Characterisation of seven medications approved for attention-deficit/hyperactivity disorder using in vitro models of hepatic metabolism

Figures & data

Figure 1. Metabolic stability of seven test compounds over 48 h in cPHHs. Metabolic stability in cPHHs of (A). ATX (5 µM), (B) GUA (5 µM), (C) L-AMPH (2.5 µM) after racemic AMPH administration, (D) dAMPH (2.5 µM) after racemic AMPH administration, (E) dAMPH after dAMPH administration (5 µM), (F) CLD (5 µM), (G) dMPH (2.5 µM) after racemic MPH administration, (H) dMPH (5 µM) after dMPH administration, and (I) L-MPH (2.5 µM) after racemic MPH administration. AMPH: D/L-amphetamine; ATX: atomoxetine; CLD: clonidine; cPHH: cryopreserved primary human hepatocytes; dAMPH: dextroamphetamine; dMPH: dexmethylphenidate; GUA: guanfacine; L-AMPH: L-amphetamine; L-MPH: L-methylphenidate; MPH: D/L-methylphenidate.

Figure 2. Formation of metabolites over 48 h in cPHHs. The formation of metabolites over 48 h in CPHHs is shown where reference standards were available. (A) Metabolite formation after ATX administration. (B) Metabolite formation after GUA administration. (C) Metabolite formation after racemic AMPH administration. (D) Metabolite formation after dAMPH administration. (E) Metabolite formation after racemic MPH administration. (F) Metabolite formation after dMPH administration. G. Metabolite formation after CLD administration. 3-OH-GUA: 3-hydroxy-guanfacine; 4-OH-AMPH: 4-hydroxy-amphetamine; 4-OH-ATX: 4-hydroxy-atomoxetine; 4-OH-ATX-O-gluc: 4-hydroxy-atomoxetine-O-glucuronide; AMPH: D/L-amphetamine; ATX: atomoxetine; CLD: clonidine; cPHH: cryopreserved primary human hepatocytes; dAMPH: dextroamphetamine; dMPH: dexmethylphenidate; MPH: D/L-methylphenidate; GUA: guanfacine; L-AMPH: L-amphetamine; L-MPH: L-methylphenidate; MPH: D/L-methylphenidate; N-desmethyl-ATX: N-desmethyl-atomoxetine.

Table 1. Substrate depletion, half-life and intrinsic clearance in cPHHs.

Substrate	Measured compound	Percent remaining after 48 h	T1/2^a estimated from exponential decay (min)	Estimated in vitro CLint^b (µL/min/million cells)
AMPH	dAMPH	97.9%	>2880	<0.185
	L-AMPH	99.1%	>2880	<0.185
dAMPH	dAMPH	No loss	>2880	<0.185
CLD	CLD	No loss	>2880	<0.185
MPH	dMPH	0%	217	>2.22
	L-MPH	0%	72.9	>2.22
dMPH	dMPH	0%	289	1.85
ATX	ATX	0%	217	>2.22
GUA	GUA	69.6%	>2880	<0.185

^aHalf-life.

^bIntrinsic clearance.

AMPH: D/L-amphetamine; ATX: atomoxetine; CLD: clonidine; dAMPH: dextroamphetamine; cPHH: cryopreserved primary human hepatocytes; dMPH: dexmethylphenidate; GUA: guanfacine; L-AMPH: L-amphetamine; L-MPH: L-methylphenidate; MPH: D/L-methylphenidate.

Table 2. Substrate loss in inhibitor-free HLMs.

Substrate	Measured compound	Percent substrate loss^a
AMPH	dAMPH	1.6
	L-AMPH	8.0
dAMPH	dAMPH	4.6
CLD	CLD	6.4
MPH	dMPH	10.5
	L-MPH	12.7
dMPH	dMPH	23.5
ATX	ATX	86.9
GUA	GUA	19.9

^aAverage percent substrate loss after 2 h in HLMs in the inhibitor-free controls (experiments run in triplicate).

AMPH: D/L-amphetamine; ATX: atomoxetine; CLD: clonidine; dAMPH: dextroamphetamine; dMPH: dexmethylphenidate; GUA: guanfacine; HLM: human liver microsomes; L-AMPH: L-amphetamine; L-MPH: L-methylphenidate; MPH: D/L-methylphenidate.

Table 3. CYP involvement in metabolism identified by specific inhibitors with HLMs.

Substrate	Measured compound^a	CYP^b
		3A4/5^c	1A2^c	2B6^c	2C9^c	2C19^c	2D6^c
AMPH	4-OH-AMPH	N.I.	N.I.	55.6	3.3	N.I.	36.8
dAMPH	4-OH-AMPH	5.7	N.I.	50.7	N.I.	6.7	≥63.1
CLD	4-OH-CLD	N.I.	11.2	31.4	N.I.	3.1	74.6
ATX	ATX	N.I.	5.7	45.0	12.4	N.I.	100
	4-OH-ATX	N.I.	11.0	29.5	1.8	17.6	86.4
	N-desmethyl-ATX	24.8	14.4	N.I.	7.6	9.1	N.I.
GUA	GUA	100	12.9	N.I.	3.6	10.4	7.8
	3-OH-GUA	54.1	N.I.	9.8	0.3	N.I.	16.9

^aA change in parent loss was only reported if at least 10% of the substrate was lost in the no solvent control condition (Table 2) and if parent loss and/or metabolite formation required active enzymes, as shown with the negative controls of no cofactor addition and no protein addition (Supplemental Table 3). For those reasons, amphetamine, dextroamphetamine, clonidine, methylphenidate, dexmethylphenidate, and ritalinic acid results are not shown.

^bPercent inhibition of parent loss and metabolite formation by chemical inhibitors after a 2-h incubation with HLMs.

^cEnzyme (Chemical Inhibitor): CYP3A4/5 (ketoconazole, 1 µM), CYP1A2 (furafylline, 10 µM), CYP2B6 (phencyclidine, 30 µM), CYP2C9 (tienilic acid, 20 µM), CYP2C19 (esomeprazole, 1 µM), and CYP2D6 (paroxetine, 5 µM).

3-OH-GUA: 3-hydroxy-guanfacine; 4-OH-AMPH: 4-hydroxy-amphetamine; 4-OH-ATX: 4-hydroxy-atomoxetine; 4-OH-CLD: 4-hydroxy-clonidine; AMPH: D/L-amphetamine; ATX: atomoxetine; CLD: clonidine; dAMPH: dextroamphetamine; GUA: guanfacine; HLM: human liver microsomes; N-desmethyl-ATX: N-desmethyl-atomoxetine; N.I.: no inhibition.

Table 4. FMO involvement in metabolism identified by heat-inactivated HLMs.

Substrate	Measured compound^a	FMO^b
AMPH	4-OH-AMPH	2.8%
dAMPH	4-OH-AMPH	N.I.
CLD	4-OH-CLD	7.9%
ATX	ATX	5.9%
	4-OH-ATX	18.9%
	N-desmethyl-ATX	29.1%
	GUA	GUA	23.6%
3-OH-GUA		48.8%

^aA change in parent loss was only reported if at least 10% of the substrate was lost in the no solvent control condition (Table 2) and if parent loss and/or metabolite formation required active enzymes, as shown with the negative controls of no cofactor addition and no protein addition (Supplemental Table 3). For those reasons, amphetamine, dextroamphetamine, clonidine, methylphenidate, dexmethylphenidate, and ritalinic acid results are not shown.

^bPositive findings are only those over 30% due to the mild loss of CYP450 activity following heat inactivation.

3-OH-GUA: 3-hydroxy-guanfacine; 4-OH-AMPH: 4-hydroxy-amphetamine; 4-OH-ATX: 4-hydroxy-atomoxetine; 4-OH-CLD: 4-hydroxy-clonidine; AMPH: D/L-amphetamine; ATX: atomoxetine; CLD: clonidine; dAMPH: dextroamphetamine; FMO: flavin-containing monooxygenase; GUA: guanfacine; HLM: human liver microsomes; N-desmethyl-ATX: N-desmethyl-atomoxetine; N.I.: no inactivation.