The Clinically-tested S1P Receptor Agonists, FTY720 and BAF312, Demonstrate Subtype-Specific Bradycardia (S1P₁) and Hypertension (S1P₃) in Rat

<div><p>Sphingosine-1-phospate (S1P) and S1P receptor agonists elicit mechanism-based effects on cardiovascular function <em>in vivo</em>. Indeed, FTY720 (non-selective S1P_X receptor agonist) produces modest hypertension in patients (2–3 mmHg in 1-yr trial) as well as acute bradycardia independent of changes in blood pressure. However, the precise receptor subtypes responsible is controversial, likely dependent upon the cardiovascular response in question (e.g. bradycardia, hypertension), and perhaps even species-dependent since functional differences in rodent, rabbit, and human have been suggested. Thus, we characterized the S1P receptor subtype specificity for each compound <em>in vitro</em> and, <em>in vivo</em>, the cardiovascular effects of FTY720 and the more selective S1P_1,5 agonist, BAF312, were tested during acute i.v. infusion in anesthetized rats and after oral administration for 10 days in telemetry-instrumented conscious rats. Acute i.v. infusion of FTY720 (0.1, 0.3, 1.0 mg/kg/20 min) or BAF312 (0.5, 1.5, 5.0 mg/kg/20 min) elicited acute bradycardia in anesthetized rats demonstrating an S1P₁ mediated mechanism-of-action. However, while FTY720 (0.5, 1.5, 5.0 mg/kg/d) elicited dose-dependent hypertension after multiple days of oral administration in rat at clinically relevant plasma concentrations (24-hr mean blood pressure = 8.4, 12.8, 16.2 mmHg above baseline <em>vs.</em> 3 mmHg in vehicle controls), BAF312 (0.3, 3.0, 30.0 mg/kg/d) had no significant effect on blood pressure at any dose tested suggesting that hypertension produced by FTY720 is mediated S1P₃ receptors. In summary, in vitro selectivity results in combination with studies performed in anesthetized and conscious rats administered two clinically tested S1P agonists, FTY720 or BAF312, suggest that S1P₁ receptors mediate bradycardia while hypertension is mediated by S1P₃ receptor activation.</p> </div

A GPBAR1 (TGR5) small molecule agonist shows specific inhibitory effects on myeloid cell activation in vitro and reduces experimental autoimmune encephalitis (EAE) in vivo.

GPBAR1 is a G protein-coupled receptor that is activated by certain bile acids and plays an important role in the regulation of bile acid synthesis, lipid metabolism, and energy homeostasis. Recent evidence suggests that GPBAR1 may also have important effects in reducing the inflammatory response through its expression on monocytes and macrophages. To further understand the role of GPBAR1 in inflammation, we generated a novel, selective, proprietary GPBAR1 agonist and tested its effectiveness at reducing monocyte and macrophage activation in vitro and in vivo. We have used this agonist, together with previously described agonists to study agonism of GPBAR1, and shown that they can all induce cAMP and reduce TLR activation-induced cytokine production in human monocytes and monocyte-derived macrophages in vitro. Additionally, through the usage of RNA sequencing (RNA-Seq), we identified a select set of genes that are regulated by GPBAR1 agonism during LPS activation. To further define the in vivo role of GPBAR1 in inflammation, we assessed GPBAR1 expression and found high levels on circulating mouse monocytes. Agonism of GPBAR1 reduced LPS-induced cytokine production in mouse monocytes ex vivo and serum cytokine levels in vivo. Agonism of GPBAR1 also had profound effects in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, where monocytes play an important role. Mice treated with the GPBAR1 agonist exhibited a significant reduction in the EAE clinical score which correlated with reduced monocyte and microglial activation and reduced trafficking of monocytes and T cells into the CNS. These data confirm the importance of GPBAR1 in controlling monocyte and macrophage activation in vivo and support the rationale for selective agonists of GPBAR1 in the treatment of inflammatory diseases

S1P receptor subtype selectivity of FTY720-P and BAF312, EC₅₀ values reported in nM.

<p>S1P receptor subtype selectivity of FTY720-P and BAF312, EC₅₀ values reported in nM.</p

Plasma (nM) concentrations of FTY720 and the phosphorylated form of FTY720 after oral administration (0.5, 1.5, 5.0 mg/kg/d) or BAF312 after oral administration (0.3, 3.0, 30.0 mg/kg/d) at 4, 8, and 24 hrs post-dose on Day 1 and at 0, 4, 8, and 24 hrs post-dose on the last day of the study; values were quantified by mass spectrometry and are shown as mean±SEM.

<p>Plasma (nM) concentrations of FTY720 and the phosphorylated form of FTY720 after oral administration (0.5, 1.5, 5.0 mg/kg/d) or BAF312 after oral administration (0.3, 3.0, 30.0 mg/kg/d) at 4, 8, and 24 hrs post-dose on Day 1 and at 0, 4, 8, and 24 hrs post-dose on the last day of the study; values were quantified by mass spectrometry and are shown as mean±SEM.</p

Chemical structure of FTY720 (A) and BAF312 (B), the latter as reported by Thompson Integrity and as referred to as BAF312 throughout the publication.

<p>Chemical structure of FTY720 (A) and BAF312 (B), the latter as reported by Thompson Integrity and as referred to as BAF312 throughout the publication.</p

Effect of FTY720 and BAF312 on mean arterial pressure (MAP) and heart rate (HR) in conscious, telemetry-instrumented, rats during 10 days of daily oral administration.

<p>At doses that elicited no significant bradycardia, FTY720 elicited dose-dependent hypertension whereas BAF312 had no affect on MAP values at any dose tested during the study. FTY720 elicited dose-dependent increases in MAP at all doses tested (0.5, 1.5, and 5.0 mg/kg/d); 24-hr mean values over the treatment period increased 8.4±0.4, 12.8±0.4, and 16.2±0.8 mmHg, respectively, (vehicle = 3.7±0.5 mmHg) and values reached statistical significance in all treated groups (*p<0.05 <i>vs.</i> vehicle). However, BAF312 elicited no significant increase in mean arterial pressure during the study; average changes in 24-mean values during 14-days of treatment in the 0.3, 3.0, and 30 mg/kg/d dose groups were 3.0±0.5, 1.1±0.5, and 0.1±0.5 mmHg, respectively.</p

Plasma concentrations of FTY720 and the phosphorylated form of FTY720, (S)-FTY720-P, after a single oral dose (0.3, 1.0, 3.0, 10.0 mg/kg) at 4, 8, 24, 48, and 72 hrs post-dose; values quantified by MS and are shown in nM (mean±SEM).

<p>Values in brain, muscle, and CSF at each timepoint in satellite rats in the 3 and 10 mg/kg dose groups are shown in the supplementary data.</p

Effect of FTY720 on mean arterial pressure (MAP, Panel A) and heart rate (HR, Panel B) in anesthetized rats.

<p>Plasma concentrations at the end of each infusion period and at the end of the post-treatment period are shown in (A) in µM. At the end of each infusion period mean arterial pressure decreased to −9±3, −22±4, and −24±4 mmHg below baseline and heart rate decreased to −71±26, −139±37, and −173±26 beats/min below baseline values; *p<0.05 <i>vs.</i> the vehicle control group.</p