Rate-Determining and Rate-Limiting Steps in the Clearance and Excretion of a Potent and Selective p21-Activated Kinase Inhibitor: A Case Study of Rapid Hepatic Uptake and Slow Elimination in Rat

Abstract

Background: Significant under-prediction of in vivo clearance in rat was observed for a potent p21-activated kinase (PAK1) inhibitor, GNE1.Objective: Rate-determining (rapid uptake) and rate-limiting (slow excretion) steps in systemic clearance and elimination of GNE1, respectively, were evaluated to better understand the cause of the in vitro-in vivo (IVIV) disconnect.Methods: A series of in vivo, ex vivo, and in vitro experiments were carried out: 1) the role of organic cation transporters (Oct or Slc22a) was investigated in transporter knock-out and wild-type animals with or without 1-aminobenzotriazole (ABT) pretreatment; 2) the concentration-dependent hepatic extraction ratio was determined in isolated perfused rat liver; and 3) excreta were collected from both bile duct cannulated and non-cannulated rats after intravenous injection.Results: After intravenous dosing, the rate-determining step in clearance was found to be mediated by the active uptake transporter, Oct1. In cannulated rats, biliary and renal clearance of GNE1 accounted for only approximately 14 and 16% of the total clearance, respectively. N-acetylation, an important metabolic pathway, accounted for only about 10% of the total dose. In non-cannulated rats, the majority of the dose was recovered in feces as unchanged parent (up to 91%) overnight following intravenous administration.Conclusion: Because the clearance of GNE1 is mediated through uptake transporters rather than metabolism, the extrahepatic expression of Oct1 in kidney and intestine in rat likely plays an important role in the IVIV disconnect in hepatic clearance prediction. The slow process of intestinal secretion is the rate-limiting step for in vivo clearance of GNE1.

1-Aminobenzotriazole: A Mechanism-Based Cytochrome P450 Inhibitor and Probe of Cytochrome P450 Biology

“…Work published in a poster supported the inference that 1-ABT does not interfere with transporters, as concentrations of 1-ABT up to 1 mM did not inhibit the MDR1, Bcrp1, and MRP2 transporters [ 76 ]. In another study, the in vivo clearance of GNE1, an inhibitor of the p21-activated kinase PAK1, was lower than predicted [ 77 ]. The use of transporter knockout rats with and without 1-ABT administration indicated that the clearance of this agent is determined by uptake transporters rather by metabolism.…”

Drug Design and Success of Prospective Mouse In Vitro–In Vivo Extrapolation (IVIVE) for Predictions of Plasma Clearance (CLp) from Hepatocyte Intrinsic Clearance (CLint)

“…The reasons for IVIVE “disconnects” are often initially unknown and might be understood only later, following mechanistic absorption, distribution, metabolism, excretion (ADME) assays, and quantification of disposition and elimination pathways in vivo. Typical examples are compounds liable to active hepatic uptake, active renal elimination, , and/or extrahepatic metabolism. , Efforts to deconvolute disconnected IVIVE are lengthy and costly, likely opposing our ambition to replace, reduce, and refine use of animal studies. Can we do better?…”

“…Figure 1 illustrates differences in chemical space between Roche's discovery compounds tested in mouse PK and a set of marketed drugs compiled by Pfizer colleagues (panels A and B). 19 While overlaps exist, increases in median MW (477 vs 371 Da), lipophilicity (log P of 2.87 vs 2.0), and polarity (TPSA of 105 vs 89 Å 2 ) are notable for Roche's discovery dataset. Moreover, Roche's chemical space is continuously evolving with the recent decade bringing many more compounds with high MW (>500 Da) and polarity (TPSA > 140 Å 2 ) (Figure 1C,D).…”

Sinusoidal Uptake Determines the Hepatic Clearance of Pevonedistat (TAK-924) as Explained by Extended Clearance Model

“…With recent advances in the drug transporter field, it has become apparent that uptake transporters at the sinusoidal membrane of hepatocytes play an important role in the hepatic clearance of drugs (Giacomini et al, 2010;Hillgren et al, 2013). Of note, organic anion transporting polypeptides (OATPs), organic anion transporter 2 (OAT2) and organic cation transporter 1 (OCT1) are shown to play an important role in the hepatic clearance of their substrate drugs, such as atorvastatin (Maeda et al, 2011), fluvastatin (Watanabe et al, 2010), cerivastatin (Shitara, 2011), repaglinide (Varma et al, 2013), bosentan (Sato et al, 2018) (OATP substrates), tolbutamide (OAT2 substrate) (Bi et al, 2018b), warfarin (OAT2 substrate) (Bi et al, 2018a) and GNE1 (OCT1 substrate) (Fan et al, 2016).…”