HK1178444B - Long-acting insulin analogue preparations in soluble and crystalline forms - Google Patents

Description

Long-acting insulin analogue formulations in soluble and crystalline form

Cross Reference to Related Applications

This application claims the benefit of pending U.S. provisional application No. 61/306,722 filed on 22/2/2010.

Statement regarding federally sponsored research or development

The invention was carried out with government support and the cooperative agreement was awarded by the national institutes of health under contract numbers NIH R01 DK40949, RO1DK069764 and R01-DK 74176. The united states government may have certain rights in the invention.

Background

Insulin potentiation therapy for the treatment of type 1 diabetes requires subcutaneous injection of an insulin formulation or insulin analog formulation. The treatment regimen may include multiple daily injections or continuous subcutaneous infusion of insulin or insulin analogs ("pump therapy"). Control of blood glucose concentration is sought during meals, after meals and between meals and throughout the wake-up cycle. Because pumps that can be continuously infused are used by only a few patients, a great deal of effort has been made to develop short, medium and long acting formulations, which are generally defined as human insulin formulations, mammalian insulin formulations or insulin analogue formulations with effective times of action of about 4, 12 and 18-24 hours, respectively, but potentially lasting as long as 7, 16 and 30 hours. Particular interest in long acting insulin formulations or long acting insulin analogue formulations has been motivated by the need to avoid nighttime hypoglycemia and/or morning hyperglycemia. The present invention relates to a process for the preparation of novel long acting insulin analogue formulations. The preparation can also be used for treating type 2 diabetes.

Insulin administration is a long established treatment for diabetes. Insulin is a small globular protein that plays an important role in vertebrate metabolism. Insulin contains two chains: an A-chain of 21 residues and a B-chain of 30 residues. The hormone acts as Zn²⁺Stable hexamers stored in pancreatic beta cells but Zn-free in the bloodstream²⁺The monomer (b) plays a role.

Insulin is the product of the single chain precursor proinsulin, a linking region (35 residues) in proinsulin that links the C-terminal residue of the B chain (residue B30) to the N-terminal residue of the A chain (FIG. 1A). Recently, as determined by nuclear magnetic resonance, the structure of proinsulin as an engineered monomer contains an insulin-like core and a disordered linker peptide, as long thought (fig. 1B). The formation of three specific disulfide bonds (A6-A11, A7-B7, and A20-B19, FIG. 1B) is thought to couple with the oxidative folding of proinsulin in the rough Endoplasmic Reticulum (ER). Upon export from the ER to the Golgi apparatus, proinsulin assembles to form soluble Zn²⁺-coordinated hexamers. Digestion of endogenous protein breakdown and conversion to insulin occurs in immature secretory granules, followed by morphological aggregation. Crystals of zinc-insulin hexamers in mature storage granules have been observed by Electron Microscopy (EM). Insulin analog formulations are described that direct the assembly of novel zinc-containing polyhexammers to alter the duration of action of subcutaneously injected active insulin analogs.

The design of insulin analogs for the treatment of diabetes utilizes the three-dimensional structure of insulin monomers, dimers, and hexamers. Insulin monomers contain three alpha-helices, two beta-turns, and two extension segments. The A-chain consists of an N-terminal alpha-helix (residue)Group A1-A8), irregular turn (A9-A12), second alpha-helix (A12-A18) and C-terminal extension (A19-A21). The B chain contains N-terminal arms (B1-B6), beta-turns (B7-B10), a central alpha-helix (B9-B19), beta-turns (B20-B23), beta-chains (B24-B28), and flexible C-terminal residues B29-B30. The two chains assemble to form a compact globular domain stabilized by three disulfide bonds (cystine A6-A11, A7-B7, and A20-B19). For Zn in various crystal lattice forms²⁺The coordinated insulin hexamer was subjected to extensive X-ray crystallography studies; multiple crystal forms define the designation T₆、T₃R^f ₃And R₆Three structural families of (a). In each case, two Zn ions were found at positions along the hexamer central axis ("axial zinc ions"), each with three histidine side chains (His)^B10) Coordination; in some crystal forms, additional low affinity or partially occupied zinc binding sites have been observed. The T-form protomer is similar in structure to insulin monomers in solution. The R-morphotropic species exhibits changes in the secondary structure of the B-chain: the central alpha-helix extends to B1 (R state) or to B3 (broken (ray) R_fState). The three families of hexamers also differ in the subtle features of side chain assembly.

Subcutaneous disassembly of insulin hexamers can be a key driver of the pharmacokinetics of injected insulin. Thus, pharmaceutical insulin formulation designs are typically based on the assembly or disassembly of zinc insulin hexamers. For example, fast acting analogs may limit the self-assembly of insulin hexamers or accelerate the disassembly of hexamers. On the other hand, long acting analogs typically slow the disintegration of the subcutaneous depot (depot), or promote precipitation and self-assembly. For example, with respect to Humalog^®And NovoLog^®The design and pharmacokinetics of (a), the constituent insulin analogs are injected as hexamers, which must be disintegrated to be absorbed into the capillaries. Substitutions in those analogues help the hexamer to disintegrate to make rapid acting insulin formulations possible. In contrast, long-acting Lantus^®Injected with initial monomer and dimer solution, and the pH of the injection solution is raised after injection due to the buffering of subcutaneous tissue and body fluidThe monomer and dimer precipitate to form an amorphous or microcrystalline reservoir. These strategies depend on a common principle-the relationship between the availability of free insulin monomer or dimer in the subcutaneous depot and the rate of absorption thereof by the capillaries. The various insulin formulations so developed provide a variety of pharmacokinetic properties. The combination of short-, medium-and long-acting insulin formulations or insulin analogue formulations allows the design of a daily regimen to suppress fluctuations in blood glucose concentration and thus optimize glycemic control. The main categories of clinical preparations are:

general insulin-formulating the rapid acting insulin preparation as a clear solution of soluble zinc insulin hexamer at neutral pH. Phenol, m-cresol or methylparaben, originally introduced as antimicrobial preservatives, also binds to hexamers to induce the T → R structural switch. R₆Hexamers exhibit a more classical T₆Higher thermodynamic and kinetic stability of the hexamer. Analogous zinc-based hexameric insulin analog formulations for the quick-acting product Humalog^®(Eli Lilly and Co.) and Novollog^® (Novo–Nordisk)。

NPH InsulinIntermediate-acting insulin preparations (NPH, neutral protamine Hagedorn) based on R₆A suspension of orthorhombic crystals of zinc insulin hexamers comprising phenol (or m-cresol) and a sub-stoichiometric concentration of protamine, which is a mixture of small basic peptides containing multiple arginine residues. X-ray crystallography studies of NPH insulin crystals indicate the position of these basic peptides in the crystals in their binding mode with zinc insulin hexamers. An otherwise similar NPH formulation, SUXIAOLIAZIPro insulin, has been developed(insulin lispro)(Humalog^®Active component(s) to allow for a mixing regimen. However, NPH insulin is difficult and expensive to produce: protamine is a number of basic peptides derived from sperm, usually bovine; the production of NPH crystals is a difficult and complex process that builds around the uniform seed crystals that are first produced. In addition, NPH insulin is prone to fibrosis.

Slow insulin (Lente Principle)Also Insulin Zinc Suspensions (IZS), by inversion into T₆An excess of zinc ions (usually 20-30 per hexamer) is added to the insulin hexamer suspension to obtain the protracted action of human insulin or animal insulin. This large excess results in binding to low affinity sites and produces amorphous precipitates (amorphous semi-slow (Semilente) or IZS) or rhombohedral zinc Ts of the zinc insulin complex₆Insulin microcrystalline suspensions (ultra-slow to crystallize (Ultralente) or IZS). Methyl paraben is generally used as a preservative and is mixed with T₆One face of the zinc insulin hexamer was bound. Ultra-slow formulations are more long acting than semi-slow formulations and intermediate time courses can be achieved by mixing amorphous and crystalline particles (slow or IZS, mixed). The following two steps are adopted in the manufacturing:

(1) precursor insulin crystalsThe first step is to form a microcrystalline seed suspension in the absence of preservatives and at a pH of 5.5, using zinc ions and a high (over physiological) concentration of chloride ions (1.2M NaCl). The precursor crystal belongs to space group R3 and contains T₃R^f ₃Zinc insulin hexamers, wherein each hexamer binds a total of four zinc ions (charge +8) and seven chloride ions (charge-7), together providing a formal charge of +1 for the hexamer. And R of the conventional formulation₆The hexamer differs in that the precursor hexamer contains only one axial zinc ion, which is located at T₆Among the trimers. The other three zinc ions are in R^f ₃And the inner and outer axis of the trimer. His (His)^B10With His^B5And two chloride ions rotate in unison to change (flip) their conformation to form tetrahedral zinc ion binding sites. These off-axis sites are adjacent to the canonical R₆The phenol in the hexamer binds to the pocket. Off-axis zinc ion binding sites inside the hexamers of the ultra-slow precursor crystals are independent of the zinc binding sites between the interfaces/hexamers of the invention.

(2) Ultra-slow insulin crystalTo obtain ultra-slow micro-crystalsSuspension, seeds are diluted into a buffer of pH 7.4 containing methylparaben, a lower concentration of chloride ion (120 mM) and a higher concentration of zinc ion. The crystal consists of T in space group R3₆Insulin hexamer composition. Due to very high zinc ion concentration in the formulation (e.g. in>5 per insulin molecule) additional zinc ions were observed in each hexamer. In addition to the normal two axial zinc ions, partial occupation of one of the two sites of atypical, weakly binding that are mutually exclusive was observed to be also located in the center of the hexamer. There is not sufficient mass electron density to analyze the bound chloride ions. Off-axis zinc ion binding sites inside the hexamer of the mature ultra-slow crystal are also independent of the zinc binding sites between the interface/hexamer of the invention.

Insulin analogues with extended B-chains are also known which form hexamers with more than 2 zinc per hexamer. Human insulin with the following substitution set forms stable complexes with 6.5, 5.3, 6.7 and 5 zinc/hexamers, respectively: GlyA 21-HisB 31-HisB 32, GlyA 21-HisB 31-HisB 32-ArgB 33, GlyA 21-AlaB 31-HisB 32-HisB 33, and GlyA 21-AlaB 31-HisB 32-HisB 33-ArgB 34. These complexes (in particular GlyA 21-AlaB 31-HisB 32-HisB 33-ArgB 34) also show long-term pharmacokinetics in dogs. This is probably due to the binding between the additional zinc ion in each hexamer and the new histidine at the a-amino and C-terminal ends of the a-chain.

Others-by subcutaneous injection of a clear acidic insulin analogue solution (insulin glargine), Lantus^®Sanofi-Aventis), wherein the isoelectric point of the insulin analogue has been shifted to between 7.0 and 7.4 by modifying the polypeptide sequence of human insulin. A long acting depot was formed by precipitation at the pH of the subcutaneous tissue (pH 7.4). Prolongation of action can also be achieved by: covalent modification of insulin (insulin detemir), Levimir, by a non-polar moiety^®The active ingredient of (a); Novo-Nordisk) to enhance its hydrophobicity in the subcutaneous depot and to bind to serum albumin to delay clearance from the blood stream. In the pastOf interest are mixtures of animal insulins (e.g. porcine and bovine) which exploit their difference in solubility.

The present innovation utilizes non-axial zinc ions (non-axial zinc ions) to extend the duration of action of the insulin analog formulations provided herein. Prior uses of zinc ions known in the art are as follows. Common insulin formulations and corresponding fast acting formulations Humalog^®And Novalog^®Zinc ions are used to guide and stabilize the assembly of insulin hexamers. The hexamer consists of three insulin dimers linked by a central three-fold axis of symmetry. Each insulin hexamer or insulin analogue hexamer contains two zinc ions located at the triplet symmetry axis of the hexamer. These "axial Zinc ions" and His^B10The imidazole ring of (b) is coordinated. At R₆In hexamers, the coordination geometry is considered to be tetrahedral; thus each zinc ion binds three symmetrically related His^B10And chloride ion occupies the fourth coordination site. The total formal charge of the hexamer is augmented by +2 by the two axial zinc ions (charge +4) and the two coordinated chloride ions (charge-2). There are no non-axial zinc ions in the structure. Single crystal X-ray diffraction studies of wild-type NPH insulin microcrystals show two axes of zinc ions in each hexamer without additional zinc ions. The crystal lattice has space group P2₁2₁2₁Which leads to a hexamer-hexamer assembly pattern that is inconsistent with the present invention (below).

Most insulin products currently used for the treatment of diabetes contain insulin analogues with a sequence different from the sequence of natural human insulin. The possible beneficial effects of amino acid substitutions in the A-and/or B-chains of insulin on the pharmacokinetics of insulin action following subcutaneous injection have been extensively studied. Examples known in the art contain substitutions that accelerate or retard the time course of absorption. The former analogs are collectively defined as "mealtime" insulin analogs since diabetics may inject the fast-acting formulation at mealtime; while delayed absorption of wild type human insulin or animal insulin (e.g. porcine or bovine insulin) makes it necessary to take 3 days before mealsThese formulations were injected for 0-45 minutes. Substitutions are designed to destabilize the zinc insulin hexamer by altering the steric or electrostatic complementarity at the subunit interface and thereby facilitate rapid dissociation of the zinc insulin hexamer following subcutaneous administration. Meal time insulin analogues were formulated as clear solutions (pH 7.4) of zinc-insulin analogue hexamers (Humalilog)^®And Novalog^®) Or as a zinc-free solution containing monomers, dimers, trimers, tetramers and hexamers in equilibrium (Apidra)^®(ii) a Sanofi-Aventis). Although Humalilog was formulated in phosphate buffered zinc solution (similar to that used for long periods in common formulations of human and animal insulin)^®And Novalog^®However, unlike previous common formulations known in the art, their assembly into zinc insulin hexamers requires the binding of phenol, m-cresol or other specific ligands to stabilize the mutant insulin hexamer. It is known in the art to replace Pro with a wide variety of amino acid substitutions (other than cysteine)^B28Destabilizing the zinc insulin hexamer to a similar extent to Asp^B28And Lys^B28Said substitution optionally including a proline substitution of B29.

Long acting insulin analogues, whose slow absorption over 12-24 hours is intended to provide basic control of blood glucose concentration, are also known in the art. Such as, but not limited to, [ Gly ]^A21, Arg^B31, Arg^B32]Insulin (insulin glargine or Lantus)^®) Extension with amino acid substitutions and/or a-or B-chains can be designed to shift the isoelectric point of the insulin analogue to between pH 7.0 and 7.4. Usually at pH<At pH 5, the analogs are formulated into clear solutions containing soluble insulin monomers, dimers, and higher order oligomers<5 conditions Zinc-mediated Assembly due to His^B10Is impaired by protonation. Prolonged absorption is achieved by aggregation and precipitation of insulin analogues in the subcutaneous tissue due to a pH shift to 7.4. With Lantus^®Insulin preparations are sold containing the active analogue [ Gly ] in a solution at pH 4 made to 0.6 mM^A21, Arg^B31, Arg^B32]-insulin(insulin glargine) prepared by adding aliquots of dilute HCl or NaOH in the presence of the inactive components m-cresol (2.7 mg/ml or 25 mM), glycerol (17 mg/ml or 185 mM), polysorbate-20 (20. mu.g/ml) and (30. mu.g zinc ions/ml or 0.52 mM). Lantus^®The U-100 solution of (b) contains 0.60 mM [ Gly ]^A21, Arg^B31, Arg^B32]-insulin. Since it is known in the art that in wild-type insulin, Asn^A21Undergoes acid-catalyzed chemical change, so Gly^A21The purpose of substitution is to avoid said chemical degradation in acidic solution.

Another type of long acting insulin analogue is exemplified by insulin detemir (trade name Levemir), also known in the art^®) Wherein the residue Thr^B30Has been deleted, C₁₄Fatty acid chain and Lys^B29Is attached to the side chain (molecular weight 5912.9 daltons). Fatty acid chains increase the hydrophobicity of the insulin molecule, which is associated with delayed absorption in the subcutaneous depot. The fatty acid chain also mediates binding of the insulin analogue to serum albumin and thus extends its circulatory life. Insulin detemir is formulated as a soluble zinc-insulin analog hexamer (14.2 mg/ml or 2.5 mM insulin monomer units, defined as U-100 solution) clear solution buffered at pH 7.4 with sodium phosphate (0.89 mg/ml disodium dihydrate) in the presence of inactive excipients sodium chloride (1.17 mg/ml), m-cresol (2.06 mg/ml), phenol (1.80 mg/ml mM), mannitol (30 mg/ml) and zinc ions (65.4 μ g/ml or 1.1 mM). The zinc ion concentration corresponds to a ratio of about 2.6 zinc ions per hexamer. The molar activity of insulin detemir is reduced by about 4-fold relative to wild-type human insulin. In the presence of zinc ions and phenol,des–Thr^B30/C₁₄–Lys^B29the crystal structure of the modified insulin analogue is similar but not identical to that present in its formulation, which describes a natural-like R filled with fatty acids between hexamers in the crystal lattice₆A hexamer. The physical state and structure of insulin detemir formed in the subcutaneous depot is not known in the art.

Therefore, there is a need for a combined substitution of insulin analogues that can combine to create new zinc-binding sites between the hexamer surface of the zinc insulin analogue and the hexamer and thereby provide long-acting subcutaneous protein depot formation.

Insulin belongs to the vertebrate superfamily of insulin-related proteins, including (in addition to insulin itself) insulin-related growth factors I and II (IGF-I and IGF-II), relaxin and relaxin-related factors. These proteins exhibit homologous alpha-helical domains and disulfide bonds. IGF is a single chain polypeptide comprising A-and B domains, an intervening linked (C) domain and a C-terminal D domain; due to proteolytic processing, insulin and relaxin-related factors contain two chains (designated a and B). However, six motif-specific cysteines and selected core residues are largely conserved throughout the vertebrate insulin-related superfamily, with other residues being restricted to specific proteins, which confer functional specificity. Insulin and IGF act as ligands for receptor tyrosine kinases, Insulin Receptor (IR) and class I IGF receptor (IGF-1R), while relaxin and related factors bind to G-protein coupled receptors (GPCRs). Insulin binds most strongly to IR, weakly to IGF-1R, and no detectable binding to GPCRs. IGF-I binds most strongly to IGF-1R, weakly to IR, and no detectable binding to the GPCR. Cross-binding of insulin to IGF-1R triggers mitotic signal transduction pathways, including those associated with cancer cell proliferation. By using insulin analogues containing amino acid substitutions which reduce the degree of said cross-binding, the long-term safety of insulin replacement therapy in the treatment of diabetes can be improved. The amino acid substitution will increase the ratio of the affinity of the insulin analogue for IR to the affinity for IGF-1R. Thus, there is a need for long-acting insulin analogue formulations wherein the active component (the insulin analogue component in monomeric form) exhibits a reduced intrinsic affinity for IGF-1R, an increased ratio of the affinity of the insulin analogue for IR to the affinity for IGF-1R, in each case relative to wild-type human insulin.

Insulin glargine and insulin-like growth factor I(IGF-I) binds more strongly to the type 1 receptor than human insulin. The receptor (IGF-1R) mediates mitotic signal transduction pathways and inhibits apoptosis. The degree of enhancement of IGF-1R binding and signal transduction is estimated to be between factors 1.4 and 14, depending on the in vitro or cell-based assay employed. The enhanced binding and signal transduction of IGF-1R is associated with increased proliferation of human cancer cell lines in culture. [ Gly ] formed under formulation conditions or in subcutaneous depots^A21, Arg^B31, Arg^B32]The physical state or molecular structure of insulin is not yet known in the art.

Over a decade ago, a safety concern for insulin analogues exhibiting increased relative or absolute affinity for IGF-1R was first raised due to Asp in cell culture studies of human cancer cell lines^B10Mitogenic Activity enhancement of insulin (relative to human insulin) and because of the use of Asp^B10Increase in the incidence of breast cancer in Sprague-Dawley rats treated with insulin (relative to treatment with human insulin), and therefore, not continuing to introduce Asp^B10Insulin as an insulin analogue formulation for clinical human use. Recently, for Lantus^®A similar concern is also raised, Lantus^®Enhanced cross-binding to IGF-1R and increased mitogen activity in human cell culture was also shown. More recently, more than 120,000 Lantus samples were treated^®Retrospective disease case studies in treated european diabetic patients indicate a dose-dependent increase in the incidence of various cancers, including breast, prostate, colon, and pancreatic cancers. Not only does an increase in the level of cross-binding to IGF-1R increase the degree of cancer risk, but also Lantus^®A decrease in affinity to IR may also increase the degree of cancer risk. Thus, in current clinical use, [ Gly ] relative to wild-type insulin or other insulin analogs^A21, Arg^B31, Arg^B32]The receptor binding selectivity (ratio of IR association constant to IGF-1R association constant) of insulin is abnormally reduced.

Human insulin itself binds to IGF-1R, but itThe affinity of the receptor for detergent-solubilized and lectin-purified in vitro is 333-fold lower than its binding to IR. Meal time insulin analogs such as Humalilog^®And Novollog^®Show similar levels of cross-binding to IGF-1R (insulin lispro has been reported to bind to IGF-1R (Humalilog)^®Active ingredient) with a slight increase in cross-binding). Epidemiological studies have shown an association of an increased prevalence of endogenous hyperinsulinemia (a compensatory response to insulin resistance in metabolic syndrome and in early stages of type 2 diabetes) and cancer, particularly colorectal cancer. Treatment of insulin resistant patients with high doses of human insulin or insulin analogs has also been associated with an increased risk of cancer, which may reflect a baseline level of cross-binding to IGF-1R. With respect to the accumulated risk of cancer for said patients, it is likely that even the baseline receptor specificity of human insulin and meal time insulin analogues is not sufficient to strictly ensure the safety of long-term therapy. Without wishing to be bound by theory, it is prudent that the receptor binding selectivity of insulin analogues designed for the treatment of diabetes should be equal to or greater than the selectivity of wild type human insulin receptor binding.

The regulation of blood glucose concentration by insulin analogues does not require the binding of precise levels of human insulin to IR. The decrease in the affinity of the analog for IR can be compensated in vivo by delaying the clearance of the analog from the blood. The compensation occurs because insulin clearance is mediated by its binding to IR. Insulin analogues with three-fold reduced affinity for IR may still exhibit similar potency in vivo as human insulin. Further decreases in affinity can be compensated by increasing the injected amount of the analog. An example of an insulin analogue with such reduced affinity is insulin glargine (Lantus)^®) And insulin detemir (Levemir)^®). Changes in the affinity of insulin analogues to IR typically reflect changes in off-rate: decreased affinity is associated with decreased residence time of the hormone at the receptor, while increased affinity is associated with increased residence time. The overall relationship between residence time and metabolic potency or residence time and mitotic signaling is not known. Has already proposedExtension of Asp^B10The residence time of insulin on the IR complex causes at least in part an increase in its mitogenic activity. Without wishing to be bound by theory, past experience teaches that insulin analogues with relative affinities to IR in vitro of between 20% and 200% of human insulin are effective in treating diabetes in mammals.

Thus, there is a need for insulin analogs that exhibit reduced cross-binding to IGF-1R and an extended duration of action while maintaining at least a portion of the biological activity of the analog in controlling blood glucose concentrations. In particular, there is a need for insulin analogs that exhibit delayed absorption from the subcutaneous depot but, once absorbed into the blood, exhibit reduced affinity for IGF-1R while also retaining at least a portion of the biological activity of the analog in controlling blood glucose concentrations. There is also a need for insulin analogues that exhibit an increase in isoelectric point towards neutral without an increase in affinity for IGF-1R, while maintaining at least part of the biological activity of the analogue, in controlling blood glucose concentrations.

The biological, physical and chemical properties of insulin analogues can be altered compared to human insulin, either due to the presence of amino acid substitutions in the a-and/or B-chain, or due to possible extension of the a-and/or B-chain to create larger molecules. Studies of insulin analogues have shown that the properties of analogues containing two or more modifications cannot be reliably predicted based on the properties of groups of analogues containing the corresponding single modification. Because amino acid substitutions or chain extensions at one site in a molecule can result in a change in the delivery of the protein conformation, kinetics, or solvation, the effect of amino acid substitutions at another site in the molecule can differ from the effect of the same substitutions in the absence of the first modification. Arg^A0A modification of the crystalline structure of insulin, which is associated with a reduction in receptor binding, provides an example of an unexpected transmission modification effect. Thus, includes Arg^A0The N-terminal extension of the A-chain of (a) changes the structural environment of residues A4, A8 and other sites. Amino acid substitutions or chain extensions generally incorporating one or more basic residues (Arg or Lys) result in isoelectric pointsAn upward excursion to neutral; the extent of shift is affected by the structure, solvation and the transmission of conformational changes that accompany the modification, so experience teaches that the properties of the amino acids alone are not well predictive of the observed pI. Without wishing to be bound by theory, empirical teaching that the combined effect of two or more modifications cannot be expected based on the nature of the analog containing the single modification. It is therefore possible that the new combination of modifications may together have properties that provide unique advantages for insulin analogues in therapeutic use for the treatment of diabetes.

Summary of The Invention

The present invention relates to insulin analogue formulations containing multiple histidine substitutions that combine to create a new zinc-binding site between the hexamer surface of the zinc insulin analogue and the hexamer and thereby enable the formation of a long acting subcutaneous protein depot. More specifically, the present invention provides insulin analogues containing paired histidine substitutions at a4 and A8, with or without substitution at a21, and provides formulations for subcutaneous administration to achieve extended duration of action. Without wishing to be bound by any particular theory of patentability, it is believed that the side chains of each of these sites project from the a-chain surface to the solvent as it assembles into the insulin hexamer, thereby providing partially new zinc-ion-binding sites that associate with complementary side chain protrusions in adjacent hexamers, such that zinc-ion-bridging interactions occur between adjacent insulin analogue hexamers. As shown in FIG. 1E, wild type T₃R^f ₃Insulin hexamers comprise the following (T)₃A trimer; rounded rectangle) and the following (R)^f ₃A trimer; pointed rectangles) in which each column contains the central axis zinc ions (grey circles). FIG. 1F provides a schematic of the stacking of variant hexamers in a lattice, believed to be due to His in formulations of the present invention^A4And His^A8Occurs. Each bridging zinc ion layer (black circles) was composed of His from each T-form protomer (not shown)^A4And His^A8And the above R^fHis of morphotropium^A4Side chain (vertical fragment) coordination. Again without wishing to be bound by any particular theory, this combination of substitutions also enhances the receptor binding selectivity of the insulin analogue and reduces the absolute affinity for IGF-1R.

Another aspect of the invention provides a long acting insulin analogue formulation at about pH 4 which forms a microcrystalline suspension when its pH shifts between 6 and 7.4. In a specific example, the formulation contains zinc ions at a relative concentration of at least about 4 zinc ions per 6 insulin analogue molecules. Thus, the formulation can be injected subcutaneously into an individual and then form a subcutaneous depot upon exposure to physiological pH. The formulation additionally exhibits reduced affinity for the IGF receptor compared to wild-type insulin of the same species, while maintaining at least 20% of the affinity of wild-type insulin for the insulin receptor of the same species.

In the natural structure of insulin, residues A1-A8 comprise an alpha-helix. This fragment is believed to contribute to the binding of insulin and insulin analogs to both IR and IGF-1R. Without wishing to limit the patentability with respect to any particular theory, it is believed that the residue Glu exposed to the solvent^A4And Thr^A8Substitutions (not conserved in IGF-I) can tolerate insulin analogue binding to IR well and still be close to the hormone-receptor interface. It is known in the art to substitute Gly for Asn when formulated under acidic conditions^A21Chemical degradation of insulin analogues can be delayed.

It would therefore be desirable to provide insulin analogues that provide a long-acting zinc-dependent subcutaneous protein depot and retain high affinity for the insulin receptor with reduced cross-binding to the type I IGF receptor. Without wishing to be bound by theory, it is also desirable to provide insulin analogues in which the two positive charges of the non-axial zinc ions incorporated in the hexamer of the insulin analogue contribute to a further shift in its assembly-dependent isoelectric point. It would also be desirable to provide insulin analogues in which the paired histidine side chains at positions a4 and A8 contribute to the zinc-ion binding site of the new interface between the insulin analogue hexamers in the lattice. Again without wishing to be bound by theory, the interfacial zinc ions may delay the disintegration of higher order contacts between and among the hexamers to prolong the duration of insulin analog action.

The a 1-A8 α -helix of insulin or insulin analogs contributes to its isoelectric point (pI) through its charged site, neutral site, α -helix dipole moment and mutual electrostatic interactions. Again without wishing to be bound by theory, by removal of the acidic residue (which occurs in the substitution of Glu with His)^A4Time) will shift the expected pI upward but not more than pH 6.5. The minor variation in pI may be associated with the insertion of histidine residues at positions a4 or A8, depending on the local pK of the substituted histidine_a(typically between 6 and 7). Again without wishing to be bound by theory, an acidic residue was observed at position a4 of human insulin. Substitution of Gly, Ala or other neutral side chain for Asn is contemplated^A21Will not result in significant changes in pI; substitution with a basic side chain (Arg or Lys) is expected to result in a further shift of pI upwards; substitution with Asp (as can occur for natural Asn upon storage in acidic solution)^A21Deamidation of the side chain) will result in a downward shift in pI. Non-axial zinc ions bound to the surface of the insulin analogue hexamer or to the interfacial sites between the zinc insulin analogue hexamers may also contribute to the overall charge of the hexamer or multi-hexamer complex and thus affect its solubility at pH 7.4 in the subcutaneous depot.

It would therefore also be desirable to provide an insulin analogue which exhibits the above-mentioned receptor binding properties and which also exhibits an upward shift in isoelectric point but not more than neutral so that the combined effect of the amino acid substitution and the additionally bound zinc ion renders the complex insoluble at pH 7.4 in subcutaneous depots when atypical zinc ions are bound at the surface of the insulin analogue hexamer or between the hexamers.

It would therefore also be desirable to provide soluble formulations of insulin analogues that are clear solutions at acidic pH, which are easy to handle when injected subcutaneously with a syringe, which facilitate accurate adjustment of the dosage, and which can be delivered in accurate doses by a metering pen (pen). It would also be desirable to provide a crystallization process as a basis for insulin analogue microcrystalline suspensions at neutral pH that imparts increased shelf life and stability at room temperature after the first use of the product.

In general, a method of treating a patient comprises administering a physiologically effective amount of an insulin analogue or a physiologically acceptable salt thereof to the patient, wherein the analogue or a physiologically acceptable salt thereof contains a modified insulin a-chain sequence, the modification being a substitution at positions a4 and A8 with a paired histidine, and possibly an additional modification at a 21. In one example, the a21 side chain is a native Asn residue. In another example, the a21 side chain is Gly. In another example, the a21 substitution can be Ala, Thr, or Ser.

Insulin analogs can be analogs of any vertebrate insulin, or insulin analogs containing a modified B-chain as known in the art to alter absorption following subcutaneous injection. In one example, the insulin analogue is a mammalian insulin analogue, e.g., a human, murine, rodent, bovine, equine or canine insulin analogue. In further examples, the insulin analog is an analog of ovine, whale, rat, elephant, guinea pig, or chinchilla insulin.

Specific insulin analogues include insulin analogues comprising an a-chain sequence as provided in any of seq. ID. numbers 4-6 or 14 and a B-chain sequence as provided in any of seq. ID. numbers 7-12. The nucleic acid may encode a polypeptide having one of these sequences. The nucleic acid may be part of an expression vector that may be used to transform a host cell.

Brief description of several views of the drawings

FIG. 1A is a schematic representation of the sequence of human proinsulin, including the A-and B-chains and the linking region (filled circles) and C-peptide (open circles) showing flanking dibasic cleavage sites.

FIG. 1B provides a structural model of proinsulin, consisting of an insulin-like moiety and a disordered connecting peptide (dashed line).

Figure 1C provides a diagram of the proposed pathway for fibrillation of insulin via partial unfolding of the monomer. The native state is protected by typical self-assembly (leftmost). The disintegration leads to a balance between natural monomers and partially folded monomers (hollow triangles and trapezoids, respectively). This partial fold can be completely unfolded (open circle) by an off-path event, or aggregated to form a core and converted to (en route to) protofilaments (far right).

FIG. 1D is a schematic representation of the sequence of human insulin, indicating the position of residue A8 in the A-chain and the substitution site in the B-chain known in the art to confer rapid uptake following subcutaneous injection.

FIG. 1E is wild type T₃R^f ₃Schematic of insulin hexamers comprising the above list (T)₃A trimer; rounded rectangle) and the following (R)^f ₃A trimer; pointed rectangles) where each column contains axial zinc ions (grey circles).

FIG. 1F is a schematic of a stack of variant hexamers in a crystal lattice, where each of the three bridging zinc ion layers (black circles) is each His of each T-morphia (rounded rectangle)^A4And His^A8And from R^fHis of morphotropic (pointed rectangle)^A4Side chain (vertical fragment) coordination.

FIG. 2a provides the wild type insulin sequence and (above group) insulin glargine (Lantus)^®Sanofi-Aventis) and (group below) analogs of the invention. Wild-type A-and B-chain sequences are shown in black and grey; disulfide bonds are indicated by black lines (A6-A11, A7-B7 and A20-B19). Insulin glargine contains a two residue extension of the B-chain (Arg)^B31And Arg^B32) And Asn^A21→ Gly substitution (red upper panel). Endogenous subcutaneous proteases may slowly remove one or two Arg residues from the insulin glargine extension B-chain, which in part moderates its enhanced mitogenic activity. The analogs of the invention compriseWith pairs of (i, i +4) substituted Glus^A4→ His and Thr^A8→ His (lower panel). Long-acting analogue insulin detemir (Levemir) ^® Novo-Nordisk) functions by attachment to an albumin-binding member (not shown).

Figure 2b provides a ribbon model of insulin monomers depicting the putative zinc-ion binding site moiety formed by the outer surface of the a 1-A8 a-helix. The A-chain and B-chain bands are indicated in black and gray, respectively.

FIG. 2c depicts wild type T₃R^f ₃Structure of insulin hexamers. Two axial zinc ions in the hexamer are aligned centrally through trimer-associated His^B10Side chains (light grey) to coordinate. The A-chain is indicated in black and the B-chain in grey (R)^fSpecific B1-B8. alpha. -helix). The structure of the wild type was obtained from the protein database (accession number 1 TRZ).

FIG. 2d depicts the variant [ His ]^A4, His^A8] T₃R^f ₃Structure of insulin hexamers. Two axial zinc ions in the hexamer are aligned centrally through trimer-associated His^B10Side chains (light grey) to coordinate. Variant hexamers at T₃The trimeric surface (surface sphere) contains three non-typical zinc ions. Shown in grey as His from the adjacent hexamer^A4、His^A8And the third His^{A4 ’}The side chain of (1). The A-chain is in each case represented in black and the B-chain (R) in grey^fSpecific B1-B8. alpha. -helix).

FIG. 2e illustrates FIG. 2F_o–F_cElectron density map (stereo pair outlined at 1 s), which shows His in T-morphotropium^A4And His^A8New zinc-ion binding sites are formed. From residue A4' (belonging to R in the adjacent hexamer)^fMorphotropic) to accomplish distorted tetrahedral coordination.

Fig. 3A depicts wild-type hexamer-hexamer assembly. (Left side of) In thatThe upper trimer in each hexamer has T₃Conformation, the following trimer being R^f ₃. Interfacial water molecules (smaller spheres) and axial zinc ions (larger spheres) adjacent to residues a4 and A8 are shown. The A-chain is shown in grey and the B-chain in black. T-and R protomers differ in the secondary structure of B1-B9, being either extended (T) or helical (R); residues B1 and B2 at "breakage" R^fDisorder in the state. (Right side) Enlargement of the left boxed area. The larger sphere near the bottom is T in the bottom hexamer₃Axial zinc ion of trimer. The arrow indicates the upper R^f ₃R in trimer^f-form residue Glu^{A4 ’}。

FIG. 3B depicts Zinc-mediated [ His ]^A4, His^A8]Hexamer-hexamer assembly of insulin: the above trimer having T₃Conformation, the following trimer being R^f ₃. Axial zinc ions and zinc ions coordinated by A4-A8-A4' are shown. The A-chain is shown in grey and the B-chain in black. (Right side) And enlarging the framed area. Three new zinc ions were observed at the hexamer-hexamer interface. Arrow indicates R^f-form side chain His^{A4 ’}(bottom trimer from the top hexamer) which completes the zinc-binding site of the interface.

FIG. 3C provides a display [ His ]^A4, His^A8]T and R of the insulin hexamer^fFace CPK model (Left and right). The view is rotated 90 deg. with respect to the group of fig. 3 b. Three non-typical zinc ions are shown with His^A4And His^A8Is combined with the side chain of (1). The white cross marks the position of the chloride ions; the other diagram is the same as that of FIG. 3B.

FIG. 3D provides stereo pairing, which shows R^fHis of the protomer^A4His of the ` and T protomers^A4–His^A8The non-typical zinc ions of interest (dark grey large spheres), chloride ions (overlapping light grey spheres) and three bound water molecules (smaller spheres). The bonded water molecule participates in R^fHydrogen bond network of (1)Involving Glu^{B4 ’}Side chain carboxyl (carboxylate), Tyr of (2)^{B26 ’}para-OH and Pro of^{B28 ’}Carbonyl oxygen (labeled).

FIG. 3E depicts the results of a competitive substitution assay that detects high affinity binding of insulin or insulin analogs to IR (three curves on the left; solid line) and low affinity cross-binding to IGF-1R (three curves on the right; dashed line). The results in each group are shown: wild type insulin (a)x) Insulin glargineAnd His^A4, His^A8-insulin。His^A4, His^A8The receptor binding selectivity of insulin is improved because its IR-binding titration shifts to the left and its IGF-1R-binding titration shifts to the right. Tables 2 and 3 provide the relative affinity and dissociation constants. The measurement was carried out in the absence of zinc ions.

Figure 3F provides the results of the in vivo assay. Using wild type insulin (x) Insulin glargine、His^A4, His^A8-insulinOr buffer control (Lilly dilution; ●) streptozotocin (streptozotocin) -induced diabetic male rats were injected subcutaneously. The dose at 0 is: 3.44 nanomole of wild type insulin (20 mg in 100- μ l injection volume), 12 nanomole of insulin glargine (equivalent to 2.0U of Lantus)^®) 13.7 nanomolar [ His ]^A4, His^A8]Insulin and 100- μ l protein free buffer (Lilly dilution). Blood glucose concentration from the tail tip was measured at the indicated time. Each of the 5 rats was testedOne analog (mean ± SEM); the experiment was repeated 2 times with similar results. Rats fed 6-8 hours after injection.

FIG. 4 provides a graphical representation of the blood glucose levels (mg/dL) as a function of time in streptozotocin-induced diabetic male rats after injection of: control dilutions of insulin (round), insulin glargine (Lantus) as controls^®Square), insulin lispro (Humalog)^®"X"), or insulin analogs containing His substitutions at positions A4 and A8 and a Humalog substitution of lispro (A4 A8-lispro + Zn, inverted triangle), otherwise as provided in figure 3f above.

Figure 5 is a schematic of the use of histidine substitutions to allow zinc-mediated protein association to produce a long-acting depot of the protein of interest.

Detailed Description

The present invention relates to the innovative use of non-axial interfacial zinc ions between insulin hexamers to prolong the duration of action of insulin analogue formulations. The present invention provides a novel system for creating an extended subcutaneous depot. It utilizes new non-axial zinc ions to bind between the insulin analogue hexamer surface and the insulin analogue hexamer and extends the time required for these analogue depots to release the monomeric insulin analogue into the bloodstream. The invention also provides insulin analogs that have reduced absolute and relative binding to type 1 IGF receptors simultaneously. This combination of properties will improve the efficacy and safety of diabetes treatment, especially in terms of the risk of cancer. To this end, the present invention provides insulin analogues containing a para-histidine amino acid substitution at positions a4 and A8 and a zinc containing formulation which is a clear solution at pH 4, or a microcrystalline suspension at about neutral pH. Pairs of A4-A8 substitutions may be combined with a substitution at position A21 (e.g., Gly, Ala, Ser, or Thr).

Insulin analogues of the invention may also contain other modifications. As used in the specification and claims, various substitutions of insulin analogs may be annotated by convention, which designates the amino acids that are substitutedFollowed by the position of the amino acid, optionally in the form of the above target. The amino acid position of interest includes the insulin A-chain or B-chain in which the substitution is made. For example, an insulin analogue of the invention may also contain a substitution of aspartic acid (Asp or D) or lysine (Lys or K) for amino acid 28 (B28), i.e. proline (Pro or P), of the B-chain, or a substitution of Pro for amino acid 29 (B29), i.e. Lys, of the B-chain, or a combination thereof. These substitutions may also be individually designated as Asp^B28、Lys^B28And Pro^B29. Similarly, the insulin of the invention may be present in the A-chain at amino acid A0 (i.e., N-terminal to Gly)^A1) Contains arginine (Arg or R), histidine (His or H), or lysine (Lys or K) as additive. These additions may be separately noted as Arg^A0、His^A0Or Lys^A0. Furthermore, the substitution may be with Phe^B1→ introduction of His. Unless otherwise or clearly indicated anywhere in the context, the amino acids referred to herein are to be considered L-amino acids.

As used herein, a "metal-spike" or "metal-zipper" is a metal-binding site formed when two or more amino acid side chains from two or more molecules or molecular complexes associate with a metal of interest. For example, one side chain from a first molecule can combine with two side chains from a second molecule to form a zinc-binding site. Thus, it is well known in the art that insulin trimers are "zinc-stapled" linked together by axial zinc ions. However, it was not previously known that zinc-bonding sites could be introduced to cause certain insulin analogue hexamers to form zinc-pins between hexamers.

The present invention provides insulin analogues that form novel insulin hexamer-complexes with "zinc-spikes", exhibiting reduced affinity for IGF-1R but retaining at least part of its affinity for IR and thus retaining biological activity. The invention also provides formulations of these analogs with high relative concentrations of zinc that form "zinc-spike" hexamer complexes, even at higher concentrations of zinc, forming slow-like crystals of these hexamer-complexes. In some embodiments, the insulin analogue contains at least 4, at least 5, at least 6, at least 7, or at least 8 zinc ions per insulin analogue hexamer.

A method for treating a patient includes administering the insulin analog to the patient. In one example, the insulin analog is an insulin analog containing an a-chain modification that concomitantly results in a shift in the isoelectric point (pI) up to neutral, allowing for the assembly of a zinc-stapled insulin hexamer. In another example, the modification also reduces the affinity of the zinc-free monomer for IGF-1R. In another example, the insulin analogue also contains a substitution at position a21, which protects the insulin analogue made under acidic conditions from chemical degradation. The insulin analogue is administered by subcutaneous injection using a syringe, a metering pen or other suitable device.

It is also envisaged that it is possible to apply the para-histidine substitutions introduced at positions a4 and A8 to analogues made with zinc ions at sufficient concentrations under acidic conditions, possibly making the analogues insoluble at pH 7.4 by two co-existing mechanisms: a shift towards a higher isoelectric point (about 6.5-6.6), mainly due to Glu^A4Removing medium negative charges; and a further shift in the net isoelectric point of the zinc insulin hexamer, which results from the binding of non-axial zinc ions in addition to typical axial zinc ions. The same substitutions at a4 and A8 introduced to reduce the solubility of insulin analogs in the subcutaneous depot may also reduce the potential risk of cancer, which has been suggested to be associated with the cross-binding of insulin and insulin analogs to the type 1 IGF receptor.

It is also envisaged that it is possible to apply the combined substitutions introduced at positions a4 and A8, with or without substitution at a21, to other kinds of insulin analogue formulations (such as but not limited to plain, NPH, semi-chronic and chronic insulin, including mixtures of these types) with the aim of achieving one or more of reduced cross-binding of the analogue to type 1 IGF receptors. In order to form a normally soluble formulation at pH 7.4, the paired histidine substitution must be combined with substitution at other positions in the A-or B-chain which remove oneOne or more positive charges or one or more negative charges added to reduce the pI sufficiently to make the solubility at pH 7.4 similar to human insulin in the presence of excipients known in the art, including but not limited to zinc chloride, phenol, m-cresol, glycerol, sodium phosphate buffer, and water for injection. Examples of substitutions that when combined with the paired histidine substitutions of a4 and A8 will reduce pI include, but are not limited to: glu (glutamic acid)^A14、Asp^A21、Glu^A21、Asp^B9、Glu^B9、Asp^B10、Glu^B10、Ala^B22、Ser^B22、Asp^B28、Asp^B28–Pro^B29、Asp^B28–Ala^B29、Ala^B29And Pro^B29Or a combination thereof.

It has been found that paired histidine substitutions at positions a4 and A8 reduce cross-binding of the insulin analogue to the type I IGF receptor and cause an upward shift in pI towards neutrality while maintaining natural affinity for the insulin receptor.

Also found was [ His ] prepared in the absence of zinc at pH 7.4^A4, His^A8]Insulin is highly soluble, whereas the addition of 4-6 zinc ions per 6 insulin analogue molecules results in the precipitation of the zinc-protein complex. Such complexes are insoluble or slightly soluble at pH 7.0-8.4, but soluble at about pH 4. Without wishing to be bound by theory, it is likely that this pH-dependent insolubility is due to in vitro isoelectric precipitation of zinc protein complexes containing both axial and non-axial bound zinc ions.

It was also found that [ His ] containing a molar ratio of 5.2 zinc ions per 6 insulin analogue molecules prepared in a non-buffered solution at pH 4.0^A4, His^A8]Insulin, which, after subcutaneous injection in male Lewis rats (diabetic with streptozotocin), will behave like Lantus^®The duration and extent of pharmacological effects of (a), lead to prolonged control of blood glucose concentration. Without wishing to be bound by theory, it is possible that this prolonged effect is due to the presence of a zinc protein complex containing both axial and non-axial bound zinc ionsCaused by subcutaneous isoelectric precipitation.

Also found [ His ]^A4, His^A8]Insulin crystals can be readily grown as zinc insulin analogue hexamers containing two axial zinc ions/hexamers and three non-axial zinc ions (incorporated between successive hexamers in the R3 lattice); the latter interfacial zinc ion appears to be composed of His in a hexamer^A4And His^A8His adjacent to hexamer^{A4 ’}And tetrahedral coordination of bound chloride ions. The three bound zinc-and chloride ions add formal charges of +6 and-3 (formal charge), respectively, to the hexamer, with a net formal charge of + 3. These additional charges are obtained by substituting Glu with His^A4While the formal charge of +6 is expanded. Without wishing to be bound by theory, it is likely that the presence of three non-axial zinc ions/hexamers leads to the above-described pH-dependent insolubility and presumably subcutaneous isoelectric precipitation of the zinc protein complex.

Generally, a vertebrate insulin analogue, or a physiologically acceptable salt thereof, comprises an insulin analogue comprising an insulin a-chain and an insulin B-chain. Insulin analogues of the invention may also contain other modifications, for example substitutions of basic amino acid extensions of B-chain residues B1 and/or B31. In one example, the vertebrate insulin analogue is a mammalian insulin analogue, e.g. a human, porcine, bovine, feline, canine or equine insulin analogue. The insulin analogues of the present invention may also contain other modifications, such as a tether (tether) between the C-terminus of the B-chain and the N-terminus of the A-chain, which is described in more detail in co-pending U.S. patent application No. 12/419,169, the disclosure of which is incorporated herein by reference.

Pharmaceutical compositions may contain the insulin analogue and in order to obtain an extended duration of action it is necessary to include zinc ions or another divalent metal ion capable of directing protein assembly and hexameric interfacial suturing (stabbing). Zinc ions may be included in the composition at a molar ratio of 4.0-7.0 or 5.0-6.0 zinc ions per insulin analogue hexamer. It may also include higher molar ratiosTo produce an even slower absorbing hexamer-complex, at said high molar ratio, except for the interface [ His ]^A4, His^A8]In addition to the relevant zinc-nail binding sites, zinc ions will also occupy weak zinc-binding sites. The concentration of insulin analogue in the formulation is typically from about 0.1 to about 0.3 mM.

Excipients may include glycerol, glycine, other buffers and salts and antimicrobial preservatives such as phenol and m-cresol; the latter preservatives are known to enhance the stability of insulin hexamers. The pharmaceutical compositions can be used to treat a patient suffering from diabetes or other medical conditions by administering a physiologically effective amount of the composition to the patient.

A nucleic acid comprising a sequence encoding a polypeptide encodes an insulin analogue comprising a sequence encoding an a-chain with a combined histidine substitution at a4 and A8, with or without an additional substitution at a 21. The specific sequence may depend on the codon usage preferred in the species into which the nucleic acid sequence is to be introduced. The nucleic acid may also encode other modifications of wild-type insulin. The nucleic acid sequence may encode a modified A-chain or B-chain sequence containing unrelated substitutions or extensions at other sites of the polypeptide or modified proinsulin analog. The nucleic acid sequence may also be part of an expression vector, which may be inserted into a host cell, e.g., a prokaryotic host cell, e.g., E.coli (E.coli)E. coli) Cell lines, or eukaryotic cell lines, e.g. Saccharomyces cerevisiae (C.cerevisiae:S. cereviciae) Or Pichia pastoris (Pischia pastoris) A strain or a cell line.

It is also envisaged that unrelated substitutions or chain extensions may be combined with the analogues of the invention to alter their isoelectric point, which is further increased by substitution at position B13 or chain extension by Arg or Lys at positions a0, a22, B0 or B31, or decreased by substitution to insert or remove a negative charge. For example, the substitution may be in combination with AlaB 31-HisB 32-HisB 33-ArgB 34, HisB 31-HisB 32, HisB 31-HisB 32-ArgB 33 or AlaB 31-HisB 32-HisB 33. In these latter cases, zinc binding within the additional hexamers complements zinc binding between the hexamers of the invention to increase the ratio of zinc to hexamer and further stabilize the hexamer-complex.

Substitutions of the invention may also be combined with B-chain modifications which increase IGF-1R cross-binding to alleviate such adverse properties, examples including the use of one or two basic residues (e.g., Arg)^B31、Lys^B31、Arg^B31–Arg^B32、Arg^B31–Lys^B32、Lys^B31–Arg^B32And Lys^B31–Lys^B32) Extending the B-chain, or substituting Asp or Glu for His^B10. An example, but not limited to, is provided of insulin glargine (Lantus)^®) It is made at pH 4, but suffers from aggregation in subcutaneous depots at physiological pH.

It is also contemplated that the paired histidine substitutions of the present invention may also be utilized in combination with any of the variations present in existing insulin analogs or modified insulins, or with various pharmaceutical formulations such as plain insulin, NPH insulin, chronic insulin, or ultraslow insulin. Insulin analogues of the invention may also contain substitutions present in human insulin analogues which, although not clinically useful, are experimentally useful, e.g. containing a substitution Asp^B10、Lys^B28And Pro^B29Or Asp^B9Substituted DKP-insulin. However, the present invention is not limited to human insulin and analogs thereof. It is also contemplated that these substitutions may also be made in animal insulins such as, by way of non-limiting example, porcine, bovine, equine and canine insulins. Furthermore, given the similarity between human and animal insulins, and the use of animal insulins in human diabetic patients in the past, it is also envisaged that other minor modifications may be introduced into the insulin sequence, particularly those substitutions which are considered "conservative" substitutions. For example, additional amino acid substitutions may be made within a group of amino acids having similar side chains without departing from the invention. These include neutral hydrophobic amino acids: alanine (Ala or A), valine (Val or V), leucine (Leu or L), isoleucine (Ile or I), proline (Pro or P), tryptophan (Trp or W), phenylalanine (Phe or F) and methionine (Met or M). Likewise, neutral polarityAmino acids can be substituted for each other within the group: glycine (Gly or G), serine (Ser or S), threonine (Thr or T), tyrosine (Tyr or Y), cysteine (Cys or C), glutamine (Glu or Q), and asparagine (Asn or N). Basic amino acids are considered to include lysine (Lys or K), arginine (Arg or R), and histidine (His or H). Acidic amino acids are aspartic acid (Asp or D) and glutamic acid (Glu or E).

The amino acid sequence of human proinsulin is provided for comparison purposes as seq. ID. number 1. The amino acid sequence of human insulin A-chain is provided as SEQ. ID. number 2. The amino acid sequence of the B-chain of human insulin is provided for comparison purposes and is SEQ. ID. number 3.

SEQ. ID. number 1 (proinsulin)

SEQ. ID. number 2 (A-chain)

SEQ. ID. number 3 (B-chain)

Insulin analogues of the invention are envisaged to have similar affinity for the insulin receptor as native insulin but exhibit reduced affinity for the type 1 IGF receptor. The activity of insulin or insulin analogs can be determined by receptor binding assays described in more detail below. The relative activity may be expressed as the hormone-receptor dissociation constant (K)_d) Is obtained by curve fitting of competitive substitution assays; or by ED₅₀Value definition, i.e. substitution of 50% of the specifically bound labeled human insulinFor example radiolabeled human insulin (e.g.¹²⁵I-labeled insulin) or a radiolabeled high affinity insulin analog. Alternatively, activity may be expressed simply as a percentage of normal insulin. Affinity for insulin-like growth factor receptors can also be determined in the same manner as the measured substitution of IGF-1R. In particular, insulin analogs are expected to have an activity of 20-200% of the activity of insulin, such as 25, 50, 110, 120, 130, 140, 150, or 200% or more of normal insulin, while having an affinity for IGF-1R that is less than or equal to 50% of normal insulin, such as 10, 20, 30, or 50% or less of normal insulin. Insulin analogs are useful in the treatment of diabetes even though in vitro receptor-binding activity is as low as 20% due to slower clearance.

Synthesis of insulin analogues.Chain association was achieved by interaction of the S-sulfonated derivatives of the A-chain (41 mg) and B-chain analogs (21 mg) in 0.1M glycine buffer (pH 10.6, 10 ml) in the presence of dithiothreitol (7 mg). CM-52 cellulose chromatography of each of the combined mixtures partially separated the protein in the hydrochloride form contaminated with free B-chain. Final purification was done by reverse phase HPLC. MALDI mass spectrometry confirmed the prediction [ His ]^A4, His^A8]-molecular weight of insulin. The final yield (6.1 mg) was similar to that obtained for the synthesis of control wild-type insulin. [ His ]^A4, His^A8]The corresponding yield of DKP-insulin was 8.8 mg.

Isoelectric focusing electrophoresis.With preformed IEF gels (125X 125 mm, 300 μm, SERVALYT from SERVA Electrophoresis GmbH, Heidelberg) of pH 3-10^® Precotes^®Obtained from credence Chemical co. Hauppauge, NY) to measure pI values of native state insulin and insulin analogs by IEF gel electrophoresis. Precotes were tested according to the manufacturer's protocol^®Placed in a horizontal IEF apparatus Multiphor II (Pharmacia Biotech). The cell was pre-cooled to 4 c with a circulating water bath (Brinkman),the PRECOTE IEF gel was then placed on an electrophoresis bed coated with light mineral oil to achieve efficient heat exchange. The gel was connected to the electrodes at both ends with strips of filter paper soaked with anolyte at pH 3 and catholyte at pH 10 (both from SERVA). Prior to spotting, the gel was pre-focused for 30 minutes using a high voltage power supply (LKB model 2197) at an initial voltage setting of 200 volts and a final setting of 500 volts. After spotting the sample and IEF standards (5-10. mu.L, loading protein concentration 5-10. mu.g), isoelectric focusing was performed at 500-2000 volts for 2 hours or until a final voltage of 2000 volts was reached, after which focusing was continued for an additional 15 minutes. After IEF, the gel was fixed with 200 ml of 20% trichloroacetic acid for 20 minutes, rinsed with 200 ml of deionized water for 1 minute, stained with SERVA Violet 17 solution and destained with 86% phosphoric acid according to the protocol of the SERVA manual. The IEF standard proteins (from SERVA) used were as follows, with their respective pI values in parentheses: horse heart cytochrome C (10.7), bovine pancreatic ribonuclease A (9.5), lentil lectin (8.3, 8.0, 7.8), equine myoglobin (7.4, 6.9), bovine red blood cell carbonic anhydrase (6.0), milk beta-lactoglobulin (5.3, 5.2), soybean trypsin inhibitor (4.5), Aspergillus niger (A)Aspergillus niger) Glucose oxidase (4.2), Aspergillus niger amyloglucosidase (3.5). The pI of the protein samples was determined by comparing linear regression curves of migration distance versus pH gradient of IEF standards.

A receptor expression plasmid.For expression of tagged epitope IR and IGF-1R, the mammalian expression vector pcDNA3.1Zeo + was obtained from InVitrogen by subcloning an in-frame oligonucleotide cassette encoding an in-frame triple FLAG M2 epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) intoBamHI AndXbaI restriction sites, to modify its C-terminal epitope tag. The respective cDNAs encoding the B-isoforms of IGF-1R and IR are as previously described (Whittaker, J. et al.Proc. Natl. Acad. Sci. USA Vol 84, pages 5237-5241 (1987)). By introducing a C-terminal Gly-Ser linker encoding an in-frame sequence at its 3' end just before the stop codon by site-directed mutagenesisBamHISites, which are modified for subcloning into the modified expression vector.

Receptors cDNA Expression of (2).DNA for transfection was prepared as described previously. Receptor cDNA was transiently expressed in PEAK fast cells using polyethyleneimine. Three days after transfection, cells were harvested when receptor expression was maximal. Lysis was accomplished in a buffer consisting of 0.15M NaCl and 0.1M Tris-Cl (pH 8.0) containing 1% (v/v) Triton X-100 and a protease inhibitor cocktail (Roche). The lysate was stored at-80 ℃ until required for the assay.

A receptor binding assay.The binding assay for each of IGF-1R and IR-B was performed by modifying the microtiter plate antibody capture assay previously described by Whittaker and coworkers. Microtiter plates (Nunc Maxisorb) were incubated with anti-FLAG IgG (100. mu.l/well of 40. mu.g/ml phosphate buffered saline) overnight at 4 ℃. Washing and blocking were performed as described previously. Detergent lysates of 293 PEAK cells transiently transfected with cDNA encoding full length IR-B or IGF-1R with a C-terminal FLAG-tag were partially purified by Wheat Germ Agglutinin (WGA) chromatography to remove receptor precursor lysates. Wheat embryo eluates were then incubated on antibody-coated plates for 1 hour at room temperature to immobilize the receptor. After extensive washing to remove unbound protein, a labeled insulin tracer is used: (¹²⁵I–[Tyr^A14]Insulin) or a labeled IGF-I tracer: (¹²⁵I–Tyr³¹-competitive binding assays of IGF-I) and unlabelled insulin analogues were performed as described. In the same set of assays, all insulin analogues were assayed using insulin or IGF-I receptor as a control ligand. The binding data of the homocompetition assay were analyzed by non-linear regression analysis using a two-site order model to obtain dissociation constants for insulin and IGF-I. Binding data from heterogeneous competition experiments were analyzed by Wang using precise mathematical expressions to describe the competitive binding of two different ligands to the receptor.

Representative binding studies of insulin analogues known in the art are summarized in table 1. Because of insulin to IR (homoIsoform B) has an affinity similar to that of IGF-I for IGF-1R (K in each case)_dAbout 0.04 nM), column 4 gives the percentage (columns 2 and 3) ratios for the respective affinities for IR and IGF-1R, providing an estimate of the absolute specificity of the insulin analogue. Normalization of specificity against human insulin (line 1) provides an estimate of relative specificity. A relative specificity greater than 1 (less than 1) indicates an increase (decrease) in receptor binding stringency. In this assay, Asp^B10Insulin shows an increased affinity for IGF-1R, but since the increase in affinity for IR is more pronounced, the relative specificity is greater than 1. Containing substituted Asn^A21Two residue extensions of the → Gly and B-chain (Arg)^B31And Arg^B32) Insulin glargine (Lantus)^®) It appears that the absolute affinity for IGF-1R is increased, the absolute affinity for IR is decreased, and the relative stringency of receptor binding is decreased. Insulin analogues of the invention exhibit the opposite properties: absolute affinity for IGF-1R decreases and the relative stringency of receptor binding increases.

Watch (A) 1

^aError from standard error of the mean.

^bThe relative affinity of wild-type insulin for IR (column 2) was defined as 100%; the relative affinity of IGF-I for IGF-1R (column 3) is also defined as 100%. The respective absolute dissociation constants are similar.

Novel combinatorial insulin A-chain analogs containing A-chain amino acid substitutions can be prepared by total chemical synthesis of variant A-chains. The wild-type B-chain is obtained from commercial human insulin preparation by oxidative sulfitation (sulfitolysis); the DKP B-chain was also prepared by general chemical synthesis. Insulin analogs were in each case obtained by combination of the insulin chains followed by chromatographic purification. In each case, the predicted molecular weight was confirmed by mass spectrometry.

Insulin analogues containing paired histidine substitutions at positions A4 and A8, with or without substitution of Gly for Asn, were synthesized^A21In the case of wild-type human B-chain (SEQ. ID. number 3), it is as shown generally in SEQ. ID. number 4. A comparison of the properties of these analogues with human insulin showed that the overall effect of the A1, A8 substitutions was a decrease in the affinity of the analogue for IGF-1R and an increase in the ratio of the affinity for IR to that for IGF-1R (Table 2).

SEQ. ID. number 4 (paired histidine substitutions A4 and A8)

Watch (A) 2

Receptor binding properties of insulin analogs^a

^aIR-B refers to isoform B of the human insulin receptor; IGF-1R refers to the human type 1 IGF receptor; SEM is standard error of the mean.

Similarly, insulin analogs having the A-chain polypeptide sequence of SEQ. ID. number 5 or 6 and 20-21 are prepared using wild-type insulin B-chain (SEQ. ID. number 3) or an insulin analog such as insulin glargine. Isoelectric focusing gel electrophoresis demonstrated an upward shift in isoelectric point (from a baseline value of 5.6 found in zinc-free human insulin) to a value of 6.6 in the absence of zinc ions. For this purpose, studies were carried out using preformed pH 3-10 IEF gels (125X 125 mm, 300 μm, SERVALYT from SERVA Electrophoresis GmbH, Heidelberg)^® Precotes^®From Crescent Chemical CoAvailable from Hauppauge, NY). Precotes were tested according to the manufacturer's protocol^®Placed in a horizontal IEF apparatus Multiphor II (Pharmacia Biotech). The cell was pre-cooled to 4 ℃ with a circulating water bath (Brinkman) and the PRECOTE IEF gel was then placed on an electrophoresis bed coated with light mineral oil to achieve efficient heat exchange. The gel was connected to the electrodes at both ends with strips of filter paper soaked with anolyte at pH 3 and catholyte at pH 10 (both from SERVA). Prior to spotting, the gel was pre-focused for 30 minutes using a high voltage power supply (LKB model 2197) with an initial voltage setting of 200 volts and a final setting of 500 volts. After spotting the sample and IEF standards (5-10. mu.L, loading protein concentration 5-10. mu.g), isoelectric focusing was performed at 500-2000 volts for 2 hours, or until a final voltage of 2000 volts was reached, after which focusing was continued for an additional 15 minutes. After IEF, the gel was fixed with 200 ml of 20% trichloroacetic acid for 20 minutes, rinsed with 200 ml of deionized water for 1 minute, stained with SERVA Violet 17 solution and destained with 86% phosphoric acid according to the protocol of the SERVA manual. The IEF standard proteins (from SERVA) used were as follows, with their respective pI values in parentheses: equine heart cytochrome C (10.7), bovine pancreatic ribonuclease a (9.5), lentil lectin (8.3, 8.0, 7.8), equine muscle myoglobin (7.4, 6.9), bovine red blood cell carbonic anhydrase (6.0), milk beta-lactoglobulin (5.3, 5.2), soybean trypsin inhibitor (4.5), aspergillus niger glucose oxidase (4.2), aspergillus niger amyloglucosidase (3.5). The pI of the protein samples was determined by comparing linear regression curves of migration distance versus pH gradient of IEF standards.

SEQ. ID. NO. 5 (His^A4, His^A8Substituted)

SEQ. ID. NO. 6 (His^A4, His^A8, Gly^A21Substituted)

SEQ. ID. NO. 7 (His^B1B-chain)

Receptor-binding assay-relative activity is defined as the ratio of dissociation constants for the wild-type and variant hormone-receptor complexes. Data were corrected for non-specific binding (at 1)The amount of radioactivity remaining in the relevant membrane in the presence of human insulin). In all assays, the percentage of bound tracer in the presence of non-competing ligand was less than 15% to avoid the artifact of ligand depletion. The relative affinity of insulin analogs for isolated insulin pan receptor (isoform B) was determined using microtiter plate antibody capture techniques known in the art. Microtiter plates (Nunc Maxisorb) were incubated with AU5 IgG (100. mu.l/well of 40. mu.g/ml phosphate buffered saline) overnight at 4 ℃. Binding data was analyzed by a two-site order model. Cross-binding to this cognate receptor was assessed using a corresponding microtiter plate antibody assay using IGF type I receptor.

Rodent assay-male Lewis rats (average body weight about 300 g) were diabetic with streptozotocin. The effect of insulin analogues on blood glucose concentration after subcutaneous injection relative to wild-type insulin or buffer only (16 mg glycerol, 1.6 mg m-cresol, 0.65 mg phenol and 3.8 mg sodium phosphate (pH 7.4); Lilly dilution) was evaluated with a clinical glucometer (Hypoguard advanced Micro-Draw meter). Wild type insulin and [ His ] in the above buffer^A4, His^A8]Insulin is zinc-free. [ His ]^A4, His^A8]-insulin and insulin glargine are also dissolved in ZnCl at a ratio of 5.2:1₂Insulin monomer, 25 mM meta-cresol and 185 mM glycerol in dilute HC (pH 4). When t = 0Each rat (which corresponds to 2 IU/kg body weight for wild type insulin) was injected subcutaneously with 3.44 nanomole of insulin or insulin analog (approximately 12-13.7 nanomole)/100 μ l buffer. For the neutral zinc-free formulation, blood was obtained from the sheared tail tip at 0 and every 10 minutes, up to 90 minutes. For the acidic zinc formulation, blood was obtained at times of 0, 1, 2, 4, 6, 10.8 and 24 hours.

The [ His ] assay was performed as follows^A4, His^A8]-crystal structure of insulin to count and visualize the number of zinc ions in each hexamer and to test whether the paired histidine substitutions at sites a4 and A8 will direct the binding of interfacial zinc ions between hexamers in the lattice. Growing crystals in the presence of zinc ions and phenol to produce T₃R^f ₃A hexamer. The structure was obtained by molecular substitution at a resolution of 1.9A (Table 3). The hexamer assembly pattern of the analogue (figure 2d) was identical to that of wild type insulin (figure 2 c). T and R^fThe respective conformations of the protomers are essentially identical to wild-type insulin. No interference of transmission occurs at the proposed receptor binding surface.

The wild-type and variant hexamers each contain two axes of Zn ions, each T₃And R^f ₃The trimers each contain one (covered central sphere in fig. 2c, 2 d). Each site is coordinated by a trimer-associated His^B10Side chains mediate, with distorted tetrahedral geometry (light grey in hexamer centers in fig. 2c, d). At R^f ₃In the trimer, the fourth ligand is chloride ion, and in T₃In trimer, this site (ratio at R)^f ₃More exposed in the trimer) appear to be partially occupied by chloride ions or bound water molecules. These features are consistent with the wild-type structure. R is also as observed in wild type crystals grown under similar conditions^f ₃The trimer contains three bound phenol molecules (not shown). Thus, substitutions of a4 and A8 do not block the TR transition, which is a classical model of insulin recombination upon receptor binding.

Variant T₃R^f ₃The T-state surface of the hexamer contains three additional trimer-associated Zn ions (purple spheres in FIGS. 2b and 2 d). These novel Zn ion moieties are His through the interface site^A4And His^A8And (4) coordination. Representative electron densities of the peripheral Zn-binding sites define the tetrahedral sites of the deformation (FIG. 2 e). By chloride ions and R belonging to adjacent hexamers^fPrime "nail" His^A4The side chain to complete the coordination (marked with a 4' in fig. 2e, and a brown arrow in fig. 3 b). T and R adjacent to the hexamer^fThe opposite view of the face is shown in fig. 3c (rotated 90 deg. in the direction shown in fig. 3 b). Also by binding to R^fA network of three water molecules on the protomer (smaller spheres paired in space in FIG. 3d) to stabilize the binding of chloride ions; substitution of R from the Zinc-binding site^fHis in^A8. Thus, T of adjacent hexamers in three non-canonical zinc ion bridged lattices₃And R^f ₃Trimers (larger spheres, fig. 3b and 3d), partially replace the water molecules originally bound at the wild-type interface (smaller spheres in fig. 3 a). N-Zn²⁺The bond length and bond angle are similar to those of axial metal-ion-binding sites. T and R^fHis between protomers^A4And His^A8Are different in side chain conformation.

Binding of hormones to IR and IGF-1R was studied to assess relative affinity and receptor binding selectivity (fig. 3e and table 2). The ligands were characterized as zinc-free monomers. Insulin glargine (solid and dashed lines, data points are shown as squares) showed a 2-fold decrease in affinity for IR and a 3-fold increase in affinity for IGF-1R, compared to the binding of human insulin to IR and IGF-IR (solid and dashed lines, respectively, data points are shown as crosses in FIG. 3 e). In contrast, [ His ]^A4, His^A8]Insulin shows a native-like affinity for IR (solid line, inverted triangle in fig. 3 e), but a 6-fold decrease in affinity for IGF-1R (dashed inverted triangle, right shift). Thus, although the receptor binding selectivity of insulin glargine is lost about 6-fold, [ His ]^A4, His^A8]Insulin was increased 7.5 (. + -. 2.5) fold. This indicates an increase in insulin glargineAt least 30 times more.

[ His ] tested in streptozotocin-induced diabetic rats^A4, His^A8]Potency and duration of action of insulin relative to insulin glargine (fig. 3 f). For glycemic control with long acting insulin analogs, the prolongation in rodents (5-10 hours) is shorter than in humans (18-24 hours), presumably due to the smaller subcutaneous depot volume. Will [ His ]^A4, His^A8]Insulin and insulin glargine dissolution (like Lantus)^®Same) in dilute HCl (pH 4.0) and Zn²⁺The molar ratio of insulin is 5.2: 1. The time course and extent of glycemic control were similar when both analogs were injected (dotted and dotted/dashed lines in fig. 3 f). A quick-acting control was provided by zinc-free human insulin in Lilly dilution (in fig. 3f, line was terminated after about 3 hours). Since rats are fed only at night, the effect of injecting daytime insulin is affected by daytime fasting; controls were provided by injecting diluent only (dashed line in figure 3 f). [ His ] in neutral Lilly dilutions without Zinc^A4, His^A8]Insulin control study with a time course similar to the wild-type insulin control (not shown). Insulin glargine was not tested at neutral pH due to its weak solubility without zinc.

X -ray crystallographyZn at 1:1.7²⁺Crystals were grown by hanging drop vapor diffusion in Tris-HCl buffer in the presence of a ratio of protein monomer and a ratio of phenol to protein monomer of 3.5: 1. The droplets contained 1. mu.l of protein solution (8 mg/ml in 0.02M HCl) mixed with 1. mu.l of stock solution (0.38M Tris-HCl, 0.1M sodium citrate, 9% acetone, 4.83 mM phenol and 0.8 mM zinc acetate, pH 8.4). Each droplet was suspended in more than 1 ml of stock solution. Crystals were obtained after two weeks at room temperature. Data was collected from single crystals placed on a rayon ring and instantaneously frozen to 100 ° K. The reflections under 32.05-1.90A were measured using a synchrotron radiation CCD detector system from Berkeley national laboratory. The data is processed with the program DTREK. The crystal belongs to space group R3, and the unit cell parameters are as follows: a = b =78.09 a, c =36.40 a, α = β =90 °, γ =120 °.The structure was determined by molecular replacement with the CNS. Thus, the model was obtained using native TR dimer (protein database (PDB) identifier 1RWE after removal of all water molecules, zinc-and chloride ions). After analyzing the data between 15.0 and 4.0 a resolution, a translation-function search was conducted with the coordinates of the best solution for the rotation function. Rigid body refinement (refinishment) corrected with all anisotropic temperature factors and bulk solvent (bulk-solvent) using the CNS for resolution data between 19.2 and 3.0A for R and R_freeThe resulting values were 0.325 and 0.344, respectively. Between refinement cycles, calculate 2 using data for 3.0A resolution _oF – _cFAnd _oF – _cFa drawing; by means of programsO(Jones et al,Acta Crystallogr. A., vol.4, pp.110-119 (1991)) built zinc and chloride ions and phenol molecules into the structure. The water molecules were calculated and examined using the DDQ program (Focco Van Akker and Wim Hol, Acta Crystal. 1999, D55, 206-218). By usingPROCHECK (Laskowski et al,J. Appl. Crystallogr., vol 26, pp 283-291 (1993)) continuously monitoring the geometry; zinc ions and water molecules were built into different figures as the refinement proceeded. The calculation of the missing map (in particular the first eight residues at the N-terminus of each monomeric B chain) was performed with the CNS and further refined, where maximum likelihood torsion angle kinetics and conjugate gradient refinement were performed.

Watch (A) 3 X -ray data collection and refinement statistics

Highest resolution (resolution shell)

Methods by which dimachi and colleagues were improved (Kohn, w. D., Micanovic, r., Myers, s. l., Vick, a. m., Kahl, s. D., Zhang, l., striffler, b. a., Li, s., Shang, j., Beals, j. m., Mayer, j. p., and dimachi, r. D).Peptides 28, 935-48 (2007)), to evaluate pH-dependent solubility of insulin analogues. Briefly, wild-type human insulin, insulin glargine or [ His ]^A4, His^A8]Insulin is made 0.60 mM in a non-buffered solution containing dilute HCl, pH 4.0; the composition in solution was similar to that used in the pharmaceutical formulation Lantus (Sanofi-Aventis), which contained 0.52 mM ZnCl₂20 mg/ml of an 85% v/v glycerol solution (to a final concentration of 185 mM) and 2.7 mg/ml m-cresol (25 mM) as an antimicrobial preservative. Each of the three proteins showed a solubility in this pH 4.0 solution of more than 0.60 mM. A series of identical aliquots (10 ml) were removed, diluted 50-fold with different pH buffers (range 5.0-9.0) to a final volume of 500 ml, and then readjusted to pH 5.0, 6.0, 7.4, 8.0, 8.5 and 9.0, respectively. The dilution consisted of 10 mM Tris-HCl and 140 mM NaCl and was adjusted in pH with dilute HCl or NaOH. The multiple samples were then mixed by inversion 20 times and centrifuged in a microcentrifuge for 5 minutes at 14,000 rpm. Then 200. mu.l of supernatant from each tube was taken in duplicate and injected into an analytical reverse phase HPLC (C4 column; 25 cm. times.0.46 cm) with an elution gradient of acetonitrile (containing 0.1% trichloroacetic acid). In each case, a single elution peak was observed, the area of which was quantified by integration with vendor software (Waters, Inc.). Wild-type insulin values at pH 7.4-9.0 provide a control for loss unrelated to solubility; percent recovery is typically in the range of 85-90%. Consistent with the results of DiMarchi and co-workers, insulin glargine was found to have a solubility of 1 to 2 at pH 7.4In the meantime. This limited solubility is similar to a zinc to analogue molar ratio of 5.2:6 (i.e. 5.2 zinc ions per hexamer) and 2.2:6 (2.2 zinc ions per hexamer), consistent with the axixing effect of zinc ions in the glycomacropolymer. At a molar ratio of 5.2 zinc ions/hexamer, [ His ] was found^A4, His^A8]Insulin solubility at pH 7.4 also 1-2。

The preparation of the invention provides Lys containing also insulin lispro (Humalilog)^B28And Pro^B29The intermediate insulin analogue of (1), which is readily prepared as a clear solution containing zinc ions and phenol at pH 4. Representative binding of insulin analogs containing lispro and histidine substitutions at positions a4 and A8 (HisA4, A8 KP-ins) to wild-type Human Insulin (HI) for human insulin receptor isoform a (hira), human insulin receptor isoform b (hira) and insulin-like growth factor receptor (IGF-1R) is provided in table 4. As can be seen in table 4, HisA4, a8 KP-ins have similar affinity for HIRA and HIRB as HI, but have a greatly reduced affinity for IGF-1R compared to HI (a greater than 4-fold reduction).

Figure 4 provides the time course of blood glucose levels in diabetic male rats under the conditions described in figure 3 f. Will [ His ]^A4, His^A8]-KP insulin, lispro insulin and insulin glargine are dissolved in (like Lantus)^®Same) dilute HCl (pH 4.0), and Zn²⁺The molar ratio of insulin is 5.2: 1. [ His ]^A4, His^A8]The time course of glycemic control by KP insulin is shorter than that of insulin glargine (Lantus)^®) But longer than insulin lispro (Humalog)^®) This indicates that the formulation provides a medium-acting insulin analogue formulation. In addition, under the conditions as provided above, crystals of HisA4, A8 KP-ins were also obtained. Without wishing to be bound by theory, it is believed that the lispro-substituted hexamer-destabilization effect is different from [ His^A4, His^A8]Substituted hexameric complexes-stabilized and at least partially [ His ]^A4, His^A8]The stabilizing effect of the substituted hexameric complex is counteracted, thereby producing an intermediate analog.

Also imagine [ His ]^A4, His^A8]Insulin analogues may also contain other substitutions, e.g. Asp^B28To obtain other intermediate-acting insulin analogue formulations. It is also envisaged that pairs of zinc-coordinated amino acid side chains, such as histidine side chains, may be introduced onto the surface of the protein structure, useful for other proteins (figure 5) to stabilise higher order structures, such as protein hexamers in insulin. More specifically, we envision that side chains from paired histidine substitutions in alpha-helix containing proteins can be complexed with complementary side chains in other polymers to form multimeric complexes. Examples of therapeutic proteins containing alpha-helices are erythropoietin and mammalian growth hormone.

Based on the foregoing disclosure, it should now be apparent that insulin analogs containing an a-chain substitution combination as provided herein, when formulated in the presence of zinc ions, will provide long lasting duration of insulin action and will concomitantly exhibit reduced absolute and relative affinity for the type I IGF receptor while maintaining at least 20% of the affinity of human insulin for the insulin receptor.

Sequence of

SEQ. ID. number 1 (proinsulin)

SEQ. ID. number 2 (A-chain)

SEQ. ID. number 3 (B-chain)

SEQ. ID. number 4 (paired histidine substitutions A4 and A8)

SEQ. ID. NO. 5 (His^A4, His^A8Substituted)

SEQ. ID. NO. 6 (His^A4, His^A8, Gly^A21Substituted)

SEQ. ID. NO. 7 (His^B1B-chain)

SEQ. ID. number 8 (insulin lispro-B-chain)

SEQ. ID. number 9 (aspart-B-chain)

SEQ ID No. ID. number 10 (AspB 10-B-chain)

SEQ ID No. ID. number 11 (DKP B-chain sequence)

SEQ. ID. number 12 (insulin glargine B-chain)

SEQ No. ID. NO 13 (insulin glargine A-chain)

SEQ. ID. NO. 14 (Arg^A0, His^A4, His^A8,Gly^A21Substituted)

Claims (10)

1. Use of a pharmaceutical formulation containing a physiologically effective amount of an insulin analogue or a physiologically acceptable salt thereof, wherein the insulin analogue or a physiologically acceptable salt thereof contains an insulin a-chain sequence containing paired histidine substitutions at a4 and A8, optionally a substitution at a21 selected from the group consisting of glycine, alanine, serine and threonine, and the pharmaceutical formulation additionally contains zinc ions at a relative concentration of at least 4 zinc ions per 6 insulin analogue molecules, for the preparation of a long acting insulin formulation for the treatment of diabetes.

2. The use of claim 1, wherein the insulin analogue or a physiologically acceptable salt thereof is a microcrystalline insulin suspension at pH 6.5-7.5.

3. The use of claim 1, wherein the insulin analogue or a physiologically acceptable salt thereof is formulated as a clear non-buffered solution at pH 3.5-5 containing zinc ions in a relative concentration of 4-6 zinc ions per 6 insulin analogue molecules, a preservative selected from phenol and m-cresol, and an excipient.

4. The use of claim 1, wherein the insulin analogue or a physiologically acceptable salt thereof is formulated as a microcrystalline insulin suspension modified to include 4-6 zinc ions per 6 insulin analogue molecules.

5. The use of any one of claims 1-4, wherein the insulin analogue or a physiologically acceptable salt thereof is modified by substitution of position A21 with glycine.

6. The use of any one of claims 1-4, wherein the insulin analogue or a physiologically acceptable salt thereof is modified by substitution of position A21 with alanine, serine or threonine.

7. The use of claim 6, wherein the insulin analogue or a physiologically acceptable salt thereof is also modified by extending the B-chain to include one or two C-terminal basic residues.

8. The use of claim 6, wherein the insulin analogue or a physiologically acceptable salt thereof is also modified by extending the B-chain to include at least one N-terminal basic residue.

9. The use of claim 6, wherein the insulin analogue or a physiologically acceptable salt thereof is also modified by extending the A-chain to include at least one N-terminal basic residue.

10. The use of claim 1, wherein the insulin analogue forms a long-acting zinc-dependent subcutaneous depot upon subcutaneous injection.