HK1149772B - Novel insulin derivatives having an extremely delayed time-action profile - Google Patents

Description

Novel insulin derivatives with ultra-delayed time-effect

The present invention relates to novel insulin analogues with basal ageing characteristics, their preparation and use.

The incidence of diabetes has increased year by year in recent years, with a near prevalence. The disease can severely shorten the life expectancy. Diabetic patients must be frequently supplemented with exogenous insulin. It is reasonable to optimize insulin therapy. Different insulins with specific pharmacological properties are now available. In fact, depending on the duration of action, different insulins can be distinguished into short-acting insulins, fast-acting insulins, long-acting insulins and mixed insulins. Long-acting insulins are also known as slow-acting insulins (slow insulins), depot insulins (depot insulins) or basal insulins (basal insulins). The active ingredient in many of these insulin products is a so-called insulin analogue, which is prepared from human insulin by substitution, deletion and/or addition of one or more amino acids. The terms "insulin analogue" and "insulin" have the same meaning herein.

Intensive insulin therapy strategies attempt to reduce health risks by targeting stable control of blood glucose levels through early administration of basal insulin. One example of a basal insulin is a drug(active ingredients: glargine ═ Gly (a21), Arg (B31), Arg (B32) human insulin). Development of novel improvementsThe general goal of basal insulins is to minimize hypoglycemic events. In this regard, the ideal basal insulin is insulin that works reliably for at least 24 hours in each patient. The ideal insulin potency should delay the onset and the aging profile should be as gradual as possible to minimize the risk of transient hypoglycemia and allow insulin to be administered even without prior intake. The basal insulin supply is good when the insulin potency lasts as long as possible at the same level, i.e. the body gets a constant amount of insulin supply. The risk of hypoglycemic events is thus reduced and patient-specific and date-specific differences are minimized. Thus, the pharmacokinetic profile of the ideal basal insulin is characterized by a delayed onset of action and a delayed action, i.e., a long-lasting and uniform action.

However, despite the therapeutic advantages that have been achieved, the slow acting insulins so far disclosed do not show the pharmacokinetic properties of the ideal basal insulin. The aging characteristics of the target insulin should be as gradual and long-lasting as possible to further reduce the risk of hypoglycemic events and date-dependent differential risk in the patient and to further extend the duration of action so that daily administration of insulin is no longer necessary under certain circumstances. This simplifies the treatment of diabetic patients, especially elderly diabetic patients and diabetic patients requiring care, who are no longer able to inject insulin themselves, and will therefore also have great economic benefits. In addition, such basal insulins are also beneficial in early stage type II diabetes. Clinicians report that many people are frightened about injections and are unable to begin insulin therapy in a timely manner. As a result, blood glucose cannot be controlled well, resulting in the late-stage insulin sequelae. Basal insulin reduces the number of insulin doses given by injection, making insulin therapy more acceptable to patients.

Kohn et al (peptides 28(2007)935-948) describe how the pharmacodynamics of insulin can be optimized by preparing insulin analogues whose isoelectric point is shifted towards basicity compared to the isoelectric point of human insulin (pI ═ 5.6) by adding lysine or arginine at the end of the B chain or at the N-terminus of the a and B chains, thus reducing solubility under physiological conditions and extending the aging profile. In this regard, compound 18 in Kohn et al (Arg (a0), Gly (a21), Arg (B31), Arg (B32) human insulin (experimentally determined pI 7.3; calculated pI 7.58) is described as the best compound ideal in context.

The design goals of the novel insulin analogues are contrary to the approach of substituting neutral amino acids of human insulin with acidic amino acids and/or adding acidic amino acids, as such substitutions and/or additions at least partially counteract the effect of introducing positively charged amino acids. However, it has now surprisingly been found that the described target basal ageing profile is obtained with an insulin analogue which is characterised by:

the B chain end consists of an amidated basic amino acid residue such as lysinamide or argininamide, and,

the N-terminal amino acid residue of the A chain of insulin is a lysine or arginine residue, and

the amino acid position A8 is occupied by a histidine residue, an

The A21 amino acid position being occupied by a glycine residue, i.e.on the amidated basic amino acid residue at the end of the B chain, the carboxyl group of the terminal amino acid being present in its amidated form, and

substitution of two neutral amino acids with acidic amino acids at positions A5, A15, A18, B-1, B0, B1, B2, B3 and B4, addition of two negatively charged amino acid residues or one such substitution and one such addition.

Since the first three features mentioned above tend to increase the pI of the corresponding insulin analogue by introducing a positive charge or eliminating a negative charge, the last mentioned substitution and/or addition of negatively charged amino acid residues has the opposite effect and can reduce the pI. Surprisingly, it is precisely this insulin analogue that has the advantage of the targeted ageing characteristics. The pI values of these compounds are lower than those of compound 18(Arg (A0), Gly (A21), Arg (B31), Arg (B32) human insulin) in Kohn et al, but still show a delayed onset of action and a longer duration of action, i.e. a very mild, long-lasting, homogeneous action profile. Thus, the risk of hypoglycemic events is significantly reduced. The delay is so significant that the efficacy can surprisingly be detected in an experimental model in rats, although by contrast the delayed action of insulin glargine cannot be clearly observed in rats. Fig. 1 shows the hypoglycemic effect of compound YKL205 of the present invention compared to that of insulin glargine. Similar results were obtained in dogs (see figure 2). Thus, a new type of basal insulin is provided which can be administered at a significantly lower frequency. In addition to these described pharmacokinetic advantages, the analogues of the invention have significantly better pharmacological properties, such as receptor specificity and in vitro mitogenicity, than insulin glargine. The claimed insulin also has physicochemical advantages.

Accordingly, the present invention relates to insulin analogues of the general formula I

Wherein

A0 corresponds to Lys or Arg;

a5 corresponds to Asp, Gln or Glu;

a15 corresponds to Asp, Glu or Gln;

a18 corresponds to Asp, Glu or Asn;

b-1 corresponds to Asp, Glu or amino;

b0 corresponds to Asp, Glu or a bond;

b1 corresponds to Asp, Glu or Phe;

b2 corresponds to Asp, Glu or Val;

b3 corresponds to Asp, Glu or Asn;

b4 corresponds to Asp, Glu or Gln;

b29 corresponds to Lys or a chemical bond;

b30 corresponds to Thr or a bond;

b31 corresponds to Arg, Lys or a chemical bond;

b32 corresponds to Arg-amide, Lys-amide or amino.

Wherein two amino acid residues of the group comprising A5, A15, A18, B-1, B0, B1, B2, B3 and B4 correspond to Asp or Glu simultaneously and independently of each other.

The invention especially relates to insulin analogues as detailed above, wherein independently of each other a0 corresponds to Arg, or wherein a5 corresponds to Glu, or wherein a15 corresponds to Glu, or wherein a18 corresponds to Asp, or wherein B-1 corresponds to amino, or wherein B0 corresponds to Glu, or wherein B1 corresponds to Asp, or wherein B2 corresponds to Val, or wherein B3 corresponds to Asp, or wherein B4 corresponds to Glu, or wherein B29 corresponds to Lys, or wherein B30 corresponds to Thr, or wherein B31 corresponds to Arg or Lys.

The present invention particularly preferably relates to insulin analogues selected from the group consisting of:

Arg(A0),His(A8),Glu(A5),Asp(A18),Gly(A21),Arg(B31),Arg(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A5),Asp(A18),Gly(A21),Arg(B31),Lys(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A15),Asp(A18),Gly(A21),Arg(B31),Arg(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A15),Asp(A18),Gly(A21),Arg(B31),Lys(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A5),Glu(A15),Gly(A21),Arg(B31),Arg(B32)–NH₂human pancreasThe number of the island elements is equal to that of the island elements,

Arg(A0),His(A8),Glu(A5),Glu(A15),Gly(A21),Arg(B31),Lys(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A5),Gly(A21),Asp(B3),Arg(B31),Arg(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A5),Gly(A21),Asp(B3),Arg(B31),Lys(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A15),Gly(A21),Asp(B3),Arg(B31),Arg(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A15),Gly(A21),Asp(B3),Arg(B31),Lys(B32)–NH₂human insulin,

Arg(A0),His(A8),Asp(A18),Gly(A21),Asp(B3),Arg(B31),Arg(B32)–NH₂human insulin,

Arg(A0),His(A8),Asp(A18),Gly(A21),Asp(B3),Arg(B31),Lys(B32)–NH₂human insulin,

Arg(A0),His(A8),Gly(A21),Asp(B3),Glu(B4),Arg(B31),Arg(B32)–NH₂human insulin,

Arg(A0),His(A8),Gly(A21),Asp(B3),Glu(B4),Arg(B31),Lys(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A5),Gly(A21),Glu(B4),Arg(B31),Arg(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A5),Gly(A21),Glu(B4),Arg(B31),Lys(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A15),Gly(A21),Glu(B4),Arg(B31),Arg(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A15),Gly(A21),Glu(B4),Arg(B31),Lys(B32)–NH₂human insulin,

Arg(A0),His(A8),Asp(A18),Gly(A21),Glu(B4),Arg(B31),Arg(B32)–NH₂human insulin,

Arg(A0),His(A8),Asp(A18),Gly(A21),Glu(B4),Arg(B31),Lys(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A5),Gly(A21),Glu(B0),Arg(B31),Arg(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A5),Gly(A21),Glu(B0),Arg(B31),Lys(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A15),Gly(A21),Glu(B0),Arg(B31),Arg(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A15),Gly(A21),Glu(B0),Arg(B31),Lys(B32)–NH₂human insulin,

Arg(A0),His(A8),Asp(A18),Gly(A21),Glu(B0),Arg(B31),Arg(B32)–NH₂human insulin,

Arg(A0),His(A8),Asp(A18),Gly(A21),Glu(B0),Arg(B31),Lys(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A5),Gly(A21),Asp(B1),Arg(B31),Arg(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A5),Gly(A21),Asp(B1),Arg(B31),Lys(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A15),Gly(A21),Asp(B1),Arg(B31),Arg(B32)–NH₂human insulin,

Arg(A0),His(A8),Glu(A15),Gly(A21),Asp(B1),Arg(B31),Lys(B32)–NH₂human insulin,

Arg(A0),His(A8),Asp(A18),Gly(A21),Asp(B1),Arg(B31),Arg(B32)–NH₂human insulin,

Arg(A0),His(A8),Asp(A18),Gly(A21),Asp(B1),Arg(B31),Lys(B32)–NH₂human insulin,

Arg(A0),His(A8),Gly(A21),Glu(B0),Asp(B1),Arg(B31),Arg(B32)–NH₂human insulin,

Arg(A0),His(A8),Gly(A21),Glu(B0),Asp(B1),Arg(B31),Lys(B32)–NH₂human insulin,

Arg(A0),His(A8),Asp(A18),Gly(A21),Asp(B3),Arg(B30),Arg(B31)–NH₂human insulin,

Arg(A0),His(A8),Asp(A18),Gly(A21),Asp(B3),Arg(B30),Lys(B31)–NH₂human insulin.

The term "human insulin" specified in the names of the mentioned insulin analogues refers to the amino acid sequence of the a-and B-chains of human insulin, all derivatives (additions, substitutions, deletions) of human insulin being indicated in the insulin analogues of the given names.

The invention further relates to a process for the preparation of an insulin analogue as described above, in particular wherein an insulin analogue precursor is recombinantly prepared, which precursor is enzymatically processed to a double-stranded insulin, which insulin analogue is isolated by coupling to argininamide in the presence of an enzyme having trypsin activity.

The invention further relates to the use of an insulin analogue as described above for the preparation of a medicament for the treatment of diabetes, in particular type I or type II diabetes. The invention also relates to the use of an insulin analogue as described above for the preparation of a medicament for assisting the regeneration of beta cells.

The invention further relates to a medicament comprising an insulin analogue and/or a physiologically acceptable salt thereof as described above.

The invention further relates to a formulation of an insulin analogue as described above, wherein the formulation is in the form of an aqueous solution comprising the dissolved insulin analogue.

The invention further relates to a formulation of an insulin analogue as described above, wherein the formulation is in powder form.

The invention further relates to a formulation as described above, wherein the insulin analogue as described above is in a crystalline and/or amorphous form.

The invention further relates to a formulation of an insulin analogue as described above, wherein the formulation is in the form of a suspension.

The invention further relates to a formulation of an insulin analogue as described above, wherein the formulation further comprises a chemical chaperone molecule.

The invention further relates to DNA encoding an insulin analogue precursor as described above, or encoding an insulin analogue a-chain or B-chain as described above.

The invention further relates to a vector comprising a DNA as described above.

The invention further relates to a host organism comprising a DNA as described above or a vector as described above.

The invention further relates to a preproinsulin analogue wherein the C-peptide carries an arginine residue at its N-terminus and two arginine residues or an arginine residue and a lysine residue at its C-terminus, and in the latter case the lysine residue forms the true C-terminus.

The present invention further relates to a formulation as described above additionally comprising glucagon-like peptide 1(GLP1) or an analogue or derivative thereof, or exendin-3 or 4 or an analogue or derivative thereof, preferably exendin-4.

The present invention further relates to a formulation as described above, wherein the analogue of exendin-4 is selected from the group consisting of:

H-desPro³⁶-exendin-4-Lys₆-NH₂，

H-des(Pro^36,37)-exendin-4-Lys₄-NH₂or

H-des(Pro^36,37)-exendin-4-Lys₅-NH_2，

Or a pharmaceutically tolerable salt thereof.

The present invention further relates to a formulation as described above, wherein the analogue of exendin-4 is selected from the group consisting of:

desPro³⁶[Asp²⁸]exendin-4(1-39),

desPro³⁶[IsoAsp²⁸]exendin-4(1-39),

desPro³⁶[Met(O)¹⁴,Asp²⁸]exendin-4(1-39),

desPro³⁶[Met(O)¹⁴,IsoAsp²⁸]exendin-4(1-39),

desPro³⁶[Trp(O₂)²⁵,Asp²⁸]exendin-2(1-39),

desPro³⁶[Trp(O₂)²⁵,IsoAsp²⁸]exendin-2(1-39),

desPro³⁶[Met(O)¹⁴Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39) and

desPro³⁶[Met(O)¹⁴Trp(O₂)²⁵,IsoAsp²⁸]exendin-4(1-39)，

or a pharmaceutically tolerable salt thereof.

The invention further relates to a formulation as described in the preceding paragraph, wherein the peptide-Lys₆-NH₂Is attached to the C-terminus of the exendin-4 analog.

The present invention further relates to a formulation as described above, wherein the exendin-4 analogue is selected from the group consisting of:

H-(Lys)₆-des Pro³⁶[Asp²⁸]exendin-4(1-39)-Lys₆-NH₂

des Asp²⁸Pro³⁶,Pro³⁷,Pro₃₈exendin-4(1-39)-NH₂,

H-(Lys)₆-des Pro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-NH₂,

H-Asn-(Glu)₅des Pro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-NH₂,

des Pro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-des Pro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-des Pro³⁶[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,

H-des Asp²⁸ Pro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵]exendin-4(1-39)-NH₂,

H-(Lys)₆-des Pro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-NH₂,

H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-NH₂,

des Pro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-des Pro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-des Pro³⁶[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,

des Met(O)¹⁴ Asp²⁸ Pro ³⁶,Pro³⁷,Pro³⁸ exendin-4(1-39)-NH₂,

H-(Lys)₆-des Pro³⁶,Pro ³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-NH₂,

H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-NH₂,

des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,

H-Asn-(Glu)₅ des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-des Pro³⁶[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,

des Asp²⁸ Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵]exendin-4(1-39)-NH₂,

H-(Lys)₆-des Pro^36,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-NH₂,

H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-NH₂,

des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-des Pro^36,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂

or a pharmaceutically tolerable salt thereof.

The invention further relates to a preparation as described above, additionally comprising Arg³⁴,Lys²⁶(N(Gamma glutamyl (N)^α-hexadecanoyl))) GLP-1(7-37) [ liraglutide]Or a pharmaceutically tolerable salt thereof.

In this regard, it will be clear to those skilled in the art that the insulin of the present invention may be a pharmaceutical product that has a beneficial effect upon administration. In this regard, the aqueous solution is the starting point. Accordingly, the other components must be miscible. The risk of viral animal contamination is minimized because the preparation should not contain any animal-derived components. It is further advantageous to add a preservative to prevent microbial contamination. Possible adverse effects of the preparation on the physiology of the tissue cells at the site of administration can be eliminated by the addition of isotonic agents. The addition of protamine may have a stabilizing effect, so that an essentially salt-free insulin preparation can be obtained by adding protamine to the formulation. The addition of a phenol component stabilizes the structure of the insulin analogue used and thus additionally brings about, among other things, a delayed action on the onset of action. Substances which stabilize the spatial structure of the slow acting insulin of the invention may also be added to the formulation and provide greater thermal stability. Such chemical chaperones may for example be short synthetic peptides which may also comprise amino acid analogues or peptide sequences including for example insulin C-peptide origin.

The insulin of the present invention can be loaded into nanoparticles to develop depot insulins. So-called sustained release formulations are also possible, wherein the slow acting insulin of the invention is reversibly bound to a polymeric carrier.

The insulin of the present invention can be administered in parallel with: fast acting insulins such asOr insulin derivatives in the research or formulations or inhaled insulin with appropriate time-lapse characteristics, or insulin in the research administered nasally or orally. It will be clear to the skilled person that a mixture of a suitably formulated fast acting insulin and a slow acting insulin of the invention may also be used for this purpose in this connection. The insulin analogues of the invention can further be used in pharmaceutical preparations comprising peptides having an activity comparable to GLP-1 (glucagon-like peptide 1) or exendin-4 or exendin-3. Examples of such peptides are GLP-1(7-37), exenatideOr peptides of which the preparation process is disclosed in patent applications WO2006/058620, WO 2001/04156, WO 2004/005342 and WO 98/08871. Particularly advantageous formulations in this regard are those containing depot forms of these peptides. Particularly advantageous types of treatment in the initial phase of type II diabetes are those that provide for parallel administration of the medicament of the invention, which increases insulin efficacy, such as metformin. Such as increasing pancreatic insulinCombinations of secreted sulfonylureas are also possible, as are combination therapies with dipeptidyl peptidase-4 inhibitors that increase incretin levels. The use of the slow acting insulins of the invention is particularly advantageous when regeneration of pancreatic beta cells derived from appropriate stem cells is initiated by administration of differentiation factors. All these applications are mentioned by way of example for the treatment of diabetes and the invention relates equally to these applications. The present invention therefore further relates to the use of the insulins according to the invention in combination with other active ingredients for the treatment of diabetes, in particular type I or type II diabetes. .

The invention further relates to a medicament comprising the insulin analogue of the invention, in particular in the form of an aqueous solution formulation or a powder.

The medicament is a pharmaceutical preparation in solution or suspension, preferably for injection purposes; characterized in that it comprises at least one insulin analogue according to the invention, and/or at least one physiologically tolerable salt thereof, in dissolved, non-crystalline and/or crystalline form, preferably in dissolved form.

The pH of the preparation is preferably between about 2.5 and 8.5, in particular between 4.0 and 8.5, and comprises suitable tonicity agents, suitable preservatives and, where appropriate, suitable sterile buffered aqueous solutions, also preferably sterile buffered aqueous solutions of a particular zinc ion concentration. All ingredients of the preparation other than the active ingredient constitute the carrier of the preparation. Examples of suitable tonicity agents are e.g. glycerol, glucose, mannitol, NaCl, calcium compounds or magnesium compounds such as CaCl₂And the like. The choice of tonicity agent and/or preservative may influence the solubility of the insulin of the present invention or a physiologically tolerable salt thereof at mildly acidic pH conditions.

Examples of suitable preservatives are phenol, m-cresol, benzyl alcohol and/or p-hydroxybenzoate.

Examples of buffering substances that can be used in particular to adjust the pH to between about 4.0 and 8.5 are sodium acetate, sodium citrate, sodium phosphate, and the like. In addition, physiologically acceptable dilute acids (typically HCl) or dilute bases (typically NaOH) are also suitable for adjusting the pH.

If the product contains zinc, a concentration of 1 to 2mg/ml, especially 1 to 200. mu.g/ml, is preferred. Surprisingly, the addition of zinc can satisfactorily affect the action profile of the insulin analogues of the present invention. This allows the production of articles with different characteristics of total duration of action, speed of onset of action and efficacy profile, thus enabling individualized stabilization of the patient. Another possibility is created by a "dual chamber insulin device" which allows to administer a formulation with a fast onset of action and/or a slow, gradual onset of action depending on the living situation.

In order to modify the active ingredient characteristics of the preparations according to the invention, unmodified insulin, preferably bovine insulin, porcine insulin or human insulin, in particular human insulin, or insulin analogues and derivatives thereof, may also be admixed. It is also possible to mix one or more exendin-4 derivatives or peptides, characterized in that their activity is comparable to GLP-1 (glucagon-like peptide 1) or corresponds directly to GLP-1. The invention also relates to such a medicament (preparation).

Preferred active ingredient concentrations are those of about 1 to 1500, more preferably about 5 to 1000, especially about 40 to 400 international units/ml.

Insulin analogues of the invention were initially prepared using biotechnology as precursors that did not yet include an amide. The skilled person is familiar with techniques for the preparation of insulin in large quantities. Host cell systems for this purpose are bacteria, yeasts and plants or plant cells which are used for the cultivation by fermentation. Expression systems with animal cells as host systems may also be used, if cost considerations permit. However, a prerequisite is to ensure that no animal viruses are present. It is therefore clear that the expression systems described using the examples represent only a small part of the host/vector systems developed for the recombinant production of proteins. For example, biotechnological methods based on yeast or plant systems such as mosses, algae or higher plants such as tobacco, pea, safflower, barley, corn or rape (oil seed rape) are not described in the present application. However, the invention likewise comprises host/vector systems and encoding DNA sequences which allow the preparation of the peptides of interest in suitable biotechnological expression systems. Thus, the host organism may in particular be selected from the kingdom Plantae, from the organisms of the phylum Schizophyta comprising Schizomycetes, bacteria or cyanobacteria, from the class Vibrio of the class Dictyophyta, from the class Vibrio of the class Dictyophyta, from the class Trichophyta, from the class Hymenomycetes, from the class Variophytes of the class VII, and from the class VII of the phylum Heterophytes.

European patent application EP-A1222207 discloses a plasmid pINT358d encoding preproinsulin comprising a modified C-peptide. The sequence encoding proinsulin can now be specifically modified by means of the Polymerase Chain Reaction (PCR) so that preproinsulin, which can be an insulin precursor according to the invention, can be expressed. The corresponding fusion protein need not be prepared intracellularly. It will be clear to the skilled person that such proteins can also be produced using bacterial expression systems which are subsequently secreted into the periplasm and/or the culture supernatant. This is disclosed by way of example in European patent application EP-A1364029. The invention also relates to proinsulin precursors that can produce the analogs of the invention.

In principle, the proinsulin thus prepared can be converted into an insulin analogue precursor which comprises lysine or arginine in position A0 and carries lysine or arginine at the C-terminus of the B chain.

If the proinsulin of the invention is expressed intracellularly in bacteria in inclusion body or soluble form, these precursors must be folded into the correct conformation using in vitro folding techniques before processing and biochemical modification can be carried out. In this regard, the disclosed fusion proteins can be folded directly after denaturation with urea or guanidine hydrochloride, and the invention also relates to folding intermediates.

The concentration of individual intermediates is carried out using biochemical methods, in particular separation methods which employ the principles published and in fact subject of textbooks. It will be clear to the skilled person that such principles may thus be combined and result in a method not previously disclosed with this result. Thus, the invention also relates to methods by which the analogs of the invention can be purified.

The invention further relates to a process for the preparation of an insulin analogue according to the invention, wherein an insulin analogue precursor is prepared by recombinant means and converted enzymatically into a double-stranded insulin precursor carrying an arginine or lysine at the N-terminus in relation to amino acid 1 of the a-chain and a lysine or arginine residue at the C-terminus of the B-chain, which is converted in the presence of an enzyme with trypsin activity into an amide with argininamide or lysinamide (lysinamide) and thus converted into the slow-acting insulin according to the invention, and wherein the insulin analogue according to the invention is then prepared in high purity by means of a biochemical purification process.

Protein "analogs" refer to proteins that differ by the substitution of at least one natural amino acid residue of the corresponding or identical natural protein with another amino acid residue and/or the addition and/or deletion of at least one amino acid residue of the corresponding or identical natural protein. In this regard, non-natural amino acid residues may also be added and/or substituted for amino acid residues.

A "derivative" of a protein refers to a protein obtained by chemically modifying certain amino acid residues of the starting protein. Chemical modifications may, for example, consist of the addition of one or more specific chemical groups to one or more amino acids.

Drawings

FIG. 1: hypoglycemic potency of novel insulin analogues in rats

FIG. 2: hypoglycemic efficacy of novel insulin analogues in dogs

FIG. 3: hypoglycemic potency of YKL205 in dogs

FIG. 4: zinc dependence of the hypoglycemic potency of YKL205 in dogs

The following examples are intended to illustrate the concept of the invention and not to limit the invention.

Example 1: preparation of derivative vector pINT3580 encoding Gly (A21) -insulin and modified C peptide with Arg Arg at C/A chain interface

European patent application EP-A1222207 discloses plasmids pINT358d, pINT91d and a primer sequence Tir. The DNA of these products was used to construct plasmid pINT 3580. Plasmid pINT358d is also characterized by a gene sequence encoding a modified C-peptide with specific properties. Three primer sequences were synthesized:

pint3580_glya21rev

5′-CAAAGGTCGACTATTAGCAGTAGTTCTCCAGCTGG-3′(SEQ ID NO：3)

this primer was used to introduce a glycine (bold, underlined) instead of an asparagine at position 21 of the a chain of the proinsulin sequence encoded by pINT358 d.

arg_cjuncf

5′-GTCCCTGCAGCGCGGCATCGTGGAGCAG-3′(SEQ ID NO：4)

This primer functions as the arg _ cjunc _ rev primer for the introduction of arginine rather than lysine at the insulin A/B chain junction.

arg_cjunc_rev

5′-CCACGATGCC GCGCTGC AGGGACCCCT CCAGCG-3′(SEQ ID NO：5)

In both primers, the arginine codon to be introduced is indicated in bold. PCR was carried out according to the method of European patent application EP-A1222207 using the DNA of primer pairs Tir/arg _ cjunc _ rev and arg _ cjuncf/pINT3580_ glya21rev and plasmid pINT358d, respectively, as templates. Aliquots of the two reaction products were combined and used in a third PCR with the primer pair Tir/pint3580_ glya21 rev. The reaction product was purified after separation of the reaction mixture by gel electrophoresis in one or the same reaction according to the manufacturer's instructions, digested with the restriction enzyme SalI/NcoI, and the reaction mixture was separated by gel electrophoresis to isolate the DNA fragment encoding the proinsulin sequence. This fragment was then inserted into the NcoI/SalI cut pINT91d vector DNA using a DNA ligase reaction.

Coli competent cells were transformed with the ligation mixture. The transformation mixture was spread on selection plates containing 25mg/l ampicillin. Plasmid DNA was isolated from the growing clones and identified by DNA sequence analysis. The correct plasmid was called pINT 3580.

Example 2: construction of a plasmid pINT3581 encoding His (A8), Gly (A21) -preproinsulin

The construction was completed with 3 polymerase chain reactions as described in example 1. The product of the third reaction was digested with NcoI/SalI and inserted into pINT91d vector DNA which was digested with NcoI/SalI. Primers Tir and pint3580_ glya21rev were used. Two additional primers were synthesized:

pint3580_Ha8f

5’-AGCAGTGCTGCAGCATCTGCTCCCTCTAC-3’(SEQ ID NO：6)

pint3580_Ha8rev

5’-GAG CAGATGCTGCAGCACTG CTCCACGATG-3’(SEQ ID NO：7)

the codon encoding the histidine 8 at position of the A chain is emphasized by bolding each letter. This construction was carried out as described in example 1. The template for PCR1 and 2 was DNA of plasmid pINT 3580. PCR1 and PCR2 were performed with primer pairs Tir/pint3580_ Ha8rev and pint3580_ Ha8f/pint3580_ glya21rev, respectively. PCR3 used the primer pair Tir/pint3580_ glya21 rev. The template for this reaction was a mixture of PCR1 and PCR2 reaction products. The correct plasmid was called pINT 3581.

Example 3: construction of the plasmid pINT3582 encoding His (A8), Glu (A5), Gly (A21) -preproinsulin

The construction was carried out using 3 polymerase chain reactions as described in examples 1 and 2. The product of the third reaction was digested with NcoI/SalI and inserted into the NcoI/SalI cut pINT91d vector DNA. Primers Tir and pint3580-glya21rev were used. Two additional primers were synthesized.

pint3581_Ea5f

5′GCATCGTGGAGTGCTGCCACAGCATCTG 3′(SEQ ID NO：8)

pint3581_Ea5rev

5’-CTGT GGCAGCACTCCACGATG CCGCGACG-3’(SEQ ID NO：9)

The codon encoding the 5 glutamic acid at position of the A chain is emphasized by bolding each letter. The construction was carried out as described in example 1. The template is DNA of a plasmid pINT 3581. The correct plasmid was called pINT 3582.

Example 4: construction of a plasmid pINT3583 encoding His (A8), Asp (A18), Gly (A21) -preproinsulin

This construction was carried out using only one polymerase chain reaction, unlike example 1. The product of this reaction was digested with NcoI/SalI and inserted into the NcoI/SalI cut pINT91d vector DNA. The primer Tir was used. An additional primer was synthesized:

pint3580_Da18rev

5′CAAAGGTCGACTATTAGCCGCAGTACTCCAGCTGGTAGAGGGAG 3′(SEQ ID NO：10)

the bold emphasizes the codon encoding the aspartic acid at position 18 of the A chain. The template was DNA of plasmid pINT 3581. The correct plasmid was called pINT 3583.

Example 5: construction of the plasmid pINT3584 encoding His (A8), Glu (A5) Asp (A18), Gly (A21) -preproinsulin

Unlike example 1, this construction method was accomplished using only one polymerase chain reaction. The product of this reaction was digested with NcoI/SalI and inserted into pINT91d vector DNA which was digested with NcoI/SalI. Primer tir. pint3580_ Da18rev (ex.4) was used. The template was DNA of plasmid pINT 3582. The correct plasmid was called pINT 3584. The preproinsulin encoded by this plasmid is a precursor of compound YKL205-1, amidated with argininamide and has the following structure:

Arg(A0),Glu(A5),His(A8),Asp(A18),Gly(A21),Arg(B31),Arg(B32)-NH₂human insulin.

Amidation with lysyl amine gives the compound YKL205-1 b:

Arg(A0),Glu(A5),His(A8),Asp(A18),Gly(A21),Arg(B31),Lys(B32)-NH₂human insulin.

Example 6: construction of the plasmid pINT3585 encoding His (A8), Glu (A15), Gly (A21) -preproinsulin

This construction was carried out using only one polymerase chain reaction, unlike example 1. The product of this reaction was digested with NcoI/SalI and inserted into the NcoI/SalI cut pINT91d vector DNA. The primer Tir was used. An additional primer was synthesized:

pint3580_Ea15rev

5′-CAAAGGTCGA CTATTAGCCG CAGTAGTTCTCCAGGTA GAGGGAGCAGATGCTG-3′(SEQ ID NO：11)

the codon encoding the glutamic acid at position 15 of the A chain is highlighted in bold. The template was DNA of plasmid pINT 3581. The correct plasmid was called pINT 3585.

Example 7: construction of a plasmid pINT3586 encoding His (A8), Glu (A15), Asp (A18), Gly (A21) -preproinsulin

Unlike example 1, this construction method was carried out using only one polymerase chain reaction. The product of this reaction was digested with NcoI/SalI and inserted into pINT91d vector DNA which was digested with NcoI/SalI. The primer Tir was used. An additional primer was synthesized:

pint3585_Ea15_Da18rev

5′-CAAAGGTCGACTATTAGCCGCAGTACTCCAGGTAGAGGGAGCAGATGCTG-3′(SEQ ID NO：12)

the codons for glutamic acid at position 15 of the A chain and aspartic acid at position A18 of the A chain are emphasized by bolding each letter. The template was DNA of plasmid pINT 3581. The correct plasmid was called pINT 3586. The preproinsulin encoded by this plasmid is a precursor of compound YKL205, amidated with argininamide and has the following structure:

Arg(A0),His(A8),Glu(A15),Asp(A18),Gly(A21),Arg(B31),Arg(B32)–NH₂human insulin.

The preproinsulin encoded by this plasmid is a precursor of compound YKL205b, amidated with lysyl amine and having the following structure:

Arg(A0),His(A8),Glu(A15),Asp(A18),Gly(A21),Arg(B31),Lys(B32)–NH₂human insulin.

Example 8: construction of the plasmid pINT3587 encoding Glu (A5), His (A8), Glu (A15), Gly (A21) -preproinsulin

Unlike example 1, this construction was carried out using only one polymerase chain reaction. The product of this reaction was digested with NcoI/SalI and inserted into the NcoI/SalI cut pINT91d vector DNA. Primer Tir and primer pint3580_ Ea15rev described in example 6 were used. The template was DNA of plasmid pINT 3582. The correct plasmid was called pINT 3587. The preproinsulin encoded by this plasmid is a precursor of compound YKL205-2, amidated with argininamide and has the following structure:

Arg(A0),Glu(A5),His(A8),Glu(A15),Gly(A21),Arg(B31),Arg(B32)–NH₂human insulin.

The preproinsulin encoded by this plasmid is a precursor of compound YKL205-2b, amidated with lysyl amine and has the following structure:

Arg(A0),Glu(A5),His(A8),Glu(A15),Gly(A21),Arg(B31),Lys(B32)–NH₂human insulin.

Example 9: construction of a plasmid pINT3588 encoding His (A8), Gly (A21), Asp (B3) -preproinsulin

The construction was completed with 3 polymerase chain reactions as described in examples 1 and 2. The product of the third reaction was digested with NcoI/SalI and inserted into the NcoI/SalI cut pINT91d vector DNA. Primers Tir and pint3580_ glya21rev were used. Two additional primers were synthesized:

pint3581_Db3f

5′-GCACGATTTGTGCAGCACCTGTGCGGC-3′(SEQ ID NO：13)

pint3581_Db3rev

5′-CACAGG TGCTGCA CAAATCGTGC CGAATTTC-3′(SEQ ID NO：14)

the codon encoding the 3 rd aspartic acid at position of the insulin B chain is emphasized by the bolding of each letter. The construction was carried out as described in example 1. The template was DNA of plasmid pINT 3581. The correct plasmid was called pINT 3588.

Example 10: construction of the plasmid pINT3589 encoding Glu (A5), His (A8), Gly (A21), Asp (B3) -preproinsulin

The reaction was carried out as described in example 9, but using DNA of the plasmid pINT3582 as template in PCR1 and PCR2, the product was plasmid pINT 3589.

The preproinsulin encoded by this plasmid is a precursor of compound YKL205-3, amidated with argininamide and has the following structure:

Arg(A0),Glu(A5),His(A8),Gly(A21),Asp(B3),Arg(B31),Arg(B32)–NH₂human insulin.

The preproinsulin encoded by this plasmid is a precursor of compound YKL205-3b, amidated with lysyl amine and has the following structure:

Arg(A0),Glu(A5),His(A8),Gly(A21),Asp(B3),Arg(B31),Lys(B32)–NH₂human insulin.

Example 11: construction of the plasmid pINT3590 encoding His (A8), Glu (A15), Gly (A21), Asp (B3) -preproinsulin

The reaction was carried out as described in example 9, but using DNA of the plasmid pINT3585 as template in PCR1 and PCR2, the product was plasmid pINT 3590. The preproinsulin encoded by this plasmid is a precursor of compound YKL205-4, amidated with argininamide and has the following structure:

Arg(A0),His(A8),Glu(A15),Gly(A21),Asp(B3),Arg(B31),Arg(B32)–NH₂human insulin.

The preproinsulin encoded by this plasmid is a precursor of compound YKL205-4b, amidated with lysyl amine and has the following structure:

Arg(A0),His(A8),Glu(A15),Gly(A21),Asp(B3),Arg(B31),Lys(B32)–NH₂human insulin.

Example 12: construction of a plasmid pINT3591 encoding His (A8), Asp (A18), Gly (A21), Asp (B3) -preproinsulin

The reaction was carried out as described in example 9, but using DNA of the plasmid pINT3586 as template in PCR1 and PCR2, the product was plasmid pINT 3591. The preproinsulin encoded by this plasmid is a precursor of compound YKL205-5, amidated with argininamide and has the following structure:

Arg(A0),His(A8),Asp(A18),Gly(A21),Asp(B3),Arg(B31),Arg(B32)–NH₂human insulin.

The preproinsulin encoded by this plasmid is a precursor of compound YKL205-5b, amidated with lysyl amine and has the following structure:

Arg(A0),His(A8),Asp(A18),Gly(A21),Asp(B3),Arg(B31),Lys(B32)–NH₂human insulin.

Example 13: construction of the plasmid pINT3592 encoding His (A8), Gly (A21), Asp (B3) -Glu (B4) -preproinsulin

The construction was carried out using 3 polymerase chain reactions as described in examples 1 and 2. The product of the third reaction was digested with NcoI/SalI and inserted into the NcoI/SalI cut pINT91d vector DNA. Primers Tir and pint3580_ glya21rev were used. Two additional primers were synthesized:

pint3581_Db3_Eb4f

5′-GCACGATTTGTGCACCTGTGCGGCTC-3′(SEQ ID NO：15)

pint3581_Db3_Eb4rev

5′-CGCACAGG TGCA CAAATCGTGC CGAATTTC-3′(SEQ ID NO：16)

the codons encoding aspartic acid at position 3 and glutamic acid at position 4 of the insulin B chain are emphasized by bolding each letter. The construction was carried out as described in example 1. The template was DNA of plasmid pINT 3581. The correct plasmid was called pINT 3592. The preproinsulin encoded by this plasmid is a precursor of compound YKL202-6, amidated with argininamide and has the following structure:

Arg(A0),His(A8),Gly(A21),Asp(B3),Glu(B4),Arg(B31),Arg(B32)-NH₂human insulin.

The preproinsulin encoded by this plasmid is a precursor of compound YKL202-6b, amidated with lysyl amine and has the following structure:

Arg(A0),His(A8),Gly(A21),Asp(B3),Glu(B4),Arg(B31),Lys(B32)–NH₂human insulin

Example 14: construction of plasmid pINT3593 encoding His (A8), Gly (A21), Glu (B4) preproinsulin

The construction was completed with 3 polymerase chain reactions as described in examples 1 and 2. The product of the third reaction was digested with NcoI/SalI and inserted into the NcoI/SalI cut pINT91d vector DNA. Primers Tir and pint3580_ glya21rev were used. Two additional primers were synthesized:

pint3581_Eb4f

5′-ACGATTTGTGAACCACCTGTGCGGCTC-3′(SEQ ID NO：17)

pint3581_Eb4rev

5′-CGCACAGG TGGTTCA CAAATCGTGC CGAATTTC-3′(SEQ ID NO：18)

the codon encoding the glutamic acid at position 4 of the insulin B chain is highlighted in bold. The construction was carried out as described in example 1. The template was DNA of plasmid pINT 3581. The correct plasmid was called pINT 3593.

Example 15: construction of the plasmid pINT3594 encoding Glu (A5), His (A8), Gly (A21), Glu (B4) -preproinsulin

The reaction was carried out as described in example 9, but using DNA of the plasmid pINT3582 as template in PCR1 and PCR2, the product was plasmid pINT 3594.

The proinsulin encoded by this plasmid is a precursor of compound YKL205-7, which is amidated with argininamide and has the following structure:

Arg(A0),Glu(A5),His(A8),Gly(A21),Glu(B4),Arg(B31),Arg(B32)–NH₂human insulin.

The proinsulin encoded by this plasmid is a precursor of compound YKL205-7b, amidated with lysyl amine and having the structure:

Arg(A0),Glu(A5),His(A8),Gly(A21),Glu(B4),Arg(B31),Lys(B32)–NH₂human insulin.

Example 16: construction of plasmid pINT3595 encoding His (A8), Glu (A15), Gly (A21), Glu (B4) -preproinsulin

The reaction was carried out as described in example 9, but using DNA of the plasmid pINT3585 as template in PCR1 and PCR2, the product was plasmid pINT 3595.

The preproinsulin encoded by this plasmid is a precursor of compound YKL205-8, amidated with argininamide and has the following structure:

Arg(A0),His(A8),Glu(A15),Gly(A21),Glu(B4),Arg(B31),Arg(B32)–NH₂human insulin.

The preproinsulin encoded by this plasmid is a precursor of compound YKL205-8b, amidated with lysyl amine and has the following structure:

Arg(A0),His(A8),Glu(A15),Gly(A21),Glu(B4),Arg(B31),Lys(B32)–NH₂human insulin.

Example 17: construction of a plasmid pINT3596 encoding His (A8), Asp (A18), Gly (A21), Glu (B4) -preproinsulin

The reaction was carried out as described in example 9, but using DNA of the plasmid pINT3586 as template in PCR1 and PCR2, the product was plasmid pINT 3596. The preproinsulin encoded by this plasmid is a precursor of compound YKL205-9, amidated with argininamide and has the following structure:

Arg(A0),His(A8),Asp(A18),Gly(A21),Glu(B4),Arg(B31),Arg(B32)–NH₂human insulin.

The preproinsulin encoded by this plasmid is a precursor of compound YKL205-9b, amidated with lysyl amine and has the following structure:

Arg(A0),His(A8),Asp(A18),Gly(A21),Glu(B4),Arg(B31),Lys(B32)–NH₂human insulin.

Example 18: construction of the plasmid pINT3597 encoding His (A8), Gly (A21), Glu (B0) -preproinsulin

The construction process was completed using 2 polymerase chain reactions. The primer pint3580_ glya21rev was used. Two additional primers were synthesized:

pint3581_Eb0f1

5′-CAACAGGAA ATTCGGCACG ATTTGTG AACCAGCACC TGTGCG-3’(SEQID NO：19)

pint3581_Eb01f2

5′-TATCGA CCAT GG CAACAACA TCAACAGGAA ATTCGGCACG A-3’(SEQ ID NO：20)

the two primers in this example are partially overlapping. Pint3581_ Eb0f2 contains the NcoI recognition sequence. The sequence is underlined. The codon encoding the glutamic acid at position 0 of the beginning of the B chain is emphasized by bolding each letter. The template for PCR1 was DNA of plasmid pINT 3581.

PCR1 was performed with the primer pair pint3581_ Eb-1f2/pint3580_ glya21 rev. The template for PCR2 is the product of PCR 1. PCR2 was performed with the primer pair pint3581_ Eb-1f2/pint3580_ glya21 rev. The product of PCR2 contained the complete preproinsulin sequence. The product of the second reaction was digested with NcoI/SalI and inserted into the NcoI/SalI cut pINT91d vector DNA. The correct plasmid was called pINT 3597. The glutamic acid codon at position B0 was replaced with an aspartic acid codon and a plasmid was generated according to this example having an aspartic acid instead of a glutamic acid at position B0.

Example 19: construction of the plasmid pINT3598 encoding Glu (A5), His (A8), Gly (A21), Glu (B0) -preproinsulin

The reaction was carried out as described in example 18, but using DNA of the plasmid pINT3582 as template in PCR1, the product was plasmid pINT 3598. The preproinsulin encoded by this plasmid is a precursor of compound YKL205-10, amidated with argininamide and has the following structure:

Arg(A0),Glu(A5),His(A8),Gly(A21),Glu(B0),Arg(B31),Arg(B32)–NH₂human insulin.

The preproinsulin encoded by this plasmid is a precursor of compound YKL205-10b, amidated with lysyl amine and has the following structure:

Arg(A0),Glu(A5),His(A8),Gly(A21),Glu(B0),Arg(B31),Lys(B32)–NH₂human insulin.

Example 20: construction of plasmid pINT3599 encoding His (A8), Glu (A15), Gly (A21), Glu (B0) -preproinsulin

The reaction was carried out as described in example 18, but using DNA of the plasmid pINT3585 as template in PCR1, the product was plasmid pINT 3599. The preproinsulin encoded by this plasmid is a precursor of compound YKL205-11, amidated with argininamide and has the following structure:

Arg(A0),His(A8),Glu(A15),Gly(A21),Glu(B0),Arg(B31),Arg(B32)–NH₂human insulin.

The preproinsulin encoded by this plasmid is a precursor of compound YKL205-11b, amidated with lysyl amine and has the following structure:

Arg(A0),His(A8),Glu(A15),Gly(A21),Glu(B0),Arg(B31),Lys(B32)–NH₂human insulin.

Example 21: construction of plasmid pINT3600 encoding His (A8), Asp (A18), Gly (A21), Glu (B0) -preproinsulin

The reaction was carried out as described in example 18, but using DNA of the plasmid pINT3586 as template in PCR1, the product was plasmid pINT 3600. The preproinsulin encoded by this plasmid is a precursor of compound YKL205-12, amidated with argininamide and has the following structure:

Arg(A0),His(A8),Asp(A18),Gly(A21),Glu(B0),Arg(B31),Arg(B32)–NH₂human insulin.

The preproinsulin encoded by this plasmid is a precursor of compound YKL205-12b, amidated with lysyl amine and has the following structure:

Arg(A0),His(A8),Asp(A18),Gly(A21),Glu(B0),Arg(B31),Lys(B32)–NH₂human insulin.

Example 22: construction of plasmid pINT3601 encoding His (A8), Gly (A21), Asp (B1) -preproinsulin

This construction was accomplished using two polymerase chain reactions. The primer pint3580_ glya21rev was used. Two additional primers were synthesized:

pint3581_Db1f1

5′-CAACAGGAA ATTCGGCACG AGTG AACCAGCACC TGTGCG-3’(SEQ IDNO：21)

pint3581_Db1f2

5′-TATCGA CCAT GG CAACAACA TCAACAGGAA ATTCGGCACG A-3’(SEQID NO：22)

in this example, the two primers overlap partially. Pint3581_ Db-1f2 contains the NcoI recognition sequence. The sequence is underlined. The codon encoding the aspartic acid at position 1 of the B chain is emphasized by the bolding of each letter. The PCR1 template was DNA of plasmid pINT 3581. PCR1 was performed with primers pint3581_ Db1f1/pint3580_ glya21 rev. The template for PCR2 is the product of PCR 1. PCR2 was performed with the primer pair pint3581_ Db1f2/pint3580_ glya21 rev. The product of PCR2 contained the complete preproinsulin sequence. The product of the second reaction was digested with NcoI/SalI and inserted into the NcoI/SalI cut pINT91d vector DNA. The correct plasmid was called pINT 3601.

Example 23: construction of plasmid pINT3602 encoding Glu (A5), His (A8), Gly (A21), Asp (B1) -preproinsulin

The reaction was carried out as described in example 22, but using DNA of the plasmid pINT3582 as template in PCR1, the product was plasmid pINT 3602. The preproinsulin encoded by this plasmid is a precursor of compound YKL205-13, amidated with argininamide and has the following structure:

Arg(A0),Glu(A5),His(A8),Gly(A21),Asp(B1),Arg(B31),Arg(B32)–NH₂human insulin.

The preproinsulin encoded by this plasmid is a precursor of compound YKL205-13b, amidated with lysyl amine and has the following structure:

Arg(A0),Glu(A5),His(A8),Gly(A21),Asp(B1),Arg(B31),Lys(B32)–NH₂human insulin.

Example 24: construction of plasmid pINT3603 encoding His (A8), Glu (A15), Gly (A21), Asp (B1) -preproinsulin

The reaction was carried out as described in example 22, but using DNA of the plasmid pINT3585 as template in PCR1, the product was plasmid pINT 3603. The preproinsulin encoded by this plasmid is a precursor of compound YKL205-14, amidated with argininamide and has the following structure:

Arg(A0),His(A8),Glu(A15),Gly(A21),Asp(B1),Arg(B31),Arg(B32)–NH₂human insulin.

The preproinsulin encoded by this plasmid is a precursor of compound YKL205-14b, amidated with lysyl amine and has the following structure:

Arg(A0),His(A8),Glu(A15),Gly(A21),Asp(B1),Arg(B31),Lys(B32)–NH₂human insulin.

Example 25: construction of plasmid pINT3604 encoding His (A8), Asp (A18), Gly (A21), Asp (B1) -preproinsulin

The reaction was carried out as described in example 22, but using DNA of the plasmid pINT3586 as template in PCR1, the product was plasmid pINT 3604. The preproinsulin encoded by this plasmid is a precursor of compound YKL205-15, amidated with argininamide and has the following structure:

Arg(A0),His(A8),Asp(A18),Gly(A21),Asp(B1),Arg(B31),Arg(B32)–NH₂human insulin.

The preproinsulin encoded by this plasmid is a precursor of compound YKL205-15b, amidated with lysyl amine and has the following structure:

Arg(A0),His(A8),Asp(A18),Gly(A21),Asp(B1),Arg(B31),Lys(B32)–NH₂human insulin.

Example 26: construction of plasmid pINT3605 encoding His (A8), Gly (A21), Glu (B0), Asp (B1) -preproinsulin

This construction was accomplished using two polymerase chain reactions. The primers pint3580_ glya21rev and pint3581_ Eb01f2 described in example 18 were used. Primer pint3597_ Db1f was synthesized:

5′-CAACAGGAA ATTCGGCACG AGTG AACCAGCACC TGTGC-3’(SEQID NO：23)

the codons encoding glutamate at position 0 and aspartate at the beginning of the B chain are emphasized by bolding each letter. The template for PCR1 was DNA of plasmid pINT 3597. PCR1 was performed with the primer pair pint3597_ Db1f/pint3580_ glya21 rev. The template for PCR2 was the PCR1 product. PCR2 was performed with the primer pair pint3581_ Eb1f2/pint3580_ glya21 rev. The product of PCR2 contained the complete preproinsulin sequence. The product of the second reaction was digested with NcoI/SalI and inserted into the NcoI/SalI cut pINT91d vector DNA. The correct plasmid was called pINT 3605. The preproinsulin encoded by this plasmid is a precursor of compound YKL205-16, amidated with argininamide and has the following structure:

Arg(A0),His(A8),Gly(A21),Glu(B0),Asp(B1),Arg(B31),Arg(B32)–NH₂human insulin.

The preproinsulin encoded by this plasmid is a precursor of compound YKL205-16a, amidated with lysyl amine and has the following structure:

Arg(A0),His(A8),Gly(A21),Glu(B0),Asp(B1),Arg(B31),Lys(B32)–NH₂human insulin.

Example 27: construction of plasmid pINT3606 encoding His (A8), Glu (A15), Asp (A18), Gly (A21), desThr (B30) -preproinsulin

The construction was carried out using 3 polymerase chain reactions as described in examples 1 and 2. Primers Tir and pint3580_ glya21rev were used. Two additional primers were synthesized:

desB30f

5′-TTCTACACACCCCGCGATGTTCCTCAGGTGG-3’(SEQ ID NO：24)

desB30rev

5’-AGG AACATCGCGC TTGGGTGTGT AGAAGAAGC-3’(SEQ ID NO：25)

the templates for PCR1 and PCR2 were DNA of plasmid pINT 3586. PCR1 was performed with primer pair desB30f/pint3580_ glya21rev and PCR2 was performed with primer pair Tir/desB30rev template. The template for PCR3 was an equimolar mixture of PCR1 and PCR2 products. The reaction was carried out with the primer pair Tir/pint3580_ glya21 rev. The product of PCR3 contained the complete preproinsulin sequence. The product of the third reaction was digested with NcoI/SalI and inserted into the NcoI/SalI cut pINT91d vector DNA. The preproinsulin encoded by this plasmid is a precursor of compound YKL205-17, amidated with argininamide and has the following structure:

Arg(A0),His(A8),Glu(A15),Asp(A18),Gly(A21),Arg(B30),Arg(B31)-NH₂human insulin.

The preproinsulin encoded by this plasmid is a precursor of compound YKL205-17b, amidated with lysyl amine and has the following structure:

Arg(A0),His(A8),Glu(A15),Asp(A18),Gly(A21),Arg(B30),Lys(B31)–NH₂human insulin

Example 28: expression of proinsulin derivatives

The expression is carried out according to the method of example 1 of European patent application EP A1222207.

Example 29: folded proinsulin derivatives

The folding is carried out essentially as disclosed in EP-A0668282.

Example 30: the folded preproinsulin is enzymatically processed to give a double-stranded Arg (a0) -insulin precursor characterized by lysine or arginine at the B chain terminal C-terminus.

The folded preproinsulin precursor is processed enzymatically, for example as disclosed in example 4 of WO 91/03550. The use of the trypsin variant disclosed in WO 2007/031187 a1 proved to have a particularly advantageous effect in this example.

Example 31: preparation of Arg (A0), His (A8), Gly (A21), Arg (B31), Arg (B32) -NH₂Human insulin.

Regardless of the location of the other acidic amino acids, the following standard reaction can be performed: 100mg of Arg (A0), Gly (A21), Arg (B31) -insulin analogue were dissolved in 0.95ml of argininamide solution (446g/L), and 0.13ml of M sodium acetate buffer (pH 5.8) and 2ml of DMF were added. The reaction mixture was cooled to 12 ℃ and the reaction was initiated by the addition of 0.094ml trypsin (0.075mg, Roche diagnostics). After 8 hours the reaction was stopped by adding TFA to lower the pH to 2.5 and HPLC analysis was performed. Arg (A0), Gly (A21), Arg (B31), Arg (B32) -NH were formed > 60%₂Human insulin. A trypsin inhibitor solution is added and the amidated analogue is then purified in a similar manner to that described in US5,656,722.

The corresponding lysylamide compounds are prepared in a similar manner. However, the starting material for this reaction was an aqueous stock solution of lysinamide containing 366g/L lysinamide in solution.

Example 32: formulating amidated insulin derivatives

To test the biopharmacological (biopharmacological) and physicochemical properties of the insulin derivatives of the present invention, solutions of the compounds were prepared as follows: the insulin derivative of the present invention was dissolved to a target concentration of 240. + -.5. mu.M in a 1mM hydrochloric acid solution containing 80. mu.g/mL of zinc (as zinc chloride).

The following composition was used as dissolution medium:

a)1mM hydrochloric acid

b)1mM hydrochloric acid, 5. mu.g/ml zinc (added as zinc chloride or hydrochloric acid)

c)1mM hydrochloric acid, 10. mu.g/ml zinc (added as zinc chloride or hydrochloric acid)

d)1mM hydrochloric acid, 15. mu.g/ml zinc (added in the form of zinc chloride or hydrochloric acid)

e)1mM hydrochloric acid, 30. mu.g/ml zinc (added as zinc chloride or hydrochloric acid)

f)1mM hydrochloric acid, 80. mu.g/ml zinc (added as zinc chloride or hydrochloric acid)

g)1mM hydrochloric acid, 120. mu.g/ml zinc (added as zinc chloride or hydrochloric acid)

For this purpose, a quantity of freeze-dried material is first weighed out, which is approximately 30% higher than the quantity required on the basis of molecular weight and target concentration. This concentration was then determined by analytical HPLC, and the solution was prepared to the volume required to achieve the target concentration using a 5mM hydrochloric acid solution containing 80. mu.g/mL zinc. The pH can be readjusted to 3.5. + -. 0.1 if necessary. After analysis by HPLC to finally confirm the target concentration of 240 ± 5 μ M, the final solution was transferred to a sterile tube using a syringe with a 0.2 μ M filter fitting, the tube being closed with a septum and a screw cap. The formulation is not optimized in short-term single testing of the insulin derivatives of the invention, for example with respect to the addition of isotonic agents, preservatives or buffer materials and the like.

Example 33: evaluation of the hypoglycemic Effect of novel insulin analogues in rats

The hypoglycemic efficacy of the selected novel insulin analogues was tested in healthy, male, normoglycemic Wistar rats. Male rats were injected subcutaneously with 9nmol/kg of insulin analogue. Blood samples were collected from animals immediately before injection of insulin analogue and at regular intervals after injection (up to 8 hours) and their blood glucose levels were measured. This experiment clearly shows (see fig. 1) that the use of the insulin analogues of the present invention significantly delays the onset of action and results in a longer and uniform duration of action.

Example 34: evaluation of the hypoglycemic efficacy of novel insulin analogues in dogs

The hypoglycemic efficacy of the selected novel insulin analogues was tested in healthy, male, beagle dog (beagle dog) with normal blood glucose levels. Male beagle dogs were injected subcutaneously with insulin analogues at a dose of 6 nmol/kg. Blood samples were collected from animals immediately prior to injection of insulin analogue and at regular intervals (up to 48 hours) after injection and their blood glucose levels were measured. This experiment clearly shows (see fig. 2) that the use of the insulin analogues of the present invention significantly delays the onset of action and results in a longer and uniform duration of action.

Example 35: evaluation of hypoglycemic efficacy in dogs at two-fold dose

The hypoglycemic efficacy of the selected novel insulin analogues was tested in healthy, male, beagle dogs with normal blood glucose levels. Male beagle dogs were injected subcutaneously with insulin analogues at doses of 6nmol/kg and 12 nmol/kg. Blood samples were collected from animals immediately prior to injection of insulin analogue and at regular intervals (up to 48 hours) after injection and their blood glucose levels were measured. This experiment clearly shows (see fig. 3) that the insulin analogue of the present invention used is dose dependent, but despite a doubling of the dose, the effect is still very mild, i.e. no significant low point (nadir) is observed. It can be concluded from this that the insulin of the present invention produces significantly fewer hypoglycemic events than known slow acting insulins.

Example 36: evaluation of hypoglycemic Effect in dogs with formulations containing varying concentrations of Zinc

The experiment was carried out as described in example 35. The results are shown in FIG. 4. According to this result, the time-potency curve of an insulin analogue of the invention can be influenced by the zinc ion content of a formulation containing the same concentration of insulin in the following manner: when the zinc content is zero or low, a rapid onset of action and maintenance of potency for 24 hours is observed, whereas when the zinc content is higher, a gradual onset of action and maintenance of insulin potency for significantly longer than 24 hours is observed.

Sequence listing

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<120> novel insulin derivatives having ultra-delayed time-effect

<130>DE2008/001

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<141>2008-01-09

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Claims (28)

1. An insulin analogue is provided which has a high insulin resistance,

it is Arg (A0), His (A8), Glu (A15), Asp (A18), Gly (A21), Arg (B31), Arg (B32) -NH₂Human insulin.

2. A process for preparing an insulin analogue according to claim 1.

3. The method of claim 2 wherein an insulin analogue precursor is recombinantly prepared, enzymatically processed to a double-stranded insulin, coupled to argininamide in the presence of an enzyme having trypsin activity, and the insulin analogue is isolated.

4. Use of an insulin analogue according to claim 1 in the manufacture of a medicament for the treatment of diabetes.

5. Use according to claim 4, for the preparation of a medicament for the treatment of type I or type II diabetes.

6. A medicament comprising an insulin analogue and/or a physiologically acceptable salt thereof according to claim 1.

7. The formulation of an insulin analogue according to claim 1, wherein the formulation is in the form of an aqueous solution containing the dissolved insulin analogue.

8. The formulation of an insulin analogue according to claim 1, wherein the formulation is in powder form.

9. The formulation of claim 8, wherein the insulin analogue of claim 1 is present in crystalline and/or amorphous form.

10. The formulation of an insulin analogue according to claim 1, wherein the formulation is in the form of a suspension.

11. The formulation of an insulin analogue according to claim 1, wherein the formulation additionally comprises a chemical chaperone.

12. Use of a DNA encoding a precursor of an insulin analogue according to claim 1 in the preparation of an insulin analogue according to claim 1.

Use of (1) DNA encoding the a-chain of an insulin analogue according to claim 1 and (2) DNA encoding the B-chain of an insulin analogue according to claim 1 for the preparation of an insulin analogue according to claim 1.

14. Use of a vector comprising DNA encoding a precursor of an insulin analogue according to claim 1 for the preparation of an insulin analogue according to claim 1.

15. Use of a vector comprising (1) DNA encoding the a-chain of an insulin analogue according to claim 1 and (2) DNA encoding the B-chain of an insulin analogue according to claim 1 for the preparation of an insulin analogue according to claim 1.

16. Use of a host organism comprising DNA encoding a precursor of an insulin analogue according to claim 1 for the preparation of an insulin analogue according to claim 1.

17. Use of a host organism comprising (1) DNA encoding the a-chain of an insulin analogue according to claim 1 and (2) DNA encoding the B-chain of an insulin analogue according to claim 1 for the preparation of an insulin analogue according to claim 1.

18. Use of a host organism comprising a vector comprising DNA encoding a precursor of an insulin analogue according to claim 1 for the preparation of an insulin analogue according to claim 1.

19. Use of a host organism comprising a vector comprising (1) DNA encoding the a-chain of an insulin analogue according to claim 1 and (2) DNA encoding the B-chain of an insulin analogue according to claim 1 for the preparation of an insulin analogue according to claim 1.

20. The formulation according to any one of claims 7-11, additionally comprising exendin-4.

21. The formulation according to any one of claims 7-11, additionally comprising an analog of exendin-4 selected from the group consisting of:

H-desPro³⁶-exendin-4-Lys₆-NH₂,

H-des(Pro^36,37)-exendin-4-Lys₄-NH₂and

H-des(Pro^36,37)-exendin-4-Lys₅-NH₂，

or a pharmacologically tolerable salt thereof.

22. The formulation according to any one of claims 7-11, additionally comprising an analog of exendin-4 selected from the group consisting of:

desPro³⁶[Asp²⁸]exendin-4(1-39),

desPro³⁶[IsoAsp²⁸]exendin-4(1-39),

desPro³⁶[Met(O)¹⁴,Asp²⁸]exendin-4(1-39),

desPro³⁶[Met(O)¹⁴,IsoAsp²⁸]exendin-4(1-39),

desPro³⁶[Trp(O₂)²⁵,Asp²⁸]exendin-2(1-39),

desPro³⁶[Trp(O₂)²⁵,IsoAsp²⁸]exendin-2(1-39),

desPro³⁶[Met(O)¹⁴Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39) and

desPro³⁶[Met(O)¹⁴Trp(O₂)²⁵,IsoAsp²⁸]exendin-4(1-39)，

or a pharmacologically tolerable salt thereof.

23. The formulation of claim 22, wherein peptide-Lys₆-NH₂Is attached to the C-terminus of the exendin-4 analog.

24. The formulation according to any one of claims 7-11, additionally comprising an exendin-4 analog selected from the group consisting of:

H-(Lys)₆-des Pro³⁶[Asp²⁸]exendin-4(1-39)-Lys₆-NH₂

des Asp²⁸Pro³⁶,Pro³⁷,Pro³⁸exendin-4(1-39)-NH₂,

H-(Lys)₆-des Pro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-NH₂,

H-Asn-(Glu)₅des Pro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-NH₂,

des Pro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-des Pro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-des Pro³⁶[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,

H-des Asp²⁸Pro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵]exendin-4(1-39)-NH₂,

H-(Lys)₆-des Pro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-NH₂,

H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-NH₂,

des Pro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-des Pro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-des Pro³⁶[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,

des Met(O)¹⁴ Asp²⁸ Pro ³⁶,Pro³⁷,Pro³⁸ exendin-4(1-39)-NH₂,

H-(Lys)₆-des Pro³⁶,Pro ³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-NH₂,

H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-NH₂,

des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,

H-Asn-(Glu)₅ des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-des Pro³⁶[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,

des Asp²⁸ Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵]exendin-4(1-39)-NH₂,

H-(Lys)₆-des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-NH₂,

H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-NH₂,

des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂，

or a pharmacologically tolerable salt thereof.

25. The formulation of any one of claims 7-11, further comprising Arg³⁴,Lys²⁶(N(Gamma-glutamyl (N)^α-hexadecanoyl))) GLP-1(7-37) [ liraglutide]Or a pharmacologically tolerable salt thereof.

26. An aqueous solution formulation of an insulin analogue according to claim 1, which contains no zinc or less than 15 μ g/ml zinc.

27. An aqueous solution formulation of an insulin analogue according to claim 1, which contains no zinc or less than 15 μ g/ml to 2mg/ml zinc.

28. The formulation of claim 27, wherein the zinc content is 200 μ g/ml.