JPH032125A - Remedy for hyperlipemia - Google Patents

Abstract

PURPOSE:To obtain a remedy for hyperlipemia, containing a genetic recombinant human monocyte-macrophage colony stimulating factor (rhM-CSF) as an active ingredient and having action of remarkably reducing blood cholesterol and neutral fat levels. CONSTITUTION:A rhM-CSF obtained by concentrating a culture solution of a human M-CSF genetic recombinant cell (CHO cell) using an ultrafiltration concentration, then desalting the resultant filtrate, adsorbing the desalted filtrate on a DEAE-cellulose, removing the unadsorbed contaminating substances, eluting the adsorbed substances with a 0.3M NaCl solution, adding NaCl to the above- mentioned eluate so as to provide 0.5M concentration thereof, preparing a solution containing a human M-CSF, adding the aforementioned solution to an antibody-bound support containing formyl-cellulofine and an anti-rhM-CSF antibody bound thereto, carrying out batch type chromatographic treatment, then purifying the treated substance and freeze-drying the purified substance. The dose of the above-mentioned active ingredient for patients suffering from hyperlipemia and arteriosclerosis is 0.4-16mg/kg body weight/day, preferably 1.6-8mg/kg body weight/day.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention relates to a therapeutic agent for hyperlipidemia containing genetically recombinant human monocyte-macrophage colony stimulating factor (hereinafter referred to as rhM-C8F) as an active ingredient. .

[Technical Background and Prior Art] Hyperlipidemia is a disease in which one or more of cholesterol, neutral fats, phospholipids, etc. increases above normal levels. In the case of Japanese people, hyperlipidemia is defined as a total cholesterol value of 220 μg or more, a neutral fat content of 130 μg or more, and a phospholipid content of 250 mg or more per 100 ml of blood. Hyperlipidemia itself is not a serious disease, but if left untreated, it can lead to arteriosclerosis, angina pectoris, and myocardial infarction.
This is a clinically significant problem. Currently, there are many drugs for the treatment of hyperlipidemia and arteriosclerosis, but clinically, probucol preparations (Akira Watanabe et al., Arteriosclerosis, Vol. 11, No. 3, p. 597, 1983) and proteolytic enzymes are used. A certain elastase (Shozo Yoshimura, Arteriosclerosis, vol. 3, p. 223, 197
5 years) is mainly used. The action of these drugs is to remove cholesterol from adhering to blood vessel walls, and to wash away cholesterol adhering to blood vessel walls.There are certain limits to their effectiveness, and they are currently being used to treat hyperlipidemia and arteriosclerosis. There are no drugs available for definitive treatment.

Among the colony-stimulating factors, which are a type of hematopoietic factor, there is a factor (M-C8F) that acts on monocyte-macrophage stem cells, and its protein and gene structure have been clarified (G, Wong etaScience
, 2. 35 (20) 1504-1508(
1987)). This human M-CSF also acts on mature human monocytes and macrophages, promoting their functional activation and the production of various cytokines (Moto7osh
iKelal, El+1. l(emxtol
, 17: 6871 (1989)).

It is also known that monocytes-macrophages are deeply involved in hyperlipidemia and arteriosclerosis.

However, the effects of rhM-C3F on monocyte-macrophage lipid metabolism and its potential as a therapeutic agent for hyperlipidemia and arteriosclerosis have remained unexamined.

[Purpose and Summary of the Invention Hyperlipidemia is a disease in which cholesterol, triglycerides, and phospholipids in the blood are higher than normal values, and if left untreated, it can lead to arteriosclerosis and induce myocardial infarction, angina, etc. It has become clear. Therefore, it is extremely important to prevent arteriosclerosis with an excellent hyperlipidemia and arteriosclerosis therapeutic agent in order to prevent myocardial infarction and angina pectoris. The present invention is based on the results of an investigation of rhM-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 hM-C3F has the effect of significantly reducing the amount of blood cholesterol and triglyceride, which are the most important factors in hyperlipidemia and arteriosclerosis.

The present invention is a therapeutic agent for hyperlipidemia and arteriosclerosis containing rhM-C8F as an active ingredient. Specifically, M-C8F is human M
- rh hi prepared from C8F genetically modified cells
-A therapeutic agent for hyperlipidemia and arteriosclerosis containing CSF as an active ingredient.

[Technical configuration of the invention] rhM-C8F related to the present invention was prepared by freeze-drying purified product by the following method. That is, anti-rhM-C3 obtained by immunizing rabbits with purified rhM-C8F.
F antibody in 0.1M phosphate buffer (p) 17. 0) and adjusted to a concentration of 20 mg/ml. The antibody solution 2
00ml was added to 100g of formylcellulofine which had been washed with distilled water and 0.1M phosphate buffer in advance, and stirred at room temperature for 2 hours, then 700mg of sodium cyanoborohydride was added and stirred for an additional 16 hours. ,
An antibody-bound support was prepared by binding 7-olmylucellofain to an anti-rhM-C8F antibody. After bonding, 0.2M
Wash with Tris-HCl buffer and add 200 ml of Tris buffer containing 500 mg of sodium cyanoborohydride.
1 was added and stirred at room temperature for 4 hours to inactivate unreacted groups. The antibody-bound support was then thoroughly washed with 0.02M phosphate buffer containing 0.5M NlCl. The antibody-bound support bound 32.6 μ of anti-C3F antibody per gram of support.

Next, 10 L of the culture solution of human M-C8F genetically modified cells (CHO cells) was concentrated using a hyperconcentrator and desalted.
DEAE- adsorbed on cellulose to remove non-adsorbed contaminants, 0. Elution was performed with a 3M NaCl solution, and sodium chloride was added to the eluate to give a concentration of 0.5M to prepare a solution containing human M-C8F. This human M-
The specific activity of C3F was 3×10 6 units/■. This solution containing human M-C3F (total Ji 1500 ml) was added to 100 g of the above antibody-bound support, and the mixture was stirred overnight at 10° C. or lower to perform batch chromatography. After stirring, the antibody-bound support was collected by filtration with a glass filter, and 0. 0,02 containing 5M NaCl
The antibody-bound support was thoroughly washed with M phosphate buffer.

After washing, 500ml of 0.2M acetate buffer (pH 2,5)
was added and stirred at 10°C for 1 hour to elute rhM-C3F. After adjusting the pH of the eluate to 7.0, it was concentrated and desalted using an ultra-I membrane to obtain a rhM-CSF fraction. This fraction was converted into H4-Pour214TP (Vydac, diameter 2
．． Human M-C3F was collected and lyophilized using high performance liquid chromatography using a linear concentration gradient of acetonitrile 0 to 100 (pH 2,0) containing 0.1 trifluoroacetic acid on a reverse phase column (2 x 25 cm) and rhM-C8F 25
mg was obtained. The specific activity of purified rhM-C8F is 1.9X1
08 units/mg, SDS-PAGE
The purity by the method was 98% or more.

rhM-CSF can usually be administered parenterally, such as intravenously, intraarterially, intramuscularly, subcutaneously, or intraperitoneally. Preparations for administration include injections and infusions, and these preparations can be prepared by methods known per se. For example, human M-C8F is added to an excipient suitable for pharmaceutical use, filtered aseptically, filled aseptically into a glass vial, sealed, and, if necessary, freeze-dried to prepare a preparation. I can do it.

The dosage of rhM-C8F for patients with hyperlipidemia and arteriosclerosis may vary depending on the patient's age and symptoms, but is usually 0.
．． 4■~16ug/kg・7 days of body weight, preferably 1.6
■~8 ug/kg, 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-C3F on hyperlipidemic animals (1) Preparation method of hyperlipidemia therapeutic agent (hereinafter referred to as water solution) of the present invention pH 7 containing 0.15M common salt, 20mM phosphate buffer, 10μ/ml of mannitol, and rhM-
CSF was prepared at a concentration of 100 ug/ml.

After sterile filtration with a nitrocellulose-based sterile 7 filtration membrane,
A therapeutic agent for hyperlipidemia and arteriosclerosis was prepared by aseptically filling 1 ml into a glass vial.

(2) Cholesterol-lowering effect of liquid medication on hyperlipidemic animals rhM-on hyperlipidemic domestic rabbits (Watanabe rabbits)
The serum cholesterol lowering effect of C8F was investigated. The test method is as follows. The test period was divided into a control administration period and a solution administration period. 2.5 ml of the buffer solution used for manufacturing the liquid preparation was subcutaneously administered to four hyperlipidemic rabbits for 7 consecutive days, and then observed for 10 days (control drug administration period). Subsequently, the liquid solution was subcutaneously administered at a rate of 50 ug/day for 7 consecutive days, followed by observation for 8 days (solution administration period).

The amount of serum cholesterol was measured over time during the control drug administration period and the solution administration period, and the reduction rate relative to the serum cholesterol amount before drug administration was calculated to examine the cholesterol-lowering effect of the solution.

As shown in FIG. 1, serum cholesterol during the control drug administration period was 97% of the control drug administration period, and no change was observed during the administration period. On the other hand, during the administration period of the liquid medication, serum cholesterol was significantly reduced to 83% to 85% of the pre-administration level. In FIG. 1, the horizontal axis shows the period expressed in days, and the vertical axis shows the serum total cholesterol amount (■/ml). These results revealed that the liquid solution is useful as a therapeutic agent for arteriosclerosis caused by hyperlipidemia.

Example 2: Cholesterol lowering effect of rhM-C8F on normal animals 5 mg of human serum Abmin obtained in the same manner as Example 1
The serum cholesterol-lowering effect in normal rabbits was investigated using a liquid solution prepared with a buffer solution containing 0.25% of the total concentration of 1.0% of the buffer solution. New Sealant White 5 rabbits with 50 kg of liquid medicine
The mice were subcutaneously administered according to their body weight for 7 consecutive days, and the amount of serum cholesterol was measured before and after administration. The results are shown in FIG. In FIG. 2, the horizontal axis represents the measurement time, and the vertical axis represents the serum total cholesterol amount (mg/n++).

As shown in Figure 2, it was observed that serum cholesterol decreased by administering the liquid medication, but the serum cholesterol-lowering effect of the liquid medication was more pronounced in domestic rabbits with high serum cholesterol levels; The effect was mild in domestic rabbits with low serum cholesterol levels before administration.

In this study, the decrease in serum cholesterol was
It was 8% (average value). These results revealed that this preparation is useful as a therapeutic agent for arteriosclerosis caused by hyperlipidemia.

[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 changes in serum total cholesterol over time after administration of the liquid medication and control drug as a ratio to that before administration, and Figure 2 is a graph showing changes in serum cholesterol levels before and after administration of the liquid medication. be. Patent Applicant: Morinaga Milk Industry Co., Ltd. Agent: Person: Kudo Ka: Percentage (%) of serum total cholesterol before administration of 1 IU Before and after administration of this product

Claims (1)

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