Drug Development and Drug Interactions | Table of Substrates, Inhibitors and Inducers

CYP Enzymes
- In vitro
- Clinical index drugs
- Examples of clinical substrates, inhibitors, and inducers
Transporters
- In vitro
  - In vitro substrates
  - In vitro inhibitors
- Examples of clinical substrates, inhibitors and inducers
  - Clinical substrates
  - Clinical inhibitors

Table 1-1: Examples of in vitro marker reactions for CYP-mediated metabolism

Enzyme	Marker reaction
CYP1A2	7-ethoxyresorufin-O-deethylation, phenacetin O-deethylation
CYP2B6	bupropion hydroxylation, efavirenz hydroxylation
CYP2C8	amodiaquine N-deethylation, paclitaxel 6α-hydroxylation
CYP2C9	diclofenac 4'-hydroxylation, S-warfarin 7-hydroxylation
CYP2C19	S-mephenytoin 4'-hydroxylation
CYP2D6	bufuralol 1'-hydroxylation, dextromethorphan O-demethylation
CYP3A4/5^(a)	midazolam 1'-hydroxylation, testosterone 6β-hydroxylation

^aRecommend the use of two structurally unrelated CYP3A4/5 substrates to evaluate in vitro CYP3A4/5 inhibition.

Abbreviations:
CYP: cytochrome P450

Table 1-2: Examples of in vitro selective inhibitors for CYP-mediated metabolism

Enzyme	Inhibitor
CYP1A2	α-naphthoflavone, furafylline^(a)
CYP2B6	clopidogrel^(a), sertraline, thiotepa^(a), ticlopidine^(a)
CYP2C8	gemfibrozil glucuronide^(a), montelukast, phenelzine^(a)
CYP2C9	sulfaphenazole, tienilic acid^(a)
CYP2C19	N-3-benzyl-nirvanol, loratadine, nootkatone, ticlopidine^(a)
CYP2D6	paroxetine^(a), quinidine
CYP3A4/5	azamulin^(a), itraconazole, ketoconazole, troleandomycin^(a), verapamil^(a)

Note: Many of these chemical inhibitors are not specific for an individual CYP enzyme. The selectivity and potency of inhibitors should be verified using the same experimental conditions with probe substrates for each CYP enzyme.

^aTime-dependent inhibitors.

Abbreviations:
CYP: cytochrome P450

Table 1-3. Examples of in vitro inducers for CYP-mediated metabolism

Enzyme	Inducer
CYP1A2	omeprazole
CYP2B6	phenobarbital
CYP2C8	rifampicin
CYP2C9	rifampicin
CYP2C19	rifampicin
CYP3A4/5	rifampicin

Abbreviations:
CYP: cytochrome P450.

Table 2-1: Examples of clinical index substrates for CYP-mediated metabolism (for use in index clinical DDI studies)

Enzyme	Sensitive index substrates unless otherwise noted
CYP1A2	caffeine, tizanidine
CYP2B6^(a)	-
CYP2C8	repaglinide^(b)
CYP2C9	tolbutamide^(c), S-warfarin^(c)
CYP2C19	lansoprazole^(c,d), omeprazole
CYP2D6	desipramine, dextromethorphan, nebivolol
CYP3A	midazolam, triazolam

Note: Index substrates predictably exhibit exposure increases due to inhibition of a given metabolic pathway and are commonly used in prospective clinical DDI studies. See section IV.A.2 of the FDA guidance for industry entitled Clinical Drug Interaction Studies —Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions (January 2020) for details. Sensitive index substrates are index drugs that demonstrate an increase in AUC of ≥5-fold with strong index inhibitors of a given metabolic pathway in clinical DDI studies. Moderately sensitive substrates are drugs that demonstrate an increase in AUC of ≥2- to <5-fold with strong index inhibitors of a given metabolic pathway in clinical DDI studies.

This table provides examples of clinical sensitive or moderately sensitive index substrates and is not intended to be an exhaustive list. Index substrates listed in this table were selected considering their sensitivity, specificity, safety profiles, and adequate number of reported clinical DDI studies with different in vivo inhibitors (≥ 3 for CYP3A or ≥ 2 for CYP1A2, 2C8, 2C9, 2C19, and 2D6). DDI data were collected based on a search of the University of Washington Metabolism and Transport Drug Interaction Database [Hachad et al. (2010), Hum Genomics, 5(1):61], and the list of references is available here.

^aWe currently do not have sensitive index substrates for CYP2B6.
^bAlso OATP1B1 substrate.
^cModerately sensitive substrates.
^dS-lansoprazole is a sensitive substrate in CYP2C19 EM subjects.

Abbreviations:
AUC: area under the concentration-time curve; CYP: cytochrome P450; DDI: drug-drug interaction; EM: extensive metabolizer; OATP1B1: organic anion transporting polypeptide 1B1.

Table 2-2: Examples of clinical index inhibitors for CYP enzymes for use in index clinical DDI studies)

Enzyme	Strong index inhibitors	Moderate index inhibitors
CYP1A2	fluvoxamine^(a)	-
CYP2B6^(b)	-	-
CYP2C8	gemfibrozil^(c)	clopidogrel^(d)
CYP2C9	-	fluconazole^(e)
CYP2C19	fluvoxamine^(a)	-
CYP2D6	fluoxetine^(f), paroxetine	Mirabegron
CYP3A	clarithromycin^(g), itraconazole^(g)	erythromycin^(g), fluconazole^(e), verapamil^(g)

Note: Index inhibitors predictably inhibit metabolism via a given pathway and are commonly used in prospective clinical DDI studies. See section IV.A.2 of the FDA guidance for industry entitled Clinical Drug Interaction Studies —Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions (January 2020) for details. Strong and moderate inhibitors are drugs that increase the AUC of sensitive index substrates of a given metabolic pathway ≥5-fold and ≥2- to <5-fold, respectively.

This table provides examples of clinical index inhibitors and is not intended to be an exhaustive list. Index inhibitors listed in this table were selected based on potency and selectivity of inhibition, safety profiles, and an adequate number of reported clinical DDI studies with different in vivo substrates [≥ 3 for CYP3A, ≥ 2 for CYP1A2, 2C9, 2C19, and 2D6, or ≥ 1 for CYP2C8 (strong inhibitors)]. DDI data were collected based on a search of the University of Washington Metabolism and Transport Drug Interaction Database [Hachad et al. (2010), Hum Genomics, 5(1):61)], and the list of references is available here.

^a Strong inhibitor of CYP1A2 and CYP2C19, moderate inhibitor of CYP3A, and weak inhibitor of CYP2D6.

^b We currently do not have index inhibitors for CYP2B6.

^c Strong inhibitor of CYP2C8 and inhibitor of OATP1B1 and OAT3.

^d Moderate inhibitor of CYP2C8 at the 75 mg dose of clopidogrel and a weak inhibitor of CYP2B6.

^e Strong inhibitor of CYP2C19 and moderate inhibitor of CYP2C9 and CYP3A.

^f Strong inhibitor of CYP2C19 and CYP2D6.

^g Inhibitor of P-gp (defined as those increasing the AUC of digoxin to ≥1.25-fold).

Abbreviations:
AUC: area under the concentration-time curve; CYP: cytochrome P450; DDI: drug-drug interaction; OATP1B1: organic anion transporting polypeptide 1B1; OAT3: organic anion transporter 3; P-gp: P-glycoprotein.

Table 2-3: Examples of clinical index inducers for CYP-mediated metabolism (for use in index clinical DDI studies)

	Strong inducers	Moderate inducers
CYP1A2	-	-
CYP2B6	-	rifampin^(a)
CYP2C8	-	rifampin^(a)
CYP2C9	-	rifampin^(a)
CYP2C19	rifampin^(a)	-
CYP3A	carbamazepine^(b), phenytoin^(c), rifampin^(a)	-

Note: Index inducers predictably induce metabolism via a given pathway and are commonly used in prospective clinical DDI studies. See section IV.A.2 of the FDA guidance for industry entitled Clinical Drug Interaction Studies — Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions (January 2020) for more details. Strong and moderate index inducers are drugs that decrease the AUC of sensitive substrates of a given metabolic pathway by ≥80 percent and ≥50 to <80 percent, respectively.

This table provides examples of clinical index inducers and is not intended to be an exhaustive list. Index inducers listed in this table were selected based on potency of induction, safety profiles, and number of reported clinical DDI studies with different in vivo substrates (≥ two substrates). DDI data were collected based on a search of the University of Washington Metabolism and Transport Drug Interaction Database [Hachad et al. (2010), Hum Genomics, 5(1):61], and the list of references is available here.

^a Strong inducer of CYP2C19 and CYP3A, and moderate inducer of CYP1A2, CYP2B6, CYP2C8, CYP2C9.
^b Strong inducer of CYP2B6 and CYP3A and a weak inducer of CYP2C9.
^c Strong inducer of CYP3A and a moderate inducer of CYP1A2 and CYP2C19.

Abbreviations:
AUC: area under the concentration-time curve; CYP: cytochrome P450; DDI: drug-drug interaction.

Table 3-1: Examples of in vitro substrates for transporters

Transporter	Gene	Substrate
P-gp	ABCB1	digoxin, fexofenadine^(a,b,c,d), loperamide, N-methylquinidine (NMQ)^(h), quinidine, talinolol, vinblastine^(c)
BCRP	ABCG2	2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), estradiol-17-beta-glucuronide^(a,c,e,h), estrone-3-sulfate^(a,b,d,f), methotrexate^(a,b,c,j), rosuvastatin^(a,b,f), prazosin(e), sulfasalazine
OATP1B1, OATP1B3	SLCO1B1, SLCO1B3	cholecystokinin octapeptide(CCK-8)^(g), estradiol-17β-glucuronide^(a,c,e,i), pitavastatin^(e,f,i), pravastatin^(b,c,f,i), rosuvastatin^(b,f,i), telmisartan^(g)
OAT1	SLC22A6	adefovir, p-aminohippurate (PAH), cidofovir, tenofovir
OAT3	SLC22A8	benzylpenicillin, estrone-3-sulfate ^(a,d,f,i), methotrexate^(a,c,i,j), pravastatin^(a,c,f,i)
MATE1, MATE-2K	SLC47A1, SLC47A2	creatinine^(j), metformin^(j), 1-methyl-4-phenylpyridinium (MPP+)^(j), tetraethylammonium (TEA)^(j)
OCT2	SLC22A2	creatinine^(j), metformin^(j), tetraethylammonium (TEA)^(j)

Note: This table provides examples of in vitro substrates for various transporters and is not intended to be an exhaustive list.

Note: The IC50 values of several OATP1B inhibitors measured using estrone-3-sulfate as a substrate were larger than those measured using estradiol-17-beta-glucuronide or pitavastatin as substrates. Thus, using estrone-3-sulfate as a substrate may underpredict the potential of a drug as an inhibitor of OATP1B.

Note: The IC50 values of several OCT2 inhibitors measured using 1-methyl-4-phenylpyridinium (MPP+) as a substrate were larger than those measured using metformin or creatinine as substrate. Thus, using MPP+ as a substrate may underpredict the potential of a drug as an inhibitor of OCT2.
^a Also a substrate of OATPs.
^b Also a substrate of OAT3.
^c Also a substrate of MRP2.
^d Also a substrate of MATEs.
^e Also a substrate of P-gp.
^f Also a substrate of NTCP.
^g Selective substrate of OATP1B3 (vs. OATP1B1).
^h Used in vesicle experiments.
ⁱ Also a substrate of BCRP.
^jSubstrate of OCTs and MATEs.

Abbreviations:
BCRP: breast cancer resistance protein; MATE: multidrug and toxin extrusion protein; MRP2: multidrug resistance-associated protein 2; NTCP: Na+-taurocholate co-transporting polypeptide; OAT: organic anion transporter; OATP: organic anion transporting polypeptide; OCT: organic cation transporter; P-gp: P-glycoprotein, also called as multidrug resistance protein 1 (MDR1).

Table 3-2: Examples of in vitro inhibitors for transporters

Transporter	Gene	Inhibitor
P-gp	ABCB1	cyclosporine^(a,b,c,d), elacridar (GF120918)^(a), ketoconazole^(a,c,e,f,g), quinidine^(c,f,g), valspodar (PSC833), verapamil^(c,f,g), zosuquidar (LY335979)
BCRP	ABCG2	elacridar (GF120918)^(h), fumitremorgin C (FTC), ko143, novobiocin^(c,e)
OATP1B1, OATP1B3	SLCO1B1, SLCO1B3	bromosulfophthalein (BSP) ^(b,d), cyclosporine^(a,b,d,,h,i), estrone-3-sulfate^(a,b,e), rifampicin^(a,d,e,h), rifamycin SV
OAT1, OAT3	SLC22A6, SLC22A8	benzylpenicillin^(j), probenecid^(c,d)
MATE1, MATE-2K	SLC47A1, SLC47A2	cimetidine^(e,f), pyrimethamine^(f)
OCT2	SLC22A2	cimetidine^(e,g), clonidine^(g)

Note: This table provides examples of in vitro inhibitors for various transporters and is not intended to be an exhaustive list.
^a Also an inhibitor of BCRP.
^bAlso an inhibitor of NTCP.
^c Also an inhibitor of OATPs.
^d Also an inhibitor of MRP2.
^e Also an inhibitor of OAT3.
^f Also an inhibitor of OCT2.
^g Also an inhibitor of MATEs.
^h Also an inhibitor of P-gp.
ⁱ Preincubation with inhibitors prior to inhibition studies causes a decrease of the Ki value.
^jSelectively inhibit OAT3 at lower concentrations. Note at the concentration inhibiting OAT3, benzylpenicillin also inhibits OATP1B3.