JP2641067B2 - Hyperlipidemia treatment - Google Patents

Description

The present invention relates to a therapeutic agent for hyperlipidemia containing a recombinant human monocyte-macrophage colony stimulating factor (hereinafter referred to as rhM-CSF) as an active ingredient. .

[Background of the Technology and Conventional Technology] Hyperlipidemia is a disease in which one or more of cholesterol, neutral fat, phospholipid, and the like increase more than normal. For Japanese, total cholesterol level is 220 mg or more, triglyceride level is 130 mg or more, and phospholipid is 25
Hyperlipidemia is defined as 0 mg or more. Hyperlipidemia itself is not a serious disease, but if left untreated, it causes arteriosclerosis, leading to angina pectoris and myocardial infarction, which is a serious clinical problem. Currently there are many drugs for the treatment of hyperlipidemia and arteriosclerosis, but clinically probucol preparations (Akira Watanabe et al.
Atherosclerosis, Volume 11, Issue 3, p. 597 (1983)) and elastase, a proteolytic enzyme (Sezo Yoshimura, Arteriosclerosis, 3
Volume, p. 223, 1975). The effects of these drugs make it difficult for cholesterol to adhere to the blood vessel wall and wash away cholesterol attached to the blood vessel wall, and their effects are limited to some extent, and are currently being cured in hyperlipidemia and arteriosclerosis. There is no cure.

There is a factor (M-CSF) that acts on monocyte-macrophage stem cells among colony stimulating factors, which is a kind of hematopoietic factor, and its protein and gene structure have been elucidated (GG Wong et al., Science, 2, 35 ( 20) 1504-1508
(1987)). This human M-CSF also acts on mature human monocytes-macrophages and promotes its function activation and production of various cytokines (Motoyoshi K et al. Exp. Hemat.
ol., 17: 68-71 (1989)).

It is also known that monocytes-macrophages are deeply involved in hyperlipidemia and arteriosclerosis. But rhM-C
The effect of SF on monocyte-macrophage lipid metabolism and its potential as a therapeutic agent for hyperlipidemia and arteriosclerosis has not been examined.

[Object and Summary of the Invention] Hyperlipidemia is a disease in which cholesterol, triglycerides and phospholipids in the blood are higher than normal levels. If left untreated, it can cause arteriosclerosis and induce myocardial infarction and angina. It is clear. Therefore, prevention of arteriosclerosis with an excellent therapeutic agent for hyperlipidemia and arteriosclerosis is extremely important in preventing myocardial infarction, angina and the like. The present invention has been studied as a therapeutic agent for hyperlipidemia and arteriosclerosis of rhM-CSF on hyperlipidemia patients and hyperlipidemia model animals, rhM-C
The present inventors have found that SF has an effect of remarkably reducing blood cholesterol and triglyceride, which are most important in hyperlipidemia and arteriosclerosis, and completed the present invention.

The present invention is a therapeutic agent for hyperlipidemia and arteriosclerosis containing rhM-CSF as an active ingredient. Specifically, M-CSF is a therapeutic agent for hyperlipidemia and arteriosclerosis containing rhM-CSF prepared from human M-CSF transgenic cells as an active ingredient.

[Technical Configuration of the Invention] rhM-CSF according to the present invention was prepared by freeze-drying a substance purified by the following method. Ie purified rhM
-An anti-rhM-CSF antibody obtained by immunizing rabbits with CSF was dialyzed in a 0.1 M phosphate buffer (pH 7.0) to a concentration of 20 mg / ml. 200 ml of the antibody solution was added with 100 g of formyl-cellulofine previously washed with distilled water and 0.1 M phosphate buffer, stirred at room temperature for 2 hours, then added with 700 mg of sodium cyanoborohydride, and further stirred for 16 hours. Then, formyl-cellulofine was bound to the anti-rhM-CSF antibody to prepare an antibody-bound support. After engagement, wash with 0.2 M Tris-HCl buffer, and further 500 mg of sodium cyanoborohydride
Was added and the mixture was stirred at room temperature for 4 hours to inactivate unreacted groups. The antibody-engaged support was then
The wells were sufficiently washed with a 0.02 M phosphate buffer containing 5 M NaCl. The antibody-bound support bound 32.6 mg of anti-CSF antibody per gram of support. Next, human M-CSF transgenic cells (C
10 L of the culture solution of HO cells) was concentrated with an ultra-superconcentrator, desalted, adsorbed on DAFE-cellulose, the unadsorbed contaminants were removed, and eluted with a 0.3 M NaCl solution. 0.5M
Sodium-M-CS
A solution containing F was prepared. The specific activity of this human M-CSF was 3 × 10 ⁶ units / mg. 100 g of the antibody binding support
To the solution containing human M-CSF (500 ml in total)
, And the mixture was stirred at 10 ° C. or less overnight, and subjected to batch chromatography. After stirring, pass through a glass filter to collect the antibody-bound support, and add 0.02M containing 0.5M NaCl.
The antibody-bound support was thoroughly washed with M phosphate buffer.
After washing, add 500 ml of 0.2 acetate buffer (pH 2.5),
After stirring for 1 hour, rhM-CSF was eluted. Adjust the pH after elution to 7.
After the concentration was reduced to 0, the mixture was concentrated and desalted with an ultraperfusion membrane to obtain an rhM-CSF fraction. This fraction was separated into Hi-Pore214TP (Vydac,
Human M-CSF was collected and lyophilized to obtain 25 mg of rhM-CSF by high-performance liquid chromatography with a linear concentration gradient of acetonitrile containing 0.1 trifluoroacetic acid 0 to 100 (pH 2.0) using a reversed phase column having a diameter of 2.2 × 25 cm). . The specific activity of the purified rhM-CSF was 1.9 × 10 ⁸ units / mg, and the purity by SDS-PAGE method was 98% or more.

rhM-CSF can be usually administered by parenteral administration such as intravenous, intraarterial, intramuscular, subcutaneous, and intraperitoneal. Formulations for administration include injections, infusions and the like,
These preparations can be prepared by a method known per se. For example, a formulation can be prepared by adding a suitable excipient as a pharmaceutical to human M-CSF, aseptically filtering, aseptically filling and sealing glass vials, and freeze-drying as necessary. .

The dosage of rhM-CSF for patients with hyperlipidemia and atherosclerosis can vary depending on the age of the patient, but is usually 0.4 μm.
g to 16 μg / kg · body weight / day, preferably 1.6 μg to 8 μg / k
g · 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 hyperlipidemic animals (1) Preparation of therapeutic agent for hyperlipidemia of the present invention (hereinafter referred to as "this agent") pH 7.2 containing 0.15M sodium chloride Mannitol 10 mg / ml and rhM-CSF were prepared at a concentration of 100 mg / ml in a 20 mM phosphate buffer. After aseptic passage with a nitrocellulose-based aseptic membrane, 1 ml was aseptically filled in a glass vial to prepare a therapeutic agent for hyperlipidemia and arteriosclerosis.

(2) Cholesterol lowering effect of this drug on hyperlipidemic animals The serum cholesterol lowering effect of rhM-CSF on hyperlipidemic rabbits (Watanabe rabbits) was examined. The test method is as follows. The test period was divided into the control administration period and this drug administration period, and the test was performed. Four hyperlipidemic rabbits were subcutaneously administered 2.5 ml of the buffer used for the production of this drug for 7 consecutive days, and then observed for 10 days (control drug administration period). Subsequently, this drug was subcutaneously administered at a rate of 50 μg / day for 7 consecutive days, and then observed for 8 days (drug administration period). Serum cholesterol levels were measured over time during the administration of the control drug and during the administration period of this drug, the rate of decrease in serum cholesterol level before drug administration was calculated, and the cholesterol lowering effect of this drug was examined.

As shown in FIG. 1, serum cholesterol during the period of administration of the control drug was 105% to 97% before administration of the control agent, and no change was observed during the administration period. On the other hand, during the administration period of this drug, serum cholesterol was markedly reduced to 83% to 85% before administration. In FIG. 1, the horizontal axis represents the period expressed in days, and the vertical axis represents the total serum cholesterol amount (mg / ml).
These results revealed that this drug is useful as a therapeutic agent for arteriosclerosis caused by hyperlipidemia.

Example 2, cholesterol-lowering effect of rhM-CSF on normal animals Human serum albumin 5 mg / obtained in the same manner as in Example 1
The serum cholesterol lowering effect of normal rabbits was examined using this drug prepared with a buffer solution containing ml. 5 New Zealand White rabbits at 50μg / kg body weight for 7 consecutive days
It was administered subcutaneously for one day and the serum cholesterol level before and after administration was measured. The result is shown in FIG. In FIG. 2, the horizontal axis represents the measurement time, and the vertical axis represents the total serum cholesterol amount (mg / m2).
l).

As shown in FIG. 2, administration of this drug reduced serum cholesterol, but the serum cholesterol lowering effect of this drug was more markedly reduced in rabbits with high serum cholesterol. The effect was mild on rabbits with low serum cholesterol levels before administration. The decrease in serum cholesterol in this study was 28% (mean) before administration. These results revealed that this formulation was useful as a therapeutic agent for arteriosclerosis caused by hyperlipidemia.

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

(2) It can treat hyperlipidemia, improve and prevent arterial effects, and provide a drug having no side effects.

[Brief description of the drawings]

FIG. 1 is a graph showing the change over time in serum total cholesterol after administration of this drug and a control drug as a ratio to that before administration, and FIG. 2 is a graph showing the change in the amount of serum cholesterol before and after administration of this drug. is there.

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Claims (1)

(57) [Claims] (1) A therapeutic agent for hypertrilipidemia comprising a recombinant human monocyte-macrophage colony stimulating factor as an active ingredient.