Differentiation of Reversible and MechanismBased Inhibitors of Human CYP MediatedReactions by an IC50 Shift Approach Zhiming Wen, Joseph Rager, Praveen Srivastava, Carol Kissa, Nicholas Oranzi, Jibin Li, Sid Bhoopathy, Albert Owen, and Chris Bode Absorption Systems, Exton, PA 19341, USA 25 -1 0 1 20 -1 1 Phenacetin 150 Acetaminophen +ESI, 152/110 2A6 Coumarin 6 7-OH Coumarin -ESI, 161/133 2B6 Bupropion 100 Hydroxybupropion +ESI, 256/238 2C9 Diclofenac 6 4’-OH Diclofenac +ESI, 312/231 2C19 S-mephenytoin 40 4’-OH Mephenytoin -ESI, 233/190 2D6 Bufuralol 7 1’-OH Bufuralol +ESI, 278/186 2E1 Chlorzoxazone 100 6-OH Chlorzoxazone -ESI, 184/120 Testosterone 75 6β-OH Testosterone +ESI, 305/269 Midazolam 1.4 1’-OH Midazolam +ESI, 342/203 75 50 25 0 -2 -1 0 1 2 -2 75 50 25 -1 0 0 -2 1 -1 0 1 0 2 0 Log C (Tranylcypromine, μM) Log C (α-Naphthoflavone, μM) CYP2C19_(+)-N-3-benzylnirvanol - NAPDH + NADPH 100 1 2 75 50 25 160 0 -1 0 1 - NAPDH + NADPH 120 80 40 2 -1 -1 0 100 - NAPDH + NADPH 80 60 40 20 1 2 -2 -1 0 1 Log C (Quinidine, μM) Log C (Paroxetine, μM) CYP2E1_4-Methylpyrazole CYP3A4_Testosterone_Ketoconazole CYP3A4_Midazolam_Ketoconazole % of control activity 75 50 25 0 125 - NAPDH + NADPH 100 75 50 25 0 -1 0 1 Log C (4-Methylpyrazole, μM) 2 % of control activity 125 - NAPDH + NADPH 100 2 - NAPDH + NADPH 100 75 50 25 0 -3 -2 -1 0 0 1 Log C (Sulfaphenazole, μM) Log C ((+)-N-3-benzylnirvanol, μM) 125 -2 -2 0 -2 25 125 -1 0 1 2 - NAPDH + NADPH 100 75 50 25 0 -2 -1 0 1 2 -2 -1 0 1 2 Log C (Metoclopramide, μM) CYP2E1_Disulfiram CYP3A4_Testosterone_Troleandomycin CYP3A4_Midazolam_Troleandomycin 75 50 25 - NAPDH + NADPH 100 75 50 25 0 -1 0 1 2 125 - NADPH 100 + NADPH 75 50 25 125 % of control activity 125 - NAPDH + NADPH 100 0 -2 -1 0 1 2 75 50 25 0 -2 Log C (Disulfiram, μM) - NAPDH + NADPH 100 -1 0 1 2 -2 Log C (Troleandomycin, μM) -1 0 1 2 Log C (Troleandomycin, μM) 3. IC50 Shift of Reversible and Mechanism-Based Inhibitors Table 2. IC50 shift values of reversible and mechanism-based inhibitors by a 30-min pre-incubation with HLMs in the presence and absence of NADPH CYP IC50 (μM) Inhibitor -NADPH +NADPH IC50 Shift (-NADPH/+NADPH) Inhibitor Type 0.14 6.0 1.6 0.49 244 5.4 209 0.82 0.79 0.73 0.79 9.1 0.12 0.39 20 1.0 6.1 5.9 0.051 >30 0.13 8.2 0.84 0.77 2.3 0. 10 190 0.42 5.5 0.51 0.12 0.94 0.16 1.7 0.83 0.40 3.2 0.99 1.2 1.3 0.14 0.72 0.25 1.9 0.16 7.7 0.71 4.9 1.3 13 38 1.6 6.5 0.78 4.9 5.2 0.14 0.98 6.3 1.1 5.0 4.6 0.36 >40 0.53 4.2 Reversible MBI Reversible MBI Reversible MBI MBI Reversible MBI Reversible MBI MBI Reversible Reversible MBI Reversible MBI MBI Reversible MBI Reversible MBI α-Naphthoflavone Furafylline Tranylcypromine 8-Methoxypsoralen Orphenadrine Thio-TEPA Phencyclidine Sulfaphenazole Tienilic Acid (+)-N-3-benzylnirvanol Ticlopidine Omeprazole Quinidine Paroxetine Metoclopramide 4-Methylpyrazole Diethyldithiocarbamate Disulfiram Ketoconazolea Troleandomycina Ketoconazoleb Troleandomycinb 1A2 2A6 2C9 2D6 3A4 : Testosterone as the probe substrate. : Midazolam as the probe substrate. a b 4. IC50 Shift in Comparison with Co-incubation and Pre-incubation (Fig. 3) 2 100 IC50 co = 0.076 μM IC50 pre = 0.16 μM IC50 shift = 0.48 75 50 25 0 -3 -2 -1 0 1 Log C (Ketoconazole, μM) 2 CYP3A4_Testosterone_Troleandomycin Co-incubation Pre-incubation 125 100 75 50 25 0 -2 IC50 co = >30 μM IC50 pre =1.5 μM IC50 shift = >20 -1 0 1 2 Log C (Troleandomycin, μM) Fig. 3 IC50 shift of ketoconazole and troleandomycin on CYP3A4 (testosterone) with co-incubation and pre-incubation. CYP3A4_Testosterone_Test Compound A 125 - NADPH + NADPH 100 75 50 25 0 -2 IC50 -NADPH = 7.9 μM IC50 +NADPH = 1.2 μM IC50 shift = 6.6 -1 0 1 Log C (Test Compound A, μM) 2 CYP3A4_Testosterone_Test Compound A 125 % of control activity Co-incubation Pre-incubation Co-incubation Pre-incubation 100 75 50 25 0 -2 IC50 co = 14 μM IC50 pre = 1.3 μM IC50 shift = 11 -1 0 1 1 Log C (Ketoconazole, μM) 2 -2 -1 0 1 Log C (Ketoconazole, μM) 2 2 Log C (Test Compound A, μM) Fig. 4 IC50 shift of test compound A on CYP3A4 (testosterone), suggesting test compound A is a MBI. CYP2D6_Paroxetine 0 -2 3 Log C (Orphenadrine, μM) CYP2D6_Quinidine % of control activity 125 % of control activity -2 50 % of control activity Fig. 1 Estimation of IC values of reversible inhibitors by a 100 100 30-min 75pre-incubation with HLMs in the presence and 75 absence50 of NADPH, following by 50the addition of the probe substrates and a 20-min incubation. 25 25 + NADPH 100 0 0 - NAPDH + NADPH % of control activity 30 CYP2C9_Sulfaphenazole 125 - NAPDH 50+ NADPH % of control activity 60 125 % of control activity % of control activity 90 % of control activity % of control activity 120 CYP2B6_Orphenadrine - NAPDH 75 3 CYP2E1_Diethyldithiocarbamate % o f co n t r o l activ it y CYP2A6_Tranylcypromine 2 Log C (Omeprazole, μM) 125 % of control activity 1. IC50 Values of Reversible Inhibitors 125 - NAPDH + NADPH 100 0 CYP3A4_Testosterone_Ketoconazole - NAPDH + NADPH 1 Log C (Ticlopidine, μM) 125 CYP1A2_α-Naphthoflavone 0 5. Application to Test Compound A using IC50 Shift Approach (Fig. 4) RESULTS 150 -1 Log C (Tienilic Acid, μM) 2E1 [1] Obach, et al., Drug Metab Dispos 2007, 35:246-255. [2] Nomeir, et al., Optimization in Drug Discovery: In Vitro Method. p245, 2004. % of control activity 25 125 - NAPDH + NADPH 100 % of control activity 125 - NAPDH + NADPH 2C19 3A4 + NADPH -2 CYP2D6_Metoclopramide 2B6 1A2 2 CYP2C19_Omeprazole 50 Table 1 Experimental Conditions LC-MS/MS (m/z) 1 CYP2C19_Ticlopidine 75 For co-incubation, substrates and inhibitors were warmed at 37˚C for 3 min with HLMs (0.25 mg/ml), and then incubated with NADPH (1 mM) for 20 min. For pre-incubation, only inhibitors were pre-incubated in the presence of NADPH for 30 min with HLMs, and continually incubated for 20 min after the addition of substrates. CYP reactions were terminated by adding 60% acetonitrile containing 0.1% formic acid. After centrifugation, the supernatant was injected for the quantitation of metabolites by LC-MS/MS. Metabolite 0 CYP2C9_Tienilic Acid Co-Incubation and Pre-Incubation [2] Concentration (μM) -1 Log C (Phencyclidine, μM) Log C (Diethyldithiocarbamate, μM) Substrate 25 0 -2 Log C (Thio-TEPA, μM) -2 CYP 25 2 Log C (8-MOP, μM) 0 CYP inhibitors were pre-incubated at 37˚C for 30 min with HLMs (0.25 mg/ml) in the presence and absence of NADPH (1 mM). CYP reactions were initiated by adding probe substrates (final concentrations were at ~Km) without and with NADPH, incubated at 37˚C for 20 min, and terminated with 60% acetonitrile containing 0.1% formic acid. After centrifugation, the supernatant was injected for the quantitation of metabolites by LC-MS/MS. 0 Log C (Furafylline, μM) 100 30-Min Pre-incubation with and without NADPH CYP2B6_Phencyclidine 0 -2 % of control activity % of control activity 40 2 0 % of control activity 60 0 -2 125 MATERIALS AND METHODS 80 % of control activity 50 CYP2B6_Thio-TEPA 125 125 Fig. 2 Estimation of IC50 values of mechanism-based inhibitors - NAPDH - NAPDH + NADPH 100 100 + NADPH by a 30-min pre-incubation with HLMs in the presence and 75 absence of NADPH, following by 75the addition of the probe substrates50and a 20-min incubation. 50 - NAPDH % of control activity 75 0 INTRODUCTION Mechanism-based inhibition (MBI) on cytochrome P450 (CYPs) reactions leads to irreversible or quasi-irreversible inactivation of CYPs, and has more clinical impact than reversible inhibition. Determining that a new chemical entity is a reversible or mechanism-based inhibitor of CYPmediated reactions is critical in drug discovery and development. In this study, known reversible and mechanism-based inhibitors of eight human CYPs (CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) were investigated with pooled human liver microsomes (HLMs) using an IC50 shift method [1]. In the IC50 shift approach, IC50 values are determined using a 30-min pre-incubation with chemical inhibitors in the presence and absence of NADPH prior to adding substrates, and then the inhibition type (reversible or mechanism-based) is estimated based on the relative IC50 values under the two incubation conditions. CYP2A6_8-MOP 100 - NAPDH + NADPH 100 % of control activity CONCLUSIONS: The IC50 shift approach can be used to differentiate reversible and mechanismbased inhibitors. CYP1A2_Furafylline 125 % of control activity METHODS: Determination of IC50 values of known reversible and mechanism-based inhibitors using human liver microsomes with a 30-min pre-incubation in the presence and absence of NADPH. 2. IC50 Values of Mechanism-Based Inhibitors % of control activity PURPOSE: To investigate the inhibitory difference between reversible and mechanism-based inhibitors on CYP reactions. % of control activity ABSTRACT CONCLUSION Mechanism-based inhibitors demonstrated a significant leftward shift in IC50 after pre-incubation in the presence of NADPH, in comparison with the IC50 after pre-incubation in the absence of NADPH. In contrast, reversible inhibitors exhibited a rightward shift or no change of IC50 after preincubation with NADPH. The IC50 shift after pre-incubation in the presence and absence of NADPH can be used to differentiate reversible (rightward IC50 shift or no change) and mechanism-based (leftward IC50 shift ≥ 4-fold) inhibitors. The unexpected findings (compared with reports in the scientific literature) that paroxetine appeared to be a reversible inhibitor of CYP2D6 and omeprazole appeared to be a mechanismbased inhibitor of CYP2C19 require further investigation.