JPH02264728A - Hyperlipemia remedy - Google Patents

Abstract

PURPOSE:To provide a hyperlipemia remedy having an action to remarkably reduce the amounts of cholesterol or neutral fatty acids in blood by using a human urine-originated monocyte-macrophage colony-stimulating factor (human M-CSF) as an active ingredient. CONSTITUTION:An agent useful for treating a hyperlipemia to improve and prevent arterial scleroses contains a human urine-originated human M-SCF prepared by freeze-drying a purified product separated from the urines of healthy men. the compound is administered in a dose of usually 0.4-16mug/kg body weight/day, preferably 1.6-8mug/kg body weight/day. The addition of a surfactant, human albumin, gelatin, etc., on the preparation of the agent permits to prevent the adsorption of the human M-CSF to glass, plastics, aseptic filtration members, etc., and further to highly improve the stability of the compound.

Description

Detailed Description of the Invention "Industrial Application Field" The present invention relates to a hyperlipidemia therapeutic agent containing human urine-derived human monocyte-macrophage colony stimulating factor (hereinafter referred to as human M-C3F) as an active ingredient. Regarding.

[Technical Background and Prior Art] Hyperlipidemia is a disease in which one or more of cholesterol, neutral fats, phospholipids, etc. increases above normal values. In the case of Japanese people, hyperlipidemia is defined as a total cholesterol level of 220 µ or higher, neutral fat level of 130 µ or higher, or phospholipid level of 250 µ or higher per 100 ml of blood. Hyperlipidemia itself is not a serious disease, but if left untreated, it can lead to arteriosclerosis, leading to angina pectoris and myocardial infarction, which is a serious clinical problem. There are many drugs for the treatment of this disease, but clinically, probucol preparations (Akira Watanabe et al., Arteriosclerosis, Vol. 11, No. 3, p. 597, 1983) and the proteolytic enzyme elastase (Shozo Yoshimura, Arteriosclerosis) are used. , Volume 3, 2
23 pages.

1975) is mainly used. The action of these drugs is to make it difficult for cholesterol to adhere to blood vessel walls, or to wash away cholesterol attached to blood vessel walls.
There are certain limits to its effectiveness, and there are currently no drugs that can radically treat hyperlipidemia and arteriosclerosis.

Among the colony-stimulating factors, which are a type of hematopoietic factor, there is a factor (M-C3F) that acts on monocyte-macrophage stem cells, and its protein and gene structure have been elucidated.
C (Japanese Unexamined Patent Publication No. 64-22899). This human M
-C3F also acts on mature human monocytes-macrophages and promotes their functional activation and production of various cytokines (Mojo7osbi K, ej gl, Exp
, Hemalol, 17:68-71 (1989
)), and clinically granulocytopenia (Mojo7osh
i K,, ej xilExp, Hematol,
14:1069-1σ75 (1986)) and bone marrow transplantation (Masxokx T,.

ef al,, Bone Marrow Tran
plantation, 3:+21-127 (
1988)), and it has great expectations as a medicine. The safety of this human M-C3F has already been confirmed in clinical trials (lloto7o
xhi K,, et tl,, 1mmu-++
obiO1og7.172; 205-212. (1
986)).

However, the possibility of using human M-C3F as a therapeutic agent for hyperlipidemia and arteriosclerosis has remained unexamined.

[Purpose and Summary of the Invention] Hyperlipidemia is a disease in which cholesterol, triglycerides, or phospholipids in the blood are higher than normal values, and if left untreated, it can lead to arteriosclerosis and induce myocardial infarction, angina pectoris, etc. has become clear. Therefore, prevention of arteriosclerosis with excellent hyperlipidemia and arteriosclerosis therapeutic agents is extremely important in preventing myocardial infarction and angina pectoris. The present invention is based on the investigation of human M-C8F as a therapeutic agent for hyperlipidemia and arteriosclerosis in hyperlipidemia patients and hyperlipidemia model animals. We have completed the present invention by discovering that it has the effect of significantly reducing the amount of cholesterol and triglyceride in the blood, which are the most important factors in arteriosclerosis.

The present invention is a therapeutic agent for hyperlipidemia containing human M-C3F as an active ingredient. Furthermore, the human M-C8F of the present invention is derived from human urine.

[Detailed description of the present invention] Human M-C3F derived from human urine related to the present invention can be obtained from urine of a healthy person using a known method (Japanese Patent Laid-Open No. 63-198700, Japanese Patent Laid-Open No. 63-250400, Kaisho 64-22
No. 899) was purified by freeze-drying. For example, it was prepared as follows using the method described in JP-A-63-198700.

That is, 1000 L of urine from a healthy person was adjusted to pH 8.5, the precipitate was removed by filtration, and concentration and desalting were performed using an ultrafiltration membrane (Amibon, Hl 0x50) with a molecular weight of 5o and ooo daltons. Next, the pH was adjusted to 7.0, and the mixture was heat sterilized at 60° C. for 10 hours in a closed container. After sterilization, centrifugal force 1m (
5000xg for 30 minutes) to remove the precipitate.
021 (phosphoric acid!! D equilibrated with buffer solution (pH 7,2)
Mixed with EAE-cellulose and adsorbed. DEAE-
After washing the cellulose with 0.02H phosphate buffer (pH 7,2) to which 0.058 NaCl was added, 0.25H NaCl was added to the cellulose.
．． 02) Elution was performed with monophosphate Wi buffer (pl+7.2). The eluate was concentrated with an ultrafiltration membrane (Amicon 810P10) and gel-filtered with 1H ammonium sulfate-added buffer (1) H7, 2) using 5ephacrYIS-30 (Pharmacia, diameter 20cJI x height 80α). . pHe obtained by equilibrating the molecular weight range 70,000 to i50.000 dalton fraction by gel filtration with the above 1H ammonium sulfate-added Mi stock solution.
It was adsorbed onto an nVI-sepharose 4 B column (manufactured by Pharmacia, diameter 10 x length 20 cm), and then eluted with a warm ladle solution containing 0.5H ammonium sulfate (pH 7.2). The eluate was filtered through an ultrafiltration model (Amicon 11P10).
) and concentrate with TSK-G3000SW (manufactured by Tosoh,
high-performance liquid chromatography at 2.5 x 60 cIn) with relative elution ff1 (Ve/Vo) 1.2-1.5.
I got both parts.

This fraction was concentrated again and Hi-Pore214 TP
(Vydac, light 2.2 x length 25 cm) reverse phase column RO, containing i% trifluoroacetic acid, acetonide! J/L10-100% <pH2, 0) (7)
The human M-C8F fraction was collected by high-performance liquid chromatography using a direct am gradient and lyophilized to obtain human M-C8F4 derived from human urine. Obtained Itg. The physicochemical properties of the obtained human M-C8F derived from human urine are as follows.

a) A homo2rf1 body consisting of subunits with the same molecular weight,
The molecular weight measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis is 7Q, 000 to 90,000 daltons, and the subunit whose biological activity has been lost by dissociating with a reducing agent was measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Molecule 5 is 35.00
It is 0 to 45°000 Daltons.

b) Amino acid sequence of subunit The subunit protein constituting the homo 2m body has the following 214 to 238 amino acid sequences, where the 122nd and 140th asparagine is asparagine (ASn)-x-threonine ( Thr)/Serine (Se
r) has a typical N-glycosidic bonding site. Here, X represents any amino acid.

GI u-G l u-Va l -3er-G 1
u-Tyr-Cys-3er-1f i 5-Het
-11e-G I y-3er-G I y-His
-Leu-G I n-3er-Leu-G I n-
Arg-Leu-T I e-Asp-3er-G l
n-)4et-G Iu-rhr-Ser-Cys
-G l n-11,e-Thr-Phe-G I u
-Phe-Val -Asp-G I n-G I
u-G I n-Leu-Lys-Asp-Pro-
Va Cys-Tyr-1, eu-Lys-Lys-
A i a-Phe-Leu-Leu-Vat-Gin
-Asp-11e-)1et-Glu-^sp-Thr
-)let-Arg-phe-Arg-831)-As
n-Thr-Pro-Asn-AIa-I 1e-Al
a-N 1e-Va I-G I n-Leu-G
I n-G lu -Leu-3er-Leu-Arg
-Leu-Lys-ser-cys-phe-rhr-
tys-85t)-Tyr-Glu-Glu-His-
Δ5p-Leu-Gln-Leu-Leu-Glu-L
ys-VaI-Lys-/1.5n-VaI-Phe
-Asn-Glu-Thr-Lys-^5n-L(+I
J-IJul-Asp-Lys-Asp-Trill-
Asn-1ie-Phe-3er-Lys-Asn-C
ys-Asn-85n-ser-phe-A la-
G I u-Cys-8e r-8er-G I n-
Asp-VaI-VaI-Thr-Lys-p
ro-Asp-eys-Δ5n-Cys-Leu-Ty
r-Pro-Lys-Ala-11, e-Pro-5e
r-8er-^5p-Pro-Ala-8er-Vat
-5er-Pro-)t+s-G I n-Pro-L
eu-A I a-Pro-8er-Net-A I
a-Pro-Va I-A l a-Gly-LeU
-Dinghr-TrD-G lu-^5p-3er-Glu
-Gly-Thr-Glu-G Iy-8er-3er
-Leu-Leu-Pro-G Iy-G 1u-G
l n-Pro-Leu-Old 5-Thr-Val-As
p-Pro-Gly-3er-8Ia-Lys-G! n
-Ar, 0-Pro-Pro-Arp-5er-Thr
-CyS-G I n-8C! r-Phe-G Iu-
Pr.

-Pro-Glu-Thr-Pro-Val-Val-
Lys-C) Isoelectric point The isoelectric point (pI) measured by polyacrylamide gel isoelectrophoresis and sucrose density gradient isoelectric focusing is 3.1 to 3.7.

d) Circular dichroism spectrum The far ultraviolet CD spectrum measured by a circular dichroism dispersion meter has a wavelength of 2.
It has minimum peaks at 08 nm and 222 nm, and contains an α-helical structure.

e) Thermal stability No loss of biological activity when heated at 60±0,5°C for 60 minutes.

t) Infrared absorption spectrum wave numbers 1680CM, 1200cm-' and 1130α
It has an infrared absorption spectrum showing strong absorption at waves ν1540 and moderate absorption at 1430 cm and 1070 cffi-1.

Margins Human IM-CSF exhibiting such physicochemical properties can usually be administered parenterally, such as intravenously, intraarterially, intramuscularly, subcutaneously, or intraperitoneally. Preparations for administration include injections, infusions, and the like, and these preparations can be prepared by methods known per se. For example, human M-CSF' can be added to a suitable buffer solution, sterilized, aseptically filled into a glass vial, sealed, and optionally lyophilized to prepare a formulation. .

・Human M-C8F has the property of adsorbing to glass, plastic, sterile filtration membranes, etc., but this adsorption can be achieved by adsorbing any single or multiple polymeric substances such as surfactants, human serum albumin, and gelatin. This can be prevented by using these polymeric substances, and at the same time, the stability of the formulation can be significantly improved by formulating it with these polymeric substances. The concentration used for each product is desirably 10/ml or more for surfactants, and 1/ml or more for human serum albumin and gelatin.

H) The dose of M-C8F may vary depending on the patient's age and symptoms, regardless of whether or not arteriosclerosis occurs, but it is usually 0.4 N to 16 μt/kg body weight 7 days, preferably 1 day.
．． 6 μg to 8 βg/kg body weight 7 days.

Examples of the present invention using human M-C8F obtained by the above method are shown below.

Example 1 Cholesterol-lowering effect of human M-C8F on hyperlipidemic patients (13) Preparation method of the hyperlipidemia therapeutic agent of the present invention (hereinafter referred to as water solution) Human M-C3F prepared as described above and the stabilizer at each concentration shown in Table 1 were added to give a concentration of human M-C8F of 10011 g/
It was prepared in ml. Filter aseptically with a nitrocellulose-based sterile filtration membrane, and aseptically place 1 mi in a glass vial.
Filled.

After freeze-drying, the mixture was sealed and a solution was prepared.

(2) Stability of the liquid formulation The stability of the liquid formulation was determined by measuring human M-C3F activity using a soft agar method using mouse bone marrow cells.

The results are shown in Table 1. The biological activity of liquid solutions prepared at a concentration of 1 hg/ml or higher for the surfactant Tween 80 and 1 hg/ml or higher for human serum albumin and gelatin was determined at the start of the test (immediately after manufacture). ) was maintained at 70% or more and was considered stable.

3) Cholesterol-lowering effect of liquid medication on hyperlipidemic patients A liquid medication prepared by adding 5 μg/ml of albumin to 2 hyperlipidemic patients was administered at 1°6 μg/kg/7 days after body weight. It was administered intravenously for 14 days. The amount of cholesterol in the serum was measured at 7-day intervals from the time of administration of the liquid medication, and the cholesterol-lowering effect of the liquid medication was investigated.

By administering the solution as shown in Figure 1, the serum cholesterol level of one hyperlipidemic patient was reduced to 395% compared to before the administration of the solution.
The concentration decreased significantly from ■/ml to 170■/ml, which became a normal value. Furthermore, as shown in FIG. 2, in the case of another patient, the serum cholesterol level decreased significantly from 280 μ/ml to 195 μ/ml. In both figures,
The horizontal axis shows the period in weeks, and the vertical axis shows the serum total cholesterol amount (■/ml). These results revealed that this drug is useful as a therapeutic agent for hyperlipidemia as well as arteriosclerosis caused by hyperlipidemia.

Example 2 Effect on lowering serum cholesterol in hyperlipidemia model animals In Example 1, 20% of Tween 80 was used as a stabilizer.
Using a solution prepared in the same manner as in Example 1 except that the concentration was adjusted to a buffer solution, the effect of the solution on reducing cholesterol and triglyceride content in hyperlipidemia model animals was investigated.

Sprague-Davle 7 rats consisting of 5 rats per group were pre-fed for one month on a high-fat diet, and then 16 μg of the liquid drug was administered.
/kg・body weight (C8F dose: 13.3pt/kg・body weight) was administered intravenously for 7 consecutive days, and the changes in the amount of cholesterol and triglycerides in serum were compared with the control group. The buffer solution used in preparing the solution was administered.

As shown in Table 2, it was observed that the amount of serum cholesterol and triglyceride decreased significantly by administration of the liquid medication, and it is clear that the liquid medication is useful as a therapeutic agent for hyperlipidemia and arteriosclerosis caused by it. became.

[Effects of the Invention] (+1) It is possible to provide a drug that significantly reduces abnormally high serum cholesterol levels and triglyceride levels and has no side effects.

(2) It is possible to provide a drug that treats hyperlipidemia, improves and prevents arteriosclerosis, and has no side effects.

[Brief explanation of drawings]

Figure 1 is a graph showing the relationship between serum cholesterol levels and administration periods for a certain hyperlipidemic patient, and Figure 2 is a graph showing the relationship between serum cholesterol levels and administration periods for other hyperlipidemia patients. . Autopsy 1 body 1 Gibe IK mouth~Sahiro~nto-EI7te'f! nJ bet a - every ωζ affirmation - sleep

Claims (1)

[Claims] (1) Hyperlipidemia therapeutic agent containing human urine-derived human monocyte-macrophage colony-stimulating factor as an active ingredient