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Hypophosphatasia andAlkaline Phosphatase –

What Are The Clinical Issues?

Center for Metabolic Bone Disease and Molecular Research,Shriners Hospital for Children;

and

Michael P. Whyte MD

Division of Bone and Mineral Diseases, Department of Internal Medicine,Washington University School of Medicine;

St. Louis, Missouri, U.S.A.

Alkaline Phosphatase:

We’ve Been Assaying It For 93 Years,But What Does It Do?

(Role Revealed in Hypophosphatasia)

DisclosureHonoraria, Travel, and

Research Grant Support;

Alexion Pharmaceuticals, Cheshire, CTasfotase alfa (Strensiq ™)*

* biologic for pediatric-onset hypophosphatasia

Robert Robison, Ph.D(1883-1941)

C. R. HaringtonObituary Notices of Fellows

of the Royal Society,Vol. 3, No. 10 (Dec., 1941), pp. 929-939

“Bone Phosphatase”

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Evidence that ALP Acts inSkeletal Mineralization

• Robison’s discovery that bone/cartilage is rich in ALP Activity

• Initial site of hydroxyapatite crystalformation is within ALP-richextracellular matrix vesicles

• Serum ALP activity reflects ratesof skeletal formation

Endochondral Bone Formation

Phase 1 Mineralization

Evidence that ALP Acts inSkeletal Mineralization

• Robison’s discovery that bone/cartilage is rich in ALP Activity

• Initial site of hydroxyapatite crystalformation is within ALP-richextracellular matrix vesicles

• Serum ALP activity reflects ratesof skeletal formation(exception=osteomalacia)

Evidence Against a Role ForALP in Skeletal Mineralization

• ALP assay not “physiologic”[alkaline pH (e.g., 10.2) withnonphysiologic substrates]

• ALP abundant in tissues that do NOTcalcify

Suggested Roles for ALPin Skeletal Mineralization

• Locally increases Pi Concentration(Robison’s Hypothesis)

• Pi Transport

• Phosphotyrosine-specificphosphoprotein phosphatase

• Ca++ Binding protein

• Ca++ ATPase

• Hydrolysis of Inhibitor

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1. Placental2. Intestinal Tissue-specific3. Germ Cell

4. Ubiquitous Tissue Nonspecific(Bone & Liver) (TNSALP)

Human Alkaline Phosphatases(ALPs)

J Biol Chem 263:12002, 1988

Homodimeric TNSALP

Bone and Liver ALPare Isoformsof TNSALP

(differ by post-translationalmodifications)

John C. Rathbun, M.D.(1915-1972)

Amer J Diseases Child 75 : 822-31, 1949

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• ~800 Literature Cases

• Affects all races

• Incidence: 1:100,000 Births

Hypophosphatasia (HPP)

• Heritable Metabolic Bone DiseaseDefective Skeletal Mineralization

Infants & Children RicketsAdults Osteomalacia

• Biochemical HallmarkLow Serum ALP Activity

• Inborn Error of MetabolismAutopsy: Global Deficiency of TNSALP Activity

(Intestinal & Placental ALP Are Normal)

Hypophosphatasia

Hypophosphatasia

• Circulating calcium, phosphate, andvitamin D levels are not low

• Hypercalcemia (severe disease)Hyperphosphatemia

Hypophosphatasia

• Greatest range of severity ofall metabolic bone diseases

• Last rickets/osteomalacia tohave a medical treatment

Weiss M, et. al (1988) Proc. Natl. Acad. Sci. USAVol. 85, pp. 7666-7669

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Loss-of-FunctionMutations of TNSALP

~ 300 different defectsworldwide

(~ 80% missense)

Inheritance

• Severe: Autosomal Recessive

• Mild: Autosomal RecessiveAutosomal Dominant

Nosologyaccording to age at Dx

“Skeletal Disease”(~ severity)

Hypophosphatasia

Clinical Forms Perinatal

Infantile

Childhood

Adult

OdontoSeverity

Perinatal

Perinatal Hypophosphatasia

• Skeletal Disease (Obvious at Birth)• Stillbirth• Deformed Limbs• Respiratory Compromise• Periodic Apnea and Bradycardia

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Infantile

• Skeletal Disease (Before Age 6 months)• Wide Fontanels• Hypotonia• Hypercalcemia• Nephrocalcinosis• Functional Craniosynostosis• Failure To Thrive• Flail Chest/Pneumonia• Seizures

Infantile Hypophosphatasia

Infantile Hypophosphatasia

~ 50% succumb

~ 50% improve, butoften with sequelaeof rickets

Childhood

• Skeletal Disease (After Age 6 months)• Premature loss of deciduous teeth

(Before age 5 years)• Short Stature• Delayed Walking• Rachitic Deformity• Craniosynostosis• Static Myopathy• Waddling Gait

Childhood Hypophosphatasia

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BONE title page

Bone 75 (2015) 229–239

Adult Hypophosphatasia

• Skeletal Disease (Typically During Middle Age)

• Premature Loss of Adult Teeth

• Osteopenia

• Recurrent Metatarsal Stress Fractures

• Femoral Pseudofractures

• Pseudogout/Chrondocalcinosis/Calcific Periarthritis

Adult Hypophosphatasia

The Am J of Med 72: 631-41, 1982

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JBMR 29(4): 929–934, 2014

J Bone Miner Res.; 29:1651-60, 2014

J Bone Miner Res.; 29:1651-60, 2014

Odontohypophosphatasia(Childhood or Adult)

• No Radiologic SkeletalAbnormalities

• Premature Loss of Teeth

“One-Tooth” Odonto-HPP

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Diagnosis

• Hyperphosphatasemiais a marker for bone orhepatobiliary disease

• Hyperphosphatasemiais a marker for bone orhepatobiliary disease

• Remarkable how oftenhypophosphatasemia is

ignored

• Remarkable how oftenhypophosphatasemia is

ignoredSource: McComb RB, Bowers GN, and Posen S 1979

Alkaline Phosphatase. Plenum Press, NY, pg 532 figure 9.4

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Causes of Hypophosphatasemia• Hypophosphatasia • Zn++ or Mg++ Deficiency• Familial Benign? • Cushing’s Syndrome• Pernicious or Profound Anemia • Milk-Alkali Syndrome• Hypothyroidism • Celiac Disease• Vitamin C Deficiency • Massive Transfusion• Osteogenesis Imperfecta, Type II* • Cleidocranial Dysostosis*• Wilson’s Disease (hemalytic anemia) • Cardiac Bypass Surgery• Vitamin D Intoxication • Improperly Collected Blood• Inappropriate Reference Range (e.g., EDTA, oxalate)• Clofibrate Therapy • Radioactive Heavy Metals• Starvation • Multiple Myeloma• Hypoparathyroidism • Achondroplasia• ERT in post-menopausal women

TNSALP (ALPL)mutation analysis

Pathogenesis

Phosphoethanolamine

(PEA)

Inorganic Pyrophosphate

(PPi)

Pyridoxal 5/-Phosphate

(PLP)

NH2CH2CH2 O P

OH

OH

O

O P

OH

OH

O P

OH

OH

N

CH2 O P

OH

OH

O

CHO

HO

H3C

O

Natural Substrates for TNSALP

Vitamin B6

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• No symptoms of Vitamin B6toxicity or deficiency

• Normal tissue levels ofVitamin B6 metabolites

• Normal urinary levels of thedegradation product, 4-pyridoxic acid

• Normal response to L-tryptophanchallenge

Normal Vitamin B6 Status inMost Hypophosphatasia Patients

PL levels arenormal in all butthe most severe

HPP (low)

Vitamin B6

Metabolism

ProteinN

C

EthN P

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PPi

PPi

Phase 2 Mineralization

Chondrocalcinosis

Calcium PyrophosphateDihydrate

Pyrophosphate Arthropathy

TREATMENT ?

Whyte et. al, J Peds 101(3):379

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Whyte et. al, J Peds 101:379, 1982

Can we increase ALP

activity directly in bone?

JBMR 18: 624-36, 2003

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JCE&M 92: 2923-30, 2007

JCE&M 92: 1203-08, 2007

11/046/04

ALP Targeted To Bone

D10 D10

TNSALP dimer

IgG1 Fc

Courtesy Dr. Marc D. McKee

Asfotase Alfa

Courtesy Dr. Marc D. McKee

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IV to Adult WT Mouse

Millan et al. (2008) J Bone Miner Res 23:781

Asfotase Alfa for Life-ThreateningHypophosphatasia

Radiographic,respiratory,

andfunctional

improvements.

PHASE I/II

March 8, 2012

N Engl J Med. 366:904-13, 2012

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Infant Study

Month 21Baseline

First Patient

Patient 2 (Infantile HPP)

Baseline 3/4 months 2 ½ months

Duration Treatment

9 Weeks

Patient 3 (Perinatal HPP)

Baseline

Age 2 years

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2 years of treatment

3 years oftreatment

7 years oftreatment

• Treatment ≥3 years well tolerated.

• Infants and children with life threatening HPP showedsignificant improvement in skeletal mineralization, onaverage at 3 months, and sustained over 3 years

• Nearly all required some respiratory support either atbaseline, or early in the study, but only a single patientrequired respiratory support (supplemental O2) at lastassessment

• Survival was 90%.

Summary

Simmons J et al. Poster presented at American College ofMedical Genetics and Genomics National Meeting, Tampa, FL.March 11, 2016

Adverse Events

Events, n

Patients, n (%)

Total events 11 (100%)URI 67 7 (64%)Craniosynostosis 12 7 (64%)

Pyrexia 20 7 (64%)

Pneumonia 14 7 (64%)

Otitis media 19 6 (55%)

Vomiting 13 6 (55%)

Constipation 7 6 (55%)

Injection site erythema 29 5 (46%)

Tooth loss 8 4 (36%)

Hemaglobin decreased 9 4 (36%)

Nasopharyngitis 6 4 (36%)

Rash 5 4 (36%)

Irritability 4 4 (36%)

Diarrhea 6 4 (36)

Dental caries 4 4 (36)

Adverse Events (in ≥4 Patients)

All patients reported≥1 AE during the study

AEs were reported byinvestigators asprimarily mild (73%)or moderate (21%) andunrelated to study drug(82%)

• Three SAEs were reported by investigators as possiblyrelated to study drug:– Craniosynostosis and conductive deafness (same patient)– Mild chronic hepatitis in 1 patient, confounded by

concomitant montelukast; montelukast was discontinuedand liver function tests returned to within normal range bylast assessment

• One patient died (septic shock, unrelated to study drug)at 7.5 months of treatment

1. Whyte MP et al. Asfotase alfa: Poster presented at the 2014Pediatric Academic Societies and Asian Society for PediatricResearch Joint Meeting, Vancouver, B.C., Canada, May 4, 2014.Abstract 752396. 2. Simmons J et al. Poster presented atAmerican College of Medical Genetics and Genomics NationalMeeting, Tampa, FL. March 11, 2016

Serious Adverse Events

SAE, serious adverse event

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PHASE II

Asfotase AlfaFOR

CHILDREN WITHHYPOPHOSPHATASIA

PHASE II

Asfotase AlfaFOR

CHILDREN WITHHYPOPHOSPHATASIA

• Phase II, open-label study; 1 site in USA, 1 site in Canada1-3

• Major inclusion criteria:1–3

• 5–12 years old

• Significant HPP-related skeletal disease

• Primary Endpoints:• Efficacy, safety and tolerability of asfotase alfa at 6 months

• Skeletal health assessed radiographically

Study design

1. ClinicalTrials.gov: NCT00952484; NCT01203826.2. Madson KL, et al. J Bone Miner Res. 2014:29 (Suppl 1).3. Madson KL, et al. Abstract presented at the Pediatric Academic Societies and Asian Society for Pediatric Research2014 Joint Meeting.

Extension Phase(n=12)

Endpoints: Radiographic and functionalimprovements,

growth/development, long-term safety

Initial dose: 3 mg/kg/weekIncreased to 6 mg/kg/week after 3-9 months

via protocol amendment

6Months

3Years

BL 2Years

1Year

12 patients continue treatment1 patient withdrew

3Months

4Years

5Years

Initial Phase(n=13)

Endpoints:Radiographic

improvement, safetyRandomized:

6 mg/kg/week (n=6)9 mg/kg/week (n=7)

• Major exclusion criteria:1–3

• Hypocalcemia or hypophosphatemia• Current other treatable rickets• Prior bisphosphonate treatment

Historical Controls (n=16) for radiographs

Secondary / Exploratory:• Physical Performance Assessments

• Transiliac Crest Bone Biopsies

• Biochemical Alterations

• Growth

Primary Outcome Objective:Improvement of Radiographic

Skeletal Disease

Primary endpoint:Radiographic Global Impression of Change (RGI-C)

• Change in ricketsseverity assessed bya 7-point scale

• Paired radiographs

• Mean score of 3independentradiologists blindedto time points afterfirst radiograph

WORSENING HEALING

0No Change

+1Minimal

+2Substantial

+3Complete or

Near

–1Mild

–2Moderate

–3Severe

Baseline 4.5 YearsRGI-C +2.0

*The primary endpoint was met (RGI-C improvement relative to historical controls at 6 months)

RGI-C scores at each visit

0

0.5

1

1.5

2

2.5

3

3 Months 6 Months 1 Year 2 Years 3 Years 4 Years 5 YearsStudy visit / Duration of treatment

12 12 12 12 8 11 12n

2.30.3

2.31.0

2.31.3

2.31.0

3.01.3

3.00.7

2.71.7

MaxMin

P < 0.01 for all time points compared with 0

+1.8

+2.0*

+1.7

+2.0 +2.0

+2.3+2.2

Med

ian

RG

I-C

Sco

re

+

+

+

+

+

+

Metaphyseal Flare

Baseline Week 24

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Physical Assessments

Hand-HeldDynamometry

• Grip• Knee flexion

• Knee extension

• Hip flexion• Hip extension

• Hip abduction

Six-Minute Walk Test BOT-2 Strength and AgilityComposite

Running Speed and Agility• 50 ft shuttle run• side step over beam• one-legged stationary hop• one-legged side hop• two-legged side hop

Strength• standing long jump• push-ups• curl-ups• wall sit• V-up

Bruininks-Oseretsky Test of Motor Proficiency, 2nd Edition(BOT-2)

Strength Test

Sit ups V-ups

Standing long jump Wall sit

Push ups

SD, standard deviationDeitz et al. Phys Occup Ther Pediatr. 2007;27:87-102

Running Speed and Agility Test

Two-legged side hop

Stepping over a balance beam

One-legged side hop

Shuttle run

Baseline

Walked 350 meters

Week 24

Walked 401 meters

6 minute walk test

Patient 4 at Baseline

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Patient 4 after 1 ½ years of Rx

Baseline

14 inches

Week 24

37 inches

Strength and Agility Composite Standard Score

119

120

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0

0.2

0.4

0.6

0.8

1

1.2

1.4

Baseline 6 Months 1 Year 2 Years 3 Years 4 Years 5 Years

Med

ian

disa

bilit

y in

dex

scor

e

Study Visit

Childhood Health Assessment Questionnaire (CHAQ):Disability Index

MCID, minimal clinically important differenceDempster H et al. Arthritis Rheum 2001;44:1768-74

2.250.0

0.50.0

2.00.0

1.750.0

1.50.0

0.880.0

1.00.0

MaxMin

P < 0.05 mean change from baseline at each time point

13 11 12 8 11 12 n123.0Maximumdisability

No disability

Decreases ≥0.13 are considered above theminimal clinically important difference (MCID) injuvenile arthritis1

Decrease: >5×MCID

8 sub-scales of age-appropriate activities of daily living

CHAQ

CHAQ and PODCI: PainPODCI

Nodiscomfort

aMean of individual patient changes from baseline compared to 0 (no change).CHAQ, Childhood Health Assessment Questionnaire; NS, non-significant; PODCI, Pediatric Outcomes Data Collection Instrument.

Safety• Transient, dose-dependent injection site

reactions in all patients which are lessprominent and less frequent with lowerdoses on the extension study

• No ENB-0040-Related SAEs

• No evidence of ectopic calcification onretinal examination or renal ultrasound

• Low Titer Anti-ENB0040 Antibodies

Lipohypertrophy

• The most common AEs were mild-to-moderate ISRs

• During treatment with asfotase alfa, improvementswere observed as early as 6 months in children for:– Strength– Running Speed and Agility– Disability– Transfer and Basic Mobility– Sports and Physical Functioning– Pain

• Improvements were sustained through 5 years oftreatment

Conclusions

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• 03 July 2015 – Japan (HPP)

• 14 August 2015 – Canada (Pediatric-Onset HPP)

• 28 August 2015 – Europe (Pediatric-Onset HPP)

• 23 October 2015– United States (Pediatric-Onset HPP)

Asfotase alfa*

*STRENSIQ™

J Bone Miner Res.; 25:2267–94, 2010.

J Bone Miner Res; 24:1132–4, 2009

J Bone Joint Surg Am 68:981-90, 1986

Adult Hypophosphatasia

J Bone Joint Surg Am 68:981-90, 1986

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JBMR 27:987-994 (2012)

JBMR 27:987-994 (2012)

January 2012 J Bone Miner Res 27(1):93-105

Thank You