Summary of Study ST002297

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001471. The data can be accessed directly via it's Project DOI: 10.21228/M8KH70 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002297
Study TitleComprehensive biotransformation analysis of phenylalanine-tyrosine metabolism reveals alternative routes of metabolite clearance in nitisinone-treated alkaptonuria (Urine metabolomic analysis)
Study TypeUrine metabolomic analysis (study 2 of 2)
Study SummaryBackground: Metabolomic analyses in alkaptonuria (AKU) have recently revealed alternative pathways in phenylalanine-tyrosine (phe-tyr) metabolism from biotransformation of homo-gentisic acid (HGA), the active molecule in this disease. The aim of this research was to study the phe-tyr metabolic pathway and whether the metabolites upstream of HGA, increased in nitisinone-treated patients, also undergo phase 1 and 2 biotransformation reactions. Methods: Metabolomic analyses were performed on serum and urine from patients partaking in the SONIA 2 phase 3 international randomised-controlled trial of nitisinone in AKU (EudraCT no. 2013-001633-41). Serum and urine samples were taken from the same patients at baseline (pre-nitisinone) then at 24 and 48 months on nitisinone treatment (patients N = 47 serum; 53 urine) or no treatment (patients N = 45 serum; 50 urine). Targeted feature extraction was per-formed to specifically mine data for the entire complement of theoretically predicted phase 1 and 2 biotransformation products derived from phenylalanine, tyrosine, 4-hydroxyphenylpyruvic acid and 4-hydroxyphenyllactic acid, in addition to phenylalanine-derived metabolites with known increases in phenylketonuria. Results: In total, we ob-served 13 phase 1 and 2 biotransformation products from phenylalanine through to HGA. Each of these products were observed in urine and two were detected in serum. The derivatives of the metabolites upstream of HGA were markedly increased in urine of nitisinone-treated patients (fold change 1.2-16.2) and increases in 12 of these compounds were directly proportional to the degree of nitisinone-induced hypertyrosinaemia (correlation coefficient with serum tyrosine = 0.2-0.7). Increases in the urinary phenylalanine metabolites were also observed across consecutive visits in the treated group. Conclusions: Nitisinone treatment results in marked increases in a wider network of phe-tyr metabolites than shown before. This network comprises alternative biotransformation products from the major metabolites of this pathway, produced by reactions including hydration (phase 1) and bioconjugation (phase 2) of acetyl, methyl, acetylcysteine, glucuronide, glycine and sulfate groups. We propose that these alternative routes of phe-tyr metabolism, predominantly in urine, minimise tyrosinaemia as well as phenylalanaemia.
Institute
University of Liverpool Institute of Life Course & Medical Sciences
DepartmentDepartment of Musculoskeletal & Ageing Science
Last NameBrendan
First NameNorman
AddressWilliam Henry Duncan Building, 6 West Derby Street, Liverpool, UK. L7 8TX
Emailbnorman@liverpool.ac.uk
Phone+447809606497
Submit Date2022-09-28
Num Groups2
Total Subjects103
Num Males65
Num Females38
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailLC-MS
Release Date2022-10-14
Release Version1
Norman Brendan Norman Brendan
https://dx.doi.org/10.21228/M8KH70
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001471
Project DOI:doi: 10.21228/M8KH70
Project Title:Metabolomic analysis in SONIA 2, a phase 3 international randomised-controlled trial of nitisinone in alkaptonuria (AKU)
Project Summary:Abstract from main SONIA 2 publication: Background Alkaptonuria is a rare, genetic, multisystem disease characterised by the accumulation of homogentisic acid (HGA). No HGA-lowering therapy has been approved to date. The aim of SONIA 2 was to investigate the efficacy and safety of once-daily nitisinone for reducing HGA excretion in patients with alkaptonuria and to evaluate whether nitisinone has a clinical benefit. Methods SONIA 2 was a 4-year, open-label, evaluator-blind, randomised, no treatment controlled, parallel-group study done at three sites in the UK, France, and Slovakia. Patients aged 25 years or older with confirmed alkaptonuria and any clinical disease manifestations were randomly assigned (1:1) to receive either oral nitisinone 10 mg daily or no treatment. Patients could not be masked to treatment due to colour changes in the urine, but the study was evaluator-blinded as far as possible. The primary endpoint was daily urinary HGA excretion (u-HGA24) after 12 months. Clinical evaluation Alkaptonuria Severity Score Index (cAKUSSI) score was assessed at 12, 24, 36, and 48 months. Efficacy variables were analysed in all randomly assigned patients with a valid u-HGA24 measurement at baseline. Safety variables were analysed in all randomly assigned patients. The study was registered at ClinicalTrials.gov (NCT01916382). Findings Between May 7, 2014, and Feb 16, 2015, 139 patients were screened, of whom 138 were included in the study, with 69 patients randomly assigned to each group. 55 patients in the nitisinone group and 53 in the control group completed the study. u-HGA24 at 12 months was significantly decreased by 99·7% in the nitisinone group compared with the control group (adjusted geometric mean ratio of nitisinone/control 0·003 [95% CI 0·003 to 0·004], p<0·0001). At 48 months, the increase in cAKUSSI score from baseline was significantly lower in the nitisinone group compared with the control group (adjusted mean difference –8·6 points [–16·0 to –1·2], p=0·023). 400 adverse events occurred in 59 (86%) patients in the nitisinone group and 284 events occurred in 57 (83%) patients in the control group. No treatment-related deaths occurred. Interpretation Nitisinone 10 mg daily was well tolerated and effective in reducing urinary excretion of HGA. Nitisinone decreased ochronosis and improved clinical signs, indicating a slower disease progression.
Institute:University of Liverpool Institute of Life Course & Medical Sciences
Department:Department of Musculoskeletal & Ageing Science
Last Name:Brendan
First Name:Norman
Address:William Henry Duncan Building, 6 West Derby Street, Liverpool, UK. L7 8TX
Email:bnorman@liverpool.ac.uk
Phone:+447809606497
Funding Source:European Commission for the Framework 7 grant award (DevelopAKUre, project number: 304985), Alkaptonuria Society (BPN is funded by the Alkaptonuria Society through a Sireau Fellowship Award).

Subject:

Subject ID:SU002383
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Species Group:Mammals

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Treatedrial arm
SA220649P03_V4Treated
SA220650P03_V1Treated
SA220651P03_V6Treated
SA220652P07_V1Treated
SA220653P07_V4Treated
SA220654P02_V6Treated
SA220655P02_V4Treated
SA220656L40_V1Treated
SA220657L40_V4Treated
SA220658L40_V6Treated
SA220659P02_V1Treated
SA220660P07_V6Treated
SA220661P11_V4Treated
SA220662P18_V6Treated
SA220663P18_V4Treated
SA220664P22_V1Treated
SA220665P22_V4Treated
SA220666P22_V6Treated
SA220667P18_V1Treated
SA220668P15_V6Treated
SA220669L35_V6Treated
SA220670P11_V6Treated
SA220671P15_V1Treated
SA220672P15_V4Treated
SA220673P11_V1Treated
SA220674L35_V1Treated
SA220675L16_V6Treated
SA220676L16_V4Treated
SA220677L18_V1Treated
SA220678L18_V4Treated
SA220679L18_V6Treated
SA220680L16_V1Treated
SA220681L14_V6Treated
SA220682L12_V4Treated
SA220683L12_V6Treated
SA220684L14_V1Treated
SA220685L14_V4Treated
SA220686L22_V1Treated
SA220687L22_V4Treated
SA220688L33_V1Treated
SA220689L30_V6Treated
SA220690L33_V4Treated
SA220691L33_V6Treated
SA220692P23_V1Treated
SA220693L30_V4Treated
SA220694L30_V1Treated
SA220695L22_V6Treated
SA220696L28_V1Treated
SA220697L28_V4Treated
SA220698L28_V6Treated
SA220699F01_V1Treated
SA220700P23_V4Treated
SA220701P48_V4Treated
SA220702P48_V1Treated
SA220703P48_V6Treated
SA220704P52_V1Treated
SA220705P52_V4Treated
SA220706P47_V6Treated
SA220707P47_V4Treated
SA220708P46_V1Treated
SA220709P46_V4Treated
SA220710P46_V6Treated
SA220711P47_V1Treated
SA220712P52_V6Treated
SA220713P55_V1Treated
SA220714P62_V6Treated
SA220715P62_V4Treated
SA220716P64_V1Treated
SA220717P64_V4Treated
SA220718P64_V6Treated
SA220719P62_V1Treated
SA220720P60_V6Treated
SA220721P55_V4Treated
SA220722P55_V6Treated
SA220723P60_V1Treated
SA220724P60_V4Treated
SA220725P44_V6Treated
SA220726P44_V4Treated
SA220727P31_V1Treated
SA220728P30_V6Treated
SA220729P31_V4Treated
SA220730P31_V6Treated
SA220731P34_V1Treated
SA220732P30_V4Treated
SA220733P30_V1Treated
SA220734P23_V6Treated
SA220735P26_V1Treated
SA220736P26_V4Treated
SA220737P26_V6Treated
SA220738P34_V4Treated
SA220739P34_V6Treated
SA220740P43_V1Treated
SA220741P43_V4Treated
SA220742P43_V6Treated
SA220743P44_V1Treated
SA220744P40_V6Treated
SA220745P40_V4Treated
SA220746P39_V1Treated
SA220747P39_V4Treated
SA220748P39_V6Treated
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Collection:

Collection ID:CO002376
Collection Summary:Serum and urine samples were collected at the three study sites; Liverpool, Paris and Piešťany. Samples collected at Paris and Piešťany were transported frozen to the Royal Liverpool University Hospital for metabolite analysis and storage at -80 °C. Urine was collected over 24-h into 2.5 L bottles containing 30 mL of 5 mol/L sulphuric acid which was subsequently aliquoted and stored at -80 °C.
Sample Type:Urine
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002395
Treatment Summary:Samples studied were from patients partaking in the SONIA 2 clinical trial of nitisinone in AKU. SONIA 2 was a 4-year, open-label, evaluator-blind, randomised, no treatment controlled, parallel-group study undertaken at three study sites; Liverpool (UK), Paris (France) and Piešťany (Slovakia). The details and outcomes of the trial are published (1). Serum and urine samples were collected from participants at baseline (pre-treatment) then at 3 months and 1, 2, 3 and 4 years on 10 mg oral daily nitisinone (Orfadin®) treatment or no treatment. The serum and urine samples from visit 1 (baseline; pre-treatment), visit 4 (2 years), visit 6 (4 years) underwent metabolomic analysis. In this study, only the data from patients with samples available for these three time points were included; 53 and 50 patients in treated and untreated groups respectively for urine, and 47 and 45 patients in treated and untreated groups respectively for serum. Reference: (1) Ranganath, L.R.; Psarelli, E.E.; Arnoux, J.B.; Braconi, D.; Briggs, M.; Bröijersén, A.; Loftus, N.; Bygott, H.; Cox, T.F.; Davison, A.S.; et al. Efficacy and Safety of Once-Daily Nitisinone for Patients with Alkaptonuria (SONIA 2): An International, Multicentre, Open-Label, Randomised Controlled Trial. Lancet Diabetes Endocrinol. 2020, 8, 762–772, doi:10.1016/S2213-8587(20)30228-X.
Treatment Protocol ID:Clinical trial ID: EudraCT no. 2013-001633-41
Treatment Compound:Nitisinone (NTBC)
Treatment Dose:10 mg daily
Treatment Doseduration:48 months total

Sample Preparation:

Sampleprep ID:SP002389
Sampleprep Summary:Prior to analysis, urine samples were thawed at room temperature before vortexing and centrifugation at 1500 ×g for 5 minutes. 150 µL of each urine sample was aliquoted into a 1 mL 96-well plate (Waters Corporation, Wilmslow, UK) and diluted with 450 µL of deionised water. Samples were mixed on a plate shaker (MTS 2/4m IKA) at 600 rpm for 10 min and sub-aliquoted into multiple replicate 96-well plates before storage at -80 °C ready for analysis. Prior to analysis, sample plates were thawed then agitated on a plate shaker (MTS 2/4m IKA) at 600 rpm for 10 min.

Chromatography:

Chromatography ID:CH002777
Chromatography Summary:Analysis of serum and urine samples was performed using a published LC-QTOF-MS acquisition method, which employed a 1290 Infinity II HPLC coupled to a 6550 QTOF-MS equipped with dual AJS electrospray ionisation source (Agilent, Cheadle, UK). Data acquisition parameters are detailed in brief below and in full in Supplementary Materials. Reversed-phase LC was performed on an Atlantis dC18 column (3×100 mm, 3 μm, Waters, Manchester, UK) maintained at 60 °C. Mobile phase composition was (A) water and (B) methanol, both with 5 mmol/L ammonium formate and 0.1 % formic acid. The elution gradient began at 5 % B 0–1 min and increased linearly to 100 % B by 12 min, held at 100 % B until 14 min, then at 5 % B for a further 5 min. MS data acquisition was performed in positive and negative ionisation polarity with mass range 50–1700 in 2 GHz mode with acquisition rate at 3 spectra/second. Sample injection volume was 1 and 2 µL in negative and positive polarities, respectively.
Instrument Name:Agilent 1290 Infinity II
Column Name:Waters Atlantis dC18 (100 x 3mm,3um)
Column Temperature:60
Flow Gradient:The elution gradient began at 5 % B 0–1 min and increased linearly to 100 % B by 12 min, held at 100 % B until 14 min, then at 5 % B for a further 5 min.
Solvent A:100% water; 0.1% formic acid; 5mM ammonium formate
Solvent B:100% methanol; 0.1% formic acid; 5mM ammonium formate
Chromatography Type:Reversed phase

Analysis:

Analysis ID:AN003752
Analysis Type:MS
Chromatography ID:CH002777
Num Factors:2
Num Metabolites:14
Rt Units:Minutes
Units:peak area (pareto-scaled, log2-transformed, 24h creatinine normalised)
  
Analysis ID:AN003753
Analysis Type:MS
Chromatography ID:CH002777
Num Factors:2
Num Metabolites:9
Rt Units:Minutes
Units:peak area (pareto-scaled, log2-transformed, 24h creatinine normalised)
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