Capillary ultra performance liquid chromatography-tandem mass spectrometry analysis of tienilic acid metabolites in urine following intravenous administration to the rat.

Capillary scale (100 mm × 150 µm id) UPLC/MS/MS, performed using reversed-phase gradient chromatography on sub 2 µm particles, has been successfully employed for the characterization of the metabolites of the drug tienilic acid (TA) excreted via the urine following oral administration to the rat. The capillary LC system provided a significant increase (range ca. 11-33-fold) in sensitivity compared with a conventional 150 mm × 2.1 mm id UPLC system. An investigation of the effect of the injection volume and sample mass loading on the capillary column on the results obtained for both endogenous metabolites and TA was performed. This demonstrated that the injection of up to 2 µL of rat urine onto the system was permitted whilst still providing excellent chromatographic results and robustness. Qualitative analysis of the urine revealed the presence of TA itself and a total of 15 metabolites of the drug, including those resulting from biotransformations such as hydroxylation or conjugation. The capillary chromatography system was shown to be robust, and capable of providing comprehensive drug metabolite profiles from small format urine samples such as those obtained from preclinical studies in rodents.

Comparative NMR-based metabonomic investigation of the metabolic phenotype associated with tienilic acid and tienilic acid isomer.

An NMR-based metabonomic approach was applied to study the systems level metabolic effects of two closely related thiophene compounds, tienilic acid (TA) and tienilic acid isomer (TAI). The metabonomic data were anchored with traditional clinical chemistry and histopathologic analyses. TA was removed from the market as a result of suspected immune-mediated hepatotoxicity, whereas TAI is an intrinsic hepatotoxin. Equimolar doses of TA and TAI were administered to Sprague-Dawley rats, and sampling was conducted at 2, 6, and 24 h post-treatment. Histopathologic analyses revealed development of a significant hepatic lesion 24 h post-TAI treatment with a parallel increase in plasma alanine aminotransferase (ALT) activity. In contrast, TA was not associated with the development of a hepatic lesion or an increase in plasma ALT activity. High-resolution NMR spectral metabolic profiles were generated for liver extracts, plasma, and urine at multiple time points. Multivariate statistical tools were applied to model the metabolic profiles and identify discriminatory metabolites that reflected both the adaptation to TA administration and the onset and progression of TAI-induced hepatotoxicity. TAI was shown to induce marked metabolic effects on the metabolome at all time points, with dramatic metabolic perturbations at 24 h post-treatment correlating with the histopathologic and clinical chemistry evidence of a hepatic lesion. The TAI-induced metabolic perturbations provided evidence for the generation of electrophilic reactive metabolites and a significant impairment of bioenergetic metabolic pathways. TA induced early metabolic perturbations that were largely resolved by 24 h post-treatment, suggesting the reestablishment of metabolic homeostasis and the ability to adapt to the intervention, with hepatic hypotaurine potentially representing a means of assessment of hepatic adaptation. This comparative metabonomic approach enabled the discrimination of metabolic perturbations that were common to both treatments and were interpreted as nontoxic thiophene-induced perturbations. Importantly, this approach enabled the identification of temporal metabolic perturbations that were unique to TAI or TA treatment and hence were of relevance to the development of toxicity or the ability to adapt. This approach is applicable to the future study of pharmacologically and structurally similar compounds and represents a refined means of identification of biomarkers of toxicity.

Involvement of cytochrome P450-mediated metabolism in tienilic acid hepatotoxicity in rats.

Tienilic acid is reported to be converted into electrophilic metabolites by cytochrome P450 (CYP) in vitro. In vivo, however, the metabolites have not been detected and their effect on liver function is unknown. We previously demonstrated that tienilic acid decreased the GSH level and upregulated genes responsive to oxidative/electrophilic stresses, such as heme oxygenase-1 (Ho-1), glutamate-cysteine ligase modifier subunit (Gclm) and NAD(P)H dehydrogenase quinone 1 (Nqo1), in rat liver, as well as inducing hepatotoxicity by co-treatment with the glutathione biosynthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO). In this study, for the first time, we identified a glutathione-tienilic acid adduct, a stable conjugate of putative electrophilic metabolites with glutathione (GSH), in the bile of rats given a single oral dose of tienilic acid (300mg/kg). Furthermore, a tienilic acid-induced decrease in the GSH level and upregulation of Ho-1, Gclm and Nqo1 were completely blocked by pretreatment with the CYP inhibitor 1-aminobenzotriazole (ABT, 66mg/kg, i.p.). The increase in the serum ALT level and hepatocyte necrosis resulting from the combined dosing of BSO and tienilic acid was prevented by ABT, despite a low hepatic GSH level. These findings suggest that the electrophilic metabolites of tienilic acid produced by CYP induce electrophilic/oxidative stresses in the rat liver and this contributes to the hepatotoxicity of tienilic acid under impaired GSH biosynthesis.

Ticrynafen-associated hepatic injury: analysis of 340 cases.

Ticrynafen, a uricosuric diuretic, was withdrawn from clinical use in the United States in 1980 after having been implicated as the cause of a number of instances of serious hepatic injury. In this report, we analyze 340 cases of ticrynafen-associated hepatic disease reported to the manufacturer from the time of initial marketing until shortly after the drug had been recalled. Jaundice was recorded in 246 of 287 patients with sufficient clinical information, and 25 (10.2%) of these icteric patients died. The high levels of serum aminotransferase and the case fatality rate are consistent with the hepatocellular injury that was evident in all of the histologic material. In three-fourths of the cases available to us for histologic study, the lesion was that of acute hepatocellular injury. In the remaining cases there was evidence of chronic active hepatitis and/or cirrhosis. Comparison of demographic characteristics of the total population exposed to ticrynafen with the subset developing hepatic injury suggests a proportionately higher risk of injury for females over the age of 60 years. The variable and unusually prolonged latent period and lack of reported eosinophilia or rash generally suggest a mechanism other than hypersensitivity. However, the recurrence of hepatic injury in 15 of the 16 patients challenged with the drug and the prominence of eosinophils in hepatic tissue in some of the cases suggests that hypersensitivity may also play a pathogenetic role. Accordingly, there is reason to incriminate both metabolic idiosyncrasy and hypersensitivity in the mechanism of injury.

[Hypertensive patients treated with tielinic acid. A retrospective study of 298 cases (author's transl)]. / Hypertendus traités par l'acide tiénilique. Etude rétrospective de 298 observations.

Two-hundred and ninety-eight hypertensive patients received tielinic acid for a period of 4 to 42 months; in 295 cases the drug was combined with a potassium-sparing diuretic. No significant changes in mean serum creatinine levels were observed in the whole group. The slight rise in serum creatinine which occurred in 17 patients cannot be ascribed with certainty to the combined treatment. Two patients developed cytolytic hepatitis, and of the 253 patients whose serum transaminase levels were systematically measured, 18 had moderately increased levels; here again, the responsibility of the drug could not be fully established. These data suggest that combining tielinic acid with a potassium-sparing diuretic carries little risk of renal impairment and that this treatment can safely be used. The risk of liver damage is unquestionable but probably small no greater than that of other commonly used hepatotoxic drugs, notably allopurinol.

Controlled inpatient study of tienilic acid in treatment of gout and hypertension.

Under inpatient controlled conditions 4 patients with gout and hypertension were treated with varying doses of tienilic acid, a new uricosuric diuretic. Plasma urate levels were reduced by an average of 50% in association with significantly increased urinary urate excretion. A twice-daily regimen was considerably more effective than a single morning dosage in reduction of plasma urate, though both regimens were equally effective in antihypertensive potency. The single daily regimen produced greater diurnal fluctuations in plasma urate and was more frequently associated with the development of acute gout attacks.

Double-blind assessment of tienilic acid in essential hypertension.

The antihypertensive effectiveness of tienilic acid in doses of 250 mg once and twice daily was compared to hydrochlorothiazide 50 mg once and twice per day in a randomized double-blind trial lasting six weeks. Blood pressure fell significantly and to a similar extent with the larger dose of both drugs. This was accompanied by a slight decrease in body weight and serum potassium concentration and a slight increase in serum creatinine and blood urea nitrogen. The magnitude of these changes was similar with both drugs. Unlike hydrochlorothiazide, however, the administration of tienilic acid was associated with a marked drop in the concentration of serum uric acid. No disturbing side effects or evidence of serious toxicity were noted during treatment with the new drug.

The hypouricaemic effect of tienilic acid: experience in patients with hyperuricaemia.

The anti-hypertensive effect of 250 mg tienilic acid was comparable to that of 50 mg hydrochlorothiazide. Unlike hydrochlorothiazide, tienilic acid caused a statistically significant decrease in the serum uric acid values. Clinically non-significant rises in blood urea nitrogen and serum creatinine occurred with both diuretics. The hypouricaemic effect of tienilic acid given in a daily dose of 125 mg was significantly greater than that of 500 mg probenecid. Glucose tolerance in patients with mild maturity onset diabetes mellitus deteriorated with both tienilic acid and hydrochlorothiazide treatments with no statistically significant difference noted between the two treatments. Tienilic acid was well tolerated and no clinically significant haematological or biochemical abnormalities were noted.