WO2012066086A1

WO2012066086A1 - PHARMACEUTICAL FORMULATION COMPRISING INSULIN GLARGINE AND SULFOBUTYL ETHER 7-ß-CYCLODEXTRIN

Info

Publication number: WO2012066086A1
Application number: PCT/EP2011/070371
Authority: WO
Inventors: Keiko Uehata; Hidetoshi Arima; Naomi Ono; John Siebert
Original assignee: Sanofi-Aventis Deutschland Gmbh
Priority date: 2010-11-17
Filing date: 2011-11-17
Publication date: 2012-05-24

Abstract

The invention relates to a pharmaceutical formulation comprising insulin glargine and Sulfobutyl Ether 7- β-cyclodextrin, its preparation and use.

Description

Pharmaceutical formulation comprising insulin

glargine and Sulfobutyl Ether 7-i -cyclodextrin

Description

The invention relates to a pharmaceutical formulation comprising insulin glargine and Sulfobutyl Ether 7- β-cyclodextrin. Insulin glargine is the first long-acting basal insulin analogue used for subcutaneous administration once daily in patients with type 1 or type 2 diabetes mellitus. To obtain the further desirable blood-glucose lowering profile of insulin glargine, in the present study, we investigated the effect of sulfate β-cyclodextrin (Sul- β-CyD) and sulfobutyl ether 7- β-cyclodextrin (SBE7- β-CyD) on pharmaceutical properties of insulin glargine and the release of insulin glargine after subcutaneous injection to rats. Sul- β-CyD and SBE7- β-CyD increased the solubility of insulin glargine in phosphate buffer at pH 9.5. SBE7- β-CyD suppressed formation of multimers and enhanced dissolution rate of insulin glargine from its precipitate, compared to that of insulin glargine alone. On the other hand, Sul- β-CyD accelerated association of the molecules and inhibited dissolution of insulin glargine from its precipitate. Furthermore, we revealed that subcutaneous administration of an insulin glargine solution with SBE7- β-CyD to rats provided an increase of bioavailability and persistence, and a decrease of the maximum level in the serum insulin glargine level. In addition, a flatter profile in the blood- glucose lowering effect of insulin glargine was observed as retaining bioavailability. These findings are possibly due to the enhancement of release from the precipitate, the inhibitory effects on the enzymatic degradation at the injection site and the modification of behaviors in the blood stream, resulting from formation of the complex with insulin glargine and SBE7- β-CyD. It indicates that SBE7- β-CyD can be a useful excipient for a peak-less profile of insulin glargine. Diabetes is a chronic disease that the pancreas does not produce enough insulin (type 1 diabetes) or the body does not respond correctly to insulin and relative insulin deficiency (type 2 diabetes). It can be a life-threatening disease and also lead to serious complications such as cardiovascular disease, kidney failure, blindness and nerve damage (Blickle et al., 2007, Patterson et al., 2009, Simo et al., 2006). The global prevalence of diabetes has been increasing in recent decades, reaching near-epidemic proportions, and is projected to more than double by 2030 (Horton, 2008). The global diabetes epidemic has devastated on not only patients and their families but also national economies.

Human insulin is a major backbone for the treatment of diabetes. Although human insulin has attributed much in clinical treatment of diabetes for long time, there are still some difficulties and challenges in hypoglycemia and short half-life. In order to overcome these drawbacks, insulin glargine (Lantus^®), an insulin analogue (C267H₄o₄N₇₂078S6, MW=6,063) was developed by replacing the asparagine at the position of 21 of the A chain with glycine, and two arginines were added to the C-terminus of the B chain in human insulin (Fig. 1 ). It has a prolonged duration of action after subcutaneous injection and therefore can provide a basal insulin level of 24 h by once daily injection (Rolla, 2008). This alteration resulted in low aqueous solubility at neutral pH (Wang et al., 2003). Insulin glargine is supplied in an acidic solution, which becomes neutralized at the injection site, leading to a formation of microprecipitates from which insulin glargine is slowly released into the circulation (Wang et al., 2003).

Cyclodextrins (CyDs) are known to form inclusion complexes with various guest molecules (Szente and Szejtli, 1999, Uekama et al., 1998). However, the low aqueous solubility of natural CyDs, especially β-CyD, has restricted their range of applications. To improve their solubility, alkylated, hydroxyl alkylated, sulfobutyl alkylated and branched CyDs have been used (Stella and Rajewski, 1997, Uekama, 2004, Uekama and Otagiri, 1987). Of these hydrophilic CyDs, maltosyl- β-CyD (G₂- β-CyD), 2-hydroxypropyl- β-CyD (HP- β-CyD) and sulfobutyl ether- β-CyD (SBE- β-CyD) have higher solubility in water and relatively low hemolytic activity, and thus have potential as pharmaceutical excipients for parenteral preparation (Uekama et ai., 1998). In fact, natural β-CyD has a toxic effect on kidney, which is the main organ for removal of CyDs from the systemic circulation and for concentrating CyDs in the proximal convoluted tubule after glomerular filtration (Irie and Uekama, 1997). On the other hand, amorphous mixtures of highly water-soluble β- CyDs such as HP- β-CyD and SBE- β-CyD have very low systemic toxicity, compared with β-CyD.

We previously reported the effects of hydrophilic β-CyDs on the aggregation of bovine insulin in aqueous solution and its adsorption onto hydrophilic surfaces (Tokihiro et al., 1996, Tokihiro et al., 1995, Tokihiro et al., 1997). Of the CyDs tested, G₂- β-CyD potently inhibited insulin aggregation in a neutral solution and its adsorption onto the surfaces of glass and polypropylene tubes. In addition, SBE- β-CyDs showed the different effects on insulin aggregation, depending on the degree of substitution (DS) of the sulfobutyl ether group: SBE4- β-CyD (DS=3.9) showed deceleration of insulin aggregation at relatively low substitution levels and SBE7- β-CyD (DS=6.2) showed acceleration at high substitution levels (Tokihiro et al., 1997). Furthermore, we reported that subcutaneous administration of insulin solution with SBE4- β-CyD to rats rapidly increased plasma insulin level and maintained higher plasma insulin levels for at least 8 h, possibly due to the inhibitory effects of SBE4- β-CyD on the enzymatic degradation and/or the adsorption of insulin onto the subcutaneous tissue at the injection site (Tokihiro et al., 2000).

Our recent study demonstrated that subcutaneous administration of an insulin glargine solution with SBE4- β-CyD to rats enhanced the bioavailability of insulin glargine and sustained the glucose lowering effect, possibly due to the inhibitory effects of SBE4- β-CyD on the enzymatic degradation at the injection site. These results suggest that SBE4- β-CyD can be a useful excipient for sustained release of insulin glargine. Sulfate- β-CyD (Sul- β-CyD) is another anionic CyD derivative having a sulfur atom in a substituted group. We previously reported that Sul- β-CyD protected against aminoglycoside-induced acute renal failure rats (Uekama et al., 1993, Shiotani et al., 1995). We also showed that Sul- β-CyD and SBE4- β-CyD interacted with the erythrocyte membrane in a different manner probably due to the difference in location of negative charged substituted groups (Shiotani et al., 1995).

In the present study, to seek further possibility in pharmaceutical application of anionic β-CyD derivatives to insulin glargine, Sul- β-CyD and SBE7- β- CyD were investigated on their effect on physicochemical properties and pharmacokinetics/pharmacodynamics of insulin glargine.

Surprisingly, we revealed that subcutaneous administration of an insulin glargine solution with SBE7- β-CyD to rats provided an increase of bioavailability and persistence, and a decrease of the maximum level in serum insulin glargine level. In addition a flatter profile in the blood-glucose lowering effect of insulin glargine was observed as retaining bioavailability. These findings indicate that SBE7- β-CyD can be a useful excipient for a peakless profile of insulin glargine.

Therefore, an embodiment of the invention is a pharmaceutical formulation comprising insulin glargine and Sulfobutyl Ether 7- β-cyclodextrin. A further embodiment of the invention is a pharmaceutical formulation as described above, additionally comprising one or more ingredients selected from a group comprising m-cresol, zinc, glycerol and polysorbate 20.

A further embodiment of the invention is a pharmaceutical formulation as described above, wherein the zinc concentration is 10 to 40 g/ml, preferably 30 /ml.

A further embodiment of the invention is a pharmaceutical formulation as described above, wherein the glycerol content per 1 ml is 10 to 30 mg/ml, preferably 20 mg/ml of a 85% glycerol solution.

A further embodiment of the invention is a pharmaceutical formulation as described above, wherein the polysorbate 20 concentration is 10 to 30 pg /ml, preferable 20 pg /ml.

A further embodiment of the invention is a pharmaceutical formulation as described above, wherein the m-cresol concentration is 2,4 to 3,0 mg/ml, preferable 2,7 mg/ml.

A further embodiment of the invention is a pharmaceutical formulation as described above, wherein the S u If o butyl Ether 7- β-cyclodextrin concentration is 10 mM to 800 mM.

A further embodiment of the invention is a pharmaceutical formulation as described above, wherein the Sulfobutyl Ether 7- β-cyclodextrin concentration is 150 to 250 mM, preferably 200 mM. A further embodiment of the invention is a pharmaceutical formulation as described above, wherein the Sulfobutyl Ether 7- β-cyclodextrin concentration is selected from a group comprising 10 mM, 100 mM and 200 mM. A further embodiment of the invention is a pharmaceutical formulation as described above, which additionally comprises a glucagon-like peptide- 1 (GLP1 ) or an analogue or derivative thereof, or exendin-3 or -4 or an analogue or derivative thereof. A further embodiment of the invention is a pharmaceutical formulation as described above, which additionally comprises exendin-4 or an analogue therof, wherein the analogue is selected from a group comprising lixisenatide, exenatide and liraglutide, H-desPro³⁶-exendin-4-Lys₆-NH₂, H- des(Pro³⁶'³⁷)-exendin-4-Lys₄-NH₂ and H-des(Pro³⁶'³⁷)-exendin-4-Lys₅-NH_2l or a pharmacologically tolerable salt thereof.

A further embodiment of the invention is a use of a pharmaceutical formulation as described above for the treatment of Type 1 or Type 2 Diabetes mellitus.

A further embodiment of the invention is the preparation of a formulation as described above by adding insulin glargine, Sulfobutyl Ether 7- β-cyclodextrin and the excipients to an aqueous solution.

Figure Legend Figure 1 . Amino acid sequence and location of intermolecular disulfide bonds of insulin glargine

Figure 2. Effect of Sul- β-CyD and SBE7- β-CyD (10 mM) on fluorescence spectrum (A), circular dichroism spectrum of insulin glargine (0.1 mM) in phosphate buffer (pH 9.5, /=0.2) at 25°C. The excitation wavelength in measurement of fluorescence spectrum was 277 nm.

Figure 3. Effect of Sul- β-CyD and SBE7- β-CyD (10 mM) on solubility of insulin glargine in phosphate buffer (pH 9.5, /=0.2) at 25°C. The concentration of insulin glargine was determined by HPLC. Each value represents the meaniS.E. of 3 experiments. ^*p<0.05, compared to insulin glargine.

Figure 4. Effect of Sul- β-CyD and SBE7- β-CyD (10 mM) on permeation of insulin glargine (0.1 mM) through ultrafiltration membrane having nominal molecular weight limit of 30,000 in phosphate buffer (pH9.5, /=0.2) at 25°C. The concentration of insulin glargine was determined by HPLC. Each value represents the meaniS.E. of 17 and 5 experiments for insulin glargine and with Sul- β-CyD or SBE7- β-CyD, respectively. *p<0.05, compared to insulin glargine. *p<0.05, compared to Sul- β-CyD.

Figure 5. Effect of Sul- β-CyD and SBE7- β-CyD (10 mM) on dissolution from isoelectric precipitation of insulin glargine in phosphate buffer (pH9.5, 1=0.2) at 25°C. The initial concentration of insulin glargine was 0.1 mM, and then precipitated at pH7.4. The concentration of insulin glargine was determined by HPLC. Each point represents the mean±S.E. of 3 experiments. *p<0.05, compared to insulin glargine. p<0.05, compared to Sul- β-CyD.

Figure 6. Effects of Sul- β-CyD and SBE7- β-CyD (5 to 20 mM) on tryptic cleavage (2 IU) of insulin glargine (0.1 mM) in phosphate buffer (pH9.5, /=0.2) at 37°C. The concentration of insulin glargine was determined by HPLC. Each point represents the mean±S.E. of 3 experiments.

Figure 7. Effects of SBE7- β-CyD (200 mM) on serum insulin glargine (A) and glucose (B) levels after subcutaneous administration of insulin glargine (2 lU/kg) to rats. Each point represents the mean±S.E. of 4-6 experiments. *p<0.05, compared to insulin glargine.

Table Legend Table 1 . Apparent Stability Constants (Kc) of Insulin Glargine/Sul- β-CyD Complexes and Glargine/SBE7- β-CyD Complexes in Phosphate Buffer (pH9.5, /=0.2) at 25 °C. Apparent (1 :1 ) stability constants were determined by measuring the changes in the fluorescence intensity (306 nm) of insulin glargine by the addition of β-CyDs at different concentrations and by analyzing the titration curves of the fluorescence intensity versus the concentration of β-CyDs using the Scott's equation. Excitation wavelength was 277 nm. The value represents the mean ±S.E. of 3 experiments. Table 2. Particle size of insulin glargine with or without Sul- β-CyD and SBE7- β-CyD (10 mM) in phosphate buffer (pH 9.5). The particle size was measured by Zetasizer Nano. The concentration of insulin glargine and β- CyDs were 0.1 mM and 10 mM, respectively. Each value represents the meanlS.E. of 5-7 experiments.

Table 3. Rate Constant (k_c) and Stability Constant (K_c) of 1 : 1 Complexes of Insulin Glargine/Sul- β-CyD and Insulin Glargine/SBE7- β-CyD under Tryptic Cleavage of Insulin Glargine in the Absence and Presence of β-CyDs in Phosphate Buffer (pH9.5, 1-0.2) at 37°C. Each value represents the meanlS.E. of 3-5 experiments.

Table 4. In vivo pharmacokinetics parameters of insulin glargine with or without SBE7- β-CyD (200 mM). 1 ) Time required to reach the maximum serum insulin glargine level. 2) Maximum serum insulin glargine level. 3) Area under the serum insulin glargine level-time curve up to 12 h post- administration. 4) Mean reduced time in plasma. Each value represents the mean ± S.E.M. of 4-6 experiments. *p < 0.05, compared to insulin glargine. Table 5. In vivo pharmacodynamics parameters of insulin glargine with or without SBE7- β-CyD (200 mM). 1 ) Time to nadir blood glucose concentration. 2) Nadir blood glucose concentration. 3) The cumulative percentage of change in serum glucose levels up to 12 h post-administration. 4) Mean reduced time in plasma. Each value represents the mean ± S.E.M. of 5-6 experiments. *p < 0.05, compared to insulin glargine.

The invention is exemplified in the following by working examples which are not indended to be limiting.

MATERIALS

Insulin glargine was a gift from Sanofi-Aventis (Paris, France). SBE7- β-CyD was provided by CyDex (Kansas, USA). Sul- β-CyD was given by Kaken Pharmaceutical Co. Ltd. (Tokyo, Japan). Recombinant trypsin (EC 3.4.21.4) of proteomics grade was purchased from Roche Diagnostics (Tokyo, Japan). All other materials were of reagent grade, and deionized double-distilled water was used.

METHODS

Spectroscopic studies

Fluorescence and circular dichroism (CD) spectra were measured at 25°C using a HITACHI fluorescence spectrophotometer F-2500 (Tokyo, Japan) and a JASCO J-720 polarimeter (Tokyo, Japan), respectively. For preparation of the phosphate buffer (pH 9.5, /=0.2), 0.1 mol/L phosphoric acid solution and 0.1 mol/L sodium hydroxide solution were mixed, which followed by addition of Sodium chloride. Solubility studies

Excess amounts of insulin glargine were shaken in phosphate buffer (pH 9.5, /=0.2) in the absence and presence of β-CyDs at 25°C. After equilibrium was attained, the solutions were filtered with Millex^® GV filter 0.22 pm and insulin glargine dissolved was determined by the high performance liquid chromatography (HPLC) with Agilent 1 100 series (Tokyo, Japan) under the following conditions: Merck Superspher^® 100 RP-18 column (4 pm, 3 mm x 250 mm, Tokyo, Japan), a mobile phase of phosphate buffer (pH 2.5) and acetonitrile and a gradient flow, increasing the ratio of the acetonitrile (25-40 %) over 30 mins, a flow rate of 0.55 mL/min, a detection of UV at 214 nm.

Ultrafiltration studies

Ultrafiltration studies were performed using stirred ultrafiltration cells model 8010 (Millipore, Tokyo, Japan) applied with YM30 ultrafiltration discs (MWCO=30,000) in phosphate buffer (pH 9.5, /=0.2) in the absence and presence of β-CyDs at 25°C under nitrogen current. Insulin glargine levels in filtrates were determined by HPLC as described above. Particle size determination

Particle sizes of insulin glargine (0.1 mM) with or without β-CyDs (10 mM) in phosphate buffer (pH 9.5, 1=0.2) were measured by Zetasizer Nano (Malvern Instruments, Worcestershire, UK).

Dissolution study of insulin glargine

Insulin glargine (0.1 mM) dissolved in phosphate buffer (pH 9.5, 1=0.2) in the absence and presence of β-CyDs (10 mM) was precipitated by a pH shift to 7.4. After centrifugation (2,500 rpm, 10 min), the supernatant was discarded and then phosphate buffer (pH 7.4, /=0.2) was newly added to the precipitate at 25°C. At appropriate intervals, an aliquot of the dissolution medium was withdrawn, centrifuged at 2,500 rpm for 10 min, and analyzed for the insulin glargine by HPLC as described in the paragraph 2.2.2. Stability of insulin glargine against tryptic cleavage

Insulin glargine (0.1 mM) in phosphate buffer (pH 9.5, /=0.2) was incubated with recombinant trypsin (0.02 mg/mL) in the absence and presence of □- CyDs at 37°C. At appropriate intervals, 5 μΙ_ of sample solution was withdrawn and determined intact insulin glargine level by HPLC. The rate constants (k_c) and stability constants (K_c) of 1 : 1 complexes of insulin glargine/ β-CyDs under the tryptic cleavage were determined by quantitative analysis according to the following equation, where k₀ and [CyD]_t stands for the rate constants without CyD and the total concentration of CyD, respectively (Ikeda et al., 1975).

[CyD], 1 1

^~ko ^~ = ko - k_c ^■ [CyD]_t + _Kc (k₀ - k_c)

2.2.7 Subcutaneous administration of insulin glargine/ Sul- β-CyD solution insulin or glargine/ SBE7- β-CyD solution to rats

Serum insulin glargine and glucose levels of rats were measured by the enzyme immunoassay and the mutarotase-glucose oxidase method. The solution (0.582 mL/kg) of the insulin glargine (2 lU/kg) in phosphate buffer (pH 9.5, /=0.2) in the absence and presence of the β-CyDs was subcutaneously injected in male Wistar rats (200-250 g), and at appropriate intervals blood samples were taken from the jugular veins. Serum insulin glargine and glucose were determined by Glyzyme Insulin-EIA Test Wako (Wako Pure Chemicals, Osaka, Japan) and Glucose-CII-Test Wako (Wako Pure Chemicals Ind., Osaka, Japan), respectively. Serum glucose levels after the administration of a solution of insulin glargine with or without the β- CyDs were expressed as a percentage of the initial glucose level before injection.

Statistical Analysis

Data are given as the mean ± S.E.M. Statistical significance of means for the studies was determined by analysis of variance followed by Scheffe's test. p-Values for significance were set at 0.05.

WORKING EXAMPLES

Example 1 : Spectroscopic studies CyDs have been claimed to interact with hydrophobic residues exposed on protein surfaces and thereby to decrease aggregation of proteins (Brewster et al., 1991 , Tavornvipas et al., 2006). We previously reported that SBE4- β- CyD inhibited the aggregation of bovine insulin in neutral solution, possibly due to the interaction of SBE4- β-CyD with aromatic side chain of insulin such as B26-tyrosine, A19-tyrosine, B1-phenylalanine and B25- phenylalanine (Tokihiro et al., 1997). Our recent study also showed that SBE4- β-CyD increased the solubility of insulin glargine and the dissolution rate from its precipitate, and inhibited its aggregation in the phosphate buffer (pH 9.5), possibly through the formation of complex with insulin glargine. In the present study, to reveal whether Sul- β-CyD and SBE7- β-CyD interact with insulin glargine, we investigated the effects of the both β-CyDs (10 mM) on the fluorescence and CD spectrum of insulin glargine (0.1 mM) (Fig. 2). To obtain the clear solution of insulin glargine (0.1 mM) in the present study, insulin glargine with β-CyDs was dissolved in phosphate buffer (pH 9.5, 1-0.2) at 25°C. The fluorescence intensity of tyrosine of insulin glargine at 306 nm was quenched remarkably by the addition of Sul- β-CyD (10 mM) while SBE7- β-CyD (10 mM) quenched slightly (Fig. 2A). As tyrosine is a hydrophobic amino acid having a phenyl group in the molecule, it indicates that those β-CyDs, particularly Sul- β-CyD, interact with those aromatic amino acid residues of insulin glargine. The apparent 1 : 1 stability constants (K_c) of the insulin glargine/Sul- β-CyD complex and insulin glargine/SBE7- β- CyD complex were determined by the titration curves of the fluorescence intensity against a concentration of β-CyDs with the Scott's equation (Ikeda et al., 1975). The stability constants of insulin glargine/Sul- β-CyD complex and insulin glargine/SBE7- β-CyD complex in phosphate buffer (pH 9.5, /=0.2) at 25°C were calculated to be 14 ± 3 M^" and 18 ± 4 M^"1 , respectively (Table 1 ). The CD spectrum of insulin glargine (0.1 mM) showed negative bands at 208 and 224 nm in phosphate buffer (pH 9.5, /=0.2) (Fig. 2B). The two negative bands assigned to a-helical (a characteristic feature of the monomer) and β-structure (a predominant feature of dimer) (Goldman and Carpenter, 1974). In the presence of Sul- β-CyD (10 mM), the CD spectrum of insulin glargine changed notably. The both negative bands at 210 and 224 nm remarkably decreased by addition of Sul- β-CyD (10 mM). These changes indicate that Sul- β-CyD decreased monomer and dimer of insulin glargine in the phosphate buffer (pH 9.5, /=0.2). It is also estimated that Sul- β-CyD changed the conformation of insulin glargine by complexation between aromatic amino residues of insulin glargine and Sul- β-CyD in phosphate buffer (pH 9.5, /=0.2). On the other hand, the CD spectrum of insulin glargine showed almost no change in the presence of SBE7- β-CyD. It suggests that the interaction mode with insulin glargine is different between Sul- β-CyD and SBE7- β-CyD.

Example 2: Solubility studies

Currently subcutaneous injection of clear solution is the main stream for administration of insulin and its analogues. However, insulin or insulin glargine is poorly soluble in aqueous solutions, in particular around the isoelectic point (pi), approximately pH 6.7, close to the physiological pH (Brange et al., 1997). Then, the effect of Sul- β-CyD and SBE7- β-CyD on the solubility of insulin glargine was examined. As shown in Fig. 3, the solubility of insulin glargine in phosphate buffer at pH 9.5 significantly increased by the addition of the both β-CyDs. It is estimated that the increase in solubility of insulin glargine was caused by interaction between those β-CyDs. Significant solubilizing effect of Sul- β-CyD was also confirmed in phosphate buffer at pH 7.4 while SBE7- β-CyD did not show a statistical significance (data not shown). These results suggest that the interaction between insulin glargine and Sul- β-CyD is predominated by electric effect more than inclusion effect whereas SBE7- β-CyD interacts insulin glargine by formation of complex with aromatic amino acid residues of insulin glargine such as tyrosine.

Example 3: Ultrafiltration studies The aggregation and self-association of insulin and its analogue are elicited by many kinds of factors such as the concentration of insulin, pH, temperature, shaking and so on (Rolla, 2008, Wang et al., 2003). Insulin glargine forms dimer, tetramer, hexamer and further soluble multimer by non-covalent interaction as proceeding in self-association (Havelund et al., 2004, Kurtzhals, 2004). Therefore, we performed ultrafiltration studies to estimate the effects of Sul- β-CyD and SBE7- β-CyD on self-association of insulin glargine using the membrane YM30 (MWCO=30,000) in phosphate buffer (pH 9.5, /=0.2). As shown in Fig. 4, insulin glargine permeated the ultrafiltration membrane by 48%. SBE7- β-CyD significantly enhanced the permeation of insulin glargine up to 67%. These results suggest that SBE7- β-CyD led to dissociation of soluble multimers of insulin glargine. On the other hand, Sul- β-CyD decreased it to 44%, though the formation of insoluble aggregation of insulin glargine was not observed under the condition of the ultrafiltration study. These results as well as the change in the CD spectrum of insulin glargine by the addition of Sul- β-CyD indicate that Sul- β-CyD enhanced the association of soluble multimer of insulin glargine from its monomer and dimer. Under the experimental condition of this study with pH 9.5 which higher than its isoelectric point, the net charge of insulin glargine is negative. And the charged and polar groups on the surface are supposed to be surrounded by water molecules via ionic hydration and/or hydrogen bondings. High concentration of sulfates such as (NH₄)₂S0₄ and Na₂S0₄ are commonly used to precipitate or crystallize protein/polypeptide by the loss of the hydration layer on the peptide surface (Nakagaki. et al., 1982). Because Sul- β-CyD has highly concentrated negative charges close to the entrance of the CyD cavity (Shiotani et al., 1994), it is thought Sul- β-CyD accelerated formation of soluble multimers of insulin glargine through the same mechanism of precipitation of polypeptides by (NH₄)₂S0₄ and Na₂S0₄. On the other hand, the sulfonate groups of SBE7- β-CyD have a proper distance from the cavity with butyl chains so that SBE7- β-CyD keeps the ability of complex formation (Stelia, 1996, Jarho. et al., 1996, Zia. et al., 1996). Thus it is supposed that SBE7- β-CyD inhibited the formation of soluble multimers of insulin glargine through the complexation with insulin glargine.

Following the ultrafiltration experiment, particle sizes of insulin glargine were determined by dynamic light scattering method in the absence and presence of Sul- β-CyD and SBE7- β-CyD (Table 2). Particle size of insulin glargine alone in phosphate buffer (pH 9.5, /=0.2) was 744 ± 82 nm. Particle size of insulin glargine in the presence of the Sul- β-CyD and SBE7- β-CyD increased significantly to 1334 ± 164 nm and 1575 ± 228 nm, respectively. The dynamic light scattering method is a technique to determine a hydrodynamic diameter of the particle with a time-dependent fluctuation in the scattering intensity caused by the Brownian movement through the Stokes-Einstein equation. With this principle, if any other molecules or solvent molecules move together with the particle targeted, a hydrodynamic diameter of the particle obtained by the dynamic light scattering method includes those molecules. It is estimated that the sulfobutyl groups of SBE7- β-CyD are strongly hydrated in aqueous solution. Therefore a hydrodynamic diameter of a complex of insulin g largine and SBE7- β-CyD determined by the dynamic light scattering method is supposed to include not only insulin glargine and SBE7- β-CyD but also water molecules hydrated with SBE7- β- CyD. With these reasons mentioned, the increased particle size observed in the solution of insulin glargine with SBE7- β-CyD was due to not aggregation of insulin glargine but formation of complexes of insulin glargine and SBE7- β-CyD.

These results suggest the potential use of SBE7- β-CyD for prevention of self-association and aggregation of insulin glargine, resulting from maintaining of the soluble form of insulin glargine through the formation of complex with insulin glargine.

Example 4: Dissolution study of insulin glargine

Insulin glargine is believed to precipitate at the physiological pH after subcutaneous injection of the solution due to pi (about pH 6.7), which is followed by a sustained release of insulin glargine over 24 h from injection site because of an extremely low solubility in aqueous solution at pH of around pi (Wang et al., 2003). In order to investigate the effects of Sul- β- CyD and SBE7- β-CyD on the sustained release of insulin glargine, the dissolution rate of insulin glargine from isoelectic precipitates formed in the absence and presence of the β-CyDs was determined (Fig. 5). Insulin glargine (0.1 mM) was dissolved in phosphate buffer (pH 9.5) in the presence and absence of the β-CyDs (10 mM), and then isoelectric precipitation of insulin glargine was obtained after pH shift from 9.5 to 7.4. Then, the release of insulin glargine was determined in the pH 7.4 phosphate buffer in the absence of β-CyDs. SBE7- β-CyD significantly increased the dissolution rate of insulin glargine after 24 h, comparing to insulin glargine alone. It is supposed that this result was due to formation of the complex between insulin glargine and SBE7- β-CyD. The enhancing effect of SBE7- β-CyD on the dissolution rate of insulin glargine from its isoelectic precipitate is consistent with the solubilizing effect (Fig. 3) and the enhancement in permeability (Fig. 4). On the other hand, Sul- β-CyD showed a tendency to decrease the dissolution rate of insulin glargine after 24 h though no statistical significance. It is supposed that the inhibitory effect of Sul- β-CyD on dissolution of insulin glargine from its isoelectric precipitation related to enhancement in the association of insulin glargine molecules more predominantly than the solubilizing effect of Sul- β-CyD on insulin glargine.

Example 5: Stability of insulin glargine against tryptic cleavage

Insulin and its analogues are digested by proteinase such as trypsin, which cleaves insulin at the carboxyl side of residues B22-Arginine and B29-Lysine, at injection site and systemic circulation (Schilling and Mitra, 1991 ). Therefore, a resistance toward enzymatic degradation is required for insulin or its analogues formulation to improve their bioavailability. Next, we investigated the effects of Sul- β-CyD and SBE7- β-CyD on stability of insulin glargine against trypsin digestion. In the case of insulin glargine, trypsin cleaves insulin glargine at the carboxyl side of three residues, B22-Arginine, B29-I_ysine and B31-Arginine which is one of the newly introduced amino acid residues in insulin glargine. In this study, insulin glargine was digested by trypsin at 2 IU of the initial concentration at pH 9.5 at 37°C in the absence and presence of the β-CyDs. As shown in Fig. 6A, the apparent degradation rate constant of insulin glargine in the absence of the β-CyDs (k₀) was 0.357 ± 0.004 h^"1. The apparent rate constant (k_obs) in the presence of Sul- β-CyD and SBE7- β-CyD decreased with the increase in the concentration of the β- CyDs. The decline in k₀bs caused by SBE7- β-CyD was more than the case of Sul- β-CyD. Table 3 shows the rate constants (k_c) and stability constants (K_c) of 1 : 1 complex calculated with the regression lines shown in the Fig. 6B. kc and K_c in the Sul- β-CyD system were 0.129 ± 0.009 h^" and 244 ± 24 M^"\ respectively. Those in the SBE7- β-CyD system were 0.137 ± 0.014 h^"1 and 182 ± 22 M^"1, respectively. These results suggest that SBE7- β-CyD is useful as a stabilizer of insulin glargine against enzymatic degradation.

Recently, it was reported that the aspartic acid residue existing in the catalytic pocket of trypsin is responsible for attracting and stabilizing positively-charged lysine and/or arginine on the substrate peptide (Leiros et al., 2004). In addition, we have reported that β-CyDs are capable of interacting with bovine insulin at B24-, B25-phenylalanines, B26-tyrosine and B28-proline (Tokihiro et al., 1997). Therefore, these results suggest that Sul- β-CyD and SBE7- β-CyD inhibited tryptic cleavage of insulin glargine through the complex formation with insulin glargine, resulting in the avoidance of trypsin to access the digestive sites of insulin glargine such as B22-B23, B29-B30 and B31 -B32.

Example 6: Subcutaneous administration of insulin glargine/Sul- β-CyD and insulin glargine/SBE7- β-CyD solutions to rats

To confirm whether Sul- β-CyD and SBE7- β-CyD are useful enhancer for insulin glargine in vivo, we evaluated the effects of these β-CyDs on pharmacokinetics and pharmacodynamics of insulin glargine after subcutaneous injection to rats. After subcutaneous administration of insulin glargine (2 lU/kg) with Sul- β-CyD (100 mM) or SBE7- β-CyD (100 mM) in phosphate buffer (pH 9.5) to rats, either of the CyDs did not change the serum glucose level-time profiles remarkably in comparison with that of insulin glargine administered alone (data not shown). Taking the positive results of SBE7- β-CyD in ultrafiltration (Fig. 4) and dissolution (Fig. 5) studies by contrast to those of Sul- β-CyD into account, further in vivo investigation was performed with higher concentration of SBE7- β-CyD.

Figure 7 A and Table 4 show the serum insulin glargine level-time profiles and pharmacokinetics parameters, respectively, after subcutaneous administration of insulin glargine (2 lU/kg) with or without SBE7- β-CyD (200 mM) in the phosphate buffer (pH 9.5) to rats. When insulin glargine alone was injected, the maximum level (C_max) of insulin glargine and the time (T_max) required to the reach C_max after injection were 150 pU/mL and 1 .00 h, respectively. In the presence of SBE7- β-CyD (200 mM), C_max significantly decreased down to 9 .60 pU/mL, although T_max did not change remarkably. The area under the serum insulin glargine level-time curve (AUC) of the SBE7- β-CyD (200 mM) system up to 12 h (687.86 (pU/mL) ' h) was significantly increased, compared to those of insulin glargine alone (582.99

(pU/ml_) - h). In addition, SBE7- β-CyD (200 mM) extended the mean reduced time (MRT) of the serum insulin glargine level significantly when comparing with that of insulin glargine alone. These results indicate that SBE7- β-CyD enhanced and retained the serum insulin glargine level, and provided a flatter profile of the serum insulin glargine level. This may be contributed to 1 ) the enhancement of dissolution of insulin glargine from its precipitate at injection site, probably due to the increase of solubility (Fig. 3) and enhancement of the dissociation of insulin glargine from multimers (Fig. 4), and 2) inhibition of enzymatic degradation (Fig. 6), through the complex formation with SBE7- β-CyD. Since the dissolution of insulin glargine from its precipitate is retained by SBE7- β-CyD, serum insulin glargine level is also maintained, resulting in high MRT level. To gain insight into the detailed mechanism, further elaborate study on the adsorption of insulin glargine in the presence of SBE7- β-CyD onto subcutaneous tissue at injection site is under estimation.

Figure 7B and Table 5 show the serum glucose level-time profiles and pharmacodynamics parameters after subcutaneous administration of insulin glargine (2 lU/kg) with or without SBE7- β-CyD (200 mM) in phosphate buffer (pH 9.5) to rats. When insulin glargine alone was administered, the minimal glucose level occurred at about 2 h after injection and then the serum glucose levels recovered within 6 h to basal level. On the other hand, in the case of administration of insulin glargine with SBE7- β-CyD (200 mM), the minimal level and the recovery in the serum glucose level were observed at about 4 h and about 9 h after injection, respectively. It indicates that SBE7- β-CyD moderated the fluctuations in the blood-sugar lowering effect of insulin glargine. T_nadir delayed significantly in the system of insulin glargine administered with SBE7- β-CyD without remarkable changes in the rest of pharmacodynamics parameters investigated. It is estimated that these results in the serum glucose level were based on the changes in the serum insulin glargine level after injection of insulin glargine with SBE7- β-CyD. Although the enhancements of the AUC and MRT in the serum insulin glargine level by SBE7- β-CyD were not directly reflected to the serum glucose level with significant differences, SBE7- β-CyD reduced fluctuations in the serum insulin glargine level.

The purpose of treatment of diabetes mellitus is to control the blood glucose concentration through the normalization of the plasma insulin profile. Endogenous insulin secretion needs a low basal level of plasma insulin during fasting and an appropriate elevation during meals (Owens and Bolli, 2008). In this context, the intensive insulin therapy is intended to give a basal level and a meal-related bolus level by means of various insulin formulations (Kramer, 1999). Neutral protamine Hagedorn insulin (NPH) was mainly used as basal insulin after its launch in 946 (Owens and Bolli, 2008). However its duration of action is not long enough to cover the entire day, typically 12 to 18 h in clinical practice (Heinemann et al., 2000, Lepore et al., 2000). And it shows a peak occurring 4 to 6 h after subcutaneous injection to diabetes patients (Heinemann et al., 2000) and this is connected to an increase of risk of hypoglycemia, particularly nocturnal hypoglycemia following bedtime injection (Fanelli et al., 2002). Insulin glargine introduced to the market in 2000 provides a longer duration action to last for 24 h at least and a nearly flat profile (Heinemann et al., 2000, Lepore et al., 2000). As shown in Fig. 7B and Table 5, subcutaneous administration of an insulin glargine solution with SBE7- β-CyD (200 mM) to rats showed a flatter profile in the blood glucose lowering effect of insulin glargine in comparison with the injection of insulin glargine alone. A peakless profile of the blood glucose level decreases risks of hypoglycemia and thus provides patients with a better glycemic control and a higher quality of life. ln conclusion, in the present study, we revealed that subcutaneous administration of an insulin glargine solution with SBE7- β-CyD to rats provided an increase of bioavailability and persistence, and a decrease of the maximum level in serum insulin glargine level. In addition a flatter profile in the blood-glucose lowering effect of insulin glargine was observed as retaining bioavailability. These findings indicate that SBE7- β-CyD can be a useful excipient for a peakless profile of insulin glargine.

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Table 1 . Apparent Stability Constants (Kc)

of Insulin Glargine/Sul-|3-CyD Complexes

and Glargine/SBE7-p-CyD Complexes in

Phosphate Buffer (pH9.5, 1=0.2) at 25 ²C

System Kc (M-¹ )

Sul-p-CyD 14±3

SBE7- -CyD 18±4

Apparent (1 : 1) stability constants were determined by measuring the

changes in the fluorescence intensity (306 nm) of insulin glargine by

the addition of β-CyDs at different concentrations and by analyzing the

titration curves of the fluorescence intensity versus the concentration

of β-CyDs using the Scott's equation. Excitation wavelength was 277

nm. The value represents the mean ± S.E. of 3 experiments.

Table 1

Table 2. Particle Size of Insulin Glargine with or without

Sul-p-CyD and SBE7- -CyD (10 mM) in Phosphate

Buffer (pH 9.5)

System Diameter (nm)

Insulin glargine 744± 82

with Sul-p-CyD 1334± 164 ^*

with SBE7-p-CyD 1575±228 ^*

The particle size was measured by Zetasizer Nano. The concentration of

insulin glargine and CyD were 0.1 mM and 10 mM, respectively. Each value

represents the mean±S.E. of 5-7 experiments. *p<0.05 versus Insulin glargine.

Table 2

Table 3. Rate Constant {k_c) and Stability Constant (K_c)

of 1 :1 Complexes of Insulin Glargine/Sul-p-CyD and

Insulin Glargine/SBE7-p-CyD under Tryptic Cleavage of

Insulin Glargine in the Absence and Presence of β- CyDs in Phosphate Buffer (pH9.5, 1=0.2) at 37^SC

System K (h-¹) K_c (M-1 )

Sul- -CyD 0.129 ±0.009 244± 24

SBE7-p-CyD 0.137±0.014 182±22

Each value represents the mean± S.E. of 3-5 experiments.

Table 3

Table 4. In vivo Pharmacokinetics Parameter of Insulin Glargine with or without SBE7-(3-CyD (200 mM)

System T max ¹⁾ (h) C_max¾ ^U/mL) AUC³> ((mU/mL)h) MRT^4¾ (h)

Insulin glargine 1 .00d bO.OO 1 50.00± 17.90 582.99±30.27 1 .83±0.08

Insulin glargine

/SBE7-p-CyD 1 .40d b0.24 91 .60± 3.04 687.86±20,57* 2.12±0.04*

1) Time required to reach the maximum plasma insulin glargine level.

2) Maximum plasma insulin glargine level.

3) Area under the plasma insulin glargine level-time curve up to 12 h post-administration.

4) Mean reduced time in plasma.

Each value represents the mean±S.E. of 5 to 6 experiments. *p<0.05 versus Insulin glargine.

Table 4 Table 5. In vivo Pharmacodynamics Parameter of Insulin Glargine with or without SBE7-p-CyD (200 mM)

System > (h) C_nadir¾ (%) AUC_G¾ (%- h) MRy> (h)

Insulin glargine 1 .60±0.16 33.14± 1 .10 544.66±31 .73 2.28±0.03

Insulin glargine *

,/SBE7-|3-CyD 3.50±0.05 32.05±4.73 612.36±40.84 2.29±0.01

1) Time to nadir blood glucose concentration. 2) Nadir blood glucose concentration.

3) The cumulative percentage of change in plasma glucose levels up to 12 h postadmimstration.

4) Mean reduced time in plasma.

Each value represents the mean±S.E. of 4 to 6 experiments. *p<0.05 versus Insulin glargine.

Tabic 5

Claims

1 . Pharmaceutical formulation comprising insulin glargine and Sulfobutyl Ether 7- β-cyclodextrin.

2. Pharmaceutical formulation according to claim 1 , additionally comprising one or more ingredients selected from a group comprising tricresol, zinc, glycerol and polysorbate 20.

3. Pharmaceutical formulation according to any of the foregoing claims, wherein the zinc concentration is 10 to 40 Mg/ml, preferably 30 yg /ml.

4. Pharmaceutical formulation according to any of the foregoing claims, wherein the glycerol content per 1 ml is 10 to 30 mg/ml, preferably 20 mg/ml of a 85% glycerol solution.

5. Pharmaceutical formulation according to any of the foregoing claims, wherein the polysorbate 20 concentration is 10 to 30 g /ml, preferable 20 yg /ml.

6. Pharmaceutical formulation according to any of the foregoing claims, wherein the m-cresol concentration is 2.4 to 3,0 mg/ml, preferable 2,7 mg/ml.

7. Pharmaceutical formulation according to any of the foregoing claims, wherein the Sulfobutyl Ether 7- β-cyclodextrin concentration is 10 mM to 800 mM.

8. Pharmaceutical formulation according to any of the foregoing claims, wherein the Sulfobutyl Ether 7- β-cyclodextrin concentration is 150 to 250 mM, preferably 200 mM.

9. Pharmaceutical formulation according to any of the foregoing claims, wherein the Sulfobutyl Ether 7- β-cyclodextrin concentration is selected from a group comprising 10 mM, 100 mM and 200 mM.

10. Pharmaceutical formulation according to any of the foregoing claims, which additionally comprises a glucagon-like peptide-1 (GLP1 ) or an analogue or derivative thereof, or exendin-3 or -4 or an analogue or derivative thereof.

1 1 . Pharmaceutical formulation according to any of the foregoing claims, which additionally comprises exendin-4 or an analogue therof, wherein the analogue is selected from a group comprising lixisenatide, exenatide and liraglutide, H-desPro36-exendin-4-Lys6-NH2, H- des(Pro36,37)-exendin-4-Lys4-NH2 and H-des(Pro36,37)-exendin-4-l_ys5- NH2, or a pharmacologically tolerable salt thereof.

12. Use of a pharmaceutical formulation according to any of the foregoing claims for the treatment of Type 1 or Type 2 Diabetes mellitus.

13. Preparation of a formulation according to any of claims 1 to 1 1 by adding insulin glargine, Sulfobutyl Ether 7- β-cyclodextrin and the excipients to an aqueous solution.