Effects of Nitisinone on Oxidative and Inflammatory Markers in Alkaptonuria: Results from SONIA1 and SONIA2 Studies

Abstract

Nitisinone (NTBC) was recently approved to treat alkaptonuria (AKU), but there is no information on its impact on oxidative stress and inflammation, which are observed in AKU. Therefore, serum samples collected during the clinical studies SONIA1 (40 AKU patients) and SONIA2 (138 AKU patients) were tested for Serum Amyloid A (SAA), CRP and IL-8 by ELISA; Advanced Oxidation Protein Products (AOPP) by spectrophotometry; and protein carbonyls by Western blot. Our results show that NTBC had no significant effects on the tested markers except for a slight but statistically significant effect for NTBC, but not for the combination of time and NTBC, on SAA levels in SONIA2 patients. Notably, the majority of SONIA2 patients presented with SAA > 10 mg/L, and 30 patients in the control group (43.5%) and 40 patients (58.0%) in the NTBC-treated group showed persistently elevated SAA > 10 mg/L at each visit during SONIA2. Higher serum SAA correlated with lower quality of life and higher morbidity. Despite no quantitative differences in AOPP, the preliminary analysis of protein carbonyls highlighted patterns that deserve further investigation. Overall, our results suggest that NTBC cannot control the sub-clinical inflammation due to increased SAA observed in AKU, which is also a risk factor for developing secondary amyloidosis.

Keywords: amyloidosis; biomarkers; disease severity; inborn errors of metabolism; inflammation; oxidative stress; protein carbonyls; rare disease; serum amyloid A (SAA).

Figure 1

SAA and BMI in SONIA2. (a) SAA serum levels according to BMI in AKU subjects classified as: U + N, underweight and normal weight (BMI < 25); OW + O, overweight and obese (BMI ≥ 25). Median values are indicated with symbols, 25–75 percentiles with vertical lines. (b) Violin plot showing the evolution of BMI in control and NTBC. Dots indicate single values; internal boxes show median and 25–75 percentiles. A Kruskal-Wallis test followed by Dunn’s test for multiple comparisons was carried out. * p ≤ 0.05; ** p ≤ 0.01 (adjusted values).

Figure 2

SAA serum levels in SONIA2 NTBC-treated subjects who developed keratopathy due to the NTBC-induced hypertyrosinemia. The graph shows the trend of serum SAA in these ten subjects for the period they could remain in the study.

Figure 3

Analysis of protein carbonyls in a small group of SONIA2 patients’ sera. Baseline and end-of-study samples were selected according to sex, treatment, and SAA levels [low (+) if < 10 mg/L, medium (++) if between 10 and 50 mg/L, and high (+++) if > 50 mg/L]. Representative images from Western blot (upper panel) underwent a semi-quantitative analysis (lower panel) by calculating end-of-study vs. baseline fold-change values on normalized band volumes. Thresholds were set at 0.5 and 2 (dotted lines) for significantly reduced or increased carbonylation, respectively. Total carbonyls and specific proteins whose molecular weight is compatible with that of ceruloplasmin (CERU) and transferrin (TRFE) are reported. For CERU, the labels “end-of-study” and “baseline” indicate carbonylation only at these specific time points; nd: not detected. Additional details on these samples are available in Supplementary Table S4.

Figure 4

ROC curve of SAA for differentiating SONIA2 alkaptonuric subjects according to quality of life and disease severity in control and NTBC-treated groups. Abbreviations AUC: area under the curve; haqdi: Health Assessment Questionnaire disability index. SF physical: physical component of Short Form-36; cAKUSSI: clinical AKU Severity Score Index.