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2012
Enzyme-replacement therapy in life-threatening Enzyme-replacement therapy in life-threatening
hypophosphatasia hypophosphatasia
Michael P. Whyte
Washington University School of Medicine in St. Louis
William H. McAlister
Washington University School of Medicine in St. Louis
et al
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Whyte, Michael P.; McAlister, William H.; and et al, ,"Enzyme-replacement therapy in life-threatening
hypophosphatasia." The New England Journal of Medicine. 366,10. 904-913. (2012).
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original article
The
new engl a nd jour nal
o f
medicine
n engl j med 366;10 nejm.org march 8, 2012
904
Enzyme-Replacement Therapy
in Life-Threatening Hypophosphatasia
Michael P. Whyte, M.D., Cheryl R. Greenberg, M.D., Nada J. Salman, M.D.,
Michael B. Bober, M.D., Ph.D., William H. McAlister, M.D., Deborah Wenkert, M.D.,
Bradley J. Van Sickle, M.D., Ph.D., Jill H. Simmons, M.D., Terence S. Edgar, M.D.,
Martin L. Bauer, M.D., Mohamed A. Hamdan, M.D., Nick Bishop, M.D.,
Richard E. Lutz, M.D., Mairead McGinn, M.D., Stanley Craig, M.D.,
Jean N. Moore, M.D., John W. Taylor, D.O., Robert H. Cleveland, M.D.,
William R. Cranley, M.D., Ruth Lim, M.D., Tom D. Thacher, M.D.,
Jill E. Mayhew, P.T., Matthew Downs, M.P.H., José Luis Millán, Ph.D.,
Alison M. Skrinar, M.P.H., Philippe Crine, Ph.D., and Hal Landy, M.D.
From the Center for Metabolic Bone Dis-
ease and Molecular Research, Shriners
Hospital for Children (M.P.W., D.W.), the
Division of Bone and Mineral Diseases,
Washington University School of Medi-
cine at Barnes–Jewish Hospital (M.P.W.),
and Mallinckrodt Institute of Radiology,
St. Louis Children’s Hospital at Washing-
ton University School of Medicine (W.H.M.)
— all in St. Louis; the University of Mani-
toba and Winnipeg Regional Health Author-
ity; Winnipeg, Canada (C.R.G.); Tawam
Hospital, Al Ain, United Arab Emirates
(N.J.S., M.A.H.); Alfred I. DuPont Hospi-
tal for Children, Wilmington, DE (M.B.B.);
Vanderbilt Children’s Hospital, Nashville
(B.J.V.S., J.H.S.); Prevea Health Clinic
(T.S.E., J.W.T.) and St. Vincent’s Hospital
(J.W.T.) — both in Green Bay, WI; Univer-
sity of Arkansas for Medical Sciences,
College of Medicine, Little Rock (M.L.B.);
Sheffield Children’s Hospital, Sheffield
(N.B.), and Royal Belfast Hospital for
Sick Children, Belfast (M.M., S.C.) — both
in the United Kingdom; University of Ne-
braska Medical Center, Omaha (R.E.L.); St.
John’s Hospital, Springfield, MO (J.N.M.);
Children’s Hospital Boston (R.H.C.), Boston
Medical Center (W.R.C.), and Massachu-
setts General Hospital (R.L.) — all in Bos-
ton; Mayo Clinic, Rochester, MN (T.D.T.);
Enobia Pharma, Montreal (J.E.M., A.M.S.,
P.C., H.L.); Statistics Collaborative, Wash-
ington, DC (M.D.); and Sanford–Burnham
Medical Research Institute, La Jolla, CA
(J.L.M.). Address reprint requests to Dr.
Whyte at Shriners Hospital for Children,
2001 S. Lindbergh Blvd., St. Louis, MO
63131, or at mwhyte@shrinenet.org.
This article (10.1056/NEJMoa1106173)
was updated on March 8, 2012.
N Engl J Med 2012;366:904-13.
Copyright © 2012 Massachusetts Medical Society.
ABSTR ACT
BACKGROUND
Hypophosphatasia results from mutations in the gene for the tissue-nonspecific iso-
zyme of alkaline phosphatase (TNSALP). Inorganic pyrophosphate accumulates
extracellularly, leading to rickets or osteomalacia. Severely affected babies often die
from respiratory insufficiency due to progressive chest deformity or have persistent
bone disease. There is no approved medical therapy. ENB-0040 is a bone-targeted,
recombinant human TNSALP that prevents the manifestations of hypophosphatasia
in Tnsalp knockout mice.
METHODS
We enrolled infants and young children with life-threatening or debilitating perinatal
or infantile hypophosphatasia in a multinational, open-label study of treatment with
ENB-0040. The primary objective was the healing of rickets, as assessed by means of
radiographic scales. Motor and cognitive development, respiratory function, and safety
were evaluated, as well as the pharmacokinetics and pharmacodynamics of ENB-0040.
RESULTS
Of the 11 patients recruited, 10 completed 6 months of therapy; 9 completed 1 year.
Healing of rickets at 6 months in 9 patients was accompanied by improvement in
developmental milestones and pulmonary function. Elevated plasma levels of the
TNSALP substrates inorganic pyrophosphate and pyridoxal 5′-phosphate diminished.
Increases in serum parathyroid hormone accompanied skeletal healing, often neces-
sitating dietary calcium supplementation. There was no evidence of hypocalcemia,
ectopic calcification, or definite drug-related serious adverse events. Low titers of
anti–ENB-0040 antibodies developed in four patients, with no evident clinical, bio-
chemical, or autoimmune abnormalities at 48 weeks of treatment.
CONCLUSIONS
ENB-0040, an enzyme-replacement therapy, was associated with improved findings on
skeletal radiographs and improved pulmonary and physical function in infants and
young children with life-threatening hypophosphatasia. (Funded by Enobia Pharma
and Shriners Hospitals for Children; ClinicalTrials.gov number, NCT00744042.)
The New England Journal of Medicine
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Enzyme-Replacement Therapy in Life-Threatening Hypophosphatasia
n engl j med 366;10 nejm.org march 8, 2012
905
H
ypophosphatasia is the inborn er-
ror of metabolism that is characterized
by low serum alkaline-phosphatase activ-
ity from loss-of-function mutations, typically mis-
sense, within the gene for the tissue-nonspecific
isozyme of alkaline phosphatase (TNSALP).
1
Nat-
ural substrates of TNSALP that accumulate in hy-
pophosphatasia include inorganic pyrophosphate,
2
an inhibitor of mineralization,
3
and pyridoxal
5′-phosphate (PLP), the principal circulating form
of vitamin B
6
.
4
High extracellular levels of inor-
ganic pyrophosphate block hydroxyapatite crystal
growth
3,5
and cause rickets or osteomalacia. Hy-
percalcemia and hyperphosphatemia can develop
in severely affected patients.
1
The deranged vi-
tamin B
6
metabolism shows that TNSALP func-
tions as a cell-surface enzyme
6
and explains why
pyridoxine-responsive seizures may occur when
TNSALP deficiency is profound.
7
The manifestations of hypophosphatasia range
from neonatal death with almost no skeletal min-
eralization to dental problems in adults without
any bone symptoms.
1,5
Such differences in expres-
sion are explained partly by autosomal recessive
and autosomal dominant patterns of inheri-
tance.
1
From a clinical nosology based on the pa-
tient’s age when skeletal disease first develops,
8
autosomal recessive inheritance explains all peri-
natal cases and nearly all infantile cases of hy-
pophosphatasia.
9-11
Perinatal hypophosphatasia is
characterized by extreme skeletal hypomineraliza-
tion, and survival is rare.
1,12
Infantile hypophos-
phatasia develops before 6 months of age with
rickets, failure to thrive, hypotonia, and myopa-
thy and is often complicated by hypercalcemia,
nephrocalcinosis, epilepsy, and craniosynosto-
sis.
7,13-18
Deciduous teeth are lost because insuf-
ficient cementum links them to the periodontal
ligament.
19
Although spontaneous improvement
sometimes occurs in infantile hypophosphata-
sia,
20
substantial bone disease and weakness
often persist.
1,5
Skeletal deterioration typically
results in death from respiratory insufficiency.
14-17
Attempts to treat patients with infantile hypo-
phosphatasia by administering intravenous infu-
sions of plasma enriched in soluble alkaline phos-
phatase from patients with Paget’s disease
14,15
or
alkaline phosphatase purified from human pla-
centas
21
have been unsuccessful, suggesting that
skeletal mineralization requires the presence of
TNSALP on osteoblasts, chondrocytes, and matrix
vesicles.
5,22,23
There is no approved medical treat-
ment for hypophosphatasia.
ENB-0040 (asfotase alfa; Enobia Pharma) is
an investigational, recombinant, fusion protein
comprising the TNSALP ectodomain, the con-
stant region of the human IgG1 Fc domain, and
a terminal deca-aspartate motif for bone target-
ing.
24
In Tnsalp knockout mice, which constitute
an animal model of infantile hypophosphata-
sia,
25
treatment with daily subcutaneous injec-
tions of ENB-0040 at doses that result in serum
levels ranging from 650 to 1000 U per liter pre-
serve the mineralization of bones and teeth.
24,26,27
A phase 1 study involving adult patients with hypo-
phosphatasia showed peak and trough ENB-0040
levels in this range after one intravenous dose of
3 mg per kilogram of body weight and weekly
subcutaneous doses of 2 mg per kilogram.
28
We
conducted a clinical trial of treatment with ENB-
0040 for at least 1 year in infants and young
children with life-threatening hypophosphatasia.
METHODS
STUDY DESIGN AND PATIENTS
In an open-label study, we evaluated the safety,
tolerability, bioavailability, pharmacokinetics,
29
pharmacodynamics, and efficacy of treatment with
ENB-0040. Efficacy assessments included skeletal
changes, as evaluated by means of radiography, and
gross motor, fine motor, and cognitive develop-
ment, as measured with the Bayley Scales of Infant
and Toddler Development, third edition (Bayley-III),
a norm-referenced instrument used to assess de-
velopmental functioning of children from 1 to 42
months of age and to identify children with de-
velopmental delay. Raw scores of successfully com-
pleted items are converted to scaled and composite
scores allowing comparison with children in a
standardized sample. Developmental age-equiva-
lent scores in months allow raw-score compari-
sons with typically developing children.
30
All pa-
tients were to receive ENB-0040 for 6 months and
then have the opportunity to enroll in an open-
label extension study.
Eligibility criteria were an age of 3 years or
less, symptoms of hypophosphatasia occurring
before the age of 6 months, hypophosphatasemia,
an elevated plasma PLP level, hypophosphatasia-
related skeletal disease as assessed radiographi-
cally, failure to thrive, rachitic chest deformity or
pyridoxine-responsive seizures, and nontraumatic
or poorly healing fractures, hypercalcemia, cranio-
synostosis, nephrocalcinosis, or respiratory com-
promise from hypophosphatasia. Patients were
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The
ne w engl and jour nal
o f
medicine
n engl j med 366;10 nejm.org march 8, 2012
906
excluded if they had a major coexisting disease,
a treatable form of rickets, previous exposure to
bisphosphonates, hypocalcemia or hypophospha-
temia, or a serum 25-hydroxyvitamin D level of
less than 20 ng per milliliter (50 nM) or if they
had received another experimental treatment for
hypophosphatasia (e.g., bone marrow transplan-
tation).
16,17
STUDY OVERSIGHT
The study design was developed through a collabo-
ration between the sponsor, Enobia Pharma, and
the authors. The protocol was approved by the
local institutional review boards, and written in-
formed consent was obtained from the patients’
parents. Safety and efficacy were monitored quar-
terly and on an ad hoc basis by an independent
data and safety monitoring board. The primary
statistical analyses were performed by Statistics
Collaborative. Other organizations involved in the
management and analysis of study data are listed
in the Methods section in the Supplementary Ap-
pendix (available with the full text of this article
at NEJM.org). All the authors evaluated the study
findings, made the decision to submit the manu-
script for publication, and vouch for the complete-
ness and accuracy of the reported data and the
fidelity of the study to the protocol. The first author
wrote the first draft of the manuscript, and the
other authors aided in its revision. The study proto-
col, including the statistical analysis plan, is avail-
able at NEJM.org.
STUDY TREATMENT
Patients received ENB-0040 (at a concentration of
40 mg per milliliter) as a single intravenous infu-
Table 1. Characteristics of the Patients at Baseline.*
Characteristic Patient 1 Patient 2 Patient 3 Patient 4 Patient 5
Form of hypophosphatasia Perinatal Infantile Infantile Infantile Perinatal
Age — mo 7.5 6 33 18 36
Sex Female Female Female Female Female
TNSALP genotype
c.881A>C p.D294A
c.547G>A
p.D183H†
c.407G>A p.Arg136His
c.1133A>T
p.Asp378Val‡
c.658G>A p.Gly220Arg
c.659G>C
p.Gly220Ala§
c.1077C>G p.Ile359Met
c.1133A>T
p.Asp378Val‡
c.98C>T p.A33V
c.98C>T
p.A33V†
Serum ALP (normal range)
— U/liter¶
20 (117–352) 8 (<390) 33 (108–317) 58 (80–425) 19 (79–267)
Plasma PLP — × ULN 10 5 4 >3 2
Severity of rickets‖ Severe Severe Extremely severe Severe Moderate
Respiratory status CPAP Progressive respiratory
deterioration
Tracheostomy and
ventilation
Tracheostomy and
ventilation
Supplemental oxygen
by nasal cannula
Failure to thrive Yes Yes Yes Yes Yes
Nephrocalcinosis Yes Yes Yes Yes No
Gross motor function —
age-equivalent in mo**
<1 No data — too fragile
to test
<1 <1 7
Feeding Tube Tube Tube Tube Oral
* ALP denotes alkaline phosphatase, CPAP continuous positive airway pressure, PLP pyridoxal 5′-phosphate, TNSALP tissue-nonspecific iso-
zyme of alkaline phosphatase, and ULN upper limit of the normal range.
† TNSALP mutation analysis was performed by Centre d’Etudes de Biologie Prénatale, Université de Versailles (Versailles, France).
‡ TNSALP mutation analysis was performed by Connective Tissue Gene Tests (Allentown, PA).
§ TNSALP mutation analysis was performed by Steven Mumm, Ph.D., of the Washington University School of Medicine (St. Louis).
¶ The normal range is for the center where the patient was treated.
‖ Extremely severe rickets was defined as the partial or complete absence of visible bones on radiographs, severe rickets as a near absence
of metaphyseal features with large radiolucent “tongues” and sclerosis, and moderate rickets as rachitic changes (metaphyseal flaring and
fraying, widening of physes, areas of subphyseal demineralization, and bowing).
** Gross motor function was assessed with the use of the Bayley Scales of Infant and Toddler Development, third edition (Bayley-III), a
norm-referenced instrument used to assess developmental functioning of children from 1 to 42 months of age and to identify children
with developmental delay. Raw scores of successfully completed items are converted to scaled and composite scores allowing comparison
with children in a standardized sample. Developmental age-equivalent scores in months allow raw-score comparisons with typically devel-
oping children.
30
The New England Journal of Medicine
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Copyright © 2012 Massachusetts Medical Society. All rights reserved.
Enzyme-Replacement Therapy in Life-Threatening Hypophosphatasia
n engl j med 366;10 nejm.org march 8, 2012
907
sion at a dose of 2 mg per kilogram, followed by
subcutaneous injections three times per week at a
dose of 1 mg per kilogram. The subcutaneous dose
could be increased up to 3 mg per kilogram if there
was worsening failure to thrive, deteriorating pul-
monary function, or no radiographic evidence of
skeletal improvement.
STATISTICAL ANALYSIS
The primary efficacy end point was a change in
the skeletal manifestations of hypophosphatasia,
including rickets, as assessed on radiography. Two
methods of assessment were used. First, three ra-
diologists rated changes from baseline to week
48 using a 7-point scale (radiographic global im-
pression of change [RGI-C]), which is based on
ratings of the characteristics of severe hypophos-
phatasia (including irregularity of the provisional
zone of calcification; physeal widening; metaphy-
seal flaring, fraying, radiolucencies, and patchy os-
teosclerosis; altered ratio of mid-diaphyseal cortex-
to-bone thickness; gracile bones; absence of some
or all bones; and recent fractures). A reduction of
3 points represented severe worsening, and an in-
crease of 3 points indicated complete healing of the
skeletal disease. For each patient, the mean score
among the radiologists was used for analysis, with
a response to treatment defined as a mean increase
of 2 or more points (i.e., substantial healing).
The proportion of patients with a response, with
the 95% exact confidence interval, was calculated
at weeks 24 and 48.
31
Two-sided Wilcoxon signed-
rank tests
32
were used to determine whether the
median scores differed from 0 (i.e., no change).
Second, a single reader rated the growth-plate
abnormalities at the wrists and knees, using a
10-point rickets-severity scale (RSS),
33
with higher
scores representing more severe rickets. All radio-
graphs were rated with masking of sequence and
patient identifiers. Changes from baseline were
calculated at weeks 24 and 48, with the use of
Wilcoxon signed-rank tests to determine whether
median changes differed from 0 (see the Supple-
mentary Appendix).
Additional efficacy studies included evaluations
of respiratory status and motor and cognitive func-
tion. Bone biopsy and densitometry were precluded
by the small size of the patients and their precari-
ous clinical status.
RESULTS
CHARACTERISTICS OF THE PATIENTS
A total of 11 patients, 7 girls and 4 boys, were en-
rolled between October 2008 and December 2009;
they ranged in age at baseline from 2 weeks to
3 years (
Table 1
). Five patients had perinatal hypo-
phosphatasia, and six had infantile hypophos-
Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Patient 11
Perinatal Perinatal Infantile Infantile Perinatal Infantile
0.6 1 30 6 2 4
Male Male Male Female Female Male
c.119C>T p.Ala40Val
c.1231A>G
p.Thr411Ala‡
c.215T>C p.Ile72Thr
c.881A>C
p.Asp294Ala‡
c.212G>A p.Arg71His
c.571G>A
p.Gly191Lys†
c.212G>A p.R71H
c.571G>A
p.E191K†
c.920C>T p.P307L
c.1171C>T
p.R391C‡
c.350A>G p.Tyr117Cys
c.1001G>A
p.Gly334Asp‡
21 (73–266) 20 (73–266) 42 (76–308) 23 (59–425) 6 (32–91) 93 (143–320)
17 8 18 14 Receiving pyridoxine 3
Extremely severe Severe Moderate Severe Severe Severe
Endotracheal intuba-
tion and ventilation
Progressive respiratory
deterioration
Ambient air Progressive respiratory
deterioration
Progressive respiratory
deterioration
Progressive respiratory
deterioration
Yes Yes Yes Yes Yes Yes
Yes Kidney stones No Yes Yes Yes
No data — too fragile
to test
<1 9 <1 1 <1
Tube Tube Oral Tube Oral Oral
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