High Throughput CYP Inhibition Assays Using RapidFire Mass Spectrometry and Conventional Drug Probe Substrates

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1 High Throughput CYP Inhibition Assays Using RapidFire Mass Spectrometry and Conventional Drug Probe Substrates David M. Stresser, Ph.D. Program Manager BD Gentest SM Contract Research Services

2 Presentation overview Why conduct cytochrome P450 inhibition testing? Strategies for high throughput screening Advantages of BD Gentest assays coupled with RapidFire MS from Biotrove Price + Speed + Reliability

3 Cytochrome P450 Inhibition Major cause of drug drug interactions Want to avoid because: Ethical considerations Competition (i.e. for me too drug) Product non-approval or withdrawal Drugs Removed from or Restricted in the U.S. Market Because of Drug Interactions Terfenadine (Seldane ) February 1998 Mibefradil (Posicor ) June 1998 Cisapride (Propulsid ) January 2000

4 Metabolism-based Drug-drug interaction - Mechanism fixed rate of metabolism (e.g. CYP3A4) Result waiting Victim Drug A Liver Metabolite of Drug A Therapeutic levels Victim with perpetrator Drug A Drug B Liver Metabolite of Drug A Metabolite of Drug B Toxic accumulation of Drug A or B

5 Cytochrome P450 inhibition testing in vitro Basic Concepts Why test in vitro? In vitro results predict in vivo result (Obach et al, 2006) Design clinical trial Avoid clinical trial SAR Test P450 metabolism of model drug with and without drug candidate ( perpetrator ) Model drug serves a surrogate for all potential victim drugs Higher fraction metabolized by a single CYP, higher likelihood of DDI Quantify model drug metabolite formation What concentration of drug candidate inhibits reaction? Percent inhibition and/or IC 50 Use information to guide decision making

6 Characteristics of robust P450 inhibition assays Reaction must be single P450 isoform-specific Use probe substrate with heterogeneous enzyme source (e.g. human liver microsomes or enzyme cocktail) Wider choice of substrate with cdna-expressed enzyme Rapid metabolism of substrate Get more metabolite, faster Better sensitivity Short incubation time Reduce inhibitor depletion (that can lead to artifacts) Improves sensitivity in detecting time-dependent inhibitors Low protein concentration 0.3 mg/ml Reduce nonspecific binding to microsomes (that can lead to artifacts) Metabolite formation linear with: Incubation time Microsomal protein concentration Scalable across discovery and development

7 Strategies for high throughput CYP inhibition screening

8 Fluorometric HT P450 inhibition assays Fluorometric Can monitor metabolite in real time Multiple reads per plate for time-dependent inhibition analysis Most do not require signal development period Decade of validation Does require understanding of potential for interference (e.g. autofluorescence of test articles) O O O HO O O CH 3 N (+) AMMC No fluorescence CYP2D6 Specific CH 3 Metabolite Fluorescent N (+)

9 Luminometry based methods Luciferin-based derivatives as P450 substrates Secondary reaction with firefly luciferase, esterase and other components to yield light Promega

10 Guidance for Industry Sept, 2006 CYP 1A2 2A6 2B6 2C8 2C9 2C19 2D6 2E1 3A4/5* Substrate Preferred phenacetin-o-deethylation coumarin-7-hydroxylation nicotine C-oxidation efavirenz hydroxylase bupropion-hydroxylation Taxol 6α-hydroxylation tolbutamide methylhydroxylation S-warfarin 7-hydroxylation diclofenac 4 -hydroxylation S-mephenytoin 4 - hydroxylation ( ± )-bufuralol 1 -hydroxylation dextromethorphan O- demethylation chlorzoxazone 6-hydroxylation midazolam 1-hydroxylation testosterone 6ß -hydroxylation Substrate Acceptable 7-ethoxyresorufin-O-deethylation theophylline-n-demethylation caffeine-3-n-demethylation tacrine 1-hydroxylation propofol hydroxylation S-mephenytoin-N-demethylation amodiaquine N-deethylation rosiglitazone para-hydroxylation flurbiprofen 4 -hydroxylation phenytoin-4-hydroxylation omeprazole 5-hydroxylation fluoxetine O-dealkylation debrisoquine 4-hydroxylation p-nitrophenol 3-hydroxylation lauric acid 11-hydroxylation aniline 4-hydroxylation erythromycin N-demethylation dextromethorphan N- demethylation triazolam 4-hydroxylation terfenadine C-hydroxylation nifedipine oxidation * Recommend use of 2 structurally unrelated CYP3A4/5 substrates for evaluation of in vitro CYP3A inhibition. If the drug inhibits at least one CYP3A substrate in vitro, then in vivo evaluation is warranted.

11 Radiometric - [ 14 C]-aldehyde release O-dealkylation Unmetabolized parent drug is separated from parent Liquid extraction * Activated charcoal Adherence to beads Dextromethorphan O-demethylation - Selective for CYP2D6 [ 14 C] Aldehyde quantified by scintillation counting Chromatography not required FDA preferred drug probes References: Riley RJ & Howbrook D, 1998; Several by AD Rodrigues * Phenacetin O-deethylation - Selective for CYP1A2

12 LC/MS/MS Selectivity permits higher throughput Use standard drug probe substrates and chromatographic separation of metabolite Use LC/MS/MS to obtain Selectivity Low/no interference from other analytes LC run times 2-4 min Label-free Stable-isotope labeled internal standards for major CYP metabolites are available and control for ion suppression MRM: for acetaminophen Desired metabolite MRM (IS): for acetaminophen-[ 13 C 3 15 N]

13 Higher Throughput using LC/MS - Selectivity permits multiple analytes in same run cassette strategy Two Cassette strategies: 1. Pool samples after individual incubations for analysis Consider effect of dilution on analytical sensitivity 3-4 analytes in a 2 min cycle? 2. Pool desired enzymes/substrates into single incubation assay Multiple considerations (see next slide) In both cases, multiple analytes to be quantified in same injection

14 Considerations for N in 1 Incubation Incubation time Must be same if N in 1 With HLM, dictated by slowest turnover substrate (S-mephenytoin for CYP2C19) Can be different with N in 2 Short for rapid turnover (e.g. amodiaquine, midazolam) Long for slow turnover probe substrates (e.g. S-meph, phenacetin) (For N in 2, can also modify protein concentration to keep incubation time same) Enzyme protein concentration Must be same if N in 1 With HLM, dictated by slowest turnover substrate (S-meph) Tailor-make HLM pool ( high CYP2C19 pool) Tailor-make cdna-expressed enzyme pool (Weaver et al, 2003; Di et al, 2007) Can be different for N in 2 (rapid and slow turnover probe substrates)

15 Ideal high throughput method Same assay method in discovery and in development Drug probes conforms to FDA guidance No pooling Maintains analytical sensitivity No need to validate the approach In a CRO: meets expectations for price, turnaround time, reliability

16 High-throughput P450 Inhibition Services thru Partnership with BioTrove Inc

17 RapidFire mass spectrometry RapidFire from BioTrove (figure at right) Replaces HPLC with a rapid sample purification system Micro scale solid-phase extraction (μspe) Raw Data: 96-well plates on Agilent well plate analyzed in under 12 minutes d4-1 OH-midazolam internal standard (100 nm) Isocratic run 5-8 sec LC/MS cycle times OH-midazolam product

18 RapidFire Mass Spectrometry System

19 RapidFire Mass Spectrometry System

20 How we conduct our assays

21 Example Assay Data Set: Diclofenac 4 -hydroxylase Resulting Data Set Linearity of metabolite formation with incubation time and HLM protein concentration K M determination 3.5 µm, 3.9 µm IC 50 and K i determination with sulfaphenazole IC 50 : 0.41 µm, 0.63 µm K i : 0.20 µm, 0.19 µm

22 Cytochrome P450 Inhibition Assay Parameters Enzyme Substrate K M Model [S] Analytical Inc time (min) HLM (mg/ml) method Competitive inhibitor Time Dependent inhibitor CYP1A2 Phenacetin 37 MM LC/MS α-naphthoflavone Furafylline CYP2B6 Bupropion 97 MM LC/MS Ketoconazole Ticlopidine CYP2C8 Amodiaquine 1.1 MM LC/MS Montelukast Gemfibrozil-gluc CYP2C9 Diclofenac 3.7 MM LC/MS Sulfaphenazole Tienilic acid CYP2C19 S-mephenytoin 43 MM LC/MS S-Benzylnirvanol S-Fluoxetine CYP2D6 Dextromethorphan 4.9 MM LC/MS Quinidine Paroxetine CYP3A4 Midazolam 2.2 MM LC/MS Ketoconazole Azamulin CYP3A4 Testosterone 65 2 Hill LC/MS Ketoconazole Azamulin 1 K s, Hill coefficient n = 1.3 Methods validated in accordance with applicable FDA guidance documents Guidance for Industry-Bioanalytical Method Validation, May 2001 Drug Interaction Studies Study Design, Data Analysis and Implications for Dosing and Labeling, Sept 2006 (Draft) Kinetically optimized to achieve linear conditions with time and protein

23 IC 50 and K i values obtained with conventional LC/MS Enzyme Substrate Inhibitor mean IC 50 (nm) mean K i (nm) Best fit model ratio IC 50 /K i CYP1A2 Phenacetin 7,8-Benzoflavone 9 3 Mixed 2.7 CYP2B6 Bupropion Ketoconazole Competitive 1.6 CYP2C8 Amodiaquine Montelukast Competitive 1.7 CYP2C9 Diclofenac Sulfaphenazole Competitive 2.7 CYP2C19 (S)-Mephenytoin (S)-Benzylnirvanol Competitive 1.2 CYP2D6 Dextromethorphan Quinidine Competitive 1.2 CYP3A4 Midazolam Ketoconazole 16 9 Mixed 1.8 CYP3A4 Testosterone Ketoconazole Competitive 0.9 Values represent means of two independent determinations; Global CV = 0.13 Mean = 1.7 Extensive experimental detail available in the following publication: Perloff et al (2009) Validation of cytochrome P450 time-dependent inhibition assays: a two-time point IC 50 shift approach facilitates kinact assay design. Xenobiotica 39:99-112

24 High-Throughput CYP inhibition with RapidFire High-Quality Data Analysis Excellent correlation with conventional LC/MS (See figure at right) Full 7-point IC 50 curves Single point, percent inhibition is risky MS with stable-labeled isotopes Individually incubated Individually analyzed GLP- Validated Methodology - in Drug Discovery! BioTrove IC50 values (um Direct Inhibition Screen Comparison unity R 2 = BD Gentest IC50 values (um) Data from 8 different enzyme/substrate pairs and 1 to 3 inhibitors for each pair. Inhibitors include ketoconazole, α-naphthoflavone, montelukast. S-benzylnirvanol, sulfaphenazole, azamulin, paroxetine, ticlopidine, S-fluoxetine, tienilic acid, verapamil and diltiazem 1 FDA Draft guidance on DDI, Sept, 2006

25 Analysis of ritonavir by IC 50 shift midazolam as substrate for CYP3A4 % remaining activity 100% 80% 60% 40% 20% w/nadph w/o NADPH IC 50 - RapidFire w/ NADPH = 11 nm w/o NADPH = 24 nm 0% Shift = 2.17 Inhibitor conc secondary inc. [µm] % remaining activity 100% 80% w/nadph w/o NADPH 60% 40% 20% 0% Inhibitor conc secondary inc. [µm] IC 50 - Conventional w/ NADPH = 11 nm w/o NADPH = 22 nm Shift = 2.04

26 Analysis of ritonavir by IC 50 shift testosterone as substrate for CYP3A4 % remaining activity 120% 100% w/nadph 80% w/o NADPH 60% 40% 20% 0% Inhibitor conc secondary inc. [µm] IC 50 - RapidFire w/ NADPH = 4.4 nm w/o NADPH = 11 nm Shift = 2.47 % remaining activity 120% 100% w/nadph 80% w/o NADPH 60% 40% 20% 0% Inhibitor conc secondary inc. [µm] IC 50 - Conventional w/ NADPH = 3.9 nm w/o NADPH = 9.4 nm Shift = 2.40

27 The BD-Biotrove Advantage Complete sample preparation and data analysis Customers provide compounds to BD, we conduct incubations, Biotrove performs analysis - BD provides completed data package to customer Rapid Turnaround Data available in 1 week Cost-effective $250 per 7 point curve Time-dependent inhibition assays available

28 BD Biosciences Full spectrum of high throughput ADME Drug Discovery services CYP inhibition by RapidFire Metabolic stability in microsomes Metabolic stability in hepatocytes Plasma protein binding (Rapid Equilibrium Dialysis) HT-reaction phenotyping with Supersomes Caco-2 or MDR1-LLC-PK1 monolayers Solubility

29 Questions? Contact information: David M. Stresser ext 2220