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.

Alkaptonuria: From Molecular Insights to a Dedicated Digital Platform

“…Serum total antioxidant capacity, protein carbonyls, and advanced oxidation protein products (AOPP) were found to be higher in AKU compared to control subjects in small cohorts of alkaptonuric subjects [ 77 , 104 ]. However, these findings were not confirmed in SONIA1 and SONIA2, where AOPP levels were almost exclusively in a non-pathological range [ 75 , 76 ]. Similarly, in SONIA1, serum thiols, S-thiolated proteins, and the Protein Thiolation Index (PTI) showed no statistically significant differences from control populations [ 76 ] despite previous contrasting evidence suggesting increased PTI values in a very-small group of alkaptonuric individuals [ 109 ].…”

“…Despite being informative, in vitro chemical studies on HGA suffer the main limitation of the lack of a complex biological context fully recapitulating the pathological setting. To overcome this limit, several in vitro and ex vivo models of AKU were investigated, showing that the presence of HGA in cells or tissues is paralleled not only by increased oxidative stress, but also persistent low-grade inflammation and impaired protein aggregation leading to secondary amyloidosis [ 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 ]. Several oxidative protein post-translational modifications (PTMs) were associated with the presence of HGA [ 85 ], including protein carbonylation [ 70 , 79 ] and the formation of high molecular weight carbonylated aggregates [ 69 ], thiol-oxidation [ 78 , 86 ], lipid peroxidation [ 55 ], and 4-HNE protein modification [ 87 ], as well as glycation and nitrate addiction [ 80 ].…”

“…A further analysis on serum proteins that could be oxidatively modified by HGA suggested alterations of metal homeostasis and protein aggregation [ 78 , 79 ]. Interestingly, differences were highlighted in HGA-induced carbonylation patterns against important anti-oxidant serum proteins such as ceruloplasmin and transferrin, whose consequences deserve investigation [ 75 ]. A comparative proteomic study on serum and plasma samples from alkaptonuric individuals showed clear alterations in the abundance of proteins involved in stress defence mechanisms, damage repair, and pathways involved in programmed death, with the expression of several apolipoproteins, glycoproteins, complement factors, and protease inhibitors compared to healthy individuals [ 77 ].…”

“…Reactive (secondary, AA) amyloidosis was recently identified in AKU as a consequence of increasing circulating serum amyloid A (SAA), an inflammatory protein. Indeed, in the majority of AKU patients, serum SAA was >10 mg/L [ 75 , 76 , 77 ]. SAA-related amyloid deposits were found in different tissues from several alkaptonuric donors, including cartilage, tendon, synovia, periumbilical fat, and salivary glands [ 71 , 81 , 83 ].…”

“…High vascularization and expression of endothelial markers were also observed in an ex vivo synovium model obtained from alkaptonuric patients, indicating neoangiogenesis [ 105 ]. This phenomenon is likely to contribute to an increased supply of oxygen, HGA, and inflammatory proteins such as serum amyloid A (SAA) [ 75 , 76 ] in proximity of ochronotic pigment patches, hence promoting oxidative stress, inflammation, aggregation, and further ochronotic pigment deposition [ 105 ] ( Figure 3 ).…”