JPH01254699A - Insulin derivative and use thereof - Google Patents

Abstract

NEW MATERIAL:An insulin derivative having a fatty acid linked to amino group of an amino acid of B1 or B29 in insulin B chain expressed by the formula (R1 and R2 are fatty acid group; X and Y are threonine or alanine; Z is isoleucine or valine). USE:A hypoglycemic agent, excellent in stability, having fat solubility and useful for diabetes. PREPARATION:For example, a fatty acid (e.g., palmitic acid) is reacted with N-hydroxysuccinimide in a solvent to provide a fatty acid N-hydroxysuccinimide ester, which is then mixed with a solution of p-methoxycarbodiimidated bovine insulin and reacted at ambient temperature for 3hr. After completing the reaction, the solvent is distilled away and trifluoroacetic acid is added to the residue and reacted while being cooled with ice. The trifluoroacetic acid is then distilled off to dissolve the residue in 1 N acetic acid and carry out purification by gel filtration and high-speed chromatography, etc., to afford the aimed compound expressed by the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Application The present invention relates to novel insulin derivatives, and more particularly to insulin derivatives useful as hypoglycemic agents in diabetes.

(Prior Art) Insulin is a peptide consisting of 51 amino acid residues secreted by the adult pancreatic Langerhans, and is a hormone that regulates the amount of glucose in the blood. If the regulation of insulin secretion from the pancreas becomes abnormal for some reason, hyperglycemia symptoms occur and diabetes is diagnosed. For diabetic patients.

If left untreated, the hyperglycemic state often complicates various diseases and leads to death.Therefore, in order to normalize this hyperglycemic state, it is necessary to administer insulin to improve qL/L. The insulin to be administered is extracted and purified from comb or pig pancreas, or genetically modified Escherichia coli is made into a human form, or pig insulin is converted into a human form by enzymes and chemicals. It is being

The difference between human insulin, bovine insulin, and pig insulin is that the amino acids in A-chains 8 and 10 (A, and A1°) of the insulin molecule expressed in the following format (1) are alanine and valine, and B-filO ( Bovine insulin consists of B*o) or alanine, and porcine insulin consists of alanine as the 30th amino acid in the B chain, and threonine and isoleucine as the amino acids AKJ8 and 1O.

In human insulin, the amino acids of A-fi8.10 are threonine and isoleucine, and the amino acid of B-chain 30 is threonine.

Such human, porcine or bovine insulin is given to the patient as an injection in the required amount subcutaneously or intramuscularly, and the tm sugar is 31
It's in order.

Patients with arterial disease must undergo insulin injections every day for the rest of their lives, and the physical pain associated with the injections, such as pain and degeneration of the injection site, is extremely great.

In order to eliminate the pain associated with such insulin injections, methods such as oral administration, nasal administration, and rectal administration are being studied.

In all of these methods, absorption enhancers, proteolytic enzyme inhibitors, etc. and insulin are formulated using a pharmaceutical technology. Examples of these include: I'l'J ;): method of combination with inhibitors (Danforth et al.: Endocri
nolagy 65.175.1978), a method of making an oil-based emulsion using an emulsifier (Uezumi et al., ^ctaDiabet
, 1. aL, 15.175.1978), Method for making liposomes (Yoshida: EP^140,08
5) Another method is to coat insulin particles with an azopolymer and release them without secreting digestive enzymes.

5aHran: Canadian J, old o
chcm,, 57. 548°1979).

Also, as insulin for continuous transdermal injection.

Glycated insulin (U.S. Pat. No. 44788:10, No. 4)
No. 478745, No. 4483792, No. 448906
No. 3, No. 4489064 and No. 4536512 specification dJ) are known. Since conventional insulin injections precipitate crystals and cannot be stored for long periods of time, various types of glycated insulin have been created.

(Problems to be Solved by the Invention) An object of the present invention is to provide an insulin derivative suitable for a stable pharmaceutically acceptable insulin preparation.

(Means for Solving the Problems) As a result 1, the present inventors have discovered a novel fat-soluble insulin as a fat-soluble insulin that exhibits a hypoglycemic effect without losing insulin activity, and have completed the present invention.

The novel insulin derivative of the present invention has the following formula (1): % formula % (wherein R3 and R2 are the same or different and represent a fatty acid group, X and Y are the same and represent threonine or alanine,
Z represents isoleucine when X and Y are threonine, and represents valine when X and Y are alanine.

Further, in the formula, Phe: phenylalanine, c: isoleucine, Val: valine, Glu: glutamic acid,
Gln: Glutamine, Cysnidistine, S
er: serine, Leu: leucine, Tyr: tyrosine, ^sn: asparagine, l1is: histidine, cry nigericine, Ala: alanine, ^'g: arginine, Thr: threonine,
Pr.

〕 represents Nibroline. It is expressed as

The compounds of the present invention are useful as hypoglycemic agents in diabetes.

The fatty-acidated insulin of the present invention comprises amino acids of one or both of the amino acids B and Bt'J of insulin Bfi as shown in the above format (1), 1. , with fatty acids bound to it.

In the present invention, any of human, porcine and bovine insulin can be used as insulin.

The fatty acids to be bound in the present invention include:

Those having about 7 to 21 carbon atoms are preferred, such as caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, valmitic acid, heptadecylic acid, stearic acid, and nonadecanoic acid.

Examples include fracic acid, undecylenic acid, oleic acid, elaidic acid, cetoleic acid, erucic acid, brassic acid, sorbic acid, linoleic acid, and liluic acid. Valmitic acid is particularly preferred.

The compounds according to the invention can be obtained, for example, in the following manner.

Step (1): Synthesis step (2) of activated ester of fatty acid
: p-Methoxybenzoxycarbonylamide (pMZ) of insulin (generation of pMZ-insulin) Step (3): Binding step of fatty acid active ester and pMZ-insulin (4): Removal of pMZ group (5): Min. The name registration process is explained as follows.

Synthesis of activated ester in step (+) is carried out in order to activate the carboxyl group of the fatty acid and increase its reactivity, since the fatty acid itself has no reactivity and does not bind to insulin as it is. - Specific examples include: N-hydroxysuccinimide ester.

The P-methoxybenzoxycarbonyl azidation of insulin in step (2) is carried out by converting the amino acid (Gly
:) In particular, since the activity of insulin itself decreases when the amino group of A is substituted with a fatty acid, pMZ conversion is performed to protect the amino group.

Step (c) is a bonding reaction between the pMZ-insulin obtained in step (2) and the active fatty acid ester of step (1), and this bonding easily proceeds by stirring at room temperature in a dimethylformamide solvent. .

In step (4), the protective group pMZ introduced in step (2) is removed with trifluoroacetic acid.

In the purification step (5), B and 1 of insulin Bfi were purified by gel filtration and then high performance liquid chromatography.
3ts with a fatty acid bound to the amino group of either one of the amino acids (RI or Ro with fatty acid bound insulin), B, and B□ with a fatty acid bound to the amino group of both amino acids (RI or Insulin in which a fatty acid is bound to R□ is obtained.

The obtained insulin derivative can be obtained as a powder by secondary freeze-drying.

(Examples and Test Examples) The present invention will be described below with reference to Examples, but the wood spring 11 is not limited thereto.

Reference example 1 Production method of fatty acid activated ester Ethyl acetate 15
After adding valmitic acid and 50mM of N-hydroxysuccinimide to 0ml, add 50mM of dicyclohexylcarbodiimide while cooling with ice and stir for 24 hours.After the reaction was completed, the reaction solution was filtered and the solvent was distilled off. after,
The residue is recrystallized from ethanol to obtain partic acid N-hydroxysuccinimide ester.

Reference example 2. PMZized ifinsulin method Bovine insulin 1mM and p-methoxybenzoxycarbonylamide 4mM ftlN-'J acid sodium hydrogen solution, water, dimethylformamide (2:3:4) dissolved in a solution and stirred at room temperature for 3 hours. 6 After the reaction, 50
% acetic acid was added and the solvent was distilled off. The residue was dissolved in ether and 1%
It was washed with acetic acid, dissolved in 50% acetic acid, and lyophilized to obtain p-methoxycarbodiimide insulin.

Example pMZ - Dissolve 1mM of insulin in dimethylformamyl, add 50mM of valmitic acid N-hydroxysuccinimide ester, and stir at room temperature for 3 hours. After the reaction, the solvent is distilled off, and the residue contains anisole and trifluoro. Add acetic acid and stir for 1 hour under water cooling.

Thereafter, trifluoroacetic acid was distilled off, ether was added to the residue, the resulting precipitate was filtered, and the residual liquid was washed with ether.

The resulting residue was dissolved in IN acetic acid and Sephadex-0
Gel filtration was performed using a column filled with 25 and the insulin fraction was concentrated.

After freeze-drying the insulin fraction, acetonitrile: 0
．． Lys-B valmitoyl insulin (pa'l-1), Pbe-B was dissolved in a 3% trifluoroacetic acid mixture (2:3) and analyzed by high performance liquid chromatography.
s-valmitoyl insulin (pal-2), Ph
e-B + -Lys-B to dipalmitoyl insulin (pal-3) was obtained.

The results of the high-speed chromatogram are shown in FIG.

The fatty acid binding site of the insulin derivative obtained above was identified by deaminating the derivative, degrading it, cleaving all peptide bonds, and decomposing it into 51 amino acids, followed by amino acid analysis. Analyzed by a meter.

Amino acid analysis values are shown in Table 1. As shown in Table 8, insulin (undenatured) has three TL# amino groups;
When this is deaminated, the amino group disappears, so it cannot be measured with an amino acid analyzer. However, if fatty acids are bound, they will not be deaminated, so when comparing raw insulin with the deaminated product, fatty acids are bound. Since only one more site appears, the binding site can be determined.

Test example (surface clogging effect) Wistar male rats were fasted for 24 hours.

The patient was fixed in the dorsal position under pendoparbital anesthesia, and the test drug was administered I.
Dissolve or suspend in N-salt sauce and inject intravenously into the femoral vein or by large injection.
It was injected intramuscularly into 11 muscles. The dose was 1 g/mouse as insulin. After administration, blood was collected from the carotid artery and the blood glucose level was measured.

The results are shown in Figure 2.

As can be seen from the figure, the insulin derivative Pa1 of the present invention
-1 and 2 significantly lower blood glucose levels by 2wJ.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the results of a high performance liquid chromatogram. FIG. 2 is a graph showing changes in blood gelcol amount after administration. Figure 1: Clear weather (min) Figure 2: Time (hr)

Claims (5)

[Claims] (1) Insulin in which a fatty acid is bound to the amino group of amino acid B_1 or B_2_9 of the insulin B chain. (2) Insulin in which fatty acids are bound to the amino groups of amino acids B_1 and B_2_9 of the insulin B chain. (3) A pharmaceutical composition containing a pharmacologically acceptable amount of the compound according to claim 1 as an active ingredient. (4) A pharmaceutical composition containing a pharmacologically acceptable amount of the compound according to claim 2 as an active ingredient. (5) The pharmaceutical composition according to any one of claims 3 and 4, which is a therapeutic agent for diabetes.