JP2006089505A - Therapeutic agent for ischemic diseases - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an effective therapy particularly for obstructive arteriosclerosis which can eliminate drawbacks with conventional therapies, such as kinesitherapy, pharmacotherapy, and revascularization, and recently proposed therapies, such as gene therapy and intramuscular transplantation of bone marrow cells. <P>SOLUTION: An effective agent for treating ischemic diseases, containing human granulocyte colony-stimulating factor (human G-CSF) as an active ingredient is disclosed. Furthermore, the therapeutic agent can be used as an agent for treating ischemic diseases, such as ischemic cerebrovascular disorder or ischemic heart. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to a therapeutic agent for ischemic disease comprising human granulocyte colony stimulating factor (hereinafter abbreviated as human G-CSF) as an active ingredient.

The present invention relates to a therapeutic agent for ischemic disease. First, obstructive arteriosclerosis, which is one of the typical ischemic diseases, will be described.
Obstructive arteriosclerosis is a disease in which arteriosclerotic lesions cause obstruction or stenosis of the main artery of the extremities, particularly the lower limbs, resulting in ischemic damage in the periphery. Clinical symptoms include coldness, numbness, intermittent claudication. It is classified as resting pain, ulcer / necrosis. It is estimated that there are approximately 100,000 patients with obstructive arteriosclerosis in Japan (Yusuke Tada: Therapeutics, Vol. 31, pp. 289-292; 1997), and will continue to increase due to an increase in the elderly population and westernization of diets. It is thought to do. Treatment for obstructive arteriosclerosis includes exercise therapy, drug therapy, and revascularization depending on symptoms and patient condition, and recently, gene therapy and intramuscular transplantation of bone marrow cells have also been attempted. ing.
Qun Shi et al. Blood vol92, 362-367; 1998 Takayuki Asahara et al. Circulation Research vol85, 221-228; 1999 Mario Peichev et al. Blood vol95, 952-958; 2000)

Although the above-mentioned therapies currently have certain results in the treatment of obstructive arteriosclerosis, the following problems exist in each therapy. In other words, although exercise therapy has been observed in mild cases with an effect of extending walking distance, the effect of this therapy is difficult to predict, and even if there is an effect of extending walking distance, patients are not satisfied with this and blood circulation reconstruction There is also a report that 30% of patients wished to have surgery (Takashi Ota: Nippon Medical Newspaper, No. 3935, pages 25-29; 1999), and this is not a very effective treatment.

  As for pharmacotherapy, prescription anti-platelet drugs are those that prevent the worsening of the pathology, and even if they are mildly developed as microcirculatory blood flow improvers or oxygen transport capacity improvers that have been developed recently In any case, there is no fundamental therapeutic agent for obstructive arteriosclerosis.

  On the other hand, revascularization is currently the most effective treatment, and percutaneous angioplasty and bypass surgery are performed depending on the patient's condition and the location and extent of the lesion. There are problems such as complications and deaths associated with surgery, and long-term survival may not be expected.

  In gene therapy, treatments using genes for angiogenic factors such as vascular endothelial growth factor and epithelial growth factor have been carried out, but they are not yet in the field of experimental treatment and are safe and effective. Since the evaluation about is not solidified, it is not popular.

Intramuscular transplantation of bone marrow cells, which has recently been reported to have a therapeutic effect, is a therapy in which bone marrow cells are transplanted into muscles near the affected area, and this is differentiated into vascular endothelial cells to form blood vessels and treat. Although it is necessary to increase the number of cases and evaluate the effect in the future, it is expected as a future treatment method because severe cases can be treated. However, this therapy also seems to have a problem that the burden on patients and medical staff accompanying bone marrow collection is large.

  According to recent research, hematopoietic stem cells that can differentiate into vascular endothelial cells not only in the bone marrow but also in peripheral blood (referred to as “endothelial cell progenitor cells” from the viewpoint of their ability to differentiate into endothelial cells). Since it has been derived from hematopoietic stem cells, it has been found that the term “hematopoietic stem cells” is used in this specification in the concept of a cell population that can also be endothelial cells, and that this is involved in angiogenesis. (Qun Shi et al. Blood vol 92, 362-367; 1998, Takayuki Asahara et al. Circulation Research vol 85, 221-228; 1999, Mario Peichev et al. Blood vol 95, 952-958; 2000). Therefore, it is expected that obstructive arteriosclerosis can be treated by collecting hematopoietic stem cells in peripheral blood and transplanting them into the muscle in the vicinity of the affected area. In this case, it is advantageous that the burden on patients and medical staff involved in collecting peripheral blood stem cells is lighter than that when transplanting stem cells in the bone marrow, but the frequency of hematopoietic stem cells in the peripheral blood is usually very low. Therefore, it is doubtful whether a sufficient amount of hematopoietic stem cells can be obtained for the treatment of obstructive arteriosclerosis.

Human G-CSF is a hematopoietic factor discovered as a differentiation and growth factor of granulocyte hematopoietic progenitor cells,
Since it promotes neutrophil hematopoiesis in vivo, it is clinically applied as a therapeutic agent for neutropenia after bone marrow transplantation and cancer chemotherapy. In addition to the above actions, human G-CSF has an action to act on hematopoietic stem cells to stimulate their proliferation and differentiation and an action to mobilize hematopoietic stem cells in the bone marrow into the peripheral blood. Based on this, peripheral blood stem cell transplantation in which peripheral blood hematopoietic stem cells mobilized by human G-CSF are transplanted for the purpose of promoting the recovery of hematopoiesis in cancer patients after undergoing powerful chemotherapy in clinical settings Yes. This hematopoietic stem cell mobilization effect of G-CSF is much stronger than GM-CSF, the same granulocyte hematopoietic factor. In addition, G-CSF has an advantage over GM-CSF in that there are few side effects.

Therefore, prior to treatment by intramuscular transplantation of bone marrow cells for patients with obstructive arteriosclerosis, administration of human G-CSF can be expected to increase the frequency of hematopoietic stem cells in the bone marrow. The number of bone marrow punctures can be reduced and the burden on the patient can be reduced. At this time, by obtaining hematopoietic stem cells to be transplanted from peripheral blood, the burden on the patient and medical staff can be further reduced. Furthermore, since hematopoietic stem cells in peripheral blood have been shown to contribute to angiogenesis, it is conceivable to promote this by increasing hematopoietic stem cells in peripheral blood by administration of human G-CSF. Therefore, it is expected that occlusive arteriosclerosis can be treated only by administering human G-CSF to a patient. This treatment of obstructive arteriosclerosis by administration of human G-CSF clearly reduces the burden on patients and medical staff in that hematopoietic stem cells are not collected or transplanted.

The treatment of obstructive arteriosclerosis using human G-CSF in the three forms described above can be expected to be effective in severe patients, which is a great gospel for patients, but promotes the differentiation and proliferation of vascular endothelial progenitor cells When used in combination with angiogenic factors such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), or gene therapy thereof Furthermore, the therapeutic effect is expected to increase. In this case, these factors or genes thereof can be administered to the patient, for example, in the vicinity of the affected area. Similarly, antiplatelet agents, vasodilators, microcirculatory improvers, anticoagulants, hyperlipidemia agents, etc. that are clinically used as pharmacotherapy for obstructive arteriosclerosis are also treated. The effect is expected to increase.

Furthermore, the G-CSF of the present invention can be applied as a therapeutic agent for the following diseases, which are the same ischemic diseases. That is, the present invention relates to trauma containing G-CSF as an active ingredient, rejection at the time of transplantation, ischemic cerebrovascular disorder (stroke, cerebral infarction, etc.), ischemic kidney disease, ischemic lung disease, infection. The present invention provides therapeutic agents for ischemic diseases, limb ischemic diseases, ischemic heart diseases (ischemic cardiomyopathy, myocardial infarction, ischemic heart failure, etc.) and the like.

As a result of the above ideas, we have reached the present invention. That is, this invention provides the ischemic disease therapeutic agent which uses human G-CSF as an active ingredient.
The present invention is described in detail below.

Human G-CSF is a protein having the amino acid sequence shown in the following formula 1, but human G-CSF used in the present invention is substituted for one or more amino acids in the protein having this sequence. Even if the mutant has been manipulated, the protein shown in Formula 1 and various modifications of the protein can be applied as long as they have G-CSF activity. “Various modifications” as used herein refer to the structural transformation / addition / deletion operation of sugar chains and the binding of inorganic or organic compounds such as polyethylene glycol and vitamin B12.

Formula 1: Amino acid sequence of human G-CSF
Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 16
Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 32
Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val 48
Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys 64
Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser 80
Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser 96
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp 112
Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro 128
Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 144
Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe 160
Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 174
Such a method for producing human G-CSF is not limited as long as the method defined in the above item is produced. Specifically, human G-CSF producing tumor, human G-CSF producing hybridoma, Manufactured using a transformed host that has been given G-CSF production ability by genetic recombination. Depending on the structure of human G-CSF to be produced, modification and various modification operations are applied as appropriate at appropriate stages of the production process. The In addition, when producing by gene recombination, the host normally used, such as Escherichia coli and an animal cell, is employ | adopted regardless of the host.

  The therapeutic agent for ischemic disease of the present invention can contain a preparation carrier and excipient necessary for taking a form as a pharmaceutical preparation, and further contains a stabilizer and an adsorption inhibitor. Depot preparations, nasal preparations, oral preparations, pulmonary preparations, transdermal preparations, transmucosal preparations, and the like can be selected, and appropriate devices can be used as necessary.

The dose and frequency of human G-CSF contained in the therapeutic agent for ischemic disease of the present invention can be determined in consideration of the pathology of the subject patient, but the dose is usually 0.1 to 500 μg / kg per adult. / day, preferably 1 to 50 μg / kg / day, and can be administered for 1 to 7 days per week. The administration method is preferably intravenous, subcutaneous or intramuscular. However, the present invention is not limited by the dose of human G-CSF, and has been used so far for antiplatelet agents, vasodilators, microcirculatory improvers, anticoagulants, hyperlipidemia therapeutic agents and the like. Drugs that can be expected to be effective against ischemic diseases, and can be used in combination with gene therapy.

Hereinafter, the present invention will be described in more detail with reference to experimental examples (pharmacological effects) and examples (formulation examples), but the present invention is not limited thereto.
Experimental Example 1 (Pharmacological effect)
The left femoral artery and vein of a nude mouse (BALB / cA Jcl-nu) was removed after ligation and a lower limb ischemia model was created. In the untreated group, 3 of 5 cases (60%) had leg dropout and 2 cases (40%) had lower extremity necrosis 2 weeks after ischemia. On the other hand, in the group administered G-CSF subcutaneously 100 μg / kg / day for 5 times from 3 days before lower limb ischemia until 1 day after surgery, 1 of 5 cases (20% ), 3 necrosis cases (60%), 1 case (20%) showed no damage and lower leg injury than the untreated group. This indicates that G-CSF has the effect of improving leg injury after ischemia, possibly through promoting angiogenesis.
Experimental Example 2 (Pharmacological effect)
A mouse (BALB / cA) was subcutaneously administered with G-CSF 100 μg / kg / day for 5 days, blood was collected, and a mononuclear cell fraction was collected using a density gradient method (d = 1.077). Nude rats (F344 / N Jcl-rnu
The left femoral artery and vein were removed, and a lower limb ischemia model was created. One day after ischemia, peripheral blood mononuclear cells derived from G-CSF-administered mice were placed in the ischemic limbs of ischemic nude rats with about 2x10 ⁷ cells / head (about 5 mL of peripheral blood)
Equivalent to intramuscular administration and transplantation. In the control group, phosphate buffer was administered intramuscularly. One week after transplantation, a lower limb tissue sample was prepared, and the capillary density was measured by alkaline phosphatase staining. As a result, the capillary density tended to be higher in the peripheral blood mononuclear cell administration group than in the control group. (Control group: 38.3 ± 1.7, peripheral blood mononuclear cell administration group; 42.3 ± 2.1 number of capillaries / five visual field groups, mean ± standard error). The results are shown in A and B of FIG.

This suggests that G-CSF promoted endothelial progenitor cell mobilization into the peripheral blood of mice, and as a result, promoted angiogenesis in rats transplanted with it. This suggests the possibility of application.
Experimental Example 3 (Pharmacological effect)
The left femoral arteriovenous of a nude rat (F344 / N Jcl-rnu) was removed, a lower limb ischemia model was created, and the capillary density was measured from alkaline phosphatase staining of a lower limb tissue specimen one week after ischemia. Group administered G-CSF subcutaneously 100 μg / kg / day from 4 days before to 1 week after ischemia (G-CSF administration group)
And the control group. In the control group, phosphate buffer was administered intramuscularly. As a result, it was shown that the capillary density was higher in the G-CSF administration group than in the control group (control group; 38.3 ± 1.7, G-CSF
Administration group: 44.7 ± 2.4 Number of capillaries / field of view 5 animals in each group, mean ± standard error). The results are shown in A and C of FIG.

This result suggests that G-CSF promotes angiogenesis at the ischemic site, and suggests the possibility of application to peripheral circulatory disorder treatment.
Example 1 (formulation example)
Polysorbate 20 (Tween20: polyoxyethylene sorbine monolaurate), a nonionic surfactant, is added to human G-CSF (10 mM phosphate buffer pH 7.0) 50 μg / mL to 0.1 mg / mL, and NaCl is added. After adjusting the osmotic pressure to 1, filter sterilize with a membrane filter having a 0.22 mm bore size. The obtained solution was filled into a sterilized vial, stoppered with a similarly sterilized rubber stopper, and subsequently wrapped with an aluminum cap to obtain a solution preparation for injection. This injectable preparation is stored in a cool dark place at 10 ° C or lower.
Example 2 (formulation example)
Polysorbate 80 (Tween 80: polyoxyethylene sorbine monooleate), a nonionic surfactant, is added to 100 μg / ml of human G-CSF (10 mM phosphate buffer pH 7.0) to a concentration of 0.1 mg / mL, NaCl. After adjusting the osmotic pressure to 1, filter sterilize with a membrane filter having a 0.22 mm bore size. The obtained solution was filled into a sterilized vial, stoppered with a similarly sterilized rubber stopper, and subsequently wrapped with an aluminum cap to obtain a solution preparation for injection. This injectable preparation is stored in a cool dark place at 10 ° C or lower.
Example 3 (formulation example)
Human G-CSF (10 mM phosphate buffer pH 7.0) 50 μg / ml, nonionic surfactant polysorbate 20 (Tween20: polyoxyethylene sorbine monolaurate) 0.1 mg / mL, HAS 10 mg / mL and mannitol 50 mg After adding and dissolving to a volume of / ml, sterilize by filtration with a membrane filter having a 0.22 mm bore size. The obtained solution was filled into a sterilized vial, and a similarly sterilized rubber stopper was half-capped and lyophilized to obtain a freeze-dried agent for injection. This lyophilisate for injection is stored at room temperature or lower and dissolved in distilled water for injection before use.

The therapeutic agent for ischemic disease comprising human G-CSF of the present invention as an active ingredient can be expected to have a therapeutic effect on a relatively severe case of obstructive arteriosclerosis as shown in Experimental Examples 1 to 3. Since this G-CSF effect is presumed to be based on the promotion of angiogenesis, other ischemic diseases such as trauma, transplant rejection, ischemic cerebrovascular disorders (stroke, cerebral infarction, etc.), Expected to have therapeutic effects on ischemic kidney disease, ischemic lung disease, ischemic disease associated with infection, limb ischemic disease, ischemic heart disease (ischemic cardiomyopathy, myocardial infarction, ischemic heart failure, etc.) . The treatment according to the present invention is simple, safe and effective as compared with conventional treatment methods.

It is a figure which shows the influence which administration (B) of G-CSF administration mouse-derived peripheral blood mononuclear cells and G-CSF administration (C) have on rat ischemic limb capillary density. The capillary density of each individual was plotted for group B, group C, and subject group (A).

Claims (2)

A therapeutic agent for peripheral circulation disorder comprising human granulocyte colony-stimulating factor as an active ingredient, wherein the stimulating factor is directly administered to a patient in need of the therapeutic agent, and hematopoietic stem cells from the patient after administration of the stimulating factor A therapeutic agent for peripheral circulatory disorder characterized in that it does not include a step of collecting and administering to a site of ischemia and is used in combination with a drug clinically used as a pharmacotherapy for the disease. The therapeutic agent according to claim 1, wherein hematopoietic stem cells increased in peripheral blood by administration contribute to angiogenesis in the affected area.

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