Evaluating the <i>In Vitro</i> Inhibition of UGT1A1, OATP1B1, OATP1B3, MRP2, and BSEP in Predicting Drug-Induced Hyperbilirubinemia

Hyperbilirubinemia may arise due to inadequate clearance of bilirubin from the body. Bilirubin elimination is a multifaceted process consisting of uptake of bilirubin into the hepatocytes facilitated by OATP1B1 and OATP1B3. Once in the hepatocytes, it is extensively glucuronidated by UGT1A1. Eventually, the glucuronide metabolite is excreted into the bile via MRP2. UGT1A1 inhibition has been previously shown to be linked with hyperbilirubinemia. However, because drug transporters also contribute to bilirubin elimination, the purpose of this work was to investigate the <i>in vitro</i> inhibition of OATP1B1, OATP1B3, MRP2, and BSEP of select test drugs known to elicit hyperbilirubinemia. Test drugs investigated in this study were atazanavir and indinavir, which are associated with hyperbilirubinemia and elevations in serum transaminase; ritonavir and nelfinavir, which are not associated with hyperbilirubinemia; and bromfenac, troglitazone, and trovafloxacin, which are associated with severe idiosyncratic hepatotoxicity exhibiting elevations in serum bilirubin and transaminase. Due to limited solubility and poor ionization of bilirubin and its glucuronide, the formation of estradiol 3-glucuronide was used as a surrogate to assess UGT1A1 activity, while the transport of pitavastatin, CDCF, and taurocholate were used as surrogate probe substrates to monitor the function of OATP1B1/OATP1B3, MRP2, and BSEP, respectively. It was assumed that any inhibition of the surrogate probe substrates by test drugs is indicative of the potential impact of test drugs to modulate the function of proteins involved in bilirubin disposition. <i>In vitro</i> inhibition was determined by calculating IC₅₀. Moreover, <i>C</i>_max and <i>C</i>_max,free were integrated with IC₅₀ values to calculate <i>R</i> and <i>R</i>_free, respectively, which represents the ratio of probe drug glucuronidation/transport in the absence and presence of test drugs. Analysis of the data showed that <i>R</i>_free demonstrated the best correlation to hyperbilirubinemia. Specifically, <i>R</i>_free was above the 1.1 target threshold against UGT1A1, OATP1B1, and BSEP for atazanavir and indinavir. In contrast, <i>R</i>_free was below this threshold for ritonavir and nelfinavir as well as for bromfenac, troglitazone, and trovafloxacin. For all test drugs examined, only minor inhibition against OATP1B3 and MRP2 were observed. These data suggest that the proposed surrogate probe substrates to evaluate the <i>in vitro</i> inhibition of UGT1A1, OATP1B1, and BSEP may be suitable to assess bilirubin disposition. For protease inhibitors, inclusion of OATP1B1 and BSEP inhibition may improve the predictability of hyperbilirubinemia

Role of P‑Glycoprotein on the Brain Penetration and Brain Pharmacodynamic Activity of the MEK Inhibitor Cobimetinib

Cobimetinib is a MEK inhibitor currently in clinical trials as an anticancer agent. The objectives of this study were to determine in vitro and in vivo if cobimetinib is a substrate of P-glycoprotein (P-gp) and/or breast cancer resistance protein (Bcrp1) and to assess the implications of efflux on cobimetinib pharmacokinetics (PK), brain penetration, and target modulation. Cell lines transfected with P-gp or Bcrp1 established that cobimetinib was a substrate of P-gp but not a substrate of Bcrp1. In vivo, after intravenous and oral administration of cobimetinib to FVB (wild-type; WT), <i>Mdr1a/b­(−/−)</i>,<i> Bcrp1 (−/−)</i>, and <i>Mdr1a/b­(−/−)/Bcrp­(−/−)</i> knockout (KO) mice, clearance was similar in WT (35.5 ± 16.7 mL/min/kg) and KO animals (22.0 ± 3.6 to 27.6 ± 5.2 mL/min/kg); oral exposure was also similar between WT and KO animals. After an oral 10 mg/kg dose of cobimetinib, the mean total brain to plasma ratio (Kp) at 6 h postdose was 0.3 and 0.2 in WT and <i>Bcrp1­(−/−)</i> mice, respectively. In <i>Mdr1a/b­(−/−)</i> and <i>Mdr1<i>a</i>/1b/Bcrp1­(−/−)</i> KO mice and WT mice treated with elacridar (a P-gp and BCRP inhibitor), Kp increased to 11, 6, and 7, respectively. Increased brain exposure in <i>Mdr1a/b­(−/−)</i> and <i>Mdr1<i>a</i>/1b/Bcrp1­(−/−)</i> KO and elacridar treated mice was accompanied by up to ∼65% suppression of the target (pErk) in brain tissue, compared to WT mice. By MALDI imaging, the cobimetinib signal intensity was relatively high and was dispersed throughout the brain of <i>Mdr1<i>a</i>/1b/Bcrp1­(−/−)</i> KO mice compared to low/undetectable signal intensity in WT mice. The efflux of cobimetinib by P-gp may have implications for the treatment of patients with brain tumors/metastases

Comparison of potency of peptide-derived caspase inhibitors in cellular lamin cleavage assay, enzymatic activity and cell permeability.

<p>nd = not determined</p><p>a = z-VEID-TFPM enzymatic IC₅₀ is less than 0.0017 due to limit of enzymatic assay detection.</p><p>*Enzymatic IC₅₀ values were determined after a 15 minute enzyme/inhibitor preincubation and 40 minute enzyme reaction.</p

Effect of peptide-based caspase inhibitors on SKNAS cells as determined using the Caspase-Glo® 6 assay.

<p>SKNAS cells were treated with Ac-VEID-CHO (•) or Ac-DEVD-CHO (▪) prior to addition of 3 µM staurosporine for 6 hours and detection of VEID-ase activity as described in Experimental Procedures. Concentration inhibition curves were performed in duplicate and represent 1 of at least 3 experiments with similar results. Concentration-response curves for each inhibitor were normalized to zero and 100% based on no staurosporine or DMSO, respectively. The mean and standard error of the mean are reported.</p

Apoptosis-mediated cleavage of lamin A/C is elevated in wild-type relative to caspase-6 KO fibroblasts.

<p>Fibroblasts derived from caspase-6 KO (▪) or wild type (•) mice were treated with the indicated concentration of staurosporine for 6 hours prior to detection of the small lamin A/C cleavage product. The assay was performed in quadruplicate two times with similar results; mean and standard error of the mean are reported.</p

Effect of peptide-based caspase inhibitors on the generation of cleaved lamin A/C in SKNAS cells.

<p>(A) SKNAS cells were treated with z-VEID-FMK (♦), z-DEVD-FMK (▪), Q-VD-OPh (•), Ac-VEID-CHO (▴) or Ac-DEVD-CHO (○) prior to addition of 3 µM staurosporine for 6 hours. (B) SKNAS cells were treated with z-ID-TFPM (•), z-EID-TFPM (▴) or z-VEID-TFPM (▪) prior to addition of 3 µM staurosporine for 6 hours. Detection of the small lamin A/C cleavage product was performed as described in Experimental Procedures. Concentration inhibition curves were performed in duplicate and represent 1 of at least 3 experiments with similar results. Concentration-response curves for each inhibitor were normalized to zero and 100% based on no staurosporine or DMSO, respectively. The mean and standard error of the mean are reported.</p

Western blot detection of lamin A/C from SKNAS neuroblastoma cells upon staurosporine treatment.

<p>(A) Schematic of the N- and C-terminal globular domains of Lamin A/C with VEID-containing central α-helical region as the site of caspase-6 proteolysis. (B) SKNAS cells were treated with DMSO control or staurosporine for 6 hours prior to cell lysis. Lysates were probed for small lamin A/C subunit (Lanes 1–2), large lamin A/C subunit (Lanes 3–4) or total lamin A/C (Lanes 5–6). (C) SKNAS cells were treated with DMSO or staurosporine for the indicated time prior to cell lysis. Lysates were probed for large lamin A/C subunit (red) or β-Actin (green).</p

Potency of peptide-derived caspase inhibitors possessing aldehyde (CHO) and fluoromethyl ketone (FMK) warheads against executioner caspases.

<p>*IC₅₀ values were determined after a 15 minute enzyme/inhibitor preincubation and 40 minute enzyme reaction. Due to the capacity for time-dependent inhibition with irreversible inhibitors the values reported can be considered “apparent IC₅₀”.</p

Effect of staurosporine on the generation of cleaved lamin A/C in SKNAS cells.

<p>SKNAS cells were treated with the indicated concentration of staurosporine for 6 hours prior to detection of the small lamin A/C cleavage product as described in Experimental Procedures. The assay was performed in triplicate one time. The mean and standard error of the mean are reported.</p

Western blot detection of recombinant GST-lamin A processing by purified caspases.

<p>(A) The indicated concentration of caspase-6 was incubated with GST-lamin A for two hours. (B) 300 Units of caspases 1–9 were incubated with GST-lamin A for two hours. Intact and cleaved lamin A were detected via western blotting using anti-GST antibody.</p