Invitro drug-drug interactions of decitabine and tetrahydrouridine involving drug transporters and drug metabolising enzymes

Figures & data

Figure 1. Structures of decitabine and tetrahydrouridine. Tetrahydrouridine is an inhibitor of CDA, an enzyme found primarily in the gastrointestinal tract and liver, that metabolises decitabine. CDA: cytidine deaminase.

Table 1. Cytochrome P450 activity assays.

Assay (Cytochrome P450 Activity)	Substrate (μM)	Protein (mg/mL)	Time (min)	Analyte	Positive control (μM)
Phenacetin O-deethylase (CYP1A2)	110	0.0625	10	Acetaminophen	Fluvoxamine (0.5)^a Furafylline (3)^b
Bupropion hydroxylase (CYP2B6)	120	0.0625	10	Hydroxybupropion	Orphenadrine (600)^a ThioTEPA (20)^b
Amodiaquine N-deethylase (CYP2C8)	1.5	0.0125	10	Desethylamodiaquine	Montelukast (0.1)^a GEM (40)^b
Diclofenac 4′-hydroxylase (CYP2C9)	6	0.1	5	4′-Hydroxydiclofenac	Sulfaphenazole (5)^a Tienilic Acid (10)^b
S-Mephenytoin 4′-hydroxylase (CYP2C19)	50	0.0625	10	4′-Hydroxymephenytoin	Nootkatone (20)^a Esomeprazole (8)^b
Dextromethorphan O-demethylase (CYP2D6)	10	0.0625	10	Dextrorphan	Quinidine (0.3)^a Paroxetine (2)^b
Testosterone 6β-hydroxylase (CYP3A4/5)	65	0.1	5	6β-Hydroxytestosterone	Ketoconazole (0.2)^a Erythromycin (100)^b
Midazolam 1′-hydroxylase (CYP3A4/5)	1.5	0.0625	5	1′-Hydroxymidazolam	Ketoconazole (0.1)^a Troleandomycin (10)^b

GEM: Gemfibrozil 1-O-β-glucuronide.

Note. The stopping solution was acetonitrile containing internal standard.

Note. Substrate concentrations were chosen to be near or slightly below experimentally determined K_m values.

Positive control for direct inhibition.

Positive control for metabolism-dependent inhibition (predilution concentration).

Figure 2. Direct inhibition of CYP enzymes by tetrahydrouridine. Shown are mean activity remaining (%) values at eight concentrations of tetrahydrouridine (0.1, 0.268, 0.720, 1.93, 5.18, 13.9, 37.3, and 100 μM). Error bars show ± SD. CYP: cytochrome P450; SD: standard deviation.

Figure 3. Metabolism-dependent inhibition of CYP enzymes by tetrahydrouridine. Shown are mean activity remaining (%) values at eight concentrations of tetrahydrouridine (0.1, 0.268, 0.720, 1.93, 5.18, 13.9, 37.3, and 100 μM). Error bars show ± SD. CYP: cytochrome P450; SD: standard deviation.

Table 2. Induction of CYP mRNA and enzyme activities by tetrahydrouridine.

	Control fold induction				Tetrahydrouridine
	mRNA level		Enzyme activity		mRNA level			Enzyme activity
Donor	Inducer^a	Non-inducer^b	Inducer^a	Non-inducer^b	Conc^c (µM)	Max Fold^d	% of positive control	Conc^c (µM)	Max Fold^d	% of positive control
CYP1A2
1	107	2.62	29.7	1.23	5	2.63	1.54	10	2.01	3.02
2	20.1	1.99	6.30	1.88	50	2.71	8.95	100	2.66	14.2
3	95.0	2.88	51.5	1.67	10	4.01	3.21	10	1.88	2.47
CYP2B6
1	43.8	2.62	9.93	1.01	2.5	2.41	3.29	10	1.02	0.190
2	27.9	1.60	6.59	1.35	10	2.33	4.95	10	1.20	2.98
3	70.7	3.27	3.45	1.15	10	2.77	2.54	10	2.81	27.7
CYP3A4/5
1	161	0.980	18.0	0.529	2.5	2.74	1.08	10	1.09	0.408
2	112	1.54	32.6	0.999	10	2.30	1.17	10	1.52	0.841
3	214	2.87	14.9	0.979	10	3.54	1.19	10	1.97	4.66

CYP: cytochrome P450.

Inducers—CYP1A2: Omeprazole (50 µM); CYP2B6: Phenobarbital (1000 µM); CYP3A4/5: Rifampicin (10 µM).

Non-inducer—Methotrexate (20 µM).

Tetrahydrouridine concentration where the maximum fold induction value was observed.

Fold induction was calculated relative to the solvent control. Value shown is maximum change observed after treatment with tetrahydrouridine (1, 2.5, 5, 10, 25, 50, and 100 µM).

Table 3. Involvement of key drug metabolising enzymes in the metabolism of tetrahydrouridine.

HLMs
	NADPH	UDPGA	HLM	CYP inhibitor	FMO inactivation	% of 0-min	% of ‘No cofactor’ control
CYP enzymes	y		y			102	101
	y		n			98.4	97.0
	n		y			101	100
	y		y	y		97.2	95.8
FMO inactivation	y		y		y	101	99.8
UGT enzymes		y	y			88.5	86.2
		y	n			103	101
		n	y			103	100
S9
	PAPS	S9				% of 0-min	% of ‘No cofactor’ control
SULT enzymes	y	y				72.3	89.1
	y	n				81.1	100
	n	y				87.9	108
Cytosol
	Cytosol			AO inhibitor	XO inhibitor	% of 0-min	% of ‘buffer only’ control
AO, XO enzymes	y					102	94.1
	n					108	100
	y			y		113	104
	y				y	103	97.8

CYP: cytochrome P450; FMO: flavin-containing monooxygenase; HLM: human liver microsomes; NADPH: nicotinamide adenine dinucleotide phosphate; UDPGA: uridine 5′-diphosphate glucuronic acid; UGT: uridine diphosphate glucuronosyltransferase; SULT: sulfotransferase; AO: aldehyde oxidase; XO: xanthine oxidase.

Table 4. Uptake of probe substrates (HEK293 cells).

Decitabine			Tetrahydrouridine
Treatment	Net uptake activity (pmol/min/mg protein) Mean^a ± SD	Percent activity remaining	Treatment	Net uptake activity (pmol/min/mg protein) Mean^a ± SD	Percent activity remaining
OAT1—^14C-para-Aminohippurate (1 μM)
Control^bc	20.8 ± 0.841	NA	Control^bc	20.8 ± 0.841	NA
Decitabine (0.5 μM)	16.3 ± 0.0976	78.3	Tetrahydrouridine (5 μM)	19.9 ± 0.393	95.7
Decitabine (5 μM)	19.5 ± 0.390	93.7	Tetrahydrouridine (50 μM)	19.9 ± 0.298	95.6
Probenecid (200 μM)	1.34 ± 0.103	6.42	Probenecid (200 μM)	1.34 ± 0.103	6.42
OAT3—^3H-Estrone-3-sulfate (1 μM)
Control^bc	7.03 ± 0.390	NA	Control^bc	7.03 ± 0.390	NA
Decitabine (0.5 μM)	6.68 ± 0.0282	95.1	Tetrahydrouridine (5 μM)	6.37 ± 0.200	90.7
Decitabine (5 μM)	7.75 ± 0.410	110	Tetrahydrouridine (50 μM)	6.05 ± 0.576	86.1
Probenecid (200 μM)	0.327 ± 0.148	4.66	Probenecid (200 μM)	0.327 ± 0.148	4.66
OCT1—^14C-Tetraethylammonium (5 μM)
Control^bc	27.7 ± 0.762	NA	Control^bc	27.7 ± 0.761	NA
Decitabine (0.5 μM)	27.1 ± 0.815	97.7	Tetrahydrouridine (5 μM)	30.6 ± 1.71	111
Decitabine (5 μM)	26.6 ± 0.161	95.8	Tetrahydrouridine (50 μM)	31.8 ± 0.666	115
Quinidine (256 μM)	1.18 ± 0.0510	4.25	Quinidine (256 μM)	1.18 ± 0.0510	4.26
OCT2—^14C-Metformin (1 μM)
Control^bc	2.10 ± 0.187	NA	Control^bc	2.10 ± 0.187	NA
Decitabine (0.5 μM)	2.91 ± 0.253	139	Tetrahydrouridine (5 μM)	3.42 ± 0.211	163
Decitabine (5 μM)	3.41 ± 0.393	163	Tetrahydrouridine (50 μM)	3.95 ± 0.185	188
Quinidine (256 μM)	0.945 ± 0.0561	45.1	Quinidine (256 μM)	0.945 ± 0.0561	45.1
OATP1B1—^3H-Estradiol-17β-D-glucuronide (0.5 μM)
Control^bc	7.07 ± 0.477	NA	Control^bc	7.07 ± 0.477	NA
Decitabine (0.5 μM)	7.12 ± 0.226	101	Tetrahydrouridine (5 μM)	5.78 ± 0.400	81.8
Decitabine (5 μM)	6.87 ± 0.226	97.2	Tetrahydrouridine (50 μM)	6.83 ± 0.357	96.6
Cyclosporine A (10 μM)	0.215 ± 0.0562	3.04	Cyclosporine A (10 μM)	0.215 ± 0.0562	3.04
OATP1B3—^3H-Cholecystokinin octapeptide (1 μM)
Control^bc	31.7 ± 1.56	NA	Control^bc	31.7 ± 1.56	NA
Decitabine (0.5 μM)	29.5 ± 1.45	93.0	Tetrahydrouridine (5 μM)	30.7 ± 1.62	96.8
Decitabine (5 μM)	29.0 ± 0.874	91.5	Tetrahydrouridine (50 μM)	28.1 ± 0.966	88.6
Cyclosporine A (10 μM)	0.521 ± 0.147	1.64	Cyclosporine A (10 μM)	0.521 ± 0.147	1.64
MATE1—^14C-Tetraethylammonium (5 μM)
Control^bc	145 ± 8.23	NA	Control^bc	145 ± 8.23	NA
Decitabine (0.5 μM)	145 ± 6.32	99.4	Tetrahydrouridine (5 μM)	138 ± 4.90	95.1
Decitabine (5 μM)	130 ± 2.97	89.5	Tetrahydrouridine (50 μM)	122 ± 5.58	83.9
Cimetidine (100 μM)	0.831^d ± NA	0.572	Cimetidine (100 μM)	0.831 ± NA	0.572
MATE2-K—^14C–Tetraethylammonium (5 μM)
Control^bc	29.4 ± 2.28	NA	Control^bc	29.4 ± 2.28	NA
Decitabine (0.5 μM)	30.6 ± 2.06	104	Tetrahydrouridine (5 μM)	21.0 ± 1.24	71.7
Decitabine (5 μM)	25.3 ± 1.00	86.2	Tetrahydrouridine (50 μM)	19.5 ± 1.90	66.5
Cimetidine (100 μM)	1.06 ± 1.27	3.60	Cimetidine (100 μM)	1.06 ± 1.27	3.60

NA: not applicable; OAT: organic anion transporter; OCT: organic cation transporter; OATP: organic anion transporting polypeptide; MATE: multidrug and toxin extrusion; SD: standard deviation.

Note. Activities were normalised with the vector control values.

a Mean of three replicates, unless noted otherwise.

b Final solvent content was 1% dimethyl sulfoxide.

c Mean of six replicates.

d Average of two replicates (one excluded as outlier).

Table 5. Permeability and efflux ratios of probe substrates (Caco-2 cells).

Treatment	Permeability (×10^−6 cm/s)		Efflux ratio^b	Percent of control
	A to B	B to A
	Mean^a ± SD	Mean^a ± SD
Decitabine
P-gp—^3H-Digoxin (1 μM)
Solvent^c	0.841 ± 0.0440	23.5 ± 3.03	27.9	100
Zosuquidar (2 μM)	4.98 ± 0.236	6.45 ± 0.656	1.29	1.10
Ko143 (1 μM)	0.691 ± 0.0556	19.9 ± 2.47	28.8	103
Decitabine (0.5 μM)	0.778 ± 0.0575	23.6 ± 2.27	30.4	109
Decitabine (5 μM)	0.738 ± 0.0539	26.0 ± 2.93	35.1	127
BCRP—^3H-Estrone-3-sulfate (0.1 μM)
Solvent^c	1.94 ± 0.131	29.0 ± 3.06	14.9	100
Zosuquidar (2 μM)	1.91 ± 0.220	23.8 ± 2.95	12.5	82.6
Ko143 (1 μM)	3.94 ± 0.157	7.29 ± 0.181	1.85	6.11
Decitabine (0.5 μM)	1.81 ± 0.0674	28.3 ± 3.88	15.7	105
Decitabine (5 μM)	1.85 ± 0.118	31.6 ± 1.06	17.1	116
Tetrahydrouridine
P-gp—^3H-Digoxin (1 μM)
Solvent^c	0.841 ± 0.0440	23.5 ± 3.03	27.9	100
Zosuquidar (2 μM)	4.98 ± 0.236	6.45 ± 0.656	1.29	1.10
Ko143 (1 μM)	0.691 ± 0.0556	19.9 ± 2.47	28.8	103
Tetrahydrouridine (5 μM)	0.865 ± 0.0460	23.7 ± 2.51	27.3	97.8
Tetrahydrouridine (50 μM)	0.733 ± 0.0290	24 ± 2.84	32.7	118
BCRP—^3H-Estrone-3-sulfate (0.1 μM)
Solvent^c	1.94 ± 0.131	29.0 ± 3.06	14.9	100
Zosuquidar (2 μM)	1.91 ± 0.220	23.8 ± 2.95	12.5	82.6
Ko143 (1 μM)	3.94 ± 0.157	7.29 ± 0.181	1.85	6.11
Tetrahydrouridine (5 μM)	1.80 ± 0.107	27.3 ± 3.16	15.2	102
Tetrahydrouridine (50 μM)	1.95 ± 0.150	31.7 ± 0.472	16.3	110
^14C Mannitol (10 µM)	1.56 ± 0.0594	1.15 ± 0.0339	0.737	–
^14C Caffeine (5 µM)	47.9 ± 0.425	28.3 ± 1.77	0.591	–

A to B: apical to basolateral direction; B to A: basolateral to apical direction; BCRP: breast cancer resistance protein; BSEP: bile salt export pump; NA: not applicable; P-gp: P-glycoprotein; SD: standard deviation.

Mean of three replicates.

The ratio of apparent permeability of B to A over A to B.

Final solvent content was 1% dimethyl sulfoxide.

Table 6. ATP-dependent uptake of probe substrates (BSEP membrane vesicles).

		Uptake activity (pmol/min/mg protein)	Signal-to-noise ratio	ATP-dependent uptake activity (pmol/min/mg protein)
Treatment	Sample	Mean^a	Mean^a ± SD	Mean^a ± SD	Percent of control
Decitabine
Control	Mg-ATP	17.6	23.7 ± 1.25	16.8 ± 0.927	NA
	Mg-AMP	0.740
Decitabine (0.5 μM)	Mg-ATP	18.1	25.8 ± 1.34	17.4 ± 0.942	104
	Mg-AMP	0.702
Decitabine (5 μM)	Mg-ATP	17.1	21.8 ± 1.94	16.3 ± 1.52	96.9
	Mg-AMP	0.784
Bosentan (200 μM)	Mg-ATP	3.87	7.61 ± 0.493	3.36 ± 0.251	20.0
	Mg-AMP	0.509
Tetrahyrdrouridine
Control	Mg-ATP	17.6	23.7 ± 1.25	16.8 ± 0.927	NA
	Mg-AMP	0.740
Tetrahyrdrouridine (5 μM)	Mg-ATP	19.1	26.4 ± 1.38	18.3 ± 0.995	109
	Mg-AMP	0.722
Tetrahyrdrouridine (50 μM)	Mg-ATP	18.0	24.3 ± 2.20	17.2 ± 1.63	103
	Mg-AMP	0.741
Bosentan (200 μM)	Mg-ATP	3.87	7.61 ± 0.493	3.36 ± 0.251	20.0
	Mg-AMP	0.509

Mg-AMP: magnesium-adenosine 5′-monophosphate; Mg-ATP: magnesium-adenosine 5′-triphosphate; NA: not applicable; SD: standard deviation.

Mean of three replicates.

Data availability statement

Data sets for the research presented in the publication are available from the corresponding author upon reasonable request.