William A' Gahl, M'D', Ph'D'

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Nitisinone Treatment for Alkaptonuria From
Darkness into Light
William A. Gahl, M.D., Ph.D.
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G152fs D153G IVS72TgtC IVS75GgtA
S189I T196fs I216T IVS9-56GgtA IVS9-17GgtA
AKU sequence variations in HGD
V300G S305F R321X W322R R330S
W97G W97R IVS51GgtA IVS51GgtT
W60G Y61P Y62C P92T
P158R Q159H G161R E168D E168K K171N E178D V181F IV
S8-2AgtC
F10fs L25P
M339fs G360R G362E M368V P370fs H371R IVS131GgtT
R225H R225L F227S P230T K248R Q258P
L119insG C120W A122V A122D F136Y E143D R145X
E3A L4S L4X IVS1-1GgtA
E42A S47L R53W R58fs S59X IVS3-2GgtA
G270R D291E H292R
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Exon
Missense Frameshift Splice/Intronic Nonsense Polym
orphism
Courtesy of M. Kayser, MD
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Alkaptonuria Clinical Findings

• Autosomal recessive 1 in 500,000
• Ochronosis Black, deteriorating connective
  tissue.
• Orthopedic Spine, hips, knees, shoulders
• Cardiac Valve, vessel calcification
• Stones Kidney, prostate

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Natural History Study of AKU

• Sixty-four individuals (33 male, 31 female), age
  4 to 80 were evaluated.
• Alkaptonuria was diagnosed before one year of age
  in 21.
• Forty-five percent were diagnosed because of
  chronic joint pain. The mean age of diagnosis for
  females was 46 years while for males it was 37
  years.

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31-40 y 41-50 y 51-60 y
61-80 y
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Schober Test of Spinal Flexion
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A
B
C
D
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Radiographic Severity Score
Age (y)
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55 y
64y
of patients not manifesting
Age(y)
Age(y)
A. Joint replacement
B. Renal stones
59 y
54 y
Age(y)
Age(y)
C. Cardiac valve involvement
D. Coronary artery calcification
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SF-36 Physical Health Survey
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Ochronosis Along the Spine in an AKU Patient with
Renal Failure
AKU nephropathy AKU sib no
nephropathy
Normally, HGA is secreted at 4-5 times the GFR.
In renal failure, this secretory capacity appears
to be impaired.
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Renal Transplant in an Alkaptonuria Patient
Pre
Post Normal AKU Cr Cl
(ml/min/1.73m2) 31 72 82-125
78 HGA (Urine-mmol/kg-d) 562 222
2 1 393 145
The renal allograft reduced daily HGA production.
Did the new kidney contribute HGA oxygenase
activity?
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Incorrect Diagnoses of Alkaptonuria

• 10 of 84 referred cases did not have
  alkaptonuria
• All were women
• Some had clinical findings, i.e., cartilage or
  bone discoloration
• Two had inaccurate or improper laboratory testing
• At least 5 had a history of long-term minocycline
  use

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MINOCYCLINE MIMICS OCHRONOSIS
31-year old
44-year old
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HGA production in AKU Patients receiving Vitamin
C or Protein Restriction

• Urinary HGA1
• Treatment N Mean SD
• None 45 3.0 1.0
• Vitamin C2 10 3.2 1.1
• Low protein 6 3.9 1.6
• 1Mmol/mmol creatinine.
• 20.25-4.0 g/d

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Alkaptonuria Nitisinone Therapy
Australian Bottlebrush

• Previously known as NTBC.
• Used since 1994 for tyrosinemia type I.
• New Drug Approval January, 2002 as OrfadinR
  (Swedish Orphan International AB)

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Phenylalanine
Phenylalanine hydroxylase
Tyrosine
Tyrosine aminotransferase
Tyrosinemia II
4-Hydroxyphenylpyruvic acid
4-hydroxyphenylpyruvic acid dioxygenase
Nitisinone
Tyrosinemia III
Homogentisic acid
Homogentisate 1,2-dioxygenase
Alkaptonuria
Maleylacetoacetate
Succinylacetoacetate
Fumarylacetoacetate
Fumarylacetoacetate hydrolase
Tyrosinemia I
Succinylacetone
Fumaric acid acetoacetic acid
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Nitisinone in Alkaptonuria Study 1 Proof of
Principle
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Nitisinone in AlkaptonuriaStudy 2 Dosage/Safety

• - Incremental dosing regimen (0.35mg, 1.05mg,
  4.0mg bid) to see how much nitisinone is required
  to lower urinary HGA to lt0.5 g/day.
• - No plasma tyrosine limit watch for corneal
  signs and symptoms for 3 months on chosen dose.
• - Mild protein restriction final week.
• - 9 patients enrolled.

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Flow Chart for First Stage of Protocol
N I T I S I N O N E
0.35 mg bid
1.05 mg bid
4 mg bid
1 3 5 7 9 11 13
15 17 19 21 23
Day
24-h Urine Plasma AA
Safety Blood Panel
Eye Exam
?
?
?
?
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Flow Chart Overview of Protocol
N I T I S I N O N E
Week
1 2 3 4 5 6 7 8 9
10 11 12 13 14 15
Continuous Impatient


Diet
1-2 Day Visit (Labs, eye exam)
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Patient 1
Day NTBC (mg/day) Urine HGA (mg/day)
Plasma Tyr (mM) 0 0 3474 62
1 0.7 958 312 3 0.7 545 529
5 0.7 944 615 8 2.1 358 662
11 2.1 220 721 13 2.1 214 757
15-21 2.1 140-194 598-844
26-77 2.1 143-375 512-958
84 (diet) 2.1 77
231
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Nitisinone Study 2 9 patients
Suwannarat P, et al., Metabolism, 2005
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Safety

• No corneal signs or symptoms.
• No significant systemic or laboratory
  abnormalities.
• 3 significant Adverse Events (AEs)
• Renal stones 53-y old with history of stones
• Aortic stenosis, coronary bypass 69-y old who
• acknowledged symptoms while on nitisinone
• gt liver enzymes 46-y old ethanol history on
  statins ALT 117 U/L reversible in 1 month
• ? Related to nitisinone?

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Conclusions

• Adult dosage is probably 1 mg bid.
• This reduces urinary HGA by 95.
• Plasma tyrosine increases to 800 mM.
• Systemic effects minimal, but nitisinone may
  cause liver toxicity.
• Is dissolution of renal (and prostatic) stones an
  outcome parameter?
• No corneal effects after 80-90 days but only 9
  patients.

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Nitisinone in AlkaptonuriaStudy 3 Controlled
Trial

• - 20 patients nitisinone, 20 no treatment age
• 30-80 variable block size randomization
• - Single blind (evaluators) urine changes color
• - Dose 2 mg daily IND 71780
• - Exam every 4 months 3 year study
• - Interim analysis at 16 months
• - Primary outcome measure total hip rotation
• - Secondary outcomes Schobers, 6-min walk,
• timed up and go, functional reach, joint
  ROM
• - Repeated measures with random coefficients

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Study 3 Baseline Data20 Patients Each

• No Treatment Nitisinone
• Age (y) 51 52
• Male 15 12
• ROM worse hip 46o 40o
• 6-min walk (ft) 1598 1336
• Schober (cm) 11.4 10.9
• Urine HGA (g/d) 4.5 4.4

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Study 3 Adverse Events

• 1 61-y old male atrial fibrillation after 2
  weeks of nitisinone off nitisinone 4 weeks at
  time of death due to myocardial infarction.
• - Probably not related.
• 2 48-y old male eye pain 6 weeks after
  nitisinone keratopathy nitisinone stopped and
  started twice more, with recurrence.
• - Definitely related.
• 3 54-y female gallstones 11 months after
  nitisinone cholecystectomy nitisinone
  restarted.
• - Probably not related.

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Study 3 Data Analysis

• - Dropouts 2 (AE 1 and AE 2)
• - Missed appointments 5 missed by 4 patients.
• More than 250 appointments kept.
• - Interim analysis 16 months after last person
• enrolled, i.e., in July, 2007.
• -- Data of 19 untreated patients
• -- Data of 18 treated patients (1
  intent-to-treat)
• -- Primary outcome Difference in rate of
• decline of total worse hip ROM.
• -- If plt0.001, stop study and treat for rest
  of 3 y.

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Future

• - Will nitisinone prove beneficial, and
• be approved by the FDA?
• - Can nitisinone prevent all the signs
• symptoms of alkaptonuria if begun
• early in life?
• - What are the long-term toxicities?
• - Can nitisinone be efficacious when
• given every other month?

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Acknowledgments

• - Wendy J. Introne, MD
• - Michael Kayser, MD
• - Pim Suwannarat, MD
• - Chanika Phornphutkul, MD
• - Kevin OBrien, NP
• - Robert Kleta, MD, PhD
• - Monique Perry, MD
• - Isa Bernardini, MEd
• - Marjan Huizing, PhD
• - Yair Anikster, MD
• - Lynn Gerber, MD

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Section on Human Biochemical Genetics MGB, NHGRI,
NIH