TABLE 8

Studies commonly done through the course of drug development to estimate f_CL and f_m

Stage	Study Type	Information Gathered	Pros and Cons
Pre-FIH/ Preclinical Development	• Preclinical Species PK/ADME	• NME cleared via metabolism or excretion in animals	Pros:
	(in vivo)	• Major metabolic pathways in animals	•Confidence from IVIVC in animals
	• Preclinical Species CL_int + metabolite profiling (in vitro)	• Metabolic pathway in vitro similar to that in vivo in animals	Cons:
			• Human routes of metabolism maybe quite different from animals
	• Human CL_int + metabolite profiling (in vitro)	• Major metabolic pathways in humans in vitro	Pros:
	• P450 and other DME identification (in vitro human matrices)	• Relative contribution of oxidation versus Conjugation	• Preliminary estimates of P450-mediated DDI risk
		• Relative contribution of P450 enzymes	Cons:
		• Metabolism involve single or multiple enzymes	• In vitro pathways may not be major pathways in vivo
FIH (SAD /MAD)	• Detailed reaction phenotyping (in vitro)	• f_m of P450 or other major enzyme involved (in vitro)	Pros:
	• Metabolite profiling in plasma and urine in human usually available; in certain cases in bile (Entero-Test)	• First look into major metabolic pathways in humans(commonly in plasma and urine; sometimes in bile)	• Usually metabolite monitoring in in vitro phenotyping studies
		• Crude estimate of f_CL,renal (if NME substantially cleared renally); quantitative metabolite information in plasma and urine in certain cases (e.g., with quantitative NMR)	• First look in humans—are metabolic pathways similar in vivo versus in vitro? Any human unique pathway not captured in vitro
		• PK linearity (understand saturable processes)	Cons:
			• All qualitative estimates; quantitative estimate in plasma and urine possible if using quantitative NMR which allows minimum estimate of f_m
			• Missing metabolites info in feces
Definitive Human Studies	Human radiolabel ADME study	• Route of CL in humans (f_CL)	Pros:
		• Quantitative metabolite profiling	• f_{CL,metabolism} + f_CL,renal + f_CL,biliary quantitatively determined
			• Metabolites determined in feces
			• Learn if f_CL pathways predicted earlier is consistent with observed human f_CL pathways
			Cons:
			• f_CL,biliary challenging after oral dose when substantial unchanged NME in feces
			• In case of poor mass balance f_CL pathways still not well defined
	DDI with potent and selective enzyme inhibitor	• Contribution of a DME toward overall NME metabolic clearance (f_m) (assumption that inhibitor completely inhibits only enzyme of interest and for orally administered drugs, maximal intestinal inhibition is achieved)	Pros:
			• f_m quantitatively determined
			Cons:
			• Wide range of f_m values when variability of PK, which can have significant impact on DDI magnitude
	PK in genotyped population	• Contribution of a polymorphic drug metabolizing enzyme toward overall NME metabolic clearance (f_m) (assumption that in null phenotype (PM), polymorphic enzyme pathway is completely absent)	Pros:
			• f_m quantitatively determined
			Cons:
			• Inaccurate f_m if residual activity of polymorphic enzyme in PM

FIH, first in human; PK, pharmacokinetics; NME, new molecular entity; ADME, absorption distribution metabolism excretion; CL_int, intrinsic clearance; CL, clearance; IVIVC, in vitro-in vivo correlation; P450, cytochrome P450; DDI, drug-drug interactions; f_CL, fraction of clearance; f_m, fraction metabolized; NMR, nuclear magnetic resonance; PM, poor metabolizer.