JP2002509079A - Modified PF4 fragment and pharmaceutical composition containing the same - Google Patents

Abstract

  (57) [Summary] The present invention relates to novel industrial products represented by the following (a) and (b). (a) is X₀₁ X₀₂Ser X₀₄ X₀₅ X₀₆ X₀₇ X₀₈ X₀₉ X_Ten X₁₁ X₁₂ X₁₃ X₁₄ X_Fifteen Gln X₁₇ X₁₈ Ser X₂₀Asp Leu Gln X_{twenty four} Y_{twenty five}Where X is₀₁, X₀₂, X₀₄, X₀₅, X₀₆, X₀₇, X₀₈, X₀₉, X_Ten, X₁₁, X₁₂, X₁₃, X₁₄, X_Fifteen, X₁₇, X₁₈, X₂₀, X_{twenty four}, Y_{Two Five}Are amino acids as defined in the specification, and (b) are non-toxic acid addition salts of those peptides. These new industrial products are useful for therapy.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD [0001] The present invention relates to a novel industrial product, which is a peptide represented by the following chemical formula 1. Further, the present invention relates to the use of the peptide for medical treatment and a pharmaceutical composition containing the peptide.

BACKGROUND OF THE INVENTION To the applicant's knowledge, the peptide represented by (Chemical Formula 1) according to the present invention is novel, and no similar peptide product is found in the prior art literature. That is, like the peptide represented by Chemical Formula 1, (a) it is active on hematopoietic cells (particularly in terms of suppressing proliferation and protecting cells), and (b) expanding hematopoietic cells. And / or no peptide products have been found to be effective in purifying the bone marrow.

DISCLOSURE OF THE INVENTION [0003] The first aspect of the present invention is a novel peptide compound, which comprises (a) a peptide represented by the following formula (1), and (b) a nontoxic acid addition salt of the peptide, A peptide compound selected from the group consisting of: ...... (Where, SEQ if Y ₂₅ is Pro ID No: 1 the references SEQ ID For Y ₂₅ is Pro-X ₂₆ No: See 2 Y ₂₅ is Pro-X ₂₆ - S for Tyr
See EQ. ID. No.3. )

[0004] The peptide represented by Chemical Formula 1 of the present invention can be used as it is or as a salt to which a mineral acid or an organic acid is added. Here, preferred mineral or organic acids include hydrochloric acid, hydrobromic acid, trifluoroacetic acid, methanesulfonic acid, and p-toluenesulfonic acid.

A second main point of the present invention is the use of the product of the present invention, wherein (a) a peptide represented by the formula (1), and (b) a non-toxic acid addition salt of the peptide, The use of a peptide compound selected from the group consisting of: (1) Hematopoietic suppressive drugs used in the treatment of myeloproliferative syndrome (2) Cell protective drugs used in the treatment of combating cytotoxic attacks (3) Treatment of bone marrow dysfunction or in the case of bone marrow transplantation A drug that stimulates hematopoietic cell expansion and / or bone marrow purification used in (4) has an inhibitory effect on hematopoietic cell differentiation and expands the least differentiated hematopoietic cells during the hematopoietic cell pre-viability expansion protocol (5) Anti-angiogenic drug used to treat abnormal angiogenesis (6) Drug to suppress viral growth used to treat viral diseases (7) To treat inflammation Anti-inflammatory drug used. Of the above, it is clear that the drugs (1) to (4) have an advantageous effect particularly on normal cells (healthy cells), but on abnormal cells, especially leukemic and cancerous cells. Has no advantageous effect.

[0006] Finally, a third aspect of the invention relates to a physiologically acceptable excipient,
A therapeutic composition comprising at least one peptide represented by the above (Chemical Formula 1) or one of nontoxic acid addition salts of the peptide.

About Abbreviations Many abbreviations are used herein for convenience. For conventional amino acid residues, (i) a one-letter code and (ii) a three-letter code recommended by IUPAC are used. Thus, B (Asx) on the one hand represents N (Asn) or D (Asp), while Z (Glx) on the other hand
Represents Q (Gln) or E (Glu). Other abbreviations and acronyms are as follows. AAS (aplastic anemia serum): dysplastic anemia serum BP (citrated bovine plasma): citrate-added bovine plasma BSA (bovine serum albumin): bovine serum albumin BFU-E (burst-forming unit-erythroblast) : Rupture-forming unit erythroid CFU (colony-forming unit): colony-forming unit CFU-GM (colony-forming unit-granulocyte / macrophage): colony-forming unit leukocyte / macrophage CFU-MK (colony- forming unit-megakaryocyte: colony-forming megakaryocyte CSF (colony-stimulating factor): colony stimulating factor FITC (fluorescein isothiocyanate): fluorescein isothiocyanate 5FU (5-fluorouracil, a reference cytotoxic product): fluorouracil: for control Cytotoxic product GM-CSF (granulocyte / macrophage colony-stimulating factor): leukocyte / macrophage colony stimulating factor HPLC (high performance liquid chromatography): high performance liquid chromatography Graphic IU (international unit): International unit 2ME (2-mercaptoethanol): 2-mercaptoethanol MEM (minimum essential medium): Minimum essential medium αMEM (modified MEM): Modified minimum essential medium MK (megakaryocyte): Unit megakaryocyte PBS (phosphate buffered saline): phosphate buffered saline TGF-β1 (transforming growth factor β1): transforming growth factor β1: control product containing apoptosis

BEST MODE FOR CARRYING OUT THE INVENTION The biological and pharmacological properties of the compounds according to the invention mainly depend on the fact that they themselves have one or more secondary structures (α-helix, β-sheet , Β-hairpins) and can adopt a particular conformation. Here, the concepts of α-helix, β-sheet, and β-hairpin are described in the literature and are well known to those skilled in the art. Particularly relevant literature is S. MARQUSEE et al., Proc. Natl. Acad. Sci. USA, 1989; 86 , pp. 52.
86-5290 and CR MATTHEWS rt al., Annu. Rev. Biochem., 1993; 62 , pp. 653-
683 can be referenced.

[0009] For peptide represented by (Formula 1), beginning with X _04, segments of peptide ending in X ₁₅ (S) has the property of forming a stable α- helix. The helicity of the segment S and the helicity of the peptide represented by Chemical Formula 1 are important, if not essential, for the expression of the biological and pharmacological properties of the product of the present invention. It is something. Hereinafter, this point will be described.

(1) According to the experiments of the applicant, in order to maintain and maintain the α-helicity of the segment, the residue X ₁ -X ₀₂ -Ser _{03 at} the N-terminal of X ₀₄ has , A few, are required. Here, partially upstream of said sequence of X _04, or if there is a total deletion, is as following (i) ~ (iii) as a result. (i) The α-helicity of the peptide represented by (Chemical Formula 1) decreases. This is based on calculations using a data bank (V. MUNOZ et al., Nature: Struct. Biol., 1994; 1 , pp. 399-409) and measurements of photodichroism and Fourier transform infrared absorption spectra. (ii) The ratio R (α-helix / β-structure) is reversed. This is based on optical dichroism and Fourier transform infrared absorption spectrum measurements. (iii) Due to the deletion, the biological properties of the peptide are substantially reduced as compared to the peptide represented by the formula (1) of the present invention.

(2) According to the knowledge obtained by the applicant, the residues at the fourth and sixth positions of the peptide represented by the formula (1), that is, the selection of _X04 and _X06 are determined by α- It is important in relation to helicity and the ratio R. Thus, in order to obtain the maximum α-helicity value and increase the ratio R, according to the present invention, (i) using Pro as residue X04, preferably using Ala or Leu, and (ii) ) Ala, Leu or Ile is preferably used as residue X06. here,
For example, setting residue X06 to Pro would, on the one hand, significantly reduce the percentage of α-helices in the segment S and the peptide containing the segment S, and on the other hand, The pharmacological activity would be reduced by a factor of 500 to 1000.

(3) Among the segments S, the peptide of the present invention has the maximum α-helicity at amino acid residues 7 to 11 (mainly at residue 9) of the sequence of (Chemical Formula 1). For these peptides, the maximum value of α-helicity is 0.12 units or more. Except for the segment S, the sequence of amino acid residues from about position 16 to about position 25 affects the activity of the peptide represented by the formula (1) of the present invention. Beyond position 25, the insertion of a few additional amino acid residues (especially 1 to 5) has no substantial effect on the activity. Therefore, (i) the C-terminus of the peptide represented by (Chemical Formula 1) can be stopped at Y ₂₅ = Pro (pentacosa peptide compound), and (i)
i) The sequence can be continued up to position 27 for final introduction of Tyr residues to make the peptide suitable as a biological reagent for diagnosis.

From an analytical point of view (particularly from the viewpoint of diagnostics and pharmacological experimental fields), it is necessary to have at least one Tyr residue in the amino acid sequence of the peptide represented by Chemical Formula 1. Recommend. In particular, Tyr residues that can be more easily detected or labeled (by radioisotope assays) than other naturally occurring amino acid residues are positions 2, 5, and 2 in the peptide represented by It can be located in any one of 7 to 15, 17, 24 or 27. Experiments have shown that Tyr is the last amino acid residue at the C-terminus, which is more practical from an analytical point of view. In other words, the Tyr residue, if present, is preferably located at position 27 in (Formula 1).

The peptide represented by the formula (1) of the present invention has the following seven essential activities (1)
-7), and these activities can be classified into the following three functional categories (AC). That is, the peptide represented by Chemical Formula 1 of the present invention is useful as the following.

Category A (hematopoietic function) (1) Hematopoietic inhibitor, more precisely an inhibitor of hematopoietic proliferation (has a valuable effect in combating myeloproliferative syndrome) (2) cytoprotective agent, In particular, an anti-apoptotic cytoprotective agent for hematopoietic stem cells (3) A product having an inhibitory effect on hematopoietic cell differentiation (4) An agent for hematopoietic expansion and / or bone marrow purification Category B (angiogenic function (5) Anti-angiogenic drugs Category C (anti-inflammatory function) (6) Drugs that suppress viral proliferation, especially those that fight against viruses and retroviruses with lipid capsid (7) Anti-inflammatory drugs

The myeloproliferative syndromes mentioned above include, inter alia, intrinsic thrombocytosis, VAQUEZ erythrocytosis, myelofibrosis, chronic myeloid leukemia. The above-mentioned agents of hematopoietic expansion are to be understood here as meaning products which act on hematopoietic cells to promote and stimulate the growth of stem cells, for example precursors of leukocytes, platelets, erythrocytes. Bone marrow purification often occurs after bone marrow enlargement. Bone marrow purification is required during bone marrow transplantation. Cell differentiation results in a delay in the loss of the very early differentiation marker (CD34) in more immature cells. This suggests that cells treated with the peptides of the invention remain undifferentiated or more immature.

Furthermore, it has been found that the peptide represented by Chemical Formula 1 has the advantage of extending the S phase (occurring during mitosis) to the cell cycle. It has also been found that anticoagulants do not inhibit the activity of the peptides of the invention.

The peptide represented by Chemical Formula 1 can be produced by a known method including conventional chemical and biological reaction mechanisms, that is, peptide synthesis or application of genetic engineering. Essentially, each compound represented by the formula (1) of the present invention has its amino acid residue 7-1
At one, (i) has a maximum α-helicity of at least 0.12, and (ii) has a ratio R of at least 2.

A number of examples of the invention and pharmacological test results are shown below. These detailed data are for explaining the present invention and do not limit the present invention. Examples 1 to 26 Table 1 shows typical examples of the present invention. These samples were produced by peptide synthesis by conventional solid phase technology and purified by HPLC column.
Further, Comparative Examples CP1 to CP3 are included in Table 1.

Test 1. Inhibition of hematopoiesis In order to evaluate the ability of the product of the present invention to inhibit hematopoietic proliferation, hematopoietic stem cells (CFU-
Hematopoietic suppression for MK, CFU-GM and BFU-E) was examined. 1.1 CFU-MK Plasma coagulation system examined MK and its progenitor cells. Rat (Balb / C
Male rats (6-8 weeks old) were sacrificed by cervical dislocation, and bone marrow was collected from the femur using 5 ml of αMEM. In a Petri dish (35 mm diameter), BSA (1%), BP (10%), 2
In 1 ml of a mixture of ME (10 ⁻⁴ M), CaCl ₂ (0.34 mg), penicillin (15 IU), streptomycin (15 μg) and AAS (10%), 2 × 10 ⁵ nucleated Bone marrow cells were cultured (at least 3 sets). Here, the AAS was collected from pig blood collected 5 days after whole body irradiation with 8 Gy (gray) of radiation. The peptide to be tested was exogenously added immediately before the plasma coagulation test. After coagulation, heparin (5 IU / dish) was added. Cultures were cultured at 37 ° C. in a humid atmosphere containing CO ₂ (5%). After culturing for 7 days, the dishes were fixed with 1% paraformaldehyde and stained with acetylcholinesterase to determine the number of CFU-MK-derived colonies. Where CF
U-MK colonies are defined as clusters of three or more cells. On the one hand, the number of MKs and their colonies and, on the other hand, the area of each MK were counted with an automatic analyzer.

Table 2 shows the results obtained for all murine bone marrow (suppression of megakaryocyte formation).
Shown in In the table, the content of CFU-MK is expressed as a percentage of the control batch as a function of the dosage. (Control animals did not receive any of the peptides under test.) From Table 2, it can be seen that the products of the Examples of the present invention were compared with the products of the Comparative Examples in terms of CFU-
It turns out that proliferation of MK is suppressed more effectively.

1.2 CFU-GM and BFU-E nucleated bone marrow cells (10 ⁵ cells / ml) were converted to methylcellulose (0.8%), AAS (10%), 2M
E (10 ^-4 M), recombinant murine SCF [ie rmSCF] (10 ng / ml) and rmGM-CSF (10 ng / ml)
ml) in a semi-solid medium. Three cultures were used for each medicinal product under test. Cultures were cultured at 37 ° C. for 5 days in a humid atmosphere containing CO ₂ (5%). Then, colonies of BFU-E (3 or more clusters composed of 20 cells) and CFU-GM (50 or more cells) were counted. Table 3 shows the obtained results (suppression of leukocytes and macrophages). In the table, CFU-GM
And the content of BFU-E is expressed as a percentage of the control batch as a function of the dosage.

1.3 Tests on murine CD34 ⁺ cells [0023] Murine bone marrow cells were placed on ice together with an anti-CD34 ⁺ murine monoclonal antibody conjugated to biotin ["RAM34" antibody marketed by PHARMINGEN]. Cultured for 0.5 hours. Cells were washed twice with PBS-BSA and stained with streptavidin conjugated to FITC (ie, streptavidin-FITC marketed by CALTAG). Cells used as controls were treated with IgG2a murine immunoglobulin [PHARMI
"R35-95" clone from NGEN] and streptavidin. Purified and viable murine CD34 ⁺ cells were collected. Replace these cells with AAS (10%)
Were placed in a tube containing, then counted and analyzed to check purity. Then, to measure CFU-MK as described above, 24,000 CD34 ⁺ cells per ml were cultured on a plasma clot system and in parallel with CF as described above.
To measure U-GM and BFU-E, 16,000 CD34 ⁺ cells per ml were cultured on a culture medium containing methylcellulose. Table 4 shows the obtained results. In the table, the contents of CFU-MK, CFU-GM and BFU-E are expressed as a percentage of the control batch as a function of the dose of the peptide under test.

2. Protection of cell apoptosis Human peripheral blood CD34 ⁺ cells were examined for anti-apoptotic cell protection of hematopoietic stem cells. Observed in the presence and absence of the product under test and the results, expressed as a percentage of apoptotic cells, show that the products of the examples of the present invention have better cells than the products of the comparative examples. It was found to show protection.

List of Amino Acid Sequences (Appendix) The list of amino acid sequences (Table 5) is as follows. (i) As described above, the product represented by Chemical Formula 1 is shown in SEQ. ID. No. 1 to No. 3. (ii) The products of Examples 1-26 are shown in SEQ. ID. Nos. 4-29. (iii) The products of Comparative Examples CP1 to CP3 are shown in SEQ. ID. No. 30 to No. 32.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Sequence list]

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] May 19, 2000 (2000.5.19)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

## STR1 ## _{X 01 X 02 Ser X 04 X} 05 X 06 X 07 X 08 X 09 X 10 X 11 X 12 X 13 X 14 X 15 Gln X 17 X 1 8 Ser X 20 Asp Leu Gln X 24 Y 25 wherein Wherein X ₀₁ is Pro, Gly or Ala, and preferred amino acid residues are Pro and Ala. X ₀₂ is an amino acid residue except Cys, preferably a His or GIx, more preferably residues His. _X04 is Pro, Gly, Thr, Ala, Ile or Leu, and preferred amino acid residues are Pro, Al
a and Leu. X ₀₅ is an amino acid residue except Cys, preferably Thr, Glx, Asx or Ala,
A more preferred residue is Thr. _X06 is Ala, Val, Gln, Leu, Ile or Thr, and a preferred amino acid residue is Ala. X ₀₇ is an amino acid residue except Cys, preferably Ala or GIx. _X08 is an amino acid residue other than Cys, and is preferably Ile, Leu or Val. _X09 is an amino acid residue other than Cys, preferably Ile, Leu or Val, and a more preferred residue is Ile. X ₁₀ is an amino acid residue except Cys, preferably Ala or Val. X ₁₁ is an amino acid residue except Cys, preferably Lys, Ser or Thr, more preferably residues are Thr. X ₁₂ is an amino acid residue except Cys, preferably Leu or Ile, more preferably residues are Leu. X ₁₃ is an amino acid residue other than Cys, preferably Lys or Ser. X ₁₄ is an amino acid residue other than Cys, preferably Asx or Glx, and a more preferred residue is Asn. X ₁₅ is an amino acid residue other than Cys, and is preferably Gly. X ₁₇ is an amino acid residue other than Cys, preferably Lys or Gln. X ₁₈ is Leu, Ile, Val, Ala or Tyr, and a preferred residue is Ile. X ₂₀ is Leu, Ile or Val, and a preferred residue is Leu. X ₂₄ is an amino acid residue other than Cys, preferably Ala, Asx, Glx or Ser,
More preferred residues are Ala and Glu. Y ₂₅ is the residue Pro, Pro-X ₂₆ or Pro-X ₂₆ -Tyr, the preferred residue being the mono-amino acid residue Pro. X ₂₆ is an amino acid residue other than Cys, preferably Arg, Ile, Leu, Val, Met or T
rp and a more preferred residue is Leu.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 35/00 A61P 39/00 39/00 43/00 105 43/00 105 107 107 107 A61K 37/02 (72 Inventor Cohen, Paul France 75003 Paris, Lyu Barbet 16 (72) Inventor Han, Zhong Kao China Tianjin 300020 Nanjing Road 288, Hospital of Blood Dizzies, Institute of Dementology Notice (72) Inventor Loram , Mohammed FR-60600 Clermont-de-Royce, Rue Emile Bousseau 2F term (reference) 4C084 AA02 AA07 BA01 BA08 BA19 BA23 MA01 NA14 ZA512 ZB112 ZB212 ZB262 ZB332 4H045 AA10 AA30 BA05 BA18 EA22 EA24 EA29 FA33

Claims (13)

[Claims] (1) a peptide represented by (a), and (b) non-toxicity of the peptide.
Acid addition salts, peptide compounds selected from the group consisting of: Embedded image X₀₁ X₀₂ Ser X₀₄ X₀₅ X₀₆ X₀₇ X₀₈ X₀₉ X_Ten X₁₁ X₁₂ X₁₃ X₁₄ X_Fifteen Gln X₁₇ X_{1 8} Ser X₂₀ Asp Leu Gln X_{twenty four} Y_{twenty five} Where X₀₁Is Pro, Gly or Ala, and preferred amino acid residues are Pro and Ala. X₀₂Is an amino acid residue other than Cys, preferably His or Glx, more preferably
The remaining residue is His. X₀₄Is Pro, Gly, Thr, Ala, Ile or Leu; preferred amino acid residues are Pro, Al
a and Leu. X₀₅Is an amino acid residue other than Cys, preferably Thr, Glx, Asx or Ala,
A more preferred residue is Thr. X₀₆Is Ala, Val, Gln, Leu, Ile or Thr, and a preferred amino acid residue is Ala.
You. X₀₇Is an amino acid residue other than Cys, preferably Ala or Glx. X₀₈Is an amino acid residue other than Cys, and is preferably Ile, Leu or Val. X₀₉Is an amino acid residue other than Cys, preferably Ile, Leu or Val, and more preferably the residue is Ile. X_TenIs an amino acid residue other than Cys, and is preferably Ala or Val. X₁₁Is an amino acid residue other than Cys, preferably Lys, Ser or Thr, and more preferably the residue is Thr. X₁₂Is an amino acid residue other than Cys, preferably Leu or Ile, more preferably
The remaining residue is Leu. X₁₃Is an amino acid residue other than Cys, preferably Lys or Ser. X₁₄Is an amino acid residue other than Cys, preferably Asx or Glx, more preferably
The remaining residue is Asn. X_FifteenIs an amino acid residue other than Cys, and is preferably Gly. X₁₇Is an amino acid residue other than Cys, preferably Lys or Gln. X₁₈Is Leu, Ile, Val, Ala or Tyr, and a preferred residue is Ile. X₂₀Is Leu, Ile or Val, and a preferred residue is Leu. X_{twenty four}Is an amino acid residue other than Cys, preferably Ala, Asx, Glx or Ser,
More preferred residues are Ala and Glu. Y_{twenty five}Is the residue Pro, Pro-X₂₆Or Pro-X₂₆-Tyr, the preferred residue is the monoamino acid residue
Group Pro. X₂₆Is an amino acid residue other than Cys, preferably Arg, Ile, Leu, Val, Met or T
rp and a more preferred residue is Leu. 2. Beginning in X _04, peptide compound according to claim 1, wherein a segment is stable α- helix that ends in substantially X _15. 3. The method according to claim 1, wherein the physiologically acceptable excipient is
A therapeutic composition comprising at least one peptide represented by Chemical formula 1) or one of non-toxic acid addition salts of the peptide. 4. The peptide according to claim 1 or 2, or one of the non-toxic acid addition salts of the peptide, for producing a medicament for suppressing hematopoietic proliferation, which is used for the treatment of myeloproliferative syndrome. Use of a peptide, characterized by using 5. The use of claim 1 or 2 for the manufacture of a cytoprotective agent for the treatment of combating cytotoxic attacks, in particular for the anti-apoptotic protection of hematopoietic cells.
Use of a peptide according to any of the preceding claims, or one of the non-toxic acid addition salts of said peptide. 6. The peptide according to claim 1 or 2, for producing a medicament for expanding and / or purifying bone marrow for use in the treatment of bone marrow dysfunction or in the case of bone marrow transplantation. Use of a peptide, characterized by using one of the non-toxic acid addition salts of the above. 7. The peptide according to claim 1 or 2, or one of the non-toxic acid addition salts of the peptide, for producing a drug for inhibiting the growth of a virus, which is used for treatment against a viral disease. Use of a peptide, characterized by using 8. Use of the peptide according to claim 1 or 2 or one of the non-toxic acid addition salts of the peptide for the manufacture of an anti-inflammatory drug for use in the treatment of combating inflammation. Usage of peptide to be. 9. Use of a peptide according to claim 1 or 2 or one of the non-toxic acid addition salts of said peptide for the manufacture of an anti-angiogenic agent for the treatment of combating abnormal angiogenesis. A method for using a peptide, comprising: 10. In order to produce a drug that differentiates healthy cells from abnormal cells,
Use of the peptide according to claim 1 or 2, or a non-toxic acid addition salt of the peptide. 11. To produce a drug that extends the S phase of the cell cycle,
Use of the peptide according to claim 1 or 2, or a non-toxic acid addition salt of the peptide. 12. The peptide compound according to claim 1, wherein the maximum value of α-helicity is 0.12 units or more for residues 7 to 11. 13. The peptide compound according to claim 1, wherein R represented by R = (α-helix) / (β-structure) is 2 or more.