Articles. Translational Investigation

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1 nture publishing group Trnsltionl Investigtion A seven-dy study of the phrmcokinetics of intrvenous levetircetm in neontes: mrked chnges in phrmcokinetics occur during the first week of life Cynthi M. Shrpe 1, Edmund V. Cpprelli 2, Andrew Mower 3, Michel J. Frrell 2, Steven J. Soldin 4 nd Richrd H. Hs 2,3 Introduction: Levetircetm (LEV) is incresingly used in the tretment of neontl seizures. The im of this study ws to determine phrmcokinetics in neontes with seizures nd to obtin preliminry sfety nd efficcy dt. Methods: Eighteen term neontes with seizures persisting fter mg/kg of phenobrbitl received intrvenous LEV for 1 wk. LEV ws dministered s or mg/kg bolus followed by 5 mg/kg/d. Phrmcokinetic dt were nlyzed using nonliner mixed-effects popultion pproch. Continuous electroencephlogrm monitoring llowed preliminry ssessment of the efficcy of LEV in this popultion. Results: LEV clernce (CL) incresed from men of 0.7 ml/ min/kg (SD 0.27 ml/min/kg) on dy 1 to 1.33 ml/min/kg (SD 0.35 ml/min/kg) by dy 7. Men hlf-life ws 18.5 h (SD 7.1 h) on dy 1 of the study nd decresed to 9.1 h (SD 2.0 h) by dy 7. The men volume of distribution ws 1.01 l/kg (SD 0.13 l/kg). No study-relted serious dverse events were observed. Discussion: CL of LEV in neontes ws higher thn expected on the bsis of immture renl function in term infnts nd incresed significntly during the first week of life. More frequent dosing of LEV is needed in term infnts to mintin serum concentrtions in the rnge seen in children nd dults. The stndrd tretments for neontl seizures re indequte (1). Current tretment relies on medictions in use between 1914 (phenobrbitl) nd 1938 (phenytoin). When used individully, ech of these gents produces seizure cesstion in <% of infnts treted (1,2). When used in combintion, the seizure cesstion rte is still <60% (3). Acute side effects of phenobrbitl nd phenytoin include hypotension, suppression of respirtory drive, crdic rrhythmi, nd sedtion. Chronic exposure to phenobrbitl my be ssocited with decresed cognitive bility (4 6). Studies on nimls suggest tht these gents my cuse ccelerted neuronl poptosis when used in immture subjects (7). Levetircetm (LEV) ((-)-(S)-α-ethyl-2-oxo-1-pyrrolidine cetmide) is very promising mediction for the tretment of neontl seizures. An intrvenous preprtion of LEV is vilble, llowing its use in neontes with seizures, who frequently cnnot be fed. LEV is chemiclly novel nticonvulsnt gent tht hs been in clinicl use for lmost decde in dults nd older children with good efficcy, n excellent sfety profile, nd ner idel phrmcokinetic chrcteristics. Studies on nimls hve shown tht LEV does not cuse neuronl poptosis in the immture brin nd shows promise s neuroprotective gent (8,9). Promising dt re emerging from recent studies regrding the efficcy of LEV in neontes (9 13). Khn et l. reported 32% rte of complete cesstion of clinicl nd electrogrphic seizure ctivity following LEV lod of mg/kg in series of 21 ptients (14). Abend et l. reported complete seizure cesstion in % of ptients 24 h following LEV lod of mg/kg (15). These preliminry efficcy dt re encourging, prticulrly considering tht LEV ws mostly dministered s secondor third-line gent in ptients with phrmcoresistnt seizures. However, the tendency of neontl seizures to resolve spontneously over time mkes the interprettion of these dt difficult. There is evidence tht off-lbel use of LEV for neontl seizures is becoming commonplce (16). This is despite pucity of bsic dt on the phrmcokinetics, sfety, nd efficcy of this drug in this popultion in the first few dys of life. These dt re needed to llow the rtionle use of this nti-epileptic drug in neontes. We therefore conducted phrmcokinetic study of LEV nd preliminry study of its sfety nd efficcy in neontes with seizures. Results Ptient Bseline Chrcteristics A totl of 18 ptients received tretment with intrvenous LEV, six t the first dosing level nd 12 t the higher dose. At enrollment ll subjects hd corrected gesttionl ge between 37 nd 41 wk nd weight between 2.5 nd 4.7 kg. In eight subjects, the underlying etiology of seizures ws hypoxic ischemic encephlopthy (HIE). Plsm cretinine did not exceed 0.09 mmol/l in ny subject. Five of these subjects received hypothermi tretment during the study period, (body cooling to 33.5 C for 72 h). Ptient demogrphics re detiled in Tble 1. 1 Deprtment of Neuroservices, Strship Children s Hospitl, Aucklnd, New Zelnd; 2 Deprtment of Peditrics, University of Cliforni, Sn Diego, L Joll, Cliforni; 3 Deprtment of Neurosciences, University of Cliforni, Sn Diego, Sn Diego, Cliforni; 4 Deprtment of Lbortory Medicine, Ntionl Institutes of Helth Clinicl Center, Bethesd, Mrylnd, Wshington, DC. Correspondence: Richrd H. Hs (rhs@ucsd.edu) Received 7 October 11; ccepted 13 Mrch 12; dvnce online publiction 9 My 12. doi:.38/pr Copyright 12 Interntionl Peditric Reserch Foundtion, Inc. Volume 72 Number 1 July 12 Peditric Reserch 43

2 Shrpe et l. Tble 1. Ptient demogrphics Subject number Gender Weight (kg) Gesttionl ge (wk) Postntl ge (d) Etiology of seizures 1 Mle Unknown b No 2 Femle Brin mlformtion (polymicrogyri, No hypoplstic brinstem nd cerebellr vermis, Dndy Wlker mlformtion) 3 Mle IVH No 4 Femle IVH No 5 Mle HIE (lminr necrosis bilterl perirolndic No cortex) 1 Mle Unknown No 2 Femle HIE Yes 3 Femle Unknown No 4 Mle HIE Yes 5 Femle Stroke No 6 Mle HIE Yes 7 Mle HIE No 8 Mle Stroke No 1 Femle HIE Yes 2 Femle HIE Yes 3 Femle HIE No 4 Femle Birth trum (skull frcture, intrcrnil No hemorrhge, nd right prietl stroke) 5 Mle Unknown No Hypothermi tretment IVH, intrventriculr hemorrhge; HIE, hypoxic ischemic encephlopthy. Postntl ge t strt of therpy. b All subjects with unknown etiology hd investigtions including brin mgnetic resonnce imging, sepsis screen with lumbr puncture, nd extensive metbolic investigtions. Phrmcokinetic Results The finl phrmcokinetic dt set contined 149 plsm concentrtion vlues from the 18 subjects. All subjects hd five or more evluble serum levels. Urine collections were completed in 18 subjects. Figure 1 shows serum drug concentrtion vs. time curves for ll subjects. The men (+SD) LEV concentrtions 1 h fter the initil doses of nd mg/kg were 18.2 ± 5.9 nd 33.0 ± 9.8 μg/ml, respectively. The concentrtions of predose LEV before the sixth dose were nd μg/ml for the 5 nd mg/kg dose levels, respectively. Figure 2 shows goodness of fit for the finl model, with popultion predicted LEV concentrtions vs. mesured LEV concentrtions. Postntl ge ws found to be significnt covrite for LEV clernce (CL) (chnge in objective function, 63.3). Serum cretinine lso ppered to be inversely relted to CL but this ssocition did not meet the model development criteri for inclusion. Sex, seizure onset dy, dosing rm, recent phenobrbitl concentrtion, nd hypothermi were not found to be significnt covrites for LEV CL. Given the limited number of subjects, intersubject vribility could be determined for CL but not for volume of distribution. The popultion typicl prmeter estimtes were well within the 95% bootstrp confidence intervls for the individul prmeters: volume of distribution, ( ) l/kg; CL on fifth dy of life, 0.97 Levetircetm concentrtion (µg/ml) Time (h) Figure 1. Serum drug concentrtion vs. time curves for ll subjects. ( ) ml/min/kg; nd ge effect (θ3) on CL ((Age/5) θ3 ), ( ) The empiric Byesin estimtes for individul subject phrmcokinetic prmeters re summrized in Tble 2. The men volume of distribution seen in neontes ws 1.01 l/kg (bootstrp 95% confidence intervl l/kg), greter thn previously reported in older children nd greter thn totl body wter. CL ws much greter thn the predicted ml/ min/kg on the bsis of kidney immturity. During the week- 44 Peditric Reserch Volume 72 Number 1 July 12 Copyright 12 Interntionl Peditric Reserch Foundtion, Inc.

3 Seven-dy phrmcokinetic study of intrvenous LEV in neontes long tretment period, CL doubled, to rech the CL seen in older children. For comprison, phrmcokinetic prmeters reported in other peditric studies re summrized in Tble 3 (17 21). Although of 12 subjects treted t the higher dosing level hd trough LEV concentrtions >6 μg/ml t 36 h, no subjects hd trough LEV concentrtions >6 μg/ml by the end of the week of tretment. Before the seventh dose, LEV trough levels verged 1.7 μg/ml (SD 1.0) on 5 mg/kg/d mintennce nd 2.4 μg/ml (SD 1.3) on mg/kg/d mintennce. Substntil LEV metbolite concentrtions were seen in both plsm nd urine with lrge rnge of vlues cross the subjects (see Tbles 4 nd 5). Plsm nd urinry UCB L057/LEV rtios were higher in subjects receiving the higher dosing regimen. The urinry UCB L057/LEV rtio showed substntil increses between the 0 12 s compred with the h collection, suggesting incresed hydrolysis. The urinry UCB L057/LEV rtio ws not predictive of LEV CL. The plsm UCB L057/LEV rtio lso filed to predict LEV CL nd ws stble throughout the therpy. Overll, these dt suggest tht both renl CL nd hydrolysis pthwys re mturing during this time frme. Efficcy in Seizure Cesstion: Preliminry Anlysis As simple mesure of LEV efficcy, 6 of the 18 subjects studied in this tril required no dditionl nti-epileptic drugs fter LEV ws commenced becuse of cesstion of both clinicl nd electrogrphic seizures. Five of the responders were mong the 12 subjects who received the higher dose of LEV (42%). There ws only one responder mong the six subjects who received the lower LEV dose. (An dditionl of three subjects hd n initil response to LEV; with temporry cesstion of seizures Mesured levetircetm concentrtion (µg/ml) 0 0 Popultion predicted concentrtion (µg/ml) Figure 2. Goodness-of-fit plot. Popultion predicted vs. mesured levetircetm drug concentrtions. documented by electroencephlogrms (EEG) s the loding dose ws given but lter hd recurrence of seizures). Detiled nlysis of EEG dt from this study demonstrting the effect of LEV on EEG-confirmed neontl seizures is under wy nd will be reported seprtely. Adverse Events nd Sfety Monitoring Ptients were ssessed dily for possible dverse clinicl events relted to LEV tretment. LEV ws well tolerted. Only one serious dverse event occurred in this study. One subject with brin mlformtion nd high phenobrbitl level required intubtion while on tretment with LEV. LEV ws not thought to be cusl of this event. Mild dverse events which resolved spontneously nd were possibly relted to tretment with LEV included mild sedtion in two subjects, feeding difficulty in three subjects, mild pne nd brdycrdi in one subject, nd decresed urine output responding to furosemide in one subject. In no cse ws LEV thought custive of n dverse event. Blood tests (complete blood count, chemistry with electrolytes, cretinine, ure, nd lnine minotrnsferse) were performed on dy 3 nd dy 7 of tretment in ll but one subject, whose dy 7 monitoring chemistry nd full blood count were not performed; however, serum cretinine ws mesured. Hemtology Three subjects developed mildly low pltelet counts (rnge ) while on tretment with LEV. All subjects hd HIE. In two of the three subjects, the pltelet count normlized by dy 7 while on tretment with LEV. Three subjects developed white-cell counts below the norml rnge for their institution. In these cses the leukopeni ws very mild (rnge ,000 cells/mm 3 ). In eight subjects, the level of hemoglobin dropped while on tretment to below the norml rnge. In four of the eight subjects, this resolved by dy 7 while still receiving tretment. One subject hd low hemoglobin before tretment (13.8 gm/ dl), nd during the first week of life the level of hemoglobin dropped further to.8 gm/dl. In three subjects mildly low levels of hemoglobin persisted (rnge gm/dl). Chemistry Serum cretinine did not increse during tretment. Smll increses in blood ure were seen in three subjects. In two subjects, with HIE nd bnorml bseline pretretment lnine minotrnsferse levels, lnine minotrnsferse incresed on tretment with LEV. In both the cses, lnine minotrnsferse Tble 2. Phrmcokinetic results Men SD Medin Minimum Mximum BS 95% CI Vd (l/kg) CL dy 1 (ml/min/kg) CL dy 7 (ml/min/kg) T½ dy 1 (h) T ½ dy 7 (h) BS, bootstrp; CI, confidence intervl; CL, clernce; T½, hlf life; Vd, volume of distribution. Copyright 12 Interntionl Peditric Reserch Foundtion, Inc. Volume 72 Number 1 July 12 Peditric Reserch 45

4 Shrpe et l. Tble 3. Comprison of phrmcokinetic prmeters from previous studies Reference This neontl study (i.v. dosing) This neontl study Merhr et l. (17) Gluser et l. (18) (orl dosing) Pellock et l. (19) (orl dosing) Fountin et l. () (orl dosing) Chhun et l. (21) (orl dosing) Age CL, ml/min/kg (CL/F for orl dosing studies) T½ (h) V/F b, l/kg Dy ± ± ± 0.13 Dy ± ± ± d 1.21 ( ) 8.9 ( ) 0.89 ( ) 2 46 mo 1.46 ± ± y 1.43 ± ± ± y 1. ± ± y 1.24 ± ± ± 0.12 All sttistics expressed s men ± SD in ll studies except Merhr et l., in which medin nd rnge were given. CL/F = pprent clernce fter orl dministrtion. b V/F = pprent volume of distribution. returned to below the pretretment bseline level by dy 7 of LEV tretment. Mild bnormlities of serum electrolytes were seen with no consistent pttern. One subject developed tretment-emergent hypontremi. This infnt ws criticlly ill with HIE nd ws receiving hypothermi nd multiple medictions. The hypontremi (126 mmol/l) resolved to norml by dy 4 of tretment nd remined norml t dy 7. Three subjects hd hypoklemi while receiving LEV tretment. In two cses this ws mild, (potssium = 3 mmol/l, 3.8 mmol/l) nd serum potssium normlized spontneously by dy 7. A third ptient received tretment with potssium chloride for serum potssium level of 2.7 mmol/l. Trnsiently rised serum potssium levels were seen in five subjects nd mild hyperchloremi ws seen in six subjects; in three chloride normlized by dy 7 of tretment. Mild hyperclcemi ws seen in five subjects (mximum clcium 2.8 mmol/l), nd mild hypoclcemi ws seen in two subjects (minimum clcium 1.9 mmol/l). Mildly low serum bicrbonte developed in three subjects (minimum serum bicrbonte 16 mmol/l). In one subject serum bicrbonte incresed while on tretment to 35 mmol/l on dy 3, then normlized spontneously. All blood test bnormlities were reviewed by n independent dt sfety monitoring bord. Mild bnormlities in blood count nd serum chemistries were thought consistent with wht would hve been expected in this ptient popultion of sick neontes. No serious or consistent tretment-emergent lbortory bnormlities were observed. Tble 4. Plsm LEV nd UCB L057 metbolite levels Plsm levels, trough before second dose, t 12 h Plsm levels, trough before seventh dose, t 132 h Men SD Medin Minimum Mximum Men SD Medin Minimum Mximum Low dose LEV level (µg/ml) Metbolite UCB L057 (μg/ml) Metbolite/LEV rtio High dose LEV level (μg/ml) LEV, levetircetm. Metbolite UCB L057 (μg/ml) Metbolite/LEV rtio UCB L057 is the nme of the min cid metbolite of levetircetm. Tble 5. Urine LEV nd UCB L057 metbolite levels Urine levels t 0 12 h collection Urine levels t h collection Men SD Medin Minimum Mximum Men SD Medin Minimum Mximum Low dose LEV level (μg/ml) Metbolite UCB L057 level (μg/ml) Metbolite/LEV rtio High dose LEV level (μg/ml) LEV, levetircetm. Metbolite UCB L057 level (μg/ml) Metbolite/LEV rtio UCB L057 is the nme of the min cid metbolite of levetircetm. 46 Peditric Reserch Volume 72 Number 1 July 12 Copyright 12 Interntionl Peditric Reserch Foundtion, Inc.

5 Seven-dy phrmcokinetic study of intrvenous LEV in neontes Discussion About two-thirds of LEV is eliminted s unchnged drug in the urine while the remining third is hydrolyzed to UCB L057, lso excreted in the urine. Bsed on glomerulr function immturity in neontes (22), we expected LEV CL in this popultion would be between 15 nd 45% of tht of older popultions. Conducting this study in sick neontes, we were obliged to err on the side of cution nd use conservtive estimtion of CL in our dosing selection. CL of LEV in neontes ws higher thn predicted nd incresed significntly during the first week of life into the rnge seen in older children (Tbles 2 nd 3) nd exceeded vlues reported in dults (23). This study illustrtes the importnce of performing phrmcokinetic studies in neontes nd the inccurcy of our best predictions extrpolting to neontes from phrmcokinetic dt in older subjects. Our dt re in greement with dt from the concurrently performed neontl study by Merhr et l. (17) nd further dvnce those dt. Becuse of the week-long durtion of our study we were ble to detect the drmtic chnge in phrmcokinetics of LEV within the first week of life. Identifying this chnge reduces interindividul vribility t ny given time point. Our nlysis of UCB L057 metbolite levels is not previously reported in neontes. Severl fctors my ccount for the greter-thn-predicted LEV CL in the neonte. LEV hs low plsm protein binding nd its renl CL is substntilly less thn glomerulr filtrtion in dults. These chrcteristics suggest tht LEV undergoes net renl tubulr rebsorption in dults. Therefore, one potentil explntion for the higher-thn-expected CL in newborns is tht in ddition to immture glomerulr function, infnts my hve reduced cpcity for tubulr rebsorption of LEV. Altered hydrolysis to UCB L057 my lso contribute. The newborn expression of the specific esterse responsible for LEV hydrolysis is unknown. Our nlysis of UCB L057 metbolite levels demonstrtes tht the ctivity of this enzyme ccounts for up to % of overll LEV CL by 36 h of life. (In children nd older dults it ccounts for pproximtely one-third of LEV CL.) The stble UCB L057 metbolite/ LEV rtios in serum over the course of the week indictes tht both elimintion pthwys increse in function during the first week of life. All 18 subjects in our study were comedicted with phenobrbitl nd 16 received mintennce therpy with phenobrbitl. LEV is metbolized by β-esterse present in serum nd liver (24). Becuse metbolism of LEV is not medited by heptic cytochrome 4 isoenzymes, incresed CL ssocited with comediction ws not expected. However, in peditric study exmining ge effects nd drug interctions with LEV, significnt % increse in LEV CL in subjects comedicted with enzyme-inducing nti-epileptic drugs such s phenobrbitl, crbmzepine, nd phenytoin ws reported (25). The uthors postulte tht this effect my be medited by comediction inducing the enzymtic hydrolysis of LEV nd note other drugs in which this process hs been documented. We therefore nlyzed phenobrbitl levels vilble in 15 subjects. The medin concentrtion of phenobrbitl ws 34 (rnge 18 49) mg/dl. Phenobrbitl concentrtion ws not significnt covrite for LEV CL during univrite nlysis (1.98 drop in model objective function from the bse model).however, s ll of the subjects were on phenobrbitl, it would be difficult for this study to detect differentil effects of phenobrbitl levels on LEV CL; therefore, this remins possible prtil explntion for the greter-thn-expected LEV CL seen. Loding with LEV mg/kg followed by mg/kg once dily dosing results in lrge drop-off in serum concentrtions over the first week of life with more thn % infnts expected to hve trough concentrtions below 5 μg/ml. With our improved knowledge of the phrmcokinetics of LEV in neontes we cn now construct dosing guideline to mintin desired trough concentrtions. In dults on stndrd therpeutic doses of LEV, trough concentrtions re typiclly in the rnge 6 μg/ml. Given the intrctbility of the seizures in mny neontes nd the sfety profile of LEV, we suggest tht the upper end of this rnge could be pplied in the neonte. Figure 3 compres medin predicted LEV serum concentrtions t different mintennce dose frequencies: the mg/ kg dily mintennce dose used in this study s compred with mg/kg mintennce dosing every 12 h nd every 8 h. Figure 4 shows the expected distribution of LEV serum concentrtions (5th to 95th percentiles) with loding dose of mg/kg followed by mg/kg mintennce dose every 8 h. These figures demonstrte tht 8-hourly mintennce dosing is required to ensure tht 95% of infnts mintin trough concentrtions greter thn μg/ml. This dosing regimen is predicted to mintin trough levels bove μg/ml for the first 3 d of tretment, when seizures re most ctive. Although this would be our recommended dosing regimen t the present time, the optiml serum level to im for in the setting of neontl sttus epilepticus is uncler. LEV hs n extremely high therpeutic index, >148, in rodents (26). No deths, orgn filure, or other irreversible toxicity ws seen Levetircetm concentrtion (µg/ml) Time (h) Figure 3. Simultions of proposed dosing. Comprison of expected medin levetircetm serum concentrtions with loding dose of mg/ kg followed by mg/kg dministered every 8 h (upper line), 12 h (middle line), or 24 h (lower line). Copyright 12 Interntionl Peditric Reserch Foundtion, Inc. Volume 72 Number 1 July 12 Peditric Reserch 47

6 Shrpe et l. Levetircetm concentrtion (µg/ml) Time (h) Figure 4. Expected distribution of levetircetm serum concentrtions with loding dose of mg/kg followed by mg/kg every 8 h. 95th percentile (upper line), medin (middle line), nd 5th percentile (lower line). fter long-term orl tretment up to doses of 1,800 mg/kg/d in the rt, 960 mg/kg/d in the mouse, nd 1,0 mg/kg/d in the dog (27). Ten yers of experience hve shown LEV to be extremely sfe in humns lso. Dose escltion studies should be performed nd this sfety mrgin should be exploited if dditionl efficcy would be obtined by using higher doses. A recent peditric study suggested tht there my be dditionl efficcy of extremely high doses of LEV. Subjects with refrctory sttus epilepticus received men dose of 228 ± 48 mg/ kg/d. The higher doses were effective in relieving sttus epilepticus where stndrd doses hd been ineffective. These high doses were well tolerted; the uthors report no significnt short-term side effects, including behviorl side effects (28). This study hs severl limittions. We hve studied smll number of subjects. The subject popultion ws lso nrrowly designed to include term infnts during the first few dys of life with reltively norml renl function for ge. Given the dynmic nture of LEV CL in our study popultion, preterm nd older term infnts or those with some renl dysfunction re likely to hve different LEV CL nd possibly ltered dosing requirements. Our preliminry efficcy dt re encourging. It should be remembered tht these dt reflect the response rte to LEV when used s second-line gent in subjects refrctory to phenobrbitl s first-line gent. As such, this response rte of 42% seen in the higher dose cohort compres fvorbly with efficcy dt for phenytoin nd phenobrbitl when used s second-line gents. Pinter et l. found tht of 4 of 17 (24%) of neontes with seizures refrctory to phenobrbitl responded to the phenytoin s the next nti-epileptic drug, nd 5 of 16 (31%) neontes with seizures refrctory to phenytoin responded to phenobrbitl s the next nti-epileptic drug (3). However, the tendency of neontl seizures to resolve spontneously over time mkes interprettion of these very preliminry dt difficult. In summry, this study hs chieved more ccurte knowledge of the phrmcokinetics of LEV in neontes. LEV ws well tolerted in this study of sick neontes. Further sfety, efficcy, nd dose escltion studies re now needed to determine the trget concentrtion for efficcy nd relted optiml dose. Methods Study Design The tril ws n open-lbel phrmcokinetic nd preliminry sfety study with LEV dded on to phenobrbitl tretment. Between August 07 nd Februry 09, eligible neontes dmitted in three prticipting neontl intensive cre units were recruited to this study. The sites were the University of Cliforni Sn Diego Medicl Center, Shrp Mry Birch Hospitl Sn Diego, nd Aucklnd City Hospitl, Aucklnd, New Zelnd. The institutionl review bord t ech center pproved the protocol nd informed consent ws obtined from the prents in ech cse. The study ws registered with the Clinicl Trils Registry (NCT004619). Study Entry Criteri Subjects were inptients in the three prticipting neontl intensive cre units. Eligibility required the study subjects to be less thn 14 d of ge with corrected gesttionl ge between 37 wk nd 44 wk nd weight of t lest 2.5 kg. To receive the study drug, subjects hd to be experiencing clinicl or electrogrphic seizures tht persisted fter receiving mg/kg loding dose of phenobrbitl. Subjects were excluded from the study if they hd serum cretinine of >1.2 mg/dl t the time of enrollment, if they were nuric or if seizures were becuse of biochemicl bnormlity such s hypoglycemi or hypoclcemi, which once rectified resulted in seizure cesstion. Ptients were lso excluded from this study if deth of the ptient seemed imminent. Intervention Subjects were recruited in two wys. Ptients recognized to be t high risk of developing neontl seizures, for exmple neontes with HIE, were recruited prospectively. Other subjects were recruited t the time of presenttion with seizures. Following recruitment nd consent, ptients were monitored by three-chnnel continuous EEG with mplitude-integrted EEG to detect seizures (29). A neurologist skilled in neontl EEG interprettion monitored the recording for the first hour nd then t lest every 8 h therefter. If the EEG confirmed persistence of seizures hlf n hour fter receiving phenobrbitl, n i.v. LEV loding dose ws dministered over 15 min. LEV mintennce dosing ws given strting 12 h fter the initil infusion nd continued every 24 h for totl of 1 wk. If EEG-confirmed seizures persisted 1 h fter completion of the LEV loding dose, ptients received further mediction following the locl hospitl protocol, typiclly further phenobrbitl or fosphenytoin. The study protocol did not mesure phenobrbitl levels nd did not require phenobrbitl mintennce tretment. The first cohort of bbies (n = 6) ws treted with mg/kg initil lod followed by 5 mg/kg/d s single dily dose (qd). Following plnned interim nlysis of the first cohort, the dose ws esclted. The second cohort (n = 12) received mg/kg s lod followed by mg/kg/d qd. The dosing selected for this tril ws bsed on LEV phrmcokinetics in older popultions nd took into ccount expected developmentl differences in term newborns. Distribution of LEV is chrcterized by low protein binding nd volume of distribution tht pproches totl body wter (0.7 l/kg). Given the high totl body wter content in infnts, it ws expected tht infnt LEV volume of distribution would be slightly lrger thn in dults. LEV is clered from the body by the kidney nd by hydrolysis to UCB L057. Bsed on immture glomerulr filtrtion in neontes nd resulting renl function only % tht of older children (22), LEV CL in infnts ws expected to be between 15 nd 45% of n older popultion with the degree of hydrolysis present in neontes n importnt unknown vrible. With these considertions, trough concentrtions on the initil (mg/kg lod, then 5mg/kg qd) nd second (mg/kg lod, then mg/kg qd) dose levels were expected to be t the low end nd middle end of the rnge typiclly seen with therpeutic doses in dults (~35 1 µmol/l or 6 μg/ml). Seril determintions of LEV concentrtions were performed to enble phrmcokinetic nlyses. Blood smples were collected before therpy, t predose troughs five times during the first week of therpy, nd 1 h post pek levels following the first nd seventh dose, to mesure 48 Peditric Reserch Volume 72 Number 1 July 12 Copyright 12 Interntionl Peditric Reserch Foundtion, Inc.

7 Seven-dy phrmcokinetic study of intrvenous LEV in neontes pek nd trough serum concentrtions of LEV nd its mjor metbolite UCB L057. Urine ws collected in two liquots: ll urine output for the first 12 h fter LEV therpy ws commenced, nd ll urine output between 12 nd 36 h. Concentrtions of serum nd urine LEV nd UCB L057 were mesured by liquid chromtogrphy tndem mss spectrometry method developed for this project () tht permitted the simultneous mesurement of prent drug nd metbolite. Phrmcokinetic nlysis ws performed using the computer progrm NONMEM ver. 6.2 (ICON, Ellicott City, MD). NONMEM employs nonliner mixed-effects modeling to nlyze dt composed of repeted mesurements in nonliner systems. This pproch nlyses ll of the ptient dt together nd determines the set of phrmcokinetic prmeters nd within nd between-subject vribility tht describe the observed dt. A one-comprtment model ws used nd popultion phrmcokinetic model ws developed using the first-order conditionl estimtion subroutine with interction. Ptient weight ws included in the model before the ssessment of other covrites. Postntl ge, serum cretinine, sex, seizure onset dy, dose level, phenobrbitl concentrtion, nd hypothermi were ssessed s potentil covrites on CL. An initil univrite screen ws performed for ech covrite nd those tht mrginlly improved the model (reduction in the objective function >4) were included in bckwrd-elimintion multivrite nlysis. A covrite tht improved the finl model by reduction >6.6 ws retined. After ssessment of covrites, vrious residul error models (proportionl, dditive, nd combined) were ssessed. A bootstrp of the finl model using 1,000 bootstrp dt sets ws performed to generte 95% confidence intervls of the prmeters estimtes using Wings for NONMEM (31). Empiric Byesin estimtes of individul subjects phrmcokinetic prmeters were generted using the popultion model prmeters s the priors. During the tretment phse, ech ptient ws cliniclly reviewed dily. Follow-up continued by phone review t 3 nd 6 d fter intrvenous dministrtion of the study drug ws completed. A follow-up visit ws conducted 1 wk fter completion of the tretment phse of the study. Sfety monitoring included mesurement of complete blood count, serum cretinine, electrolytes, nd liver enzymes t bseline, between 48 nd 72 h of tretment nd t completion of 7 d of tretment. ACKNOWLEDGMENTS We grtefully cknowledge the vluble ssistnce with dt nd sfety monitoring provided by Bruce Brshop nd Frnk Mnnino, nd the ssistnce of Mrk Nespec, the neontl fculty nd stff t University of Cliforni Sn Diego Medicl Center neontl intensive cre unit (NICU), the NICU t the Strship Hospitl Aucklnd (in prticulr Mlcolm Bttin), nd the fculty nd stff t Shrp Mry Birch Hospitl, Sn Diego (in prticulr Mynrd Rsmussen). STATEMENT OF FINANCIAL SUPPORT This work ws supported by grnt from the Thrsher Foundtion (Thrsher wrd no ). Levetircetm nd UCB057 for ssy development were provided by UCB Phrm. REFERENCES 1. Snkr R, Pinter MJ. Neontl seizures: fter ll these yers we still love wht doesn t work. Neurology 05;64: Scher MS, Alvin J, Gus L, Minnigh B, Pinter MJ. Uncoupling of EEGclinicl neontl seizures fter ntiepileptic drug use. Peditr Neurol 03;28: Pinter MJ, Scher MS, Stein AD, et l. Phenobrbitl compred with phenytoin for the tretment of neontl seizures. 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Comput Methods Progrms Biomed 1999;59: Copyright 12 Interntionl Peditric Reserch Foundtion, Inc. Volume 72 Number 1 July 12 Peditric Reserch 49