Four-Year Prospective Clinical Trial of Agalsidase Alfa in Children withFabry Disease

Raphael Schiffmann, MD, Rick A. Martin, MD, Tyler Reimschisel, MD, Karen Johnson, MD, MPH, Victoria Castaneda, MD,

Y. Howard Lien, MD, PhD, Gregory M. Pastores, MD, Christoph Kampmann, MD, Markus Ries, MD, PhD, MHSc,

and Joe T. R. Clarke, MD, PhD

Objectives To investigate a 4-year prospective clinical trial of agalsidase alfa in children with Fabry disease, anX-linked metabolic disorder caused by a deficiency of the lysosomal enzyme a-galactosidase A.Study design Seventeen (16 boys, 1 girl; age range, 7.3 to 18.4 years) of the 24 children who completeda 6-month, open-label agalsidase alfa study enrolled in a 3.5-year extension study that investigated the safetyand potential efficacy of long-term treatment. All 17 patients completed the initial 6-month study, and 10 patients(9 boys) completed the extension study.Results Agalsidase alfa was well tolerated. In treated boys, there were sustained, statistically-significant improve-ments in the clinical features of Fabry disease, including reduced plasma globotriaosylceramide levels, reducedpain severity assessed by the Brief Pain Index, and improved heart rate variability. Mean urine globotriaosylcera-mide levels were reduced to normal range (P < .05 compared with baseline during 1.5 to 4 years). Kidney functionand left ventricular mass indexed to height remained stable throughout.Conclusions This clinical trial demonstrates that treatment with agalsidase alfa was well tolerated and associatedwith improvement of Fabry disease–related features. (J Pediatr 2010;156:832-7).

See related article, p 828

Fabry disease is a chronic, progressive, X-linked disorder caused by a deficiency of the lysosomal enzyme, a-galactosidase A.1 The cellular accumulation of the enzyme substrate globotriaosylceramide (Gb3) results inprogressive, multi-organ pathology. The signs and symptoms emerge in childhood and adolescence, beginning

with episodes of neuropathic pain and the appearance of angiokeratomas.2-4 The incidence of cardiac involvement, renal

From the Institute of Metabolic Disease, Baylor ResearchInstitute (R.S.), Dallas, TX; Developmental and MetabolicNeurology Branch (R.S., M.R.), National Institute ofNeurological Disorders and Stroke, National Institutes ofHealth, Bethesda, MD; Washington University School ofMedicine (R.A.M., T.R.), St Louis, MO; University ofTennessee Health Science Center (K.J.), Memphis, TN;East Tennessee Children’s Hospital (V.C.), Knoxville, TN;Arizona Health Sciences Center (Y.L.), Tucson, AZ; NewYork University School of Medicine (G.M.P.), New York,NY; University Children’s Hospital (C.K.), Mainz,Germany; and Hospital for Sick Children (J.T.R.C.),Toronto, Ontario, Canada

The study described in this report was a clinical trialconducted by Shire Human Genetic Therapies, Inc.(HGT), which designed the study in collaboration with theprincipal investigators and was responsible for the col-lection and analysis of the data. R.S. has received re-search funding, consultancy fees, and/or speaker’s feesfrom Shire HGT, Genzyme, and Amicus Therapeutics.J.T.R.C. has received research funding, consultancyfees, and/or speaker’s fees from Shire HGT, Genzyme,ExSAR, and Actelion. M.R. is currently an employee ofShire HGT. R.A.M. received research funding and con-sultancy fees from Shire HGT and Genzyme during thecourse of this study and is now employed by Shire HGT.G.M.P. received honoraria, travel grants, and/or researchsupport from Shire HGT, Genzyme, Protalix, and Bio-marin. C.K. has received research funding, consultancyfees, and/or speaker’s fees from Shire HGT and Gen-zyme Corporation. Y.H.L. has received research grantsfrom Shire HGT and Genzyme Corporation. The other

dysfunction, and cerebrovascular problems increases with age and are thesource of major morbidity and premature mortality.5,6

Two forms of enzyme replacement therapy are available for the treatment ofFabry disease: agalsidase alfa, which is manufactured in a human cell-line bygene activation,7,8 and agalsidase beta, which is manufactured in Chinese ham-ster ovary cells.9 In clinical trials, both drugs have been associated with biochem-ical and clinical benefit.9-13

The experience with enzyme replacement therapy in pediatric patients withFabry disease is less extensive compared with that in adults. Agalsidase betahas been the subject of a single, 48-week study in 14 boys and 2 girls with Fabrydisease.14 In boys, plasma Gb3 levels were reduced, and superficial skin capillaryendothelial cells were cleared of Gb3 deposits. Gastrointestinal symptoms alsoimproved. Agalsidase alfa has been studied in 2 separate pediatric clinical trials.Ramaswami et al15 treated 9 boys and 4 girls for 24 weeks and found reducedplasma and urine sediment Gb3 levels in boys and improvement in pain in theentire study population. Ries et al16 treated 19 boys and 5 girls for 6 monthsand found reductions in plasma Gb3 levels in patients with elevated baselinelevels and a significant reduction in the use of pain medications for chronic neu-ropathic pain. In addition, they documented improvement in heart rate variabil-ity in boys. Here we report on the long-term follow-up of the children in thelatter cohort.

authors declare no conflicts of interest. The authors areresponsible for the interpretation presented in this report.R.S., G.M.P., Y.H.L., and J.T.R.C. contributed to the firstdraft of the manuscript. All authors have approved thefinal version of the manuscript and are fully responsiblefor its content.

0022-3476/$ - see front matter. Copyright � 2010 Mosby Inc.

All rights reserved. 10.1016/j.jpeds.2009.11.007

BPI Brief Pain Index

eGFR Estimated glomerular filtration rate

Gb3 Globotriaosylceramide

LVM Left ventricular mass

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Vol. 156, No. 5 � May 2010

Methods

This study was an open-label extension of a Phase III,4-center clinical trial of the patient cohort previouslydescribed.16 All patients who completed the original 6-month,open-label trial were eligible for enrollment in this study.Fabry disease was confirmed by deficient enzyme activityin boys and by GLA mutation analysis in girls. The studyprotocol and related documents were reviewed and ap-proved by the institutional review board or independentethics committee at each site. Each patient and their par-ents or legal guardian provided written consent to partici-pate in the study.

All patients were treated with agalsidase alfa (Replagal,Shire Human Genetic Therapies, Inc., Cambridge, Massa-chusetts), administered every other week at a dose of 0.2mg/kg infused intravenously over 40 minutes. Premedicationwas not routinely administered.

Exploratory Efficacy AssessmentsEfficacy assessments were performed at study baseline andevery 6 months thereafter. Changes in plasma and urinesediment Gb3 were measured as described previously.7

Kidney function was assessed by estimating glomerular fil-tration rate (eGFR) using the Counahan-Barratt equation17

and by measuring the 8-hour albumin excretion (expressedas mg/24 hours). Cardiac structure and function was as-sessed by echocardiography and electrocardiogram. In ad-dition, heart rate variability was assessed quantitativelyfrom 2-hour ambulatory electrocardiographic monitoringas previously described.16 Pain was assessed with the BriefPain Index.18 Sweat function was measured by the quanti-tative sudomotor axon reflex test (QSART)16 at a singlestudy site.

SafetySafety of the study drug was assessed continuously throughthe study by monitoring adverse events observed by investi-gators or reported by patients. In addition, vital signs, phys-ical and neurologic examinations, clinical laboratorymeasurements, and the incidence of anti-agalsidase alfa anti-bodies were evaluated at each study visit. Anti–agalsidase alfaantibodies were measured in serum using a validated plateenzyme-linked immunosorbent assay as described previ-ously.10 An absolute absorbance >0.04 U combined witha time point/baseline ratio $2 was considered a positiveresponse.

Analysis of ResultsThe primary objective of this study was to assess the safety ofagalsidase alfa at a dose of 0.2 mg/kg administered everyother week in pediatric patients with Fabry disease. All safetyevaluations are presented descriptively. Exploratory efficacyvariables were analyzed as change from baseline, with base-line being defined as the last measurement before initiatingtreatment with agalsidase alfa. The change from baseline at

each time point was evaluated with either a paired t test fornormally distributed data or with a Wilcoxon signed-ranktest for data that were not normally distributed. A 2-tailedP value <.05 was considered to be statistically significant.All computations were done using SAS version 8.2 (SASInstitute, Cary, North Carolina).

Results

Seventeen (16 boys, 1 girl; age range, 7.3 to 18.4 years) of the24 patients who completed the initial 6-month study enrolledin the extension study. The baseline characteristics of theoriginal patient population have been previously described.16

All 17 patients completed 6 months of the extension study (1year of agalsidase alfa treatment), 12 patients (11 boys) com-pleted 12 months of the extension study (1.5 years of treat-ment), and 10 patients (9 boys) completed 3.5 years of theextension study (4 years of treatment). One study site(Mainz) was closed shortly after the initiation of the exten-sion study when agalsidase alfa became commercially avail-able in Germany, accounting for 6 patients (including 4girls) who did not enroll in the extension study. At thetime of data cut-off, 11 patients remained in the trial, butonly 10 had completed 4 years of treatment because of stag-gered entry into the study.

SafetyDuring the 4 years of treatment including the original6-month study and this long-term extension study, a totalof 908 adverse events were reported for the 24 patients.More than 93% were mild or moderate in severity, and lessthan 10% were deemed by the investigator to be possiblyrelated (6.5%) or probably related (3.4%) to study treatment.No life-threatening, treatment-related adverse events ordeaths were reported. Treatment-emergent adverse eventsthat were possibly or probably related to treatment were ob-served in 11 and 4 patients, respectively. The adverse eventsthat occurred in 2 or more patients are listed in the Table. In-fusion reactions, which typically consist of rigors, flushing,nausea, and/or pyrexia, with or without headache, occurredduring 4.2% of the infusions (62 events/1472 infusions)over the 4-year period. None of these infusion reactionswere considered severe and were managed effectively by pre-medication with an antihistamine and/or corticosteroids, orby lengthening the duration of the infusions. One 16-year-old boy who entered the study with a history of small-vesselstrokes continued to experience strokes during the study.One male patient tested positive for IgG anti–agalsidasealfa antibodies during the study. No increase in the incidenceof adverse events was apparent for this patient. No IgE anti-bodies were observed.

Exploratory Efficacy MeasuresPlasma Gb3. Plasma Gb3 levels were elevated in boys atbaseline but not in girls (normal = <3.0 nmol/mL).16 MeanGb3 levels in boys declined from 7.91 � 3.09 nmol/mL

833

Table. Incidence of treatment-emergent adverse eventsoccurring in 2 or more patients

No. of patients(% of patients)

experiencing adverse event

System organclass/preferred term

Possibly relatedto treatment

Probably relatedto treatment

Any system organ class 11 (45.8%) 4 (16.7%)Nervous system disorder 5 (20.8%) 2 (8.3%)

Headache 3 (12.5%) 2 (8.3%)Dizziness 3 (12.5%) 0 (0%)

Vascular disorders 2 (8.3%) 1 (4.2%)Flushing 2 (8.3%) 1 (4.2%)

Respiratory, thoracic,and mediastinal disorders

4 (16.7%) 0 (0%)

Cough 2 (8.3%) 0 (0%)Dyspnea NOS 3 (12.5%) 0 (0%)

Gastrointestinal disorders 1 (4.2%) 2 (8.3%)Nausea 1 (4.2%) 2 (8.3%)

General disorders 8 (33.3%) 4 (16.7%)Pyrexia 2 (8.3%) 4 (16.7%)Chest pain 5 (20.8%) 0 (0%)Rigors 1 (4.2%) 4 (16.7%)

Figure 1. The effect of agalsidase alfa on urine sediment Gb3in boys with Fabry disease. *P < .05, **P < .01, ***P < .001compared with baseline (paired t test). The dashed and dottedlines represent the mean and range of values reported fora population of 1140 subjects without Fabry disease.31

THE JOURNAL OF PEDIATRICS � www.jpeds.com Vol. 156, No. 5

(n = 19) at baseline to 4.21 � 1.33 nmol/mL at 6 months(P < .001) and remained significantly reduced throughoutthe extension study. After 4 years of treatment, mean Gb3was 4.03� 0.887 nmol/mL (P < .05 compared with baseline).The boy who developed IgG antibodies during the study hada baseline plasma Gb3 level of 4.85 nmol/mL at baseline anda final plasma Gb3 level of 4.36 nmol/mL.

Urine Gb3. Urine sediment Gb3 levels were elevated inboys at baseline. Agalsidase alfa administration decreasedurine sediment Gb3 levels, with the changes becoming signif-icantly different from baseline values by 18 months of therapy(Figure 1). Urine Gb3 levels were not elevated in girls at base-line.16 The boy who developed IgG antibodies during thestudy failed to demonstrate a reduction in urine sedimentGb3.

Cardiac Structure and Function. Individual left ventric-ular mass (LVM) indexed to height2.7 was within the normalrange for children (<51 g/m2.7 in boys and <48 g/m2.7 ingirls). No significant changes in mean LVM were observedduring the 4 years of treatment and no trends toward increas-ing or decreasing LVM were observed (data not shown).Mean electrocardiographic measures (ie, heart rate, PR inter-val, QRS duration, and QTc) were within normal range atbaseline19 and remained within the normal range duringthe 4 years of treatment. During the first 6 months of agalsi-dase alfa, all indices of heart rate variability were significantlyimproved in boys.16 These improvements were sustainedduring up to 4 years of treatment, as illustrated in Figure 2(available at www.jpeds.com). Baseline mean indices of heartrate variability in girls with Fabry disease were not differentthan those seen in the non-Fabry pediatric population.16,20

Kidney Function. Mean eGFR was 121.0 � 24.3 mL/min/1.73 m2 at baseline and after 4 years was 121.7� 25.0 mL/min/

834

1.73 m2. Five boys ages 8.4 to 16.2 years had baseline eGFR>135 mL/min/1.73 m2, which indicates hyperfiltration. After1 year of agalsidase alfa, the abnormally high eGFR decreasedfrom 143.4 � 6.8 to 121.3 � 5.6 mL/min/1.73 m2 (P < .05,paired t test) in this subgroup. One 16.8-year-old boy, whohad a baseline eGFR below 90 mL/min/1.73 m2 at baseline(85.5 mL/min/1.73 m2), demonstrated an increase into thenormal range ($90 mL/min/1.73 m2) after 6 months of agal-sidase alfa (110 mL/min/1.73 m2) and remained above 90 mL/min/1.73 m2 for the remainder of the study. This patient didnot exhibit proteinuria during the study.

Nine of the 12 patients who completed at least 1 year of theextension study had a quantitative baseline albuminuriameasurement. In this group, average albumin excretion atbaseline was 7.6 � 5.2 mg/24 hours (range, 1.7 to 17.6 mg/24 hours). In the 10 patients who completed 4 years of agal-sidase alfa, final average albumin excretion was 11.6 � 7.7mg/24 hours (range, 4.9 to 27.0 mg/24 hours). Two of these10 patients exhibited single albuminuria measurementsslightly in excess of 30 mg/24 hours (the threshold for micro-albuminuria) during the first year of treatment (40 mg/24hours in a 16.2-year-old boy and 31 mg/24 hours in a 10.3-year-old boy). Both patients were within the normal rangeat 4 years. The 4 children in the original study who demon-strated microalbuminuria at baseline16 did not participatein this extension study.

Pain. In the 17 boys who had baseline Brief Pain Index(BPI) pain assessments, the mean ‘‘pain at its worst’’ scorewas 6.05 � 3.29 at baseline. After 1 year of agalsidase alfa,the mean ‘‘pain at its worst’’ score declined to 3.19 � 3.21(P < .001, paired t test). In this group, the ‘‘pain at its worst’’score was significantly reduced at nearly all assessments dur-ing the study (Figure 3). Similar reductions were seen in theBPI ‘‘average pain’’ score. The mean BPI ‘‘average pain’’ score

Schiffmann et al

Figure 3. The effect of agalsidase alfa on Brief Pain Index‘‘pain at its worst’’ score in boys with Fabry disease. *P < .05,**P < .01, ***P < .001 compared with baseline.

May 2010 ORIGINAL ARTICLES

in boys was 2.96 � 2.16 at baseline, was significantly im-proved to 1.70 � 1.87 after 1 year of agalsidase alfa(P < .05, paired t test), and remained significantly reducedthrough 4 years.

Sweat Measurement. Average baseline sweat volume was0.54 � 0.47 mL/mm2 in the 12 boys and 1 girl who partici-pated in QSART testing. At 6 months, the measured sweatvolume was 0.72 � 0.68 mL/mm2 (n = 13) and at 12 monthswas 0.57 � 0.57 mL/mm2 (n = 13). At the final measurementat 3.5 years, the volume was 0.11 � 0.12 (n = 6).

Height and Weight. The growth rate in boys treated withagalsidase alfa was normal when compared with boys in thenon-Fabry population (Figure 4).21 Several boys appearedto experience an increased rate of weight gain comparedwith their non-Fabry counterparts, but no overall trendwas observed. The single girl who was followed long-term ex-perienced no effect on growth but did appear to gain weightfaster than expected during treatment with agalsidase alfa(data not shown).

Discussion

We report an evaluation of enzyme replacement therapy ina clinical trial setting in children with Fabry disease. The re-sults indicate that agalsidase alfa was generally well toleratedduring 4 years of treatment at 0.2 mg/kg every other week.In addition, the analysis of the exploratory efficacy variablesrevealed significant improvement in pain and heart rate var-iability and reductions in plasma and urine Gb3 levels occur-ring within 6 to 12 months and sustained through 4 years.Importantly, the presence of IgG antibodies directed at agal-sidase alfa was low, and no IgE antibodies were detected.

The low incidence of anti–agalsidase alfa IgG antibodies inthe present study is similar to other experience with agalsi-

Four-Year Prospective Clinical Trial of Agalsidase Alfa in Children

dase alfa in children. In a 6-month, open-label study con-ducted by Ramaswami et al, 1 of 9 treated boys developedIgG antibodies directed against agalsidase alfa. The low im-munogenicity of agalsidase alfa at the typically administereddose in boys is in contrast to the results reported for boystreated with agalsidase beta. Wraith et al14 treated 14 boysand 2 girls with Fabry disease by administering agalsidasebeta at 1.0 mg/kg every other week for a period of 48 weeksand reported that 11 of 14 boys developed anti–agalsidasebeta IgG antibodies. The potential impact of IgG antibodieson the response to enzyme replacement therapy for Fabrydisease remains unresolved. In adults (and in the one IgG-positive boy in the present study), IgG antibodies appear toattenuate the reduction in urinary Gb3 levels11,22,23 but no ef-fect on clinical outcomes has been demonstrated.11,23

The sustained improvement in the time-domain indices ofheart rate variability in boys is important because it suggestsa long-term improvement in cardiac autonomic innervation.Reduced heart rate variability is seen in adults with diabetesand is associated with a 2-fold increase in cardiac mortalitythat may be caused by sudden death.24 The effect of reducedheart rate variability in adults with Fabry disease has not beeninvestigated, but it is possible that it contributes to the in-creased cardiac mortality seen in this population.5

Chronic neuropathic pain associated with Fabry diseasetypically has its onset in childhood2-4,25 and was present inthis study population. A BPI ‘‘pain at its worst score’’ above5 is considered to be associated with pain interference withactivities of daily living (eg, physical activity, mood, sleep,and social activities).26 In the present study, agalsidase alfareduced the mean BPI ‘‘pain at its worst score’’ from aboveto well below this threshold after 1 year. Importantly, thisimprovement was sustained through 4 years in boys. Im-provement in BPI pain scores during 6 months of agalsidasealfa therapy was also observed in the study by Ramaswamiet al.15

Average LVM did not increase during 4 years of treatmentwith agalsidase alfa in the present study. Although boys andgirls with Fabry disease appear to have larger LVM than chil-dren without Fabry disease,20 recent cross-sectional studieswould suggest that left ventricular hypertrophy is unusualin children with Fabry disease.25,27 The constancy of LVMduring the present study could be interpreted as an effectof agalsidase alfa, but the rate of increase in cardiac wallthickness in untreated children with Fabry disease is un-known. Long-term follow-up should clarify whether earlyinitiation of treatment with agalsidase alfa reduces the rateof incidence of cardiac complications.

Although renal manifestations of Fabry disease usually oc-cur in adulthood, children with Fabry disease may presentwith mild renal dysfunction including hyperfiltration, smallreduction in GFR, and microalbuminuria.14,16 Hyperfiltra-tion and microalbuminuria may be the initial signs of kidneydysfunction in Fabry disease. eGFR was normalized in the 5children with baseline hyperfiltration as well as in the singlepatient with a small baseline deficit of eGFR during 4 yearsof agalsidase alfa. As for microalbuminuria, only 2 children

with Fabry Disease 835

Figure 4. Change in height and weight in boys during treat-ment with agalsidase alfa. Longitudinal data for individualpatients are connected by lines. The average �2 standarddeviations in the non-Fabry populations are shown by solidand dotted lines.21

THE JOURNAL OF PEDIATRICS � www.jpeds.com Vol. 156, No. 5

had transient microalbuminuria during the first year of treat-ment. However, the fact that after 4 years of treatment all pa-tients demonstrated normal eGFR and urine albumin levelssuggests that early initiation of agalsidase alfa may have sta-bilized kidney function. A longer-term study would be neces-sary to confirm this.

The ability to sweat is often impaired in children withFabry disease,2,15 and QSART has been used to demonstratethis deficiency.2 In the first 6 months of treatment with agal-sidase alfa, the QSART results suggested an improvement insweat function in the present study population.2 In this ex-tension study, the initial improvement did not appear to besustained. A possible reason for this lack of long-term re-sponse is the poor follow-up testing in which only 6 patientswere evaluated at treatment times exceeding one year. In ad-dition, the reproducibility of QSART may be too low to reli-ably detect small, clinically important improvements infunction.28 The utility of this assessment in the long-termfollow-up of Fabry disease patients is unknown.

836

As noted above, agalsidase beta has been the subject ofa single clinical trial in pediatric patients.14 Superficial capil-lary endothelial Gb3 deposits were cleared at week 24 andplasma Gb3 was reduced in boys. Gastrointestinal symptomstended to decrease during the study. Body mass index was be-tween the 6th and 97th percentile at baseline and between the5th and 75th percentile at study end. Infusion reactions oc-curred in 38% of patients (6 boys), despite the use of routinepremedication with an antipyretic.

Studies conducted in adults suggest that enzyme replace-ment therapy can slow the progression of Fabry diseaseprovided that it is initiated before irreversible organ dam-age has occurred.11,29 Tissue accumulation of Gb3 beginsduring fetal development,30 but it takes years before signsand symptoms emerge. It is attractive to speculate thatenzyme replacement therapy initiated in childhood mightdelay or prevent major organ damage later in life, but itwill be critically important to demonstrate that such anapproach can be safely undertaken.

This study was conducted in a population that was re-ferred to tertiary care centers and may be different fromone encountered in a primary care setting. The patients,reflecting the broad phenotype of pediatric Fabry disease,were not selected for specific signs and symptoms. Aconcurrently followed placebo group would have elevatedthe level of evidence of our findings, but the commercialavailability of enzyme replacement therapy for Fabry diseasewould have made the implementation of a placebo-con-trolled trial difficult. The study was primarily designed asa safety study. n

The authors acknowledge Edward Weselcouch, PhD, of PharmaWrite,for providing editorial assistance, which was funded by Shire HGT. Theauthors acknowledge the other physicians who provided care and mon-itoring of patients in this study, including Alison Whelan, MD (Wash-ington University School of Medicine, St Louis, MO), Yoko Broussard,MD (CHRISTUS St Patrick Hospital, Lake Charles, LA), Leslie Car-roll, MD (All Children’s Hospital, St Petersburg, FL), Lynda Bideau,MD (Children’s Physicians Group, Palm Beach Gardens, FL), andMichael Cohen, MD (Stafford, VA).

Submitted for publication Jun 15, 2009; last revision received Sep 3, 2009;

accepted Nov 4, 2009.

Reprint requests: Dr Raphael Schiffmann, Institute of Metabolic Disease,

Baylor Research Institute, 3812 Elm Street, Dallas, TX 75226. E-mail: Raphael.

Schiffmann@baylorhealth.edu.

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with Fabry Disease 837

Figure 2. Effect of agalsidase alfa on measures of heart rate variability in boys with Fabry disease. *P < .05, **P < .01 comparedwith baseline (paired t test). SDNN, standard deviation of the normal beat to normal beat intervals over the entire 2-hour recordingperiod; SDNN-I, mean of the SD of all of the filtered RR intervals for all 5-minute intervals of the analysis; SDANN-I, SD of themean of all filtered RR intervals for all 5-minute segments of the analysis; r-MSSD, square root of the mean of the sum of squaresof differences between adjacent filtered RR intervals during the 2-hour recording period; pNN50, the percentage of adjacentfiltered RR intervals that are >50 ms for the entire 2-hour analysis period. Baseline values are mean � SEM.

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