Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• X 10 is a non-conservative exchange of proline; or Xg and X 10 are substituted by a single amino acid;
• X 14 Js D, E, I, L or V; Xi 5 is C, A, K, ⁇ -amino- ⁇ -bromobutyric acid or homocysteine (hoc) provided that either X 6 or X 15 is C or hoc.
• X 6 is C
• N terminal and end (C terminal) of the described individual peptide sequences up to five amino acids may be removed and/or added. It is self-evident that size is not of relevance as long as the peptide function is preserved. Furthermore, please note that individual peptide sequences that might be too short to enfold their activity as monomers usually function as agonists upon dimerisation. Such peptides are thus preferably used in their dimeric form.
• peptides selected from the group consisting of SEQ ID NOS 2, 4-9 given below. Especially preferred is a peptide with a K in position 10 and a K in position 17 as is the case in SEQ ID NO 2.
• peptide dimers or multimers are formed on the basis of the monomers according to SEQ ID NO 2 and 4 to 9 as given above or modifications thereof.
• the peptides described herein can e.g. also be modified by a conservative exchange of single amino acids, wherein preferably, not more than 1, 2 or 3 amino acids are exchanged.
• This embodiment of the invention allows the custom-made design of a suitable linker by molecular modeling in order to avoid distortions of the bioactive conformation.
• a linker composed of 3 to 5 amino acids is especially preferred.
• the linker between the functional domains (or monomeric units) of the final bivalent or multivalent peptides can be either a distinct part of the peptide or can be composed - fully or in parts - of amino acids which are part of the monomeric functional domains.
• the glycine residues in amino acid positions 1 and 2 and 19 and 20 can form part of the linker. Examples are given with Seq. 11 to 14.
• linker is thus rather defined functionally than structurally, since an amino acid might form part of the linker unit as well as of the monomeric subunits.
• This sequence presents a continuous bivalent peptide according to the invention harboring two slightly different (heterogeneous) binding domains. Such bivalent peptides would not be accessible economically with a prior art dimerization approach (see above). Also these binding domains can be applied as a monomer as
• a further example is
• the peptide optionally carries an additional amino acid, preferably one with a reactive side chain such as cysteine at the N-terminus such as e.g. in the following sequences C-GGTYSCHFGKLTWVCKKQGG-GGTYSCHFGKLTWVCKKQGG
• the first sequence depicts a serine in position X 7 . It was found that a new hydrogen bridge is created through the introduction of the hydroxyl group when this sequence is incorporated in a dimer. The use of a serine in position X 7 is thus especially favourable for dimers since the bioactive conformation is stabilised.
• This can include:
• the dimeric molecules known in the state of the art provide merely one target respectively receptor binding unit per dimer. Thus only one receptor complex is generated upon binding of the dimeric compound thereby inducing only one signal transduction process.
• two monomeric EPO mimetic peptides are connected via PEG to form a peptide dimer thereby facilitating dimerisation of the receptor monomers necessary for signal transduction (Johnson et. al., 1997).
• the supravalent compounds according to the invention comprise several already di- or multimeric respective receptor binding units.
• an appropriate carrier unit is a homobifunctional polymer, of for example polyethylene glycol (bis-maleimide, bis-carboxy, bis-amino etc.).
• the attachment can occur e.g. via a reactive amino acid of the peptide units e.g. lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine or the N- terminal amino group and the C-terminal carboxylic acid.
• a reactive amino acid of the peptide units e.g. lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine or the N- terminal amino group and the C-terminal carboxylic acid.
• Acylating groups which react with the amino groups of the protein, for example acid anhydride groups, N-acylimidazoIe groups, azide groups, N-carboxy anhydride groups, diketene groups, dialkyl pyrocarbonate groups, imidoester groups, and carbodiimide-activated carboxyl-groups. All of the above groups are known to react with amino groups on proteins/peptides to form covalent bonds, involving acyl or similar linkages;
• ester and amide forming groups which react with a carboxyl group of the protein, such as diazocarboxylate groups, and carbodiimide and amine groups together;
• the compound according to the invention may be made by - optionally - first modifying the polymer chemically to produce a polymer having at least one chemical group thereon which is capable of reacting with an available or introduced chemical group on the peptide unit, and then reacting together the - optionally - modified polymer and the peptide unit to form a covalently bonded complex thereof utilising the chemical group of the - if necessary - modified polymer.
• the coupling of the peptide units to the polymeric carrier unit is performed using reactions principally known to the person skilled in the art. E.g. there are number of PEG and HES attachment methods available to those skilled in the art (see for example WO 2004/100997 giving further references, Roberts et al., 2002; US 4,064,118; EP 1 398 322; EP 1 398 327; EP 1 398 328; WO 2004/024761 ; all herein incorporated by reference).
• one embodiment of the present invention teaches the use of a polymeric carrier unit that is composed of at least two subunits.
• the polymeric subunits are connected via biodegradable covalent linker structures.
• the molecular weight of the large carrier molecule for example 40 kD
• several small or intermediate sized subunits for example each subunit having a molecular weight of 5 to 1OkD
• the molecular weights of the modular subunits add up thereby generating the desired molecular weight of the carrier molecule.
• the biodegradable linker structures can be broken up in the body thereby releasing the smaller carrier subunits (e.g. 5 to 1OkD).
• the small carrier subunits show a better renal clearance than a polymer molecule having the overall molecular weight (e.g. 4OkD).
• An example is given in Fig. 16.
• the linker structures are selected according to known degradation properties and time scales of degradation in body fluids.
• the breakable structures can, for instance, contain cleavable groups like carboxylic acid derivatives as amide/peptide bonds or esters which can be cleaved by hydrolysis (see e.g. Roberts, 2002 herein incorporated by reference).
• PEG succinimidyl esters can also be synthesized with various ester linkages in the PEG backbone to control the degradation rate at physiological pH (Zhao, 1997, herein incorporated by reference).
• Other breakable structures like disulfides of benzyl urethanes can be cleaved under mild reducing environments, such as in endosomal compartments of a cell (Zalipsky, 1999) and are thus also suitable.
• This embodiment has the advantage that a supravalent composition is created due to the first carrier which is however, very durable due to the presence of the second carrier, which is constituted preferably by PEG units of 3 to 5 or 1OkD.
• the whole entity is very well degradable, since the first carrier (e.g. HES) and the peptide units are biodegradable and the second carrier, e.g. PEG is small enough to be easily cleared from the body.
• cleavage solution 50 ⁇ l of the cleavage solution was added to each well and the cleavage was performed for 10 min, this procedure was repeated three times.
• the cleaved peptide was eluted with 200 ⁇ l cleavage solution by gravity flow into the deep well plate.
• the deprotection of the side chain function was performed for another 2.5 h within the deep well plate.
• the peptide was precipitated with ice cold ether/hexane and centrifuged.
• the peptides were solved in neutral aqueous solution and the cyclization was incubated over night at 4° C.
• the peptides were lyophilized.
• the working-up was performed via ultra filtration and freeze-drying
• TNBS 2,4,6-trinitrobenzole sulphonic acid
• a cysteine containing peptide was used which had either a free (Pep-IA) or a biotinytated (Pep-IB) N-term.
• a 4:1 mixture of Pep-IA/B was converted over night in excess (approx. 6 equivalents with MaIPA-HES in phosphate-buffer, 50 mM, pH 6.5/DMF 80:20; working up occurred with ultra filtration and freeze-drying.

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• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicinal Preparation (AREA)

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• 2005
  • 2005-11-10 KR KR1020077012937A patent/KR20070092706A/ko not_active Application Discontinuation
  • 2005-11-10 WO PCT/EP2005/012075 patent/WO2006050959A2/en active Application Filing
  • 2005-11-10 AU AU2005303887A patent/AU2005303887A1/en not_active Abandoned
  • 2005-11-10 EP EP05819212A patent/EP1812461A2/en not_active Withdrawn
  • 2005-11-10 JP JP2007540582A patent/JP2008519589A/ja active Pending
  • 2005-11-10 CA CA002586915A patent/CA2586915A1/en not_active Abandoned
• 2007
  • 2007-06-05 NO NO20072856A patent/NO20072856L/no not_active Application Discontinuation

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