HK1128419B - Treatment of ischemic disease using erythropoietin - Google Patents

Abstract

A method for the regeneration of a blood vessel, comprising the steps of: (a) administering erythropoietin to a subject; (b) collecting a peripheral blood mononuclear cell from the subject; and (c) administering the collected peripheral blood mononuclear cell to a desired site in the subject. When erythropoietin is administered to a subject, a peripheral blood mononuclear cell (particularly a CD34-positive one) is recruited into the peripheral blood of the subject. The method is useful for the treatment of an ischemic disease such as peripheral vascular disorder.

Description

Treatment of ischemic diseases using erythropoietin

Technical Field

The present invention relates to methods of revascularization, methods of treating ischemic diseases, and compositions for use in these methods.

Background

Peripheral vascular disorder is a disease in which peripheral tissues become ischemic due to reduced peripheral arterial blood flow caused by, for example, narrowing of the lumen of a blood vessel, formation of blood clots, blockage of blood vessels, vasculitis, vasoconstriction or increased blood viscosity. In recent years, it has been found that when bone marrow-derived mononuclear cells are transplanted to an ischemic site, angiogenesis is enhanced, and transplantation of bone marrow-derived mononuclear cells is considered as a potential method for treating peripheral vascular disorders. However, transplantation of bone marrow-derived mononuclear cells requires collection of bone marrow from a patient, which adds a substantial burden to the patient.

In order to reduce the burden on patients, there have been attempts to replace bone marrow-derived monocytes with peripheral blood-derived monocytes; however, this method is not successful in providing effective treatment due to the small number of monocytes present in the peripheral blood.

Erythropoietin (also known as EPO) is an acidic glycoprotein hormone that promotes differentiation and proliferation of erythroid progenitor cells and is produced primarily in the kidney. Red blood cells (the largest number of cells in the blood) function for a certain period of time and then are destroyed mainly in the spleen (the average life span in humans is about 120 days). Normally, the total peripheral red blood cell count is kept constant continuously by a continuous supply from the bone marrow. EPO plays an important role in this homeostasis of erythrocytes in the body. In clinical settings, EPO is used to treat anemia and for preoperative and postoperative management.

In addition, EPO has been reported to have angiogenesis-promoting activity and to be effective as a therapeutic agent for The treatment of ischemic diseases (Besarab A et al, The New England Journal of medicine, 339(9), 584 590, (1998), Heeschen C et al, Blood, 102(4), 1340-1346, (2003), Bahlmann F H et al, Blood, 103(3), 921-926, (2004), Smith K J et al, Cardiovasular Research, (59), 538-548, (2003), Bahlmann H et al, Kidney International, 64, 1648-1652, (2003)). EPO has also been reported to promote mobilization of vascular endothelial progenitor cells into peripheral Blood (Heeschen C et al, Blood, 102(4), 1340-1346, (2003), Bahlmann F H et al, Blood, 103(3), 921-926, (2004)).

However, it is not clear whether the cells mobilized by EPO into the peripheral blood are effective for the treatment of ischemic diseases such as peripheral vascular disorders.

Summary of The Invention

The present inventors have discovered that the mobilization of monocytes into peripheral blood is facilitated by the administration of EPO, and that monocytes so mobilized by EPO are particularly useful for the treatment of ischemic diseases.

The present invention provides a composition for mobilizing monocytes for the treatment of ischemic diseases or for stimulating revascularization into peripheral blood, wherein the composition comprises erythropoietin as an active ingredient. Preferably, the mobilized monocytes are collected from a subject and then administered to the same subject.

The composition of the present invention is preferably administered to the subject 3-12 days before peripheral blood is collected from the subject.

The present invention also provides a composition for treating ischemic diseases and a composition for stimulating revascularization, comprising monocytes as an active ingredient isolated from peripheral blood of a subject previously administered erythropoietin. In such compositions, erythropoietin is preferably administered to the subject 3-12 days prior to collection of peripheral blood.

The present invention also provides a composition for mobilizing CD34 positive cells for the treatment of ischemic diseases or for stimulating revascularization into peripheral blood, comprising erythropoietin as an active ingredient.

In another aspect, the present invention provides a method for preparing monocytes for stimulating revascularization and for treating ischemic diseases comprising isolating monocytes from peripheral blood of a subject previously administered erythropoietin.

In another aspect, the present invention provides a method of treating an ischemic disease or stimulating revascularization in a subject, comprising the steps of:

(a) administering erythropoietin to a subject;

(b) collecting peripheral blood mononuclear cells from a subject; and

(c) administering the collected peripheral blood mononuclear cells to a target site of the subject.

Brief description of the drawings

FIG. 1 shows the results of FACS analysis measuring erythropoietin mobilized hematopoietic stem cells;

FIG. 2 shows the results of measurement of the number of colonies of hematopoietic stem cells mobilized by the administration of erythropoietin; and

FIG. 3 shows angiograms of a patient receiving erythropoietin (case 3) after one month of peripheral blood mononuclear cell transplantation.

Preferred embodiments of the invention

Mobilization of peripheral blood mononuclear cells by erythropoietin

One aspect of the present invention relates to a composition for mobilizing monocytes for the treatment of ischemic diseases or for stimulating revascularization into peripheral blood, wherein the composition comprises erythropoietin as an active ingredient. The terms "mobilising" monocyte "and" mobilisation of monocytes "mean the stimulation of differentiation and proliferation of pluripotent stem cells present in various organs, and the release of monocytes comprising pluripotent stem cells into the blood. Monocytes mobilized in this manner can be collected from the peripheral blood of a subject and administered to the subject for the purpose of treating ischemic disease or stimulating revascularization. The subject is preferably a patient suffering from an ischemic disease or a patient in need of revascularization therapy.

The present invention is not particularly limited with respect to the number of doses of erythropoietin to be administered to a subject prior to collection of peripheral blood mononuclear cells, but generally 1-3 doses. In the case of administration of 3 doses, for example, the first administration of erythropoietin is given systemically (e.g., subcutaneously or intravenously) to the subject about 2 weeks prior to collection of peripheral blood mononuclear cells. Approximately one week later, a second erythropoietin administration is given systemically after blood supply (draw). After another week, a third administration of erythropoietin is administered topically and the desired peripheral mononuclear cells can then be collected from the subject's peripheral blood. In the case of 2 doses, for example, the first erythropoietin administration is given systemically to the subject about one week before peripheral blood mononuclear cells are collected, and the second erythropoietin administration is given locally about one week after, and then the desired peripheral mononuclear cells can be collected from the peripheral blood. In the case of a single administration, for example, erythropoietin is administered systemically to the subject and the desired peripheral mononuclear cells can be collected about one week later.

A unit dose of erythropoietin generally comprises 1000U/human to 100000U/human, preferably 3000U/human to 12000U/human (e.g., 6000U/human). The dosage for an individual subject will be determined by the attending physician, taking into account factors such as the age, weight and condition of the subject and the route of administration. Therefore, the dose of erythropoietin in the present invention is not limited to the above dose.

Typically erythropoietin is administered by parenteral routes, e.g., it may be administered in injections (e.g., subcutaneous, intravenous, intramuscular, or intraperitoneal) or by transmucosal, transdermal, nasal, or pulmonary routes. It may also be administered orally.

Peripheral blood mononuclear cells are mononuclear cells present in peripheral blood. Monocytes (monuclar cells), also known as monocytes, are cells which are predominantly present in the blood and reside in the stage of differentiation into macrophage-type cells. Hematopoietic stem cells are differentiated in bone marrow into monocytes and promonocytes, and then differentiated into monocytes, which are released into the blood. Upon migration into tissue, monocytes differentiate into, for example, macrophages, dendritic cells, and tissue cells.

With respect to mobilization of monocytes into peripheral Blood using EPO, it is believed that two weeks are required after EPO administration to mobilize monocytes into peripheral Blood (Bahlmann F H et al, Blood, 103(3), 921-926, (2004)). However, it was found in accordance with the present invention that, at about one week after administration of EPO, monocytes are mobilized into the peripheral blood in an amount sufficient for the treatment of ischemic diseases or for stimulating revascularization.

Accordingly, the present invention relates to a method of obtaining monocytes or a method of preparing monocytes for transplantation comprising the steps of:

(a) administering erythropoietin to a subject; and

(b) about one week after erythropoietin administration, peripheral blood mononuclear cells are collected from the subject.

In the context of the present invention, the phrase "about one week" is generally 3 to 12 days, preferably 5 to 9 days (e.g., 6 to 8 days or 7 days).

In another aspect, the present invention relates to compositions for mobilizing CD34 positive cells into the peripheral blood for the treatment of ischemic diseases or for stimulating revascularization, comprising erythropoietin as an active ingredient.

CD34 is a blood stem cell (blood stem cell) antigen. It is expressed on blood stem cells, and also in, for example, vascular endothelial cells, vascular endothelial progenitor cells, and stromal cells. Since CD34 positive cells such as vascular endothelial progenitor cells are known to be involved in angiogenesis, in order to obtain a better therapeutic effect on ischemic diseases or a better revascularization effect, it is necessary to increase the proportion of CD34 positive cells in the transplanted monocyte population. The monocyte population mobilized into the peripheral blood according to the methods of the invention typically has a titer (titer) positive for CD34 sufficient to obtain a therapeutic effect on ischemic disease or a revascularization effect. In transplantation for the purpose of treating ischemic diseases or stimulating revascularization, it is particularly advantageous to increase the proportion of CD 34-positive cells by isolating or concentrating CD 34-positive cells from the monocyte population obtained by the method of the present invention.

In the present invention, a high proportion of CD34 positive cells in monocytes means that the CD34 positive cell count represents at least 1%, preferably at least 2%, more preferably at least 3% of the monocytes. The upper limit of the proportion of CD34 positive cells in monocytes may be, for example, 99.99%, 99.9% or 99%, and theoretically 100%.

The CD34 positive cells present in the monocytes obtained according to the invention may also be CD45 positive. CD45 is a leukocyte common antigen and is a key membrane glycoprotein of hematopoietic cell types.

Peripheral blood mononuclear cells can be collected from a subject by a common method. For example, monocytes can be obtained by directly recovering blood-derived monocytes obtained by plasmapheresis (blood apheresis) of blood, removing most of erythrocytes, granulocytes and platelets by centrifugation necessary for capturing peripheral blood leukocytes, and then washing the obtained peripheral blood leukocytes by, for example, centrifugation.

The peripheral blood mononuclear cells obtained in this way can optionally be further processed by addition, isolation or purification. For example, desired cells such as CD34 positive cells and/or vascular endothelial progenitor cells can be further concentrated or isolated from the collected peripheral blood mononuclear cells for administration. Substantially pure CD34 positive cells can be prepared using standard techniques. For example, peripheral blood mononuclear cells are reacted with anti-CD 34 antibody, and then CD34 positive cells are attached to magnetic beads carrying anti-mouse IgG antibody. The CD34 positive cell reactions bound to the magnetic beads were collected with a sheet magnet and CD34 positive cells were subsequently released from the magnetic beads by enzymatic treatment. Alternatively, CD34 positive cells were bound to anti-CD 34 antibodies labeled with a fluorescent dye and collected using a fluorescent cell sorter.

Treatment of ischemic diseases and revascularization

Peripheral blood mononuclear cells prepared according to the present invention can be administered to a subject for the purpose of treating an ischemic disease. Thus, another aspect of the invention relates to a method of treating an ischemic disease in a subject, comprising the steps of:

(a) administering erythropoietin to a subject;

(b) collecting peripheral blood mononuclear cells from a subject; and

(c) administering the collected peripheral blood mononuclear cells to a target site of the subject.

Ischemic disease is a disease in which tissues are trapped in an ischemic state due to a decrease in blood flow in the vasculature caused by various factors such as narrowing of the lumen of a blood vessel, formation of blood clots, occlusion of a blood vessel, vasculitis, constriction of a blood vessel, or an increase in blood viscosity. Ischemic diseases include peripheral vascular disorders, ischemic heart diseases (e.g., ischemic cardiomyopathy, myocardial infarction, ischemic heart failure), ischemic cerebrovascular diseases, ischemic renal diseases, ischemic lung diseases, and ischemic diseases associated with infectious diseases.

Peripheral vascular disorder is a disease in which peripheral tissues are trapped in an ischemic state due to reduced peripheral arterial blood flow caused by, for example, narrowing of the lumen of a blood vessel, formation of blood clots, blockage of blood vessels, vasculitis, vasoconstriction or increased blood viscosity. Diseases associated with peripheral vascular disorders include chronic arterial occlusive diseases such as arteriosclerosis obliterans and thromboangiitis obliterans (Buerger's disease), as well as progressive systemic sclerosis, systemic lupus erythematosus (systemic lupus erythematosus), Raynaud's disease, vibrational syndrome, aneurysms and vasculitis. Peripheral vascular disorders are preferred target diseases for the therapeutic agents of the present invention, with arteriosclerosis obliterans and thromboangiitis obliterans being particularly preferred targets.

In addition, peripheral blood mononuclear cells prepared according to the present invention can be administered to a subject to stimulate revascularization. Thus, another aspect of the invention relates to a method of stimulating revascularization in a subject, comprising the steps of:

(a) administering erythropoietin to a subject;

(b) collecting peripheral blood mononuclear cells from a subject; and

(c) administering the collected peripheral blood mononuclear cells to a target site of the subject.

As used herein, revascularization means the stimulation of angiogenesis and/or the growth and development of blood vessels. The method of the invention can be used to stimulate the neogenesis, growth and development of any type of blood vessel, preferably arteries, and particularly preferably peripheral arteries. Angiogenesis can be monitored by techniques known to those skilled in the art, for example, measuring capillary density using alkaline phosphatase dyes.

As used herein, a target site generally refers to a site where ischemia occurs and a site where angiogenesis is required. In the case where transplantation of peripheral blood mononuclear cells to another site causes angiogenesis to occur at a site requiring angiogenesis, the target site may refer to such another site. Specific examples of sites of local administration include lower limb skeletal muscle, upper limb skeletal muscle, and heart (cardiac muscle).

The number of collected monocytes to be administered or transplanted to the target site is not particularly limited, but is generally 1.0X 10⁷-1.0×10¹²Individual cells, preferably 1X 10⁹-1×10¹¹And (4) cells.

Topical administration is a method that enables effective application of cells to the affected area without significant systemic effects. Topical administration can be carried out by using, for example, a conventional syringe, needle, or positioning needle.

When administering the collected monocytes to the target site, the monocytes may be administered alone or in combination with another substance. The substance to be co-administered with the monocytes is not particularly limited, but is preferably a substance capable of improving the activity of regenerating blood vessels.

Erythropoietin

The present invention may employ any type of EPO, but preferably high purity EPO, and preferably EPO having substantially the same biological activity as mammalian EPO, particularly human EPO.

The EPO used in the present invention can be prepared by any one of methods, for example, it may be natural human EPO obtained by purification from an extract of human origin (see, for example, examined Japanese patent application laid-open No. Hei 1-38800), or it may be human EPO produced by genetic engineering techniques in Escherichia coli, yeast, Chinese hamster ovary cells (CHO cells), C127 cells, COS cells, myeloma cells, BHK cells or insect cells, and then extracted, isolated and purified by any one of various methods. The EPO used in the present invention is preferably EPO produced by genetic engineering techniques, and preferably EPO produced using mammalian cells, particularly CHO cells (see, for example, examined Japanese patent application laid-open No. Hei 1-44317, Kenneth Jacobs et al, Nature, 313,806-810 (1985)).

EPO obtained by gene recombination techniques may have the same amino acid sequence as EPO of natural origin, or may have the same biological activity as EPO of natural origin with deletion, substitution or addition of one or more amino acids in the amino acid sequence. The deletion, substitution or addition of amino acids can be introduced by methods known in the art. For example, one skilled in the art can prepare polypeptides functionally equivalent to EPO by introducing appropriate mutations in the amino acid sequence of EPO using site-directed mutagenesis (Gotoh, T. et al (1995) Gene 152, 271-275; Zoller, M.J. and Smith, M. (1983) Methods enzymol.100, 468-500; Kramer, W. et al (1984) Nucleic Acids Res.12, 9441-9456; Kramer, W. and Fritz, H.J. (1987) Methods enzymol.154, 350-367; Kunkel, T.A. (1985) Proc. Natl. Acad. Sci.USA.82, 488-492; Kunkel (1988) Methods enzymol.85, 2763-2766). Amino acid mutations may also occur naturally. In general, an amino acid residue is preferably substituted with another amino acid residue having similar amino acid side chain properties. Properties of amino acid side chains include, for example, 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 with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl functional side chains (S, T, Y), amino acids with sulfur-containing side chains (C, M), amino acids with carboxylic acid-or amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), and amino acids with aromatic side chains (H, F, Y, W) (the single letter codes of the amino acids are given in parentheses). Polypeptides having a modified amino acid sequence resulting from deletion, addition or substitution of one or more amino acid residues are known to retain their biological activity (Mark, D.F. et al, Proc. Natl. Acad. Sci. USA (1984)81, 5662-.

Fusion proteins of EPO with another protein may also be used. For example, a fusion protein can be constructed by ligating a DNA encoding EPO, a DNA encoding another protein in frame, inserting the DNA into an expression vector, and expressing the expression vector in a host. There is no particular limitation on the other protein fused to the EPO of the present invention.

Chemically modified EPO may also be used in the present invention. Chemically modified EPO includes EPO linked to inorganic or organic compounds such as polyethylene glycol or vitamin B12. The EPO used in the present invention may also be EPO derivatives.

As used herein, an EPO derivative refers to EPO having amino acid modifications in the EPO molecule, or EPO having sugar chain modifications in the EPO molecule. The sugar chain modification in the EPO molecule includes addition, substitution or deletion of sugar chains. The sugar chain modification preferred in the present invention is the removal of sialic acid from EPO molecules.

The obtained EPO produced by the recombinant animal cells or the EPO derived from urine is generally an EPO composition comprising various types of EPO having different sugar chain structures. The number of sialic acids attached to an EPO molecule in an EPO composition will vary with the EPO molecule, but generally, 11-15 sialic acids are attached to each individual EPO molecule. Non-sialylated EPO (asialo-EPO) may be prepared by removal of sialic acid. There is no particular limitation on the number of sialic acids removed during non-sialylation; all sialic acids may be removed, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 sialic acids may be removed. Preferred asialo-EPO molecules for use in the present invention contain no more than 10 sialic acids attached to the EPO molecule, more preferably no more than 5, and especially preferably no more than 2. The number of sialic acids mentioned herein refers to the average number of sialic acids on the EPO molecule contained in the EPO composition. The average sialic acid per molecule can be measured by methods known to those skilled in the art (see, e.g., EP 0428267).

EPO (asialo-EPO) from which sialic acid has been removed can be produced by methods known to those skilled in the art. For example, EPO can be prepared by treatment with an enzyme such as sialidase. Commercially available sialidases can be used for this purpose (see, for example, national publication of translation version 2005. sup. 507426; Nobuo Imai et al, Eur. J. biochem.194, 457. sup. sup. (1990)).

Examples of amino acid modifications in the EPO molecule include carbamoylation, biotinylation, amidination (amidination), acetylation, and guanidation (guanidation). Carbamylation is a preferred mode of amino acid modification in the present invention.

The amino acid residue to be modified is not particularly limited, and may include lysine, arginine, glutamic acid and tryptophan. Lysine is a preferred modified amino acid of the present invention.

Thus, EPO containing carbamoylated lysine is a particularly preferred embodiment of amino acid modified EPO of the present invention (see, e.g., Marcel L. et al, Derivatives of Erythropoietin thaat tissue protective but not erythropolitic. science, 2004; 305: 239; Fiordaliso E. et al, A nonerythropolitic derivative of erythropoli protectones protects of the myocarpidium free-perfusion input. PNAS, 2005; 102: 2046). Methods of EPO carbamylation include, for example, carbamylation by reaction with cyanate ions, alkylcarbamylation by reaction with alkyl isocyanates, and arylcarbamylation by reaction with arylisocyanates.

Pharmaceutical preparation

EPO can be formulated according to techniques known in the art by appropriately adding, for example, suspending agents, solubilizers, stabilizers, isotonic agents (isotonizers), preservatives, adsorption inhibitors, surfactants, diluents, excipients, pH adjusters, soothing agents, buffers, sulfur-containing reducing agents, antioxidants, and the like.

Suspending agents include methylcellulose, polysorbate 80, hydroxyethyl cellulose, acacia, powdered tragacanth, sodium carboxymethylcellulose, and polyoxyethylene sorbitan monolaurate.

Solubilizers include polyoxyethylene hydrogenated castor oil, polysorbate 80, niacinamide, polyoxyethylene sorbitan monolaurate, polyethylene glycol, and ethyl esters of castor oil fatty acids.

Stabilizers include dextran 40, methylcellulose, gelatin, sodium sulfite, and sodium metabisulfite.

Specific amino acids may also be added as stabilizers (see, for example, Japanese patent application laid-open No. Hei 10-182481). Amino acids added as stabilizers include, for example, free amino acids and salts thereof (e.g., sodium, potassium and hydrochloride salts). A single amino acid or a combination of two or more amino acids may be added. The amino acid to be added as the stabilizer is not particularly limited, but preferable amino acids in this respect are, for example, leucine, tryptophan, serine, glutamic acid, arginine, histidine and lysine.

Isotonic agents include D-mannitol and sorbitol.

Preservatives include methyl paraben, ethyl paraben, sorbic acid, phenol, cresol and chlorocresol.

Adsorption inhibitors include human serum albumin, lecithin, dextran, ethylene oxide-propylene oxide copolymers, carboxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenated castor oil, and polyethylene glycol.

Representative examples of the surfactant are nonionic surfactants, for example, nonionic surfactants having an HLB of 6 to 18, for example, sorbitan fatty acid esters such as sorbitan monocaprylate, sorbitan monolaurate and sorbitan monopalmitate, glycerin fatty acid esters such as glycerin monocaprylate, glycerin monomyristate and glycerin monostearate, polyglycerin fatty acid esters such as decaglycerin monostearate, decaglycerin distearate and decaglycerin monolinoleate, polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate and polyoxyethylene sorbitan tristearate, polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan tetrastearate and polyoxyethylene sorbitan tetraoleate Polysaccharide fatty acid esters, such as polyoxyethylene glycerolPolyoxyethylene glycerin fatty acid esters of oil monostearate, polyethylene glycol fatty acid esters such as polyethylene glycol distearate, polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether, polyoxyethylene-polyoxypropylene alkyl ethers such as polyoxyethylene-polyoxypropylene glycol, polyoxyethylene-polyoxypropylene propyl ether and polyoxyethylene-polyoxypropylene cetyl ether, polyoxyethylene alkylphenyl ethers such as polyoxyethylene nonylphenyl ether, polyoxyethylene castor oil and polyoxyethylene hardened castor oil (polyoxyethylene hydrogenated castor oil), polyoxyethylene/beeswax derivatives such as polyoxyethylene sorbitol beeswax, polyoxyethylene/lanolin derivatives such as polyoxyethylene lanolin, and polyoxyethylene fatty acid amides such as polyoxyethylene stearamide. Representative examples of additional surfactants are anionic surfactants, e.g., sodium lauryl sulfate and oleyl sulfate having C_10-18Alkyl sulfates of alkyl groups, with an average of 2-4 moles of ethylene oxide addition and having C, such as sodium polyoxyethylene lauryl sulfate_10-18Polyoxyethylene alkyl ether sulfate of alkyl group, and a compound having C such as sodium lauryl sulfosuccinate_8-18Alkyl sulfosuccinates of alkyl groups. Representative examples of additional surfactants are natural surfactants, e.g., lecithin, glycerophospholipids, sphingomyelins such as sphingomyelins, and mixtures with C_12-18Sucrose fatty acid esters of fatty acids. A single one of these surfactants or a combination of two or more of these surfactants may be added to the formulation of the present invention. Polyoxyethylene sorbitan fatty acid esters such as polysorbates 20, 40, 60 and 80 are preferred surfactants, with polysorbates 20 and 80 being particularly preferred. Polyoxyethylene-polyoxypropylene glycols such as poloxamers (e.g., Pluronic F-68 (registered trademark)) are also preferred.

Sulfur-containing reducing agents include, for example, N-acetylcysteine, N-acetylhomocysteine, thiolactic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid (thioglycolic acid) and salts thereof, sodium thiosulfate, glutathione and C_1-7Process for preparing thioalkanoic acidsA reducing agent containing a mercapto group.

Antioxidants include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α -tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbyl stearate, sodium bisulfite, sodium sulfite, tripentyl gallate and propyl gallate, and chelating agents such as disodium Ethylenediaminetetraacetate (EDTA), sodium pyrophosphate and sodium metaphosphate.

The components that may be suitably added also include inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate and sodium bicarbonate, and organic salts such as sodium citrate, potassium citrate and sodium acetate.

The contents of all patents and references explicitly cited in the specification of the present application are hereby incorporated by reference in their entirety. In addition, the contents of the specification and drawings of Japanese patent application No. 2006-158998, which is the basis of the priority claims of the present application, are incorporated herein in their entirety by reference.

The present invention is described in detail by the following examples, but the present invention is not limited by these examples.

Example 1

Mobilization of hematopoietic cells by EPO

Recombinant human EPO (Epogin (registered trademark)) was subcutaneously administered to mice (C57BL/6, mouse age: 9 weeks) once a day at a dose of 100. mu.g/kg/day for 4 days. Control mice were similarly administered the vehicle alone subcutaneously. On day 5, mice were sacrificed and peripheral blood cells (0.6-1mL) were collected. Erythrocyte lysis buffer (SIGMA) containing 8.3g/L ammonium chloride in 0.01M Tris-HCl buffer (pH 7.5. + -. 0.2) was added as a hemolytic agent, and left to stand at room temperature for 5 to 10 minutes, followed by washing with 2% BSF + PBS (-). At a ratio of 1. mu.g/10⁶Cell amount A non-specific reaction inhibiting antibody against CD16/CD32(Fc γ III/II receptor)(reaction blocking antibody) (BD-Pharmingen, San Diego, Calif.) and kept on ice for 30 minutes. A mixture of FITC-conjugated anti-mSca-1 antibody (BD Pharmingen, San Diego, CA), PE-conjugated anti-mCD 34 antibody, APC-conjugated anti-mc-Kit antibody (BD Pharmingen, San Diego, CA), and APC-Cy 7-conjugated anti-mCD 3, CD4, CD8a, CD45R/B220, Ly6G (Gr-1), CD11B (Mac-1. alpha. chain), and Ter119 antibody was used as lineage marker (linkage marker). After antibody staining, cells were analyzed by FACSVantage SE (Becton Dickinson, Mountain View, Calif.).

Fig. 1 shows the analysis results. By administration of EPO, c-Kit, as shown in R3 of FIG. 1⁺Sca-1⁺The number of Lin-cells increased approximately 13-fold. c-Kit⁺Sca-1⁺Lin-cells are markers for CFU-S-like cells in mice (see, e.g., Blood, 1992 Dec.15; 80 (12): 3044-50). The results reported above indicate that CFU-S-like cells are mobilized into the peripheral blood by administration of EPO.

CFU-S colony assay

Peripheral blood cells from C57/b6 mice that had been administered EPO5 days subcutaneously were used as donor cells. 10⁴-10⁵Cells/100. mu.L of donor cells were transplanted via the tail vein of recipient C57/b6 mice, and recipient C57/b6 mice had received lethal doses of gamma-radiation (from Hitachi Medicio) using a radiation irradiation device (Hitachi Medicio)¹³⁷950cGy for Cs). Spleens were excised 8-12 days after transplantation, and splenic colonies (number of colonies formed in the spleen) were analyzed by Bouin's staining.

FIG. 2 shows the results of the analysis in agreement with the results of the FACS analysis, and the mobilization of CFU-S-like cells into peripheral blood was observed.

Example 2

Angiogenesis treatment by peripheral blood mononuclear cell transplantation

The study was designed to investigate whether hematopoietic stem cells could be mobilized into the peripheral blood by administration of erythropoietin preparations in 5 patients with severe limb ischemic disease. Clinical studies have also been conducted for angiogenesis therapy by autologous transplantation of peripheral monocytes. The overall design of clinical studies employs an open study protocol.

Two weeks prior to the planned cell transplantation, the patient received 6000U of EPO by subcutaneous injection (first administration). One week prior to cell transplantation, blood was taken for autologous blood supply purposes (about 400mL) and received 6000U of EPO by subcutaneous injection (second administration). In the morning of the surgery, 6000U of EPO were injected (third administration) and approximately 10U of EPO was isolated from peripheral blood by apheresis of the blood⁹The peripheral mononuclear cell of (4). Peripheral blood samples were collected before EPO administration, after the second administration and just before apheresis, and blood tests (WBC, CRP, CK) and CD34 positive cell counts were performed.

Isolated peripheral monocytes are injected into the muscle of one or more limbs of a patient's ischemia at 50-100 sites. The effect of angiogenesis therapy was evaluated before cell transplantation, one day after administration, one week after administration, two weeks after 1 month, 2 months after and 6 months after, based on observations and measurements for the following items:

QOL: pain was assessed by Visual Analogue Scale (VAS), 0 for no pain and 10 for the most intense pain

Blood sample collection

Blood testing

Angiography

And (3) pedal test: absolute distance walked or distance at which pain occurred was measured in a horizontal condition at 2.4 km/h

Objective assessment of skin and ulcer lesions: ankle brachial index (ABPI), digital plethysmography, pedal test (ambulation), thermography, laser Doppler perfusion imager (LDP)I) And transdermal oxygen partial pressure (TdPO)₂) Measuring

Alteration of CD34 positive cells

A summary of cases, transplant cell counts, and CD34 positive cell counts is given in table 1 below. Normalized to 100% before erythropoietin administration, the proportion of CD34 positive cells in peripheral blood showed an increase of 82-245% (158% on average) when blood was drawn one week after the first erythropoietin administration. The growth rate was 88-175% (139% average) just before apheresis after another week. These results indicate that one week after the first administration of erythropoietin, the CD34 positive cell count (during exsanguination) is the same or greater than the cell count two weeks after (just prior to apheresis).

CD34 positive cells accounted for 0.02-0.1% (0.06% on average) of the monocytes recovered by plasmapheresis of blood.

Table 1.

Cases of disease

Sex

Age (age)

Cause of disease

Graft site

Total number of cells transplanted

CD34 positive cell

1

For male

77

Arteriosclerosis obliterans

Lower right limb

0.5×109

0.1×106(0.2×104Cells/kg)

2

For male

66

Arteriosclerosis obliterans

Left lower limb

16.7×109

5.0×106(8×104Cells/kg)

3

For male

48

Thromboangiitis obliterans

Double upper limbs

9.2×109

9.2×106(14×104Cells/kg)

4

For male

48

Thromboangiitis obliterans

The right upper limb

6.9×109

5.5×106(9×104Cells/kg)

Clinical evaluation of cell transplantation

Subjective symptom and angiographic evaluation

Table 2.

Cases of disease	Observation period	Situation(s)	VAS	Fontaine fractionation	Angiography
						1	11 month	Numbness and pain of rest	7→7	III→II	Without change

2	3 month	Numbness and pain of rest	2→0	III→II	Without change
						3	3 month	Ulcer of stomach	6→3	Not gradable	—
4	1 week	Ulcer of stomach	3→1	Not gradable	Has not been carried out

From a subjective point of view, improvement in pain was observed in 3 out of 4 cases.

In addition, revascularization was observed in the angiograms of case 31 month after transplantation (fig. 3).

Objective evaluation

Table 3.

Cases of disease

Observation period

ABPI

Volume tracing of finger (toe)

Thermogram

LDPI

TdPO2

1

11 month

0.24→0.4

Improvements in or relating to

Improvement in moderate degree

Without change

Without change

2

3 month

0.49→0.66

Improvements in or relating to

Without change

Without change

Without change

3

3 month

Has not been carried out

Improvements in or relating to

Improvement in moderate degree

Improvement in moderate degree

Improvements in or relating to

4

1 week

Has not been carried out

Improvements in or relating to

Has not been carried out

Has not been carried out

Has not been carried out

ABPI and TdPO were noted in some cases₂The improvement of (1). In case 2, a significant improvement in the distance traveled from 160m to 915m in the pedal test was seen.

The above results indicate that peripheral occlusive arterial disease can be treated by an angiogenic therapy in which erythropoietin is administered to mobilize hematopoietic stem cells into the peripheral blood, and peripheral monocytes are transplanted into ischemic skeletal muscle.

Also, the content of CD 34-positive cells in peripheral blood mononuclear cells recovered after one week of the first EPO administration was equal to or higher than the content of CD 34-positive cells in mononuclear cells recovered after two weeks, indicating that the mononuclear cells recovered after one week of EPO administration can provide a therapeutic effect of angiogenesis of the mononuclear cells recovered after two weeks or more.

Industrial applicability

The methods and compositions of the invention are useful for treating ischemic conditions such as peripheral vascular disorders.

Claims (6)

1. Use of erythropoietin in combination with monocytes for the preparation of a medicament for the treatment of an ischemic disease, wherein the erythropoietin is used to mobilize monocytes into peripheral blood, and wherein the monocytes are collected from the peripheral blood of a subject 3-12 days after erythropoietin administration.

2. Use of erythropoietin in combination with monocytes for the preparation of a medicament for stimulating revascularization, wherein the erythropoietin is used to mobilize monocytes into peripheral blood, and wherein the monocytes are collected from the peripheral blood of a subject 3-12 days after erythropoietin administration.

3. Use of mononuclear cells isolated from peripheral blood of a subject previously administered erythropoietin for the preparation of a medicament for transplantation to treat an ischemic disease, wherein the erythropoietin is administered 3-12 days before peripheral blood is collected from the subject.

4. Use of mononuclear cells isolated from peripheral blood of a subject previously administered erythropoietin for the preparation of a medicament for transplantation to stimulate revascularization, wherein the erythropoietin is administered 3-12 days before peripheral blood is collected from the subject.

5. Use of erythropoietin for the preparation of a medicament for transplantation for the treatment of an ischemic disease, wherein the erythropoietin is administered to a subject 3-12 days before peripheral blood is collected from the subject, wherein peripheral blood mononuclear cells are collected from the subject, and wherein the collected peripheral blood mononuclear cells are administered to a target site of the subject.

6. Use of erythropoietin for the preparation of a medicament for transplantation to stimulate revascularization in a subject, wherein the erythropoietin is administered to the subject 3-12 days prior to collecting peripheral blood from the subject, wherein peripheral blood mononuclear cells are collected from the subject, and wherein the collected peripheral blood mononuclear cells are administered to a target site of the subject.