Physiologically Based Pharmacokinetic Modeling Framework for Quantitative Prediction of an Herb Drug Interaction

Transcription

1 Originl Article Cittion: CPT Phrmcometrics Syst. Phrmcol. (24) 3, e7; doi:.38/psp ASCPT All rights reserved /4 Physiologiclly Bsed Phrmcokinetic Modeling Frmework for Quntittive Prediction of n Herb Drug Interction SJ Brntley, BT Gufford 2, R Du, DJ Fediuk, TN Grf 3, YV Scrlett 4, KS Frederick 5, MB Fisher 6, NH Oberlies 3 nd MF Pine 2 Herb drug interction predictions remin chllenging. Physiologiclly bsed phrmcokinetic (PBPK) modeling ws used to improve prediction ccurcy of potentil herb drug interctions using the semipurified milk thistle preprtion, silibinin, s n exemplr herbl product. Interctions between silibinin constituents nd the probe substrtes wrfrin (CYP2C9) nd midzolm (CYP3A) were simulted. A low silibinin dose (6 mg/dy 4 dys) ws predicted to increse midzolm re under the curve (AUC) by %, which ws corroborted with externl dt; higher dose (,65 mg/dy 7 dys) ws predicted to increse midzolm nd (S)-wrfrin AUC by 5% nd 4%, respectively. A proof-of-concept clinicl study confirmed miniml interction between highdose silibinin nd both midzolm nd (S)-wrfrin (9 nd 3% increse in AUC, respectively). Unexpectedly, (R)-wrfrin AUC decresed (by 5%), but this is unlikely to be cliniclly importnt. Appliction of this PBPK modeling frmework to other herb drug interctions could fcilitte development of guidelines for quntittive prediction of cliniclly relevnt interctions. CPT Phrmcometrics Syst. Phrmcol. (24) 3, e7; doi:.38/psp.23.69; published online 26 Mrch 24 Herbl products represent n ever-incresing component of Western phrmcotherpy due in prt to the perception tht nturl equtes with sfety. Current regultory oversight of herbl products in mny Western countries, including the United Sttes, does not include evlution of drug interction libility prior to mrketing. Consequently, systemtic pproches for quntittive prediction of both the mgnitude nd likelihood of herb drug interctions re nonexistent. Drug interction libility ssessment of herbl products is more chllenging thn for conventionl drugs becuse unlike most drug products, herbl products typiclly re mixtures of bioctive constituents tht vry substntilly between preprtions. 3 Compounding this complexity is the often scnt knowledge of specific custive constituents or systemic exposure of such constituents. Due to incomplete bsorption nd extensive presystemic clernce, herbl product constituents my rech sufficient concentrtions in the intestine nd liver to inhibit only first-pss extrction of sensitive substrtes. 4 Consequently, trditionl (sttic) prediction pproches frequently do not trnslte to the clinicl setting for herb drug interctions. Recent drug drug interction guidelines suggest dynmic modeling nd simultion pproches to predict complex interctions. 5,6 Extension of this pproch to herb drug interctions is logicl step to fcilitte prospective evlution of these interctions. As with drug drug interctions, 7,8 physiologiclly bsed phrmcokinetic (PBPK) modeling my be used to improve in vitro to in vivo extrpoltion of herb drug interctions. Well-chrcterized herbl products re needed to develop quntittive frmework. Milk thistle preprtions re top-ten selling herbl products in the United Sttes. 9 The crude extrct, silymrin, contins t lest seven flvonolignns nd one flvonoid. The semipurified extrct, silibinin, contins roughly : mixture of the flvonolignns silybin A nd silybin B nd represents n exemplr herbl product for initil model development. First, silybin A nd silybin B hve been purified in quntities sufficient to recover requisite in vitro prmeters.,2 Second, in vitro studies hve demonstrted both reversible nd mechnism-bsed inhibition of the key drug metbolizing enzymes CYP2C9 3 5 nd CYP3A4. 3,4,6 Third, in vitro to in vivo extrpoltion hs been inconsistent. 7 9 Bsed on these observtions, the objective of this work ws to dvnce the mechnistic understnding of this herb drug interction using PBPK modeling nd simultion with wrfrin nd midzolm s probe substrtes. The models were evluted through proof-of-concept clinicl study in helthy volunteers. Results could help develop guidelines for prospective evlution of herb drug interction libility. RESULTS Modeling nd simultion PBPK model genertion nd initil evlution. Simulted probe substrte concentrtions closely pproximted previously published concentrtion time profiles for both wrfrin 2 nd midzolm 2 under bseline conditions (dt not shown). Model-predicted primry endpoints (re under the curve (AUC) nd for (S)-wrfrin nd midzolm) were within the prespecified criterion (3%) for cceptble model performnce (Tble ). Eshelmn School of Phrmcy, The University of North Crolin t Chpel Hill, Chpel Hill, North Crolin, USA; 2 College of Phrmcy, Wshington Stte University, Spokne, Wshington, USA; 3 Deprtment of Chemistry nd Biochemistry, The University of North Crolin t Greensboro, Greensboro, North Crolin, USA; 4 School of Medicine, The University of North Crolin t Chpel Hill, Chpel Hill, North Crolin, USA; 5 Boehringer Ingelheim Phrmceuticls, Inc., Ridgefield, Connecticut, USA; 6 ProPhrm Services, LLC, Oxford, Connecticut, USA. Correspondence: MF Pine (mry.pine@wsu.edu) Received 2 August 23; ccepted 28 October 23; dvnce online publiction 26 Mrch 24. doi:.38/psp.23.69

2 2 PBPK Modeling Frmework for Quntittive Prediction of n Herb Drug Interction Tble Comprison of previously published nd model-predicted phrmcokinetic outcomes Outcome (R)-Wrfrin (5 mg) c Previously published Model predicted b Accurcy (%) (hour) 42 (8) (.5 2).6 8 (µmol/l).7 (22) AUC inf (µmol/l hour) 93 (2) 9 99 Cl/F (l/hour).8 (2).8 (S)-Wrfrin (5 mg) c (hour) 32 (26) (.5 4).5 75 (µmol/l) 2. (29) 2. 5 d AUC inf (µmol/l hour) 65 (3) 7 8 d Cl/F (l/hour).25 (3) Midzolm (5 mg) e (hour) 2.9 (4) (.25.5).6 2 (nmol/l) 88 (44) 7 8 d AUC inf (nmol/l hour) 22 (33) 2 95 d Cl/F (l/hour) 7 (33) 72 Midzolm (8 mg) f (hour) 4.2 (29) (hour) (men (SD)).47 (5).6 28 (µmol/l) (49) AUC inf (nmol/l hour) 3 (44) 34 3 Cl/F (l/hour) 95 (35) Silybin A (92.8 mg) g,h (hour) ( 2).3 (µmol/l).84 (89).27 AUC 8 (µmol/l hour).3. Cl/F (l/hour) 5 7 Silybin B (28 mg) g,h (hour)..4.5 (.5 2). (µmol/l).27 (2).6 AUC 8 (µmol/l hour) Cl/F (l/hour) 95 4 AUC inf, re under the concentrtion time curve from time zero to infinity; AUC 8, AUC from 8 hours;, mximl concentrtion; Cl/F, pprent orl clernce;, terminl hlf-life;, time to mximl concentrtion. Geometric or rithmetic mens nd coefficients of vrition (%) unless indicted otherwise. b Point estimtes. c,e g Previously published outcomes from refs. 2,2,8,26, respectively. d Model predictions were considered ccurte if the primry outcomes ((S)-wrfrin nd midzolm AUC nd )) were within 3% of previously published outcomes. h Due to the sprse nture of the previously published dt, modified Biler method (vilble in Phoenix WinNonlin) ws used to recover, AUC 8, nd Cl/F; ccurcy ws not clculted bsed on the sprse dt nd the 3% criterion being pplicble only to the victim drugs. Midzolm (nmol/l) 2 4 Time (hour) Figure Men concentrtion time profile ( 6 hours) of midzolm in 9 helthy volunteers following n 8 mg orl midzolm dose given lone (open symbols) or following 4-dy tretment with milk thistle product (solid symbols). 8 Lines denote physiologiclly bsed phrmcokinetic model simultions of the midzolm concentrtion time profile when given lone (blck) or with milk thistle (green). The dotted green line denotes incorportion of reversible inhibition of CYP3A, wheres the dshed green line denotes incorportion of mechnism-bsed inhibition of CYP3A. Symbols nd error brs denote observed mens nd SDs, respectively, nd were obtined from ref. 8. Prediction of silibinin drug interction mgnitude. Simultions of previously reported milk thistle-midzolm interction, ssuming reversible CYP3A inhibition solely due to silybin A nd silybin B, demonstrted negligible chnges in the midzolm concentrtion time profile (Figure ). The milk thistle product tested, silymrin, contined mg of silybin A nd 8 mg of silybin B nd ws dministered dily for 4 dys. 8 Simultions ssuming mechnism-bsed CYP3A inhibition predicted 3% nd 6% increse in midzolm nd AUC, respectively; increses of 6% nd 3% in midzolm nd AUC, respectively, were reported 8 (Figure ). Simultions of the silibinin wrfrin interction with higher dose of silibinin (,65 mg/dy, or 72 mg silybin A plus 93 mg silybin B/dy; see below), ssuming reversible CYP2C9 inhibition only, predicted negligible chnges (<5%) in ll phrmcokinetic outcomes (Figure 2; Tble 2). Simultions of the high-dose silibinin midzolm interction ssuming reversible CYP3A inhibition predicted no chnge in midzolm nd 5% increse in both nd AUC (Figure 2b; Tble 2). Simultions ssuming mechnismbsed CYP3A inhibition predicted 2-, 5-, nd.5-fold increse in, AUC, nd, respectively (Tble 2). Proof-of-concept clinicl evlution Silibinin content in test product. A single lot (#349) of Siliphos cpsules, lbeled to contin 6 mg silibinin, ws selected. The cpsules were overfilled consistently, contining 69. ± 4.28 mg silibinin represented s 3.3 ±.88 mg silybin A nd 38.9 ± 2.39 mg silybin B. The cpsules lso contined minor mounts of the regioisomers isosilybin A (.55 ±.9 mg) nd isosilybin B (.94 ±.6 mg). Study subjects. All enrolled subjects completed the study (Supplementry Tble S2). The study drugs nd silibinin generlly were well tolerted. One subject experienced mild 6 CPT: Phrmcometrics & Systems Phrmcology

3 PBPK Modeling Frmework for Quntittive Prediction of n Herb Drug Interction 3 b Concentrtion (nmol/l) c Concentrtion (µmol/l) Concentrtion (µmol/l) Time (hour) Time (hour) (R)-Wrfrin (control) (S)-Wrfrin (control) (R)-Wrfrin (tretment) (S)-Wrfrin (tretment) Midzolm (control) Midzolm (tretment) Silybin A Silybin B Time (hour) Figure 2 Geometric men concentrtion time profile of () wrfrin, (b) midzolm, nd (c) silibinin in 2 helthy volunteers following mg orl dose of wrfrin or 5 mg orl dose of midzolm given lone (open symbols) or following 7-dy tretment with silibinin (solid symbols). Lines in nd b denote physiologiclly bsed phrmcokinetic (PBPK) model simultions of the concentrtion time profiles when the probe substrtes were given lone (blck) or with silibinin (green). Blue nd ornge lines in c denote PBPK model simultions of the concentrtion time profiles of silybin A nd silybin B, respectively. Symbols nd error brs denote observed geometric mens nd upper limits of the 9% confidence intervl, respectively. gstrointestinl upset following the first dose of silibinin. The effect ws deemed likely to be drug relted by the study physicin but ws trnsient nd did not limit the subject s continued prticiption. During Clinicl nd Trnsltionl Reserch Center (CTRC) visits, four subjects (two in both phses) reported mild hedches ttributed to cffeine withdrwl. No interntionl normlized rtio elevtions from bseline were observed following wrfrin dministrtion. CYP2C9 genotyping. All enrolled subjects consented to genotyping. Ten subjects were homozygous for the reference CYP2C9* llele. Two subjects crried one copy of the reference llele nd either the CYP2C9*2 or CYP2C9*3 llele. Effects of high-dose silibinin on wrfrin nd midzolm phrmcokinetics. The effects of high-dose silibinin (,65 mg/dy) were compred to bseline orl phrmcokinetics of wrfrin nd midzolm. Due to the reported mechnism-bsed inhibition of CYP3A in vitro, 4,6 silibinin ws dministered three times dily for 6 dys prior to dministrtion of the probe substrtes. Silibinin constituents were not expected to ccumulte during the dministrtion period due to short reported hlf-lives (<4 hours). 22 One subject demonstrted poor goodness-of-fit sttistics for the of both wrfrin enntiomers in both phses (R 2 <.85). Accordingly, dt from this subject were excluded from nlysis of AUC 48 nd. Wrfrin enntiomers were bsorbed rpidly during both study phses, with medin occurring t.25 nd.5 hours for (R)- nd (S)-wrfrin, respectively (Figure 2). Codministrtion with silibinin did not lter medin (S)- wrfrin but delyed medin (R)-wrfrin by 5 minutes. Reltive to control (bseline), silibinin decresed (R)-wrfrin geometric men by 7% (Figure 3; Tble 2) nd AUC 48 by 5% (Figure 3b; Tble 2). Silibinin decresed geometric men (S)-wrfrin by 2% (Figure 3c; Tble 2). Geometric men AUC 48 of (S)-wrfrin incresed by 3% (Figure 3d; Tble 2), with three subjects lying outside the predefined no effect rnge (.75.33). The 9% confidence intervls for the (S)-wrfrin primry endpoints ( nd AUC) ly within the predefined no effect rnge (Tble 2). The rpid bsorption of midzolm ws unltered by codministrtion with silibinin, with medin occurring t.5 hours (Figure 2b). Reltive to control, silibinin incresed midzolm geometric men by 2% (Figure 3e; Tble 2) nd AUC inf by 9% (Figure 3f; Tble 2). Except for one subject (2.3-fold increse), tretment/control rtios of AUC inf ly within the predefined no effect rnge (Figure 3f). The 9% confidence intervl for midzolm tretment/control rtio of extended bove, wheres tht of AUC inf ly within, the predefined no effect rnge (Tble 2). The smpling strtegy ws not optimized for recovery of silybin A nd silybin B phrmcokinetic outcomes; s such, these outcomes were interpreted for qulittive rther thn quntittive purposes. The medin of silybin A nd silybin B following the initil dministrtion of silibinin (3 nd 3.5 hours, respectively) nerly coincided with the second dministrtion of silibinin (Figure 2c). Geometric men for silybin A ws more thn double tht for silybin B (Tble 2). Geometric men of both silybin A nd silybin B ws ~5 hours (Tble 2). DISCUSSION Although herbl product usge continues to increse, current regultory guidelines in severl Western countries do not request premrket evlution of herb drug interction libility. Investigtions into such libilities re frught with inconsistent results due to the lck of stndrd system for evlution, high compositionl vrition between herbl products, nd uncertinty bout custive constituents. Unlike conventionl drug products, the reltive composition of herbl products my vry substntilly depending on wether conditions, product collection nd storge

4 4 PBPK Modeling Frmework for Quntittive Prediction of n Herb Drug Interction Tble 2 Comprison of proof-of-concept clinicl study outcomes to physiologiclly bsed phrmcokinetic model predictions Outcome Observed Predicted (reversible inhibition) Predicted (mechnism-bsed inhibition) Geometric men (CV %) Geometric men Geometric men / / control rtio (9% CI) control rtio / control rtio (R)-Wrfrin (µmol/l).92 (3).6 (29).83 (.77.9) AUC 48 (µmol/l hour) 55. (24) 47. (23).85 (.8.9) (hour) 52. (28) 6.2 (27).4 (.96.36) (S)-Wrfrin (µmol/l) 2. (32).97 (27).98 (.9.5) b AUC 48 (µmol/l hour) 37.4 (4) 42.3 (34).3 (..26) b (hour) 29.6 (25) 3.3 (2).97 (.84.2) Midzolm (nmol/l) 74.2 (43) 89.5 (39).2 (.96.5) b AUC inf (nmol/l hour) 98 (42) 26 (36).9 (.93.25) b ,7 5.5 (hour) 5.7 (36) 4.9 (48).95 (.82.) Silybin A (µmol/l).97 (9).76 (hour) 5. (34).4 Silybin B (µmol/l).4 ().43 (hour) 5. (56).4 AUC 48, re under the concentrtion time curve from to 48 hours; AUC inf, AUC from to infinite time;, mximl concentrtion;, terminl elimintion hlf-life. Evluble for subjects; ll other outcomes were evluble for 2 subjects. b The predefined no effect rnge ws for the primry endpoints (S)-wrfrin nd midzolm tretment/control rtio for nd AUC). methods, nd processing procedures. 4 Accurte predictions of herb drug interction libility require not only identifiction nd quntifiction of custive constituents, but lso mesures of exposure in orgns with metbolic cpbility. Silibinin ws selected s n exemplr herbl product due to well-chrcterized composition, vilbility of inhibitory kinetic prmeters from individul constituents, nd disprte impct of milk thistle products on victim drug phrmcokinetics in previous clinicl studies. 7 9 A PBPK modeling nd simultion pproch ws used to ddress the chllenges inherent to investigtion of herb drug interction libility. Wrfrin is widely used orl nticogulnt with nrrow therpeutic window. Wrfrin is ssocited with notoriously complicted phrmcotherpy due in prt to myrid drugs nd herbl products tht lter the metbolism or nticogulnt ctivity of wrfrin. As the clernce of the more phrmcologiclly ctive (S)-enntiomer is medited primrily by CYP2C9, inhibition of this enzyme cn led to incresed risk of bleeding. Silymrin ws shown previously to increse systemic exposure to the CYP2C9/3A substrte losrtn, 9 prompting evlution of the interction potentil between milk thistle nd wrfrin. Of the milk thistle constituents whose CYP2C9 interction libility hs been evluted in vitro, silybin A nd silybin B were the most potent. 5 These observtions led to the selection of silibinin, which consists primrily of these two constituents, for clinicl evlution. Reltive to control, silibinin unexpectedly decresed both the geometric men nd AUC lst of (R)-wrfrin. Clinicl mnifesttion of the previously reported CYPA2 induction by milk thistle extrct 23 is consistent with this decrese in exposure. In contrst to the doubling of losrtn exposure following dministrtion of silymrin, highdose silibinin did not increse geometric men (S)-wrfrin exposure to cliniclly relevnt extent. However, increses bove 33% were observed in three subjects, indicting tht the CYP2C9 interction potentil of silibinin cnnot be disregrded completely. Consistent with the expected decrese in (S)-wrfrin clernce, the subject crrying the reduced function CYP2C9*3 llele demonstrted prolonged wrfrin exposure, which ws not cptured in the 48-hour smpling window. As such, the AUC nd for this subject were excluded from nlysis. Modeling nd simultion of the silibinin wrfrin interction demonstrted tht the rpid clernce of the silibinin constituents precluded mrked inhibition of wrfrin clernce. Sensitivity nlysis of this interction potentil demonstrted tht -fold increses in silybin A or silybin B inhibition potency (reversible K i ) would led to roughly 5% increses in (S)-wrfrin AUC (Supplementry Figure S). Extensive intestinl nd heptic conjugtion of silybin A nd silybin B followed by rpid elimintion likely would limit the interction potentil to first-pss clernce of sensitive substrtes. Wrfrin is not sensitive to first-pss elimintion nd CPT: Phrmcometrics & Systems Phrmcology

5 PBPK Modeling Frmework for Quntittive Prediction of n Herb Drug Interction 5 is clered only upon subsequent pssges through the liver, t which time ny reversible inhibition of CYP2C9 by silybin A nd silybin B would be bted. In contrst, losrtn hs low biovilbility (33%) tht is ttributed, in prt, to first-pss elimintion. 24 This observtion, coupled with the differences in study popultion nd herbl product tested, could explin the difference between the reported interction with losrtn 9 nd the lck of interction with wrfrin in the present study. Collectively, these observtions suggest exmintion of other CYP2C9 substrtes sensitive to first-pss elimintion, such s fluvsttin, to understnd fully the milk thistle- CYP2C9 interction potentil. Midzolm is gold stndrd CYP3A probe substrte metbolized extensively by intestinl nd heptic enzymes. Inhibition of CYP3A t either site cn increse systemic exposure to midzolm; inhibition of heptic CYP3A lso cn increse. Milk thistle constituents, including silybin A nd silybin B, hve been shown to be reversible nd mechnism-bsed inhibitors of CYP3A ctivity in both humn liver microsomes nd expressed enzyme systems. 3,4,6 Previous clinicl interction studies with midzolm 7,8 hve demonstrted limited interction libility with the milk thistle product silymrin, lbeit the doses dministered were not sufficient to determine the difference between reversible nd mechnism-bsed inhibition of CYP3A (Figure ). The c (µmol/l) (µmol/l) e (µmol/l) Figure 3 Effects of silibinin (,65 mg/dy for 7 dys) on (,c,e) nd (b,d,f) AUC of (,b) (R)-wrfrin, (c,d) (S)-wrfrin, nd (e,f ) midzolm in 2 helthy volunteers following orl dministrtion of wrfrin ( mg) nd midzolm (5 mg). Open symbols connected by solid lines denote individul vlues. Solid symbols connected by dshed lines denote geometric mens. b AUC 48 (µmol/l h) d AUC 48 (µmol/l h) f AUC inf (nmol/l h) suprtherpeutic silibinin dose in the current study ws selected to provide lrge rnge between the predicted interction bsed on reversible nd mechnism-bsed inhibition of CYP3A nd to mximize the bility to observe clinicl interction. The lck of n interction observed in ll but one subject indicted tht the CYP3A interction libility for silibinin is low nd is more consistent with reversible thn mechnism-bsed inhibition (ssuming inhibition indeed occurred). The current work represents nother exmple of potentil mechnism-bsed inhibitor identified in vitro 6 tht does not mnifest cliniclly. Modeling nd simultion of the silibinin midzolm interction indicted tht the low interction potentil is due, in prt, to the lower inhibition potency of the silibinin constituents towrd CYP3A compred to CYP2C9 (Tble 3). Ten-fold increses in inhibition potency of silybin A nd silybin B towrd CYP3A ctivity incresed midzolm exposure by roughly 25% (Supplementry Figure S nd Supplementry Mterils nd Methods). These observtions indicted tht t the predicted exposures, the constituents would need to be -fold more potent to demonstrte Tble 3 Physiologiclly bsed phrmcokinetic model input prmeters Prmeter (R)- Wrfrin Victim compound Perpetrtor compound (S)- Silybin Silybin Wrfrin Midzolm A B Physicochemicl/binding Moleculr weight Frction bsorbed b.77 b k (hour ) 3. c 3. c.7 d.5 b.5 b Blood/plsm rtio. e. e.8 f.58 b.58 b Unbound frction in plsm.6 g.6 g.2 f.4 h.4 h Metbolism Intestinl K m (µmol/l) 3.7 i 22 c 8.5 c Intestinl V mx (µmol/, i 2,7 c 2,6 c hour) Heptic K m (µmol/l) 6.5 e 6. i 54 c 57 c Heptic V mx (µmol/hour) 26 c 8, i 2,3 c 2,7 c Heptic Cl int (l/hour) 3.4 c Inhibition CYP2C9 K i (µmol/l) 6.5 j k 4.8 k CYP2C9 α 5 k 8 k CYP3A4 K i (µmol/l) 26.5 l 3.5 l CYP3A4 k inct (minute ).22 l.5 l CYP3A4 K I (µmol/l) l 89 l See Methods for detiled informtion on model prmeteriztion. Cl int, intrinsic clernce; k, bsorption rte constnt; K i, reversible inhibition constnt; α, ffinity chnge of the enzyme substrte nd enzyme inhibitor complexes; K I, concentrtion required to chieve hlf-mximl rte of enzyme inctivtion (k inct ). Assumed. b Predicted bsed on physicochemicl properties using ADMET Predictor (Simultions Plus). c Obtined by fitting the model to clinicl dt (ref. 26). d Ref. 35. e Ref. 34. f Ref. 3. g Ref. 33. h Ref. 4. i Extrpolted from in vitro dt. j Ref. 34. k Ref. 5. l Obtined from recombinnt dt in ref. 24.

6 6 PBPK Modeling Frmework for Quntittive Prediction of n Herb Drug Interction ny cliniclly relevnt interction with CYP3A. The lrge predicted increse in midzolm exposure incorporting mechnism-bsed inhibition further supported the hypothesis tht products with limited systemic exposure (first posited with fruit juices) 25 need to be mechnism-bsed inhibitors of CYP enzymes to perpetrte cliniclly relevnt interctions. One limittion to the current work is tht silybin A nd silybin B clernce prmeters were recovered by fitting the model to dt obtined from heptitis C ptients dministered product (silymrin) tht contined dditionl constituents not present in silibinin. 26 In vitro determintion of silibinin clernce prmeters would provide true bottomup modeling pproch nd reduce complexities inherent to phrmcokinetic dt from ptients with heptic disese. Alterntively, disese-relted prmeters could be used to develop heptitis C virtul popultion before fitting the PBPK model with the observed phrmcokinetic dt, fcilitting recovery of disese-independent silibinin clernce prmeters In summry, prospective evlution of herb drug interctions, consistent with tht for drug drug interctions, lrgely hs been ignored due to substntil compositionl vribility inherent to herbl products, multiple inhibitory constituents, vrying inhibition mechnisms, nd reltive lck of regultory oversight. The PBPK interction model developed in the current work incorported in vitro inhibition kinetic prmeters nd systemic exposure estimtes of individul constituents for the exemplr herbl product, silibinin. Simultions of the silibinin wrfrin nd silibinin midzolm interctions ccurtely predicted miniml clinicl interction libility. This work demonstrted the utility nd predictive power of PBPK modeling nd simultion, which could be extended to investigte scenrios (e.g., wide dosing rnges, tissue exposure ssessment, nd herbl product composition vrition) nd ptient popultions (e.g., peditric, geritric, nd pregnnt women) not menble to clinicl investigtion. Refinement of the PBPK model by recovering disese-independent silibinin clernce prmeters nd incorporting lternte victim drugs, including losrtn, will enhnce confidence in model predictions nd generlizbility. This frmework represents n initil step to estblishing systemtic pproch tht cn be pplied to other combintions of herbl products nd conventionl drugs under vrious clinicl scenrios to identify potentil cliniclly significnt herb drug interctions, predict the extent of those interctions, nd ultimtely help guide phrmcotherpeutic decisions. METHODS PBPK model development. The bse model structure ws dpted from the literture 3 (Figure 4), incorporting physiologic prmeters obtined from the Interntionl Commission on Rdiologicl Protection. 3 Wrfrin prtition coefficients (K p s) 32 nd binding prmeters 33 were obtined from the literture (Tble 3); bsorption rte constnts (k s) nd clernce prmeters were obtined by fitting the PBPK model to previously reported plsm concentrtion time Arteril Orl dose Stomch PSP Intestine CI Lungs Brin Hert Skin Adipose Kidneys Muscle Reminder Liver Figure 4 Bse physiologiclly bsed phrmcokinetic model structure. Model structure ws modified from the literture. 3 Orgn weights nd blood flows were obtined from the Interntionl Commission on Rdiologicl Protection. 3 Following orl dministrtion, drug trnsfer from dosing comprtment to intestine is driven by the orl bsorption rte constnt (k ). Drug clernce (Cl) is medited by metbolic processes in the intestine nd liver. The pncres nd spleen were combined into hybrid orgn designted s PSP. profiles. 2 The reversible inhibition constnt (K i ) of (R)- wrfrin towrd CYP2C9 ctivity ws obtined from the literture. 34 Midzolm K p s nd k were obtined from the literture 3,35 ; intestinl nd heptic clernce prmeters were extrpolted from in vitro dt 6 s described 36,37 (Tble 3). Silybin A nd silybin B K p s were predicted from physicochemicl properties 38 using GstroPlus (version 8.; Simultions Plus, Lncster, CA). Silibinin binding prmeters were obtined from the literture 39 ; clernce prmeters were generted by fitting the PBPK model to plsm concentrtion time dt from heptitis C ptients receiving silymrin 26 (Tble 3). Silybin A nd silybin B mechnismbsed (K I, k inct ) nd reversible inhibition kinetic prmeters were obtined from the literture. 5,6 Mechnism-bsed inhibition of CYP2C9 ws not considered bsed on previous publiction showing no IC 5 shift using (S)-wrfrin s the probe substrte. 5 PBPK interction model simultions. PBPK models were developed for midzolm, (R)-wrfrin, (S)-wrfrin, silybin A, nd silybin B using Berkeley Mdonn (version 8.3; University of Cliforni t Berkeley, Berkeley, CA) with code compiled in MEGen 4 (version.5; UK Helth & Sfety Lbortory, Buxton, UK) (Supplementry Mterils nd Methods). The PBPK model for perpetrtor (silybin A nd silybin B) nd victim (wrfrin or midzolm) compounds were linked through the reversible or mechnism-bsed CI Venous CPT: Phrmcometrics & Systems Phrmcology

7 PBPK Modeling Frmework for Quntittive Prediction of n Herb Drug Interction 7 inhibition of victim probe substrte. Initil simultions used doses of probe substrtes nd milk thistle products reported in previous studies. Simultions were considered ccurte if the predicted primry phrmcokinetic outcomes (AUC nd for (S)-wrfrin nd midzolm) were within 3% of observed outcomes. Following initil model evlution, simultions were conducted with higher dose of silibinin (,65 mg/dy) to determine whether cliniclly importnt interction is possible. Phrmcokinetic outcomes from the simulted profiles were recovered vi noncomprtmentl nlysis using Phoenix WinNonlin (version 6.3; Phrsight, Cry, NC). Anlysis of silibinin product. Siliphos cpsules (n = 28) (Thorne Reserch, Dover, ID) were nlyzed using modifiction of previously described methods 4,42 to ensure purity nd content. Briefly, the contents of ech cpsule were weighed nd extrcted twice with 2 ml cetone. The extrct ws vortex mixed nd centrifuged (3,g for 2 minutes); the superntnt ws trnsferred to clen vil. Milk thistle constituents were quntified using n Acquity UPLC system with n HSS-T3.8 µm (2. mm) Acquity column nd Empower 3 softwre (Wters, Milford, MA). Stndrds nd Siliphos cpsule extrcts were nlyzed using grdient from 3:7 to 55:45 methnol:wter (.% formic cid) over 5. minutes t flow rte of.6 ml/minute t 5 C; peks were detected t 288 nm. Proof-of-concept clinicl study. Helthy volunteers (six men nd six nonpregnnt women) were enrolled in n openlbel, fixed sequence crossover study conducted t the UNC CTRC. The study protocol ws pproved by the UNC Office of Humn Reserch Ethics Biomedicl Institutionl Review Bord nd the CTRC Advisory Committee. Eligibility to prticipte ws bsed on screening evlution nd inclusion/exclusion criteri (Supplementry Tble S). Written informed consent ws obtined from ech subject prior to enrollment. The first (control) phse consisted of dministrtion of mg wrfrin (Coumdin; Bristol Meyers Squibb, Princeton, NJ), mg vitmin K (Mephyton; Aton Phrm, Lwrenceville, NJ), nd 5 mg midzolm syrup (Rnbxy; Jcksonville, FL). A negtive pregnncy result ws required before drug dministrtion to women of childbering potentil. Vitl signs (blood pressure, temperture, respirtory rte, pulse, nd oxygen sturtion) were obtined t bseline nd every 5 minutes for the first 2 hours. All subjects underwent n interntionl normlized rtio with prothrombin time. Blood (7 ml) ws collected through n intrvenous line before nd from.25 2 hours following drug dministrtion. Subjects continued to fst until fter the 4-hour blood collection, when mels nd sncks, devoid of fruit juices nd cffeinted beverges, were provided. Subjects returned to the CTRC 24 nd 48 hours post-drug dministrtion for blood collection. Optiml study design simultions 43 of previously reported clinicl dt 2 demonstrted tht 48-hour collection ws n ccurte surrogte of totl systemic exposure (AUC inf ) for wrfrin. Plsm ws collected nd stored t 8 C pending nlysis by high-performnce liquid chromtogrphy tndem mss spectrometry (HPLC/ MS-MS). Following t lest 4-dy wshout, subjects received 48 mg silibinin (bsed on lbeled content) to self-dminister three times dily for 7 dys. Ech subject received his/ her silibinin in blister pck nd ws sked to complete pill diry documenting the time of dministrtion. Subjects were contcted t lest twice during the week of silibinin selfdministrtion to monitor complince nd dverse events, which were grded using vlidted Adverse Events Scle. Subjects returned to the CTRC on dy 7 for concomitnt dministrtion of silibinin, wrfrin, vitmin K, nd midzolm. Plsm ws collected nd stored s described for the first phse. Anlysis of plsm for wrfrin enntiomers, midzolm, silybin A, nd silybin B. Concentrtions of ll nlytes were quntified using Sciex (Frminghm, MA) API4 Qtrp HPLC-MS/MS triple qudrupole mss spectrometer fitted with Turbo ionspry interfce operted in the positive ion mode. Plsm ws treted with cetonitrile (6 volumes) contining the internl stndrd, wrfrin-d 5 (Toronto Reserch Chemicls; Toronto, Cnd) or -hydroxymidzolm-d 4 (Cerillint, Round Rock, TX), nd centrifuged (3,g). The superntnt ws injected into the HPLC-MS/ MS system. Wrfrin enntiomers were seprted on Supelco Astec Chirobiotic V 5 cm 2. mm 5 micron chirl column (Sigm Aldrich; St Louis, MO) nd eluted with n isocrtic mixture consisting of 75% 5 mmol/l mmonium cette contining.% (v/v) formic cid nd 25% cetonitrile (flow rte,.4 ml/minute). Midzolm ws eluted with binry grdient mixture consisting of mmol/l mmonium formte contining % (v/v) isopropyl lcohol nd.% (v/v) formic cid nd methnol on Vrin Polris C8-A 2 cm 2. mm 5 micron column (Agilent, Snt Clr, CA) (flow rte,.65 ml/minute). Silybin A nd silybin B were eluted with n isocrtic mixture consisting of 44% wter, 56% methnol, nd.% (v/v) formic cid on n Agilent Zorbx XDB C8 5 cm 3. mm 3.5 micron column (Agilent) (flow rte,.7 ml/minute). Anlyte concentrtions were determined by interpoltion from liner stndrd curve with n ssy dynmic rnge of.5, nmol/l (wrfrin enntiomers) or.5 5, nmol/l (midzolm, silybin A, silybin B). Anlyticl methods were vlidted ccording to US Food nd Drug Administrtion guidelines. 44 Inter- nd intrdy vribility for ll nlytes ws less thn %. Phrmcokinetic nlysis. Phrmcokinetic outcomes were recovered by noncomprtmentl nlysis using Phoenix WinNonlin. Concentrtions below the limit of quntifiction were excluded. The terminl elimintion rte constnt (λ z ) ws estimted by liner regression of the terminl portion of the log-trnsformed concentrtion time profile using t lest three dt points. The terminl hlf-life ( ) ws clculted s ln2/λ z. The mximum observed concentrtion ( ), time to rech ( ), nd lst mesured concentrtion (C lst ) were obtined directly from the concentrtion time profile. AUC from time zero to C lst (AUC lst ) ws determined using the

8 8 PBPK Modeling Frmework for Quntittive Prediction of n Herb Drug Interction trpezoidl method with liner up/log down interpoltion. The AUC from time zero to infinity (AUC inf ) ws clculted s the sum of AUC lst nd the rtio of C lst to λ z. Genotyping for common CYP2C9 vrints. CYP2C9*2 nd *3 polymorphisms were determined using previously published polymerse chin rection restriction frgment length polymorphism ssy. 45 Sttisticl nlysis. All sttisticl nlyses were conducted using SAS (version 9.2; SAS Institute, Cry, NC). The smple size for the proof-of-concept study (n = 2 evluble subjects) ws clculted bsed on 8% power to detect 25% chnge in the primry endpoints with type I error of.5; the primry endpoints were the tretment/control rtios of logtrnsformed AUC 48 ((S)-wrfrin) or AUC inf (midzolm) nd ((S)-wrfrin nd midzolm), nd the predefined no effect rnge ws ,6 Intrindividul vribility in midzolm nd wrfrin AUC nd were ssumed to be ~2% Secondry outcomes, nd, were evluted using pired two-tiled Student s t-test on log-trnsformed dt or Wilcoxon signed-rnk test s pproprite, with 9% confidence intervls nd rnges reported for nd, respectively. A P vlue <.5 ws considered sttisticlly significnt. Acknowledgments. The uthors thnk Dr. Vness González-Pérez nd the CTRC stff for their ssistnce in the conduct of the clinicl study; Grrett Ainslie for his ssistnce with the physiologiclly bsed phrmcokinetic modeling; Dr. Crig Lee nd Kimberly Vendrov for their ssistnce with the CYP2C9 genotyping; nd Drs. Dn Jons, Kren Weck, nd Ky Cho for providing the CY- P2C9 positive controls. This work ws supported by the Ntionl Institutes of Helth through wrd number RGM77482 from the Ntionl Institute of Generl Medicl Sciences nd wrd number ULTR83 from the Ntionl Center for Advncing Trnsltionl Sciences. The content is solely the responsibility of the uthors nd does not necessrily represent the officil views of the Ntionl Institutes of Helth. R.D. nd D.J.F. were supported by Clinicl Phrmcokinetics/Phrmcodynmics fellowship sponsored by Quintiles nd GlxoSmithKline, respectively. Phoenix WinNonlin softwre ws generously provided to the Division of Phrmcotherpy nd Experimentl Therpeutics, UNC Eshelmn School of Phrmcy, by Certr s member of the Phrsight Acdemic Center of Excellence Progrm. M.F.P. dedictes this rticle to Dr. Dvid P Pine. Author contributions. S.J.B., B.T.G., N.H.O., nd M.F.P. wrote the mnuscript. S.J.B., R.D., D.J.F., Y.V.S., nd M.F.P. designed the reserch. S.J.B., B.T.G., R.D., D.J.F., T.N.G., nd Y.V.S. performed the reserch. S.J.B., B.T.G., D.J.F., K.S.F., M.B.F., N.H.O., nd M.F.P. nlyzed the dt. T.N.G., K.S.F., M.B.F., nd N.H.O. contributed new regents/ nlyticl tools. Conflict of interest. The uthors declred no conflict of interest. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? 33 Despite incresing recognition of herb drug interctions in clinicl prctice, robust informtion bout the custive ingredients nd mechnisms underlying these interctions remins limited. Consequently, evidence-bsed recommendtions bout dding herbl products to existing phrmcotherpeutic regimens virtully re nonexistent. WHAT QUESTION DID THIS STUDY ADDRESS? 33 This study ddressed the utility of PBPK modeling pproch to predict the drug interction libility of n herbl product. This pproch ws tested using the exemplr herbl product silibinin nd the widely used cytochrome P45 probe substrtes wrfrin nd midzolm. 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Quntifiction of 3-month intrindividul vribility nd the influence of sex nd menstrul cycle phse on CYP3A ctivity s mesured by phenotyping with intrvenous midzolm. Clin. Phrmcol. Ther. 64, (998). 47. Khrsch, E.D., Jubert, C., Senn, T., Bowdle, T.A. & Thummel, K.E. Intrindividul vribility in mle heptic CYP3A4 ctivity ssessed by lfentnil nd midzolm clernce. J. Clin. Phrmcol. 39, (999). 48. Ycobi, A. et l. Who needs individul bioequivlence studies for nrrow therpeutic index drugs? A cse for wrfrin. J. Clin. Phrmcol. 4, (2). CPT: Phrmcometrics & Systems Phrmcology is n open-ccess journl published by Nture Publishing Group. This work is licensed under Cretive Commons Attribution- NonCommercil-NoDerivtive Works 3. License. To view copy of this license, visit Supplementry informtion ccompnies this pper on the CPT: Phrmcometrics & Systems Phrmcology website (