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J O U R N A L O F T H E AM E R I C A N C O L L E G E O F C A R D I O L O G Y VO L . 7 1 , N O . 1 0 , 2 0 1 8

ª 2 0 1 8 B Y T H E AM E R I C A N C O L L E G E O F C A R D I O L O G Y F O UN DA T I O N

P U B L I S H E D B Y E L S E V I E R

Etidronate for Prevention ofEctopic Mineralization in Patients WithPseudoxanthoma Elasticum

Guido Kranenburg, MD,a Pim A. de Jong, MD, PHD,b Jonas W. Bartstra, MD,a Suzanne J. Lagerweij, MD,b

Marnix G. Lam, MD, PHD,b Jeannette Ossewaarde-van Norel, MD, PHD,c Sara Risseeuw, MD,c

Redmer van Leeuwen, MD, PHD,c Saskia M. Imhof, MD, PHD,c Harald J. Verhaar, MD, PHD,d Job J. de Vries, BSC,b

Riemer H.J.A. Slart, MD, PHD,e Gert Luurtsema, PHD,e Annemarie M. den Harder, MD,b Frank L.J. Visseren, MD, PHD,a

Willem P. Mali, MD, PHD,b Wilko Spiering, MD, PHDa

ABSTRACT

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BACKGROUND In pseudoxanthoma elasticum (PXE), low pyrophosphate levels may cause ectopic mineralization,

leading to skin changes, visual impairment, and peripheral arterial disease.

OBJECTIVES The authors hypothesized that etidronate, a pyrophosphate analog, might reduce ectopic mineralization

in PXE.

METHODS In the Treatment of Ectopic Mineralization in Pseudoxanthoma Elasticum trial, adults with PXE and leg

arterial calcifications (n ¼ 74) were randomly assigned to etidronate or placebo (cyclical 20 mg/kg for 2 weeks every

12 weeks). The primary outcome was ectopic mineralization, quantified with 18fluoride positron emission tomography

scans as femoral arterial wall target-to-background ratios (TBRfemoral). Secondary outcomes were computed tomography

arterial calcification and ophthalmological changes. Safety outcomes were bone density, serum calcium, and phosphate.

RESULTS During 12 months of follow-up, the TBRfemoral increased 6% (interquartile range [IQR]: �12% to 25%) in the

etidronate group and 7% (IQR: �9% to 32%) in the placebo group (p ¼ 0.465). Arterial calcification decreased 4%

(IQR: �11% to 7%) in the etidronate group and increased 8% (IQR: �1% to 20%) in the placebo group (p ¼ 0.001).

Etidronate treatment was associated with significantly fewer subretinal neovascularization events (1 vs. 9, p ¼ 0.007).

Bone density decreased 4% � 12% in the etidronate group and 6% � 9% in the placebo group (p ¼ 0.374). Hypocal-

cemia (<2.20 mmol/l) occurred in 3 versus 1 patient (8.1% vs. 2.7%, p ¼ 0.304). Eighteen patients (48.6%) treated with

etidronate, compared with 0 patients treated with placebo (p < 0.001), experienced hyperphosphatemia (>1.5 mmol/l)

and recovered spontaneously.

CONCLUSIONS In patients with PXE, etidronate reduced arterial calcification and subretinal neovascularization

events but did not lower femoral 18fluoride sodium positron emission tomography activity compared with placebo,

without important safety issues. (Treatment of Ectopic Mineralization in Pseudoxanthoma elasticum; NTR5180)

(J Am Coll Cardiol 2018;71:1117–26) © 2018 by the American College of Cardiology Foundation.

N 0735-1097/$36.00 https://doi.org/10.1016/j.jacc.2017.12.062

m the aDepartment of Vascular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands;

epartment of Radiology and Nuclear Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands;

epartment of Ophthalmology, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands; dDepartment of

riatric Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands; and the eMedical Imaging

nter, Department of Nuclear Medicine and Molecular Imaging, University of Groningen, University Medical Center Groningen,

oningen, the Netherlands. This study was supported by the Dutch Innovation Fund of Health Insurers (Innovatiefonds Zorg-

rzekeraars), Dutch Foundation PXE Fund, Dutch Eye Association, and Foundation Friends of University Medical Center Utrecht.

I-Pharma Kleon Tsetis Pharmaceutical Laboratories SA (Greece) provided all etidronate and placebo capsules for free, as

nufacturer of the finished product (OSTOPOR hard capsules, 400 mg/capsule). UNI-Pharma SA was not involved in the design,

execution, the analysis, or the reporting of the Treatment of Ectopic Mineralization in Pseudoxanthoma Elasticum trial. The

thors have reported that they have no relationships relevant to the contents of this paper to disclose.

nuscript received December 14, 2017; accepted December 19, 2017.

ABBR EV I A T I ON S

AND ACRONYMS

18F-NaF PET = 18fluoride

sodium positron emission

tomography

ALT = alanine transaminase

AST = aspartate transaminase

BCVA = best corrected visual

acuity

CT = computed tomography

eGFR = estimated glomerular

filtration rate

IMT = intima-media thickness

IQR = interquartile range

PPi = inorganic pyrophosphate

PWV = pulse wave velocity

PXE = pseudoxanthoma

elasticum

SF-36 = Short Form 36

TBRfemoral = femoral arterial

wall target-to-background

ratio

VEGF = vascular endothelial

growth factor

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Etidronate for Prevention of Ectopic Mineralization M A R C H 1 3 , 2 0 1 8 : 1 1 1 7 – 2 6

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P seudoxanthoma elasticum (PXE,OMIM #264800) is an autosomalrecessive systemic calcification disor-

der. PXE is characterized by skin involve-ment (e.g., yellowish papules/plaques), eyeinvolvement (e.g., angioid streaks), andvascular involvement (arterial calcification),and has a considerable morbidity, includingsevere visual impairment and blindness, pe-ripheral arterial disease, ischemic stroke,and vascular dementia (1,2). The prevalenceis approximately 1:25,000 to 100,000 (2). Toprevent progression of visual impairmentcaused by choroidal neovascularization inpatients with PXE, injections with antivascu-lar endothelial growth factor (VEGF) are used(3); however, there is no specific and preven-tive treatment available for patients withPXE.

SEE PAGE 1127

PXE is caused by mutations in the ABCC6gene and associated with ectopic minerali-zation of elastic fibers in the skin, the Bruch

membrane beneath the retina, and the medial layer ofarteries (4). Recently, major steps have been made indeciphering the etiology of PXE. ABCC6 mutationshave been shown to result in reduced adenosinetriphosphate secretion in the liver causing low levelsof inorganic pyrophosphate (PPi) (5). PPi is a stronginhibitor of ectopic mineralization (6–8). Thedecreased levels of PPi in PXE may therefore causethe ectopic mineralization in PXE (9).

Bisphosphonates, well-established drugs for thetreatment of osteoporosis and bone metastases, arestable PPi analogs and could thus stimulate theinhibitory effects on ectopic mineralization (10). Infact, bisphosphonates have been shown to reduce softtissue calcifications in rats even before their effect onbone resorption was known (11). Of the currentlyavailable bisphosphonates, etidronate may have thelargest potential to delay ectopic mineralization givenits predominant inhibition of calcium precipitationand hydroxyapatite binding. This is different fromnewer bisphosphonates, such as alendronate, whichpredominantly inhibit osteoclasts (12,13).

Several nonrandomized and uncontrolled reportsdescribe beneficial effects of etidronate in patientswith rare diseases with ectopic mineralizationresulting from a deficiency in PPi homeostasis. Inpatients with basal ganglia calcifications or primarybrain calcifications (OMIM #213600), treatment withetidronate alleviates neurological symptoms (12,14).In generalized arterial calcification of infancy (OMIM

#208000) etidronate treatment reduces arterialcalcification and is associated with improved survival(15,16). Generalized arterial calcification of infancycan be seen as an aggressive form of PXE with aconsiderable overlap in genotype and phenotype(17,18). Treatment with etidronate in PXE mousemodels results in prevention of ectopic mineraliza-tion and in alterations in bone microarchitecture(19,20). The effectiveness of etidronate remains to beestablished in patients with PXE in a randomized,placebo-controlled trial.

We therefore hypothesized that synthetic PPisupplementation with etidronate treatment couldreduce ectopic mineralization in patients with PXE.To be able to investigate this hypothesis in a ran-domized, placebo-controlled trial, we used femoralarterial wall 18

fluoride sodium positron emissiontomography (18F-NaF PET) activity and computedtomography (CT)-based femoral calcium scores asmarkers of ectopic mineralization (21–25). Imagingwith 18F-NaF PET may be more sensitive to changes inectopic mineralization compared with traditional CTbecause it is believed to be able to visualize the activeand ongoing calcification process and discriminatebetween the active and the more indolent calcifica-tions (22,26).

Here, we report the results of the Treatment ofEctopic Mineralization in Pseudoxanthoma elasticum(TEMP) trial in which we set out to investigate theeffectiveness and safety of 1 year’s treatment withetidronate (cyclical 20 mg/kg for 2 weeks every12 weeks) on ectopic mineralization among partici-pants with PXE.

METHODS

TRIAL DESIGN AND STUDY POPULATION. The TEMPtrial was a single-center, randomized, double-blind,placebo-controlled trial conducted in the PXE exper-tise center at the University Medical Center Utrecht,the Netherlands. Participants eligible for participationhad a confirmed clinical diagnosis of PXE, were $18years of age, and had evidence of arterial calcificationon a CT scan of the legs that was acquired in all patientsduring the first visit in our center. PXE was diagnosedif 2 of the following were present: skin involvement(e.g., yellowish papules/plaques), eye involvement(e.g., angioid streaks), and genetically confirmation(biallelic ABCC6mutations) (1). Exclusion criteria weresevere renal impairment, known abnormality of theesophagus, known sensitivity to etidronate, use ofbisphosphonates during the past 5 years, osteoma-lacia, chronic diarrhea, pregnancy, claustrophobia,hypocalcemia (calcium <2.20 mmol/l), and vitamin D

FIGURE 1 Flowchart Eligibility, Randomization, and Follow-Up

77 Patients were assessed foreligibility

74 Underwent randomization

37 Were assigned to etidronate(cyclical 20 mg/kg for 2 weeks

every 12 weeks)

1 Discontinued intervention (after second treatment period)

37 were included in the(primary endpoint) analysis

36 were included in the(primary endpoint) analysis

1 Refused further participation(after ophthalmological event at

3 months of follow-up)

37 Were assigned to placebo(for 2 weeks every 12 weeks)

3 were ineligible or declinedto participate- 1 Declined to participate- 1 Did not meet PXE criteria- 1 Had claustrophobia

Seventy-seven patients were screened for eligibility and 74 patients underwent

randomization. Eventually, 37 patients in the etidronate group and 36 patients in the

placebo group were included in the (primary endpoint) analysis. PXE ¼ pseudoxanthoma

elasticum.

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deficiency (25-OH vitamin D <35 nmol/l). TheTEMP trial was approved by the institutional reviewboard of the University Medical Center Utrecht (num-ber 15/522). All participants provided written informedconsent.

RANDOMIZATION AND INTERVENTION. Eligibleparticipants were randomized in a 1:1 ratio to either acyclical regime of 2 weeks 20 mg/kg etidronate withoral administration of 400 mg capsules every12 weeks or to an identical treatment regime andcapsules without the active pharmacological sub-stance (i.e., placebo). Thus, patients were given20 mg/kg every day for 2 weeks. Etidronate (OSTO-POR hard capsules, 400 mg/capsule) and placebocapsules were provided by Uni-Pharma Kleon TsetisPharmaceutical Laboratories SA (Athens, Greece).Randomization with random permuted blocks for sexwas performed using a random number generator atthe pharmacy department, where the randomizationlist was stored during the entire duration of the trial.

PROCEDURES. Full body 18F-NaF PET/CT scans wereperformed at baseline and after 12 months of follow-up on a Siemens Biograph 40 scanner (SiemensHealthcare, Erlangen, Germany). Images were recon-structed according to the European Association ofNuclear Medicine Research Ltd recommendations(27). Ninety minutes before imaging, an intravenousinjection of 2.0 MBq/kg 18F-NaF (maximum dosage:200 MBq) was administered. A conventional full-bodyCT scan was performed after 6 months of follow-up.

Measurements of carotid intima-media thickness(IMT) and carotid-femoral pulse wave velocity (PWV)were performed at baseline and after 12 months offollow-up. IMT was measured with ultrasound(Esaote, Florence, Italy) in the left and right carotidarteries. PWV was measured using applanationtonometry with a micromanometer (Millar In-struments Inc., Houston, Texas) in combination withSphygmocor software (Atcore Medical Pty. Ltd., Syd-ney, New South Wales, Australia).

Ophthalmological examination took place at base-line and after 3, 6, 9, and 12 months. The frequency ofsubretinal neovascularization events was monitoredwith optical coherence tomography scans and fundusphotography during all visits. The frequency of anti-VEGF injections in the year before the baseline visitand during the trial was obtained. If needed, medicalinformation was retrieved from other hospitals. Bestcorrected visual acuity (BCVA) was measured atbaseline and after 12 months follow-up using EarlyTreatment Diabetic Retinopathy Study letter charts(Online Appendix) (28). Quality of life was assessed atbaseline and after 12 months of follow-up using the

validated Dutch version of the 36-Item Short Formsurvey (SF-36) questionnaire (29).

Laboratory measurements for serum calcium andphosphate were performed at baseline, and after 3, 6,9, and 12 months of follow-up. Estimated glomerularfiltration rate (eGFR calculated using the ChronicKidney Disease Epidemiology Collaboration formula),aspartate transaminase (AST), and alanine trans-aminase (ALT) were measured at baseline, after6 months, and after 12 months of follow-up.

OUTCOMES. The primary outcome was change infemoral 18F-NaF PET activity. The femoral arterytarget-to-background ratio (TBRfemoral) as a measureof femoral 18F-NaF PET activity was determinedblinded for treatment status using previouslydescribed methods (Online Appendix) (24,30).

Change in arterial calcification on conventional CTscan was a secondary outcome. Calcifications in boththe left and right femoral arteries were quantifiedblinded for treatment status using the femoral artery

TABLE 1 Patient Characteristics of the Study Population at Baseline

Etidronate(n ¼ 37)

Placebo(n ¼ 37)

Clinical characteristics

Age, yrs 57 � 9 57 � 8

Male 19 (51) 19 (51)

Diabetes mellitus 3 (8) 1 (3)

Systolic blood pressure, mm Hg 142 � 20 138 � 18

Diastolic blood pressure, mm Hg 81 � 9 80 � 11

Body mass index, kg/m2 26.7 � 4.6 25.5 � 3.5

Non-HDL cholesterol, mmol/l 3.6 � 1.1 3.8 � 1.2

Femoral 18F-NaF PET activity

TBR 2.80 � 1.04 2.59 � 0.90

Arterial calcification

Femoral calcium mass, mg 405 (35-1,797) 1,035 (282-1,563)

Arterial wall characteristics

Carotid IMT, mm 0.72 � 0.11 0.79 � 0.14

Carotid-femoral PWV, m/s 11.0 � 3.1 9.7 � 1.9

Ophthalmological status

Total number of anti-VEGF injections used in past year 0 (0-8) 0 (0-9)

Best corrected visual acuity worst eye, ETDRS letters 26 (5-79) 26 (10-79)

Best corrected visual acuity best eye, ETDRS letters 78 (35-86) 72 (37 to 83)

Laboratory measurements

25-OH vitamin D, nmol/l 93 � 45 70 � 45

Calcium, mmol/l 2.40 � 0.09 2.37 � 0.07

Phosphate, mmol/l 1.05 � 0.11 1.05 � 0.14

eGFR, ml/min/1.73 m2 90 (86-90) 90 (83-90)

AST, U/l 29 � 6 28 � 6

ALT, U/l 27 � 13 23 � 10

Bone density

CT bone density, HU 139 � 50 128 � 34

Values are mean � SD, n (%), or median (interquartile range).

ALT ¼ alanine transaminase; AST ¼ aspartate transaminase; CT ¼ computed tomography; ETDRS ¼ EarlyTreatment Diabetic Retinopathy Study; eGFR ¼ estimated glomerular filtration rate using the Chronic KidneyDisease Epidemiology Collaboration formula; 18F-NaF PET ¼ 18

fluoride sodium positron emission tomography;HDL ¼ high-density lipoprotein; HU ¼ Hounsfield units; IMT ¼ intima-media thickness; PWV ¼ pulse wavevelocity; TBR ¼ target-to-background and ratio; VEGF ¼ vascular endothelial growth factor.

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calcification mass (Online Appendix). Changes in IMTand PWV between month 12 and baseline were addi-tional secondary outcomes that were assessed blin-ded for treatment status.

Ophthalmological changes were a secondaryoutcome. These changes were monitored blinded fortreatment status by the occurrence of subretinalneovascularization events, the frequency of anti-VEGF administration and BCVA. A subretinal neo-vascularization event was defined as any of thefollowing that provided an indication to start orintensify anti-VEGF injections to prevent (further)visual impairment: 1) retinal bleeding suspected to becaused by subretinal neovascularization (if necessaryconfirmed by fluorescein angiography); 2) a signifi-cant increase in subretinal or intraretinal fluid; and/or3) growth of a subretinal neovascular complex.During the TEMP trial, these events were scored

by $2 investigators, including at least 1 board-certified ophthalmologist, who were blinded fortreatment status.

Transformed SF-36 scores between 0 and 100 onthe 9 quality-of-life domains were used: physicalfunction, physical role functioning, pain, generalhealth, mental health, social function, social rolefunctioning, vitality, and health changes.

SAFETY MONITORING. Safety assessments werechanges in bone density, as measured with CT scansat baseline, month 6, and month 12 (OnlineAppendix); changes in serum calcium and phos-phate; the occurrence of hypocalcemia or hyper-phosphatemia; and changes in eGFR, AST, and ALT,and recording of adverse events (Online Appendix).

A data and safety monitoring board, consisting of 3independent members, monitored safety data(serious adverse events, bone mineral density, cal-cium levels, ophthalmological events, frequency ofanti-VEGF injections) and performed interim analysesafter the first 20 participants reached 3 and 6 months’follow-up.

STATISTICAL ANALYSIS. Under the assumption of ameanTBRfemoral of 1.96 and a standarddeviation of 0.58and anticipating 6 dropouts or loss to follow-up, 74participants were required to detect a 20% change inTBRfemoral with 80% power and a 2-sided a of 5% (24).

Descriptive data were presented as normallydistributed continuous (mean � SD), non-normallydistributed continuous (median, interquartile range[IQR]) or as categorical variables (n, %). Intention-to-treat analyses were performed comparing the inter-vention and placebo group using independentsample Student’s t-tests, Mann-Whitney U tests, orchi-square tests when appropriate. In our primaryanalysis, we compared the percentage differencein femoral TBR between the etidronate and theplacebo groups using a Mann-Whitney U test. Allanalyses were performed blinded before definitivebreaking of the blinding, comparing treatment groupA versus B. A p value <0.05 was regarded as statisti-cally significant. Analyses were performed withR version 3.3.2.

RESULTS

TRIAL POPULATION. A total of 77 patients werescreened between July 2015 and June 2016. Threepatients were ineligible; the remaining 74 partici-pants were enrolled between October 7, 2015, andJune 8, 2016. One participant in the placebo groupdeclined further participation at month 3, having

TABLE 2 Primary and Secondary Outcomes

Etidronate Placebo p Value

Femoral arterial wall 18F-NaF PET activity

Change in target-to-background ratio, % 6 (�12 to 25) 7 (�9 to 32) 0.465

CT arterial calcification

Change in femoral calcification mass(month 12 vs. baseline), %

�4 (�10 to 7) 8 (�1 to 20) 0.001

Arterial wall characteristics

Change in carotid IMT, mm 0.00 � 0.09 0.02 � 0.09 0.530

Change in carotid-femoral PWV, m/s 0.6 � 2.2 0.6 � 2.1 0.990

Ophthalmological status

Subretinal neovascularization events during follow-up 1 (2.7) 9 (24.3) 0.007

Change in frequency of VEGF inhibitors 0 (�2 to 0) 0 (�1 to 1) 0.137

Change in best corrected visual acuityworst eye, ETDRS letters

�1 � 5 �1 � 7 0.900

Change in best corrected visual acuitybest eye, ETDRS letters

�2 � 4 �3 � 7 0.460

Values are median (interquartile range), mean � SD, or n (%). Treatment groups were compared using inde-pendent sample Student’s t-tests and Mann-Whitney U tests when appropriate. Change is displayed as % forfemoral arterial wall 18F-NaF PET activity and femoral calcium mass.

Abbreviations as in Table 1.

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experienced severe uveitis following an anti-VEGFinjection. One participant in the etidronate groupdiscontinued treatment because of a hypersensitivityskin reaction at month 6 but remained in the study(Figure 1). Baseline characteristics are presented inTable 1. The mean age in both groups was 57 years;both groups were 51% male.

EFFICACY. The TBRfemoral increased with 6%(IQR: �12% to 25%) in the etidronate group and with7% (IQR: �9% to 32%) in the placebo group in the12 months of follow-up (p ¼ 0.465) (Table 2, Figure 2).During the 12 months of follow-up, arterial calcifica-tion decreased 4% (IQR: �11% to 7%) in the etidronategroup and increased 8% (IQR: �1% to 20%) in theplacebo group (p ¼ 0.001) (Figure 2).

Carotid IMT increased 0.00 � 0.09 mm in the eti-dronate group and 0.02 � 0.09 mm in the placebogroup (p ¼ 0.523). The PWV increased with 0.6 �2.2 m/s in the etidronate group and 0.6 � 2.1 m/s inthe placebo group (p ¼ 0.990).

During follow-up, 1 patient (2.7%) in the etidronategroup had a subretinal neovascularization eventcompared with 9 patients (24.3%) in the placebogroup (p ¼ 0.007). The median number of anti-VEGFinjections administered per year did not change inthe etidronate group (0; IQR: �2 to 0) or in theplacebo group (0; IQR: �1 to 1) (p ¼ 0.137). The BCVAin LogMAR number of letters of the worse eyedecreased with 1 � 5 letters in the etidronate groupand with 1 � 7 letters in the placebo group (p ¼ 0.900).The BCVA of the best eye decreased with 2 � 4 lettersin the etidronate group and with 3 � 7 letters in theplacebo group (p ¼ 0.460).

No significant differences between the etidronateand placebo group were found in quality of life do-mains of the SF-36 questionnaire (Online Table 1).

SAFETY. From baseline to 12 months of follow-up,the bone mineral density changed with �4 � 12%(�7 � 16 HU) in the etidronate group comparedwith �6 � 9% (�9 � 16 HU) in the placebo group(p ¼ 0.374) (Table 3).

No significant differences were found between theetidronate and placebo group when comparing base-line with values after 12 months of follow-up of cal-cium, phosphate, eGFR, AST, and ALT (Table 3). Duringfollow-up, hypocalcemia (calcium <2.20 mmol/l)occurred in 3 patients (8.1%) in the etidronate groupand in 1 (2.7%) in the placebo group (p ¼ 0.304).Hyperphosphatemia (phosphate >1.5 mmol/l)occurred in 18 patients (48.6%) in the etidronate groupcomparedwith 0 (0%) in the placebo group (p<0.001).All cases were monitored and recovered without anymedical intervention.

Gastrointestinal complaints (reflux, stomach pain,diarrhea) presented in 2 patients (5.4%) in the eti-dronate and 2 patients (5.4%) in the placebo group(p ¼ 1.000). A hypersensitivity dermatological reac-tion occurred in 1 patient (2.7%) in the etidronategroup compared with 0 (0%) in the placebo group(p ¼ 0.304).

DISCUSSION

The TEMP trial, a randomized double-blind, placebo-controlled trial including 74 PXE participants,showed that etidronate did not lower femoral arterialwall 18F-NaF PET activity compared with placebo af-ter 12 months of follow-up (Central Illustration);however, etidronate significantly decreased ectopicmineralization as quantified by arterial calcificationcompared to placebo. Also, etidronate treatment inPXE was associated with significantly lower rates ofsubretinal neovascularization events (1 vs. 9 pa-tients). There were no important safety issues of eti-dronate treatment during the year of follow-up.Although in these dosages etidronate is known toincrease the risk of bone-related adverse events andhypocalcemia, no significant differences wereobserved between the etidronate and placebo group.Eighteen patients in the etidronate group comparedwith 0 in the placebo group experienced hyper-phosphatemia (>1.5 mmol/l), but all patients recov-ered without any medical intervention.

The body has several inhibiting pathwaysfor ectopic mineralization including vitaminK–dependent pathways, the Klotho protein, Fetuin-A,

FIGURE 2 Effects on Femoral Mineralization

Placebo (n = 36)

–3

–2

–1

Delta

Fem

oral

Tar

get-

to-B

ackg

roun

d Ra

tio (M

12-B

asel

ine)

Femoral 18F-NaF PET Activity

0

1

2

A B3

Etidronate (n = 37) Placebo (n = 36)

–1000

–500

0

p < 0·01

Delta

CT

Fem

oral

Cal

cium

Mas

s (M

12-B

asel

ine,

mg)

Femoral CT Calcium Mass

500

1000

1500

Etidronate (n = 37)

(A) Differences in femoral target-to-background ratio stratified for treatment group between baseline and 12 months of follow-up.

(B) Differences in femoral calcium mass stratified for treatment group between baseline and 12 months of follow-up. The bottom and top

of the boxes represent the first and third quartiles. The end of the whiskers represent the lowest and highest value still within 1.5 times

the interquartile range. 18F-NaF PET ¼ 18fluoride sodium positron emission tomography; CT ¼ computed tomography.

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and PPi (6). In PXE, ABCC6 mutations havebeen shown to result in low levels of PPi (5). Thedecreased levels of PPi may cause the ectopicmineralization in PXE (9). Increasing the concentra-tion of ectopic mineralization inhibitors such as PPimay result in ectopic mineralization preventionand subsequent inhibition of disease progression(9,12,14–16,19,20,31–33).

We hypothesized that etidronate could delayectopic mineralization and subsequent disease pro-gression in patients with PXE. The TEMP trialconfirmed this hypothesis with an observed decreasein ectopic mineralization, quantified with conven-tional CT femoral calcium scores in the etidronategroup compared with the placebo group. The effectson CT arterial calcification in the TEMP trial are in linewith findings from etidronate trials in chronic kidney

disease, diabetes mellitus, and hypercholesterolemiapatients (34–37).

The observed decrease in subretinal neo-vascularization events in the etidronate comparedwith the placebo group further confirmed our hy-pothesis. This finding is in line with findings fromintervention studies in non-PXE populations,showing preventive effects of bisphosphonates onsubretinal neovascularization in age-related maculardegeneration and pathological myopia (38). Etidro-nate has been found to reduce the expression ofseveral angiogenic factors in human retinal pigmentepithelial cells, which likely play a key role in neo-vascularization (39). Similar to the vascular effects,these ophthalmological effects of etidronate treat-ment in PXE may be attributed to inhibition of ectopicmineralization by etidronate. Bruch membrane

TABLE 3 Safety Measurements and Adverse Events

Etidronate Placebo p Value

Bone mineral density

%D CT bone density, HU �4 � 12 �6 � 9 0.374

Laboratory measurements*

Hypocalcemia (calcium <2.20 mmol/l) 3 (8.1) 1 (2.7) 0.304

Hyperphosphatemia (phosphate >1.5 mmol/l)† 18 (48.6) 0 (0.0) <0.001

Renal impairment (eGFR �20%) 0 (0.0) 0 (0.0) 1.000

Liver impairment (AST/ALT þ50%)† 1 (2.7) 0 (0.0) 0.304

D Calcium, mmol/l �0.03 � 0.08 �0.01 � 0.07 0.438

D Phosphate, mmol/l �0.01 � 0.14 �0.03 � 0.14 0.707

D eGFR, ml/min/1.73 m2 0 (0 to 0) 0 (�1 to 0) 0.864

D AST, U/l 0 � 5 0 � 4 0.841

D ALT, U/l �1 � 9 2 � 8 0.147

Adverse events

Gastrointestinal complaints 2 (5.4) 2 (5.4) 1.000

Hypersensitivity dermatological reaction‡ 1 (2.7) 0 (0.0) 0.304

Posterior vitreous detachment 1 (2.7) 0 (0.0) 0.304

Suspected uveitis after anti-VEGF injection 0 (0.0) 1 (2.7) 0.304

Blurry sight after anti-VEGF injection,disappearing within a day

0 (0.0) 1 (2.7) 0.304

Drop in visual acuity not relatedto neovascularization

0 (0.0) 1 (2.7) 0.304

Hospital admission§ 0 (0.0) 1 (2.7) 0.304

Values are mean � SD, n (%), or median (interquartile range). Treatment groups were compared using inde-pendent sample Student’s t-tests, chi-square tests, and Mann-Whitney U tests when appropriate. *Calcium andphosphate were measured at baseline and after 3, 6, 9, and 12 months of follow-up. Renal function and liverfunction were measured at baseline and after 6 and 12 months of follow-up. †All cases were followed andrecovered without any medical intervention. ‡Skin rash arising during second treatment period, after which thestudy medication was discontinued and the rash disappeared. Restart of study medication again resulted in rash,after which this participant continued the study off study medication. §Hospital admission because of (planned)hip operation.

D Delta ¼ month 12 minus baseline value; other abbreviations as in Table 1.

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mineralization in PXE may impede the transport ofoxygen and waste products across Bruch membrane.Inhibition of this mineralization might thus preventthe growth or recurrence of activity of a choroidalneovascularization (3).

We assumed that 18F-NaF PET activity wouldvisualize the active calcification process and thuswould be sensitive to small changes in arterial calci-fication, providing an efficient marker of ectopicmineralization in the TEMP trial (22). The TEMP trialis the first interventional trial that reports on femoral18F-NaF PET activity as an outcome. The absence ofan effect of etidronate treatment on femoral arterialwall 18F-NaF PET activity is remarkably discrepantwith the observed effects of etidronate on CT arterialcalcification and subretinal neovascularizationevents. This discrepancy questions our assumptionon the value of femoral 18F-NaF PET activity as anintermediate outcome in such an interventional trial.Although femoral 18F-NaF PET activity relates tocardiovascular risk scores (24), regional 18F-NaF PETactivity and regional conventional CT arterial calcifi-cation scores are not correlated, suggesting that thebiological processes measured with 18F-NaF PETscans are not exactly known (40,41). Recent evidenceshows that arterial wall 18F-NaF PET activity coloc-alize closely and preferentially bind to pathologicalmineralization, but also that the increased surfacearea of microcalcifications relative to macro-calcifications resulted in increased tracer uptake and,therefore, does not necessarily represent activecalcification (41). Our assumptions on the 18F-NaFPET measurements as a reflection of disease activityproved to be incorrect as a result. Another explana-tion for the discrepancy between effects of etidronateon 18F-NaF activity and CT arterial calcification scoremay be that etidronate treatment increases dissolu-tion of existing mineral deposits while not loweringnew calcium deposition; however, this remainsspeculative and potential biological mechanisms areunknown. Taking the effects of etidronate treatmenton conventional CT arterial calcification into account,even in the absence of an effect on the primaryoutcome, the TEMP trial showed that etidronatetreatment can delay ectopic mineralization in PXE.

Based upon our findings, all patients with PXEshould be considered for etidronate treatment. Pref-erably, all patients with PXE treated with etidronateshould be carefully monitored in a research settingfor long-term efficacy and safety. Being one of theoldest bisphosphonates, etidronate has been on themarket for almost 40 years and is easily available atlow cost. Given the large effect on quality of life ofophthalmological and vascular involvement in PXE,

etidronate treatment may eventually enhance qualityof life in patients with PXE (42). Other potentialtreatment strategies considered in patients with PXEshould be weighed against the efficacy and safetyprofile of etidronate.

The TEMP trial provides proof of the concept thatbisphosphonates can reduce arterial calcificationsand may reduce subsequent vascular risk. Pop-ulations other than patients with PXE may benefitfrom this potential of bisphosphonates. Medial arte-rial calcification is a prevalent process occurring inrenal failure, diabetes mellitus, and aging, which re-lates to an increased vascular risk (43,44). In-vestigations on the effects of bisphosphonates inthese populations, associated with a high residualvascular risk, seem warranted.

STUDY STRENGTHS AND LIMITATIONS. The TEMP trial isthe first randomized placebo-controlled trial thatinvestigates the efficacy and safety of bisphospho-nates in patients with PXE. Strengths of the TEMP trialinclude the relatively large number of patients withPXE and its success to achieve a low dropout rate.Limitations include the limited treatment and

CENTRAL ILLUSTRATION PXE: Treatment of Ectopic Mineralization

Kranenburg, G. et al. J Am Coll Cardiol. 2018;71(10):1117–26.

PXE is characterized by ectopic mineralization of elastic fibers in the skin, the Bruch membrane beneath the retina, and the medial layer of arteries. PXE is caused by a

biallelic mutation in the ABCC6 genes, causing reduced levels of inorganic pyrophosphate and ectopic mineralization. Clinical manifestations are present in the skin,

eyes, and vascular system. In the TEMP trial, 74 adult patients with PXE with leg arterial calcifications were randomly assigned to etidronate or placebo (cyclical

20 mg/kg for 2 weeks every 12 weeks) during 1 year. 18F-NaF PET ¼ 18fluoride sodium positron emission tomography; CT ¼ computed tomography;

Events ¼ subretinal neovascularization events; PXE ¼ pseudoxanthoma elasticum; TEMP ¼ Treatment of Ectopic Mineralization in Pseudoxanthoma Elasticum.

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PERSPECTIVES

COMPETENCY IN MEDICAL KNOWLEDGE: Compared with

placebo, etidronate can prevent progression of arterial

calcification and subretinal neovascularization in patients with

PXE without significant adverse effects.

TRANSLATIONAL OUTLOOK: Further research is needed to

assess the long-term efficacy and safety of etidronate in patients

with PXE and to evaluate the potential therapeutic utility of

bisphosphonates on arterial calcification and vascular risk in

patients with diabetes mellitus and renal failure.

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follow-up period. With this limited follow-up time, wewere unable to show effects of etidronate treatment onother outcomes besides CT arterial calcification andsubretinal neovascularization events. Also, no safetyinformation for etidronate treatment in patients withPXE for$1 year is available. The lack of generalizabilityto patients with PXE not eligible for the TEMP trial,such as patients without leg arterial calcification orwith osteomalacia, is another limitation.

CONCLUSIONS

In patients with PXE, etidronate treatment did notchange femoral arterial wall 18F-NaF PET activitycompared with placebo after 12 months of follow-up;however, etidronate significantly reduced arterialcalcification and the number of subretinal neo-vascularization events compared to placebo. Noimportant safety issues of etidronate treatmentoccurred during the year of follow-up. Furtherresearch in the long-term effects and safety of eti-dronate seems warranted.

ACKNOWLEDGMENTS The authors gratefullyacknowledge the contribution of all participantsand the contributions of C.A.M. Joosten, I.P. Klaassen,E.T. Koene, and I. Janse-Seip (research nurses);A. Lalmohamed, PhD (pharmacist); G. Berkelmans,MD,

and C. Koopal, MD, PhD (subinvestigators); J.Westerink, MD, PhD (independent study physician);M.J.C Eijkemans, MD, PhD; G.K. Hovingh, MD, PhD;and R.O. Schlingemann, MD, PhD (members of thedata and safety monitoring board).

ADDRESS FOR CORRESPONDENCE: Dr. WilkoSpiering, Department of Vascular Medicine,University Medical Center Utrecht, PO Box85500, 3508 GA Utrecht, the Netherlands. E-mail:W.Spiering@umcutrecht.nl.

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KEY WORDS arterial calcification,bisphosphonates, etidronate, PXE

APPENDIX For an expanded Methods sectionas well as a supplemental table, please see theonline version of this paper.