Development of a Physiologically Based Pharmacokinetic Model of Ethionamide in the Pediatric Population by Integrating Flavin-Containing Monooxygenase 3 Maturational Changes Over Time.

Currently, ethionamide is the most frequently prescribed second-line antituberculosis drug in children. After extensive metabolism by flavin-containing monooxygenase (FMO) isoform 3 in the liver, the drug may exert cytotoxic effects. The comparison of children in different age groups revealed a significant age-related increase in ethionamide elimination in vivo. However, to date, the exact mechanism underlying this dynamic increase in ethionamide elimination has not been elucidated. We hypothesized that the age-dependent changes in ethionamide elimination were predominantly a result of the progressive increases in the expression and metabolic capacity of FMO3 during childhood. To test this hypothesis, a full physiologically based pharmacokinetic (PBPK) model of ethionamide was established and validated in adults through incorporation of comprehensive metabolism and transporter profiles, then expanded to the pediatric population through integration of FMO3 maturational changes over time. Thus, a good prediction PBPK model was validated successfully both in adults and children and applied to demonstrate the critical contribution of FMO3 in the mechanistic elimination pathway of ethionamide. In addition, a significant correlation between clearance and age was observed in children by accounting for ethionamide maturation, which could offer a mechanistic understanding of the age-associated changes in ethionamide elimination. In conclusion, this study underlines the benefits of in vitro-in vivo extrapolation and a quantitative PBPK approach for the investigation of transporter-enzyme interplay in ethionamide disposition and the demonstration of FMO3 ontogeny in children.

Plasma drug activity in patients on treatment for multidrug-resistant tuberculosis.

Little is known about plasma drug concentrations relative to quantitative susceptibility in patients with multidrug-resistant tuberculosis (MDR-TB). We previously described a TB drug activity (TDA) assay that determines the ratio of the time to detection of plasma-cocultured Mycobacterium tuberculosis versus control growth in a Bactec MGIT system. Here, we assess the activity of individual drugs in a typical MDR-TB regimen using the TDA assay. We also examined the relationship of the TDA to the drug concentration at 2 h (C2) and the MICs among adults on a MDR-TB regimen in Tanzania. These parameters were also compared to the treatment outcome of sputum culture conversion. Individually, moxifloxacin yielded superior TDA results versus ofloxacin, and only moxifloxacin and amikacin yielded TDAs equivalent to a -2-log killing. In the 25 patients enrolled on a regimen of kanamycin, levofloxacin, ethionamide, pyrazinamide, and cycloserine, the C2 values were found to be below the expected range for levofloxacin in 13 (52%) and kanamycin in 10 (40%). Three subjects with the lowest TDA result (<1.5, a finding indicative of poor killing) had significantly lower kanamycin C2/MIC ratios than subjects with a TDA of ≥1.5 (9.8 ± 8.7 versus 27.0 ± 19.1; P = 0.04). The mean TDAs were 2.52 ± 0.76 in subjects converting to negative in ≤2 months and 1.88 ± 0.57 in subjects converting to negative in >2 months (P = 0.08). In Tanzania, MDR-TB drug concentrations were frequently low, and a wide concentration/MIC range was observed that affected plasma drug activity ex vivo. An opportunity exists for pharmacokinetic optimization in current MDR-TB regimens, which may improve treatment response.

Pharmacokinetics of ethionamide in children.

Ethionamide (ETH), a second-line antituberculosis drug, is frequently used in treating childhood tuberculosis. Data supporting ETH dose recommendations in children are limited. The aim of this study was to determine the pharmacokinetic parameters for ETH in children on antituberculosis treatment including ETH. ETH serum levels were prospectively assessed in 31 children in 3 age groups (0 to 2 years, 2 to 6 years, and 6 to 12 years). Within each age group, half received rifampin (RMP). Following an oral dose of ETH (15 to 20 mg/kg of body weight), blood samples were collected at 0, 1, 2, 3, 4, and 6 h following 1 and 4 months of ETH therapy. The maximum serum concentration (C(max)), time to C(max) (T(max)), and area under the time-concentration curve from 0 to 6 h (AUC(0-6)) were calculated. Younger children were exposed to lower ETH concentrations than older children at the same mg/kg body weight dose. Age correlated significantly with the AUC after both 1 month (r = 0.50, P = 0.001) and 4 months (r = 0.63, P = 0.001) of therapy. There was no difference in the AUC or C(max) between children receiving concomitant treatment with RMP and those who did not. Time on treatment did not influence the pharmacokinetic parameters of ETH following 1 and 4 months of therapy. HIV infection was associated with lower ETH exposure. In conclusion, ETH at an oral dose of 15 to 20 mg/kg results in sufficient serum concentrations compared to current adult recommended levels in the majority of children across all age groups. ETH levels were influenced by young age and HIV status but were not affected by concomitant RMP treatment and duration of therapy.

Development and validation of a highly sensitive LC-MS/MS method for simultaneous quantitation of ethionamide and ethionamide sulfoxide in human plasma: application to a human pharmacokinetic study.

A highly sensitive and specific LC-MS/MS method has been developed for simultaneous quantification of ethionamide and ethionamide sulfoxide in human plasma (300 µL) using prothionamide as an internal standard (IS). Solid-phase extraction was used to extract ethionamide, ethionamide sulfoxide and IS from human plasma. The chromatographic separation of ethionamide, ethionamide sulfoxide and IS was achieved with a mobile phase consisting of 0.1% acetic acid : acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a Peerless Basic C(18) column. The total run time was 3.5 min and the elution of ethionamide, ethionamide sulfoxide and IS occurred at 2.50, 2.18 and 2.68 min, respectively. A linear response function was established for the range of concentrations 25.7-6120 ng/mL (r > 0.998) for ethionamide and 50.5-3030 ng/mL (r > 0.998) for ethionamide sulfoxide. The intra- and inter-day precision values for ethionamide and ethionamide sulfoxide met the acceptance as per FDA guidelines. Ethionamide and ethionamide sulfoxide were stable in battery of stability studies, viz. bench-top, autosampler and freeze-thaw cycles. The developed assay was applied to a pharmacokinetic study in humans.

Population pharmacokinetics of ethionamide in patients with tuberculosis.

SETTING: Three US referral hospitals. OBJECTIVE: Determine the population pharmacokinetic (PK) parameters of ethionamide (ETA) following multiple oral doses. DESIGN: Fifty-five patients with tuberculosis (TB) participated. Patients received multiple oral doses of ETA as part of their treatment. They also received other anti-tuberculosis medications based upon in vitro susceptibility data. Serum samples were collected over 12 h post-dose, and concentrations were determined using a validated high-performance liquid chromatography (HPLC) assay. Concentration-time data were analyzed using population methods. RESULTS: ETA areas under the concentration-versus-time curve (AUCs) increased linearly with increasing oral doses from 250 to 1000 mg. Compared to the population pattern, delayed absorption was seen at least once in 15% of patients. ETA PK parameter estimates were independent of age, weight, height, gender, and creatinine clearance. TB patients appeared to have larger volumes of distribution (3.22 l/kg) and clearance values (1.88 l/h/kg) compared to previously studied healthy volunteers. This resulted in lower AUC values (3.95 mcg h/ml) in the TB patients. ETA displayed a short elimination half-life (1.94 h). The effect of different dosing strategies on calculated pharmacodynamic parameters was explored. Simulated doses of 250 mg BID to TID failed to achieve serum concentrations above the MIC. CONCLUSION: ETA PK parameters differed between TB patients and healthy volunteers, possibly due to differences in the completeness of absorption. Doses of at least 500 mg appear to be required to achieve serum concentrations above the typical ETA MIC. Additional research is needed to determine the optimal dosing of ETA.

High-performance liquid chromatographic-tandem mass spectrometric method for the determination of ethionamide in human plasma, bronchoalveolar lavage fluid and alveolar cells.

We have developed and validated an accurate, sensitive, and rapid high-performance liquid chromatographic-tandem mass spectrometric method (HPLC-MS-MS) for the determination of ethionamide in plasma, bronchoalveolar fluid (BAL) and alveolar cells (AC). The retention times for ethionamide, clemastine fumarate (internal standard for plasma), promethazine (internal standard for plasma) and propranolol (internal standard for BAL and AC) were approximately 2.62, 1.21, 2.14, and 2.22 min, respectively, with a total run time of 3.2 min. Ethionamide detection for plasma was carried out on a PE Sciex API III (Perkin-Elmer, Foster City, CA, USA). BAL and cell pellets and some plasma specimens were analyzed on a Micromass Quattro LC (Micromass Co., Manchester, UK). The detection limits for ethionamide were 0.05 microg/ml for plasma, and 0.005 microg/ml for BAL supernatants and alveolar cell suspensions.

Ion interaction reagent reversed-phase high-performance liquid chromatography determination of anti-tuberculosis drugs and metabolites in biological fluids.

New methods of ion interaction reagent (IIR) RP-HPLC are presented for the determination of anti-tuberculosis drugs and their metabolites, singly or in multi-component mixtures, in biological fluids. The following analytes are considered: isoniazid, ethionamide, pyrazinamide, morphazinamide, p-aminosalicylic acid, nicotinic and isonicotinic acids. Aqueous solutions of three different ion interaction reagents are alternatively or comparatively used as the mobile phases, namely: (A) 5.00 mM octylamine at pH 3.00 for o-phosphoric acid, (B) 5.00 mM octylamine at pH 8.00 for o-phosphoric acid, and (C) 5.00 mM 1,6 diaminohexane at pH 6.00 for o-phosphoric acid. The response linearity between peak area and analyte concentration is verified for all the analytes in the concentration range within the determination limits and 2.00 mg/l. Detection limits are always lower than 82 microg/l for standard solutions; in the analysis of samples of rat serum, rat plasma and human serum, the matrix effect is negligible, the detection limits are always lower than 94 microg/l and the average recovery yield is always greater than 96%.

Effects of AIDS and gender on steady-state plasma and intrapulmonary ethionamide concentrations.

Ethionamide, 250 mg every 12 h for a total of nine doses, was administered to 40 adult volunteers (10 men with AIDS, 10 healthy men, 10 women with AIDS, and 10 healthy women). Blood was obtained for drug assay prior to administration of the first dose, 2 h after the last dose, and at the completion of standardized bronchoscopy and bronchoalveolar lavage, which were performed 4 h after the last dose. Ethionamide was measured in epithelial lining fluid (ELF) and alveolar cells (AC) using a new mass spectrometric method. The presence of AIDS or gender was without significant effect on the concentrations of ethionamide in plasma, AC, or ELF. Plasma concentrations (mean +/- standard deviation [SD]) were 0.97 +/- 0.65 and 0.65 +/- 0.35 microg/ml at 2 and 4 h after the last dose, respectively, and both values were significantly greater than the concentration of ethionamide in AC (0.38 +/- 0.47 microg/ml) (P < 0. 05). The concentration of ethionamide was significantly greater in ELF (5.63 +/- 3.8 microg/ml) than in AC or plasma at 2 and 4 h and was approximately 10 to 20 times the reported MIC for ethionamide-susceptible strains of Mycobacterium tuberculosis. For all 40 subjects, the ELF/plasma concentration ratios (mean +/- SD) at 2 and 4 h were 8.7 +/- 11.7 and 9.7 +/- 5.6, respectively. We conclude that the absorption of orally administered ethionamide, as measured in this study, was not affected by gender or the presence of AIDS. Ethionamide concentrations were significantly greater in ELF than in plasma or AC, suggesting that substantial antimycobacterial activity resides in this compartment.

Therapeutic monitoring of antituberculosis drugs by direct in-line extraction on a high-performance liquid chromatography system.

A direct in-line pre-column extraction technique in which guanidinium and ammonium sulfate are used, followed by column switching, was employed to analyze serum, plasma and cerebrospinal fluid samples of patients treated for tuberculous meningitis. Resolution of a wide range of polar to non-polar xenobiotics was obtained on a C8 silica column by using a linear gradient from a binary system consisting of solvent A (0.05 M KH2PO4) and solvent B (acetonitrile-isopropanol, 4:1, v/v). Apart from the antituberculosis drugs (isoniazid, pyrazinamide, ethionamide and rifampicin) the patients received up to sixteen different medicines for prevention of complications and the treatment of symptoms. Qualitative resolution of all the drugs was obtained by the chromatographic system. Quantitation of pyrazinamide and ethionamide was achieved with high precision and low inter-sample variation.

Improved high-performance liquid chromatographic assay for the determination of ethionamide in serum.

A solid-phase extraction (SPE) method was developed to simplify the preparation of human serum prior to high-performance liquid chromatography of ethionamide (ETA). Octadecyl SPE columns were used. Serum constituents were removed from the column with water, and ETA was eluted with methanol. Samples were evaporated to dryness, reconstituted in mobile phase, and assayed. The method is reproducible, with a recovery of ETA of 64%, comparable to the more tedious liquid-liquid extraction method for ETA.

Pharmacokinetic evaluation of ethionamide suppositories.

The absorption and elimination of ethionamide (ETA) after oral tablets and rectal suppositories were determined in 12 healthy, adult male volunteers. A randomized, double-blind, double-dummy, crossover design was used. Treatments compared 250-mg ETA tablets and a placebo suppository to a 500-mg ETA suppository and two placebo tablets, given 7 days apart. Blood samples were collected at predetermined intervals for 12 hours after the dose. Serum concentrations of ETA were determined using high-performance liquid chromatography. The area under the serum concentration-time curve was used to compare the relative bioavailability of ETA from the two preparations. Relative bioavailability after rectal administration was 57.3% of that after oral administration. The maximum serum concentration after rectal administration was 33% of that after oral administration. Higher doses of ETA and serum concentration monitoring are recommended whenever the suppositories are used.

A comparison of the blood levels and urinary excretion of ethionamide and prothionamide in man.

The blood levels and urinary excretion of the anti-mycobacterial drugs ethionamide and prothionamide have been compared after oral dosage in man. High pressure liquid chromatographic methods were used to determine the two closely related thioamides and their microbiologically active sulphoxide metabolites after the ingestion of both single and combined doses of the two drugs. Both drugs were rapidly eliminated from the body, the half-life for the urinary excretion and removal from the plasma of prothionamide being slightly less than that of ethionamide. Less than 0.1% of the orally administered doses were excreted unchanged in the faeces. Plasma concentrations of ethionamide and its sulphoxide metabolite were substantially higher than those of prothionamide and prothionamide sulphoxide. The implications of these findings for the use of ethionamide or prothionamide in the treatment of lepromatous leprosy are discussed.

The binding of antituberculous drugs to normal and kwashiorkor serum.

The protein binding of 6 antituberculous drugs--ethambutol, ethionamide, isoniazid, para-aminosalicylic acid, rifampicin and streptomycin--to normal and kwashiorkor serum has been investigated. The binding of these drugs was mildly decreased in kwashiorkor serum, but not to such an extent as to be of therapeutic importance, except for streptomycin and possibly para-aminosalicylic acid (PAS). With streptomycin there was a 15% increase in the free component in kwashiorkor serum, while with PAS there was a 12% increase in the free component. Of interest is the observation that rifampicin is predominantly bound to the gamma-globulin fraction, both in normal and in kwashiorkor serum. Secondary binding, predominantly to the alpha 1-, alpha 2-and gamma-globulin fractions, was seen quite commonly in kwashiorkor serum in association with diminished albumin binding.