Direct and quantitative evaluation of the major human CYP contribution (fmCYP) to drug clearance using the in vitro Silensomes™ model

Figures & data

Table 1. Conditions for CYP-Silensomes incubations.

CYP	Substrates	Substrates concentration (µM)	Metabolites	HLM protein concentration (mg/ml)	Incubation time (min)
(A) Initial rate incubation conditions
1A2	Phenacetin	4.5	Acetaminophen	1	15
2B6	Bupropion	50	Hydroxybupropion	0.5	15
2C8	Paclitaxel	4	6α-Hydroxypaclitaxel	0.5	15
2C9	Diclofenac	4	4’-Hydroxydiclofenac	0.05	10
2C19	Omeprazole	5	5-Hydroxyomeprazole	0.5	15
	(S) mephenytoin	10	4’-Hydroxymephenytoin	1	30
2D6	Dextromethorphan	5	Dextrorphan	1	15
2E1	Chlorzoxazone	40	6-Hydroxychlorzoxazone	0.5	20
3A4	Testosterone	30	6β-Hydroxytestosterone	0.5	30
3A4	Midazolam	0.5	1’-Hydroxymidazolam	0.1	15
3A4	Nifedipine	10	Oxidized nifedipine	0.5	15
(B) Saturating incubation conditions
1A2	Phenacetin	220	Acetaminophen	0.5	30
2B6	Bupropion	100	Hydroxybupropion	0.5	30
2C8	Amodiaquine	20	Desethylamodiaquine	0.5	30
2C9	Diclofenac	200	4’-Hydroxydiclofenac	0.5	30
2C19	(S) mephenytoin	60	4’-Hydroxymephenytoin	0.5	30
2D6	Dextromethorphan	100	Dextrorphan	0.5	30
2E1	Chlorzoxazone	200	6-Hydroxychlorzoxazone	0.5	30
3A4	Testosterone	75	6β-Hydroxytestosterone	0.5	30
3A4	Midazolam	100	1’-Hydroxymidazolam	1	5
3A4	Nifedipine	50	Oxidized nifedipine	0.5	30

HLM: human liver microsomes.

Table 2. Characteristics of the mechanism-based inhibitors selected.

CYP	MBI selected (pre-incubated concentration selected)	Substrate	KI (µM)	Kinact (min^−1)	References	Other CYPs inhibited (IC50)	% initial MBI concentration in target CYP-silensomes^a
3A4	Azamulin (5 µM)	Midazolam	0.17	0.68	Perloff et al. (2009)	CYP3A5 (0.38)CYP3A7 (0.34)CYP2J2	11
1A2	Furafylline (10 µM)	Phenacetin	4	0.6	Perloff et al. (2009)	–	4
2B6	ThioTepa (15 µM)	Bupropion	3.8	0.16	Richter et al. (2005)	CYP2A6 (6.6)	9
2C8	Gemfibrozil glucuronide (30 µM)	Amodiaquine	29	0.072	Baer et al. (2009)	–	8
2C9	Tienilic acid (10 µM)	Diclofenac	1.6	0.201	Mori et al. (2009)	–	4
2D6	Paroxetine (50 µM)	Dextromethorphan	1.96	0.08	Uttamsingh et al. (2015)	CYP2B6 (1.03)	46

–, No other CYP inhibited or IC₅₀ >30 µM (data are extracted from DIDB database htpp:/www.druginteractioninfo.org).

^a % initial total MBI concentration minus the irreversible fraction of MBI bound to CYP. K_I, Inhibition constant. K_inact, Maximal inactivation rate constant; MBI, Mechanism-based inhibitor.

Table 3. Estimated CYP contribution.

		Estimated cytochrome contribution (%)							Literature data
Drugs	Models	1A2	2C8	2C9	2C19	2B6	2D6	3A4	Inhibitor used in DDI study	Reference
Bupropion	Silensomes	–	–	–	nd	89	–	–
	rh RAF (a)	–	–	–	nd	100	–	–
	rh RAF (b)	–	–	––	nd	100	–	–
	In vivo					Major*
Carvedidol	Silensomes	–	–	–	nd	–	52	–
	rh RAF (a)	17	–	12	–	–	63	6.1
	rh RAF (b)	15	–	11	–	–	59	13
	In vivo						60		Paroxetine (10 mg BID)	Stout et al. (2010))
Celecoxib	Silensomes	–	–	81	nd	–	–	–
	rh RAF (a)	1	1.7	78	2.7	1	15	0
	rh RAF (b)	1.5	1.5	77	2.4	1	15	2
	In vivo			57					Fluconazole (200 mg)	NDA #020998
Dextromethorpan	Silensomes	–	–	–	nd	–	83	–
	rh RAF (a)	3.9	–	–	–	–	96	–
	rh RAF (b)	–	–	–	–	–	100
	In vivo						95		Quinidine (200 mg)	Bosilkovska et al. (2014)
Domperidone	Silensomes	–	36	–	nd	–	–	65
	rh RAF (a)	–	19	4.2	4.4	–	5	67
	rh RAF (b)	–	6.7	–	–	–	0.57	93
	In vivo							67	Itraconazole (200 mg)	Yoshizato et al. (2012)
Glimepiride	Silensomes	–	–	75	nd	–	–	–
	rh RAF (a)	–	–	100	–	–	–	–
	rh RAF (b)	–	–	100	–	–	–	–
	In vivo			58					Fluconazole (200 mg)	Niemi et al. (2001)
Losartan	Silensomes	–	–	55	nd	–	–	–
	rh RAF (a)	–	–	94	–	–	–	4.5
	rh RAF (b)	–	–	90	–	–	–	8.6
	In vivo			47					Fluconazole (200 mg)	Kazierad et al. (1997)
Ibuprofen	Silensomes	–	–	90	nd	–	–	–
	rh RAF (a)	–	–	100	nd	–	–	–
	rh RAF (b)	–	–	100	nd	–	–	–
	In vivo			53					Fluconazole (200 mg)	Hynninen et al., 2006)
Lope ramide	Silensomes	–	40	–	nd	–	–	53
	rh RAF (a)	–	30	–	–	–	3.8	67
	rh RAF (b)	–	5.5	–	–	–	1.2	93
	In vivo		54						Gemfibrozil (1200 mg)	Niemi et al. (2006)
								74	Itraconazole (200 mg)
Melatonin	Silensomes	91	–	–	nd	–	–	–
	rh RAF (a)	100	–	–	–	–	–	–
	rh RAF (b)	100	–	–	–	–	–	–
	In vivo	89							Fluvoxamine (50 mg)	Härtter et al. (2000)
Mirtazapine	Silensomes	41	–	–	nd	–	30	24
	rh RAF (a)	24	6.3	13.3	18	–	33	5
	rh RAF (b)	15	3.6	3.7	10	–	38	30
	In vivo						15		Paroxetine (40 mg)	Ruwe et al. (2001)
								33	Ketoconazole (400 mg)	NDA #020415
Omeprazole	Silensomes	–	–	–	nd	–	–	37
	rh RAF (a)	–	–	–	81	–	–	19
	rh RAF (b)	–	–	–	52	–	–	–
	In vivo				82				Fluvoxamine (25 mg)	Yasui-Furukori et al. (2004)
								27	Ketoconazole (200 mg)	Böttiger et al. (1997)
Pioglitazone	Silensomes	–	69	–	nd	–	–	–
	rh RAF (a)	–	94	5.9	nd	–	–	1.4
	rh RAF (b)	–	93	5.8	nd	–	–	–
	In vivo		60						Gemfibrozil (1200 mg)	Aquilante et al. (2013)
Propranolol	Silensomes	–	–	–	nd	–	58	–
	rh RAF (a)	10	1	–	3.3	–	86	–
	rh RAF (b)	9	2	–	1.7	–	88	–
	In vivo						58		Quinidine (50 mg)	Zhou et al. (1990)
Ramelteon	Silensomes	77	–	–	nd	–	–	–
	rh RAF (a)	81	7.5	1.7	8.4	–	–	1
	rh RAF (b)	66	1	7.3	18	–	–	5.7
	In vivo	99							Fluvoxamine (100 mg BID),	NDA #021782
				58^c	58^c			58	Fluconazole (200 mg)	NDA #021782
								45	Ketoconazole (200 mg)	NDA #021782
Rasagiline	Silensomes	50	–	–	nd	–	–	–
	rh RAF (a)	59	–	–	–	41	–	–
	rh RAF (b)	59	–	–	–	41	–	–
	In vivo	43							Ciprofloxacine (500 mg BID)	NDA #021641
Répaglinide	Silensomes	–	80	–	nd	–	–	–
	rh RAF (a)	–	94	0.4	nd	–	–	4.5
	rh RAF (b)	–	90	0.4	nd	–	–	9.3
	In vivo		78						Gemfibrozil (900 mg)	Honkalammi et al. (2011)
Sertraline	Silensomes	–	–	–	nd	58	–	–
	rh RAF (a)	7.5	4.3	2.6	nd	70	10	5.1
	rh RAF (b)	7.5	4.3	2.6	nd	69	10	5.8
	In vitro			11	15	59		16		Obach et al. (2005)
Sibutramide	Silensomes	–	–	–	nd	31	–	–
	rh RAF (a)	2.8	3.2	2.9	7.3	64	2.8	17
	rh RAF (b)	2.6	3.0	2.7	6.8	59	2.6	23
	In vivo					56			Clopidogrel (75 mg)	(Bae et al., 2011)
Tamsulosin	Silensomes	–	–	–	nd	–	29	55
	rh RAF (a)	–	–	–	–	–	45	37
	rh RAF (b)	–	–	–	–	–	48	52
	In vivo						39		Paroxetine (20 mg)	Troost et al. (2011)
								64	Ketoconazole (400 mg)
Tasimelteon	Silensomes	46	–	–	nd	–	–	–
	rh RAF (a)	40	–	6.3	54	–	–	–
	rh RAF (b)	41	–	8.7	46	–	–	3.9
	In vivo	85							Fluvoxamine (50 mg)	Ogilvie et al. (2015)
								31	Ketoconazole (400 mg)

*: from FDA, 2017. RAF: relative activity factor. (a) RAF nifedipine. (b) RAF testosterone. nd: not determined. c: fmCYP could not be fully attributed to CYP2C9 or CYP2C19 since fluconazole is known as a CYP2C9 and CYP2C19 inhibitor in clinic.

Figure 1. Specificity of the inhibition of CYP activities in CYP1A2-, CYP2B6-, CYP2C8-, CYP2C9-, CYP2D6-Silensomes at initial rate (K_m) and saturating conditions (V_max) of substrates. CYP activities at K_m and V_max concentration of substrates in CYP1A2-Silensomes (A, B), CYP2B6-Silensomes (C, D), CYP2C8-Silensomes (E, F), CYP2C9-Silensomes (G, H), CYP2D6-Silensomes (I, J), and corresponding control-Silensomes were compared using the following CYP probe substrates: phenacetin for CYP1A2, bupropion for CYP2B6, paclitaxel or amodiaquine for CYP2C8, diclofenac for CYP2C9, omeprazole or S-mephenytoin for CYP2C19, dextromethorphan for CYP2D6, chlorzoxazone for CYP2E1 and midazolam (a), nifedipine (b) and testosterone (c) for CYP3A4 used at either concentrations close to K_m (non-saturating conditions) and V_max (saturating conditions) (see details in Tables 1A and B). Inhibition is expressed as a % activity in control-Silensomes, mean ± SEM of experiments performed in triplicate on two CYP-Silensomes batches (saturating and non-saturating conditions) except CYP1A2 (three batches at non-saturating conditions) and CYP2B6 (one batch at non-saturating conditions), independent CYP-Silensomes batches in triplicate. (1) is the result of Anova tests comparing non-target CYP inhibition to be different from target CYP inhibition; (2) corresponds to statistically significant inhibition different from 0 (ns 0 = non significantly different from 0).

Figure 2. Correlation between observed in vivo and predicted contribution to drug metabolism using a direct quantitative method with CYP-Silensomes (A) or indirect rhCYP approach with the relative activity factors (RAFs) using nifedipine (B) and testosterone (C) as substrates for the CYP3A4 activity. All contribution values are shown in Table 3. The solid line indicates the line of perfect correlation and the dotted line ± 15% error interval.

Figure 3. Determination of the fm_CYP2D6 of Carvedidol (A) and both fm_CYP2D6 and fm_CYP3A4 of tamsulosin (B) using CYP2D6-, CYP2B6- (A, B) and CYP3A4-Silensomes (B) and their respective homologous control-Silensomes. Drugs were incubated at 0.1 µM and the disappearance of the parent compound was measured over 60 min. Percentages of remaining substrates are mean ± SEM of three independent experiments and were linearly fitted to determine clearance.

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