JPH07107080B2 - Glycosylated insulin and therapeutic agent for diabetes containing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to the preparation of glycosylated insulin. More particularly, the present invention relates to the preparation of glycosylated insulin and novel intermediates used in the preparation of glycosylated insulin.

[0002]

BACKGROUND OF THE INVENTION Various systems have been proposed for the administration of insulin to diabetic patients with a high degree of response to the patient's needs.

Bionic solutions are directed to the design of insulin infusion pumps. Many diabetic patients are currently using external battery operated pumps. Pumps continuously infuse insulin through a needle attached to a catheter that is inserted intravenously or subcutaneously. The flow rate can be adjusted automatically if the amount of insulin required changes. The unit is usually mounted on a belt or belted to the legs.

[0004]

Pumps that deliver an amount of insulin that is accurately measured by a detector that measures glucose levels in blood are still in the experimental stage. Despite the successful developments in this area, these pumps are still heavy and cannot be portable. Another drawback is that the system has a device for continuous sampling of blood, an analyzer for rapid and continuous determination of glucose levels in the blood, and analysis of the results to determine the optimal insulin dose. It requires a computer and an infusion pump that delivers insulin intravenously in a manner similar to that secreted by the beta cells of the pancreas. Efforts are being made to reduce the size of the system and extend its detector life. Elliot J. Am. Med. Assoc 241, 2, about "best pocket" type, a system sized for a cigarette case that includes a glucose detector, power supply, computer, insulin reservoir and pump .
23 (1979).

Another obstacle at the present time is the lack of a precise implantable electrode for detecting the concentration of glucose in the blood. Long-term skin connection to the patient's bloodstream poses problems of infection risk and coagulation. or,
Condensed insulin presents considerable problems in that it condenses or crystallizes from its solution state, thus reducing the biological efficacy of insulin in the insulin reservoir. Moreover, the condensed insulin may remain in the needle, blocking the flow of insulin from the infusion system to the diabetic patient.

Therefore, it is an object of the present invention to prepare semi-synthetic insulin which does not coagulate as rapidly as bioinsulin and therefore has a longer shelf life.

It is also an object of the present invention to prepare new intermediate compounds for use in the preparation of non-flocculating semisynthetic insulin.

Yet another object of the present invention is to prepare a glycosylated insulin having significant biological efficacy in controlling blood sugar levels.

[0009]

These and other objects can be achieved by a novel glycosylated insulin having one of the following general formulas 3, 4, and 5.

[0010]

[Chemical 3]

[0011]

[Chemical 4]

[0012]

[Chemical 5]

Wherein m is an integer from 1 to 3, X and Z are different and are selected from the group consisting of --H and --OH, and --Q-- is a dicarboxylic acid spacer group having the formula is there. In the formula of Chemical formula 6, n is an integer of 2 to 6, and is preferably 2 or 3.

[0014]

[Chemical 6]

[0015]

Example: Bronley et al., Science, 206.22
It is known that insulin can be combined with maltose, as disclosed in 3 (1979). However, it has been found that the disaccharide and insulin derivatives do not have any significant biological efficacy in lowering blood glucose levels.

In the present invention, the prepared intermediates for binding insulin all consist of glucose or mannose monosaccharides linked to a spacer group. The spacer group is derived from acids of dicarboxylic acids, acid anhydrides or phenylamines or combinations thereof. The intermediate has the general structural formula:

[0017]

[Chemical 7]

Wherein Y is H, a member selected from the group consisting of Chemical formula 8 or Chemical formula 9; and X is --H, --OH or Chemical formula 1.
0; is a member selected from the group consisting of 0; and Z is a member selected from the group consisting of —H or —OH.

[0019]

[Chemical 8]

[0020]

[Chemical 9]

[0021]

[Chemical 10]

However, when Y is -H, Z must also be -H and X must be X is-
When OH, Z must be -H and Y must be

[0023]

[Chemical 11]

[0024]

[Chemical 12]

[0025]

[Chemical 13]

When Z is --OH, X must be --H and Y must be ## STR14 ##

[0027]

[Chemical 14]

[0028]

[Chemical 15]

In the formula (7), n is an integer of 2 to 6. n is an integer of 2 or 3, and the spacer compound 16 is obtained from succinic acid or glutaric anhydride.

[0030]

[Chemical 16]

The intermediates described in the structural formula above can be divided into two sub-groups.

The first sub-group is a glucosamine derivative, where Z is --H, Y is --H, and X is --H.
Is a chemical formula 17 wherein n is an integer from 2 to 6.

[0033]

[Chemical 17]

The second sub-group is N-succinyl or N-glutaryl-amido-phenyl-α-D-gluco and mannopyranoside and p-isothiocyanotophenyl-α-D-gluco and mannopyra. Noxide, wherein X and Z are different and are selected from the group consisting of —H and —OH, Y is a member selected from the group consisting of formula 18 and formula 19, and n is an integer from 2 to 6. Is.

[0035]

[Chemical 18]

[0036]

[Chemical 19]

The starting materials used to prepare the sugar plus spacer glycolated intermediate are glucosamine and p-nitrophenyl-α-D-gluco and mannopyranoside, which are commercially available.

Glucosamine can react directly with acid anhydrides. Suitable spacers are moieties of succinyl and glutaryl, so the rest of the description will be directed to these derivatives. However, four corresponding derivatives from adipic acid, pimelic acid and suberic acid are also available by suitable synthesis.

The p-nitrophenyl-α-D-glucose and mannopyranoside are first treated to reduce the nitro group to the amino group. These can then be reacted with succinic and glutaric anhydrides to produce the corresponding N-succinyl- and N-glutaryl derivatives.

The p-aminophenyl-α-D-glucos and mannopyranoside can also react with thiophosgene to form the corresponding p-isothiocyanotophenyl-α-D-glucos and mannopyranoside. The synthesis of these products is described in detail in the examples which follow.

The glycosylated intermediates that follow are representative of novel pyranosides that can be used to bind insulin.

[0042]

[Chemical 20]

N = 2 N-succinyl glucosamine,
m. p. 174-175 ° C n = 3 N-glutaryl glucosamine, m.p. p. 19
5-196 ° C

[0044]

[Chemical 21]

N = 2 p- (succinylamido) -phenyl-α-D-glucopyranoside, m.p. p. 178
-180 ° C n = 3 p- (glutarylamide) -phenyl-α-
D-glucopyranoside, m. p. 167-168 ° C

[0046]

[Chemical formula 22]

N = 2 p- (succinylamido) -phenyl-α-D-mannopyranoside, m.p. p. 65-
66 ° C. n = 3 p- (glutarylamide) -phenyl-α-
D-mannopyranoside, m. p. 134-136 ° C

[0048]

[Chemical formula 23]

P-isothiocyanotophenyl-α-D-
Glucopyranoside

[0050]

[Chemical formula 24]

P-isothiocyanotophenyl-α-D-
Manno pyranoside

The structure of the insulin molecule is well known. Its structure consists of two polypeptide chains, A and B, linked together by a disulfide bond of cysteine.
The N-terminal group of the A fraction is glycine (Gly A-1) and B
The N-terminal group of the fraction is phenyl alanine (Phe B-
1). Both N-terminal positions contain unreacted α-amino groups. Free ε adjacent to the C end group of the B fraction
-A lysine with an amino group is present. These free amino groups are believed to be involved in the coagulation problem of insulin molecules by their final precipitation.

It was believed that by blocking these groups with the aforementioned glycosylation intermediates, the biological potency of insulin was not significantly affected and that coagulation could be significantly inhibited or prevented. In addition, other properties that glycosylated insulin contribute to the chemical resistance release mechanism that provides insulin to diabetics in a form that directly responds to changes in blood glucose levels without the need for external or implantable detection devices. Is believed to have.

The reaction of the intermediate shown above was carried out by converting the carboxylic acid at the end of the spacer into a mixed anhydride through reaction with an alkyl chloroformate and reaction of the mixed anhydride with bioinsulin.
The mixed anhydrides are combined with one or more A-1, B-1 or B-29 on insulin to produce mono, di or triglycosylated insulin via an amide bond.
Reacts with the free amino groups of. The degree of substitution depends on the reaction conditions including the molar ratio of intermediate to insulin and the pH value. Generally, the molar ratio of intermediate to insulin is 2 to 1.
Change to 0. For the purpose of the reaction, a pH of about 8 to 9.5
A value range is preferred.

Due to the complexity of the reaction it will be rare to produce glycosylated insulin as a mono-, di- or tri-substituted derivative. Rather, a mixture will be obtained, as shown in the examples below.

For purposes of illustration, glycosylated insulin can be divided into three categories.

The first class has the spacer of formula
Insulin prepared from glucosamine having the general structural formula:

[0058]

[Chemical 25]

[0059]

[Chemical formula 26]

In the formula, n is an integer of 2 to 6, m is an integer of 1 to 3, and the glycosyl group is A-1 glycine, α-amino group of B-phenylalanine or B-29 lysine of insulin molecule. It is attached to insulin through one or more ε-amino groups of the moiety.

Representative insulins are (glucosamide succinyl-) _m insulin and (glucosamide glutaryl-) _m insulin.

The second class includes gluco- and mannopyranoside linked to a Formula 27 spacer having the general structural formula of Formula 28.

[0063]

[Chemical 27]

[0064]

[Chemical 28]

In the formula, X and Z are different, and --H and --OH
N is an integer from 2 to 6, m is an integer from 1 to 3 and each glycosyl group is A
-1 Glycine, α-amino group of B-1 phenylalanine or ε-amino group of B-29 lysine moiety of insulin molecule is attached to insulin through one or more amide bonds.

Representative compounds include [p- (α-D-glucopyranosyloxy) -phenyl-N-succinamil] insulin; [p- (α-D-glucopyranosyloxy).
-Phenyl-N-glutaramyl] _m insulin; [p
-(Α-D-mannopyranosiloxy) -phenyl-N-
Succinamil] _m insulin; [p- (α-D-mannopyranosiloxy) -phenyl-N-glutaramyl] _m
Contains insulin.

The third class includes gluco- and mannopyranoside linked to the spacer of formula 29 having the general structural formula of formula 30:

[0068]

[Chemical 29]

[0069]

[Chemical 3]

In the formula, Z and X are different, and --H and --OH
M is an integer of 1 to 3 and each glycosyl group is an α-amino group of A-1 glycine, one of the ε-amino groups of the B-29 lysine moiety of the insulin molecule, or Through the above, it is attached to insulin by a thioamide bond.

Representative compounds include [p- (α-D-glucopyranosyloxy) -phenyl-thiocarbamoyl-] _m insulin and [p- (α-D-mannopyranosiloxy) -phenyl- Thiocarbamoyl-] _m insulin.

The glycosylated insulin prepared according to the present invention can be administered to diabetic patients by any conventional method, ie subcutaneous, intramuscular or intraperitoneal injection. The dose is IU
The unit (international unit) can be equal to free or living insulin. No attempt will be made to range the dosage as the dosage will vary widely depending on the patient's requirements. This dose determination will be left to the discretion of the patient's attending physician. Generally, a 60 kg person is required to administer a dose of 2 mg of insulin per day.

The following examples demonstrate the biological potency and ability of insulin to prepare intermediate compounds, to prepare glycosylated insulin, and to inhibit or prevent clotting.

Example I : Preparation of N-succinyl glucosamine. Glucosamine hydrochloride (0.05 m , 10.78 g) was dissolved in 15 ml double distilled water and 0.05 m triethylamine (6.95 ml). 37.5 ml of acetone by stirring in this solution
Succinic anhydride (0.05 m , 5.705 g) in was added. The resulting mixture was separated into two layers and sufficient water was added to bring both layers into one solution. The solution is left at room temperature for 4 hours to complete the reaction, then
The solution was placed in a vacuum chamber and evaporated until a thick viscous yellowish concentrated solution was obtained. The concentrate was measured and diluted with 3 volumes of glacial acetic acid resulting in the formation of a white precipitate of N-succinyl glucosamine. The product was separated from the acetic acid solution by filtration and washed with ethanol and then petroleum ether. The resulting product yield was 39%. The melting point of the product is 174-175.
At ℃, the molecular weight is 279.26 2.5% of the calculated molar weight
It was inside. Its structure and molecular weight are IR, NMR and MS
/ GC spectrum confirmed.

Example II : Preparation of N-glutaryl glucosamine. The method of Example I was followed using glutaric anhydride. The product yield was 41%. The melting point was 195-196 ° C. The calculated molar weight was 293.27. IR and NM
The structure was confirmed using the R spectrum.

Example III : Preparation of p- (succinylamido) -phenyl-α-D-glucopyranoside. In the first step, p-nitrophenyl-α-D-glucopyranoside (14 mmol, 4.214 g) was added to ammonium formate (56 mmol, 3.54 g) and 25 g of palladium on carbon particles in 350 ml of methanol. Reduced by mixing at ° C. The whole system was flushed with nitrogen for 4 hours, then filtered and the filtrate evaporated under reduced pressure. The crude p-aminophenyl-α-D-glucopyranoside was purified by recrystallisation in an ethanol-water (50: 1) mixture. The yield was 71%. The melting point was 169-170 ° C. Structure and molecular weight were confirmed by IR and MS / GC spectra. The measured molecular weight is 2.7 of the calculated molar weight of 271.27.
It was within%.

P-Aminophenyl-α-D-glucopyranoside was reacted with succinyl anhydride according to the method of Example I to give a 53% yield of p- (succinylamide)-.
Phenyl-α-D-glucopyranoside was produced. The melting point of the product was 178-180 ° C. The structure and molecular weight were confirmed by IR, NMR and MS / GC spectra. The measured molecular weight was within 2% of the calculated molar weight of 371.34.

Example IV : Preparation of p- (glutarylamide) -phenyl-α-D-glucopyranoside. The method of Example III was repeated using glutaric anhydride instead of succinyl anhydride.
p- (Glutarylamide) -phenyl-α-D-glucopyranoside was produced with a yield of 63% and its melting point was 167.
It was -168 degreeC. The structure was confirmed by IR spectrum. The calculated molar weight was 385.37.

Example V : Preparation of p- (succinylamido) -phenyl-α-D-mannopyranoside. First, p-nitrophenyl-α-D-mannopyranoside was reduced to p-aminophenyl-α-D-mannopyranoside using the method outlined in Example III. The product yield was 91% and the product was 1
Melted at 50-153 ° C. The structure was confirmed by IR spectrum.

The p-aminophenyl-α thus obtained
-D-mannopyranoside was reacted with succinyl anhydride by the method described in Example III to give a 67% yield, mp 6.
P- (succinylamido) -phenyl-α at 5-66 ° C
-D-mannopyranoside was produced. The structure and molecular weight were confirmed using IR, MNR and MS / GC spectra. The measured molecular weight is 2% of the calculated molecular weight of 371.34.
It was within.

Example VI : Preparation of p- (glutarylamide) -phenyl-α-D-mannopyranoside. The glutaric anhydride was replaced with succinyl anhydride according to the method outlined in Example V. The resulting p- (glutarylamide) -phenyl-α
-D-mannopyranoside was produced with a melting point of 134-136 ° C and a yield of 75%. The calculated molecular weight is 385.
It was 37. The structure was confirmed by IR spectrum.

Example VII : Preparation of p-isothiocyanotophenyl-α-D-glucopyranoside. An excess mole of thiophosgene (CSCI ₂ ) was added to a solution of p- (aminophenyl) -α-D-glucopyranoside in 80% aqueous ethanol. The reaction was carried out at room temperature and was completed in a few minutes. A crystalline product was obtained. The calculated molecular weight was 313.3.

Example VIII : Preparation of p-isothiocyanotophenyl-α-D-mannopyranoside. p-isothiocyanotophenyl-α-D
The method of Example VII can be applied to replace p- (aminophenyl) -α-D-mannopyranoside with the corresponding glucopyranoside to produce -mannopyranoside.

Glucosamine or p-aminophenyl-
The following tests were utilized to confirm the synthesis of the aforementioned combinations of α-D-glucose and mannopyranoside with succinylic acid and glutaric anhydride. An infrared spectrophotometer (Beckman Microlab 620MX Computing Infrared Spectrophotometer) was used to determine the reaction between the amino group and the anhydride by detecting the presence of an amide bond. Samples were prepared as 0.5% (w / w) KBr pellets. The presence of the amino group before the reaction is 1650.
Detected by the NH stretching band at -1580 cm ^-1 . The p-aminophenyl derivative prepared by reduction of the corresponding p-nitrophenyl derivative showed N at 1580 cm ⁻¹ and 1330 cm ⁻¹ , indicating that the reduction reaction was complete.
It showed no -O stretch zone. 16 amide bond formation
This was indicated by the presence of the C = O stretching zone at 60 cm- ¹ and the NH bending mode at 1600 cm- ¹ . The normal dimer carboxyl C = O extension zone is also about 1725 cm ^-1
It was seen nearby. These data confirm a distinct amide band indicating the completion of the coupling reaction between the amino and dicarboxylic anhydride reactants.

DEC PDP 11/34 Computer, LKB 9000S in interface
Molecular weights were formed in the MS-GC spectrum using a MS / GC spectrophotometer. The volatility of carbohydrate derivatives was enhanced using trimethylsilyl derivatives of hydroxyl and carboxylic acid groups. The observed molecular weight of the trimethylsilyl derivative in all examples was within 2.5 ± 0.5 of the calculated theoretical value.

[0086]

[Chemical 31]

The presence of the chemical formula 31 is JOEL JNM-
Confirmed by proton MNR spectrum using FX270 Furia Transform NMR spectrophotometer. The sample was dissolved in D ₂ O and sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DS
S) was used as an internal reference component. For example, in p- (succinylamide-α-D-glucopyranoside), the proton signal of the methylene group in the succinyl moiety was observed as a triplet at δ = 2.71. The peak area was proportional to the number of protons representing the four methylene protons in the succinyl moiety. The melting point was measured by the capillary melting point method.

The yields of the above-mentioned glucose and mannose derivatives utilizing dicarballylic anhydride are about 39 to 9
It fluctuated between 1%. The variation in yield is believed to be due to the use of limited solvent (ethanol-water mixture) for recrystallization. The yield should be increased by choosing the appropriate solvent for the recrystallization method.

The recombining action of insulin with the above-mentioned derivative of glycosylamidocarboxylic acid is carried out through the mixed anhydride method so that the mixed anhydride is not separated from the reaction mixture. The glycosyl amidocarboxylic acid is converted to a mixed anhydride by reaction with isobutyl chloroformate, and the resulting mixed anhydride reacts with free amino groups from the insulin molecule to form an amide bond. This method is generally described by Erlanger et al . , J. Biol. Chem. 228, 713 (1957) and Arekatu et al., J. Immunal., 97 , 858 (196).
6).

There are three primary sites available on the insulin molecule for the reaction of glycosylamidocarboxylic acid derivatives with insulin, and insulin can be bound by one, two or three of these derivatives. These available sites include glycine (Gly A-
1) α-amino group, phenylalanine (Phe B-
1) and lysine of insulin molecule (Lys B-2
9-) ε-amino group is included. PKap of these groups
The p-values are 8.0 for Gly A-1, 6.7 for Phe B-1 and 11.2 for Lys B-29.

Since insulin is denatured when the pH value is too high, the conjugation reaction between glycosyl-amide-carboxylic acid and insulin is performed in order to maintain the ε-amino group of the Lys B-29 moiety in a less reactive protonated state. P
The H value was chosen between 7.5 and 10, preferably 9.5. Therefore, the α-amino group at the Gly A-1 and Phe B-1 positions is considered as the primary reaction site. However, the tri-substituted glycosylated insulin was added to Lys B-29 by using the highly nucleophilic tri-N-butylamine added to complex the HCl formed during the anhydride formation with isobutyl chloroformate. Since the free ε-amino group from the moiety can be produced by deprotonation, it can also be produced by the method described above. Also,
A pH value of 9.5 based on the Henderson-Hasselbach equation
And about 2% of the ε-amino groups of Lys B-29 are in equilibrium in the free or deprotonated form. Therefore, a considerable amount of tri-substituted glycosylated insulin can be prepared.
However, the selected pH value, namely 9.5 and its pH
Due to the highly reactive free amino groups of Gly A-1 and Phe B-1 by value, glycosylated insulin will predominantly be a mixture of di- and tri-substituted derivatives. Some monosubstitution may also be present.

In the following example, unreacted insulin is removed from glycosylated insulin by affinity chromatography using a column with Sehalose microspheres bound to Con-A (concanavalin-A).

It is known that Con-A has a binding affinity for saccharides. Therefore, the more glycosyl moieties are attached to insulin, the more glycosylated insulin the Co in the chromatography column.
The amount bound to n-A increases. Therefore, the unreacted insulin would be the first to elute through the column followed by mono-, di- and tri-glycosylated insulin in this order.

This is generally true. However, some glycosylated derivatives may elute from the column along with unreacted insulin.

The following example is representative of a method for separating unreacted insulin from glycosylated insulin by affinity chromatography with Con-A.

Example IX : Preparation of N-succinyl glucosamine. Bound insulin (glucosaminosuccinyl insulin). Bovine insulin (87.77 micromol 500 mg) was added to distilled water and dimethylformamide (DM).
Dissolved in 200 ml of an equal volume mixture of F), 0.1 N
The pH value was adjusted to 9.5 with sodium hydroxide and then cooled in an ice bath. N-succinyl glucose amine, (8
(00 micromol) was dissolved in a solution of DMF containing 800 micromol of each tri-N-butylamine and isobutyl chloroformate and kept at 0 ° C. for 20 minutes. 1.6 mmol of tri-N-butylamine was added to this solution and then mixed by stirring to give an insulin solution. The reaction mixture thus purified was adjusted to a pH value of 9.5 with 0.1 N sodium hydroxide and kept at 0 ° C. for 1 hour. The mixture was then kept at room temperature overnight and then dialyzed through a semipermeable membrane against distilled water for 2 days to remove unreacted N-succinylglucosamine. Distilled water was kept at 4 ° C. and replaced every 4 hours.

The glycosylated insulin remaining on the inside of the dialysis membrane was lyophilized and dissolved in the Tris-buffer solution described below. The resulting solution was sterilized by filtration to remove the bacteria present in it.

The sterilized product was treated with Sepharose 4B.
Placed on a 2.5 x 60 cm column containing commercially available Con A (Concanavalin-A) microspheres bound to (Sigma Chemical Co., St. Louis, MO). Unreacted insulin is 1 mm MnCl ₂ , 1 mm
0.0, which also contains CaCl ₂ and 0.5 m NaCl
It was removed from the column using a buffer eluent - 2 m Tris. The eluent had a pH value of 7.4 and was kept at 4 ° C. The flow rate was maintained at 72 ml / hr and 7.0 ml fractions were collected and analyzed by UV spectroscopy at A276 nm for the presence of insulin. Colorimetric measurement of sugars at 480 nm utilizing the phenol-sulfuric acid test also showed the presence of some N-succinyl glucosamine linked insulin.

As shown in FIG. 1, after about 105 minutes, all unreacted insulin (component 1) was UV at 276 nm.
It was collected and monitored by the spectrum. At that time, 0.1 m α-methyl-D-mannopyranoside was added to the Tris-buffer solution as an eluent and the flow rate was maintained at 72 ml / h. About 200 minutes later N-
All of component 2, consisting of succinyl glucosamine linked insulin, were assembled as also shown in FIG.

It was believed that the inherent low avidity of the glucosamine moiety for Con-A was due to a mixed eluent of free insulin and glycosylated insulin within component 1. The degree of substitution could not be determined due to the low adsorption of glycosylated insulin in component 2 at 480 nm.

The glycosylated insulin in component 2 was lyophilized to determine its ability to lower blood glucose levels. The corresponding N-glutaryl glucosamine linked insulin (glucosamino glutaryl insulin) was prepared in a similar manner.

Example X : Preparation of p- (succinylamido) -phenyl-α-D-glucopyranoside linked insulin. [P- (α-D-glucopyranosyloxy) -phenyl-
N-succinamil insulin].

The procedure of Example IX is the same as that of Example III
The (succinylamido) -phenyl-α-D-glucopyranoside was supplemented to react with bovine insulin. The result is shown in FIG. Component 1 in FIG. 2 is 480 nm
It was composed of free insulin and some glycosylated insulin demonstrated by the phenol-sulfuric acid method in. Ingredients 2 and 3 are collected and 2 for insulin
As with 76 nm, it was examined by the phenol sulphate method for the presence of glycosyl groups. Due to the large amount of eluent required to separate component 3, it can be expected that component 3 contained more glycosyl groups on insulin than component 2. The areas under the curves for components 2 and 3 are each 58.9.
% And 41.1%. Component 2 was predominantly a diglycosyl-substituted insulin and component 3 was predominantly a triglycosyl-substituted derivative. Thus combined component 2
And the average number of glycosyl derivatives on insulin contained in 3 and 0.589 × 2 + 0.411 ×
3 = 2.411 was obtained. This degree of substitution was unique to the phenol sulfate test, which showed 2.3 glycosyl groups per insulin molecule. For more information on the phenol-sulfuric acid test, please contact Jupoir and other analytical chemists.
Lee 28, 350 (1956).

After the assembled components 2 and 3 were combined and dialyzed to remove the eluent, the purified product α-methyl-
D-mannopyranoside was lyophilized for biological testing.

Corresponding p- (glutarylamide) -phenyl-α-D-glucopyranoside linked insulin [p-α-D-glucopyranosyloxy)-by the same method.
Phenyl-N-glutaramyl insulin] was prepared.

Example XI : Preparation of p- (succinylamido) -phenyl-α-D-mannopyranoside-coupled insulin. [P- (α-D-mannopyranosiloxy) -phenyl-
N-succinamil insulin]. The method outlined in Example X was performed and the eluent geometry is shown in FIG. Component 1 was unreacted free insulin because the phenol-sulfuric acid test was negative. The average degree of glycosyl groups attached to insulin due to the combination of components 2 and 3 was 2.5 by the phenol-sulfuric acid test. Ingredients 2 and 3
The area under the curve for shows a mean degree of substitution of 2.66 which is closely compared to the above test results at 34% and 66% respectively.

The purified lyophilized product was retained for the hypoglycemic test.

Corresponding p- (glutarylamide) -phenyl-α-D-mannopyranoside linked insulin]
p- (α-D-mannopyranosiloxy) -phenyl-N
-Glutaramyl insulin] was prepared and purified by the method described above.

Example XII : Preparation of p- (α-D-glucopyranosyloxy) -phenyl-thiocarbamoyl insulin. P- (Isothiocyanotophenyl) -α-D-glucopyranoside (355.08 micromoles) obtained from Example VII was dissolved in a solution of 3 parts pyridine and 1 part water at 5 ° C. The pH value was adjusted to 8.0 with 0.1 NaOH. Bovine insulin (177.54 μmol,
1 gm) was prepared using a pyridine-water solvent and combined with the glucopyranoside solution. The combined solution at 5 ° C
The pH value was maintained at 8.0 for 1 hour and then left overnight at room temperature. Next, p- (α-D-glucopyranosyloxy)-
The reaction product consisting of phenyl-thiocarbamoyl insulin was lyophilized as in Example IX to remove unreacted p- (isothiocyanotophenyl) -α-D-glucopyranoside and the remaining product was lyophilized. And dissolve in Tris buffer, and Con-
Provided for affinity chromatography on an A Sephorase 4B column. The flow rate is 26 ml / hour at 4 ° C,
A 5.0 ml fraction was collected. The properties of the eluent are shown in FIG. Component 1 contains both free and glycosylated insulin, and component 2 has p- (α-D- with an average value of 1.5 glycosyl groups per insulin molecule.
It was composed of glucopyranosyloxy) -phenyl-thiocarbamoyl insulin.

The product from component 2 was dialyzed to remove the α-methyl-D-mannopyranoside eluent and then lyophilized for biological testing.

Example XIII : Coagulation Test One of the problems associated with free or bioinsulin is that insulin tends to coagulate and eventually crystallize out of solution, thus losing its biological potency. It is a point. In the case of glycosylated insulin, this trend is due to Gly A-1, Phe B- in insulin.
It is significantly reduced because portions of 1 and Lys B-29's active aminosites are blocked by the glycosyl linking reaction.

Studies on bulk aggregation with free insulin by comparison with glycosylated insulin were performed by two methods. In the study on agglomeration, various aqueous solutions and a glycosylated insulin solution containing 0.1 mg / ml of insulin were stirred at 1555 rpm until agglomeration was visible or for 2 weeks. In another test, a solution containing the same insulin concentration was deposited on polyurethane (Biomer) and microscopically observed for coagulation. The results are shown in Table 1.

[0113]

[Table 1]

It is clear from the above results that glycosylated insulin is more stable to coagulation than free insulin and therefore has a better shelf life.

Example XIV : Biological activity of glycosylated insulin. The biological activity of the glycosylated insulin described herein was measured by the hypoglycemic test and compared to commercial insulin formulations and control solutions. In this test, normal laboratory rats were modeled and fasted for 20 hours. After measuring the baseline blood glucose level, either 1 mg / kg of free or glycosylated insulin was infused via the intraperitoneal route. The blood glucose level in each rat was measured by a colorimeter 20 minutes after the injection. The results are shown in Table 2.

[0116]

[Table 2]

As described herein, it is clear from the above description that all seven types of prepared glycosylated insulins have remarkable biological effect of lowering blood glucose level.

[Brief description of drawings]

FIG. 1 is a diagram of the elution profile from the chromatographic test reported and described in Example IX.

FIG. 2 is a diagram of the elution profile from the chromatographic test reported and described in Example X.

FIG. 3 is a diagram of the elution profile from the chromatographic test reported and described in Example XI.

FIG. 4 is a diagram of the elution profile from the chromatographic test reported and described in Example XII.

Claims (7)

[Claims] Claims: Glycosylated insulin having the structural formula of Chemical formula 1. [Wherein Z and X are different, selected from the group consisting of -H and -OH, m is an integer of 1 to 3, and each glycosyl group is A-1 glycine, B-1 phenyl alanine α-amino. It is attached to insulin by a thioamide bond through one or more groups or through the ε-amino group of the B-29 lysine moiety of the insulin molecule. ] 2. The glycosylated insulin according to claim 1, wherein Z is —H and X is —OH. 3. The glycosylated insulin according to claim 1, wherein Z is —OH and X is —H. 4. A compound represented by the formula: A therapeutic agent for diabetes comprising an effective amount of glycosylated insulin having the structural formula of Formula 2 in a pharmaceutically acceptable carrier. [Wherein Z and X are different, selected from the group consisting of -H and -OH, m is an integer of 1 to 3, and each glycosyl group is A-1 glycine or B-1 phenylalanine α-amino. B-2 of one or more groups or insulin molecules
It is attached to insulin through a thioamide bond through the ε-amino group of the 9-lysine moiety. ] 5. The therapeutic agent for diabetes according to claim 4, wherein the effective amount of glycosylated insulin is determined based on the amount required by the patient. 6. The therapeutic agent for diabetes according to claim 5, wherein Z is —H and X is —OH. 7. The therapeutic agent for diabetes according to claim 5, wherein Z is —OH and X is —H.