Sumaira Umbreen

Regulation of Peroxisome Proliferator–Activated Receptor γ Activity by Losartan Metabolites

Two active metabolites of the angiotensin type 1 (AT1) receptor blocker losartan have been described previously, EXP3174 and EXP3179. Whereas EXP3174 is the main antihypertensive AT1 receptor–blocking metabolite, the role of EXP3179 is widely unknown. Recently, a subgroup of AT1 receptor blockers has been identified as ligands for the peroxisome proliferator–activated receptor &ggr; (PPAR-&ggr;). Here we characterize the PPAR-&ggr;–activating properties of the 2 active losartan metabolites. PPAR-&ggr; activity was measured with a chimeric Gal4-DNA–binding domain–hPPAR&ggr;-ligand–binding domain (LBD) fusion protein on a Gal4-dependent luciferase reporter system. EXP3179 prominently induced the activation of the PPAR-&ggr;–LBD reaching a maximum at 100 &mgr;mol/L with a 7.1±1-fold induction (P<0.05 versus vehicle-treated cells). Maximum PPAR-&ggr;–LBD activation by EXP3179 reached 51% of the maximum response induced by the full PPAR-&ggr; agonist pioglitazone, identifying EXP3179 as a partial PPAR-&ggr; agonist. EXP3174 did not induce PPAR-&ggr;–LBD activation. EC50 values were calculated for PPAR-&ggr;–LBD activity (pioglitazone EC50: 0.88 &mgr;mol/L; EXP3179 EC50: 17.1 &mgr;mol/L; losartan EC50: >50 &mgr;mol/L). Consistent with the activation of PPAR-&ggr;, EXP3179 potently induced 3T3-L1 adipocyte differentiation, a typical PPAR-&ggr;–dependent cell function, and markedly stimulated PPAR-&ggr; target gene expression. EXP3174 failed to regulate differentiation or PPAR-&ggr; target gene expression. The present study characterizes the active losartan metabolite EXP3179 as a partial PPAR-&ggr; agonist. PPAR-&ggr; activation by EXP3179 may help us to understand the beneficial metabolic effects of losartan observed in clinical trials.

Regulation of peroxisome proliferator-activated receptor gamma activity by losartan metabolites.

Two active metabolites of the angiotensin type 1 (AT1) receptor blocker losartan have been described previously, EXP3174 and EXP3179. Whereas EXP3174 is the main antihypertensive AT1 receptor–blocking metabolite, the role of EXP3179 is widely unknown. Recently, a subgroup of AT1 receptor blockers has been identified as ligands for the peroxisome proliferator–activated receptor &ggr; (PPAR-&ggr;). Here we characterize the PPAR-&ggr;–activating properties of the 2 active losartan metabolites. PPAR-&ggr; activity was measured with a chimeric Gal4-DNA–binding domain–hPPAR&ggr;-ligand–binding domain (LBD) fusion protein on a Gal4-dependent luciferase reporter system. EXP3179 prominently induced the activation of the PPAR-&ggr;–LBD reaching a maximum at 100 &mgr;mol/L with a 7.1±1-fold induction (P<0.05 versus vehicle-treated cells). Maximum PPAR-&ggr;–LBD activation by EXP3179 reached 51% of the maximum response induced by the full PPAR-&ggr; agonist pioglitazone, identifying EXP3179 as a partial PPAR-&ggr; agonist. EXP3174 did not induce PPAR-&ggr;–LBD activation. EC50 values were calculated for PPAR-&ggr;–LBD activity (pioglitazone EC50: 0.88 &mgr;mol/L; EXP3179 EC50: 17.1 &mgr;mol/L; losartan EC50: >50 &mgr;mol/L). Consistent with the activation of PPAR-&ggr;, EXP3179 potently induced 3T3-L1 adipocyte differentiation, a typical PPAR-&ggr;–dependent cell function, and markedly stimulated PPAR-&ggr; target gene expression. EXP3174 failed to regulate differentiation or PPAR-&ggr; target gene expression. The present study characterizes the active losartan metabolite EXP3179 as a partial PPAR-&ggr; agonist. PPAR-&ggr; activation by EXP3179 may help us to understand the beneficial metabolic effects of losartan observed in clinical trials.

EXP3179 Inhibits Collagen-Dependent Platelet Activation via Glycoprotein Receptor-VI Independent of AT1-Receptor Antagonism Potential Impact on Atherothrombosis

Objective—Thrombus formation after atherosclerotic plaque rupture critically involves the platelet collagen receptor glycoprotein (GP) VI. We investigated the impact of EXP3179, an active metabolite of the angiotensin II type 1 (AT1)-receptor antagonist Losartan (LOS) on GPVI-dependent platelet activation. Methods and Results—EXP3179 and LOS but not EXP3174—the major AT1-receptor blocking metabolite of LOS—dose-dependently inhibited collagen-I (P<0.01) and GPVI-dependent platelet aggregation (P<0.01) analyzed by optical aggregometry. Platelet activation was further determined by flow cytometry measuring the expression of platelet PAC-1, an epitope of the activated fibrinogen-receptor complex. EXP3179 and LOS inhibited collagen-I (P<0.01) and GPVI-dependent PAC-1 expression (P<0.01). EXP3179 and LOS but not EXP3174 decreased the adhesion of GPVI-receptor expressing Chinese hamster ovarian cells on collagen-I under arterial shear conditions determined by flow chamber analysis (P<0.01 and P<0.05). EXP3179 also reduced human atherosclerotic plaque material-induced platelet aggregation (P<0.01) in vitro and murine platelet adhesion after acute vessel injury in vivo as determined by intravital microscopy (P<0.01). Conclusion—EXP3179 acts as a specific inhibitor of the platelet collagen receptor GPVI independent of AT1-receptor antagonism. Further investigations may clarify its individual potential as a novel pharmacological approach to specifically inhibit atherothrombotic events by GPVI-receptor blockade.

Combined Effect of Proteasome and Calpain Inhibition on Cisplatin-Resistant Human Melanoma Cells

Resistance of tumor cells to cisplatin is a common feature frequently encountered during chemotherapy against melanoma caused by various known and unknown mechanisms. To overcome drug resistance toward cisplatin, a targeted treatment using alternative agents, such as proteasome inhibitors, has been investigated. This combination could offer a new therapeutic approach. Here, we report the biological effects of proteasome inhibitors on the parental cisplatin-sensitive MeWo human melanoma cell line and its cisplatin-resistant MeWo(cis1) variant. Our experiments show that proteasome inhibitor treatment of both cell lines impairs cell viability at concentrations that are not toxic to primary human fibroblasts in vitro. However, compared with the parental MeWo cell line, significantly higher concentrations of proteasome inhibitor are required to reduce cell viability of MeWo(cis1) cells. Moreover, whereas proteasome activity was inhibited to the same extent in both cell lines, IkappaBalpha degradation and nuclear factor-kappaB (NF-kappaB) activation in MeWo(cis1) cells was proteasome inhibitor independent but essentially calpain inhibitor sensitive. In support, a calpain-specific inhibitor impaired NF-kappaB activation in MeWo(cis1) cells. Here, we show that cisplatin resistance in MeWo(cis1) is accompanied by a change in the NF-kappaB activation pathway in favor of calpain-mediated IkappaBalpha degradation. Furthermore, combined exposure to proteasome and calpain inhibitor resulted in additive effects and a strongly reduced cell viability of MeWo(cis1) cells. Thus, combined strategies targeting distinct proteolytic pathways may help to overcome mechanisms of drug resistance in tumor cells.

Determinants of Steady-State Torasemide Pharmacokinetics Impact of Pharmacogenetic Factors, Gender and Angiotensin II Receptor Blockers

AbstractBackground: Torasemide is frequently used for the treatment of hypertension and heart failure. However, the determinants of torasemide pharmacokinetics in patients during steady-state conditions are largely unknown. We therefore explored the impact of genetic polymorphisms of cytochrome P450 (CYP) 2C9 (CYP2C9) and organic anion transporting polypeptide (OATP) 1B1 (SLCO1B1), gender, and the effects of losartan and irbesartan comedication on the interindividual variability of steady-state pharmacokinetics of torasemide. Patients and methods: Twenty-four patients receiving stable medication with torasemide 10 mg once daily and with an indication for additional angiotensin II receptor blocker (ARB) treatment to control hypertension or to treat heart failure were selected. Blood samples were taken before torasemide administration and 0.5, 1, 2, 4, 8, 12 and 24 hours after administration. After this first study period, patients received either irbesartan 150 mg (five female and seven male patients aged 69 ± 8 years) or losartan 100 mg (two female and ten male patients aged 61 ± 8 years) once daily. After 3 days of ARB medication, eight blood samples were again collected at the timepoints indicated above. The patients’ long-term medications, which did not include known CYP2C9 inhibitors, were maintained at a constant dose during the study. All patients were genotyped for CYP2C9 (*1/*1 [n = 15]; *1/*2 [n = 4]; *1/*3 [n = 5]) as well as for SLCO1B1 (c.521TT [n = 13]; c.521TC [n = 11]). Results: Factorial ANOVA revealed an independent impact of the CYP2C9 genotype (dose-normalized area under the plasma concentration-time curve during the 24-hour dosing interval at steady state [AUC24,ss/D]: *1/*1 375.5 ± 151.4 μg · h/L/mg vs *1/*3 548.5 ± 271.6 μg · h/L/mg, p = 0.001), the SLCO1B1 genotype (AUC24,ss/D: TT 352.3 ± 114 μg · h/L/mg vs TC 487.6 ± 218.4 μg · h/L/mg, p < 0.05) and gender (AUC24,ss/D: males 359.5 ± 72.2 μg · h/L/mg vs females 547.3 ± 284 μg · h/L/mg, p < 0.01) on disposition of torasemide. Coadministration of irbesartan caused a 13% increase in the AUC24,ss/D of torasemide (p = 0.002), whereas losartan had no effect. Conclusion: This study shows that the CYP2C9*3 and SLCO1B1 c.521TC genotype and female gender are significant and independent predictors of the pharmacokinetics of torasemide. Coadministration of irbesartan yields moderate but significant increases in the torasemide plasma concentration and elimination half-life.

Regulation of Peroxisome Proliferator-Activated Receptor Activity by Losartan Metabolites

Two active metabolites of the angiotensin type 1 (AT1) receptor blocker losartan have been described previously, EXP3174 and EXP3179. Whereas EXP3174 is the main antihypertensive AT1 receptor–blocking metabolite, the role of EXP3179 is widely unknown. Recently, a subgroup of AT1 receptor blockers has been identified as ligands for the peroxisome proliferator–activated receptor &ggr; (PPAR-&ggr;). Here we characterize the PPAR-&ggr;–activating properties of the 2 active losartan metabolites. PPAR-&ggr; activity was measured with a chimeric Gal4-DNA–binding domain–hPPAR&ggr;-ligand–binding domain (LBD) fusion protein on a Gal4-dependent luciferase reporter system. EXP3179 prominently induced the activation of the PPAR-&ggr;–LBD reaching a maximum at 100 &mgr;mol/L with a 7.1±1-fold induction (P<0.05 versus vehicle-treated cells). Maximum PPAR-&ggr;–LBD activation by EXP3179 reached 51% of the maximum response induced by the full PPAR-&ggr; agonist pioglitazone, identifying EXP3179 as a partial PPAR-&ggr; agonist. EXP3174 did not induce PPAR-&ggr;–LBD activation. EC50 values were calculated for PPAR-&ggr;–LBD activity (pioglitazone EC50: 0.88 &mgr;mol/L; EXP3179 EC50: 17.1 &mgr;mol/L; losartan EC50: >50 &mgr;mol/L). Consistent with the activation of PPAR-&ggr;, EXP3179 potently induced 3T3-L1 adipocyte differentiation, a typical PPAR-&ggr;–dependent cell function, and markedly stimulated PPAR-&ggr; target gene expression. EXP3174 failed to regulate differentiation or PPAR-&ggr; target gene expression. The present study characterizes the active losartan metabolite EXP3179 as a partial PPAR-&ggr; agonist. PPAR-&ggr; activation by EXP3179 may help us to understand the beneficial metabolic effects of losartan observed in clinical trials.