EP1812461A2 - Molecules which promote hematopoiesis - Google Patents

Molecules which promote hematopoiesis

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Publication number
EP1812461A2
EP1812461A2 EP05819212A EP05819212A EP1812461A2 EP 1812461 A2 EP1812461 A2 EP 1812461A2 EP 05819212 A EP05819212 A EP 05819212A EP 05819212 A EP05819212 A EP 05819212A EP 1812461 A2 EP1812461 A2 EP 1812461A2
Authority
EP
European Patent Office
Prior art keywords
peptide
amino acid
groups
peptides
unit
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05819212A
Other languages
German (de)
English (en)
French (fr)
Inventor
Hans-Georg Frank
Franz-Peter Bracht
Udo Haberl
Andreas Rybka
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AplaGen GmbH
Original Assignee
AplaGen GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US11/041,207 external-priority patent/US7589063B2/en
Application filed by AplaGen GmbH filed Critical AplaGen GmbH
Priority to EP05819212A priority Critical patent/EP1812461A2/en
Publication of EP1812461A2 publication Critical patent/EP1812461A2/en
Withdrawn legal-status Critical Current

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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

  • the present invention relates to peptides as binding molecules for the erythro ⁇ poietin receptor, methods for the preparation thereof, medicaments containing these peptides, and their use in selected indications, preferably for treatment of various forms of anemia and stroke.
  • the hormone erythropoietin is a glycoprotein constituted by 165 amino acids and having four glycosylation sites.
  • the four complex carbohydrate side chains comprise 40 percent of the entire molecular weight of about 35 kD.
  • EPO is formed in the kidneys and from there migrates into the spleen and bone marrow, where it stimulates the production of erythrocytes. In chronic kidney diseases, reduced EPO production results in erythropenic anemia.
  • recombinant EPO prepared by genetic engineering, anemias can be treated effectively. EPO improves dialysis patients' quality of life. Not only renal anemia, but also anemia in premature newborns, inflammation and tumor-associated anemias can be improved with recombinant EPO.
  • a high dosage chemotherapy can be performed more successfully in tumor patients.
  • EPO improves the recovery of cancer patients, if administered within the scope of radiation therapy.
  • Another important issue associated with the treatment with recombinant EPO is the danger, that patients develop antibodies to recombinant EPO during treatment. This is due to the fact, that recombinant EPO is not completely identical to endogenous EPO. Once antibody formation is induced, it can lead to antibodies, which compromise the activity of endogenous erythropoietin as well. It frequently increases the dosage of recombinant EPO needed for treatment. Especially if such antibodies compromise the activity of endogenous EPO, this effect can be interpreted as a treatment-induced autoimmune disease. It is especially undesired e.g. in case of dialysis patients undergoing renal transplantation after months or years of EPO-treatment.
  • Synthetic peptides mimicking EPO's activity are subject of the international laid open WO96/40749. It discloses mimetic peptides of 10 to 40 amino acids of a distinct consensus preferably containing two prolines at the position commonly referred to as position 10 and 17, one of which is considered to be essential.
  • prolines are considered indispensable to the effectiveness of the peptides.
  • proline at position 17 this has been substantiated by interactions with the receptor, while the proline at position 10 was thought to be necessary for the correct folding of the molecule (see also Wrighton et al. 1996, 1997).
  • the correct folding, supported by the specific stereochemical properties of proline, is usually a necessary precondition of biological activity.
  • proline is a structure- forming amino acid which is often involved - as in this case - in the formation of hairpin structures and beta turns. Due to this property, inter alia, it is a frequent point of attack for post-proline-specific endopeptidases which destroy proline- containing peptides/proteins.
  • a number of endogenous peptide hormones are inactivated by such "single-hit" post-proline cleavage.
  • Half-life time of proline-containing EPO- mimetic peptides is thus shortened by the activity of these frequent and active enzymes.
  • Such peptides can be produced chemically and do not need recombinant pro- duction, which is much more difficult to control and to yield products with defined quality and identity. Chemical production of peptides of such small size can also be competitive in terms of production costs. Moreover, chemical production allows defined introduction of molecular variations such as glycosylation, pegylation or any other defined modifications, which can have a known potency to increase biological half-life. However, so far there has been no approval of any therapy with existing EPO mimetic peptides.
  • EPO mimetic peptides which exhibit at least essential parts of the biological activity of the native EPO and thus provide alternative means for efficient therapeutic strategies, in particular for the treatment of anemia or stroke.
  • a peptide of at least 10 amino acids in length capable of binding to the EPO receptor and comprising an agonist activity.
  • the peptide thus depicts EPO mimetic properties.
  • EPO mimetic peptides according to this invention do not comprise proline in the position commonly referred to as position 10 of EPO mimetic peptides, but a positively charged amino acid (for numbering please refer e.g. to Johnson et al, 1997 describing the ancestral sequence of EMP 1 ).
  • Said proline at position 10 is located in an amino acid motif which is characteristic for a certain folding structure, namely the beta-turn motif (please refer to Johnson, 1997). Said beta-turn structure forms upon receptor binding.
  • the EPO mimetic peptides according to the invention thus do not comprise a proline in the beta-turn motif at position 10 but a positively charged amino acid. Examples are K, R, H or respective non-natural amino acids such as e.g. homoarginine.
  • peptide which comprises the following sequence of amino acids:
  • each amino acid is selected from natural or unnatural amino acids and
  • X 6 is C, A, E, ⁇ -amino- ⁇ -bromobutyric acid or homocysteine (hoc);
  • X 7 is R 1 H, L, W or Y or S;
  • X 8 is M, F, I, homoserinmethylether (hsm) or norisoleucine;
  • X 9 is G or a conservative exchange of G
  • X 10 is a non-conservative exchange of proline; or Xg and X 10 are substituted by a single amino acid;
  • X 11 is independently selected from any amino acid; X 12 is T or A;
  • X 13 is W, 1-nal, 2-nal, A or F;
  • 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.
  • the length of the described peptide consensus is preferably between ten to forty or fifty or sixty amino acids. Peptides of above sixty amino acids in length, even though technically suitable, are not necessarily preferred since with increasing length of the peptide synthesis is usually getting more complicated and thus costly.
  • the peptide consensus depicts a length of at least 10, 15, 18 or 20 amino acids. Of course they can be embedded respectively be comprised by longer sequences.
  • the described peptide sequences can be perceived as binding domains for the EPO receptor. As EPO mimetic peptides they are capable of binding to the EPO receptor.
  • the peptides according to the invention do exhibit EPO mimetic activities although one or - according to some embodiments - even both prolines may be replaced by other natural or non-natural amino acids.
  • the peptides according to the invention have an activity comparable to that of proline- containing peptides.
  • the amino acids substituting proline residues do not represent a conservative exchange but instead a non- conservative exchange.
  • a positively charged amino acid such as basic amino acids such as K, R and H and especially K is used for substitution.
  • the non- conservative amino acid used for substitution can also be a non-natural amino acid and is preferably one with a positively charged side chain.
  • respective analogues of the mentioned amino acids are also comprised.
  • a suitable example of a non- natural amino acid is homoarginine.
  • the peptide carries a positively charged amino acid in position 10 except for the natural amino acid arginine.
  • the praline 10 is thus substituted by an amino acid selected from K, H or a non-natural positively charged amino acid such as e.g. homoarginine.
  • the peptides depict a lysine or homoarginine in position 10.
  • the proline in position 17 might be replaced by a non-conservative amino acid.
  • said non-conservative amino acid is one with a positively charged side chain such as K, R, H or a respective non-natural amino acid such as e.g.
  • the peptide carries a positively charged amino acid in position 17 except for the natural amino acid arginine.
  • the proline 17 is thus substituted by an amino acid selected from K, H or a non- natural positively charged amino acid such as homoarginine. It is preferred that the peptides depict a lysine or homoarginine in position 17.
  • sequences can have N-terminal and/or C-terminal acetylations and amidations. Some amino acids may also be phosphorylated.
  • a peptide that binds to the erythropoietin receptor and comprises a sequence of the following amino acids:
  • X 6 is C
  • X 8 is M, F or I; Xg is G or a conservative exchange of G;
  • X 10 is a non-conservative exchange of proline
  • X 11 is independently selected from any amino acid
  • X12 is T
  • X 7 can be serine
  • X 8 can be hsm or norisoleucine
  • X 13 can also be 1-nal, 2-nal, A or F.
  • the length of the peptide consensus is preferably between ten to forty or fifty or sixty amino acids. In preferred embodiments, the peptide consensus comprises at least 10, 15, 18 or 20 amino acids.
  • the peptides according to the invention may comprise besides. L-amino acids or the stereoisomeric D- amino acids, unnatural/unconventional amino acids, such as e.g. alpha, alpha-disubstituted amino acids, N-alkyl amino acids or lactic acid, e.g.
  • N-methylglycine sarcosine
  • AcG N-acetylglycine
  • peptides which are retro, inverso and retro/inverso peptides of the defined peptides and those peptides consisting entirely of D-amino acids.
  • the present invention also relates to the derivatives of the peptides, e.g. oxidation products of methionine, or deamidated glutamine, arginine and C-terminus amide.
  • the peptides do have a single amino acid substituting the amino acid residues Xg and X-io.
  • both residues may be substituted by one non-natural amino acid, e.g. 5-aminolevulinic acid or aminovaleric acid.
  • the peptides according to the invention comprise the consensus sequence
  • X ⁇ to Xi 5 have the above meaning and wherein X 4 JsY; X 5 is independently selected from any amino acid and is preferably A, H, K, L, My S, T or I.
  • the peptides according to the invention may be extended and may comprise the consensus sequence
  • X3X4X5X6X7X8X9X10X11Xi2X13X14X15X16 ⁇ 17X18 wherein X 4 to Xi 5 have the above meaning and wherein X 3 is independently selected from any amino acid, preferably D, E, L, N, S, T or V; X-16 is independently selected from any amino acid, preferably G, K, L, Q, R, S or T, more preferred K, R, S or T;
  • Xi 7 is independently selected from any amino acid, preferably A, G, P, R, K, Y or a non-natural amino acid with a positively charged side chain, more preferred K or Har;
  • X- 1 8 is independently selected from any amino acid.
  • the peptides comprise X 6 as C, E, A or hoc, preferably C and/or X 7 as R, H or Y or S and/or Xs as F or M and/or Xg as G or A, preferably G and/or Xio as K or Har and/or Xn as V, L, I, M, E, A 1 T or norisoleucine and/or X 12 as T and/or Xi 3 as W and/or Xi 4 as D or V and/or Xi 5 as C or hoc, preferably C and/or Xi 7 as P, Y or A or a basic natural or non-natural amino acid. It is, however, also preferred that Xi 7 is K or a non-natural amino acid with a positively charged side chain such as e.g. homoarginine.
  • Fig. 19 discloses further novel and suitable peptide sequences depicting EPO mimetic activity. Further peptides depict the following sequences:
  • 5-AIs Also disclosed are peptides having a binding capacity to the receptor of the hormone erythropoietin and depicting an agonist activity which are characterised in that the peptides do not depict a proline. As described above, these peptides preferably do not comprise a proline in the positions commonly referred to as 10 and 17 but a different natural amino acid or 5-aminolevulinic acid. They preferably depict a lysine in position 17. Also disclosed are nucleic acids coding for respective peptides.
  • One or more conservative amino acid substitutions can be carried out within the amino acid sequence of the polypeptides according to this invention, wherein the substitution occurs within amino acids having unpolar side chains, the natural or non-natural uncharged D- or L amino acids with polar side chains, amino acids with aromatic side chains, the natural or non-natural positively charged D- or L- amino acids, the natural or non-natural negatively charged D- or L amino acids as well as within any amino acids of similar size and molecular weight, wherein the molecular weight of the original amino acid should not deviate more than approximately +/- 25% of the molecular weight of the original amino acid and the binding capacity to the receptor of the hormone erythropoietin with agonistic effect is maintained.
  • no more than 1 , 2 or 3 amino acids are substituted. Sequence variants wherein no proline is introduced at the positions
  • the peptide sequences described herein can be used as suitable monomeric peptide units which constitute binding domains for the EPO receptor. They can be used in there monomeric form since they bind to the EPO receptor. As described herein, they are preferably used as dimers since it was shown that the capacity to induce dimerisation of the EPO receptor and thus biological activity is enhanced by dimerisation of the monomeric binding units.
  • 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.
  • the present invention also includes modifications of the peptides and defined peptide consensuses by conservative exchanges of single amino acids. Such exchanges alter the structure and function of a binding molecule but only slightly in most cases. In a conservative exchange, one amino acid is replaced by another amino acid within a group with similar properties.
  • amino acids having non-polar side chains A, G, V, L, I, P, F, W, M
  • uncharged amino acids having polar side chains S, T, G, C, Y, N, Q
  • amino acids of similar size or molecular weight wherein the molecular weight of the replacing amino acids deviates by a maximum of +/- 25% (or +/- 20%, +/- 15%, +/- 10%) from the molecular weight of the original amino acid.
  • the groups also include non-natural amino acids with the respective side chain profile such as e.g. homoarginine in case of the group depicting positively charged side chains.
  • a proline 10 substituting molecule such as e.g. a non-natural amino acid cannot be clearly assigned to one of the above groups characterized by their side-chain properties, it should usually be perceived as a non-conservative substitution of proline according to this invention.
  • the classification aid according to the molecular weight might be helpful.
  • 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.
  • these peptides are modified as to AcG at the N-terminus and MeG at the C-terminus.
  • the described peptides of the invention can be regarded as monomeric binding domains recognizing the binding site of the erythropoietin receptor.
  • two of these binding domains are generally needed in order to homodimerize the receptor and to induce signal transduction.
  • a combination of two of these binding domains in one single molecule enhanced activity considerably, leading to the result that peptides with one single binding domain showed the same qualitative pattern of activity while two of the binding domains joint together show a much lower ED50 (Effect Dose 50%, a measure of activity).
  • Peptides harboring two binding domains are specified as being bivalent or dimeric peptides within the context of this description and are particularly preferred.
  • binding domains are first synthesized separately as monovalent or monomeric peptides, which can be modified e.g. by attachment of reactive groups in preparation for step b b) in a second reaction step, two - in most cases identical - binding domains are joined together in separate dimerization reaction, which can also include linker molecules usually being interposed between the two dimerised domains.
  • dimers are examples of bivalent peptides and exhibit essentially the same biological functions as the monomers. Usually, they show enhanced biological activity in case of EPO mimetic peptides.
  • Monomers can be dimerized e.g. by covalent attachment to a linker.
  • a linker is a joining molecule creating a covalent bond between the polypeptide units of the present invention.
  • the polypeptide units can be combined via a linker in such a way, that the binding to the EPO receptor is improved (Johnson et al. ,1997; Wrighton et al. 1997).
  • the linker comprises NH-R-NH wherein R is a lower alkylene substituted with a functional group such as carboxyl group or amino group that enables binding to another molecule moiety.
  • the linker might contain a lysine residue or lysine amide.
  • PEG may be used a linker.
  • the linker can be a molecule containing two carboxylic acids and optionally substituted at one or more atoms with a functional group such as an amine capable of being bound to one or more PEG molecules.
  • a peptide monomer or dimer may further comprise at least one spacer moiety.
  • spacer connects the linker of a monomer or dimer to a water soluble polymer moiety or a protecting group, which may be e.g. PEG.
  • the PEG has a preferred molecular weight of at least 3 kD, preferably between 20 and 60 kD.
  • the spacer may be a C1-12 moiety terminated with -NH-linkages or COOH- groups and optionally substituted at one or more available carbon atoms with a lower alkyl substituent.
  • a particularly preferred spacer is disclosed in WO 2004/100997. All documents - WO 2004/100997 and WO 2004/101606 - are incorporated herein by reference.
  • the PEG modification of peptides is disclosed in WO 2004/101600, which is also incorporated herein by reference.
  • the core concept of this strategy refrains from synthesizing the monomeric peptides forming part of the multi- or bivalent peptide in separate reactions prior dimerization or multimerization, but to synthesize the final bi- or multivalent peptide in one step as a single peptide; e.g. in one single solid phase reaction. Thus a separate dimerization or multimerization step is no longer needed.
  • This aspect provides a big advantage, i.e. the complete and independent control on each sequence position in the final peptide unit.
  • the method allows to easily harbor at least two different receptor-specific binding domains in a peptide unit due to independent control on each sequence position.
  • the sequence of the final peptide between the bind ⁇ ing domains (which is the "linker region") is composed of amino acids only, thus leading to one single, continuous bi- or multivalent EPO mimetic peptide.
  • the linker is composed of natural or unnatural amino acids which allow for a high conformational flexibility.
  • glycine residues as linking amino acids, which are known for their high flexibility in terms of torsions.
  • other amino acids such as alanine or beta-alanine, or a mixture thereof can be used. The number and choice of used amino acids depend on the respective steric facts.
  • 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.
  • each sequence position within the final peptide is under control and thus can be precisely determined it is possible to custom- or tailor make the peptides or specific regions or domains thereof, including the linker.
  • This is of specific advantage since it allows the avoidance of distortion of the bioactive conformation of the final bivalent peptide due to unfavorable intramolcular interactions.
  • the risk of distortions can be assessed prior to synthesis by molecular modeling. This especially applies to the design of the linker between the monomeric domains.
  • the continuous bivalent/multivalent peptides according to the invention show much higher activity then the corresponding monomeric peptides and thus confirm the observation known from other dimeric peptides that an increase of efficacy is associated with bivalent peptide concepts.
  • the continuous bivalent/multivalent peptides can be modified by e.g. acetylation or amidation or be elongated at C- terminal or N-terminal positions.
  • the prior art modifications for the monomeric peptides (monomers) mentioned above including the attachments of soluble moieties such as PEG, starch or dextrans are also applicable for the multi- or bivalent peptides according to the invention.
  • linker All possible modifications also apply for modifying the linker.
  • soluble polymer moieties to the linker such as e.g. PEG, starch or dextrans.
  • the synthesis of the final multi- or bivalent peptide according to the invention favorably can also include two subsequent and independent formations of disulfide bonds or other intramolecular bonds within each of the binding domains. Thereby the peptides can also be cylized.
  • the bivalent structures according to the invention are favorably formed on the basis of the peptide monomers reported herein.
  • SEQ ID NO 10 (based on SEQ ID NO 2):
  • the linker in these bivalent structures is custom-made by molecular modelling to avoid distortions of the bioactive conformation (fig. 1 ).
  • the linker sequence can be shortened by one glycine residue. This sequence is also an example for a linker composed by glycine residue forming at the same time part of the binding domain (see SEQ ID NO 2).
  • binding domains can also be used as a monomer sequence (SEQ ID NO 13)
  • 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.
  • the second sequence depicting the non-natural amino acid homoarginine is especially suitable for use in a pharmaceutical composition for veterinary purposes. It was generally found that peptide sequences carrying an amino acid with a long positively charged side chain such as for example homoarginine in positions 10 and/or 17 depict a strong binding capacity to EPO receptors such as e.g. the mouse/dog receptor. They are thus especially suitable for use in veterinary products, however, their use is not limited thereto.
  • the reactive side chains may serve as a linking tie e.g. for further modifications.
  • the peptides furthermore optionally comprise intramolecular disulfide bridges between the first and second and/or third and fourth cysteine.
  • the linker/spacer between the monomers can contain a diketopiperazine unit.
  • a preferred GIy-GIy diketopiperazine scaffold can be achieved by activating the C-terminal glycine monomer. This principle can also be use for forming a C-terminal dimerization.
  • the monomer (SEQ ID NO 17) can also be applied as a EPO mimetic peptide.
  • the resulting dimers on the basis of SEQ ID NO 2 elongated at the N-Terminus by one glycine residue contain hexanedioyl unit as linker/spacer (fig. 6):
  • the dimerization can be achieved by using a octanedioyl unit as linker/spacer (fig. 7):
  • This can include:
  • Respective assembly methods as described above can also be used for the preparation of multimers.
  • binding domains respectively peptides described herein either alone or as a part of a bivalent/multivalent peptide can also be used in a monomeric form and/or can be combined with one or more other either identical or different peptide domains in order to form respective homo- or heterogenous bi- or multivalent peptides.
  • the peptides can be modified by e.g. acetylation or amidation or be elongated at the C-terminal or N-terminal positions. Extension with one or more amino acids at one of the two termini, e.g. for preparation of an attachment site for a polymer often leads to a heterodimeric bivalent peptide unit which can best be manufactured as a continuous peptide.
  • the compounds of the present invention can advantageously be used for the preparation of human and/or veterinarian pharmaceutical compositions.
  • EPO mimetics they depict the basically the same qualitative activity pattern as erythropoietin. They are thus generally suitable for the same indications as erythropoietin.
  • Erythropoietin is a member of the cytokine superfamily. Besides the stimulating effects described in the introduction, it was also found that erythropoietin stimulates stem cells. The EPO mimetics described herein are thus suitable for all indications caused by stem cell associated effects.
  • Non-limiting examples are the prevention and/or treatment of diseases associated with the nerve system. Examples are neurological injuries, diseases or disorders, such as e.g. Parkinsonism, Alzheimer's disease, Huntington's chorea, multiple sclerosis, amyotrophic lateral sclerosis, Gaucher's disease, Tay-Sachs disease, a neuropathy, peripheral nerve injury, a brain tumor, a brain injury, a spinal cord injury or a stroke injury.
  • neurological injuries, diseases or disorders such as e.g. Parkinsonism, Alzheimer's disease, Huntington's chorea, multiple sclerosis, amyotrophic lateral sclerosis, Gaucher's disease, Tay-Sachs disease, a neuropathy, peripheral
  • the EPO mimetic peptides according to the invention are also usable for the preventive and/or curative treatment of patients suffering from, or at risk of suffering from cardiac failure.
  • cardiac infarction coronary artery disease, myocarditis, chemotherapy treatment, alcoholism, cardiomyopathy, hypertension, valvar heart diseases including mitral insufficiency or aortic stenosis, and disorders of the thyroid gland, chronic and/or acute coronary syndrome.
  • EPO mimetics can be used for stimulation of the physiological mobilization, proliferation and differentiation of endothelial precursor cells, for stimulation of vasculogenesis, for the treatment of diseases related to a dysfunction of endothelial precursor cells and for the production of pharmaceutical compositions for the treatment of such diseases and pharmaceutical compositions comprising said peptides and other agents suitable for stimulation of endothelial precursor cells.
  • diseases are hypercholesterolaemia, diabetis mellitus, endothel-mediated chronic inflammation diseases, endotheliosis including reticulo-endotheliosis, atherosclerosis, coronary heart disease, myocardic ischemia, angina pectoris, age-related cardiovascular diseases, Raynaud disease, pregnancy induced hypertonia, chronic or acute renal failure, heart failure, wound healing and secondary diseases.
  • the peptides according to the invention are suitable carriers for delivering agents across the blood-brain barrier and can be used for respective purposes and/or the production of respective therapeutic conjugation agents capable of passing the blood-brain barrier.
  • the peptides described herein are especially suitable for the treatment of disorders that are characterized by a deficiency of erythropoietin or a low or defective red blood cell population and especially for the treatment of any type of anemia or stroke.
  • the peptides are also suitable for increasing and/or maintaining hematocrit in a mammal.
  • Such pharmaceutical compositions may optionally comprise pharmaceutical acceptable carriers in order to adopt the composition for the intended administration procedure. Suitable delivery methods as well as carriers and additives are for example described in WO 2004/101611 and WO 2004/100997.
  • dimerization of the monomeric peptides to dimers or even multimers usually improves the EPO mimetic agonist activity compared to the respective monomeric peptides. However, it is desirable to further enhance activity. For example, even dimeric EPO mimetic peptides are less potent than the EPO regarding the activation of the cellular mechanisms.
  • a further embodiment of the present invention provides a solution to that problem.
  • a compound is provided that binds target molecules and com ⁇ prises
  • each peptide unit comprises at least two domains with a binding capacity to the target
  • bivalent as used for the purpose of the present invention is defined as a peptide comprising two domains with a binding capacity to a target, here in particular the EPO receptor. It is used interchangeably with the term “dimeric”. Accordingly, a “multivalent” or “multimeric” EPO mimetic peptide has several respective binding domains for the EPO receptor. It is self-evident that the terms “peptide” and “peptide unit” do not incorporate any restrictions regarding size and incorporate oligo- and polypeptides as well as proteins.
  • 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.
  • the EPO mimetic peptide units used in this embodiment can be either homo- or heterogenic, meaning that either identical or differing peptide units are used.
  • the bi- or multivalent peptide units bound to the carrier unit bind the same receptor target. However, they can of course still differ in their amino acid sequence.
  • the monomeric binding domains of the bi- or multivalent peptide units can be either linear or cyclic.
  • a cyclic molecule can be for example created by the formation of intramolecular cysteine bridges (see above).
  • the polymeric carrier unit comprises at least one natural or synthetic branched, linear or dendritic polymer.
  • the polymeric carrier unit is preferably soluble in water and body fluids and is preferably a pharmaceutically acceptable polymer.
  • Water soluble polymer moieties include, but are not limited to, e.g. polyalkylene glycol and derivatives thereof, including PEG, PEG homopolymers, mPEG, polypropyleneglycol homopolymers, copolymers of ethylene glycol with propylene glycol, wherein said homopolymers and copoloymers are unsubstituted or substituted at one end e.g.
  • acylgroup polyglycerines or polysialine acid
  • cellulose and cellulose derivatives including methylcellulose and carboxymethylcellulose
  • starches e.g. hydroxyalkyl starch (HAS), especially hydroxyethyl starch (HES) and dextrines, and derivatives thereof
  • dextran and dextran derivatives including dextransulfat, crosslinked dextrin, and carboxymethyl dextrin
  • heparin and fragments of heparin polyvinyl alcohol and polyvinyl ethyl ethers
  • polyvinylpyrrollidon a,b-poly[(2-hydroxyethyl)-DL- aspartatamide; and polyoxyethylated polyols.
  • an appropriate carrier unit is a homobifunctional polymer, of for example polyethylene glycol (bis-maleimide, bis-carboxy, bis-amino etc.).
  • the polymeric carrier unit can have a wide range of molecular weight due to the different nature of the different polymers that are suitable in conjunction with the present invention. There are thus no size restrictions. However, it is preferred that the molecular weight is at least 3 kD, preferably at least 1OkD and approximately around 20 to 500 kD and more preferably around 30 to 150 or around 60 or 80 kD.
  • the size of the carrier unit depends on the chosen polymer and can thus vary. For example, especially when starches such as hydroxyethylstarch are used, the molecular weight might be considerably higher. The average molecular weight might then be arranged around 100 to 4.000 kD or even be higher.
  • the size of the carrier unit is preferably chosen such that each peptide unit is optimally arranged for binding their respective receptor molecules.
  • a carrier unit comprising a branching unit.
  • the polymers as for example PEG, are attached to a branching unit thus resulting in a large carrier molecule allowing the incorporation of numerous peptide units.
  • branching units are glycerol or polyglycerol.
  • dendritic branching units can be used as for example taught by Haag 2000, herein incorporated by reference.
  • the polymeric carrier unit is connected to the peptide units.
  • the polymeric carrier unit is connected to the peptide units via a covalent or a non-covalent (e.g. a coord inative) bond.
  • 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.
  • the polymeric carrier unit does not possess an appropriate coupling group
  • several coupling substances can be used in order to appropriately modify the polymer in order that it can react with at least one reactive group on the peptide unit.
  • Suitable chemical groups that can be used to modify the polymer are e.g. as follows:
  • 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;
  • alkylating groups which react with sulfhydryl (mercapto), thiomethyl, imidazo or amino groups on the peptide unit, such as halo-carboxyl groups, maleimide groups, activated vinyl groups, ethylenimine groups, aryl halide groups, 2- hydroxy 5-nitro-benzyl bromide groups; and aliphatic aldehyde and ketone groups together with reducing agents, reacting with the amino group of the peptide;
  • ester and amide forming groups which react with a carboxyl group of the protein, such as diazocarboxylate groups, and carbodiimide and amine groups together;
  • disulfide forming groups which react with the sulfhydryl groups on the protein, such as 5,5'-dithiobis (2-nitrobenzoate) groups and alkylmercaptan groups (which react with the sulfhydryl groups of the protein in the presence of oxidizing agents such as iodine);
  • dicarbonyl groups such as cyclohexandione groups, and other 1 ,2-diketone groups which react with the guanidine moieties of the peptide;
  • 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.
  • a targeted approach for attaching the peptide units to the polymeric carrier unit In order to generate a defined molecule it is preferred to use a targeted approach for attaching the peptide units to the polymeric carrier unit. In case no appropriate amino acids are present at the desired attachment site, appropriate amino acids can be incorporated in the dimeric EPO mimetic peptide unit. For site specific polymer attachment a unique reactive group e.g. a specific amino acid at the end of the peptide unit is preferred in order to avoid uncontrolled coupling reactions throughout the peptide leading to a heterogeneous mixture comprising a population of several different polyethylene glycol molecules.
  • 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).
  • PEGylation is usually undertaken to improve the biopharmaceutical properties of the peptides.
  • the most relevant alterations of the proteine molecule following PEG conjugation are size enlargement, protein surface and glycosylation function masking, charge modification and epitope shielding.
  • size enlargement slows down kidney ultrafiltration and promotes the accumulation into permeable tissues by the passive enhance permeation and retention mechanism.
  • Protein shielding reduces proteolysis and immune system recognition, which are important routes of elimination.
  • the specific effect of PEGylation on protein physicochemical and biological properties is strictly determined by protein and polymer properties as well as by the adopted
  • dosage intervals in a clinical setting are triggered by loss of effect of the drug.
  • Routine dosages and dosage intervals are adapted such that the effect is not lost during dosage intervals.
  • peptides are usually PEGylated with very large PEG-moieties ( ⁇ 20-40kD) which thus show a slow renal elimination.
  • PEG-moieties ⁇ 20-40kD
  • the peptide moiety itself undergoes enzymatic degradation and even partial cleavage might suffice to deactivate the peptide.
  • 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.
  • hydroxyalkylstarch and preferably HES is used as polymeric carrier unit.
  • HES has several important advantages. First of all, HES is biodegradable. Furthermore, the biodegradability of HES can be controlled via the ratio of ethyl groups and can thus be influenced. 30 to 50 % ethyl groups are well suitable for the purpose of the present invention. Due to the biodegradability, accumulation problems as described above in conjunction with PEG do usually not occur. Furthermore, HES has been used for a long time in medical treatment e.g. in form of a plasma expander. Its innocuousness is thus approved.
  • HES-peptide conjugates can be hydrol ⁇ sed under conditions under which the peptide units are still stable. This allows the quantification and monitoring of the degradation products and allows evaluations and standardisations of the active peptides.
  • a first type of polymeric carrier unit is used and loaded with peptide units.
  • This first carrier is preferably easily biodegradable as is e.g. HES.
  • not all attachment spots of the first carrier are occupied with peptide units but only e.g. around 20 to 50%.
  • several hundred peptide units can be coupled to the carrier molecule.
  • the rest of the attachment spots of the first carrier are occupied with a different carrier, e.g. small PEG units having a lower molecular weight than the first carrier.
  • 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.
  • the monomers constituting the binding domains of the peptide units recognize the homodimeric erythropoietin receptor.
  • the latter property of being a homodimeric receptor differentiates the EPO-receptbr from many other cytokine receptors.
  • the peptide units comprising at least two EPO mimetic monomeric binding domains as described above bind the EPO receptor and preferably are able to di- respectively multimerise their target and/or stabilize it accordingly thereby creating a signal transduction inducing complex.
  • the present invention also comprises respective compound production methods, wherein the peptide units are connected to the respective carrier units.
  • the present invention furthermore comprises respective compound production methods, wherein the peptide units are connected to the respective polymeric carrier units.
  • the compounds of the present invention can advantageously be used for the preparation of human and/or veterinarian pharmaceutical composi ⁇ tions. They can be especially suitable for the treatment of disorders that are char- acterized by a deficiency of erythropoietin or a low or defective red blood cell population and especially for the treatment of any type of anemia and stroke. They are also usable for all indications described above.
  • Such pharmaceutical compositions may optionally comprise pharmaceutical acceptable carriers in order to adopt the composition for the intended administration procedure. Suitable delivery methods as well as carriers and additives are for example described in WO 2004/100997 and WO 2004/101611 , herein incorporated by reference. EXAMPLES
  • Fig. 13 shows an example of a simple supravalent molecule according to the invention.
  • Two continuous bivalent EPO mimetic peptides are connected N- terminally by a bifunctional PEG moiety carrying maleimide groups. Cysteine was chosen as reactive attachment site for the PEG carrier unit.
  • supravalent molecules can comprise more than two continuous bi- or multivalent peptide units.
  • Fig. 14 gives an example that is based on a carrier unit with a central glycerol unit as branching unit and comprising three continuous bivalent peptides. Again cysteine was used for attachment.
  • Fig. 20 shows an example using HES as polymeric carrier unit. HES was modified such that it carries maleimide groups reacting with the SH groups of the peptide units. According to the example, all attachment sites are bound to peptide units. However, also small PEG units (e.g. 3 to 10 kD) could occupy at least some of the attachment sites.
  • the supravalent concept can also be extended to polyvalent dendritic polymers wherein a dendritic and/or polymer carrier unit is connected to a larger number of continuous bivalent peptides.
  • the dendritic branching unit can be based on polyglycerol (please refer to Haag 2000, herein incorporated by reference).
  • a supravalent molecule based on a carrier unit with a dendritic branching unit containing six continuous bivalent peptides is shown in Fig. 15.
  • supravalent molecules comprise carrier units with starches or dextrans, which are oxidized using e.g. periodic acid to harbor a large number of aldehyde functions.
  • many bivalent peptides are attached to the carrier unit and together form the final molecule.
  • many bivalent peptide units can be coupled to the carrier molecule, which is e.g. HES.
  • Fig. 16 demonstrates the concept of a simple biodegradable supravalent molecule.
  • Two continuous bivalent EPO mimetic peptides are connected N-terminally by two bifunctional PEG moieties that are connected via a biodegradable linker having an intermediate cleavage position. The linkers allow the break up of the large PEG unit in the subunits thereby facilitating renal clearance.
  • the synthesis is carried out by the use of a Discover microwave system (CEM) using PL-Rink-Amide-Resin (subtitution rate 0.4mmol/g) or preloaded Wang- Resins in a scale of 0.4mmol.
  • CEM Discover microwave system
  • Removal of Fmoc-group is achieved by addition of 30ml piperidine/DMF (1 :3) and irradiation with 100W for 3x30sec.
  • Coupling of amino acids is achieved by addition of 5fold excess of amino acid in DMF PyBOP/HOBT/DIPEA as coupling additives and irradiation with 5OW for 5x30sec. Between all irradiation cycles the solution is cooled manually with the help of an ice bath.
  • the synthesis is carried out by the use of an Odyssey microwave system (CEM) using PL-Rink-Amide-Resins (subtitution rate 0.4mmol/g) or preloaded Wang- Resins in a scale of 0.25mmol.
  • CEM Odyssey microwave system
  • Removal of Fmoc-groups is achieved by addition of 10ml piperidine/DMF (1 :3) and irradiation with 100W for 10x10sec.
  • Coupling of amino acids is achieved by addition of 5fold excess of amino acid in DMF PyBOP/HOBT/DIPEA as coupling additives and irradiation with 50W for 5x30sec. Between all irradiation cycles the solution is cooled by bubbling nitrogen through the reaction mixture.
  • peptides were purified using a Nebula-LCMS-system (Gilson). The crude material of all peptides was dissolved in acetonitrile / water (1/1) and the peptide purified by RP-HPLC (Kromasil 100 C18 10 ⁇ m, 250x4.6mm). The flow rate was 20ml/min and the LCMS split ratio 1/1000.
  • Solutioni 10mg of the peptide are dissolved in 0.1 % TFA/acetonitrile and diluted with water until a concentration of 0.5mg/ml is reached. Solid ammonium bicarbonate is added to reach a pH of app. 8.
  • Solution 2 In a second vial 10ml 0.1% TFA/acetonitrile are diluted with 10ml of water. Solid ammoniumbicarbonate is added until a pH of 8 is reached and 1 drop of a 0.1 M solution of K 3 [(FeCN 6 )] is added.
  • peptides were purified using a Nebula-LCMS-system (Gilson). The crude material of all peptides was dissolved in acetonitrile/water (1/1) or DMSO and the peptide was purified by RP-HPLC (Kromasil 100 C18 or C8 10 ⁇ m, 250x4.6mm). The flow rate was 20ml/min and the LCMS split ratio 1/1000.
  • TF-1 Cells in logarithmic growth phase (-2.10 5 - 1.10 6 cells/ml; RPMI medium; 20% fetal calf serum; supplemented with Penicillin, streptomycin, L-Glutamine; 0.5ng/ml lnterleukin 3) are washed (centrifuge 5 min. 1500 rpm and resuspend in RPMI complete without IL3 at 500.000 cells/ml) and precultured before start of the assay for 24 h without IL-3. At the next day the cells are seeded in 24- or 96- well plates usually using at least 6 concentrations and 4 wells per concentration containing at least 10.000 cells/well per agent to be tested.
  • Each experiment includes controls comprising recombinant EPO as a positive control agent and wells withoug addition of cytokine as negative control agent.
  • Peptides and EPO- controls are prediluted in medium to the desired concentrations and added to the cells, starting a culture period of 3 days under standard culture conditions (37 0 C, 5% carbon dioxide in the gas phase, atmosphere saturated with water) . Concentrations always refer to the final concentration of agent in the well during this 3-day culture period. At the end of this culture period, FdU is added to a final concentration of 8ng/ml culture medium and and the culture continued for 6 hours.
  • BrdU bromodeoxyuridine
  • dCd (2-deoxycytidine
  • the cells are washed once in phosphate buffered saline containing 1.5% BSA and resuspended in a minimal amount liquid. From this suspension, cells are added dropwise into 70% ethanol at -20 0 C. From here, cells are either incubated for 10min. on ice and then analysed directly or can be stored at 4°C prior to analysis.
  • cells Prior to analysis, cells are pelleted by centrifugation, the supernatant is dis ⁇ carded and the cells resuspended in a minimal amount of remaining fluid. The cells are then suspended and incubated for 10min. in 0.5 ml 2M HCI/ 0.5% triton X-100. Then, they are pelleted again and resuspended in a minimal amount of remaining fluid, which is diluted with 0.5ml of 0.1 N Na 2 B 4 0 7 , pH 8.5 prior to immediate repelleting of the cells. Finally, the cells are resuspended in 40 ⁇ l of phosphate buffered saline (1.5% BSA) and divided into two reaction tubes containing 20 ⁇ l cell suspension each.
  • phosphate buffered saline (1.5% BSA
  • 2 ⁇ l of anti-Brd U-FITC (DAKO, clone Bu20a) are added to one tube and 2 ⁇ l control mlgG1-FITC (Sigma) are added to the second tube starting an incubation period of 30min. at room temperature. Then, 0.4ml of phosphate buffered saline and 10 ⁇ g/ml Propidium Iodide (final concentration) are added. Analysis in the flow cytometer refers to the fraction of 4C cells or cells with higher ploidy and to the fraction of BrdU-positive cells, thus determining the fraction of cells in the relevant stages of the cell cycle.
  • TF-1 Cells in logarithmic growth phase (-2.10 5 - 1.10 6 cells/ml; RPMI medium; 20% fetal calf serum; supplemented with Penicillin, streptomycin, L-Glutamine; 0.5ng/ml lnterleukin 3) are washed (centrifuge 5 min. 1500 rpm and resuspend in RPMI complete without IL3 at 500.000 cells/ml) and precultured before start of the assay for 24 h without IL-3. At the next day the cells are seeded in 24- or 96- well plates usually using at least 6 concentrations and 4 wells per concentration containing at least 10.000 cells/well per agent to be tested.
  • Each experiment includes controls comprising recombinant EPO as a positive control agent and wells without addition of cytokine as negative control agent.
  • Peptides and EPO- controls are prediluted in medium to the desired concentrations and added to the cells, starting a culture period of 3 days under standard culture conditions (37 0 C, 5% carbon dioxide in the gas phase, atmosphere saturated with water). Concentrations always refer to the final concentration of agent in the well during this 4-day culture period.
  • a dilution series of a known number of TF- 1 cells is prepared in a number of wells (0/2500/5000/10000/20000/50000 cells/well in 100 ⁇ l medium). These wells are treated in the same way as the test wells and later provide a calibration curve from which cell numbers can be determined. Having set up these reference wells, MTS and PMS from the MTT proliferation kit (Promega, CellTiter 96 Aqueous non-radioactive cell proliferation assay) are thawed in a 37 0 C waterbath and 100 ⁇ l of PMS solution are added to 2ml of MTS solution.
  • MTT proliferation kit Promega, CellTiter 96 Aqueous non-radioactive cell proliferation assay
  • the synthesis is carried out by the use of a Liberty microwave system (CEM) using Rink-Amide-Resin (subtitution rate 0.19mmol/g) in a scale of 0.25mmol.
  • CEM Liberty microwave system
  • Removal of Fmoc-groups is achieved by double treatment with 10ml piperidine/DMF (1 :3) and irradiation with 5OW for 10x10sec.
  • Coupling of amino acids is achieved by double treatment with a of 4fold excess of amino acid in DMF PyBOP/HOBT/DIPEA as coupling additives and irradiation with 5OW for 5x30sec. Between all irradiation cycles the solution is cooled by bubbling nitrogen through the reaction mixture.
  • the resin After deprotection and coupling, the resin is washed 6 times with 10ml DMF. After the double coupling cycle all unreacted amino groups are blocked by treatment with a 10fold excess of N-(2- Chlorobenzyloxycarbonyloxy)succinimide (0.2M solution in DMF) and irridation with 5OW for 3x30sec. After deprotection of the last amino acid, the peptide is acetylated by incubation with 0.793ml of capping-solution (4.73ml acetic anhydride and 8.73ml DIEA in 100ml DMSO) for 5 minutes. Before cleavage the resin is then washed 6 times with 10ml DMF and 6 times with 10ml DCM.
  • Solution A Acetonitrile / water (1 / 1 ) containing 0.1 % TFA. The pH is adjusted to 8.0 by the addition of ammoniumbicarbonate.
  • cyclic AGEM11 The purification parameters for cyclic AGEM11 are given in fig. 10 and 11 (scheme: Purification of cyclic AGEM11 , Kromasil 100 C18 10 ⁇ m, 250x4.6mm, gradient from 5% to 35% acetonitrile (0.1 % TFA) in 50 minutes).
  • TF1 cells in logarithmic growth phase (2.10 5 - 1.10 6 cells/ml grown in RPMI with
  • FCS fetal calf serum
  • IL-3 0.5 ng/ml IL-3
  • Pretreated (starved) cells were centrifuged (5 min. 1500 rpm) and resuspended in RPMI with 5% FCS at a concentration of 200 000 cells per ml. Fifty ⁇ l of cell suspension (containing 10 000 cells) was added to each well. Note that due to the addition of the cells the final concentrations of the substances in the wells were half that of the original dilution range. Plates were incubated for 72 h at 37 0 C in 5% CO 2 .
  • a dilution range of known amounts of TF-1 cells into wells was prepared: 0/2500/5000/10000/20000/50000 cells/well were pipetted (in 100 ⁇ l RPMI + 5% FCS) in quadruplicate.
  • MTT reagent Promega, CellTiter 96 Aqueous One Solution Cell Proliferation Assay
  • 20 ⁇ l of MTT reagent was added, and plates were incubated at 37 0 C in 5% CO 2 for another 1-2 h.
  • Twentyfive ⁇ l of a 10% SDS solution was added, and plates were measured in an ELISA reader (Genios, Tecan). Data were processed in spreadsheets (Excel) and plotted in Graphpad.
  • the peptides were synthesized as peptides amides on a LIPS-Vario synthesizer system.
  • the synthesis was performed in special MTP-synthesis Plates, the scale was 2 ⁇ mol per peptide.
  • the synthesis followed the standard Fmoc-protocol using HOBT as activator reagent.
  • the coupling steps were performed as 4 times coupling. Each coupling step took 25 min and the excess of amino acid per step was 2.8.
  • the cleavage and deprotection of the peptides was done with a cleavage solution containing 90% TFA, 5% TIPS, 2.5% H 2 O and 2.5% DDT.
  • the synthesis plate containing the finished peptide attached to the resin was stored on top of a 96 deep well plate.
  • 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.
  • Figure 19 gives an overview over the synthesised and tested peptides monomers.
  • the peptides were tested for their EPO mimetic activity in an in vitro proliferation assay.
  • the assay was performed as described under V. On each assay day, 40 microtiter plates were prepared for measuring in vitro activity of 38 test peptides, 1 reference example, and EPO in parallel. EPO stocks solutions were 20 nM.
  • the aim of the described method is the production of a derivative of a starch, according to this example HES, which selectively reacts with thiol groups under mild, aqueous reaction conditions. This selectivity is reached with maleimide groups.
  • HES is functionalised first with amino groups and converted afterwards to the respective maleimide derivative.
  • the reaction batches were freed from low molecular reactants via ultra membranes.
  • the product, the intermediate products as well as the educts are all poly-disperse.
  • Hydroxyethylstarch (Voluven®) was attained via diafiltration and subsequent freeze-drying. The average molar weight was approximately 130 kDa with a substitution grade of 40 %.
  • the synthesis was performed according to the synthesis described for amino dextran in the dissertation of Jacob Piehler, ,,Modatty von Oberflachen fur die thermodynamische und kinetician purmaschine biomolekularer Erkennung mitmmis Transducem", 1997, herein incorporated by reference. HES was activated by partial, selective oxidation of the diolic hydroxyl groups to aldehyde groups with sodium periodate as described in Floor et. al (1989). The aldehyd groups were converted via reductive amination with sodiumcyanoborhydride (NaCNBHa) in the presence of ammonia to amino groups (Yalpani and Brooks, 1995).
  • NaCNBHa sodiumcyanoborhydride
  • the working-up was performed via ultra filtration and freeze-drying
  • TNBS 2,4,6-trinitrobenzole sulphonic acid
  • 3-maleimidopropione acid-N-hydroxysuccinimidester (MaIPA-OSu) was used in excess (10-fold) (50 mM phosphate buffer, pH 7, 20 % DMF, over night), working-up via ultra filtration and freeze-drying.
  • 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.
  • the UV-absorption was determined at 280 nm and the remaining content of maleimide groups was determined with GSH/DNTB.
  • Recombinant human EPO (rhuEPO) was used as a control.
  • rhuEPO Recombinant human EPO
  • the numerical values depicted in Fig. 22 represent the %cpm of the total counts used in the IP. A serum is assessed as positive when the %cpm value is > 0.9. 100% cpm represents the amount of the overall used counts (the radioactive tracer), presently the radioactively labelled EPO.
  • the assay demonstrates that the EPO mimetic peptides according to the invention depict advantageously no cross-reactivity to anti-EPO antibodies.
  • the EPO mimetic peptides described herein should thus depict a therapeutic effect even in patients who developed antibodies against rhuEPO. Furthermore, it is expected, that antibodies against EPO mimetic peptides should not bind erythropoietin.
  • EPO mimetic peptides according to this invention are thus preferably also characterised in that they show no significant cross-reactivity with anti-EPO antibodies.

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