In vivo pharmacokinetic study and oral glucose tolerance test of sulfoxide analogs of GPR40 agonist TAK-875
INTRODUCTION
Type 2 diabetes accounts for approximately 90% of all cases of diabe- tes. Type 2 diabetes commonly affects older adults, but it is increasingly seen in children, adolescents and younger adults due to the rising levels of obesity, lack of exercise and unhealthy diet.
An estimated 463 million adults aged 20–79 years are currently living with diabetes. This repre- sents 9.3% of the world’s population in this age group. The total number is predicted to rise to 578 million (10.2%) by 2030 and to 700 million (10.9%) by 2045 (“IDF Diabetes Atlas, 9th edn,” 2019).
With growing needs for novel antidiabetic drugs, G protein- coupled receptor 40 (GPR40) has been developed as a novel and potential therapeutic target for type 2 diabetes (Choi, Shin, & Lee, 2014).
GPR40, also known as FFA1 receptor (Free Fatty Acid Receptor 1), is abundantly expressed in the insulin-expressing beta cells of pancreas and can mediate the effects of medium-(C6-C3) and long-(C14-24) chain saturated and unsaturated fatty acids on insulin secretion (Briscoe et al., 2003; Itoh et al., 2003; Latour et al., 2007; H. Lu et al., 2013; Tikhonova et al., 2007).
Free fatty acids (FFAs) pro- vide an important energy source and also act as signaling molecules in a variety of cellular processes, including insulin secretion by activating GPR40 (Bharate, Nemmani, & Vishwakarma, 2009; Itoh et al., 2003).
GPR40 activation not only can stimulate the Gαq-PLC signaling pathway, causing release of endoplasmic reticulum (ER) Ca2+, but also can stimulate the L-type calcium channels (LTCC) in the presence of extracellular Ca2+ and elevated glucose, resulting in further increase in intracellular Ca2+ concentration and glucose-stimulated insulin secre- tion (GSIS) (Shapiro, Shachar, Sekler, Hershfinkel, & Walker, 2005).
Because the insulinotropic effects via GPR40 activation are dependent on glucose concentration (Fujiwara, Maekawa, & Yada, 2005; Itoh et al., 2003; Shapiro et al., 2005), a selective GPR40 agonist has the advantage of low risk of hypoglycemia over other anti- diabetic agents (Tan et al., 2008).
At present, a number of agonists for GPR40 have been reported (Defossa & Wagner, 2014; Li et al., 2019; Li et al., 2020; Negoro et al., 2010; Negoro et al., 2012; Takano et al., 2015), several of which have entered the clinic in recent years, such as TAK-875, AMG 837, LY2881835 (Figure 1a) (Defossa & Wagner, 2014). TAK-875, or fasiglifam (Figure 1a), is an orally available, potent and selective partial agonist of GPR40, which reached phase III clinical trials for the treat- ment of type 2 diabetes (Negoro et al., 2010; Negoro et al., 2012).
In the extracellular loops of GPR40, two arginine residues Arg183 and Arg258 could form “ionic locks” with Glu145 and Glu172 respectively to lock GPR40 in the inactive state (Ho et al., 2018; Lin et al., 2012; J. Lu et al., 2017; Luckmann et al., 2019; Sum, Tikhonova, Costanzi, & Gershengorn, 2009; Yabuki et al., 2013). Through interacting with Arg183 and Arg258 with its carboxyl group (Figure 1b), TAK-875 could break the “ionic locks” to allosterically active GPR40 in cooperativity with endogenous FFAs (Srivastava et al., 2014; Sum et al., 2009; Yabuki et al., 2013).
According to the hGPR40-TAK-875 co-complex (Srivastava et al., 2014), the sulfonyl of TAK-875 was sol- vent exposed (Figure 1b), so we presumed that structure transforma- tion of sulfonyl of TAK-875 to sulfinyl would not dramatically change its binding mode with GPR40. Besides, due to the lower lipophilicity of sulfinyl compared with sulfonyl, we expected our sulfoxide analogs would exhibit less undesirable off-target activities (Waring, 2010) and better pharmacokinetic (PK) profiles (Christiansen et al., 2012) relative to its parent compound TAK-875.
In the previous study, we successfully synthesized the TAK-875 sulfoxide analog 2, which was further separated to optically pure com- pounds 3 and 4 through chiral HPLC separation (Figure 1a). In vitro biological evaluation revealed that the GPR40 agonistic potency of epimeric mixture 2 (EC50 = 77.5 nM) and its two optically pure epi- mers 3 (EC50 = 76.1 nM) and 4 (EC50 = 114.0 nM) were comparable to that of TAK-875 (EC50 = 84.3 nM) (Yan, Chen, Yang, Xu, & Zhang, 2017).
In order to further evaluate the druglikeness of TAK- 875 sulfoxide analogs, the in vivo pharmacokinetic properties of com- pounds 2, 3, and 4 in rats were investigated and compared with that of TAK-875.
Moreover, asymmetric synthesis was carried out to get enough amount of sulfoxides for the following oral glucose tolerance test (OGTT) in rats.
RESULTS AND DISCUSSION
Asymmetric synthesis
Then, asymmetric synthesis was carried out to obtain large amount of 3 and 4 for further in vivo evaluation. As shown in Scheme 1, the starting material M1 obtained as previously described (Yan et al., 2017) was further oxidized by CHPO (80%) in the presence of chiral ligand D-DET and L-DET to get the intermediates M2 and M3, respectively.
M2 and M3 were hydrolyzed to get the target com- pounds, respectively. Our asymmetric synthesis yielded two sulfox- ides 5 (S, S, 66.4% de) and 6 (R, S, 71.0% de) with moderate de values according to the analytical chiral HPLC analysis.
Oral glucose tolerance test (OGTT)
Considering that the (S)-sulfoxide 3 was the major component of 5 and the (R)-sulfoxide 4 was the major component of 6 (Figure 3), we believed that the in vivo activity of 5 and 6 could somewhat indicated the in vivo potency of sulfoxides 3 and 4, respectively.
Therefore, compounds 5 and 6 were evaluated in an oral glucose tolerance test (OGTT) in the type 2 diabetic rat model with TAK-875 (compound 1) as the positive control. In brief, the type 2 diabetic rats were orally given compounds (5 mg/kg) or vehicle once a day for 21 days, then OGTT was performed.
At the Day 21, all animals received an oral glucose load (1 g/kg) 1 hr after drug or vehicle administration. Blood glucose levels were measured before drug or vehicle administration (time point: −60 min), before glucose load (time point: 0 min) and after glucose load (time points: 30, 60, and 120 min). Results in Figure 4 showed that the fasting blood glucose level (time point: −60 min) in rats treated with compound 5 was the lowest among all the treatment groups.
It is clear that compound 5 showed more potent glucose-lowering effect than 1, while 6 was less potent than 1. Besides, the activity comparison of 2, 5, and 6 revealed that the more proportion of (S)-sulfoxide 3 they contained, the more potent in vivo activity they exhibited, which could be partly rationalized by the superior pharmacoki- netic property of 3 (Tables 1 and 2).
CONCLUSION
In our previous study, two TAK-875 sulfoxide analogs 3 (S, S,100.0% de) and 4 (R, S, 100.0% de) were developed and showed comparable GPR40 agonist activity to TAK-875. In the present study, in vivo phar- macokinetic study revealed that sulfoxide and sulfone could be converted into each other in different degrees.
Interestingly, (S)- sulfoxide 3 could be metabolized to sulfone 1 more easily than its epi- mer (R)-sulfoxide 4. What is more, 3 exhibited better pharmacokinetic profiles characterized by higher drug exposure and longer period of effectiveness.
In order to further investigate the in vivo glucose- lowering potency of sulfoxide analogs of TAK-875, asymmetric syn- thesis was carried out and led to two sulfoxides with moderate de values, 5 (S, S, 66.4% de) and 6 (R, S, 71.0% de).
The following oral glu- cose tolerance test (OGTT) in rats showed that 5 (S, S, 66.4% de) had stronger glucose-lowering effect than 6 (R, S, 71.0% de) and TAK-875, which could be partly rationalized by the superior pharmacokinetic property of (S)-sulfoxide 3 (the main component of 5) relative to (R)- sulfoxide 4 (the main component of 6) and TAK-875.
Taken together, our research showed that transformation of the sulfone group of TAK-875 to sulfoxide group could dramatically change its in vivo pharmacokinetic property and glucose-lowering effect. Interestingly, the TAK-875 analog with S-configuration of sulfoxide group showed more favorable druglikeness, which deserves further research and development as an antidiabetic agent.
EXPERIMENTAL SECTION
Pharmacokinetic study in rats
All experiments involving laboratory animals were performed with the approval of the Shandong University Laboratory Animal Center ethics committee. Wistar male rats were obtained from Center for New Drug Evaluation of Shandong University. Compounds 1, 2, 3 and 4 were respectively tested in 6 Wistar male rats at an oral dose of 3 mg/kg. After oral administration, blood samples were collected.
The blood samples were centrifuged to obtain the plasma fraction. Aceto- nitrile was added in the plasma samples. After centrifugation, the compound concentrations in the supernatant were measured by a liq- uid chromatograph/mass spectrometer (LC/MS). The PK profiles were calculated with DAS 2.0.
Chemistry
Unless otherwise noted, materials were obtained from commercial suppliers and used without further purification. All reactions were monitored by thin-layer chromatography on 0.25 mm silica gel plates (60GF-254) and visualized with UV light.
Chromatographic purifica- tion was carried out on silica gel columns (300–400 mesh HaiYang) and on Combiflash Rf 200 (Teledyne Isco) instruments. Melting points were determined on an uncorrected electrothermal melting point apparatus. The IR spectra were recorded by means of KBr plate on a Nicolet 6,700 FT-IR Spectrometer. NMR was recorded on Bruker DRX spectrometers at room temperature.
High-resolution mass spec- tra were conducted by Shandong Analysis and Test Center in Ji’nan, China. ESI-MS spectra were recorded on an API 4000 spectrometer.
Analytical chiral HPLC was performed on a Shimadzu 20A HPLC instrument using a CHIRALPAK IA column (250 mm × 4.6 mm, 5 μm), with the mobile phase of 75% n-hexane/10% ethanol/15% EtOAc/0.1% TFA and the flow rate of 0.8 ml/min. Optical rotations were determined on a MCP200 polarimeter.
(S)-2-(6-((20,60-dimethyl-40-(3-[methylsulfonyl]propoxy)-[1,10- biphenyl]-3-yl)methoxy)-2,3-dihydrobenzofuran-3-yl)acetic acid (1), 2-((3S)-6-((20,60-dimethyl-40-(3-[methylsulfinyl]propoxy)-[1,10-biphe- nyl]-3-yl)methoxy)-2,3-dihydrobenzofuran-3-yl)acetic acid (2), 2-((S)- 6-((20,60-dimethyl-40-(3-((S)-methylsulfinyl)propoxy)-[1,10-biphenyl]-3-yl)methoxy)-2,3-dihydrobenzofuran-3-yl)acetic acid (3) and 2-((S)- 6-((20,60-dimethyl-40-(3-((R)-methylsulfinyl)propoxy)-[1,10-biphenyl]-3-yl)methoxy)-2,3-dihydrobenzofuran-3-yl)acetic acid (4) were obtained as previously described (Yan et al., 2017).
Oral glucose tolerance test (OGTT)
Male Wistar rats fed high-fat and high-sugar foods (contained 30% lard, 10% sucrose and 3% yolk in the general food) for 28 days. Then the rats fasted for 12 hr, received intraperitoneal injection of streptozotocin (STZ, 35 mg/kg body weight, dissolved in 0.1 mol/L citrate buffer, pH = 4.2, avoided light and kept in ice bath).
Fasting blood glucose levels were measured by glucometer 5D-1 (Yicheng, China) after 72 hr. Rats with fasting plasma glucose concentrations ≥13.9 mmol/L and stable for more than 7 days, were considered the type 2 diabetic rat model successfully.
The type 2 diabetic rats were divided in to the control and treatment groups. They were orally given vehicle (5% tween-80 aqueous solution) or compounds (suspended in vehicle with a dose of 5 mg/kg) once a day for 21 days. Then OGTT was performed in the five groups of rats.
All animals received an oral glucose load (1 g/kg) 1 hr after drug or vehicle administration. Blood samples were collected from tail vein before drug or vehicle administration (time point: −60 min), before glucose load (time point: 0 min) and after glucose load (time points: 30, 60, and 120 min).
Blood glucose levels were measured. The statistical significances versus control were assessed by the Tukey test. Fasiglifam