JP5549912B2 - Drugs for polycythemia vera or essential thrombocythemia - Google Patents

Description

The present invention relates to a growth inhibitor of fibrillarin-carrying cells.

In the research on Antheraea yamamai of the Saturniidae family, the present inventors have an amino acid sequence, aspartic acid-isoleucine-leucine-arginine-glycine (DILRG), and the C-terminus is amidated. In addition, a novel peptide (DILRG-NH ₂ ) having a molecular weight of 570.959 has been found, and a patent has been obtained by clarifying the dormancy control action and cancer cell growth inhibition action of this peptide (patent document) 1, 2). This peptide (DILRG-NH ₂ ) is also named “Yamamarin” by the present inventors.

  In addition, in order to improve the cell permeability of the peptide, the present inventors proceeded with research on peptide derivatives and marked liver cancer caused by a conjugate with palmitic acid (named “C16-Yamamarin”). Cell growth inhibition activity has been reported (Non-patent Document 1).

  However, although it has been clarified in previous studies that yamamarine and C16-yamamarin exhibit the above-mentioned activity against cancer cells, no specific investigation has been made on the activity against cells other than cancer cells. . At present, the mechanism of action on cancer cells by Yamamarine and C16-Yamamarin has not been clarified, and the cell growth inhibitory activity by Yamamarine and C16-Yamamarin is a unique effect on cancer cells. It was not known at all whether it would exert any effect on other cells.

Patent No. 3023790 Patent No. 3571971 JP 2009-72186 A

Yang et al. (2007): A palmitonyl pentapeptide of insect origin induces growth arrest in mammalian cells and insect embryos. J. Insect Biotechnol. Sericology 76, 63-69. T. Schimmang et al., A yeast nucleolar protein related to mammalian is associated with amall nucleolar RNA and is essential for viability, EMBO J. 8 (1989) 4015-4024 K. Newton et al., Fibrillarin is essential for early development and required for accumulation of an intron-encoded small nucleolar RNA in the mouse, Mol.Cell Biol. 23 (2003) 8519-8527. M. Abdullahel Amin, et al., Fibrillarin, a nucleolar protein, is required for normal nuclear morphology and cellular growth in HeLa cells, BBRC 360 (2007) 320-326. Jansen RP (1991) J. Cell Biol. 113, 715-729 Reichow SL (2007) Nucleic Acids Res 35, 1452-1464 Tollervey D (1997) Curr Opin Cell Biol. 9, 337-342 Yang et al., Growth Suppression of Rat Hepatoma Cells by a Pentapeptide from Antheraea yamamai, J. Insect Bio. Seri, 2004, 73, 7-13

The present invention, as described above has been made in view of the circumstances, and an object of the invention to provide a Yamamarin (DILRG-NH ₂₎ and Yamamarin novel uses of derivatives.

  This time, the present inventors proceeded with research on substances that bind to yamamarin and C16-yamamarin in cells, and obtained the new knowledge that yamamarin and yamamarin derivatives bind to fibrillarin (fibrillarin), thereby completing the present invention. I came to let you.

  Fibrillarin is a component of snoRNP (nuclear small ribonucleic acid protein) that is localized in the nucleolus in the cell nucleus, and its role is involved in rRNA methylation. It is known to perform methylation of precursors.

  Furthermore, in yeast, Fibrillarin is essential for cell survival (Non-Patent Document 2), mouse Fibrillarin-deficient mutants are embryonic lethal at an early stage of development (Non-Patent Document 3), and in Hela cells Has also been reported to exhibit a cell growth inhibitory effect by knocking down Fibrillarin by RNAi (Non-patent Document 4).

  It has also been demonstrated that the deficiency of the Fibrillarin gene in yeast can be functionally complemented by introducing the human Fibrillarin gene (Non-patent Document 5). Furthermore, the amino acid sequences of many vertebrate fibrillarins are known, and their functions are known to be involved in ribosome biosynthesis, biosynthesis of small nucleolar ribonucleic acid proteins and mRNA processing ( Non-Patent Documents 6 and 7).

  As shown in FIG. 1, it can be seen that Fibrillarin is widely conserved among eukaryotes in the evolutionary phylogeny. Fibrillarin derived from human is a protein composed of 321 amino acids, and has a homology of about 90% or more in amino acid sequence in mammals such as monkeys, cows, dogs, rats, and mice. Even when Fibrillarin derived from non-mammals such as fish, Drosophila, nematode, Arabidopsis thaliana, and yeast is included, it is confirmed that there is a high homology of about 75% or more in amino acid sequence (Patent Document 3). ).

Under such circumstances, the present invention provides the following cell growth inhibitor, therapeutic agent and cell growth suppression method in order to solve the above problems.
<1> A therapeutic drug for polycythemia vera caused by proliferation of erythroblast progenitor cells or essential thrombocythemia caused by proliferation of megakaryocyte progenitor cells, which contains DILRG-NH ₂ .
<2> DILRG-NH ₂ is the first therapeutic agent having an acyl group at the N-terminus.
<3> The second therapeutic agent, wherein the acyl group has 6 to 28 carbon atoms.

  According to the present invention, it is possible to suppress the growth of fibrillarin-carrying cells, and as a therapeutic agent for diseases caused by the proliferation of cells having fibrillarin, the present inventors have reported a cancer cell proliferation inhibitor that has been conventionally reported. In addition, for example, there are therapeutic agents for chronic myeloproliferative diseases, specifically polycythemia vera caused by proliferation of erythroid progenitor cells, essential thrombocythemia caused by proliferation of megakaryocyte progenitor cells, etc. Provided.

  In addition, in the medium containing Fibrillarin-bearing cells, it is possible to increase the efficiency of substance production of cultured cells. Furthermore, by suppressing the growth of cultured cells, the cells in the medium can be adjusted to a desired number.

It is a figure which shows the evolution systematic classification of Fibrillarin. A) Western blotting using C16-yamamarin binding protein confirmed by ABB after electrophoresis and CBB staining, and B) Fibrillarin antibody. It is a figure which shows the proliferation inhibitory effect of the mouse | mouth bone marrow cell by C16-yamamarin. It is a figure which shows the growth inhibitory effect of BaF3-Bcr / Abl cell by C16-yamamarin. It is a figure which shows the proliferation cell ratio in each cell cycle of the Drosophila embryo origin cell (S2 Cell) which added C16-yamamarin. FIG. 5 (A) shows the cell growth inhibitory effect after 96 hours when C16-yamamarin is added. FIG. 5 (B) shows the ratio of cells in the cell cycle of G0 / G1 phase in the S2 cells of FIG. 5 (A). FIG. 5 (C) shows the proportion of cells in the S-phase cell cycle in the S2 cells of FIG. 5 (A). FIG. 5 (D) shows the rate of proliferation of cells in the G2 / M phase cell cycle in the S2 cells of FIG. 5 (A). In addition, the sum total of the ratio of figure (B), (C), (D) becomes 100%.

The fibrillarin-carrying cell growth inhibitor of the present invention is a peptide called Yamamarin, which has been found by the inventors' research, that is, “having aspartic acid-isoleucine-leucine-arginine-glycine and having a C-terminus. “Amidated peptide” (hereinafter referred to as “DILRG-NH ₂ ”). The chemical formula of this peptide is shown below.

DILRG-NH _2, for example, larvae from the isolation of wild silkworm (Antheraea yamamai), can either be used after purification, it is also possible to use those produced by known peptide synthesis methods. Although it can be obtained by other methods, taking into consideration economic efficiency, mass productivity, etc., acquisition by a peptide synthesis method is preferable.
Even more preferably, the cell growth inhibitor of the present invention may contain an N-terminal acylated DILRG-NH _2. N-terminal acylated DILRG-NH _2, the cell permeable compared to DILRG-NH ₂ is high, to exert a more pronounced cytostatic effect. The chemical formula of N-terminal acylated DILRG-NH ₂ is shown below.

In the compound, R in the formula represents an acyl group. N-terminal acylated DILRG-NH ₂ can be synthesized by introducing an acyl group into the N-terminus of DILRG-NH ₂ . The introduction of the acyl group can be carried out by a known method, and the carbon number of the acyl group in N-terminal acylated DILRG-NH ₂ is preferably 6 to 28 from the viewpoint of cell permeability. In particular, a palmitoyl group having 16 carbon atoms is preferable. N-terminal acylated DILRG-NH ₂ can also be used as a mixture with the above-mentioned DILRG-NH ₂ .

And since most cells in eukaryotes have a nucleus, and Fibrillarin exists in the nucleolus in the nucleus as described above, the cytostatic agent of the present invention is effective against many eukaryotic cells. And exerts an inhibitory effect on proliferation. Further, N-terminal acylated DILRG-NH ₂ is excellent in cell permeability, it reaches into the nucleus, further exert remarkable cytostatic effect. In addition, for example, when the Fibrillarin gene is introduced into cells using an expression vector, it is also considered to suppress the proliferation of cells with an increased amount of Fibrillarin. Therefore, the “Fibrillarin-carrying cells” in the present invention are not necessarily limited to eukaryotic cells.

The cell growth inhibitor of the present invention can be blended with other substances depending on the application as long as it does not interfere with the action of DILRG-NH ₂ and / or N-terminal acylated DILRG-NH _2. .

  In addition, as described in Patent Document 2, the cell growth inhibitory activity in the present invention is to suppress cell proliferation by shortening the S phase corresponding to the DNA replication phase and extending the quiescent phase. . That is, the present invention does not suppress cell growth by causing apoptosis, but suppresses cell growth by controlling the cell cycle.

Thus, since DILRG-NH ₂ and / or N-terminal acylated DILRG-NH ₂ suppresses the growth of fibrillarin-bearing cells, the present invention suppresses the growth of cancer cells that the inventors have created. In addition to agents, for example, treatment of chronic myeloproliferative diseases, specifically polycythemia caused by proliferation of erythroid progenitor cells, essential thrombocythemia caused by proliferation of megakaryocyte progenitor cells, etc. Also provide medicine. The present invention is effective as a therapeutic agent as long as it is a disease caused by the proliferation of fibrillarin-carrying cells, and is not limited to the above-mentioned diseases.

In this case, the therapeutic agent is prepared by mixing with any pharmaceutically acceptable carrier, excipient or stabilizer in addition to the above-mentioned DILRG-NH ₂ and / or N-terminal acylated DILRG-NH _2. Saved. Acceptable carriers, excipients, or stabilizers can be appropriately selected depending on the dosage form and administration route, provided that they are non-toxic to patients at the dosages and concentrations used. For example, buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride Phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; serum albumin , Gelatin, or protein such as immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; amino acid such as glycine, glutamine, asparagine, histidine, arginine, or lysine; Monosaccharides, disaccharides, including lucose, mannose, or dextrin, and other carbohydrates such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg, Zn − Protein complexes) or TWEEN (trade name), PLURONICS (trade name), and nonionic surfactants such as polyethylene glycol (PEG).

  In addition, the drug administered into the body must be sterile. This is easily accomplished by filtration through sterile filtration membranes. In addition, sustained-release preparations may be prepared. A suitable example of a sustained release formulation is a semi-permeable matrix (eg, in the form of a film or microcapsule) of a solid hydrophobic polymer. Examples of sustained release matrices are polyester hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polyactides (US Pat. No. 3,773,919), L-glutamic acid and γ-ethyl-L-glutamate. , Non-degradable ethylene-vinyl acetate, LUPRON DEPOT (trade name) (injectable globules of lactic acid-glycolic acid copolymer and leuprolide acetate), poly- (D) -3-hydroxy Butyric acid and the like.

  The drug thus formulated can be systemically administered, for example, orally, locally, or intravenously, depending on the type and symptoms of the disease. The dose can be determined according to the patient's body weight, symptoms, etc., and can be about 0.1 to 50 mg / Kg body weight per dose, for example.

In addition, the cell growth inhibitor of the present invention can be used to suppress the growth of fibrillarin-bearing cells, mainly eukaryotic cells in culture. That is, by adding the above-mentioned DILRG-NH ₂ and / or N-terminal acylated DILRG-NH ₂ to a medium containing Fibrillarin-carrying cells at a concentration depending on the cells, the growth of Fibrillarin-carrying cells in the medium is suppressed. be able to.

In general, in the production of useful substances using cultured cells, the cell cycle of each cell does not match, and thus the problem that production of useful substances is inefficient is pointed out. However, the medium contains DILRG-NH _2. By adding N-terminal acylated DILRG-NH ₂ , the growth of fibrillarin-carrying cells can be suppressed and the cell cycle can be synchronized, so that the material production of cultured cells can be made more efficient. .

Further, in the production of cultured cells, the above-mentioned DILRG-NH ₂ and / or N-terminal acylated DILRG-NH ₂ can be used to control the number of cells to a predetermined number by suppressing cell growth.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples at all.

<Example 1> Drosophila embryo-derived cell (S2 Cell) culture and protein extraction Drosophila embryo-derived cell S2 Cell is cultured in 1,000 mL of serum-free medium ExpressFive (R) SFM (manufactured by Invitrogen) with 200 mM L-glutamic acid. 90 mL of the solution and 1 mL of a penicillin streptomycin mixed solution (ICN Biomedicals: 16-700-49) were added. After culturing for 3-4 days and the cells have reached confluence, transfer the cells together with the medium to a centrifuge tube, and centrifuge the cells by centrifugation at 1,000 ° C for 3 minutes at 4 ° C using a centrifuge (H-9R, Kokusan Co., Ltd.). The cells were collected and washed 3 times with ice-cold PBS. Next, add 5 times the amount of HEPES buffer {50 mM HEPES-KOH (pH 7.8), 420 mM KCl, 0.1 mM EDTA, 5 mM MgCl ₂ , 20% glycerol}, a zwitterion buffer, to the collected cells. After freezing and thawing three times, the cells were disrupted and the protein was extracted, followed by centrifugation at 4 ° C., 16,000 × g for 10 minutes, and the supernatant was recovered to obtain a soluble protein.

<Example 2> N synthetic peptide synthesizer terminal acylation _{DILRG-NH 2 (PSSM-8} , ( Ltd.) manufactured by Shimadzu Corporation) using a peptide by conventional methods, aspartic acid - isoleucine - leucine - arginine - glycine was synthesized _{-NH 2 (DILRG-NH 2)} . Subsequently, after palmitoylation on the resin, it was subjected to a cleaving reaction to obtain C16-DILRG-NH ₂ having the following chemical structure.

  The purification was carried out by connecting a reverse phase column Develosil-ODS HG-5 (20 mm × 250 mm, manufactured by Nomura Chemical Co., Ltd.) to an HPLC system (Gulliver Corporation JASCO). Elution is carried out at a flow rate of 4 ml / min using an acetonitrile concentration gradient (0 to 100% from 0 to 120 minutes) in the presence of 0.1% trifluoroacetic acid (TFA) to elute the active fraction. It was. Absorbance was measured at 220 nm. The peptide was mixed with an equal amount of matrix (saturated with 40% acetonitrile / 0.1% TFAα-CHCA) on the sample plate, dried, and structured by MALDI-TOF MS (Discovery, manufactured by Shimadzu Corporation). confirmed.

As a method other than the above, after obtaining free DILRG-NH ₂ , palmitoyl group having 16 carbon atoms is bonded to the N-terminus thereof to introduce a palmitoyl group (acylation), and C16- DILRG-NH ₂ can also be synthesized.

Hereinafter, C16-DILRG-NH ₂ (C16-yamamarin) is used as an example, but the present invention is not limited to this example.

<Example 3> Preparation of affinity column As a preliminary step of affinity column preparation, CNBr activated sepharose 4B powder (manufactured by GE Healthcare) was suspended in 1 mM HCl, swollen at room temperature for 15 minutes, and glass filter (pore size 30 μm). ) Was added to the attached column. The carrier after addition to the column was washed by adding 3 bed amount of 1 mM HCl, sealing the column closed and suspending, and this operation was repeated twice. Next, in order to couple the ligand of the binding protein in S2 Cell to the carrier, a 3-bed amount of 25% ethanol was allowed to flow through the column, and then a 3-bed amount of 50% ethanol was allowed to flow to equilibrate the column.

The ligand used was Yamamarin (DILRG-NH ₂ ) obtained in Example 2, dissolved in 50% ethanol so as to be 2 μmol / 1 mL-Gel, mixed with the gel, and slowly stirred with a rotator. The reaction was carried out overnight at 4 ° C. for coupling. The carrier after the coupling reaction was washed by sequentially flowing 3 bed volumes of 50% ethanol, then 3 bed volumes of 25% ethanol, and 3 bed volumes of MQ. In order to block the remaining reactive groups, the carrier after ligand binding was added with 3 bed volumes of 0.1 M Tris-HCl (pH 8.0) and allowed to react overnight at 4 ° C. while slowly stirring with a rotator. Finally, 3 bed volumes of wash buffer <1> (0.1 M acetic acid / sodium acetate [pH 4.0] containing 0.5 M NaCl), followed by 3 bed volumes of wash buffer <2> (0.1 M Tris-HCl [pH 8.0] The column was washed by repeating this operation three times in total.

<Example 4> Affinity purification The affinity column prepared as described above was placed in an incubator at 4 ° C, and a 3-bed equilibration buffer {50 mM HEPES-KOH (pH 7.8), 20% glycerol} was passed through the column. Equilibrated. Then, to react the ligand with the binding protein, add the soluble protein sample extracted from the S2 Cell to the column, suspend the carrier and sample, and then react overnight at 4 ° C while stirring gently with a rotator. It was. Washing of the carrier after the reaction was performed by sequentially flowing 3 bed amounts of equilibration buffer, equilibration buffer containing 0.5 M NaCl, equilibration buffer, equilibration buffer containing 25% ethylene glycol, and equilibration buffer. The bound protein was eluted from the carrier after washing by adding 0.1 M Glycine-HCl (pH 2.5), and immediately after collection, an appropriate amount of 2.5 M Tris-HCl (pH 8.8) was added for neutralization.

<Example 5> Electrophoresis of C16-yamamarine binding protein The binding protein obtained by affinity purification was concentrated by ultrafiltration (Microcon YM-3, manufactured by Millipore) and then adjusted with a sample buffer for electrophoresis. Electrophoresis was performed on a% polyacrylamide gel. After electrophoresis, C16-yamamarin binding protein was confirmed by CBB staining (FIG. 1A). The left lane in FIG. 1A shows a low molecular marker.

<Example 6> In-gel protease digestion with trypsin of target band In order to identify C16-yamamarin binding protein, in-gel protease digestion with trypsin was performed, and peptide fragments were recovered. LC-MS / MS (HCTultra ESI-ion-trap mass spectrometer (Bruker Daltonics, Leipzig, Germany) equipped with an Agilent 1100 CapLC system (Agilent, Wilmington, DE, USA)}} It was performed using.

Next, based on the analysis data, Mascot Search
(http://www.matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH=PMF), and the C16-yamamarine binding protein is rRNA 2'-O-methyltransferase fibrillarin with a molecular weight of 34673 consisting of 344 amino acids Was identified.

<Example 7> Confirmation by Western Blotting To confirm that the C16-yamamarin-binding protein is Fibrillarin, the antibody {Fibrillarin antibody (ab4566)} (purchased from abcam) was used to perform Western blotting on an affinity purified product. As a result, a signal was detected (FIG. 1B). Therefore, it was confirmed that the C16-yamamarine binding protein is Fibrillarin. The left lane in FIG. 1B shows a low molecular marker.

<Example 8> Mouse bone marrow progenitor cell assay One of the femurs of a ddY mouse (SLC, Shizuoka) that had been deeply anesthetized with ether was removed, and a 21-gauge injection needle of a 5 ml syringe was inserted to culture human lymphocytes. The medium RPMI 1640 medium (Invitriogen, Carlsbad, California) was injected and bone marrow cells were pushed from the other into a clean petri dish. Absorbed bone marrow cells were obtained by aspiration into a syringe and extrusion. Bone marrow cells are fibrillarin-bearing cells.

Bone marrow cells were washed twice with RPMI 1640 medium, and the cell concentration was adjusted to 2 × 10 ⁵ / ml. 0.4 ml of bone marrow cell solution was added to 4 mL of MethoCult (registered trademark) (StemCell Technologies, Vancouver, BC, Canada) containing rm IL-3, rm SCF, rh IL-6, rh EPO, fetal bovine serum. After mixed well using a vortex, syringe by sucking MethoCult (TM), aliquoted 1.1mL in 35mm culture Deisshu min (final cell concentration 2x10 ^{4 /} 35mm culture Deisshu). The cells were cultured for 12 days in an incubator (37 ° C, 5% CO ₂ , 95% humidity). Therefore, the number of colonies was measured with an inverted microscope. Generally, the number of hematopoietic progenitor cells contained in mouse bone marrow cells 2 × 10 ⁴ is around 50. Each of the progenitor cells is stimulated by a stimulating factor for 12 days, and differentiates and proliferates to form a colony consisting of 50 to 1000 cells. C16-DILRG-NH ₂ (0 to 50 μM, DMSO final concentration 0.5%) dissolved in DMSO was added to this measurement system, and the inhibitory effect was compared.
As a result, as shown in FIG. 3, it was found that C16-DILRG-NH ₂ suppresses differentiation / proliferation of bone marrow progenitor cells in a concentration-dependent manner.

<Example 9> BaTT3-Bcr / Abl MTT assay
BaF3 cells (ATCC, Manassas, VA 20108, USA) are acute lymphocytic leukemia cell lines developed from BalbC mice and possess Fibrillarin. To obtain a high titer helper-free retrovirus, Bosc23 cells were infected with a full-length Bcr-Abl plasmid (pSRa-p210Bcr-Abl) responsible for human chronic myeloid leukemia, and the supernatant was obtained 48 hours later. Was recovered. BaF3 cells were mixed with this supernatant in the presence of rm IL-3, and human Bcr-Abl was incorporated into BaF3 cells. When the cells were stabilized, cell lines were obtained that grew without rmIL-3 except for rmIL-3. Whether these cells (BaF3-Bcr / ABl) expressed p210 (molecular weight: 210 KD), which is a transcription product of Bcr / Abl, was confirmed by Western blot using a monoclonal antibody of human Abl.

  BaF3 cells require rm IL = 3 for growth, but BaF3-Bcr / ABl cells do not. That is, it is thought that it grows in the same system as the growth signal of chronic myelogenous leukemia.

BaF3-Bcr / ABl cells 96-well microplates on seeded (2.5x10 ⁵ / 200μl / well), 10% fetal bovine serum under 24, 48 and 72 hours, 37 ° C, of 5% CO _2, 95% Cultivated in a humid incubator. Three hours after adding MTT, the microplate was centrifuged, flicked to remove the supernatant, and a known document (Denizot, F. And Lang, R. (1986) Rapid colorimetric assay for cell growth and survival. Modifications to the The number of viable cells was measured by the MTT assay method according to the description of tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods, 89: 271-277.

  Then, C-16 yamamarine was added, and the growth inhibitory effect of BaF3-Bcr / Abl cells was compared.

  As a result, as shown in FIG. 4, it was found that C-16 yamamarin suppresses the growth of BaF3-Bcr / Abl cells in a concentration-dependent manner.

<Example 10> Comparative test of cell proliferation in each cell cycle by S2 Cell When using Drosophila embryo-derived cells (S2 Cell) cultured by the culture method of Example 1 and adding C16-yamamarine synthesized in Example 2 The proportion of proliferating cells in each cell cycle was analyzed (FIG. 5). As a comparative example, palmitic acid (concentration: 12.5 uM and 25 uM) was used, and C16-yamamarine was used as the experimental group, and the concentrations were 12.5 uM and 25 uM. The protocol followed the protocol shown in Non-Patent Document 8. FIG. 5 (A) confirms the cell growth inhibitory effect after 96 hours when C16-yamamarin is added. S2 cells to which palmitic acid was added showed that after 96 hours, the cells had grown, but the growth of S2 cells to which C16-yamamarin had been added almost stopped. Hereinafter, the cell cycle state of the S2 cell in FIG. FIG. 5 (B) shows the ratio of cells in the cell cycle of G0 / G1 phase in the S2 cells of FIG. 5 (A). FIG. 5 (C) shows the proportion of cells in the S-phase cell cycle in the S2 cells of FIG. 5 (A). FIG. 5 (D) shows the rate of proliferation of cells in the G2 / M phase cell cycle in the S2 cells of FIG. 5 (A). According to FIGS. 5B to 5D, the addition of C16-yamamarin does not stop the cell growth at a specific cycle of the insect cell cycle, but stops the cell growth at any cell cycle, while the cell growth is stopped. It shows that the suppression effect is shown. In addition, the sum total of the ratio of (B), (C), (D) is 100%.

  The number of cells in each cell cycle was measured using a flow cytometer (BDFACSCanto (registered trademark), Becton Dickinson) from the DNA histogram obtained from the cultured cells.

  As described above, it has been clarified that the Yamamarine derivative (C-16 Yamamarin) binds to Fibrillarin, and if it is a cell having Fibrillarin, the Yamamarine derivative (C-16 Yamamarin) can be used even in cells other than cancer cells. ) Revealed that growth could be suppressed in any cell cycle.

Claims (3)

A therapeutic agent for polycythemia vera caused by proliferation of erythroblast progenitor cells or essential thrombocythemia caused by proliferation of megakaryocyte progenitor cells, which comprises DILRG-NH ₂ A therapeutic agent comprising: DILRG-NH ₂ Has an acyl group at the N-terminus. The therapeutic agent according to claim 2, wherein the acyl group has 6 to 28 carbon atoms.