JPH0463053B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION In general, when various hormones, enzymes, and other physiologically active substances containing polypeptides are administered to a living body, they are processed by various proteases in the living body.
They are quickly degraded or inhibited by various inhibitors, so their effects are only exerted for a short period of time.

For this reason, when considered as pharmaceuticals, many of them have difficulty achieving the desired effect.

Therefore, the present inventors made the effect more reliable by sustaining the physiological activity in vivo, and
As a result of various studies on substances that can reduce the dosage, the above objective was achieved by binding polyoxyethylene-polyoxypropylene copolymer (hereinafter simply referred to as copolymer) to a physiologically active substance. I discovered that.

The present invention is a sustained-effect composition characterized by binding a polyoxyethylene-polyoxypropylene copolymer to a human-derived physiologically active polypeptide or glycoprotein.

From another point of view, the present invention can also be described as a method for sustaining the efficacy of the polypeptide or glycoprotein by binding the above copolymer to the polypeptide or glycoprotein.

Polyoxyethylene-polyoxypropylene copolymers are commercially available and have an average molecular weight of
1000 to 14000, i.e. the ratio of polyoxyethylene to polyoxypropylene is 4:16, 196:67 or
Approximately 33 types of ratios between 256:54 are mainly used. However, the molecular weight of the above copolymer is
Bound products with physiologically active substances created using over 10,000 copolymers have reduced activity expression due to the active groups being wrapped in the copolymer, and also remain in the body for an extremely long time. This is an undesirable side effect caused by doing so.

Therefore, in the present invention, a copolymer having a molecular weight of about 1,000 to about 10,000 is preferably used. The copolymer has hydroxyl groups at both ends, but the hydrogen of one of the hydroxyl groups may be substituted with an alkyl group or an acyl group. Preferred examples of the alkyl group are a methyl group and an ethyl group, and examples of the acyl group are an acetyl group and a propionyl group. Substitution of these groups can be performed by known methods.

In the present invention, polypeptides or glycoproteins derived from humans, ie, extracted from or derived from human urine, placenta, or blood components, or produced by tissue culture of human cells, are used. Examples include chorionic gonadotropin (HCG), menopausal urinary gonadotropin (HMG), growth hormone (HGH), epidermal growth factor (EGF), neuronal growth factor (NGF),
Colony formation stimulating factor (CSF), urokinase (UK), plasminogen (PLG), kallikrein, erythropoietin, thymodin, interferon alpha, interferon beta, interferon gamma, interleukin 1, interleukin 2, interleukin 3. Urinary trypsin inhibitor, urinary thiol protease inhibitor, placental arylsulfatase, urinary lysozyme, urinary asparakinase, and the like.

In the present invention, the above copolymer is combined with a bioactive polypeptide or glycoprotein. The linkage is carried out using a linking agent that crosslinks the terminal hydroxyl groups of the copolymer and the amino groups of the active substance. As the binder, one having at least one functional group capable of reacting with a hydroxyl group and an amino group, such as 2,4,6-trichloro-S-
Examples include triazine, dibromosuccinic anhydride, maleic anhydride, and the like.

For example, in the presence of an alkali, the copolymer is
When the reactive copolymer obtained by reacting with 4,6-trichloro-S-triazine is reacted with the above-mentioned active substance, the N-terminal primary amino group of the active substance or the lysine residue in the polypeptide is activated. The copolymer is bonded to the ε-amino group at one or more locations.

The above-mentioned bonding reaction can be carried out by a known method depending on the terminal hydroxyl group of the copolymer, the amino group of the active substance, and the reactivity of the binder used.

The composition of the present invention has the effect of significantly extending the efficacy duration of the active substance in vivo by 10 to 20 times or more.

In addition, the combination of a copolymer and a physiologically active substance is less susceptible to the action of proteases in vivo.
Moreover, since it is not affected by the effects of various blood inhibitors, it has a remarkable effect on sustainability and expression of activity.

The composition of the present invention can be administered orally or parenterally depending on the type of physiologically active substance. Parenteral administration optionally takes the form of intravenous, intramuscular, or subcutaneous injection.

The dose is proportional to the known dose of the physiologically active substance, but in the composition of the present invention, the active unit of the active substance tends to decrease slightly, so the single dose should be increased by that amount. is desirable. However, as mentioned above, the long-term effect is significant, so that the active substance itself, for example, can be administered at intervals of several days or more, even though it should be administered each time.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 5.5 g (30 m.mole) of 2,4,6-trichloro-S-triazine (cyanuric chloride) was added to 400 ml of anhydrous benzene containing 10 g of anhydrous sodium carbonate.
In addition to methoxy-polyoxyethylene-
Polyoxypropylene glycol (average molecular weight
5000, monomethylated product of Pluronic F-38 manufactured by Asahi Denka Kogyo Co., Ltd., EO:PO:EO=46:16:46) 50
g (10 m.mole) was added thereto, and the mixture was stirred at room temperature overnight.

Next, 5 times the amount of petroleum ether was added to the liquid from which insoluble matter had been removed by filtration to precipitate the activated product of the copolymer produced, and the resulting product was collected. Further, the desired activated copolymer is obtained by repeating the redissolution and reprecipitation twice using benzene and petroleum ether.
51.5g was obtained.

Next, add 3 million units of purified urokinase to 0.1M at 4°C.
- Dissolve in 30 ml of phosphate buffer pH 7.0, add 600 mg of the above activated copolymer, and react with stirring at 4°C for 3 hours.

Next, after stopping the reaction by lowering the pH to below 5.0,
Gel filtration is performed using Sephadex G-100 equilibrated with 0.1M phosphate buffer PH5.0 to remove unreacted activated copolymer.

The average molecular weight of the modified urokinase obtained is
150,000 and the activity was determined by fibrin plate method.
40% remained, and 70% remained in the fluorescent synthetic substrate method.

In addition, the blood half-lives of unmodified urokinase and modified urokinase were measured using rabbits, and they were found to be 5 minutes and 120 minutes, respectively, which was a 24-fold difference in half-life. The measurements were carried out as follows.

That is, 50,000 units of unmodified urokinase and modified urokinase per 1 kg of rabbits weighing approximately 2.0 kg were administered over time through the ear vein on the opposite side of the ear from which they were collected.

Blood was collected by connecting a syringe to an indwelling needle placed in the auricular artery. Additionally, urokinase administration 10
−30 minutes before administration of heparin sodium at a rate of 1000 units/Kg intravenously.

Before sample administration, immediately after administration, 2 minutes, 5 minutes, 10 minutes after administration
2 ml of blood was collected at 30 minutes, 20 minutes, 30 minutes, 40 minutes, 60 minutes, 120 minutes, and 240 minutes, the blood was immediately centrifuged (3000 rpm, 5 minutes), plasma was collected, and the titer was measured. The above results were obtained.

The fibrin plate method for measuring urokinase titer in this example is described by PL Walton, Clin.
Chem. Acta 13 (5) 680-684 (1966).

In addition, the fluorescent synthetic substrate method T. Morita et al., J.
Biochem. 82 1495 (1977).

Example 2 Human urine kallikrein purified to almost pure product
100,000 units in 0.1M phosphate buffer pH7.0 at 4℃,
Dissolve in 50 ml of ethoxy-polyoxyethylene-polyoxypropylene glycol (average molecular weight
3400, EO:PO:EO=19:30:19 Asahi Denka Kogyo
0.7 g of a copolymer activated in the same manner as in Example 1 was added to the copolymer (P-65) manufactured by Pluronik Co., Ltd., and reacted at 4° C. for 3 hours with stirring.

Next, after reducing the pH to 5.0 or less and stopping the reaction,
Unreacted activated copolymer was removed by dialysis overnight at 4° C. using 0.1M phosphate buffer PH 0.5 as an external solution.

The average molecular weight of the obtained modified kallikrein is
100,000, and the activity is 50 in the blood pressure lowering method using dogs.
%, and 80% remained in the fluorescent synthetic substrate method using Pro-Phe-Arg-MCA.

In addition, blood was collected from rabbits in the same manner as in Example 1, and the blood half-lives of unmodified kallikrein and modified kallikrein were subtracted, and the results were 7 minutes and 110 minutes, respectively, which was 15 times the half-life. There was a difference.

The blood pressure lowering method using dogs for the kallikrein titer measurement method in this example is described in J.Biochem.58, 201.
(1965).

In addition, the fluorescent synthetic substrate method is described in J.Biochem.82,
1495 (1977).

Example 3 Activation used in Example 1 by dissolving 100 million units of human leukocyte interferon (specific activity 2 x 10 ⁷ units/mg-protein) in 22 ml of 0.1M phosphate buffer pH 7.0 at 4°C Add 220 mg of polymer and react with stirring at 4°C for 3 hours. Next, adjust the pH to 5.0 or less to stop the reaction, and then use 0.1M phosphate buffer pH 5.0 as an external solution and react at 4°C overnight. After removing unreacted activated polymer by dialysis, the activity of the obtained modified interferon α remained 40% compared to the activity before modification.The cells used for activity measurement were FL- cells (human amniotic cells (Fogh & Lund Strain)), and the challenge virus is VSV (Vesicular
Stomatitis Virus), CPE (Cytopathogen Effect) using the microplate method was evaluated using the dye-uptake method of Phenone Red (NBFinter: J.General
Virology 5 , 419 (1969)), blood was collected from rabbits in the same manner as in Example 1, and the blood half-lives of unmodified interferon α and modified interferon α were measured. 5 minutes and 80 minutes, a 16-fold difference in half-life.

Claims (1)

[Scope of Claims] 1 Hormones or enzymes that are human-derived physiologically active polypeptides or glycoproteins, or their inhibitors with an average molecular weight of about 1000
10,000 polyoxyethylene-polyoxypropylene copolymer bonded to a polyoxyethylene-polyoxypropylene copolymer using a binder that crosslinks between the amino groups of the former and the hydroxyl groups of the latter. 2. The sustained-effect composition according to claim 1, wherein urokinase, kallikrein, or interferon is bonded to a polyoxyethylene-polyoxypropylene copolymer. 3. The long-acting composition according to claim 1, wherein the binder is 2,4,6-trichloro-S-triazine, dibromosuccinic anhydride or maleic anhydride.