JP3156236B2 - Remedies for improving memory impairment - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a kind of cytokine,
The present invention relates to a prophylactic, ameliorating, and therapeutic agent for Alzheimer's-type memory impairment, which contains an erythropoietin (hereinafter abbreviated as EPO) protein as a hematopoietic factor as an active ingredient.

[0002]

2. Description of the Related Art EPO is a highly glycosylated protein having a molecular weight of 30 to 40 kDa and has been discovered as a hematopoietic factor which promotes erythropoiesis, and its entire amino acid sequence has already been revealed [Jacobs et al, Nature, 313, 806-810 (198
Five)〕. In addition, human EPO is produced in large quantities in high purity by genetic engineering means and has already been applied as a therapeutic agent for anemia [Urabe et al, Int. J. Cell Cloling,
6, 179-191 (1988)]. The action of EPO is carried out through the EPO receptor present on the surface of the target cell of EPO, and it has been clarified that the EPO receptor belongs to the cytokine superfamily [D'Andrea et a.
l, J. Clin. Invest., 86, 681-687 (1990)]. From the viewpoint of repairing central nervous system disorders, studies on the effects of cytokines as neurotrophic factors have been actively conducted in recent years.
3 enhances choline acetyltransferase (ChAT activity) in cultured rat septal area neurons and PC-12 cells derived from cholinergic neurons [Takeshi Tahira et al., Human Science Research Report 1990, 3rd Field 3-4
-1-A, 288-302 (1991)], and that IL-6 promotes the development of dendrites in PC-12 cells [Kunio Torii, Metabolism, 2
6, Extra Issue, Brain Metabolism and Its Abnormalities, 195-201 (1989)].

On the other hand, in Alzheimer's disease patients,
A remarkable decrease in ChAT activity in the brain and cerebrospinal fluid is observed [Nakamura Shigenobu, Protein Nucleic Acid Enzyme, 36, 30-40 (1991)]. That is, in central degenerative diseases such as Alzheimer's dementia, degeneration of cholinergic neurons in the basal forebrain (septal area, blocker diagonal zone and basal ganglia of Meinert) causes the neurotransmitter acetylcholine. Is not produced and a significant decrease is caused. Decreased acetylcholine is thought to be the cause of impaired memory learning in Alzheimer's dementia [Whiteahouse et al, Science, 215, 123].
7-1239 (1982); Coyle et al, Science, 219, 1184-119.
0 (1983)]. Experimentally, it has been reported that destruction of cholinergic neurons in the basal forebrain of rats causes a decrease in acetylcholine levels and memory impairment [Helper e
tal, J Neurosci, 5, 866-873 (1985)]. The 1980s, the correlation of LTP and learning and memory of hippocampal been noted, the brain by a rise in intracellular calcium to induce LTP through a calmodulin-dependent <br/> kinase of stone C kinase [Hachiro Nakagawa, Metabolism, 26, extra edition, Brain metabolism and its abnormalities, 37-44
(1989)].

[0004] From the above, drugs that act on cholinergic neurons to increase ChAT activity (eg, NGF)
And substances that increase the concentration of intracellular calcium are thought to have preventive and therapeutic effects on central degenerative diseases such as Alzheimer's dementia. In recent years,
Among the senile diseases that increase rapidly with aging, especially senile Alzheimer's dementia, there is no effective treatment,
It has become a social problem, and the development of an effective therapeutic drug is eagerly desired.

[0005]

SUMMARY OF THE INVENTION The present invention relates to the application of a drug acting on nerve cells to the prevention, amelioration and treatment of Alzheimer's-type memory disorders. That is, an object of the present invention, preventing Alzheimer's type memory impairment, is to provide an agent for improving or treating.

[0006]

In view of the above technical background, the present inventors have focused on EPO, which is an erythropoietic factor among cytokines, and have developed a human neuroblast cell line (derived from cholinergic neurons) PC- When the effect of EPO on 12 cells was closely examined, EPO acted on PC-12 cells,
The present inventors have found that they have an effect of increasing the intracellular Ca ²⁺ concentration, and have completed the present invention.

[0007] That is, the present invention provides a method for preventing Alzheimer-type memory impairment containing an EPO protein as an active ingredient.
It relates to improvement and therapeutic agents. The EPO protein may be any substance as long as it has an EPO activity, that is, a differentiation / proliferation action of erythroid progenitor cells. Such an EPO protein may be a natural human urine-derived EPO protein, or may be an EPO protein produced by genetic engineering techniques,
Alternatively, mutein may be used. Examples of the EPO mutein include a mutein described in JP-A-3-151399. As the EPO Mi Interview Te in, there is one Asn of the peptide chain of the original glycoprotein mutated to Gln, number of bonds N-linked sugar chains bound is mutated. In addition, amino acid mutations include those described in JP-A-2-59.
No. 599 and JP-A-3-72855. That is, as long as the action characteristics of EPO to the EPO receptor are not lost, amino acid mutation, deletion,
Any number may be added.

[0008] The EPO of the present invention has already been clinically applied as a therapeutic agent for anemia, and no notable side effects have been reported. In this way, the therapeutic agent has low toxicity and can be used safely. EPO acts on PC-12 cells to increase the acetylcholine content in the cells, so it acts on brain nerve cells to increase the amount of acetylcholine, and is used for prevention, amelioration and treatment of Alzheimer's-type memory impairment. be able to. Alternatively, it acts on brain nerve cells to regulate Ca ²⁺ concentration in nerve cells to induce long-term potentiation. This therapeutic agent can be prepared by adding and dispersing 10,000 to 100,000 units in a pharmaceutically acceptable liquid according to a known pharmaceutical manufacturing method. Further, the obtained solution may be freeze-dried and dissolved in a physiological saline solution at the time of use to obtain a solution for use at the time of use. Furthermore, it can be used stably by adding a general drug used for a known biological preparation as a stabilizer, for example, human serum albumin or sugar. Further, amino acids and fatty acids can be added as other compounding agents.

The present agent for ameliorating and treating a memory disorder can be ameliorated and treated for an Alzheimer-type memory disorder by administering it directly into brain tissue as a method of administration. It is desirable that the injection site is directly injected into the septum of the brain. In addition, the dose can be appropriately changed depending on the degree of the disorder.
Administer about 00 IU.

Next, the present invention will be described more specifically with reference to examples and experimental examples. However, the present invention is not limited to these Examples and Experimental Examples.

Example 1 Manufacture of a preventive, ameliorating and therapeutic agent for Alzheimer's type memory disorder EPO derived from human urine 6,000,000 IU Human serum albumin 2,500 mg The total volume was adjusted to 1 liter with distilled water for injection. 1 ml of this composition
Filled into ampoules, sterilized, and EPO 6000
An IU injection was made.

[0011]

Example 2 Preparation of Agent for Prevention / Improvement / Treatment of Alzheimer's-type Memory Impairment 120,000,000 IU Genetically Modified EPO Human Serum Albumin 2,500 mg The total volume is made up to 1 liter with distilled water for injection. This composition was filtered through a Millipore filter, and 1 ml was placed in a sterile vial.
Each was dispensed, lyophilized and sealed. By dissolving the contents of this vial in 1 ml of physiological saline, 12
A 000 IU injection was made.

[0012]

Example 3 Production of Preventive / Improvement / Therapeutic Agent for Alzheimer's-type Memory Disorder 2.44 ml (2,10
(0.00IU) 20% human serum albumin 7.75 ml Distilled water for injection 144.81 ml After mixing and dissolving the whole amount, the mixture was filtered through a Millipore filter, filled into sterilized vials in 0.5 ml portions, freeze-dried and sealed. This vial corresponds to 6,000 IU.

Hereinafter, experimental examples using the therapeutic agent of the present invention will be described.

EXPERIMENTAL EXAMPLE 1 Measurement of EPO Receptor in PC-12 Cells Derived from Nerve Cells The receptor assay and the measurement of the molecular size of the receptor were performed according to the method of Sasaki et al. [Eur. J. Biochem., 168, 43-48.
(1987)]. PC-12 cells (ATCC
CRL-1721) 1-8 × 10 ⁶ cells / 150 μl,
The reaction was performed at 15 ° C. for 2 to 3 hours at an EPO concentration of 0.5 to 10 nM. E of EPO receptor on PC-12 cells
The binding affinity constant for PO was calculated as Kd = 8 nM, and the number of receptors was calculated to be about 1000 per cell. Also, SD
The molecular size of the EPO receptor by S electrophoresis is
It was calculated to be about 66 kDa.

[0014]

EXPERIMENTAL EXAMPLE 2 Characteristics of EPO Receptor of PC-12 Cells As in Experimental Example 1, the affinity of EPO for the EPO receptor of PC-12 cells and the inhibition of the binding of sugars, chelating agents, cytokines and hormones were measured. did. Measurement is ¹²⁵
The ability to inhibit this specific binding was measured using I-rHuEPO. The specific binding was represented by a relative rate, where the binding inhibition rate of ¹²⁵ I-EPO without addition was 100. ¹²⁵ I for smaller numbers
-Significant inhibition of binding between EPO and PC-12 cells. That is, the added substance has a strong binding affinity to the EPO receptor.
The characteristics of the EPO receptor of PC-12 cells thus measured are shown in Tables 1 to 3. The EPO receptor is
Human EPO, mutant EPO lacking the N-linked sugar chain binding site, and mouse EPO were recognized (Table 1). R shown in Table 1
HuEPO is a genetically modified EPO, rHuEPO
(N-glycanase treatment) was performed by using the recombinant EPO by the method of Tsuda et al. (Tsuda et. Al., J. Biochem, vol. 188, 405-41).
NQ123 was prepared according to the method disclosed in JP-A-3-151399 and lacked three N-linked sugar chains per molecule.
PO Mutein. In addition, various other cytokines (Table 3) and sugars (Table 2) did not affect the binding of EPO to the receptor. That is, the EPO receptor of PC-12 cells specifically bound to EPO.

[0015]

[Table 1] Specificity of various EPO binding to PC-12 cells Additive amount ¹²⁵ I-rHuEPO (ng in 150 μl) Specific binding (%) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ {No addition} 100 rHuEPO 15 16 rHuEPO (N-glycanase treatment) 15 29 NQ123 (rHuEPO mutein lacking N-linked sugar chain binding site) 15 21 rat EPO 15 23} ━━━━━━━━━━━━━━━━━━━━━━━━ * rHuEPO: Recombinant human erythropoietin * ¹²⁵ I-rHuEPO shows specific binding to ¹²⁵ I- It was expressed as a relative value of the amount of binding between EPO and PC-12 cells. The smaller the value, the stronger the binding affinity of the added substance to the EPO receptor.

[0016]

[Table 2] Influence of sugar and chelating agent on binding between PC-12 cells and EPO ━━━━ Additive amount (mM) Specific binding of ¹²⁵ I-rHuEPO (%) ━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━Nano── 100 N-acetylgalactosamine 10 105 galactose 10 95 EDTA 40 98 (ethylenediaminetetraacetate) EGTA 4091 (ethylene glycol bis beta aminoethyl ether NN N'N ' -4 acetate) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ * ¹²⁵ I-rHuEPO and PC-12 cells Of the specific binding of
It was represented by a relative value of 0. The smaller the value, the greater the inhibition of binding. That is, the additive has a strong binding affinity to the EPO receptor.

[0017]

[Table 3] Binding specificity of various cytokines and hormones to PC-12 cells Volume ¹²⁵ I-rHuEPO (ng in 150 μl) Specific binding (%) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ No addition 100 rHuEPO 125 16 human IL-2 50 102 monkey IL-3 50 117 human IL-6 200 92 mouse G-CSF 500 92 human GM-CSF 500 82 human EDF 500 100 human TNF 500 99 human HGF 500 102 mouse NGF 500 91 human EGF 500 97 human insulin 1000 102 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━ * ¹²⁵ specific binding of I-rHuEPO is At the time of addition of ^{125 I-rHuE}
It was expressed as a relative value of the amount of binding between PO and PC-12 cells. The smaller the value, the stronger the binding affinity of the added substance to the EPO receptor.

[0018]

[Experimental Example 3] Measurement of calcium-increasing activity of rHuEPO in PC-12 cells
Measurement of constant intracellular calcium concentration is described by Grynkiewiez et al.) J. Bi
o. Chem., 260, 3440-3450 (1985))
-12 cells were performed at 5 × 10 ⁶ cells / ml. EPO
The 20 unit / ml increase in PC-12 intracellular calcium concentration was comparable to the effect of adding 10 μM of bradykinin. Table 4 shows the amount of EPO added and the intracellular calcium concentration.

[0019]

TABLE 4 Effect of EPO on PC-12 cell calcium levels Additives Amount of addition Intracellular calcium concentration Relative increase in the number of measurements (%) (vs. basal level) ━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━ EPO 0.3 unit / ml 143 ± 40 12 〃 3 〃149 ± 46 12 １０10 〃 160 ± 48 12 ２０20 〃 169 ± 45 7 EPO (20 units / ml) addition Thereafter, EGTA (1 mM) 126 ± 93 was added. Bradykinin 10 μM 169 ± 40 10 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━ results of the above experiments, EPO was confirmed on gel intracellular calcium concentration of nerve cells.

[0020]

Industrial Applicability The preparation containing the EPO protein of the present invention as an active ingredient acts as a neurotrophic factor on nerve cells and has preventive, ameliorating, and therapeutic effects on Alzheimer's-type memory impairment. -It can be advantageously used as an improvement / therapeutic agent.

────────────────────────────────────────────────── ─── Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) A61K 38/00-38/58 BIOSIS (DIALOG) CA (STN) MEDLINE (STN)

Claims (3)

(57) [Claims] 1. A preventive, ameliorating, or therapeutic agent for Alzheimer's-type memory disorder, comprising an erythropoietin protein as an active ingredient. 2. The preventive, ameliorating or therapeutic agent according to claim 1, wherein the erythropoietin protein is erythropoietin or erythropoietin mutein protein produced by a genetic engineering technique. 3. The preventive, ameliorating, or therapeutic agent according to claim 1, wherein the dosage form is a direct injection into the septum in the brain.