WO2015071368A1

WO2015071368A1 - Long-acting insulin glargine analogue

Info

Publication number: WO2015071368A1
Application number: PCT/EP2014/074508
Authority: WO
Inventors: You-Ping Chan; Corine Vialas; Maximilien BLAS
Original assignee: You-Ping Chan
Priority date: 2013-11-14
Filing date: 2014-11-13
Publication date: 2015-05-21
Also published as: FR3013049B1; FR3013049A1

Abstract

The present invention relates to an insulin glargine analogue which is Lys^B29(N^ε-His)Gly^A21Arg^B31Arg^B32-human insulin.

Description

LONG-ACTING INSULIN GLARGINE ANALOGUE

FIELD OF INVENTION The invention relates to a new basal insulin glargine analogue for the treatment of diabetes, its preparation and use.

TECHNICAL BACKGROUND The prevalence of diabetes poses a major health burden around the world and this is expected to increase significantly in the future. Analogues of insulin have been engineered with the aim of improving the treatment. Rapid acting, intermediate acting and long acting (basal) insulins have been developed and approved for human use. The goal of the treatment is to mimic prandial and/or basal insulin. The most common side effect of insulin therapy is hypoglycemia when too much insulin is present as a result of either too much insulin injected or peak concentration due to variability of absorption rate from subcutaneous tissue. The latter is particularly true for intermediate or long acting insulins that form a depot or are in crystalline form at the injection site. Insulin is one of the most studied proteins in terms of structural properties and chemical modifications, and implications on physiological properties. Human insulin is composed of two chains of 21 (A-chain) and 30 (B-chain) aminoacids linked through two inter-chain and one intra-chain disulfide bonds. Human insulin has a molecular weight of 5807 and isoelectric point at around 5.3. In solution at neutral pH, insulin displays a concentration dependent self-assembly into dimers and hexamers: see Brange in Galenics of Insulin Springer Verlag 1987. The hexameric form is the most stable form but it is the monomer that is biologically active.

Currently, the most efficacious insulin products used are insulin analogues (see Oiknine at al. Drugs 2005, 65, 325-340 for a review). Rapid acting insulins are those based on analogues in which the dimer and/or hexamer structures are unfavorable, so the monomeric form is rapidly available after administration. These are commercialized under trade names Humalog® (insulin lispro), Novolog® (insulin aspart) and Apidra^® (insulin glulisine); the common name of the insulin analogue is given in parenthesis. Long acting basal insulins, Lantus^® (insulin glargine), has an isoelectric point at around 7 and precipitates on the site of injection leading to slow release of the monomers from the injection site, and Levemir^® (detemir), has a fatty acid grafted on the B-chain and this leads to a prolonged action due to adsorption to circulating albumin. Both these long acting insulins do not provide full 24-hour coverage in all patients. Also, in the case of detemir the potency of the insulin has been reduced by a factor of four due to the chemical modification thus requiring more protein for the same effect: see Kurtzhals et al. Diabetes, 2000, 49, 999-1005. The need for better long acting insulins, namely a flat profile (peakless) to reduce nocturnal hypoglycemic events is still highly desirable to improve glyceamic control and is an active field of research.

Recently, Tresiba^® (insulin degludec) has been approved in Europe and Japan. This insulin has one hexadecanedioic acid grafted to Lys B29 via a gamma-L-glutamyl spacer. This modification allows for the formation of multi-hexamers in subcutaneous tissues resulting in slow insulin release over time and also has strong affinity to albumin binding. Overall, the duration of action is greater than 24 hours. Synthesis and description of this molecule are given in US patent 7,615,532 and Jonassen et al. Pharm. Res. 2012, 29, 2104-21 14.

Particularly relevant to the invention is insulin glargine (active protein of Lantus^®) that is described in US patent 5,656,772. The insulin glargine molecule has the A21 asparagine replaced by glycine and two arginines have been added to the terminal B30. The molecule is termed Gly^A21-Arg^B31-Arg^B32-human insulin or more commonly insulin glargine. It is formulated at pH 4 and precipitates at neutral pH, its isoelectric point being about 7.0. The protein is made by site-directed mutagenesis in E. Coli. Other approaches to obtain long-acting insulins have been described in the literature but none have led to commercial products so far. Indeed, the pK profile requirement for a truly basal insulin that meets the patients need is very stringent. Some examples based on insulin modifications are given hereafter. US patent 6,221 ,837 describes insulin analogues with 1 to 5 histidines at the B30 position. These products are able to bind more zinc and provide delayed action profile after subcutaneous injection. The extension of duration up to 14 hours in dogs requires excess of zinc (typically 80 mg/mL for an insulin formulation of 40 lU/mL). The duration for a zinc free formulation of the same compounds is similar to human insulin and is about 6 to 8 hours. In a similar manner, US patent 6,686,177 describes insulin analogues with 1 to 5 histidines at the B0 position to increase zinc binding and the duration is extended to 16 hours in dog.

Philipps et al. J. Biological Chemistry 2010, 285, 1 1755-1 1759 describe insulin analogues in which two histidines are present at A4 and A8. The goal being to create zinc stapled hexamers and thus improve the time action profile. In a rat study, the duration was found to be similar to Lantus®.

In another publication by Kohn et al. Peptides 2007, 28, 935-048, the authors describe a strategy to extend the duration by increasing the isoelectric point of insulin by addition of positively charged lysines and/or arginines. Promising results are obtained when one arginine is added to the N-terminal of the A chain and two arginines to the C-terminal of the B-chain. The isoelectric point is measured at 7.3 compared to 7.0 for insulin glargine. However their biopotencies are much lower compared to insulin glargine. The molecule is also described in US patent application 2006/0217290.

US patent 2005/0014679 describes the addition of basic aminoacids such as arginine or lysine to insulin in order to provide long acting formulations. Products described, for example Arg ^A0Gly^A21Lys-(N^£-Arg)^B29 - insulin have a time action profile similar to Lantus^®.

In this context, modification of insulins by various ways (mainly fatty acid grafting and sequence changes of the A and/or B chain to increase isoelectric point) may lead to improved properties such as albumin binding or pH induced precipitation but sometimes at the expense of losing potency and/or inducing changes in pharmacological properties that exclude lifelong clinical use. Obtaining true basal insulin with very low peak and trough ratios with one or less than one injection per day to improve patient treatment remains a challenge.

SUMMARY OF THE INVENTION The inventors have found that the pharmacokinetic properties of insulin glargine (active protein of Lantus^®) can be significantly improved by having a single histidine grafted to the N- epsilon lysine residue at position B29 of insulin glargine via an amide bond termed Lys^B29(N^£- His) insulin glargine or Lys^B29(N^£-His)Gly^A21Arg^B31Arg^B32-human insulin hereafter. In terms of structural consideration, this new molecule differs from prior art molecules described above in which one or several histidines are inserted in the main insulin A- or B-chain or the end chains. With this new insulin analogue, long acting formulations have been obtained without the need for excess zinc or converting to crystalline suspensions. The addition of one histidine as described herein does not have any impact on the structural features and the isoelectric point of insulin glargine precursor. The improvement contemplated is a longer and flatter release profile compared to Lantus^® and thereby leads to a reduced number of hypoglycemic and hyperglycemic events with only one injection per day or less.

Consequently a first aspect of the invention relates to an insulin glargine analogue which is Lys^B29(N^£-His)Gly^A21Arg^B31Arg^B32-human insulin.

In a second aspect, the invention relates to a pharmaceutical composition comprising insulin glargine analogue which is Lys^B29(N^£-His)Gly^A21Arg^B31Arg^B32-human insulin. The present invention also relates to an insulin glargine analogue or a pharmaceutical composition according to the invention for use in a therapeutic treatment in human or animals. In particular, the present invention relates to the insulin glargine analogue or the pharmaceutical composition as described above for use in the treatment of diabetes and/or hyperglycemia.

The present invention also relates to a process for the manufacture of the insulin glargine analogue according to the invention, comprising the step of grafting of a protected histidine to insulin glargine at pH 10 to 1 1 , followed by a deprotection step.

DETAILED DESCRIPTION Definitions

As used herein and unless specified otherwise, the term "amino acid" refers to natural or unnatural amino acids in their D and L stereoisomers for chiral amino acids. It is understood to refer to both amino acids and the corresponding amino acid residues, such as are present, for example, in peptidyl structure. Natural and unnatural amino acids are well known in the art. Common natural amino acids include, without limitation, alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (lie), leucine (Leu), Lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). In naturally occurring proteins, the amino acids are all L stereoisomers.

Insulin glargine analogue The present invention relates to insulin glargine analogue which is Lys^B29(N^£- His)Gly^A21Arg^B31Arg^B32-human insulin. Lys^B29(N^£-His)Gly^A21Arg^B31Arg^B32-human insulin is also called hereinafter Lys^B29(N^£-His) insulin glargine.

By insulin glargine analogue, it is meant an analogue of insulin glargine or more precisely, in the context of the invention, a chemically modified insulin glargine.

As mentioned above insulin glargine is a well-known compound which is described in US patent 5,656,772 and marketed under the brand Lantus^®.

The insulin glargine molecule has the A21 asparagine replaced by glycine and two arginines have been added to the terminal B30 when compared to native human insulin. The molecule is termed Gly^A21-Arg^B31-Arg^B32-human insulin or more commonly insulin glargine. Lys (N^£-His) means that a histidine aminoacid is grafted to the N-epsilon amine group of the lysine residue in the B29 position of insulin glargine (or Gly^A21Arg^B31Arg^B32-human insulin) via an amide bond.

In particular, the insulin glargine analogue of the invention does not comprise any arginine or equivalent aminoacid residue at the AO position.

In one embodiment, the histidine of (N^£-His), that is to say the histidine aminoacid which is grafted to the N-epsilon amine group of the lysine residue in the B29 position of insulin glargine, is selected from the group consisting of L-histidine, D-histidine and D,L-histidine. Preferably, the histidine of (N^£-His) is L-histidine.

Pharmaceutical composition

In a second aspect, the present invention relates to pharmaceutical composition comprising the insulin glargine analogue as described above.

The insulin glargine analogue is formulated to provide a pharmaceutical composition that is clinically acceptable to be administered to a human or an animal. Accordingly, the pharmaceutical composition comprises the insulin glargine analogue and at least one pharmaceutically acceptable excipient.

Pharmaceutical vehicle or excipients are known to those skilled in the art. These most typically would be standard vehicles or excipients for administration of compositions to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions could also be administered orally, intramuscularly, subcutaneously, or in an aerosol form. The compositions may be administered according to standard procedures used by those skilled in the art. Pharmaceutical excipients can include thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Descriptions of some of these pharmaceutically acceptable excipients or vehicles may be found in The Handbook of Pharmaceutical Excipients, published by the American Pharmaceutical Association and the Pharmaceutical Society of Great Britain. Remington: the Science and Practice of Pharmacy 20th edition (2000), describes compositions and formulations suitable for pharmaceutical delivery of the insulin glargine analogues of the invention, in the form of aqueous solutions, lyophilized or other dried formulations. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like. Pharmaceutical compositions can also include one or more additional active ingredients. In a preferred embodiment, the pharmaceutical composition is an aqueous solution. Typically, the pharmaceutically acceptable excipient is water. In a preferred embodiment, the insulin glargine analogue is at a concentration from 1 mg/mL to 20 mg/mL. It is assumed that the potency of the compound is similar to the potency of Lantus®. In that case, 100 IU will be equivalent to 600 nmol or 3.7 mg. It is contemplated that pharmaceutical formulations can be in the range of 40 to 500 lU/mL which would be equivalent to 1 .49 to 18.6 mg of protein respectively. Preferably the formulation is in the range of 40 to 300 lU/mL or for example between 3.5 and 4 mg/mL.

The pharmaceutical composition may typically comprise divalent salt, base, acid, isotonicity agent, preservatives and/or sterile water.

In a preferred embodiment, the pharmaceutical composition comprises divalent metal ions selected from the group consisting of zinc, magnesium, copper and calcium ions. Preferably, the divalent metal ions are zinc ions. The zinc salts are preferably zinc chloride, zinc sulfate, zinc oxide or zinc acetate. The concentration of zinc ions is preferably from 0.3 to 3 equivalents per mole of insulin glargine analogue, more preferably from 0.5 to 1 .5 equivalents per mole of insulin glargine analogue.

In a preferred embodiment, the pharmaceutical composition has a pH from 3.5 to 4.5. Formulated at pH 3.5 to 4.5, the formulation is a clear liquid that is injectable through small 30 to 32 G needles. Preferably, the pH is about 4. Acids and bases used to adjust pH may be, for example, sodium hydroxide and hydrochloric acid, and for ensuring buffering properties, phosphate, acetate or citrate may be used.

The pharmaceutical composition may further comprise a preservative. The preservative which is necessary for multiple-use vials or cartridges may be selected from the group consisting of phenol derivatives such as phenol, m-cresol, chlorocresol, and benzylalcohol and mixture thereof. Preferably, the preservative may be selected from the group consisting of phenol, m-cresol and a mixture thereof.

Phenol and m-cresol mixture or m-cresol alone is preferred.

The pharmaceutical composition may further comprise an isotonicity agent. For example, isotonicity agent may be glycerol (glycerin), sodium chloride, trehalose, mannitol or dextrose. For subcutaneous injection, the osmolality is usually adjusted to about 290 to 330 mOsm/kg. The amount ranges from about 1.6 to 2.5 % wt/wt. Isotonicity agent is preferably glycerol or sodium chloride, more preferably glycerol. Additionally a surfactant to reduce or prevent surface adsorption may be added as described in prior art: Development and manufacture of protein pharmaceuticals Eds. Nail et al. Chapter 2, 2002, Kulwer Academic/plenum publishers, NY. In a specific embodiment, the pharmaceutical composition comprises:

- an insulin glargine analogue at a concentration between 3.5 and 4.0 mg/mL, preferably 3.7 mg/mL,

- zinc ions at a concentration of 20 to 50 μg/mL,

- m-cresol at a concentration of 25 to 30 mmol/L,

- glycerol at about 1 .6 to 2.5 % wt/wt,

and pH is about 4.

Method of treatment

Another object of the invention relates to the insulin glargine analogue or the pharmaceutical composition as described above for use in a therapeutic treatment in human or animals.

Another object of the invention relates to a method of treatment comprising administering to a subject in need thereof a therapeutically effective amount of the insulin glargine analogue or the pharmaceutical composition as described above.

Another object of the invention relates to a use of insulin glargine analogue or the pharmaceutical composition as described above in the manufacture of a medicament.

In particular, the present invention relates to the insulin glargine analogue or the pharmaceutical composition as described above for use in the treatment of diabetes and/or hyperglycemia. In particular, the present invention relates to a method for treating diabetes and/or hyperglycemia comprising administering to a subject in need thereof a therapeutically effective amount of the insulin glargine analogue or the pharmaceutical composition as described above. In particular, the present invention relates to a use of insulin glargine analogue or the pharmaceutical composition in the manufacture of a medicament for the treatment of diabetes and/or hyperglycemia.

Process for the manufacture

Another object of the invention relates to a process for the manufacture of the insulin glargine analogue as described above comprising the step of grafting of a protected histidine to insulin glargine at pH 10 to 1 1 , followed by a deprotection step. The insulin glargine analogue of the invention is conveniently made by grafting a protected histidine derivative onto insulin glargine followed by deprotection. The techniques that can be used are similar to those used for acylation of insulin or insulin analogues and are known to those skilled in the art. For example, US patent 5,646,242 teaches acylation preferably at position B29 (epsilon-lysine position) at pH above 10. The histidine used to perform this reaction has its carboxylate function activated by an N-hydroxysuccinimide (NHS activated ester) and the active nitrogens protected by t-butyloxycarbonyl (Boc) functions. After the acylation reaction, the Boc are removed using trifluoroacetic acid. The Boc-His(Boc)-N- hydroxysuccinimide ester or similar compounds can be prepared easily according to procedures describe for example in Greene's Protecting Groups in Organic Synthesis, Wuts et al. John Wiley and Sons 4^th Ed. 2007. The histidine may be L-histidine, D-histidine or a racemic mixture.

The insulin glargine used can be produced using standard recombinant techniques for example in E. Coli, saccharomyses cerevisae or pichia pastoris. Synthesis of insulin glargine is described for example in US patent 5,656,772 and US patent applications 201 1/0236925 and 2012/0214965.

The invention will be further illustrated by the following figures and examples. However, the examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES Figure 1 : Figure 1 shows a gel after isoelectric focusing to determine Ip of Insulin (well 1 ), LysB²⁹(N^£-His) insulin glargine (well 2), insulin glargine (well 3) and Lantus® Insulin glargine (well 4). M: Ip Marker.

Figure 2: Figures 2A and 2B show thermograms of Insulin glargine and LysB²⁹(N^£-His) Insulin glargine formulations. Figure 2A shows a thermogram of a composition wherein Insulin glargine is at 3.6 mg/mL and pH is 4. Figure 2B shows a thermogram of a composition wherein LysB²⁹(N^£-His) Insulin glargine is at 3.6 mg/mL and pH is 4.

Figure 3: Figures 3A and 3B show near-UV spectra of Insulin glargine and LysB²⁹(N^£-His) Insulin glargine formulations. Figure 3A shows near-UV spectra of a composition wherein Insulin glargine is at 3.6 mg/mL and pH is 4. Figure 3B shows near-UV spectra of a composition wherein LysB²⁹(N^£-His) Insulin glargine is at 3.6 mg/mL and pH is 4. Figure 4: Figure 4 shows mean PK profiles after SC single administration of formulation FT-2 as described in example 4 below and Lantus®.

Examples:

Example 1 : Synthesis of Lys^B29(N^£-His) insulin glargine

About 600 mg of insulin glargine (Biocon) were dissolved in a sodium carbonate buffer (50 mM, pH 10.2) to obtain a protein concentration of 20 g/L. Boc-L-His(Boc)-N- hydroxysuccinimide ester (1 equivalent) was dissolved in acetonitrile (30 mL) and then added to the protein solution. The solution was left stirring for 30 min at room temperature and the reaction was quenched by addition of ethanolamine (12 equivalents, 6.1 mL adjusted to pH 9.0 with HCI 0.1 N) and 120 mL of water. The conversion to the desired mono-acylated protein was estimated at about 67 % by RP-HPLC. The reaction mixture was then freeze- dried and dissolved in pure trifluoroacetic acid (12 mL) for deprotection of the BOC moieties. After 1 hour at room temperature, water (108 mL) was slowly added and the medium was freeze-dried, redissolved in HCI 0.01 N (100 mL) and freeze-dried again. The product was then dissolved to 50 mg/mL in HCI 0.01 N and purified by preparative RP-HPLC employing a Chromolith® Prep RP-18e column (100 x 25 mm, 3 μηη, 120 A). The mobile phase consisted of 0.1 % TFA v/v in distilled water (eluent A) and acetonitrile containing 0.1 % TFA v/v (eluent B). The mobile phase was run with a linear gradient from 25 to 28% eluent B for 6 min then 28% eluent B for 6 min at a flow rate of 20 mL/min and the UV absorbance was monitored at 215 and 280 nm. The fractions containing the desired product were pooled and freeze-dried. Finally the insulin glargine analogue was desalted by dialysis against HCI 0.01 N and freeze- dried. The RP-HPLC purity of the compound was 95% with a yield of 33%.

Example 2: Structural analysis and properties

Mass spectrometry. The molecular mass of Lys^B29(N^£-His) insulin glargine was determined by LC/ESI-MS TOF in positive mode (Mass spectrometer, LC/MSD TOF from Agilent, fitted with an electrospray ion source and a Time-of-Flight analyzer, hyphenated with an HPLC system HP1 100 from Agilent). A main peak at 6200.0 Da was measured (theoretical value is 6200.1 ). Peptide mapping. A peptide mapping of Lys^B29(N^£-His) insulin glargine has been performed using a protease from Staphylococcus aureus strain V8 (Glu-C). Four fragments are expected from this protease digestion which selectively cleaves peptide bonds on the carboxyl side of aspartic and glutamic acid residues. LC/ESI-MS TOF analysis of the digestate confirms that the fragment III (B22 -B32) has been grafted with one histidine while the other fragments remained unchanged as compared to an insulin glargine digestate. The measured molecular mass of the fragment III containing the grafted histidine is 1565.8 Da (theoretical value is 1565.9 Da). The mass spectrometry results of compound 1 and its digestates confirmed the position of the grafting and the chemical structure.

Zinc content. The determination of zinc in the final freeze-dried powder of Lys^B29 (N^£-His) insulin glargine has been carried out by the spectrophotometric method designed by Sabel et al Anal. Biochem. 2010, 397, 218-226. This method allows the determination of low level of zinc ions in aqueous solution using the colorimetric reagent Zincon (2-carboxy-2'-hydroxy-5'- sulfoformazylbenzene). The Zn²⁺-zincon complex formed is stable and present a strong absorbance at 620 nm. The Lys^B29(N^£-His) insulin glargine powder, obtained after synthesis and purification steps, contains a negligible amount of zinc (<0.002% w/w).

Zinc binding. The maximum binding of zinc onto Lys^B29(N^£-His) insulin glargine in comparison with insulin glargine has been assessed via the determination of the related adsorption isotherms. Experiments were performed where the concentration of zinc, as zinc chloride, was increased while the protein concentration (insulin glargine or Lys^B29(N^£-His) insulin glargine) was kept constant at 1 .0 mg/g of solution. The formulations Zn²7protein (insulin glargine or Lys^B29 (N^£-His) insulin glargine) were first prepared at 3.5 mg/mL and pH 4.0 and then diluted in a Tris-HCI buffer (25 mM, pH 7.4) by adding 300 μΙ_ of each sample in 700 μΙ_ of buffer. A precipitate was formed. After 1 h rest at room temperature, free zinc was assayed by spectrophotometry after ultrafiltration of the supernatant (to separate the free from the bound Zn²⁺), as described previously.

A maximum binding of about 1 .5 zinc per Lys^B29(N^£-His) insulin glargine monomer and a maximum binding of about 0.75 zinc per insulin glargine monomer molecules are measured.

Isoelectric point. Isoelectric focusing (IEF) was performed to evaluate the isoelectric point (Ip) of Lys^B29(N^£-His) insulin glargine in comparison with recombinant human Insulin and insulin glargine (raw material precursor and Lantus®). The Ip was determined using Novex IEF gel of pH 3-10 (Invitrogen, #EC6655) and comparison with Ip of known markers from 4.45 to 9.6. After electrophoresis, the proteins were visualized by gel incubation in coloration solution (Coomassie blue 0.04 % and Crocein scarlet 0.05% in acetic acid 10% and isopropanol 27%). The isoelectric point of the Lys^B29(N^£-His) insulin glargine was measured at 7.0 and at 7.1 for insulin glargine. The isoelectric point of human insulin was measured at 6.0. The results show that the addition of the histidine moiety to insulin glargine does not meaningfully change its isoelectric point. The results are shown in figure 1 . Microcalorimetry. The melting temperature of the proteins insulin glargine and Lys (Ν^ε- His) insulin glargine, in solution at pH 4, were measured by microcalorimetry (VP-DSC differential scanning microcalorimeter from Microcal, LLC). The solutions were prepared at a concentration of 3.6 mg/mL by suspending the powder in water and bringing the solutions to pH 4 with HCI/NaOH 1 N. Glycerol (2.3%) and m-Cresol (25 mM) were also added. The amount of Zn²7 monomer was 0.75. The scan rate was 1 °C/min and an excess pressure of 28 psi was applied. Both products exhibited a monomodal melting transition at 73-74°C. This temperature suggests that both proteins are in the dimeric state as for human insulin under the same conditions (Huus, K.; et al. Biochemistry. 2005, 44, 1 1 171 -1 1 177). Additionally this melting temperature was found to be not dependent on the amount of zinc in the range of 0 to 1.1 for Lys^B29(N^£-His) insulin glargine in solution, further suggesting that there is no zinc binding at pH 4.0. The results are shown in figure 2.

Circular Dichroism. Near-UV analysis (spectra recorded from 250 to 310 nm) were performed in a 1 cm path length quartz cuvette at 20°C using a speed of 50 nm/min, a response time of 2s and a bandwidth of 1 nm. Each spectrum was the result of an averaging of 3 repeated scans background corrected with the corresponding buffer spectrum. The CD signals were then converted to molar ellipticity. The identical spectral shape and molar ellipticity of both insulin glargine and Lys^B29 (N^£-His) insulin glargine, when formulated at pH 4, and 3.64 mg/mL with an amount of 0.75 Zn²7monomer, imply that the three-dimensional structure of both compounds are the same. The results are shown in figure 3.

Example 3: ln-vitro solubility test An in vitro test was developed to evaluate the capacity of the analogue of the invention to precipitate at a neutral pH (step A) and then its ability to dissociate to soluble form upon dilution (step B). Formulations were prepared at pH 4 and at a concentration of 3.5 mg/mL and further containing 30 μg/mL Zn²⁺ (corresponding to 0.75 Zn²7monomer), 2.3% glycerol and 25 mM m-cresol. This formulation was compared to insulin glargine prepared under the same conditions and which has the same composition as commercial Lantus®. The formulations were then added to a pH 7.4 25 mM Tris-HCI buffered medium (150 μί in 350 L respectively) and allowed to rest for one hour at room temperature. Each suspension was then centrifuged and the percentage of solubilized protein in the supernatant was measured by RP-HPLC. To evaluate the dissociation ability, the suspensions were diluted 50 times in the same buffer and treated as before (centrifugation and HPLC dosing) after one hour rest at room temperature. The results comparing Lys^B29(N^£-His) insulin glargine and insulin glargine are given in the table below. Compound % soluble protein: step A % soluble protein: step B

Lys^B29(N^£-His) insulin glargine < limit of detection (0.5 %) < limit of detection (5 %)

Insulin glargine < limit of detection (0.5 %) 25 %

The new anologue precipitates quantitatively at pH 7.4 as insulin glargine and upon dilution releases no soluble protein compared to 25 % release for insulin glargine. A slower release profile in vivo is thus anticipated for the new analogue.

Example 4; ln-vitro properties ln-vitro Receptor affinity. The affinity of Lys^B29(N^£-His) insulin glargine for insulin receptor was measured by a competitive binding assay.

Microtiter 96-well plates (Meso Scale Discovery, #L15XA-3) were coated with a monoclonal antibody (clone 83-7, Millipore, #MAB1 138) directed against the soluble alpha subunit of the insulin receptor (25μΙ per well of 0.1 μg ml solution in Phosphate Buffered Saline (PBS): 10 mM sodium phosphate, pH 7.4, 137 mM NaCI, 2.7 mM KCI). The plate was incubated for 3 hours at room temperature (22°C) before the saturation step with BSA 5% in PBS during 1 hour with shaking at room temperature. A suitable dilution of the purified soluble extracellular fragment of the insulin receptor (R&D Systems, #1544-IR) was then added in binding buffer (100 mM HEPES, pH 8.0, 100 mM NaCI, 10 mM Mg CI2, 0.02% Triton X-100) and incubated for 2 hours with shaking at room temperature. After 3 washes with cold binding buffer, the binding was performed by incubating for 16 hours at +5°C with 50 μΙ of cold binding buffer containing a mix of biotinylated Human Insulin (IBT Systems, #BiolNS100) at 0.5 nM and different concentrations of unlabeled Human Insulin, Insulin glargine or Lys^B29(N^£-His) Insulin glargine. After 3 washes with cold binding buffer to eliminate unbound peptides, 25 μΙ of streptavidine conjugated to Sulfo-tag (Meso Scale Discovery, #R32AD) at 0.2 μg ml in Diluent 3 from Meso Scale Discovery (#R51 BA) was added in each well to reveal bound biotinylated insulin. Plates were again washed 3 times with cold binding buffer before adding of 150 μΙ of Read buffer 4X (Meso Scale Discovery, #R92TC) diluted 2-fold with water. The quantity of bound biotinylated insulin was measured after electroluminescence signal reading with SECTOR^® Imager 2400 from Meso Scale Discovery. The binding data were fitted using a 4-parameter logistic non-linear regression analysis to determine the EC50 for the different reference peptides. In each assay, relative insulin receptor affinity was calculated by comparing the insulin analogue EC50 with human insulin EC50. The results comparing Lys^B29(N^£-His) Insulin glargine and insulin glargine are given in the table below.

These results show that the affinity for the insulin receptor is very similar between insulin glargine and Lys^B29(N^£-His) insulin glargine.

In-vitro Mitogenic properties The mitogenic potency of Lys^B29(N^E-His) insulin glargine was determined by measuring proliferation of human mammary epithelial cells (HMEC) in culture. HMEC were obtained from Lonza (Basel, Switzerland), as cryopreserved cells at passage 7 and were expanded and frozen at passage 8. A fresh ampoule was used for all experiments which were conducted at passage 10. The HMEC cells were maintained in culture according to supplier instructions in mammary epithelial growth medium (MEGM SingleQuot Kit, provided by Lonza ref CC- 4136) containing insulin, bovine pituitary extract, hydrocortisone, human epidermal growth factor, gentamicin and amphotericin-B. To maintain the cell culture, the growth medium was changed every other day and cells were inspected daily. For a growth experiment, the assay medium was growth medium without insulin. For mitogenicity experiments, cells were seeded at a density of 4>< 10³ cells/well in 96-well plates and incubated for about 72 hours in assay medium with graded doses of Lys^B29(N^E-His) insulin glargine, insulin glargine, recombinant human insulin and IGF-1 from 0 to between 1000 and 5000 nM final concentration. After about 72 hours incubation, 20μί of MTS (3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt) was added to each well and Optical Densities were read after 3 hours and 15 minutes ± 15 minutes at 490 nm.

Concentration-response data were fitted by four-parameters Marquadt regression (Magellan® software). Relative mitogenic potency (EC50 ratio) was determined by comparing Lys^B29(N^E- His) insulin glargine to recombinant human insulin within each experiment and the average of the relative potencies obtained in different experiments was calculated. The results are given in the table below.

The results show that Lys (N^E-His) insulin glargine and insulin glargine have equivalent mitogenic potencies. Example 5: Preparation of formulations for animal study

Formulations of Lys^B29(N^£-His) insulin glargine were prepared by mixing stock solutions of all ingredients to achieve the desired amounts. All formulations contain approximately 600 nmol of protein per ml_, 25 mM of m-cresol, 2.3 % of glycerol and zinc added as zinc chloride. The pH was adjusted by solutions of hydrochloric acid and sodium hydroxide. The pH, osmolality and zinc content are given in the table below. These formulations have been prepared to be very similar to the Lantus® preparation according to public data. Only the zinc content is varied; for comparison the FT-2 formulation contains about the same amount of zinc per protein as in Lantus®. All formulations have been sterile filtered and are clear and colorless. All formulations showed no change of protein content after storage at one month at both 25 and 5 °C.

[1] Composition given in prescribing information of Lantus®.

[2] The figure in bracket indicates the amount of zinc per monomer (mol/mol ratio). Example 6: Rat pK study

The three prototypes were used in a parallel sparse-sampling PK study in male streptozitocin induced diabetic rat. A total of 40 animals (10 rats by group, divided in 2 sub-goups of 5 rats) were given one of the three prototypes or Lantus® by single subcutaneous injection of 30 lU/kg.

At tO, all rats used in the study had a glycaemia upper or equal to 300 mg/dL. The induction of diabetes in the animals was done by the administration of a streptozotocin (STZ) solution at 40 mg/kg by the intravenous route 3 days before the beginning of the study.

Blood was collected from jugular vein at following time-points: 0 (pre-dose), 4h, 12h, 24h, 36h, 72h post-dose for one sub-group, 0 (pre-dose), 1 h, 8h, 18h, 30h, 48h, 96h post-dose for the other sub-group. Plasma samples were analyzed using an ELISA assay developed for the determination of insulin analogues based on Mercodia #10-1 128-01 iso-insulin ELISA kit. It has been verified that both insulin glargine and Lys^B29(N^£-His) insulin glargine have the same reactivity with the Iso-lnsulin ELISA kit. The PK parameters were determined using non-compartmental analysis (on a Phoenix WinNonlin 6.3 system) and are summarized in the table below.

The mean PK parameters and into bracket for concentrations: CV% and the number of time- points used for calculation. When it is not specified, n=5.

Cmax AUC₀-36h C1 h C8h C18h C24h C36h

Group

(mlU/L) (h*mlU/L) (mlU/L) (mlU/L) (mlU/L) (mlU/L) (mlU/L)

137 (46,

FT-1 2190 (21 ) 14728 2190 (47) 574 (28) 200 (15) 85 (n=1 ) n=4)

1 1 1 (23,

FT-2 532 (24) 1 1055 532 (54) 395 (47) 319 (66) 220 (37)

n=3)

132 (26,

FT-3 741 (41 ) 1 1969 377 (79) 315 (33) 387 (39) 21 1 (38)

n=4)

Lantus® 1783 (36) 16561 1783 (80) 563 (47) 370 (69) 95 (n=1 ) 98 (n=1 )

Values that are less than 80mlU/L (LLOQ) are not taken into account and n is then less than 5.

The FT-2 and FT-3 formulations led to lower Cmax values and longer mean PK profiles than Lantus^®. The insulin concentrations 18h after administration were very close but were higher 24h and 36h after administration. In figure 3, the pK curves for FT-2 and Lantus® are illustrated for comparison. FT-1 which has less zinc content than in Lantus® displays similar profile to the latter. It is thus clear that longer duration of exposure in human can be expected with the new analogue. REFERENCES

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Oiknine R, Bernbaum M, Mooradian AD. A critical appraisal of the role of insulin analogues in the management of diabetes mellitus. Drugs. 2005;65(3):325-40.

Kurtzhals P, Schaffer L, S0rensen A, Kristensen C, Jonassen I, Schmid C, Trub T. Correlations of receptor binding and metabolic and mitogenic potencies of insulin analogs designed for clinical use. Diabetes. 2000 Jun;49(6):999-1005.

Jonassen I, Havelund S, Hoeg-Jensen T, Steensgaard DB, Wahlund PO, Ribel U. Design of the novel protraction mechanism of insulin degludec, an ultra-long-acting basal insulin. Pharm Res. 2012 Aug;29(8):2104-14. Epub 2012 Apr 7.

Phillips NB, Wan ZL, Whittaker L, Hu SQ, Huang K, Hua QX, Whittaker J, Ismail-Beigi F, Weiss MA. Supramolecular protein engineering: design of zinc-stapled insulin hexamers as a long acting depot. J Biol Chem. 2010 Apr 16;285(16):1 1755-9. Epub 2010 Feb 24.

Kohn WD, Micanovic R, Myers SL, Vick AM, Kahl SD, Zhang L, Strifler BA, Li S, Shang J, Beals JM, Mayer JP, DiMarchi RD. pl-shifted insulin analogs with extended in vivo time action and favorable receptor selectivity. Peptides. 2007 Apr;28(4):935-48. Epub 2007 Jan 25.

Development and manufacture of protein pharmaceuticals Eds. Nail et al. Chapter 2, 2002, Kulwer Academic/plenum publishers, NY

Green's Protecting Groups in Organic Synthesis, John Wiley and Sons 4^th Ed. 2007

Sabel CE, Neureuther JM, Siemann S A spectrophotometric method for the determination of zinc, copper, and cobalt ions in metalloproteins using Zincon. Anal Biochem. 2010 Feb 15;397(2):218-26. Epub 2009 Oct 2

Huus K, Havelund S, Olsen HB, van de Weert M, Frokjaer S. Biochemistry. Thermal dissociation and unfolding of insulin. 2005 Aug 23;44(33):1 1 171 -7.

Claims

1 . An insulin glargine analogue which is Lys (N^£-His)Gly Arg Arg -human insulin.

2. A pharmaceutical composition comprising the insulin glargine analogue according to claim 1 and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 2 comprising divalent metal ions selected from the group consisting of zinc, magnesium, copper and calcium ions.

4. The pharmaceutical composition according to claim 3 wherein

said divalent metal ions are zinc ions and

the concentration of zinc ions is from 0.3 to 3 equivalents per mole of insulin glargine analogue.

5. The pharmaceutical composition according to any of claims 2 to 4 having a pH from 3.5 to 4.5.

6. The pharmaceutical composition according to any of claims 2 to 5 wherein said insulin glargine analogue is at a concentration from 1 mg/mL to 20 mg/mL.

7. The pharmaceutical composition according to any of claims 2 to 6, further comprising a preservative selected from the group consisting of phenol, m-cresol and a mixture thereof.

8. The pharmaceutical composition according to any of claims 2 to 7, further comprising an isotonicity agent being glycerol or sodium chloride.

9. The pharmaceutical composition according to any of claims 2 to 8 comprising:

a. said insulin glargine analogue at a concentration comprised between 3.5 and 4.0 mg/mL, for example 3.7 mg/mL

b. zinc ions at a concentration comprised between 20 and 50 μg/mL

c. m-cresol at a concentration comprised between 25 and 30 mmol/L

d. glycerol between 1 .6 and 2.5 % wt/wt, for example 2.3 % wt/wt

e. and pH is about 4.

10. The insulin glargine analogue according to claim 1 or the pharmaceutical composition according to any of claims 2 to 9 for use in a therapeutic treatment in human or animals.

1 . The insulin glargine analogue according to claim 1 or the pharmaceutical composition according to any of claims 2 to 9 for use in the treatment of diabetes and/or hyperglycemia.

2. A process for the manufacture of the insulin glargine analogue according to claim 1 comprising the step of grafting of a protected histidine to insulin glargine at pH comprised between 10 to 1 1 followed by a deprotection step.