JPH06217772A - Preparation of polypeptide type product having inhibitive action against excessive production of tumor necrosis factor - Google Patents

Abstract

PURPOSE: To obtain the subject product having no protein decomposition action and capable of reducing the serum concentration of TNF induced with bacterial endotoxins when administered per oral to mice by treating to dialyze a pepsin formulation under a specific condition and lyophilizing the dialyzed product.

CONSTITUTION: This production method for a polypeptide type product having inhibiting action to the excessive production of a tumor necrosis factor is to dissolve a pepsin formulation in deionized water to prepare a solution having 50-200 mg/ml concentration, fill in a Visking type dialyzer having a 6-14 Kd molecular length, dialyze with running water at a room temperature for 2-6 days, then, dialyze using deionized water of a volume 50-1000 times as much as the volume of the liquid to be dialyzed at a temp. of 4-10°C for 4-20 hours, similarly dialyze with distilled water for 8-30 hours, lyophilize the dialyzate and obtain the dried product.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to tumor necrosis factor.
The present invention relates to a method for producing a polypeptide-type molecular product having an inhibitory effect on excessive production of rosis factor (TNF), and therefore to a method for producing a product which is useful for the production of a pharmaceutical preparation.

[0002]

BACKGROUND OF THE INVENTION Advances in scientific knowledge have established the involvement of TNF in numerous pathological processes. TNF is usually produced by monocytes and / or macrophages. This cytokin was initially characterized by its tumor necrosis effect, and later on by the checkin (cackin).
hectin) and is now included in the group of substances known as inflammatory cytokins, that is, included in the group of substances known as cytokins that are produced at the site of inflammation by infiltrating mononuclear cells. (Jan Vilcek and Tae
H Lee, J. Biol. Chem, 266, 7313-7316.
Page (1991)). This document describes the involvement of cytokins in regulating the expression of polymorphonuclear leukocyte adhesion antigen. The state of overproduction of TNF-α has been described in numerous pathologies such as cachexia, septic shock, rheumatoid arthritis due to other autoimmune diseases, parasite infection, viral infection and the like. It is therefore important to have available a product that is pharmacologically equipped with the ability to suppress the overproduction of TNF. In this sense, suppression of TNF production has been described in various experimental models. For example, J. Exp. Me by E. Sampaio et al.
d. 173, pp. 699-703 (1991), describes that thalidomide suppresses TNF production by human monocytes stimulated in vitro by lipopolysaccharide (LPS); M. Gardina et al. J. Exp. Med. 173: 1305-1310 (1991) describes the effect of chlorpromazine, which consists in lowering the serum concentration of TNF in LPS-excited animals; S. B.
illy et al. Int. J. Immunopharm, 12: 31-36 (19
91) describes the inhibitory effect of quinolones on the production of TNF by human monocytes. Similar effects have been described for indomethacin. The literature describes the pharmacological properties of intravenously administered pepsin and its unique or proteolytic effect. These properties have an effect on plaque formation (HG Scheider, E. Goebbels and K.
Puechner, Stomatol. DDR Vol. 36, pp. 433-438 (1986)), Effect of Masushi on nephritis (H. Oh
Nishi, Journal of Japanese Pharmacology 83, 105-114 (19
84)), the effect on autoimmune diseases in a rat model (H. Ohnishi, Life Sciences, Vol. 33, 1641-16).
48 (1983)) and effects on glomerulonephritis such as those caused by immune complexes (H. Ohnishi, Life Sciences,
33, pp. 671-677 (1983)).

The present invention is a polypeptide type which has no proteolytic activity and which can partially interfere with the serum concentration of TNF induced by bacterial endotoxin (LPS) when orally administered to Meiss in an appropriate manner. It relates to a method for producing a product from pepsin.

[0004]

It is an object of the present invention,
Tumor necrosis factor (TNF) overproduction
The method for producing a polypeptide-type product having an inhibitory effect on the product comprises a thorough and continuous dialysis treatment of an aqueous solution of a pepsin preparation, said aqueous solution having a constant concentration, and said dialysis treatment comprising three steps. The time, type and volume of dialysate, and temperature are defined for each, followed by a drying process by freeze-drying.

During processing under controlled conditions as described herein, the source protein is modified by autolysis and denaturation without losing its original proteolytic activity, and has new clinical applications. Possible pharmacological properties are obtained. The steps of the treatment are described below.

(A) In the first step, a solution of the pepsin preparation is prepared in deionized water, and the solution is 50 to 200 mg /
It has a final concentration of ml and the solution is filtered if necessary.

(B) In the second step, the solution obtained in the above step is subjected to Visking (Viski) having a molecular length of 6 to 14 Kd.
ng) type dialysis container and then a first dialysis is performed with running water at room temperature over a period of 2 to 6 days.

(C) In the third step, deionized water having a volume of 50 to 1000 times the volume of the liquid to be dialyzed is used for 4 to 4 times.
A second dialysis is performed at a temperature of 4-10 ° C. for a period of 20 hours.

(D) In the fourth step, distilled water having a volume of 50 to 1000 times the volume of the liquid to be dialyzed is used for 8 to 30 times.
A third dialysis is performed at a temperature of 4-10 ° C. for a period of time.

(E) In the fifth step, the dialysate is freeze-dried (l
freeze and dry.

(F) In the sixth step, the dried product obtained in the above step is used to produce a medicinal drug product suitable for oral administration or intravenous administration, and the medicinal product has various biological forms. Formulation used for physical testing. Upon oral administration to produce a solid mixture containing 1-5% of _{CaH (PO 4) · 2H 2} O. For intravenous (IV) administration, an extemporaneous solution with an active ingredient concentration of 2-5 μg / ml in isotonic salt solution is prepared.

Example 1 In the first step, an amount of 0.1 to 2.5 g of 1: 3000 commercial grade pepsin was weighed and dissolved in deionized water having a volume of 10 to 200 ml. If the resulting solution is not clear, filter through a 0.45μ cellulose filter.

In the second step, the aqueous pepsin solution is charged into a Visking type dialysis container having a molecular length of 10 to 14 Kd, which is first washed with water at a temperature of 60 ° C. and then rinsed thoroughly with distilled water. Is. The flask is filled with running water, charged into a dialysis vessel and the mouth of the flask is placed under a slow flow rate of running water, eg 20-40 ml / min, thus ensuring a continuous renewal of dialysate. This step is performed at room temperature and continues for a period of 2-6 days.

In the third step, the dialysis container containing the dialyzed pepsin solution from the previous step is dialyzed against distilled water in an amount of 1-10 liters at a temperature of 4-10 ° C. for a period of 4-20 hours. .

In the fourth step, the dialysis container containing the dialyzed pepsin solution from the previous step is dialyzed again, but this time over a period of 8-30 hours at a temperature of 4-10 ° C. of 1-10 l. Dialyze with an amount of distilled water.

In the fifth step, the dialyzed pepsin solution obtained in the previous step was frozen and freeze-dried, thus obtaining a hygroscopic white powder.

In the sixth step, a pharmaceutical dosage form product for pharmacological testing was prepared. For oral administration, 200 mg of the lyophilized product was weighed and mixed with an amount of 10 g CaH (PO ₄ ) .2H ₂ O. This mixing is achieved by mechanical stirring. Upon intravenous (IV) administration, solutions with component concentrations of 2-5 μg / ml in isotonic saline were prepared.

The products obtained after the fifth step, more particularly the products described in the above examples, have novel biochemical and pharmacological properties which differ from the starting compounds. The properties are shown in Table I below.

[0019] The product which is the object of the present invention and which has been obtained after carrying out the sixth step of the treatment has the following biological effects: 1) in the form of a 2% mixture with phosphate and 150 mg /
Balb / c at a dose of kg for 6 consecutive days
The product when administered to Mice was the IV of LPS administered 2.5 hours after the last dose of the product was administered.
75% of serum concentration of TNF as induced by injection
Can be suppressed.

2) Corticosterone is the product in the form of a 2% solids mixture with phosphate and administered at a dosage of 150 mg / kg to Balb / c mice for 1 day or 4 consecutive days. Is estimated to be 2-3 times above basal concentration 5 hours after the last dose.

3) 0.05 m of product in the form of isotonic salt solution
g / kg intravenously administered to swiss mais, and no abnormal body weight was observed on the 7th or 14th day after the administration, and the liver, spleen, kidney or mesenteric lymph gland tissue No abnormality was found.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Giuan Pablo Pivuel Ranieri Spain. 28014-Madrid. Gutenberg. 8

Claims (6)

[Claims] 1. A method for producing a polypeptide type product having an inhibitory effect on the excessive production of tumor necrosis factor (TNF), comprising: a) preparing an aqueous pepsin solution having a constant concentration; and b) the first dialysis. In the step, the pepsin aqueous solution is dialyzed with running water under constant temperature and time conditions and with a specific type of dialysis membrane, and c) in the second dialysis step, the solution dialyzed in the above step is subjected to constant temperature and time conditions. And dialyzing with deionized water with a specific volume of dialysate, d) in the third dialysis stage, again with distilled water under constant conditions of temperature and time and with a specific volume of dialysate, e) the step D) drying the dialysate obtained in step f) to prepare a pharmaceutical dosage form suitable for oral administration after dilution of the solid phase with calcium salt or a suitable dosage form for intravenous administration by dilution with an isotonic salt solution. A polypeptide-type product characterized by Manufacturing method. 2. The pepsin used is of type 1: 2500 to type 1: 6 because the aqueous solution used in the dialysis treatment has a pepsin concentration of 10 to 125 mg / ml.
A method according to claim 1 which is pepsin of type 30,000, preferably type 1: 3000. 3. The conditions for the first dialysis step are as follows; the dialysate is running water at a flow rate of 20-40 ml per minute; the time is 4-20 hours; The method of claim 1, wherein the temperature is room temperature. 4. The conditions for the second dialysis step are as follows; the dialysate is distilled water, the volume of the dialysate is 50 to 1000 times the volume of the liquid to be dialyzed; the dialysis time. Is 4 to 20 hours; the temperature during processing is 4 to 10 ° C. 5. The conditions of the third dialysis step are as follows; the dialysate is distilled water, the volume of the dialysate is 50 to 1000 times the volume of the liquid to be dialyzed; the dialysis time Is 8 to 30 hours; the temperature during the treatment is 4 to 10 ° C. 6. The method according to claim 1, wherein the dialysate obtained at the end of the dialysis treatment is pharmacologically active when lyophilized to dryness and orally administered in the form of a solid mixture with a calcium salt.