Hepatic, Intestinal, Renal, and Plasma Hydrolysis of Prodrugs in Human, Cynomolgus Monkey, Dog, and Rat: Implications for In Vitro–In Vivo Extrapolation of Clearance of Prodrugs

Abstract

Hydrolysis plays an important role in metabolic activation of prodrugs. In the current study, species and in vitro system differences in hepatic and extrahepatic hydrolysis were investigated for 11 prodrugs. Ten prodrugs in the data set are predominantly hydrolyzed by carboxylesterases (CES), whereas olmesartan medoxomil is also metabolized by carboxymethylenebutenolidase (CMBL) and paraoxonase. Metabolic stabilities were assessed in cryopreserved hepatocytes, liver S9 (LS9), intestinal S9 (IS9), kidney S9 (KS9), and plasma from human, monkey, dog, and rat. Of all the preclinical species investigated, monkey intrinsic hydrolysis clearance obtained in hepatocytes (CL int,hepatocytes ) were the most comparable to human hepatocyte data. Perindopril and candesartan cilexetil showed the lowest and highest CL int,hepatocytes , respectively, regardless of the species investigated. Scaled intrinsic hydrolysis clearance obtained in LS9 were generally higher than CL int,hepatocytes in all species investigated, with the exception of dog. In the case of human and dog intestinal S9, hydrolysis intrinsic clearance could not be obtained for CES1 substrates, but hydrolysis for CES2 and CMBL substrates was detected in IS9 and KS9 from all species. Pronounced species differences were observed in plasma; hydrolysis of CES substrates was only evident in rat. Predictability of human hepatic intrinsic clearance (CL int,h ) was assessed for eight CES1 substrates using hepatocytes and LS9; extrahepatic hydrolysis was not considered due to high stability of these prodrugs in intestinal and kidney S9. On average, predicted oral CL int,h from hepatocyte data represented 20% of the observed value; the underprediction was pronounced for highclearance prodrugs, consistent with the predictability of cytochrome P450/conjugation clearance from this system. Prediction bias was less apparent with LS9, in particular for high-clearance prodrugs, highlighting the application of this in vitro system for investigation of prodrugs.

Phase II Metabolism in Human Skin: Skin Explants Show Full Coverage for Glucuronidation, Sulfation,N-Acetylation, Catechol Methylation, and Glutathione Conjugation

“…The intrinsic hepatic in vitro clearances were scaled up to a ml·min -1 ·kg -1 body weight basis as described previously (Camenisch and Umehara, 2012). The following scaling factors from the literature were applied: 52.5 mg of protein/g of liver for liver microsomes (Iwatsubo et al, 1997), 80.7 mg of protein/g of liver for cytosol (Houston and Galetin, 2008), 121 mg of protein/g of liver for S9 fraction (Nishimuta et al, 2014), and 25.7 g of liver/kg of body weight for the liver weight (Davies and Morris, 1993). Calculation of Systemic Organ Clearances.…”

Key to Opening Kidney for In Vitro–In Vivo Extrapolation Entrance in Health and Disease: Part I: In Vitro Systems and Physiological Data

“…It is evident from Table I that human kidney subcellular fractions (microsomes, S9) and recombinant enzyme expression systems are the most commonly applied sources of kidney drug-metabolising enzymes in in vitro assays (7,(14)(15)(16). Subcellular fractions require supplementation with appropriate cofactors lost during the preparation procedure and these are highlighted in Table I.…”

Pharmacokinetics and Metabolism of Streptochlorin and Its Synthetic Derivative, 5-Hydroxy-2′-isobutyl Streptochlorin, in Mice

“…18,19) Fractions unbound in microsomes ( f u,mic ) and S9 ( f u,S9 ) were measured in triplicate by equilibrium dialysis described previously. 20) f u,mic values of streptochlorin and HIS were 0.33±0.04 and 0.09±0.02, respectively.…”