JP3954004B2 - Drugs for preventing and treating diseases caused by brain dysfunction - Google Patents

Description

  The present invention relates to a therapeutic agent used for preventing and treating a disease caused by cerebral dysfunction, and more specifically, comprising erythropoietin, granulocyte colony stimulating factor or macrophage colony stimulating factor as an active ingredient. The present invention relates to a prophylactic / therapeutic agent for diseases caused by cerebral dysfunction.

  Diseases caused by cerebral dysfunction include Alzheimer's disease that is inherited and progresses rapidly, Alzheimer's senile dementia that develops as an elderly person and progresses slowly, and ischemic disorders such as cerebral infarction and cerebral hemorrhage Also known are cerebrovascular dementias such as memory impairment, intelligence impairment, reduced motivation, and reduced attention, which are thought to be associated with cerebral circulation disorders. In these diseases, the major feature is that the symptoms of memory impairment are prominent in the early stages of onset. This is because cholinergic neurons in the basal ganglia are relatively selectively degenerated in the early stages of disease progression. It is thought to be caused by dropping out.

  Diseases caused by such brain dysfunction are becoming a social problem especially in the recent aging population, and the pharmaceutical industry is urgently required to develop therapeutic drugs to fundamentally prevent and treat these diseases. It is said that.

  Therefore, conventionally, the development of therapeutic agents as well as the mechanism of the onset of such diseases has been studied from various aspects, and attempts have been made to develop several therapeutic agents. For example, Alzheimer-type senile dementia is accompanied by a decrease in the function of the cholinergic nervous system in the brain, that is, a decrease in the amount of acetylcholine in the brain. In addition, use of an acetylcholinesterase inhibitor that inhibits the activity of acetylcholinesterase, which is an acetylcholine degrading enzyme, has been proposed. In fact, the use of physostigmine, tetrahydroaminoacridine and the like as acetylcholinesterase inhibitors has been proposed. However, these drugs have problems such as insufficient therapeutic effects on diseases caused by cerebral dysfunction such as Alzheimer-type senile dementia, and undesirable side effects.

In addition, recently, the purpose is to increase the amount of acetylcholine in the brain as described above, but it does not utilize the acetylcholinesterase inhibitory action described above, but activates the activity of acetylcholine transferase (ChAT), which is an acetylcholine synthase. The use of a substance having an acetylcholine transferase activation action (ChAT activation action) has been proposed, and actually a therapeutic / preventive agent for senile dementia comprising interleukin 3 (IL-3) as an active ingredient [Japanese Patent Laid-Open No. 3-93,728 Publication, Kamegai et al., Neuron, 4 , 429-436 (March 1990), Kamegai et al., Brain Research, 532 , 323-325 (1990)) and nerve growth factor (NGF) Acts on nerves, sensory nerves, forebrain cholinergic neurons, etc. to promote their differentiation and maturation, and is effective for survival and function maintenance (Dev. Brain Res. 9 , 45-52 (1983)), Granulocyte-Macro It has been reported that phage colony-stimulating factor (GM-CSF) has an action as a neurotrophic factor in vitro [Kamegai et al., Brain Research, 532 , 323-325 (1990)].
Japanese Unexamined Patent Publication No. 3-93,728 Kamegai et al., Neuron, 4, 429-436 (March 1990) Kamegai et al., Brain Research, 532, 323-325 (1990) Dev. Brain Res. 9, 45-52 (1983) Kamegai et al., Brain Research, 532, 323-325 (1990)

  Therefore, the present inventors have focused on the fact that cholinergic neurons in the basal ganglia are relatively selectively degenerated and dropped out, characterized by a disorder caused by cerebral dysfunction, which is accompanied by a symptom of memory impairment early in the onset. As a result of extensive research on the development of therapeutic agents that can prevent and treat cholinergic neuronal degeneration, hematopoietic factors such as erythropoietin (EPO), granulocyte colony-stimulating factor (G- CSF) and macrophage colony stimulating factor (M-CSF) have excellent cell survival prolonging action and acetylcholine transferase activation action, and are found to be effective in the prevention and treatment of diseases caused by cerebral dysfunction. did.

Accordingly, an object of the present invention is to provide a prophylactic / therapeutic agent effective for the prevention and treatment of various diseases caused by cerebral dysfunction.
Another object of the present invention is to provide a prophylactic / therapeutic agent effective in the prevention and treatment of diseases in which the cell survival prolonging action and / or acetylcholine transferase activation action is effective. Furthermore, an object of the present invention is to provide a prophylactic / therapeutic agent effective for the prevention and treatment of Alzheimer's disease, Alzheimer type senile dementia or cerebrovascular dementia.

  The present invention is a prophylactic / therapeutic agent for diseases caused by cerebral dysfunction, which contains one or more hematopoietic factors selected from erythropoietin, granulocyte colony stimulating factor and macrophage colony stimulating factor as active ingredients.

  The hematopoietic factors erythropoietin, granulocyte colony stimulating factor and macrophage colony stimulating factor used as active ingredients in the present invention may be produced by any method as long as they have essential effects as hematopoietic factors. it is conceivable that. That is, it may be extracted from nature or may be produced by a gene recombination technique. In this case, the transformed cells may be either prokaryotic cells or eukaryotic cells.

That is, for erythropoietin (EPO), for example, it corresponds to natural human EPO (Japanese Patent Publication No. 1-38,800) obtained by extraction from urine of a patient with aplastic anemia and the amino acid sequence of human EPO. Messenger RNA (mRNA) is collected, and a recombinant DNA body is prepared using the mRNA, and then a suitable host (for example, fungi such as E. coli, yeast, plant cell lines, COS cells, Manufactured by genetic recombination technology produced in Chinese hamster ovary cells (CHO), mouse cell lines such as mouse C-127 cells, etc.) [eg, Japanese Patent Publication No. 1-44,317, Kenneth Jacobs et al., Nature, 313 , 806-810 (1985)] and the like. Specific examples include natural human urine EPO and EPO such as rhEPO / CHO, which is a recombinant human EPO produced using Chinese hamster ovary cells (CHO) as a host.

  As for granulocyte colony-stimulating factor (G-CSF), for example, natural human G-CSF obtained by culturing human G-CSF-producing cells and extracting, separating and purifying them from the culture supernatant (specifically, No. 1-44,200), or a product obtained by transforming a host such as Escherichia coli or animal cells by gene recombination and isolating or purifying it or chemically modifying it. (For example, Japanese Patent Publication Nos. 2-5,395, JP-A 62-129,298, JP-A 62-132,899, JP-A 62-236,488, and JP-A 64-85,098). Specifically, for example, natural human G-CSF, rhG-CSF / CHO, which is a recombinant human G-CSF produced using Chinese hamster ovary cells (CHO) as a host, E. coli. G-CSF such as rhG-CSF / E. Coli, which is a genetically modified human G-CSF produced using as a host.

  Further, for macrophage colony stimulating factor (M-CSF), for example, natural human M-CSF obtained by extraction, separation and purification from a biological sample such as human urine (for example, JP-A-64-22,899). JP-A-3-17,021) and those produced by gene recombination technology (for example, JP-A-62-501,607 and JP-A-1-502,397) . Specifically, for example, natural human M-CSF, rhM-CSF / CHO, which is a recombinant human M-CSF produced using Chinese hamster ovary cells (CHO) as a host, E. coli (E. coli). And M-CSF such as rhM-CSF / E. Coli, which is a recombinant human M-CSF produced using as a host.

  Such administration routes of EPO, G-CSF or M-CSF include intracerebral administration in which a drug is surgically administered directly into the brain and intracerebral spinal fluid administration in which the drug is directly injected into the cerebrospinal fluid. In addition, the possibility of intravenous injection is also expected.

  The dose of EPO, G-CSF or M-CSF can be appropriately determined in consideration of the target disease or its disease state, etc. The dose is usually 1 for adults. 0.1 to 500 μg per person, preferably 5 to 100 μg, and G-CSF is usually 0.1 to 1,000 μg, preferably 1 to 700 μg per adult, and M-CSF. Is usually 0.1 to 1,000 μg, preferably 1 to 700 μg per adult.

  Furthermore, for preparations containing EPO, G-CSF or M-CSF of the present invention as an active ingredient, suspending agents, solubilizers, stabilizers, isotonic agents, as necessary, depending on the administration method and dosage form. Agents, preservatives, adsorption inhibitors and the like can be 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. Polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinic acid amide, polyoxyethylene sorbitan monolaurate, tuna gorgol, castor oil fatty acid ethyl ester and the like can be mentioned as stabilizers such as human serum albumin, dextran 40, Examples thereof include methylcellulose, gelatin, sodium sulfite, and sodium metasulfite. Examples of isotonic agents include D-mannitol and sorbitol. Examples thereof include methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, phenol, cresol, chlorocresol, and the like, and examples of the adsorption inhibitor include human serum albumin, lecithin, dextran, ethylene oxide / propylene. Examples thereof include an oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenated castor oil, and polyethylene glycol.

Next, experimental examples for confirming the effects of the present invention will be shown below.
[1] Preparation of primary cultured cells A septal area including the forebrain base was obtained from BALB / mouse (Sankyo Experimental Service, Tokyo) on the 15th day of fetal period. Tissues were excised and minced in Hanks' balanced salt solution (HBSS solution, pH 7.4) and treated with HBSS solution (pH 6.5) containing 0.03% trypsin at 37 ° C. for 3 minutes. . After filtration through a 63 μm nylon mesh, the suspension was resuspended in serum-free medium. The culture was started at 6 × 10 5 cells / ml. At the start of cell culture, mouse βNGF (mβNGF, Sigma, MO) 100 ng / ml, recombinant human macrophage colony stimulating factor (rhM-CSF) (Genzyme, Massachusetts, USA) 10 CFU / ml, 50 CFU / ml or 100 CFU / ml ml, recombinant human granulocyte colony stimulating factor (rhG-CSF) (manufactured by Chugai Pharmaceutical Co., Ltd.) 10 CFU / ml, 50 CFU / ml or 100 CFU / ml, or recombinant human erythropoietin (rhEPO) (manufactured by Chugai Pharmaceutical Co., Ltd.) 1 IU / ml, 5 IU / ml, or 10 IU / ml, and after 3 days, the culture medium was changed. On the 5th day, the cells were collected with a rubber policeman (a device fitted with rubber at the end of a glass tube). The choline acetyltransferase (ChAT) activity was measured by the method.
The results are shown in Table 1.

The serum-free medium is Dulbecco's modified Eagle's medium (DMEM) (Gibco, New York, USA) containing 4.5 g / l glucose and Ham's F12 supplemented with the following components (Gibco) ( 1: 1 mixture with pH 7.4). That is, 15 mM HEPES buffer, 30 nM sodium selenate, 1% penicillin-streptomycin solution (Gibco), 100 μg / ml human transferrin, 25 μ / ml bovine crystallized insulin, 20 nM progesterone, 20 nM hydrocortisone -21-phosphate, 10 mM L-carnitine, 30 nM 3,3 ′, 5-triiodo-L-thyronine, 7 ng / ml tocopherol, 7 ng / ml retinol, 1 μM thioctic acid, and 1 μl / ml Mineral mixture [Hutchings et al., PNAS, 75 , 901-904 (1978)]. Unless otherwise indicated, the compounds used were those manufactured by Sigma (Louisiana or Missouri, USA).

[2] Preparation of cultured cells of SN6.10.2.2 SN6.10.2.2 cells stored at the University of Chicago, USA and provided by Dr. Wainer of this university are mouse septal area neurons and mouse neuroblasts. It is a subclone of SN6 cells [Hanmond et al., Science, 234 , 127-1240 (1986)] established as a hybrid cell with the tumor N18TG2. Cells were maintained in DMEM containing 10% fetal bovine serum. Prior to use, 2 × 10 5 cells / ml were washed twice with HBSS solution (pH 7.4) and once with serum-free medium. After culturing for 2 days in a 35 mm dish (Becton Dickinson, NJ, USA) in a test medium containing rhM-CSF, rhG-CSF or rhEPO, the cells were collected with a rubber policeman on the 3rd day, and the following method was used. The ChAT activity was measured.
The results are shown in Table 2.

[3] Preparation of in vivo brain model by cutting Fimbria-Fornix (brain arch) Wister-type albino male rats (CLEA, Tokyo, Japan) weighing 300-350 g (30-50 mg / kg pentobarbital sodium) Sodium pentobarbital) was anesthetized, and the head was fixed to a brain stereotaxic device (Narishige Kikai Co., Ltd., Tokyo). Next, Fimbria-Fornix cutting was performed according to the following procedure. That is, a 3 mm square portion having two perpendicular sides of a line 0.5 mm behind and a median line from Bregma of the left skull was excised, and the left dorsal side Fimbria and Fornix were removed together with the cortex. A hole was drilled 0.2 mm forward from the Bregma of the right skull and 1 mm to the right of the midline, and a 1 mm diameter cannula (Kunii, Tokyo) was inserted into this hole.

  Through this cannula, 125 I.U./15 .mu.l / day or 12.5 I.U./15 .mu.l / day of 2 dose rhEPO was administered continuously for 4 days to an experimental group consisting of 3 rats each. As a control group, 5 μg / 15 μl / day of β-NGF or 15 μl / day of physiological saline was administered under the same conditions as described above.

Rats 14 days after surgery were intramuscularly administered with 2 mg / kg diisopropyl fluorophosphate, and 4 hours later, 100 ml saline and 300 ml 4% paraformaldehyde and 0.1% It was perfused with a chilled 0.1 M phosphate buffer containing glutaraldehyde. The brain was removed and fixed with 2% Zamboni solution for 4 days, and then left overnight in a 10% sucrose solution at 4 ° C. A coronal section having a thickness of 20 μm was prepared and stained by a modification of the method of Butcher et al. [Butcher et al. Neuron, 7 , 197-208 (1991)].

Image analysis of acetylcholinesterase (AchE) positive cells, that is, nerve cells with a minimum diameter of 12 μm along the main axis of the medial septum, according to Gage et al. [Gage et al. Neuroscience, 19 , 241-255 (1986)] The software (Olympus Optical Co., Tokyo) was used for investigation.

  The number of AchE positive cells was counted for both fimbria-Fornix transcutaneous and contralateral regions of individual animals, and the number of AchE positive cells present on the cut side was divided by the number of cells present on the opposite side. The percentage was defined as the survival rate of acetylcholinergic neurons.

The results are shown in FIG.
The column in the figure is Mean ± 1S.D., ** indicates p <0.01, EPO-H is the 125I.U./15 μl / day administration group, EPO-L is 12 Each of the 5I.U./15 μl / day administration groups is shown.

[4] Measurement of choline acetyltransferase (ChAT) activity and protein quantification ChAT activity was determined based on the method of Fonnum [F. Fonnum, J. Neurochem., 24 , 407-409 (1975)]. That is, the primary cultured cells or SN6.10.2.2 cultured cells described above were collected with a rubber policeman, homogenized in 30 μl of 10 mM EDTA solution, and further the final concentration of 0.5% (v / v) Triton-X100 was obtained. What was added was used as the enzyme source. The enzyme reaction solution was 300 mM NaCl, 50 mM sodium phosphate buffer (pH 7.4), 20 mM EDTA, 0.2 mM [1-14C] acetyl-CoA (purchased from Amersham Japan) and 8 mM Choline bromide as substrate. 0.1 mM Physostigmine is added as an acetylcholinesterase inhibitor. 5 μl of this reaction solution was placed in a 1.5 ml microtube (Eppendorf, Germany), 2 μl of enzyme solution sample was added, and the mixture was gently shaken and reacted at 37 ° C. for 15 minutes. The reaction was stopped by cooling with ice, the microtube was placed in a liquid scintillation vial, and the reaction mixture was washed with 5 ml of 10 mM Phosphate buffer. To this vial, 2 ml of acetonitrile containing 10 mg / ml of potassium tetraborate and 10 ml of toluene scintillator were added and shaken gently for 1 minute. After the vial was allowed to stand for 10 minutes, the amount of acetylcholine labeled with 14C was determined by a liquid scintillation counter to determine ChAT activity.

Proteins were quantified according to the Lowry method [Lowry et al., J. Biol. Chem., 193 , 265-275 (1951)]. For this purpose, 10 μl of the enzyme solution sample described above was used.
Based on these values, specific activity, total protein amount, and total ChAT activity were calculated. A t-test was performed for ChAT activity, protein concentration and viability.

  As apparent from the results shown in Table 1 above, the ChAT ratio after 5 days when rhM-CSF, rhG-CSF or rhEPO of the present invention was added to the primary culture system of septal area neurons in the dose shown in the table In the case of rhM-CSF, the results of investigating the activity, the total protein amount and the total ChAT activity were 25% and 44% respectively for the total protein amount at Dose: 10 CFU / ml, 50 CFU / ml and 100 CFU / ml with respect to Control. In the case of rhG-CSF, Dose: 10 CFU / ml, 50 CFU / ml and 100 CFU / ml increase the total protein amount by 12%, 24% and 18%, respectively, and rhEPO In this case, the total protein amount was increased by 31%, 25% and 38%, respectively, with Dose: 1 IU / ml, 5 IU / ml and 10 IU / ml with respect to Control. These values show a result equivalent to or more than 23% of the effect of mβNGF (100 ng / ml) tested as a control. The hematopoietic factors used in the present invention are similar to the neurotrophic factor mβNGF. It was proved that the survival of primary cultured neurons was promoted.

  As for the ChAT specific activity, rhM-CSF was increased by 17% with respect to Control at Dose: 50 CFU / ml and 100 CFU / ml, and rhG-CSF was increased at Dose: 10 CFU / ml, 50 CFU / ml and 100 CFU / ml. Increased by 1%, 25% and 14%, respectively, and rhEPO increased by 20% and 18% at Dose: 5 IU / ml and 10 IU / ml, respectively. Furthermore, regarding the total ChAT activity, rhM-CSF was increased by 20%, 70%, and 37% at dose: 10 CFU / ml, 50 CFU / ml, and 100 CFU / ml, respectively, and rhG-CSF was dose: 10 CFU. increase by 14%, 55% and 33% at 50 CFU / ml and 100 CFU / ml, respectively, and rhEPO is 19%, 49% and 62% at Dose: 1 IU / ml, 5 IU / ml and 10 IU / ml, respectively. Increased. From these results, it was found that the hematopoietic factor, rhM-CSF, rhG-CSF or rhEPO used in the present invention exerts an excellent effect also in the ChAT activation action.

  Further, Table 2 shows the effects of rhM-CSF, rhG-CSF and rhEPO of the present invention on the ChAT specific activity, total protein amount and total ChAT activity of SN6.10.2.2 cells. As can be seen from the fact that the amount is almost unchanged between the case of Control and the case of adding the hematopoietic factor, the influence only on the differentiation traits of the hematopoietic factors can be observed alone. As is apparent from the results of this experiment, rhM-CSF (50 CFU / ml), rhG-CSF (50 CFU / ml) and rhEPO (10 IU / ml) increased the ChAT specific activity by 61%, 68% and 80%, respectively. And increased total ChAT activity by 80%, 58% and 74%, respectively.

In addition, as shown in FIG. 1, in the rhEPO-administered group, survival of AchE-positive cells in the septal area of rats unilaterally cut by Fimbria-Fornix compared to the control group (saline, 0.1% BSA). The rate was significantly improved.
The in vivo effects of rhEPO on acetylcholinergic neurons in rat septal areas that were unilaterally cut by Fimbria-Fornix are significant in considering actual clinical development.
As is clear from the above experiments, rhM-CSF, rhG-CSF and rhEPO of the present invention are hematopoietic factors that were originally discovered to have an effect on blood cell systems, However, it was found to have the same activity as mβNGF. That is, in the primary culture of mouse septal area neurons in vitro, the total protein amount is remarkably increased and ChAT specific activity and total ChAT activity are increased, and the mouse septal area cholinergic nerve cell line SN6.10.2 .2 also increases ChAT specific activity and total ChAT activity in cells. Therefore, these hematopoietic factors, rhM-CSF, rhG-CSF and rhEPO, exhibit excellent cell survival prolongation and ChAT activation. Two effects are demonstrated.

  Furthermore, the effect of supporting the survival of acetylcholinergic neurons was also observed in in vivo experiments, that is, Fimbria-Fornix nerve pathway disruption systems. In this system, the supply of NGF produced in the hippocampus and transported axons retrogradely is disrupted by cleavage, and the acetylcholinergic neurons in the septum are unable to receive the supply and die. Therefore, it was revealed that rhEPO has NGF-like Neurotrophic Factor activity in vivo.

  Therefore, rhM-CSF, rhG-CSF and rhEPO of the present invention are effective against diseases such as Alzheimer's disease and Alzheimer-type senile dementia, etc., in which cholinergic neurons degenerate and drop and cause memory impairment. In addition to being effective, cerebral ischemic disorders such as cerebral infarction and cerebral hemorrhage, and memory disorders, intelligence disorders, decreased motivation, and reduced attention, etc. It is valid.

  However, the adaptation diseases of the hematopoietic factor, rhM-CSF, rhG-CSF and rhEPO of the present invention are not limited to the diseases involving cholinergic neurons as described above. That is, the action of mβNGF on the central nervous system is limited to peripheral sympathetic nerves or sensory nerves and cholinergic nerves of the brain and has a relatively narrow cell specificity, whereas rhM-CSF and rhG- CSF and rhEPO are originally factors for blood cell lineage and are thought to have the potential to act as survival promoting factors for a wider variety of neuronal cells in the brain. It is thought that it is a factor to exert. For this reason, rhM-CSF, rhG-CSF and rhEPO of the present invention are not limited to diseases involving cholinergic neurons. For example, dopaminergic neurons in the basal ganglia nigra are degenerated. As a prophylactic agent for diseases caused by cerebral dysfunction, such as Parkinson's disease that occurs as a result, and Huntington's chorea caused by degeneration of GABAergic neurons in the striatum (caudate nucleus, putamen) of the basal ganglia, Or it is promising as a therapeutic agent.

  The prophylactic / therapeutic agent containing erythropoietin, granulocyte colony stimulating factor or macrophage colony stimulating factor of the present invention as an active ingredient has an excellent cell survival prolonging action and ChAT stimulating action, and has low side effects. It is useful as a pharmaceutical applied to various diseases caused by cerebral dysfunction such as Alzheimer's disease, Alzheimer type senile dementia or cerebrovascular dementia.

Hereinafter, based on the Example regarding a formulation, suitable embodiment of this invention is described concretely.
Example 1
Erythropoietin 8μg
2ml in distilled water for injection
A solution was prepared aseptically at the above composition ratio, dispensed into vials, and sealed.

Example 2
Erythropoietin 8μg
2ml in distilled water for injection
A solution was prepared aseptically at the above composition ratio, dispensed into vials, lyophilized and sealed.

Example 3
Erythropoietin 16μg
2ml in distilled water for injection
A solution was prepared aseptically at the above composition ratio, dispensed into vials, and sealed.

Example 4
Erythropoietin 16μg
2ml in distilled water for injection
A solution was prepared aseptically at the above composition ratio, dispensed into vials, lyophilized and sealed.

Example 5
Erythropoietin 8μg
Human serum albumin 5mg
2ml in distilled water for injection
A solution was prepared aseptically at the above composition ratio, dispensed into vials, and sealed.

Example 6
Erythropoietin 8μg
Human serum albumin 5mg
2ml in distilled water for injection
A solution was prepared aseptically at the above composition ratio, dispensed into vials, lyophilized and sealed.

Example 7
Erythropoietin 16μg
Human serum albumin 5mg
2ml in distilled water for injection
A solution was prepared aseptically at the above composition ratio, dispensed into vials, and sealed.

Example 8
Erythropoietin 16μg
Human serum albumin 5mg
2ml in distilled water for injection
A solution was prepared aseptically at the above composition ratio, dispensed into vials, lyophilized and sealed.

Examples 9-12
An injection was prepared in the same manner as in Examples 5 to 8 except that 5 mg of dextran 40 was used instead of human serum albumin in Examples 5 to 8.

Example 13
An aqueous solution was prepared by aseptically dissolving 5 g of D-mannitol, 1 mg of erythropoietin and 100 mg of human serum albumin in 100 ml of distilled water for injection, dispensed 1 ml into vials, lyophilized and sealed.

Example 14
Aseptically dissolve 0.5 mg of erythropoietin and 1 g of sorbitol in 50 ml of 0.05M phosphate buffer at pH 7.0 to prepare an aqueous solution, dispense 0.5 ml into vials, and freeze-dry And sealed. Separately, a 0.1% -methylcellulose aqueous solution was prepared aseptically, and 1 ml was dispensed into ampoules to obtain a solution for dissolution.

Example 15
75 μg / ml of purified human G-CSF (10 mM phosphate buffer pH 7.0) and 15 mg / ml of D-mannitol were dissolved in distilled water for injection to make 0.3 ml, and then 0.22 μm The solution was sterilized by filtration through a membrane filter having a pore size. The obtained solution was filled into a sterilized vial, a rubber stopper similarly sterilized was half-capped, and then wound around an aluminum cap to obtain a solution preparation for injection. The solution preparation for injection is stored in a cool dark place at 10 ° C. or lower.

Example 16
50 μg / ml of purified human G-CSF (10 mM phosphate buffer pH 7.0) was adjusted with NaCl to an osmotic pressure ratio of 1, and then sterilized by filtration through a membrane filter having a pore size of 0.22 μm. The obtained solution was filled into a sterilized vial, and a similarly sterilized rubber stopper was struck, and subsequently wrapped around an aluminum cap to obtain a solution preparation for injection. The solution preparation for injection is stored in a cool dark place at 10 ° C. or lower.

Example 17
100 μg / ml of purified human G-CSF (10 mM phosphate buffer pH 7.0) was adjusted with NaCl to an osmotic pressure ratio of 1, and then sterilized by filtration through a membrane filter having a pore size of 0.22 μm. The obtained solution was filled into a sterilized vial, and similarly sterilized rubber stoppers were plugged, and subsequently wound around an aluminum cap to obtain a solution preparation for injection. The solution preparation for injection is stored in a cool dark place at 10 ° C or lower.

Example 18
After dissolving by adding HSA concentration 10 mg / ml and D-mannitol concentration 50 mg / ml to 50 μg / ml of purified human G-CSF (10 mM phosphate buffer pH 7.0), 0.22 μm The solution was sterilized by filtration through a membrane filter having a pore size. The obtained solution was filled into a sterilized vial, a rubber stopper similarly sterilized was half-capped, and then wound around an aluminum cap to obtain a solution preparation for injection. This solution preparation for injection is stored at room temperature or lower and diluted with distilled water for injection before use.

Example 19
After adding and dissolving to 100 μg / ml of purified human G-CSF (10 mM phosphate buffer pH 7.0) to a gelatin concentration of 10 mg / ml and D-mannitol concentration of 50 mg / ml, 0.22 μm The solution was sterilized by filtration through a membrane filter having a pore size. The obtained solution was filled into a sterilized vial, a rubber stopper similarly sterilized was half-capped, and then wound around an aluminum cap to obtain a solution preparation for injection. This solution preparation for injection is stored at room temperature or lower and diluted with distilled water for injection before use.

Example 20
75 μg / ml of purified human M-CSF (10 mM phosphate buffer pH 7.0) and 15 mg / ml of D-mannitol were dissolved in distilled water for injection to make 0.3 ml, and then 0.22 μm The solution was sterilized by filtration through a membrane filter having a pore size. The obtained solution was filled into a sterilized vial, a rubber stopper similarly sterilized was half-capped, and then wound around an aluminum cap to obtain a solution preparation for injection. The solution preparation for injection is stored in a cool dark place at 10 ° C. or lower.

Example 21
50 μg / ml of purified human M-CSF (10 mM phosphate buffer pH 7.0) was adjusted with NaCl to an osmotic pressure ratio of 1, and then sterilized by filtration through a membrane filter having a pore size of 0.22 μm. The obtained solution was filled into a sterilized vial, and a similarly sterilized rubber stopper was struck, and subsequently wrapped around an aluminum cap to obtain a solution preparation for injection. The solution preparation for injection is stored in a cool dark place at 10 ° C. or lower.

  The prophylactic / therapeutic agent containing erythropoietin, granulocyte colony stimulating factor or macrophage colony stimulating factor of the present invention as an active ingredient has an excellent cell survival prolonging action and ChAT stimulating action, and has low side effects. It is useful as a pharmaceutical applied to various diseases caused by cerebral dysfunction such as Alzheimer's disease, Alzheimer type senile dementia or cerebrovascular dementia.

FIG. 1 is a graph showing the cell survival prolonging effect of rhEPO in the in vivo model of the brain obtained in Experimental Example [3]. In the figure, the vertical axis represents the relative cell viability and the horizontal axis. Indicates experimental groups, respectively.

Claims (3)

A prophylactic / therapeutic agent for brain dysfunction containing granulocyte colony-stimulating factor as an active ingredient. Brain dysfunction, an agent for the prophylaxis or treatment of brain dysfunction according to claim 1, wherein cell viability prolongation and / or acetylcholine transferase activation action is a valid dementia. Brain dysfunction, Alzheimer's disease, an agent for the prophylaxis or treatment of brain dysfunction according to claim 1, wherein the Alzheimer's type senile dementia or cerebrovascular dementia.

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