Derived insulin, soluble prolonged pharmaceutical composition, method of prolonged hypoglycemic effect in the treatment of diabetes

 

(57) Abstract:

Described is a new derivative of insulin having the amino acid sequence of SEQ ID NO : 2, where XAA at positions A21 and B3 are independently any amino acid residue which can be encoded by the genetic code except Lys, Arg, Cys; XAA at position B1 is Phe or is deleted; XAA at position B30 is (a) any amino acid residue which can be encoded by the genetic code except Lys, Arg, Cys, and in this case - amino group of LysWhas lipophilic Deputy or (C) is removed, and in this case - amino group of LysWhas lipophilic Deputy; and any of its complexes with Zn2+provided that when XAA at position B30 both are Thr, Ala, XAA at position A21 and B3 are both Asn, XAA at position B1 is Phe, then the derived insulin is a Zn2+complex. Also disclosed soluble prolonged pharmaceutical composition based on it and the way prolonged hypoglycemic effect. The invention can be used in medicine for treatment of diabetes. 3 S. and 17 C.p. f-crystals, 3 tab., 3 Il.

This invention relates to new derivatives of human insulin, coorientations songs containing them, and to the use of such derivatives of insulin in the treatment of diabetes.

Many patients with diabetes treated with multiple daily injections of insulin by the scheme, including one or two daily injections of long-acting insulin, which cover basal needs, supplemented by injection loading dose of fast-acting insulin, which covers the needs associated with food intake.

Composition of prolonged insulin is well known in the art. So, one basic type of prolonged insulin compositions includes injectisome aqueous suspension of crystalline insulin or amorphous insulin. In these compositions commonly used compounds of insulin are Protamine-insulin-zinc-insulin or Protamine-zinc-insulin.

There are certain disadvantages associated with the use of insulin suspensions. So, in order to maintain accurate dosing, insulin particles must be homogeneous suspended by gently shaking before a certain amount of suspension will be selected from a vial or removed from the cartridge. In addition, to avoid the formation of wsdw Elah, than for solutions of insulin.

Although previously believed that Protamine neimenovani, now it turned out that Protamine can be immunogenic to humans, and that their use for medical purposes can lead to the formation of antibodies (Samuel et al., Studies on the immunogenecity of protamines in humans and experimental animals by means of amicro-compliment fixation test, Clin. Exp. Immunol. 33. pp. 252-260 (1978)).

In addition, there is evidence that protaminensulin complex itself immunogene (Kurtz et al., circulating IgG antibody to protamine in patients treated with protamin-insulins. Diabetologia, 25, pp. 322-324 (1983)). Therefore, some patients should avoid prolonged use of the compositions of insulin.

Another type of prolonged insulin compositions are solutions having a pH below physiological pH, at which insulin may be deposited due to the increase in pH when the solution is injected. The disadvantage of these solutions is that the distribution of particle size of the precipitate formed in the tissue when injected, and therefore the timing of medication depends on blood flow to the site of injection and other parameters in a somewhat unpredictable way.

WO 91/12817 (Novo Nordisk A. S.) reveals prolonged, soluble insuracne and bioavailability is reduced.

Human insulin has three primary amino groups: N-terminal A-chain and B-chain and the amino group of LysB29. There are several derivatives of insulin in which one or more of these groups substituted. So, Pat. USA N 3528960 (Eli Lilly) refers to the N-carboxyethyl insulin in which one, two or three primary amino groups of the insulin molecule are carboxyaniline group. In particular, it is not disclosed NB29-substituted insulin.

According to U.S. Pat. UK N 1492997 (Nat. Res. Dev. Corp.) found that insulin with karbonilnym substitution at the NB29has an improved profile hypoglycemic effect.

In lined with the application for Pat. Japan N 1-254699 (Kodama Co., Ltd) discloses insulin, in which the fatty acid is linked to the amino group of PheB1or with the-amino group of LysB29or with both these groups. Purpose for obtaining the derivatives of insulin is to obtain pharmacologically acceptable, stable insulin product.

Insulin, which in position B30 have an amino group having at least five carbon atoms, which optionally can be encoded by a triplet of nucleotides that are described in the posted application for Pat. Japan N 57-067548 (Shionogi). Zayavlyayutsya insulinresistant due to the generation of antibodies to bovine or porcine insulin.

The term "derived insulin" as used here, see compound having a molecular structure similar molecular structure to human insulin, including disulfide bridges between CysA7and CysB7and between CysA20and CysB19and an internal disulfide bridge between CysA6and CysA11and which has the activity of insulin.

There is still the need for prolonged injectively insulin compositions, which would represent the solutions and which contained insulin, which remain in solution after injection and have minimal inflammatory and immunogenic properties.

One of the purposes of the present invention is to develop a derivative of human insulin, with a prolonged profile of action, which are soluble at physiological pH values.

Another objective of the present invention is to develop a pharmaceutical composition comprising a derivative of human insulin according to this invention.

The next objective of the present invention is to develop a method of obtaining derivatives of human insulin.

THE ESSENCE IZOBRETENIYA has lipophilic Deputy, have prolonged profile of action and are soluble at physiological pH values.

Thus, in its broadest aspect, the present invention relates to an insulin derivative having the sequence N 1, where Xaa at position A21 and B3, independently represents any amino acid residue which can be encoded by the genetic code except Lys, Arg and Cys;

Xaa at position B1 is Phe or is deleted;

Xaa at position B30 is

(a) non-coded, lipophilic amino acid having from 10 to 24 carbon atoms, and in this case, the acyl group of the carboxylic acid with up to 5 carbon atoms linked with the-amino group of LysB29,

(b) any amino acid residue which can be encoded by the genetic code except Lys, Arg and Cys, and in this case-amino group of LysB29has lipophilic Deputy or

(c) is deleted, and in this case-amino group of LysB29has lipophilic Deputy; and any Zn2+complexes, provided that when Xaa at position B30 is Thr or Ala, Xaa at positions A21 and B3 are both Asn and Xaa at position B1 is Phe, then the insulin derivative predstavlennomu human insulin, in which the B30 amino acid residue is deleted or is any amino acid residue which can be encoded by the genetic code except Lys, Arg and Cys; A21 and B3 amino acid residues, independently, are any amino acid residues, which can be encoded by the genetic code except Lys, Arg and Cys; PheB1can be removed; -amino group of LysB29has lipophilic Deputy, which includes at least 6 carbon atoms; and 2-4 Zn2+ions can be associated with each hexameron insulin taking into account the fact that when B30 is Thr or Ala and A21 and B3 are both Asn and PheB1not removed, then 2-4 Zn2+ions associated with each hexameron insulin derivative.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the B30 amino acid residue is deleted or is any amino acid residue which can be encoded by the genetic code except Lys, Arg and Cys; A21 and B3 amino acid residues, independently, are any amino acid residues, which can be encoded by the genetic code except Lys, Arg and Cys, with the understanding that if the B30 amino acid residue is Al LysB29has lipophilic Deputy, which includes at least 6 carbon atoms.

In another variant embodiment of the invention, the invention relates to a derivative of human insulin in which the B30 amino acid residue is deleted or is any amino acid residue which can be encoded by the genetic code except Lys, Arg and Cys; A21 and B3 amino acid residues, independently, are any amino acid residues, which can be encoded by the genetic code except Lys, Arg and Cys; PheB1can be removed; -amino group of LysB29has lipophilic Deputy, which includes at least 6 carbon atoms; and 2-4 Zn2+ions can be associated with each hexameron insulin.

In another preferred variant of embodiment, the invention relates to a derivative of human insulin in which the B30 amino acid residue is deleted.

In another preferred variant of embodiment, the invention relates to a derivative of human insulin in which the B30 amino acid residue is Asp.

In another preferred variant of embodiment, the invention relates to a derivative of human insulin,s refers to a derivative of human insulin, in which the B30 amino acid residue Thr.

In another preferred variant of embodiment, the invention relates to a derivative of human insulin in which the B30 amino acid residue is a lipophilic amino acid having at least 10 carbon atoms. In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the B30 amino acid is lipophilic-amino acid having from 10 to 24 carbon atoms.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the B30 amino acid is an unbranched (chain) saturated, aliphatic-amino acid having from 10 to 24 carbon atoms.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the B30 amino acid is D - or L - N-dodecanolide.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the B30 amino acid is mindcanvas acid.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the B30 aminobutane refers to a derivative of insulin, in which the B30 amino acid is aminododecanoic acid.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the B30 amino acid is aminotadalafil acid.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the B30 amino acid is aminotetraline acid.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the B30 amino acid is aminoundecanoic acid.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the B30 amino acid is aminohexanoic acid.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the B30 amino acid is amino acid.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the A21 amino acid residue is Ala.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the A21 amino is raised to the derived insulin, where A21 amino acid residue is Gly.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the A21 amino acid residue is Ser.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which B3 amino acid residue is Asp.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which B3 amino acid residue is Gln.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which B3 amino acid residue is Thr.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the amino group of LysB29has lipophilic Deputy, who is an acyl group corresponding carboxylic acid having at least 6 carbon atoms.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the amino group of LysB29has lipophilic Deputy, who is an acyl group, a branched or prazwell is another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the amino group of LysB29has lipophilic Deputy, who is an acyl group corresponding to a fatty acid having at least 6 carbon atoms.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the amino group of LysB29has lipophilic Deputy, who is an acyl group corresponding to a linear, saturated carboxylic acid having 6-24 carbon atoms.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the amino group of LysB29has lipophilic Deputy, who is an acyl group corresponding to a linear, saturated carboxylic acid having 8-12 carbon atoms.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the amino group of LysB29has lipophilic Deputy, who is an acyl group corresponding to a linear, saturated carboxylic acid having 10 to 16 carbon atoms.

In another preferred variant of embodiment, the invention relates to PR the Wallpaper oligoarticular group, containing up to 10, preferably up to 5, oxyethylene links.

In another preferred variant of embodiment, the invention relates to a derivative of insulin, in which the amino group of LysB29has lipophilic Deputy, which is oligoastrocytoma group containing up to 10, preferably up to 5, oxypropylene links.

In another preferred variant of embodiment, the invention relates to a derivative of human insulin, in which each hexamer insulin binds 2 Zn2+ion.

In another preferred variant of embodiment, the invention relates to a derivative of human insulin, in which each hexamer insulin binds 3 Zn2+ion.

In another preferred variant of embodiment, the invention relates to a derivative of human insulin, in which each hexamer insulin binds 4 Zn2+ion.

In another preferred variant of embodiment, the invention relates to the use of a derivative of human insulin according to the invention for obtaining a medicinal product for the treatment of diabetes.

In another preferred is the case for the need of such treatment, comprising a therapeutically effective amount of a derivative of human insulin according to the invention together with a pharmaceutically acceptable carrier.

In another preferred variant of embodiment, the invention relates to pharmaceutical compositions for the treatment of diabetes patients, in case of need of such treatment, comprising a therapeutically effective amount of a derivative of human insulin according to the invention in mixture with an insulin or an insulin analogue which has a rapid onset of action, together with a pharmaceutically acceptable carrier.

In another preferred variant of embodiment, the invention relates to pharmaceutical compositions comprising a derivative of human insulin according to the invention, which are soluble at physiological pH values.

In another preferred variant of embodiment, the invention relates to pharmaceutical compositions comprising a derivative of human insulin according to the invention, which is soluble at pH values in the range of from about 6.5 to about 8.5.

In another preferred variant of embodiment, the invention relates to prolonged pharmaceutical to the equipment variant embodiment, the invention relates to a pharmaceutical composition which is a solution containing from about 120 nmol/ml to about 1200 nmol/ml, preferably about 600 nmol/ml derived human insulin according to the invention.

In another preferred variant of embodiment, the invention relates to a method of treating diabetes in a patient, in case of need of such treatment, comprising administration to the patient a therapeutically effective amount of a derivative of insulin according to the invention pharmaceutically acceptable carrier.

In another preferred variant of embodiment, the invention relates to a method of treating diabetes in a patient, in case of need of such treatment, comprising administration to the patient a therapeutically effective amount of a derivative of insulin according to this invention, in mixture with an insulin or an insulin analogue which has a rapid onset of action, together with a pharmaceutically acceptable carrier.

Examples of preferred derivatives of human insulin according to the present invention, in which there are no associated Zn2+ions are the following:

NB29-tridecanol des(B30) human and the(B30) human insulin,

NB29-dodecanol des(B30) human insulin,

NB29-tridecanol GlyA21des(B30) human insulin,

NB29the deletion of GlyA21des(B30) human insulin,

NB29-decanoyl GlyA21des(B30) human insulin,

NB29-dodecanol GlyA21des(B30) human insulin,

NB29-tridecanol GlyA21GlnB3des(B30) human insulin,

NB29the deletion of GlyA21GlnB3des(B30) human insulin,

NB29-decanol GlyA21GlnB3des(B30) human insulin,

NB29-dodecanol GlyA21GlnB3des(B30) human insulin,

NB29-tridecanol AlaA21des(B30) human insulin,

NB29the deletion of AlaA21des(B30) human insulin,

NB29-decanoyl AlaA21des(B30) human insulin,

-dodecanol AlaA21des(B30) human insulin,

NB29-tridecanol AlaA21GlnB3des(B30) human insulin,

NB29the deletion of AlaA21GlnB3des(B30) human insulin,

NB29-decanoyl AlaA21GlnVinculin,

NB29-tridecanol GlnB3des(B30) human insulin,

NB29the deletion of GlnB3des(B30) human insulin,

NB29-decanoyl GlnB3des(B30) human insulin,

NB29-dodecanoyl GlnB3des(B30) human insulin,

NB29-tridecanol GlyA21human insulin,

NB29the deletion of GlyA21human insulin,

NB29-decanoyl GlyA21human insulin,

NB29-dodecanoyl GlyA21human insulin,

NB29-tridecanol GlyA21GlnB3human insulin,

NB29the deletion of GlyA21GlnB3human insulin,

NB29-decanoyl GlyA21GlnB3human insulin,

NB29-dodecanoyl GlyA21GlnB3human insulin,

NB29-tridecanol AlaA21human insulin,

NB29the deletion of AlaA21human insulin,

NB29-decanoyl AlaA21human insulin,

NB29-dodecanoyl AlaA21human insulin,

NB29-tridecanol AlaA21Ulin,

NB29-decanoyl AlaA21GlnB3human insulin,

NB29-dodecanoyl AlaA21GlnB3human insulin,

NB29-tridecanol GlnB3human insulin,

NB29the deletion of GlnB3human insulin,

NB29-decanoyl GlnB3human insulin,

NB29-dodecanoyl GlnB3human insulin,

NB29-tridecanol GluB30human insulin,

NB29the deletion of GluB30human insulin,

NB29-decanoyl GluB30human insulin,

NB29-dodecanoyl GluB30human insulin,

NB29-tridecanol GlyA21GluB30human insulin,

NB29the deletion of GlyA21GluB30human insulin,

NB29-decanoyl GlyA21GluB30human insulin,

NB29-dodecanoyl GlyA21GluB30human insulin,

NB29-tridecanol GlyA21GlnB3GluB30human insulin,

NB29the deletion of GlyA21GlnB3GluB30human insulin,

N is nail GlyA21GlnB3GluB30human insulin,

NB29-tridecanol AlaA21GluB30human insulin,

NB29the deletion of AlaA21GluB30human insulin,

NB29-decanoyl AlaA21GluB30human insulin,

NB29-dodecanoyl AlaA21GluB30human insulin,

NB29-tridecanol AlaA21GlnB3GluB30human insulin,

NB29the deletion of AlaA21GlnB3GluB30human insulin,

NB29-decanoyl AlaA21GlnB3GluB30human insulin,

NB29-dodecanoyl AlaA21GlnB3GluB30human insulin,

NB29-tridecanol GlnB3GluB30human insulin,

NB29the deletion of GlnB3GluB30human insulin,

NB29-decanoyl GlnB3GluB30human insulin,

NB29-dodecanoyl GlnB3GluB30human insulin.

Examples of preferred derivatives of human insulin according to this invention, in which two Zn2+ion associated with hexameron the Insa is

( NB29the deletion of des(B30) human insulin)6, 2Zn2+,

( NB29-decanoyl des(B30) human insulin)6, 2Zn2+,

( NB29-dodecanoyl des(B30) human insulin)6, 2Zn2+,

( -tridecanol GlyA21des(B30) human insulin)6, 2Zn2+,

( NB29the deletion of GlyA21des(B30) human insulin)6, 2Zn2+,

( NB29-decanoyl GlyA21des(B30) human insulin)6, 2Zn2+,

( NB29-dodecanoyl GlyA21des(B30) human insulin)6, 2Zn2+,

( NB29-tridecanol GlyA21GlnB3des(B30) human insulin)6, 2Zn2+,

( NB29the deletion of GlyA21GlnB3des(B30) human insulin)6, 2Zn2+,

( NB29-decanoyl GlyA21GlnB3des(B30) human insulin)6, 2Zn2+,

( NB29-dodecanoyl GlyA21GlnB3des(B30) human insulin)6, 2Zn2+,

( NB29-tridecanol AlaA21des(B30) human insulin)6, 2Zn2+,

( NB29the deletion of AlaA21des(B30) human PI2+,

( NB29-dodecanoyl AlaA21des(B30) human insulin)6, 2Zn2+,

( NB29-tridecanol AlaA21GlnB3des(B30) human insulin)6, 2Zn2+,

( NB29the deletion of AlaA21GlnB3des(B30) human insulin)6, 2Zn2+,

( NB29-decanoyl AlaA21GlnB3des(B30) human insulin)6, 2Zn2+,

( NB29-dodecanoyl AlaA21GlnB3des(B30) human insulin)6, 2Zn2+,

( NB29-tridecanol GlnB3des(B30) human insulin)6, 2Zn2+,

( NB29the deletion of GlnB3des(B30) human insulin)6, 2Zn2+,

( NB29-decanoyl GlnB3des(B30) human insulin)6, 2Zn2+,

( NB29-dodecanoyl GlnB3des(B30) human insulin)6, 2Zn2+,

( NB29-tridecanol human insulin)6, 2Zn2+,

( NB29the deletion of the human insulin)6, 2Zn2+,

( NB29-decanoyl human insulin)6, 2Zn2+,

( NB29-dodecanoyl human insulin)6, 2Zn
B29the deletion of GlyA21human insulin)6, 2Zn2+,

( NB29-decanoyl GlyA21human insulin)6, 2Zn2+,

( NB29-dodecanoyl GlyA21human insulin)6, 2Zn2+,

( NB29-tridecanol GlyA21GlnB3human insulin)6, 2Zn2+,

( NB29the deletion of GlyA21GlnB3human insulin)6, 2Zn2+,

( NB29-decanoyl GlyA21GlnB3human insulin)6, 2Zn2+,

( NB29-dodecanoyl GlyA21GlnB3human insulin)6, 2Zn2+,

( NB29-tridecanol AlaA21human insulin)6, 2Zn2+,

( NB29the deletion of AlaA21human insulin)6, 2Zn2+,

( NB29-decanoyl AlaA21human insulin)6, 2Zn2+,

( NB29-dodecanoyl AlaA21human insulin)6, 2Zn2+,

( NB29-tridecanol AlaA21GlnB3human insulin)6, 2Zn2+,

( NB29the deletion of AlaA21GlnB3human insulin)6, 2Zn2+,
B29-dodecanoyl AlaA21GlnB3human insulin)6, 2Zn2+,

( NB29-tridecanol GlnB3human insulin)6, 2Zn2+,

( NB29the deletion of GlnB3human insulin)6, 2Zn2+,

( NB29-decanoyl GlnB3human insulin)6, 2Zn2+,

( NB29-dodecanoyl GlnB3human insulin)6, 2Zn2+,

( NB29-tridecanol GluB30human insulin)6, 2Zn2+,

( NB29the deletion of GluB30human insulin)6, 2Zn2+,

( NB29-decanoyl GluB30human insulin)6, 2Zn2+,

( NB29-dodecanoyl GluB30human insulin)6, 2Zn2+,

( NB29-tridecanol GlyA21GluB30human insulin)6, 2Zn2+,

( NB29the deletion of GlyA21GluB30human insulin)6, 2Zn2+,

( NB29-decanoyl GlyA21GluB30human insulin)6, 2Zn2+,

( NB29-dodecanoyl GlyA21GluB30human insulin)6, 2Zn2+,

( N>29the deletion of GlyA21GlnB3GluB30human insulin)6, 2Zn2+,

( NB29-decanoyl GlyA21GlnB3GluB30human insulin)6, 2Zn2+,

( NB29-dodecanoyl GlyA21GlnB3GluB30human insulin)6, 2Zn2+,

( NB29-tridecanol AlaA21GluB30human insulin)6, 2Zn2+,

( NB29the deletion of AlaA21GluB30human insulin)6, 2Zn2+,

( NB29-decanoyl AlaA21GluB30human insulin)6, 2Zn2+,

( -dodecanoyl AlaA21GluB30human insulin)6, 2Zn2+,

( NB29-tridecanol AlaA21GlnB3GluB30human insulin)6, 2Zn2+,

( NB29the deletion of AlaA21GlnB3GluB30human insulin)6, 2Zn2+,

( NB29-decanoyl AlaA21GlnB3GluB30human insulin)6, 2Zn2+,

( NB29-dodecanoyl AlaA21GlnB3GluB30human insulin)6, 2Zn2+,

( NB29-tridecanol GlnB3GluB30human is)6, 2Zn2+,

( NB29-decanoyl GlnB3GluB30human insulin)6, 2Zn2+,

( NB29-dodecanoyl GlnB3GluB30human insulin)6, 2Zn2+.

Examples of preferred derivatives of human insulin according to this invention, in which three Zn2+ion associated with hexameron insulin, are the following:

( NB29-tridecanol des(B30) human insulin)6, 3Zn2+,

( NB29the deletion of des(B30) human insulin)6, 3Zn2+,

( NB29-decanoyl des(B30) human insulin)6, 3Zn2+,

( NB29-dodecanoyl des(B30) human insulin)6, 3Zn2+,

( NB29-tridecanol GlyA21des(B30) human insulin)6, 3Zn2+,

( NB29the deletion of GlyA21des(B30) human insulin)6, 3Zn2+,

( NB29-decanoyl GlyA21des(B30) human insulin)6, 3Zn2+,

( NB29-dodecanoyl GlyA21des(B30) human insulin)6, 3Zn2+,

( NB29-tridecanol GlyA21GlnB3des(B30) human insulin)6, 3Zn2+,

( NB29-decanoyl GlyA21GlnB3des(B30) human insulin)6, 3Zn2+,

( NB29-dodecanoyl GlyA21GlnB3des(B30) human insulin)6, 3Zn2+,

( NB29-tridecanol AlaA21des(B30) human insulin)6, 3Zn2+,

( NB29the deletion of AlaA21des(B30) human insulin)6, 3Zn2+,

( NB29-decanoyl AlaA21des(B30) human insulin)6, 3Zn2+,

( NB29-dodecanoyl AlaA21des(B30) human insulin)6, 3Zn2+,

( NB29-tridecanol AlaA21GlnB3des(B30) human insulin)6, 3Zn2+,

( NB29the deletion of AlaA21GlnB3des(B30) human insulin)6, 3Zn2+,

( NB29-decanoyl AlaA21GlnB3des(B30) human insulin)6, 3Zn2+,

( NB29-dodecanoyl AlaA21GlnB3des(B30) human insulin)6, 3Zn2+,

( NB29-tridecanol GlnB3des(B30) human insulin)6, 3Zn2+,

( NB29the deletion of GlnB3des(B30) human insulin)>/BR>( NB29-dodecanoyl GlnB3des(B30) human insulin)6, 3Zn2+,

( NB29-tridecanol human insulin)6, 3Zn2+,

( NB29the deletion of the human insulin)6, 3Zn2+,

( NB29-decanoyl human insulin)6, 3Zn2+,

( NB29-dodecanoyl human insulin)6, 3Zn2+,

( NB29-tridecanol GlyA21human insulin)6, 3Zn2+,

( NB29the deletion of GlyA21human insulin)6, 3Zn2+,

( NB29-decanoyl GlyA21human insulin)6, 3Zn2+,

( NB29-dodecanoyl GlyA21human insulin)6, 3Zn2+,

( NB29-tridecanol GlyA21GlnB3human insulin)6, 3Zn2+,

( NB29the deletion of GlyA21GlnB3human insulin)6, 3Zn2+,

( NB29-decanoyl GlyA21GlnB3human insulin)6, 3Zn2+,

( NB29-dodecanoyl GlyA21GlnB3human insulin)6, 3Zn2+,

( NB29-tridecanol AlaA21 insulin)6, 3Zn2+,

( NB29-decanoyl AlaA21human insulin)6, 3Zn2+,

( NB29-dodecanoyl AlaA21human insulin)6, 3Zn2+,

( NB29-tridecanol AlaA21GlnB3human insulin)6, 3Zn2+,

( NB29the deletion of AlaA21GlnB3human insulin)6, 3Zn2+,

( NB29-decanoyl AlaA21GlnB3human insulin)6, 3Zn2+,

( NB29-dodecanoyl AlaA21GlnB3human insulin)6, 3Zn2+,

( NB29-tridecanol GlnB3human insulin)6, 3Zn2+,

( NB29the deletion of GlnB3human insulin)6, 3Zn2+,

( NB29-decanoyl GlnB3human insulin)6, 3Zn2+,

( NB29-dodecanoyl GlnB3human insulin)6, 3Zn2+,

( NB29-tridecanol GluB30human insulin)6, 3Zn2+,

( NB29the deletion of GluB30human insulin)6, 3Zn2+,

( NB29-decanoyl GluB30human insulin)6, 3ZnB29-tridecanol GlyA21GluB30human insulin)6, 3Zn2+,

( NB29the deletion of GlyA21GluB30human insulin)6, 3Zn2+,

( NB29-decanoyl GlyA21GluB30human insulin)6, 3Zn2+,

( NB29-dodecanoyl GlyA21GluB30human insulin)6, 3Zn2+,

( NB29-tridecanol GlyA21GlnB3GluB30human insulin)6, 3Zn2+,

( NB29the deletion of GlyA21GlnB3GluB30human insulin)6, 3Zn2+,

( NB29-decanoyl GlyA21GlnB3GluB30human insulin)6, 3Zn2+,

( -dodecanoyl GlyA21GlnB3GluB30human insulin)6, 3Zn2+,

( NB29-tridecanol AlaA21GluB30human insulin)6, 3Zn2+,

( NB29the deletion of AlaA21GluB30human insulin)6, 3Zn2+,

( NB29-decanoyl AlaA21GluB30human insulin)6, 3Zn2+,

( NB29-dodecanoyl AlaA21GluB30human insulin)6, 3Zn2+
,

( NB29the deletion of AlaA21GlnB3GluB30human insulin)6, 3Zn2+,

( NB29-decanoyl AlaA21GlnB3GluB30human insulin)6, 3Zn2+,

( NB29-dodecanoyl AlaA21GlnB3GluB30human insulin)6, 3Zn2+,

( NB29-tridecanol GlnB3GluB30human insulin)6, 3Zn2+,

( NB29the deletion of GlnB3GluB30human insulin)6, 3Zn2+,

( NB29-decanoyl GlnB3GluB30human insulin)6, 3Zn2+,

( NB29-dodecanoyl GlnB3GluB30human insulin)6, 3Zn2+.

Examples of preferred derivatives of human insulin according to this invention, in which four Zn2+ion associated with examples of the preferred derivatives of human insulin according to this invention, in which four Zn2+ion associated with hexameron insulin, are the following:

( NB29-tridecanol des(B30) human insulin)6, 4Zn2+,

( NB29the deletion of des(B30) human+
,

( NB29-dodecanoyl des(B30) human insulin)6, 4Zn2+,

( NB29-tridecanol GlyA21des(B30) human insulin)6, 4Zn2+,

( NB29the deletion of GlyA21des(B30) human insulin)6, 4Zn2+,

( NB29-decanoyl GlyA21des(B30) human insulin)6, 4Zn2+,

( NB29-dodecanoyl GlyA21des(B30) human insulin)6, 4Zn2+,

( NB29-tridecanol GlyA21GlnB3des(B30) human insulin)6, 4Zn2+,

( NB29the deletion of GlyA21GlnB3des(B30) human insulin)6, 4Zn2+,

( NB29-decanoyl GlyA21GlnB3des(B30) human insulin)6, 4Zn2+,

( NB29-dodecanoyl GlyA21GlnB3des(B30) human insulin)6, 4Zn2+,

( NB29-tridecanol AlaA21des(B30) human insulin)6, 4Zn2+,

( NB29the deletion of AlaA21des(B30) human insulin)6, 4Zn2+,

( NB29-decanoyl AlaA21des(B30) human insulin)6, 4Zn2+,

( NB29AlaA21GlnB3des(B30) human insulin)6, 4Zn2+,

( NB29the deletion of AlaA21GlnB3des(B30) human insulin)6, 4Zn2+,

( NB29-decanoyl AlaA21GlnB3des(B30) human insulin)6, 4Zn2+,

( NB29-dodecanoyl AlaA21GlnB3des(B30) human insulin)6, 4Zn2+,

( NB29-tridecanol GlnB3des(B30) human insulin)6, 4Zn2+,

( NB29the deletion of GlnB3des(B30) human insulin)6, 4Zn2+,

( NB29-decanoyl GlnB3des(B30) human insulin)6, 4Zn2+,

( NB29-dodecanoyl GlnB3des(B30) human insulin)6, 4Zn2+,

( NB29-tridecanol human insulin)6, 4Zn2+,

( NB29the deletion of the human insulin)6, 4Zn2+,

( NB29-decanoyl human insulin)6, 4Zn2+,

( NB29-dodecanoyl human insulin)6, 4Zn2+,

( NB29-tridecanol GlyA21human insulin)6, 4Zn2+,

( NB29the deletion of Glythe Lin)6, 4Zn2+,

( NB29-dodecanoyl GlyA21human insulin)6, 4Zn2+,

( NB29-tridecanol GlyA21GlnB3human insulin)6, 4Zn2+,

( NB29the deletion of GlyA21GlnB3human insulin)6, 4Zn2+,

( NB29-decanoyl GlyA21GlnB3human insulin)6, 4Zn2+,

( NB29-dodecanoyl GlyA21GlnB3human insulin)6, 4Zn2+,

( NB29-tridecanol AlaA21human insulin)6, 4Zn2+,

( NB29the deletion of AlaA21human insulin)6, 4Zn2+,

( NB29-decanoyl AlaA21human insulin)6, 4Zn2+,

( NB29-dodecanoyl AlaA21human insulin)6, 4Zn2+,

( NB29-tridecanol AlaA21GlnB3human insulin)6, 4Zn2+,

( NB29the deletion of AlaA21GlnB3human insulin)6, 4Zn2+,

( NB29-decanoyl AlaA21GlnB3human insulin)6, 4Zn2+,

( NB29-dodecanoyl AlaA21Glnnsulin)6, 4Zn2+,

( NB29the deletion of GlnB3human insulin)6, 4Zn2+,

( NB29-decanoyl GlnB3human insulin)6, 4Zn2+,

( NB29-dodecanoyl GlnB3human insulin)6, 4Zn2+,

( NB29-tridecanol GluB30human insulin)6, 4Zn2+,

( NB29the deletion of GluB30human insulin)6, 4Zn2+,

( NB29-decanoyl GluB30human insulin)6, 4Zn2+,

( NB29-dodecanoyl GluB30human insulin)6, 4Zn2+,

( NB29-tridecanol GlyA21GluB30human insulin)6, 4Zn2+,

( NB29the deletion of GlyA21GluB30human insulin)6, 4Zn2+,

( -decanoyl GlyA21GluB30human insulin)6, 4Zn2+,

( NB29-dodecanoyl GlyA21GluB30human insulin)6, 4Zn2+,

( NB29-tridecanol GlyA21GlnB3GluB30human insulin)6, 4Zn2+,

( NB29the deletion of GlyA21GlnB3GluB30human insulin)6, 4Zn2+,

( NB29-dodecanoyl GlyA21GlnB3GluB30human insulin)6, 4Zn2+,

( NB29-tridecanol AlaA21GluB30human insulin)6, 4Zn2+,

( NB29the deletion of AlaA21GluB30human insulin)6, 4Zn2+,

( NB29-decanoyl AlaA21GluB30human insulin)6, 4Zn2+,

( NB29-dodecanoyl AlaA21GluB30human insulin)6, 4Zn2+,

( NB29-tridecanol AlaA21GlnB3GluB30human insulin)6, 4Zn2+,

( NB29the deletion of AlaA21GlnB3GluB30human insulin)6, 4Zn2+,

( NB29-decanoyl AlaA21GlnB3GluB30human insulin)6, 4Zn2+,

( NB29-dodecanoyl AlaA21GlnB3GluB30human insulin)6, 4Zn2+,

( NB29-tridecanol GlnB3GluB30human insulin)6, 4Zn2+,

( NB29the deletion of GlnB3GluB30human insulin)6, 4Zn2+,

( NB29-decanoyl Gln30 human insulin)6, 4Zn2+.

Brief description of drawings

The present invention is illustrated in more detail by the following drawings.

Fig. 1 illustrates the construction of plasmids pEA5.3.2;

Fig. 2 illustrates a plasmid pEA108; and

Fig. 3 illustrates the plasmid reas.

Terminology

Three letter codes and one letter codes for amino acid residues, used here, are such as are installed in. Viol. Chem. 243, p. 3558 (1968).

In DNA sequences, A - adenine, C - cytosine, G - guanine T - thymine.

Use the following acronyms:

DMSO (DMSO) dimethyl sulfoxide, DMF DMF for dimethylformamide, SIDE (Boc) for tert-butoxycarbonyl, OF GHUR (RP-HPLC) liquid chromatography high-resolution reversed-phase, X-OSu is a N-hydroxysuccinimide ether, X - acyl group, and TFA (TFK) for triperoxonane acid.

Getting lipophilic derivatives of insulin

The proposed derivatives of insulin can be obtained as described below:

1. Insulin derivatives with amino acid residue in position B30, which can be encoded by the genetic code, such as threonine (human insulin) or laativat Boc-reagent (for example, di-tert-BUTYLCARBAMATE) with the formation of (A1,B1)-divas human insulin, i.e., human insulin, in which the N-terminal ends of both circuits are protected Boc-group. After optional purification, for example by using GHUR, enter the acyl group to the amino group of LysB29exposing the product to interact with the N-hydroxysuccinimide ether of the formula X-OSu, where X is the acyl group, which is injected. At the final stage, to remove the Boc group using TFA and the product, NB29-X human insulin, emit.

1.2 Based on single-chain precursor of insulin.

Single-chain precursor of insulin, extended in position B1 extension (Ext), which is associated with B1 by arginine residue and in which the bridge from B30 to A1 is an arginine residue, i.e., the compound of General formula Ext-Arq-B(1-30)-Arg-A(1-21), can be used as the starting material. When the acylation of this original substance N-hydroxysuccinimide ether of General formula X-OSu, where X is the acyl group introduced acyl group, X is an amino group of LysB29in the N-terminal amino group of the precursor. When processing this acylated precursor of the formula (NB29-X), X-Ext-Arg-B(1-30)-ArgF), DMSO (DMSO) or a lower alcohol, receive intermediate of formula (NB29-X), ArgB31the insulin. Treatment of this intermediate carboxypeptidase B gives the desired product, (NB29-X) insulin.

2. Derivatives of insulin without amino acid residue in position B30, i.e., des(B30) insulin.

2.1 on the Basis of human insulin or porcine insulin.

When processing carboxypeptidase A in ammonium buffer human insulin and pork insulin both give des(B30) insulin. After optional purification, des(B30) insulin treated with Boc-reagent (for example, di-tert-BUTYLCARBAMATE) with the formation of (A1, B1)-divas des(B30) insulin, i.e., des(B30) insulin, in which the N-terminal ends of both circuits are protected Boc-group. After optional purification, for example by using GHUR, enter the acyl group to the amino group of LysB29exposing the product to interact with the N-hydroxysuccinimide ether of the formula X-OSu, where X is the acyl group, which is injected. At the final stage, to remove the Boc group using TFA and the product (NB29-X) des(B30) insulin, emit.

2.2 on the Basis of single-stranded precursor of human insulin.

Single-stranded precursor human is of STATCOM and which has a bridge from B30 to A1, can be useful source material. Preferably, the bridge is a peptide of the formula Yn-Arg, where Y is encoded by amino acids, except lysine and arginine, and n is zero or an integer between 1 and 35. When n>1, Y-and can define different amino acids. Preferred examples of the bridge from B30 to A1 are: AlaAlaArg, SerArg, SerAspAspAlaArg and Arg (European Patent N 163529). Processing of this precursor of the General formula Ext-Arg-B(1-30)-Yn-Arg-A(1-21) lysyl-endopeptidase, for example, Achromobacter lyticus protease, gives Ext-Arg-B(1-29)-Thr-Yn-Arg-A(1-21) des(B30) insulin. The acylation of the intermediate N-hydroxysuccinimide ether of General formula X-OSu, where X is acyl group, introduces the acyl group X-amino group of LysB29in the N-terminal amino group of the A-chain and B-chain with obtaining (NB29-X) X-Ext-Arg-B(1-29)-X-Thr-Yn-Arg-A(1-21) des(B30) insulin. Treatment of this intermediate with trypsin in a mixture of water and a suitable organic solvent, for example DMF (DMF), DMSO (DMSO) or a lower alcohol, gives the desired derivative, (NB29-X) des (B30) human insulin.

Data for NB29modified insulin.

Some experimental data for NB29modified insulin is tn.
measure on Lichrosorb OF (5 µm, 250 x 4 mm) GHUR column with the isocratic elution at 40oC, using a mixture of (A) 0.1 M sodium phosphate buffer, pH 7.3, containing 10% acetonitrile, and B) 50% acetonitrile in water as eluents. The elution is monitored by subsequent UV absorption of the eluate at 214 nm. Dead time, t0determine by injection of 0.1 mm sodium nitrate. Retention time for human insulin, tINHand bring it at least up to 2t0varying the relation between the solutions A and B. k'Rel= (tp- t0)/(tINH- t0).

The degree of prolongation hypoglycemic effect (reduction in the level of glucose in the blood) study on rabbits. Each derived insulin experience with subcutaneous injections 12 nmol each of the six rabbits in the test deceleration times a day. Sampling of blood glucose is carried out before injection and 1, 2, 4 and 6 hours after injection. The values of glucose expressed as a percentage of the original values. Index deceleration, which is calculated from the values of glucose in the blood, is a graded Index deceleration (renewal), see p. 211 Markussen et al., Protein Engineering 1 (1987) Twestival insulin Actrapid (ActrapidR) (Novo Nordisk A/S, 2880 Bagsvaerd, Denmark).

Derivatives of insulin, are presented in table 1, is introduced into the solutions containing 3Zn2+on hexamer insulin, with the exception of derivatives, specifically noted as containing no Zn.

For very prolonged analogues rabbit model is inadequate because the reduction of glucose level in the blood from the start too little in order to set the index of the slow (prolongation). The prolongation of such analogues are better characterized by the disappearance of the dose in pigs. T50%represents the time when 50% of Tyr A14 (125I) analogue will disappear from the site of injection, as measured using an external counter (Ribel, U. et al. The Pig as a Model for Subcutaneous Absorption in Man. In: M. Serrano-Rios and P. J. Lefebre (Eds): Diabetes 1985; Proceedings of the 12th Congress of the International Diabetes Federation, Madrid, Spain, 1985 (Excerpta Medica, Amsterdam, (1986) 891-96).

Table 2 presents T50%the values of a number of very prolonged insulin analogues. Analogues were introduced into a solution containing 3Zn2+on hexamer insulin.

Solubility

The solubility of all NB29modified insulins, are presented in table 1, which contain 3Zn2+ion on hexamer insulin exceeds 600 CME is e preservative and 1.6% glycerol to achieve isotonicity. 600 nmol/ml is the concentration of human insulin installed 100 IU (M. E.)/ml of the compositions commonly used in the clinical setting.

-B29 amino group can be a component of the amide bond, sulfonamidnuyu communication, carbamide, thiocarbamide or carbamate. Lipophilic Deputy, who is-B29 amino group may be an alkyl group.

Pharmaceutical compositions containing a derivative of human insulin according to the present invention, can be administered to the patient parenterally, for the need of such treatment. Parenteral administration can be carried out by subcutaneous, intramuscular or intravenous injection with a syringe, optionally with a syringe-like handle. Alternatively, parenteral administration can be carried out using an infusion pump. An additional choice is a composition, which may be a powder or liquid for introduction of derivative of human insulin in the form of a nasal spray.

Injectable compositions human insulin of this invention can be obtained by using conventional techniques of the pharmaceutical industry which involves dissolving and shmesani is, proizvodnje human insulin was dissolved in the amount of water which is somewhat less than the final volume of the composition that you want to receive. Add isotonic agent, a preservative and a buffer, as needed, and bring the pH of a solution - if necessary - using acid, for example hydrochloric acid, or base, for example aqueous sodium hydroxide, if necessary. Finally, the volume of the solution was adjusted with water, obtaining the desired concentration of the ingredients.

Examples of the isotonic agent include sodium chloride, mannitol and glycerin.

Examples of preservatives are phenol, m-cresol, methyl p-hydroxybenzoate and benzyl alcohol.

Examples of suitable buffers include sodium acetate and sodium phosphate.

Composition for nasal introduction derived insulin according to the invention can, for example, be obtained as described in Heb. Pat. N 272097 (Novo Nordisk A/S).

Compositions of insulin of the present invention can be used in the treatment of diabetes. The optimal dose level for any patient will depend on a number of factors, including the effectiveness of a specific derivative of human insulin, age, body weight, Fizicheskaya diabetes. It is recommended that the daily dose derived human insulin of this invention for each individual patient was determined by experts in the field of technology in the same way as it is done for known insulin compositions.

Where appropriate, derivative of human insulin of this invention can be used in a mixture with other types of insulin, such as human insulin or porcine insulin or insulin analogs with a more rapid onset of action. Examples of such insulin analogues are described, for example, in applications to the European patent with publication N EP 214826 (Novo Nordisk A/S), EP 375437 (Novo Nordisk A/S) and EP 383472 (Eli Lilly & Co.

The invention is illustrated in more detail by the following examples, which, however, should not be construed as limiting the scope of its protection. The characteristics disclosed in the above description and the following examples, both separately and in any combination, can serve as a material for realizing the invention in its various forms.

Examples

Plasmids and DNA substance

All expression plasmids are cPOT type. Such plasmids are described in application EP patent N 171142 and otlichie. A plasmid containing the POT gene available from the deposited E. Coli strain (ATCC 39685). In addition, the plasmid containing the S. cerevisiae triose phosphate isomerases promoter and terminator (PTP1and TTP1). They are identical pMT742 (Egel. Mitani, M., et al., Gene 73 (1988) 113 - 120) (see Fig. 1), with the exception of the region defined ECoRI-XbaI restriction sites surrounding the coding region of the signal/leader/product.

Synthetic DNA fragments synthesized on an automatic DNA synthesizer (Applied Biosystems model 380A), using phosphoramidite chemistry and commercially available reagents (Beaucage, S. L. and Caruthers, M. H., Tetrahedron Letters 22 (1981) 1859-1869).

All other used methods and materials well known in the art (see for example, Sambrook, J. , Fritsch, E. F. and Maniatis, J., Molecular Cloning: A Laborital Manual, Cold Spring Harbor Laboratory Press, New York, 1989).

Analytical methods

The molecular weight of the obtained insulin obtained with MS (mass spectroscopy), or by using the method PDMS (PDMS) (mass spectroscopy using plasma desorption), using Bio-Ion 20 installation (Bio-Ion Nordic AB, Uppsala, Sweden) or by using ESMS (electrospray mass spectrometry), using an API III Biomolecular Mass Analyzer (Perkin-Elmer Science Instruments, Thornhill, Canada).

Example 1

The synthesis of the precursor AlaA21Asp 2).

Following Polymerase Chain Reaction (PCR PCR) performed using the Gene Amp PCR reagent kit (Perkin Elmer, 761 Main Avewalk, CT 07859, USA), according to manufacturer's instructions. In all cases, the PCR mixture coated with 100 μl mineral oil (Sigma Chemical Co., St. Louis, MO, USA).

to 2.5 μl of the oligonucleotide #98 (2,5 pmol)

to 2.5 μl of the oligonucleotide #128 (2,5 pmol)

10 ál of 10X PCR buffer

16 μl dNTP mix

a 0.5 μl Taq enzyme

58,5 ál of water

Spend one cycle: 94oC for 45 sec., 49oC for 1 min, 72oC for 2 min.

Subsequently, 5 μl of oligonucleotides #16 and #126 add and spend 15 cycles: 94oC for 45 sec., 45oC for 1 min, 72oC for 1.5 min PCR mixture load on a 2.5% agarose gel and subjected to electrophoresis using standard techniques (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). The obtained DNA fragment cut out from agarose gel and was isolated using Gene Clean kit (Bio 101 Inc., PO BOX 2284, Lajolla, CA 92038, USA) according to manufacturer's instructions. The purified PCR DNA fragment was dissolved in 10 μl water and buffer for their endonuclease enzyme and digested with restriction endonucleases NcoI and XbaI according to standard techniques, performed on a 2.5% agarose gel, and purified using the coI fragment, encoding the synthetic yeast signal/leader gene LaC212spx3 (described in example 3 of WO 89/02463), followed by synthetic NcoI/XbaI fragment encoding an insulin precursor M15, which has SerAspAspAlaLys the bridge linking the B29 and the A1 amino acid residues (see SEQ ID NOS 14, 15 and 16) included in the EcoRI/XbaI fragment of the vector (fahmida) pBlueScript IISK (+/-) (Stratagene, USA). Plasmid pAK188 shown in Fig. 1.

Plasmid pAK188 also cleaved by restriction endonucleases NcoI and XbaI and the fragment of the vector length 3139 bp emit. Two DNA fragment are ligated together using T4 DNA ligase and standard conditions (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). The ligation mixture transformed into competent E. coli strain (R-, M+), followed by selection for resistance to ampicillin. Plasmids are isolated from the resulting E. coli colonies using standard DNA minipreparation technique Sambrook et al. , Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989), checking the appropriate restriction endonucleases, i.e., EcoRI, XbaI, NcoI and HpaI. As shown by DNA sequencing (Sequenase, U.S. Biochemical Corp. selected plasmid contains the correct sequence of the precursor AlaA21AspB3human insulin, and it is denoted pEA5.3.

Plasmid pKFN1627 pray DNA sequence against the course of transcription from the unique XbaI site. In pKFN1003, this sequence is a fragment of a length of 178 bp, encoding a synthetic Aprotinin gene fused in frame read (fused in-frame with the signal/leader sequence of the yeast sparavalo factor alpha 1. In pKFN1627, the corresponding sequence length 184 bp encodes an insulin precursor M15 (GluB1GluB28) (i.e., B(1-29, GluB1GluB28)-SeeAspAspAlaLys-A(1-21), fused in frame with the sequence sparavalo factor alpha 1 (see SEQ ID NOS 17, 18 and 19). Vector pKFN1627 shown in Fig. 1.

pEA5.3 were cleaved by restriction endonucleases EcoRI and XbaI and isolate the resulting DNA fragment of a length of 412 bp. Yeast expression vector pKFN1627 cleaved by restriction endonucleases NcoI and XbaI and NcoI and EcoRI and isolated DNA fragment length 9273 bp from the first digestion, and the isolated DNA fragment length 1644 bp from the second digestion. Then EcoRI/XbaI fragment length 412 bp are ligated with the other two fragments, i.e. NcoI/XbaI fragment length 9273 bp NcoI/EcoRI fragment length 1644 bp, using standard techniques.

The ligation mixture transformed into E. coli as described above. The plasmids from the resulting E. coli isolated using standard techniques, and checked with the appropriate endonucleases RES is abortion practices, as described by the manufacturer, U. S. Biochemical) selected plasmid contains the correct DNA sequence of the precursor AlaA21AspB3human insulin and has to be included after DNA coding LaC212spx3 signal/leader DNA. Plasmid denote pEA5.3.2 and it is shown in Fig. 1. The DNA sequence encoding the complex LaC212spx3 signal/leader/predecessor AlaA21AspB3human insulin and amino acid sequences are SEQ ID NOS 20, 21 and 22. Plasmid pEA5.3.2 transformed into S. cerevisiae strain MT663, as described in application EP with the publication N 214826, and the resulting strain indicate yEA002.

Example 2

The synthesis of the precursor AlaA21ThrB3human insulin from yeast strain yEA005 using LaC212spx3 signal/leader. (Sequence No. 3).

DNA encoding the precursor AlaA21ThrB3human insulin, designed in the same manner as described for the DNA that encodes the precursor AlaA21AspB3human insulin in example 1. The DNA sequence encoding the complex LaC212spx3 signal/leader/predecessor AlaA21ThrB3human insulin and amino acid sequences are SEQ ID NOS 23, 24 is 663, as described in example 1 and the resulting strain indicate yEA005.

Example 3

Synthesis of precursor GlyA21AspB3human insulin from yeast strain yEA007 using LaC212spx3 signal/leader. (Sequence No. 4).

DNA encoding the precursor GlyA21AspB3human insulin, designed in the same manner as described for the DNA that encodes the precursor AlaA21AspB3human insulin in example 1. The DNA sequence encoding the complex LaC212spx3 signal/leader/predecessor GlyA21AspB3human insulin and amino acid sequences are SEQ ID NOS 26, 27 and 28. Plasmid pEA1.5.6., as shown, contains the desired sequence, transformed into S. cerevisiae strain MT663 as described in example 1 and the resulting strain indicate yEA007.

Example 4

Synthesis of precursor GlyA21ThrB3human insulin from yeast strain yEA006 using LaC212spx3 signal/leader. (Sequence No. 5).

DNA encoding the precursor GlyA21ThrB3human insulin, designed in the same manner as described for the DNA that encodes the precursor AlaA21and AspB3chelovechest>A21ThrB3human insulin and amino acid sequences are SEQ ID NOS 29, 30 and 31. Plasmid pEA4.4.11., as shown, contains the desired sequence, transformed into S. cerevisiae strain MT663 as described in example 1 and the resulting strain indicate yEA006.

Example 5

Synthesis of single-stranded precursor ArgB1ArgB31human insulin with N-terminal extension (GluGluAlaGluAlaGluAlaArg) from yeast strain yEA113 using alpha factor leader. (Sequence No. 6).

Following Polymerase Chain Reaction (PCR PCR) performed using the Gene Amp PCR reagent kit (Perkin Elmer, 761 Main Avewalk, CT 06859, USA), according to manufacturer's instructions. In all cases, R mixture coated with 100 μl mineral oil (Sigma Chemical Co., St. Louis, MO, USA). Plasmid pAK220 (which is identical to pAK188) consists of a DNA sequence by the length of 412 bp that encodes a synthetic yeast signal/leader LaC212spx3 (described in example 3 of WO 89/02463), followed by insulin precursor M15 (see SEQ ID NOS 14, 15 and 16), inserted into a vector (fahmida) pBlueScript IISK(+/-) (Stratagene, USA)

5 μl of the oligonucleotide #220 (100 pmol)

5 μl of the oligonucleotide #263 (100 pmol)

10 ál of 10X PCR buffer

16 μl dNTP mix

a 0.5 ál Taq EN zymes who are 16 cycles, each cycle consists of 1 minute at 95oC; 1 minute at 40oC and 2 min at 72oC. Then PCR mixture load on a 2% agarose gel and subjected to electrophoresis using standard techniques. The obtained DNA fragment cut out from agarose gel and isolated using the Gene Clean kit (Bio 101 Inc. 101 Inc., PO BOX 2284, La jolla, CA 92038, USA) according to manufacturer's instructions. The purified PCR DNA fragment was dissolved in 10 μl water and buffer for their endonuclease enzyme and digested with restriction endonucleases HindIII and XbaI according to standard techniques. HinsIII/XbaI DNA fragment, using the Gene Clean kit.

Plasmid pAK406 consists of a DNA sequence with a length of 520 bp, including EcoRI/HindIII fragment obtained from pMT636 (described in WO 90/10075), encoding a yeast alpha factor leader and part of the insulin precursor, legirovannye with HindIII/XbaI fragment from pAK188, encoding the remainder of the insulin precursor M15 (see SEQ ID NOS 32, 33 and 34) inserted into the vector cPOT. Vector pAK406 shown in Fig. 2.

Plasmid pAK233 consists of a DNA sequence by the length of 412 bp that encodes a synthetic yeast signal/leader LaC212spx3 (described in example 3 of WO 89/02463), followed by gene insulin precursor B(1-29)-GluLysArg-A(1-21) (A21-Gly) (Aut the restriction endonucleases NcoI and XbaI and the fragment of the vector length 9273 bp isolate. Plasmid pAK406 cleaved by restriction endonucleases NcoI and HindIII and the vector fragment length 2012 bp isolate. These two DNA fragment are ligated with HindIII/XbaI PCR fragment using T4 DNA ligase and standard conditions. Then the ligation mixture transformed into competent E. coli strain (R-, M+), followed by selection for resistance to ampicillin. Plasmids are isolated from the resulting E. coli colonies using standard DNA minipreparation technique and checked with appropriate restriction endonucleases, i.e., EcoRI, XbaI, NcoI and HindIII. Selected plasmid, as shown by analyses on DNA sequencing, contains the correct sequence for DNA single-stranded precursor ArgB31human insulin, and which should be included after the DNA encoding the S. cerevisiae alpha factor DNA. Plasmid denote pEA108 and it is shown in Fig. 2. The DNA sequence encoding the complex alpha factor leader/single-stranded precursor ArgB31human insulin and amino acid sequences are SEQ ID NOS 38, 39 and 40. Plasmid pEA108 transformed into S. cerevisiae strain MT663 as described in example 1 and the resulting strain indicate yEA108.

Following the Polymerase Zeem manufacturer. In all cases, the PCR mixture coated with 100 μl mineral oil (Sigma Chemical Co., St. Louis, MO, USA).

5 μl of the oligonucleotide #220 (100 MPa)

5 μl of the oligonucleotide #307 (100 pmol)

10 µl 10X dNTP, buffer

16 μl dNTP mix

a 0.5 μl Taq enzyme

0.2 µl pEA108 plasmid as template (0.1 ág DNA)

63 μl of water

In General carried out by 16 cycles, where each cycle consists of 1 minute at 95oC; 1 minute at 40oC and 2 min at 72oC. Then PCR mixture load on a 2% agarose gel and subjected to electrophoresis using standard techniques. The obtained DNA fragment cut out from agarose gel and isolated using the Gene Clean kit (Bio 101 Inc., PO BOX 2284, La Jolla, CA 92038, USA) according to manufacturer's instructions. The purified PCR DNA fragment was dissolved in 10 μl water and buffer for their endonuclease enzyme and digested with restriction endonucleases NcoI and XbaI, according to standard techniques. NcoI/XbaI DNA fragment is purified using the Gene Clean kit.

Plasmid pAK401 consists of a DNA sequence by the length of 523 bp, including EcoRI/NcoI fragment derived from pMT636 (described in WO 90/10075) (constructed by introducing a NcoI site at the 3'-end of the alpha leader using site directed mutagenesis), encoding the alpha factor is 43), inserted into the vector (fahmida) pBlueScript IISK(+/-) (Stratagene, USA). Plasmid pAK401 shown in Fig. 3.

Plasmid pAK401 cleaved by restriction endonucleases NcoI and XbaI and the fragment of the vector length 3254 bp isolated and are ligated with NcoI/XbaI PCR fragment. Then the ligation mixture transformed into competent E. coli strain and the plasmid isolated from the obtained E. coli. colonies using standard DNA minipreparation technique and checked with appropriate restriction endonucleases, i.e., EcoRI, XbaI, NcoI. Selected plasmid, designated p113A (shown in Fig. 3), digested EcoRI and XbaI and the fragment length of 535 bp isolated.

Plasmid pAK233 cleaved by restriction endonucleases NcoI and XbaI, and EcoRI/NcoI fragments of length 9273 and 1644 bp isolate. These two DNA fragment are ligated with EcoRI/XbaI fragment from p113A using T4 DNA ligase and standard conditions. Then the ligation mixture transformed into competent E. coli strain (R-, M+), followed by selection for resistance to ampicillin. Plasmids are isolated from the resulting E. coli colonies using standard DNA minipreparation technique and checked with appropriate restriction endonucleases, i.e., EcoRI, XbaI, Ncol and Hindlll. Selected plasmid, as shown by analyses on Sequeira theB31human insulin with N-terminal extension GluGluAlaGluAlaGluAlaArg and which should be included after the DNA encoding the S. cerevisiae alpha factor DNA. Plasmid denote pEA113 and it is shown in Fig. 3. A DNA sequence encoding a (complex) alpha factor leader/single-stranded precursor ArgB-1ArgB31human insulin with N-terminal extension (GluGluAlaGluAlaGluAlaArg), and its amino acid sequence are SEQ ID NOS 44, 45 and 46. Plasmid pEA113 transformed into S. cerevisiae strain MT663 as described in example 1 and the resulting strain indicate yEA113.

Example 6

Synthesis of single-stranded precursor ArgB-1ArgB31human insulin with N-terminal extension (GluGluAlaGluAlaGluAlaGluArg) from yeast strain yEA136 using alpha factor leader (Sequence No. 7).

The following PCR (PCR) carried out using the Gene Amp PCR reagent kit

5 μl of the oligonucleotide #220 (100 pmol)

5 μl of the oligonucleotide #389 (100 pmol)

10 ál of 10X PCR buffer

16 μl dNTP mix

a 0.5 μl Taq enzyme

2 μl of pEA113 plasmid as template (0.5 μg DNA)

63 μl of water

In General carried out 12 cycles, where each cycle consists of 1 minute at 95oC; 1 minute at 37oC and 2 monotonic human insulin, with N-terminal extension (GluGluAlaGluAlaGluAlaGluArg), design in the same manner as described for the DNA encoding the alpha factor leader/ArgB-1ArgB31single-stranded precursor of human insulin with N-terminal extension (GluGluAlaGluAlaGluAlaArg) in example 5. Plasmid denote pEA136. The DNA sequence encoding the alpha factor leader/ArgB-1ArgB31single-stranded precursor of human insulin with N-terminal extension (GluGluAlaGluAlaGluAlaGluArg), and its amino acid sequence are SEQ ID NOS 47, 48 and 49. Plasmid pEA136 transformed into S. cerevisiae strain MT663 as described in example 1 and the resulting strain indicate yEA136.

Example 7

Synthesis of (A1,B1)-divas human insulin

5 g free from zinc human insulin was dissolved in a 41.3 ml of DMSO. To the solution add 3,090 ml of acetic acid. The reaction is carried out at room temperature and initiate the addition of 565 mg of di-tert-butyl pyrocarbonate dissolved in 5,650 ml DMSO. The reaction continued for 5 1/2 hours and then stopped by the addition of 250 ml of ethanolamine. The product is precipitated by the addition of 1500 ml of acetone. The precipitate was separated by centrifugation and dried in vacuum. Get 6,85 g of substance.

(A1,B1)-divas insulae in water, bring pH to 3.0 with HCl and injected into a column (5 cm diameter, 30 cm height) filled with particles octadecylsilyl-substituted silicon dioxide (average particle size 15 μm, pore size 100 ) and balanced eluting buffer. Elution is performed using a mixture of ethanol and 1 mm aqueous HCl, 0.3 M KCl at a flow of 2 l/h Insulin elute by increasing the ethanol content from 30% to 45%. The appropriate fraction was diluted with 20% ethanol and precipitated at pH 4.8. The precipitated substance produce by centrifugation and dried in vacuum. So get 1,701 g (A1,B1)-divas human insulin with the purity of 94.5%.

Example 8

Synthesis ( NB29-benzoyl human insulin)6, 3Zn2+.

400 mg of (A1, B1)-divas human insulin was dissolved in 2 ml of DMSO (DMSO). To the solution add 748 μl of a mixture of N-methylmorpholine and DMSO (1:9, V/V). The reaction is carried out at 15oC and initiated by the addition of 14.6 mg N-hydroxysuccinimide ester of benzoic acid dissolved in 132 μl DMF. The reaction is stopped after 2 hours by adding 100 ml of acetone. The precipitated substance was separated by centrifugation and dried in vacuum. Collect 343 mg of the substance.

Boc protecting group helps eliminate by adding 4 the dock is separated by centrifugation and dried in vacuum.

NB29-benzoyl human insulin purified using GHUR with reversed phase, as described in example 7. Obtain 230 mg of the substance. By recrystallization from 15% aqueous ethanol containing 6 mm Zn2+and 50 mm citrate at pH 5.5 to get the crystals of these compounds, which emit by centrifugation and dried in vacuum. The output of 190 mg.

Molecular weight determined by MS: 5911, theory: 5911.

Example 9

Synthesis (NB29-lithocholyl human insulin)6, 3Zn2+< / BR>
400 mg of (A1, B1)-divas human insulin was dissolved in 2 ml of DMSO (DMSO). To the solution add 748 μl of a mixture of N-methylmorpholine and DMSO (1:9, V/V). The reaction is carried out at 15oC and initiated by adding 31,94 mg N-hydroxysuccinimide ether lithocholic acid, dissolved in 300 μl of DMF. The reaction is stopped after 2 hours by adding 100 ml of acetone. The precipitated substance was separated by centrifugation and dried in vacuum. Collect 331 mg.

Boc protecting group helps eliminate by adding 4 ml of TFA. The dissolved substance is incubated for 30 minutes and then precipitated by adding 50 ml of acetone. The precipitate was separated by centrifugation and dried in vacuum. Exit 376 MK described in example 7. Obtain 67 mg of the substance when the purity of 94%. By recrystallization from 15% aqueous ethanol containing 6 mm Zn2+and 50 mm citrate at pH 5.5 to get the crystals of these compounds, which emit by centrifugation and dried in vacuum. Exit 49 mg.

Molecular weight determined by MS: 6160, theory: 6166.

Example 10

Synthesis (NB29-decanoyl human insulin)6, 3Zn2+< / BR>
400 mg of (A1, B1)-divas human insulin was dissolved in 2 ml of DMSO (DMSO). To the solution add 748 μl of a mixture of N-methylmorpholine and DMSO (1:9, V/V). The reaction is carried out at 15oC and initiated by the addition of 18.0 mg N-hydroxysuccinimide ether decanoas acid dissolved in 132 μl DMF. The reaction is stopped after 60 minutes and the product precipitated by adding 100 ml of acetone. The precipitated substance was separated by centrifugation and dried in vacuum. Collect 420 mg of the intermediate product.

Boc protecting group helps eliminate by adding 4 ml of TFA. The dissolved substance is incubated for 30 minutes and then the product is precipitated by adding 50 ml of acetone. The precipitate was separated by centrifugation and dried in vacuum. The yield of the crude product is 420 mg of Crude product cleaned the screens 96,1%. By recrystallization from 15% aqueous ethanol containing 6 mm Zn2+and 50 mm citrate at pH 5.5 to get the crystals of these compounds, which emit by centrifugation and dried in vacuum. The output is 217 mg.

Molecular weight determined by MS: 5962, theory: 5962.

Example 11

Synthesis of des(B30) human insulin

Synthesis of des(B30) human insulin as described Markussen (Methods in diabetes research, Vol. 1, Laboratory methods, part B, 404-410. Ed: J. Larner and S. Phol, John Wiley & Sons, 1984). 5 g of human insulin was dissolved in 500 ml of water, while the pH of the solution support to 2.6 by the addition of 0.5 M sulfuric acid. Subsequently, insulin vymalivayut by adding 100 g of ammonium sulfate and the precipitate was separated by centrifugation. The residue is dissolved in 800 ml of 0.1 M ammonium hydrogen carbonate and the pH of the solution was adjusted to 8.4 with 1 M ammonia.

50 mg of bovine carboxypeptidase A are suspended in 25 ml of water and separated by centrifugation. The crystals are suspended in 25 ml of water and add 1 M ammonia until will not get a clear solution when the final pH of 10. A solution of carboxypeptidase added to a solution of insulin in conducting a reaction within 24 hours. As conservat by gradually adding 80 g of sodium chloride under stirring of the solution. Then the pH was adjusted to 8.3 and conduct crystallization for 20 hours with gentle stirring. The crystals separated on a 1.2 μm filter, washed with 250 ml of cooled with ice 2-propanol and finally dried in vacuum.

Example 12

Synthesis of (A1,B1)-divas des(B30) human insulin

The named compound is synthesized in a manner similar to the method described in example 7, using as the starting material des(B30) porcine insulin. The crude product is precipitated with acetone and dried in vacuum. (A1,B1)-divas-des(B30) human insulin purified using GHUR with reversed phase, as described in example 7.

Example 13

Synthesis of NB29-decanoyl des(B30) human insulin

400 mg of (A1, B1)-divas-des(B30) human insulin is used as the starting material for the synthesis of NB29-decanoyl des(B30) human insulin, following the procedure described in example 10. The crude product is precipitated with acetone, dried in vacuum and remove the protection using TFA. The resulting product is then precipitated with acetone and dried in vacuum. Then NB29-decanoyl des(B30) human insulin purified using GHUR with reversed phase, as described in example 10.

Molecular weight, human insulin

a. Immobilization of A. lyticus protease

13 mg A. Lyticus protease dissolved in 5 ml of aqueous 0.2 M NaHCO3buffer, pH 9,4, mixed with 4 ml of settled gel MiniLeakRMedium, which was washed with the same buffer (MiniLeak represents Sepharose CL 6B, activated by diphenylsulfone received from KemEnTec, Copenhagen). The gel is incubated in suspension with gentle stirring for 24 hours at room temperature. Then the gel was separated by filtration, washed with water and suspended in 20 ml of 1 M ethanolamine buffer, pH 9,4, and incubated in suspension for 24 hours at room temperature. Finally, the gel was washed with water, and then 0.1 M acetic acid and stored at 4oC. the enzyme Activity in the filtrate was 13% of the activity of the enzyme in the original solution, which indicates that the yield of the reaction immobilization is about 87%.

b. Immobilization of porcine trypsin.

Porcine trypsin immobilized on MiniLeakRLow degree of substitution of 1 mg per ml of gel, using conditions described above for the immobilization of A. lyticus.

c. Synthesis of Glu(GluAla)3Arg-B(1-29), ThrArg-A(1-21) insulin using immobilized A. lyticus protease.

To 200 mg of precursor Glu(GluAla)3Arg-B(1-29), ThrArg-A(1-21) of ADNOC the th immobilized A. lyticus protease. After keeping the gel in suspension in the reaction mixture for 6 hours at room temperature, the hydrolysis is complete, receiving Glu(GluAla)3Arg-B(1-29), ThrArg-A(1-21) human insulin (reaction traced GHUR with reversed phase). After hydrolysis, the gel is removed by filtration. To the filtrate add 5 ml of ethanol and 15 ál of 1 M ZnCl2and bring the pH to 5.0 using HCl. The precipitated product is formed upon standing overnight at 4oC with gentle stirring. The product is separated by centrifugation. After one washing, 1 ml of a cooled ice 20% ethanol and drying in vacuo the yield was 190 mg.

d. Synthesis of NA1NB1NB29-tridecanol Glu(GluAla)3Arg-B(1-29), Thr-Arg-A(1-21) human insulin, using N-hydroxysuccinimide ether dodecanol acid

190 mg (30 mmol) Glu(GluAla)3Arg-B(1-29), ThrArg-A(1-21) insulin dissolved in 1 ml DMSO and 1.05 ml 0,572 M solution of N,N-diisopropylethylamine in DMF. The solution is cooled to 15oC and added 36 mg (120 mmol) N-hydroxysuccinimide ether dodecanol acid, dissolved in 0.6 ml DMSO. The reaction is complete within 24 hours. Lipophilic named connection is not isolated.

e. Synthesis of NB29-DoD is isopropylethylene, dilute to 10.6 ml of 50 mm glycine buffer containing 20% ethanol, and the pH adjusted to 10 with NaOH. After standing for 1 hour at room temperature was added 1 ml of MiniLeak gel carrier 1 mg of immobilized trypsin in 1 ml of gel. The reaction mixture was gently stirred for 48 hours at room temperature. In order to isolate the desired product, the reaction mixture is subjected to chromatographicaliy on column GHUR with reversed phase (5 cm in diameter, height 30 cm) filled with particles octadecylsilyl-substituted silicon dioxide (average particle size 15 μm, pore size 100 ). For elution using 20 mm Tris/HCl buffers, brought to a pH of 7.7 and including increasing the ethanol concentration from 40% to 44% (V/V), with a rate of 2000 ml/h Main peak elution at about 43-44% ethanol contained a named connection. Fractions containing the main peak, combine, add water to reduce the concentration of ethanol up to 20% (V/V) and pH adjusted to 5.5. The solution is stored overnight at -20oC, the product precipitates. The precipitate was separated by centrifugation at -8oC and dried in vacuum. The output of these compounds is 90 mg Molecular weight, determined by MS: 5892, theory: 5890.

oC and added 85 mg of N-myristoyl-Glu(OBut) N-hydroxysuccinimide ether, dissolved in 2.5 ml DMSO/DMF 1/1 (V/V). After 30 minutes the reaction mixture is poured into 60 ml of water, pH adjusted to 5 and the precipitate allocate by centrifugation. The precipitate is dried in vacuum. The dried reaction mixture is dissolved in 25 ml of TFA and the solution is kept for 30 minutes at room temperature. TFA is removed by evaporation in a vacuum. Gel-like residue is dissolved in 60 ml of water and pH was adjusted to 11.2 using concentrated ammonia. The named compound is crystallized from this solution by setting the pH to 8.5 using 6N HCl. The product is separated by centrifugation, once washed with 10 ml of water and dried in vacuum. Exit 356 mg. Purity according to GHUR 94%.

The product of this example is a human insulin in which the amino group of LysB29has the Deputy of the following structure: CH3(CH2)12CONHCH(CH2CH2COOH)CO-.

Molecular weight determined by MS: 6146, theory: 6148.

Example 16

Synthesis of NB29-undecanoyl des(B30) human insulin

The named compound is synthesized analogously to N

Molecular weight determined by MS: 5876, theory: 5876.

Example 17

Synthesis of NB29-tridecanol Deux(30) human insulin

The named compound is synthesized analogously to NB29-dodecanoyl des(B30) human insulin as described in example 14, using N-hydroxysuccinimide ether tridecanoic acid instead of N-hydroxysuccinimide ether dodecanol acid.

Molecular weight determined by MS: 5899, theory: 5904.

Example 18

Synthesis of NB29-myristoyl Deux(30) human insulin

The named compound is synthesized analogously to NB29-dodecanoyl des(B30) human insulin as described in example 14, using N-hydroxysuccinimide ester of myristic acid instead of N-hydroxysuccinimide ether dodecanol acid.

Molecular weight determined by MS: 5923, theory: 5918.

Example 19

Synthesis of NB29-Palmitoyl Deux(30) human insulin

The named compound is synthesized analogously to NB29-dodecanoyl des(B30) human insulin as described in example 14, using N-hydroxysuccinimide ester of palmitic acid instead of N-hydraxis is.

Example 20

Synthesis of NB29-suberoyl-D-thyroxine human insulin

a. Obtaining N-(succinimidylester)-D-thyroxine.

Disuccinimidyl suberate (1.0 g, Pierce) was dissolved in DMF (50 ml), and add D-thyroxine (2.0 g, Aldrich) with stirring at 20oC. Thyroxine slowly dissolves, and after 20 hours the solvent is removed by evaporation in a vacuum. The oily residue is crystallized from 2-propanol, receiving 0.6 g of N-(succinimidylester)-D-thyroxine, so pl. 128-133oC.

b. The reaction of (A1,B1)-divas human insulin with N-(succinimidylester)-D-thyroxine.

(A1, B1)-divas human insulin was dissolved in dry DMF (10 ml) and add triethylamine (20 μl) at room temperature. Then add N-(succinimidylester)-D-thyroxine (80 mg). Reaction control using GHUR with reversed phase, and when the reaction is finished at about 90%, the solvent is removed in vacuum. To the residue after evaporation add anhydrous triperoxonane acid (5 ml) and the solution was incubated for 1 hour at room temperature. After removal triperoxonane acid in vacuo, the residue dissolved in a mixture of 1 M acetic acid (5 ml) and acetonitrile (1.5 ml), purified using preparative GHW Olina 50 mg.

The product of this example is a human insulin in which the amino group of LysB29has the Deputy of the following structure: Tirex-CO(CH2)6CO-, in which Tirex is thyroxine, which is associated with octanedionato acid part through amide bond with him-amino group.

Molecular weight determined by MS: 6724, theory: 6723.

Example 21

Synthesis of NB29-(2 succinylamino)myristic acid human insulin

a. Obtaining methyl ester-aminopyridine acid, HCl.

To a methanol (5 ml, Merck) at -10oC, dropwise with vigorous stirring, thionyl chloride (0.2 ml, Aldrich). Then add-aminopyridinium acid (0.7 g, obtained from a-bromo acid by reacting with ammonia. The reaction mixture was stirred at room temperature overnight, and then evaporated to dryness. The crude product (0.7 g) used directly in stage b.

b. Obtain methyl ester of N-succinoyl--aminopyridine acid

Methyl ether-aminopyridine acid, HCl (0.7 g) is dissolved in chloroform (25 ml, Merck). Add triethylamine (0.35 ml, Fluka), then the anhydride of succinic acid (0.3 g, Fluka). will crystallised from a mixture of ethyl acetate/petroleum ether (1/1). Output: 0,8 g

c. Obtain methyl ester of N-(succinimidylester)--aminopyridine acid

Methyl ester of N-succinoyl--aminopyridine acid (0.8 g) dissolved in dry DMF (10 ml, Merck, dried over 4 a molecular sieve). Add dry pyridine (80 μl, Merck), and di(N-Succinimidyl)carbonate (1.8 g, Fluka), and the reaction mixture was stirred over night at room temperature. The residue after evaporation is purified using flash chromatography on silica gel 60 (Merck), and recrystallized from a mixture of 2-propanol/petroleum ether (1/1). The yield of methyl ester of N-(succinimidylester)--amino-myristic acid: 0,13 g, so pl. 64-66oC.

b. Interaction (A1,B1)-divas human insulin with methyl ester of N-(succinimidylester)--aminopyridine acid

The reaction is carried out as in example 20b., but using the methyl ester of N-(succinimidylester)--aminopyridine acid (16 mg) instead of N-(succinimidylester)-D-thyroxine. After removal triperoxonane acid in vacuo, the residue after evaporation is treated with 0.1 M sodium hydroxide at 0oC, amylea methyl ether. When the saponification according to the control by using GHUR with reversed phase is complete, the pH of the solution was adjusted to column output NB29-(2 succinylamino)myristic acid human insulin is 39 mg.

The product of this example is a human insulin in which the amino group of LysB29has the Deputy of the following structure: CH3(CH2)11CH(COOH)NHCOCH2CH2CO.

Molecular weight determined by MS: 6130, theory: 6133.

Example 22

Synthesis of NB29-octyloxybiphenyl human insulin

Synthesis is carried out as in example 20 b., but using n-octyloxybiphenyl N-hydroxysuccinimide (9 mg) was obtained from n-octyl of chloroformate (Aldrich) and N-hydroxysuccinimide), instead of N-(succinimidylester)-D-thyroxine. Output-octyloxybiphenyl human insulin was 86 mg.

The product of this example is a human insulin in which the amino group of LysB29has the Deputy of the following structure: CH3(CH2)7OCO-.

Molecular weight determined by MS: 5960, theory: 5964.

Example 23

Synthesis of NB29-(2 succinylamino)palmitic acid human insulin

a. Obtain methyl ester of N-(succinimidylester)- -aminoalkanoic acid.

b. Interaction (A1, B1)-divas human insulin with methyl ester of N-(succinimidylester)--aminoalkanoic acid

The reaction is carried out as in example 21 (d., but using the methyl ester of N-(succinimidylester)--aminoalkanoic acid instead of methyl ester of N-(succinimidylester)- -aminopyridine acid, receiving NB29-(2 succinylamino)palmitic acid human insulin.

The product of this example is a human insulin in which the amino group of LysB29has the Deputy of the following structure: CH3(CH2)13CH(COOH)NHCOCH2CH2CO.

Example 24

Synthesis of NB29-(2 Coccinellidae)palmitic acid human insulin

a. Obtain methyl ester of N-(succinimidylester)-2-aminoethoxy palmitic acid

This connection receive as described in example 21 a. - c. using 2-aminoethoxy palmitic acid (synthesized according to conventional methods described R. TenBrink, J, Org. Chem. 52 (1987) 418-422) instead-amino myristic acid.

b. Interaction (A1, B1)-divas human insulin with methyl ester of N-(succinimidylester)-2-aminoethoxyethanol to tracibility acid instead of methyl ester of N-(succinimidylester)--aminopyridine acid, receiving NB29-(2 Coccinellidae)palmitic acid human insulin.

The product of this example is a human insulin in which the amino group of LysB29has the Deputy of the following structure: CH3(CH2)13CH(COOH)NHCH2CH2OCOCH2CH2CO.

Example 25

Synthesis of NB29-lithocholyl--glutamyl des(B30) human insulin

Synthesis is carried out as in example 13, using N-hydroxy-succinimide ether N-lithocholyl-L-glutamic acid, tert-butyl ester instead of N-hydroxysuccinimide ether decanoas acid.

The product of this example is des(B30) human insulin, in which the amino group of LysB29has the Deputy of the following structure: lithocholyl-NHCH(CH2CH2COOH)CO-.

Molecular weight determined by MS: 6194, theory: 6193.

Example 26

Synthesis of NB29-3,3',5,5'-tetraiodothyronine human insulin

Synthesis is carried out as in example 10, using N-hydroxysuccinimidyl ether 3,3',5,5'-tetraiodothyronine acid instead of N-hydroxysuccinimide ether decanoas acid.

The molecular weight of the set is on

Synthesis is carried out as in example 10, using Boc-L-thyroxine N-hydroxysuccinimidyl ester instead of N-hydroxysuccinimide ether decanoas acid.

Molecular weight determined by MS: 6572, theory: 6567.

Example 28

Pharmaceutical composition comprising 600 nmol/ml NB29-decanoyl des(B30) human insulin, 1/3Zn2+in the solution.

NB29-decanoyl des(B30) human insulin (1,2 mmol) dissolved in water (0.8 ml) and the pH was adjusted to 7.5 by addition of 0.2 M sodium hydroxide. Add 0.01 M zinc acetate (60 ml) and a solution containing 0.75 percent phenol and 4% glycerol (0.8 ml). The pH of the solution was adjusted to 7.5 using 0.2 M sodium hydroxide and the solution volume was adjusted with water to 2 ml

The resulting solution is sterilized by filtration and aseptically transferred into catridge or bubble.

Example 29

Pharmaceutical composition comprising 600 nmol/ml NB29-decanoyl human insulin, 1/2Zn2+in the solution.

1,2 mmol these compounds are dissolved in water (0.8 ml) and the pH was adjusted to 7.5 by addition of 0.2 M sodium hydroxide. Add a solution containing 0.75 percent phenol and 1.75% sodium chloride (0.8 ml). Meant the resulting solution is sterilized by filtration and aseptically transferred into catridge or bubble.

Example 30

Pharmaceutical composition comprising 600 nmol/ml NB29-lithocholyl human insulin in solution

1,2 mmol these compounds are suspended in water (0.8 ml) and dissolved, bringing the pH of the solution to 8.5, using 0.2 M sodium hydroxide. Then to the solution was added 0.8 ml source for cultivation of a solution containing 0.75 percent cresol and 4% glycerin in water. Finally, the pH again adjusted to 8.5 and the solution volume was adjusted with water to 2 ml

The resulting solution is sterilized by filtration and aseptically transferred into catridge or bubble.

2. Additional experimental data

Example 31

Pharmaceutical composition comprising a solution of 600 nmol/ml of NB29-hexadecanoyl human insulin, 1/3 of zinc ions of the insulin monomer, 16 mm m-cresol, 16 mm phenol, 1.6% glycerol, 10 mm NaCl and 7 mm of sodium phosphate.

1.2 µmol NB29-hexadecanoyl human insulin was dissolved in water (0.5 ml), the addition of 0.2 M sodium hydroxide pH adjusted to 8.0 and add 40 ál of 0.01 M zinc acetate. Further to the solution add 100 ál of 0.32 M phenol, 200 μl of 0.16 M-cresol, 800 ál of 4% glycerol, 33,3 ál of 0.6 M sodium chloride and 140 μl of 0.1 M of fastata soda is 2">

Example 32

Determination of the solubility of NB29the deletion of des(B30) human insulin and NB29-hexadecanoyl human insulin in various compositions containing them.

Test NB29the deletion of des(B30) human insulin and NB29-hexadecanoyl human insulin in various compositions. The composition is prepared as described in example 31 with the necessary number of components. Zinc acetate is either not added or added in an amount corresponding to 1/3 of Zn2+the insulin monomer. Sodium chloride is used in amounts giving a final concentration of 5, 25, 50, 75, 100 or 150 mm sodium chloride, respectively. Insulin, not containing zinc ions, added to the number, giving the final concentration in the composition 1000 nmol/ml In some cases a precipitate. The resulting solutions or suspensions stored at 4oC during the week and measure the concentration of insulin in the solution of each composition using the method by size exclusion liquid chromatography high resolution compared to the standard human insulin (column: Waters ProteinPak h mm, eluent: 2.5 M acetic acid, 4 mm arginine, 20% acetonitrile, korostil. 3.

The data presented in the table show that the soluble acylated insulin increases with the addition of zinc. This is contrary to the data published for human, porcine and bovine insulin (J. Brange: Galenics of insulin, page 19, Springer Verlag (1987); J. Marcussen et al. Protein Engineering 1 (1987) 205-213).

Example 33

Preparative solution containing zinc NB29the deletion of des(B30) human insulin.

10 g of NB29the deletion of des(B30) human insulin was dissolved in 120 ml of 0.02 M NH4Cl buffer, brought to a pH of 9.0 NH3in a mixture of ethanol/water (1: 4, V/V). Careful stirring maintain during the entire crystallization process. Crystallization was initiated by adding 20 ml of 2.5 M NaCl, dissolved in a mixture of ethanol/water (1:4, V/V). The solution appears a slight darkening. Next, add 20 ml of 2.5 M sodium chloride, dissolved in a mixture of ethanol/water (1:4, V/V) at a constant speed of 5 ml/h, which causes a slow crystallization. To reduce the solubility of insulin is then adjusted pH to 7.5 using 1 n hydrochloric acid. In the end the temperature was lowered to 4oC and stirring is continued over night. The crystals are collected by filtration, washed twice what ptx2">

The weight of the wet filter cakes is 19,33,

The weight of the dried filter cakes is 9,71,

Example 34

Synthesis of LysB29(N-[Nthe deletion-Glu-Gly] ) des(B30) human insulin.

500 mg of (A1,B1)-di-Boc human insulin was dissolved in a mixture of 186 μl of 4-methylmorpholine and 1314 ál DMSO. The reaction is initiated by adding 144 mg deletion-Glu(-Atrebatic)-Gly-OSu dissolved in 1000 μl of DMF. The reaction is carried out at 15oC and stopped after 4.5 hours by adding 100 ml of acetone. The reaction product is precipitated by adding a few drops of concentrated HCl, then separated by centrifugation. The precipitate is then suspended in 100 ml of acetone, separated by centrifugation and dried in vacuum. Receive 637 mg of product.

Boc-protecting group is removed by adding 5 ml TFUCK. The dissolved product was incubated for 30 min and then precipitated by adding 100 ml of acetone and a few drops of concentrated HCl. The precipitate is then suspended in 100 ml of acetone and separated by centrifugation. The precipitated product is dissolved in 200 ml of 25% ethanol at a pH of 8, obtained by the addition of NH4OH, and purified using GHUR with reversed phase. Rosamistero of silica gel (average particle size 15 μm, pore size 100 ) and balance of 0.02 M bis-Tris, 30% ethanol, pH, increased to 7.3 with hydrochloric acid, at a temperature of 40oC. Elution carried out with a mixture of 70% ethanol in water and bis-Tris buffer. The flow rate is 2 l/h. Insulin elute, increasing the ethanol content from 30% to 50% and the effluent is fixed by its absorption in the UV at 280 nm. The appropriate fractions were diluted to 20% ethanol, pH 4.5 and frozen at -20oC. Precipitated material emit trim the sample at the 1oC and subsequent centrifugation at the same temperature. The precipitate is dried in vacuum. Get 292 mg of the target compound with a purity of 95.5 percent.

Molecular weight determined by MS: 6102+6, tiora.: 6103.

The lipophilicity of target compound relative to human insulin k'Rel.determined according to methods described above, is 20.

The value of T50%determined according to the method described on page 36 of the description specified in the connection header with subcutaneous injections pigs is to 11.9 hours Using a composition similar to that described in table 2.

Example 35

Synthesis of LysB29(Nthe deletion-Glu) des(B30) human insulin.

Get 356 mg of the target compound with a purity of 94,1%.

Molecular weight determined by MS: 6053+6, tiora.: 6046.

The lipophilicity of target compound relative to human insulin k'Rel.determined according to methods described above, is 24.

The value of T50%determined according to the method described on page 36 of the description specified in the connection header with subcutaneous injections pigs is 8.8 hours Using a composition similar to that described in table 2.

Example 36

Synthesis of LysB29(N-[Nthe deletion-Glu(-)-OH]) human insulin.

400 mg of (A1,B1)-di-Boc human insulin was dissolved in a mixture of 232 μl of ethyldiethanolamine, 1880 μl DMSO and 2088 μl of 1-methyl-2-pyrrolidone. The reaction is initiated by addition of 138 mg of N-deletion-Glu(OSu)-O-tert-butyl dissolved in 800 μl of 1-methyl-2-pyrrol described in example 34. The protective group is removed from the intermediate product by the addition of TN to clean GHUR with reversed phase and the target product are precipitation and vacuum drying. Obtain 222 mg of the target compound with a purity of 95.5 percent.

Molecular weight determined by MS: 6150+6, tiora.: 6147.

The lipophilicity of target compound relative to human insulin k'Rel.determined according to methods described above, is 21.

The value of T50%determined by the method described above, for the specified header connection with subcutaneous injections pigs is 8.0 hours Using a composition similar to that described in example 31.

Example 37

Synthesis of LysB29(N-[N-hexadecanoyl-Glu(-)-OH]) human insulin.

400 mg of (A1,B1)-di-Boc human insulin was dissolved in a mixture of 232 μl of ethyldiethanolamine, 880 μl DMSO and 2088 μl of 1-methyl-2-pyrrolidone. The reaction is initiated by adding 73 mg-hexadecanoyl-Glu(OSu)-O-tert-butyl dissolved in 800 μl of DMF. The reaction is carried out at 15oC and stopped after 4.5 hours.

All other stages of the method were carried out as described in example 34.

Get 476 mg of the intermediate product the target product are precipitation and vacuum drying.

Obtain 222 mg of the target compound with a purity of 81.2 per cent.

Molecular weight determined by MS: 6179+6, tiora.: 6175.

The lipophilicity of target compound relative to human insulin k'Rel.determined by the method presented above is 67.

The value of T50%determined by the method described above, for the specified header connection with subcutaneous injections pigs is 13 hours Using a composition similar to that described in example 31.

Example 38

Synthesis of LysB29( N-[ N-octadecanoyl-Glu(-)-OH]) des(B30) human insulin.

400 mg of (A1,B1)-di-Boc des(B30) human insulin was dissolved in a mixture of 232 μl of ethyldiethanolamine, 3000 ál DMSO and 268 μl of dimethylformamide. The reaction is initiated by addition of 114 mg of N-octadecanoyl-Glu(OSu)-O-tert-butyl dissolved in 500 μl of DMF. The reaction is carried out at 15oC and stopped after 4.5 hours. All other stages of the method were carried out as described in example 34. Obtain 420 mg of the intermediate product Protective group is removed from the intermediate product by adding TFUK to clean GHUR with reversed phase and the target product are precipitation and vacuum drying.

The lipophilicity of target compound relative to human insulin k'Rel.determined according to methods described above, is 185.

The value of T50%determined by the method described above, for the specified header connection with subcutaneous injections pigs of 9.7 hours Using a composition similar to that described in example 31.

Example 39

Synthesis of LysB29(N-[Nthe deletion-Glu(-)-OH] ) des(B30) human insulin.

400 mg of (A1,B1)-di-Boc des(B30) human insulin was dissolved in a mixture of 232 μl of ethyldiethanolamine and 3000 μl of DMSO. The reaction is initiated by addition of 138 mg of N-deletion-Glu(OSu)-O-tert-butyl dissolved in 768 μl DMF. The reaction is carried out at 15oC and stopped after 4.5 hours. All other stages of the method were carried out as described in example 34. Get 505 mg of the intermediate product. The protective group is removed from the intermediate product by adding TFUK to clean GHUR with reversed phase and the target product are precipitation and vacuum drying.

Obtain 237 mg of the target compound with a purity of 96.7% of.

Molecular weight determined by MS: 6053+6, tiora.: 6046.

The value of T50%determined by the method described above, for the specified header connection with subcutaneous injections pigs is 12.8 hours Using a composition similar to that described in example 31.

Example 40

Synthesis of LysB29( N-[ N-hexadecanoyl-Glu(-)-OH]) des(B30) human insulin.

400 mg of (A1,B1)-di-Boc des(B30) human insulin was dissolved in a mixture of 232 μl of ethyldiethanolamine, 3000 ál DMSO and 400 μl of dimethylformamide. The reaction is initiated by adding 73 mg N-hexadecanoyl-Glu(Osu)-O-tert-butyl dissolved in 400 μl of DMF. The reaction is carried out at 15oC and stopped after 4.5 hours. All other stages of the method were carried out as described in example 34. The protective group is removed from the intermediate product by adding TFUK to clean GHUR with reversed phase and the target product are precipitation and vacuum drying.

Obtain 153 mg of the target compound with a purity of 95.2 per cent.

Molecular weight determined by MS: 6073+6, tiora.: 6074.

The lipophilicity of target compound relative to human insulin k'Rel.determined according to methods described above, is 67.deposits in the subcutaneous injection to pigs is 18,0 PM Use a composition similar to that described in example 31.

Example 41

Synthesis of LysB29(N- N-lithocholyl-Glu(-)-OH]) des(B30) human insulin.

400 mg of (A1,B1)-di-Boc des(B30) human insulin was dissolved in a mixture of 148 μl of 4-methylmorpholine and 3452 ál DMSO. The reaction is initiated by adding 132 mg N-lithocholyl-Glu(OSu)-O-tert-butyl dissolved in 400 μl of DMF. The reaction is carried out at 15oC and stopped after 4.5 hours. All other stages of the method were carried out as described in example 34. Get 493 g of the intermediate product. The protective group is removed from the intermediate product by adding TFUK to clean GHUR with reversed phase and the target product are precipitation and vacuum drying.

Obtain 209 mg of the target compound with a purity of 97.4%.

Molecular weight determined by MS: 6185+6, tiora.: 6194.

Example 42

Synthesis of LysB29(N-[Nthe deletion-Aad(-)-OH] ) des(B30) human insulin.

Aad is a 5-aminohexanoic acid. 347 mg (A1,B1)-di-Boc des(B30) human insulin was dissolved in a mixture of 129 μl 4-methylmorpholine and 2645 ál DMSO. The reaction is initiated by addition of 58 mg of N-deletion-Aad(OSu)-O-t is the procedure of obtaining derivatives of asparginase acid (L. Benoiton: Can J. Chem. 40 570-72 (1962); R. Roeske: J. Jrg. Chem. 28 1251-93 (1963)). The reaction is carried out at 15oC and stopped after 4.5 hours.

All other stages of the method were carried out as described in example 34. The protective group is removed from the intermediate product by adding TFUK to clean GHUR with reversed phase and the target product are precipitation and vacuum drying.

Obtain 149 mg of the target compound with a purity of 97.9% of.

Molecular weight determined by MS: 6061+6, tiora.: 6060.

The lipophilicity of target compound relative to human insulin k'Rel.determined according to methods described above, is 21.

The value of T50%determined by the method described above, for the specified header connection with subcutaneous injections pigs $ 16.1 hours Use composition, podobnie described in example 31.

Example 43

Synthesis of LysB29(N-[ Nthe deletion--carboxy-Glu) des(B30) human insulin.

400 mg of (A1,B1)-di-Boc des(B30) human insulin was dissolved in a mixture of 190 μl of triethylamine and 3000 μl of DMSO. The reaction is initiated by adding 83 mg-carboxy-Glu-N-tetradecane -, di-O-tert-butyl--(OSu) (for example (twico)the stop after 4.5 hours. All other stages of the method were carried out as described in example 34. The protective group is removed from the intermediate product by adding TFUK to clean GHUR with reversed phase and the target product are precipitation and vacuum drying.

Obtain 63 mg of the target compound with a purity of 95.2 per cent.

Molecular weight determined by MS: 6090+6, tiora.: 6091.

The lipophilicity of target compound relative to human insulin k'Rel.determined by the method presented above is 10.

Example 44

Multi-purpose, in uncontrolled conditions, outdoor titration test NB29the deletion of des(B30) human insulin containing 2 ion Zn2+on hexamer insulin (code: NN304) which enter in the mode of the basal dose/bolus to patients suffering from diabetes Type I.

The first objective of experiment: to obtain clinical data using NN304 to define the main directions in determining the dose required for the treatment of patients suffering from diabetes Type I, receiving NPH insulin in mode bolus/basal dose (once or twice a day) for their mode using NN304 (once a day).

In the us/evening dose within 4 to 14 days (measured as the profile of glucose in the blood on 9 targets) with the subsequent 2-week optimization mode bolus/NPH. Monitor the incidence of hypoglycemia during treatment mode bolus/evening dose NN304.

Evaluation method.

Multi-purpose, in uncontrolled conditions, outdoor titration analysis of the experimental group patients with diabetes Type I.

Test subjects

Twenty-two patients (men and women) between 25 and 54 years old, has a negative index on the C-peptide suffering from Type I diabetic for 24 months or more receiving treatment NPH insulin in accordance with the mode bolus/basal (lowest possible dose) once or twice a day.

Treatment method

Individually titrate with 100 U/ml NN304. As basal insulin during test use NPH-insulin. Patients for test use is compatible with food insulin in accordance with individual assignments. Insulin injected by the subcutaneous injection of basal insulin in the thigh, insulin, compatible with food, in the abdominal cavity.

Method test

The test consists of a 2-week+3 days treatment period in mode bolus/basal dose of NPH (once or twice a day) with the subsequent stage tiralongo study the live mode bolus/evening dose NN304 within 2 days and return to the mode bolus/basal dose of NPH (once or twice a day).

The total duration of treatment NN304 is a minimum of 6 days and a maximum of 16 days.

Defined parameters

Efficiency: Express-analysis of glucose in blood, the profile of blood glucose on 9 indicators, the profile of insulin on 9 indicators, HbA1c.

Security: physical condition, patient vital signs, ECG, profile of glucose in the blood, Hematology, biochemistry, fat, urine analysis, pregnancy test, adverse side effects and frequency of hypoglycemia.

Discussion of findings on effectiveness.

Treatment NN304 reduces the level of glucose in the blood to levels that are achieved in the treatment of NPH and only one patient was observed adverse effects in the treatment of NN304.

In General, the required increase in the average dose NN304 2.2 times compared to the dose of NPH to obtain stable glucose levels in the blood, regardless of the day of insulin. However, changes in the dose largely depends on the individual patient and for one of the studied patients required dose NN304 less than the dose of NPH. Observed no differences in average levels of Glu is times a day. However, the introduction of once a day NN304 patients treated with twice-daily NPH, requires increased bolus insulin to obtain metabolic control. Treatment NN304 patients treated with NPH once daily, is effective in relation to metabolic control at slightly reduced bolus insulin. Observed only minor changes in mean levels of blood glucose in patients receiving NPH once daily, in the treatment of NN304 compared with the treatment of NPH. On the basis of obtained data we can conclude about the almost complete lack of differences in the treatment NN304 and treatment of NPH.

1. Derived insulin having the following sequence shown in the graphics part,

where Xaa at positions A21 and B3 are independently any amino acid residue which can be encoded by the genetic code except Lys, Arg and Cys;

Xaa at position B1 is Phe or is deleted; Xaa at position B30 is (a) any amino acid residue which can be encoded by the genetic code except Lys, Arg and Cys, and, in this case-amino group of LysB29has lipophilic Deputy; or (C) is removed, and in this case-amino group of LysB29has lipophilic Deputy; and any of Her Asn, and Xaa at position B1 is Phe, then the derived insulin is a Zn2+complex.

2. Derived insulin p. 1, where Xaa at positions A21 and B3 are independently any amino acid residue which can be encoded by the genetic code except Lys, Arg and Cys; Xaa at position B1 is Phe or is deleted; Xaa at position B30 is deleted or is any amino acid residue which can be encoded by the genetic code except Lys, Arg and Cys, and the-amino group of LysB29has lipophilic Deputy, which includes at least 6 carbon atoms.

3. Derived insulin p. 2, wherein Xaa at position B30 is deleted.

4. Derived insulin p. 2, wherein Xaa at position B30 is Asp, Glu or Thr.

5. Derived insulin p. 2, in which the lipophilic Deputy associated with the-amino group of LysB29is an acyl group derived from carboxylic acids having from 6 to 29 carbon atoms.

6. Derived insulin p. 5, in which the acyl group that may be branched, has a main chain comprising from 8 to 24 carbon atoms.

7. Derived insulin p. 5, in which the acyl group is autoram acyl group is the acyl group of a linear unsaturated carboxylic acid, having from 6 to 24 carbon atoms.

9. Derived insulin p. 5, in which the acyl group is chosen from the group comprising dodekanisou acid, tridecane acid and tetradecanoic acid.

10. Derived insulin p. 1 wherein Xaa at position A21 is Ala, Gln, Gly or Ser.

11. Derived insulin p. 1, in which Xaa at position B3 is Asp, Gln or Thr.

12. Derived insulin p. 1, in which Xaa at position B1 is removed.

13. Derived insulin p. 2, which represents the NB29the deletion of des (B30) human insulin.

14. Derived insulin p. 2, which is a Zn2+complex NB29the deletion of des (B30) human insulin.

15. Derived insulin p. 2, which is a Zn2+complex NB29the deletion of des (B30) human insulin containing 2, 3 or 4 ion Zn2+on hexamer insulin.

16. Derived insulin p. 2, which represents the NB29(lithocholyl-glutamyl) des (B30) human insulin.

17. Derived insulin p. 2, which is a Zn2+complex NB29-(litho is a Zn2+complex NB29(lithocholyl-glutamyl) des (B30) human insulin containing 2, 3 or 4 ion Zn2+on hexamer insulin.

19. Soluble prolonged pharmaceutical composition comprising a derivative of insulin together with a pharmaceutically acceptable carrier, characterized in that as a derivative of insulin it includes a derived under item 1 in a therapeutically effective amount for the treatment of diabetes.

20. The way prolonged the hypoglycemic effects in the treatment of diabetes comprising the administration to a patient a therapeutically effective amount of a derivative of insulin p. 1 together with a pharmaceutically acceptable carrier.

Priorities for items:

17.09.93 on PP.1 - 20;

02.02.94 on PP.1 - 20 (clarification of the claims).

 

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