JP2008519589A - Molecules that promote hematopoiesis - Google Patents

Abstract

The present invention relates to EPO mimetic peptides and special synthetic methods for the production of multivalent and / or supervalent peptides. According to one aspect of the invention, there is provided a peptide of at least 10 amino acids in length that can bind to an EPO receptor and comprises agonist activity. Thus, the peptide exhibits EPO mimetic properties. The EPO mimetic peptide of the present invention contains a positively charged amino acid without a proline at the position generally referred to as position 10 of the EPO mimetic peptide.

Description

  The present invention relates to peptides as binding molecules for erythropoietin receptors, methods for their preparation, pharmaceuticals containing these peptides, and preferably for treatment of various forms of anemia and stroke in selected instructions. Of their use.

  The hormone erythropoietin (EPO) is a glycoprotein composed of 165 amino acids and having four glycation sites. The four complex carbohydrate side chains constitute 40 percent of the total molecular weight of about 35 kD. EPO is formed in the kidneys and travels from there to the spleen and bone marrow where it stimulates the production of red blood cells. In chronic kidney disease, reduced EPO production results in erythrocytic anemia. Anemia can be effectively treated using recombinant EPO prepared by genetic engineering. EPO improves the quality of life of dialysis patients. In addition to renal anemia, anemia, inflammation and tumor-related anemia in premature newborns can also be ameliorated by recombinant EPO. EPO allows better high-dose chemotherapy in tumor patients. Similarly, EPO improves cancer patient recovery when administered within the scope of radiation therapy.

  In treatment with EPO, the problem is that the required dosage regimen is based on frequent or continuous intravenous or subcutaneous application, since the protein is degraded relatively rapidly in the body. Thus, the development of recombinant EPO-derived molecules is towards selective modification of glycoproteins, for example by further glycation or pegylation, in order to increase stability and thus biological half-life.

  Another important issue associated with treatment with recombinant EPO is the risk that the patient will develop antibodies to recombinant EPO during treatment. This is because recombinant EPO is not completely identical to endogenous EPO. Once antibody formation is induced, this can lead to antibodies that also impair the activity of endogenous erythropoietin. This often increases the dose of recombinant EPO required for treatment. This effect can be interpreted as a treatment-induced autoimmune disease, especially when such antibodies are disorders of endogenous EPO. This is not particularly desirable, for example, in the case of dialysis patients who receive a kidney transplant after months or years of EPO treatment. At this time, the antibody may interfere with the activity of endogenous EPO produced by the transplantation, and thus may interfere with the erythropoietic activity of the transplanted organ. Currently, it is an open question whether modifications introduced in recombinant EPO to increase biological half-life exacerbate or ameliorate this problem. In general, a wide range of modifications and longer half-lives are expected to exacerbate this problematic property.

  An alternative strategy is the preparation of synthetic peptides from amino acids that do not share sequence homology or structural relationships with erythropoietin. It has been shown that peptides that are significantly smaller than erythropoietin and unrelated to the sequence of EPO can act as agonists (Non-patent Document 1). The same author showed that such peptides can be truncated to become the smallest peptide of 10 amino acids in length that is still active.

  Synthetic peptides that mimic the activity of EPO are the subject of US Pat. This discloses a well-defined consensus mimetic peptide of 10-40 amino acids that contains two prolines, preferably at positions considered to be essential, commonly referred to as positions 10 and 17.

  Thus, to date, all small peptide-based agonists of the EPO receptor have been defined, usually numbered 10 and 17 with reference to positions in the highly active erythropoietin mimetic peptide EMP1 It has a structure containing at least one proline and often two proline residues at positions (Patent Document 1, Non-Patent Document 1, Non-Patent Document 2).

これらのプロリンは、ペプチドの効果に不可欠であると考えられている。１７位のプロリンに関しては、これは受容体との相互作用によって実証されているが、１０位のプロリンは分子の正しい折りたたみに必要であると考えられた（非特許文献２、非特許文献３も参照のこと）。プロリンの特異的立体化学的特性によって支持される、正しい折りたたみは、通常、生物学的活性の、必要な前提条件である。一般に、プロリンは、（この場合のように）しばしばヘアピン構造およびβターンの形成に関係する、構造形成アミノ酸である。この特性のために、とりわけ、これは、プロリン含有ペプチド／タンパク質を破壊するポストプロリン特異的エンドペプチダーゼの頻繁な攻撃の点である。多数の内在性ペプチドホルモン（アンギオテンシンＩおよびＩＩ、ウロテンシン、チレオリベリン、他の分泌刺激因子等）が、かかる「シングルヒット」ポストプロリン切断によって不活性化される。したがって、プロリン含有ＥＰＯ模倣ペプチドの半減期は、これらの頻繁で活性のある酵素の活性によって短縮される。

These prolines are thought to be essential for the effect of the peptide. Regarding the 17th proline, this is demonstrated by the interaction with the receptor, but the 10th proline was considered necessary for the correct folding of the molecule (Non-Patent Documents 2 and 3). See Correct folding, supported by the specific stereochemical properties of proline, is usually a necessary prerequisite for biological activity. In general, proline is a structure-forming amino acid that is often (as in this case) involved in the formation of hairpin structures and β-turns. Because of this property, among other things, this is the point of frequent attack of postproline-specific endopeptidases that destroy proline-containing peptides / proteins. A number of endogenous peptide hormones (angiotensins I and II, urotensin, thyreoliberin, other secretory stimulators, etc.) are inactivated by such “single hit” post-proline cleavage. Thus, the half-life of proline-containing EPO mimetic peptides is shortened by the activity of these frequent and active enzymes.

Such peptides can be produced chemically and do not require recombinant production, which is more difficult to control and produce products with defined quality and identity. The chemical production of such small size peptides can also be competitive from a production cost standpoint. In addition, chemical generation allows the introduction of defined molecular changes, such as glycation, pegylation, or any other defined modification that can have a known ability to increase biological half-life. To do. However, to date, no therapy using existing EPO mimetic peptides has been approved.
International Publication No. 96/40749 Pamphlet Whitton NC, Frell FX, Chang R, Kashyap AK, Barbone FP, Mulcahy LS, Johnson DL, Barret RW, Jolliffe LK, Dower WJ (1996) Small Petr. Science 273: 458-463 Johnson, D.C. L. , F.A. X. Farrell, et al. (1997). “Amino-terminal dimerization of an erythropoietin mimetic peptide results in increased erythropoietic activity.” Chemistry and Biology 4: 939-9. Whitton NC, Balabramanian P, Barbone FP, Kashapap AK, Frell FX, Jolliffe L, Barrett RW, and Dower WJ (1997) Nature Biotechnology 15: 1261-1265

  Accordingly, it is an object of the present invention to provide alternative synthetic peptides that exhibit at least an essential part of the biological activity of natural EPO, and thus efficient therapy, particularly for the treatment of anemia or stroke To provide an alternative means of strategy.

  According to one aspect of the invention, there is provided a peptide of at least 10 amino acids in length that can bind to an EPO receptor and comprises agonist activity. Thus, the peptide exhibits EPO mimetic properties. The EPO mimetic peptides of the present invention do not contain a proline at the position generally referred to as position 10 of the EPO mimetic peptide, and contain positively charged amino acids (for example, Johnson et al. Described for the ancestral sequence of EMP1). , 1997).

  The proline at position 10 is located in the amino acid motif characteristic of certain folding structures, ie, the β-turn motif (see Johnson, 1997). The β-turn structure occurs during receptor binding. Thus, the EPO mimetic peptides of the present invention do not contain a proline in the β-turn motif at position 10, but contain a positively charged amino acid. Examples are K, R, H, or the respective unnatural amino acid such as, for example, homoarginine.

  In addition, peptides comprising the following amino acid sequences are provided.

配列中、各アミノ酸は、天然または非天然アミノ酸から選択され、
Ｘ_６はＣ、Ａ、Ｅ、α−アミノ−γ−ブロモ酪酸もしくはホモシステイン（ｈｏｃ）であり、
Ｘ_７はＲ、Ｈ、Ｌ、ＷもしくはＹもしくはＳであり、
Ｘ_８はＭ、Ｆ、Ｉ、ホモセリンメチルエーテル（ｈｓｍ）もしくはノルイソロイシンであり、
Ｘ_９はＧ、もしくはＧの保存的交換であり、
Ｘ_１０はプロリンの非保存的交換であるか、
またはＸ_９およびＸ_１０は単一のアミノ酸によって置換され、
Ｘ_１１は任意のアミノ酸から独立して選択され、
Ｘ_１２はＴもしくはＡであり、
Ｘ_１３はＷ、１−ｎａｌ、２−ｎａｌ、ＡもしくはＦであり、
Ｘ_１４はＤ、Ｅ、Ｉ、ＬもしくはＶであり、
Ｘ_１５はＣ、Ａ、Ｋ、α−アミノ−γ−ブロモ酪酸もしくはホモシステイン（ｈｏｃ）であり、
ただしＸ_６もしくはＸ_１５のいずれかはＣもしくはｈｏｃである。

In the sequence, each amino acid is selected from natural or unnatural amino acids,
X ₆ is C, A, E, α-amino-γ-bromobutyric acid or homocysteine (hoc);
X ₇ is R, H, L, W or Y or S;
X ₈ is M, F, I, homoserine methyl ether (hsm) or norisoleucine,
X ₉ is G or a conservative exchange of G,
X ₁₀ is a non-conservative exchange of proline,
Or X ₉ and X ₁₀ are replaced by a single amino acid;
X ₁₁ is independently selected from any amino acid;
X ₁₂ is T or A;
X ₁₃ is W, 1-nal, 2-nal, A or F;
X ₁₄ is D, E, I, L or V;
X ₁₅ is C, A, K, α-amino-γ-bromobutyric acid or homocysteine (hoc);
However, either X ₆ or X ₁₅ is C or hoc.

  The length of the peptide consensus described is preferably between 10-40 or 50 or 60 amino acids. Although the 60 amino acid long peptides described above are technically suitable, increasing the length of the peptides usually makes the synthesis more complex and therefore expensive, which is not always preferred. In preferred embodiments, the peptide consensus exhibits a length of at least 10, 15, 18 or 20 amino acids. Of course, these can each be embedded and included in a longer array. The described peptide sequence can be seen as the binding domain of the EPO receptor. As EPO mimetic peptides, they can bind to the EPO receptor.

  It is very surprising that one or even both prolines (according to some embodiments) can be replaced by other natural or non-natural amino acids, but the peptides of the invention just exhibit EPO mimetic activity. Was that. Indeed, the peptides of the present invention have activity comparable to that of proline-containing peptides. However, it is noteworthy that the amino acid replacing the proline residue does not represent a conservative exchange but rather a non-conservative exchange. Preferably, positively charged amino acids, such as basic amino acids such as K, R and H, and in particular K are used for substitution. Non-conservative amino acids used for substitution can also be unnatural amino acids, preferably those with positively charged side chains. Also included are analogs of each of the mentioned amino acids. A suitable example of an unnatural amino acid is homoarginine. According to certain embodiments, the peptide has a positively charged amino acid at position 10, except for the natural amino acid arginine. Thus, according to this embodiment, proline 10 is replaced by an amino acid selected from K, H, or an unnatural positively charged amino acid such as, for example, homoarginine. It is preferred that the peptide exhibits lysine or homoarginine at position 10. As mentioned above, the proline at position 17 can also be replaced by a non-conservative amino acid. In this regard, it is also preferred that the non-conservative amino acid has a positively charged side chain, such as each unnatural amino acid such as K, R, H or homoarginine. According to a sub-embodiment of this embodiment, the peptide has a positively charged amino acid at position 17, except for the natural amino acid arginine. Thus, according to this embodiment, proline 17 is replaced by an amino acid selected from non-natural positively charged amino acids such as K, H, or homoarginine. It is preferred that the peptide exhibits lysine or homoarginine at position 17.

  Furthermore, the sequence can have N-terminal and / or C-terminal acetylation and amidation. Some amino acids can also be phosphorylated.

  According to the present invention there is also provided a peptide that binds to the erythropoietin receptor and comprises the following amino acid sequence:

配列中、各アミノ酸は標準的文字省略形によって示され、
Ｘ_６はＣであり、
Ｘ_７はＲ、Ｈ、ＬまたはＷであり、
Ｘ_８はＭ、ＦまたはＩであり、
Ｘ_９はＧ、またはＧの保存的交換であり、
Ｘ_１０はプロリンの非保存的交換であり、
Ｘ_１１は任意のアミノ酸から独立して選択され、
Ｘ_１２はＴであり、
Ｘ_１３はＷであり、
Ｘ_１４はＤ、Ｅ、Ｉ、ＬまたはＶであり、
Ｘ_１５はＣである。

In the sequence, each amino acid is indicated by a standard letter abbreviation,
X ₆ is C,
X ₇ is R, H, L or W;
X ₈ is M, F or I;
X ₉ is G, or a conservative exchange of G,
X ₁₀ is a non-conservative exchange of proline,
X ₁₁ is independently selected from any amino acid;
X ₁₂ is T;
X ₁₃ is W;
X ₁₄ is D, E, I, L or V;
X ₁₅ is C.

Further, X ₇ can be serine, X ₈ can be hsm or norisoleucine, and X ₁₃ can also be 1-nal, 2-nal, A or F. The length of the peptide consensus is preferably between 10-40 or 50 or 60 amino acids. In preferred embodiments, the peptide consensus comprises at least 10, 15, 18 or 20 amino acids.

  In addition to L-amino acids or stereoisomeric D-amino acids, the peptides of the present invention include non-natural / non-conservative amino acids such as α, α-disubstituted amino acids, N-alkyl amino acids or lactic acid, such as 1-naphthylalanine. 2-naphthylalanine, homoserine-methyl ether, β-alanine, 3-pyridylalanine, 4-hydroxyproline, O-phosphoserine, N-methylglycine (sarcosine), homoarginine, N-acetylserine, N-acetylglycine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, nor-lysine, 5-aminolevulinic acid or aminovaleric acid can be included. Specifically, the use of N-methylglycine (MeG) and N-acetylglycine (AcG) at the terminal position is particularly preferred. Also within the scope of the invention are peptides that are defined as retro, inverso, and retro / inverso peptides, as well as peptides consisting entirely of D-amino acids.

  The present invention also relates to peptide derivatives, such as methionine oxidation products, deamidated glutamine, arginine and C-terminal amides.

According to one embodiment of the invention, the peptide has a single amino acid replacing amino acid residues X ₉ and X ₁₀ . In this embodiment, both residues can also be substituted by one unnatural amino acid, such as 5-aminolevulinic acid or amivaleric acid.

  In a further embodiment, the peptide according to the invention comprises a consensus sequence

を含み、
配列中、Ｘ_６〜Ｘ_１５は上記の意味を有し、
Ｘ_４はＹであり、
Ｘ_５は任意のアミノ酸から独立して選択され、好ましくはＡ、Ｈ、Ｋ、Ｌ、Ｍ、Ｓ、ＴまたはＩである。

Including
In the sequence, X _{6 to} X ₁₅ have the above meanings,
X ₄ is Y,
X ₅ is independently selected from any amino acid and is preferably A, H, K, L, M, S, T or I.

  The peptides of the invention can be extended and the consensus sequence

を含むことができ、
配列中、Ｘ_４〜Ｘ_１５は上記の意味を有し、
Ｘ_３は任意のアミノ酸から独立して選択され、好ましくはＤ、Ｅ、Ｌ、Ｎ、Ｓ、ＴまたはＶであり、
Ｘ_１６は任意のアミノ酸から独立して選択され、好ましくはＧ、Ｋ、Ｌ、Ｑ、Ｒ、ＳまたはＴ、より好ましくはＫ、Ｒ、ＳまたはＴであり、
Ｘ_１７は任意のアミノ酸から独立して選択され、好ましくはＡ、Ｇ、Ｐ、Ｒ、Ｋ、Ｙ、または正に荷電した側鎖を有する非天然アミノ酸、より好ましくはＫまたはＨａｒであり、
Ｘ_１８は任意のアミノ酸から独立して選択される。

Can include
In the sequence, X _{4 to} X ₁₅ have the above meanings,
X ₃ is independently selected from any amino acid, preferably D, E, L, N, S, T or V;
X ₁₆ is independently selected from any amino acid, preferably G, K, L, Q, R, S or T, more preferably K, R, S or T;
X ₁₇ is independently selected from any amino acid, preferably A, G, P, R, K, Y, or a non-natural amino acid having a positively charged side chain, more preferably K or Har;
X ₁₈ is independently selected from any amino acid.

In a further embodiment of the invention, the peptide is C, E, A or hoc, preferably X ₆ as C, and / or X ₇ as R, H or Y or S, and / or as F or M. X ₈ and / or G or A, preferably X ₉ as G and / or X ₁₀ as K or Har, and / or as V, L, I, M, E, A, T or norisoleucine X ₁₁ , and / or X ₁₂ as T, and / or X ₁₃ as W, and / or X ₁₄ as D or V, and / or C or hoc, preferably X ₁₅ as C, and / or P, Y or A, or X ₁₇ as a basic natural or unnatural amino acid. However, it is also preferred that X ₁₇ is K, or a non-natural amino acid having a positively charged side chain such as, for example, homoarginine.

  FIG. 19 discloses additional novel suitable peptide sequences that exhibit EPO mimetic activity. Further peptides exhibit the following sequence:

５−アミノレブリン酸（５−Ａｌｓ）は、

5-aminolevulinic acid (5-Als) is

である。

It is.

  Also disclosed is a peptide that has the ability to bind to the receptor for the hormone erythropoietin, exhibits agonist activity, and the peptide does not exhibit proline. As mentioned above, these peptides preferably do not contain proline at positions commonly referred to as positions 10 and 17, but contain different natural amino acids or 5-aminolevulinic acid. These preferably represent lysine at position 17. Nucleic acids encoding the respective peptides are also disclosed.

  One or more conservative amino acid substitutions can be made within the amino acid sequence of the polypeptides of the invention, where the substitution is an amino acid having a non-polar side chain, a natural or non-natural, charged side chain having a polar side chain. No D- or L-amino acids, amino acids with aromatic side chains, natural or non-natural positively charged D- or L-amino acids, natural or non-natural negatively charged D- or L-amino acids, and A receptor for the hormone erythropoietin that occurs within any amino acid of smaller size and molecular weight, where the molecular weight of the original amino acid should not deviate more than approximately ± 25% of the molecular weight of the original amino acid, with an agonistic effect The ability to bind to is maintained. Preferably, only 1, 2 or 3 amino acids are substituted. Sequence changes that do not introduce proline at positions 10 and 17 are preferred.

  The peptide sequences described herein can be used as suitable monomeric peptide units that constitute the binding domain to the EPO receptor. Since they bind to the EPO receptor, they can be used in their monomeric form. As described herein, it has been shown that the ability to induce dimerization of the EPO receptor and thus biological activity is facilitated by dimerization of monomer binding units. These are preferably used as dimers.

  Thus, it is clear that many different peptides are within the scope of the present invention. However, the sequence

は、ある欠点を有し、したがって本発明によると好ましくないことがわかっている。

Have certain disadvantages and are therefore found to be undesirable according to the invention.

  Up to 5 amino acids can be removed and / or added at the beginning (N-terminus) and end (C-terminus) of the individual peptide sequences described. It is self-evident that size is not directly related as long as peptide function is preserved. Furthermore, it should be noted that individual peptide sequences that may be too short to encompass monomeric activity usually function as agonists during dimerization. Accordingly, such peptides are preferably used in their dimeric form. Accordingly, each truncated and / or elongated embodiment is also within the spirit of the invention.

  In the present invention, single letter abbreviations as capital letters are from standard polypeptide nomenclature and are extended by the addition of unnatural amino acids.

上述のように、本発明はまた、単一のアミノ酸の保存的交換による、ペプチドおよび定義されたペプチドコンセンサスの修飾を含む。かかる交換は、結合する分子の構造および機能を変化させるが、ほとんどの場合、変化はわずかでしかない。保存的交換では、あるアミノ酸が、同様の特性を有するグループ内の別のアミノ酸によって置換される。

As mentioned above, the present invention also includes modification of peptides and defined peptide consensus by conservative exchange of single amino acids. Such an exchange changes the structure and function of the molecule to which it binds, but in most cases there is little change. In a conservative exchange, one amino acid is replaced by another amino acid in a group with similar properties.

Examples of corresponding groups are
-Amino acids with non-polar side chains: A, G, V, L, I, P, F, W, M
-Uncharged amino acids with polar side chains: S, T, G, C, Y, N, Q
-Amino acids with 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 acid deviates by up to ± 25% (or ± 20%, ± 15%, ± 10%) from the molecular weight of the original amino acid.

  It is self-evident that the group also includes unnatural amino acids having their respective side chain profiles, such as homoarginine in the case of groups exhibiting positively charged side chains. In the case of proline 10, the substituting molecule, such as an unnatural amino acid, for example, cannot be clearly assigned to one of the above groups characterized by side chain properties, and is generally non-conservative substitution of proline according to the invention Should be accepted as. Classification aids by molecular weight may help to classify these unusual amino acids.

  More specifically, Wrightton et al. (US Pat. No. 5,773,569 and related patents) used phage display technology to examine in detail which amino acids can be substituted while retaining activity. They also examined and published data on possible truncations, ie the minimum length of a given EPO mimetic peptide. However, the proline near the central Gly residue appeared to be the only possibility to obtain an active peptide.

  According to an embodiment of the present invention, there is provided a peptide selected from the group consisting of SEQ ID NOs: 2, 4 to 9 below. As in the case of SEQ ID NO: 2, a peptide having K at position 10 and K at position 17 is particularly preferred.

ある実施形態によると、ペプチド二量体または多量体は、上記の配列番号２および４〜９またはそれらの修飾による単量体に基づいて形成される。本明細書中に記載されるペプチドは、例えば単一のアミノ酸の保存的交換によっても修飾されることができ、好ましくは、１、２または３個より多くないアミノ酸が交換される。

According to certain embodiments, peptide dimers or multimers are formed based on monomers as described above in SEQ ID NOs: 2 and 4-9 or modifications thereof. The peptides described herein can also be modified, for example, by conservative exchange of single amino acids, preferably no more than 1, 2, or 3 amino acids are exchanged.

  Preferably, these peptides are modified with respect to N-terminal AcG and C-terminal MeG.

  As outlined above, the described peptides of the invention can be thought of as a monomer binding domain that recognizes the binding site of the erythropoietin receptor. However, as pointed out by Wrightton et al. (Wrightton, 1997), two of these binding domains are generally required to homodimerize the receptor and induce signal transduction. Thus, the combination of two of these binding domains in one single molecule significantly increases the activity, and peptides with one single binding domain show the same qualitative pattern of activity but are bound together It was not very surprising that two of the domains led to results that showed a fairly low ED50 (50% effective dose, a measure of activity). Peptides comprising two binding domains are identified and particularly preferred as being bivalent or dimeric peptides within the context herein.

One well-known technical solution for combining two monomer binding domains is dimerization. All the solutions that follow this approach have so far been
a) The binding domain is first synthesized separately as a monovalent or monomeric peptide, which can be modified in the preparation of step b, for example by attachment of reactive groups,
b) In the second reaction step, two (mostly identical) binding domains are linked in separate dimerization reactions, which usually comprise a linker molecule sandwiched between the two dimerization domains It is characterized by being able to.

  Such dimers are examples of bivalent peptides and exhibit essentially the same biological function as the monomer. These usually show enhanced biological activity in the case of EPO mimetic peptides.

  Several techniques for monomer dimerization or oligomerization, which can also be applied in accordance with the teachings of the present invention, are known to those skilled in the art. Monomers can be dimerized, for example, by covalent attachment to a linker. A linker is a linking molecule that creates a covalent bond between polypeptide units of the invention. Polypeptide units can be combined through linkers to improve binding to the EPO receptor (Johnson et al., 1997, Wrightton et al., 1997). This further refers to multimerization of monomeric biotinylated peptides by non-covalent interactions with protein carrier molecules described by Wrightton et al. (Wrightton, 1997). It is also possible to use the biotin / threptavidin system, ie biotinylating the C-terminus of the peptide and subsequently incubating the biotinylated peptide with streptavidin. Alternatively, it is also known to achieve dimerization by forming a diketopiperazine structure. Methods known to those skilled in the art are described in detail, for example, in Cavelier et al. (Peptides: The wave of the Future, in Michael Labl and Richard A. Houghten (eds.), American Peptide Society, 2001). The disclosures of these documents regarding dimerization and noncovalent multimerization are hereby incorporated by reference. Another alternative method of obtaining peptide dimers, known from the prior art, is as a reactive precursor of the later linker moiety, which forms the final dimeric peptide by reacting with the N-terminal amino group. The use of bifunctional activated dicarboxylic acid derivatives (Johnson et al., 1997). Monomers can also be dimerized by covalent attachment to a linker. Preferably, the linker comprises NH-R-NH, where R is lower alkylene substituted with a functional group such as a carboxyl group or amino group that allows attachment to another molecular moiety. The linker can contain a lysine residue or lysine amide. Similarly, PEG can be used as a linker. A linker can be a molecule that contains two carboxylic acids and any one or more atoms substituted with a functional group such as an amine that can be attached to one or more PEG molecules. A detailed description of possible steps of peptide oligomerization and dimerization using linking moieties is also given in WO 2004/101606.

  The disclosures of these documents regarding dimerization / multimerization are incorporated herein by reference.

  The peptide monomer or dimer can further comprise at least one spacer moiety. Preferably, such spacers link a monomeric or dimeric linker to a water-soluble polymer moiety, or a protecting group that can be, for example, PEG. PEG has a preferred molecular weight of at least 3 kD, preferably between 20-60 kD. The spacer can be a C1-12 moiety ending with a —NH— bond or a COOH group, and one or more available carbon atoms can be optionally substituted with a lower alkyl substituent. Particularly preferred spacers are disclosed in WO 2004/100997. All documents, WO 2004/100997 and WO 2004/101606 are hereby incorporated by reference. PEG modifications of peptides are disclosed in WO 2004/101600, which is also incorporated herein by reference.

  Although functionally sufficient and therefore can be used in accordance with the teachings of the present invention, the prior art approach to the synthesis of dimeric molecules may have several drawbacks.

  It was recognized that one possible drawback is that the monomers to be linked must first be synthesized separately. Heterodimeric bivalent / multivalent peptides (selectively and intentionally) in this approach for dimerization, a stochastic combination of monomeric peptides during each multimerization reaction It is particularly difficult to get. At the very least, this leads to a large loss of special and intentional heterodimer yields. A bivalent or multivalent peptide containing two or more slightly different monomer binding domains can stabilize the interaction in the final bivalent peptide due to its heterodimeric nature 2 This is highly desirable because it allows the introduction of special interactions between the two domains. However, due to the high yield loss associated with the prior art “estimative dimerization reaction”, this is usually not an economically attractive approach.

  Therefore, applying the prior art approach to dimerization is technically suitable, but there are several economics to provide these peptides with heterologous binding domains as described. Has drawbacks. However, the present invention also advantageously teaches a much more efficient strategy for obtaining highly active multivalent or bivalent peptides that can also include heterologous binding domains.

  The central concept of this strategy is to cut off the synthesis of monomeric peptides that form part of a multivalent or divalent peptide in a separate reaction prior to dimerization or multimerization, but in a single step. As a peptide, for example, the final divalent or multivalent peptide is synthesized in one single solid phase reaction. Thus, separate dimerization or multimerization steps are no longer necessary. This aspect provides a great advantage, ie complete and independent control over each sequence position in the final peptide unit. This method allows easy inclusion of at least two different receptor-specific binding domains in the peptide unit for independent 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 only of amino acids, and thus one single, continuous, bivalent or multivalent EPO mimetic peptide Leads to. In a preferred embodiment of the invention, the linker is composed of natural or non-natural amino acids that allow for high structural flexibility. In this regard, it may be advantageous to use a glycine residue, known for its high flexibility with respect to twist, as the linking amino acid. However, other amino acids such as alanine or β-alanine, or mixtures thereof can also be used. The number or selection of amino acids used depends on the respective steric fact. This embodiment of the present invention allows tailor-made design of suitable linkers by molecular modeling to avoid distortion of the bioactive structure. A linker composed of 3 to 5 amino acids is particularly preferred.

  The linker between the functional domain (or monomer unit) of the final bivalent or multivalent peptide is a separate part of the peptide or part of the monomer functional domain, either completely or partially It is worth noting that it can be either composed of amino acids that are For example, glycine residues at amino acids 1 and 2 and 19 and 20 can form part of the linker. Examples include sequences 11-14. Thus, the term “linker” is defined functionally rather than structurally because an amino acid can form a linker unit as well as part of a monomeric subunit.

  As mentioned above, during synthesis of the bivalent / multivalent peptide, each sequence position within the final peptide is controlled and can therefore be accurately determined so that the peptide or specific region thereof, including the linker Alternatively, the domain can be made specially or tailored to the purpose. This is particularly advantageous as it makes it possible to avoid distortion of the bioactive structure of the final bivalent peptide due to unfavorable intramolecular interactions. The risk of distortion can be assessed by molecular modeling prior to synthesis. This applies in particular to the design of linkers between monomer domains.

  The continuous bivalent / multivalent peptides of the present invention show significantly higher activity than the corresponding monomeric peptides and are therefore known from other dimeric peptides that increased efficiency is associated with the bivalent peptide concept. Make sure the observations being made.

  For monomeric and dimeric peptides, sequential bivalent / multivalent peptides can be modified, for example, by acetylation or amidation, or can be extended at the C-terminal or N-terminal position. Prior art modifications of the above-mentioned monomeric peptides (monomers) involving the attachment of soluble moieties such as PEG, starch or dextran also apply to the multivalent or divalent peptides of the present invention.

  All possible modifications also apply to the linker modifications. In particular, it may be advantageous to attach a soluble polymer moiety such as PEG, starch or dextran to the linker.

  The synthesis of the final multivalent or bivalent peptide of the present invention can also advantageously involve the formation of two, subsequent, independent, disulfide bonds or other intramolecular bonds within each of the binding domains. . Thereby, the peptide can also be cyclized.

  The divalent structure of the present invention is advantageously formed based on the peptide monomers reported herein.

  Some examples of peptide units suitable for dimerization of the EPO receptor are given below. The bar above the binding domain represents a preferred intramolecular disulfide bridge that is optional.

これらの二価構造中のリンカーは、分子モデリングによって、生物活性構造のゆがみを避けるように特別に作製する（図１）。

The linkers in these divalent structures are specially made by molecular modeling to avoid distortion of the bioactive structure (Figure 1).

SEQ ID NO: 12
The linker sequence can be shortened by one glycine residue. This sequence is also an example of a linker composed of glycine residues that simultaneously form part of a binding domain (see SEQ ID NO: 2).

結合ドメインはまた、単量体単位として使用され得る（配列番号１３）。

The binding domain can also be used as a monomer unit (SEQ ID NO: 13).

この配列は、２つのわずかに異なる（異種の）結合ドメインを含む、本発明の連続した二価ペプチドを提供する。かかる二価ペプチドは、従来技術の二量体化アプローチ（上記参照）では、経済的に入手できない。これらの結合ドメインもまた、単量体

This sequence provides a continuous bivalent peptide of the invention comprising two slightly different (heterologous) binding domains. Such bivalent peptides are not economically available with prior art dimerization approaches (see above). These binding domains are also monomeric

として適用することができる。

Can be applied as

  Further examples are

である。

It is.

  According to a further embodiment, the peptide optionally has an additional amino acid, preferably an amino acid having a reactive side chain, such as the N-terminal cysteine, for example in the following sequence or the like.

さらなるペプチドの例は、以下のアミノ酸配列を示す。

Further peptide examples show the following amino acid sequences:

第１の配列は、Ｘ_７位にセリンを示す。この配列が二量体に組み込まれると、水酸基の導入を介して新しい水素架橋が生じることがわかった。したがって、生物活性構造が安定化されるので、Ｘ_７位のセリンの使用は、二量体に特に有利である。

The first sequence shows a serine to X ₇ positions. It was found that when this sequence was incorporated into the dimer, a new hydrogen bridge was formed through the introduction of a hydroxyl group. Therefore, since the bioactive structure is stabilized, the use of X ₇ serine is particularly advantageous in the dimer.

  The second sequence representing the unnatural amino acid homoarginine is particularly suitable for use in pharmaceutical compositions for veterinary purposes. A peptide sequence having an amino acid with a long positively charged side chain, such as homoarginine, at positions 10 and / or 17 may exhibit a strong binding capacity for an EPO receptor such as a mouse / dog receptor. Generally known. Thus, they are particularly suitable for use in veterinary products, but their use is not limited thereto.

  The reactive side chain can function as a linking bond, for example for further modification. The peptide further optionally includes an intramolecular disulfide bridge between the first and second and / or third and fourth cysteines.

  It is important to note that monomeric peptides as exemplified by SEQ ID NOs: 2, 4-9 and 12, 13 and 15, 15a are advantageously combined with the continuous bivalent peptides of the present invention. However, the prior art methods of dimerization of these monomers can be applied as well. Examples of these prior art approaches applied to monomeric peptides falling within the scope of the present invention include (but are not limited to) the following:

  1. Dimerization via a C-terminal to C-terminal linkage in which one C-terminus of the monomeric peptide is covalently linked to the C-terminus of the other peptide. The linker / spacer between the monomers can include diketopiperazine units. A preferred Gly-Gly diketopiperazine skeleton can be achieved by activating the C-terminal glycine monomer. This principle can also be used to form C-terminal dimerization.

  The following equations and examples represent four custom examples optimized by molecular modeling.

  (A) Dimer based on SEQ ID NO: 2 (dimer structure is shown in FIG. 2):

（ｂ）グリシン１つ分短縮されたリンカーを有する、配列番号２に基づく二量体。構造を図３に示す。

(B) A dimer based on SEQ ID NO: 2 with a linker shortened by one glycine. The structure is shown in FIG.

（ｃ）グリシンがβ−アラニンによって置換された、配列番号２に基づく二量体（図４）。単量体（配列番号１６）もまた、ＥＰＯ模倣ペプチドとして適用可能である。

(C) Dimer based on SEQ ID NO: 2 with glycine replaced by β-alanine (FIG. 4). Monomers (SEQ ID NO: 16) are also applicable as EPO mimetic peptides.

（ｄ）代替的グリシンがβ−アラニンによって置換された、配列番号２に基づく二量体（図５）。単量体（配列番号１７）もまた、ＥＰＯ模倣ペプチドとして適用される。

(D) Dimer based on SEQ ID NO: 2 with an alternative glycine replaced by β-alanine (FIG. 5). Monomer (SEQ ID NO: 17) is also applied as an EPO mimetic peptide.

。

.

  2. One N-terminus of said monomeric peptide is covalently linked to the N-terminus of the other peptide, whereby the spacer unit preferably comprises a dicarboxylic acid building block via an N-terminal to N-terminal linkage. Dimerization.

  (A) In an embodiment, the dimer (SEQ ID NO: 18) obtained on the basis of SEQ ID NO: 2, extended by one glycine residue at the N-terminus, contains hexanedioyl units as linker / spacer ( (Fig. 6):

（ｂ）代替的な実施形態において、二量体化は、リンカー／スペーサーとしてオクタンジオイル単位を用いることによって達成することができる（図７）：

(B) In an alternative embodiment, dimerization can be achieved by using octane oil units as linker / spacers (FIG. 7):

。

.

3. Via a side chain, by including a suitable spacer molecule to link two peptide monomers, so that one amino acid side chain of the monomer peptide is covalently bonded to the amino acid side chain of the other peptide Dimerization. this is,
(A) Linkage via amide bond

（ｂ）またはジスルフィド架橋を介した連結

(B) or via a disulfide bridge

を含むことができる。

Can be included.

  X symbolizes the backbone center of each amino acid involved in the formation of each linking bond.

  Each of the assembly methods described above can also be used for the preparation of multimers.

  To form each heterologous or homologous bivalent or multivalent peptide, all of the binding domains, each peptide described herein, either alone or as part of a bivalent / multivalent peptide. It is pointed out that either can be used in monomeric form and / or combined with any of one or more other identical or different peptide domains.

  The peptides can be modified, for example by acetylation or amidation, or can be extended at the C-terminal or N-terminal position. For example, for the preparation of a polymer binding site, extension with one or more amino acids at one of the two ends often leads to a heterodimeric bivalent peptide unit that can best be produced as a contiguous peptide. .

  The compounds according to the invention can advantageously be used for the preparation of human and / or veterinary pharmaceutical compositions. As EPO mimetics, they show essentially the same qualitative activity pattern as erythropoietin. They are therefore generally suitable for the same indications as erythropoietin.

  Erythropoietin is a member of the cytokine superfamily. In addition to the stimulatory effects described in the introduction, erythropoietin is also known to stimulate stem cells. Thus, the EPO mimetics described herein are suitable for all indications caused by stem cell related effects. A non-limiting example is the prevention and / or treatment of diseases associated with the nervous system. Examples include, for example, Parkinson's syndrome, Alzheimer's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, Gaucher's disease, Tay-Sachs disease, neuropathy, terminal nerve injury, brain tumor, brain injury, spinal cord injury or A neurological injury, disease or disorder, such as a stroke injury. The EPO mimetic peptides of the present invention can also be used for prophylactic and / or therapeutic treatment of patients suffering from or at risk of suffering from heart failure. Examples include myocardial infarction, coronary artery disease, myocarditis, chemotherapy treatment, alcoholism, cardiomyopathy, hypertension, valvular heart disease including mitral regurgitation or aortic stenosis, and thyroid disorders, chronic and / or Or acute coronary syndrome.

  In addition, EPO mimetics may be used to treat endothelial precursor cell physiological mobilization, proliferation and differentiation stimulation, angiogenesis stimulation, treatment of diseases associated with endothelial precursor cell dysfunction, and pharmaceutical compositions for the treatment of such diseases. And a pharmaceutical composition comprising said peptide and other agents suitable for stimulation of endothelial progenitor cells. Examples of such diseases are hypercholesterolemia, diabetes mellitus, endothelium-mediated chronic inflammatory diseases, endothelium including reticuloendotheliosis, atherosclerosis, coronary heart disease, myocardial ischemia, angina, aging Cardiovascular disease, Lehno's disease, gestational hypertension, chronic or acute renal failure, heart failure, wound healing and secondary diseases.

  Furthermore, the peptides of the present invention are suitable carriers for delivering an agent across the blood brain barrier, and for each purpose and / or production of each therapeutic complex capable of crossing the blood brain barrier. Can be used.

  The peptides described herein are particularly suitable for the treatment of disorders characterized by erythropoietin deficiency or a low or incomplete red blood cell population, and in particular for the treatment of any type of anemia or stroke. The peptides are also suitable for increasing and / or maintaining hematocrit in mammals. Such pharmaceutical compositions can optionally include a pharmaceutically acceptable carrier in order to employ the composition for the intended administration procedure. Suitable delivery methods and carriers and additives are described, for example, in WO 2004/101611 and WO 2004/100997.

  As outlined above, dimerization of monomeric peptides into dimers or even multimers usually improves EPO mimetic agonist activity compared to the respective monomeric peptide . However, it is desirable to further promote activity. For example, even dimeric EPO mimetic peptides are less capable than EPO in activating cellular mechanisms.

  In the prior art, several approaches have been taken to increase the activity of peptides, for example by altering the amino acid sequence to identify more capable candidates. So far, however, it is still desirable to further increase the activity of peptides, particularly EPO mimetic peptides, in order to improve biological activity.

Further embodiments of the present invention provide a solution to this problem. Here, it binds to the target molecule,
i) at least two peptide units, each peptide unit comprising at least two domains capable of binding to a target;
ii) There is provided a compound comprising at least one polymer carrier unit, wherein the peptide unit is attached to the polymer carrier unit.

  Surprisingly, combinations of two or more of the divalent or multivalent peptides of the invention on a polymer support are not only additive but also in excess, bivalent (or even more) to their bound receptors. It has been found to greatly increase the efficiency of (multivalent) peptides. Therefore, a synergistic effect is observed.

  The term “bivalent”, as used for the purposes of the present invention, is defined as a peptide comprising two domains capable of binding to a target, specifically to the EPO receptor. The This is used interchangeably with the term “dimer”. Thus, a “multivalent” or “multimeric” EPO mimetic peptide has several individual binding domains to the EPO receptor. It is self-evident that the terms “peptide” and “peptide unit” do not incorporate any restrictions on size, but incorporate oligos and polypeptides and proteins.

  A compound comprising two or more divalent or multivalent peptide units attached to a polymeric carrier unit is referred to as “supervalent” in the context of this embodiment. These supervalent molecules are very different from the dimeric or multimeric molecules known in the state of the art. The state of the art is simply combining monomeric EPO mimetic peptides to produce dimers. In contrast, supervalent molecules arise by already linking (at least) a divalent peptide unit to a polymer carrier unit, thereby producing a supervalent molecule (examples are given in FIGS. 13-15). Thereby, the overall activity and efficiency of the peptide is greatly facilitated, thus reducing the EC50 dose.

  So far, the reason for the great ability of supervalent molecules compared to molecules known in the state of the art is not fully understood. This may be because dimeric molecules known in the state of the art simply provide one target per dimer, each receptor binding unit. Thus, only one receptor complex occurs upon binding of the dimeric compound, thereby inducing only one signal transduction process. For example, two monomeric EPO mimetic peptides are linked via PEG to form a peptide dimer, thereby facilitating dimerization of the receptor monomer required for signal transduction ( Johnson et al., 1997). In contrast, the supervalent compounds of the present invention comprise several, already divalent or multivalent, receptor binding units. This allows several receptor complexes to occur on the cell surface per compound molecule, thereby inducing some signal transduction, thereby over-adding the activity of the peptide unit. Rise. Binding of supervalent compounds can result in crowding of receptor complexes at the cell surface.

  The EPO mimetic peptide units used in this embodiment can be either homologous or heterogeneous, meaning that either the same or different peptide units are used. The same applies to the binding domains (monomeric peptides as described above) of peptide units that may be homologous or heterogeneous. Bivalent or multivalent peptide units that bind to the carrier unit bind to the same receptor target.

  However, these can of course still differ in their amino acid sequences. The monomer binding domain of a divalent or multivalent peptide unit can be either linear or cyclic. Cyclic molecules can be generated, for example, by formation of intramolecular cysteine bridges (see above).

  The polymeric carrier unit comprises at least one natural or non-natural, branched, linear or dendritic polymer. The polymeric carrier unit is preferably a polymer that is soluble in water and body fluids, preferably pharmaceutically acceptable. Examples of the water-soluble polymer portion include PEG, PEG homopolymer, mPEG, polypropylene glycol homopolymer, a copolymer of ethylene glycol and propylene glycol (wherein the homopolymer and copolymer are not substituted, Or polyalkylene glycol and derivatives thereof, including polyglycerol or polysialic acid, methylcellulose and carboxymethylcellulose, starch (eg, hydroxyalkyl starch), including at one end, eg substituted with an acyl group (HAS), especially hydroxyethyl starch (HES) and dextrin and their derivatives), dextran sulfate, cross-linked dextrin and carboxymethyl dextrin. Dextran and dextran derivatives, heparin and heparin fragments, polyvinyl alcohol and polyvinyl ethyl ether, polyvinyl pyrrolidone, a, b-poly [(2-hydroxyethyl) -DL-aspartic acid amide, and polyoxyethylated polyols. However, it is not limited to these. Of course, other biologically inert aqueous polymer can also be used. Simple but still preferred examples of suitable carrier units are homobifunctional polymers, for example of polyethylene glycol (bis-maleimide, bis-carboxy, bis-amino, etc.).

  The polymeric carrier unit can have a wide range of molecular weights due to the different properties of different polymers suitable in connection with the present invention. Therefore, there is no size limit. However, it is preferred that the molecular weight be at least 3 kD, preferably at least 10 kD and approximately about 20-500 kD, more preferably about 30-150 or about 60 or 80 kD. The size of the carrier unit depends on the polymer selected and can therefore vary. For example, the molecular weight can be much higher, especially when using starch such as hydroxyethyl starch. In this case, the average molecular weight can be as high as about 100-4,000 kD or even higher. The size of the carrier units is preferably selected so that each peptide unit is optimally aligned for binding to their respective receptor molecule. To facilitate this, certain embodiments of the present invention use a carrier unit that includes a branching unit. According to this embodiment, a polymer, such as PEG, is attached to the branch unit, thus resulting in a large carrier molecule that allows the incorporation of multiple peptide units. Examples of suitable branching units are glycerol or polyglycerol. Similarly, dendritic branching units can also be used, for example, as taught in Haag, 2000, which is incorporated herein by reference.

  Preferably, after the peptide units are generated by combining monomers (either head-to-head, head-to-tail, or tail-to-tail), the polymer carrier unit is linked to the peptide unit. The polymer carrier unit is linked to the peptide unit via a covalent or non-covalent (eg coordination) bond. However, the use of covalent bonds is preferred. Coupling can occur via, for example, reactive amino acids of peptide units such as lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, or the N-terminal amino group and the C-terminal carboxylic acid. it can.

  If the polymer carrier unit does not have a suitable linking group, several linking substances can be used to appropriately modify the polymer in order to be able to react with at least one reactive group on the peptide unit. . Suitable chemical groups that can be used to modify the polymer are, for example:

  Acylated groups that react with amino groups of proteins, such as acid anhydride groups, N-acylimidazole groups, azide groups, N-carboxyanhydride groups, diketene groups, dialkyl pyrocarbonate groups, imide ester groups and carbodiimide-activated carboxyls Group. All of the above groups are known to react with amino groups of proteins / peptides to form covalent bonds including acyl or similar bonds.

A sulfhydryl of a peptide unit such as a halo-carboxyl group, a maleimide group, an activated vinyl group, an ethyleneimine group, a halogenated aryl group, a 2-hydroxy5-nitro-benzyl bromide group, together with a reducing agent that reacts with the amino group of the peptide Alkylated groups that react with (mercapto), thiomethyl, imidazo or amino groups, and aliphatic aldehyde and ketone groups,
Ester and amide-forming groups that react with carboxyl groups of proteins, such as diazocarboxylic acid groups, and carbodiimide and amine groups,
Disulfide-forming groups that react with protein sulfhydryl groups, such as 5,5′-dithiobis (2-nitrobenzoic acid) groups and alkyl mercaptan groups (reacting with protein sulfhydryl groups in the presence of oxidizing agents such as iodine)
Dicarbonyl groups, such as cyclohexanedione groups, and other 1,2-diketone groups that react with the guanidine moiety of the peptide,
A diazo group that reacts with the phenolic group of the peptide,
A reactive group from the reaction of cyanogen bromide with a polysaccharide that reacts with the amino group of the peptide.

  Thus, in summary, the compounds of the present invention (at least) can first chemically modify the polymer to react with available or introduced chemical groups on the peptide unit. Producing a polymer having a chemical group, and then reacting the modified polymer with the peptide unit together (if necessary) and utilizing the chemical group of the modified polymer (if necessary) It can be produced by forming a covalently bound complex.

  If the coupling occurs via a free SH-group of the peptide (eg a cysteine group), the use of a maleimide group in the polymer is preferred.

  In order to generate a defined molecule, it is preferable to use a targeted approach to attach peptide units to polymer carrier units. If the appropriate amino acid is not present at the desired binding site, the appropriate amino acid can be incorporated into the dimeric EPO mimetic peptide unit. In order to avoid uncontrolled binding reactions throughout the peptide leading to a heterogeneous mixture containing several different populations of polyethylene glycol molecules due to site-specific polymer coupling, a unique reactive group such as the end of a peptide unit The specific amino acids are preferred.

  Conjugation of peptide units to polymer carrier units such as PEG or HES is mainly performed using reactions known to those skilled in the art. For example, there are many PEG and HES conjugation methods available to those skilled in the art (eg, WO 2004/100997, Roberts et al., Which is incorporated herein by reference in its entirety). 2002, U.S. Pat. No. 4,064,118, EP 1398322, EP 1398327, EP 1398328, WO 2004/024761).

  It is important to understand that the concept of supervalence described herein differs from the known concept of PEGylation or HESylation. In the state of the art, for example, PEGylation is only used to generate peptide dimers or to improve pharmacokinetic parameters. However, as outlined above, conjugation of two or more at least divalent peptide units to, for example, PEG as a polymer carrier unit also greatly enhances efficiency (and thus reduces EC50 dose). Thus, the inventive concept has a strong effect not only on pharmacokinetic parameters but also on pharmacodynamic parameters as in the case of PEGylation concepts known in the state of the art. However, of course, the incorporation of eg PEG as a polymer carrier unit also has known advantages with respect to pharmacokinetics.

  PEGylation is usually undertaken to improve the biopharmaceutical properties of peptides. The most relevant changes in protein molecules after PEG conjugation are size expansion, protein surface and glycation function masking, charge modification and epitope masking. Specifically, size enlargement slows down kidney ultrafiltration and promotes accumulation in permeable tissues by a passive facilitated infiltration and retention mechanism. Protein shielding reduces proteolysis and immune system recognition, which are important pathways of excretion. The specific effect of PEGylation on the physicochemical and biological properties of a protein is strictly determined by the properties of the protein and polymer and the PEGylation strategy employed.

  However, the use of PEG or other non-biodegradable polymers as supervalent molecular support units can lead to new problems.

  During in vivo application, dosing intervals in the clinical environment are caused by loss of drug effect. Regular administrations and dosing intervals are adapted so that no effect is lost between dosing intervals. Peptides attached to large non-biodegradable polymer units (eg, PEG moieties) may degrade faster than the support molecule and be excreted by the body, thus creating a risk of accumulation of carrier units. The risk of such accumulation always occurs because the effective half-life of the drug is shorter than the elimination half-life of the drug itself or its components / metabolites. Thus, accumulation of carrier molecules should be avoided, as peptides usually exhibit slow renal excretion due to being PEGylated with very large PEG moieties (about 20-40 kD). The peptide moiety itself undergoes enzymatic degradation and partial cleavage may be sufficient to deactivate the peptide.

  In order to find a solution to this problem, certain embodiments of the present invention teach the use of a polymer support unit composed of at least two subunits. The polymer subunits are linked via a biodegradable covalent linker structure. According to this embodiment, the molecular weight of a large carrier molecule (eg 40 kD) has several small or intermediate sized subunits (eg 5-10 kD molecular weight) linked via a biodegradable linker. Produced by each subunit). The molecular weight of the modular subunit is large, thereby producing the desired molecular weight of the carrier molecule. However, biodegradable linker structures can be degraded in the body, thereby releasing smaller carrier subunits (eg, 5-10 kD). Smaller carrier subunits show better renal clearance than polymer molecules with an overall molecular weight (eg 40 kD). An example is shown in FIG.

  The linker structure is selected according to known degradation characteristics and duration of degradation in body fluids. A degradable structure can include a cleavable group, such as a carboxylic acid derivative, eg, as an amide / peptide bond or ester that can be cleaved by hydrolysis (eg, incorporated herein by reference). Roberts, 2002). PEG succinimidyl esters can also be synthesized with various ester linkages in the PEG backbone to control degradation rates at physiological pH (Zhao, 1997, incorporated herein by reference). . Other degradable structures such as benzylurethane disulfide can be cleaved in a mild reducing environment, such as in the endosomal compartment of cells (Zalipsky, 1999) and are therefore equally suitable. Other selection criteria for suitable linkers are selections for fast (often enzymatic) degradation or slow (often non-enzymatic degradation) degradation. A combination of these two mechanisms in bodily fluids is also feasible. This highly advantageous concept is not limited to the specific peptide units described or referred to herein, but also to other pharmaceutical molecules attached to large polymer units such as PEG molecules that cause the same accumulation problems. It is clear that it applies.

  According to an embodiment, hydroxyalkyl starch and preferably HES are used as polymer carrier units. HES has several important advantages. First, HES is biodegradable. Furthermore, the biodegradability of HES can be controlled via the proportion of ethyl groups and can therefore be influenced. 30-50% ethyl groups are well suited for the purposes of the present invention. Due to biodegradability, the above accumulation problems in complex with PEG usually do not occur. In addition, HES has been used for many years in medicine, for example in the form of plasma substitutes. Therefore, its harmlessness has been proven.

  In addition, derivatives of HES hydrolysates can be detected by gas chromatography. The HES-peptide complex can be hydrolyzed under conditions where the peptide units remain stable. This allows for the quantification and monitoring of degradation products and allows the evaluation and normalization of active peptides.

  According to a further embodiment, a first type of polymer carrier unit is used and loaded with peptide units. This first carrier is preferably readily biodegradable, for example HES. However, not all the binding sites of the first carrier are occupied by peptide units, for example only about 20-50%. Depending on the size of the polymer used, several hundred peptide units can be attached to the carrier molecule. The remaining attachment sites of the first carrier are occupied by different carriers, eg small PEG units having a lower molecular weight than the first carrier. This embodiment results in a supervalent composition for the first carrier, which is very durable due to the presence of the second carrier, preferably composed of 3-5 or 10 kD PEG units. It has the advantage of being. However, since the first carrier (eg HES) and the peptide unit are biodegradable and the second carrier, eg PEG, is small enough to be easily removed from the body, the overall entity is very degradable. High nature.

  The monomer that constitutes the binding domain of the peptide unit recognizes the homodimeric erythropoietin receptor. The latter property of being a homodimeric receptor distinguishes the EPO receptor from many other cytokine receptors. Peptide units comprising at least two EPO mimetic monomer binding domains, as described above, bind to EPO receptors, preferably bivalent, multimerize and / or stabilize their targets, respectively. Can thus produce signal transduction thereby inducing the complex.

  The present invention also includes each compound production method wherein peptide units are linked to respective carrier units. The present invention further includes respective compound production methods wherein peptide units are linked to respective polymer carrier units. The compounds according to the invention can advantageously be used for the preparation of human and / or veterinary pharmaceutical compositions. They are particularly suitable for the treatment of disorders characterized by erythropoietin deficiency or a small or incomplete red blood cell population, and in particular for the treatment of any type of anemia and stroke. They can also be used for all the instructions mentioned above. Such pharmaceutical compositions can also optionally contain a pharmaceutically acceptable carrier in order to employ the composition in the intended administration procedure. Suitable delivery methods and carriers and additives are described, for example, in WO 2004/100997 and WO 2004/101611, which are incorporated herein by reference.

  The concept of supervalent molecules is illustrated by examples. FIG. 13 shows an example of a simple supervalent molecule of the present invention. Two consecutive bivalent EPO mimetic peptides are linked at the N-terminus by a bifunctional PEG moiety having a maleimide group. Cysteine was selected as the reactive binding site for the PEG carrier unit.

  However, a supervalent molecule can contain more than two consecutive bivalent or multivalent peptide units. FIG. 14 gives an example based on a carrier unit having a central glycerol unit as a branching unit and comprising three consecutive bivalent peptides. Again, cysteine was used for conjugation. FIG. 20 shows an example using HES as the polymer carrier unit. The HES was modified to have a maleimide group that reacts with the SH group of the peptide unit. According to the examples, all binding sites are bound to peptide units. However, small PEG units (eg 3-10 kD) could also occupy at least part of the binding site.

  As explained above, the supervalent concept can also be extended to multivalent dendritic polymers in which dendritic and / or polymer carrier units are linked to a larger number of consecutive divalent peptides. For example, dendritic branching units can be based on polyglycerol (see Haag, 2000, incorporated herein by reference).

  An example of a supervalent molecule based on a carrier unit having a dendritic branching unit containing six consecutive bivalent peptides is shown in FIG.

  Other examples of supervalent molecules include carrier units with starch or dextran that are oxidized using, for example, periodic acid and contain multiple aldehyde functional groups. In the second step, many divalent peptides are bound to the carrier unit and together form the final molecule. It should be noted that as many as several hundred (eg 50-1000, preferably 150-800, more preferably 250-700) peptide units can be bound to a carrier molecule, eg HES.

  FIG. 16 illustrates the concept of a simple biodegradable supervalent molecule. Two consecutive bivalent EPO mimetic peptides are linked at the N-terminus by two bivalent PEG moieties linked via a biodegradable linker with an intermediate cleavage position. The linker allows the degradation of large PEG units in the subunit, thereby facilitating renal clearance.

I. Monomeric peptide synthesis Manual synthesis PL-Rink-amide-resin (displacement rate 0.4 mmol / g) or Discover microwave system (CEM) using pre-treated Wang-resin on a scale of 0.4 mmol The synthesis is performed by using Removal of the Fmoc group is achieved by addition of 30 ml piperazine / DMF (1: 3) and irradiation with 100 W for 3 × 30 seconds. Amino acid binding is achieved by addition of a 5-fold excess of amino acid in DMF PyBOP / HOBT / DIPEA as a binding additive and 50 W irradiation for 5 × 30 seconds. During every irradiation cycle, the solution is cooled manually with the help of an ice bath. After deprotection and binding, the resin is washed 6 times with 30 ml DMF. After deprotection of the last amino acid, some peptides are acetylated by incubation for 5 min with 1.268 ml capping solution (4.73 ml acetic anhydride and 8.73 ml DIEA in 100 ml DMSO). The resin is then washed 6 times with 30 ml DMF, 6 times with 30 ml DCM and then cleaved. Cleavage of the crude peptide is achieved by treatment with 5 ml TFA / TIS / EDT / H ₂ O (94/1 / 2.5 / 2.5) for 120 minutes under inert atmosphere. This solution is filtered into 40 ml of cold ether. The precipitate is dissolved in acetonitrile / water (1/1) and the peptide is purified by RP-HPLC (Kromasil 100 C18 10 μm, 250 × 4.6 mm).

Automated synthesis The synthesis is carried out by use of an Odyssey microwave system (CEM) with PL-Rink-amide-resin (substitution rate 0.4 mmol / g) or pretreated Wang-resin on a scale of 0.25 mmol. Removal of the Fmoc group is achieved by addition of 10 ml piperazine / DMF (1: 3) and irradiation with 100 W for 10 × 10 seconds. Amino acid binding is achieved by addition of a 5-fold excess of amino acid in DMF PyBOP / HOBT / DIPEA as a binding additive and 50 W irradiation for 5 × 30 seconds. During all irradiation cycles, the solution is cooled by blowing nitrogen bubbles through the reaction mixture. After deprotection and binding, the resin is washed 6 times with 10 ml DMF. After deprotection of the last amino acid, some peptides are acetylated by incubation for 5 minutes with 0.793 ml capping solution (4.73 ml acetic anhydride and 8.73 ml DIEA in 100 ml DMSO). The resin is then washed 6 times with 10 ml DMF, 6 times with 10 ml DCM and then cleaved. Cleavage of the crude peptide is achieved by treatment with 5 ml TFA / TIS / EDT / H ₂ O (94/1 / 2.5 / 2.5) for 120 minutes under inert atmosphere. This solution is filtered into 40 ml cold ether, the precipitate is dissolved in acetonitrile / water (1/1) and the peptide is purified by RP-HPLC (Kromasil 100 C18 10 μm, 250 × 4.6 mm).

Purification All peptides were purified using the Nebula-LCMS-system (Gilson). All peptide crudes were dissolved in acetonitrile / water (1/1) and the peptides were purified by RP-HPLC (Kromasil 100 C18 10 μm, 250 × 4.6 mm). The flow rate was 20 ml / min and the LCMS split ratio was 1/1000.

II. Formation of intramolecular disulfide bridges Cyclization with K ₃ [(FeCN ₆ ) Solution 1: 10 mg 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 until a pH of approximately 8 is reached.

Solution 2: In a second vial, 10 ml of 0.1% TFA / acetonitrile is diluted with 10 ml of water. Solid ammonium bicarbonate is added until a pH of 8 is reached, and one drop of a 0.1 M solution of K ₃ [(FeCN ₆ )] is added.

  Solutions 1 and 2 are added dropwise over 3 hours to a mixture of acetonitrile / water (1/1, pH = 8). Incubate the mixture at room temperature overnight and concentrate and purify the mixture by LCMS.

Cyclization with CLEAR-OX ™ resin Solid ammonium bicarbonate is added to 100 ml acetonitrile / water (1/1, 0.1% TFA) until a pH of 8 is reached. Degas from this solution by blowing a bubble of argon for 30 minutes. Now add 100 mg of CLEAR-OX ™ resin. After 10 minutes, 10 mg of peptide is added as a solid. After 2 hours incubation, the solution is filtered, concentrated and purified by LCMS.

Purification of cyclic peptides All peptides were purified using the Nebula-LCMS system (Gilson). All peptide crudes were dissolved in acetonitrile / water (1/1) or DMSO and the peptides were purified by RP-HPLC (Kromasil 100 C18 or C8 10 μm, 250 × 4.6 mm). The flow rate was 20 ml / min and the LCMS split ratio was 1/1000.

III. Monomeric in vitro assay Proliferation assay in TF-1 cells by BrdU incorporation TF-1 cells in logarithmic growth phase (approximately 2 × 10 ⁵ to 1 × 10 ⁶ cells / ml, RPMI medium, 20% fetal calf Serum, penicillin, streptomycin, L-glutamine, supplemented with 0.5 ng / ml interleukin 3) washed (centrifuged at 1500 rpm for 5 minutes and resuspended in RPMI medium without IL3 at 500,000 cells / ml) Pre-incubate for 24 hours without IL-3 before starting the assay. The next day, cells are seeded in 24- or 96-well plates using 4 wells containing at least 6 concentrations per agent to be tested and at least 10,000 cells / well per concentration. Each experiment includes a control containing recombinant EPO as a positive control agent and wells without addition of cytokines as a negative control agent. Peptide and EPO controls are pre-diluted in the medium to the desired concentration, added to the cells, and a 3 day culture period is established under standard culture conditions (37 ° C., 5% carbon dioxide in gas phase, water saturated atmosphere). Start. Concentration always refers to the final concentration of agent in the well during this 3 day culture period. At the end of this culture period, FdU is added to a final concentration of 8 ng / ml medium and the culture is continued for 6 hours. BrdU (bromodeoxyuridine) and dCd (2-deoxycytidine) are then added to their final concentrations (10 ng / ml BrdU, 8 ng / ml dCD, final concentration in the medium) and the culture is continued for another 2 hours.

  At the end of this incubation and culture period, the cells are washed once with phosphate buffered saline containing 1.5% BSA and resuspended in a minimal amount of liquid. From this suspension, the cells are added dropwise in 70% ethanol at -20 ° C. From here, cells can be incubated on ice for 10 minutes 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 are resuspended in a minimal amount of remaining liquid. The cells are then suspended in 0.5 ml of 2M HCl / 0.5% triton X-100 and incubated for 10 minutes. They are then pelleted again, resuspended in a minimal amount of remaining liquid and diluted with 0.5 ml of 0.1 N Na ₂ B ₄ O ₇ , pH 8.5 prior to the subsequent re-pelleting of cells. Finally, cells are resuspended in 40 μl phosphate buffered saline (1.5% BSA) and split into two reaction tubes each containing 20 μl cell suspension. 2 μl of anti-BrdU-FITC (DAKO, clone Bu20a) is added to one tube and 2 μl of control mlgG1-FITC (Sigma) is added to the second tube to start a 30 minute incubation period at room temperature. Then 0.4 ml phosphate buffered saline and 10 μg / ml propidium iodide (final concentration) are added. Analysis in the flow cytometer refers to the fraction of 4C cells or cells with higher ploidy and the fraction of BrdU positive cells, thus determining the fraction of cells at the relevant stage of the cell cycle.

Proliferation assay in TF-1 cells by MTT TF-1 cells in logarithmic growth phase (approximately 2 × 10 ⁵ to 1 × 10 ⁶ cells / ml, RPMI medium, 20% fetal calf serum, penicillin, streptomycin, L- Glutamine, supplemented with 0.5 ng / ml interleukin 3) is washed (centrifuged at 1500 rpm for 5 minutes and resuspended in RPMI complete medium without IL3 at 500,000 cells / ml) and IL before the start of the assay Pre-culture for 24 hours without -3. The next day, cells are seeded in 24 or 96 well plates, with 4 wells containing at least 6 concentrations per agent to be tested and at least 10,000 cells / well per concentration. Each experiment includes a control containing recombinant EPO as a positive control agent and wells without addition of cytokines as a negative control agent. Peptide and EPO controls are pre-diluted in the medium to the desired concentration, added to the cells, and a 3 day culture period is established under standard culture conditions (37 ° C., 5% carbon dioxide in gas phase, water saturated atmosphere). Start. Concentration always refers to the final concentration of agent in the well during this 4 day culture period.

  On day 4, before the start of the analysis, a known number of dilution series of TF-1 cells are prepared in several wells (0/2500/5000/10000/20000/50000 cells / well in 100 μl medium). ). These wells are processed in the same way as test wells and are later provided with a calibration curve that can determine the number of cells. These reference wells were constructed to thaw MTS and PMS from the MTT proliferation kit (Promega, CellTiter 96 aqueous non-radioactive cell proliferation assay) in a 37 ° C. water bath, and 100 μl of PMS solution into 2 ml of MTS solution. Added. Add 20 μl of this mixture to each well of the assay plate and incubate at 37 ° C. for 3-4 hours. Prior to the measurement of E492 in an ELISA reader, 25 μl of 10% sodium dodecyl sulfate in water is added to each well.

  The following EC50 values were determined based on the MTT assay data using a graphical evaluation as shown in FIGS. 17 and 18 based on dose response relationship calculations using the program GraphPad.

The following table shows EC ₅₀ values for some exemplary peptides.

。

.

IV. Synthesis of divalent EPO mimetic peptides Automated synthesis of linear SEQ ID NO: 11 (AGEM11) Liberty microwave system (CEM) using Rink-amide-resin (substitution rate 0.19 mmol / g) with a scale of 0.25 mmol The synthesis is performed by using Removal of the Fmoc group is achieved by dual treatment of 10 ml piperazine / DMF (1: 3) and 10 × 10 seconds of 50 W irradiation. Amino acid binding is achieved by a double treatment of a 4-fold excess of amino acid in DMF PyBOP / HOBT / DIPEA as a binding additive and 50 W irradiation for 5 × 30 seconds. During every irradiation cycle, the solution is cooled manually by bubbling nitrogen through the reaction mixture. After deprotection and binding, the resin is washed 6 times with 10 ml DMF. After the double bond cycle, all untreated amino groups were treated with a 10-fold excess of N- (2-chlorobenzyloxycarbonyloxy) succinimide (0.2 M DMF solution) and 3 × 30 seconds of 50 W irradiation. Block by. After deprotection of the last amino acid, some peptides are acetylated by incubation for 5 minutes with 0.793 ml capping solution (4.73 ml acetic anhydride and 8.73 ml DIEA in 100 ml DMSO). The resin is then washed 6 times with 10 ml DMF, 6 times with 10 ml DCM and then cleaved. Cleavage of the crude peptide is achieved by treatment with 5 ml TFA / TIS / EDT / H ₂ O (94/1 / 2.5 / 2.5) for 120 minutes under inert atmosphere. This solution is filtered into 40 ml of cold ether, the precipitate is dissolved in acetonitrile / water (1/1) and the peptide is purified by RP-HPLC (Kromasil 100 C18 10 μm, 250 × 4.6 mm).

  Therefore, a purification schematic of linear AGEM11, Kromasil 100 C18 10 μm, 250 × 4.6 mm and the gradient used was shown in FIGS. 8 and 9 from 5% to 50% acetonitrile (0.1% TFA) in 50 minutes. Show.

  Cyclization of AGEM11

３０ｍｇの直鎖状ペプチドを６０ｍｌの溶液Ａに溶解する。この溶液および６０ｍｌのＤＭＳＯを、６０ｍｌの溶液Ａに滴下する（添加の合計時間は３時間）。４８時間後、蒸発によって溶媒を除去し、残った残渣を３０ｍｌのＤＭＳＯ／水（１／１）に溶解する。３０ｍｌの酢酸および１７ｍｇのヨウ素（ＤＭＳＯ／水（１／１）に溶解）を添加し、溶液を９０分間室温で混合する。その後、２０ｍｇのアスコルビン酸を添加し、蒸発によって溶媒を除去する。この粗混合物をアセトニトリル／水（２／１）に溶解し、ＲＰ−ＨＰＬＣ（Ｋｒｏｍａｓｉｌ １００ Ｃ１８ １０μｍ、２５０×４．６ｍｍ）によってペプチドを精製する。

30 mg of 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 time of addition is 3 hours). After 48 hours, the solvent is removed by evaporation and the remaining residue is dissolved in 30 ml DMSO / water (1/1). 30 ml acetic acid and 17 mg iodine (dissolved in DMSO / water (1/1)) are added and the solution is mixed for 90 minutes at room temperature. Then 20 mg ascorbic acid is added and the solvent is removed by evaporation. This crude mixture is dissolved in acetonitrile / water (2/1) and the peptide is purified by RP-HPLC (Kromasil 100 C18 10 μm, 250 × 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 shown in FIGS. 10 and 11 (Schematic: Purification of cyclic AGEM11, Kromasil 100 C18 10 μm, 250 × 4.6 mm, gradient from 5% to 35% acetonitrile (0.1% TFA) in 50 minutes. ).

V. In vitro proliferation assay to determine EPO activity TF1 cells in logarithmic growth phase (approximately 2 × 10 ⁵ to 1 × 10 ⁶ cells / ml, in RPMI with 20% fetal calf serum (FCS) and 0.5 ng / ml IL3 (Proliferation) was counted and the number of cells needed to perform the assay was centrifuged (1500 rpm for 5 minutes) and resuspended in RPMI without IL-3 at 300,000 cells / ml with 5% FCS. Cells were pre-cultured for 48 hours in this IL-3 free (starved) medium. Prior to starting the assay, the cells were counted again.

  Just before starting the assay, a stock solution of peptide and EPO was prepared. Peptides were weighed and dissolved in RPMI with 5% FCS to a concentration of 1 mM, 467 μM or 200 μM. The EPO stock solution was 10 nM or 20 nM. 292 μl of these stock solutions were pipetted into one well of a 96 well culture plate and one plate was taken for each substance to be tested. 200 μl of RPMI containing 5% FCS was pipetted into 17 other wells of each plate. 92 μl of stock solution was pipetted into wells containing 200 μl of medium. The contents were mixed and 92 μl from this well was transferred from side to side. In this way, a dilution series of each substance (18 dilutions) was prepared so that the concentration in each successive well was 1: √10 of the concentration in the previous well. From each well, 3 × 50 μl was transferred to 3 empty wells. In this way, each concentration of the substance was measured in quadruplicate. Note that the top and bottom rows of wells in each plate remained empty.

Pretreated (starved) cells were centrifuged (1500 rpm for 5 minutes) and resuspended in RPMI containing 5% FCS at a concentration of 200,000 cells per ml. 50 μl of cell suspension (containing 10,000 cells) was added to each well. Note that due to the addition of cells, the final concentration of the substance in the well was half of the original dilution range. Plates were incubated for 72 hours at 37 ° C. in 5% CO ₂ .

  Prior to starting the evaluation, a known amount of dilution range TF-1 cells in wells were prepared. 0/2500/5000/10000/20000/50000 cells / well were pipetted in quadruplicate (in 100 μl RPMI + 5% FCS).

To determine the number of viable cells per well, off-the-shelf MTT reagent (Promega, CellTiter 96 aqueous one solution cell proliferation assay) was thawed in a 37 ° C. water bath. 20 μl of MTT reagent was added per well and the plates were incubated for an additional 1-2 hours at 37 ° C. in 5% CO ₂ . 25 μl of 10% SDS solution was added and the plates were measured in an ELISA reader (Genios, Tecan). Data was processed in a spreadsheet (Excel) and plotted on Graphpad.

  The data is summarized in FIG.

ED50 (nM)
EPO 0.0158
BB49 (monomer, SEQ ID NO: 2) 4113
AGEM11 (divalent) 36.73.

VI. Extended Peptide Assay In the extended assay, approximately 200 peptide sequences were tested for their EPO mimetic activity.

Peptides were synthesized as peptide amides on a LIPS-Vario synthesizer system. The synthesis was performed in a special MTP synthesis plate and the scale was 2 μmol per peptide. The synthesis followed the standard Fmoc protocol using HOBT as the activation reagent. The coupling step was performed as 4 couplings. Each binding step took 25 minutes and the excess amino acid per step was 2.8. Peptide cleavage and deprotection were performed with a cleavage solution containing 90% TFA, 5% TIPS, 2.5% H ₂ O and 2.5% DDT. A synthetic plate containing the finished peptide bound to the resin was stored on top of a 96 deep well plate. 50 μl of cleavage solution was added to each well, cut for 10 minutes, and this procedure was repeated three times. The cleaved peptide was eluted with 200 μl of cleavage solution by gravity flow to a deep well plate. Further, the side chain functional groups were deprotected in the deep well plate for 2.5 hours. Thereafter, the peptide was precipitated with ice-cold ether / hexane and centrifuged. The peptide was dissolved in a neutral aqueous solution and cyclization was incubated overnight at 4 ° C. The peptide was lyophilized.

  FIG. 19 shows an overview of the peptide monomers synthesized and tested.

  The peptides were tested for their EPO mimetic activity in an in vitro proliferation assay. The assay was performed as described in V below. On each assay day, 40 microtiter plates were prepared to measure in vitro activity of 38 test peptides, 1 reference example, and EPO in parallel. The EPO stock solution was 20 nM.

  FIG. 19 shows the result. As can be seen from the results, the tested peptides that did not meet the consensus of the present invention did not show EPO mimetic activity.

VII. Synthesis of peptide HES-complex A schematic diagram of the principle reaction is shown in FIG.

  The aim of the described method is the production of starch derivatives that react selectively with thiol groups under mild aqueous reaction conditions according to the HES of this example. This selectivity is achieved with maleimide groups.

  HES is first functionalized with an amino group and then converted to the respective maleimide derivative. Low molecular weight reactants were removed from the reaction batch through an ultrafiltration membrane. The product, intermediate product and extract are all polydisperse.

Synthesis of Amino-HES (AHES) Hydroxyethyl starch (Voluven®) was obtained via diafiltration followed by lyophilization. The average molecular weight was approximately 130 kDa, 40% substitution grade.

Jacob Piehler's paper, which is incorporated herein by reference. . HES was inactivated by partial selective oxidation of hydroxyl groups of diols to aldehydes with sodium periodate as described in Floor et al. (1989). The aldehyde group was converted to an amino group via reductive amination with sodium cyanoborohydride (NaCNBH ₃ ) in the presence of ammonia (Yalpani and Brooks, 1995).

  Periodate onset: Periodate usage indicates a 20% number of glucose building blocks (180 g / mol glucose building block mass applied, DS = 0,4). Work-up was performed by ultrafiltration and lyophilization.

Reductive amination with MH ₄ Cl / Na [BH ₃ CN] (excess) Production via precipitation and diafiltration Analysis Qualitative: Ninhydrin reaction (Kaiser test)
Quantitative: using 2,4,6-nitrobenzole sulfonic acid (TNBS) compared to aminodextran. The resulting substitution grade was about 2.8%. This results in a molar mass of one building block with one amino group of approximately 6400 g / mol.

Synthesis of maleimidopropionyl-amino-hydroxyethyl starch (“MalPA-HES”) Synthesis Using 3-maleimidopropionic acid-N-hydroxysuccinimide ester (MalPA-OSu) in excess (10-fold) (50 mM phosphate buffer, pH 7, Work-up was performed by 20% DMF, overnight), ultrafiltration and lyophilization.

Analysis The reaction of the amino group was demonstrated with ninhydrin and TNBS.

The number of maleimide groups introduced was indicated by reaction of glutathione (GSH) and detection of excess thiol groups via 700 MHz- ¹ H-NMR spectroscopy with Elman reagent (DNTB).

  The resulting substitution grade is about 2%, corresponding to 8500 g / mol per maleimide building block (180 g / mol glucose building block mass, DS = 0,4).

Peptide-hydroxyethyl starch-complex (Pep-AHES)
Synthesis Cysteine-containing peptides with either free (Pep-IA) or biotinylated (Pep-IB) N-terminus were used. A 4: 1 mixture of Pep-IA / B was converted overnight with MalPA-HES in phosphate buffer, 50 mM, pH 6.5 / DMF 80:20 in excess (approximately 6 equivalents), by ultrafiltration and lyophilization. Post-processing was performed.

Analysis UV absorption was measured at 280 nm and the remaining contents of the maleimide group were measured with GSH / DNTB.

  Peptide yield was almost quantitative. There were almost no detectable free maleimide groups.

VIII. Antibody Cross-Reactivity Assay As described in the introduction of this application, patients sometimes develop antibodies against rhuEPO. This leads to the serious consequences described in the introduction.

  To further investigate the properties of the peptides of the present invention, it was analyzed whether the peptides actually cross-react with anti-EPO antibodies.

  Rabbit and human sera containing anti-EPO antibodies were used for testing. These sera were pretreated with either EPO or the following EPO mimetic peptides.

異なる濃度のエリスロポイエチンおよびＥＰＯ模倣ペプチドを解析で使用した。血清中に存在する抗ＥＰＯ抗体を吸着させるための試験物質での血清の前処理の後、血清を放射活性標識エリスロポイエチンで処理した。前吸着工程の後に血清中に残っている抗体はエリスロポイエチンによって結合し、再度免疫沈降する。この試験に用いたプロトコルは、参照によって本明細書中に援用される、Ｔａｃｅｙら、２００３年に記載されている。

Different concentrations of erythropoietin and EPO mimetic peptides were used in the analysis. After pretreatment of the serum with a test substance to adsorb the anti-EPO antibody present in the serum, the serum was treated with radioactively labeled erythropoietin. The antibody remaining in the serum after the pre-adsorption step is bound by erythropoietin and immunoprecipitated again. The protocol used for this study is described in Tacey et al., 2003, which is incorporated herein by reference.

  The results of pre-adsorption with anti-EPO antibody-containing serum performed using either EPO or the EPO mimetic peptides of the present invention are disclosed in FIG.

  When the serum was pretreated with the EPO mimetic peptide, the serum was subsequently positive when contacted with radioactively labeled erythropoietin. Therefore, anti-EPO antibody was detected in serum despite pretreatment. This means that the EPO mimetic peptide could not bind to the anti-EPO antibody during pretreatment. Without binding activity, the anti-EPO antibody is not excluded from the serum along with the EPO mimetic peptide and therefore remains in the serum. The anti-EPO antibody could not recognize the EPO mimetic peptide and therefore could not bind to the EPO mimetic peptide.

  Recombinant human EPO (rhuEPO) was used as a control. When the serum was pretreated with erythropoietin, the antibody was already bound and eliminated by pretreatment with erythropoietin, so in most cases, the detectable antibody in subsequent assays incorporating radiolabeled erythropoietin. There was no.

  The absolute values shown in FIG. 22 represent% cpm of the total count used in IP. Serum is rated positive if the% cpm value is greater than 0.9. 100% cpm represents the total count used (radioactive tracer), currently radioactively labeled EPO.

  The assay shows that the EPO mimetic peptides of the invention advantageously do not show cross-reactivity to anti-EPO antibodies. Thus, the EPO mimetic peptides described herein also show a therapeutic effect in patients who have developed antibodies against rhuEPO. Furthermore, antibodies against EPO mimetic peptides are not expected to bind to erythropoietin. Accordingly, the EPO mimetic peptides of the present invention are also preferably characterized in that they do not show significant cross-reactivity with anti-EPO antibodies.

  (References)

。

.

FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. FIG. 22.

Claims (55)

  A peptide of at least 10 amino acids in length that can bind to the EPO receptor and has agonist activity, wherein the EPO mimetic peptide is free of proline at a position called position 10 of the EPO mimetic peptide and is positively charged A peptide comprising an amino acid.   The EPO mimetic peptide has an amino acid motif (β-turn motif) characteristic of a folded structure, and the peptide does not contain a proline in the β-turn motif at position 10, and contains a positively charged amino acid, preferably K The peptide according to claim 1, wherein 9- and 10-positions are 5-aminolevulinic acid (5-Als)

The peptide according to claim 1, characterized in that it is occupied by   4. Peptide according to one of claims 1 to 3, characterized in that it has a positively charged amino acid at position 17, preferably K or Har. In particular, it can bind to the EPO receptor and has the following amino acid sequence:

Including
In the sequence, each amino acid is selected from natural or unnatural amino acids,
X ₆ is C, A, E, α-amino-γ-bromobutyric acid or homocysteine (hoc);
X ₇ is R, H, L, W or Y or S;
X ₈ is M, F, I, homoserine methyl ether or norisoleucine,
X ₉ is G or a conservative exchange of G,
X ₁₀ is a non-conservative exchange of proline,
Or X ₉ and X ₁₀ are replaced by a single amino acid,
X ₁₁ is selected from any amino acid;
X ₁₂ is T or A,
X ₁₃ is W, 1-nal, 2-nal, A or F;
X ₁₄ is D, E, I, L or V;
X ₁₅ is C, A, K, α-amino-γ-bromobutyric acid or homocysteine (hoc);
However, either X ₆ or X ₁₅ is C or hoc,
peptide. The following amino acid sequences:

Features
In the sequence, each amino acid is indicated by a standard letter abbreviation,
X ₆ is C,
X ₇ is R, H, L or W;
X ₈ is M, F or I,
X ₉ is G or a conservative exchange of G,
X ₁₀ is a non-conservative exchange of proline,
X ₁₁ is independently selected from any amino acid;
X ₁₂ is T,
X ₁₃ is W,
X ₁₄ is D, E, I, L or V;
X ₁₅ is C or
Or X ₉ and X ₁₀ are replaced by a single amino acid,
Or the following amino acid sequence:

Features
In the sequence, X ₆ is C,
X ₇ is R, H, L or W;
X ₈ is M, F, I or hsm (homoserine methyl ether);
X ₉ is G or a conservative exchange of G,
X ₁₀ is a non-conservative exchange of proline,
X ₁₁ is independently selected from any amino acid;
X ₁₂ is T,
X ₁₃ is W,
X ₁₄ is D, E, I, L or V, 1-nal (1-naphthylalanine) or 2-nal (2-naphthylalanine);
X ₁₅ is C,
The peptide according to claim 5. X ₁₀ is an amino acid having a positively charged side chain, preferably R, K or the respective unnatural amino acid, preferably Har, or X ₉ and X ₁₀ are a single amino acid, preferably 5-aminolevulinic acid 7. Peptide according to one of claims 5 or 6, characterized in that it is substituted by (Als) or aminovaleric acid. A peptide according to one of claims 1 to 7,
The following amino acid sequence:

Including
In the sequence, X _{6 to} X ₁₅ have the meanings described in claims 1 to 7,
X ₄ is Y,
X ₅ is independently selected from any amino acid, preferably A, H, K, L, M, S, T or I.
peptide. A peptide according to claim 8,
The following amino acid sequence

Including
In the sequence, X _{4 to} X ₁₅ have the meaning of claim 8,
X ₃ is independently selected from any amino acid, preferably D, E, L, N, S, T or V;
X ₁₆ is independently selected from any amino acid, preferably G, K, L, Q, R, S or T;
X ₁₇ is independently selected from any amino acid, preferably A, G, P, R, K, Y, or a non-natural amino acid having a positively charged side chain, preferably homoarginine;
X ₁₈ is independently selected from any amino acid,
peptide. X ₆ is C, E, A or hoc, preferably C, and / or X ₇ is R, S, H or Y, and / or X ₈ is F or M, and / or X ₉ Is G or A, preferably G and / or X ₁₀ is K or Har and / or X ₁₁ is V, L, I, M, E, A, T or norisoleucine, and / or Or X ₁₂ is T and / or X ₁₃ is W and / or X ₁₄ is D or V and / or X ₁₅ is C or hoc, preferably C and / or X The peptide according to one of claims 1 to 9, wherein ₁₇ is P, Y, A or K or Har.

An amino acid sequence selected from the group consisting of: 5-aminolevulinic acid (Als),

The peptide according to claim 1 or 5, wherein

Comprising an amino acid sequence selected from the group consisting of
The peptide according to at least one of claims 1 to 11, wherein in the sequence, X is 1-naphthylalanine and U is 2-naphthylalanine.   13. Peptide according to at least one of claims 1 to 12, characterized in that it is modified by conservative exchange of single amino acids, preferably no more than 1, 2 or 3 amino acids are exchanged.   14. Peptide according to one of the claims 1 to 13, characterized in that it does not cross-react with anti-EPO antibodies.   Said peptide is modified, said modification preferably being N-terminal and / or C-terminal acetylation (Ac) and / or amidation (Am), preferably intramolecular cyclization via intramolecular disulfide bridge, and / or phosphorus Particularly preferred is a modification of the C-terminal glycine as N-methylglycine (meG) and a modification of the N-terminal glycine as N-acetylglycine (AcG) selected from the group consisting of oxidation and / or the peptide is in the polymer moiety 15. A peptide according to at least one of claims 1 to 14, wherein said peptide is linked and said moiety is preferably selected from the group consisting of polyethylene glycol, dextran and starch.   16. A peptide according to at least one of claims 1-15, which forms either a monomer, dimer or multimer of the peptide sequence defined in claims 1-15.   The dimer or multimer is either a homomer or a heteromer having a branched or unbranched structure, and the monomeric peptide units are mutually N-terminal to N-terminal, C-terminal to C-terminal, or N-terminal The peptide according to claim 16, which is linked from to the C-terminus.   18. A peptide according to one of claims 15 or 17, comprising a linker and / or a spacer unit.   A synthetic peptide having a continuous peptide chain, comprising at least two domains capable of binding to a receptor, said domains comprising the amino acid sequence defined in claims 1-14 and 55.   20. A synthetic peptide according to claim 19, comprising at least two heterologous binding domains.   21. A synthetic peptide according to claim 19 or 20, comprising a linking moiety (linker) of natural or unnatural amino acid residues.   The synthetic peptide according to one of claims 19 to 21, wherein the linking moiety comprises 3 to 5 glycine and / or alanine residues or derivatives thereof.   23. A synthetic peptide according to one of claims 19 to 22, wherein the linker is provided by an amino acid forming part of a binding domain.

Or comprising a peptide sequence selected from one of the peptide sequences defined in claims 1 to 14 and having an additional amino acid, preferably an amino acid optionally having a reactive side chain such as an N-terminal cysteine , If present in each sequence, requires an intramolecular disulfide bridge between the first cysteine and the second cysteine and / or an intramolecular disulfide bridge between the third cysteine and the fourth cysteine 20. The synthetic peptide of claim 19, comprising according to At least the following:
a. First peptide b. A second peptide and c. Preferably, a linking moiety (linker) linking the first and second peptides
Including
At least one of said peptides comprises a peptide unit comprising an amino acid sequence as defined in one of claims 1-14, 24 or 55,
Peptide dimer or multimer.   The C-terminus of the first peptide is covalently bound to the N-terminus of the second peptide, or the C-terminus of the peptide is covalently bound to the C-terminus of the second peptide, or the first peptide 26. The peptide dimer or multimer according to claim 25, wherein the N-terminus of said peptide is covalently linked to the N-terminus of said second peptide.   27. Peptide dimer or multimer according to claim 25 or 26, wherein the linker comprises a sequence of natural and / or unnatural amino acids, preferably glycine, alanine, or derivatives thereof.   28. A peptide dimer or multimer according to claims 25 to 27, wherein the linker / spacer unit comprises a diketopiperazine unit.   27. A peptide dimer or multimer according to claim 26, wherein the linker is a divalent diacyl building block, preferably a diacyl building block derived from an aliphatic dicarboxylic acid.   26. The peptide dimer or multimer of claim 25, wherein the amino acid side chain of the first peptide is covalently bound to the amino acid side chain of the second peptide.   31. A peptide according to any of claims 1 to 30, further comprising a water soluble polymer covalently bound to the peptide, preferably a water soluble polymer selected from the group consisting of polyethylene glycol, dextran or starch.   32. Peptide according to claim 31, wherein the water-soluble moiety is PEG, preferably PEG having a molecular weight of at least 10 kD, most preferably between 20 and 60 KD. (I) at least two peptides, each peptide unit comprising at least two domains capable of binding to a target, wherein said binding domain comprises an amino acid sequence as defined in any one of claims 1-14 or 55 unit;
(Ii) comprising at least one polymer carrier unit;
A compound that binds to a target molecule, wherein the peptide unit is bound to the polymer carrier unit.   Said carrier unit is or comprises at least one natural or synthetic branched, dendritic or linear polymer, preferably selected from the group consisting of polyglycerol, polysialic acid, dextran, starch or polyethylene glycol 34. The compound of claim 33, wherein the compound is derived from or is derived from other biologically inert water soluble polymers.   35. A compound according to claim 33 or 34, wherein the carrier unit comprises a branching unit.   36. The compound of claim 35, wherein the branching unit comprises glycerol or polyglycerol.   37. At least according to claim 33-36, wherein the carrier molecule has a molecular weight of at least 5 kD, preferably 20-200 or 4000 kD, and has a molecular weight of 20-80 kD when a smaller carrier such as polyethylene glycol is used. A compound according to one paragraph.   38. A compound according to at least one of claims 33 to 37, wherein said carrier unit comprises at least two polymer subunits, said polymer subunits being linked to each other via at least one biodegradable covalent linker structure.   39. A compound according to at least one of claims 33 to 38, comprising a first biodegradable carrier unit, wherein a peptide unit and a second polymer carrier unit are attached to said first polymer carrier unit.   Approximately 20-50% of the binding sites of the first carrier unit, wherein the second carrier unit has a lower molecular weight than the first carrier unit, preferably a hydroxyalkyl starch such as HES, is preferred. 40. The compound of claim 39, wherein is occupied by the second carrier unit which is a polyethylene glycol having a molecular weight of about 3-10 kD.   41. A compound according to at least one of claims 33 to 40, wherein a modified polymeric carrier unit is used. The peptide unit is bonded to the polymer carrier unit via a covalent bond, and the bond occurs via a reactive amino acid, an N-terminal amino group and / or a C-terminal carboxylic acid of the peptide unit, and the reactive amino acid is Preferably selected from the group consisting of lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine and tyrosine,
If the polymer does not have a suitable reactive binding group, a binding substance is used to modify the polymer carrier unit,
Said binding substance is preferably selected from the group consisting of acylating groups that react with amino groups of peptide units, sulfhydryls (mercapto), thiomethyl, imidazo or peptide groups of alkylating groups that react with amino groups, most preferably maleimide groups Sulfhydryls of peptide units such as ester and amide forming groups, 5,5′-dithiobis (2-nitrobenzoic acid) groups and alkyl mercaptan groups, dicarbonyl groups such as cyclohexanedione groups, which react with carboxyl groups of peptide units Disulfide-forming groups that react with groups and other 1,2-diketone groups that react with the guanidine moiety of the peptide unit; diazo groups that react with the phenolic group of the peptide; polymers and cyanogen bromide that react with the amino group of the peptide unit Consisting of reactive groups derived from the reaction with Selected from the group,
42. The compound of claim 41.   43. The compound of claim 42, wherein the reactive amino acid is cysteine and the linking group is maleimide.   44. A compound according to at least one of claims 33 to 43, wherein the binding domains of the peptide units are linked internally via a linker structure.   45. The compound of claim 44, wherein the linker is a continuous peptide linker.   Use of a peptide and / or compound according to at least one of claims 1-45 or 55 for the preparation of a pharmaceutical composition.   For the prevention or treatment of disorders characterized by erythropoietin deficiency or a small or incomplete red blood cell population, or treatable by administration of erythropoietin, and especially for the treatment of any type of anemia or stroke Use of a peptide and / or compound according to at least one of claims 1-45 or 55 for the preparation of a pharmaceutical composition of   For the prevention or treatment of disorders characterized by erythropoietin deficiency or a small or incomplete red blood cell population, or treatable by administration of erythropoietin, and especially for the treatment of any type of anemia or stroke 56. Use of a peptide and / or compound according to at least one of claims 1-45 or 55.   56. A pharmaceutical composition comprising a compound according to at least one of claims 1-45 or 55 and optionally a pharmaceutically acceptable carrier.   For the prevention or treatment of disorders characterized by erythropoietin deficiency or a small or incomplete red blood cell population, or treatable by administration of erythropoietin, and especially for the treatment of any type of anemia or stroke 50. The pharmaceutical composition of claim 49. (I) generating at least two peptide units, each peptide unit comprising at least two domains capable of binding to a receptor;
(Ii) producing at least one polymer carrier unit;
46. A method of producing a compound according to at least one of claims 33 to 45, comprising (iii) attaching the peptide unit to the polymer carrier unit.   52. The method of claim 51, wherein the peptide units are synthesized as a continuous peptide chain.   Reacting with available chemical groups on the peptide unit and then reacting the reactive polymer carrier unit with the peptide unit together using the chemical group of the polymer carrier unit to form a covalently bonded complex thereof 53. A method according to claim 51 or 52, wherein a polymer support unit having at least one chemical group capable of   The nucleic acid which codes the peptide as described in any one of Claims 1-14 and 16.   A peptide characterized in that it is an inverso and / or retro / inverso peptide of the peptide according to at least one of claims 1 to 14, or a peptide consisting entirely of D-amino acids.

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