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Clinical T e apeutic /V lume 32, Num e 3, 2010

556 V lume 32 Num e 3

Accepted for publication October 29, 2009.doi:10.1016/j.clinthera.2010.03.0080149-2918/$ - see front matter

2010 Excerpta Medica Inc. All rights reserved.

AbsTrACT Background: Mycophenol te ofetil (MMF), pro-drug of ycophenolic cid (MPA), is n i unosuppres-sive gent indic ted for the prophyl xis of org n rejectionin llogeneic kidney, he rt, or liver tr nspl nt recipients.The Europe n regul tory uthorities require bioequiv -lence studies for the rketing of generic products.

Objective: The i of this study w s to ssess thebioequiv lence of generic (test) nd br nded (refer-ence) for ul tion of MMF 500 g nd MPA.

Methods: This single-center, single-dose, r ndo ized,open-l bel, 4-w y crossover study w s conducted tAn ph r s Clinic l Rese rch F cility, Qubec, Qubec,C n d . He lthy volunteers ged 18 to 55 ye rs wereeligible. Subjects were ssigned to receive, in r ndo -ized order, single dose of the test nd reference for-

ul tions of MMF 500 g under f sting conditions.Bec use the study design w s 4-w y replic te, therewere 2 test periods nd 2 reference periods. The4 study periods were e ch sep r ted by 14-d yw shout period. Blood s ples were collected over period of 12 hours fter d inistr tion for the deter-

in tion of MMF ph r cokinetic properties, ndover 48 ( 0.5) hours, for MPA properties. Con-centr tions of the n lytes were deter ined by reverseLC nd detected using LC-MS/MS. Ph r cokineticp r eters were c lcul ted fro MMF nd MPAconcentr tion d t using nonco p rt ent l n lysis.C x nd AUC 0t were the pri ry ev lu tion criteri ,while AUC 0 w s second ry p r eter. The drugswere to be considered bioequiv lent if the 90% CIsfor the test/reference r tios of n tur l log rith tr nsfor ed v lues of these p r eters (obt ined using

ANOVA) were between 80% nd 125%, per Europe nregul tions for bioequiv lence. Toler bility w s oni-tored using physic l ex in tion, including vit l sign

e sure ents, l bor tory n lysis, nd dverse-events(AE) onitoring (including p tient interview).

Results: A tot l of 103 subjects were enrolled(64 en, 39 wo en; 101 white, 2 bl ck; e n [SD]

ge, 38 [10] ye rs; weight, 68.2 [9.1] kg). The 90%CIs were s follows: MMF, C x , 85.94% to 106.63%;AUC0t, 91.94% to 102.20%; nd AUC 0, 93.15%to 105.48%; MPA, C x , 92.03% to 105.82%;AUC

0t, 97.42% to 100.59%; nd AUC

0, 96.96%

to 100.90%. These v lues et with the regul torydefinition of bioequiv lence. A tot l of 148 AEs werereported (68 in subjects who received the test tre t-

ent nd 80 in subjects who received the referencetre t ent). The ost co only reported AEs wereprocedur l p in (13/102 [12.7%] nd 10/101 [9.9%]with the test nd reference for ul tions, respectively),procedur l site re ction (12 [11.8%] nd 4 [4.0%]),

nd so nolence (7 [6.9%] nd 14 [13.9%]).Conclusions: The generic nd br nded for ul -

tions of MMF 500 g et the Europe n regul torycriteri for ssu ing bioequiv lence, b sed on the r te

nd extent of bsorption of single dose under f stingconditions. Both for ul tions were well toler ted inthese he lthy volunteers. ( Clin Ther. 2010;32:556574) 2010 Excerpt Medic Inc.

Mycophenolate Mofetil 500-mg Tablet Under FastingConditions: Single-Dose, Randomized-Sequence, Open-Label,Four-Way Replicate Crossover, Bioequivalence Study in

Healthy SubjectsSusana Almeida, MSc 1,2 ; Augusto Filipe, MSc 1; Rita Neves, MSc 1;Ana Cristina Franco Spnola, MSc 1; Mario Tanguay 3; Jordi Ortuo 4; Anna Farr, MSc 4;and Alex Torns 4

1Medical Department, Grupo Tecnimede, Sociedade Tecnico-Medicinal S.A., Sintra, Portugal; 2Department of Pharmacology and Therapeutics, Universidad Autnoma de Barcelona, Barcelona, Spain;3 Anapharm,Montreal, Qubec, Canada; and 4 Anapharm Europe S.L., Barcelona, Spain

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terohep tic recircul tion, second ry incre ses in pl s- MPA concentr tions ight be observed t ~4 to

12 hours fter the d inistr tion of MMF. 3 A negli-gible ount of the initi l dose is excreted s MPA inthe urine. 1 The e n pp rent t 1/2 of MPA r nges

between 11 nd 19 hours.2

The Europe n regul torycriteri require testing for bioequiv lence of genericnd br nded for ul tions for the rketing pprov l

of generic for ul tions.The present study i ed to ssess the bioequiv lence

of generic (test) nd br nded (reference) for ul -tion of MMF 500 g nd its ctive et bolite.

sUbJECTs AND METhoDsstudy P t c l

An independent ethics co ittee (Institution l

Review Bo rd Services, Auror , Ont rio, C n d )pproved the clinic l study protocol, nd letter of no objection w s obt ined fro C n di n uthorities.This single-center, single-dose, r ndo ized, open-l bel, 4-w y crossover study w s conducted in ccor-d nce with the Decl r tion of Helsinki 4 nd theGuideline for Good Clinic l Pr ctice, 5 nd writteninfor ed consent w s obt ined fro e ch p rticip ntbefore study co ence ent. The clinic l p rt of thestudy w s conducted t An ph r s Clinic l Rese rchF cility (Qubec, Qubec, C n d ) nd the bio n -lytic p rt t An ph r Europe S.L. f cility (B rcelon ,Sp in).

su jectHe lthy volunteers were recruited fro the co -

unities of Qubec, Montr l, nd Trois-Rivires,C n d . Men nd wo en ged 18 to 55 ye rs with

body ss index (BMI) between 20 nd 27 kg/ 2

were eligible.Volunteers who s oked >9 cig rettes per d y, were

un ble to provide consent, h d illness or surgerywithin 4 weeks before d inistr tion of the study

edic tion; h d bnor l findings on ECG or vit lsign e sure ent, or l bor tory testing for hep titisB or C or HIV t screening; h d history of signifi-c nt use of lcohol or drugs within 1 ye r before thescreening visit; h d history of n llergic re ction tofood or drugs or used drugs known to induce or in-hibit hep tic drug et bolis within 14 d ys before

d inistr tion of the study edic tion; or h d used prescription or over-the-counter edic tion within

7 d ys before d inistr tion of the study edic tion

Key words: AUC, bioequiv lence, C x , ph r co-kinetic, ycophenol te ofetil, ycophenolic cid,MMF, MPA.

INTroDUCTIoNMycophenol te ofetil (MMF) is n i unosuppres-s nt indic ted for the prophyl xis of org n rejectionin llogeneic kidney, he rt, or liver tr nspl nt recipi-ents. The reco ended dos ge r nges between 1 nd1.5 g BID (tot l d ily dose, 23 g). 1 Pl s drug con-centr tions y be onitored s dee ed ppropri tefor the purpose of dose djust ents.

The ctive et bolite of MMF, ycophenolic cid(MPA), is potent, selective, unco petitive nd re-versible inhibitor of inosine onophosph te dehydro-

gen se. MPA inhibits the de novo p thw y of gu no-sine nucleotide synthesis without incorpor tion intoDNA. Bec use T- nd B-ly phocytes re critic lly de-pendent for their prolifer tion on de novo synthesis of purines, where s other cell types c n use s lv ge p th-w ys, MPA h s ore potent cytost tic effects on ly -phocytes th n on other cells. 1

Following or l d inistr tion, MMF is bsorbednd undergoes presyste ic et bolis to the ctiveet bolite, MPA. As evidenced by suppression of cute rejection following ren l tr nspl nt tion, the

i unosuppress nt ctivity of MMF is correl ted withMPA concentr tion. The e n bsolute bio v il bili-ty of or l MMF (expressed in ter s of MPA exposure)is 94%. 1 As per the product infor tion, 1 MMF isnot e sur ble syste ic lly in pl s following or l

d inistr tion. However, the develop ent of oresensitive bio n lytic ss y llows the detection of p r-ent co pound in pl s , even though the concentr -tions re

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Clinical T e apeutic

( croscopic ex in tion, pH, specific gr vity,protein, glucose, ketones, bilirubin, occult blood ndcells, nitrite, urobilinogen, nd leukocytes), nd i-croscopic ex in tion (perfor ed on bnor l find-ings). For seru nd urin ry pregn ncy testing,

G -Dyn c re Medic l L bor tories (Qubec,Qubec, C n d ) w s used s the clinic l l bor toryf cility. The re ining di gnostic tests were conducted

t An ph r Clinic l L bor tory (Qubec, Qubec,C n d ). Both clinic l l bor tories re certified forintern l nd extern l qu lity control by independentorg niz tions (College of A eric n P thologists,Northfield, Illinois; Socit qubcoise de biologieClinique, Qubec, Qubec, C n d ; nd L bor toirede s nt publique, Qubec, Qubec, C n d ).

Se ted blood pressure (BP) nd he rt r te were

e sured before study drug d inistr tion nd ~24nd 48 hours fter d inistr tion in e ch study pe-riod. Vit l sign e sure ents were repe ted t le stonce s soon s possible fter the initi l scheduled

e sure ent under the following conditions: systol-ic BP (SBP) 140 Hg, di stolic BP (DBP)90 Hg, nd/or he rt r te 100 be ts/ in.

study D u Admini t ati nSubjects were enrolled by the qu lified investig tor

or the edic l subinvestig tor. On rriv l t the clini-c l f cility for the first study period, subjects were

ssigned nu bers th t corresponded to r ndo iz -tion code gener ted by An ph r (C n d ) using SASversion 8.2 (SAS Institute Inc., C ry, North C rolin ).The co puter-gener ted sequence ensured equ l dis-tribution of tre t ents t ultiples of 4, the blocksize, fro the list of subjects ssign ents, fter r n-do definition of the st rting v lue. The r ndo iz -tion sche e w s un v il ble to the Bio n lytic l Divi-sion of An ph r Europe S.L. until co pletion of theclinic l nd n lytic ph ses. The sche e w s sep r t-ed into 3 groups by the clinic l st ff to ccount fortechnic l restrictions. The study design w s 4-w yreplic te (2 test nd 2 reference periods). The investi-g tor nd clinic l st ff were blinded to tre t ent s-sign ent until fter subjects qu lified s eligible forthe study. The ph r cist w s unblinded to the r n-do iz tion sche e but h d no role in the conduct of the study.

After supervised overnight f st of 10 hours, t6 am nd 7:10 am, subjects were d inistered, per the

were excluded. Volunteers were lso excluded if theyh d history of g strointestin l bnor lities or un-resolved sy pto s; liver or kidney dise se; neurologic,c rdiov scul r, pul on ry, he tologic, i uno-logic, psychi tric, or endocrine/ et bolic dise se; n

ctiv ted renin- ngiotensin syste ; history or pres-ence of zote i nd/or oliguri , nuri , bili ry ob-struction, gout, or uric c lculi; n untre ted heredit rydeficiency of hypox nthine-gu nine-phosphoribosyltr nsfer se such s Lesch-Nyh n syndro e; or his-tory of l tent or ctive tuberculosis or exposure toende ic re s within 8 weeks before purified proteinderiv tive (PPD) skin testing t screening. A PPDtest result indic ting possible tuberculosis infection(5 ) w s lso c use for exclusion. Volunteerswere excluded if they h d received depot injection or

n i pl nt of ny drug within 3 onths before thed inistr tion of study edic tion, or i uniz tionwith live ttenu ted v ccine 1 onth before dosingor pl nned v ccin tion during the course of the study;h d ny ctive infection; or h d history of or ctive

lign ncy.Subjects were considered eligible for enroll ent in

this study b sed on edic l nd edic tion histories,de ogr phic nd clinic l d t (including sex, ge,r ce, ethnicity, weight, height, nd BMI), vit l sign

e sure ents, 12-le d ECG, physic l ex in tion,urin ry drug screen (E-Z Split Key cup kit [Innov con,Inc., S n Diego, C liforni ]), urin lysis for phet -

ine, b rbitur tes, benzodi zepines, coc ine, ecst sy,riju n , eth done, eth phet ine, opi te, nd

phencyclidine, seru pregn ncy testing (fe le sub-jects), nd clinic l l bor tory testing (he tology,bioche istry, urin lysis, HIV, hep titis C virus ntibod-ies, nd hep titis B surf ce ntigen).

study De i nThe study design w s 4-w y replic te (ie, 2 test

periods nd 2 reference periods). Clinic l l bor toryn lysis w s conducted t the ti e of the screeningnd poststudy procedures. He tology nd urin ry

pregn ncy testing were lso conducted before studyperiods 2, 3, nd 4. L bor tory testing included he-

tology (co plete blood count with differenti l,he oglobin, nd he tocrit), bioche istry ( lbu in,

lk line phosph t se, l nine inotr nsfer se, sp r-t te inotr nsfer se, blood ure nitrogen, c lciu ,chloride, glucose, phosphorus, pot ssiu , cre tinine,sodiu , tot l bilirubin, nd tot l protein), urin lysis

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10 inutes t 4C. Pl s w s sep r ted into 2 liquots,which were fl sh-frozen t 80C nd subsequentlytr nsferred to 80C (65C to 90C) freezer,pending ship ent to the n lytic f cility.

At the end of the study, the frozen pl s liquots

(1 of 2) fro the clinic l f cility, together with n in-ventory list nd sufficient dry ice to int in the li-quots in frozen st te for 72 hours, were sent to thebio n lytic f cility. The second set of liquots w skept in the clinic l f cility nd w s to be sent only onrequest fro the bio n lytic f cility. The pl s li-quots were received t the n lytic f cility in goodcondition nd still frozen.

MMF nd deuter ted n logue of MMF s inter-n l st nd rd (MMF-d4, Toronto Rese rch Che ic ls,Toronto, Ont rio, C n d ) nd MPA nd cyclopro-

p ne n logue of MPA s intern l st nd rd (MPAcycloprop ne n logue, Synfine Rese rch, Rich ondHill, Ont rio, C n d ) were e sured using reverseLC-MS/MS.

MMF w s extr cted fro n liquot of hu nEDTA pl s using solid-ph se extr ction. Pl ss ples were vortex- ixed nd centrifuged t 3000 rp(1900 g ) for 5 inutes t 4C. Aliquots of pl s werecollected into borosilic ted tubes in s ples of 200 Le ch; 200 L of Milli-Q w ter (Millipore, Billeric , M s-s chusetts) cont ining intern l st nd rd nd 800 Lof cetic cid were dded. S ples were ixed for~30 seconds nd extr cted using solid-ph se extr c-tion with MCX o sis pl tes (W ters Corpor tion,Milford, M ss chusetts). The extr cted s ples wereeluted using 400 L of cetonitrile/ oniu hy-droxide 95/5 vol/vol s elution solution nd ev po-r ted co pletely under stre of nitrogen t 60Con TurboV p LV concentr tor (Zy rk Corpor -tion, Hopkinton, M ss chusetts). The residues werereconstituted in 200 L of reconstitution solution( obile ph se) ( oniu for te 1 M [pH 3]/

eth nol [40/60 vol/vol]). MMF nd its intern l st n-d rd were e sured using LC-MS/MS. The sep r tionw s perfor ed on reversed-ph se colu n (Zorb xSB-C18, 4.6 50 , 5- p rticle size; Agilent Tech-nologies, S nt Cl r , C liforni ). The chro togr phicsep r tion w s isocr tic lly perfor ed t roo te -per ture t flow r te of 1 L/ in.

MPA w s extr cted fro n liquot of hu n EDTApl s using solid-ph se extr ction. Pl s s pleswere vortex- ixed nd centrifuged t 3000 rp (1900 g )for 5 inutes t 4C. Aliquots of pl s were col-

r ndo iz tion sche e, single dose of the test*(b tch no. 4210701; expir tion, Nove ber 2009) orreference (b tch no. M1764; expir tion, April 2010)for ul tion of MMF 500 g (1 or l fil -co ted t b-let) with 240 L of w ter. A outh check w s per-

for ed for co pli nce verific tion per the protocol.Subjects f sted subsequently for period of 4 hours.S oking w s prohibited fro 2 hours before to 4 hours

fter d inistr tion nd w s controlled nd docu-ented during e ch study period to ensure th t sub-

jects who were light s okers did not exceed the d ilyqu ntity of cig rettes uthorized by the study proto-col (ie,

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cur cy nd precision r nged fro 90.60% to 95.39%nd 0.95% to 6.49%, respectively. The qu lity controle ns of recovery of MMF were 90.57%, 86.99%,nd 89.14%, nd for intern l st nd rd MMF-d4, thee n recovery w s 87.12%. Both n lyses were con-

ducted using robotic liquid h ndling syste (Multi-PROBE II EX, PerkinEl er Inc., W lth , M ss chu-setts). No signific nt interferences were observed intested trices for MMF nd MMF-d4.

The ethod for the deter in tion of MPA concentr -tions showed good line rity ( r 0.9945) nd llowedthe qu ntific tion of MPA in hu n EDTA pl swithin r nge of 49.99 pg/ L to 19,955.00 ng/ L.The LLOQ w s set t 19.95 ng/ L, with sign l-to-noise r tio of 124, in which ccur cy nd precisionwere 102.72% nd 2.99%, respectively, when n -

lyzed 6 ti es. Between-run ccur cy nd precisionr nged fro 92.64% to 102.35% nd 2.36% to 3.10%,respectively. Within-run ccur cy nd precision were89.24% to 101.14% nd 1.65% to 3.82%, respec-tively. The qu lity control e ns of recovery of MPAwere 81.32%, 84.23%, nd 84.76%, nd for intern lst nd rd (MPA cycloprop ne n logue), the e nrecovery w s 104.46%. Both n lyses were conductedusing the MultiPROBE II EX robotic liquid h ndlingsyste . No signific nt interferences were observedin tested trices for MPA nd MPA cycloprop ne

n logue.

P a mac inetic and bi equivalence Analy eT x , AUC 0, k e, nd t 1/2 were deter ined for MMF

nd MPA for infor tion l purposes. The e n (SD),%CV, r nge, edi n, nd interqu rtile r nge (IQR) of the pl s MMF nd MPA concentr tions were c lcu-l ted for the AUC 0t, AUC 0, C x , residu l re , T x ,eli in tion t 1/2, k e, ti e t which ln-line r k e c lcul -tion beg n, nd ti e of l st qu ntifi ble concentr -tion. Ph r cokinetic c lcul tions were de usingBioequiv version 3.50, propriet ry softw re developed

nd tested for bioequiv lence studies t An ph r ,which perfor s nonco p rt ent l n lyses of ph r-

cokinetic p r eters nd st tistic l n lyses (vi SASrele se 8.02, SAS Institute Inc.) ccording to the FDA, 2

He lth Product nd Food Br nch of He lth C n d , 7

nd guid nce fro the Europe n Agency for theEv lu tion of Medicin l Products. 8,9

Bioequiv lence w s ssessed fro the 90% CIs of thegeo etric r tios of the le st squ res e ns, obt ined us-ing ANOVA of the ln-tr nsfor ed C x nd AUC 0t

lected into borosilic ted tubes in s ples of 50 Le ch. One hundred icroliters of Milli-Q w ter con-t ining intern l st nd rd nd 900 L of buffer solution(phosph te trisodiu , 100 M, pH 12) w s dded.S ples were fin lly ixed for ~10 seconds nd ex-

tr cted using solid-ph se extr ction with MAX o sispl tes (W ters Corpor tion). The extr cted s pleswere eluted using 400 L of cetonitrile/for ic cid(95/5 vol/vol) s elution solution nd then ev por tedco pletely under stre of nitrogen t 60C on TurboV p LV concentr tor (Zy rk Corpor tion).The residues were reconstituted in 400 L of reconsti-tution solution ( oniu for te 1 M / eth nol[32/68 vol/vol], for ic cid 0.15%). Concentr tionsof MMA nd its intern l st nd rd (MMA cyclopro-p ne n logue) were e sured using LC-MS/MS. The

sep r tion w s perfor ed on reversed-ph se col-u n (Zorb x SB-C18, intern l di eter, 4.6 50 ;5- p rticle size; Agilent Technologies). The o-bile ph se w s oniu for te 1 M/ eth nol(32/68 vol/vol), for ic cid 0.15%. The chro togr phicsep r tion w s isocr tic lly perfor ed t roo te -per ture t flow r te of 1 L/ in.

For both co pounds (MMF nd MPA), the odu-l r liquid chro togr phic syste w s de up of nHTC-PAL utos pler (CTC An lytics AG, Zwingen,Switzerl nd), high-pressure bin ry pu p 1200 series(Agilent Technologies), nd n API4000 spectro eter(MDS Sciex, Concord, Ont rio, C n d ).

The s ple n lysts were blinded to the r ndo i-z tion sche e. Bio n lysis w s perfor ed under theGuideline for Good L bor tory Pr ctice 6 nd the bio-

n lytic l process v lid ted ccording to US Food ndDrug Ad inistr tion (FDA) Guid nce for IndustryBio n lytic l Method V lid tion, 2 therefore t king intoconsider tion the study of line rity, precision, nd c-cur cy intr - ss y nd inter ss y, selectivity, lowerli it of qu ntit tion (LLOQ), recovery, trix effect,ionic suppression, dilution integrity, nd st bility un-der different conditions.

The ethod for MMF deter in tion showed goodline rity ( r 0.9975) nd llowed the qu ntific tionof MMF in hu n EDTA pl s within the r ngeof 20.02 to 4004.00 pg/ L. The LLOQ w s set t19.90 pg/ L, with sign l-to-noise r tio of 35, whereprecision nd ccur cy were 102.86% nd 4.77%,respectively, when n lyzed 6 ti es. The between-run

ccur cy nd precision r nged fro 95.14% to 96.09%nd 5.14% to 6.60%, respectively. The within-run c-

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After the first or l d inistr tion, the e n (SD)of MPA C x v lues were 14,965.96 (6407.22) nd15,884.98 (6380.70) ng/ L for the test nd referencefor ul tions, respectively. The edi n (IQR) T xv lues were 0.667 (0.333) nd 0.500 (0.333) hours.Me n AUC 0t nd AUC 0 v lues were 29,712.19(8301.24) nd 33,111.54 (10,317.02) ng/ L/h withthe test for ul tion nd 29,888.52 (7982.68) nd33,187.01 (9945.36) ng/ L/h with the reference for-

ul tion ( Table III ).After the second or l d inistr tion of MMF

500 g, e n (SD) MPA C x v lues were 14,549.20(6101.50) nd 14,683.17 (6671.67) ng/ L with the

2376.53 (1711.93) pg/ L/h with the test for ul tionnd 2225.99 (1552.87) nd 2375.66 (1756.72) pg/ L/h

with the reference for ul tion were tt ined ( Table II ).After the second d inistr tion of or l MMF

500 g, e n (SD) C x v lues of 3151.25 (3846.64)nd 3037.23 (3104.52) pg/ L with the test nd refer-

ence for ul tions, respectively, were tt ined t e-di n (IQR) T x v lues of 0.500 (0.333) nd 0.667(0.333) hours. Me n AUC 0t nd AUC 0 v lues were2405.59 (1825.76) nd 2462.04 (1547.46) pg/ L/hwith the test for ul tion nd 2394.89 (1674.35) nd2691.21 (2156.06) pg/ L/h with the reference for u-l tion (Table II ).

Discontinuedtreatment (n = 2)

Catheter could not beinserted (n = 1)

Infection (n = 1)


Catheter could not be

inserted (n = 1)


Loss to follow-up(n = 2)

AE (n = 1)Personal reasons (n = 1)

Received referenceformulation

(n = 52)

Received testformulation

(n = 51)


(n = 49)

Received testformulation and

completed the study

(n = 47)


Personal reasons(n = 1)


Cough (n = 1)

Personal reasons(n = 1)


AE (n = 1)Loss to follow-up

(n = 1)

P e r

i o d 2

P e r

i o d 1

P e r

i o d 3

P e r

i o d 4

Assessed for eligibility (N = 194)

Excluded (n = 91)Did not meet inclusion

criteria (n = 44)Refused to participate

(n = 2)Other reasons (n = 45)

Randomized(n = 103)


(n = 51)


(n = 49)


(n = 48)

Received referenceformulation and

completed the study

(n = 44)

E n r o

l l m e n

t

Figure 1. Disposition of the subjects in this bioavailability study of a 500-mg dose of a generic formulation(test; manufactured by Grupo Tecnimede, Sintra, Portugal) and a branded formulation (reference;trademark: CellCept , Roche Registration Ltd., Welwyn Garden City, United Kingdom) of mycophe-nolate mofetil. AE = adverse event.

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both the MMF nd MPA profiles. In nother subject, l g ti e in bsorption w s found fter the first d-inistr tion of the reference for ul tion nd the

second d inistr tion of the test for ul tion. Second-ry elev tion of MPA pl s concentr tion t ~4 to

12 hours, which h s been ttributed to enterohep ticrecircul tion, 10 w s shown in th t subjects pl sconcentr tionti e curve. A ultiple-pe k profile w sevident in 1 subjects MMF nd MPA curves ( Figure 4 ).

bi equivalenceThe e n r tios nd the 90% CIs of the ln-tr ns-

for ed v lues of C x , AUC 0t , nd AUC 0 re pre-sented in Table IV . For MMF, these v lues were95.73 pg/ L (85.94106.63), 96.93 pg/ L/h (91.94102.20), nd 99.12 pg/ L/h (93.15105.48), respec-

tively. For MPA, these v lues were 98.68 ng/ L(92.03105.82), 98.99 ng/ L/h (97.42100.59), nd98.91 ng/ L/h (96.96100.90). These CIs were withinthe regul tory r nges for bioequiv lence.

T le a ility A tot l of 148 tre t ent-e ergent AEs (TEAEs)

were reported in 66 of the 103 subjects (64.1%) in-cluded in the toler bility popul tion. The bre kdownby tre t ent group is s follows: 68 TEAEs reportedin 43 of 102 subjects (42.2%) who received 1 doseof the test for ul tion, nd 80 TEAEs were reportedin 48 of 101 subjects (47.5%) who received 1 doseof the reference for ul tion ( Table V ).

The ost co only reported AEs were procedur lp in (13/102 [12.7%] nd 10/101 [9.9%] with thetest nd reference for ul tions, respectively), proce-dur l site re cton (12 [11.8%] nd 4 [4.0%]), ndso nolence (7 [6.9%] nd 14 [13.9%]).

Of the 148 TEAEs reported, 125 were r ted s ildnd 23 were r ted s oder te. The rel tionships of

54 TEAEs were considered possibly rel ted, 8 s re-otely possibly rel ted, nd 86 s unrel ted to the

study drug.No de ths or serious AEs were reported during

this study. Clinic lly signific nt AEs were cystitis, her-pes si plex, cellulitis, nd respir tory tr ct infection(1 subject [1%] e ch). The he lth of these subjects w snot considered to be t risk during the study.

Vit l sign e sure ents nd l bor tory testing onconclusion of the clinic l portion of the study foundno signific nt ch nges in ny subjects st te of he lth.

test nd reference for ul tions, respectively. The edi n(IQR) T x v lues were 0.667 (0.283) nd 0.667 (0.500)hours. Me n AUC

0tnd AUC

0were 29,444.85

(8075.52) nd 32,195.74 (10,077.77) ng/ L/h withthe test for ul tion nd 29,910.48 (8292.20) nd33,375.82 (10,007.80) ng/ L/h with the reference for-

ul tion ( Table III ). The residu l re obt ined withboth the test nd reference products for the p rent co -pound w s

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nd MPA both s highly v ri ble drugs bec use fterthe 4-w y replic te study, the intr subject %CV forC x w s >30% with both n lytes (63.83% nd33.47%, respectively). 14

MPA ph r cokinetic v ri bility p tterns h vepreviously been described in dult kidney tr nspl ntrecipients. 10 D t fro the present study were consis-tent with those fro th t report 10 nd extend tohe lthy individu ls, supporting th t the ph r co-kinetic properties of MMF ight be err tic, s indi-c ted by the intr subject %CVs for C

xnd by the

individu l concentr tionti e curves.The concentr tionti e curve bsorption ph se of

the study design w s considered well ch r cterized,nd the s pling sche e w s considered dequ te for

the deter in tion of AUC 0t nd C x (ie, of sufficientdur tion to fully ssess the eli in tion ph se of MMF

nd MPA) given th t the residu l re with both the testnd reference products w s

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ever, the he lth of these subjects w s not considered toh ve been t risk during the study.

Bioequiv lence studies with replic te design resc rce b sed on PubMed se rch, using the keyter s bioequivalence nd replicate . This study design

llowed the ssess ent of true intr subject %CVsof the test or reference for ul tion when using 4-period crossover sche e, nd the ssess ent of true within-subject %CV of both for ul tions, whenthe full replic te design w s pplied. Despite the f ctth t this study design reduced the nu ber of sub-jects exposed to the drug, by incre sing the dur tionof exposure, it w s ore ti e consu ing th n thest nd rd 2-w y crossover ppro ch, involved l rgervolu e of blood s ples t ken per subject, ndincre sed the dropout r te (with longer studyperiods). 15,16

The f cts th t the study edic tion w s d inis-tered s single dose nd the dose d inistered w s500 g (co p red with the 1-g dose reco endedby the nuf cturer) ight expl in the low r tes of

19 hours), 10 reinforcing the dequ te concentr tionti e curve ch r cteriz tion nd opti u selection of the w shout period of 14 d ys, which w s considered

dequ te to llow co plete eli in tion of the drug be-fore subsequent dosing, voiding c rryover effects.Hence, no predose c rryover concentr tions were ob-served in periods 2, 3, or 4.

The study design w s considered dequ te to detect signific nt difference between the test nd reference

for ul tions in c ses in which the 90% CIs obt inedfor AUC

0t, AUC

0, nd C

xwere within the Euro-

pe n regul tory require ents for bioequiv lence,80% to 125%. 8,9 No st tistic lly signific nt differ-ences were found between the 2 for ul tions in thetested popul tion. No sequence, period, or tre t enteffects were verified for AUC 0t , AUC 0, or C x . The2 for ul tions et the Europe n regul tory criteri forbioequiv lence in ter s of r te nd extent of

bsorption. 8,9

No de ths or serious AEs were reported during thisstudy. Three clinic lly signific nt AEs occurred; how-

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Figure 3. Mean plasma mycophenolic acid (MPA) concentrations obtained after single 500-mg dose adminis-tration of a generic formulation (test; manufactured by Grupo Tecnimede, Sintra, Portugal) and abranded formulation (reference; trademark: CellCept , Roche Registration Ltd., Welwyn GardenCity, United Kingdom) of the inactive prodrug mycophenolate mofetil.

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M ar c h 2

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Table III. Pharmacokinetic properties of generic (test)* and branded (reference)

oral formulations of mycophenolisecond administration in healthy subjects under fasting conditions.

Test Ref

No. of No. of Parameter Patients Mean (SD) Median %CV IQR Patients Mean (SD)

First administrationAUC0t , ng/mL/h 100 29,712.19 (8301.24) 28,984.99 27.94 10,162.76 100 29,888.52 (7982.68) 28,155AUC0, ng/mL/h 82 33,111.54 (10,317.02) 31,507.91 31.16 11,998.93 80 33,187.01 (9945.36) 31,31Residual area, % 82 10.09 (7.93) 7.98 78.58 6.66 80 9.43 (6.09) Cmax, ng/mL 100 14,965.96 (6407.22) 14,459.65 42.81 6681.55 100 15,884.98 (6380.70) 15,68

T max, h 100 0.667 0.333 100 t1/2 , h 82 16.33 (6.33) 15.50 38.79 7.65 80 16.29 (6.31) ke, 1/h 82 0.0486 (0.0177) 0.0447 36.45 0.0231 80 0.0478 (0.0162)

Second administrationAUC0t , ng/mL/h 95 29,444.85 (8075.52) 28,792.60 27.43 12,024.80 93 29,910.48 (8292.20) 29,18AUC0, ng/mL/h 82 32,195.74 (10,077.77) 12,024.80 31.30 13,612.31 80 33,375.82 (10,007.80) 32,34Residual area, % 82 8.77 (5.16) 7.62 58.87 6.12 80 9.57 (6.31) Cmax, ng/mL 95 14,549.20 (6101.50) 14,003.26 41.94 7913.76 93 14,683.17 (6671.67) 14,42T max, h 95 0.667 0.283 93 t1/2 , h 82 15.88 (5.86) 14.51 36.87 7.02 80 16.19 (5.64) ke, 1/h 82 0.0492 (0.0168) 0.0478 34.13 0.0232 80 0.0484 (0.0187) 0

IQR = interquartile range. * Manufactured by Grupo Tecnimede, Sintra, Portugal. Trademark: CellCept (Roche Registration Ltd., Welwyn Garden City, United Kingdom).

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Test, second administrationReference, second administration

Figure 4. (A and B) Two selected individual plasma concentrationtime curves after single 500-mg doseadministration of a generic formulation (test; manufactured by Grupo Tecnimede, Sintra, Portugal)and branded formulation (reference; trademark: CellCept , Roche Registration Ltd., WelwynGarden City, United Kingdom) of mycophenolate mofetil (MMF).

(continued)

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15105 20 40 50353025 45

Figure 4 (continued). Two selected individual plasma concentrationtime curves after single 500-mg doseadministration of a generic formulation (test; manufactured by Grupo Tecnimede, Sintra, Portugal)and branded formulation (reference; trademark: CellCept , Roche Registration Ltd., Welwyn GardenCity, United Kingdom) of (C) mycophenolate mofetil (MMF) and of its active metabolite, (D) myco-phenolic acid (MPA).

(continued)

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M ar c h 2

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Table IV. Mean treatment ratios for the pharmacokinetic proper ties of generic (test)* and branded (reference) orlate mofetil (MMF) 500 mg and its active metabolite, mycophenolic acid (MPA), after first and second admsubjects under fasting conditions.

MMF MPA

WS %CV WS %CV

Parameter Test Reference Ratio (90% CI), % Test Reference AUC0t 28.90 pg h/mL 25.44 pg h/mL 96.93 pg h/mL (91.94102.20) 9.51 ng h/mL 9.49 ng h/mL 98

AUC0 20.18 pg h/mL 29.04 pg h/mL 99.12 pg h/mL (93.15105.48) 11.07 ng h/mL 9.30 ng h/mL 98

Cmax 63.83 pg/mL 61.04 pg/mL 95.73 pg/mL (85.94106.63) 33.47 ng/mL 48.83 ng/mL 98

WS = within subject. * Manufactured by Grupo Tecnimede, Sintra, Portugal. Trademark: CellCept (Roche Registration Ltd., Welwyn Garden City, United Kingdom). Values calculated using natural logarithmtransformed data. Intrasubject values.

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Table V. Characterization of the adverse events after the administration of generic (test)* andbranded (reference) oral formulations of mycophenolate mofetil (MMF) 500 mg inhealthy subjects under fasting conditions. Values are number (%) of subjects.

Test ReferenceSystem Organ Class/Preferred Term (n = 102) (n = 101)

Cardiac disorders 1 (1.0)Palpitations 0 1 (1.0)

Gastrointestinal disorders 2 (2.0) 4 (4.0)Nausea 1 (1.0) 2 (2.0)Diarrhea 1 (1.0) 1 (1.0)Upper abdominal pain 0 1 (1.0)Oral pain 0 1 (1.0)

General disorders and administration site conditions 1 (1.0) 2 (2.0)Chest discomfor t 1 (1.0) 0Fatigue 0 2 (2.0)

Infections and infestations 5 (4.9) 1 (1.0)Cellulitis 1 (1.0) 0Herpes simplex 1 (1.0) 0Oral herpes 1 (1.0) 0Respiratory tract infection 1 (1.0) 0Rhinitis 1 (1.0) 0Cystitis 0 1 (1.0)

Injury, poisoning, and procedural complications 26 (25.5) 19 (18.8)Procedural pain 13 (12.7) 10 (9.9)Procedural site reaction 12 (11.8) 4 (4.0)Postprocedural hematoma 3 (2.9) 4 (4.0)

Postprocedural swelling 1 (1.0) 2 (2.0)Skin laceration 1 (1.0) 2 (2.0)Thermal burn 1 (1.0) 1 (1.0)Procedural dizziness 1 (1.0) 0Animal bite 1 (1.0) 0Contusion 0 1 (1.0)

Vital signs and laboratory parameters 5 (4.9) 6 (5.9)Blood pressure decreased 2 (2.0) 4 (4.0)Heart rate decreased 1 (1.0) 1 (1.0)Heart rate increased 1 (1.0) 1 (1.0)White blood cell count increased 1 (1.0) 0

Musculoskeletal and connective tissue disorders 1 (1.0) 3 (2.9)Back pain 1 (1.0) 1 (1.0)Musculoskeletal pain 0 2 (2.0)

Nervous system disorders 11 (10.8) 22 (21.8)Somnolence 7 (6.9) 14 (13.9)Headache 4 (3.9) 8 (7.9)Dizziness 1 (1.0) 0Hypoesthesia 0 1 (1.0)Neuralgia 0 1 (1.0)

(continued)

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http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformat ion/Guidances/UCM070107.pdf. Accessed October 7, 2009.

3. Franco Spinola AC, Almeida S, Filipe A, Neves R. Limita-tions of Non-Magic Bullets Compounds in BioequivalenceAssessment. How Can This Enhance Knowledge Towards

the Development of Generic Products. The MycophenolateMofetil Case. http://www.ehrlich-2008.org/Programm_final.pdf. Accessed October 7, 2009.

4. World Medical Association (WMA). World Medical As-sociation Declaration of HelsinkiEthical Principles for Medical Research Involving Human Subjects. http://www.wma.net/en/30publications/10policies/b3/index.html.Accessed October 7, 2009.

5. European Medicines Agency for the Evaluation of Medici-nal Products (EMEA). ICH Topic E 6 (R1). Guideline for Good Clinical Practice. http://www.emea.europa.eu/pdfs/human/ich/013595en.pdf. Accessed October 7, 2009.

6. The Spanish Agency of Medicinal and Sanitary Products.Royal Decree 822 of May 28, 1993, that Establishes thePrinciples of Good Laboratory Practice and Chemical Ap-plication in the Accomplishment of Nonclinical Studies onSubstances and Products. http://www.aemps.es/actividad/legislacion/espana/docs/RCL_1993_1646Vigente.pdf.Accessed March 4, 2010.

7. Health Canada. Guidance for Industry: Conduct andAnalysis of Bioavailability and Bioequivalence StudiesPart A: Oral Dosage Formulations Used for SystemicEffects. http://www.hc-sc.gc.ca/dhp-mps/prodpharma/

g strointestin l disturb nces nd blood dyscr si sfound in this study.

CoNCLUsIoNsIn this open-l bel study in he lthy subjects, the test

nd reference for ul tions of MMF 500- g t bletset the Europe n regul tory definition of bioequiv -

lence b sed on the r te nd extent of bsorption of single dose under f sting conditions. Both for ul -tions were well toler ted.

ACkNowLEDgMENTsAll of the uthors re e ployees or vendors of GrupoTecni ede, the nuf cturer of the test product usedin this study. The uthors h ve indic ted th t theyh ve no other conflicts of interest reg rding the con-tent of this rticle.

rEFErENCEs1. The European Agency for the Evaluation of Medicinal

Products (EMEA). European Public Assessment Report.Mycophenolate Mofetil, Cellcept. http://www.emea.europa.eu/humandocs/PDFs/EPAR/Cellcept/emea-combined-h82en.pdf. Accessed October 7, 2009.

2. US Dept of Health and Human Services, Food andDrug Administration, Center for Drug Evaluation andResearch (CDER), Center for Veterinary Medicine (CVM).Guidance for Industry. Bioanalytical Method Validation.

Respiratory, thoracic, and mediastinal disorders 1 (1.0) 2 (2.0)Pharyngolaryngeal pain 1 (1.0) 2 (2.0)

Skin and subcutaneous tissue disorders 2 (2.0) 2 (2.0)Erythema 1 (1.0) 1 (1.0)Skin lesion 1 (1.0) 0Pruritus 0 1 (1.0)

Vascular disorders 1 (1.0) 1 (1.0)Hot flush 1 (1.0) 1 (1.0)

Total 43 (42.2) 48 (47.5)

* Manufactured by Grupo Tecnimede, Sintra, Portugal.

Trademark: CellCept

(Roche Registration Ltd., Welwyn Garden City, United Kingdom). Each subject was counted only once in each adverse-event category, regardless of the number of occurrences. Some patients had >1 adverse event. Medical Dictionary for Regulatory Activities. 11

Table V (continued).

Test ReferenceSystem Organ Class/Preferred Term (n = 102) (n = 101)

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l 32 3


lency assessment, pros and cons:Bioavailabilities of the antidiabeticdrugs pioglitazone and glimepiridepresent in a fixed-dose combina-tion formulation. J Clin Pharmacol .2007;47:806816.

EUFEPS BABP Network open discus-sion: Revised European guidelineson bioequivalence. Bonn, Germany:

January 1415, 2009.16. Karim A, Zhao Z, Slater M, et al.

Replicate study design in bioequiva-

applic-demande/guide-ld/bio/bio-a-eng.php. Accessed October 7,2009.

8. Committee for Proprietary MedicinalProducts (CPMP). Draft Guideline onthe investigation of bioequivalence(CPMP/EWP/QWP/1401/98R).http://www.emea.europa.eu/pdfs/human/ewp/056095en.pdf. Ac-cessed October 7, 2009.

9. Therapeutics Goods Administration(TGA). CPMP GuidelineAs Adopt-ed in Australia by the TGAWithAmendment. Note for Guidance onthe Investigation of Bioavailability and Bioequivalence (CPMP/EWP/QWP/1401/98). http://www.tga.gov.au/DOCS/pdf/euguide/ewp/140198entga.pdf. Accessed October 7, 2009.

10. Staatz CE, Tett SE. Clinical pharma-cokinetics and pharmacodynamicsof mycophenolate in solid organtransplant recipients. Clin Pharmaco-kinet . 2007;46:1358.

11. US Food and Drug Administration.Medical Dictionary for Regulatory Activities. http://www.meddramsso.com/MSSOWeb/index.htm. AccessedOctober 7, 2009.

12. Armstrong VW, Tenderich G, Ship-kova M, et al. Pharmacokineticsand bioavailability of mycophenolicacid after intravenous administrationand oral administration of myco-phenolate mofetil to heart transplantrecipients. Ther Drug Monit . 2005;27:315321.

13. Levesque E, Benoit-Biancamano MO,Delage R, et al. Pharmacokineticsof mycophenolate mofetil and itsglucuronide metabolites in healthy volunteers. Pharmacogenomic s. 2008;9:869879.

14. Blume HH, Midha KK. Bio-International 92, conference onbioavailability, bioequivalence, andpharmacokinetic studies. J Pharm Sci.1993;82:11861189.

15. Spnola AC, Almeida S, Filipe A, NevesR. Highly variable drugs and highly variable drug products. Briefing onregulatory and scientific perspectives.

Address correspondence to: Sus n Al eid , Medic l Dep rt ent,Grupo Tecni ede, Socied de Tecnico-Medicin l S.A., Zon Industri l dAbrunheir , R. d T p d Gr nde, n.2 Abrunheir , 2710-089 Sintr ,Portug l. E- il: d ed.ct@tecni ede.pt

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