During nearly a decade of research dedicated to the study of sphingosine signaling pathways, we identified sphingosine-1-phosphate lyase (S1PL) as a drug target for the treatment of autoimmune disorders. S1PL catalyzes the irreversible decomposition of sphingosine-1-phosphate (S1P) by a retro-aldol fragmentation that yields hexadecanaldehyde and phosphoethanolamine. Genetic models demonstrated that mice expressing reduced S1PL activity had decreased numbers of circulating lymphocytes due to altered lymphocyte trafficking, which prevented disease development in multiple models of autoimmune disease. Mechanistic studies of lymphoid tissue following oral administration of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI) 3 showed a clear relationship between reduced lyase activity, elevated S1P levels, and lower levels of circulating lymphocytes. Our internal medicinal chemistry efforts discovered potent analogues of 3 bearing heterocycles as chemical equivalents of the pendant carbonyl present in the parent structure. Reduction of S1PL activity by oral administration of these analogues recapitulated the phenotype of mice with genetically reduced S1PL expression.

Sphingosine 1-phosphate lyase (S1PL) has been characterized as a novel target for the treatment of autoimmune disorders using genetic and pharmacological methods. Medicinal chemistry efforts targeting S1PL by direct in vivo evaluation of synthetic analogues of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI, 1) led to the discovery of 2 (LX2931) and 4 (LX2932). The immunological phenotypes observed in S1PL deficient mice were recapitulated by oral administration of 2 or 4. Oral dosing of 2 or 4 yielded a dose-dependent decrease in circulating lymphocyte numbers in multiple species and showed a therapeutic effect in rodent models of rheumatoid arthritis (RA). Phase I clinical trials indicated that 2, the first clinically studied inhibitor of S1PL, produced a dose-dependent and reversible reduction of circulating lymphocytes and was well tolerated at dose levels of up to 180 mg daily. Phase II evaluation of 2 in patients with active rheumatoid arthritis is currently underway.

2-Acetyl-4(5)-tetrahydroxybutyl imidazole (THI) has been shown to reduce rodent peripheral blood lymphocytes through increasing lymphoid sphingosine 1-phosphate (S1P) by inhibiting S1P lyase. The objective of this study was to characterize the relationship between systemic THI exposure, splenic S1P concentrations, and lymphopenia in rats. Following the oral administration of 10 and 100 mg kg(-1) THI to male rats, THI was rapidly absorbed and reached a plasma peak level at 1 h post-dosing. Splenic S1P increased and reached the peak level at 24 h. Blood lymphocyte count decreased as the splenic S1P level increased. THI plasma concentration was linked to splenic S1P concentration using an indirect model incorporated with a four-step signal transduction model. In turn, the S1P level was directly coupled with blood lymphocyte number. The integrated model simultaneously captured the splenic S1P and blood lymphocyte responses. This pharmacokinetic-biomarker-pharmacodynamic model resolved the remarkable discrepancy between plasma THI concentration and the pharmacological response and quantitatively described the relationship of THI exposure, S1P, and lymphopenic response.

Abstract:In vitro determination of metabolic stability is routinely used to assess the overall metabolic liability of compounds and for prioritization for in vivo studies. If in vitro metabolic stability data could be used to reliably predict in vivo clearance (CL), it would add significant value in the selection of compounds for in vivo pharmacokinetic and pharmacology studies. We have evaluated the utility of our in vitro metabolic stability screening assay to estimate in vivo CL in the mouse. The in vitro mouse clearances (CL in vitro ) of 146 structurally diverse compounds with metabolic stabilities > 30 %, were compared to mouse in vivo CL data. Approximately 45 % of the compounds showed agreement between in vivo CL and predicted CL in vitro within a 2-fold error criteria. The correlation appeared worse when correction for the extent of incorporation of plasma protein binding or both plasma and S9 bindings (i.e. ~14 % and~ 28 % agreement, respectively). Classification of the compounds into three groups based on in vivo CL (<30 mL/min/kg, 30-70 mL/min/kg, and >70 mL/min/kg) did not show any improvement between in vivo CL and predicted CL in vitro . The percentage of compounds falling within the 2-fold error criteria for low CL, moderate CL and high CL groups were 54, 31 and 24 %, respectively. In conclusion, our analysis suggests that in vitro metabolic stability data, as routinely obtained in early ADME screening protocols, does not demonstrate a strong correlation with or predictivity for, absolute in vivo CL in the mouse.