JPS62252729A - Promote for recovering hematopoietic function - Google Patents

Abstract

PURPOSE:The titled drug for treating leukemia, etc., showing recovering effects on hematopoietic function after bone-marrow transplantation, containing a human granular colony stimulation factor as an active ingredient. CONSTITUTION:A promoter for recovering hematopoietic function containing a human granular colony stimulation factor (G-CSF for short) as an active ingredient having the following physical and chemical properties. Molecular weight: 19,000+ or -1,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. Isoelectric point: having at least three isoelectric points of pI 5.5+ or -0.1, 5.8+ or -0.1 and 6.1+ or -0.1. UV: having maximum absorption at 280nm and minimum absorption at 250nm. Amino acid sequence: having amino acid sequence shown by formula I from N end to 21-th residue. The human G-CSF contains a polypeptide shown by formula II (X is Leu, etc.; n is 0 or 1) or part thereof. 0.1-500mug promotor for recovering hematopoietic function is usually administered to a patient having hematopoietic disorder 1-7 times/week in order to promote recovery hematopoietic function after bone-marrow transplantation as a treating method for patients.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to human granulocyte colony stimulating factor (hereinafter referred to as G-C3F).
The present invention relates to an agent for promoting recovery of hematopoietic function after bone marrow transplantation, which contains as an active ingredient

(Prior Art) Bone marrow transplantation (hereinafter referred to as BMT) refers to the transplantation of healthy bone marrow from another person or from one's own body to a patient exhibiting a congenital or acquired hematopoietic disorder.

In recent years, BMT has been increasingly used as a treatment for blood diseases such as leukemia and malignant lymphoma, as well as certain types of cancer. And the effects are increasing. [Clinical and Research, Vol. 61, 1480-1487
．． (1984); 2 [Experimental Hematology (Exp, Tlematol), 12205-
215 (1984)] However, this treatment method also has some clinical problems.

[Problem that the invention seeks to solve]

The main causes include infection immediately after BMT, interstitial pneumonia (IP), and graft-versus-host disease (GVHD).

Of these, infection occurs during the non-hematopoietic period immediately after BMT.
As a countermeasure against this, sterile room therapy is currently being used. However, it takes about three weeks for the patient's hematopoietic function to recover, and in some cases it may take more than a month.

During this time, the patient will be treated in a sterile room.
This treatment is expensive and has the problem that it not only imposes an excessive financial burden on the patient, but also requires a great deal of effort on the part of the doctor.

IP often develops after transplantation. As a countermeasure against IP, sulfamethoxylizole-trimethoprim (
Sulfamethoxazole-rrimett
Prophylactic administration of a so-called ST combination drug (oprim) has been carried out, but since the ST combination drug has a myelosuppressive effect,
Can only be used in patients whose hematopoietic function has fully recovered [Clinical Immunology Vol. 15, No. 9, 700-707 (1983)
, Clinical Immunology Vol. 15, No. 9, 687-699 (19
83) ;Exp, flema [ol, Vol. 12, 2
05-215 (1984)). In addition, acute GVHD that occurs after graft survival is something to be wary of;
TX) also causes bone marrow suppression, and cyclosporine A (C3A), which has recently been used, has the problem of strong nephrotoxicity (Clinical and ω (Research, Vol. 61, No. 5). No. 1480-1487 (1984)).In any case, under these circumstances, early recovery of hematopoietic function after BMT is strongly desired.

However, at present, there is no appropriate drug to address this issue, so patients are left waiting for the patient's hematopoietic function to recover naturally.

[Means for solving problems]

As a result of repeated studies to overcome this situation, we have developed pure human G-C8F, which the applicant had previously applied for and successfully manufactured.
I came up with the idea of using this, and further advanced my ω1 research to realize this idea.

In addition, C3F is a factor that acts on granulocytic stem cells of human or animal bone marrow cells to induce proliferation and differentiation into monocytes, macrophages, and neutrophils.
alt etc; Experimental hematology (Exp
, Hematol, )1, 185. (1973
), h]~-○ SF have already been reported in large numbers. (Stanley et al., Federal Proceedings (Fed, Proc.) 35.
2272 (1975); Burgas et al., Bfood 49.573 (1977), and many others] However, the above-mentioned C3F is not completely purified, and there is no method for obtaining pure and homogeneous C3F that can be mass-produced. It had not been established.

In order to develop a drug that promotes the recovery of hematopoietic ability after BMT, it is necessary to obtain a large amount of uniform G-C3F, and the present applicant's previous success was achieved (Japanese Patent Application No. 59-153273@).

Patent Application No. 1983-220450, Patent Application No. 1983-26945
No. 5, patent application No. 60-269456@, patent application No. 60-27
No. 0838, Japanese Patent Application No. 60-270839) has made this possible.

Therefore, the present inventor found that when the human G-C3F was continuously administered to mice, blood function was enhanced (see Experimental Example 1). Promote functional recovery^
When examining whether or not it could be used as II, an increase in CFU-S was observed in the G-C3F throwing group. This confirmed that G-C3F has an effect of promoting hematopoietic function of BMT 1!2. (See Experimental Example 2) In addition, in an experimental system where recovery of hematopoietic function is slow, it was confirmed that the survival rate was 33% in the control group, while the survival rate was as high as 75% in the G-C3F administration group. Ta. This is G-
This is an expression of the hematopoietic function promoting effect of C3F. (See Experimental Example 3) The present inventor completed the present invention based on the above knowledge.

The present invention provides an agent for promoting recovery of hematopoietic function after 3MT, which contains human hG-C5F as an active ingredient.

Hi-1-G-C3F, which is the active ingredient of the present invention, is II! Highly isolated human G-csr: Although its origin does not matter, the following human G-C3F obtained by the method previously filed by the present applicant is particularly preferably used.

(1) Human G-C3F having the following physical and chemical properties.

■19000±1ooo as measured by intermolecular nidodecyl sulfate J thorium-polyacrylamide gel electrophoresis.

■Isoelectric point: PI=5.5±0.1, pI=5
．． 8±0.1°p)=8.1±o, i has at least one of the three isoelectric points.

■Ultraviolet absorption: Maximum absorption at 280 nm, 250 nm
It has a minimum value in nm.

(4) The amino acid sequence from the N-terminus to the 21st is as follows.

112N-rhr-Pro-Leu-Gly-Pro-
^1a-3er-3er-LeLl-PrO-G l
n-3er-Phe-Leu-Leu-Lys-Cys
-Leu-G I u-G I n-Va I -(2
) Human G-C3F having a polypeptide represented by the following amino acid sequence or a part thereof.

(Net)n Thr Pro Leu Gly P
ro Ala Ser Ser Ser LeuPro Gl
n Ser Phe Leu Leu Lys C
ys Leu Clu Ginvat^rg Lys
Ile Gln Qly Asp Gly Ala A
la LeuGln Glu Lys X Cy
s Ala Thr Tyr Lys Leu
Cys old s Pro Glu C1u Leu
Vat Leu Leu Gly 1li
s 5erLeu Gly Ile Pro Trp
^Ia Pro Leu Scr CysPr
o Ser Gln 八la Leu Gln
Leu Ala GIyCVS LeuSer G
ln Leu His Ser Gly Leu Ph
e Leu Tyr GlnGly Leu Leu
Gln Ala Lcu Glu Gly Ile
Ser Pr.

Glu Leu Gly Pro 'rhr Leu
Asp Tt+r Leu Gln Leu 83
vat Ala Asp Phe Ala
rhr Thr Ile rrp GlnGl
n Het Qlu Glu Leu Gly Met
Ala Pro Aha LeuGln Pro
Dinghr Gln Gly Ala Met
Pro Ala Phe ^1aSer Al
a Phe Gln＾rg＾rg＾Ia Gly Gl
y Vat LeuVat^Ia Ser tlis
Leu Gln Ser PhOLou c+u Va
tSer ryr Arg Val Leu Arg
1lis Leu^Ia Gln Pr.

(In the formula, X is teu or 1.eu-Vat-3er-Gl
(1 represents O or 1) In addition, the above-mentioned H1~G-C3F in the form of a glycoprotein having a sugar chain moiety is most preferable.

The above (1) G-C3F can be produced by the production method described in Japanese Patent Application No. 59-153273 or Japanese Patent Application No. 1987-220450.

The former includes the cell line C[-1U-1(
(C, N, C, H,) Deposit? ftft-315) from the culture supernatant;
-2 ((C,N,C,M,) Deposit No. ■-483) is produced from the culture supernatant.

Please refer to the respective specifications for details.

Moreover, G-C3F in (2) is patent application No. 60-2694559.
Patent Application No. 1983-269456, Patent Application No. 1983-27083
8 and Japanese Patent Application No. 60-270839. The methods described in each of these specifications are methods based on the so-called Daobutsu recombinant technology.

The first two articles disclose a method using E. coli'9 prokaryotes as host cells, and the second 2ft discloses a method using animal cells as hosts, so please refer to the respective specifications for details. Please refer to it.

In addition, G in the form of a glycoprotein that has the aforementioned sugar chain moiety
-C3F can be produced by using animal cells as a host.

1q human G-C3F can be stored frozen or by removing moisture by freeze drying, vacuum drying, or the like.

Furthermore, if desired, Hj-G-C3F can be dissolved in a suitable buffer solution and then sterile filtered using a Millipore filter or the like to prepare an injection agent.

Furthermore, the hematopoietic function recovery promoting agent of the present invention can contain a pharmaceutical carrier and excipients necessary for taking the form of a pharmaceutical preparation, as well as a stabilizer and an adsorption prevention agent.

Human G-C3F contained in the hematopoietic function recovery promoter of the present invention
The dosage and frequency of administration can be determined by taking into consideration the condition of the patient with the target disease, but it is usually administered at a dosage of o, i~ per adult.
500 μ9, preferably 5 to io.

μri-no-hi] ~ G-C3F-containing preparations once per week
Can be administered 7 times. However, the present invention
It is not limited by the content of 3F.

〔Example〕

The present invention will be described below as a reference example (manufacturing example of G-C8F) and an experimental example (
Pharmacological effects) and Examples (formulation examples) will be described, but the present invention is not limited thereto.

Reference Example [Example of production of human G-C8F using animal cells (mouse C127 cells)] A PTN-V2 plasmid was obtained by the method described in Examples 1 to 12 of Japanese Patent Application No. 60-269456, and this is treated with Barrll-11. That is, pT N
-v2 plasmid 20 μ (J 1 nm 8 Tri
s-t 1c, l1 (II 8.0>, 7
l1lN M (JCl, 100 mM
NaC,l! , 2mH2-mercaptoethanol B
Dissolved in SA100 ufl and treated with 20 units of BamtiI (manufactured by Takara Shuzo Co., Ltd.), followed by phenol treatment, ether treatment, and ethanol precipitation.

On the other hand, mouse C127 cells were prepared using 10% beef tallow serum (GIB).
DLIIbQCCO'S minimal including CO)
Grow in essential medium. C127 cells grown on 5 cm diameter plates were treated with the above prepared DNA at a ratio of 10 μ9 per plate using the phosphate-calcium method (Haynes, J & Weissmann, C (1
983) Nucleic Acid Res, 11,
887-706), treated with grillerol, and incubated at 37°C for 12 hours.

Next, the cells were transferred to three new plates with a diameter of 5 cm, and the medium was exchanged twice a week.

The parts that formed Foci (agglomerates) on the 16th day were transferred to new plates, subcultured in the above-mentioned medium, and G-
Clones with high CSF-producing ability were selected. the result~
There was a level of GC'SF production of 1 mg/ρ.

Regarding recovery, purification, and testing methods, please refer to the above patent application filed in 1983.
0-269456 specification was used as disclosed in the relevant examples.

Experimental example 1 Relationship between G-CSF daily investment function (mouse) G
CSF Rimple 0.1 d (G- from CI-IU-2
A mouse (C57 [3
L-8W, male) were administered E1 once a day, and the number of CFtJ-C in the spleen, CFtJ-
The number of S and peripheral neutrophils were measured, and control "Ripple 0.17 (n-propatool 1%, syngeneic mouse serum 10
compared with those of mice administered with saline (containing % saline). The results are shown in Table-1, Table-2 and Table-3. cr
: u-s refers to red blood cells, neutrophils, megakaryocytes, eosinophils,
It is a stem cell that has the ability to differentiate into monocytes and produce 1q.
FU-C refers to stem cells that have the ability to differentiate into neutrophils, monocytes (macrophages), and in some cases, eosinophils.

As is clear from Tables 1, 2, and 3, when G-CSF is administered to mice on a daily basis, hematopoietic function is enhanced.

Table-1 p(Risk rate) ***<0.001<**<0.01Table-2 p(Risk rate)***<0.001<*Book<0.01Table-
3 Number of peripheral neutrophils per m3 of peripheral blood (number of measurements n = 4) p
(Risk rate) ***<Q, QQl<*Umbrella<0. Ql<*<
Q, 05 Experimental Example 2 G C3F m-blood function 1 promotion effect after BMT A mouse (C57 [3L 8W, male) was irradiated with 95OR X-rays whole body, and 2 x 10 bone marrow cells of a syngeneic mouse were immediately injected from the tail vein. Moved 416 times. For this mouse, 5 days after transplantation [IJ, control used in Experiment 1 or G
-C3F-rimple 0.1 f was administered every day, and the number of CFU-3 in the glen and bone marrow was counted on the 6th and 12th day after the start of administration.

The results are shown in Tables 4 and 5.

Experimental Example 3 Survival rate in experimental system for recovery of hematopoietic ability Mice (C57 [31-8W1 male)
Immediately after whole body irradiation with 1A, syngeneic mouse bone marrow cells 7.5
X10 cells were transplanted from the tail vein. From day 5 after transplantation, these mice were treated with Control 1 to Roll used in Experimental Example 1 or G-C3F Rimple 0. IIIIIIl every day 11E
The survival rate (400 days after irradiation was observed) was as shown below.

Survival rate C0ntr”01 group 33.3% (n=12) G-C3
The remarkable improvement in the survival rate (75.0% (n=12) in the F-decapitation group is presumed to be due to the effect of G-C8F on promoting recovery of the hematopoietic system.

Example 1 (Formulation Example) Human I-G-C3F obtained in Reference Example was sterilized and then frozen at -20°C to prepare an injection.

Example 2 (Formulation example) Hi1~G-C8F obtained in Example 1 and Example were filled into 10 m vials for 5rd time using aseptic operation, freeze-dried at -20°C, and then capped with a rubber stopper. It was made into an injection. .

〔Effect of the invention〕

The hematopoietic function recovery promoter of the present invention has the effect of promoting hematopoietic function recovery after bone marrow transplantation, which is a treatment for patients with hematopoietic disorders, and thereby is effective in promoting hematopoietic function recovery in patients with many intractable diseases such as leukemia. Hopes for a cure rose.

Claims (1)

[Scope of Claims] 1. A hematopoietic function recovery promoter after bone marrow transplantation, which contains human granulocyte colony stimulating factor as an active ingredient. 2. The hematopoietic function recovery promoter according to claim 1, wherein the human granulocyte colony stimulating factor has the following physicochemical properties. (1) Molecular weight: 19,000 ± 100 as measured by sodium dodecyl sulfate-polyacrylamide glucose electrophoresis
0. (2) Isoelectric point: PI=5.5±0.1, PI=5.8±
0.1, PI=6.1±0.1. (3) Ultraviolet absorption: maximum absorption at 28 nm, 250
It has a minimum value in nm. (4) The amino acid sequence from the N-terminus to the 21st residue is as follows. [There is an amino acid sequence] 3. Hematopoietic function recovery according to claim 1, characterized in that the human granulocyte colony stimulating factor has a polypeptide represented by the following amino acid sequence or a part thereof. Accelerator. [There is an amino acid sequence] (In the formula, X is Leu or Leu-Val-Ser-Glu
, and n represents 0 or 1. )