JPWO2006075587A1 - CSF-containing therapeutic agent for low dose administration - Google Patents

Abstract

A therapeutic agent for low-dose administration comprising CSF as an active ingredient, wherein the therapeutic agent is used as one or more therapeutic agents selected from a blood flow increasing agent, an angiogenesis promoting agent, and an ischemic disease therapeutic agent.

Description

  The present invention relates to a therapeutic agent for low dose administration comprising CSF as an active ingredient. The therapeutic agent of the present invention is useful as a blood flow increasing agent, an angiogenesis promoter or an ischemic disease therapeutic agent.

  Human granulocyte colony-stimulating factor (G-CSF) is a hematopoietic factor discovered as a differentiation-inducing factor for granulocyte hematopoietic stem cells, and promotes neutrophil hematopoiesis in vivo. Therefore, after bone marrow transplantation and cancer chemotherapy It has been clinically applied as a treatment for neutropenia. In addition to the above actions, human G-CSF has an action of acting on stem cells to stimulate differentiation and proliferation, and an action of mobilizing stem cells in bone marrow into peripheral blood. Based on the latter action, peripheral blood stem cells transplanted with peripheral hematopoietic stem cells mobilized by human G-CSF with the aim of promoting hematopoietic recovery in cancer patients after powerful chemotherapy in clinical practice Transplantation has been performed.

  It has also been reported that G-CSF has an angiogenesis-promoting action and is useful as a therapeutic agent for ischemic diseases (WO02 / 22163; Bussolino F. et al., Nature, 337: 471-473, 1989). Until now, when using G-CSF for the purpose of stem cell mobilization, leukocytosis, angiogenesis promotion, etc., it is common to administer a relatively high dose of 10-20 μg / kg / day subcutaneously. there were.

However, since G-CSF has a wide variety of activities, when a relatively high dose of 10 to 20 μg / kg / day is administered, effects other than the original purpose of promoting angiogenesis appear. .
WO02 / 22163 Bussolino F. et al., Nature, 337: 471-473, 1989

  An object of the present invention is to provide a therapeutic agent containing a colony stimulating factor (CSF) such as G-CSF at a low dose and exhibiting angiogenesis promoting action.

  The present inventors have found that even with a very low dose of G-CSF compared to the conventional dose, it is possible to obtain the desired effect by local administration, and completed the present invention. .

That is, the present invention provides the following.
(1) A therapeutic agent for low-dose administration comprising CSF as an active ingredient, wherein the therapeutic agent is used as one or more therapeutic agents selected from a blood flow increasing agent, an angiogenesis promoting agent, and an ischemic disease therapeutic agent.
(2) The therapeutic agent according to (1) above, which is locally administered to an ischemic site.
(3) The therapeutic agent according to (1) or (2) above, wherein the CSF is G-CSF.
(4) The therapeutic agent according to any one of (1) to (3), which is used as a blood flow increasing agent.
(5) The therapeutic agent according to any one of (1) to (3), which is used as an angiogenesis promoter.
(6) The therapeutic agent according to any one of (1) to (3), which is used as a therapeutic agent for ischemic disease.
(7) The therapeutic agent according to the above (6), wherein the ischemic disease is peripheral circulation disorder.
(8) The therapeutic agent according to the above (6), wherein the ischemic disease is ischemic heart disease.

  According to the present invention, administration of CSF such as G-CSF at a low dose can be used to treat increased blood flow, promote angiogenesis, or ischemic disease without causing undesirable effects such as increased circulating leukocytes. it can.

Diagram of electrophoresis examining the expression of G-CSF receptor (G-CSFR) mRNA on human umbilical vein endothelial cells (HUVEC), human dermal microvascular endothelial cells (HMVEC), and human coronary artery endothelial cells (HCAEC) It is. It is a figure of the microscope picture which shows the result of having tested the influence which rhG-CSF has on the migration activity of HUVEC. It is a graph which shows the result of having tested the influence which rhG-CSF has on the migration activity of HUVEC. It is a graph which shows the result of having tested the influence which rhG-CSF has on the cell proliferation of HUVEC. It is a figure of the microscope picture which shows the result of having tested the influence which rhG-CSF has on the endothelial network formation of HUVEC. It is a graph which shows the result of having tested the influence which rhG-CSF has on the endothelial network formation of HUVEC. FIG. 3 shows an experimental protocol using an in vivo angiogenesis model. It is a figure (white blood cell count of peripheral blood) which shows the influence which rhG-CSF has on the circulating white blood cell count of a rat. It is a figure which shows the influence which rhG-CSF exerts on the hindlimb blood flow of a rat. It is the figure (upper figure) which shows the result of having quantified the figure (upper figure) of the micrograph which shows the influence which rhG-CSF has on the tissue capillary density of a rat (upper figure), and the number of capillaries. It is a figure of the microscope picture which shows the influence which rhG-CSF has on the angiogenesis using a mouse ear and a subcutaneous blood vessel observation model.

  As CSF used in the therapeutic agent or method of the present invention, in addition to G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) interleukins are powerful hematopoietic cytokines. 3 (IL-3) and interleukin 5 (IL-5). Most preferred is G-CSF.

  In the present invention, low-dose administration means that CSF is usually administered at a dose of 0.01 μg / kg / day or more and less than 10 μg / kg / day, preferably 0.1 μg / kg / day or more and 10 μg / kg / day. Less than 1 μg / kg / day, more preferably less than 5 μg / kg / day, and even more preferably 1 μg / kg / day to 3 μg / kg / day. It is to be administered at a dose of less than. In addition, a dose of 0.01 μg / kg / day or more and less than 1 μg / kg / day, for example, 0.1 μg / kg / day or more and less than 1 μg / kg / day is also included in the low dose administration of the present invention. The dosage of the therapeutic agent of the present invention for each patient is determined by a doctor in consideration of the patient's age, weight, symptoms, route of administration and the like.

  In the case of low dose administration in the present invention, a sustained release preparation may be used. When a sustained-release preparation is used, a single dose may exceed the above-mentioned low dose, and the CSF released into the body from the sustained-release preparation during one day only needs to be the above-mentioned low dose. .

  In the therapeutic agent or method of the present invention, CSF is preferably administered locally. The site to be locally administered is not particularly limited, and can be administered to a site (tissue, organ, etc.) where angiogenesis is promoted or blood flow is increased or a site ischemic. Specific examples of the local administration site include lower limb skeletal muscle, upper limb skeletal muscle, and heart (myocardium).

  The local administration is not particularly limited as long as it can efficiently administer CSF to the affected area without greatly affecting the whole body, and can be administered locally using a normal syringe, needle, local needle, etc. is there.

  In the present invention, topical administration usually refers to administration of CSF directly to a site where angiogenesis is promoted or blood flow is increased, or ischemic, or the like. You may administer to the blood vessel directly connected to the site | part which wants to occur, the site | part which is in an ischemic state, etc. (For example, when a liver is an ischemic site | part, when administering CSF to a hepatic artery, etc.).

  The therapeutic agent of the present invention is useful as a blood flow increasing agent, an angiogenesis promoter or an ischemic disease therapeutic agent.

  In the present invention, the increase in blood flow means that the blood flow at a site where CSF is locally administered or at a site located more peripheral than the site is increased compared to before administration, or the blood flow reaching the ischemic tissue is increased. Means that. Whether or not an effect of increasing blood flow has been obtained can be measured by a method known to those skilled in the art, and can be quantified using, for example, radioactive microspheres by a flow technique (Am. J Physiol. 243: H371-H378, (1982)). Furthermore, it can also be measured and confirmed by methods such as skin temperature, thermography, laser blood flow meter, lower limb / upper limb blood pressure ratio, tissue oxygen partial pressure.

  In the present invention, promotion of angiogenesis refers to promotion of angiogenesis and / or promotion of blood vessel growth / development. Although the blood vessel whose neoplasm, growth, development, etc. are promoted by CSF is not particularly limited, an artery is preferable, and a peripheral artery is particularly preferable. Whether or not an angiogenesis-promoting action has been obtained can be measured by a method known to those skilled in the art, for example, by measuring capillary density using alkaline phosphatase staining or the like. Furthermore, it can be measured and confirmed by a method such as angiography.

  In addition, by using the method of the present invention, not only is blood vessel formation, growth, development, etc. promoted, but also lymphangiogenesis, growth, development, nutrition to peripheral nerves, improvement of neuropathy due to angiogenesis, etc. It can also be promoted.

  The therapeutic agent of the present invention is also useful for the treatment of ischemic diseases. Ischemic disease is caused by various factors such as narrowing of blood vessel lumen, thrombus formation, blood vessel occlusion, vasculitis, blood vessel contraction, increased blood viscosity, etc. It is a medical condition that falls. Examples of ischemic diseases include peripheral circulation disorder, ischemic heart disease (ischemic cardiomyopathy, myocardial infarction, ischemic heart failure, etc.), ischemic cerebrovascular disorder, ischemic kidney disease, ischemic lung disease, infection Related to ischemic disease, etc. A preferred disease that is a target of the therapeutic agent of the present invention is peripheral circulation disorder or ischemic heart disease.

  Peripheral circulatory disorder is a condition in which peripheral arterial blood flow decreases due to stenosis of blood vessel lumen, thrombus formation, blood vessel occlusion, vasculitis, blood vessel contraction, blood viscosity increase, etc., and peripheral tissues fall into ischemic state It is. Diseases with peripheral circulatory disturbance include obstructive arteriosclerosis, chronic arterial occlusion such as Buerger's disease, progressive systemic sclerosis, systemic lupus erythematosus, Raynaud's disease, vibration disease, aneurysm, vasculitis, etc. it can.

  When the CSF used in the therapeutic agent or method of the present invention is G-CSF, any G-CSF can be used, but it is preferably highly purified G-CSF, more specifically Are those having substantially the same biological activity as mammalian G-CSF, particularly human G-CSF.

  The G-CSF used in the present invention may be produced by any method. For example, G-CSF obtained by culturing human tumor cells or human G-CSF-producing hybridoma cell lines, and then extracting, purifying, and purifying the cells by various methods. It is produced in Escherichia coli, yeast, Chinese hamster ovary cells (CHO cells), C127 cells, COS cells, myeloma cells, BHK cells, insect cells, etc. by CSF or genetic engineering techniques, extracted, separated and purified by various methods. G-CSF or the like can be used. The G-CSF used in the present invention is preferably G-CSF produced by a genetic engineering technique, and G-CSF produced using mammalian cells (particularly CHO cells) is preferred (for example, JP-B-1- No. 44200, Japanese Patent Publication No. 2-5395, Japanese Patent Laid-Open No. 62-129298, Japanese Patent Laid-Open No. 62-132899, Japanese Patent Laid-Open No. 62-236488, Japanese Patent Laid-Open No. 64-85098).

  G-CSF obtained by a genetic recombination method has the same amino acid sequence as that of naturally-occurring G-CSF, or one obtained by deleting, substituting, or adding one or more amino acids in the amino acid sequence. Those having the same biological activity as naturally-derived G-CSF may be used. Amino acid deletion, substitution, addition and the like can be performed by methods known to those skilled in the art. For example, those skilled in the art will recognize site-directed mutagenesis (Gotoh, T. et al. (1995) Gene 152, 271-275; Zoller, MJ and Smith, M. (1983) Methods Enzymol. 100, 468- 500; Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456; Kramer, W. and Fritz, HJ (1987) Methods Enzymol. 154, 350-367; Kunkel, TA (1985) Proc. Natl. Acad. Sci. USA. 82, 488-492; Kunkel (1988) Methods Enzymol. 85, 2763-2766) and the like, by introducing appropriate mutations in the amino acids of G-CSF, Functionally equivalent polypeptides can be prepared. Amino acid mutations can also occur in nature. In general, the amino acid residue to be substituted is preferably substituted with another amino acid that preserves the properties of the amino acid side chain. For example, as the properties of amino acid side chains, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids having aliphatic side chains (G, A, V, L, I, P), amino acids having hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains Amino acids (C, M) having carboxylic acids and amide-containing side chains (D, N, E, Q), amino groups having base-containing side chains (R, K, H), aromatic-containing side chains (H, F, Y, W) can be mentioned (all parentheses represent single letter amino acids). It is already known that a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution with other amino acids of one or more amino acid residues to a certain amino acid sequence maintains its biological activity. (Mark, DF et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666; Zoller, MJ & Smith, M. Nucleic Acids Research (1982) 10, 6487-6500; Wang, A et al., Science 224, 1431-1433; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413).

  It is also possible to use a fusion protein of G-CSF and another protein. In order to prepare a fusion polypeptide, for example, a DNA encoding G-CSF and a DNA encoding another protein are linked so that the frames coincide with each other, introduced into an expression vector, and expressed in a host. Good. Other proteins to be subjected to fusion with G-CSF of the present invention are not particularly limited.

  It is also possible to use chemically modified G-CSF. As an example of chemically modified G-CSF, for example, G-CSF subjected to sugar chain structural conversion / addition / deletion operation, polyethylene glycol / vitamin B12, and other compounds such as inorganic or organic compounds were bound. Examples include G-CSF.

  The therapeutic agent of the present invention includes a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a sea surface activator, a diluent, an excipient, if necessary. A pH adjuster, a soothing agent, a buffering agent, a sulfur-containing reducing agent, an antioxidant and the like can be appropriately added.

  Examples of the suspending agent include methyl cellulose, polysorbate 80, hydroxyethyl cellulose, gum arabic, tragacanth powder, sodium carboxymethyl cellulose, polyoxyethylene sorbitan monolaurate and the like.

  Examples of the solubilizer include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinic acid amide, polyoxyethylene sorbitan monolaurate, Magrogol, castor oil fatty acid ethyl ester, and the like.

  Examples of the stabilizer include dextran 40, methylcellulose, gelatin, sodium sulfite, and sodium metasulfite.

  Examples of the isotonic agent include D-mannitol and sorbate.

  Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, phenol, cresol, chlorocresol and the like.

  Examples of the adsorption inhibitor include human serum albumin, lecithin, dextran, ethylene oxide / propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenated castor oil, polyethylene glycol and the like.

  Examples of the sulfur-containing reducing agent include N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, carbon Examples thereof include those having a sulfhydryl group such as thioalkanoic acid having 1 to 7 atoms.

  Examples of the antioxidant include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salt, L-ascorbyl palmitate, L-ascorbic acid stearate, sodium bisulfite, Chelating agents such as sodium sulfite, triamyl gallate, propyl gallate or disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, sodium metaphosphate and the like can be mentioned.

  Furthermore, normally added components such as inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate and sodium bicarbonate; organic salts such as sodium citrate, potassium citrate and sodium acetate May be included.

  When the present invention is carried out as a sustained-release preparation, the sustained-release preparation can be prepared by a known method, for example, a method using a polymer as a base. The polymer used as the base is preferably a polymer that is decomposed in vivo.

  The present inventors investigated the influence of recombinant human G-CSF (rhG-CSF) on the angiogenic ability of mature endothelial cells in vitro, as shown in the Examples below. Of local administration of a low dose of rhG-CSF to ischemic tissue, which does not cause the increase in leukocyte proliferation, promotes angiogenesis using a unilateral hindlimb ischemic rat model (Murohara T. et al. al., J. Clin. Invest. 105: 1527-1536, 2000). As a result, it was confirmed that even with a very low dose of G-CSF, blood flow was increased and angiogenesis-promoting action was achieved without causing a marked increase in circulating leukocytes. Furthermore, in the study using the mouse ear subcutaneous blood vessel observation model, formation of microaneurysms was observed in VEGF-treated mice, whereas such microaneurysms were not observed in G-CSF-treated mice. , Confirmed to form clean blood vessels.

  The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. Various changes and modifications can be made by those skilled in the art, and these changes and modifications are also included in the present invention.

  In the statistical analysis in the following examples, all data are expressed as an average value ± SE. Comparisons between groups were made by ANOVA followed by Fisher's test. Significant differences were defined at p <0.05.

Example 1: Confirmation of G-CSF receptor mRNA expression on various human endothelial cells Using reverse transcription PCR (RT-PCR) analysis, human umbilical vein endothelial cells (HUVEC), human dermal microvascular endothelial cells ( The expression of G-CSF receptor (G-CSFR) mRNA on HMVEC) and human coronary artery endothelial cells (HCAEC) was examined. The cells were purchased from Sanko Junyaku Co., Ltd. Total RNA was extracted from these cells using a guanidinium isothiocyanate-phenol chloroform solution (TRIzol reagent, Invitrogen), quantified by measuring absorption at 260/280 nm, and subjected to RT-PCR analysis. First, 1 μg of total RNA of each sample was reverse-transcribed using an oligo dT primer and Rnase H-reverse transcriptase (Superscript II; Invitrogen).

The following were used as primers for human G-CSFR.
Sense strand: 5'-tcctcaccctgatgaccttga-3 '(SEQ ID NO: 1)
Antisense strand: 5'-gaagttgagcagtggcccaaa-3 '(SEQ ID NO: 2)
RT-PCR of G-CSFR yields three PCR products of sizes 620 bp (transmembrane type, GenBank M59818), 701 bp (cytoplasmic domain inserted type, GenBank M59820), and 548 bp (soluble type, GenBank M59819) It was expected.

It is known that human glyceryl aldehyde-3-phosphate dehydrogenase (GAPDH) is induced and produced in vascular endothelial cells exposed to a hypoxic environment. In order to confirm whether GAPDH was also detected in various endothelial cells used in this example, GAPDH RT-PCR was performed using the following primers.
Sense strand: 5'-cttcaccaccatggaggagg-3 '(SEQ ID NO: 3)
Antisense strand: 5'-tgaagtcagaggagaccacc-3 '(SEQ ID NO: 4)
The PCR product obtained is predicted to be 557 bp by RT-PCR of GAPDH. As a positive control, G-CSF receptor was expressed on bone marrow mononuclear cells (BM-MNC).

The results of subjecting the obtained PCR product to agarose gel electrophoresis are shown in FIG. Expression of G-CSF receptor on HUVEC, HMVEC and HCAEC was confirmed.

Example 2: Cell migration assay
In order to examine the effect of rhG-CSF, a migration activity assay of HUVEC was performed. The migration ability assay was performed using a modified Boyden Chamber device (Neuroprobe, Gaithersburg, Maryland). A polyvinylpyrrolidone (PVP) free porous (8 mm diameter) polycarbonate filter was coated with 0.1% gelatin and 50 mg / mL fibronectin and allowed to dry in air for 1 hour. Medium supplemented with 1% FBS and G-CSF (1,10.100 ng / mL) was placed in the lower chamber of the apparatus (rhG-CSF was a gift from Chugai Pharmaceutical Co., Ltd.). As a positive control, rhVEGF (10 ng / mL) was placed in the lower chamber. Subconfluent HUVEC cultures were washed and trypsinized for the minimum time required for cell detachment. HUVEC (1 × 10 ⁴ cells) suspended in Medium-199 (50 mL) containing 1% FBS (without G-CSF) after placing coated PVP-free polycarbonate filter between upper and lower chambers ) In the upper room. The entire apparatus was incubated for 3 hours at 37 ° C. in a humidified incubator with 95% air and 5% CO _{2 to} allow HUVEC to move through the membrane. After the incubation, the filter was taken out, and non-migration HUVEC on the upper surface of the filter was scraped off with a rubber spatula. The filters were then fixed with methanol for 10 minutes and stained with May-Gruenwald solution (Merck, Darmstadt, Germany) and Giemsa stain (SIGMA). For each chamber, the migratory HUVEC adhering to the lower surface of the filter was measured in a randomly selected microscopic field (× 200). All experiments were performed 6 times and data were expressed as number of migrating cells / field of view.

The obtained results are shown in the photograph of FIG. 2 and the graph of FIG. It was revealed that rhG-CSF promotes HUVEC migration through the microchemotactic membrane at a concentration of 100 ng / mL. These in vitro findings indicate that rhG-CSF promotes migration of mature endothelial cells.

Example 3: Cell proliferation assay
The colorimetric MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, which was previously confirmed for the proliferation activity of HUVEC ) Assay and analysis using the electron binding reagent phenazine methosulfate (CellTiter 96 AQ; Promega, Madison, Wis.). HUVEC were spread in 96-well plates in Medium-199 (0.2 ml) containing 2% FBS (with or without G-CSF (0.1, 1, 10, 100, 1000 ng / mL)) (1000 cells / hole). MTS / phenazine methosulfate mixture (20 μl) was added to each well on day 3 of culture and incubated at 37 ° C. for 4 hours. Next, the absorbance at 490 nm / 655 nm was measured using an ELISA plate reader (Model 680 Microplate Reader, Bio-Rad). As a positive control, 10 ng / ml VEGF (vascular endothelial growth factor) was used.

The obtained results are shown in FIG. The positive control, 10 ng / ml VEGF, significantly stimulates HUVEC proliferation, whereas rhG-CSF does not promote HUVEC proliferation at concentrations of 0.1, 1, 10, 100 and 1000 ng / mL. It was revealed.

Example 4: In vitro angiogenesis model: Endothelial network formation on substrate gel culture The influence of rhG-CSF on endothelial network formation, an angiogenic reaction on a culture dish, was examined. 4 × 10 ⁴ HUVECs suspended in Medium-199 (1 mL) containing 2% FBS (with or without G-CSF (0.1, 1, 10, 100, 1000 ng / mL)) are growth factors Cultured on reduced Matrigel (Becton Dickinson, San Jose, CA). After incubation for 12 hours in humidified air at 37 ° C., 5% CO ₂ , endothelium network formation was examined and photographed with randomly selected fields in each well (× 40). For quantitative evaluation, the length of network formation in each image was calculated on a computer by the WIN ROOF program (Mitani corporation, Tokyo, Japan). All experiments were performed 4 times and data were expressed as network formation length / field of view.

The obtained results are shown in the photograph of FIG. 5 and the graph of FIG. Incubation with rhG-CSF (100 ng / mL) for 12 hours showed a significant stimulatory effect on endothelial tube formation. Although there was a tendency to stimulate endothelial network formation at low doses of rhG-SCF (1 or 10 ng / mL), the effect was not statistically significant.

Example 5: In vivo angiogenesis model
The in vivo experimental protocol is shown in FIG. Surgical unilateral hindlimb ischemia was caused by complete resection of the left femoral artery and vein of male nude rats (F344 / N rnu / rnu). The rats in the rhG-CSF group received rhG-CSF (2, 10 or 20 μg / kg / day) on the ischemic adductor muscle (n = 8 in each administration group) for 6 days from the first day after surgery. Gave. On the other hand, control rats (n = 4) were given physiological saline on the ischemic adductor muscle for 6 days.

All rats were given normal food and drinking water. All experimental protocols were approved by the Animal Experiment Committee in Nagoya University School of Medicine.
(1) Effect of G-CSF on circulating white blood cell count in rats
Total white blood cell count was examined as a sign of the mobilization effect of rhG-CSF. A blood sample (0.2 mL of each animal) was collected from the tail vein before and after surgery and on the 3rd and 7th days for the measurement of circulating total white blood cell count. Complete blood cell counts were performed with Sysmex KX-21 (Sysmex, Kobe, Japan).

The obtained result is shown in FIG. On the third day after surgery, in the rhG-CSF 10 and 20 μg / kg groups, the number of WBC measurements slightly increased to 1.4 and 1.3 times as compared with the control group, respectively. On day 7, the number of measurements decreased to the first reference level (no significant difference compared to the baseline).
(2) Laser Doppler blood flow analysis The ratio of ischemia / normal hindlimb blood flow was measured using a laser Doppler blood flow (LDBF) analyzer (moorLDI, Moor Instrument). Two consecutive laser scans were performed on the sites of interest (limbs and feet) at seven pre-determined time points (preoperative and postoperative days 1, 3, 7, 14, 21, 28). The average perfusion of ischemic and non-ischemic paws was calculated based on color pixel histograms related to the degree of blood flow. In order to minimize fluctuations due to room light, blood flow was expressed as an ischemia / normal limb LDBF ratio.

The obtained results are shown in FIG. On the 14th, 21st, and 28th postoperative days, the rhG-CSF treated group showed a significant increase in the ischemia / normal hindlimb blood flow ratio in a dose-dependent manner compared with the saline treated group. It became clear by analysis.
(3) Tissue capillary density analysis The effect of rhG-CSF (or physiological saline) administration on angiogenesis was examined by measuring the number of capillary endothelial cells under an optical microscope. Day 28 adductor skeletal muscle was embedded in OCT compound (Miles) and snap frozen in liquid nitrogen. Sections were stained with alkaline phosphatase to detect capillary endothelial cells. The number of endothelial cells was counted under a light microscope (× 200), and the capillary density was determined. Capillary density was measured by randomly selecting 5 fields from muscle samples of each animal.

  The obtained result is shown in FIG. In FIG. 10, the upper photograph shows alkaline phosphatase staining of a femoral adductor skeletal muscle section 28 days after lower limb ischemia surgery. Endothelial cells are stained blue-violet. A: Control saline administration group, B: rhG-CSF (2 μg / kg / day) administration group, C: rhG-CSF (10 μg / kg / day) administration group, D: rhG-CSF (20 μg / kg / day) ) Administration group. In B, C, and D, it is observed that the density of endothelial cells is higher than that in A.

In addition, in FIG. 10, the graph in the lower diagram shows quantitative data based on the number of capillary endothelial cells per visual field. From these results, it was revealed that the rhG-CSF group had a significantly increased capillary density (p <0.01 each) compared to the saline control group.

Example 6: Mouse Ear Effect of G-CSF on Angiogenesis Using a Subcutaneous Blood Vessel Observation Model On the first day of the study, mice (C57BL / 6J, each group: 3 mice, obtained from Clea Japan Co., Ltd.) Nembutal (10 times) The ear hair was treated with a hair removal cream after anesthesia with a dilution of 0.3-0.5 ml) and the recombinant protein (rhG-CSF or rhVEGF) was administered subcutaneously. Administration was performed for 3 consecutive days using a 29G needle with recombinant protein 150 ng / 15 μl (PBS). From the second day, the skin was lightly anesthetized with ether and administered subcutaneously. Separately, a control saline administration group was prepared.

  Tissue collection was performed on the fourth day from the start of administration. Nembutal diluted 10-fold was injected intraperitoneally with 0.3 to 0.5 ml. As seen from the ventral side of the tail, there is an artery in the midline, and veins on both sides of the artery. .

  Two to three minutes later, the thoracotomy was performed, and PBS containing about 1% paraformaldehyde (about 20 ml) was allowed to flow from the left ventricle to fix the blood vessels. It was washed with PBS alone.

  Subsequently, after euthanasia, the auricle was collected and divided into two layers on the cartilage surface. At that time, try not to leave as much fat and muscle as possible on the cartilage surface, and peel off carefully because there are many connective tissues especially when inflammation is induced. This was prepared as a preparation and fluorescently stained with a con-focal microscope and observed from the dermis side.

The obtained micrograph is shown in FIG. Angiogenesis was observed in rhVEGF-administered mice and rhG-CSF-administered mice as compared to the control saline-administered group. The formation of a microaneurysm was observed in the rhVEGF-treated mice, but the formation of such a microaneurysm was not observed in the rhG-CSF-treated mice, forming clean blood vessels.

Claims (8)

A therapeutic agent for low-dose administration comprising CSF as an active ingredient, wherein the therapeutic agent is used as one or more therapeutic agents selected from a blood flow increasing agent, an angiogenesis promoting agent, and an ischemic disease therapeutic agent. The therapeutic agent according to claim 1, which is locally administered to an ischemic site. The therapeutic agent according to claim 1 or 2, wherein the CSF is G-CSF. The therapeutic agent according to any one of claims 1 to 3, which is used as a blood flow increasing agent. The therapeutic agent according to any one of claims 1 to 3, which is used as an angiogenesis promoter. The therapeutic agent according to any one of claims 1 to 3, which is used as a therapeutic agent for an ischemic disease. The therapeutic agent according to claim 6, wherein the ischemic disease is peripheral circulation disorder. The therapeutic agent according to claim 6, wherein the ischemic disease is ischemic heart disease.