JPS61178926A - Chemically modified peptide hormone - Google Patents

Abstract

PURPOSE:The titled hormone obtained by bonding directly a polyoxyethylene group to at least one primary amino group in the molecule. CONSTITUTION:A group shown by the formula (R is terminal oxygen-protecting group such as alkyl, alkanoyl, etc.; n is positive number) is directly bonded to at least one primary amino group in the molecule, to give a chemically modified peptide hormone. Peptide hormone is a general term of a substance which obtained by bonding two or more amino acids by peptide bond, has <=100 amino acids, and has activity such as metabolic regulation, antiviral action, antitumor action, etc. For example, insulin, ACTH, gastrin, calcitonin, etc. may be cited. The peptide hormone of this invention has 500-10,000, espe cially 3,000-8,000mol.wt. The group shown by the formula has <=25,000, espe cially 250-6,000mol.wt. EFFECT:The peptide hormone has retarded clearance and shows effectively its activity for a long period and has low toxicity and antigenecity.

Description

[Detailed description of the invention] The present invention relates to chemically modified peptide hormones.

BACKGROUND OF THE INVENTION In recent years, with the development of genetic recombination technology and organic peptide synthesis methods, it has become possible to synthesize peptide hormones in large quantities. However, it is known that the in vivo clearance of peptide hormones administered to a living body is very fast, on the order of the first class. Furthermore, if the peptide hormone is obtained from a different species of animal and has a slightly different structure, there is a risk that antibodies may be produced and serious symptoms may occur. Therefore, when using these drugs as medicines, it is desired to develop a technique that delays clearance and further attenuates their antigenicity while retaining their activity. Chemically modifying peptide hormones is an extremely effective means to achieve this objective. In other words, by chemical modification,
Delayed clearance and attenuation of antigenicity in vivo as described above.

Furthermore, enhancement of physiological activity is expected, and the practical significance of chemical modification of peptide hormones is extremely large.

The first problem to be solved by the invention is the chemical modification of physiologically active peptides, which requires a method that can chemically modify them while retaining their physiological activity. Polyethylene glyco-μ methyl ether is used for the chemical modification of proteins and peptides because it is thought that it itself has no antigenicity. The method using is first class. However, thea chloride y, which is introduced as a binding group at the same time, has safety problems in itself, and the safety of its decomposed products in vivo has not been elucidated, so it is necessary to be careful when using it. be. Furthermore, the reaction requires an alkaline pH, and the final method cannot be applied to proteins and peptides that are alkaline and easily deactivated.

Also, U.S. Pat. No. 4,002,5a1 discloses a method for preparing an enzymatic heptanoalkyl polyethylene glycol l'v rust conductor using sodium borohydride at a pH of 8.5 as disclosed therein. If this method is applied to peptide hormones, there is a risk of deactivating their physiological activity, so it cannot be an effective production method.Furthermore, the patent document does not even suggest the effect of delaying the clearance of enzyme derivatives in vivo, It is unknown.

In addition, homeoapdehyde is added to bioactive proteins.

A method of introducing low-molecular aldehydes such as acetaldehyde, benzaldehyde, and pyridoxal in the presence of a boron-based reducing agent [Method in Engemology. Volume 47, pp. 469-478 (1977):) - Japanese Patent Publication No. 5
No. 8-154596] is known. However, even if this method is applied to a peptide hormone, it will not be effective and delay in reaction will not be achieved, and a decrease in antigenicity will not be expected. May confer immunogenicity.

In order to solve these drawbacks, the present inventors conducted extensive research and completed the present invention.

Means for Solving the Problem The present invention provides at least one primary amino group in the molecule,
The object of the present invention is to provide a chemically modified peptide hormone formed by directly bonding R10-0M2-CH2 coating group (I:R is a positive integer that protects the terminal oxygen and xln can be arbitrarily changed).

As used herein, peptide hormones are defined as two or more amino acids linked together by peptide bonds, have less than 100 amino acids, and are used for metabolic regulation (memory, sleep, blood sugar levels, blood pressure, immunity), antibacterial, antiviral, and antitumor , anti-insect, poison,
Taste, enzyme activity inhibition.

A general term for substances that have activities such as promoting conjugation of microorganisms.

In other words, peptide hormones are genetically engineered products.

Substances derived from various animals including humans, synthetic products, etc. may be used, and substances having similar structures and similar physiological activities are also included.

Among them, the number of amino acids is 2 to 50, divided into 10 to 30.
peptide hormones are preferred.

Specifically, for example, insulin, ACTH, gastrin, cytonin, dindorphin, glucagon, somatostatin, urogastrone, growth hormone releasing factor (
GRF), corticotropin releasing factor (CRF), and derivatives thereof.

The peptide hormone in the present invention has a molecular weight of 500
~to,ooo, especially a,ooo~8.000 is preferred.

The N-terminal α-
Examples include amino groups and C-amino groups of lysine residues.

Regarding the group represented by (I) above, examples of the protecting group for the terminal oxygen represented by R include alkylμ, alkanoylρ, and the like. , ethyl, propy, 1-propyl,
Preferred are lower (C-ni, )alky, such as butyl, 1-buty, 5-buty, t-buty μ. Specifically, Akanoi μ includes those of C0-8, and 9 kinds of lower (C knee 8) Akanoi y such as holmi μ, aceti~, propioni, butyri~, 1-butyri p, and kaburoi μ. preferable.

The positive integer represented by n must be less than or equal to 500.
120 is preferred.

The molecular weight of the group represented by formula CI) is 25,000 or less,
) is preferably 850 to 6000. 8 to 150% of the molecular weight of the peptide hormone, preferably 5 to 10%, from the viewpoint of maintaining physiological activity and delaying clearance effect.
Formula (I) with a molecular weight of 0%, especially from 10 to 86%
Examples include groups represented by:

The chemically modified peptide hormone of the present invention has the formula (
It has a group represented by I).

When it has only an N-terminal α-amino group as a partial amino group, it has a group represented by formula CI) directly bonded to the amino group. In addition, when the peptide hormone molecule has one or more lysines, a part of its ε-amino groups preferably contains 15 to 80 lysines of the ε-amino groups.
% (on average) has a directly bonded group represented by formula (I), in which case the N-terminal α-amino group is
It may or may not have a directly bonded group represented by formula (I).

The chemically modified peptide hormone of the present invention is, for example, a peptide hormone and R(-0-CH2CH20-CI(,,C
It can be produced by reacting an aldehyde represented by HO (II: R and n have the same meanings as above) in the presence of a reducing agent.

The boron-based reducing agent used in this reaction includes sodium borohydride, sodium cyanoborohydride, etc. Among them, sodium cyanoborohydride is more effective in terms of reaction selectivity and ability to conduct the reaction near neutrality. preferable.

In the reaction, aldehyde (If) may be used in an amount of 1 to 10,000 times the molar amount of the peptide hormone, and boron-based reducing agent may be used in a molar amount of 1 to 7 times the amount of the aldehyde (n). The degree of modification can be arbitrarily selected by increasing or decreasing the molar ratio of aldehyde (n).

The solvent used in the reaction may be any solvent as long as it does not interfere with the reaction, and examples include buffers such as phosphate buffer and borate buffer. In addition, lower alkali-1v (
Examples, methanol, ethanol, 1-gluvano I'v)
, acetonitrile μ, or the like may be added. The pH of the reaction can be in a wide range of 3 to 14, but it is preferably around neutrality (pH 6.5 to 7.5). The reaction temperature is θ″
Although any temperature may be used as long as the peptide hormone is not deactivated within the range of 0.06 to 80.degree. C., the range of 0.06 to 50.degree. C. is more preferable. Reaction time is 0.5-72 hours, usually 3-30 hours
It's about half an hour. The reaction solution was subjected to dialysis, salting out, ion exchange chromatography, and gel filtration.

The desired chemically modified peptide hormone can be obtained by purification using conventional protein purification methods such as high performance liquid chromatography and fluoro-1 electrophoresis. Furthermore, the degree of modification of amino groups can be calculated by, for example, performing amino acid analysis after acid decomposition. .

The above aldehyde (If) is, for example, R+0-CH2
CH, ioH (II: R and n have the same meanings as above)
It can be produced from ethylene glycol derivatives represented by the following, but the method described below is an advantageous production method because it produces less by-product of the corresponding hydroboxylic acid.

That is, compound (1[) was mixed with methylene chloride, chloropho
Oxidize with pyridinium chlorochromate in a munatohalogenated solvent. In this case, pyridinium chlorochromate is used in an amount of 1 to 37μ relative to compound (II), and -1
The reaction is carried out at 0° to 50°C, preferably at room temperature, for 1 to 30 hours.

In addition, after treating the compound (■, n-1) with potassium t-butoxide in t-butanol, the bromoacetate
The corresponding aldehyde (I[ ) can be manufactured. In this case, firstly, potassium t-butoxide is added to the above compound (I[) from 10 to aO mo/'v'! Add and dissolve bromoaceter in an amount of 8 to 157 to compound (III), and dissolve at 10° to 80°C to dissolve 0.5
The reaction mixture is allowed to react for 5 hours, and after post-treatment by a conventional method, the mixture is dissolved in a dilute aqueous solution of the above acid and heated for 5 minutes to 2 hours.

All of the above reaction solutions were extracted, concentrated, and recrystallized.

It can be purified by conventional chemical treatments such as reprecipitation, chromatography, and distillation.

The chemically modified peptide hormone of the present invention has the same useful physiological activity as the corresponding known unmodified peptide hormone, and is useful as a pharmaceutical or the like.

The chemically modified peptide hormone of the present invention not only exhibits delayed clearance in the body and effective long-term activity as compared to the corresponding known unmodified peptide hormone, but also has low toxicity and antigenicity, and has low toxicity and antigenicity. It can be safely used for the same purposes and in the same way as peptide hormones.

The chemically modified peptide hormone of the present invention is usually administered to mammals orally or parenterally as a suitable pharmaceutical composition using known carriers, diluents, etc.

It can be administered to dogs, pigs, rabbits, mice, and humans.

For example, when the chemically modified insulin of the present invention is used as a hypoglycemic agent, it is preferable to administer 10 to 100 units by intramuscular injection once a day for adults.

In MIIF of the present invention, when amino acids are indicated by abbreviations, IUPAC-IUB (Commission of
Biological NomeQlstWr4)
It is based on the abbreviation .

Effects and Examples The present invention will be explained in more detail by the following Examples and Reference Examples, but the present invention is not limited thereto.

Example 1. B1 - Production of polyethylene glycol methyl ether modified insulin (1) 150 porcine insulin was suspended in 20 g/water, and normal hydrochloric acid was added drop by drop to dissolve the insulin. After that, 0.4M! J acid buffer (pH 7
, 0) was added to make a final 0.2M phosphate buffer. To this was added polyethylene glycol methyl engineered aldehyde (average molecular weight 5000) obtained in Reference Example 1 (1).
) 1.125F was added, then 100 caps of sodium cyanoborohydride was added, and the mixture was stirred at 37°C for 24 hours. The reaction solution was dialyzed against water for 12 hours, and the contents were then filtered onto a column of poxymethyl cellulose (8,0×2).
0c11). After washing the column with water, add water (5
00yt) and 0.2M ammonium acetate buffer (pH
6, 8) was eluted using a logarithmic gradient. The main elution fraction (320-400 g/) was collected and lyophilized. Then, it was subjected to PIOGE and JIFF-AO's Ge/L/P filter, and 0.
It was developed with 1N acetic acid. Main elution fraction (110-150d
) were collected and freeze-dried. Amino acid analysis in acid decomposition product (6N hydrochloric acid, 110°C, 24 hours) fli: Lys, 0.92 (1); His,
2.12(2).

Arg, 1. 08(1); Asp, 8.
20(a) Thr, 2. 11(2);S
er, 2.86(a); Glu, 7.88(7)
; Pro, 1.11(1) iGly, 8.78 (
4); Ala, 2.16(2): Half Cy
e.

6, 08 (6); Val, a, 67 (4); Engineering 1e, 1.78 (2) iLeu, 6.17 (6);
Tyr, 4.04 (4); Pha, 2.09 (
2) Porcine insulin originally has three Phes, but the N-terminal Phe of the B chain is modified with polyethylene glyco-ρ methyl ether μ, resulting in one fewer Phe. In addition, the glucose-lowering effect was about 50% that of porcine insulin.

(11) Average molecular weight 1900 and 75 obtained in Reference Example 1
Porcine insulin was treated in the same manner using polyethylene glycol methyl ether adehyde of 0, and as in (1) above, the α-amino group of the N-terminal Phe of the B chain was treated with polyethylene glyco-μ methyl ether having an average molecular weight of 1900 and 750. An insulin derivative modified with was obtained. Acid decomposition product of polyethylene glyco-μ methyl ether modified inswan having an average molecular weight of 1900 (6N hydrochloric acid, 100°C,
Amino acid analysis during 24 hours ill: Lys, 0
．． 98(1); Hls, 2.09(2); Arg,
1.09(1); Asp, 3.17(a): T
hr, 2.04(2); Ser, 2.89(a)
;Gln.

7, 60 (7) + Pro, 1.09 (1); Gl
y, 3.76(4); Ala, 2.0a(2);
Half Cys, a, 9a(6); Mal.

;3.27(4); Engineering Is, 1.54(2); I
,eu, 5.87(6)iTyr, ,(,8B(4
); Phe, 2.00 (2) Amino acid analysis value in acid decomposition product (in 6N hydrochloric acid, 110°C, 24 hours) of polyethylene glyco-μ methyl ether modified Inswan with average molecular weight t750: I, ys, 1.08 (1); Hls,
2.18(2); Arg, 1.12(1): A
sp, a, aO(a); Thr, 2.15(2)
: Sar.

a, 1a(a): Glu, 7.88(7); P
ro, 1.17(1) iGly, 4.04(4);
Ala, 2.24(2); Half Cye.

5.48(6) Tsuke Val, 3.26(4) Electrician 1e,
1.58(2)iLeu, 6.30(6)+Ty
r, 4.0a(4); Phe, 2.23(2)(
iii) The glucose-lowering effect was determined by the method described in the literature (2, Anatactic Chemistry, Vol. 252, p. 224 (1970)). 85% of pig insulin.

The average molecular weight polyethylene glycol methyl ether modified insulin of 750 was 100%.

(1v) Measurement of blood clearance Leptozotocin <50W/Ni) was intravenously injected into SD flat male, 7 weeks old), and 3 days later, porcine insulin, average molecular weight 500, was intraperitoneally injected into rats that had fasted for 20 hours.
0 and 1900 polyethylene glycol methyl ether-modified insulin (O1a units/k as insulin)
g) was administered, blood was collected from the tail vein, and changes in blood sugar over time were measured. The results are shown in Figure 1.

Example 2. B1-Acanoy ~ Production of polyethylene glycol-modified insulin Porcine insulin was reacted in the same manner as in Example 1 using acetyl polyethylene glycol aldehyde with an average molecular weight of 1500 obtained in Reference Example 2, and α- of the N-terminal Phe of the B chain was An insulin derivative whose amino group is modified with acetyl polyethylene glycol having an average molecular weight of t1500 is obtained.

9 Example 1 Synthesis of polyethylene glycol methyl ether aldehyde (i) Polyethylene glycol methyl ether (5
f, average molecular weight 5,000) in methylene chloride (100 g
/), pyridinium chlorochromate (330 ~
) and stirred at room temperature for 12 hours. The reaction solution was diluted with twice the amount of methylene chloride, and then transferred to a Florigi p column (6X
The column was washed with methylene chloride and then with chloroformte, and then eluted with methanol-chloroform (1:9). Fractions positive in the 2,4-dinitrophenylρhydrofusine test were collected, and 'f4n was distilled off under reduced pressure to obtain a crystalline wax. Accommodation ff11.5f (
30%), #Layered Romatogoufy: Rf=0.08(
Chloroform:methanol:acetic acid=9:1:0.5. silikage/l/), “96.2Ppl by C-NMR” (
Absorption of aldehyde groups was observed in the hydrated form <-CH(OH)2).

(1t) Polyethylene glyco-ρ methyl ether (
10! ! ', average molecular weight 5,000) tertiary butano-/
L/(dissolved in 100g/litre, potassium tert-butoxy)
'(4,17y) and then bromoacetal (2
, 56g/) and stirred at 40°C for 2 hours.

Tertiary butanol μ was distilled off under reduced pressure, water was added to the residue, and then extracted with chloroform (200 ml×2).

It was washed with water and dried over anhydrous sodium sulfate.

Chloroform was distilled off under reduced pressure, petroleum benzine was added to the residue, and the resulting crystalline residue was collected by filtration and washed with ether to obtain the corresponding polyethylene glycol methyl ether diethyl aceter A/9.5F (95%). It was done. Of these, 51 were taken, 0.05M)! It was dissolved in 50 gt of J-fluoroacetic acid, treated in boiling water for 30 minutes, and then lyophilized to obtain polyethylene glycol methanol ether aldehyde, which has a longer chain length by -0-CH2CH2 than that obtained in (i). .

(iii) Polyethylene glycol methyl ether (5.7f, average molecular weight 1,900) was mixed with methylene chloride (
Pyridinium chlorochromate (970 il) was added to the mixture, and the mixture was stirred at room temperature for 12 hours.

Dilute the reaction solution with methylene chloride and apply it to a Florisil column (
After washing the column with methylene chloride and then chloroform, add 10% methanol.
Eluted with /L//chloroform. Fractions positive in the 2.4-dinitrophenyl p-hydrofusine test were collected and the solvent was distilled off to yield a crystalline wax. Adult fish 1.8f (8
0%), thin layer chromatography: Rf=0.10 (chloroform:methanol-1v:acetic acid=9:l:0.5.!
/Rikage/v)96.2 at 13cmNMR)'l'/
The absorption of aldehyde groups was investigated in the hydrated form <-CH(O)l)).

= 2 (iv) Polyethylene glycol methyl engineering μ(
19,5F, average molecular weight 1900) to tertiary butano-A/
(100m+/), potassium tertiary butkind (
10,4F), then bromoacetal (6,4g+
/) was added and stirred at 40°C for 2 hours.

Tertiary butano was distilled off under reduced pressure, water was added to the residue, and then extracted with chloroform (200g/X2). The reaction solution was washed with water and then dried over anhydrous sodium sulfate. Chloroform was distilled off under reduced pressure, petroleum benzine was added to the residue, and the resulting crystalline residue was collected by filtration and washed with etherμ to obtain aceter/L/8.5'1 (89.5%). Konouchi a
f 0.05M)! Dissolved in μ-fluoroacetic acid, treated in boiling water for 80 minutes, and then freeze-dried to obtain a polyethylene glyco--methoxyether with a longer chain length of -0-CH2CH2- than that obtained in (iii). An aldehyde was obtained.

Cv) Polyethylene glycol methylate μt with average molecular weight R750,550,150 - The corresponding aldehyde was obtained in the same manner as above.

Reference Example 27 Synthesis of Kanoi-Polyethylene Glymer μA-dehyde (i) Polyethylene glycine with average molecular weight m1500
/'1540 (Wako Tsumugi Pharmaceutical Co., Ltd.) 15f was added to pyridine 5 (J
ml, and added 1.85 liters of acetic anhydride, and reacted with stirring at 40"C for 2 hours and then at room temperature for 16 hours. After the reaction, the solvent was distilled off under reduced pressure. Dissolved in chloroform, washed with water, and dissolved in chloroform The layer was dried over anhydrous sodium sulfate, and the chloroform was distilled off under reduced pressure.The residue was dissolved in a small amount of chloroform, and petroleum benzine ether/L/(2:
1) Add the mixture and release to form crystalline wax 14F.
(90%). 1.4F of this was dissolved in methylene chloride, pyridinium chlorochromate aoo
Iq was added and the mixture was reacted at room temperature for 18 hours with stirring.

The reaction solution was passed through a Silikage/L/C-200 (Wako Tsumugi Co., Ltd.) column (lX503), washed with 5% methanol-chloroform (200 l), and then 10% methanol-chloroform.
Remove R with chloroform. 2.4-dinitropheny/
The drazine test positive fractions were collected and the solvent was distilled off under reduced pressure to obtain crystalline wax.

Yield: 580q (41%) (ii) Polyethylene glycol/l/100G (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) 201 with an average molecular weight of 211,000 was dissolved in 50ml of methylene chloride (1K), and 5.15F of n-caproic anhydride was added thereto at 70°C for 2 hours. Made it react. The solvent was distilled off and washed with Silikage/S/C-200 (aX50aIl). Purified using A and eluting with ethyl acetate (methanol-PC4: 1) to give an oil of 14.9160% that solidifies in the refrigerator.
) was obtained. Oxidation was performed with pyridinium chlorochromate in the same manner as in (i) to obtain an aldehyde.

Effects of the Invention The chemically modified peptide hormone of the present invention maintains physiological activity as a peptide hormone, has delayed clearance in the living body, and has reduced antigenicity.

shows the effect of delaying the clearance of ■ and ■ are the average molecular weight of ff11900 obtained in Example 1, respectively.
■ indicates the measurement results of porcine insulin as a control.

Figure 1'j l 2 34 J I)
(time

Claims (1)

[Claims] R-(OC
H_2CH_2)-_n group (R is a terminal oxygen protecting group, n
is a positive integer that can be changed arbitrarily).