JPS6051115A - Preparation of complex of blood coagulation factor viii and phospholipid - Google Patents

Abstract

PURPOSE:The titled complex useful remedying hemorrhage of a patient of hemophilia, having raised preservation stability of the factor VIII and purification degree, obtained by bringing phospholipid into contact with blood coagulation factor. CONSTITUTION:For example, phospholipid (e.g., phosphatidylcholine, or phosphatidic acid) is dissoved in a solvent (e.g., CHCl3, ethanol, etc.), the solvent is distilled away, so that a thin film of phospholipid is formed in the inner face of a container. The formed film is shaked, stirred, and destroyed to prepare particles. The particles are preferably treated with ultrasonic wave, and adjusted to <=0.3mu particle diameters. The particles are blended and brought into contact with blood coagulation factor VIII, to give a complex of blood coagulation factor VIIIand phospholipid. The complex has 2-25 times as much activity as that of the factor VIII, and 88-96wt% recovery ratio. It has also high preservation stability. Intravenous injection is made possible by adjusting the complex to proper particle diameter.

Description

DETAILED DESCRIPTION OF THE INVENTION The invention relates to a method for producing a complex of blood coagulation factor V (hereinafter referred to as Veil factor) and phospholipid.

Factor Vm, also called antihemophilic factor A, is the most important nrL/V involved in the formation of n1 endogenous thromboplastin.
j, one of the coagulation factors. Currently, the most rational and effective treatment for hemorrhage in hemophilia A patients is the administration of deficient 7Cm 1ift factor.

However, factor 1) exists only in trace amounts in plasma;
It is not easy to recover factor V111 from human plasma because it is unstable and its activity rapidly decreases from S. The present inventors have conducted various studies with the aim of stabilizing factor V111. As a result, it was found that phospholipids and factor V111 have a pulmonophilic property, and the complex 7 is formed. Takasukoshichi,
The complex can be used as a medicine, and by adjusting the particle size of the complex, it can also be administered intravenously.

The present invention consists of phospholipid, factor
Formation one +! [Characteristics] This is a method for producing factor V111/phospholipid complex.

The non-inventive complex may be produced by, for example, forming a thin film of phospholipid, breaking it into particles, and adjusting the particle size by ultrasonication if necessary.
■ Manufactured by mixing the factors to form a complex.

Phospholipids are non-toxic phospholipids that are physiologically tolerated and metabolized. Examples include phosphatidylcholine, phosphatidylserine, phosphatidic acid, and phosphatidylglycerin. ,
A mixture of phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, etc.
(eg, soybean phospholipids, egg yolk phospholipids), etc. are used.

Phospholipids are dissolved in a solvent such as chloroform or ethanol, thoroughly mixed, and the container is dried under reduced pressure to remove the solvent. Shape the film and make it explode. In this case, it is preferable to use an antioxidant, for example tocopherol (vitamin E), for the stabilization of the phospholipids, preferably with a weight attack on the phospholipids of ~6.5% (w/w). w) Addition amount/SO The formed film is immediately shaken or stirred to break it and form particles/X 'g +! :60 particles should be treated with ultrasonic waves? applied, particle size is 0,
Desired to be 3μ or less - T/8° These particles are then injected into -C1 factor Ⅰ-containing solution (for example, human plasma fraction) kpH 5.5-8
Preferably, it is added to a buffer solution adjusted to pH 6 to 7 (e.g., quenching buffer, phosphate buffer, acetate buffer, physiological saline bath, etc.) and dissolved in this m solution. Let's touch the particles,
Quickly fry or stir Tゐ0 here's the first 'j! The amount of the factor I-containing solution (plasma fraction) added is 0.01 to 10 parts by weight per 1 part by weight of the phospholipid used as protein to form the film.

As for the number of baths to be used for the first factor, it is best to use a relatively Japanese-made bath. Crude factor V is free from contaminants such as factor Examples include the drawing method (US Pat. No. 3,652,530) and the anion converter treatment method (Japanese Patent Publication No. 12,890/1989), etc. Thus, a factor II/phospholipid complex is formed. Separation and purification can be performed by known means such as centrifugation.

Preferably, the complex is co-located as a medicament in the form of a dry formulation, such a formulation being prepared, for example, as follows. That is, after washing the complex several times by centrifugation, addition of known stabilizers and solvent M agents, sterilization filtration, dispensing, freeze-drying, and concentration drying of the first factor H/phospholipid complex. I'm pregnant with Y4 drug. The thus obtained composite preparation was used by Hiroshi Hara, and the specific activity r increased δ by 2 to 25 times compared to the fC resistance factor, and the recovery rate was 88 to 96%. Furthermore, as shown in the experimental example described later, Mizuko's unexploded 1 complex had an extremely high storage stability of factor V.

Practical Example Under the following conditions, Example 1''1: The obtained dimeric solution and factor Ⅰ solution were stored in a color bottle to check their stability, and the results are shown in Table 1. Ta.

Storage conditions: ■ Storage at 4-10°C ■ Temperature storage ■ Freezing (-20°C) Storage Below margins Example 1 ■ Soybean Resitia containing 7 phosphatidyl ymlO% (w/w) ■ Containing 80% phosphatidyl seri Soybean lecithin containing 50% (w/w) K of Dingi beef brain extract, ■ Shoulder Dingi Bovine brain extract containing 8% of phosphatidyl serif?150
% (W/W) Tori Zophos 7 Atidylcoli 710% (W/W)
W) Soybean lecithin containing 'C, or ■Phos7 atidyl 9F80 Brain extract? 14 0 0m9'l knee,) co7 erol 0
．． 1% (W/WJ added) was dissolved in 7j100 tri of chloroform or methanol and mixed, and the container was dried under reduced pressure to remove chloroform or ethanol. A phospholipid film was formed on the inner surface of the container. liquid(
pH 7) was added and shaken rapidly to form particles. Then, the particle size io. 31/-r
Upon contact with the particles, the container 2''' was shaken rapidly. Due to this shaking, factor V was bound to the surface of the particles and formed a complex. The complex was centrifuged as a precipitate, and the particles were separated several times by centrifugation using 0.02M citrate buffer (pH 7, 0) for 0 minutes. After washing, dissolve in 0.01M trishydroxymethylaminomethane-0.01M sodium citrate-0.01M sodium chloride solution (pH 7.4J' (r), filter to remove sterilization, dispense, and freeze. Drying 2
The dried preparation of factor V111/phospholipid complex (
4 as the amount of factor vm activity.

000 units 41] Portrait) r Obtainable. The response rate was 96% (there were 4 results).

In addition, the unit is based on the thromboplasty/generation test [Pritish Journal Og Hematology, 5,
17 (1957) l).

Patent applicant Midori + Te Co., Ltd. Representative Patent Attorney Takashima

Claims (1)

[Claims] 1. A method for producing a blood coagulation factor Ill-phospholipid complex, characterized by bringing the phospholipid into contact with blood coagulation factor V1ff.