BRPI0611745A2 - supravalent compounds - Google Patents

Abstract

SUPRAVALENT COMPOUNDS. The present invention relates to supravalent peptide compounds having enhanced efficacy. Supravalent compounds comprise at least several bivalent peptide units that bind to a target receptor and are attached to a large polymeric carrier unit.

Description

Patent Descriptive Report for "SUPRAV ALENT COMPOUNDS".

The present invention relates to peptide compounds.

With the development of protein research, a large number of peptides having various biological and pharmaceutical activities was found. Several of these peptides perform their actions via binding to target molecules such as receptor molecules (such as cytokine receptors).

Hematopoietic growth factors (HGFs) have been proven to be clinically successful therapeutic products; however, its size (15-70kDa), conformational instability, susceptibility to proteolytic degradation, poor membrane penetration, antigenicity, high cost of unfavorable pharmacokinetic production can make the same drug candidates less than ideal. In addition, the poor availability of native proteins requires that they be administered parenterally. It is therefore advantageous to develop small molecule agonists (and antagonists) of HGF receptors that are equipotent to their polypeptide counterparts but lack some of the inherent deficiencies of large proteins. Identification and examination of smaller peptides that bind to and activate cytokine receptors also provides a better understanding of ligand-receptor interactions. This information is used to design orally available small molecule cytokine mimetics in a rational manner. Activation of transmembrane receptors by ecitocin growth factors occurs when a ligand binds to a specific domain on the receptor, thereby inducing a conformational change and triggering dimerization or oligomerization of receptor chains. When ligand ligation, several class I cytokine receptor members are homodimers, including the erythropoietin receptor (EPOFt), the thrombopoietin receptor (TPOR) 1, degranulocyte colony stimulating factor receptor (G-CSFR), growth hormone (GHR) and prolactin receptor (PrP). Several studies have been reported that address the discovery of precise details of the dimerization interfaces and the degree to which unbound receptors exist as dimers. The results of these studies have shown structural and functional similarities between class I cytokine receptors. Studies have also shown that receptor dimerization alone, although necessary for intracellular signaling, is not sufficient to produce signal transduction. Recent receptors have shown that small molecules and peptides can bind to homodimeric cytokine activators by acting as agonists and mimicking the effects of natural proteins (see Laber, EG (2004). Small-Molecule and Peptide Agonists: A Literature Review. Hematopoietic GrowthFactors in Oncoloqy - Basic Science and Clinical Therapeutics (G. Morstyn, M. Foote and GJ Lieschke: 65-80). However, their biological activity is often inferior to natural molecules. Consequently, attempts are made to improve the biological activity of mimetic molecules.

Successful examples of such peptides include erythropoietin receptor-binding peptides that mimic the function of erythropoietin and thrombopoietin receptor-binding peptides that mimic the function of thrombopoietin.

Erythropoietin hormone (EPO) is a glycoprotein consisting of 165 amino acids and having four glycosylation sites. It stimulates mitotic divisions and differentiation of erythrocyte precursor cells and thus ensures the production of erythrocytes. Since the use of EPO or recombinant EPO has several disadvantages, including immunogenic reactions, synthetic peptides are used which do not share any sequence homology or structural relationship with EPO but anyway bind and interact like EPO-R. (see, for example, Wrighton et al., 1996). Synthetic peptides that mimic EPO activity ("EPO mimetic peptides") are, for the time being, well known in the art (see, for example, WO 96/40772; WO 96/40749; WO 01/38342; WO 01/091780; WO 2004/101611; WO 2004/100997; WO 2004/101600; WO 2004/101606).

EPO and EPO mimetic peptides activate the EPO receptor by binding receptor extracellular domains and presumably dimerizing two receptor monomers to a receptor complex, thereby initiating signal transduction (Johnson et al., 1997). ). The crystal structure of the EMP1-linked EPO receptor (a well-known EPO mimetic peptide) revealed the formation of a receptor-peptide complex consisting of two peptides attached to two receptor monomers. Thus, it was not really surprising that the combination of exactly two of these binding domains in a single molecule greatly enhanced activity, leading to the result that common peptides in the single binding domain showed the same qualitative pattern of activity, whereas two of the binding domains joined together show a much smaller ED50 (50% dose to effect, a measure of activity).

Methods of preparation for the respective peptide dimers of, for example, EPO or TPO mimetic peptides are also well known in the art and range, for example, from dimerization via PEGylation, disulfide bridges or lysine side chains (see, e.g. WO 96/40772; WO 96/40749; WO 01/38342; WO 01/091780; WO 2004/101611; WO 2004/100997; WO 2004/101600; WO 2004/101606, Wrighton et al. 1997; Johnson et al., 1997; WO 98/25965). All of these methods combine monomeric peptides via a ligand structure to obtain the desired dimeric or even multimeric molecules, which enhance the formation of the active dimeric or mesmultimeric receptor complex.

A similar concept for combining monomeric units is also known for other binding molecules (see, for example, WO 2004/014951). In order to generate a molecule that is capable of interacting with the respective di- or multimeric receptor complex, this patent application teaches to use a small support structure in order to connect the monomeric receptor binding domains in a relative relationship. spatial that allows interaction with the respective receptor complexes (and, for example, induce receptor di- or trimerization).

However, even though dimerization (or even multimerization in the case of a multimeric receptor) of the monomeric peptide units usually improves activity compared to the respective monomeric peptides, it is still desirable to intensify the activity. For example, even dimeric EPO mimetic peptides are still far more potent than the EPO molecule in activating cellular mechanisms.

It is also known in the art to couple one or more hydrophilic carrier moieties (such as, for example, PEG) to a peptide.

When peptides are derivatized with a hydrophilic polymer, their solubility and circulating half-life are found to be increased and their immunogenicity is decreased (see, for example, WO 98/25965). However, it has also been reported that the fixation of such a hydrophilic vehicle may decrease biological activity. An increase in biological activity has not been reported.

Several approaches have been made to increase peptide activity, for example by varying the amino acid sequence to identify more potent candidates. However, so far no suitable solution for enhancing the activity of peptides, especially EPO or TPO mimetic peptides, is known in the art.

Thus, it is an object of the present invention to provide peptide compounds which bind to a receptor target and have increased activity.

The goal is solved by a compound that binds to the target molecules and comprises:

(i) at least two peptide units, wherein each peptide unit comprises at least two domains with a target deletion capacity (and therefore at least two monomeric linker units);

ii) at least one polymeric carrier unit;

wherein each of said peptide units is attached to said polymeric carrier unit.

Surprisingly, it has been found that combining two or more bi- or multivalent peptides on a polymeric support greatly increases the effectiveness of bivalent (or even multivalent) peptides to bind to their target, which is usually a receptor molecule not only additively, but even super additively. Thus, an allergic effect on binding efficacy is observed.

The term "bivalent" as used for purposes of the present invention is defined as a peptide comprising two domains having a target binding capacity which is usually a receptor (this term will be used hereinafter ). The term "bivalent" is used interchangeably with the term "dimeric". Accordingly, a "multivalent" or "multimeric" peptide has several respective binding domains and thus monomeric binding units. It is self-evident that the terms "peptide" and peptide unit — do not incorporate any size restrictions and incorporate oligo- and polypeptides as well as proteins. However, it is preferred that the unit-coupled peptide units have a length of about 200 amino acids or less or about 150 amino acids or less, more preferably about 100 amino acids or even 50 amino acids and less.

Compounds comprising two or more bis or multivalent peptide moieties attached to a polymeric carrier moiety are termed "supravalent" in the context of the present invention. Such supervalent molecules differ greatly from the dimeric or multimeric molecules known in the art. The prior art combines various merely monomeric peptide units to create a dimer or multimer. In contrast, supravalent molecules are generated by connecting already divalent (at least) peptide units to a polymeric carrier moiety, thereby creating a supravalent molecule bringing several di- or multimeric peptide units (illustrated examples of this concept are provided). 13 to 15). Thus, the overall activity and efficacy of di- or multimeric peptides are greatly enhanced thereby decreasing the ED50 dose.

To date, the reasons for the higher potency of superprivalent molecules compared to bi- or multimeric molecules known in the prior art are not fully understood. It could be due to the fact that the known dimeric molecules in the state of the art (e.g., dimeric EPO mimetic peptides) provide a target, respectively, a receptor-binding unit per compound molecule. Thus, only one (dimeric) receptor complex is generated upon binding of the dimeric compound, thereby inducing only a signal transduction process in the cell. For example, two monomeric EPO mimetic peptides are linked via PEG to form a peptide dimer, thereby facilitating the dimerization of receptor monomers required for signal transduction (Johnson et al., 1997).

In contrast, the supravalent compounds according to the invention comprise several already di- or multimeric receptor binding units. Supravalent compounds according to the present invention thus carry several receptor binding units (bi- or multivalent). Ca- of the vehicle-coupled di- or multimeric peptide unit represents a receptor binding unit. This could allow the generation of multiple receptor complexes on the cell surface per compound molecule, thereby inducing (or blocking, in the case of an antagonist) various signal transductions and thereby enhancing the activity of the super peptide units. additively. Binding of supravalent compounds could result in a clustering of receptor complexes on the cell surface.

The peptide units according to the invention are homo- or heterogeneous, meaning that identical or different peptide units are coupled to the polymeric carrier. The same applies to the deletion domains of peptide units which may be homo- or heterogeneous. Homogeneous binding domains (monomers) are preferred where a target receptor is bound, which is composed of identical protein subunits (such as, for example, the homodimeric EPO receptor).

However, the amino acid sequence of the homogeneous binding domains may still vary even though they bind to the same receptor target (and thus are functionally homogeneous). Heterogeneous binding domains (monomers) are preferred if a target receptor is bound, which is composed of different protein subunits (such as, for example, heterodimeric interleukin receptors). Preferably, the bi- or multivalent peptide units attached to the carrier unit bind to the same receptor target. However, they may, of course, still differ as to their amino acid sequence. The monomeric binding units of the bi- or multivalent peptide units may be linear or cyclic. A cyclic molecule can be, for example, created by forming intramolecular cysteine bridges.

The polymeric carrier unit comprises at least one polymeric polymer —I ί η and the natural- or synthetic drit ro uter. The polymeric carrier unit is preferably soluble in water and body fluids and preferably -rency is a pharmaceutically acceptable polymer. Water soluble polymeric moieties include, but are not limited to, for example, polyalkylene glycol derivatives thereof, including PEG1 PEG homopolymers, mPEG, homopolymers of polypropylene glycol homopolymers, ethylene glycol copolymers with propylene glycol, wherein said homopolymerose copolymers are unsubstituted or substituted at one end, for example by an acyl group; polyglycerines or polysalic acid; carbohydrates, polysaccharides, cellulose and cellulose derivatives, including methylce-cellulose and carboxymethyl cellulose; starches (e.g. hydroxyalkyl starch (HAS), especially hydroxyethyl starch (HES) and dextrins, same derivatives; dextran and dextran derivatives including dextran sulfate, cross-linked dextrin and carboxymethyl dextrin; chitosan (a li-near polysaccharide), heparin and heparin fragments; polyvinyl alcohol and polyvinyl ethyl ethers; polyvinylpyrrolidone; alpha, beta-poly [(2-hydroxyethyl) -DL-aspartamide; and polyoxyethylated polyols. An example of a carrier unit is a ho-multifunctional polymer, for example polyethylene glycol (bis-maleimide, bis-carboxy, bis-amino, etc.).

The polymeric carrier unit to which it is coupled to the peptide units according to the present invention may have a broad molecular weight range because of the different nature of the different polymers which are suitable in conjunction with the present invention. Thus, there are no size restrictions. However, it is preferred that the molecular weight is at least 3 kD, preferably at least 10 kD and about 20 to 500 kD and more preferably about 30 to 150 or about 60 or more. 80 kD. The size of the vehicle unit depends on the polymer chosen and thus may vary. For example, especially when starches such as hydroxyethyl starch are used, the molecular weight could be considerably higher. The average molecular weight could then be distributed around 100 to 4,000 kD or even higher. However, it is preferred that the molecular weight of the HES molecule be about 100 to 300kD, preferably about 200kD. The size of the carrier unit is preferably chosen such that each peptide unit, respectively, can be optimally distributed for ligation of their respective receptor molecules.

In order to facilitate this, one embodiment of the present invention is a vehicle unit comprising a branch unit. According to this embodiment, polymers, such as PEG, are attached to a branch unit, thus resulting in a large carrier molecule allowing the incorporation of numerous peptide units. Examples of suitable branching units are glycerol or polyglycerol. Also, dendritic branch units may be used, for example, as taught by Haag 2000, incorporated herein by reference. Also, the HES vehicle may be used in a branched form. This, for example, if it is obtained in a high proportion from amylopectin.

Preferably, after the peptide units are created by combining monomeric linker units (either head to tail, head to tail or tail to tail) to the peptide units of the polymeric carrier unit is attached / coupled to the peptide units. The polymeric carrier unit is connected to the peptide units via a covalent or non-covalent bond (e.g., a coordinative). However, the use of a covalent bond is preferred.

Fixation may occur, for example, via an amino acid reactive peptide units, for example lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine or an N-terminal amino group and carboxylic acid C-terminal. In case the peptide does not have a corresponding reactive amino acid, such amino acid may be introduced in the amino acid sequence. The coupling should be chosen such that binding to the target is not at least as little as possible prevented. Depending on the conformation of the peptide unit, the reactive amino acid is at the beginning, end or within the peptide sequence.

In case the polymeric carrier unit lacks an appropriate coupling group, various coupling substances / binders may be used to appropriately modify the polymer so that it can react with at least one reactive group on the peptide unit to form the supravalent compound. . Suitable chemical groups which may be used to modify the polymer are, for example, as follows: Acylation groups which react with amino groups of protein, for example acid anhydride groups, N-acylimidazole groups, azide groups, N groups carboxy anhydride, diketene groups, dialkyl pyrocarbonate groups, imidoester groups and carbodiimide-activated carboxyl groups.

All of the above groups are known to react with overprotein / peptide amino groups to form covalent bonds, involving acyl bonds or the like;

alkylating groups which react with sulfhydryl (mercapto), thiomethyl, imidazo or starch groups on the peptide unit such as halo-carboxyl groups, maleimide groups, activated vinyl groups, ethyleneenoimine groups, aryl halide groups, 2-bromide groups -hydroxy-5-benzyl; aliphatic aldehyde and ketone groups together with reducing agents, which react with the amino group of the peptide;

ester and amide forming groups which react with a carboxyl peptide group such as diazocarboxylate groups and carbodiimide and amine groups together disulfide forming groups which react with sulfhydryl groups on the protein such as 5.5 '-dithiobis (2-nitrobenzoate), ortho-pyridyl disulfides and alkyl mercaptan groups (which react with the peptide sulfhydryl groups 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 protein;

diazo groups, which react with phenolic groups on opeptide;

reactive groups from the reaction of cyanogen bromide with polysaccharide, which react with amino groups on the protein.

Thus, in summary, the compound according to the invention may be optionally made first by modifying the polymeric carrier chemically to produce a polymeric carrier having at least one chemical group upon which it is capable of reacting. with an available or introduced chemical group on the peptide unit and then reacting the optionally modified polymer and the peptide unit to form a covalently bonded complex thereof using the modified polymer chemical group if necessary.

In case coupling occurs via a peptide free SH group, the use of a maleimide group in the polymer is preferred.

In order to generate a defined molecule, it is preferred to use an objectified approach for attaching the peptide units to the polymeric carrier unit. Where no appropriate amino acid is present at the desired attachment site, appropriate amino acids should be incorporated into the peptide unit. For site-specific polymer attachment, a single reactive group, for example, a specific amino acid at the end of the peptide moiety is preferred so as to avoid uncontrolled coupling reactions across the peptide, leading to a heterogeneous mixture comprising a population of several different polymeric molecules.

Coupling of the peptide units to the polymeric carrier unit, for example, PEG or HES, is accomplished using reactions known to those skilled in the art. For example, there are a number of PEG and HES fixation methods available to those of skill in the art (see, for example, WO 2004/100997 which provides other references, Roberts et al., 2002; US 4,064,118; EP 1 398 322). EP 1 398327; EP 1 398 328; WO 2004/024761; all incorporated herein by reference).

It is important to understand that the concept of supravalence described herein is different from the known concept of PEGylation or HESylation. In the prior art, for example, PEGylation is used only to produce peptide dimers or to improve pharmacokinetic parameters through attachment of one or more PEG units to a peptide. -Gofituated — and as outlined above, attachment of two or more at least bivalent peptide units, for example, HES as a polymeric single unit, also greatly enhances efficacy ( thus decreasing the EC50 dose). The concept of the present invention thus has strong effects on pharmacodynamic parameters and not only on pharmacokinetic parameters, as is the case with the concepts of PEGylation or HESylation known in the prior art. However, of course, incorporation, for example, of PEG or HES as a polymeric carrier unit also has the known advantages over pharmacokinetics.

Pegylation is usually understood to improve the biopharmaceutical properties of peptides. The most relevant alterations of the protein molecule after PEG conjugation are size enlargement, protein surface and disguise of glycosylation function, charge modification and epitope protection. In particular, size enlargement decreases renal ultrafiltration and promotes accumulation in permeable tissues by intensifying the permeation and passive retention mechanism. Protein protection reduces proteolysis and recognition by the immune system, which are important pathways of elimination. The specific effect of PEGylation on the physicochemical and biological properties of the protein is strongly determined by the properties of the protein and polymer, as well as the PEGylation strategy adopted.

However, the use of PEG or other non-biodegradable polymers could lead to new problems.

During in vivo applications, dosing intervals in a clinical setting are triggered by the loss of drug effect. Routine dosages and dosing intervals are adapted such that the effect is not lost during the dosing intervals. Due to the fact that peptides attached to a large non-biodegradable polymer unit (eg, a PEG moiety) can be degraded faster than the support molecule can be eliminated from the body, a risk of vehicle unit accumulation may arise. Such a risk of accumulation always occurs, since the half-life for effect is shorter than the half-life for elimination of the drug itself or its components / metabolites. Thus, accumulation of the carrier molecule should be avoided, especially in long-term treatments, because the peptides are usually PEGylated with very large (~ 20-40kD) PEG moieties which thus show slow renal elimination. The peptide portion itself undergoes enzymatic degradation and even partial cleavage could be sufficient to deactivate the peptide.

In order to find a solution to this potential problem, one embodiment of the present invention teaches the use of a polymeric carrier unit which is composed of at least two subunits. The polymeric subunits are connected via biodegradable covalent ligand structures. According to this embodiment, the molecular weight of the large carrier molecule (e.g., 40 kD) is created by several small or intermediate sized subunits (e.g., each subunit has a molecular weight of 5 to 10 kD), which are connected. via biodegradable binders. The molecular weights of the modular subunits added in this way generate the desired molecular weight of the carrier molecule. However, biodegradable ligand structures can be disrupted in the body, thereby releasing smaller vehicle subunits (eg, 5 to 10 kD).

Small carrier subunits show better renal elimination than a polymeric molecule having an overall molecular weight (e.g. 40 kD). An illustrative example is given in figure 6.

Binding structures are selected according to known degradation properties and degradation time scales in body fluids. Breakable structures may, for example, contain cleavable groups such as carboxylic acid derivatives, such as amide / peptide bonds or esters which may be cleaved by hydrolysis (see, for example, Roberts, 2002, incorporated herein by PEG succinimidyl esters can also be synthesized with various ester linkages in the major part of PEG to control the rate of degradation at physiological pH (Zhao, 1997, incorporated herein by reference). Other breakable structures, such as benzyl urethane disulfides, can be cleaved under mildly reducing environments, such as in single cell endosomal compartments (Zalipsky, 1999) and are thus also suitable. Other criteria for selection of appropriate ligands are the selection of rapid degradation (often enzymatic) or slow degradation (often non-enzymatic decomposition). Combination of these two mechanisms in body fluids is also possible. It is clear that this highly advantageous concept is not limited to the specific peptide units described or mentioned herein, but also applies to other pharmaceutical molecules that are attached to larger polymeric units, such as PEG molecules, where the same buildup problems arise.

According to one embodiment, hydroxyalkyl starch and preferably HES is used as a polymeric carrier unit. HES has several important advantages. First of all, HES is biodegradable. In addition, the biodegradability of HES can be controlled via the proportion of hydroxyethyl groups and thus can be influenced. A molar degree of substitution of 0.4-0.8 (on average 40-80% of glucose units contain a hydroxyethyl group) is well suited for purposes of the present invention. Due to biodegradability, accumulation problems described above in conjunction with PEG usually do not occur. In addition, HES has been used for a long time in medical treatment, for example as a plasma expander. Its innocuous character is thus approved.

In addition, derivatives of HES hydrolysis products are detectable by gas chromatography. HES-peptide conjugates may be hydrolyzed under conditions under which the peptide units are still stable. This allows quantification and monitoring of degradation products and allows evaluations and standardization of active peptides.

According to another embodiment, a first type of polymeric carrier unit is used and loaded with peptide units. This first vehicle is preferably readily biodegradable as well as, for example, Or-HES. However, not all first-point attachment points are occupied with peptide units, but only, for example, around 20 to 50%. Depending on the size of the polymer used, several hundred peptide units may be coupled to the carrier molecule. However, depending on the peptide used, usually fewer peptide units (2a 50, 2 to 20, 2 to 10 or 3 to 5) are coupled. The rest (or at least some) of the remaining attachment points are occupied with a different vehicle, for example small PEG units having a lower molecular weight than the first vehicle. This embodiment has the advantage that a supravalent composition is created by virtue of the first vehicle which is, however, very durable by virtue of the presence of the second vehicle, which is preferably comprised of 3 PEG units. at 5 or 10 kD. However, the whole entity is very well degradable since the first vehicle (eg HES) and peptide units are biodegradable and the second vehicle eg PEG1 is small enough to be easily eliminated from the body. .

The monomers constituting the binding domains of the peptide units recognize a binding site of a target. The term binding domain refers to the binding portion of the monomeric peptide that is involved in target binding. Depending on the peptide, the deletion domain may be composed of different structural motifs of the peptide (e.g., beta-leaves, alpha helices, beta turns) that define the deletion domain in the three-dimensional conformation of the peptide.

According to one embodiment, the peptide unit binds to a transmembrane receptor. Activation of growth factor and cytokine transmembrane receptors generally occurs when a ligand selects a specific domain over the receptor, thereby inducing conformational alteration and / or triggering dimerization or oligomerization of receptor chains. When binding to the ligand, several class I cytokine receptor members form homodimers, including the erythropoietin receptor (EPOR), thrombopoietin receptor (TPOR), granulocyte colony-stimulating receptor (G-CSFR), hormone receptor. --Grow Growth- (GHR) and prolactin receptor (PrR). These class I cytokine receptors show structural and functional similarities with each other. According to one embodiment, the peptide units are chosen such that they bind to these cytokine daclass I receptors.

As outlined, a homodimeric receptor is any biological target protein being composed of two non-covalently associated identical protein subunits. Such receptors are usually only functional if both subunits are associated in homodimeric form. The latter property of being a homodimeric receptor differentiates the EPO receptor and, for example, the related TPO receptor from many other cytokine receptors. In most cases of cytokine receptors, the receptor is a heterodimer (many interleukin receptors) or even a heterotrimer (for example, IL-2).

The peptide units according to the invention, comprising at least two monomeric binding domains, bind to each other and preferably are able to dimultimerize the target respectively and / or thereby stabilize it, thereby creating signal transduction. that induces the complex. The peptide units preferably have a heterodimeric target molecule which is preferably a decytocin receptor (see above). As outlined above, the peptide units for creation of supravalent molecules bind to the target receptor. According to one embodiment, peptide units act as receptor agonists. The term agonist refers to a biologically active peptide which binds to its complementary biologically active receptor (target) and activates the latter to cause a biological response at the receptor or to enhance the pre-existing receptor (target) biological activity. According to a different embodiment, the peptide unit acts as a receptor antagonist. An antagonist also binds to his complementary biologically active receptor (al-vo). However, an antagonist does not induce or intensify receptor (target) biological activity.

Various methods are known in the art for dimerization or multimerization of monomeric peptide units. These methods can be used to create the peptide units according to the invention. The solutions that prevail following the dimerization approach are characterized by:

(a) the fact that the binding domains are first synthesized separately as monovalent or monomeric peptides, which may be modified, for example, by fixing reactive groups in preparation for step b,

b) in a second reaction step, two - in most identical cases - binding domains are joined together in a distinct dimerization reaction, which may also include ligand molecules usually being interposed between the two dimerized domains.

Such dimers are examples of bivalent (dimeric) peptides and exhibit essentially the same biological functions as monomers, but show enhanced biological activity due to improved receptor interaction.

Various techniques are known to those skilled in the art to dimerize or oligomerize the monomers which may also be applied in accordance with the teachings of the present invention. Monomers may be dimerized, for example, by covalent attachment to an ligand. A ligand is a coupling molecule that creates a covalent bond between the polypeptide units of the present invention. Polypeptide units may be combined via a ligand in such a way that EPO receptor binding is improved (Johnson et al. 1997; Wrighton et al. 1997). In addition, multimerization of monomeric biotinylated peptides by non-covalent interaction with a protein molecule described by Wrighton et al. (Wrighton, 1997) is mentioned. It is also possible to use a biotin / streptavidin system, that is, C-terminus biotinylation of the peptides and a subsequent incubation of the streptavidin biotinylated peptides. Alternatively, dimerization is known to be obtained by forming a diketopiperazine structure. This method-known to those skilled in the art-is described in detail, for example, in Cavelier et al. (In: Peptides: The Wave of the Future; MichalLebl and Richard A. Houghten (eds); American Peptide Society, 2001). The specification of such documents with respect to dimerization and non-covalent multimerization is incorporated herein by reference. Another alternative way to obtain peptide dimers known from the prior art is to use bifunctional activated dicarboxylic acid derivatives as reactive precursors of the last linker moieties, which react with N-terminal amino groups, thereby forming the final dimeric peptide (Johnson and others, 1997). Monomers may also be dimerized by covalent attachment to a binder. Preferably, the ligand comprises NH-R-NH, wherein R is a lower alkylene substituted by a functional group, such as a carboxyl group or amino group that allows attachment to the other molecular moiety. The binder could contain a lysine residue or lysine amide.

Also, PEG may be used as a binder. The binder may be a molecule containing two carboxylic acids and optionally substituted on one or more atoms by a functional group, such as an amide, capable of being attached to one or more PEG molecules. A detailed description of the possible steps for oligomerization and dimerization of peptides with a binding moiety is also provided in WO 2004/101606.

While sufficiently functional and thus usable according to the teachings of the present invention, prior art approaches of dimeric molecule synthesis could have some disadvantages for some peptides.

A potential deficiency could be perceived by the fact that the monomers to be connected must first be synthesized separately. Because of the stochastic pairing of monomeric peptides during the dimerization reaction, it is particularly difficult to obtain (selectively and intentionally) heterodimeric bivalent peptides with this approach. At least this would lead to greater yield losses of a desired special heterodimer. Bivalent peptides harboring two or more slightly different monomeric binding domains are very desirable, since due to their heterodimeric nature, special interactions between the two domains, which are capable of stabilizing their alteration in the final bivalent peptide, may be introduced. while weekly or even enhances binding to the homodimeric receptor. However, because of the high yield losses associated with prior art "stochastic dimerization reactions", this is usually not an economically attractive approach.

Application of the prior art approaches to dimerization - albeit technically adequate - thus has some economic disadvantages for providing these peptides with heterogeneous binding domains as described. Thus, preferably, a more efficient strategy is to use to obtain highly reactive bivalent peptide units, which could even contain heterogeneous binding domains.

The central concept of this dimerization method is to refrain from synthesizing the monomeric peptides that form part of the divalent peptide in distinct reactions prior to dimerization or multimerization, but to synthesize the final peptide unit having at least two binding domains in one step as follows. a single peptide; for example, in a single solid phase reaction. Thus, a distinct dimerization or multimerization step, as taught by the state of the art, is no longer required. This aspect provides a great advantage, that is, complete and independent control of each sequence position in the final peptide unit. The method allows easily harboring at least two different receptor-specific binding domains in a peptide unit by virtue of indepen- dent control over each sequence position.

According to this embodiment, the sequence of the final peptide between the binding domains (which is the "linker region") is composed of α-mino acids only, thus leading to a single continuous peptide unit.

In a preferred embodiment of the invention, the binder is composed of natural or unnatural amino acids which allow for high conformational flexibility. In this regard, it may be advantageous to use glycine-amino acid binding residues, which are known for their high flexibility in terms of twisting. Also other amino acids, such as alanine or beta-alanine, or a mixture of the same. same, they can be used. The number and choice of amino acids used depends on their spherical factors. This embodiment allows the standard design of an appropriate binder through molecular modeling to avoid distortion of the bioactive conformation and thus allows perfect combination with receptor units. A binder composed of 3 to 5 amino acids is especially preferred.

It is noteworthy that the linker between the functional binding domains (also referred to as monomeric units) of the peptide units may be a distinct part of the peptide or may be composed - wholly or in part - of amino acids which are part of the monomeric binding domains. Thus, the term "binder" is functional rather than structurally defined, since an amino acid could form part of the binder moiety as well as the monomeric binding subunits.

Since - as mentioned above - during the bivalent peptide synthesis each sequence position within the final peptide is under control and thus can be precisely determined, it is possible to standardize or make configurations on the peptide units or specific regions or domains thereof. including the binder. This is of particular advantage as it allows the creation of polymer-specific attachment sites and avoids distortion of the bioactive conformation of peptide units due to unfavorable intramolecular interactions. The risk of distortions can be assessed prior to synthesis through molecular modeling. This applies especially to the design of the ligand between monomeric domains.

The continuous bivalent / multivalent peptides according to the invention show much greater activity than the corresponding monomeric peptides and thus confirm the known observation of other dimeric peptides that an increase in efficacy is associated with bivalent peptide concepts.

According to a preferred embodiment, all peptide units (wherein each peptide unit is considered to be a receptor binding moiety) bind to the same target receptor. However, they may be heterogeneous, thus differing in their amino acid sequence.

According to a preferred embodiment, said peptide units bind to the EPO receptor, thereby dimerizing the EPO receptor complex. Preferably, they induce desinal transduction and are thus EPO receptor agonists. Peptide units, respectively, the monomeric binding domains that create the peptide units, may be selected from the group of EPO mimetic peptides. Suitable EPO mimetic peptides are well known in the art and may be used in connection with the present invention (please refer, for example, to WO 96/40772; WO 96/40749; WO 01/38342; WO 01/091780 WO 2004/101611; WO 2004/100997; WO 2004/101600; WO 2004/101606).

Other suitable EPO mimetic peptide units that may be used in accordance with the present invention comprise binding domains of at least 10 amino acids in length that bind to the EPO receptor. They preferably do not comprise proline in the position commonly referred to as position 10 of the EPO mimetic peptide (for numbering, please refer to Wrighton et al., 1996 and Johnson, 1998), but a positively charged amino acid. These EPO mymetic peptides bring a characteristic amino acid motif to a beta-gyrus structure (Wrighton et al.), Wherein the peptide moiety binding domain according to this embodiment does not comprise a proline in said beta-gyrus motif in position. 10, but a positively charged amino acid, preferably K or R. Also, other basic amino acids, especially unnatural amino acids, such as homoarginine, could be used. Positions 9 and 10 of the EPO mimetic binding domain may be occupied by 5-aminolevulinic acid (5-Als). The domain of the peptide may also bring R in position 17.

According to one embodiment, at least one of the EPO receptor-binding domains of the peptide units comprises the following amino acid sequence:

X6XyXeXgX10X11 Xi 2X13X14X15

wherein each amino acid is selected from natural or unnatural amino acids and:

X 6 is C, A, E, α-amino-Y-bromobutyric acid or homocysteine (hoc);

X7 is R, H, L, W or Y or S;

Xe is M, F, I, homoseromethylether (hsm) or norisoleucine;

Xg is G or a conservative exchange of G;

X10 is a non-conservative proline exchange;

or Xg and X10 are replaced by a single amino acid;

X11 is independently selected from any amino acid;

X12 is T or A;

X 13 is W, 1-nal, 2-nal, A or F;

X14 is D, Ε, I, L or V;

X15 is C, A, K, α-amino-Y-bromobutyric acid or homocysteine (hoc)

as long as X6 or X15 is C or hoc.

The length of a binding domain of said peptide unit is preferably between ten to forty or 50 to 60 amino acids. In preferred embodiments, the consensus peptide represents a length of at least 10, 15, 18 or 20 amino acids. Of course, they can be encrusted, respectively, comprised of longer sequences. The described monomeric peptide sequences may be perceived as binding domains for the EPO receptor. As EPO peptide mimetics, they are capable of binding to and activating the EPO receptor.

It was very surprising that these peptides exhibited EPO mimetic activity although one or - according to some modalities - even both prolines could be replaced by other natural or unnatural amino acids. In fact, peptides according to -HFivention4 have an activity-comparable to that of proline-containing peptides. However, it is noteworthy that amino acids that replace proline residues do not represent a conservative exchange but rather a non-conservative exchange. Preferably, 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 may also be an unnatural amino acid and is preferably one with a positively charged side chain. They will also include respective analogs of the mentioned amino acids. A suitable example of an unnatural amino acid is homoarginine. In one embodiment, the peptide has a positively charged amino acid at position 10, except for the natural amino acid arginine. According to this embodiment, proline 10 is thus substituted by a selected amino acid of Κ, H or a unnatural positively charged amino acid such as, for example, homoarginine. It is preferred that the peptides represent a lysine or homoarginine at position 10. As described above, also proline at position 17 could be replaced by a non-conservative amino acid. In this regard, it is also preferred that said non-conservative amino acid is one with a positively charged side chain, such as K, R, H or a non-natural amino acid such as, for example, homoarginine. According to a submodality of this embodiment, the peptide carries a positively charged amino acid at position 17, except for the natural amino acid arginine. According to this embodiment, proline 17 is thus replaced by a selected amino acid of Κ, H or a unnatural positively charged amino acid such as homoarginine. It is preferred that the peptides represent a lysine or homoarginine at position 17.

The EPO-R binding domain may further comprise a sequence of the following amino acids:

X6X7X8X9X1 0X11 Xi 2X13X14X15

where each amino acid is indicated by abbreviation for the standard letter:

Ββ is C;

X7eR, H1LouW;

X8 is M, F or I;

X9 is G or a conservative exchange of G;

X10 is a non-conservative proline exchange;

X11 is independently selected from any amino acid;

X12 is T;

XiséW;

X14 is D, Ε, I, Lou V;

X15 ° C.

In addition, X7 may be serine, Xe may be hsm or norisoleucine and X2 may also be 1-nal, 2-nal, A or F. The length of the consensus peptide is preferably between ten to forty or fifty. sixty amino acids. In preferred embodiments, the consensus peptide comprises at least 10, 15, 18 or 20 amino acids.

Other EPO mimetic peptides which may be used to create the peptide units according to the present invention are defined by the following consensus peptide sequences:

A peptide capable of EPO receptor binding selected from the group consisting of:

- peptides comprising the following amino acid consensus sequence:

ΧβΧγΧεΧθΧι 0X11 Xi 2X13X14X15 wherein each amino acid is selected from a natural or unnatural amino acid and:

X6 is an amino acid with a functionally capable side chain forming a covalent bond or A or α-amino-γ-bromobutyric acid;

X7 is R, H, L, W1 You S;

X8 is M, F, I, homoseramethylether or norisoleucine;

Xg is G or a conservative exchange of G;

X10 is a non-conservative proline exchange (or, according to another embodiment, proline or a conservative proline exchange) —or X9 and X10 are replaced by a single amino acid;

X11 is selected from any amino acid;

X12 is an uncharged polar amino acid or A;

X13 is W, 1-nal, 2-nal, A or F;

X14 is D, Ε, I, Lou V;

X15 is an amino acid with a side chain functionally capable of forming a covalent bond or A or α-amino-γ-bromobutyric acid; and

- functionally equivalent peptide fragments, derivatives and variants defined by the above consensus sequence, which represent an EPO mimetic activity and have an amino acid at position X10 which constitutes a non-conservative proline exchange (or, according to another embodiment , proline or a conservative proline exchange) or wherein Xge X10 are replaced by a single amino acid.

According to the first modality consensus sequence, Xe and X1S represent amino acids with a side chain functionally capable of forming a covalent bond. These amino acids are thus capable of forming a bridging moiety. According to one embodiment, the amino acids at position Xe and X15 are chosen such that they are capable of forming an intramolecular bridging bond within the peptide by forming a covalent bond between them. Formation of an intramolecular bridging can lead to cyclization of the peptide. Examples of suitable bridging moieties are disulfide bridging and diselidated bridging. Suitable examples of amino acids representing such bridging functionalities in their laterase chains are, for example, cysteine and cysteine derivatives such as homocysteine or selenocysteine, but also thiolysin. The formation of a diselide bridging bond, for example between two selenocysteine residues, even has advantages over a cysteine bridging bond. This is because a selenium jumper is more stable in reducing environments. The conformation of the peptide is thus preserved even under difficult conditions.

However, it is evident that amino acids are also suitable at position X6 and X15, representing a side chain having a functionality that allows the formation of different covalent bonds such as, for example, an amide bond between an amino acid having a positively charged chain-side ( for example, proteinogenic amino acids,, H, R or ornithine, DAP or DAB) and an amino acid having a negatively charged chain-side (e.g. proteinogenic amino acids D or E). Other examples are amide and thioether bridges.

A peptide of at least 10 amino acids capable of EPO receptor binding is selected from the following alternatives:

(a) a peptide comprising the following central amino acid sequence:

X9X10X11X12X13

wherein each amino acid is selected from unnatural or natural amino acids and wherein:

Xg is G or a conservative exchange of G;

X10 is a non-conservative proline exchange (or, according to another embodiment, proline or a conservative proline exchange), or Xg and Χ10 are replaced by a single amino acid;

X11 is selected from any amino acid;

X12 is an uncharged polar amino acid or A;

X13 is naphthylalanine.

(b) a peptide capable of EPO receptor binding comprising the following amino acid sequence:

X6X7X8X9X10X1 1X12X13X14X15 wherein each amino acid is selected from natural or unnatural amino acids and:

X6 is C1 A, E, α-amino-Y-bromobutyric acid or homocysteine (hoc);

X7 is R1-H1 LrW or -Y- or R1 H, L1W1Y or S;

X8 is M, F11, homoseramethylether or norisoleucine;

X9 is G or a conservative exchange of G;

X-10 is a non-conservative proline exchange;

or X9 and X10 are replaced by a single amino acid;

Xn is selected from any amino acid;

X12 is T or A;

X13 is 1-nal, 2-nal

Χ4 is D, Ε, I, L or V;

X15 is C, A, K1 α-amino-Y-bromobutyric acid or homocysteine (hoc)

provided that Xe or X15 is C or hoc;

(c) functionally equivalent fragments, derivatives and variants of the peptides defined by the above consensus sequences that represent an EPO mimetic activity and have an amino acid at position X10 which constitutes a non-conservative proline exchange (or, according to another modality, proline or a conservative proline exchange) or wherein X9 and X10 are replaced by a single amino acid and naphthylalanine in the X13 position.

A peptide of at least 10 amino acids in length, capable of binding to the EPO receptor and comprising an agonist activity, selected from the group consisting of:

- peptides comprising at least one of the following central amino acid sequences:

XgX10X11X12X13;

X9X10X11X12X13X14X15X16X17

or

X9X10X11X12X13X14X15X16X17X18X19

wherein each amino acid is selected from natural or unnatural amino acids and wherein at least one of the positions X10, X17 or X19 is a negatively charged amino acid and wherein:

X9 is G or a conservative exchange of G;

Re-selected X11 of any amino acid;

X12 is an uncharged polar amino acid or A; preferably threonine, serine, asparagine or glutamine;

X 13 is W, 1-nal, 2-nal, A or F;

X14 is D, Ε, I, L or V;

X15 is an amino acid with a side chain functionally capable of forming a covalent bond or A or Î ± -amino-γ-bromobutyric acid;

X 16 is independently selected from any amino acid, preferably G, K, L, Q, R, S1 Har or T;

X18 is independently selected from any amino acid, preferably L or Q;

functionally equivalent fragments, derivatives and variants of the peptides defined by the above consensus sequences, which represent an EPO mimetic activity and wherein at least one of the positions X10, X17 or X19 is a negatively charged amino acid.

The EPO mimetic peptide having a negatively charged amino acid at at least one of the positions X10, X17 or X19 can also be described by the following extended consensus sequence:

ΧβΧγΧβΧθΧ10X11X12X13X14X15X10Χ17X18X19

wherein each amino acid is selected from natural or unnatural amino acids and wherein:

Xe is an amino acid with a functionally capable side chain forming a covalent bond or A or α-amino-γ-bromobutyric acid;

X7 is R, H, L, W or Y or S;

Xe is M, F1 I, Υ, H, homoserinamethylether or norisoleucine;

Xg is G or a conservative exchange of G;

In the case where X10 is not a negatively charged amino acid, X10 is proline, a conservative proline exchange or a non-conservative proline exchange or X9 and X10 are replaced by a single amino acid;

X11 is selected from any amino acid;

X-12 is an uncharged polar amino acid or A; preferably threonine, serine, asparagine or glutamine;

X 13 is W, 1-nal, 2-nal, A or F;

X14 is D1-E1 I, Lou V;

X15 is an amino acid with a side chain functionally capable of forming a covalent bond or A or α-amino-γ-bromobutyric acid;

X16 is independently selected from any amino acid, preferably G, K1 L, Q, R, S, Har or T;

in the case where X 17 is not a negatively charged amino acid, X 17 is selected from any amino acid, preferably A, G, Ρ, You is a naturally occurring, unnatural or positively charged derivatized amino acid, preferably K, R, H, ornithine or homoarginine;

X18 is independently selected from any amino acid, preferably L or Q;

where X19 is not a negatively charged amino acid, X19 is independently selected from any amino acid, preferably a positively charged amino acid such as K, R, H, ornithine or homoarginine;

provided that at least one of Χ10, Xi70uX19 is a negatively charged amino acid.

A peptide of at least 10 amino acids in length, capable of binding to the EPO receptor and comprising an agonist activity, selected from the following group of peptides:

(a) a peptide comprising the following central amino acid sequence:

X9X10X11X12X13, · X9X10X11X12X13X14X15X16X17

or

X9X10X11X12X13X14X15X16X17X18X19

wherein each amino acid is selected from natural or unnatural amino acids, and wherein:

X9 is G or a conservative exchange of G;

Xn is selected from any amino acid;

X12 is an uncharged polar amino acid or A; preferably threonine, serine, asparagine or glutamine;

X13 is W, naphthylalanine, A or F;

X14 is D, Ε, I, Lou V;

X15 is an amino acid with a functionally capable side chain forming a covalent bond or A or α-amino-γ-bromobutyric acid,

as well as functionally equivalent fragments, evariant derivatives of the peptides defined by the above consensus sequence, which represent an EPO1 mimetic activity.

wherein at least one of positions X10, X16, X17 or X19 represents a positively charged non-proteinogenic amino acid having a side chain which is elongated compared to lysine;

(b) a peptide, especially one being capable of EPO receptor binding comprising the following amino acid sequence:

X6X7X8X9X10X11X12X13X14X15

wherein each amino acid is selected from natural or unnatural amino acids and: X6 is C, A, E1 α-amino-Y-bromobutyric acid or homocysteine (hoc);

X7 is R, H, L1WouYou S;

X8 is M, F, I, homoseramethylether or norisoleucine;

X9 is G or a conservative exchange of G;

X10 is Har

X11 is selected from any amino acid;

X12 is T or A;

X 13 is W, 1-nal, 2-nal, A or F;

X14 is D, E1 I1LouV;

X15 is C1 A1 K1 α-amino-Y-bromobutyric acid or homocysteine (hoc)

provided that X6 or X15 is C or hoc;

(c) a peptide comprising the following amino acid sequence:

X3X4X5X6X7X8X9X10X11 Xi 2X13X14X15X1 βΧι 7X18in which X6 to Xi5 have the above meaning of variant (b) and where:

X 3 is independently selected from any amino acid, preferably D 1 E 1 L 1 N 1 S 1 T or V;

X4 is Y;

X5 is independently selected from any amino acid, preferably A, Η, K, L, M, S, T or I.

X 16 is independently selected from any amino acid, preferably G, K, L, Q 1 R, S or T;

X17 is homoarginine;

X18 is independently selected from any amino acid. These peptides may also be described by the following central amino acid sequence:

X6XyX8XgX10X11 Xl 2X13X14X15X16X17X18Χ19 where each amino acid is selected from natural or unnatural amino acids and in which:

X6 is an amino acid with a functionally capable side chain forming a covalent bond or A or α-amino-γ-bromobutyric acid;

X7 is R1 H1 L1WouY or S;

X 8 is M, F1 I, Υ, H, homoseramethylether or norisoleucine;

Xg is G or a conservative exchange of G;

where X10 is not a positively charged non-proteinogenic amino acid having a side chain which is elongated compared to lysine, X10 is proline, a conservative proline exchange or a non-conservative proline exchange or Xg and X10 are replaced by a single amino acid;

X11 is selected from any amino acid;

-X12 is an uncharged polar amino acid or A; preferably threonine, serine, asparagine or glutamine;

X 13 is W, 1-nal, 2-nal, A or F;

X14 D, Ε, I, Lou V;

X15 is an amino acid with a side chain functionally capable of forming a covalent bond or A or α-amino-γ-bromobutyric acid;

where X 16 is not a positively charged non-proteinogenic amino acid having a side chain which is elongated compared to lysine, X 16 is independently selected from any amino acid, preferably G, K, L, Q, R, S or T;

where Xi7 is not a positively charged non-proteinogenic amino acid having a side chain which is elongated compared to lysine, Xi7 is selected from any amino acid, preferably A, G, Ρ, Y or a natural, unnatural or positively charged derivatized, preferably K, R, H or ornithine;

X18 is independently selected from any amino acid, preferably L or Q;

where X19 is not a positively charged non-proteinogenic amino acid having a side chain which is elongated compared to lysine, X19 is independently selected from any amino acid, preferably a positively charged amino acid such as K, R, H or ornithine;

as long as at least one of X10, X16. X17 or X17 is a positively charged non-proteinogenic amino acid having a yeast chain which is elongated compared to lysine.

Monomeric EPO mimetic peptide units, at least two of which are constructed from a peptide unit, could comprise a single amino acid substituting amino acid residues X9 and Χ10 · Preferably, both residues are replaced by an unnatural amino acid, for example, 5-aminolevulinic acid or 5-aminovaleric acid.

In another embodiment, the binding domains used in the peptide units comprise the following sequence:

X4X5X6X7X8X9X10X11 Xi 2X13X14X15

where Χβ to X15 have the above meaning and where:

X4 is Y;

X5 is independently selected from any amino acid and is preferably A, Η, K, L, M, S, T or I.

Binding domains can be extended and can comprise the consensus sequence:

XaX4X5XeXTXeXgXi 0X11 Xi 2X13X14X15X1 βΧι 7X18

wherein X4 to Xi5 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 1 Q, R, S or T, more preferably K, R, S or T;

X17 is independently selected from any amino acid, preferably A, G, P, R,,, Y or a non-natural amino acid as a positively charged side chain, more preferably R, K or a non-natural amino acid such as homoarginine;

X-18 is independently selected from any amino acid.

In another embodiment of the invention, it is preferred that the peptides comprise X6 as C, Ε, A or hoc, preferably C and / or X7 as R, H or Y and / or X8 as F or M and / or X9 as G or A, preferably Ge / or X10 as K and / or Xn as V, L, I, Μ, Ε, A, T or norisoleucine and / or X12 as T and / or X2 as W and / or X14 as D or V and / or X15 as C or hoc, preferably C and / or X17 as Ρ, Y or A. However, it is also preferred that X17 is K or a unnatural amino acid with a positively charged side chain, such as homoarginine.

Figure 19 further describes suitable new peptide monomers representing EPO mimetic activity. In conjunction with the present invention, they may be used as suitable binding domains (monomers) for the creation of peptide units according to the present invention. In addition dissoreles may be used as monomeric or multimeric EPO mimetic peptides as described above.

At the beginning (end) and end (end) of the ligation monomers, up to five amino acids may be removed and / or added.

Since only the functional characteristics of the peptide are de-cisivative - here the ability to bind to and, in the case of an EPO receptor agonist, to activate the EPO receptor, the amino acid sequence precise peptide unit may vary. Above only suitable examples of EPO mimetic peptides are provided to support the general concept. However, also other EPO mimetic peptides with a different amino acid sequence may be used in conjunction with the present invention.

According to another embodiment, said peptide units bind to the TPO receptor, thereby dimerizing the TPO doreceptor complex. Preferably, they induce signal transduction and are thus TPO receptor agonists. Peptide units, respectively, the monomeric binding domains that create the peptide units, may be selected from the group of TPO mimetic peptides. Suitable TPO mimetic peptides are well known in the art and may be used with respect to to the present invention. Suitable examples are, for example, described in WO 98/25965, US5932546, W00024770, Cwirla, S.E.,. Balasubramanian, D.J. Duffin, C.R. Wags-trom, C.M. Gates, S.C. Singer, A.M. Davis, R.L. Tansik, L.C. Mattheakis1C. M. Boytos, P. J. Schatz, D. P. Baccanari, N. C. Wrighton, R. W. Barrett eW. J. Dower (1997). "Agonist peptide of the thrombopoietin receptor as well as the natural cytokine." Science 276 (5319): 1696-1699, US6083913, US6465430, US5659451, US6121238, US6251864, Dower, W.J., S. E.Cwirla, P. Balasubramanian, P.J. Schatz, D. P. Baccanari and R. W. Barrett (1998). "Peptide agonists of the thrombopoietin receptor." Stem Cells 16 Supl2: 21-29, W005023834, W00024770, the description of all documents with respect to the structure / amino acid sequence of TPO mimetic peptides is fully incorporated by reference.

Peptide units according to the invention may comprise, in addition to stereoisomeric L-amino acids or D-amino acids, non-natural / unconventional α-amino acids such as alpha-disubstituted amino acids, N-alkyl amino acids or lactic acid, for example 1-naphthylalanine, 2-naphthylalanine, homoserine methyl ether, β-alanine, 3-pyridylalanine, 4-hydroxyproline, O-phospho-serine, N-methylglycine (sarcosine), N-acetyl serine, N-acetylglycine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, nor-lysine, 5-aminolevulinic acid or 5-aminovaleric acid. The use of N-methylglycine (MeG) and N-acetylglycine (AcG) is especially preferred, particularly in a terminal position. Also within the scope of the present invention are peptides which are retro, inverse / inverse peptides of the defined peptides and those peptides consisting entirely of D-amino acids.

Also, peptide derivatives may be used, for example, the oxidation products of methionine or deamidated glutamine, ar-ginine and C-terminus amide.

Here, the abbreviations for the one-letter code as uppercase letters are those of the standard peptide nomenclature, extended by the addition of unnatural amino acids. Amino Acid Code

L-Alanine

V L-Valine

L L-Ieucine

I L-Isoleucine

M L-methionine

F L-phenylalanine

Y L-tyrosine

W L-Tryptophan

H L-Histidine

S L-Serine

T L-threonine

C L-cysteine

N L-Asparagine

Q L-Glutamine

D L-Aspartic Acid

And L-glutamic acid

K L-Iysine

R L-arginine

P-proline

G glycine

Ava, 5-Ava 5-Aminovaleric acid

Als1 5-Als 5-Aminolevulinic Acid

MeG N-methylglycine

AcG N-acetylglycine

Hsm homoserine methyl ether

Har homoarginine

1nal 1 -naphtylalanine

2nal 2-naphthylalanine

3Ala beta-alanine

hoc / hcy homocysteineAc acetylated

Am amid

Dap diamino propionic acid

Dab diamino butyric acid

Aad adipic alpha-amino acid

Asu alpha-amino-suberic acid

Adi adipic acid

Glr Glutaric Acid

Selenocysteine Sec

Peptide units may be modified, for example, by conservative single amino acid exchanges. Such exchange alters the structure and function of a binding molecule, but slightly in most cases. In a conservative exchange, one amino acid is substituted for another amino acid within a group with similar properties.

Examples of corresponding groups are:

- 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, Y1N1Q

- amino acids having aromatic side chains: F, Y, W

- positively charged amino acids: K, R, H

- negatively charged amino acids: D, E

- amino acids of similar size or molecular weight, wherein the molecular weight of the substituting amino acids deviates by a maximum of +/- 25% (or +/- 20%, +/- 15%, +/- 10%) from the molecular weight of the original amino acid.

More specifically, Wrighton et al. (US Patent 5,773,569 and associated patents) have examined in detail, using phagedisplay techniques, which amino acids of an EPO mimetic peptide can be replaced while maintaining activity. They also investigated and published data on possible truncation, that is, the minimum length of a given monomeric peptide.

According to one embodiment of the invention, selected monomers of the group consisting of SEQ ID NOs: 2, 4-9 provided below are used for the formation of at least one bivalent peptide unit. Good activity shows a peptide with K at position 10 and K at position 17 according to SEQ ID NO: 2.

SEQ ID NO 2: GGTYSCHFGKLTWVCKKQGG

SEQ ID NO 4 GGTYSCHFGKLTWVCKPQGG

SEQ ID NO 5 GGTYSCHFGRLTWVCKPQGG

SEQ ID NO 6 GGTYSCHFGRLTWVCKKQGG

5-Aminolevulinic Acid Incorporation (Als):

<formula> formula see original document page 38 </formula>

SEQ ID NO 7 GGTYSCHF- (Als) -LTWVCKPQGG

SEQ ID NO: 8 GGTYSCHF- (Als) -LTWVCKKQGG

SEQ ID NO 9 GGTYSCHFGKLT-InaI-VCKKQRG

According to one embodiment, the peptide units are monomer-based according to SEQ ID NO 2 and 4 to 9 as provided above or modifications thereof. The peptides may, for example, be modified by a conservative exchange of a single amino acid wherein preferably no more than 1, 2 or 3 amino acids are exchanged. Preferably, these peptides are modified as AcG at the N-terminus and MeG at the C-terminus.

Some examples of peptide units suitable for EPO receptor dimerization are listed below. The above link domain bars symbolize optional but preferred intramolecular disulfide bridging. SEQ ID NO 10 (based on SEQ ID NO 2):

GGTYSCHFGKLTWVCKKQGG - GGTYS CHFGKLTWCKKQGG

SEQ ID NO: 11

ao — GGTYSCHFGKLTWVCKKQGG - GGTYS CHFGKLTWVCKKQGG-cowt The binder in these bivalent structures is made standardized through molecular modeling to avoid bioactive conformation distortions (Figure 1).

SEQ ID NO 12

The Binding sequence may be shortened to a glycine residue. This sequence is also an example of a ligand composed of a glycine residue that forms part of the binding domain at the same time (see SEQ ID NO: 2).

<formula> formula see original document page 39 </formula>

This sequence has a continuous bivalent peptide unit harboring two slightly different (heterogeneous) binding domains. Such divalent peptides would not be economically accessible with a prior art dimerization approach (see above). The advantage of this heterodimeric molecule lies in the fact that the shunting amino acids (presently K and D at the C-terminus) interact with each other thereby stabilizing the dimer. Thus, it is advantageous to incorporate the respective stabilization modifications into the molecule through molecular modeling.

Another example is:

GGTYSCHFGKLT-1 nal-VCKKQRG-GGTYSCHFGKLT-1 nal-VCKKQRG

In another embodiment, the peptide optionally carries an additional amino acid, preferably one with a reactive side chain, such as cysteine, at the N-terminus such as, for example, in the following sequences:

C-GGTYSCHFGKLTWVCKKQGG-GGTYSCHFGKLTWVCKKQGG

C-GGTYSCHFGKLT-1 nal-VCKKQRG-GGTYSCHFGKLT-1 nal-VCKKQRG

The reactive cysteine side chain may serve as a ligation loop, for example for attachment of the polymeric carrier unit. The peptides, furthermore, optionally comprise intramolecular disulfide bridging between the first and second and / or third and quartcysteines.

Monomeric units, as exemplified by SEQ ID2, 4-9 and 12, 13 and 15, 15a, may be favorably combined with peptide units.

Examples for dimerization methods being applied to peptide unit monomers include (but are not limited to):

1. Dimerization via C-terminus to N-terminus connection, wherein the C-terminus of one of said monomeric peptides is covalently linked to the C-terminus of the other peptide. The binder / spacer between monomers may contain a diketopiperazine unit. A preferred Gly-Gly dedicated piperazine base can be obtained by C-terminal glycine domomer activation. This principle can also be used for forming a C-terminal dimerization.

<formula> formula see original document page 40 </formula>

The following formulas and examples represent four standardized examples which are optimized through molecular modeling:

(a) dimer based on SEQ ID NO 2 (dimer conformation is shown in figure 2): <formula> formula see original document page 41 </formula>

(b) dimer based on SEQ ID NO 2 with a shortened ligand in a glycine; the conformation is shown in figure 3.

<formula> formula see original document page 41 </formula>

(c) dimer based on SEQ ID NO 2 with a beta-alanine-substituted glycine (Figure 4). The monomer (SEQ ID NO 16) is also applicable as an EPO mimetic peptide.

<formula> formula see original document page 41 </formula>

(d) dimer based on SEQ ID NO 2 with an alternative beta-alanine-substituted glycine (Figure 5). The monomer (SEQ ID NO 17) may also be applied as an EPO mimetic peptide.

<formula> formula see original document page 41 </formula>

2. Dimerization via N-terminus to N-terminus connection, wherein the N-terminus of one of said monomeric peptides is covalently linked to the N-terminus of the other peptide, whereby the spacer unit contains preferably a dicarboxylic acid building block.

<formula> formula see original document page 0 </formula>

(a) In one embodiment, the resulting dimers based on SEQ ID NO 2 elongated at the N-terminus by a glycine residue (SEQ ID NO

18) contain a hexanedioyl unit as binder / spacer (Figure 6): GGGTYSCHFGKLTWVCKKQGG

<formula> formula see original document page 42 </formula>

(b) In an alternative embodiment, dimerization may be achieved using an octane diyl unit as binder / spacer (Figure 7):

<formula> formula see original document page 42 </formula>

3. Dimerization via side chains, wherein an amino acid side chain of one of said morionic peptides is covalently linked to an amino acid side chain of the other peptide with the inclusion of a suitable spacer molecule connecting the two monomers. peptides.

<formula> formula see original document page 43 </formula>

This may include:

(a) the connection via an amide bond:

<formula> formula see original document page 43 </formula>

(b) or the connection via a disulfide jumper:

<formula> formula see original document page 43 </formula> <formula> formula see original document page 44 </formula>

X symbolizes the nucleus of a major part of the respective amino acid that participates in the formation of the respective peptide bond.

According to a different strategy, the co-valent bridging of the peptide monomers to one another, thereby forming the peptide moiety, is formed between the C-terminal amino acid side chains of the first monomeric peptide unit and the N-terminal amino acid. second peptide monomer. Accordingly, it is preferred, in accordance with this dimerization strategy, that the monomeric peptides to be dimerized carry an amino acid with a bridging functionality, either at the N- or C-terminus, thereby allowing the formation of a bond. covalent between the last amino acid of the first peptide and the first amino acid of the second peptide. The bond that creates the dimer is preferably covalent. Suitable examples of respective point bonds are, for example, disulfide bridging and diseligate bridge bonding. However, also, for example, positively and negatively charged inter-amino acid amide bonds or other covalent bonds, such as thioether bonds, are suitable as bonding moieties. Preferred amino acids suitable for forming the respective bridging bonds between monomeric binding units to form the final peptide unit are, for example, cysteine, cysteine derivatives such as homocysteine or selenocysteine or thiolysin. They form disulfide bridges or, in the case of selenium-containing amino acids, disulfide bridges.

Suitable examples for respectively created peptide unit dimers are provided below using EPE mimetic peptides as examples:

<formula> formula see original document page 45 </formula> <formula> formula see original document page 46 </formula>

According to another development, the N- or C-terminus peptide dodimer (and hence of the respective monomeric peptide units being located at the beginning or end of the dimer) comprises an extra amino acid, allowing vehicle coupling. such as HES to create the supravalent compound. Accordingly, the introduced amino acid brings about its coupling functionality, such as, for example, an SH- group. A common example for such an amino acid is cysteine. However, other amino acids common to the functional group which allow the formation of a covalent bond (eg all negatively and positively charged amino acids) are suitable.Ac-C (tBu) -GGTYSCSFGKLT-Nal-VCK-Har-QG-Cys-Am

I

s

<formula> formula see original document page 47 </formula>

Bars on peptide monomers represent covalent intramolecular bridging bonds; in this case, disulfide bridges.

According to another development, the C and / or N-terminus amino acid involved in the formation of a covalent bridging linkage of monomeric units to a dimer represents a fully charged group such as the COO "or NH3 + Essa group. This characteristic leads to a favorable stabilization of the intermolecular jumper structure:

<formula> formula see original document page 47 </formula>

4. Dimerization by formation of continuous bi- or multivalent peptides has already been outlined above.

<formula> formula see original document page 47 </formula>

The central concept of this strategy refrains from synthesizing the monomeric peptides that form part of the multi- or bivalent peptide in distinct reactions before dimerization or multimerization, but synthesizing the final bi- or multivalent peptide in one step as a single peptide, for example in a single peptide. solid phase reaction. Thus, a separate demerization or multimerization step is no longer required. This aspect provides a great advantage, that is, complete and independent control of each sequence position in the final peptide unit. The method allows easily harboring at least two different receptor-specific binding domains in a peptide unit by virtue of indepen- dent control of each sequence position.

As for di- or multimeric monomers and peptides, continuous bivalent / multimeric peptides can be modified, for example, by acetylation or amidation or lengthened to positions.

C-terminals or N-terminals.

All possible modifications also apply to binder modification. In particular, it would be advantageous to attach polymeric soluble portions to the binder such as, for example, PEG1 starch or dextrans.

The synthesis of the final multi or bivalent peptide according to the invention may also include two subsequent and independent formations of disulfide bonds or other intramolecular bonds within each of the binding domains. In this way the peptides can be cyclized.

The reactive side chains of the peptides may serve as a binding loop, for example for other modifications. The peptide units optionally comprise intramolecular pore linkages between amino acids having a bridging-forming side chain functionality such as, for example, cysteines.

The peptides may be modified, for example, by acetylation or amidation or elongated at the C-terminal or N-terminal positions. Extension with one or more amino acids at one end (Nou C), for example, for preparation of a polymer vehicle attachment site often leads to a bivalent heterodimeric peptide unit which can be manufactured as a continuous peptide.

Several reactive amino acids are known in the art to couple vehicles to proteins and peptides. A preferred coupling amino acid is cysteine, which may be coupled to the term N or C or introduced into the peptide sequence. However, the coupling direction can make a considerable difference and thus should be carefully chosen for the peptide unit. This would be demonstrated based on the following examples of an EPO mimetic peptide: The following two dimers are used:

AGEM400C6C4

1 2 3 4 4100001

Ac-GGTYSCHFGkLT-1 -Na 1 -VCKKQFGGGTYSCHFGKLT-] -Nal-VCKKORG-Cys (tBu) -NH21 _—-- j I -; -: - ι

AGEM40C6C4

1 2 3 4 4

yooooo __ ____ ___ _

Ac-Cys (tBu) -GGTYSCHFGKLT-17Nal-VCKKQRGGGTYSCHFGKLT-1 -Nal-VCKKQRG-NH2

I _......... ι I .................. T w .....- ...... "..... "........ * ...... ~

1-Nal: 1-Naphthylalanine

Cys (tBu): Protected L-cysteine S-tert.-butyl

The 41 mere AGEM400C6C4 and AGEM40C6C4 have the same central sequence. Amino acids 1-40 of AGEM40C6C4 are the same as amino acids 2-41 of AGEM40C6C4. The only difference is the position of the tBu-protected cysteine. This amino acid is not involved in the receptor / drug interaction, but is intended to function as the vehicle-polymer linking group in the final conjugate. In the case of AGEM400C6C4, tBu-protected cysteine is attached to the terminus, in the case of AGEM40C6C4 it is attached to the terminus. Connection bars represent cysteine bridges.

There are two advantages of AGEM400C6C4 over AGEM40C6C4.

The first advantage is its synthetic accessibility. A-GEM400C6C4 can be isolated in higher overall yields than AAGEM40C6C4. In the case of the linear sequence synthesis of AGEM40C6C4, a CIZ-22 mer (CIZ-RGGGTYSCHFGKLT-1-Nal-VCKKQRG-NH2, CIZ: 2-Chlorobenzyloxycarbonyl group) is observed as a byproduct. During linear sequence purification with reverse phase high pressure liquid chromatography (RP-HPLC), it exhibits chromatographic behavior similar to the linear precursor of AGEM40C6C4 and thus becomes difficult to separate, leading to a loss in overall yield of the desired product. . In the case ofAGEM400C6C4, no analogous compounds are found.

The second advantage of AGEM400C6C4 over A-GEM40C6C4 lies in the easier implementation of a final conjugate analysis of the unprotected peptide with a polymeric carrier. One strategy for the analysis of a peptide conjugate is the selective degradation of the conjugate by endoprotease cleavage. Ideally, the entire peptide is released from the polymeric carrier during enzymatic hydrolysis. These peptide fragments can be identified and quantified by standard analytical techniques such as HPLC with MS or UV detection, etc.

In the case of AGEM400C6C4, cleavage can be performed with trypsin - an endoprotease that is known to cleave highly selective C-resting bonds of the loaded amino acids arginine and lysine (F. Lottspeich, H. Zorbas (Hrsg.), "Bioanalytik" , Spectrum Akademischer Verlag, Heidelberg Berlin, 1998). Applied to the DE400C6C4 conjugates, this will set free fragments covering 38 of 41 amino acids of the original peptide bound to the carrier molecule. In the case of A-GEM40C6C4, fragments of only 21 of 41 amino acids are released through tryptic digestion:

Conjugate of AGEM400C6C4

Ac-GGTYSCHFGKLT-1-Nal-VCKKQRGGGTYSCHFGKLT-1 -Nal-VCKKQRG — AGEM40C6C4 Cys-Polymeroconjugate

(Ac) Cys-GGTYSCHFGKLT-1-Nal-VCKKQRG-NH2 polymer

Fragments that are set free and can be detected by further analysis are marked in gray.

Since the analysis of an Active Pharmaceutical Ingredient is a key issue during its development, AGEM400C6C4 has a clear advantage over AGEM40C6C4.

Thus, in the case of a positively charged amino acid being located at respective positions, it is highly preferred to incorporate the binding amino acid (here cysteine) into the C-terminus because an almost complete peptide fragment can be generated since the cleavage site is by virtue of arginine at position X-19 of the monomer is typically very right before the polymer. Respective mounting methods as described above can also be used for the preparation of multimers with peptide moieties.

It is emphasized that all binding domains described herein, either alone or as part of a bivalent peptide, may also be combined with one or more other identical or different peptide domains to form respective bi- or multivalent peptide units. - or heterogeneous.

Peptide units are optionally modified as AcG at the N-terminus and MeG at the C-terminus.

Peptide units may be modified, for example, by acetylation or amidation or may be elongated at α-terminal or N-terminal positions. Extension with one or more amino acids in just one of two terms, especially for the preparation of attachment of the last carrier unit, often leads to a bivalent heterodimeric peptide unit which can be manufactured as a continuous peptide (see above).

The synthesis of the final multi-or bivalent peptide according to the invention may also favorably include two subsequent and independent formations of disulfide bonds or other intramolecular bonds within each of the binding domains.

The present invention further comprises respective compound production methods, wherein the peptide units are connected to the respective vehicle units. The compounds of the present invention may advantageously be used for the preparation of human and / or veterinary pharmaceutical compositions. Indications depend on the peptide units attached to them.

In the case of coupling EPO mimetic peptides, the compounds according to the present invention are especially suitable for the same indications as erythropoietin. Thus, the present invention also provides a method for treating a patient suffering from a disorder that is susceptible to treatment with a deeritropoietin agonist comprising administering to the patient a therapeutically effective dose or amount of a compound of the present invention comprising a peptide unit comprising a deeritropoietin agonist activity.

Erythropoietin is a member of the cytokine superfamily (see above). In addition to stimulating effects described in the introduction, erythropoietin has also been found to stimulate stem cells. The EPO mimetics described here are thus suitable for all indications caused by stem cell effects. Nonlimiting examples are the prevention and / or treatment of diseases associated with the nervous system. Examples are neurological lesions, diseases or disorders, such as, for example, Parkinsonism, Alzheimer's disease, Huntington's chorea, multiple sclerosis, amyotrophic lateral sclerosis, Gaucher disease, Tay-Sachs disease, a neuropathy, peripheral nerve injury, a tumor. spinal cord injury or a stroke injury. 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 heart failure. Examples are cardiac infarction, coronary artery disease, myocarditis, chemotherapy treatment, alcoholism, cardiomyopathy, hypertension, valvular heart disease, including mitral regurgitation or aortic stenosis, and thyroid gland disorders, chronic and / or acute coronary syndrome. In addition, EPO mimetics may be used to stimulate physiological mobilization, proliferation and differentiation of endothelial precursor cells, to stimulate vasculogenesis, to treat diseases related to endothelial precursor cell dysfunction, and to treat endothelial cell dysfunction. producing pharmaceutical compositions for treating such diseases and pharmaceutical compositions comprising said peptides and other agents suitable for stimulating endothelial precursor cells. Examples of such diseases are hypercholesterolemia, diabetes mellitus, endothelium-mediated chronic inflammation diseases, endothelium, including reticulum endotheliosis, atherosclerosis, coronary heart disease, myocardial ischemia, angina pectoris, age-related cardiovascular disease, Raynaud's disease1 pregnancy-induced hypertonia, chronic or acute renal failure, heart failure, wound healing, and secondary diseases.

The compounds bearing EPO mimetic peptide units are especially useful for the treatment of disorders which are characterized by an erythropoietin deficiency or a low or defective red blood cell population and especially for the treatment of any type of anemia and stroke. Such pharmaceutical compositions may optionally comprise pharmaceutically acceptable carriers in order to adopt the composition for the intended administration procedure. Suitable delivery methods, as well as vehicles and additives, are described, for example, in WO 2004/101611, incorporated herein by reference.

In the event that the compound carries PTTP mimetic peptide units (having an agonist activity), the compounds may be used for all indications as to thrombopoietin. They are thus useful for the prevention and treatment of TPO-mediated diseases such as, for example, haematological disorders, including thrombocytopenia, granulocytopenia, and anemia, and treatment of haematological malignancies. Thus, the present invention also provides a method for treating a patient suffering from a disorder that is susceptible to treatment with a detrombopoietin agonist comprising administering to the patient a therapeutically effective dose or amount of a compound of the present invention comprising a peptide unit comprising an agonist activity. detrombopoietin.

EXAMPLES

A. Illustration of the concept of supravalent compounds

The concept of supravalent molecules will be explained by half examples. Figure 13 shows an example of a simple supravalent molecule according to the invention. Two continuous bivalent peptides are N-terminally connected via a bifunctional PEG moiety carrying maleimide groups. Cysteine was chosen as the reactive attachment site for the PEG vehicle unit.

However, supravalent molecules may comprise more than two continuous bi- or multivalent units. Fig. 14 provides an example which is based on a carrier unit with a central glycerol unit as the branch unit and comprising three continuous bivalent peptides. Again, cysteine was used for fixation. Figure 20 shows an example using HES as a polymeric carrier unit. HES has been modified so that it brings maleimide groups that react with the SH groups of peptide units. According to the example, all attachment sites are linked to peptide units. However, also small PEG units (eg, 3 to 10 kD) could occupy at least some of the fixation sites.

As explained above, the supravalent concept may also be extended to polyvalent dendritic polymers in which a dendritic and / or polymeric unit of vehicle is connected to a large number of continuous bivalent peptides. For example, the denitic branch unit may be glycerol based (please refer to Haag 2000, incorporated herein by reference).

An example for a supravalent molecule based on a carrier unit with a dendritic branch unit containing six continuous bivalent peptides is shown in Figure 15.

Other examples of supravalent molecules comprise starch or dextran carrier units, which are oxidized using, for example, periodic acid to harbor a large number of aldehyde functions. In a second step, many bivalent peptides are attached to the carrier unit and together form the final molecule. Please note that even hundreds (e.g., 50 to 1000, preferably 150 to 800, more preferably 250 to 700) peptide units may be coupled to the carrier moiety which is, for example, HES. However, also, fewer peptide moieties may be attached to the HES molecule as shown in the figures, especially if EPO mimetic peptides are coupled. The average number of peptide units to be coupled may be chosen from about 2 to 1000.2 at 500, 2 to 100, 2 to 50, preferably 2 to 20, and more preferably 2 to 10, depending on the peptide and the linked aser receptor (s).

Figure 16 demonstrates the concept of a simple biodegradable supravalent molecule. Two continuous bivalent peptides are N-terminally connected via two bifunctional PEG moieties which are connected via a biodegradable ligand having an intermediate cleavage position. The ligands allow rupture of the large PEG unit in the subunits, thereby facilitating renal elimination.

The advantages of the supravalence effect were very surprising and unexpected. Initially, it was feared that conjugation to a macromolecule could reduce efficacy. This expectation was based on the presumed disadvantages in binding rate due to reduced diffusion rates with larger molecules. Another expectation was that of the various peptide APIs attached to a vehicle, not all of them could potentially bind to the receptor because of steric binding problems or because of the number of receptors which may be affected by macromolecular vehicle extensions, to be limited. and possibly below the number of peptide APIs. Thus, a potency increase of the API (Active Pharmaceutical Ingredient) peptide as observed with the supravalence concept of the present invention was not expected.

On the other hand, because of the significant pharmacokinetic changes that a macromolecular carrier is capable of introducing, the inventive potency could have been enhanced due to the longer half-life of the entire peptide / carrier complex. This phenomenon also has the effect that a supravalence effect is difficult to determine in vivo since it is a pharmacodynamic entity which has to be determined separately. In vitro assays, therefore, are not only sufficient, but could be the only useful way to clearly demonstrate the effect of supremacy.

The supravalence effect as described in the present invention can be demonstrated by comparing molar amounts of peptide API (conjugated to a carrier vs. unconjugated).

An experiment was performed in a standard TF-1 cell assay as recommended by the European Pharmacopoeia for the determination of EPO-like activity in vitro (please see also below). Basically, TF-1 cells (their proliferation being dependent presence of EPO-like activity) are grown in the presence of various concentrations of EPO or EPO mimetic substances. The resulting cell numbers are quantified using the MTT colorimetric assay and photometric measurements. Based on these data, it is possible to determine normalized dose-response ratios for each given substance.

In this assay, EPO and AGEM40 peptide (see below), the latter being a continuous bivalent peptide with known EPO mimetic activity, were used.

AGEM40 was used as an unconjugated peptide and as a comopeptide conjugated to a macromolecular carrier (in this case, 130 kD average molecular weight hydroxyethyl starch; the commercial source is Pharma-cay, Voluven as a plasma substitute). The building block size of this conjugate is approximately 40 kD, which means that the HES molecule has on average about 2-5, preferably 3 to 4 peptide portions. Also, a HES 200 / 0.5 can be used. After modification of the 130 kD HEES, approximately 4 AGEM 40 peptides were conjugated (molecular weight of the conjugated molecule: 150 kD). When an HES having a molecular weight of 200 kD was used, this totals approximately 5 HES-conjugated peptide units (molecular weight of the conjugated molecule: 220 kD).

The comparison shown in Figure 33 is based on molar comparison of peptide concentration, whether the peptide is conjugated or not. Surprisingly, the potency is increasing (EC50 decreases and the dose / response curve is to the left of the unconjugated peptide), therefore, demonstrating a positive pharmacodynamic influence of oligovalent conjugation to a macromolecular vehicle.

Thus - regardless of the expected pharmacokinetic improvements - the concept of conjugation according to the invention clearly increases the potency of the global active pharmaceutical ingredient (API) and thus its effectiveness.

This is a novel mechanism, which can certainly be used for EPO receptor-directed peptides, but potentially also for other membrane-bound pharmacological targets, especially other cytokine receptors, such as those for thrombopoietin, G-CSF, Interleukins and others ( look above).

B. Concepts for Conjugation of Peptide Units to Hydroxyethyl Starch

As outlined above, according to one embodiment, polysaccharides, such as hydroxyalkyl starch and preferably hydroxyethyl starch, are used as a polymeric vehicle for peptide units. In order to be able to conjugate the peptide units to the vehicle, it is possible. appropriate linker groups can be introduced into the starch molecule to facilitate coupling. According to one embodiment, amino groups are introduced onto the starch moiety (hereinafter described under the example hydroxyethyl starch). There are different strategies that can be followed. Three of them are explained in more detail here below; An overview of these methods is provided in Figure 34.

1. A two-step process: Aldehyde groups are introduced through oxidation and followed by reductive amination.

Oxidation of the HES molecule can be accomplished by various oxidation reagents, for example sodium periodate (NaIO4) and 2-lodoxybenzoic acid (IBX). Oxidation with NaIO4 is long and well known and leads to aldehydes through opening of saccharide rings.

<formula> formula see original document page 57 </formula> can be used stoichiometrically to convert primary alcohol groups to aldehydes without opening saccharide rings (see Figure 36, for review, see: VV Zhdankin, Current Organic Synthesis, 2005, 2,121-145 cited articles). Derivatives that are more water soluble are described in literature (Thottumkara, A. P.; Vinod. T. K., Tetrahedron Lett., 2002, 43 (4), 569). ;

According to a different approach, the carbohydrate (preferably a starch molecule such as HAS) is oxidized by contacting the carbohydrate-containing starting material (preferably a starch molecule such as as HAS) with a reagent that produces an o-xoammonium ion in the presence of an oxidizing agent or through contact of the starting material directly with the reactive species, the oxoammonium ion.

The oxidizing agent is, for example, a chemical oxidizing agent, such as a hypohalide, for example hypochlorite and hypobromite, disodium or hydrogen peroxide. Alternatively, the oxidative enzyme may be used as an oxidizing agent (see, for example, WO 99/23240, incorporated herein by reference).

The oxoammonium ion-producing reagent is preferably a nitroxyl compound, more preferably a di-tert-nitroxyl compound such as 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) or the resp. asset derived from it. Oxidation with catalytic amounts of TEMPO In the presence of stoichiometric amounts of suitable co-oxidation reagent, ie sodium hypochlorite (NaOCI), leads mainly to the oxidation of primary alcohol groups in aldehydes (see Figure 35 for HES, position 6 or the C-terminal atom of the hydroxyethyl group is converted to an aldehyde) without opening the saccharide rings (lit: PL Bragd, H. van Bek-kum, AC Besemer, Topics in Catalysis, 2004, 27, 1-4; Review: W. Adam, CR Saha-Moller, PA Ganeshpure, Chem. Feb. 2001, 101, 3499-3548 and cited articles, Nooy AEJ, AC Besemer, H. v. Bekkum, Carbohydrate Research, 1995, 269, 89, EP 1 093 467, EP 1 173 409, WO 00/50621, EP 1077 221, EP 1 149 846).

Alternatively, instead of catalytic amounts of TEM-PO, stoichiometric amounts of the active species - the oxo-ammonium compound - may be used (lit. J. M. Bobbitt, N. Merbouh, Organic Synthe- ses, 2005, 82, 80). Other derivatives of TEMPO (i.e. 4-acetamido-, 4-hydroxy-TEMPO) are also suitable, especially with regard to reaction pH or water solubility.

After oxidation, the obtained aldehyde groups are converted to amines by reductive amination. As reducing agents, for example, sodium cyanoborohydride or a borane-dimethylamine complex (or other complex borane compounds) may be used. As an amine compound, for example, ammonium chloride or diamines, such as 1,3-diaminopropane, 1,3-diaminopropan-2-ol or lysine, may preferably be used at slightly acidic pH values. The use of diamines enhances the spacer length between the main part of HES and the peptide drug and the yield of reductive amination.

Unconverted aldehyde groups will be reduced back to the starting primary alcohol.

A modification of the oxidation with IBX can be made in DMSO in the presence of N-hydroxy sucinimide (Figure 39). In this case, the corresponding activated ester of uronic acid is directly formed. Such a species may be directly converted, for example, with diamines, for example 1,3-diaminopropane, to an aminated HES (lit. R. Mazitschek M.MJbaier, A. Gi-annis, Angew. Chem. 2002, 114, 21 , 4216-4218; A. Schulze, A, Giannis, Adv. Synth. Cat. 2004, 346, 252-256).

2. A two-step process where HES is activated by an "a-copying" agent and reacted with excess of a bisfunctional amine.

Several methods are described for the activation of polysaccharides, i.e. 1,1'-carbonyldiimidazole (CDI) (lit. GS Bethell, JS Ayers, J. TW Hearn, WS Hancock, J. of Chromatography, 1981, 219, 361-372), epi-bromoidrin (1-bromo-2,3-epoxypropane, alternatively epichlorohydrin, respectively 1-chloro-2,3-epoxypropane) (lit: H. Dóbeli, E. Huchuli, Patent 0253303 B1), chloride 2,2,2-trifluoroethanesulfonyl (tresyl chloride) (lit., P. P. Jennissen, J. Mol. Recogn., 1995, 8, 116-124), bromocyanide (BrCN) (lit: GS Bethell, JS Ayers, J. TW Hearn J. WS Hancock, J. of Chromatography, 1981, 219, 361-372; P. P. Jennissen, J. Moi Recogn., 1995, 8,116-124, H. Dobeli, E. Huchuli , EP0253303 B1). All reagents have in common the fact that they introduce functional groups, which are highly reactive and can be reacted in a common second step in excess of bifunctional nucleophiles, such as amines, ie deamonium chloride (not suitable with all activation reagents). ) or diamines, i.e. 1,3-diaminopropane, 1,3-diaminopropan-2-ol or lysine.

3. A one-step process where HES is activated by the addition of a suitable amine precursor.

Suitable amine precursors are, for example, haloalkylamines (i.e. as their salts, i.e. bromoethylammonium bromide) or reactive aza-rings, i.e. aziridines, i.e. lithium L-aziridine-2-carboxylate.

All three of the described strategies have in common that upon introduction of the amino groups into the hydroxyethyl starch, these amino groups are converted to maleimides as an example of an appropriate linker. This can be carried out, for example, by reaction with activated ω-maleimido carboxylic acids, ie N-hydroxy succinimide 3- (maleimido) propionic acid ester or N-hydroxy succinimide 4- (maleimidate) acid ester ) butyric. The resulting maleimides represent the final active functional groups for coupling with peptides bearing a free group thiol.

1. A two-step process: Aldehyde groups are introduced through oxidation and followed by reductive amination.

1.1. Oxidation of primary alcohols in aldehydes

Through direct oxidation of the primary alcohols in hydroxyethyl starch, more precisely the C6-OH glucose groups and hydroxyethyl groups, aldehyde groups can be formed. Such oxidation products are obtained with commercially available oxidizing agents such as TEMPO or IBX (e.g., Sigma-Aldrich or Acros).

a) Periodate oxidation

This method is described in more detail in the experiential section under C (see below).

b) Oxidation with TIME

Using 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) or derivatives thereof, for example 4-acetamido-TEMPO or 4-hydroxy-TEMPO and co-oxidants such as sodium hypochlorite in a mixture with bromide After potassium (molar ratio of TIME: NaOCI: KBr, eg 1:40:20), primary alcohols may be oxidized in short reaction times around 60 min in a phosphate buffer in a pH range between 6-8, whereby a higher pH increases the reaction rate. With a different molecular concentration of the oxidation mixture, especially the co-oxidant, the number of aldehydes formed can be controlled. Consequently, the amount of anchor groups, and thus the amount of peptide drug on the vein, can be controlled through this first step.

Optimization was monitored with Purpald's reagent which forms a purple aldehyde-only adduct and the redox titration of hypochlorochloride with an iodine / starch complex.

Processing was performed by ultrafiltration techniques using a PES membrane of different molecular weight sections, followed by lyophilization (literature: PL Bragd, H. van Bekkum, AC Bese-mer, Topics in Catalysis, 2004, 27, 1-4; review: W. Adam, CR Saha-Moller, PA Ganeshpure, Chem., Feb. 2001, 101, 3499-3548 and quoted articles; AEJ of Nooy, AC Besemer, H. v. Bekkum, Carbohydrate Research , 1995, 269, 89).

Fig. 35 provides an illustrative overview of the TEMPO-mediated oxidation mechanism of primary alcohols. Additional oxidation to carboxylates occurs only through the use of an excess oxidation reagent.

c) Oxidation with IBX

Using the 2-lodoxybenzoic acid oxidation reagent (IBX) or its derivatives, HES can be oxidized to DMSO as a solvent. After a 1-2 h reaction time, IBX can be removed by the addition of water (10 times) and precipitated IBX is removed by filtration. The processing was performed by ultrafiltration techniques using a PES membrane of different molecular weight sections, followed by lyophilization.

With a molar concentration other than IBX, the number of formeddehydes can be controlled. Through this concentration, the amount of anchor groups and thus the amount of peptide drug on the vehicle can be controlled as well.

Optimization was monitored with Purpald's reagent which forms a purple aldehyde-only adduct (for a review see: V. V. Zhdankin, Current Organic Synthesis, 2005, 2, 121-145 and cited articles).

Figure 36 provides a schematic view of the oxidation of TEMPO or IBX primary alcohols, followed by a reductive amination.

Figure 37 illustrates the introduction of maleimide groups and conjugation with a peptide drug.

1.2 Reductive Amination

a) Reductive ammonium chloride demining

This method is described in more detail in Experimental section C (see below).

b) Reductive Amination with Diamine Ligand

In order to enhance the spacer length between the HES main portion of a peptide drug and the reductive amination yield, the reductive amination reaction may be performed with a diamine such as 1,3-diaminopropane, 1, 3-diaminopropan-2-ol or lysine as amine source and different reducing agents, i.e. sodium cyanoborohydride or borane dimethylamine complex.

Example: Reductive Amination with 1,3-Diaminopropane

Reductive amination of oxidized HES is performed in a 1 M phosphate buffer, pH = 5, with a 10-fold excess of 1,3-diaminopropane compared to the oxidizing agent used in the previous step. After equilibration for approximately 90 min, an excess of sodium cyanoborohydride (Na [CN] BH3) is added and several portions. The processing was performed by ultrafiltration techniques using a FT membrane of different molecular weight sections, followed by lyophilization. Of the functionalized HES derivatives, only the massamolar range greater than 100 kDa was used.

Fig. 38 provides an illustrative overview of reductive amination with diamines, such as 1,3-diaminopropane, followed by the introduction of maleimide groups through a periodate-oxidized HES.

Oxidation of primary alcohols directly in activated esters

Through oxidation of the primary alcohols in hydroxyethyl starch, more precisely the C6-OH groups of glucose and hydroxyethyl. For such oxidation, commercially available IBX oxidizing agents (eg, Sigma-Aldrich or Acros) in the presence of N-hydroxy succinimide (HOSu), the oxidized alcohol to OSu-ester, which can be directly converted to an amine. using diamines.

Oxidation with IBX in the presence of HOSu - direct conversion to amine

Using the 2-lodoxybenzoic acid (IBX) oxidation reagent or its derivatives in the presence of N-hydroxy succinimide, HES may be oxidized to DMSO as a solvent. After 1-2h, the oxidation is the 10u-formed excess O-ester of diamine is added, for example 1,3-diaminopropane.

Processing was carried out by ultrafiltration techniques using a FOOT membrane of different molecular weight sections, followed by lyophilization. Of the optimized HES derivatives, only molar mass greater than 100 kDa was used (for literature see: R.Mazitschek, M. MJbaier1 A. Giannis, Angew. Chem. 2002, 114, 21, 4216-4218; A. Schulze, A. Giannis, Adv. Synth. Cat. 2004, 346, 252-256)

Figure 39 illustrates the oxidation of primary alcohols to the OSu ester, followed by direct conversion with a diamine.

2. A two-step process where HES is activated by a "coupling" reagent and reacted with an excess of a bisfunctional amine.

Several alternatives may be applied to introduce groupsamine over the major part of HES. Some examples: a) Modification with carbonyldiimidazole (CDI)

Dry HES is suspended in dry acetone for 1 h. CDI is added and the mixture is stirred for 1h. Alternatively, some salts, such as potassium iodide, as activator / co-nucleophiles may be added. The HES is centrifuged (2000 U / min,> 10 min). After decantation, novaacetone is added and the HES is centrifuged again. After 3 times washing with acetone, HES is taken up in 1M carbonate buffer, pH 10 and 1,3-diaminopropane (pH 11) is added and the mixture is stirred for 1h. HES is processed by ultracentrifugation (MWCO 100 kD), followed by lyophilization.

Figure 40 illustrates modification with carbonyldiimidazole, followed by a diamine to introduce an amino group.

(b) Modification with epibromoidrin (Epi)

HES is dissolved in some DMF and epibromoidrin is added (alternatively, some salts, for example potassium iodide, as activator / co-nucleophiles may also be added) and the mixture is stirred overnight. HES is diluted with water (10 times) and processed by ultracentrifugation (MWCO 50 or 100 kD), followed by lyophilization. The product is dissolved in 1 M phosphate buffer, pH = 7 and 1,3-diaminopropane is added and the mixture is stirred for 1 h. HES is processed by ultracentrifugation (MWCO 50 or 100 kD), followed by lyophilization.

Fig. 41 illustrates the modification with epibromoidrine, followed by a diamine to introduce an amino group.

3. A one-step process where HES is activated by the addition of a suitable amine precursor.

a) Modification with 2-aminoethyl bromide hydrobromide

HES is dissolved in DMSO and 2-bromoethylamine bromide hydrobromide is added (alternatively, some salts, eg potassium iodide, as activator / co-nucleophiles may also be added) and stirred overnight (also some heating can be used). HES is diluted with water (10-fold) and processed by ultra-centrifugation (MWCO 50 or 100 kD), followed by lyophilization. Figure 42 illustrates the modification with 2-aminoethyl bromide hydrobromide to introduce an amino group.

b) Modification with lithium L-aziridine-2-carboxyate

HES is dissolved in DMSO or an aqueous buffered solution and 2-lithium L-aziridine-2-carboxylate is added (alternatively, some salts, eg potassium iodide, as activator / co-nucleophiles may also be added) and stirred overnight. . The mixture is diluted with water (10 times) and processed by ultracentrifugation (MWCO 50 or 100 kD), followed by lyophilization.

Figure 43 illustrates modification with L-aziridine-2-carboxylate delithium to introduce an amino group.

C. Detailed Examples of Embodiments of the Present Invention

I. Peptide Synthesis of Monomers

Manual Overview

Synthesis is performed using a Discover micron-das (EMC) system using Rink-PL Amide Resin (0.4 mmol / g replacement rate) or pre-loaded Wang Resins on a full scale. 0.4 mmol. Removal of the Fmoc group is achieved by the addition of 30 ml depiperidine / DMF (1: 3) and 100 W irradiation for 3 χ 30 sec. Amino acid coupling is achieved by the addition of a 5-fold excess of amino acid in PyBOP / HOBT / DIPEA DMF as coupling and 50 W irradiation additives for 5 χ 30 sec. Between all irradiation cycles, the solution is manually cooled with the aid of an ice bath. After deprotection and coupling, the resin is washed 6 times with 30 ml of DMF. After deprotection of the last amino acid, some peptides are acetylated by incubation with 1.268 ml of coating solution (4.73 ml of acetic anhydride and 8.73 ml of DIEA in 100 ml of DMSO) for 5 minutes. Prior to cleavage, the resin is then washed 6 times with 30 ml of DMF and 6 times with 30 ml of DCM. Cleavage of crude peptides is obtained by treatment with 5 ml TFA / TIS / EDT / H2 O (94/1 / 2.5 / 2.5) for 120 minutes under inert atmosphere. This solution is filtered through 40 ml of frozen ether. The precipitate is dissolved in acetonitrile / water (1/1) and the peptide is epurified by RP-HPLC (Kromasil 100 C18 IOpm1 250 χ 4.6 mm).

Automatic Overview

Synthesis is performed using an Odyssey microon-das (EMC) system using Rink-PL Amide Resins (0.4 mmol / g substitution rate) or preloaded Wang Resins on a full scale. of 0.25 mmol. Removal of Fmoc groups is achieved by adding 10 ml piperidine / DMF (1: 3) and 100 W irradiation for 10x10 sec. Amino acid coupling is obtained by the addition of a 5-fold excess of amino acid in PyBOP / HOBT / DIPEA DMF as coupling additives and 50 W irradiation for 5 χ 30 sec. Between all irradiation cycles, the solution is cooled by bubbling nitrogen through the reaction mixture. After deprotection and coupling, the resin is washed 6 times with 10 ml of DMF. After deprotection of the last amino acid, some peptides are acetylated by incubating with 0.793 ml of coating solution (4.73 ml of acetic anhydride and 8.73 ml of DIEA in 100 ml of DMSO) for 5 minutes. Prior to cleavage, the resin is then washed 6 times with 10 ml DMF and 6 times with 10 ml DCM. Cleavage of crude peptides is obtained by treatment with 5 ml TFA / TIS / EDT / H2 O (94/1 / 2.5 / 2.5) for 120 minutes under an inert atmosphere. This solution is filtered into 40 ml ice cold ether, the precipitate dissolved in acetonitrile / water (1/1) and the peptide purified by RP-HPLC (Kromasil 100C18 10 Mm, 250x4.6 mm).

Purification

All peptides were purified using a Nebu-la-LCMS system (Gilson). Crude material from all peptides was dissolved in acetonitrile / water (1/1) and the peptide purified by RP-HPLC (Kromasil100 C18 10 μπι, 250 χ 4.6 mm). The flow rate was 20 ml / min and the LCMS split ratio 1/1000.

II. Intramolecular disulfide bridgingKaCl cycling (FeCNp)

Solution 1: 10 mg of the peptide is dissolved in 0.1% TFA / acetonitrile and diluted with water until a concentration of 0.5 mg / ml is reached. Solid ammonium bicarbonate is added to reach a pH of ap. 8

Solution 2: In a second vial, 10 ml 0.1% TFA / acetonitrile is diluted with 10 ml water. Solid ammonium bicarbonate is added until a pH of 8 is reached and 1 drop of a 0.1 M solution of K3 [(FeCN6)] is added.

Solution 1 and 2 are added dropwise over a period of 3 hours until an acetonitrile / water mixture (1/1; pH = 8). The mixture is incubated at room temperature overnight and the mixture is concentrated and purified by LCMS.

CLEAR-OX® Resin Cycling

To 100 ml of acetonitrile / water (1/1; 0.1% TFA), solid deammonium bicarbonate is added until a pH of 8 is reached. This solution is degassed by bubbling argon for 30 minutes. Now 100 mg of CLEAR-OX ™ resin is added. After 10 minutes, 10 mg of the peptide is added as a solid. After 2 h incubation, the solution is filtered, concentrated and purified by LCMS.

Purification of cyclic peptides:

All peptides were purified using a Nebu-la-LCMS system (Gilson). Crude material from 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 μπι, 250 χ 4.6 mm). The flow rate was 20 ml / min and the LCMS split ratio was 1/1000.

III. Tests in wYrocom Monomers

TF-1 cell proliferation assay by incorporation of BrdUCell TF-1 cells into the logarithmic growth phase (~ 2.105 - 1.106 cells / ml; RPMI medium; 20% fetal calf serum; supplemented with Pe-niciline, streptomycin, L 0.5 ng / ml Interleukin 3) is washed (centrifuged 5 min. At 1500 rpm and resuspended in complete RPMI semIL3 at 500,000 cells / ml) and precultured prior to the start of the assay for 24 h without IL -3. The next day, cells are cultured in 24- or 96-well slides using at least 6 concentrations and 4 wells per concentration containing at least 10,000 cells / well per test agent. Each experiment includes controls comprising recombinant EPO as a positive control agent and wells without the addition of decytocin as a negative control agent. EPO peptides and controls are prediluted in medium to the desired concentration and added to the cells, starting a 3-day culture period under standard culture conditions (37 ° C, 5% gaseous carbon dioxide, atmospheresaturated with Water). Concentrations always refer to the final agitation concentration in the well during this 3-day culture period. At the end of this culture period, FdU is added to a final concentration of 8 ng / ml culture medium and the culture continued for 6 hours. Then BrdU (bromodeoxyuridine) and dCd (2-deoxycytidine) are added to their final concentrations (10 ng / ml BrdU; 8 ng / ml dCD; final concentrations in culture medium) and continued culture for an additional 2 hours. .

At the end of this incubation and culture period, the cells are washed once in phosphate buffered saline containing 1.5% BSA and resuspended in a minimum amount of liquid. From this suspension, cells are added dropwise in 70% ethanol at -20 ° C. Thereafter, cells are incubated for 10 min on ice and then analyzed directly or stored at 4 ° C prior to analysis.

Prior to analysis, the cells are pelleted by centrifugation, the supernatant is discarded and the cells resuspended in a minimum amount of fluid remaining. The cells are then suspended and incubated for 10 min. in 0.5 ml 2 M HCl / 0.5% triton X-100. They are then pelleted again and resuspended in a minimal amount of fluid remaining, which is diluted with 0.5 ml of 0.1 N NaaB4 O7, pH 8.5 before direct repelling of the cells. Finally, the cells are resuspended in 40 μl phosphate buffered saline (1.5% BSAa) and divided into two reaction tubes containing 20 μΙ cell suspension each. 2 μl of anti-BrdU-FITC (DAKO, clone Bu20a) is added to one tube and 2 μl of control IgGI-FITC (Sigma) is added to the second tube, starting a 30 min incubation period at room temperature. environment. Then 0.4 ml phosphate buffered saline and 10μg / ml propidium iodide (final concentration) are added. Flow nocitometer analysis refers to the fraction of 4C cells or cells with majorploidy and the fraction of BrdU-positive cells, thereby determining the fraction of cells at the relevant stages of the cell cycle.

TF-1 cell proliferation assay by MTT

TF-1 cells at logarithmic growth phase (~ 2.105 - 1.106 cells / ml; RPMI medium; 20% fetal calf serum; supplemented with Pe-niciline, streptomycin, L-Glutamine; 0.5 ng / ml Interleukin 3 ) are washed (centrifuged 5 min. at 1500 rpm and resuspended in complete RPIVII semIL3 at 500,000 cells / ml) and precultured prior to the assay for 24 h semIL-3. The next day, cells are cultured on 24- or 96-well slides 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 re-combining EPO as a positive control and wells without addition of cytokine as a negative control agent. EPO peptides and controls are prediluted in medium to desired concentrations and added to cells, beginning a 3-day culture period under standard culture conditions (37 ° C, 5% gaseous carbon dioxide, saturated atmosphere with water). Concentrations always refer to the initial concentration of agent in the well during this 4 day culture period.

On day 4, prior to the start of the analysis, a dilution series of a known number of TF-1 cells is prepared in a series of wells (0/2500/5000/10000/20000/50000 cells / well in 100 μΙ medium). . These wells are treated the same as the test wells and then provided with a calibration curve from which cell numbers can be determined. Having adjusted these reference wells, MTS ePMS from the MTT Proliferation Kit (Promega, CeIITiter 96 Aqueous Non-Radioactive Cell Proliferation Assay) is thawed in a water bath at 37 ° C and 100 μΙ of PMS solution are added. 2 ml of MTS solution. 20 μl of this mixture is added to each well of the assay slides and incubated at 37 ° C for 3-4 h. 25 μΙ of 10% sodium dodecyl sulfate in water is added to each well before measuring 49492 in an ELISA reader.

Using graphical assessments as shown in Figures 17 and 18 based and dose-response relationship calculations using the GraphPad program, the following EC50 values were determined based on the MTT assay data.

The following table shows the EC50 values of some exemplary peptide monomers:

SEQ ID NO 2: GGTYSCHFGKLTWVCKKQGG 3284 nmols / l

SEQ ID NO 4: GGTYSCHFGKLTWVCKPQGG 4657 nmols / l

SEQ ID NO 5: GGTYSCHFGRLTWVCKPQGG 5158 nmols / l

SEQ ID NO 6: GGTYSCHFGRLTWVCKKQGG 4969 nmols / l

SEQ ID NO 7: GGTYSCHF- (Als) -LTWVCKPQGG 5264 nmols / l

SEQ ID NO: 8 GGTYSCHF- (Als) -LTWVCKKQGG 4996 nmols / l

GGTYSCHFGPLTWVCKKQGG 2518 nmols / l

GGTYSCHFAKLTWVCKKQGG 5045 nmols / l

GGTYSCHFGGLTWVCKPQGG no detectable activity

IV. Synthesis of bivalent EPO mimetic peptide unitsAutomatic synthesis of linear SEQ ID NO 11 (AGEM11)

The synthesis is performed using a Liberty micro-das (EMC) system using Rink Amide Resin (0.19 mmol / g substitution rate) on a 0.25 mmol scale. Removal of Fmoc groups is achieved by double treatment with 10 ml piperidine / DMF (1: 3) and 50 W irradiation for 10x10 sec. Amino acid coupling is obtained by double treatment with a 4-fold excess of amino acid in DMF PyBOP / HOBT / DIPEA as 50 W coupling and irradiation additives for 5 χ 30 sec. Between all irradiation cycles, the solution is cooled by bubbling nitrogen through the reaction mixture. After deprotection and coupling, the resin is washed 6 times with 10 ml DMF. After the double coupling cycle, all unreacted amino groups are blocked by treatment with a 10-fold excess of N- (2-Chlorobenzyloxycarbonyloxy) succinimide (solution). 0.2 M in DMF) and 50 W irradiation for 3 χ 30 sec. After deprotection of the last amino acid, the peptide is acetylated by incubation with 0.793 ml of coating solution (4.73 ml of acetic anhydride and 8.73 ml of DIEA in 100 ml of DMSO) for 5 minutes. Prior to cleavage, the resin is then washed 6 times with 10 ml DMF and 6 times with 10 ml DCM. Cleavage of crude peptides is obtained by treatment with 5 ml of TFA / TIS / EDTVH2O (94/1 / 2.5 / 2.5) for 120 minutes under an inert atmosphere. This solution is filtered into 40 ml of ice ether, the precipitate dissolved in acetonitrile / water (1/1) and the peptide is purified by RP-HPLC (Kromasil 100C18 10 pm, 250 χ 4.6 mm).

The linear AGEM11 purification scheme, Kromasil 100 C1810 pm, 250 χ 4.6 mm and the gradient used, are therefore represented in Figures 8 and 9 from 5% to 50% acetonitrile (0.1% TFA). in 50 minutes.

Linear AGEM11 Cycling

ac-GGTYSCHFGKLTWVCKKQGG - GGTYSCHFGKLTWVCKKQGG-meet,

30 mg of the linear peptide is dissolved in 60 ml of solution A. This solution and 60 ml of DMSO are added dropwise to 60 ml of solution A (total addition time: 3 h). After 48 h, the solvents are removed by evaporation and the remaining residue dissolved in 30 ml DMSO / water (1/1). 30 ml of acetic acid and 17 mg of iodine (dissolved in DMSO / water (1/1)) are added and the solution is mixed for 90 minutes at room temperature, whereupon 20 mg of ascorbic acid is added and the solvents are added. removed by evaporation The crude mixture is decomposed into acetonitrile / water (2/1) and the peptide is purified by RP-HPLC (Kromasil 100 C18 10 pm, 250 χ 4.6 mm).

Solution A: Acetonitrile / water (1/1) containing 0.1% TFA. The pH is adjusted to 8.0 by the addition of ammonium bicarbonate.

Purification parameters for cyclic AGEM11 are given in Figures 10 and 11 (Scheme: Cyclic AGEM11 Purification, Kromasil100 C18 10 μm, 250 χ 4.6 mm, 5% to 35% acetonitrile gradient (TFA 0.1) %) in 50 minutes).

V. In vitro Proliferation Assay to determine EPQ activity

TF1 cells in the logarithmic growth phase (2.105 - 1.106 cells / ml grown in RPMI with 20% fetal calf serum (FCS) and 0.5ng / ml IL-3) were counted and the number of cells required to perform An assay was centrifuged (5 min. 1500 rpm) and resuspended in RPMI with 5% FCS without IL-3 at 300,000 cells / ml. Cells were precultured in this medium (deprivation) without IL-3 for 48 hours. Before starting the assay, cells were counted again.

Just before the start of the assay, depeptide and EPO stock solutions were prepared. Peptides were weighed and dissolved in RPMI with 5% FCS to a concentration of 1 mM, 467 μΜ or 200μΜ. EPO stock solutions were at 10 nM or 20 nM. Two hundred and two μΙ of these stock solutions were pipetted into a well of a 96-well culture slide - one slide was taken for each substance to be tested. Two hundred μΙ RPMI with 5% FCS were pipetted into seventeen other wells on each slide. The contents were mixed and 92 μΙ of this well was transferred to the next and so on. Thus, a series of dilutions (18 dilutions) of each substance was prepared so that, in each consecutive well, the concentration was 1: V10 of the concentration in the well before it. From each well, 3 χ 50 μΙ were transferred to three empty wells. Thus, each substance concentration was measured in quadruplicate. Note that the row of cavities higher and lower each blade has been left empty.

Pretreated (private) cells were centrifuged (5 min. 1500rpm) and resuspended in RPMI with 5% FCS at a concentration of 200,000 cells per ml. Fifty μΙ cell suspension (containing 10,000 cells) was added to each well. Note that due to cell addition, the final concentration of substances in the wells was half the original dilution range. The slides were incubated for 72h at 37 ° C in 5% CO 2.

Prior to commencing evaluation, a dilution range of known quantities of TF-1 cells in wells was prepared: 0/2500/5000/10000/20000/50000 cells / well were pipetted (in 100 μΙ RPMI + FCS at 5 ° C). %) in quadruplicate.

To measure the number of living cells per well, ready-to-use MTT reagent (Promega, CeIITiter 96 Aqueous One Solution Cell Pro-liferation Assay) was thawed in a 37 ° C water bath. Per well, 20 μl of MTT reagent was added and slides were incubated at 37 ° C in 5% CO 2 for an additional 1-2 h. Twenty-five μΙ of a 10% SDS solution was added and slides were measured on an ELISA reader (Genius, Tecan). Data were processed in spreadsheets (Excel) and plotted on Graphpad.

The data are summarized in figure 12.

ED50 (nM):

EPO 0.0158

BB49 (monomer, SEQ. ID NO 2) 4113

AGEM11 (Bivalent) 36.73

SAW. Extended peptide assays

In a prolonged trial, approximately 200 peptide sequences were tested for their EPO mimetic activity.

The peptides were synthesized as peptide amides on a LIPS-Vario synthesizer system. The synthesis was performed on special MTP desynthesis slides, the scale being 2 μηιοίβε per peptide. The synthesis followed the standard protocol with Fmoc using HOBT as the activating reagent. The coupling steps were performed as coupling 4 times. Each coupling step took 25 min and the excess amino acid per step was 2.8. Cleavage and deprotection of the peptides were done with a cleavage solution containing 90% TFA, 5% TIPS, 2.5% H2O and 2.5% DTT. The synthesis slide containing the final peptide attached to the resin was stored above a 96-well deep slide. 50 μΙ cleavage solution was added to each well and the cleavage was performed for 10 min, this procedure being repeated three times. Cleaved opeptide was eluted with 200 μΙ of cleavage solution by gravity deflux in the deep cavity slide. Deprotection of the side chain defect was performed for an additional 2.5 h inside the slide with deep cavity. Thereafter, the peptide was precipitated with ice cold ether / hexane and centrifuged. The peptides were decomposed in neutral aqueous solution and the cyclization was incubated overnight at 4 ° C. The peptides were lyophilized.

Figure 19 provides an overview of the synthesized and test monomerospeptides.

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 day of the assay, 40 microtiter slides were prepared for in vitro activity measurement of 38 test peptides, 1 reference example and parallel EPO. EPO stock solutions were at 20 nM.

The results are given in Figure 19. As can be seen from the results, test peptides that do not fulfill the consensus of the present invention do not represent EPO mimetic activity.

VII. Synthesis of HES-peptide conjugates using periodate oxidation

The initial reaction scheme is represented in figure 21.

The objective of the described method is the production of a deamid derivative according to this example HES which selectively reacts with thiol groups under mild aqueous reaction conditions. This selectivity is obtained with maleimide groups.

HES is first functionalized with amino groups and then converted to the respective maleimide derivative. The reaction batches were released from the low molecular weight reagents via ultrafiltration membranes. The product, the intermediate products as well as the educts are all polydisperse. Modified HES synthesis

Hydroxyethyl starch (Voluven® 130 / 0.4 or Serumwerk Bernburg200 / 0.5) was obtained via diafiltration and subsequent lyophilization. The average molecular weight was approximately 130 kDa, with a degree of molar substitution of 0.4, respectively, 200kD, MS = 0.5.

The synthesis was performed according to the synthesis described for amino dextran in Jacob Piehler's dissertation, "Modifizierung vonOberflãchen für thermodynamische und kinetische Charakterisierung Erkennung mit optischen Transducern", 1997, incorporated herein by reference. HES was activated by selective partial oxidation of diol hydroxyl groups in sodium periodate aldehyde groups, as described in Floor et al. (1989). The aldehyde groups were converted via reductive sodium cyanoborohydride (NaCNBH3) amino acids in the presence of ammonia in amino groups (Yalpani and Brooks, 1995).

1.1 Oxidation of primary alcohols in aldehydes

a) Periodate oxidation / opening

Through gentle oxidation of the 1,2-diols in the saccharide through sodium periodate in water, aldehyde groups are introduced. Using different molar concentrations of the oxidizing agent, the number of available anchor groups and thus the amount of peptide drug on the vehicle can be controlled. To optimize the protocol, oxidation was monitored with Purpald's reagent, which forms a purple feathery adduct with aldehydes. Reaction time can be reduced to 8-18 h. The amount of periodate used represents 20% of the number of glucose building blocks (applying a mass of the glucose building block of 180 g / mol, DS = 0.4). Processing was performed via ultra filtration and lyophilization. The purification of each polymeric product was performed by ultrafiltration techniques using a FOP membrane of different molecular weight cuts, followed by lyophilization. Of the HES optimized derivatives, only the molar mass range greater than 100 kDa was used.

Aldehyde Analysis

Qualitative / Semiquantitative: Purpald reaction of available aldehyde groups

1.2 Reductive Amination

a) Reductive ammonium chloride termination

In the next step, the introduced aldehyde groups were converted to amines by reductive amination in a saturated ammonium chloride solution at a slightly acidic pH value with sodium cyanoborohydride.

To optimize the protocol, the aldehyde groups of the starting material were accompanied by Purpald's reagent and the amines formed with TNBS. These experiments showed that the formation of the imine intermediate is in equilibrium after an initial period and the added reducing agent prefers the imines over the aldehyde. Thus, it may be found that the optimal reaction is accomplished by various additions of the reducing agent with a total reaction time of 24 h.

Processing via product precipitation or ultrafiltration.

Amine Analysis

Qualitative: reaction with nihydrin (Kaiser test)

Quantitative: with 2,4,6-trinitrobenzol sulfonic acid (TNBS) compared to an amino dextran.

The degree of substitution obtained was around 2.8%. This results in a molar mass of a building block bringing an amino group of approximately 6400 g / mol.

Synthesis of maleimidopropionyl amino hydroxyethyl starch ("MalPA-HES")

After the amino groups are introduced (there are several different strategies, for example, see above), the maleimide anchor group is introduced with N-hydroxy succinimide esters of α (male) alkyl (or aryl) α-maleimide acid.

Synthesis

The final introduction of the maleimide groups into the HES is carried out with N-hydroxy succinimide 3-maleimidopropionic acid ester (MaIPA-OSu). When using an excess (5 to 10 times) in a slightly acidic buffer, the conversion is quantitative ( 50 mM phosphate buffer, pH 6 or 7, 20% DMF overnight). The ultrafiltered and lyophilized product is stored at -18 ° C.

Analyze

The amino group reaction was verified with nihydrin and TNBS. The number of introduced maleimide groups is demonstrated by glutathione reaction (GSH) and detection of excess thiol groups with Ellmans reagent (DNTB) and via 700 MHz-1 H-NMR spectroscopy.

The degree of substitution obtained was around 2% and corresponds to 8500 g / mol per maleimide building block (180 g / mol glucose building block mass, MS = 0.4).

Figure 22 shows a 1 H-NMR spectrum (D 2 O, 700 MHz) of maleimide-modified HES. The ratio of maleimide protons (6.8ppm) to anomeric CH (4.8-5.6 ppm) provides a building block size of approximately 6,900g / mol (compared: testecom GSH / DNTB provided 7,300g / moi ).

The number of maleimide groups and thus the size of the building block can be measured by GSH saturation and DNTB reaction. The yellow color formed is significant and can be easily quantified. These values provide reliable building block sizes of between 5,000 and 100,000 g / mol, depending on the starting material used, respectively, and the amount of periodate in the oxidation step. This method was validated by 1 H-NMR spectroscopy of the product. In NMR, the content of maleimide groups can be quantified from the proportion of all anomeric C-H signals and maleimide ring protons.

Amount of Periodate (15 Step) Building Block Sizes (eq) _ Maleimide (g / mol) _

<table> table see original document page 77 </column> </row> <table>

Table 1: Examples for the size of the virtual building block obtainable from the anchor group in the major part of HES via periodate oxidation. Peptide-hydroxyethyl starch conjugate (Pep-AHES)

Synthesis

A cysteine-containing peptide was used which had either a free biotinylated N-terminus (Pep-IA) or a (Pep-IB). A 4: 1 mixture of Pep-IA / B was converted to excess overnight (approx. 6 equivalents with MaIPA-HES in 50 mM phosphate buffer, pH 6.5 / DMF 80:20; processing took place). with ultrafiltration and lyophilization.

Analyze

UV absorption was determined at 280 nm and the remaining content of maleimide groups was determined with GSH / DNTB.

Peptide yield was almost quantitative. Almost no free maleimide group was detectable.

For peptide / drug conjugation, a peptide domain:

Ac-GGTYSCHFGKLT-NaI-VCKKQRG-Am (BB68)

is used to create a peptide unit by introducing a free thiol group (for example by introducing a cysteine residue at the N-terminus) as in:

Ac-C (tBu) -GGTYSCHFGKLT-Na1 -VCKKQRG-GGTYSCHFGKLT-NaI -VCKKQRG-Am (AGEM40)

10-50% excess of the unprotected peptide is conjugated to a slightly acidic buffer for 1-2 h. Conditions were optimized to ensure, on the one hand, that the major part of HES, maleimide groups and disulfide bridging are stable and, on the other hand, to observe quantitative conversion. Using different maleimized-functionalized HES compounds, ApIaGen was able to synthesize a series of supravalent EPO mimetic peptides which showed an invaluable supravalent effect. Some examples are provided below. <table> table see original document page 79 </column> </row> <table>

Table 2: A-GEM40 supravalent EPO mimetic peptide conjugates with different peptide contents.

An easy chemical analysis of the supravalent EPO mimetic peptide conjugates was performed in two steps. First, the peptide content was quantified by HPLC after slight hydrolysis of the main part of HES, and second, the amount of polysaccharide was measured by a phenol colorimetric test after complete hydrolysis by sulfuric acid. .

Figure 23 shows an HPLC chromatogram (Shimadzu H-PLC) of TFA / water hydrolysis of the supravalent EEM mimetic peptide conjugates AGEM40-AHES A2. After a certain time, the UV absorbance of all peptide-containing species is constant at a maximum value and, by comparison with the free peptide, a 37% peptide content can be calculated (theoretical value: 39%).

VIII. Other in vitro experiments

Many of the experiments described below have already been described above. However, the following details provide a brief overview of the described tests and results. Predominantly, human cell culture and bone marrow assays are discussed.

On the one hand, rapid cell line-based assays were used to verify the potency of peptide sequences optimized by all stages of optimization. These cell culture assays are still valid as rapid efficacy tests of a new peptide or a new batch. The two endpoints which were used for the TF-1 cell line (human cells) are proliferation (here usually determined as the number of living cells at defined time points) and differentiation such as marked hemoglobin production in TF cells. -1.

In addition, primary cells (human bone marrow stem cells) were used for CFU1 assays which are very close to the in vivo situation. They provide responses to the erythropoietic activity caused by the use of EPO mimetic peptides as peptide units in a much more similar manner to in vivo. However, they have to be handled more sophisticated and need more time per assay than cell culture assays.

Assays using human TF-1 cells

TF-1 is a human erythroleukemia cell line that proliferates only in response to certain cytokines such as IL3 or EPO. In addition, TF-1 cells can differentiate between an erythroid phenotype in response to EPO. TF-1 cells were obtained from DSMZ (Braunschweig, Germany). A product sheet is available on the DSMZ web site, dsmz.de. TF-1 'is the recommended cell line for EPO activity evaluation by the European Pharmacopoeia.

The internal culture protocol for culture maintenance:

Medium: RPMI + P / S + AmphoB + L-Glut. + 20% FCS + h-IL-3

1. - 500 ml RPMI + 5 ml P / S + 5 ml AmphoB

2. - 200 ml RPMI + PS / AmphoB + 2.5 ml L-Glutamine + 50 ml FCS = complete medium (1 month at 4 ° C)

3. - 45 ml complete medium + 22.5 ul h-IL-3 (1 week at 4 ° C) Culture: Maintain between 200,000 and 1,000,000 cells / ml for 3 days at 2 χ105 / m I

• For 2 days at 3 χ 105 / ml

• For 1 day at 5 χ 105 / ml

Design of a TF-1 Proliferation Assay

In a TF-1 proliferation assay, TF-1 cells are seeded for several days at varying concentrations of EPO or EPO mimetic peptides on a multiwell slide.

For optimal results, TF-1 cells should be cultured for two days in the absence of any cytokine (deprivation) prior to initiating the assay. Three days after starting the assay, cell proliferation is measured indirectly by assessing the number of viable cells.

A tetrazolium reagent called MTS is added which is reduced to colored formazan. This reaction depends on NADH and NADPH, in other words, it depends on mitochondrial activity. The amount of formazan is measured spectrophotometrically. Using a range of known cell numbers for calibration, it is possible to determine the absolute number of viable cells present under each condition. The main design is also illustrated in figure 24.

The activity of a particular agent in this assay is determined by:

1. whether this agent causes an increase in the number of viable cells at a given concentration and

2. at which concentration this agent has a half-maximal effect (EC50 determination). Results from TF-1 proliferation assays

As a general feature, it should be mentioned that all EPO mimetic peptides (EMP1 and the proline-modified peptides described above) behave in this assay as partial agonists, ie the maximum response is weaker than that observed with EPO.

However, the assay can be used to determine left / right deviation in normalized plots and thus determine optimal results.

The first graph represents this effect in absolute response without normalization. All other graphs show normalized plots, which allow the determination of EC50 values from the curves.

Two reference substances were used in the trials:

1) EMP1, a published peptide sequence with known EPO mimetic properties (Johnson et al., 1997).

2) Recombinant Human Erythropoietin (EPO), was purchased in pharmacy as Ortho Biotech's Epoietin alfa product (Trademark in Germany: ErypoR)

Plots of these substances are provided as black, continuous lines for EPO and dotted for EMP1.

Proline-modified EPO mimetic peptides are shown in the following figures as colored solid lines. These modified peptides represent the following sequence:

1) BB49

Ac-GGTYSCHFGKLTWVCKKQGG

Shows efficiency and power in the same range as EMP1

2) BB68

Ac-GGTYSCHFGKLT-NaI -VCKKQRG-Am is even more effective than EMP1 and BB49

3) AGEM40,

Ac-C (tBu) -GGTYSCHFGKLT-Na1 -VCKKQRG-GGTYSCHFGKLT-NaI -VCKKQRG-Am

which is a bivalent continuous peptide which was designed based on the BB68 sequence representing improved characteristics.

4) AGEM40_HES,

which is an advanced, highly effective and potent HESi-Ia peptide (AGEM40) according to the principle of supravalence of the present invention.

These sequences were used as examples, inter alia, to illustrate the benefits of the principle of supravalence.

Figure 25 depicts the results of monomeric EPO mimetic peptides compared to EPO. Figure 25 includes a plot of actual absorbance data documenting the absolute difference between peptides in general and EPO in this assay.

Figure 26 provides the EC50 values calculated from adapted standard plots.

Figure 27 shows the improved effect of BB68 compared to BB49. Using the optimized BB68 as a building block for the creation of a peptide unit in accordance with the present invention, the effect was enhanced by two additional orders of magnitude. This is documented in figure 27 and the corresponding table shown in figure 28.

The dimeric peptide units were then coupled to the HES macromolecular vehicle at an optimized density. The resulting API is at least equipotent to EPO in a molar comparison and very close to EPO in a mass comparison (see figure 29 and figure 30 below).

Figures 30 and Figures and Tables earlier clearly demonstrate the greatest power of the concept of supravalence. Maintaining the precision which can be obtained with a cell culture assay, the obtained API is at least equipotent to EPO in vitro. Thus, it is superior to any known EPO mimetic peptide API not employing the concept of supravalence.

Bone Marrow Assays

Bone marrow contains hematopoietic stem cells with a potential for self-renewal and develop in all types of blood cells. In addition, the bone marrow contains compromised progenitor cells capable of developing in one or more blood cell lines. Among these progenitor cells, some may develop into erythrocytes (erythroid progenitors).

Progenitor cells can be demonstrated by placing bone marrow cells in a semi-solid methylcellulose-based medium. In the presence of an appropriate cytokine cocktail, progenitor cells proliferated and differentiated to provide a cell colony of a given lineage. Myeloid progenitors develop into granulocytic colonies (derived from a CFU-G), monocytic colonies (from a CFU-M) or mixed granulocytic-monocytic colonies (from a CFU-GM).

Erythroid progenitors develop in a colony of erythrocytes (red cells). Depending on the size of the erythroid colony, the progenitor cells are called BFU-E (providing colonies of 200 cells or more) or CFU-E (providing colonies of less than 200 cells). Progenitor cells at an earlier stage of impairment may develop into mixed granulocytic-erythroid-monocytic-megakaryocytic colonies. These early parents are called CFU-GEMM.

EPO stimulates the development of erythroid colonies of BFU-E or CFU-E1 if certain cytokines are present as well. Without EPO1 no erythroid colony can develop. The growth of erythroid colonies from a homogeneous batch of bone marrow cells in methylcellulose, therefore, is a measure with respect to EPO activity.

Since the above mentioned processes are very similar, if not identical, to the processes which occur in the inventive bone marrow, they are an excellent predictor of EPO-like activity.

Bone Marrow Assay Desian

Human bone marrow cells (commercially available from Cryosystems, serologically verified) are thawed from cryovials and placed in methyl cellulose medium with a given cytokine base (but without EPO) at a fixed cell density. EPO or EPO mimetic peptide is added in varying concentrations. The cultures are incubated for 12-14 days at 37 ° C. Then the numbers of myeloid and erythroid colonies are enumerated by microscopic inspection.

Bone Marrow Assay End Points:

1. Assumptions: Non-EPO cultures should provide only myeloid (white) but not erythroid (red) colonies. Cultures with EPO would provide a concentration-dependent increase in red cell colonies and a concentration-dependent increase in red cell colonies.

2. A peptide shows EPO mimetic activity if it causes a concentration-dependent increase in red cell colonies and a concentration-dependent increase in red cell colony sizes. However, a peptide should not interfere with the myeloid colony numbers obtained. Bone Marrow Assay Results

The proline-modified EPO mimetic peptides described above do not stimulate the formation of myeloid colonies, but have shown significant activity on the formation of red colonies. Qualitatively, this is shown in figure 31 in a photograph of a culture slide, while colony counting is documented in figure 32.

Reference Listing

Wrighton NC, Balasubramanian P1 Barbone FP, Kashyap AK, Farrell FX, Jolliffe L, Barrett RW, Dower WJ (1997) Increased potency of anerythropoietin peptide mimetic through covalent dimerization. Nature Biotechnology 15: 1261-1265

Wrighton NC1 Farrell FX1 Chang R, Kashyap AK, Barbone FP, Mulcahy LS, Johnson DL, Barrett RW, Jolliffe LK, Dower WJ (1996) SmallPeptides as Potent Mimetics of the Protein Hormone Erythropoietin. Science273: 458-463

Johnson, D.L., F. X. Farrell et al. (1997). "Amino-terminalization of an erythropoietin mimetic peptide results in increased eryt-hropotietic activity." Chemistry and Biology 4: 939-950.Haag R, Sunder A, Stumbe JF, J. Am. Chem. Sound. (2000), 122, 2954.

Roberts, M.J., M.D. Bentley, and others. (2002). "Chemistry forpeptide and protein PEGylation." Advanced Drug Deliverv Review 54 (4): 459-476.

ZaIipsky S, Qazen, S, Walker Il JA, Mullah N1 Quinn YP, (1999) "New detachable poly (ethylene glycol) conjugates: Cysteine-cleavable natural regenerating phospholipid Ipo-polymers, diacyl phosphatidylethanolami-ne, Bioconjug. Chem. 10: 703-707.

Zhao, X. et al. (1997), "Novel Degradable Poly (ethylene glycol) esters for drug delivery." In Poly (ethylene glycol) chemistry and biological applications; Harris JM, Zalipsky, S. Eds.; ACS Symposium Series 680; American Chemical Society: Washington DC, 1997; 458-472.

Claims (32)

A targeting molecule binding compound comprising: (i) at least two peptide units, wherein each peptide unit comprises at least two domains with a targeting ability, (ii) at least one polymeric carrier unit; said peptide units are attached to said polymeric carrier unit. A compound according to claim 1, wherein said target molecule is a receptor molecule, preferably a transmembrane receptor. A compound according to claim 2, wherein the peptide units bind to said receptor and act as receptor antagonists or as receptor agonists. A compound according to any one of claims 1 to 3, wherein said peptide moiety is about 200 amino acids or less, about 150 amino acids or less, about -100 amino acids or less, or about 100 amino acids or less. of 50 amino acids or less. A compound according to any one of claims 1 to 4, wherein said polymeric carrier unit comprises about 50 peptide units or less, 25 peptide units or less, 15 or less peptide units, 10 or less peptide units, and preferably , between 2 and 6 peptide units. A compound according to any one of claims 1 to 5, wherein said carrier moiety is or comprises at least one synthetic or natural dendritic or linear polymerised, and is preferably selected from the group consisting of polyglycerins, polysaline acid , dextrans, polysaccharides, starches or polyethylene glycol or other biologically inert water soluble polymers. A compound according to at least one of the preceding claims 1 to 6, wherein said polymeric carrier unit comprises a branching unit, wherein said branching unit preferably comprises glycerol or polyglycerol. A compound according to at least one of the preceding claims 1 to 7, wherein said carrier molecule has a peptide of at least 5 kD, preferably from 20 to 200 or 4000 kD and from 20 to 80 kD where smaller carriers are present. , such as polyethylene glycol, are used. A compound according to at least one of claims 1 to 8, wherein said carrier unit is composed of at least two polymeric subunits, wherein said polymeric subunits are connected via at least one biodegradable covalent linker structure. A compound according to at least one of the preceding claims, comprising a first biodegradable carrier unit, wherein peptide units and second polymeric carrier units are attached to said first polymeric carrier unit. A compound according to claim 10, wherein said second vehicle unit has a lower molecular weight than said first vehicle unit and wherein approximately 20 to 50% of the attachment sites of said first vehicle unit are preferably HES, are occupied with said second vehicle units which are preferably PEGs having a molecular weight of about 3 to 10 kD. A compound according to at least one of the preceding claims, wherein a modified polymeric carrier unit is used. A compound according to claim 12, wherein said peptide unit is attached via a covalent bond to said polymeric unit and the attachment occurs via a reactive amino acid, the N-terminal α-min group and / or the acid. C-terminal carboxylic acid of said peptide units, wherein said reactive amino acid is preferably selected from the group consisting of lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine and wherein, where said polymeric carrier unit does not have an appropriate reactive coupling group, a coupling unit is used for modification of the polymeric carrier unit, wherein said coupling unit is preferably selected from the group consisting of acylation groups. which react with the amino groups of said peptide unit, alkylating groups which react with sulfhydryl (mercapto), thiomethyl, imidazo or amino groups above said peptide unit, more preferably maleimide groups, ester and amide forming groups which react with a carbohydrate group of the protein, disulfide forming groups which react with sulfhydryl groups on said peptide unit such as 5,5'-dithiobis (2-nitrobenzoate), ortho-pyridyl and alkylmercapetane disulfide groups, dicarbonyl groups such as cyclohexandione groups and other 1,2-diketone groups which react with the guanidine moieties of said peptide unit; diazo groups which react with phenolic groups on said peptide; reactive groups of the cyanogen bromide reaction with areferida polymeric carrier unit, which react with amino groups on said peptide unit. The compound of claim 13, wherein said reactive amino acid is cysteine and wherein said coupling group is maleimide. A compound according to at least one of the preceding claims 1 to 14, wherein at least one of said peptide units binds to a homo-, di- or multimeric receptor, preferably to a class I cytokine receptor member. including the deeritropoietin receptor (EPOR), thrombopoietin receptor (TPOR), granulocyte colony stimulating factor receptor (G-CSFR) 1 growth hormone receptor (GHR) and prolactin receptor (PrR). A compound according to at least one of the preceding claims 1 to 14, wherein at least one of said peptide units binds to a hetero-, di- or multimeric receptor, such as an interleukin receptor. A compound according to at least one of the preceding claims 1 to 14, wherein said peptide units bind and activate the EPO receptor and are selected from the group consisting of EPO mimetic empeptides. The compound of claim 17, wherein at least one of said peptide units comprises a deletion domain characterized by the following amino acid sequence: wherein each amino acid is selected from natural or unnatural amino acids and: X6 is C1 A , E, α-amino-Y-bromobutyric acid or homocysteine (hoc); X7eR, H, L1WouYou S; X8 is M1 F, I, homoseromethylether or norisoleucine; X9 is G or a conservative exchange of G; non-conservative proline exchange; or X9 and X10 are replaced by a single amino acid; X11 is selected from any amino acid; X12 is T or A; X13 is W, 1-nal, 2-nal, A or F; X14 is D E1 I1 L or V; X15 is C, A, K, α-amino-Y-bromobutyric acid or homocysteine (hoc) as long as X6 or X15 is C or hoc. The compound of claim 17, wherein at least one of said peptide units comprises a deletion domain characterized by the following amino acid sequence: queβΧγΧδΧθΧΐ 0X11 Xi 2X13X14X15 wherein each amino acid is indicated by the standard abbreviation with one letter e: X6 is C; X7 is R, H, L or W X8 is M1 F or I. X9 is G or a conservative exchange of G X10 is a non-conservative proline exchange X11 is independently selected from any amino acid X12 is T X13 is W; X14 is D, Ε, I, Lou V; X15 is C; A compound according to at least one of the preceding claims 1 to 19, wherein said binding domains (monomeric linkers) of said peptide units are internally connected via a linker structure, preferably a continuous peptide linker. 21. A compound according to at least one of claims 17 to 20 tions preceding claims, wherein at least one of said unidadespeptídicas comprises a binding domain selected from the following peptides GRU-po: GGTYSCHMGKLTXVCKKQGGGGTYTCHFGKLTXVCKKLGGGGLYSCHFGKITXVCKKQGGGGLYSCHMGKLTWVCRKQGGGGLYSCHFGKLTXVCQKQGGGGTYSCHFGKLTWVCQKQRGGGTYSCHFGKLTXVCKKQRGGGLYACHFGKLTWDCQKQGGGGTYTCHFGKLTUVCKKQGGGGTYSCHFGKLTUVCKKLGGGGTYSCHFGKITXVCKKQGGGGLYSCHFGKLTUVCKKLGGGGLYACHFGKLTUVCKKQGGGGLYSCHMGKLTWLCKKLGGGGTYSCRFGKLTWVCKKQGGGGTYTCHFGKITUVCKKQGGGGLYSCHFGKLTXVCKKQGGGGLYACHFGKLTULCKKQGGGGLYSCHFGKLTWVCKKQRGGGTYTCHFGKITXVCKKQGGGGTYTCHMGKLTWVCKKQRGGGLYSCHFGKLTXVCKKQRGGGTYTCHFGKLTXVCKKQGGGGLYSCHFGKITUVCKKQGGGGLYSCHFGKLTXVCRKQGGGGTYACH FG KLTXVC KKLG GGG LY ACHFGK LTXVC R KQG GG AC GTY H FG FG KLTXVC KKQG GGGLYSCHMGKLTXVCRKQGGGGLYSCHFGKLTUVCKKQRGGGLYSCHMGKLTXVCKKQGGGGTYTCHMGKLTXVCKKQGGGGLYSCHFGKLTXVCRKQRGGGTYSCHFGKLTXVCKKQGGGGTYSCHFGKLTWVCKKQRGGGTYACHFGKLTWVCKKQRGGGLYSCHFGKLTWVCQKQRGGGTYTCH KLTXVCKKQR GGGTYSCHFGKLTWVCKKQGGGGTYSCHFGKLTWVCKPQGGGGTYSCHFGRLTWVCKPQGGGGTYSCHFGRLTWVCKKQGGGGTYSCHF- (ALS) -LTWVCKPQGGGGTYSCHF- (ALS) -LTWVCKKQGGGGTYSCHFGKLT-Inai-VCKKQRGGGTYSCHFGKLTWVCKKQGG GGTYSCHFGKLTWVCKKQGGGGTYSCHFGKLT-1-Nal-VCKKQRG GGTYSCHFGKLT-1-Nal-VCKKQRGem -naftilalanina wherein X is 1 and U is 2-naftalinae where 5-aminolevulinic acid ( AIs) is: <formula> formula see original document page 91 </formula> Use of a compound as defined in at least one of claims 1 to 21 for the preparation of a pharmaceutical composition. Use of a compound as defined in at least one of claims 1 to 21, bringing a peptide unit binding to EPO receptor activation for the preparation of a pharmaceutical composition for the prevention or treatment of a disorder characterized by a deficiency. erythropoietin or a low or defective red blood cell population and especially for the treatment of any type of anemia and stroke. Use of a compound as defined in at least one of claims 1 to 21, bringing a peptide unit binding and TPO receptor activation for the preparation of a pharmaceutical composition for the prevention or treatment of a disorder characterized by a deficiency. thrombopoietin and especially for the treatment of haematological disorders including thrombocytopenia, granulocytopenia and anemia. A pharmaceutical composition comprising the compound as defined in at least one of claims 1 to 21, and optionally a pharmaceutically acceptable carrier. A method for producing a compound as defined in at least one of claims 1 to 21, comprising: (i) generating at least two peptide units wherein each peptide unit comprises at least two domains having a binding capacity. (ii) generating at least one polymeric carrier unit, (iii) attaching said peptide units to said polymeric carrier unit. A method according to claim 26, wherein said peptide units are synthesized as a continuous peptide chain, A method according to claim 26 or 27, wherein a polymeric carrier unit is used which has at least one group thereof which is capable of reaction with an available chemical group about said peptide unit and then reaction together. of the reactive polymeric vehicle unit and the peptide unit to form a covalently bonded complex thereof using the chemical group of the polymeric vehicle unit. A method according to claims 26 to 28, wherein said polymeric carrier is a modified starch molecule which has been oxidized and wherein an amino group has been introduced into the main part of the starch and wherein said amino group is modified by introducing a linker group, preferably a maleimide group. The method of claim 29, wherein the oxidation is performed by contacting the starch molecule with a reagent that produces an oxoammonium ion in the presence of an oxidizing agent or by contacting the starting material directly with the starch. reactive species, the oxoammonium ion. The method of claim 30, wherein the oxidizing agent is a chemical oxidizing agent, such as a hypohalide such as sodium hypochlorite and sodium hypobromite or hydrogen peroxide or an oxidative enzyme. The method of claim 30 or 31, wherein the oxoammonium ion producing agent is preferably a denitroxyl compound, more preferably a di-tert-nitroxyl compound such as 2,2,6,6. -Tetramethylpiperidine-1-oxyl (TIME) or a derivative thereof.