WO2011144674A2

WO2011144674A2 - PHARMACEUTICAL FORMULATION COMPRISING INSULIN GLARGINE AND SBE4-ß-CYD

Info

Publication number: WO2011144674A2
Application number: PCT/EP2011/058081
Authority: WO
Inventors: Hidetoshi Arima; Naomi Ono; John Siebert; Keiko Uehata
Original assignee: Sanofi-Aventis Deutschland Gmbh
Priority date: 2010-05-20
Filing date: 2011-05-18
Publication date: 2011-11-24
Also published as: WO2011144674A3

Abstract

Pharmaceutical formulation comprising insulin glargine and SBE4-β-CyD The invention relates to a pharmaceutical formulation comprising insulin glargine and SBE4-β-CyD, its preparation and use.

Description

Pharmaceutical formulation comprising insulin glargine and SBE4- -CyD The invention relates to a pharmaceutical formulation comprising insulin glargine and SBE4- -CyD.

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 bioavailability and the sustained glucose lowering effect of insulin glargine, in the present study, we investigated the effect of sulfobutyl ether^-cyclodextrin

(SBE4^-CyD), with the degree of substitution of sulfobutyl ether group of 3.9, on pharmaceutical properties of insulin glargine and the release of insulin glargine after subcutaneous injection to rats. SBE4^-CyD increased the solubility and suppressed aggregation of insulin glargine in phosphate buffer at pH 9.5, probably due to the interaction of SBE4^-CyD with aromatic amino acid residues such as tyrosine of insulin glargine. In addition, SBE4^-CyD accelerated the dissolution rate of insulin glargine from its precipitates, compared to that of insulin glargine alone. Furthermore, we revealed that subcutaneous administration of an insulin glargine solution with SBE4^-CyD to rats enhanced the bioavailability of insulin glargine and sustained the glucose lowering effect, possibly due to the inhibitory effects of SBE4^-CyD on the enzymatic degradation at the injection site. These results suggest that SBE4^-CyD can be a useful excipient for sustained release 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

(C26₇H₄o₄N₇2O₇8S6, MW=6,063) with a prolonged duration of action after subcutaneous injection, 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 ) (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^-CyD (G₂^-CyD), 2-hydroxypropyl-B-CyD (ΗΡ-β-CyD) and sulfobutyl ether-B-CyD (SBE^-CyD) have higher solubility in water and relatively low hemolytic activity, and thus have potential as pharmaceutical excipients for parenteral preparation (Uekama et al., 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 ΗΡ-β-CyD and

SBE^-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^-CyD potently inhibited insulin aggregation in a neutral solution and its adsorption onto the surfaces of glass and polypropylene tubes. In addition, SBE^-CyDs showed the different effects on insulin aggregation, depending on the degree of substitution (DS) of the sulfobutyl ether group: SBE4^-CyD (DS=3.9) showed deceleration of insulin aggregation at relatively low substitution levels and SBE7^-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^-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^-CyD on the enzymatic degradation and/or the adsorption of insulin onto the subcutaneous tissue at the injection site (Tokihiro et al., 2000). However, it is still unknown whether SBE4^-CyD shows the sustained-glucose lowering effects for insulin analogues. Of various insulin analogues, only a few experiments on pharmaceutical application of insulin glargine were performed. Therefore, in the present study, to evaluate the potential use of SBE4^-CyD on not only bioavailability of insulin glargine but also the

sustained-glucose lowering effect, we examined the effects of SBE4^-CyD on physicochemical properties and pharmacokinetics/ pharmacodynamics of insulin glargine.

Surprisingly, we revealed that SBE4^-CyD enhanced both bioavailability and a persistence of the blood-glucose loweing effect of insulin glargine after subcutaneous injection to rats, probably due to the inhibitory effects of SBE4^-CyD on the enzymatic degradation at the injection site, resulting from the interaction with insulin glargine molecule.

Therefore, an embodiment of the invention is a pharmaceutical formulation comprising insulin glargine and SBE4- -CyD. A further embodiment of the invention is a pharmaceutical formulation according to claim 1 , 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 Mg/nnl, preferably 30 g /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 g /ml, preferable 20 g /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 SBE4- -CyD concentration is 10 mM to 800 mM. A further embodiment of the invention is a pharmaceutical formulation as described above, wherein the SBE4- -CyD concentration is 150 to 250 mM, preferably 200 mM.

A further embodiment of the invention is a pharmaceutical formulation as described above, wherein the SBE4- -CyD 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 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(Pro^36,37)-exendin-4-Lys5-NH₂, or a pharmacologically tolerable salt thereof. A further embodiment of the invention is the 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, SBE4- -CyD and the excipients to an aqueous solution.

Figure Legend

Figure 1 . Secondary chemical structure of insulin glargine Figure 2. Effect of SBE4^-CyD (10 mM) on fluorescence spectrum (A) and circular dichroism spectrum (B) of insulin glargine (0.1 mM) in phosphate buffer (pH 9.5, 1=0.2) at 25°C. The excitation wavelength in measurement of fluorescence spectrum was 277 nm. Figure 3. Effect of SBE4^-CyD (10 mM) on solubility of insulin glargine in phosphate buffer (pH 9.5, 1=0.2) at 25°C. The concentration of insulin glargine was determined by HPLC. Each value represents the mean ± S.E.M. of 3 experiments. ^*p < 0.05, compared to insulin glargine. Figure 4. Effect of SBE4^-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 (pH 9.5, 1=0.2) at 25°C. The concentration of insulin glargine was determined by HPLC. Each value represents the mean ± S.E.M. of 17 and 5 experiments for insulin glargine and with SBE4^-CyD, respectively. ^*p < 0.05, compared to insulin glargine.

Figure 5. Effect of SBE4^-CyD (10 mM) on the dissolution rate from isoelectric precipitation of insulin glargine in phosphate buffer (pH 9.5, 1=0.2) at 25°C. The initial concentration of insulin glargine was 0.1 mM, and then precipitated at pH 7.4. The concentration of insulin glargine was determined by HPLC. Each point represents the mean ± S.E.M. of 3 experiments. ^*p < 0.05, compared to insulin glargine. Figure 6. Effects of SBE4^-CyD (5 to 20 mM) on tryptic cleavage (2 IU) of insulin glargine (0.1 mM) in phosphate buffer (pH 9.5, 1=0.2) at 37°C. The concentration of insulin glargine was determined by HPLC. Each point represents the mean ± S.E.M. of 3 experiments.

Figure 7. Effects of SBE4^-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.M. of 7-11 experiments. ^*p < 0.05, compared to insulin glargine.

Table Legend:

Table 1 . Particle size of insulin glargine with or without SBE4^-CyD (10 mM) in phosphate buffer (pH 9.5). The particle size was measured by Zetasizer Nano. The concentration of insulin glargine and SBE4^-CyD were 0.1 mM and 10 mM, respectively.

Table 2. In vivo pharmacokinetics parameters of insulin glargine with or without SBE4^-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 9 h post-administration. Each value represents the mean ± S.E.M. of 7-11 experiments. ^*p < 0.05 , compared to insulin glargine. Table 3. In vivo pharmacodynamics parameters of insulin glargine with or without SBE4^-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 9 h post-administration. Each value represents the mean ± S.E.M. of 7-11 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 obtained from Sanofi-Aventis (Paris, France). SBE4^-CyD was provided by CyDex (Lenexa, KS). 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. Solubility studies

Excess amounts of insulin glargine were shaken in phosphate buffer (pH 9.5, 1=0.2) in the absence and presence of SBE^-CyDs at 25°C. After equilibrium was attained, the solutions were filtered with Millex^® GV filter 0.22 μιτι and insulin glargine dissolved was determined by the high performance liquid chromatography (HPLC) with Agilent 1100 series (Tokyo, Japan) under the following conditions: Merck Superspher^® 100 RP-18 column (4 μιτι, 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%), 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, 1=0.2) in the absence and presence of SBE4^-CyD 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 SBE4^-CyD (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 SBE4^-CyD (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, 1=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 before.

Stability of insulin glargine against tryptic cleavage

Insulin glargine (0.1 mM) in phosphate buffer (pH 9.5, 1=0.2) was incubated with recombinant trypsin (0.02 mg/nnL) in the absence and presence of SBE4^-CyD 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/SBE4^-CyD 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]_t 1 1

= [CyD]_t +

k₀ - kobs k₀ - kc Kc^■ (k₀ - k_c)

Subcutaneous administration of insulin glargine/SBE4^-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 SBE4^-CyD (200 mM) 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 insulin glargine/SBE4^-CyD solutions 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^-CyD inhibited the insulin aggregation in neutral solution, possibly due to the interaction of SBE4^-CyD with aromatic side chain of insulin such as B26-tyrosine, A19-tyrosine,

B1 -phenylalanine and B25-phenylalanine (Tokihiro et al., 1997). In the present study, to reveal whether SBE4^-CyD interacts with insulin glargine, we investigated the effects of SBE4^-CyD (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 spectroscopic studies, insulin glargine with SBE4^-CyD 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 by the addition of SBE4^-CyD (Fig. 2A). As tyrosine is a hydrophobic amino acid having a phenyl group in the molecule, SBE4^-CyD interacts with those aromatic amino acid residues of insulin glargine. The apparent 1 :1 stability constant (K_c) of the insulin glargine/SBE4^-CyD complexes was determined by the titration curves of the fluorescence intensity against a concentration of SBE4^-CyD with the Scott's equation (Ikeda et al., 1975). The stability constant of insulin glargine/SBE4^-CyD complex in phosphate buffer (pH 9.5, 1=0.2) at 25°C were calculated to be 20 ± 5 M^~1. The CD spectrum of insulin glargine (0.1 mM) showed negative bands at 210 and 220 nm in phosphate buffer (pH 9.5, 1=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).

However, CD spectrum of insulin glargine in the presence of SBE4^-CyD (10 mM) was changed only very slightly, compare to that of insulin glargine alone, suggesting that SBE4^-CyD did not induce a conformational change in insulin glargine in the phosphate buffer (pH 9.5, 1=0.2). These results suggest that SBE4^-CyD interacts with insulin glargine without topological change of insulin glargine in the phosphate buffer (pH 9.5, 1=0.2).

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 SBE4^-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 was significantly increased by the addition of SBE4^-CyD. It is estimated that the increase in the solubility of insulin glargine was caused by the complexation between the SBE4^-CyD and aromatic amino acid residues of insulin glargine such as tyrosine. This solubilizing effect of SBE4^-CyD was also confirmed in phosphate buffer at pH 7.4 (data not shown). These results suggest that SBE4^-CyD potentially enhances the solubility of insulin glargine in phosphate buffer. Example 3: Ultrafiltration studies

The aggregation of insulin and its analogue is 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 SBE4^-CyD on aggregation of insulin glargine using the membrane YM30 (MWCO=30,000) in phosphate buffer (pH 9.5, 1=0.2). As shown in Fig. 4, insulin glargine permeated the ultrafiltration membrane by approximately 50%. SBE4^-CyD significantly enhanced the permeation of insulin glargine up to almost 70%. These results suggest that SBE4^-CyD leads to dissociation of soluble multimer of insulin glargine. Following ultrafiltration experiment, particle sizes of insulin glargine were determined in the absence and presence of the SBE4^-CyD (Table 1 ). Particle size of insulin glargine alone in phosphate buffer (pH 9.5, 1=0.2) was 744 ± 82 nm. On the other hand, the addition of SBE4^-CyD significantly increased in the particle size to 1724 ± 275 nm. These results suggest the potential use of SBE4^-CyD for an aggregation-inhibitor for 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 SBE4^-CyD on the sustained release of insulin glargine, the dissolution rate of insulin glargine from isoelectic precipitates formed in the absence and presence of SBE4^-CyD was determined (Fig. 5). Insulin glargine (0.1 mM) was dissolved in the phosphate buffer (pH 9.5) in the presence and absence of SBE4^-CyD (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 SBE4^-CyD. SBE4^-CyD significantly increased the dissolution rate of insulin glargine after 3 h through 24 h, compare to insulin glargine alone. This enhancing effects of SBE4^-CyD is consistent with the solubilizing effect as shown in Fig. 3. These results suggest that SBE4^-CyD increases dissolution of insulin glargine from its precipitate.

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 B29-Lysine and B22-Arginine, 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 the SBE4^-CyD on stability of insulin glargine against trypsin digestion. 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 SBE4^-CyD. As shown in Fig. 6A, the apparent degradation rate constant of insulin glargine in the absence of the SBE4^-CyD (k₀) was 0.357 ± 0.004 h^~1. Furthermore, the apparent rate constant (k₀bs) in the presence of the SBE4^-CyD decreased with the increase in the concentration of SBE4^-CyD. The rate constants (kc) and stability constants (K_c) of 1 :1 complex calculated with the regression lines shown in the Fig. 6B were 0.145 ± 0.012 h^"1 and 144 ± 18 M^"1, respectively. These results suggest that the inhibition of tryptic cleavage of insulin glargine by SBE4^-CyD was caused by a formation of complex with insulin glargine, resulting from decreasing the free insulin glargine to be easily digested by trypsin. 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 (Leiros et al., 2004). Therefore, the insulin glargine/SBE4^-CyD complex is supposed to ameliorate the interaction between the negatively-charged aspartic acid in the catalytic pocket of trypsin and positively-charged lysine and/or arginine mentioned, since SBE4^-CyD has negative charge originated from sulfobutyl groups. This hypothesis is supported by the report that the aromatic amino acid residues in insulin glargine is capable of interacting with β-CyDs (at B24-, B25-phenylalanines, B26-tyrosine and B28-proline) locate near the three digestive sites by trypsin (B22-B23, B29-B30 and B31 -B32)

(Tokihiro et al., 1997). These results suggest that SBE4^-CyD acts as a stabilizer of insulin glargine against enzymatic degradation due to interaction with insulin glargine.

Example 6: Subcutaneous administration of insulin glargine/SBE4^-CyD solution to rats

To confirm whether SBE4^-CyD is useful enhancer for insulin glargine in vivo, we evaluated the effects of SBE4^-CyD on pharmacokinetics and pharmacodynamics of insulin glargine after subcutaneous injection to rats. Figure 7A and Table 2 show the serum insulin glargine level-time profiles and pharmacokinetic parameters, respectively, after subcutaneous administration of insulin glargine (2 lU/kg) with or without

SBE4^-CyD (200 mM) in the phosphate buffer (pH 9.5) to rats. When insulin glargine was injected, the time (T_max) required to reach maximum level (C_max) of insulin glargine was at 1 h after injection, and then the serum insulin glargine level decreased to the basal level. Although, T_max in the SBE4^-CyD system was the same as that of insulin glargine alone, SBE4^-CyD significantly sustained the serum insulin glargine level. The area under the serum insulin glargine level-time curve (AUC) of the SBE4^-CyD system (AUC=934.39 (μυ/mL)· h) was significantly increased, compared to those of insulin glargine alone (AUC=786.31 (μυ/mL)· h). Figure 7B and Table 3 show the serum glucose level-time profiles and pharmacodynamics parameters after subcutaneous administration of insulin glargine (2 lU/kg) with or without SBE4^-CyD (200 mM) in the phosphate buffer (pH 9.5) to rats. When insulin glargine alone was injected, 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, insulin glargine administered with SBE4^-CyD significantly sustained the hypoglycemic effect. We confirmed that this sustained the blood-glucose lowering effects of SBE4^-CyD was in a dose-dependent manner (data not shown). Further, the area under serum glucose level-time curve (AUCG) was significantly increased by the addition of SBE4^-CyD (Table 3). These results may be contribute to; 1 ) the inhibitory effects of SBE4^-CyD on the enzymatic degradation of insulin glargine (Fig. 6), 2) the enhancement of solubility and the dissolution rate of insulin glargine by SBE4^-CyD (Figs. 3-5). To gain insight into the mechanism, further elaborate study on the adsorption of insulin glargine in the presence of SBE4^-CyD onto subcutaneous tissue at injection site is under estimation. Furthermore, the improving effects of the other β-CyD derivatives including SBE7^-CyD on pharmaceutical properties of insulin glargine are under investigation. In conclusion, in the present study, we revealed that SBE4^-CyD enhanced both bioavailability and a persistence of the blood-glucose loweing effect of insulin glargine after subcutaneous injection to rats, probably due to the inhibitory effects of

SBE4^-CyD on the enzymatic degradation at the injection site, resulting from the interaction with insulin glargine molecule. These findings indicate that SBE4^-CyD can be a useful excipient for sustained release of insulin glargine.

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Claims

Claims:

1 . Pharmaceutical formulation comprising insulin glargine and SBE4- -CyD.

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

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

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.

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

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.

Pharmaceutical formulation according to any of the foregoing claims, wherein the SBE4- -CyD concentration is 10 mM to 800 mM.

8. Pharmaceutical formulation according to any of the foregoing claims, wherein the SBE4- -CyD concentration is 150 to 250 mM, preferably 200 mM.

9. Pharmaceutical formulation according to any of the foregoing claims, wherein the SBE4" -CyD 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(Pro^36,37)-exendin-4-Lys5-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 11 by adding insulin glargine, SBE4- -CyD and the excipients to an aqueous solution.