JPH082796B2 - Hyperlipidemia treatment - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial application] The present invention is a therapeutic agent for hyperlipidemia, which comprises human urine-derived human monocyte-macrophage colony stimulating factor (hereinafter referred to as human M-CSF) as an active ingredient. Regarding

[Background of the Technology and Prior Art] Hyperlipidemia is a disease in which one or more of cholesterol, triglyceride, phospholipid and the like increase above a normal value. In the case of Japanese, hyperlipidemia is defined as a total cholesterol level of 220 mg or more, neutral fat of 130 mg or more, or phospholipid of 250 mg or more per 100 ml of blood. Although hyperlipidemia itself is not a serious disease, if left untreated, it causes arteriosclerosis and induces angina and myocardial infarction, which is a serious clinical problem. Currently, there are many drugs for the treatment of hyperlipidemia and arteriosclerosis, but clinically there are probucol preparations (Akira Watanabe et al., Arteriosclerosis, Vol. 11, No. 3, pp. 597,
1983) and the proteolytic enzyme elastase (Shozo Yoshimura, Atherosclerosis, Vol. 3, pp. 223, 1975) are mainly used. The action of these drugs is to make it difficult for cholesterol to adhere to the blood vessel wall or to wash away cholesterol adhering to the blood vessel wall, but there is a certain limit to its effect, and it currently causes hyperlipidemia and arteriosclerosis. There is no cure for this.

Among colony stimulating factors, which is a type of hematopoietic factor, there is a factor (M-CSF) that acts on monocyte-macrophage stem cells, and its protein and gene structure have been clarified (Japanese Patent Laid-Open No. 64-22899). . This human M-CSF also acts on mature human monocyte-macrophage to promote its functional activation and production of various cytokines (Motoyosh
i K. et al., Exp. Hematol., 17: 68-71 (1989)) and clinically granulocytopenia (Motoyoshi K., et al., Exp. Hema
tol.14: 1069-1075 (1986)) and bone marrow transplantation (Masaoka T.,
et al., Bone Marrow Transplantation. 3: 121-127 (198
The utility for 8)) has been clarified, and there are great expectations as a medicine. This human M-CSF has already been clinically tested,
Its safety has been confirmed (Motoyoshi K., et al., Im
Munobiology, 172; 205-212, (1986)).

However, the applicability of human M-CSF to the treatment of hyperlipidemia and arteriosclerosis has been left unexamined.

[Object and Summary of the Invention] Hyperlipidemia is a disease in which blood cholesterol, triglyceride, or phospholipid is higher than the normal value, and if left untreated, arteriosclerosis may occur, causing myocardial infarction, angina, etc. Has become clear. Therefore, it is extremely important to prevent arteriosclerosis with an excellent therapeutic agent for hyperlipidemia and arteriosclerosis in order to prevent myocardial infarction and angina. In the present invention, human M-CSF was examined as a therapeutic agent for hyperlipidemia and arteriosclerosis in hyperlipidemia patients and hyperlipidemia model animals. The present invention has been completed by discovering that it has the effect of significantly reducing blood cholesterol level and triglyceride level, which are the most important factors in arteriosclerosis.

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

[Detailed Description of the Present Invention] Human urine-derived human M-CSF relating to the present invention is a known method from urine of a healthy person (Japanese Patent Laid-Open No. 63-198700 and Japanese Laid-Open Patent Publication No. Sho 63-198700).
Those purified according to JP-A 63-250400 and JP-A No. 64-22899) were lyophilized and adjusted. For example 63-1987
The following preparation was carried out by the method described in Japanese Patent Publication No. 00. That is, after adjusting 1000 L of urine of a healthy person to pH 8.5, the precipitate is filtered off,
Ultrafiltration membrane with a molecular weight of 50,000 daltons (Amicon, H10
× 50) and concentrated and desalted. Next, the pH was adjusted to 7.0 and heat sterilization was performed in a closed container at 60 ° C for 10 hours. After sterilization, centrifuge (5000 xg for 30 minutes) to remove the precipitate, then 0.02M
After mixing with DEAE-cellulose equilibrated with phosphate buffer (pH7.2), the adsorbed DEAE-cellulose was washed with 0.02M phosphate buffer (pH7.2) containing 0.05M sodium chloride, and then 0.25M sodium chloride. Elution was performed with a 0.02 M phosphate buffer (pH 7.2). The eluate was concentrated with an ultrafiltration membrane (H10P10 manufactured by Amicon) to obtain Seph
acryl S-30 (Falmacia, diameter 20 cm x height 80 cm)
Gel filtration was performed using 1M ammonium sulfate addition buffer (pH 7.2).
Phenyl-Sepharos obtained by equilibrating the molecular weight range of 70,000 to 150,000 Dalton by gel filtration with the above 1 M ammonium sulfate-added buffer.
It was adsorbed on an e4B column (Diameter 10 × length 20 cm, manufactured by Pharmacia), and then eluted with 0.5 M ammonium sulfate-added buffer (pH 7.2). The eluate was concentrated with an ultrafiltration membrane (H1P10 manufactured by Amicon) and subjected to high performance liquid chromatography with TSK-G3000SW (manufactured by Tosoh, diameter 2.5 x 60 cm), and the relative elution amount (Ve / V
o) 1.2-1.5 fractions were obtained. Concentrate this fraction again and
-Pore214TP (Bidadsk Co., Ltd., 2.2 x 25 cm long) reverse phase column containing 0.1% trifluoroacetic acid and subjected to high-performance liquid chromatography with a linear concentration gradient of 0-100% acetonitrile (pH 2.0) to give human M- The CSF fractions were collected and lyophilized to obtain 4 mg of human M-CSF derived from human urine. The physicochemical properties of the obtained human M-CSF derived from human urine are as follows.

a) A homodimer consisting of identical molecular weight subunits, which has a molecular weight of 70,000 to 90,000 daltons measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis and is dissociated with a reducing agent to eliminate the biological activity. The molecular weight measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis is 35,000-45,000 daltons.

b) Amino acid sequence of subunit The subunit protein that constitutes the homodimer has the amino acid sequence of 214 to 238 shown below, and the 122nd and 140th amino acids.
The second asparagine is Asparagine (Asn)-
It has a typical N-glycoside binding site represented by x-threonine (Thr) / serine (Ser). Here, x represents an arbitrary amino acid.

Glu-Glu-Val-Ser-Glu-Tyr-Cys-Ser-His-Met-
Ile-Gly-Ser-Gly-His-Leu-Gln-Ser-Leu-Gln-
Arg-Leu-Ile-Asp-Ser-Gln-Met-Glu-Thr-Ser-
Cys-Gln-Ile-Thr-Phe-Glu-Phe-Val-Asp-Gln-
Glu-Gln-Leu-Lys-Asp-Pro-Val-Cys-Tyr-Leu-
Lys-Lys-Ala-Phe-Leu-Leu-Val-Gln-Asp-Ile-
Met-Glu-Asp-Thr-Met-Arg-Phe-Arg-Asp-Asn-
Thr-Pro-Asn-Ala-Ile-Ala-Ile-Val-Gln-Leu-
Gln-Glu-Leu-Ser-Leu-Arg-Leu-Lys-Ser-Cys-
Phe-Thr-Lys-Asp-Tyr-Glu-Glu-His-Asp-Lys-
Ala-Cys-Val-Arg-Thr-Phe-Tyr-Glu-Thr-Pro-
Leu-Gln-Leu-Leu-Glu-Lys-Val-Lys-Asn-Val-
Phe-Asn-Glu-Thr-Lys-Asn-Leu-Leu-Asp-Lys-
Asp-Trp-Asn-Ile-Phe-Ser-Lys-Asn-Cys-Asn-
Asn-Ser-Phe-Ala-Glu-Cys-Ser-Ser-Gln-Asp-
Val-Val-Thr-Lys-Pro-Asp-Cys-Asn-Cys-Leu-
Tyr-Pro-Lys-Ala-Ile-Pro-Ser-Ser-Asp-Pro-
Ala-Ser-Val-Ser-Pro-His-Gln-Pro-Leu-Ala-
Pro-Ser-Met-Ala-Pro-Val-Ala-Gly-Leu-Thr-
Trp-Glu-Asp-Ser-Glu-Gly-Thr-Glu-Gly-Ser-
Ser-Leu-Leu-Pro-Gly-Glu-Gln-Pro-Leu-His-
Thr-Val-Asp-Pro-Gly-Ser-Ala-Lys-Gln-Arg-
Pro-Pro-Arg-Ser-Thr-Cys-Gln-Ser-Phe-Glu-
Pro-Pro-Glu-Thr-Pro-Val-Val-Lys-c) Isoelectric point The isoelectric point (pI) measured by polyacrylamide gel isoelectric focusing method and sucrose density gradient isoelectric focusing method is
It is 3.1 to 3.7.

d) Circular dichroism spectrum The deep UV CD spectrum measured by the circular dichroism disperser has a wavelength of 20.
It has minimum peaks at 8 nm and 222 nm, respectively, and contains an α-helix structure.

e) Thermal stability No biological activity is lost by heating at 60 ± 0.5 ° C for 60 minutes.

f) Infrared absorption spectrum wavenumber 1680 cm ^-1, having an infrared absorption spectrum shown intensity absorbed in 1200 cm ^-1 and 1130 cm ^-1, wave number 1540 cm ^-1, a moderate absorption in 1430 cm ^-1 and 1070 cm ^-1.

Human M-CSF showing such physicochemical properties is usually
It can be administered by parenteral administration such as intravenous, intraarterial, intramuscular, subcutaneous, and intraperitoneal administration. Examples of the preparation for administration include injections and infusions, and these preparations can be prepared by a method known per se. For example, human M-CSF can be prepared by adding human M-CSF to an appropriate buffer, aseptically filling, aseptically filling in a glass vial, sealing, and freeze-drying if necessary.

Human M-CSF has the property of being adsorbed on glass, plastic, sterile membrane, etc., but this adsorption uses any one or a plurality of macromolecules such as surfactants, human serum albumin and gelatin. This can be prevented, and at the same time, the stability thereof can be remarkably improved by formulating with these polymer substances. The concentration of each used is preferably 10 μg / ml or more in the case of a surfactant and 1 mg / ml or more in the case of human serum albumin and gelatin per formulation.

The dose of human M-CSF may vary depending on the age and symptoms of the patient regardless of whether arteriosclerosis is accompanied or not, but is usually 0.4 μg to 16 μg / kg body weight / day, preferably 1.6 μg to
8 μg / kg / body weight / day.

Examples of the present invention using human M-CSF obtained by the above method will be described below.

Example 1 Cholesterol-lowering effect of human M-CSF on hyperlipidemia patients (1) The therapeutic agent for hyperlipidemia of the present invention (hereinafter referred to as "this agent")
Preparation method of human M-CSF prepared as described above and stabilizers at respective concentrations shown in Table 1 were added to a 20 mM phosphate buffer solution having a pH of 7.2, and the concentration of human M-CSF was 100 μg / ml. Was prepared. Aseptic sterilization was performed with a nitrocellulose-based aseptic membrane, and 1 ml was aseptically filled in a glass vial. This product was prepared by freeze-drying and sealing.

(2) Stability of this drug The stability of this drug was determined by measuring the human M-CSF activity by a soft agar method using mouse bone marrow cells. As shown in Table 1, the biological activity of this agent prepared at a concentration of 10 μg / ml or more for the surfactant Tween 80 and 1 mg / ml or more for human serum albumin and gelatin at the start of the test (immediately after production) is shown in Table 1. of
It was maintained at 70% or more and was considered stable.

(3) Cholesterol-lowering effect of this drug on hyperlipidemic patients The drug prepared by adding 5 mg / ml of albumin to 2 hyperlipidemic patients was infused intravenously for 14 days at 1.6 μg / kg / body weight / day. did. The amount of cholesterol in serum was measured at intervals of 7 days after administration of this drug, and the cholesterol lowering effect of this drug was examined.

By administering this drug as shown in Fig. 1, the serum cholesterol level in one hyperlipidemic patient was 395m before administration of this drug.
It decreased remarkably from g / ml to 170 mg / ml, and became a normal value. Further, as shown in FIG. 2, in the case of the other one patient, the amount of serum cholesterol was remarkably reduced from 280 mg / ml to 195 mg / ml. In both figures, the horizontal axis represents the period expressed in weeks, and the vertical axis represents the total serum cholesterol level (mg / ml). From these results, it became clear that this drug is useful as a hyperlipidemia and as a therapeutic agent for arteriosclerosis caused by hyperlipidemia.

Example 2 Effect on serum cholesterol lowering in hyperlipidemia model animal In Example 1, Tween 80 20 μm was used as a stabilizer.
Using this drug in the same manner as in Example 1 except that the concentration was changed to g / ml, the effect of this drug on the reduction of cholesterol and neutral fat in hyperlipidemic model animals was examined.

Sprague-Dawley rats consisting of 5 animals per group were preliminarily fed with a high-fat diet for 1 month, and then 16 μg / kg body weight (CS
F dose of 13.3 μg / kg / body weight) was intravenously administered for 7 consecutive days, and changes in serum cholesterol and triglyceride levels were compared with the control group. The control group was administered with the buffer solution used in the preparation of this drug.

As shown in Table 2, administration of this drug markedly reduced serum cholesterol and triglyceride content, demonstrating that this drug is useful as a therapeutic agent for hyperlipidemia and its associated arteriosclerosis. became.

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

(2) Treat hyperlipidemia, improve / prevent arteriosclerosis,
And a drug without side effects can be provided.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the serum cholesterol level of a hyperlipidemic patient and the administration period, and FIG. 2 is a graph showing the relationship between the serum cholesterol level of another hyperlipidemic patient and the administration period. is there.

Claims (1)

[Claims] 1. A therapeutic agent for hypertriglyceridemia, which comprises human urine-derived human monocyte-macrophage colony stimulating factor as an active ingredient.