concentration (2 mg/mL) required for this procedure was equivalent to that used for the PEGylation procedure described above in step J). Isopropyl alcohol (approximately 15%) was used as a co-solvent. Approximately 2.2 equivalents of aminooxy lipid were pre-dissolved. The protein was dissolved in a 10OmM sodium acetate 10OmM aniline buffer having pH 4.6.
L) PEGylation procedure for Example 115. i.e., [2-(ethoxyimmo(2-carbamate-20K branched PEGΪIacetyl-Glv1. Asn59lhIGF-l(l-70VOH (SEO ID NO: 106^)
Approximately 13 mg of unfolded (Ser-Gly1, Asn59)hIGF- 1(1 -7O)-OH (SEQ ID NO:116) was weighed in a 1.5-mL eppendorf tube. The protein was transferred into a 15 mL conical tube and dissolved in 13 mL of folding buffer. To this, 26 μL of a 250μM copper (II) bromide solution was added. The reaction was allowed to proceed for 6 hours with vigorous shaking with the occasional refreshing of the headspace air. The reaction was quenched with 13 mL of 10% TFA water. The folded protein was purified using a SemiPrep-HPLC (Vydac 218TP101510, C18, 10-15 μm, 10 x 250 mm column) with a gradient of 0-60% acetonitrile (0.1% TFA) and a flow rate of 5 mL/min for a 40-minute period. The product was then lyophilized yielding 4.1 mg of folded (Ser 1, Asn59)hIGF-l (0-7O)-OH.
The folded IGF-I analogue was then dissolved in 2 mL of imidazole buffer. Approximately 37 μL of a 13 mg/mL solution of sodium periodinate was added. The reaction was allowed to proceed for 5 minutes, and was quenched thereafter by the addition of 85 μL of ethylene glycol. The reaction was allowed to rest for 15 minutes. Approximately 6 mL of 0.1% TFA water was used to dilute the solution. A SemiPrep-HPLC (Vydac 218TP101510, C18, 10-15 μm, 10 x 250 mm column) with a gradient of 5-60% acetonitrile (0.1% TFA) and a flow rate of 5 mL/min was employed for 40 minutes to purify the product which was analyzed by HPLC and MS. The remaining peptide was lyophilized, re-dissolved in 4 mL of 0.1% TFA water, divided into four equal 1.5-mL eppendorf tubes, and lyophilized again.
Approximately 12.4 mg of 2OK branched PEG oxyamine (NOF, Tokyo, Japan) was weighed in a 1.5-mL eppendorf tube. 0.575 mL of PEGylation buffer (10OmM sodium acetate, 10OmM aniline, 10% ethanol, pH 4.6) was used to dissolve the peptide and the dissolved peptide solution was then transferred with an aliquot of (glyoxylyl-Gly1, Asn59)hIGF-l(l -7O)-OH (1.15 mg by weight) to a new vessel. The reaction was allowed to proceed overnight. The reaction mixture was diluted with 9 mL of 0.1% TFA water. The sample was purified using a SemiPrep-HPLC (Vydac 218TP101510, C18, 10-15 μm, 10 x 250 mm) with a gradient of 5-95% acetonitrile (0.1% TFA) and a flow rate of 5 mL/min for 40 minutes. The product was lyophilized, weighed, and analyzed for QC (HPLC, MALDI- TOF MS).
M) Lipidation procedure for Example 117. i.e.. f2-(n-octadecoxyimino)acetyl-Gly1. Asn591hIGF- If 1-7O)-OH (SEO ID NO: 109)
A 3.2 mg portion of octadecyloxyamine, i.e., CH3 (CH2)i70NH2, was dissolved in 1.6 mL of isopropyl alcohol. Concurrently, 0.8 mg of (glyoxylyl-Gly1, Asn59)hIGF-l(l-70)- OH (SEQ ID NO: 115) was dissolved in 184 μL of 10OmM sodium acetate / 10OmM aniline buffer having pH 4.6. Approximately 32 μL of an aminooxy lipid/isopropyl alcohol solution was transferred to the protein solution. The reaction was monitored by HPLC and quenched/diluted after 30 minutes with 0.1% TFA water to a volume of 10 mL. The reaction was purified using a SemiPrep-HPLC (Vydac 218TP101510, C18, 10-15 μm, 10 x 250 mm) with a gradient of 5-80% acetonitrile (0.1% TFA) and with a flow rate of 5mL/min for 40 minutes. The product was lyophilized. The protein content was determined by re-dissolving the product in 0.1% TFA water then measuring the absorbance at 280 nm (NanoDrop NDlOOO Spectrophotometer). It was also analyzed for QC (HPLC, MS).
Other peptides of the invention can be prepared by a person of ordinary skill in the art using synthetic procedures analogous to those disclosed in the foregoing examples. Physical data for the compounds exemplified herein are given in Table 1.
Table 1
Figure imgf000028_0001
Functional Assays
A) In Vitro IGF-I Receptor Binding Assay
Membranes were prepared for radioligand binding studies by homogenization of human MCF-7 cells expressing the native IGF-I receptor in 20 ml of ice-cold 5OmM Tris- HCl with a Brinkman Polytron (Westbury, NY, USA) (setting 6, 15 sec). The homogenates were washed twice by centrifugation (39,000 g / 10 minutes) and the final pellets were resuspended in 5OmM Tris-HCl containing 2.5mM MgCl2 and 0.1% BSA.
For the assay, aliquots were incubated with 0.05nM [125I]IGF-I. Unlabeled competing test peptides were sometimes included. The final assay volume was 0.25 ml. After a 120-minute incubation (20 0C) period, the bound [125I]IGF-I (-2000 Ci/mmol, Perkin Elmer Life Sciences, Boston, MA, USA) was separated from the free radioactive particles by centrifugation at 3000 rpm for 10 minutes. The supernatant was decanted and the radioactive particles trapped in the pellet was counted by gamma spectrometry (Wallac LKB, Gaithersburg, MD, USA). Specific binding was defined as the total [125I]IGF-I bound minus that bound in the presence of 10OnM IGF-I .
In vitro IGF-I receptor binding data (i.e. Ki values) for the compounds exemplified herein are given in Table 2. B) In Vitro IGF-I Bioactivitv Assay
Mouse 3T3/R cells (obtained from Dr. E. Rozengurt at UCLA in Los Angeles, CA, USA) were cultured on a 24-well plate (DMEM + 10% FCS) and maintained for 2 days in culture.
For the assay, the media was removed and washed once with serum-free DMEM. The serum was then starved for 24 hours. After starvation, [3H]thymidine and IGF-I peptides were added. The cells were then incubated for 24 hours at 37 0C.
At the end of the incubation, the media was aspirated. The cells were then washed with an ice-cold 0.9% NaCl solution. An ice-cold 5% TCA solution was then added for a 30- minute incubation at 4 0C. The TCA was aspirated and the wells were incubated with 95% ethanol for 4 hours. The media was then transferred to a liquid scintillation vial for radioactivity counting.
In vitro IGF-I bioactivity data (i.e., EC50 values) for the compounds exemplified herein are also given in Table 2.
C) In Vitro Screening of IGF-I Peptides for Insulin Receptor Cross Reactivity in U2QS Cells
U2OS cells (Catalog # 93-0466C3, DiscoveRX Corporation, Fremont, CA, USA) were plated at 6 x 105 cells/mL in a 96-well poly-D-lysine plate 16 hours prior to assay in serum-free assay media. The wild-type insulin (Catalog # 10908, Sigma, St. Louis, MO, USA), the wild-type IGF-I (Increlex®, Tercica, Inc., Brisbane, CA, USA ), or a test IGF-I peptide disclosed in the instant application was added at a dose range of lOμM (micromolar) to 0.15nm (nanomolar), and incubated for 3 hours at 370C with 5% CO2. PathHunter™ reagent (Catalog # 93-001, DiscoveRX) was prepared according to manufacturer's instructions, and added to each well. Plates were incubated at room temperature for 1 hour. Luminescence was read on an Envision 2104 multi-label plate reader (PerkinElmer, Inc., Waltham, MA, USA). Activity of each test peptide was analyzed and reported as
maximum/minimum (max/min) values.
In vitro insulin receptor cross reactivity data (i.e., max/min values) for the compounds exemplified herein are also given in Table 2.
Many of the compounds exemplified herein were found to be significantly more potent than the wild-type IGF-I which has Ki value of 4.59 nM, EC50 value of 3.75 nM, and the Max/Min value of 2.1. It was also found that pegylated and lipidated IGF-I analogues retain receptor binding affinity. Table 2
Figure imgf000030_0001
Administration
The analogues of IGF-I of this invention can be provided in the form of pharmaceutically acceptable salts. Examples of such salts include, but are not limited to, those formed with organic acids (e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, methanesulfonic, toluenesulfonic, or pamoic acid), inorganic acids (e.g., hydrochloric acid, sulfuric acid, or phosphoric acid), and polymeric acids (e.g., tannic acid, carboxymethyl cellulose, polylactic, polyglycolic, or copolymers of polylactic-glycolic acids).
A typical method of making a salt of a peptide of the present invention is well known in the art and can be accomplished by standard methods of salt exchange. For instance, the TFA salt of a peptide of the present invention (the TFA salt results from the purification of the peptide by using preparative HPLC eluting with TFA containing buffer solutions) was converted into another salt, such as an acetate salt, by dissolving the peptide in a small amount of 0.25 N acetic acid aqueous solution. The resulting solution is applied to a SemiPrep HPLC column (Zorbax, 300 SB, C-8). The column is eluted with (1) 0.1N ammonium acetate aqueous solution for 0.5 hours, (2) 0.25N acetic acid aqueous solution for 0.5 hours, and (3) a linear gradient (20% to 100% of solution B over 30 min) at a flow rate of 4 ml/min (solution A is a 0.25N acetic acid aqueous solution, and solution B is a 0.25N acetic acid in acetonitrile/water, with a 80:20 ratio). The fractions containing the peptide are collected and lyophilized to dryness.
The dosage of active ingredient in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained. The selected dosage depends on the desired therapeutic effect, on the route of administration, and on the duration of the treatment. Dosing is easily determined by the skilled, competent medical practitioner.
The compounds of this invention can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual, or topical routes of administration, and can be formulated with pharmaceutically acceptable carriers to provide dosage forms appropriate for each route of administration.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch. Such dosage forms can also comprise, as is normal practice, additional substances other than such inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
Liquid dosage forms for oral administration include, without limitation, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, and the like, containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents.
Preparations according to this invention for parenteral administration include, without limitation, sterile aqueous or non-aqueous solutions, suspensions, emulsions, and the like. Examples of non-aqueous solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Preparations may be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, and/or by heating the compositions. Pharmaceutical compositions containing the novel IGF-I analogues described herein can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water or some other sterile injectable medium immediately before use.
Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as coca butter or a suppository wax.
Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.
Further, a compound of this invention can be administered in a sustained release composition such as those described in the following patents and patent applications. U.S. Patent No. 5,672,659 teaches sustained release compositions comprising a bioactive agent and a polyester. U.S. Patent No. 5,595,760 teaches sustained release compositions comprising a bioactive agent in a gelable form. U.S. Patent No. 5,821,221 teaches polymeric sustained release compositions comprising a bioactive agent and chitosan. U.S. Patent No.5,916,883 teaches sustained release compositions comprising a bioactive agent and cyclodextrin. PCT publication WO99/38536 teaches absorbable sustained release compositions of a bioactive agent. PCT publication WO00/04916 teaches a process for making microparticles comprising a therapeutic agent such as a peptide in an oil-in-water process. PCT publication WO00/09166 teaches complexes comprising a therapeutic agent such as a peptide and a phosphorylated polymer. PCT publication WO00/25826 teaches complexes comprising a therapeutic agent such as a peptide and a polymer bearing a non-polymerizable lactone.
Further, the invention disclosed in U.S. Pat. No. 7,258,864 features a method for treating a subject having insulin-like growth factor- 1 deficiency (IGFD) comprising administering to a human pediatric subject an effective amount of the unmodified IGF-I wherein the subject is characterized as follows: a) at the time of treatment or prior to initial treatment with IGF-I, has or had a height at least about 2 standard deviations (SD) below a normal mean for a corresponding age and gender, and b) at the time of treatment or prior to initial treatment with IGF-I, has or had a blood level of IGF-I at least about -1 SD below normal mean levels wherein the subject does not have Laron syndrome or partial growth hormone insensitivity syndrome, and wherein said administering is effective to treat IGFD in the subject.
Similarly, the invention disclosed in WO 2006/130769 features a method for treating a subject having idiopathic short stature (ISS) comprising administering to a human pediatric subject suffering from ISS characterized by partial endogenous growth hormone activity or signaling, an amount of IGF-I effective to promote growth in the subject, wherein the subject is further characterized as follows: a) at the time of treatment or prior to initial treatment with IGF-I, has or had a height at least about 2.0 standard deviations (SD) below the normal mean height for a subject of the same age and gender, and b) has blood levels of GH and IGF-I that are at least normal for a subject of the same age and gender.
Further, the novel analogues disclosed herein are able to be administered alone or in combination with another therapeutic agent as determined by a skilled medical practitioner.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, all publications, patent applications, patents and other references mentioned herein are hereby incorporated by reference, each in its entirety.

Claims

CLAIMS What is claimed is:
1. An amino-terminal pegylated IGF-I conjugate of formula (I),
W-B
(I)
wherein,
O
/X^ IT
PEG-linker Nk^ -
W is wherein X is O or NH; and
B is the wild-type IGF-I or an analogue of IGF-I;
or a pharmaceutically acceptable salt thereof.
2. An amino-terminal pegylated IGF-I conjugate according to claim 1, wherein said linker is alkyl, aryl, alkyl-aryl, substituted alkyl, substituted aryl, or substituted alkyl-aryl; or a pharmaceutically acceptable salt thereof.
3. An amino-terminal pegylated IGF-I conjugate according to claim 2, wherein
said linker is
Figure imgf000033_0001
wherein p is an integer from 1 to 10 inclusive;
or a pharmaceutically acceptable salt thereof.
4. An amino-terminal pegylated IGF-I conjugate according to claim 3, wherein said p is 1; or a pharmaceutically acceptable salt thereof.
5. An amino-terminal pegylated IGF-I conjugate according to any one of claims 1 -4, wherein said PEG is linear; or a pharmaceutically acceptable salt thereof.
6. An amino-terminal pegylated IGF-I conjugate according to claim 5,
Figure imgf000033_0002
wherein said is
Figure imgf000034_0001
or a pharmaceutically acceptable salt thereof.
7. An amino-terminal pegylated IGF-I conjugate according to claim 6, wherein said mPEG has average molecular weight of from about 1 to about 50 kDa; or a pharmaceutically acceptable salt thereof.
8. An amino-terminal pegylated IGF-I conjugate according to claim 7, wherein said mPEG has average molecular weight of from about 5 to about 40 kDa; or a pharmaceutically acceptable salt thereof.
9. An amino-terminal pegylated IGF-I conjugate according to claim 8, wherein said mPEG has average molecular weight of about 5, 10, 20, 30, or 40 kDa; or a pharmaceutically acceptable salt thereof.
10. An amino-terminal pegylated IGF-I conjugate according to claim 9, wherein said mPEG has average molecular weight of about 10, 20, or 30 kDa; or a pharmaceutically acceptable salt thereof.
11. An amino-terminal pegylated IGF-I conjugate according to any one of claims 1-4, wherein said PEG is branched; or a pharmaceutically acceptable salt thereof.
12. An amino-terminal pegylated IGF-I conjugate according to claim 11, wherein said
Figure imgf000034_0002
or a pharmaceutically acceptable salt thereof.
13. An amino-terminal pegylated IGF-I conjugate according to claim 12, wherein said mPEG has average molecular weight of from about 0.5 to about 30 kDa; or a pharmaceutically acceptable salt thereof.
14. An amino-terminal pegylated IGF-I conjugate according to claim 13, wherein said mPEG has average molecular weight of from about 1 to about 20 kDa; or a pharmaceutically acceptable salt thereof.
15. An amino-terminal pegylated IGF-I conjugate according to claim 14, wherein said mPEG has average molecular weight of from about 5 to about 20 kDa; or a pharmaceutically acceptable salt thereof.
16. An amino-terminal pegylated IGF-I conjugate according to claim 15, wherein said mPEG has average molecular weight of about 10 or 20 kDa; or a pharmaceutically acceptable salt thereof.
17. An amino-terminal pegylated IGF-I conjugate according to any one of claims 1-16, wherein said analogue of IGF-I has formula (LA),
W-A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-Au-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21- A22-A23-A24-A25-A26-A27-A28-A29-A30-A31-A32-A33-A34-A35-A36-A37-A38-A39-A40-A41-A42-A43- A44-A45-A46-A47-A48-A49-A50-A5l-A52-A53-A54-A55-A56-A57-A58-A59-A60-A61-A62-A63-A64-A65- A66-A67-A68-A69-A70-A7I-R1,
(IA)
wherein:
A1 is GIy, Ala, Asn, Asp, GIn, GIu, or deleted;
A2 is Pro, Ala, Arg, Asp, GIn, GIu, Lys, or deleted;
A3 is GIu, Ala, Asp, GIn, or deleted;
A4 is Thr, Ala, Asn, Asp, GIn, GIu, Ser;
A5 is Leu, Ace, Ala, lie, or VaI;
A6 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A7 is GIy, Ala, Asn, Asp, GIn or GIu;
A8 is Ala, Arg, Asn, Asp, GIn, GIu, or Lys;
A9 is GIu, Ala, Asp, or GIn;
A10 is Leu, Ace, Ala, Be, or VaI;
A11 is VaI, Ala, He, or Leu; A12 is Asp, Ala, Arg, Asn, GIn, GIu, or Lys;
A13 is Ala, Asn, Asp, GIn, GIu, He, Leu, or VaI;
A14 is Leu, Ace, Ala, lie, or VaI;
A15 is GIn, Ala, Asn, Asp, or GIu;
A16 is Phe, Ala, Asn, Asp, GIn, GIu, Trp, or Tyr;
A17 is VaI, Ala, lie or Leu;
A18 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A19 is GIy, Ala, Asn, Asp, GIn, or GIu;
A20 is Asp, Ala, Asn, GIn, or GIu;
A21 is Arg, Ala, Asn, Asp, GIn, GIu, or Lys;
A22 is GIy, Ala, Asn, Asp, GIn, or GIu;
A23 is Phe, Ala, Trp, or Tyr;
A24 is Tyr, Ala, Phe, or Trp;
A25 is Phe, Ala, Trp, or Tyr;
A26 is Asn, Ala, Asp, GIn, GIu, Ser, or Thr;
A27 is Lys, Ala, Arg, Asn, Asp, GIn, GIu, or Pro;
A28 is Pro, Ala, Arg, or Lys;
A29 is Thr, Ala, Asn, Asp, GIn, GIu, or Ser;
A30 is GIy, Ala, Asn, Asp, GIn, or GIu;
A31 is Tyr, Ala, Phe, or Trp;
A32 is GIy, Ala, Asn, Asp, GIn, or GIu;
A33 is Ser, Ala, Thr, or VaI;
A34 is Ser, Ala, Asn, Asp, GIn, GIu, or Thr;
A35 is Ser, Ala, Asn, Asp, GIn, GIu, or Thr;
A36 is Arg, Ala, Asn, Asp, GIn, GIu, or Lys;
A37 is Arg, Ala, Asn, Asp, GIn, GIu, or Lys;
A38 is Ala, Asn, Asp, GIn, or GIu;
A39 is Pro, Ala, Arg, or GIu;
A40 is GIn, Ala, Asn, Asp, or GIu;
A41 is Thr, Ala, Asn, Asp, GIn, GIu, or Ser;
A42 is GIy, Ala, Arg, Asn, Asp, GIn, GIu, or Lys;
A43 is He, Ala, Arg, Asn, Asp, GIn, GIu, or Lys;
A44 is VaI, Ala, Arg, Asn, Asp, GIn, GIu, lie, Leu, or Lys;
A45 is Asp, Ala, Arg, Asn, GIn, GIu, or Lys;
A46 is GIu, Ala, Arg, Asn, Asp, GIn, or Lys; A47 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A48 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A49 is Phe, Ala, Arg, lie, Leu, Lys, Ser, Thr, Tip, Tyr, or VaI;
A50 is Arg, Ala, Lys, Ser, or Thr;
A51 is Ser, Aib, Ala, Arg, Lys, or Thr;
A52 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A53 is Asp, Ala, Arg, Asn, GIn, GIu, Lys, Ser, or Thr;
A54 is Leu, Ace, Ala, Arg, He, Phe, or VaI;
A55 is Arg, Ala, lie, Leu, Lys, Phe, Trp, Tyr, or VaI;
A56 is Arg, Ala, Asn, Asp, GIn, GIu, or Lys;
A57 is Leu, Ace, Ala, lie, Phe, or VaI;
A58 is GIu, Ace, Ala, Arg, Asn, Asp, GIn, or Lys;
A59 is Met, Ace, Ala, Arg, Asn, Asp, GIn, GIu, lie, Leu, Lys, NIe, Ser, D-Ser, Thr, Trp, Tyr, or VaI;
A60 is Tyr, Ala, Arg, lie, Phe, or Trp;
A61 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A62 is Ala, Arg, Asn, Asp, GIn, GIu, lie, Leu, or VaI;
A63 is Pro, D-Pro, Ala, Ser, Thr, or deleted;
A64 is Leu, D-Leu, des-Leu, Ala, Arg, Ee, Phe, VaI, or deleted;
A65 is Lys, D-Lys, des-Lys, Ala, Arg, lie, Leu, VaI, or deleted;
A66 is Pro, D-Pro, Ala, or deleted;
A67 is Ala, D-AIa, Aib, Arg, or deleted;
A68 is Lys, D-Lys, Ala, Arg, lie, Leu, VaI, or deleted;
A69 is Ser, D-Ser, Aib, Ala, Arg, Thr, or deleted;
A70 is Ala, D-AIa, Asn, Asp, GIn, GIu, or deleted;
A71 is Asn, Ala, Asp, GIn, GIu, Lys, Ser, Thr, or deleted; and
R1 is OH or NH2;
provided that the side-chains of residue pairs A6 and A48, A47 and A52, and A18 and A61, each form a disulfide bond; and
further provided that when A59 is either Leu, lie, NIe, Thr, or VaI, then the analogue contains at least one additional amino acid substitution or addition;
or a pharmaceutically acceptable salt thereof.
18. An amino-terminal pegylated IGF-I conjugate according to claim 17, wherein
A1 is GIy, GIu, or deleted;
A2 is Pro, GIu, Lys, or deleted;
A3 is GIu or deleted;
A4 is Thr or GIu;
A5 is Leu;
A6 is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A7 is GIy or GIu;
A8 is Ala, Arg or GIu;
A9 is GIu;
A10 is Leu;
A11 is VaI or Leu;
A12 is Asp;
A13 is Ala or GIu;
A14 is Leu;
A15 is GIn or GIu;
A16 is Phe or GIu;
A17 is VaI or Leu;
A18 is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A19 is GIy or GIu;
A20 is Asp or GIu;
A21 is Arg or GIu;
A22 is GIy or GIu;
A23 is Phe;
A24 is Tyr;
A25 is Phe;
A26 is Asn, GIu, or Thr;
A27 is Lys, Arg, GIu, or Pro;
A28 is Pro or Lys;
A29 is Thr or GIu;
A30 is GIy or GIu;
A31 is Tyr;
A32 is GIy or GIu;
A33 is Ser;
A34 is Ser or GIu;
A35 is Ser or GIu; A36 is Arg or GIu;
A37 is Arg or GIu;
A38 is Ala or GIu;
A39 is Pro;
A40 is GIn or GIu;
A41 is Thr or GIu;
A42 is GIy, Arg, or GIu;
A43 is lie, Arg, or GIu;
A44 is VaI, Arg, or GIu;
A45 is Asp, Arg, GIn, or GIu;
A46 is GIu, Arg, or GIn;
A47 is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A48 is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A49 is Phe, Arg, Leu, or Thr;
A50 is Arg or Ser;
A51 is Ser, Aib, Arg, or Thr;
A52 is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A53 is Asp, Arg, or Ser;
A54 is Leu, A6c, Arg, or Phe;
A55 is Arg, Tyr, or VaI;
A56 is Arg or GIn;
A57 is Leu or Phe;
A58 is GIu, A6c, or Arg;
A59 is Met, A6c, Arg, Asn, Asp, GIn, GIu, lie, Leu, NIe, Ser, D-Ser, Trp, or Tyr;
A60 is Tyr, Arg, He, or Phe;
A61 is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A62 is Ala, Arg, or Asn;
A63 is Pro, D-Pro, Thr, or deleted;
A64 is Leu, D-Leu, des-Leu, Arg, Phe, or deleted;
A65 is Lys, D-Lys, des-Lys, Arg, VaI, or deleted;
A66 is Pro, D-Pro, or deleted;
A67 is Ala, D-AIa, Aib, Arg, or deleted;
A68 is Lys, D-Lys, Arg, VaI, or deleted;
A69 is Ser, D-Ser, Aib, Arg, Thr, or deleted;
A70 is Ala, D-AIa, Arg, GIu, or deleted; and
A71 is Ser, Asp, GIu, Lys, or deleted;
or a pharmaceutically acceptable salt thereof.
19. An amino-terminal pegylated IGF-I conjugate according to claim 18, wherein said analogue of IGF-I is:
(Asn59)hIGF-l(l-70)-OH; (SEQ ID NO:1)
(Asn59)hIGF- 1 ( 1 -62)-OH; (SEQ ID NO:2)
(AsniS)hIGF-l (4-7O)-OH; (SEQ ID NO:3)
(Pro27, Lys28, Asn59)hIGF-l (1-7O)-OH; (SEQ ID NO:4)
(Pro", Lys28, Asn5y)hIGF-l(l-62)-OH; (SEQ ID NO:5)
(Ser53, Asn59)hIGF-l(l-70)-OH; (SEQ ID NO:6)
(Leu .1"7-)hIGF-l(l-70)-OH; (SEQ ID NO:7)
(Asn59, Thr63, des-Leu64, des-Lys65, Glu70)hIGF-l (1 -7O)-OH; (SEQ ID NO:8) (Tyr55, Asn59)hIGF-l(l-70)-OH; (SEQ ID NO:9)
(Thr49, Asn59)hIGF-l(l-70)-OH; (SEQ ID NO: 10)
(Asn59' 62)hIGF-l(l-70)-OH; (SEQ ID NO: 11)
(Asn59, Phe60)hIGF-l(l -7O)-OH; (SEQ ID NO: 12)
(Ser50, Asn59)hIGF-l(l -7O)-OH; (SEQ ID NO: 13)
(GIn50, Asniy)hIGF-l(l-70)-OH; (SEQ ID NO: 14)
(Asn59, D-Pro63)hIGF-l(l-70)-OH;
(Asn59, D-Leu^hlGF-Ul -7O)-OH;
(Asn59, D-Lys65)hIGF-l(l-70)-OH;
(Asn59, D-Pro66)hIGF-l(l -7O)-OH;
(Asn59, D-Ala67)hIGF-l(l-70)-OH;
(Asn59, D-Lys68)hIGF-l(l -7O)-OH;
(Asn59, D-Ser69)hIGF-l(l -7O)-OH;
(Asn59, D-Ala70)hIGF-l(l-70)-OH;
(Arg27' 65' 68, Leu59)hIGF-l(l-70)-OH; (SEQ ID NO: 15) (Leu59, Arg65'68)hIGF-l(l-70)-OH; (SEQ ID NO:16)
(Leu49' 59)hIGF-l(l -7O)-OH; (SEQ ID NO: 17)
(β-Me-Cys52, Leu59)hIGF- 1(1 -7O)-OH; (SEQ ID N0:18)
(β-Me-Cys47, Leu59)hIGF-l (1 -7O)-OH; (SEQ ID NO: 19)
(LeiΛ Glu")hIGF-l(l-71)-OH; (SEQ ID NO:20)
(Leu5y, Asp")hIGF-l(l-71)-OH; (SEQ ID NO:21)
(Leu59, Lys7l)WGF-l(l-71)-OH; (SEQ ID NO:22)
(Leu59, Ser71)hIGF-l(l-71)-OH; (SEQ ID NO:23)
(Leu59, Thr69)hIGF-l(l-7O)-OH; (SEQ IDNO:24)
(Thr51, Leu59)hIGF-l(l-7O)-OH; (SEQ IDNO:25)
(N-Me-Cys47, Nle59)hIGF-l(l-7O)-OH; (SEQ ID NO:26)
(NIe59, Aib69)hIGF-l(l-70)-OH; (SEQ IDNO:27)
(N-Me-Cys48, Nle59)hIGF-l(l-7O)-OH; (SEQ ID NO:28)
(NIe59, Aib67)hIGF-l(l-7O)-OH; (SEQ IDNO:29)
(hCys52, Nle59)hIGF-l(l-70)-OH; (SEQ ID NO:30)
(Aib51, Nle59)hIGF-l(l-7O)-OH; (SEQ IDNO:31)
(Pen52, Nle59)hJGF-l(l-7O)-OH; (SEQ IDNO:32)
(NIe59, Pen61)hIGF-l(l-70)-OH; (SEQ ID NO:33) (A6c54, Nle59)hIGF- 1(1 -7O)-OH; (SEQ IDNO:34)
(Arg53, πe59)hIGF-l(l -7O)-OH; (SEQ IDNO:35)
(Arg49, πe59)hIGF-l(l -7O)-OH; (SEQ ID NO:36)
(Arg51, πe59)hIGF-l(l -7O)-OH; (SEQ ID NO:37)
(Arg58, nes>K3F-l(l -7O)-OH; (SEQIDNO:38)
(A6c59)hIGF- 1 ( 1 -7O)-OH; (SEQ IDNO:39)
(Asp59)hIGF- 1 ( 1 -7O)-OH; (SEQ ID NO:40)
(Trp^hlGF- 1 ( 1 -7O)-OH; (SEQIDNO:41)
(Ser59)hIGF-l(l -7O)-OH; (SEQ IDNO:42)
(Tyr59)hIGF-l(l -7O)-OH; (SEQ IDNO:43)
(GlιT)hΙGF-l(l -7O)-OH; (SEQ ID NO:44)
(Gln59)hIGF-l(l -7O)-OH; (SEQ IDNO:45)
(Arg59)hIGF-l(l-70)-OH; (SEQ IDNO:46)
(Glu')hΙGF-l(l -7O)-OH; (SEQ IDNO:47)
(Glu2)hIGF-l(l-70)-OH; (SEQ ID NO:48)
(Glu4)hIGF-l(l-70)-OH; (SEQ IDNO:49)
(Glu7)hIGF-l(l -7O)-OH; (SEQ IDNO:50)
(Glu8)hIGF-l(l-70)-OH; (SEQIDNO:51) (GluI3)hIGF-l(l-70)-OH; (SEQ ID NO:52)
(Glu15)hIGF-l(l-70)-OH; (SEQ ID NO:53)
(Glu16)hIGF-l(l-70)-OH; (SEQ ID NO:54)
(Glu19)hIGF-l (1-7O)-OH; (SEQ ED NO:55)
(Glu20)hIGF-l(l-70)-OH; (SEQ ID NO:56)
(Glu21)hIGF-l(l-70)-OH; (SEQ ED NO:57)
(Glu22)hIGF-l(l -7O)-OH; (SEQ ED NO:58)
(Glu26)hIGF- 1(1 -7O)-OH; (SEQ ED NO:59)
(Glu27)hIGF- 1(1 -7O)-OH; (SEQ ED NO:60)
(Glu29)hIGF-l(l-70)-OH; (SEQ ED NO:61)
(Glu30)MGF-l(l -7O)-OH; (SEQ ED NO:62)
(Glu32)hIGF- 1(1 -7O)-OH; (SEQ ED NO:63)
(Glu34)hIGF-l(l-70)-OH; (SEQ ED NO:64)
(Glu35)hIGF- 1(1 -7O)-OH; (SEQ ED NO:65)
(Glu36)WGF-l(l-70)-OH; (SEQ ED NO:66)
(Glu37)hIGF-l(l-70)-OH; (SEQ ED NO:67)
(GlO .38 hIGF-I(I -7O)-OH; (SEQ ED NO:68)
(Glu40)hIGF-l(l -7O)-OH; (SEQ ED NO:69)
(Glu41)hIGF-l(l-70)-OH; (SEQ ED NO:70) (Glu42)hIGF-l(l -7O)-OH; (SEQ ID NO:71)
(Glu43)hIGF-l(l-70)-OH; (SEQ ID NO:72)
(GlOhIGF-I(I -7O)-OH; (SEQ ID NO:73)
(Glu45)hIGF-l(l -7O)-OH; (SEQ ID NO:74)
(Pro27, Lys28)hIGF-l(l -7O)-OH; (SEQ ID NO:75)
(Lys2)hIGF-l(l-70)-OH; (SEQ ID NO:76)
(Thr26)hIFG-l(l-70)-OH; (SEQ ID NO:77)
(Val65)hIGF-l(l -7O)-OH; (SEQ ID NO:78)
(Val68)hIGF-l(l -7O)-OH; (SEQ ID NO:79)
(VaI65' 68)hIGF-l(l -7O)-OH; (SEQ ID NO:80)
(Arg8)hIGF-l(l-70)-OH; (SEQ ID NO:81)
(Arg42)hIGF-l(l -7O)-OH; (SEQ ID NO:82)
(ATg44^IGF-I(I -7O)-OH; (SEQ ID NO: 83)
(Arg45)hIGF-l(l-70)-OH; (SEQ ID NO:84)
(Arg46)hIGF-l(l-70)-OH; (SEQ ID NO:85)
(Arg43)hIGF-l(l-70)-OH; (SEQ ID NO:86)
(Gln45)hIGF- 1 ( 1 -7O)-OH; (SEQ ID NO: 87)
(Gln46)hIGF-l(l-70)-OH; (SEQ ID NO:88)
(Leu1 ')hIGF-l(l -7O)-OH; (SEQ ID NO:89)
(Leu49)hIGF-l(l -7O)-OH; (SEQ ID NO:90)
(πe60)hIGF-l(l -7O)-OH; (SEQ ID NO:91)
(Phe54)hIGF- 1(1 -7O)-OH; (SEQ ID NO:92)
(Phe57)hIGF-l(l -7O)-OH; (SEQ ID NO:93) (Phe^hlGF-l (1-7O)-OH; (SEQ ID NO:94)
(Arg54)hIGF-l(l-70)-OH; (SEQ ID NO:95)
(Arg60)hIGF-l(l-70)-OH; (SEQ ID NO:96)
(Arg62)hIGF-l(l -7O)-OH; (SEQ ID NO:97)
(Arg^hlGF-Ul -7O)-OH; (SEQ ID NO:98)
(Arg65)hIGF-l(l-70)-OH; (SEQ ID NO:99)
(Arg67)hIGF-l (1-7O)-OH; (SEQ ID NO: 100)
(Arg68)hIGF-l (1-7O)-OH; (SEQ ID NO: 101)
(Arg69)hIGF-l(l -7O)-OH; or (SEQ ID NO: 102)
(Arg70)hIGF-l(l-70)-OH; (SEQ ID NO: 103) or a pharmaceutically acceptable salt thereof.
20. An amino-terminal pegylated IGF-I conjugate according to claim 19, wherein said conjugate is:
[2-(ethoxyimino(2-carbamate-10K linear PEG))acetyl-Gly\ Asn59]hIGF-l(l -7O)-OH;
(SEQ ID NO: 104)
[2-(ethoxyimino(2-carbamate-20K linear PEG))acetyl-Gly\ Asn59]hIGF-l(l -7O)-OH;
(SEQ ID NO: 105)
[2-(ethoxyimino(2-carbamate-20K branched PEG))acetyl-Gly1, Asn59]hIGF-l(l-70)- OH;
(SEQ ID NO: 106)
[2-(ethoxyimino(2-carbamate-30K linear PEG))acetyl-Gly', Asn59]hIGF-l(l -7O)-OH; or
(SEQ ID NO: 107)
[2-(ethoxyimino(2-carbamate-40K branched PEG))acetyl-Gly\ Asn59]hIGF-l(l-70)- OH;
(SEQ ID NO: 108)
or a pharmaceutically acceptable salt thereof.
21. An amino-terminal lipidated IGF- 1 conjugate of formula (II),
Y-B
(ID
wherein,
Figure imgf000046_0001
wherein Z is alkyl, aryl, alkyl-aryl, substituted alkyl, substituted aryl, or substituted alkyl-aryl; and
B is the wild-type IGF-I or an analogue of IGF-I ;
or a pharmaceutically acceptable salt thereof.
22. An amino-terminal lipidated IGF-I conjugate according to claim 22, wherein Z is CH3-(CH2)m-, wherein m is an integer from 1 to 30 inclusive; or a pharmaceutically acceptable salt thereof.
23. An amino-terminal lipidated IGF-I conjugate according to claim 22 or claim 18, wherein said analogue of IGF-I has formula (ILA),
Y-A1-A2-A3-A4-A5-A6-A7-A8-A9-A1O-AU-A12-A13-AI4-A15-A16-A17-A18-A19-A2O-A21- A22-A23-A24-A25-A26-A27-A28-A29-A30-A31-A32-A33-A34-A35-A36-A37-A38-A39-A40-A41-A42-A43- A44-A45-A46-A47-A48-A49-A50-A51-A52-A53-A54-A55-A56-A57-A58-A59-A60-A61-A62-A63-A64-A65- A66-A67-A68-A69-A70-A71-R1,
(ILA)
wherein:
A1 is GIy, Ala, Asn, Asp, GIn, GIu, or deleted;
A2 is Pro, Ala, Arg, Asp, GIn, GIu, Lys, or deleted;
A3 is GIu, Ala, Asp, GIn, or deleted;
A4 is Thr, Ala, Asn, Asp, GIn, GIu, Ser;
A5 is Leu, Ace, Ala, lie, or VaI;
A6 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A7 is GIy, Ala, Asn, Asp, GIn or GIu;
A8 is Ala, Arg, Asn, Asp, GIn, GIu, or Lys;
A9 is GIu, Ala, Asp, or GIn;
A10 is Leu, Ace, Ala, He, or VaI;
A" is VaI, Ala, lie, or Leu;
A12 is Asp, Ala, Arg, Asn, GIn, GIu, or Lys;
A13 is Ala, Asn, Asp, GIn, GIu, lie, Leu, or VaI;
A14 is Leu, Ace, Ala, lie, or VaI;
A15 is GIn, Ala, Asn, Asp, or GIu;
A16 is Phe, Ala, Asn, Asp, GIn, GIu, Trp, or Tyr; A17 is VaI, Ala, lie or Leu;
A18 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A19 is GIy, Ala, Asn, Asp, GIn, or GIu;
A20 is Asp, Ala, Asn, GIn, or GIu;
A21 is Arg, Ala, Asn, Asp, GIn, GIu, or Lys;
A22 is GIy, Ala, Asn, Asp, GIn, or GIu;
A23 is Phe, Ala, Trp, or Tyr;
A24 is Tyr, Ala, Phe, or Trp;
A25 is Phe, Ala, Trp, or Tyr;
A26 is Asn, Ala, Asp, GIn, GIu, Ser, or Thr;
A is Lys, Ala, Arg, Asn, Asp, GIn, GIu, or Pro;
A28 is Pro, Ala, Arg, or Lys;
A29 is Thr, Ala, Asn, Asp, GIn, GIu, or Ser;
A30 is GIy, Ala, Asn, Asp, GIn, or GIu;
A31 is Tyr, Ala, Phe, or Trp;
A32 is GIy, Ala, Asn, Asp, GIn, or GIu;
A33 is Ser, Ala, Thr, or VaI;
A34 is Ser, Ala, Asn, Asp, GIn, GIu, or Thr;
A35 is Ser, Ala, Asn, Asp, GIn, GIu, or Thr;
A36 is Arg, Ala, Asn, Asp, GIn, GIu, or Lys;
A37 is Arg, Ala, Asn, Asp, GIn, GIu, or Lys;
A38 is Ala, Asn, Asp, GIn, or GIu;
A39 is Pro, Ala, Arg, or GIu;
A40 is GIn, Ala, Asn, Asp, or GIu;
A41 is Thr, Ala, Asn, Asp, GIn, GIu, or Ser;
A42 is GIy, Ala, Arg, Asn, Asp, GIn, GIu, or Lys;
A43 is lie, Ala, Arg, Asn, Asp, GIn, GIu, or Lys;
A44 is VaI, Ala, Arg, Asn, Asp, GIn, GIu, Ue, Leu, or Lys;
A45 is Asp, Ala, Arg, Asn, GIn, GIu, or Lys;
A46 is GIu, Ala, Arg, Asn, Asp, GIn, or Lys;
A47 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A48 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A49 is Phe, Ala, Arg, lie, Leu, Lys, Ser, Thr, Trp, Tyr, or VaI;
A50 is Arg, Ala, Lys, Ser, or Thr; A51 is Ser, Aib, Ala, Arg, Lys, or Thr;
A52 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A53 is Asp, Ala, Arg, Asn, GIn, GIu, Lys, Ser, or Thr;
A54 is Leu, Ace, Ala, Arg, He, Phe, or VaI;
A55 is Arg, Ala, He, Leu, Lys, Phe, Tip, Tyr, or VaI;
A56 is Arg, Ala, Asn, Asp, GIn, GIu, or Lys;
A57 is Leu, Ace, Ala, lie, Phe, or VaI;
A58 is GIu, Ace, Ala, Arg, Asn, Asp, GIn, or Lys;
A59 is Met, Ace, Ala, Arg, Asn, Asp, GIn, GIu, He, Leu, Lys, NIe, Ser, D-Ser, Thr, Trp, Tyr, or VaI;
A60 is Tyr, Ala, Arg, He, Phe, or Trp;
A61 is Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen, or D-Pen;
A62 is Ala, Arg, Asn, Asp, GIn, GIu, lie, Leu, or VaI;
A63 is Pro, D-Pro, Ala, Ser, Thr, or deleted;
A64 is Leu, D-Leu, des-Leu, Ala, Arg, lie, Phe, VaI, or deleted;
A65 is Lys, D-Lys, des-Lys, Ala, Arg, He, Leu, VaI, or deleted;
A66 is Pro, D-Pro, Ala, or deleted;
A67 is Ala, D-AIa, Aib, Arg, or deleted;
A68 is Lys, D-Lys, Ala, Arg, He, Leu, VaI, or deleted;
A69 is Ser, D-Ser, Aib, Ala, Arg, Thr, or deleted;
A70 is Ala, D-AIa, Asn, Asp, GIn, GIu, or deleted;
A71 is Asn, Ala, Asp, GIn, GIu, Lys, Ser, Thr, or deleted; and
R1 is OH or NH2;
provided that the side-chains of residue pairs A6 and A48, A47 and A52, and A18 and A61, each form a disulfide bond; and
further provided that when A59 is either Leu, He, NIe, Thr, or VaI, then the analogue contains at least one additional amino acid substitution or addition;
or a pharmaceutically acceptable salt thereof.
24. An amino-terminal lipidated IGF-I conjugate according to claim 23, wherein
A1 is GIy, GIu, or deleted;
A2 is Pro, GIu, Lys, or deleted;
A3 is GIu or deleted;
A4 is Thr or GIu;
A5 is Leu; A6 is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A7 is GIy or GIu;
A8 is Ala, Arg or GIu;
A9 is GIu;
A10 is Leu;
A11 is VaI or Leu;
A12 is Asp;
A13 is Ala or GIu;
A14 is Leu;
A15 is GIn or GIu;
A16 is Phe or GIu;
A17 is VaI or Leu;
A18 is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A19 is GIy or GIu;
A20 is Asp or GIu;
A21 is Arg or GIu;
A22 is GIy or GIu;
A23 is Phe;
A24 is Tyr;
A25 is Phe;
A26 is Asn, GIu, or Thr;
A27 is Lys, Arg, GIu, or Pro;
A28 is Pro or Lys;
A29 is Thr or GIu;
A30 is GIy or GIu;
A31 is Tyr;
A32 is GIy or GIu;
A33 is Ser;
A34 is Ser or GIu;
A35 is Ser or GIu;
A36 is Arg or GIu;
A37 is Arg or GIu;
A38 is Ala or GIu;
A39 is Pro;
A40 is GIn or GIu;
A41 is Thr or GIu;
A42 is GIy, Arg, or GIu; A43 is lie, Arg, or GIu;
A44 is VaI, Arg, or GIu;
A45 is Asp, Arg, GIn, or GIu;
A46 is GIu, Arg, or GIn;
A47 is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A48 is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A49 is Phe, Arg, Leu, or Thr;
A50 is Arg or Ser;
A51 is Ser, Aib, Arg, or Thr;
A52 is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A53 is Asp, Arg, or Ser;
A54 is Leu, A6c, Arg, or Phe;
A55 is Arg, Tyr, or VaI;
A56 is Arg or GIn;
A57 is Leu or Phe;
A58 is GIu, A6c, or Arg;
A59 is Met, A6c, Arg, Asn, Asp, GIn, GIu, lie, Leu, NIe, Ser, D-Ser, Trp, or Tyr;
A60 is Tyr, Arg, He, or Phe;
A61 is Cys, hCys, β-Me-Cys, N-Me-Cys, or Pen;
A62 is Ala, Arg, or Asn;
A63 is Pro, D-Pro, Thr, or deleted;
A64 is Leu, D-Leu, des-Leu, Arg, Phe, or deleted;
A65 is Lys, D-Lys, des-Lys, Arg, VaI, or deleted;
A66 is Pro, D-Pro, or deleted;
A67 is Ala, D-AIa, Aib, Arg, or deleted;
A68 is Lys, D-Lys, Arg, VaI, or deleted;
A69 is Ser, D-Ser, Aib, Arg, Thr, or deleted;
A70 is Ala, D-AIa, Arg, GIu, or deleted; and
A71 is Ser, Asp, GIu, Lys, or deleted;
or a pharmaceutically acceptable salt thereof.
25. An amino-terminal lipidated IGF-I conjugate according to claim 24, wherein wherein said analogue of IGF-I is:
(Asn59)hIGF- 1 ( 1 -7O)-OH; (SEQ ID NO: 1 )
(Asn3>IGF-l(l-62)-OH; (SEQ ID NO:2) (Asn59)hIGF-l (4-7O)-OH; (SEQ ID NO:3)
(Pro27, Lys28, Asn59)hIGF-l (1-7O)-OH; (SEQ ID NO:4)
(Pro27, Lys28, Asn59)hIGF-l (1 -62)-OH; (SEQ ID NO:5)
(Ser53, Asn59)hIGF-l(l-70)-OH; (SEQ ID NO:6)
(Leu17)hIGF-l(l-70)-OH; (SEQ ID NO:7) (Asn59, Thr63, des-Leu64, des-Lys65, Glu70)hIGF-l(l -7O)-OH; (SEQ ID NO:8)
(Tyr55, Asn59)hIGF-l(l-70)-OH; (SEQ ID NO:9)
(Thr49, Asn59)hIGF-l(l-70)-OH; (SEQ ID NO:10)
(Asn59' 62)hIGF-l(l-70)-OH; (SEQ DD NO: 11)
(Asn59, Phe60)hIGF-l(l-70)-OH; (SEQ ID NO: 12)
(Ser50, Asn59)hIGF-l(l-70)-OH; (SEQ ID NO: 13)
(GIn56, Asn59)hIGF-l(l-70)-OH; (SEQ ID NO:14)
(Asn59, D-Pro6>IGF-l(l -7O)-OH;
(Asn59, D-LeuM)hIGF-l(l -7O)-OH;
(Asn59, D-Lys65)hIGF-l(l -7O)-OH;
(Asn59, D-Pro66)hIGF-l(l -7O)-OH;
(Asn59, D-Ala67)hIGF-l(l -7O)-OH;
(Asn59, D-Lys68)hIGF-l(l-70)-OH;
(Asn59, D-Ser69)hIGF-l(l -7O)-OH;
(Asn59, D-Ala70)hIGF-l(l-70)-OH;
(Arg27' 65' 68, Leu59)WGF-l(l-70)-OH; (SEQ ID NO: 15)
(Leu59, Arg65' 68)hIGF-l(l-70)-OH; (SEQ ID N0:16)
(Leu49' 59)hIGF-l(l-70)-OH; (SEQ ID NO: 17)
(β-Me-Cys52, Leu59)hIGF-l(l-70)-OH; (SEQ DD NO: 18)
(β-Me-Cys47, Leu59)hIGF-l(l-70)-OH; (SEQ DD NO: 19)
(Leu5", Glu")hIGF-l(l-71)-OH; (SEQ DD NO:20) (Leu59, Asp71)hIGF-l(l-71)-OH; (SEQ ID NO:21)
(Leu59, Lys71)hIGF-l(l-71)-OH; (SEQ ID NO:22)
(Leu59, Ser71)hIGF-l(l-71)-OH; (SEQ ID NO:23)
(Leu59, Thr69)hIGF-l(l-70)-OH; (SEQ ID NO:24)
(Thr51, Leu59)hIGF-l(l-70)-OH; (SEQ ID NO:25)
(N-Me-Cys47, Nle59)hIGF- 1(1 -7O)-OH; (SEQ ID NO:26)
(NIe59, Aib69)hIGF-l(l -7O)-OH; (SEQ ID NO:27)
(N-Me-Cys48, Nle59)WGF-l(l -7O)-OH; (SEQ ID NO:28)
(NIe59, Aib67)hIGF-l(l-70)-OH; (SEQ ID NO:29)
(hCys52, Nle59)hIGF-l(l -7O)-OH; (SEQ ID NO:30)
(Aib51, Nle59)hIGF-l(l-70)-OH; (SEQ ID NO:31)
(Pen52, Nle59)hIGF-l(l -7O)-OH; (SEQ ID NO:32)
(NIe59, Pen61)hIGF-l(l -7O)-OH; (SEQ ID NO:33)
(A6c54, Nle59)hIGF-l(l-70)-OH; (SEQ ID NO:34)
(Arg53, πe59)hIGF-l(l-70)-OH; (SEQ ID NO:35)
(Arg49, πe59)hIGF-l(l -7O)-OH; (SEQ ID NO:36)
(Arg51, πe59)hIGF-l(l -7O)-OH; (SEQ ID NO:37)
(Arg58, πe59)hIGF-l(l -7O)-OH; (SEQ ID NO:38) (A6c59)hIGF-l(l-70)-OH; (SEQ J0 NO:39)
(Asp59)hIGF-l(l-70)-OH; (SEQ IDNO:40)
(Tφ59)MGF-l(l-7O)-OH; (SEQ ID NO:41)
(Ser59)hIGF-l(l-70)-OH; (SEQ IDNO:42)
(Tyr59)hIGF-l(l-70)-OH; (SEQ ID NO:43)
(Glu59)hIGF-l(l-7O)-OH; (SEQ ID NO:44)
(Gln59)hIGF-l(l-70)-OH; (SEQ ID NO:45)
(Arg59)hIGF-l(l-7O)-OH; (SEQ IDNO:46)
(Glu')MGF-l(l-7O)-OH; (SEQ IDNO:47)
(Glu2)hIGF-l(l-70)-OH; (SEQ ID NO:48)
(Glu4)hIGF-l(l-7O)-OH; (SEQ ID NO:49)
(Glu7)MGF-l(l-70)-OH; (SEQIDNO:50)
(Glu8)hIGF-l(l-70)-OH; (SEQ IDNO:51)
(Glu13)WGF-1(1-7O)-OH; (SEQ IDNO:52)
(Glu15)hIGF-l(l-70)-OH; (SEQ ID NO:53)
(Glu16)hIGF-l(l-70)-OH; (SEQ ID NO:54)
(Glu19)hIGF-l(l-70)-OH; (SEQ IDNO:55)
(Glu20)hIGF-l(l-7O)-OH; (SEQ IDNO:56) (Glu21)hIGF-l(l-70)-OH; (SEQ ID NO:57)
(Glu22)hIGF-l(l-70)-OH; (SEQ ID NO:58)
(Glu26)hIGF-l(l-70)-OH; (SEQ ID NO:59)
(Glu27)hIGF-l(l -7O)-OH; (SEQ ID NO:60)
(Glu29)hIGF-l(l -7O)-OH; (SEQ ID NO:61)
(Glu30)hIGF-l(l-70)-OH; (SEQ IDNO:62)
(Glu32)hIGF-l(l-7O)-OH; (SEQ IDNO:63)
(Glu34)hIGF-l(l-70)-OH; (SEQ ID NO:64)
(Glu35)hIGF-l(l-7O)-OH; (SEQ IDNO:65)
(Glu36)hIGF-l(l-7O)-OH; (SEQ IDNO:66)
(Glu37)hIGF-l(l-7O)-OH; (SEQ ID NO:67)
(Glu38)hIGF-l(l-70)-OH; (SEQ IDNO:68)
(Glu40)hIGF-l(l-70)-OH; (SEQ IDNO:69)
(Glu41)hIGF-l(l-7O)-OH; (SEQ IDNO:70)
(Glu42)hIGF-l(l-7O)-OH; (SEQ IDNO:71)
(Glu43)hIGF-l(l-7O)-OH; (SEQ ID NO:72)
(Glu^hlGF-Ul-7O)-OH; (SEQ ID NO:73)
(Glu45)hIGF-l(l -7O)-OH; (SEQ ID NO:74)
(Pro27, Lys28)hIGF- 1(1 -7O)-OH; (SEQ ID NO:75) (Lys2)hIGF-l(l -7O)-OH; (SEQ ID NO:76)
(Thr26)hIFG-l(l-70)-OH; (SEQ ID NO:77)
(Val65)hIGF-l(l-70)-OH; (SEQ ID NO:78)
(Val68)hIGF-l(l -7O)-OH; (SEQ ID NO:79)
(VaI65' 68)hIGF-l(l -7O)-OH; (SEQ ID NO:80)
(Arg8)hIGF-l(l-70)-OH; (SEQ ID NO:81)
(Arg42)hIGF-l(l -7O)-OH; (SEQ ID NO: 82)
(ATg4^hIGF-I(I -7O)-OH; (SEQ ID NO:83)
(Arg45)hIGF-l(l-70)-OH; (SEQ ID NO:84)
(Arg46)hIGF-l(l -7O)-OH; (SEQ ID NO: 85)
(Arg43)hIGF-l(l-70)-OH; (SEQ ID NO:86)
(Gln45)hIGF- 1 ( 1 -7O)-OH; (SEQ ID NO: 87)
(Gln46)hIGF-l(l -7O)-OH; (SEQ ID NO:88)
(Leu1 ')hIGF-l(l -7O)-OH; (SEQ ID NO:89)
(Leu49)hIGF-l(l-70)-OH; (SEQ ID NO:90)
(He60)hIGF-l(l-70)-OH; (SEQ ID NO:91)
(Phe54)hIGF-l(l -7O)-OH; (SEQ ID NO:92)
(Phe57)hIGF-l(l-70)-OH; (SEQ ID NO:93)
(Phe^hlGF-Kl -7O)-OH; (SEQ ID NO:94)
(Arg54)hIGF- 1(1 -7O)-OH; (SEQ ID NO:95)
(Arg60)hIGF-l(l-70)-OH; (SEQ ID NO:96)
(Arg62)hIGF-l(l-70)-OH; (SEQ ID NO:97)
Figure imgf000055_0001
(Arg65)hIGF-l(l-70)-OH; (SEQ ID NO:99)
(Arg67)hIGF-l(l-70)-OH; (SEQ ID NO: 100)
(Arg68)hIGF-l(l -7O)-OH; (SEQ ID NO: 101)
(Arg69)hIGF-l(l-70)-OH; or (SEQ ID NO: 102) (Arg70)hIGF-l(l-70)-OH; (SEQ ID NO: 103) or a pharmaceutically acceptable salt thereof.
26. An amino-terminal lipidated IGF-I conjugate according to claim 25, wherein said conjugate is:
[2-(n-octadecoxyimino)acetyl-Gly1, Asn59]hIGF-l(l-70)-OH; (SEQ ID NO: 109) or a pharmaceutically acceptable salt thereof.
27. A pharmaceutical composition comprising an effective amount of an analogue of any one of claims 1-26.
28. A pharmaceutical composition according to claim 27, further comprising a pharmaceutically acceptable carrier.
29. A method of eliciting an agonist effect from an IGF-I receptor in a subject in need thereof, which comprises administering to said subject a therapeutically effective amount of an analogue of any one of claims 1-26 or a pharmaceutical composition of claim 27 or claim 28.
30. A method for treating conditions or diseases mediated by IGF-I receptor binding, comprising the step of administering to a subject in need thereof a therapeutically effective amount of an analogue of any one of claims 1-26 or a pharmaceutical composition of claim 27 or claim 28.
31. A method for treating conditions or diseases mediated by insulin receptor binding, comprising the step of administering to a subject in need thereof a therapeutically effective amount of an analogue of any one of claims 1-26 or a pharmaceutical composition of claim 27 or claim 28.
32. A method for treating conditions or diseases mediated by inhibiting the binding of an insulin-like growth factor to one or more insulin-like growth factor binding proteins, comprising the step of administering to a subject in need thereof a therapeutically effective amount of an analogue of any one of claims 1-26 or a pharmaceutical composition of claim 27 or claim 28.
33. The method according to any one of claims 30-32, wherein said condition or disease is selected from the group consisting of short stature, obesity, weight loss, cachexia, anorexia, neurodegenerative disorders, fibrosis-linked conditions, cartilage disorders, bone diseases, inflammatory disorders, intestinal disorders, insulin resistance, diabetes, diabetic ketoacidosis, Rabson-Mendenhall syndrome, retinopathy, acromegaly, fibromuscular hyperplasia, and cardiac disorders.
34. The method of claim 33, wherein said subject in need of treating short stature is a human pediatric subject having insulin-like growth factor- 1 deficiency (IGFD), wherein said administering is effective to treat IGFD in the human pediatric subject.
35. The method of claim 34, wherein said human pediatric subject is characterized as follows:
the subject, at the time of treatment or prior to initial treatment with an analogue of any one of claims 1-26 or a pharmaceutical composition of claim 27 or claim 28, has or had a height at least about 2.0 standard deviations (SD) below the normal mean height for a subject of the same age and gender, and
the subject, at the time of treatment or prior to initial treatment with an analogue of any one of claims 1-26 or a pharmaceutical composition of claim 27 or claim 28, has or had a blood level of IGF-I at least about -1 SD below normal mean levels for a human pediatric subject of the same age and gender.
36. A method for treating a subject having idiopathic short stature (ISS) comprising administering to a human pediatric subject suffering from ISS characterized by partial endogenous growth hormone activity or signaling, an amount of an analogue of any one of claims 1-26 or a pharmaceutical composition of claim 27 or claim 28 effective to promote growth in the subject, wherein the subject is further characterized as follows:
the subject, at the time of treatment or prior to initial treatment with an analogue of any one of claims 1-26 or a pharmaceutical composition of claim 27 or claim 28, has or had a height at least about 2.0 standard deviations (SD) below the normal mean height for a subject of the same age and gender, and
the subject, at the time of treatment or prior to initial treatment with an analogue of any one of claims 1-26 or a pharmaceutical composition of claim 27 or claim 28, has blood levels of GH and IGF-I that are at least normal for a subject of the same age and gender.
37. The method of claim 33, wherein said neurodegenerative disorder is selected from the group consisting of ALS, multiple sclerosis, muscular dystrophy, diabetic neuropathy, demyelinating peripheral neuropathies, Parkinson's disease, Alzheimer's disease, and a sequela of traumatic spinal cord lesions.
38. The method of claim 33, wherein said cartilage disorder is osteoarthritis.
39. The method of claim 33, wherein said bone disease is osteoporosis.
40. The method of claim 33, wherein said inflammatory disorder is rheumatoid arthritis.
41. The method of claim 33, wherein said diabetes is either type I diabetes or type II diabetes.
42. The method of claim 33, wherein said cardiac disorder is atherosclerosis.
43. The method of claim 33, wherein said fibrosis-linked condition is cirrhosis, systemic sclerosis, or thyroid eye disease.
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