PT98124A - PROCESS FOR THE PREPARATION OF NEW PROLONGED ACID INSULIN ANALOGS AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN THEM - Google Patents

Description

" PROCEDURE FOR THE PREPARATION OF NEW PROLONGED INSULIN ANALOGS AND PHARMACEUTICAL POSITIONS < "EU OS CONTAINS"

Field of the Invention The present invention relates to novel long-acting insulin analogues, a process for the preparation of such insulin analogues and injectable solutions containing the novel insulin analogues.

Background of the Invention

Insulin analogs with a prolonged insylin action have been described previously in European Patents EP 0194 864 A and EP 0255 516.

European Patent Specification EP-A-1 describes human prolonged insulin analogues in which the C-terminal carboxyl group of the B chain is blocked with an amido or ester residue and the amino acid residue at the A- t, Al7, B13 and B21 may be replaced by Gla. A human insulin of the same type described in European patent EP O9M + 86A is described in EP patent specification EP G25 * f516A but further containing a modification at position A21,

Some of the insulin analogs may, however, exhibit too low biological potency or a degree of prolongation that may be too short for the purposes in view. It is an object of the present invention to provide novel insulin analogs with prolonged insulin action which do not exhibit the aforementioned drawbacks.

Summary of the Invention

According to the present invention, it has been surprisingly found that the introduction of a positive charge at the N-terminal end of the B chain gives rise to insulin analogues with a very prolonged insulin action, when compared to human insulin, and still with great potency in vivo.

In its broadest aspect, the present invention is therefore related to novel human insulin analogues in which at least one of the amino acid residues of B 1 to B 6 has been replaced by a residue of a basic amino acid, i.e. a lysine residue or an arginine residue (Lys or Arg).

In order to achieve improved stability of the novel insulin analogues, the asparagine at position A21 (Asn) can be further substituted by another amino acid residue.

An additional positive charge may also be introduced by blocking the C-terminal carboxyl group at the B30 position, preferably by means of an amido or ester group.

If the present insulin analogs are prepared by the method termed transpeptidation (for details see below) it should still be an advantage that the amino group attached to the C-terminal end of the substituent residue Lys or Arg is a proline residue. 3

The invention further relates to a method for the preparation of the novel insulin analogues by means of which a biosynthetic precursor of the insulin analog converts to insulin analogues by enzymatic and chemical conversion and relates to insulin solutions which contain the new insulin analogues. The term " insulin analogs " as used herein means a compound having a molecular structure similar to insulin including dissolution bridges. C® e falkyl and Cys^ e and Cys ^ e and an internal disulfide bond between Cys and Cys and which have insulin activity.

Detailed description of the present invention

The present insulin analogues are represented by the following general formula rs_sn

A (1-6) -Cys-A (8-19) -Cys-Z

I I (s) s

I I s s

(20-29) -Y in which Z represents Asn or another natural amino acid X-1 represents Phe, Lys or Arg X 1 represents H 2, R 2, R 2, R 2, R 2, R 2, R 2, R 2, R 2, R 2, R 2, and Y represents a threonine residue in which the carboxyl group is optionally blocked by an ester or amido group, with the proviso that at least one of Xp X2 > X1, X2, X1 and X2 are Lys or Arg.

Compared with human insulin, carbamate modification is obtained by the substitution of one or more amino acid residues at the positions of B 1 to B 6 by an arginine or lysine residue. In addition, the C-terminal carboxyl group of the -B chain can be blocked with an ester or amido group.

If Z does not represent asparagine, it may represent a neutral amino acid, for example, valine, glutamine, isoleucine, leucine, phenylalanine, guanidine, guanidine, or preferably glycyrrhizin, threonine or alanine, and may be an acidic amino acid such as glutamic acid or aspartic acid, or a basic amino acid, such as, for example, lysine, arginine or histidine. Preferably, Z represents glycine, alanine or serine. Examples of groups which block the C-terminal carboxyl group at the amino acid residue at the B30 (threonine) position are ester groups, such as lower alkoxy, preferably not more than 8 carbon atoms, advantageously less than 5 atoms. carbon. Preferred alkoxy groups are methoxy, ethoxy and tert-butoxy groups. 1

The blocking group may also be an amine group of the general formula -NR 4 wherein R 2, which may be the same or different, each represents a hydrogen atom or an alkyl group, preferably having up to 8 carbon atoms. Preferably, 1 and each represents a hydrogen atom.

Since the compounds of Formula I can be applied clinically in the form of solutions having a prolonged action, a decrease in immunogenicity may occur as compared to commonly used suspensions of porcine or human insulin. The degree of prolongation can be increased and controlled by the addition of zinc ions.

Parameters which can control the degree of prolongation of the insulin effect are the concentration of zinc and the choice of the compound of formula I. Preferred values for the zinc content are from 0 to about 2 mg / ml, from preferably between Θ and 200 æg / ml, preferably between about 20 and 200 æg / ml, in a preparation containing about 2 æmoles of a compound of formula I per ml. When other concentrations of the compound of formula I are used, the zinc content should be adjusted accordingly. The prolonged action of the solutions of the general formula I compounds in the presence of zinc ions is related to the low solubility of these compounds at neutral pH. The pH of the injectable solution of the present invention should preferably be less than the physiological pH, the upper limit being the pH value at which precipitation occurs. No 6

At the physiological pH value, the compounds of the general formula I of the invention have low solubility. There were obtained standard solutions containing about 2-40 nmoles / ml of the compounds of formula I at a pH of about 5%. The upper limit depends on the components of the solution, i.e. isotonic reagent, preservative agent and zinc concentration, and finally the compound of formula I chosen. There is no lower pH limit for the solutions and the chemical stability of the compounds of the general formula I in which Z does not represent an asparagine residue is large even at pH 3. The preferred pH range for the injectable solutions of this invention The pH ranges from about 2.5 to 8.5, preferably from about 5.5 to 8. pH ranges of from about 2.5 to 5> 5 g. more preferably from about 3 to about 5,

A further aspect of this invention is to provide improved flexibility for patients. With two aqueous solutions, one containing a compound of the general formula I and the other containing a zinc salt, the patient may obtain the desired long-acting prgd and the desired profile by mixing the two solutions appropriately. Thus, the patient has, by using two stock solutions, the possibility of choosing an action and scheme for the morning injection and one or more action and another scheme for the evening injection. Preferably, the zinc solution of this invention contains about 2 μg to 20 mg of zinc per ml.

Alternatively, both stock solutions may contain zinc, either in equal or different concentrations and / or both stock solutions may contain a compound of formula I, either with the same compound or with a different compound.

Preferably, the injectable solutions of this invention have a concentration of between about 60 and 6,000 nmoles of a compound of formula I per ml.

Examples of the novel insulin analogs according to this invention are:

ArgB3, SerA2?

ArgB3, ProB2, SerA2, Thr2, NEb, human insulin

ArgB3, GlyA2, Thr2 + NH3, human insulin

ArgB3, ProB2, Gly2, ThrB3B-NH2, human insulin

ArgB 2, Ser 2 ', 1', ThrB 3 '-NH 2 human insulin

ArgB2, ProB3, SerA2? ThrB3B-NH2 human insulin

ArgB2, GlyA2, ThrB3, -NH2 human insulin

ArgB2, ProB3, GlyA21, ThrB30-NH2 human insulin

ArgB2, ArgB3, SerA2, ThrB3, -NH2 human insulin

ArgB2, ArgB3, SerA2, human insulin

ArgB? ProB3, SerA2?, ThrB3? -NH2 human insulin

ArgB ?, ArgB 3, ProB 2, Gly 2 '1', ThrB 3 'human insulin

ArgB3, GlyA2? ThrB3? -NH2 human insulin

ArgB3, SerA2 ", ThrB3 → -NH2 human insulin

ArgB? GlyA2?, ThrB3? -NH2 human insulin

ArgB2, SerA2, *, ThrB3 → -NH2 human insulin

ArgB? ProB2, GlyA2? ThrB3? -NH2 human insulin.

The novel insulin analogs according to the present invention may be prepared by altering the

proinsulin by the substitution of codon or codons at the appropriate site in the natural human proinsulin gene, by codon or codons encoding the desired amino acid residue (s) as the substituent (s) or by synthesis of the complete DNA sequence encoding the desired insulin analogue. The gene encoding the desired insulin analog is then inserted into an appropriate expression vector which when transferred to a suitable host organism, for example, for example. coli, Bacillus or yeast, the desired product. The expressed product is then isolated from the cells or culture broth, depending on whether the expressed product is secreted from the cells or not.

The novel insulin analogs may also be prepared by chemical synthesis by methods analogous to the method described by Márki et al., Hoooe-Sevleis Z. Physiol. Chem., 60, (1979) 1619-1632. They may also be formed from the A and B chains, prepared in vitro separately containing the appropriate amino acid residue substitutions, after which the modified A and B chains are linked to one another by formation of disulfide bridges according to methods (See, for example, Chance et al., In: Rick, DH, Gross, E., (eds.) Peptides: Synthesis - Structure - Function Proceedings of the Seventh American Peptide Svmposium, Illinois, pp. 721- 728)

Insulin analogues may further be prepared by a method analogous to the method described in EP 0 167 6 299, the disclosure of which is hereby incorporated by reference.

the By this method, a human insulin precursor in which Lys3 binds to Gly2 by means of a peptide bond or peptide chain of variable length with disulfide bridges at the correct positions is expressed and secreted by yeast and thereafter converted into human insulin by the so-called transpeptidation reaction. The transpeptidation reaction is described in U.S. Patent No. 4,343,898, the disclosure of which is incorporated herein by reference. In this reaction, the peptide bond or the peptide chain linking Lys® to Gly® is excised and a threonine ester group or a threonine amido group B29 is coupled to the C-terminal end of Lys.

Thus, the novel insulin analogs may be prepared by a method which comprises reacting an insulin biosynthesis precursor of the general formula wq '' r " S-S- |

Al-A (2-6) -Cys-A (8-19) -Cys-Z (II)

(8-18) -Cys-B (20-28) -B29 in which Q represents a peptide chain with q amino acid residues, representing q zero or an integer from 1 to 33; T represents Lys or Arg; r represents zero or the integer 1; and X 1, X 2, X 2, X 2, X 2, X 2, X 2, and Z are as defined above, with a compound of the general formula HY (III) in which Y represents a protected residue of the amino acid threonine having a carboxyl group protecting group; amidoj using trypsin or trypsin-like enzymes as catalysts in a mixture of water and an organic solvent. The ester or amide protecting group may then be cleaved by acidic or basic hydrolysis.

Preferred compounds of formula III are Thr-N-1, Lys (Boc) -N,, Thr (Bufc) -OB e and Thr-OBu &.

The insulin preparations of the present invention are obtained by dissolving a compound of Formula I in an aqueous medium under slightly acidic conditions, for example at a concentration of 240 or 600 nmoles / ml. The aqueous medium is rendered isotonic, for example with sodium chloride or glycerol. In addition, the aqueous medium may contain zinc ions at a concentration of from about 30 æg Zn per mole of a compound of formula I, buffering agents such as, for example, acetate, citrate or histidine and preserving agents, such as, for example, m-cresol, nipagin or phenol. The pH value of the final insulin preparation depends on the number of charges which have been modified in the compound of formula I, the concentration of the zinc ions, the concentration of the compound of formula I and the chosen compound of formula. The pH value is adjusted to a convenient value for administration, such as, for example, from 2.5 to 5.5 g. preventing precipitation. The insulin preparation is sterilized by sterilizing filtration.

The insulin preparations of this invention may be used similarly to the other known insulin preparations.

Terminology-

The substances used for amino acids are those set forth in J. Biol. Chem. (1968), 3558. The amino acids have the L configuration. Except where otherwise specified, the insulin species referred to herein are human. In the test that follows the designation B (1-29) which represents a shortened B-chain of the human insulin of

* D " | -OpQ from Phe to Lys and A (1-21) represents an A chain of human insulin. A or substitutions made on the human insulin molecule according to the practice of this invention are indicated with a prefix referenced to human insulin. As an example, Arg-insulin human represents an analogue of human insulin in which V 1a was replaced by Arg at the 2-position in the B chain. Arg-2, B (1-29) -Ala-Ala-Lys-A (1-21) human insulin represents a precursor for an insulin analogue, cited above, wherein V 1a was replaced by Arg at the 2-position in the B shortened and wherein the Bili-29) 12 'chain and the A (1-21) chain are linked by an Ala-Ala-Lys peptide sequence. Unless otherwise specified, it is to be understood that the B chain (1-29) and the A chain (1-21) are linked by disulfide bridges between Cys 2 and Cys 2, and between Cys 2 and Cys 2, respectively, and that the A chain contains an internal disulfide bridge between CysA2 and CysA1, as in human insulin.

Experimental part

Example 1

ArgB2, ProB3, SerA21, ThrB3C-NH2 human insulin, ArgB2, SerA2, ThrB3B-NH2, human insulin,

ArgBlf, GlyA21, ThrB3 ° -NH2 human insulin, and ArgB2, ProB3, GlyA21, ThrB3, T human insulin, ArgB3, GlyA21, ThrB3 ° -NH2 human insulin

The above-mentioned compounds are synthesized from the following corresponding precursors:

ArgB2, ProB3, SerA21, B (1-29) -Ala-Ala-Lys-A (1-21) human insulin,

(1-21) ArgB1a, SerA21, B (1-29) -Ala-Ala-Lys-A (1-21) human insulin, ArgB1 +, GlyA2B (1-29) -Ala-Ala-Lys- human Insulin, ArgB2, ProB3, Gly1, B (1-29) -Als-Ala-Lys-A (1-21), and

ArgB3, GluA1, B (1-29) -Ala-Ala-Lys-A (1-21) human insulin, ia

by means of tryptic transpeptidation in organic / aqueous solution of Thr-NEb, as described in EP 0198 8 + +, Examples 6 and 6, Table 1 shows the yields, charges relative to human insulin, migration rates relative to insulin in electrospray on BISC PAGE, pH 8.9 and deviations in the amino acid compositions relative to human insulin.

The insulin precursors were produced by a method analogous to the method described in EP 0163529A.

Insulin precursors were collected from the fermentation broths by adsorption of LiChroprep RP-18, in the manner described in Example 7 of EP-A-03529A. Column precursors were eluted with 0.2M potassium chloride, clor hydrochloric acid, ΟΟΙΜ in 33% (v / v) ethanol. The insulin precursors are crystallized from the mixture by successive additions of water (1 volume by volume of the mixture), solid trisodium citrate to give 0.0% molarity and finally zinc acetate to obtain molarity Ο, ΟΟβΜ. The pH was adjusted to 6.8 and the mixture was left overnight at 4 ° C. The crystals were isolated by centrifugation, washed with water and dried under vacuum.

Protected amino acids and protected peptides were prepared for enzyme semi-synthesis according to standard methods or were purchased (usual synthesis) from Nova Biochem or Bachem, Switzerland.

Wed-

I

• Φ «Ο Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η + The compound according to claim 1, wherein R 1 and R 2 are as defined in claim 1, wherein R 1 and R 2 are independently selected from the group consisting of: C0 03 \ rH rH H rH HH rH CM ιο co + 1 + 1 + 1 α Η Η Η Η Η ΟΟΉΗΟ ΟΟΉΗΟ ΟΟΉΗΟ ΟΟΉΗΟ ΟΟΉΗΟ ΟΟΉΗΟ ΟΟΉΗΟ ΟΟΉΗΟ ΟΟΉΗΟ ΟΟΉΗΟ ΟΟΉΗΟ ΟΟΉΗΟ ΟΟΉΗΟ Η Η Η Η Η Η Η Η Η Η Η Η Η Η d 00 rH <D <0 <0 <0 <0 <4 <4 <4 <4 <4 <4 <4 <4 '' '' '' '' 'Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό + UA UA • Η ο> \ \ \ \ \ j O O O O O 0 0 0 0 0---------------------Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η 1 + »C0 C0 H d W Φ Η Η Η Η H H H OJ OJ OJ OJ OJ OJ OJ OJ OJ OJ OJ +

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

O-p d Cv. -d ~ 00 [X CM -d &quot; UA Ό d φ 03 * K i- | H = (4) = 4 &lt; 4 &gt; &lt; / RTI &gt; &lt; tb &gt; CM CM CM Φ CM H H H Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ -P -P -P -P -P (1) (1) (1) (1): wherein R 1 is as defined in formula (I) <4 EH <4 EH <4 EH <4 EH 11 c%

Injectable sterile solutions of the above-mentioned compounds for the determination of the degree of prolonged action were prepared using isotonic agent 1.6% (w / v) glycerol and as a preservative, phenol, 0.26% (w / v) . The concentration of the zinc ion was 8, 80 or 160æg / ml. The pH values of the solutions were adjusted sufficiently far enough away from the isoelectric point of the analogs to maintain the clear solutions after storage at the temperature. The solutions contained 240 nmoles / ml of the test compounds. The concentration of 2x + 0 nmole / ml was checked by HPLC.

Injection solutions containing 2 .mu.mol / ml of the compounds shown in Table 2 were then prepared with a pH of 3 and an established zinc content. Prolongation of the hypoglycemic effect was verified according to the British Pharmacopeia. 19Ô0, A l * + 2 in fed rabbits. Each test solution was administered subcutaneously at a dose of 11.3nmole per rabbit in 12 animals weighing between 3 and 4 kg, and the behavior of hypoglycaemia was followed for 6 hours. For comparison of the fast-acting preparation, human insulin in Actrapid was included in the tests. The test results are shown in Table 2 as a percentage of glucose after 1, 2, 5 and 6 hours.

The results of the determination of the biological power, by the Assay of. Mouse Blood Glucose (MBG) and Free Fat Cell Test (FFC) are shown in Table 3,

Table 2

Compound Zn Glucose at baseline

Replacements in human insulin + ¹H + hr 6h ArgB5, SerA21, ThrB3 ° -NH2 8 59 6½ 96 9 * + ArgB2, SerA2B, ThrB3 ° -NH2 80 85 92 97 95 ArgB2, SerA21, ThrB3 ° -NH2166 76 89 96 9 * + ArgB2, ProB3, Se / 21, ThrB3 ° -NH2 8 58 67 73 72 ArgB2, ProB3, SerA21, ThrB3 ° -NH2 80 73 66 6 * 61 TM Actrapid, human insulin 7 53 53 92 97.

Wed-

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The ri TJ o *

1Z

Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό -d &quot; Cv tx S d UA A &quot; v \ • H or CU ►d Uw or Ixi

CD R Cd Cd * H • H J &gt; r-1 ϋ H P IO Ρ +5 «D CD d CD P Η -H g Ο Φ P O, d, ctí Λ vO or uv ir \ ON ua

yCD cg j-H-4 I rH J-i-I < <1 or Φ Π3 C0 ΙΧΛ ΟΌ ΟΌ ΟΌ ΟΌ ΟΌ ri ri ri 00 00 cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd cd P • H &gt; iH · * ϋ -H p cd P P P Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ

The relative biological potency of the compound of the formula (I) is given by the following formula: ## STR1 ## in which R 1 is hydrogen, H $ CD d CD N N N N N Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ H Φ d W d td cd rd P cd co CU Pu & d w p d 1 • H • H • Η • Η o #x o rH P P H H c cg cg rn P • H W p p q p q p. pq pq w d P Ui fafl w &gt; d bfl d C pq p d d d X d xi • H v- 'ca -η <4 <4 EH <4 H

ArgB, GlyA21, ThrB3 ° -NHp 66 60-72 ° C 38 -seI

Claims (6)

18 A process for the preparation of analogues of human insulin of general formula A (1-6) -Cvs-A (3-19) -Cys-z ' 1 (I): -X2 -X3 -X4 -X5 -X6 -Cys-B (3-18) -J- (20.29) Y wherein Z is Asn or another natural amino acid; represents Phe, Lys or Arg; X 2 is Val, Pro, Lys or Arg; X 3 represents Asn, Lys, Arg or Pro; X4 represents Gin, Lys, Arg or Pro? X5 represents His, Lys, Arg or Pro; X6 represents Lys, Arg, Leu or Pro; Y represents a threonine moiety in which the carboxyl group is optionally blocked by an ester or amido group; with the proviso that at least one of X2, X2, X2, X2, X2 and X2 is Lys or Arg, characterized in that a precursor of the formula Al-A (2-6) -CVS-A (8-19) -CVS-Z1Ss (II) ss X1-X2-X3-X4-X5-X6-cys-3 (8-18) -Cys -3 (20-28) -329 in which Qg represents a chain of peptides with 9 amino acid residues representing zero or an integer of 1 to 33; T represents Lys or Arg; r is zero or the integer 1; and And X are as defined above with a compound of the general formula HY (III) in which Y represents a protected moiety of the amino acid threonine having the protecting group of the carboxyl group ester or starch group using trypsin or other similar enzymes as the catalyst in a mixture of water and an organic solvent and then optionally the protective group is cleaved by acidic or alkaline hydrolysis. 2. A process according to claim 1 for the preparation of human insulin analogues of formula I in which Phe is represented by the use of correspondingly substituted starting compounds. 3. A process according to claim 1 or 2, for the preparation of human insulin analogs of general formula I in which Z is Gly, Ala or Ser, characterized in that correspondingly substituted starting compounds are used. 4. A process according to claim 1, the preparation of human insulin analogs of Formula I wherein X1 is Phe, X2 is Arg or Lys, X3 is Pro, X4 is Gin, X1 is His, X2 is Leu, Z is Gly, Ala or Ser and Y represents Thr-NHâ, "or Thr, characterized in that correspondingly substituted starting compounds are used. 5. A process according to claim 1 for the preparation of human insulin analogs of formula I wherein X1 is Phe, X2 is Val, X3 is Asn, X4 is Gin, X5 is His, Xg is Arg or Lys, Z represents Gly, Ala or Ser and Y represents Thr-NH 2 or Thr, characterized in that the starting materials are suitably substituted. 6. A process for the preparation of pharmaceutical compositions having a long-acting insulin action, characterized in that a therapeutically effective amount of a compound of formula I prepared by the process according to claim 1 is converted into an injectable solution. , in association with auxiliary agents such as buffers, preservatives and agents which allow the solution to be isotonised. Official Agent of Indusrial Property 1