DE2256574A1 - INSULINE DERIVATIVES - Google Patents

Description

DR. MÜLLER-BORE DIPL.-PHYS. DR. MANITZ DIPL.-CHEM. DR. DEUFEL DiPL.-ING. FINSTERWALD DIPL.-ING. GRÄMKOW

Munich, I 7. Hoy, 1272

Lo / Sv - N 1090

NATIONAL.BESEARCS DEVELOPMENT COEPOBATIOjJ 66-74- Victoria Street, London S.W.1., England

Insulin derivatives

Priorities: Great Britain on November 18, 1971?

No. 53622/71 - '

Great Britain November 18, 1971? No. 53623/71

The invention relates to insulin derivatives.

Having more comprehensive methods of diagnosing diabetes mellitus have been introduced and as normal life expectancy increases, the observed incidence of this disease decreases constantly to. Current treatment consists of diet control, usually in combination with insulin injections or with an oral anti-diabetic agent Injections are often required once or twice daily for the life of the patient. Self with such treatment, blood sugar levels will vary

303322/1163

ues patient considerably from normal, creating a strict Diet is required. Oral remedies are only mild Suitable for cases of diabetes. In addition, it is assumed today that they exhibit certain undesirable side effects · In addition to the deficiencies listed above, a current one Treatment produces a part of the diabetic antibodies Insulin and becomes increasingly resistant to its effects.

Therefore, it would be desirable to manufacture therapeutic agents that provide better control of blood sugar levels than the means used in current treatments. Investigations were carried out for this purpose carried out on properties of insulin derivatives. This is however, an area in which, despite the efforts of many researchers, very few conclusions have been drawn so far could be drawn, mainly because of the lack of separating the individual components of the complex mixture and adequately identify which stems from the acylation and other reactions to which parent insulins are subjected became. There have therefore been extensive research on the development have undertaken a number of derivatives in which the combination of substituent groups is such that an increase to one a better profile of the hypoglycemic effect is achieved. Around To solve this problem, the type of substitution on the primary amino residues of the insulin molecule is of the greatest importance Importance.

The object of the invention is to produce an N-substituted insulin, in which the profile of the hypoglycemic effect can be modified, but in which the desired one Retained activity of parent insulin to a significant extent will. Furthermore, the substitution of the molecule of the N-substituted Insulins are also being researched to temporarily mask the hypoglycemic effects. The substitution of the amino residues also modifies the antigenic ones Properties of the molecule and can reduce the occurrence of immunological resistance.

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■. ■ 225657V

The object according to the invention, namely the fulfillment of the requirements given above, is achieved to a remarkable extent by an insulin _{derivative, with at least one of A x} , (glycine), B. (phenylalanine) and Bpg (Ly sin) -Ami no residues through a carbamyl radical, HHpCO-, or a substituted carbamyl radical, R _x -RpNOO-, in which R _x and Hp can be hydrogen, an alkyl, aryl or another radical, is blocked. Among the compounds in which R _x , hydrogen and R ^ is a lower alkyl radical with 1 to 4 carbon atoms, those with the 2- ethylcarbamyl radical as the blocking radical are of particular interest.

The introduction of carbamyl radicals or substituted carbamyl radicals according to the invention can suitably be carried out by adding the insulin with an alkali met all cyanate, e.g. KCM), for the carbamylation and with an alkyl isocyanate, _{e.g. C 2} H ₅ IfOO or PhNCO, for IT '-substituted carbolization is allowed to react.

The blocking of one or more amino groups as mentioned above, suitably including the group in the A _x . (Glycine) position and preferably all three residues has been found to be a necessary condition for the production of compounds which have the desired properties. For example, it has been found that If, N ¹ , N "-tricarbamyl insulin has superior properties and its effectiveness in guinea pigs, as determined by the effect on blood glucose due to the response of the peak and the area under the curve, is twice that of not modified insulin As already described, at least one of the N-blocking residues must be a carbamyl residue, but other possibilities are open for the other N-blocking residues.

Modification of the hypoglycemic effect of the N-substituted en insuline can be achieved through additional substitution, namely at one or more tyrosine hydroxyl sites

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22S8S7A

B ^ ₆ , B ₂₆ ), and insulin derivatives which have G-ayl trisite in one or more tyrosine positions in addition to the N-carbamyl or N-substituted carbamyl residues also belong to the derivatives according to the invention. Such 0-substituted derivatives are of greatest interest in the case of von'I-trigutoetittdeiftea Inaulinen. Since the O-acylation almost inevitably results in an inactive derivative, it is essential for the activity to be expressed that the O-blocking agent must be removed in vivo. For these reasons, for example, acetyl and glutaryl residues are preferred to succinyl residues · In addition, there are four tyroein positions in the insulin molecule and both partial and complete blocking of tyrosine is possible with the derivatives according to the invention. Acylation of! Tyrosine or other forms of blocking can easily can be monitored by measuring the decrease in the characteristic tyrosine absorption peak at approximately 275 mm. For example, an N-trisubstituted derivative can gradually become progressively acylated and the reaction terminated when the decrease in absorbance indicates that the desired degree of substitution has occurred.

Various reagents can be used to carry out the acylation be used. Suitable active systems comprising carboxylic acid or functional derivatives thereof include those Acid in the presence of garbodiimide reagents, the acid chloride or anhydride or activated esters such as those with p-nitrophenol and hydroxysuccinic acid imide

Other possibilities than acylation are possible for blocking the hydroxyl radicals, e.g. carbamylation or the N-abatituted carbamylation. The methods and remnants which are applied, are partly due to the observance of the sin Quality of insulation, but mainly of products 4

309822 / 11C3

In view of the ease of removal of the residue or residues in vivo. The latter property can be determined by hydrolysis experiments in vitro, in a simple manner that the physiological conditions simulating ^one reindeer, is made possible so that the adjustment of the final product to the needs of the particular patient.

It has been found that certain of the "H-" carbamylated, O-acylated derivatives of the invention are caused by naturally occurring Esterases are attacked, so that another Change in the nature of the 0-acyl radical may be necessary in certain cases. So can O-Ohloracetylderivate with With the help of chloroacetic anhydride and ithoxycarbonyl derivatives with the help of ityl chloroformate. Longer-chain substituent residues, which are derived from stearic acid,

may be derived from octadecylsuccinic acid or liaphthoic acid, / by Reaction with a suitable anhydride or "by coupling." of the acid in the presence of carbodiimide reagents.

As with the parent insulins, zinc can be present in some forms of the insulin derivatives according to the invention, for example iiälT, lI ', l' ^II -tricarbamyl derivative.

The insulin derivatives according to the invention can be formulated as pharmaceutical preparations in the same way as the parent insulins or starting insulins, and they. can clinically in veig. calibratable, larger or smaller dose values can be used. For example, the normal daily insulin dose is 20 to 80 IU / day for adults and for patients who are already used to it, more than 200 U and in special cases more than 500 U of the standard insulin. The derivatives according to the invention can be produced as solutions, suspensions or as freeze-dried preparations. A typical approach is a neutral solution or a solution with a physiological pH value, which is 0.136 w / v. -% sodium acetate, 0.7 w / v% sodium chloride and 0 »1 w / v% hydroxybenzoate in pyrogen-free water.

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_6_ 225657A

The invention is applicable to all types of insulin and in particular on insulins from pigs or binders, which have been used clinically for many years in the treatment of diabetes and other diseases are used.

The invention is explained in more detail below with the aid of examples.

Example 1 : Preparation of N, N ¹ , N "-tricarbamyl insulin

(1) Pig zinc insulin / ~ 100 mg, Burroughs Wellcome Ltd., 1 χ crystallized, moisture content »7.09%» zinc content = 0.336 % (not adjusted for loss on drying), biological activity ■ 23.0 E. / mg (not corrected for the loss on drying) detected by convulsions on the mouse_ "J in a suspension in

5 ml of 0.2 M y) aminomethane buffer (Tris buffer) with a pH of 8.5 is mixed with a solution of 5 ml of 1M potassium cyanate. The solution becomes clear within 2 minutes and the incubation is ^{continued at 30 0} O for 8 hours.

The desalting is carried out by removing the reaction mixture on a 30 cm χ 2.3 cm column with a three-dimensionally cross-linked polysaccharide, which by cross-linking the linear Macromolecules obtained from dextran was given (Sephadex G-25) which was treated with 0.01M N-ethylmorpholine acetate with pH »9 was operated as an eluent. The protein containing Fractions were pooled, stored at room temperature for at least 1 hour and then lyophilized. The booty of tricarbamyl insulin is 95 - 100% based on the measurement the absorption at 280 mn.

(2) In a second manufacturing method, a small amount of dicarbamyl derivatives was separated from the tricarbamylineulin. The carbamylation mixture was as described above desalinated and a gradient elution on one

309822/1163

changed gemiö & naa & « received on

225BS74

100 egg χ 1 oil column with three-dimensionally cross-linked saccharide, which was obtained by cross-linking the linear Mato? Ö ~ molecules of dextran (Sephadex Gh2 $) with a starting buffer solution of Ö, O1M Tris (hydröxymethyi) «aniinomethanac etatpuff er with pH-« ± 7.1, which contained 8H urea, into which the same buffer, which contained 8M urea, Ο, ΟΙΜ Tri £ i (hydroxymethyl) -ajainoffiethanabetatbuffer with pH = 7> Ί 'and 0.2M NaOL, flowed. The volume of the mixing tank was 200 ml. The protein in the liquid flowing out of the column was determined by measuring the absorbance E280, and the larger, eluted peak representing more than 80 % of the starting insulin was pooled and desalted. The trieasoimyl insulin was stored in a lyophilized state.

The iricarbamyide derivative is soluble at least at pH β 4.0, and desalination can take place by 'precipitation at pH = 4.0 be carried out by gel filtration *

The characterization of the derivative by analyzing the UHp residues shows that both the A ^ -glycine and the IL-phenylalanine were quantitatively carbamylated. The B ^ q amino residue (liysine) is carbamylated, as was determined by the degradation of the derivative with trypsin (enzyme substrate ratio = 1:20) at pH 9 and 37 ° 0 for 4 hours * Less than 0.04 * ^ Best of the alanine is of the CÖÖH-end position of the B-chain is released ■■ "the derivative is compared to ninhydrin without Keaktion, this bestätigtj that the three NHo residues of insulin are blocked * the derivative gives electrophoresis Aerylsffiid-in at pH = 8.5 s a single band in 8M urea.

changed according to
received on ..

225657A

Example 2 - Preparation of (i) NA ^ -Monocarbamyl Insulin,

(ii) N-B ^ monocarbamyl insulin and (iii) Njli '

200 mg of zinc-free insulin are dissolved in 10 ml of 0.1M sodium phosphate dissolved at pH 7 »0, then 16.8 mg of potassium cyanate are added and the reaction mixture is kept at 30 ° C. for 3 hours. The mixture of the carbamylated derivatives is on a 30 χ 3 cm Pillar with three-dimensional networked! Polysaccharide (Sephadex G-25) using 0.05M N-ethylmorpholine as the eluent desalinated and lyophilized. The mixture is then analyzed by chromatography on a 55 x 1j5 cm column with three-dimensional networked! Polysaccharide (Sephadex G-25) using 0.09M tris (hydroxymethyl) aminomethane.HCl buffer (tris-HOl) separated in 8M urea as eluent, until 250 ml are collected, 0.12M tris (hydroxymethyl) aminomethane.HCl buffer is then added used in 8M urea. The first peak that emerges between 98 and 130 ml contains unreacted insulin, while the subsequent peaks at 149-204 ml, 373 to 414 ml and 428 to 507 ml of N-B ^ monocarbamyl insulin, N-A ^ monocarbamyl insulin or Ν, Ν'-Α ^, Β ^ -dicarbamyl insulin. Each of these factions is networked on three-dimensional Polysaccharide (Sephadx G-25) desalted and lyophilized.

Example 3 - Preparation of N, N ', IP-Triäthylcarbamylinsulini

100 mg insulin are dissolved in 20 ml 0.05M phosphate buffer with pH «8, which contains 3M urea and where, the urea previously at pH 3 during 30 piled to the destruction of any

X · c ^ itTwytbenanaelt was
existing --fliAfrtjfc To the stirred solution, three portions of 10 μl of 20% ethyl isocyanate each are added at 15 minute intervals, the solution being scanned in the range of 25 ° - 30 ° nm to check that no significant tyrosine modification occurs The resulting solution is desalted by passing it through a 40 χ 2 cm column of three-dimensionally crosslinked polysaccharide (Sephadex G-25) using 1 mM M-ethylmorpholine as the eluent 3 ml fractions determined, indicated changed according to Eu iu * »- s8 0 9822/1163

by absorption at 275 im · These fractions are combined and lyophilized to give a white solid which is dissolved in 0.09M tris (hydroxymethyl) aminomethane hydrochloride containing 8M urea at pH 7jO and reduced to a 16 χ 1 , 5 cm column with three-dimensionally crosslinked polysaccharide (Sephadex A-25) is added. The column is first eluted with the same buffer at a flow rate of 10 ml / h and then, after 250 ml of eluate have been collected, a gradient elution is carried out on the column, starting with the same buffer and starting with a 0.36 (iris ( hydroxymethyl) aminomethane hydrochloride buffer, which contains 8M urea, the elution is terminated at pH 7.0, the size of the mixing container being 200 ml. The IT, N ¹ , N '^ - triethylcarbamyl insulin is eluted from the column after 110 ml Eluate "were collected, the eluent then being 0.18M tris (hydroxymethyl) aminomethane hydrochloride. The fractions containing this derivative are combined with one another on three-dimensionally crosslinked polysaccharide (Sephadex G-25) using -1 mM IT-ethylmorpholine as the eluent desalted and lyophilized, 31 ing Ν, Η ¹ , N "-Triäthylcarbamylinsulin are obtained.

Example 4- - Preparation of N, M ', li ^lf -iDricarbamyltetra-O-aeetylinsulin -

60 mg Ν, ϋί ¹ , N ^ -Iricarbamylinsulin in the form of a freeze-dried powder are dissolved in 4 ml 3M 2! Ris (hydroxymethyl) -aminomethane acetate buffer (fEris-acetate buffer) with pH 9.2, and the solution is added in small volumes treated by acetic anhydride in dioxane (1s1; dioxane freed from peroxides by passing through aluminum oxide). The addition is carried out until a complete reaction on the cryosin hydroxyl residues is achieved, as indicated by regular monitoring of the absorption spectrum of the reaction mixture and a total volume of 1> 2 ml of the acetic anhydride / dioxane mixture usually required

according to & 6 inputs,

is. The resulting solution is desalted by being passed through a column with three-dimensionally cross-linked polysaccharide (Sephadex G-25) at 4 ⁰ G using 0.1 #dgem pyridine as eluant, and the eluate is lyophilized to give N, N ', N' ^ Tricarbamyl-tetra-O-acetylinsulin. Is obtained.

If this procedure is modified, the desalting will be used carried out by lowering the pH of the solution to 4 is, the precipitate is centrifuged off, this is washed with distilled water, the residue at pH « dissolved and the resulting solution is lyophilized.

Example 5

The procedure of Example 4 was repeated, except that the reaction was not carried out to completion, but rather, in the intermediate stages of acetylation, as indicated by monitoring the spectra was displayed has ended. This procedure was carried out three times, with 25%, 50% and 75% acetylation was canceled, this corresponds to the mono-, di- and tri-O-acetyl derivatives.

Example 6

The procedure of Examples 4 and 5 was repeated with Ν, Ν *, Ν "-Triäthylcarbamylinsulin and acetic anhydride as reactants, wherein N ₁ N ¹ , ^ '- triethylcarbamyl-ioono-, -di, -tri- and -tetra-O acetylinsulin were obtained.

Example 7 - Preparation of N, N ', N ^M -Tricarbamyl ~ tetra-Q-glutarylin sulini

29 mg of N, N ¹ , N "-tricarbamyl insulin were added to 1.5 ml of 1K Iris (hydroxy-

JmUL / 1 ι

methyl) aminomethane hydrochloride buffer (TriaWuffer) at pH resolved by 9.5. 110 mg of powdered glutaric anhydride were added in portions with vigorous stirring and the spectrum

309822711 ·)

of the mixture recorded in 1 mm cuvettes. At the end of the reaction, the mixture was cross-linked on a column with three-dimensional! Polysacdbaride (Sephadex G-25) "was desalted at M- ⁰ O , the product being eluted with 0.1M pyridine acetate (pH " -6.5). The eluate was lyophilized, an almost quantitative yield of the letraglutaryl derivative being obtained.

In a modification of this procedure, the reaction was carried out in the presence of 8M urea, which the tertiary structure of the molecule Opens. As a result, less glutaric anhydride was consumed to complete the reaction. In this case, there is little or no substitution on serine, which is undesirable.

Example 8

The procedure of Example 7 was repeated, except that the response due to the monitoring of the spectra in the intermediate stages the glutarylation was terminated instead of by Finalize * The procedure. was carried out three times, with 25%, 50% and 75% glutarylation in each case ■ canceled wunfe, this corresponds to the mono-, dl- and 1JrI-SUsubstituted Derivatives.

Example 9

The procedure of Examples y and 8 was ^{repeated with Ν, Ν ', Ιϊ 11} -triethylcarbamylinsulin and glutaric anhydride as reactants, with K, N ¹ , Ν "- triethylcarbamylmono-, -di, -tri- and -tetra-0-glutarylinsulin were obtained.

Example 10 - O-carbamylation of N, N ¹ , N "-tricarbamyl insulin:

· Ν, Ιί ¹ , N "-Tricarbamylinsulin are dissolved in an aqueous 8M urea solution. Potassium cyanate is added in a concentration of 2M. The reaction is carried out at Zimmer-

»0 98 2 2/1163

- .i

temperature is allowed to proceed for one hour at pH = 8.0, 4 hours at pH = 7.0, 2 hours at pH = 6.5 and then 1 hour at pH 6.0, the pH being kept at the desired value during the progress of the experiment is maintained by automatic addition of acetic acid. The reaction mixture is then acidified to pH = 4.5 by adding hydrochloric acid, diluted 5 times with water and left to stand at 4 ^° C. overnight. The precipitate formed is spun off and redissolved in 0.1 tiger aqueous pyridine acetate at pH 6.0. The desalting is effected with a 40 χ 2.5 cm column with three-dimensionally crosslinked polysaccharide (Sephadex G-25) using 0.1% pyridine acetate at pH 6.0 as the eluent, the elution at 4 ⁰ G is performed. Immediately after the elution of the protein from the column is complete, the fractions are combined and the product is isolated in dry form by lyophilization. The extent of carbamylation is on the order of 50% as confirmed by measuring the optical density at 280 nm. The half-life of the decarbaniylation of the product at pH = 7.4 and 37 ° C. in a physiological buffer is 75 minutes with full recovery of the tyrosine absorption of the tricarbamyl insulin.

When changing this way of working, products are obtained that which show both higher and lower levels of carbamylation.

Example 11

The procedure of Example 10 'fetches -Iriätfaylcarbamylinsulin / KaliuiBcyanat as fieakt ions subscriber again ^, where 0-carbamylated H ₁ S ^1, H "with M, H ^1, H -Triäthylcarbamy! Insuline be obtained.

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Example 12 - Preparation of K, liSl ^ll -irica2? Bamyl-te-t; 3? AiO - ethylearbamy! Insulin;

20 mg of NjN ¹ , N "-Sricarbamy! Insulin are dissolved in 2 ml of O, IH phosphate buffer containing 8M urea at pH = 8.0. The stirred solution is treated in portions with 100» 1 parts of ethyl isocyanate, The pH value is automatically maintained at 8.0 by adding iron sodium hydroxide solution. Pie additions of ethyl isocyanate are continued until there is no further decrease in the absorption of the solution at 280 nm, whereby the addition of four portions of 1 is usually required The pH value of the solution

then reduced to 6.0 and the solution allowed to stand until no further change / absorption occurs to hydrolyze the carbamylhistidine derivative. The solution is desalted by sifting it through a 20 χ 1 cm column with three-dimensionally crosslinked polysaccharide (Sephadex G-25) using Imfi N-iithylniQrphoiin as Elutiqnsmittel, whereby the protein fractions are combined and lyophilized, 18 mg Ν, Ν ¹ , N ^ -fricarbamyl-tetra-O-ethyl-carbamyl insulin obtained. '. .-

Example 1B '-'

The procedure of example 12 is carried out with Ν, ΪΓ ¹ _? N "-iDriäthylcarbamylinsulin and Ä'thylisocyanat repeated as Eeaktignteilnehmer, N, NSW" - ^ ^r iäthylcarbamyl-tetra-0-Ä1bhylearbamylinsulin is obtained.

Example 14 ; -

If the preceding examples are modified, other N-substituted carbamyl radicals are used instead of the carbamyl radical or of the N-ethylcarbamyl radical, e.g. N-methyl- and N-phenylcarbamyl radicals, the respective Beagenzieb.

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Are N-methyl and N-phenyl isocyanate. The binding of these remnants as substituents on the amino and / or Hydrpxylresti achieved using the previously described, experiment © 34 #n working methods.

Example 15 '

A typical, pharmaceutical preparation containing insulin derivatives according to the invention is a preparation either in JOrm of a solution or a suspension at a neutral or physiological pH, which is 0.136 w / v. -% üiatriujnacetat, 0.7 w / v. -% sodium chloride and 0.1 w / v% methyl hydroxybenzoate in pyrogen-free water contains. Such a preparation can be administered with a daily dose of 20 to 80 IU standard insulin for adult patients and more than 200 U and even more than 500 U for patients who are already used to it.

: ■ - ■ .)

Claims (1)

Claims 1. insulin derivative, characterized in that at least one of A ^ - (glycine), B ^ - (phenylalanine) and Bpq- (lysine) amino acid units by a carbamyl radical or a substituted carbamyl radical, E ^ RpNGO-, wherein Ry. and Ep is hydrogen, an alkyl group, an aryl group or another group, is blocked. - 2. insulin derivative according to claim 1, characterized in that g e k e η η ζ ei chnet that all three amino residues through one Oarbamyl radical or a U-substituted carbamyl radical are blocked. 3. insulin derivative according to claim 1 or 2, characterized g e k e η η ζ ei chnet that the blocking radicals are the carbamyl or the N-ethylcarbamyl radical. 4. NjW ¹ ,! ^ " 5. N, N ¹ , ^ "- triethylcarbamyl insulin. 6. NA ^ monocarbamyl insulin.
7 NB ^ monocarbamyl insulin. 8. Ν, Ν'-Α, -, Β.-dicarbamyl insulin. ' 9 · Insulin derivative according to claim 1, 2 or 3> characterized g e - <indicates that in addition at least one of the {dyrosine hydroxyl radicals is blocked by a substituent is that in vivo with restoration of the free hydroxyl residue can be separated. '30982271163 10. isulin derivative according to claim 9, characterized in that that the blocking radical is an acyl radical. 11. insulin derivative according to claim 10 »thereby g e k e η η '" ζ ei c h η e t that the acylregt the acetyl or is. 12. N ,! ¹ , !! "- triearbamyliaono-, -di, -tri- or -tetra-O-acetyl- insulin. 13. N, H ¹ , IP-xriätbylcarbaißyli & ono-, -di, -tri- or -tetra-O-acetylinsulin. 14. Ν, ϊί ·, H ^w -TricarbiDiylmono- | -di, -tri- or -tetra-O-glutarylinsulin. 15.H, N · ', N ^w -! I? RiethylcarbamylmQno- »-di, -tri- or -tetra-0-glutary! Insulin. 16. Insulin derivative according to claim 9 »characterized in that the blocking radical is a carbamyl radical or a substituted parba» yl radical, R ^ HgNGO-, wherein B, is. and E _{2 have} the meaning given in claim 1. 17 · Insulin derivative according to claim 16, characterized in that the blocking radical is the carbamyl radical or which is N-Ethylcarbaiiylreet. 18. N, W, N ^w -tricarbamylmono-, -di, -tri- qder -tetra-O-carbanylinsulin. 19. li _l li ^I , N ^M -Triäthylcarbaiaylmono-, -dl-, -tri- or -tetra-O-cerbamy! Insulin. 309122/1163 20. N, H'Vli ^ri -Iricarbamylmono-, -di, -tri- or ^: -tetra-CK ethylcarbamylinsulin. .Ν, ΪΓ, ^ '- Iriathylcarbamylmono-, -di, -tri- or -tetra-0-ethy-1G arb amy linsuün :. 22. Pharmaceutical products suitable for parenteral administration Preparation, characterized in that it is a derivative according to one of the preceding as an active component Contains claims. 23. A method for producing an Ihsulin derivative according to claim 1, characterized in that 'at least' one of the A ^, B _x , and B ^ q amino radicals of insulin is carbamylated or N-substituted carbamylated * 24. The method according to claim 23, characterized in that an alkali metal cyanate is used as the reagent for the Garbamylation and an alkyl isocyanate for the N-substituted Carbamylation is used. 25.Verfahren according to claim 23 or 24, characterized in that at least two amino radicals by one Carbamyl radical or an N-substituted carbamyl radical will. - - " 26. The method according to claim 25 »characterized geke η η ζ ei ch η et that all three amino radicals are substituted by an orbamyl radical or a Ή- substituted carbamyl radical. 27 · Method according to claim 26, characterized in that η η ζ eic h net that a further substitution on one or more hydroxyl radicals is carried out by ^ a substituent which can be split off in vivo. 309822/1163 28. The method according to claim 27, characterized in that the further substitution is carried out by acylation will. 29 · The method according to claim 27 »characterized in that the further substitution by carbamyIation
or N-substituted carbamylation is carried out. 309822/1163