Global Urine Metabolomics in Patients Treated with First-Line Tuberculosis Drugs and Identification of a Novel Metabolite of Ethambutol

Abstract

Population level variation of drug metabolism phenotype (DMP) has great implications in treatment outcome, drug-related side effects, and resistance development. In this study, we used a gas chromatography-time of flight-mass spectrometry (GC-TOF-MS)-based untargeted urine metabolomics approach to understand the DMP of a tuberculosis (TB) patient cohort (n ‫؍‬ 20) from Tripura, a state in the northeastern part of India. Urine samples collected at different postdose time points (2 h, 6 h, 12 h, 24 h, 36 h, and 48 h) from these newly diagnosed TB patients receiving first-line anti-TB drugs were analyzed, and we have successfully detected three of the four first-line drugs, viz., isoniazid (INH), ethambutol (ETB), and pyrazinamide (PZA). The majority of their known metabolites, acetyl-isoniazid (AcINH), isonicotinic acid (INA), isonicotinuric acid (INTA), 2,2=-(ethylenediimino)-dibutyric acid (EDBA), 5-hydroxypyrazinamide (5OH-PZA), pyrazinoic acid (POA), and 5-hydroxypyrazinoic acid (5OH-POA), were also detected. Analyzing the variation in abundances of drugs and their known metabolites and calculating the metabolic ratios in these samples, we offer comprehensive DMP information on this small patient cohort that represents Tripura, India. The majority (75%) of these patients are found to be slow acetylators of INH. The average metabolic ratios of POA/PZA and 5OH-POA/POA are 3.16 ؎ 3.03 and 6.09 ؎ 6.15, respectively. Employing correlation analysis of the metabolomics metadata and a manual prediction of drug catabolism, we have proposed 2-aminobutyric acid (AABA) as a novel metabolite of ETB. These observations indicate the usefulness of GC-MS-based metabolomics to characterize the DMP at a population level and also to identify novel drug metabolites.

Time-Dependent Changes in Urinary Metabolome Before and After Intensive Phase Tuberculosis Therapy: A Pharmacometabolomics Study

“…Metabolites originating directly from the ingested TB drugs PZA, and one of its major metabolites, pyrazinoic acid (POA), formed through the de-amination of the parent drug (Ramappa and Aithal, 2013) and excreted through the kidneys (Saukkonen et al, 2006), were identified as significant markers and remained elevated throughout the first 4 weeks of therapy (Table 1), confirming observations made by Das et al (2016). PZA is a sterilizing drug that inhibits actively growing M. tuberculosis after being converted to its active form, POA, through the bacterial enzyme, pyrazinamidase.…”

“…This observed trend was further investigated in a follow-up study by specifically exploring the known urinary metabolites of INH, RIF, PZA, and EMB, and how the concentrations of these drug metabolites changed over time. A novel drug metabolite of EMB was identified with this approach (Das et al, 2016).…”

Urine metabolome of tuberculosis patients receiving intensive phase of treatment show diurnal variations

“…Earlier reported methods were used in extracting urine analytes and subsequent derivatization. 23 An attempt was made to optimize methods to acquire data using commonly available quadrupole GC-MS in place of a multidimensional GC-MS. We observed that 100 ng of ribitol, as a spike-in standard, provides enough signal in urine metabolite analysis (Figure 2A, Figure S1). From the different GC-MS methods used for data acquisition (GC1-GC6), GC1 (50 °C for 1 min, ramp at 10 °C/min to 150 °C, hold for 3 min, ramp at 7 °C/ min to 300 °C and hold for 3 min) yielded maximum urine analyte details (Figure 2B, 2C, Figure S2).…”

Vaccine and Therapeutic Options To Control Chikungunya Virus

“…In one example, computer-aided design of molecules that would "dock" onto the nsP2 protein and serve as protease inhibitors was performed. These designer molecules were synthesized and evaluated for their ability to inhibit protein activity (104). Several of these unnamed compounds, all having common core structures, were shown to inhibit nsP2 functionality in vitro, demonstrating the potential utility of targeted designer molecules based on in silico characteristics.…”