JPH01106827A - Neurergic agent containing human b cell differentiation factor - Google Patents

Abstract

PURPOSE:To obtain a neurergic agent containing human B cell differentiation factor as an active ingredient, having differentiation-inducing function of nerve cell and useful in nervous system function disorder such as senile dementia. CONSTITUTION:A human B cell differentiation factor (human BCDF) having structure of amino acid sequence of polypeptide obtained by adding one A1a to amino acid sequence or N end amino acid sequence of natural human B cell differentiation factor expressed by the formula is contained as an active ingredient and as necessary stabilizer such as albumin, artificial cerebrospinal fluid and diluent such as mannitol are added thereto and the mixture is formulated to provide the aimed agent. The human BCDF is contained in the agent in amounts of 0.0001-100wt.%, preferably 0.1-1.0wt.%. The agent is administered to brain or ventricle using osmotic pump, etc. As the human BCDF, a human BCDF produced and purified by a known method or obtained by cultivating, producing and purifying a strain obtained by transforming a gene by a vector is used.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a neuroactive drug that targets nervous system dysfunction such as senile dementia.

[Prior art]

nerve growth factor
r; NGF) is essential for perception during the embryonic period, promoting the differentiation and growth of sympathetic nerve cells, and maintaining the survival and function of mature sympathetic nerve cells, and it has been shown in rats that NGF is synthesized and functions in the brain. , it has been revealed (Experimental Medicine, 4 57.1986) that N is also present in the human brain.
It has been reported that GF mRNA is high in the cerebral cortex and hippocampus, but low in the cerebellum, septum, and magnocellular basal ganglia (Goedert,
M., etalMol Brain Res, Vol. 85-92, 1986), suggesting that NGP may act as a trophic factor for cholinergic neurons in the basal forebrain in the human brain as well. . On the other hand, disorders of the cholinergic nervous system due to degeneration of the basal ganglia of Meynert are assumed to be the cause of Alzheimer's dementia (
Marx, J.

L,, 5science 232231-232.1
986), if NGF functions as a trophic factor in the brain, especially in the magnocellular cholinergic nervous system, it is expected to have potential as a therapeutic agent. Recently, it has been reported that intracerebral administration of NGF ameliorated cholinergic nervous system atrophy and memory impairment in old rats (Fishe
r.

Wetal, , Nature, 329 65-6
8.1987), which affirms the potential of this factor as a therapeutic agent for Alzheimer's dementia.

[Problem that the invention seeks to solve]

The object of the present invention is to provide a neuroactive drug containing a new factor different from NGF that acts on the nervous system.

[Means for solving the problem] H) The structure of BCDF has been determined as a factor that induces differentiation of B cells and enhances antibody production (Natural
e324 73 1986). This BCDF is currently
BSF-2 (B cell stimulating
factor-2) or IL-6 (Inter Le
It is called ukin-6).

As a result of extensive research, the present inventors discovered that a drug containing human BCDF as an active ingredient induces the differentiation of nerve cells and is effective against nervous system disorders such as senile dementia. completed.

That is, the present invention is a neuroactive drug containing human BCDF as an active ingredient. BCDF according to the present invention has, for example, the following amino acid sequence (I) or (II).

Amino acid sequence (■): Pro Val Pro Pro Gly Glu A
sp Ser Lys Asp Vat 8I Al
a Pro His Arg Gln Pr
o Leu Thr Ser 5erGlu
Arg Ile Asp Lys Gin
Tie Arg Tyr Ile LeuA
sp Gly lie Ser Ala L
eu Arg Lys Glu Thr C
ysAsn Lys Ser Asn Met
Cys Glu Ser Ser Lys
GluAlaLeuAlaGlu
Asn Asn Leu Asn Leu
Pro LysMet Ala Glu Lys
Asp Gly Cys Phe Gin Se
r GlyPhe Asn Glu Glu Thr
Cys Lea Vat Lys Ile l1
eThr Gly Leu Leu Glu Phe
Glu Val Tyr Leu Glu Tyr
Leu Gin Asn Arg Phe Glu
Ser Ser Glu GluGin Ala
Arg Ala Val Gln Met S
er Thr Lys ValLeu lie G
ln Phe Leu Gin Lys Lys
Ala Lys AsnLeu Asp Ala
lie Thr Thr Thr Pro Asp Pro
Thr Thr Asn Ala Ser Leu
Leu Thr Lys Leu Gin A
la GinAsn Gln Trp Leu Gi
n Asp Met Thr Thr ) Iis Le
ulle Leu Arg Ser Phe Lys
Glu Phe Leu Gin 5erSer L
eu Arg Ala Leu Arg G
in Met or amino acid sequence (■): Ala Pro Val Pro Pro
Gly Glu Asp Ser Lys
AspVal Ala Ala Pro His Ar
g Gin Pro Leu Thr 5erSer
Glu Arg lie Asp Lys G1. &
Ile Arg Tyr TleLeu Asp
Gly lie Ser Ala Leu
Arg Lys 'Glu ThrCys A
sn Lys Ser Asn Met C
ys Glu Ser Ser LysGlu
Ala Leu Ala Glu Asn
Asn Leu Asn Leu Pr.

Lys Met Ala Glu Lys Asp G
ly Cys Phe Gln 5erGly Ph
e Asn Glu Glu Thr Cy
s Leu Val Lys l1elie
Thr Gly Leu Leu Glu Phe G
lu Val Tyr LeuGlu Tyr Leu
Gln Asn Arg Phe Glu Ser
Ser GluGlu Gin Ala Arg Al
a Val Gin Met Ser Thr Lys
Val Leu Ile Gln Phe Leu G
in Lys Lys Ala Lys Asn Leu
Asp Ala lie Thr Thr Pro
Asp Pro Thr Thr Asn Ala
Ser Leu Leu Thr Lys
Leu Gln AlaGln Asn G
ln Trp Leu Gln Asp M
et Thr Thr
Leu Arg Ser Phe Lys Glu P
he Leu G1n5er Ser Leu Arg
Ala Leu Arg Gln Met amino acid sequence) is a natural human BCDF, and the amino acid sequence (
II) is a polypeptide in which one Ala is added to the N-terminus of natural human BCDF (hereinafter referred to as human Ala-BCDF). However, human BCDF used in the present invention does not necessarily have to have the structure shown in the above amino acid sequence (I) or (II).

In other words, natural type BCDF has a structure in which one or more amino acids are added to the N-terminus and/or C-terminus of BCDF, and natural type BCDF has one or more amino acids added to the N-terminus and/or C-terminus of BCDF. Human BCDF having a structure substituted with an amino acid can also be used as the human BCDF of the present invention as long as it has human BCDF activity. Preferably, natural human BCDF or human Ala-BCDF is used. The content of human BC leaf according to the present invention is 0.0001 to 100% by weight, preferably 0.0001 to 100% by weight in the neuroactive drug.
．． It is 1 to 1.0% by weight.

In addition, the neuroactive drug containing human BCDF as an active ingredient of the present invention includes a stabilizer such as serum albumin, an artificial brain, and cerebrospinal fluid.
Further, excipients such as mannitol may be included.

This neuroactive drug is osmotic in the brain or ventricles.
It is administered using pump etc.

Now, the human BC cells used in the present invention are obtained using known methods (Proc.

Natl, Acad, Sci, USA, 82.
5490 (1985)) can be transformed into a suitable host such as Escherichia coli, yeast, monkey cells (CO5 cells), hamster cells, etc. with the gene encoding human BC using an appropriate vector. Human BCDF produced by culturing or further purified may also be used. The production of Hi1-BC leaves will be explained again in Examples.

The present inventors discovered that Hi1-BC leaf induces differentiation of nerve cells. Due to this action, human BCDF has been found to be able to exhibit therapeutic effects on nervous system dysfunctions such as senile dementia.

Previously, it was known that NGF exhibited similar effects, but we discovered that human BCDF acts on a different receptor than NGF, has a slower effect, and has a different action function than NGF. .

[Effects] H) The drug of the present invention containing BCDF as an active ingredient has the function of inducing differentiation of nerve cells, and is therefore effective against nervous system dysfunction such as senile dementia.

Hereinafter, the present invention will be explained in more detail based on examples.

[Example 1: Production of human BCDF and human Ala-BC leaves] First, natural human BCDF and human Ala-B
The method for manufacturing e-leaves will be explained. In addition, unless otherwise specified, human BCDF means natural human Bc.
Let me show you the leaf.

(1) Manufacture of ΔHIL-2-BCDF Human BCDF and human 81a-BCDF were obtained by patent application in 1988-
Produced by the method described in No. 302699. In other words, human IL-
Plasmid pTBCD containing part of 2cDNA
HB 101 strain harboring F-12 (FERMp-90
62) at 25μg/streptomycin and 25μg
g/ml ampicillin (1% bactodryptone, 0.5% yeast extract, 0.5% NaCj2.
0.1% glucose, pH 7,5) 37 °C in 10 d
- Late in life. Next, 5 d of the culture suspension was mixed with M9-casamino acid medium (O, 6% NazllPO, '12HzO,
0,3%KH2PO4,0,05%Na(/!, 0.1
%No, cf, 0.05%MgSO4.71hO.

0.00147% CaC1z, 0.2% glucose, 0
．． 2% casamino acids, 0.02% L-leucine, 0.02
% L-Proline, 0.0002% Thiamine Hydrochloride, 1
00μg/1ydl ampicillin, 25μg/rrdl
Streptomycin, pH 7,4) was inoculated at 28°C.
The cells were cultured for 3 hours. After that, it seems to be 25μg/ml3
- Add indole acrylic acid (IAA) and at 23 °C
The cells were induced to be cultured for 21 hours. The cultured cells were collected by centrifugation, and 201M7ris-HCf buffer (pH 7.5") containing 30mM NaCl2 was added to make the cells concentrated 10 times. After suspension, lysozyme 1/d, EDT was added thereto.
After adding AO, 05M and stirring, the mixture was left in water for 1 hour. Next, the bacterial cells were destroyed by ultrasonic crushing, and 10
．． The granules were collected by centrifugation at 00 rpm for 5 minutes.

The granules were solubilized with 6M guanidine hydrochloride and ΔHIL-
The concentration was adjusted so that the 2-BCDFI degree was 100 μg/d and the 2M guanidine hydrochloride solution, and
Oxidized glutathione 1mM and reduced glutathione 10mM
M- was added and left at pH 8.0 and room temperature for 10-16 hours. Next, guanidine hydrochloride was removed by gel filtration using 5ephadex G-25, and at the same time, a fraction corresponding to human IL-2-BCDF (hereinafter referred to as Δold L-2-BC leaf equivalent fraction), which became a buffer solution for kallikrein reaction, was removed. ) was obtained. When this substance was subjected to 5OS-polyacrylamide gel electrophoresis, the molecular weight was almost the same as the value calculated from the amino acid composition, and the amino acid sequence on the N-terminal side was assayed using a protein sequencer. It was confirmed that the sequence was

(2) Cleavage with kallikrein 50mM Tris-H containing 113mM NaCl
(f! Δold L −2− obtained in buffer, pH 7,8
BCDF 21.4■ and human plasma kallikrein (
After reacting 73.5 μg (manufactured by Sigma) at 37° C. for 116 hours, approximately 55% acetonitrile and 0.1% TFA, which correspond to human Ala-BCDF, were fractionated using reverse phase liver LC. Analysis of the amino acid sequence near the N-terminus using a protein sequencer revealed that ΔHIL-2-BCD
F is human Ala-BCDF! It was confirmed that it was converted to white. The amount of human Ala-BCDF recovered was 18.03
mg, and the recovery rate was 84%. In addition, “human Ala-B
"C-Ko" means Ala 1 at the N-terminus of natural human BCDP.
This is what the individual added.

(3) Removal of N-terminal Ala by aminopeptidase P.
ol.

19, 521 (1970).

Add the human Ala-BC leaf solution obtained in (2) to 0.4 m
MMnCJ2. 50mM Tris-HCj! 5ephadex G equilibrated with buffer (pH 8,0)
A fraction corresponding to human Ala-BCDF was obtained by gel filtration using -25. Human 8Ia-BCDF thus obtained
2. Add aminopeptidase P to 02■ and heat at 37°C.
After 16 hours of reaction, a fraction corresponding to human BC leaves was collected using reverse phase 1 (PLC).Furthermore, as a result of analyzing the N-terminal amino acid sequence using a protein sequencer, human Ala-
It was confirmed that BCDF was quantitatively converted into human BC leaf. The amount of human BCDF recovered was 2.0 ■. The activities of human BCDP and human Ala-BC leaf are shown in Table 1. Activity units are Proc, Natl, Ac
ad, Sci, USA+ Dan 2. 5490
(1985).

Table 1 (4) Formulation of human BCDF human 1-BCDF or human Ala-BCDF IIPL
Fraction C was left at -2°C overnight, and the upper layer of acetonitrile was removed. The lower layer was subjected to gel filtration or dialysis using 5ephadex G-25 to remove residual acetonitrile and TFA, and replaced with a PBS solution. Dilute this, add human serum albumin or artificial brain as necessary, add cerebrospinal fluid or mannitol, and then sterile filter it to make human BC.
A DF formulation and a human Ala-BCDP formulation were used.

[Example 2: Induction of nerve cell differentiation by Hi1-BC leaf]
Neurite outgrowth was induced in the same manner as in the presence of

In the case of human BC leaves, this morphological change was shown 2-3 days after addition, but this was delayed compared to the case of NGF (~12 hours).

The effect of human BCDF on neuronal differentiation was investigated using changes in the number of sodium channels as an indicator.

PC cells were cultured in the presence of human BCDF (20 ng/d) for 11 days. The culture medium was replaced every two days. The binding of saxitoxin (a neurotoxin that specifically binds to sodium channels) is caused by the binding of [3H]-labeled saxitoxin (Amer
sham 68 Ci mmole-') (20μ
M). (311) Specific binding of labeled saxitoxin was determined by the difference in values in the presence and absence of tetrodotoxin (a neurotoxin that specifically binds to sodium channels at the same location as saxitoxin) (1 μM).

As shown in Table 2, human BCDF increased the number of sodium channels by about 7 times.

Table 2: Enhancement by BCDF of saxitoxin binding, a neurotoxin that specifically binds to thorium channels (-) 3.81±1.19 (n
= 5)NGF (50μg/In1) 2
2.2±1.88” (n=4) human BCDF (20
μg/ml) 26.3 ± 5.59” (n
=4) Values indicate the standard error of the mean of the number of experiments (n=4-5).

* indicates a significant difference (p<0.01) compared to (-).

[Example 3: Proof of the presence of human BCDF receptor in nerve cells] The presence of human BCDF receptor on PCI2 cells was investigated using 125■-labeled human BCDF. Binding was measured according to a conventional method. Figure 1 shows 5catch
Showing the results of ardanalysis, human BCDF
The dissociation constant of the receptor was 3.7 Xl0-9M, revealing the presence of approximately 2,500 receptors per cell.

In addition, a competition experiment was conducted using human BCDF and NGF for the binding of 125■-labeled human BC leaves to PCI2 cells.

That is, 125I-labeled human BCDF (9,7X 10-
Specific binding of 'mole/70 μl was determined in the presence of various concentrations of human) BCDF or NGF. The data are +2 in the absence of human BCDF or NGF.
Expressed as a percentage of binding of 51-labeled human BCDF. 450c for 2.5 x 106 cells
125I-labeled human BC leaf of pm was bound.

As shown in Figure 2, Hi1-BC leaf binding was specific and not inhibited by NGF.

[Brief explanation of the drawing]

Figure 1 shows PC1 using +25■ labeled human BCDF.
2 is a drawing showing binding to two cells. FIG. 2 is a diagram showing that the binding of 125I-labeled human BCDF to PC12 cells is specifically inhibited only by human BCDF rather than NGF. □□=== 1 to 1 human BCDF bound (moj2ecul, e/c
euQX10)]

Claims (3)

[Claims] (1) Neuroactive drug containing human B cell differentiation factor (hereinafter referred to as BCDF) as an active ingredient (2) Neuroactive drug amino acid sequence (I) according to claim 1, in which human BCDF has the following amino acid sequence (I): [There is a gene sequence] (3) Neuroactive drug amino acid sequence (II) according to claim 1, in which human BCDF has the following amino acid sequence (II): [There is a gene sequence]