Background Alkaptonuria (AKU) is a serious genetic disease characterised by premature spondyloarthropathy. Homogentisate-lowering therapy is being investigated for AKU. Nitisinone decreases homogentisic acid (HGA) in AKU but the dose-response relationship has not been previously studied. Methods Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1) was an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study. The primary objective was to investigate the effect of different doses of nitisinone once daily on 24-h urinary HGA excretion (u-HGA₂₄) in patients with AKU after 4 weeks of treatment. Forty patients were randomised into five groups of eight patients each, with groups receiving no treatment or 1 mg, 2 mg, 4 mg and 8 mg of nitisinone. Findings A clear dose-response relationship was observed between nitisinone and the urinary excretion of HGA. At 4 weeks, the adjusted geometric mean u-HGA₂₄ was 31.53 mmol, 3.26 mmol, 1.44 mmol, 0.57 mmol and 0.15 mmol for the no treatment or 1 mg, 2 mg, 4 mg and 8 mg doses, respectively. For the most efficacious dose, 8 mg daily, this corresponds to a mean reduction of u-HGA₂₄ of 98.8% compared with baseline. An increase in tyrosine levels was seen at all doses but the dose-response relationship was less clear than the effect on HGA. Despite tyrosinaemia, there were no safety concerns and no serious adverse events were reported over the 4 weeks of nitisinone therapy. Conclusions In this study in patients with AKU, nitisinone therapy decreased urinary HGA excretion to low levels in a dose-dependent manner and was well tolerated within the studied dose range. Trial registration number EudraCT number: 2012-005340-24. Registered at ClinicalTrials.gov: NCTO1828463.

Background: Alkaptonuria is a rare debilitating autosomal recessive disorder of tyrosine metabolism, where deficiency of homogentisate 1,2-dioxygenase results in increased homogentisic acid. Homogentisic acid is deposited as an ochronotic pigment in connective tissues, especially cartilage, leading to a severe early onset form of osteoarthritis, increased renal and prostatic stone formation and hardening of heart vessels. Treatment with the orphan drug, nitisinone, an inhibitor of 4-hydroxyphenylpyruvate dioxygenase has been shown to reduce urinary excretion of homogentisic acid. Method: A reverse phase liquid chromatography tandem mass spectrometry method has been developed to simultaneously analyse serum homogentisic acid, tyrosine and nitisinone. Using matrix-matched calibration standards, two product ion transitions were identified for each compound (homogentisic acid, tyrosine, nitisinone) and their respective isotopically labelled internal standards ( ¹³ C ₆ -homogentisic acid, d ₂ -tyrosine, ¹³ C ₆ -nitisinone). Results: Intrabatch accuracy was 94–108% for homogentisic acid, 95–109% for tyrosine and 89–106% for nitisinone; interbatch accuracy (n = 20) was 88–108% for homogentisic acid, 91–104% for tyrosine and 88–103% for nitisinone. Precision, both intra- and interbatch were <12% for homogentisic acid and tyrosine, and <10% for nitisinone. Matrix effects observed with acidified serum were normalized by the internal standard (<10% coefficient of variation). Homogentisic acid, tyrosine and nitisinone proved stable after 24 h at room temp, three freeze–thaw cycles and 24 h at 4℃. The assay was linear to 500μmol/L homogentisic acid, 2000μmol/L tyrosine and 10μmol/L nitisinone; increased range was not required for clinical samples and no carryover was observed. Conclusions: The method developed and validated shows good precision, accuracy and linearity appropriate for the monitoring of alkaptonuria patients, pre- and post-nitisinone therapy.