Transfer of Expertise on Rare Metabolic Diseases in Adults

ALKAPTONURIA

Pathophysiology Signs and symptoms Old treatment Prognosis New treatment?

Harold W de Valk, endocrinologist Zaira M. Barrientos, researcher University Medical Centre Utrecht The Netherlands Parts of the presentation

Background of the disease • Biochemical pathophysiology • Pathology • Genetics Signs and symptoms Complications and prognosis The Dutch survey Current treatment New treatment • How to test effectivity, safety and toxicity in orphan drugs • What about NTBC? Research & Development; outcome analysis Conclusions Background of the disease

Deficiency of the enzyme homogentisic acid oxidase Accumulation of homogentisic acid in blood, tissue and urine Rest activity can vary from person to person Biochemical pathway

Phenylalanine Tyrosine Tyrosine amino transaminase Phenylalanine hydroxylase 4 Hydroxyphenylpyruvate 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) Homogentisic acid Deficiency in Homogentisate 1,2-dioxygenase Alkaptonuria Maleylacetoacetate Maleylacetoacetate Porphobilinogen isomerase Fumarylacetoacetate Deficiency in Hereditary Fumarylacetoacetase tyrosinaemia I Fumarate + Acetoacetate Tricyclic acid cycle Genetic defect Pathophysiologic pathway Pathogenesis Excess HGA Excess HGA Excess HGA in plasma in urine

Polyphenol Polymers Binding to oxidase in tissue sulhydrylgroups

BQA Binding to collagen

Free radical damage

Cartilage damage Joint disease Genetics

Autosomal recessive disease Many DNA-mutations Detected hot spots: • a valley in Slovakia • Dominican republic − geographically and socially secluded area − non-paternity Prevalence: 1/500.000 to 1/1.000.000 • France (80 million): 80-160 patients • Les Pays Bas (16 million): 16-32 patients • European union (350 million): 350-700 patients Pathology

Dark coloration of the cartilage

Loose fragments Synovium involvement

Intra- and extracellular deposits

Joint destruction Abnormal collagen Culture system

Human articular cartilage 4 days culture DMEM + AHS + PSG Treatment

Homogentisic Homogentisic acid acid and vit C Analysis

Cartilage matrix synthesis by proteoglycan synthesis measured by 35 SO4 incorporation rate during the last 4 hours of culture Results

Propotional changes of the synthesis with regard to the controlgroup

160

140

120

100

% 80

0 0 30 10 100 concentration homogentisic acid (ug/ml) Signs, symptoms and complications (1)

Discoloration of fluids and tissues • Discoloration urine • Discoloration of sclerae • Discoloration of cerumen • Discoloration of sweat • Discoloration of cartilage Joint disease at an early age − pain − limited mobility − inflammatory episodes Signs, symptoms and complications (2)

Renal calculi Prostate calculi Cardiac valve abnormalities Conduction hearing loss Rupture of the Achilles tendon Clinical evolution of alkaptonuria A life’s time

Urine

Sclerae

Urinary Calculi

Joints

Valvular disease The Dutch survey

Research questions: • What is the prevalence • What are the clinical signs and symptoms • What about radiographic evaluation of the skeletal system • What about ultrasound evaluation of cardiac valves and function • What about the current and previous therapy • First estimation of incidence of complications • Start of a (larger) follow-up study The Dutch survey

Recruitment of patients: • Contacting known professionals dealing with inborn errors of metabolism • Contacting laboratories • Contacting siblings and other family members • Chance (1) The Dutch survey

Research protocol: • Written questionnaire completed by the patient • Physical examination • Radiographic evaluation: • spine • hip • knee • shoulder • Ultrasound evaluation of cardiac valves and function The Dutch survey

Results: • Number of patients: 40 (32 adults; 23 presented) • Prevalence: 1/400.000 subjects • Sex (n,%): 18/5 (78/22) • Age (years): 40 (16-72) • Early-diagnosed (n,%): 14 (61) • Late-diagnosed (n,%): 9 (39) • Joint replacement (n): 3 • Spinal surgery (n): 1 • Cardio-vascular disease (n): 1 Age at diagnosis

15 r e b 10 m Nu 5

0 5 40 45 50 55 60 65 15 20 25 30 35 10 0- 6- 36- 41- 46- 51- 56- 61- 11- 16- 21- 26- 31- Age at diagnosis (years) Age at evaluation

15 r e b 10 m Nu 5

0 60 80 40 20 0- 41- 61- 21- Age at evaluation (years) The Dutch survey

Results: • Spinal pain: 16/23 patients 70% • Joint pain: 15/23 patients 65% − shoulder 8/23 patients 35% − hip 11/23 patients 48% − knee 11/23 patients 48% The Dutch survey

Results: • 8 patients stopped working or changed job because of the disease • No patients with renal calculi • 1 patient with prostate calculi • 2 patients with an Achilles tendon rupture • 5 patients with mild hearing loss • 1 patient with a valve replacement • No patients with coronary heart diseasetient Painful joint(s)

20 s ent

i 15 t a p

f No joints 10 Yes joints er o 5 mb u N 0 80 40 60 20 0- 61- 21- 41- Age at evaluation (years) Pain according to joint involved

100 s t n e i 80 t

pa Shoulder f 60 Hip o Knee ge 40 a

t Spine n e 20 c r e

P 0 60 80 40 20 0- 41- 61- 21- Age at evaluation (years) First symptom large joints

100

80 e 60 tag n e c r 40 Pe

0 0 50 60 70 80 10 20 30 40 Age (years) First symptom spine

100

80 e 60 tag n e c r 40 Pe

0 0 50 60 70 80 10 20 30 40 Age (years) First symptom joints & spine

100

80 e 60 tag Spine n e Joints c r 40 Pe

0 0 50 60 70 80 10 20 30 40 Age (years) Difference between start of joint and spine problems

8 r e 6 b m 4 Nu

0 0 1 2 3 4 5 6 7 8 9 0 -9 -8 -7 -6 -5 -4 -3 -2 -1 10 -1 Difference in years First symptom joints according to time of diagnosis

100

80 e 60 tag Early n e Late c r 40 Pe

0 0 50 60 70 80 10 20 30 40 Age (years) First symptom spine according to time of diagnosis

100

80 e 60 tag Early n e Late c r 40 Pe

0 0 50 60 70 80 10 20 30 40 Age (years) First symptom joints with early diagnosis according to diet

100

p=0.3 80 e 60 tag Yes diet n e No diet c r 40 Pe

0 0 50 60 70 80 10 20 30 40 Age (years) First symptom joints with early diagnosis according to vitamin C

100

p=0.7 80 e 60 tag Vit C n e No Vit C c r 40 Pe

0 0 60 70 80 10 20 30 40 50 Age (years) First symptom spine with early diagnosis according to diet

100

p=0.7 80 e 60 tag Yes diet n e No diet c r 40 Pe

0 0 50 60 70 80 10 20 30 40 Age (years) First symptom spine with early diagnosis according to vitamin C

100

p=0.8 80 e 60 tag Vit C n e No Vit C c r 40 Pe

0 0 60 70 80 10 20 30 40 50 Age (years) The Dutch survey

Profession: • Construction 6 • Student/teacher 3 • Administration 3 • Logistical support 3 • Medical/paramedical 2 • Security 1 • Theatre 1 • Dentist 1 • Retired 3

Similar age-relations with construction workers The Dutch survey

Ultrasound evaluation: • Valve replacement in 1 patient • No abnormalities in all the others The Dutch survey

New events: 5 joint replacements in 2 years The Dutch survey

Treatment: • Protein restriction at any time: 44% • Vitamin C at any time: 42% Current treatment

Protein restriction High doses of vitamin C Genetic defect Site of intervention PR Pathogenesis Excess HGA Excess HGA Excess HGA in plasma in urine

Polyphenol Polymers Binding to oxidase in tissue sulhydrylgroups

BQA Vit C Binding to collagen

Free radical damage

Cartilage damage Joint disease Current treatment

Protein restriction • Difficult to maintain at higher ages and less effective Dietary manipulations in alkaptonuria

4 ) l / g

YOUNG 2 OLD HGA ( y a n i r U

0 Usual diet Protein restriction Liberal protein intake Diet Current treatment

Protein restriction • Difficult to maintain at higher ages and less effective

High doses of vitamin C • High doses may even be pro-oxidative

→ What is the evidence → Does it justify the burden of treatment? Case report

• A male (FZ) born 28-8-1963 • Referred by paediatrician from the AMC Amsterdam • First diagnosis made within the first year: discoloration of urine • Period of treatment with vitamin C and protein restriction until 12th year • Surgery left knee (meniscus) • Second diagnosis made in 1998 (35 year) with the first operation because of a lumbar hernia nuclei pulposi Case report

• No other complications related to alkaptonuria • Physical examination: dark cerumen, darkened helices • Limited mobility in all directions of the shoulder joints • Increased kyphosis of the spine • No other abnormalities • Radiographic examinination: – Cervical spine: little exophytes – Thoracic spine: no abnormalities – Lumbo-sacral spine: discopathy all levels with subchondral sclerosis –SI-joints normal – Hips: no abnormalities – Knee: some narrowing of the joint space • Ultrasound: no evidence of valve abnormalities Case report

• Urine: large excretion of homogentisic acid • Referral to the dept. of Neurology: radicular syndrome, no substrate? • MRI: multiple sequesters • 2001: Surgery • No great improvement in symptoms. • His questions: – What does the future hold? – What is the best treatment? – Which professions would be advisable? New treatment

NTBC (Nitisinone)

• Initially tested as a herbicide • Inhibits competitively an upstream enzyme: 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) • Inhibits the overproduction of homogentisic acid • Lower plasma homogentisic acid levels and no urinary homogentisic acid production • Recently obtained the orphan drug designation form EMEA (European Medicine Evaluation Agency) Site of action of NTBC

Fumarylacetoacetase

Fumarate + Acetoacetate Tricyclic acid cycle Genetic defect Site of intervention NTBC Pathogenesis PR Excess HGA Excess HGA Excess HGA in plasma in urine

Polyphenol Polymers Binding to oxidase in tissue sulhydrylgroups

BQA Vit C Binding to collagen

Free radical damage

Cartilage damage Joint disease (Orphan) drug development

Phase 1: • Testing in the laboratory to get some estimation of the drug doses required • Toxicity: reproductive system • Testing in healthy volunteers whether it is not directly lethal Phase 2: • A: Dose finding study in patients • B: Longer term exposure in patients Phase 3: • Testing against usual care in a randomized trial Phase 4: • Treatment in larger patients groups and/or for longer periods (post-marketing surveillance) (Orphan) drug development

Aim of the procedures: • Development of a pharmacological agent • Known effectiveness • Known risks: − adverse events (specific, non-specific) − reproductive toxicity (female, male) − teratogenic risk • Costs of use (final price at the counter) NTBC development (1)

Phase 1: • Testing in the laboratory to get some estimation of the drug doses required Data from the tyrosinaemia experience? 1 mg/kg? Less? • Toxicity Reproductive system? Ocular abnormalities: partially depending on the degree of elevation of the plasma tyrosine level (>500-600 μmol/l). Reversible. Animals: in rats and beagle, but not in mice or rabbits. NTBC development (2)

Phase 1: • Safety Not lethal in man • Pharmacology: Non-competitive inhibitor T½ nitisinone: 54 hours T½ tyrosine: 7 days! NTBC development (3)

Phase 2: • Proper dose finding in patients: 2003 • Extension study with multiple dosing 2003 Phase 3: • Proper randomized, placebo-controlled clinical trial 2004 Phase 4

Compassionate use: with consent of the patient in cases of a mortal disease A clinical research effort in alkaptonuria

Nice idea, but….. • Alkaptonuria is a rare disease • Complications in alkaptonuria develop slowly • Do we use NTBC and/or other strategies? • Where does the funding come from? • How about collaboration with industry?

Hypercholesterolaemia? After statins Erectile dysfunction? After viagra Fabry? After α- or β-galactosidase Partir (from Paris) c’est mourir un peu, mais…... ne pas rêver c’est être complètement mort Short outline of the clinical research programme

• Description of the currently known patients (survey), initially in the Netherlands with later a European extension • Follow-up to describe prognosis and event rate: outcome analysis of unselected patients • Pre-clinical testing of NTBC • Clinical testing of NTBC Why is outcome analysis in unselected patient groups so extremely important? Design

• Inclusion of all Dutch women with type 1 diabetes mellitus becoming pregnant in a period of 12 months • Participation of all Dutch hospitals (118) – Internist – Gynaecologist – Diabetes-nurse – (Patients themselves) • Completing questionnaires (internist, gynaecologist, paediatrician, and patient) • Study duration of 4 years Preconceptional care and knowledge

• 84% of pregnancies are planned • 70% start with folic acid before conception • HbA1c at 10 weeks pregnancy: 72% < 7.0% • Problempopulation: lower education, foreign extraction HbA1c at 10 weeks 0 8 0 6 0

N 4 0 02 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 % Frequency and risk factors for severe hypoglycaemia (SH) in the first trimester

Preconceptional 1e trimester

Frequency SH (episodes/4 mts) SH (all episodes) 0.9 ± 2.4 2.6 ± 6.3 * Hypoglycaemic coma 0.3 ± 1.3 0.7 ± 3.7 *

Proportion of women: SH (all) 25% 41% * Uncomplicated SH 16% 22% * Coma 9% 19% *

* p<0.001 N=278 Complications (1) congenital abnormalities

• Major 4.9% • Minor 3.4% • Total 8.3% (95% CI 5.3-11.3%; 2%, P<0.05)

• Unplanned vs. planned pregnancies: major 10.4% vs. 3.8% (P=0.05) HbA1c at 10 weeks and congenital abnormalities

10 9,4

8 7,5 % CA 6

4,2 4

0 4.0-6.0% 6.1-7.0% >7.0% Complications (2)

• Perinatal mortality: 2.8% (95% CI 1.0-4.6%; 0.9%, P<0.05) – CM (n=4), IUVD (n=2), PE (n=2), asphyxia (n=1) – 2 late neonatal mortality

• Maternal mortality: n=2 (0.6%; 0.01%, P<0.05) – Severe hypoglycaemia at 17 weken – Amnion fluid embolism durante partu

• Macrosomia (>p90) 56.1% (>p97.7) 30.4% Birth weight children of mothers with type 1 diabetes

20 % 15

0 < 2.3 2.3-10 10-25 25-50 50-75 75-90 90-97.7 >97.7 geboortegewicht in percentielen Conclusions

1. Periconceptional preparation often good 2. HbA1c in the majority of women in the ‘safe’ area 3. Still increased frequency of congenital abnormalities, also within the ‘safe’ area (HbA1c 6-7%) 4. Very high frequency of macrosomia 5. High frequency of neonatal morbidity 6. High frequency of maternal severe hypoglycaemia 7. Strongly increased maternal mortality So what now?

1. Is an HbA1c between 6-7% really safe? 2. Is HbA1c a good index of the complexity of glycaemic control: Glucose-variability 3. Should ‘defensive’ forces be strengthened (anti-oxidantia?) 4. Should we give different information? 5. Type 1 diabetes and pregnancy is still not a won race. 6. The goals of the St. Vincentdeclaration are not met, may be this is simple not, and who knows never possible. ‘Safe’ HbA1c: 6.0-7.0% (n=5)

20,0 20,0 20,0

15,0 15,0 15,0 ) L L) L) / / / l l l o o o m m m m m m 10,0 10,0 10,0 on ( on ( on ( i i i t t t a a a r r r t t t n n n e e e nc nc nc o o o C C C e e e s s s o o 5,0 5,0 o 5,0 uc uc uc l l l G G G

0,0 0,0 0,0

-5,0 -5,0 -5,0 12:00 AM 4:00 AM 8:00 AM 12:00 PM 4:00 PM 8:00 PM 12:00 AM 12:00 AM 4:00 AM 8:00 AM 12:00 PM 4:00 PM 8:00 PM 12:00 AM 12:00 AM 4:00 AM 8:00 AM 12:00 PM 4:00 PM 8:00 PM 12:00 AM

Time Time Time

20,0 20,0

15,0 15,0 ) ) L L / / l l o o m m m (m

10,0 n 10,0 on ( o i t a ati r r t t n e cen nc n o o C e e C s os o c 5,0 c 5,0 u u l l G G

0,0 0,0

-5,0 -5,0 12:00 AM 4:00 AM 8:00 AM 12:00 PM 4:00 PM 8:00 PM 12:00 AM 12:00 AM 4:00 AM 8:00 AM 12:00 PM 4:00 PM 8:00 PM 12:00 AM

Time Time Outcome analysis in unselected patient groups is so extremely important because it

it provides adequate information on the results of the current therapy it can provide new insights leading to new ideas it can lead to the development of or use of new pharmacological or non-pharmacological treatments it can discard myths it can give valuable information of the life situation of patients It can identify patients groups at highest risk Conclusions

Alkaptonuria may be more frequent than 1/500.000 No current effective treatment Impact on life after the end of the third decade Further clinical epidemiologic studies are required with outcome analysis and assessment of event rate European collaboration is wanted NTBC offers a new and exciting pharmacological treatment possibility But it remains a ‘normal’ drug with possible serious side effects, used in a non-life-threatening disease European collaboration is wanted LE FIN This report was produced by a contractor for Health & Consumer Protection Directorate General and represents the views of the contractor or author. These views have not been adopted or in any way approved by the Commission and do not necessarily represent the view of the Commission or the Directorate General for Health and Consumer Protection. The European Commission does not guarantee the accuracy of the data included in this study, nor does it accept responsibility for any use made thereof.