The NEW ENG LAN D
`JOURNAL of M E D IC I NE
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`1503 A Common MUC5B Promoter Polymorphism
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`1513 Systemic Administration of PRO0Sl in Duchenne's
`Muscular Dystrophy
`N.M. Goemans and Others
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`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`The NEW ENGLAND JOURNAL of MEDICINE
`
`l~l __________ o_R_1_G_1_N_A_ L_A_R_T_1c_L_E _________ ~JI
`
`Systemic Administration of PRO0Sl
`in Duchenne's Muscular Dystrophy
`
`Nathalie M. Goemans, M.D., Mar Tulinius, M.D., Ph.D.,
`Johanna T. van den Akker, Ph.D., Brigitte E. Burm, Ph.D., Peter F. Ekhart, M.Sc.,
`Niki Heuvelmans, Tjadine Holling, Ph.D., Anneke A.Janson,
`GerardJ. Plate~liurg, M.Sc.,Jessica A. Sipkens, M.Sc.,J.M. Ad Sitsen, M.D., Ph.D.,
`Annemieke Aartsma-Rus, Ph.D., Gert:Jan B. van Ommen, Ph.D.,
`Gunnar Buyse, M.D., Ph.D., Niklas Darin, M.D., Ph.D.,
`JanJ. Verschuuren, M.D., Ph.D., Giles V. Campion, M.D.,
`SjefJ. de Kimpe, Ph.D., and Judith C. van Deutekom, Ph.D.
`
`ABSTRACT
`
`BACKGROUND
`Local intramuscular administration of the antisense oligonucleotide PRO0Sl in pa(cid:173)
`tients with Duchenne's muscular dystrophy with relevant mutations was previously
`reported to induce the skipping of exon 51 during pre-messenger RNA splicing of the
`dystrophin gene and to facilitate new dystrophin expression in muscle-fiber mem(cid:173)
`branes. The present phase 1-2a study aimed to assess the safety, pharmacokinetics,
`and molecular and clinical effects of systemically administered PRO0Sl.
`
`METHODS
`We administered weekly abdominal subcutaneous injections of PRO0Sl for 5 weeks
`in 12 patients, with each of four possible doses (0.5, 2.0, 4.0, and 6.0 mg per kilogram
`of body weight) given to 3 patients. Changes in RNA splicing and protein levels in the
`tibialis anterior muscle were assessed at two time points. All patients subsequently
`entered a 12-week open-label extension phase, during which they all received PRO0Sl
`at a dose of 6.0 mg per kilogram per week. Safety, pharmacokinetics, serum creatine
`kinase levels, and muscle strength and function were assessed.
`
`RESULTS
`The most common adverse events were irritation at the administration site and,
`during the extension phase, mild and variable proteinuria and increased urinary
`a 1-microglobulin levels; there were no serious adverse events. The mean terminal
`haif-life of PROOSl in the circulation was 29 days. PRO0Sl induced detectable,
`specific exon-51 skipping at doses of2.0 mg or more per kilogram. New dystrophin
`expression was observed between approximately 60% and 100% of muscle fibers in
`10 of the 12 patients, as measured on post-treatment biopsy, which increased in a
`dose-dependent manner to up to 15.6% of the expression in healthy muscle. After the
`12-week extension phase, there was a mean (±SD) improvement of35.2±28.7 m (from
`the baseline of 384±121 m) on the 6-minute walk test.
`
`CONCLUSIONS
`Systemically administered PRO0Sl showed dose-dependent molecular efficacy in
`patients with Duchenne's muscular dystrophy, with a modest improvement in the
`6~minute walk test after 12 weeks of extended treatment. (Funded by Prosensa
`Therapeutics; Netherlands National Trial Register number, NTR1241.)
`
`From the Department of Pediatric Neu(cid:173)
`rology, University Hospitals Leuven, Leu(cid:173)
`ven, Belgium (N.M.G., G.B.); the De(cid:173)
`partment of Pediatrics, University of
`Gothenburg, Queen Silvia Children's
`Hospital, Gothenburg, Sweden
`(M .T.,
`N.D.); ClinPharMed, Ermelo (J .M.A.S.);
`Prosensa Therapeutics, Leiden (J.T.A,
`B.E.B., P.F.E., N.H., T.H., A.A.J., G.J.P.,
`J.A.S., G.V.C., S.J.K., J.C.D.); and the De(cid:173)
`partment of Human Genetics, Center for
`Human and Clinical Genetics (A.A.-R.,
`G.-j.B.O.), and the Department of Neu(cid:173)
`rology (J.J.V.), Leiden University Medical
`all in the Netherlands.
`Center, Leiden -
`Address reprint requests to Dr. van
`Deutekom at Prosensa, P.O. Box 281,
`2300 AG Leiden, the Netherlands, or at
`j.vandeutekom@prosensa.nl.
`
`This article (10.1056/NEJMoal011367) was
`published on March 23, 2011, at NEJM.org
`
`N EnglJ Med 2011;364:1513-22.
`Copyright© 2011 Massachusetts Medical Society.
`
`N ENGLJ MED 364;16 NEJM.ORG APRIL 21, 2011
`
`1513
`
`

`

`The NEW ENGLAND JOURNAL of MEDICINE
`
`D UCHENNE'S MUSCULAR DYSTROPHY IS
`
`an X-linked recessive muscle disorder, af-.
`fecting 1 in 3500 newborn boys.1 Patients
`have severe, progressive muscle wasting, leading
`to early death. 2 •3 The disease is caused by muta(cid:173)
`tions in the dystrophin gene (DMD), 4•5 leading to
`disruption of the open reading frame, dystrophin
`deficiency at the myofiber membrane, and con(cid:173)
`tinued fiber degeneration.6 -8 Mutations in the
`same gene cause Becker's muscular dystrophy,
`but the open reading frame is maintained, per(cid:173)
`mitting the production of semifunctional dystro(cid:173)
`phin proteins and a typically milder phenotype
`and longer life span. 6 -9
`A promising therapeutic strategy involves anti(cid:173)
`sense oligonucleotides that induce specific exon
`skipping during pre-messenger RNA (mRNA)
`splicing,10 aimed at reading-frame correction and
`production of transcripts like those in patients
`with Becker's muscular dystrophy.11 Although the
`functionality of the resulting protein may vary,
`this treatment could delay or even stop disease
`progression and improve function in the remain(cid:173)
`ing muscle.12•13 The antisense oligonucleotides are
`chemically modified to resist nucleases and pro(cid:173)
`mote RNA binding and are designed to have high
`sequence specificity. In studies in the mdx mouse
`model, oligonucleotides with chemical properties
`similar to those of 2'-0-methyl phosphorothioate
`RNA were taken up in dystrophin-deficient mus(cid:173)
`cle up to 10 times as much as in healthy muscle
`tissue, most likely owing to increased permeabil(cid:173)
`ity of the muscle myofiber membrane.14 In addi(cid:173)
`tion, 4 to 8 weeks' subcutaneous delivery of the
`oligonucleotides resulted in a steady increase in
`oligonucleotide levels, exon skipping, and dystro(cid:173)
`phin levels.14
`Exon skipping provides a mutation-specific,
`and thus potentially persona,lized, therapeutic ap(cid:173)
`proach for patients with Duchenne's muscular
`dystrophy. Since mutations cluster around exons
`45 to 55 of DMD, the skipping of one specific exon
`may be therapeutic for patients with a variety of
`mutations. The skipping of exon 51 affects the
`largest subgroup of patients (approximately 13%),
`including those with deletions of exons 45 to 50,
`48 to 50, 50, or 52.15
`PRO0Sl, a 2'-0-methyl phosphorothioate oli(cid:173)
`goribonucleotide that induces exon 51 skipping,
`was previously tested in patients with Duchenne's
`muscular dystrophy by means of local intramus(cid:173)
`cular administration of a single dose.16 The com-
`
`pound produced sarcolemmal dystrophin in 64
`to 97% of myofibers. The amount of dystrophin
`ranged from 17 to 35% of control levels. The cur(cid:173)
`rent dose escalation and follow-up extension study
`assessed the safety, tolerability, pharmacokinetics,
`and molecular and cli.nical effects of subcutane(cid:173)
`ously administered PRO051.
`
`METHODS
`
`PATIENTS
`We recruited patients with Duchenne's muscular
`dystrophy who were 5 to 16 years of age and had
`mutations that could be corrected by means of
`inducing exon 51 skipping. Inclusion and exclu(cid:173)
`sion criteria were similar to those in the previous
`study.16 Briefly, patients with no evidence of dys(cid:173)
`trophin in 5% or more of fibers on previous diag(cid:173)
`nostic muscle biopsy were eligible to participate in
`the study. Concurrent glucocorticoid treatment was
`permitted. Eligibility criteria also included an esti(cid:173)
`mated life expectancy of 6 months or more, no seri(cid:173)
`ous preexisting medical conditions, and no depen(cid:173)
`dency on assisted ventilation (or a forced expiratory
`volume in 1 second or forced vital capacity of 60%
`or less of the predicted value). Additional details are
`given in the Supplementary Appendix (available
`with the full text of this article atNEJM.org). Writ(cid:173)
`ten informed consent was obtained from all pa(cid:173)
`tients over 12 years of age or, for younger patients,
`from their parents.
`
`STUDY DESIGN
`In this open-label, dose-escalation, phase 1- 2a
`study, 12 patients were to receive weekly abdom(cid:173)
`inal subcutaneous injections of PRO051 (from 0.5
`to 10 mg per kilogram of body weight, with 3 pa(cid:173)
`tients receiving each dose) for 5 weeks. The spe(cid:173)
`cific increases in dose were determined after
`analysis of safety and dystrophin levels in muscle(cid:173)
`biopsy specimens. Since early increases in dys(cid:173)
`trophin levels were observed in patients receiving
`0.5, 2.0, and 4.0 mg per kilogram of body weight
`(3 patients in each dose cohort), the maximum
`study dose was set at 6.0 mg per kilogram of
`body weight (which was the dose the last cohort
`of 3 patients received).
`Assessments of safety (the primary outcome)
`and pharmacokinetics and molecular and clinical
`effects (secondary outcomes) were made at regu(cid:173)
`lar intervals. Tibialis anterior muscle biopsy was
`performed at baseline and 2 weeks after the last
`
`1514
`
`N ENGL) MED 364;16 NEJM.ORG APRIL 21, 2011
`
`

`

`PRO051 IN DUCHENNE'S MUSCULAR DYST ROPHY
`
`dose of PRO051 in the 0.5-mg group and at 2 and
`7 weeks after the last dose in the three other
`groups. After an interval of 6 to 15 months after
`the last dose, each patient restarted treatment at
`6.0 mg per kilogram of body weight per week,
`with close monitoring of safety and clinical-effi(cid:173)
`cacy measures. The current report includes data
`through 12 weeks of restarted treatment (with
`biopsy not conducted at 12 weeks). No formal
`statistical testing was performed, owing to the
`small number of patients. Data are presented for
`individual patients' and are also summarized.
`The study was sponsored by Prosensa Thera(cid:173)
`peutics (Leiden, the Netherlands) and performed
`in compliance with Good Clinical Practice guide(cid:173)
`lines, the provisions of the Declaration of Hel(cid:173)
`sinki, the European Directive 2001/20/EC, and lo(cid:173)
`cal regulations in Belgium and Sweden. The
`studies were approved by the local independent
`ethics committees and authorized by the Com(cid:173)
`petent Authorities of Belgium and Sweden. The
`study was conducted in accordance with the pro(cid:173)
`tocol (available at NEJM.org). All authors con(cid:173)
`tributed to the study design, participated in the
`collection and analysis of the data, had complete
`and free access to the data, jointly wrote the
`manuscript, and vouch for the completeness and
`accuracy of the data and analyses presented.
`
`STUDY DRUG
`The antisense oligonucleotide PRO0Sl (GSK2402968)
`(5'-UCAAGGAAGAUGGCAUUUCU-3') with full(cid:173)
`length 2'-0-methyl-substituted ribose moieties
`and phosphorothioate internuckotide linkages,16
`was provided in 0.5-ml glass vials in sodium phos(cid:173)
`phate-buffered saline (100 mg pet; milliliter). Non(cid:173)
`clinical safety data are provided in the Supplemen(cid:173)
`tary Appendix.
`
`globulin G antibodies against dystrophin, serum
`samples obtained before and 120 days after treat(cid:173)
`ment were analyzed.17
`
`PHARMACOKINETIC ASSESSMENTS
`We assessed plasma levels of PRO051 during the
`dose-escalation phase of the study, by using a
`validated hybridization ligation assay adapted
`from Yu and colleagues18 (see the Supplementary
`Appendix).
`
`ASSESSMENTS OF RNA AND PROTEIN
`Details of the RNA and protein analyses are giv(cid:173)
`en in the Supplementary Appendix. To detect
`exon-51 skipping, total RNA was isolated from
`10 to 15 mg of muscle tissue and analyzed by
`means of reverse-transcriptase-polymerase-chain(cid:173)
`reaction (RT-PCR) assay and sequencing, as re(cid:173)
`ported previously.16•19•2° For detection of new dys(cid:173)
`trophin expression, immunofluorescence analysis
`of serial 8-µ,m cross sections and Western blot
`analysis of total protein extracts isolated from
`20 to 30 mg of muscle tissue were performed ac(cid:173)
`cording to methods described previously.16,20
`
`CLINICAL ASSESSMENTS
`Muscle strength assessments included both quan(cid:173)
`titative testing of 10 muscle groups according
`to the quantitative measuring system of the Co(cid:173)
`operative International Neuromuscular Research
`Group21•22 and manual testing according to the
`averaged Medical Research Council score of 34
`muscle groups. In addition, timed functional tests
`(10-m walk, 4-stair climb, and time to rise from
`floor), the 6-minute walk test, and pulmonary(cid:173)
`function tests were performed.
`
`RESULTS
`
`SAFETY AND TOLERABILITY
`Safety was monitored as described previously.1 6
`Changes in aspartate aminotransferase and ala(cid:173)
`nine aminotransferase levels were interpreted in
`relation to changes in creatine kinase levels for
`the evaluation of hepatotoxicity. Urine was mon(cid:173)
`itored for a 1-microglobulin, proteinuria, and he(cid:173)
`maturia. Creatinine clearance was not measured,
`since plasma creatinine levels change with changes
`in muscle mass in patients with Duchenne's mus(cid:173)
`cular dystrophy. Complement activation, coagula(cid:173)
`tion profiles, and inflammatory responses were
`monitored. For detection of putative iminuno-
`
`PATIENTS
`Twelve patients were prescreened with the use of
`an in vitro cell-based PRO051 assay.16 The specific
`mutation and a positive response to PRO051 were
`confirmed by means of RNA and sequence analy(cid:173)
`sis. The 12 patients had a mean age of 9.2 years
`(range, 5 to 13). All 12 met the inclusion criteria,
`received PRO051 treatment, completed the dose(cid:173)
`escalation phase, and entered the extension phase.
`For 7 of the 12 patients, a prestudy diagnostic bi(cid:173)
`opsy was available, showing less than 5% "rever(cid:173)
`tant" (dystrophin-positive) muscle fibers. Baseline
`characteristics of the 12 patients are presented in
`
`N EN G LJ M ED 3 64;16
`
`N EJM.ORG A PRIL 2 1, 2011
`
`1515
`
`

`

`The NEW ENGLAND JOURNAL of MEDICINE
`
`Table 1. Adverse Events That Occurred in More Than
`2 Patients during the 12-Week Extension Phase.
`
`Event
`
`Proteinuria
`
`Elevated urinary a 1-microglobulin levels
`
`Injection site
`
`Erythema and inflammation
`
`Hematoma or bruising
`
`Tenderness
`
`Irritation or itching
`
`Moderate pain during injection
`
`Common cold
`
`Gastroenteritis
`
`Pain*
`
`No. of Patients
`
`12
`
`11
`
`9
`
`6
`
`5
`
`3
`
`4
`
`4
`
`4
`
`3
`
`* Pain was in the stomach in 1 patient, in the foot in 1, and
`in the arm after immunization in 1.
`
`Table 1 in the Supplementary Appendix. All pa(cid:173)
`tients had been receiving a stable dose of glucocor(cid:173)
`ticoids for at least 1 year at the time of enrollment.
`
`SAFETY AND ADVERSE EVENTS
`No patients withdrew from the dose-escalation or
`extension phases of the study, and no serious ad(cid:173)
`verse events were reported. After the 12 weeks of
`extended treatment with PRO051 (6.0 mg per kilo(cid:173)
`gram of body weight per week in all 12 patients), a
`total of 120 adverse events of mild or moderate
`intensity were reported. The most common events
`(Table 1) considered to be definitely or probably
`causally related to the study drug were mild reac(cid:173)
`tions at the injection site and increased urinary
`a 1-microglobulin levels. Proteinuria, defined as a
`protein level above the upper limit of the normal
`range of 0.15 g per liter, was observed in all 12
`patients (mean [±SD] protein level, 0.078±0.038
`at baseline and 0.206±0.119 at week 12 of the
`extension phase). This may represent an adaptive
`process within renal tubules, which may absorb
`oligonucleotides; thus, this finding warrants fur(cid:173)
`ther monitoring. Pain in the lower leg, exanthema,
`dry skin, and stomach pain were also reported.
`None of these events led to changes in the injec(cid:173)
`tion schedule or treatment discontinuation.
`No clinically significant changes were ob(cid:173)
`served on physical examination, in vital signs, or
`on electrocardiograms, as compared with base(cid:173)
`line data. No drug-related decreases in platelet
`counts or prolonged activated partial-thrombo-
`
`plastin time values were observed. None of the
`patients showed liver-enzyme changes suggesting
`hepatotoxicity. No dystrophin antibodies were de(cid:173)
`tected in serum samples.
`
`PHARMACOKINETIC PROFILE
`PRO051 was rapidly absorbed and distributed, with
`peak levels occurring between 2 and 3 hours af(cid:173)
`ter administration (Fig. lA and 1B in the Supple(cid:173)
`mentary Appendix) and a decline in plasma levels
`to less than 15% of the maximal level observed
`at 24 hours. In contrast to peak plasma levels, the
`predosing trough levels increased with increasing
`numbers of injections, as anticipated.23•24 The
`
`overall terminal plasma half-life, as ascertained
`over the 13-week period after the end of the
`5-week dose-escalation phase, ranged from 19 to
`56 days (geometric mean, 29 days) (Fig. lC in the
`Supplementary Appendix).
`
`EFFECTS ON RNA
`Muscle-biopsy samples were analyzed at 2 weeks
`and 7 weeks after the end of the dose-escalation
`phase. No effect of PRO051 on RNA level was
`detected in any of the three patients receiving a
`dose of0.5 mg per kilogram of bodyweight (Fig.
`lA). In the higher-dose cohorts, however, exon-
`51 skipping was observed at both time points in
`one patient receiving 2.0 mg per kilogram of
`body weight (Fig. lB) and in all six patients re(cid:173)
`ceiving 4.0 or 6.0 mg per kilogram of body
`weight, albeit at variable levels (Fig. lC and lD).
`Exon-51 skipping was still detectable in these
`seven patients at 7 weeks after the dose-escala(cid:173)
`tion phase. The specificity of exon-51 skipping
`was confirmed by means of sequence analysis.
`No unanticipated drug-induced splicing events
`were detected in overlapping RT-PCR fragments
`throughout the full-length DMD transcript.
`
`EFFECTS ON PROTEIN EXPRESSION
`Essentially no dystrophin expression was observed
`on immunofluorescence analysis of muscle-tissue
`sections obtained at baseline in the group receiv(cid:173)
`ing 0.5 mg per kilogram of body weight, although
`two patients showed a few dystrophin-positive
`("revertant") fibers 2s-27 (Fig. 2A). In all three pa(cid:173)
`tients in this group, new dystrophin expression
`was first observed at 2 weeks after the end of
`treatment, with 20 to 88% of fibers positive for
`dystrophin and slightly higher dystrophin signal
`intensities than seen in baseline samples (Table 2).
`
`1516
`
`N ENGLJ MED 364;16 NEJM.ORG APRIL 21, 2011
`
`

`

`PRO051 IN DUCHENNE'S MUSCULAR DYSTROPHY
`
`Figure 1. Effect of PRO0Sl on RNA Processing
`at 2 Weeks and 7 Weeks after the Last Administration
`in the Dose-Escalation Phase.
`Results of reverse-transcriptase (RT)-polymerase(cid:173)
`chain-reaction (PCR) analysis of RNA isolated from
`muscle-biopsy specimens from the patients are shown
`for one patient per dose cohort: Patient 1 (Panel A)
`and Patient 4 (Panel B), whose mutations result in the
`deletion of exon 52 in the dystrophin gene (DMD); and
`Patient 9 (Panel C) and Patient 12 (Panel D), whose
`mutations result in the deletion of exons 45 to 50 in
`DMD. A positive control specific to each patient was
`derived from the in vitro PRO051 prescreening proce(cid:173)
`dure (Pt-ctrl). The arrows indicate the transcript frag(cid:173)
`ment anticipated if exon 51 were skipped during splic(cid:173)
`ing in the given patient. In the negative-control
`samples, no reverse transcriptase was added (-RT).
`DNA size-marker samples (M) are also shown. Be(cid:173)
`cause of the small amount of the patients' samples,
`high-sensitivity PCR conditions were used, which renders
`inaccurate the quantification of skipping efficiencies.
`
`Both the average number of dystrophin-positive
`fibers and the average dystrophin signal inten(cid:173)
`sity increased with increasing dose, with similar
`values at 2 weeks and 7 weeks after treatment
`(Fig. 2A and Table 2). The proportions of dystro(cid:173)
`phin-expressing fibers were between 80 and
`100% in six patients and between 56 and 75%
`in four others; the remaining two patients had
`muscle-biopsy specimens of relatively poor qual(cid:173)
`ity, in which only up to 20% of positive fibers were
`seen, which hindered accurate dystrophin signal
`detection. The average dystrophin signal intensity
`was highest in the groups receiving 4.0 mg per
`kilogram and 6.0 mg per kilogram at 2 weeks
`after the end of treatment, with a maximal signal
`of 15.6% of that observed in a control sample
`(Table 2). Plotting the average of the dystrophin
`signal intensities (vs. control), detected in the
`three patients per cohort, pooled per visit (base(cid:173)
`line, week 2 or week 7 post-treatment), showed a
`dose-dependent effect of PRO0Sl (Fig. 2B).
`Imrnunofluorescence findings were confirmed
`by analyses of total muscle-protein extracts on
`Western blotting (Fig. 2B and 2C and Table 2).
`In biopsy specimens from patients receiving
`0.5 mg per kilogram of body weight, low levels
`of dystrophin were detected at baseline (Fig.
`2C), consistent with the small numbers of dys(cid:173)
`trophin-positive ("revertant") fibers visualized on
`immunofluorescence analyses for two patients.
`No increase in dystrophin levels was observed
`in either of these patients at 2 weeks after the
`
`A Patient 1, 0.5 mg/kg
`-:k-~e,
`~e;
`~
`
`B Patient 4, 2.0 mg/kg
`
`~
`
`'::+-"I,
`~,Ii
`
`C Patient 9, 4.0 mg/kg
`
`~
`
`'::+-"I,
`~,Ii
`
`D Patient 12, 6.0 mg/kg
`
`'::+-"I,
`~,Ii
`
`~
`
`"
`
`~
`
`.
`
`•
`t
`'
`~~,--
`
`-..-
`
`..
`
`.
`
`-
`
`last dose of PRO0Sl during the escalation phase.
`In the higher-dose groups, the dystrophin-signal
`intensities at 2 weeks and 7 weeks after the last
`dose during the escalation phase were typically
`greater than the average intensity among the
`three baseline specimens from the lowest-dose
`group (Fig. 2C). Quantitative analysis of signal
`intensity, normalized for the variable levels of
`muscle-fiber content (as represented by dysferlin
`levels), suggested that the patients receiving the
`two highest doses of PROOSl (4.0 and 6.0 mg per
`kilogram) had dystrophin expression that was 1.5
`times to 8.2 times greater, respectively, than base(cid:173)
`line levels (i.e., the average signal intensity of 2.5
`with the dose of 0.5 mg per kilogram) (Table 2).
`
`N EN G L ) M ED 364;1 6 NEJ M .O RG APR IL 2 1, 2 0 11
`
`1517
`
`

`

`Th, NEW ENGLAND JOURNAL of MEDICINE
`
`Plotting the dystrophin-signal intensities detected
`on average in the three patients per group per
`visit indicated a dose-dependent effect of PRO051
`on dystrophin expression (Fig. 2B), similar to
`findings from immunofluorescence studies.
`
`CLINICAL FINDINGS
`In the dose-escalation phase, 5 weeks of treat(cid:173)
`ment with PRO051 resulted in increased dystro(cid:173)
`phin levels but did not induce clear, clinically
`relevant differences in muscle strength, timed
`functional tests, and pulmonary-function tests,
`either between or within the dose groups. The
`average distance walked in 6 minutes (Table 2
`and Fig. 3A), the distance walked per minute,
`and creatine kinase levels-were variable, consis(cid:173)
`tent with historical data for the age group of our
`patients. 29 However, after 12 weeks of treatment
`in the extension phase, there was improvement
`in the distance walked in 6 minutes (mean [±SD]
`improvement, 35.2±28.7 m); three patients (Pa(cid:173)
`tients 1, 2, and 7) showed an improvement qf
`65 m or more (Table 2 and Fig. 3B). This contrasts
`with the mean 37-m decrease in the 6-minute
`walking distance seen between the start of the
`dose-escalation study and the start of the exten(cid:173)
`sion study (time interval, 6 to 15 months) and to
`the expected decline in these patients during this
`study period (based on a previous report of a de(cid:173)
`cline ofll5 mover a total of52 weeks in a 7-year
`period30). No increase in specific muscle force
`was observed. There was minimal effect on se(cid:173)
`rum levels of creatine kinase, but the sample size
`was small, and the clinical disease stage was het(cid:173)
`erogeneous.
`
`DISCUSSION
`
`Our phase 1-2a study, consisting of a dose-esca(cid:173)
`lation phase and a follow-up extension phase,
`reports the molecular and clinical effects of sys(cid:173)
`temic weekly administration of an antisense oli(cid:173)
`gonucleotide in patients with Duchenne's mus(cid:173)
`cular dystrophy. No serious adverse events were
`reported, but receipt of PRO051 was associated
`with local skin reactions of mild to moderate in(cid:173)
`tensity, although none led to treatment discon(cid:173)
`tinuation. All patients had elevated urinary a 1 -
`microglobulin levels by week 12 of therapy, and
`all had variable proteinuria. However, given the
`decreased muscle mass in these patients, urinary
`protein:creatinine ratios are difficult to assess.
`Given the presence of dystrophin isoforms and
`
`Figure 2 (facing page). Effect of PRO051 on Dystrophin
`Expression Levels.
`Panel A shows the results ofimmunofluorescence
`analysis involving staining with the MANDYS106 dys(cid:173)
`trophin antibody,28 with increased dystrophin expres(cid:173)
`sion found in the membranes ofthe muscle fibers af(cid:173)
`ter treatment with PRO051 in all patients (only one
`patient per group is shown here). Few dystrophin-posi•
`tive "revertant" fibers 2
`s-27 were observed in biopsy
`specimens obtained at baseline, illustrated here for Pa(cid:173)
`tient 2. Panel B shows the dystrophin signal intensity
`in cross sections of muscle-tissue specimens, averaged
`for the three patients in each dose group at each time
`point after the end of treatment. Intensities are shown
`in two ways: as measured by the percentage of the con(cid:173)
`trol value (set at 100%) for the immunofluorescence
`analysis (after correction for the phosphate-buffered-(cid:173)
`saline, with results also averaged across three to six
`nonoverlapping images per cross section) and as mea(cid:173)
`sured with the use of Western blotting of protein ex(cid:173)
`tracts after normalization for the varying density and
`quality of muscle fiber (as indicated by the signal in(cid:173)
`tensities for reference protein dysferlin, calculated
`against average baseline sample intensities and report(cid:173)
`ed in arbitrary units [AU]). In addition, the percentage
`of dystrophin-positive fibers averaged across three to
`six nonoverlapping images per cross section is indicat(cid:173)
`ed across the top of the graph. Panel C (top) shows re(cid:173)
`sults of Western blot analysis (involving the dystrophin
`monoclonal antibody NCL-DYSl) of total protein ex(cid:173)
`tracts (300 to 500 µg loaded, depending on tissue
`quality) isolated from the patients' biopsy specimens
`(with results shown for one patient per dose). Patient 2
`has a positive dystrophin signal in the baseline biopsy
`specimen, consistent with the presence of few rever(cid:173)
`tant fibers. Panel C (top) also shows, for comparison ,
`blotting of 1 to 10 µg of total protein from a healthy
`gastrocnemius muscle-tissue sample. All samples were
`cohybridized with a dysferlin antibody to normalize for
`the variable levels of muscle fiber content (see bot(cid:173)
`tom). Because of the relatively high total-protein load(cid:173)
`ing required for dystrophin signal detection in our pa(cid:173)
`tients, quantitative comparison with a control sample
`was not considered accurate.
`
`revertant fibers, the risk for cell-mediated im(cid:173)
`munity to the new dystrophin was considered
`limited but should be evaluated further.
`Our pharmacokinetic studies indicate that
`PRO051 is rapidly absorbed and distributed,
`which limits the peak plasma levels and the po(cid:173)
`tential for acute adverse reactions. The fact that
`plasma trough · levels increased with repeated
`oligonucleotide administration suggests that tis(cid:173)
`sue, including muscle, gradually increases levels
`of PRO051. The terminal elimination half-life
`(ranging from 19 to 56 days) is similar to that of
`other second-generation phosphorothioate oligo(cid:173)
`nucleotides. 23•24 The half-life range suggests that
`
`1518
`
`N ENGLJ MED 364;16 NEJM . ORG APRIL21, 2011
`
`

`

`Th, NEW ENGLAND JOURNAL of MEDICINE
`
`Plotting the dystrophin-signal intensities detected
`on average in the three patients per group per
`visit indicated a dose-dependent effect of PRO051
`on dystrophin expression (Fig. 2B), similar to
`findings from immunofluorescence studies.
`
`CLINICAL FINDINGS
`In the dose-escalation phase, 5 weeks of treat(cid:173)
`ment with PRO051 resulted in increased dystro(cid:173)
`phin levels but did not induce clear, clinically
`relevant differences in muscle strength, timed
`functional tests, and pulmonary-function tests,
`either between or within the dose groups. The
`average distance walked in 6 minutes (Table 2
`and Fig. 3A), the distance walked per minute,
`and creatine kinase levels· were variable, consis(cid:173)
`tent with historical data for the age group of our
`patients. 29 However, after 12 weeks of treatment
`in the extension phase, there was improvement
`in the distance walked in 6 minutes (mean [±SD]
`improvement, 35.2±28.7 m); three patients (Pa(cid:173)
`tients 1, 2, and 7) showed an improvement qf
`65 m or more (Table 2 and Fig. 3B). This contrasts
`with the mean 37-m decrease in the 6-minute
`walking distance seen between the start of the
`dose-escalation study and the start of the exten(cid:173)
`sion study (time interval, 6 to 15 months) and to
`the expected decline in these patients during this
`study period (based on a previous report of a de(cid:173)
`cline of115 mover a total of52 weeks in·a 7-year
`period30). No increase in specific muscle force
`was observed. There was minimal effect on se(cid:173)
`rum levels of creatine kinase, but the sample size
`was small, and the clinical disease stage was het(cid:173)
`erogeneous.
`
`DISCUSSION
`
`Our phase 1-2a study, consisting of a dose-esca(cid:173)
`lation phase and a follow-up extension phase,
`reports the molecular and clinical effects of sys(cid:173)
`temic weekly administration of an antisense oli(cid:173)
`gonucleotide in patients with Duchenne's mus(cid:173)
`cular dystrophy. No serious adverse events were
`reported, but receipt of PRO051 was associated
`with local skin reactions of mild to moderate in(cid:173)
`tensity, although none led to treatment discon(cid:173)
`tinuation. All patients had elevated urinary a 1 -
`microglobulin levels by week 12 of therapy, and
`all had variable proteinuria. However, given the
`decreased muscle mass in these patients, urinary
`protein:creatinine ratios are difficult to assess.
`Given the