Mibefradil is a Ca 2ϩ channel antagonist that inhibits both Ttype and high-voltage-activated Ca 2ϩ channels. We previously showed that block of high-voltage-activated channels by mibefradil occurs through the production of an active metabolite by intracellular hydrolysis. In the present study, we modified the structure of mibefradil to develop a nonhydrolyzable analog, (1S, 2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride , that exerts a selective inhibitory effect on T-type channels. The acute IC 50 of NNC 55-0396 to block recombinant ␣ 1 G T-type channels in human embryonic kidney 293 cells was ϳ7 M, whereas 100 M NNC 55-0396 had no detectable effect on high-voltage-activated channels in INS-1 cells. NNC 55-0396 did not affect the voltage-dependent activation of T-type Ca 2ϩ currents but changed the slope of the steady-state inactivation curve. Block of T-type Ca 2ϩ current was partially relieved by membrane hyperpolarization and enhanced at a high-stimulus frequency. Washing NNC 55-0396 out of the recording chamber did not reverse the T-type Ca 2ϩ current activity, suggesting that the compound dissolves in or passes through the plasma membrane to exert its effect; however, intracellular perfusion of the compound did not block T-type Ca 2ϩ currents, arguing against a cytoplasmic route of action. After incubating cells from an insulin-secreting cell line (INS-1) with NNC 55-0396 for 20 min, mass spectrometry did not detect the mibefradil metabolite that causes L-type Ca 2ϩ channel inhibition. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of Ltype Ca 2ϩ channels, thus rendering it more selective to T-type Ca 2ϩ channels.Voltage-gated Ca 2ϩ channels are transmembrane proteins involved in the regulation of cellular excitability and intracellular Ca 2ϩ signaling. Calcium channels have been divided into various categories based on functional and pharmacological criteria. High-voltage-activated (HVA) channels, which have been further subdivided into L-, N-, P/Q-, and R-types, require strong depolarizations for activation, whereas lowvoltage-activated or T-type channels activate over a much more negative voltage range and exhibit unique inactivation and deactivation kinetics (Armstrong and Matteson, 1985;Catterall, 1998;Perez-Reyes, 1998). The main structural component of the voltage-gated calcium channel is the ␣ 1

Here, we describe the molecular engineering of insulin icodec to achieve a plasma half-life of 196 h in humans, suitable for once-weekly subcutaneously administration. Insulin icodec is based on re-engineering of the ultra-long oral basal insulin OI338 with a plasma half-life of 70 h in humans. This systematic re-engineering was accomplished by (1) further increasing the albumin binding by changing the fatty diacid from a 1,18-octadecanedioic acid (C18) to a 1,20-icosanedioic acid (C20) and (2) further reducing the insulin receptor affinity by the B16Tyr → His substitution. Insulin icodec was selected by screening for long intravenous plasma half-life in dogs while ensuring glucose-lowering potency following subcutaneous administration in rats. The ensuing structure–activity relationship resulted in insulin icodec. In phase-2 clinical trial, once-weekly insulin icodec provided safe and efficacious glycemic control comparable to once-daily insulin glargine in type 2 diabetes patients. The structure–activity relationship study leading to insulin icodec is presented here.

6-Chloro-3-alkylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide derivatives were synthesized and characterized as activators of adenosine 5'-triphosphate (ATP) sensitive potassium (K(ATP)) channels in the beta-cells by measuring effects on membrane potential and insulin release in vitro. The effects on vascular tissue in vitro were measured on rat aorta and small mesenteric vessels. Selected compounds were characterized as competitive inhibitors of [(3)H]glibenclamide binding to membranes of HEK293 cells expressing human SUR1/Kir6.2 and as potent inhibitors of insulin release in isolated rat islets. 6-Chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (54) was found to bind and activate the SUR1/Kir6.2 K(ATP) channels in the low nanomolar range and to be at least 1000 times more potent than the reference compound diazoxide with respect to inhibition of insulin release from rat islets. Several compounds, e.g., 3-propylamino- (30), 3-isopropylamino- (34), 3-(S)-sec-butylamino- (37), and 3-(1-methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (53), which were found to be potent and beta-cell selective activators of K(ATP) channels in vitro, were found to inhibit insulin secretion in rats with minimal effects on blood pressure and to exhibit good oral pharmacokinetic properties.

Recently, the first basal oral insulin (OI338) was shown to provide similar treatment outcomes to insulin glargine in a phase 2a clinical trial. Here, we report the engineering of a novel class of basal oral insulin analogues of which OI338, 10, in this publication, was successfully tested in the phase 2a clinical trial. We found that the introduction of two insulin substitutions, A14E and B25H, was needed to provide increased stability toward proteolysis. Ultralong pharmacokinetic profiles were obtained by attaching an albumin-binding side chain derived from octadecanedioic (C18) or icosanedioic acid (C20) to the lysine in position B29. Crucial for obtaining the ultralong PK profile was also a significant reduction of insulin receptor affinity. Oral bioavailability in dogs indicated that C18-based analogues were superior to C20-based analogues. These studies led to the identification of the two clinical candidates OI338 and OI320 (10 and 24, respectively).

Fibroblast growth factors (FGF) 19, 21 and 23 are characterized by being endocrinely secreted and require co-receptor α-klotho or β-klotho (BKL) for binding and activation of the FGF receptors (FGFR). FGF15 is the rodent orthologue of human FGF19, but the two proteins share only 52% amino acid identity. Despite the physiological role of FGF21 and FGF19 being quite different, both lower blood glucose (BG) when administered to diabetic mice. The present study was designed to clarify why two human proteins with distinct physiological functions both lower BG in mice and if the mouse orthologue FGF15 has similar effect to FGF19 and FGF21. Recombinant human FGF19, -21 and a mouse FGF15 variant (C110S) were expressed and purified from While rhFGF19 (recombinant human fibroblast growth factor 19) and rhFGF21 (recombinant human fibroblast growth factor) bound FGFRs in complex with both human and mouse BKL, rmFGF15CS (recombinant mouse fibroblast growth factor 15 C110S) only bound the FGFRs when combined with mouse BKL. Recombinant hFGF21 and rhFGF19, but not rmFGF15CS, increased glucose uptake in mouse adipocytes, while rhFGF19 and rmFGF15CS potently decreased expression in rat hepatocytes. The lack of effect of rmFGF15CS on glucose uptake in adipocytes was associated with rmFGF15CS's inability to signal through the FGFR1c/mouse BKL complex. In mice, only rhFGF19 and rhFGF21 decreased BG while rmFGF15CS and rhFGF19, but not rhFGF21, increased total cholesterol. These data demonstrate receptor- and species-specific differential activity of FGF15 and FGF19 which should be taken into consideration when FGF19 is used as a substitute for FGF15.

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