JPH05125097A - New protein acting on nerve cell and glia cell and medicine containing the same protein - Google Patents

Abstract

PURPOSE:To obtain the subject new protein having specific partial amino acid sequences and molecular weight and axon extension action and glia cell proliferation-suppressing action, useful as treating agent for cerebrospinal diseases such as dementia and antitumor agent for cerebral tumor. CONSTITUTION:A tumor lump of neurofibroma obtained from a patient of Von Recklinghausen's disease is homogenized in 20mM tris-hydrochloric acid buffer solution (pH7.5) containing a protease inhibitor and 1mM dithiothreitol and then centrifuged to separate supernatant, and the supernatant is passed through an anionic column and the absorbed ingredients are eluted with a concentration gradient of sodium chloride. Fractions having glia cell proliferation- suppressing action are collected and passed through a hydrophobic chromatography column and then purified with a hydroxyapatite column chromatography to provide the objective protein having about 50kd molecular weight, and containing partial amino acid sequences, MXR, FER, AGE, formula I, formula II, VAAAXDD, RGTPWL, GTPWLR, formula III, formula IV, ALQEAXV and formula V.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a protein having a molecular weight of about 50 kd, which has both a neurite outgrowth effect and a glial cell growth inhibitory effect, and a pharmaceutical containing the protein as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Cells constituting the brain are roughly classified into nerve cells and glial cells. A nerve cell has a cell body and a protrusion. There are two types of processes: axons that transmit nerve information to other nerve cells and dendrites that receive information from other nerve cells. Nerve information is transmitted from one nerve cell to the next nerve cell through the adhesion part (called synapse) of the protrusion of the nerve cell. On the other hand, glial cells support the function of nerve cells. Specifically, it protects and supports nerve cells, forms a myelin sheath, forms a blood-brain barrier, supplies nutrients to nerve cells, metabolism of neurotransmitters, and functions of proliferation and differentiation of nerve cells during brain development. Control etc. are considered. Glial cells include a wide variety of cells. In the central nervous system there are astrocytes, oligodendrocytes, microglia, etc., and in the peripheral nervous system there are Schwann cells, mantle cells, etc.,
Ependymal cells present in the endothelium of the ventricles are also glial cells.

Proliferation and differentiation of nerve cells occurs only before birth and not after birth. On the other hand, proliferation and differentiation of glial cells are actively performed even after birth. recent years,
Many humoral factors involved in the differentiation, proliferation and growth of nerve cells and glial cells have been found. When classifying currently known factors based on the difference in the target cells, they are unidirectional factors that target either one of nerve cells or glial cells and both nerve cells and glial cells. There are two types of bidirectional factors.
As unidirectional factors that act only on nerve cells, nerve growth factor (NGF, molecular weight approximately 13.5 kd (human)), brain-derived neurotrophic factor (BDNF, molecular weight approximately 13.5 kd (human)), neurotrophin 3 ( NT3, molecular weight about 13.5
kd (human)), neuroblastoma growth factor (NGIF, molecular weight approximately 5 kd (human)), glial-derived nerve growth factor (molecular weight approximately 500 kd and approximately 110 kd (both are rat)), Schwann cell Derived nerve growth factor (SchNTF, molecular weight 8 kd or more (rat)) and neurofibroma-derived nerve growth factor (NFdNTF, molecular weight about 4 kd (human)) are known. In addition, as a unidirectional factor that acts only on glial cells, neuroblastoma cell-derived glial cell growth inhibitory factor (NBdGGIF, molecular weight 33
~ 40.5kd and 31-34kd (both are mice)), glial cell-derived glial cell growth inhibitory factor (GdGGIF, molecular weight 100 kd or more (rat))
And platelet-derived growth factor (PDGF, molecular weight about 32k
d (human)) is known. On the other hand, as a bidirectional factor, glial-derived neurite outgrowth factor (GdNT), which has both neurite outgrowth promoting action and glial cell growth promoting action
F, molecular weight about 43 kd (rat), neurite outgrowth promoting action and glial cell growth factor (GMF, molecular weight about 16.5 kd (bovine)) that promotes proliferation and differentiation of glioblasts, ganglion cell survival maintaining action, Ciliary nerve growth factor (CNTF, molecular weight of about 23), which has sympathetic ganglion cell growth inhibitory action and differentiation promoting action, and astrocyte differentiation promoting action
kd (rabbit and rat)) and fibroblast growth factor (FGF, molecular weight of about 16 to 17 kd (human)) having growth-promoting activity on nerve cells and astrocytes (for details, protein nucleic acid). Enzyme, 36 (N
o.7), 260 (1991)).

More recently, epidermal growth factor (EG
F, molecular weight about 6 kd (human), insulin-like growth factor (IGF-I and IGF-II, molecular weight about 7.
6 kd and about 7.5 kd (both human), interleukin-3 (IL-3, molecular weight about 28 kd (mouse)), interleukin-6 (IL-6, molecular weight about 2)
1 kD (human)), protease nexin I (molecular weight of about 38 kd (species unknown)), alpha ₂ - macroglobulin (molecular weight of about 180 kd (human)), S-100 protein (molecular weight of about 20 kd (human)), neuron-specific Enolase (molecular weight of about 44 kd (bovine)) has also been shown to have a survival-maintaining action on nerve cells (for details, see Cranial nerve, 43 (1), 5 (1991)).

[0005]

Most recently, some of the inventors have discovered a new unidirectional factor that acts only on glial cells. That is, Von Reck
We found that a new human glial cell growth inhibitor (hereinafter abbreviated as NFdGGIF) is present in neurofibroma tissue obtained from a patient with linghausen's disease (multiple neurofibromas), Partial purification was performed. This NFdGGIF was partially purified from the crude extract of neurofibroma using affinity chromatography, anion exchange column, hydrophobic chromatography column, and hydroxyapatite column. The GGIF is estimated to have a molecular weight of about 100 kd by a gel filtration method, and is subjected to acid treatment (pH 2.3), heat treatment (100 ° C., 10 minutes) and papain treatment (37).
It is considered to be a protein factor because it loses its biological activity at 5 ° C for 5 hours. Furthermore, the biological activity of this factor tended to be gradually lost during the purification process.
Dithiothreit, a basic antioxidant
The activity could be maintained by adding ol).

The present NFdGGIF is used for various glial cells such as rat glioma cells (C6), rat schwannoma (354A) and human glioblastoma (GB).
-1, T98G), human astrocytoma (NAC-
1, NAC-2) and the like, but promoted the decrease of DNA synthesis (that is, suppression of proliferation), but had no effect on nerve cells such as human neuroblastoma cells (GOTO, TGW). In addition, the present NFdGGIF, in the coexistence of a factor (GMF) that promoted both proliferation and differentiation of glial cells,
, But the differentiation promoting effect was not inhibited. For details of this NFdGGIF, see 19
It is described in the 33rd Annual Meeting of Neurochemical Society, 1990, p. 272 (1990).

[0007]

OBJECTS OF THE INVENTION The present inventors have found that in the same tumor mass in which the NFdGGIF was found, namely in the neurofibroma tissue of patients with Recklinghausen's disease, the NFdGGIF
In addition to this, we thought that there may be humoral factors involved in nerve cells and glial cells, and conducted extensive research into its discovery. As a result, they have found a novel bidirectional factor having a molecular weight of about 50 kd, which has both a neurite outgrowth effect and a glial cell growth inhibitory activity. Furthermore, they succeeded in determining the partial amino acid sequence of the factor and completed the present invention. To date, no bidirectional factor that has both neuronal process extension and antiproliferative activity on glial cells has been known, and in terms of molecular weight, it is completely different from known unidirectional factors. It is considered to be a novel factor.

[0008]

The present invention comprises a partial amino acid sequence, MXR,
FER, AGE, DGPALSPGPQSR, ALCSG
SPAER, VAAXDD, RGTPWL, GTPW
LR, AREQEELLAPADGXVE, GMDLE
ETXVL, ALQEAXV and ALPLALVLH
ELGAG (In the formula, each alphabet is IUPAC-I
Represents the one-letter code for an amino acid by UB. ), Having a molecular weight of about 50 kd (hereinafter referred to as neurotrophin-NF (abbreviated as NT-NF)),
And a pharmaceutical containing the protein as an active ingredient.

In this specification, including the claims,
Each amino acid in an amino acid sequence shall follow the "one-letter code for representing an amino acid sequence" determined by IUPAC-IUB. According to the rule, for example, A
Is alanine, C is cysteine, D is aspartic acid, E
Is glutamic acid, F is phenylalanine, G is glycine, H is histidine, L is leucine, M is methionine,
P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, V is valine, W is tryptophan, and X is unknown or other.

As a result of analysis by Edman degradation, the NT-NF of the present invention necessarily contains the partial sequence of the 12 fragments shown above. It is unclear at present whether or not it corresponds. The molecular weight of this NT-NF is SDS-P.
The result of AGE is about 50 kd, but assuming that the protein of the present invention has not undergone chemical modification such as sugar addition, nucleic acid addition, or lipid addition, the number of amino acids constituting the present NT-NF is about 500. Calculated. from these things,
In the present invention, about one-sixth of the total sequence has been determined, at least by estimation.

Therefore, the NT-NF of the present invention is sufficiently and clearly defined by the partial amino acid sequence and molecular weight of the 12 fragments shown above. From the viewpoint of biological activity, the NT-NF of the present invention has the following various properties. That is, the NT-NF of the present invention has both the neurite outgrowth action of nerve cells and the proliferation inhibitory action on glial cells. These biological activities are due to acid treatment (pH 2.
3), heat treatment (100 ° C., 10 minutes) and papain treatment (37 ° C., 5 hours) have been confirmed to eliminate.

The NT-NF of the present invention can be isolated and purified by the following method. That is, (1) a tumor mass of a neurofibroma obtained from a Recklinghausen's disease patient was homogenized in a 20 mM Tris-hydrochloric acid buffer containing a protease inhibitor and dithiothreitol, and then centrifuged, and (2) obtained. The supernatant was equilibrated with an anion exchange column (for example, DEAE-Seph) equilibrated with 20 mM Tris-HCl buffer (hereinafter abbreviated as buffer A).
acel®) and 0.5 M sodium chloride concentration in buffer A from 0% initial to 10% final.
A hydrophobic chromatographic column that was eluted with a sodium chloride concentration of 0.15 M to 0.3 M was collected by elution with changes to 0% over time, and (3) the recovered component was equilibrated with buffer A containing 4 M sodium chloride. (For example, High Load Phenyl Sepharose (registered trademark)) was adsorbed, and 4M sodium chloride concentration in buffer A was changed with time from an initial 100% to a final 0% to elute, and eluted near a sodium chloride concentration of 0.2 M. Collect the ingredients,

(4) A hydroxyapatite column (for example, HCA) in which the recovered components are equilibrated with buffer A
-Column A-7610 (registered trademark)),
In buffer A, 0.5 M sodium phosphate buffer was eluted with time-dependent changes from the initial 0% to the final 100%, and the components eluted immediately after the passing-through peak were collected, (5)
The recovered components were adsorbed on an anion exchange column equilibrated with Buffer A (for example, Mono Q (registered trademark)), and the concentration of 1 M sodium chloride in Buffer A was changed from 0% at the initial stage to 100% at the final stage with time. It is possible to isolate and purify by recovering the components that have been eluted and eluted near the sodium chloride concentration of 0.2 M. In each column purification step, the NT-NF of the present invention is used.
The contained fraction should be traced using the biological activity, for example, the growth inhibitory activity on glial cells as an index.

[0014]

INDUSTRIAL APPLICABILITY The NT-NF of the present invention has both the neurite outgrowth action of nerve cells and the proliferation inhibitory action on glial cells. These effects were confirmed by the following experiments. (1) Neurite Outgrowth Action of Normal Rat Cerebral Cortex Neurons From the fetal brain of a normal rat (Wister system) on day 17, the cerebral cortex was separated. Subdivide the cerebral cortex,
After washing three times with Dulbecco's modified Eagle's medium (DMEM), it was digested with papain at 37 ° C for 15 minutes. After repeated digestion twice, DMEM containing 10% fetal bovine serum
Was added and the remaining tissue was loosened by pipetting. After removing large lumps with a lens paper filter, the cells were washed twice with DMEM containing 10% fetal bovine serum, and the number of cells was counted.
It was spread on a polylysine-coated 48-well plate at 5 × 10 ⁴ cells / well (300 μl / well). After culturing in a carbon dioxide incubator at 37 ° C for 24 hours,
The NT-NF of the present invention (final concentration 5 ng / ml) was added, and the culture was continued for further 48 hours. The evaluation is based on the present invention group (NT-
The neurite outgrowth after the culture was compared with that of the control group (NF-added group). Micrographs showing the neurite outgrowth effect in the present invention group and the control group are shown in FIGS. 1 (a) and 1 (b), respectively. As is clear from FIG. 1, the NT-NF of the present invention showed sufficient neurite outgrowth action at a concentration of 5 ng / ml.

(2) Inhibitory effect on proliferation of glial cells Experimental method The inhibitory activity on proliferation of glial cells was measured by using the inhibitory activity on proliferation of rat astrocytoma cells (C6) as an index. That is, 1 × 10 ⁵ C6 cells are suspended in 100 μl of a culture medium (F-10 containing 10% fetal calf serum), and the suspension is spread on a 96-well microtiter plate (Nunc) at 37 ° C. for 4 hours. Incubated. To this, 10 μl of the NT-NF of the present invention was added, tritium thymidine (20 nCi / culture) was further added, and the culture was continued for another 16 hours. Then, the cells were collected on glass fiber filter paper using a multi-cell harvester (manufactured by Labomash), dried, and then the amount of tritium thymidine incorporated into the polymer fraction was measured using a liquid scintillation counter. The results are shown in Table 1 below.

[0016]

As is apparent from the table, the NT-NF of the present invention
It was found that at a concentration of 5 ng / ml, the proliferation of glial cells was suppressed to about 1/20 as compared with the control. on the other hand,
The toxicity of the NT-NF of the present invention is extremely low, and it is considered to be sufficiently safe for use as a medicine. Since the NT-NF of the present invention has both a strong nerve cell neurite outgrowth action and a growth inhibitory action on glial cells, it can be used as a therapeutic and / or prophylactic agent for cerebrospinal diseases and an antitumor agent. ..

It can be said that the NT-NF of the present invention is isolated and purified from a tumor mass and obtained from a natural NT-NF-containing tissue. On the other hand, a substance called NT-NF produced by gene recombination technology is considered. This is NT-NF producing cells (eg tumor cells)
The gene DNA corresponding to NT-NF is isolated from the gene of the above-mentioned gene, this is inserted into a vector to obtain a recombinant DNA, the host cell is transformed with the DNA, and the transformant is cultured to obtain the desired NT-. NF is obtained. The NT-NF thus obtained has the same amino acid sequence as that of the natural NT-NF.

In general, the natural type and the in-vivo active substance produced by the gene recombination technique have the same biological activity. The reason for this is that the main body of activity is believed to be in the protein part. In fact, in bioactive substances such as tissue plasminogen activator (t-PA), erythropoietin (EPO), interferon-α and interferon-β (IFN-α and IFN-β), the natural amino acid sequence is exactly the same. It has been confirmed that the type and the genetically modified type have exactly the same biological activity. From these facts, it is easily predicted that the recombinant NT-NF also has the strong neurite outgrowth action of nerve cells and the growth inhibitory activity against glial cells, like the natural type NT-NF. Therefore, the active ingredients of the therapeutic and / or prophylactic agent for cerebrospinal disease and the antitumor agent constituting the present invention include not only natural type but also recombinant NT-NF.

Here, the cerebrospinal diseases are (1) neuron damage associated with bleeding, ischemia, trauma, surgery, etc., and (2) senile dementia, Alzheimer's disease, vascular dementia, Parkinson's disease, Tenkan, spinal cord Dementia due to injury or neurodegenerative disease in general. Although it is effective as an antitumor agent against all solid cancers, it is particularly a brain tumor, particularly glioma cells such as astrocytoma, glioblastoma, meningioma, oligodendroma, and Schwann cell tumor. Useful for treating and / or preventing tumors.

When the NT-NF of the present invention is used for the above purpose, it is usually administered systemically or locally orally or parenterally, but preferably intravenously or intracerebroventricularly. The dose varies depending on age, weight, symptoms, therapeutic effect, administration method, treatment time, etc.
It is orally administered once to several times a day in the range of 1 mg to 10 mg at a time, or 10 μ / day for each adult.
Parenteral administration (preferably intravenous administration or intraventricular administration) is carried out once to several times a day in the range of g to 1 mg. Of course, since the dose varies depending on various conditions as described above, a dose smaller than the above dose may be sufficient in some cases, or a dose exceeding the range may be required in some cases. When the compound of the present invention is administered, it is used as a solid composition, a liquid composition and other compositions for oral administration, an injection for parenteral administration, an external preparation, a suppository and the like.

Solid compositions for oral administration include tablets,
It includes pills, capsules, powders, granules and the like. In such solid compositions, the one or more active substances are at least one inert diluent (lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, aluminometasilicate). It is used as a mixture with magnesium). These compositions are prepared according to a conventional method by using additives other than inert diluents such as lubricants (magnesium stearate, etc.), disintegrating agents (calcium fibrin gluconate, etc.), solubilizing agents (arginine, aspartic acid, etc.). ) And stabilizers (human serum albumin, lactose, etc.)
May be contained. If necessary, the tablets or pills may be coated with a film of a gastric or enteric substance (sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.). Capsules include hard capsules and soft capsules.

Liquid compositions for oral administration include solutions, emulsions, suspensions, syrups and elixirs. Such a liquid composition contains a generally used inert diluent (purified water, ethanol, etc.). These compositions may contain, in addition to the inert diluent, auxiliary agents such as wetting agents and suspending agents, sweeteners, flavors, aromatics and preservatives. Other compositions for oral administration include spray formulations which contain one or more active substances and are formulated by conventional methods. The spray agent may contain a stabilizer (sodium sulfite, etc.) and a buffering agent (sodium chloride, sodium citrate, citric acid, etc.) for imparting isotonicity, in addition to the inert diluent. The method described in US Pat. Nos. 2,868,691 and 3,095,355 can be used for producing the spray agent, for example.

Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. In such an injection, one or more active substances are at least one inert aqueous diluent (distilled water for injection, physiological saline, etc.) or an inert non-aqueous diluent (propylene glycol, Polyethylene fricol, olive oil, ethanol, polysorbate 80
(Registered trademark) and the like). These injections further contain adjuvants such as preservatives, wetting agents, emulsifiers, dispersants, stabilizers (human serum albumin, lactose, etc.), solubilizing agents (arginine, aspartic acid, polyvinylpyrrolidone, etc.). You may have. These are usually sterilized by filtration (bacterial retention filter, etc.), blending of a bactericide or irradiation, or after these treatments, a solid composition is prepared by a method such as freeze-drying, and sterile water is added immediately before use. , Or by adding a sterile injectable diluent.

[0025]

The present invention will be described in more detail with reference to the following examples, but these do not limit the scope of the present invention. Example 1 Isolation / Purification of NT-NF of the Present Invention A tumor mass (100 g) of a neurofibroma obtained from a Recklinghausen's disease patient was treated with a protease inhibitor (1).
0 μM leupeptin, 10 μM pepstatin A, 0.2 m
M phenylmethanesulfonyl fluoride) and 1 m
Homogenate in 20 mM Tris-hydrochloric acid buffer (pH 7.5) (200 ml) containing M dithiothreitol and then centrifuge (25,000 xg, 1 hour) to obtain a supernatant (240 m).
l) was collected. The resulting supernatant was divided into 4 portions (60 ml
Each)) and purified by column chromatography shown below.

That is, the obtained supernatant (60 ml) was adsorbed on an anion column (DEAE-Sephacel (registered trademark), manufactured by Pharmacia, 32 × 80 mm), and 20 mM Tris-hydrochloric acid buffer solution (pH 7.5) ( Less than,
It is abbreviated as buffer A '. ), 0.5 M sodium chloride concentration was changed with time from the initial 0% to the final 100% with time to elute (1 ml / min). The glial cell growth inhibitory activity (hereinafter abbreviated as GGIF activity) of each fraction was measured (the measuring method will be described later), and the main active ingredient had a sodium chloride concentration of 0.15 M to
It eluted at 0.3 M (see Figure 2). The active fractions were collected, sodium chloride was added thereto to a concentration of 4 M, and then the column was equilibrated with a buffer A ′ containing 4 M sodium chloride in advance (Hyload Phenyl Sepharose (registered trademark), manufactured by Pharmacia, 1.6 x 1
It was adsorbed (00 ml), and 4 M sodium chloride concentration in buffer A'was eluted from the initial 100% to the final 0% with time (3 ml / min). The active fraction eluted near 0.2 M (see FIG. 3). This fraction was concentrated to 0.5 ml with Centriprep (manufactured by Amicon), and a Hydroxy Apatite column (HCA-Co) was used.
lumn A-7610 (registered trademark), manufactured by Mitsui Toatsu Co., Ltd., 7.6
X 100 mm) and 0 for buffer A '.
A 5M sodium phosphate buffer (pH 6.8) was eluted with 0% initial change to 100% final change (1 m
l / min). The active fraction eluted immediately after the flow-through peak (see Figure 4).

The GGIF activity was measured using the growth inhibitory activity of rat astrocytoma cells (C6) as an index. That is, 1 × 10 ⁵ C6 cells were suspended in 100 μl of a culture medium (F-10 containing 10% fetal bovine serum) to give 96 cells.
The cells were spread on a well microtiter plate (Nunc) and incubated at 37 ° C. for 4 hours. 10 μl of test sample was added to this, and tritiated thymidine (2
0nCi / culture) was added and the culture was continued for another 16 hours. After that, cells were collected on glass fiber filter paper using a multi-cell harvester (manufactured by Labomash), dried, and then the amount of tritium thymidine incorporated into the polymer fraction was measured using a liquid scintillation counter.

Next, the active fractions obtained from the hydroxyapatite column (4 ml in total of 4 times) were collected and divided into 2 portions (2 ml each) to an anion exchange column (Mono).
Q (registered trademark) PC 1.6 / 5, manufactured by Pharmacia, 1.6
X 5 mm), and the elution was carried out by changing the concentration of 1 M sodium chloride in the buffer A ′ from the initial 0% to the final 100% with time (20 μl / min). The active fraction eluted around 0.2 M (see FIG. 5).

Example 2: Measurement of molecular weight and assay of purity The purified NT-NF (3 μg) obtained in Example 1 was loaded with the same amount of loading buffer (250 mM Tris-hydrochloric acid buffer (pH 6.8), 4% sodium). Dodecyl sulphate (SDS), 18% glycerol, 1.4 M 2-mercaptoethanol and 0.01% bromphenol blue) were mixed and heated at 100 ° C. for 3 minutes, then SD
Separation by S-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis, using 7.5% homogeneous gel). Coomassie blue stain gives a molecular weight of 50
A single band was observed at kd (see Figure 6). In the figure,
The band on the right side (CBB side) is NT-obtained in Example 1.
NF, the band on the left is of the molecular weight marker. From this, the present invention N obtained in Example 1
It was confirmed that T-NF was completely purified and was a single product.

Example 3: Determination of partial amino acid sequence Purified NT-NF (about 15 μg) was placed in a Spectrapore dialysis tube (molecular weight 10,000 cut, manufactured by Spectrum Medical Industry), and 6M guanidine hydrochloride and 2 were added.
0.25 M Tris-HCl buffer containing mM EDTA (pH
8.5) was dialyzed overnight. After dialysis, collect the internal solution (about 500 μl) in a polyethylene tube and
Dithiothreitol (mg) was added, the atmosphere was replaced with nitrogen for about 30 seconds, and the mixture was left overnight at 37 ° C. Next, iodoacetic acid (2.4 mg) was added, and the mixture was allowed to react for 20 minutes in the dark at room temperature. After completion of the reaction, the solution was dialyzed against 0.1 M ammonium bicarbonate solution (pH 7.8) for one day using the dialysis tube.

The dialysis solution was recovered, and TPCK-trypsin [L- (1-tosylamino) -2-phenylethyl) chloromethylketone-treated trypsin, manufactured by Worthington Co.] was used as an enzyme: substrate = 1: 100 (w / w), and after reacting at 37 ° C for 1 hour, add the same amount of TPC.
K-trypsin was added, and the mixture was reacted at 37 ° C for 8 hours. The trypsinized fragment was immediately purified by water equilibration with 0.14% trifluoroacetic acid in a pore reverse phase HPLC, ie, C18 reverse phase column (μRPC C2 / C18.SC2
/ 10, 2.1 mm × 100 mm, manufactured by Pharmacia LKB), and acetonitrile was eluted from a 0.1% trifluoroacetic acid solution while changing the initial 0% to the final 60% with time. The chromatography system is a SMART system (Pharmacia LKB), the flow rate is 150 μl / min, and the eluent is 21.
Manual fractionation into polyethylene tubes with monitoring at 4 nm (see Figure 7). The amino acid sequence of the isolated fragment was determined by Edman degradation using ABI gas phase protein sequencer model 470A (manufactured by Applied Biosystems) connected to ABI model 120A online phenylthiohydantoin analyzer. The determined amino acid sequence is shown in Table 2.

[0032]

Note 1) In the case of a partial amino acid sequence fragment originally obtained by trypsin treatment, the C-terminal of the fragment is lysine (K) or arginine (R). In the above table,
... means that K or R at the C-terminal could not be analyzed because the sample amount was small, and identification was not possible on the way. Note 2) Fractions 25 and 59 were a mixture of two types of fragments, respectively, but since the content ratios were significantly different, both types could be identified. Note 3) "X" in the amino acid sequence indicates that it could not be identified. Note 4) Cysteine (C) was identified as S-carboxymethyl cysteine.

Formulation Example 10 mg of NT-NF of the present invention was sufficiently dissolved in 500 ml of physiological saline, and the resulting solution was sterilized by a conventional method,
By filling 5 ml each into an ampoule and freeze-drying, 100 injections containing 100 μg of active ingredient per ampoule can be obtained.

[Brief description of drawings]

FIG. 1 is a micrograph [(a) present group, (b) control group] showing the morphology (extension of neurites) of an organism when culturing normal rat cerebral cortical neurons.

2 is an anion exchange column (DE
It is a chart of AE-Sephacel).

FIG. 3 is a chart of the hydrophobic chromatographic column used in Example 1.

FIG. 4 is a chart of a hydroxyapatite column used in Example 1.

5 is an anion exchange column (Mo
It is a chart of no Q).

6 is a pattern of SDS-PAGE performed in Example 2. FIG.

FIG. 7 is a pore reverse phase HPLC chart performed in Example 3.

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[Procedure amendment]

[Submission date] November 19, 1991

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0003

[Correction method] Change

[Correction content]

Proliferation and differentiation of nerve cells occurs only before birth and not after birth. On the other hand, proliferation and differentiation of glial cells are actively performed even after birth. In recent years, many humoral factors involved in differentiation, proliferation and growth of nerve cells and glial cells have been found . When classifying currently known factors based on the difference in the target cells, they are unidirectional factors that target either one of nerve cells or glial cells and both nerve cells and glial cells. There are two types of bidirectional factors. As unidirectional factors that act only on nerve cells, nerve growth factor (NGF, molecular weight about 13.5 kd (human)), brain-derived neurotrophic factor (BDNF, molecular weight about 1)
3.5kd (human), neurotrophin 3 (NT
3, molecular weight of about 13.5 kd (human), neuroblastoma growth inhibitory factor (NGIF, molecular weight of about 5 kd (human)), glial-derived nerve growth factor (molecular weight of about 500 kd and about 110 kd (some. Rat)), Schwann cell-derived nerve growth factor (SchNTF, molecular weight 8 kd)
The above (rat)) and neurofibroma-derived nerve growth factor (NFdNTF, molecular weight about 4 kd (human)) are known. In addition, as a unidirectional factor that acts only on glial cells, neuroblastoma cell-derived glial cell growth inhibitory factor (N
BdGGIFF with a molecular weight of 33-40.5 kd and 31
, Which is ~ 34 kd (both are mice), and glial cell-derived glial cell growth inhibitory factor (GdGGIF, molecular weight 100 kd or more (rat)) and platelet-derived growth factor (PDGF, molecular weight approximately 32 kd (human)) are known. There is. On the other hand, as bidirectional factors, glial-derived neurite outgrowth factor (GdNTF, molecular weight of about 43 kd (rat)), which has both neurite outgrowth promoting action and glial cell growth promoting action, neurite outgrowth promoting action and glioblast Glial cell growth factor (GMF, molecular weight of about 16.5 kd (bovine)) that promotes proliferation and differentiation, ganglion cell survival maintenance action, sympathetic ganglion cell growth inhibition and differentiation promotion action, and astrocyte differentiation promotion action Ciliary nerve growth factor (CNTF, molecular weight of about 23 kd (rabbit and rat)) and fibroblast growth factor (FGF, molecular weight of about 16 to 17 kd (human)) having growth-promoting activity on nerve cells and astrocytes ) Is known (for details, see Protein Nucleic Acid Enzyme, 36 (No. 7), 260).
(1991)).

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0005

[Correction method] Change

[Correction content]

[0005]

Most recently, some of the present inventors have developed a new unidirectional factor that acts only on glial cells.
I found it. That is, Recklinghausen (VonR
neurofibroma obtained from a patient with Ecklinghausen's disease (multiple neurofibroma)
ma) into the tissue, a new human glial cell growth inhibitory factor (hereinafter abbreviated as NFdGGIF.) found that there was performed a partial purification. This NFdGGIF was partially purified from the crude extract of neurofibroma using affinity chromatography, anion exchange column, hydrophobic chromatography column, and hydroxyapatite column. The GGIF
Is estimated to have a molecular weight of about 100 kd by the gel filtration method, and its biological activity is lost by acid treatment (pH 2.3), heat treatment (100 ° C, 10 minutes) and papain treatment (37 ° C, 5 hours). It is believed that there is. Furthermore, although the biological activity of this factor tended to be gradually lost during the purification process, the activity could be maintained by adding dithiothreitol, which is an SH group antioxidant.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0007

[Correction method] Change

[Correction content]

[0007]

OBJECTS OF THE INVENTION The present inventors have proposed the above-mentioned NFdGGIF
In the same tumor mass as found , namely in the neurofibroma tissue of patients with Recklinghausen's disease, the NFdGGIF
In addition to this, we thought that there may be humoral factors involved in nerve cells and glial cells, and conducted extensive research into its discovery. As a result, a novel bidirectional factor having a molecular weight of about 50 kd was found , which has both nerve cell process extension activity and glial cell growth inhibitory activity. Furthermore, they succeeded in determining the partial amino acid sequence of the factor and completed the present invention. To date, no bidirectional factor that has both neuronal process extension and antiproliferative activity on glial cells has been known, and in terms of molecular weight, it is completely different from known unidirectional factors. It is considered to be a novel factor.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0021

[Correction method] Change

[Correction content]

When the NT-NF of the present invention is used for the above purpose, it is usually administered systemically or locally orally or parenterally, preferably intravenously or intracerebroventricularly. The dose varies depending on the age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but is usually orally administered once a day in the range of 1 mg to 100 mg per adult once a day. Or, it is parenterally administered (preferably intravenously or intraventricularly) once a day to several times in a range of 10 μg to 1 mg per adult.
Of course, since the dose varies depending on various conditions as described above, a dose smaller than the above dose may be sufficient in some cases, or a dose exceeding the range may be required in some cases.
When the compound of the present invention is administered, it is used as a solid composition, a liquid composition and other compositions for oral administration, an injection for parenteral administration, an external preparation, a suppository and the like.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0026

[Correction method] Change

[Correction content]

That is, the obtained supernatant (60 ml) was adsorbed on an anion exchange column (DEAE-Sephacel (registered trademark), Pharmacia, 32 × 80 mm), and 20 mM Tris-hydrochloric acid buffer solution (pH 7.5). (Hereinafter, abbreviated as buffer A '.) 0.5M sodium chloride concentration was eluted with time varying from 0% in the initial stage to 100% in the final stage (1 ml / min). For each fraction, glial cell proliferation inhibitory activity (hereinafter referred to as GGI
Abbreviated as F activity. ) (The measuring method will be described later), the main active ingredient was eluted at a sodium chloride concentration of 0.15 M to 0.3 M (see FIG. 2). The active fractions were collected, sodium chloride was added thereto to a concentration of 4 M, and then the column was equilibrated with a buffer A ′ containing 4 M sodium chloride in advance (Hyload Phenyl Sepharose (registered trademark), manufactured by Pharmacia, 1.6 x 100 mm), and add 4M sodium chloride in buffer A'to 100% of the initial concentration.
To 0% to the final 0%.
/ Min). The active fraction eluted around 0.2 M (see FIG. 3). This fraction was concentrated with Centriprep (Amicon) to 0.5 ml, hydroxy Ruapa <br/> Thai preparative column (HCA-Column A-7610
(Registered trademark), manufactured by Mitsui Toatsu Co., Ltd., 7.6 × 100 mm), and 0.5 M sodium phosphate buffer (pH 6.8) was added to the buffer A ′ at an initial 0% to a final 100.
%, And elution was carried out by changing over time to 1% (1 ml / mi
n). The active fraction eluted immediately after the flow-through peak (see Figure 4).

Claims (6)

[Claims] 1. A partial amino acid sequence, MXR, FER, A
GE, DGPALSGPQSR, ALCSGSPAE
R, VAAAXDD, RGTPWL, GTPWLR, A
REQEELLAPADGXVE, GMDLEETXV
L, ALQEAXV and ALPLALVLHELGA
A protein having a molecular weight of about 50 kd, which has G (in the formula, each alphabet represents a one-letter symbol of an amino acid according to IUPAC-IUB). 2. A partial amino acid sequence, MXR, FER, A
GE, DGPALSGPQSR, ALCSGSPAE
R, VAAAXDD, RGTPWL, GTPWLR, A
REQEELLAPADGXVE, GMDLEETXV
L, ALQEAXV and ALPLALVLHELGA
A therapeutic agent for cerebral spinal cord diseases containing a natural or gene recombinant protein of about 50 kd in molecule having G (in the formula, each alphabet represents one letter symbol of amino acid according to IUPAC-IUB) and / or
Or prophylactic agent. 3. The therapeutic and / or prophylactic agent according to claim 2, wherein the cerebrospinal disease according to claim 2 is neuron damage. 4. The therapeutic and / or prophylactic agent according to claim 2, wherein the cerebrospinal disease according to claim 2 is dementia. 5. A partial amino acid sequence, MXR, FER, A
GE, DGPALSGPQSR, ALCSGSPAE
R, VAAAXDD, RGTPWL, GTPWLR, A
REQEELLAPADGXVE, GMDLEETXV
L, ALQEAXV and ALPLALVLHELGA
An antitumor agent containing a natural or gene recombinant protein of about 50 kd molecule having G (in the formula, each alphabet represents one letter symbol of amino acid according to IUPAC-IUB) as an active ingredient. 6. The antitumor agent according to claim 5, wherein the tumor according to claim 5 is a brain tumor.