JPS59172425A - Novel blood coagulation factor derivative, its preparation and blood coagulation promoting agent containing the same - Google Patents

Abstract

NEW MATERIAL:A blood coagulation factor derivative (composed of a polyalkylene glycol having a molecular weight of 200-20,000, containing optionally an alkyl or alkanoyl group as a substituent group and bonded through a coupling agent to the amino acid side chain of a blood coagulation factor). EXAMPLEMonomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified factor IX (PEG5000-IX-4.2mM). USE:Blood coagulation promoting agent. PREPARATION:The objective blood coagulation factor derivative can be prepared by bonding a polyalkylene glycol (e.g. polyethylene glycol) having a molecular weight of 200-20,000 and optionally containing an alkyl group or alkanoyl group as a substituent group through a coupling agent (e.g. cyanuric halide) to the amino acid side-chain of a blood coagulation factor.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a chemically modified derivative of a blood coagulation factor, a method for producing the same, and a blood coagulation promoter containing the same.

Hemophilia is one of the diseases in which blood coagulation activity is reduced. In hemophilia, there is a congenital deficiency or malfunction of coagulation factors, resulting in symptoms such as large hematomas occurring spontaneously or due to minor trauma, or prolonged hemostasis. There is no other way than to supplement the missing factors. However, even with this replacement therapy, the blood half-lives of coagulation factor (2), which is deficient in hemophilia A or 7-on Willebrand disease, and coagulation factor (2), which is deficient in hemophilia B, are 15 hours and 25 hours, respectively. ,
Administration through frequent injections has been required to maintain blood coagulation activity. Moreover, these coagulation factors are unstable and easily degraded in the gastrointestinal tract, so oral administration is not expected to be effective, and intravenous injection or drip has been the only choice. As a result, treatment for this disease continues for the rest of the patient's life, which places a heavy burden not only on the patient, but also on doctors and family members, interfering with the patient's social life.

As a result of repeated studies to reduce the burden on patients with these blood coagulation factor deficiency or malfunctioning diseases, as well as their doctors and families, the present inventors have found that the duration of activity in the blood can be extended, and oral We have discovered a novel blood coagulation factor derivative that can be administered and completed the present invention.

Therefore, an object of the present invention is to provide a novel blood coagulation factor derivative in which a polyalkylene glycol derivative is bonded to the amino acid side chain of a coagulation factor that maintains coagulation activity in blood.

Another object is to provide a method for producing the above-mentioned novel blood coagulation factor derivatives.

Still another object is to provide a blood coagulation promoter that contains the above novel blood coagulation factor derivative and can be administered orally.

The novel blood coagulation factor derivative of the present invention is a polyalkylene with a molecular weight of 200 to 2Q and OOO, which may contain an alkyl group or an alkanoyl group as a substituent on the amino acid side chain of the blood coagulation factor via a coupling agent. 4, which is made by bonding glycol.

The term "blood coagulation factors" as used herein also includes factors (1) to (X) derived from human plasma and necessary for completing the blood coagulation cascade, as well as their active forms (Ia-XIIIa), fragments, and complexes.

Examples of the polyalkylene glycol include polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, etc., and polyethylene glycol is preferred.

Further, examples of the alkyl group as a substituent of the polyalkylene glycol include methyl group, ethyl group, stearyl group, etc., and examples of the alkanoyl group include acetyl group, glopionyl group, etc., but methyl group is particularly preferred.

The molecular weight of polyalkylene glycol is 200~
1.000 to 10,000 out of 2,000 is preferred.

The coupling agent is not particularly limited as long as it can bind to polyalkylene glycol and further bind to the amino group of the amino acid side chain of a blood coagulation factor, but examples include cyanuric halides such as cyanuric chloride and cyanuric fluoride, and dibromoal succinic anhydride. The derivatives are further reacted with poly6≠lene glycol and chloroacetic acid ethyl ester, then treated with hydrazine, and the resulting hydrazide is treated with nitrous acid to obtain acylnitrobenzyl chloride, p-nitrophenylglyceryl ether, etc. Poly-chill, 3
Examples include diazonium salts obtained by reducing the nitro group with -(p-nitronenoxy)-2-hydroxybrobyl ether and then diazotizing it, with cyanuric halides being particularly preferred. Further, in the case of this cyanuric halide, after binding the polyalkylene glycol and blood coagulation factor, the remaining halogen atoms may be converted into a hydroxyl group iCit.

The novel blood coagulation factor derivative of the present invention is produced with a molecular weight of 200 to 20,000 and may contain an alkyl group or an alkanoyl group as a substituent. The reaction must be carried out under conditions that minimize the decrease in coagulation activity of blood coagulation factors. That is, 24 hours without adjusting the pH with a buffer or the like is preferable. The pg of the buffer solution can be arbitrarily selected as long as the blood coagulation factors are not completely deactivated, but is preferably in the range of 5 to 9, and is also determined depending on the type of coupling agent. The amount of the polyalkylene glycol coupling agent conjugate is determined depending on the desired degree of modification.

That is, if an attempt is made to increase the degree of modification, the reagent concentration is increased at a high pH and the reaction is carried out for a long time, and if an attempt is made to decrease the degree of modification, the reagent concentration is reduced at a low pH and the reaction is carried out for a short time.

By appropriately combining these modification conditions, after the reaction of the conjugate of stable polyalkylene glycol and coupling agent is completed, unreacted reagents can be removed using biochemical methods known per se that do not reduce blood coagulation factor activity. Too late,
It can be removed by methods such as dialysis, 7-exchange chromatography, and affinity chromatography, either alone or in combination.The produced new blood coagulation factor derivatives can be stored in a state containing a buffer solution or salt, or frozen as a single product. The activity of preservation by freezing or freeze-drying varies depending on the type of conjugate of polyalkylene glycol and coupling agent used and the degree of modification. For example, when blood coagulation factor The one modified at 10mM with a molecular weight of 15,000 was 11% and 8% of the unmodified factor
showed that. These also contain factors X1 and ■, so when their activity was measured in the same way as factor
15.1%, factor ■ activity 115.5.10&1ch,
Factor ■ activity showed 104.9.855〇 On the other hand,
When modifying polyethylene glycol with no terminal substitution, the activity of factor The activity of factor X was 715 chi. The terminal is stearyl ether and the molecular weight is 3.20.
[The activity of factor Factor X activity is 157.5
%, factor ■ activity is 11 [L3CH, factor ■ activity is 89
It was 4chi. The terminal end is stearyl ester and the molecular weight is 2.
The activity of factor there were.

Next, the relationship between modification reagent concentration and activity will be shown.

For example, 91.2% of unmodified factor
At 4.2 mM, each showed 113 chi. These factor X activities are 104.4.105.5.
203%, factor ■ activity is 10a4.11 & 3.10
4.7%, factor X activity 87.4.74.7.102
．． It was 5%.

Furthermore, when the novel blood coagulation factor derivative modified with polyalkylene glycol obtained in the present invention was orally administered to dogs, its transfer into blood was observed. For example, the above-mentioned average molecular weight s, oo.

O monomethyl ether polyethylene glycol-4,6
-dichloro-1,5,5-)modified with riazine reagent concentration 4
When 2 mM modified factor That is, after oral administration 1°2.4
, 6. When blood was collected and citrated plasma was separated at the 8th interval, partial thromboplastin time (PTT) and prothrombin time (F
T) were all shortened, and the coagulation time correction effect of factor X-deficient plasma was also remarkable. Furthermore, since the activities of factors ①, ②, and X continued, it is considered that these coagulation factors coexisting in the factor In addition, when unmodified factor X was orally administered, the correction effect was within the range of physiological fluctuations. In addition, as a control experiment, we also orally administered only monomethyl ether polyethylene glycol-4,6-dichloro-1,5,5-) riazine with an average molecular weight of 5000, which is a modification reagent, and compared the concentration of the reagent used in the experiment. In an oral administration experiment of modified factor It can be seen that a certain level of modification is required.

Next, the blood coagulation factor preparation was mixed with 2-1 nnM of monomethyl needle polyethylene glycol-4,6-dichloro-i,5.5-) riazine with an average molecular weight of 5,000, 2
When modified at -94mM and 4.2mM concentrations and administered orally to dogs as a mixture, transfer of the primary shoulder factor into the blood was observed. That is, the plasma of the treated dog had a PTT lower than that before administration.

Both FT was shortened, and the coagulation time correction effect of factor Ⅰ-deficient plasma was also remarkable. Moreover, these effects were observed even 8 hours after administration. On the other hand, as a control experiment, when unmodified factor ① was orally administered, it was found that it was an excellent blood coagulation promoter with a transient coagulation time correction effect that lasted for 2 hours.

When the novel blood coagulation factor derivative of the present invention is used as a medicine, the factor Ⅰ derivative is used for hemophilia A or 7-on Willebrand disease, the factor X derivative is used for hemophilia B, It is also used to treat decreased coagulation factor biosynthesis or consumptive bleeding, and administration methods include intravenous injection, drip, and oral administration. Currently, commercially available blood coagulation factor preparations contain amino acids and salts for the purpose of adjusting pH and osmotic pressure, but the novel blood coagulation factor of the present invention
There is no problem in adding these or other known additives, excipients, stabilizers, etc.

Further, as a dosage form for oral administration, additives and excipients can be added to make powders, granules, tablets, capsules, etc. In addition, it is more preferable to use an enteric-coated dosage form.

EXAMPLES Next, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto in any way.

Furthermore, these modification methods can be introduced into any manufacturing process of blood coagulation factors.

Example 1 Monomethyl ether polyethylene glycol-4,6-
Dichloro-r,3.5-triazine: polyethylene glycol monomethyl ether (average molecular weight 5,000) 25. Of ((LD05 mol) was dissolved in 200 d of dry benzene by heating, then anhydrous carbonic acid was added)
IJum 5.0? and cyanuric chloride 2.75f (
0.015 mol was added and stirred overnight at room temperature. After the reaction was completed, it was filtered, and 600ml of petroleum ether was added to the solution.
A precipitate was obtained by suction filtration and washed with a small amount of petroleum ether. Reprecipitation with dry benzene-petroleum ether f: Purification was repeated three times to quantitatively obtain a white powder of monomethyl ether polyethylene glycol-4,6-dichloro-1,5,5-triazine. After hydrolysis, this product
A qualitative reaction of chloride ions (AgN03) was shown.

Similarly, average molecular weight 550,700, 2.000°10
．． 000 and 15,000 polyethylene glycol monomethyl ether, average molecular weight 3.200 polyethylene glycol monostearyl ether, average molecular weight 2
, 700 and reacted with cyanuric chloride to obtain monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-) liazine of each average molecular weight.

Monostearyl ether polyethylene glycol-4,
6-dichloro-1,3,5-)riazine, monostearyl ester polyethylene glycol-4,6-dichloro-1,6°5-triazine was quantitatively obtained.

Example 2 Polyethylene glycol-4-chloro-6-hydroquine-1,5,5-triazine: polyethylene glycol (
Average molecular weight is, 000) 33.6r was dissolved under heating in 150° of dry benzene, and then 1.62% of anhydrous sodium carbonate and 742% of cyanuric chloride α were added and stirred overnight at room temperature. Water 1.
After adding Od, the mixture was stirred at room temperature for 6 hours and further at 40° overnight. - Insoluble matter was removed by centrifugation (2,000 rpm, 10 minutes), and the supernatant was distilled off under reduced pressure. The residue was dissolved in benzene while heating and evaporated. This operation was repeated two more times. The residue was dried under reduced pressure to quantitatively obtain a white waxy solid of polyethylene glycol-4-chloro-6-hydroxy-1,5,5-triazine. Similarly, average molecular weight 1,00
0, s, and sso polyethylene glycols were reacted with cyanuric chloride to quantitatively obtain polyethylene glycol-4-chloro-6-hydroxy-1,5,5-triazine having each average molecular weight.

Examples & Monomethyl ether polyethylene glycol-4,6-
dichloro-1,3,5-) riazine-modified factor (PE)
G5, ooo-■-42mM): 400 units of a lyophilized preparation of factor Dialysis was performed for 30 minutes. After dialysis, the contents were diluted to 10 μm with the same buffer solution, and 1.66 ml of aprotinin (“Levalcin”, manufactured by Kado Pharmaceutical Co., Ltd.) was added.
and monomethyl ether polyethylene glycol-4,6-dichloro-1,5,5-)riazine z44sv with an average molecular weight of 5,000 prepared in Example 1 (final concentration 4
．． 2mM)i was added. React for 3 hours under stirring with water cooling,
D° in the same buffer for 1 hour, then θ° in α45% saline,
Dialysis was performed for 1 hour. After dialysis, the contents were transferred to a vial and lyophilized to obtain a white powder of monomethyl ether polyethylene glycol-4゜6-dichloro-1,3,5-).
Lyazine-modified factor ■ (PKG 5,000-■-4,
2mM) and PEG5.0 without aprotinin.
0 rhof) (-4.2mM was prepared., :pK
The factor ■ activity of G 5,000-IK-4, 2mM (without aprotinin) was 113 times that of unmodified factor ■, as measured by the factor ■-deficient plasma clotting time correction effect.

Example 4 Monomethyl ether polyethylene glycol-4,6-
Dichloro-1,3,5-) riazine-modified factor (PK)
05,00 D-IX-1,72mM and -2,4
4mM): 400 units of factor
,5,5-triazine 100mqc final concentration 1.72m
PEG5,000-f) (-1,72 mM
and -2,44mM were prepared.

When factor Ⅰ activity was measured in the same manner as in Example 6, PE1G
91.2 at 5,000-■-1,72mM, -2
, 97-9% at 44mM.

Example 5 Monomethyl ether polyethylene glycol-4,6-
Dichloro-1,3,5-triazine modified cylinder factor (P'
BG5,000-■-2.1mM, -2.94mM and -4.2mM): 500 units of lyophilized preparation of factor ■ ("Nee-Eight", manufactured by Cutter) was mixed with 10π of distilled water.
The mixture was dissolved in αI M phosphate buffer (pH 7O) and dialyzed for 30 minutes under water cooling. After dialysis, the contents were divided into 3 equal parts, and each volume was diluted to 4.28 cm with the same buffer. Monomethyl ether polyethylene glycol-4,6-dichloro-1,5, prepared in Example 1 and having an average molecular weight of 5.000.
5-triazine 45■ (final concentration 2.1 mM)
, 66~ (2.94mM) and 90■ (4.2mM)
M) was added. The reaction was allowed to proceed for 6 hours under water-cooled stirring, and each was dialyzed in the same buffer at 0° for 1 hour and then in 0.4 s % saline at 0° for 1 hour. After dialysis, the contents were transferred to a vial and lyophilized with azine-modified factor (PEG5,000)
-■-Z1mM, -2.94rr, λ (and -4,2
(mM) was obtained.

Examples & Monomethyl ether polyethylene glycol-4,6-
Dichloro-1,5,5-) riazine-modified thrombin (P
KG 5,000-'I[a-2, 1mM, -λ9
4 mM and -4.2 mM): In the same manner as in Example 4, instead of factor ①, 500 units of thrombin ("Trombin-Midori", manufactured by Midori Juji) was divided into three equal parts, and Example 1 was added.
Monomethyl ether polyethylene glycol-4,6-dichloro-1,5 with average molecular weight s, ooo prepared in
, 5-triazine 45■ (final concentration 2.1mM), 63
■ (2.94mM) and 9011f (2.94mM)
) and reacted with PEG5+000-1[a-2,1
mM5-2.94mM and -4.2mM were obtained.

Example 7 Polyethylene glycol-4-chloro-6-hydroxy-1,5,5-) riazine modified factor (PKG6,0
00-■-! i0mM): Factor X (“Cornine”,
(manufactured by Cutter) 400 units of lyophilized preparation in distilled water
Dissolve αIMlj in fnA in acid buffer (pH 7,0
) for 60 minutes under water cooling. After dialysis, the contents were diluted to a 20-mm volume with the same buffer and divided into 10 equal parts.

Of these, 2- has an average molecular weight of 6, prepared in Example 2,
000 polyethylene glycol-4-chloro-6-hydroxy-1,5,5-triazine 60η (final concentration 5
．． 0 mM) was added. The reaction was allowed to proceed for 6 hours under water-cooled stirring, and the reaction was stopped by adding 0.1M KH2PO42-.
A clear aqueous solution of PFiG6.000-K-45, OmM was obtained. Polyethylene glycol-4-chloro-6-hydroxy- with an average molecular weight of 3.550 was prepared in the same manner.
1,5,5-triazine 6551ny (final concentration 5.0
PEG3,3'50-■-5.0mM was obtained.

When factor Ⅰ activity was measured in the same manner as in Example 3, PEG
71.8 at 6,000-5,0mM, PEG
3.350-1) (77.5 at -5.0mM).

Example a Monomethyl ether polyethylene glycol-4,6-
Dichloro-1,3,5-triazine modified factor (PE)
G550-1) (10,0mM and P B G
15t 000- ■-2,0mM % monomethyl ether polyethylene glycol-4,ts-dichloro-1°3.5-1=riazine 1tOay (final concentration 10.0mM) and 6aOrq (2-
OmM ) to PEG550-■-IQ, QmM and PEG15,000- (K -2,0mM
I got it.

When factor Ⅰ activity was measured in the same manner as in Example 6, PEG5
50-■-1 [LOmM 1158chi, P E G
15.000-■-2-OmM was 66.6.

Example 2 Monostearyl ether polyethylene glycol-4,
6-dichloro-1,5,5-) riazine modified factor
piG3,2oo-IK-50 mM): semiuniform molecule-TtL 3r 20 in the same manner as in Example 6
0 monostearyl ether polyethylene glycol-
4,6-dichloro-1,5,5-) riazine 1θ2 cape (
PEG3,200-DC-3 (final concentration 5.0mM)
,0mM was obtained. Factor Ⅰ activity was measured in the same manner as in Example 3 and was found to be 74.0%.

Example IQ.

monostearyl ester polyethylene glycol-4,
6-dichloro-1,3,5-triazine modified factor
PEG2.700-■-5,0mM): In the same manner as in Example 6, monostearyl ester polyethylene glycol-4,6-dichloro-1,5,5-)riazine 2s0 ( Final depth
PEG2,700-■-! 10mM
I got it.

When factor Ⅰ activity was measured in the same manner as in Example 5, 9
The amount was 1 piece.

Example 11 Oral administration experiment of PEG5,000-1: (-4.2mM) Approximately 4 ml of blood was collected from a healthy pegle dog (female, 1oKr).Next, PEG 5,000 prepared using Example 3Q
0-■-4-40 units of 2mR lyophilized product approximately 10
It was dissolved in 0 trt milk and administered orally. After administration 1,
2. ．． After 4 and 6.8 hours, blood was collected at approximately 4-hour intervals. 1/9 volume for each blood! L8% sodium citrate solution was added as an anticoagulant, and the plasma was fractionated by centrifugation.

Cephaloplastin and calcium ions were added to plasma to measure partial thromboplastin time (PTT), and tissue thromboplastin and calcium ions were added to measure prothrombin time (FT). Furthermore, the coagulation time correction effect of factor-deficient plasma was measured by the Quick injection method. The correction effect of plasma before administration is assumed to be 100, and its ' / 2
g, 1/4 amount of correction effect n50chi, 25 respectively
%, a calibration curve was created on a double logarithmic graph, and the correction effect of plasma obtained by collecting blood at each time was determined by relative chi. In addition, P'FjG5.000-IX-1 prepared in Example 4,
Similar experiments were conducted with 72mhLi) and -2.44mM. Furthermore, as a control experiment, an experiment of oral administration of unmodified factor 12゜PEG5,000-■: 177 units of PEG5,000-■-2.1mM prepared in Example 5, PEG5,000-■- 177 units of 2,94mM and 1 of P,EG5.000-■-4,2mM
77 units were mixed and orally administered to dogs in the same manner as in Example 10, blood was collected over time and plasma was analyzed.

Furthermore, as a control experiment, unmodified factor

[Brief explanation of drawings]

FIG. 1 shows the PTT of the animal experiment conducted in Example 10. Kano-102 Unmodified factor ■ (no aprotinin) administration hole-----: PEG5,0nO-IK-4,2m
M (without aprotinin) administration hole r--1: PEG5,
Figure 2 shows the FT of the animal experiment conducted in Example 10.
FIG. 3 shows factor X activity in the animal experiment conducted in Example 10. FIG. 4 shows factor X activity in the animal experiment conducted in Example 10. FIG. 5 shows factor X activity in the animal experiment conducted in Example 10. FIG. 6 shows the PTT of the animal experiment conducted in Example 11. ←0: Unmodified factor ■ (no aprotinin) injection hole ←−
-: PEG5,000-■-2,1mM, -2,94
Figure 7 shows the FT of the animal experiment conducted in Example 11. FIG. 8 shows factor X activity in the animal experiment conducted in Example 11. 11th bird beforeel 2 4 6 8 (hr
) before 2 4 6 8
(hr) Figure 3 beforeel 2 4 6 8 (
hr) No. 41” before 2 4 6 8 (
hr) Figure 6 before L 2 4 6 8 (hr) Figure 71]

Claims (1)

Scope of Claims: (1) A polyalkylene glycol with a molecular weight of 200 to 2G, 000, which may contain an alkyl group or an alkanoyl group as a substituent on the amino acid side chain of a blood coagulation factor via a coupling agent. A novel blood coagulation factor derivative formed by combining (2) The novel blood coagulation factor derivative according to claim 1, wherein the polyalkylene glycol is polyethylene glycol. (5) Polyalkylene glycol is polyethylene
The novel blood coagulation factor derivative according to claim 1 or 2, which is glycol monomethyl ether. (4), the molecular weight of lJ fullkylene glycol is 1.00
Claims 1 to 0 to 11111.000
3. A novel blood coagulation factor derivative according to any one of Item 3. (5) The novel blood coagulation factor derivative according to any one of claims 1 to 4, wherein the coupling agent is a cyanuric halide. (6) Blood coagulation factors are factor ■, factor ■, factor ■,
The novel blood coagulation factor derivative according to any one of claims 1 to 5, which is either factor X or factor X. (7) A blood coagulation factor and a molecular weight of 200 to 2, which may contain an alkyl group or an alkanoyl group as a substituent.
0.000 polyalkylene glycol and a coupling agent, which contains an alkyl group or an alkanoyl group as a substituent in the amino acid side chain of a blood coagulation factor via a coupling agent. The molecular weight may be 200 to 2. Q. A method for producing a novel blood coagulation factor t conductor formed by bonding 000 polyalkylene glycols. (8) The amino acid side chain of the blood coagulation factor may contain an alkyl group or an alkanoyl group as a substituent via a coupling agent, with a molecular weight of 200 to 20.0.
A blood coagulation promoter characterized by containing a novel blood coagulation factor derivative formed by bonding 00 polyalkylene glycol. (9) The blood coagulation promoter according to claim 8, wherein the administration route is oral.