Predicting human tissue exposures to xenobiotics using a bottom-up physiologically-based biokinetic model

Advances in physiologically-based biokinetic (PBK) modelling, in vitro-to-in vivo extrapolation (IVIVE) methodologies, and development of permeability-limited biokinetic models have allowed predictions of tissue drug concentrations without utilizing in vivo animal or human data. However, there is a lack of in vivo human tissue concentrations to validate these models. Herein, we validated the performance of our previously published bottom-up rosuvastatin (RSV) PBK model with clinical data from a recently published study that made use of positron emission tomography (PET) imaging to quantify the hepatic concentrations of [11C]RSV drug-drug interaction (DDI) with cyclosporine A (CsA). Simulated RSV area under the plasma concentration-time curve (AUC0h-t) and maximum plasma concentration (Cmax) before and after DDI were within 1.5-fold of the observed data. Simulated AUC0-30min and Cmax ratios in the DDI setting matched the observed ratios closely (within 1.1-fold). To predict RSV hepatic concentrations, the model inputs were modified to account for RSV in the bile canaliculi after biliary excretion. The model recapitulated the observed hepatic concentrations before DDI and the decrease in hepatic concentrations after DDI. Simulated area under the liver concentration-time curve (AUC0-30min,liver), maximum liver concentration (Cmax,liver), AUC0-30min,liver ratio and Cmax,liver ratios were predicted within 1.5-fold of the observed data. In summary, we validated the ability of bottom-up PBK modelling to predict RSV hepatic concentrations with and without DDI with CsA. Our findings confirm the importance to account for drug distributed within the bile canaliculi for accurate prediction of hepatic tissue drug levels when compared against in vivo liver PET scan data.

Bottom-up physiologically-based biokinetic modelling as an alternative to animal testing.

There is a growing need for alternatives to animal testing to derive biokinetic data for evaluating both efficacy and safety of chemicals. One such alternative is bottom-up physiologically-based biokinetic (PBK) modeling which requires only in vitro data. The primary objective of this study is to develop and validate bottom-up PBK models of 3 HMG-CoA reductase inhibitors: rosuvastatin, fluvastatin and pitavastatin. Bottom-up PBK models were built using the Simcyp® Simulator by incorporating in vitro transporter and metabolism data (Vmax, Jmax, Km, CLint) obtained from the literature and proteomics-based scaling factors to account for differences in transporters expression between in vitro systems and in vivo organs. Simulations were performed for single intravenous, single oral and multiple oral dose of these chemicals. The results showed that our bottom-up models predicted systemic exposure (AUC0h-t), maximum plasma concentration (Cmax), plasma clearance and time to reach Cmax (Tmax) within two-fold of the observed data, with the exception of parameters associated with multiple oral pitavastatin dosing and single oral fluvastatin dosing. Additional middle-out simulations were performed using animal distribution data to inform tissue-to-plasma equilibrium distribution ratios for rosuvastatin and pitavastatin. This improved the predicted plasma-concentration time profiles but did not significantly alter the predicted biokinetic parameters. Our study demonstrates that quantitative proteomics-based mechanistic in vitro-to-in vivo extrapolation (IVIVE) could account for downregulation of transporters in culture and predict whole organ clearances without empirical scaling. Hence, bottom-up PBK modeling incorporating mechanistic IVIVE could be a viable alternative to animal testing in predicting human biokinetics.

Metabolomic Characterizations of Liver Injury Caused by Acute Arsenic Toxicity in Zebrafish.

Arsenic is one of the most common metalloid contaminants in groundwater and it has both acute and chronic toxicity affecting multiple organs. Details of the mechanism of arsenic toxicity are still lacking and profile studies at metabolic level are very limited. Using gas chromatography coupled with mass spectroscopy (GC/MS), we first generated metabolomic profiles from the livers of arsenic-treated zebrafish and identified 34 significantly altered metabolite peaks as potential markers, including four prominent ones: cholic acid, glycylglycine, glycine and hypotaurine. Combined results from GC/MS, histological examination and pathway analyses suggested a series of alterations, including apoptosis, glycogenolysis, changes in amino acid metabolism and fatty acid composition, accumulation of bile acids and fats, and disturbance in glycolysis related energy metabolism. The alterations in glycolysis partially resemble Warburg effect commonly observed in many cancer cells. However, cellular damages were not reflected in two conventional liver function tests performed, Bilirubin assay and alanine aminotransferase (ALT) assay, probably because the short arsenate exposure was insufficient to induce detectable damage. This study demonstrated that metabolic changes could reflect mild liver impairments induced by arsenic exposure, which underscored their potential in reporting early liver injury.

Metabolic profiling of CHO-AßPP695 cells revealed mitochondrial dysfunction prior to amyloid-ß pathology and potential therapeutic effects of both PPARγ and PPARα Agonisms for Alzheimer's disease.

In this study, we performed gas chromatography time-of-flight mass spectrometry (GC-TOFMS)-based extracellular metabolic profiling on AßPP-transfected CHO cells (CHO-AßPP695) and its wildtype. Orthogonal partial least squares discriminant analysis (OPLS-DA) was then used to identify discriminant metabolites, which gave clues on the effects of AßPP transgene on cellular processes. To confirm the hypotheses generated based on the metabolic data, we performed biochemical assays to gather further evidence to support our findings. The OPLS-DA showed a robust differentiation following 24 h of incubation (Q2(cum) = 0.884) and 15 discriminant metabolites were identified. In contrast, extracellular Aß42 was identified to increase significantly in CHO-AßPP695 only after incubation for 48 h. The observed 24-h metabolic fluxes were associated with increased mitochondrial AßPP and reduced mitochondrial viabilities, which occurred before extracellular Aß accumulation. We also investigated the therapeutic potential of peroxisome proliferator-activated receptor gamma (PPARγ) agonists, namely rosiglitazone (RSG) and pioglitazone (PIO), by employing the same approach to characterize the metabolic profiles of CHO-AßPP695 treated with RSG and PIO, with or without their respective receptor blockers. Treatment with PIO was found to reduce the perturbation of the discriminant metabolites in CHO-AßPP695 to a larger extent than treatment with RSG. We also attributed the PIO effects on the lowering of Aß42, and restoration of mitochondrial activity to PPARγ and PPARα agonism, respectively. Taken together, PIO was demonstrated to be therapeutically superior to RSG. Our findings provide further insights into early disease stages in this AßPP model, and support the advancement of PIO in AD therapy.

Applications of monolithic materials for sample preparation.

Recent advances in monolithic columns have made them an alternative to traditional packed columns used in liquid chromatography as well as capillary electrochromatography (CEC). The monolithic columns have been extensively studied and shown to possess several advantages that make them a promising and potential substitute for the particle packed columns. A large number of papers relating to monolithic columns have been published every year, focusing on different preparation techniques, characteristic evaluations as well as applications. This review highlighted the latest development of monoliths for other modes of analytical chemistry. In particular, this review will highlight the application of monoliths for sample preparation which is an important step of the entire analysis.

Characterization of the human tear metabolome by LC-MS/MS.

The tear film overlying the epithelial cells of the eye's surface is vital to visual function, and its composition is reflective of ocular surface health. The ultrasmall volume of tears poses challenges in its analysis, contributing to the limited number of reports on the tear metabolome. In addition, using a standard clinical method of tear collection posed some confounding factors in metabonomic analysis. We sought to establish an analytical platform for the global characterization of human tear metabolites. Following information dependent acquisition (IDA) directed liquid chromatography-tandem mass spectrometry (LC-MS/MS), isotope pattern matched peak mining was performed using Extracted Ion Chromatogram (XIC) manager within the PeakView software. Sixty metabolites representing diverse compound classes were identified in human tears, most of which have not been previously reported. Selected metabolites were verified using pure standards. Unsupervised chemometric analysis showed good separation between tear samples and blanks (PC1 = 87%, R(2) = 0.91, Q(2) = 0.87). The results demonstrated the potential of our platform for untargeted metabonomic studies of eye diseases.

Extraction of ketamine from urine using a miniature silica monolithic cartridge followed by quantification with liquid chromatography tandem mass spectrometry (LC-MS/MS).

The high surface area monolith with reactive hydroxyl group on its surface enables it to function as a miniature solid-phase extraction (SPE) cartridge in size of 1 cm in diameter and 0.5 cm in length. The prepared silica monolith was characterized by Brunauer-Emmett-Teller method, scanning electron microscopy, X-ray diffraction and Fourier transform infrared (FTIR) spectroscopy. Ketamine was selected as model analyte to validate the extraction efficiency of the prepared cartridge. The extracted ketamine from urine sample was quantitated by liquid chromatography tandem mass spectrometry (LC-MS/MS) using positive electrospray ionization. The limit of detection and quantification for ketamine was found to be 0.5 and 1.6 ng/mL, respectively. The analysis exhibited linearity in the range of 10-500 ng/mL with coefficient of correlation >0.99. The recovery was found to be in the range of 89-107% with relative standard deviation (RSD) less than 10%. The prepared cartridge was found robust in extracting ketamine efficiently and repeatedly without any significant deterioration in its performance. Moreover, the batch-to-batch variations in the performance of the prepared cartridges in terms of % ion suppression of the extracts and recoveries of samples were small, suggesting the consistency in the properties of the monolith.

Metabolic profiling of 3-nitropropionic acid early-stage Huntington's disease rat model using gas chromatography time-of-flight mass spectrometry.

3-Nitropropionic acid (3-NP), a potent irreversible inhibitor of mitochondrial complex II enzyme, leads to mitochondrial dysfunction and oxidative stress in Huntington's disease (HD) rat model. In this study, biochemical assays were used to demonstrate the presence of oxidative stress and mitochondrial dysfunction in 3-NP early stage HD rat models. Gas chromatography time-of-flight mass spectrometry (GC/TOFMS) was applied to analyze metabolites in brain and plasma of 3-NP-treated and vehicle-dosed rats. The orthogonal partial least-squares discriminant analysis (OPLS-DA) model generated using brain metabolic profiles robustly differentiated the 3-NP early stage HD rat model from the control. Metabonomic characterization of the 3-NP HD rat model facilitated the detection of biomarkers that define the physiopathological phenotype of early stage HD and elucidated the treatment effect of galantamine. Brain marker metabolites that were identified based on the OPLS-DA model were associated with altered glutathione metabolism, oxidative stress, and impaired energy metabolism. The treatment effect of galantamine in early stage HD could not be concluded mechanistically using the brain metabotype. Our study confirmed that GC/TOFMS is a strategic and complementary platform for the metabonomic characterization of 3-NP induced neurotoxicity in the early stage HD rat model.

A sensitive LC/MS/MS bioanalysis assay of orally administered lipoic acid in rat blood and brain tissue.

A sensitive liquid chromatography tandem mass spectrometry (LC/MS/MS) bioanalytical method was developed and validated to analyze lipoic acid (LA) in rat blood and brain samples. Ten mobile phase combinations were investigated during method development. Mobile phase combination of 0.1% acetic acid (pH 4 adjusted with ammonia solution)/acetonitrile was most optimum in terms of sensitivity and peak shape of LA and the internal standard, valproic acid. Sample extraction method was explored using liquid-liquid extraction and protein precipitation methods. Protein precipitation yielded the highest recovery of the analytes from blood and brain ranging from 92 to 115%. The lower limit of quantitation (LLOQ) of LA was 0.1ng/mL (0.485nM) in both blood and brain while on-column lower limit of detection (LLOD) was 0.03pg. The precision (% R.S.D.) ranged from 1.49 to 26.39% and 1.49 to 10.89% for intra- and inter-day assays, respectively. The accuracy ranged from 91.2 to 116.17% for intra-day assay and 102.68 to 114.33% for inter-day assay.

Metabolic profiling of rat brain and cognitive behavioral tasks: potential complementary strategies in preclinical cognition enhancement research.

In this study, the correlation between the metabolic profiles of rats undergoing cognition enhancement drug therapy and their associated cognitive behavioral outcomes were investigated. Male Lister Hooded rats were administered either with donepezil, galantamine, or vehicle and subjected to Atlantis watermaze training and novel object recognition tests. An UPLC/MS/MS method was developed to profile 21 neurologically related metabolites in the rat brains. Pharmacologically induced behavioral changes were compared subsequently with the metabolic fluctuations of neurologically related metabolites from multiple neurotransmitter pathways using multivariate and univariate statistical analyses. Significant improvements in cognitive behavioral outcomes were demonstrated in the rats administered with donepezil and galantamine using both AWM training (P < 0.05) and NOR (P < 0.05) tests as compared to those dosed with the vehicle. This corroborated with the significant elevation of eight prominent biomarkers after the cognitive enhancement therapy. An orthogonal partial least-squares discriminant analysis model generated using only the 8 metabolites identified as discriminating the drug-dosed rats from the vehicle-dosed rats gave a Q(2) = 0.566, receiver operator characteristic (ROC) AUC = 1.000, using 7-fold cross validation. Our study suggests that metabolic profiling of rat brain is a potential complementary strategy to the cognitive behavioral tasks for characterizing neurobiological responses to cognition enhancement drug testing.

Underlying mitochondrial dysfunction triggers flutamide-induced oxidative liver injury in a mouse model of idiosyncratic drug toxicity.

Flutamide, a widely used nonsteroidal anti-androgen, but not its bioisostere bicalutamide, has been associated with idiosyncratic drug-induced liver injury. Although the susceptibility factors are unknown, mitochondrial injury has emerged as a putative hazard of flutamide. To explore the role of mitochondrial sensitization in flutamide hepatotoxicity, we determined the effects of superimposed drug stress in a murine model of underlying mitochondrial abnormalities. Male wild-type or heterozygous Sod2(+/-) mice were injected intraperitoneously with flutamide (0, 30 or 100 mg/kg/day) for 28 days. A kinetic pilot study revealed that flutamide (100 mg/kg/day) caused approximately 10-fold greater exposure than the reported therapeutic mean plasma levels. Mutant (5/10), but not wild-type, mice in the high-dose group exhibited small foci of hepatocellular necrosis and an increased number of apoptotic hepatocytes. Hepatic GSSG/GSH, protein carbonyl levels, and serum lactate levels were significantly increased, suggesting oxidant stress and mitochondrial dysfunction. Measurement of mitochondrial superoxide in cultured hepatocytes demonstrated that mitochondria were a significant source of flutamide-enhanced oxidant stress. Indeed, mitochondria isolated from flutamide-treated Sod2(+/-) mice exhibited decreased aconitase activity as compared to vehicle controls. A transcriptomics analysis using MitoChips revealed that flutamide-treated Sod2(+/-) mice exhibited a selective decrease in the expression of all complexes I and III subunits encoded by mitochondrial DNA. In contrast, Sod2(+/-) mice receiving bicalutamide (50 mg/kg/day) did not reveal any hepatic changes. These results are compatible with our concept that flutamide targets hepatic mitochondria and exerts oxidant stress that can lead to overt hepatic injury in the presence of an underlying mitochondrial abnormality.

Pharmaceutical metabolite profiling using quadrupole/ion mobility spectrometry/time-of-flight mass spectrometry.

The use of hybrid quadrupole ion mobility spectrometry time-of-flight mass spectrometry (Q/IMS/TOFMS) in the metabolite profiling of leflunomide (LEF) and acetaminophen (APAP) is presented. The IMS drift times (T(d)) of the drugs and their metabolites were determined in the IMS/TOFMS experiments and correlated with their exact monoisotopic masses and other in silico generated structural properties, such as connolly molecular area (CMA), connolly solvent-excluded volume (CSEV), principal moments of inertia along the X, Y and Z Cartesian coordinates (MI-X, MI-Y and MI-Z), inverse mobility and collision cross-section (CCS). The correlation of T(d) with these parameters is presented and discussed. IMS/TOF tandem mass spectrometry experiments (MS(2) and MS(3)) were successfully performed on the N-acetyl-p-benzoquinoneimine glutathione (NAPQI-GSH) adduct derived from the in vitro microsomal metabolism of APAP. As comparison, similar experiments were also performed using hybrid triple quadrupole linear ion trap mass spectrometry (QTRAPMS) and quadrupole time-of-flight mass spectrometry (QTOFMS). The abilities to resolve the product ions of the metabolite within the drift tube and fragment the ion mobility resolved product ions in the transfer travelling wave-enabled stacked ring ion guide (TWIG) demonstrated the potential applicability of the Q/IMS/TOFMS technique in pharmaceutical metabolite profiling.

Development and validation of a gas chromatography/mass spectrometry metabonomic platform for the global profiling of urinary metabolites.

This paper presents a simple and reliable gas chromatography/mass spectrometry (GC/MS) method for the metabonomic analysis of human urine samples. The sample preparation involved the depletion of excess urea via treatment with urease and subsequent protein precipitation using ice-cold ethanol. An aliquot of the mixture was separated, dried, trimethylsilyl (TMS)-derivatized and 1.0 microL of the derivatized extract was injected into the GC/MS system via split injection (1:10). Approximately 150 putative metabolites belonging to different chemical classes were identified from the pooled human urine samples. All the identified metabolites were selected to evaluate precision and stability of the GC/MS assay. More than 95% of the metabolites demonstrated good reproducibility, with intra-day and inter-day precision values below 15%. Metabolic profiling of 53 healthy male and female urine samples in combination with pattern recognition techniques was performed to further validate the GC/MS metabolite profiling assay. Principal component analysis (PCA) followed by orthogonal partial least squares analysis (OPLS) revealed differences between urinary metabolite profiles of healthy male and female subjects. This validated GC/MS metabolic profiling method may be further applied to the metabonomic screening of urinary biomarkers in clinical studies.

Investigation of the drug-drug interaction between alpha-lipoic acid and valproate via mitochondrial beta-oxidation.

PURPOSE: To investigate the potential drug-drug interaction (DDI) between lipoic acid (LA) and valproate (VA) via the mitochondrial beta-oxidation pathway in rats. METHODS: In vitro mitochondrial assays were performed to compare the biotransformation of VA to valproyl-CoA (VA-CoA), in the absence and presence of LA. In vitro microsomal and protein binding assays were performed to elucidate their potential DDI at the microsomal metabolism and distribution levels. A pharmacokinetic study was conducted in Lister Hooded rats to ascertain the in vivo DDI between LA and VA. RESULTS: LA was shown to decrease significantly (p < 0.05) the in vitro formation of VA-CoA in a concentration-dependent manner. Our in vitro assay results confirmed that there was minimal interaction between LA and VA in microsomal metabolism and protein binding. Based on the pharmacokinetic data, the absolute bioavailability of VA was determined to be 1.3 in the presence of LA. CONCLUSIONS: Our study demonstrated for the first time that there is a potential DDI between LA and VA at the mitochondrial beta-oxidation level. While further clinical study is essential, our preliminary finding suggested that medical practitioners need to be prudent when managing epileptic patients who are co-administered with both VA and LA.

Prevention of acetaminophen (APAP)-induced hepatotoxicity by leflunomide via inhibition of APAP biotransformation to N-acetyl-p-benzoquinone imine.

Acetaminophen (APAP) is safe at therapeutic levels but causes liver injury via N-acetyl-p-benzoquinone imine (NAPQI)-induced oxidative stress when overdose. Recent studies indicated that mitochondrial permeability transition (mPT) plays a key role in APAP-induced toxicity and leflunomide (LEF) protects against the toxicity through inhibition of c-jun NH2-terminal protein kinase (JNK)-mediated pathway of mPT. It is not clearly understood if LEF also exerts its protective effect through inhibition of APAP bioactivation to the toxic NAPQI. The present work was undertaken to study the effect of LEF on the bioactivation of APAP to NAPQI. Mechanism-based inhibition incubations performed in mouse and human liver microsomes (MLM and HLM) indicated that inhibition of APAP bioactivation to NAPQI was observed in MLM but not in HLM. Furthermore, LEF but not its active metabolite, A77-1726, was shown to be the main inhibitor. When APAP and LEF were incubated with human recombinant P450 enzymes, CYP1A2 was found to be the isozyme responsible for the inhibition of APAP bioactivation. Species variation in CYP1A2 enzymes probably accounted for the different observations in our MLM and HLM studies. We concluded that inhibition of NAPQI formation is not a probable pathway that LEF protects APAP-induced hepatotoxicity in human.

Evaluation of BEH C18, BEH HILIC, and HSS T3 (C18) column chemistries for the UPLC-MS-MS analysis of glutathione, glutathione disulfide, and ophthalmic acid in mouse liver and human plasma.

Glutathione (GSH), glutathione disulfide (GSSG), and ophthalmic acid (OA) are important biological oxidative stress biomarkers to be monitored in pathological and toxicological studies. With the advent of liquid chromatography tandem mass spectrometry (LC-MS-MS) technology, sensitive and selective analysis of these biomarkers in biological samples is now being performed routinely. Due to the hydrophilic and polar natures of GSH and its endogenous derivatives, achieving good retention, resolution, and peak shape is often a chromatographic challenge. In this study, three ultra-performance (UP) LC column chemistries (namely, BEH C18, BEH HILIC, and HSS T3 [C18]) are evaluated for the UPLC-MS-MS analysis of GSH, GSSG, and OA extracted from mouse liver and human plasma samples. The chromatographic parameters evaluated are retentivity, tailing factor, MS sensitivity, and resolution of the three analytes. Based on the optimized method for each column chemistry, our results indicate that the HSS T3 (C18) column chemistry affords the best retention and separation of these analytes when operated under the ultra high-pressure chromatographic conditions.

Oxidative bioactivation and toxicity of leflunomide in immortalized human hepatocytes and kinetics of the non-enzymatic conversion to its major metabolite, A77 1726.

We used immortalized human hepatocytes to study the bioactivation of leflunomide and the metabolic degradation to its major metabolite, A77 1726. Both leflunomide and A77 1726 caused a time- and concentration-dependent increase in LDH release. The cytotoxicity of leflunomide, but not that of A77 1726, was prevented by the pan-CYP inhibitor, 1-aminobenzotriazole, indicating that an oxidative metabolite(s) was responsible for the cell injury. LC/MS/MS analysis revealed that leflunomide was rapidly degraded in hepatocytes biphasically (t((1/2))(a) = 1.5 h, t((1/2)) >24 h), but much slower in cell-free medium (t((1/2)) >24 h). In contrast, the generation of A77 1726 occurred at a similar rate in cells and cell-free systems. In conclusion, leflunomide was rapidly metabolized in human hepatocytes to A77 1726, but its toxicity was dependent on other, CYP-dependent intermediates.

Pharmacokinetic study of intraperitoneally administered troglitazone in mice using ultra-performance liquid chromatography/tandem mass spectrometry.

A rapid and sensitive ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed and validated for the determination of troglitazone in mouse plasma. Troglitazone and its internal standard (IS), rosiglitazone, were separated on an ACQUITY UPLC BEH C(18) column (1.7 microm particle size, 50 x 2.1 mm i.d.) by gradient elution with water and methanol at a flow rate of 0.5 mL/min. The cycle time of each analysis was 2.5 min. Rosiglitazone and troglitazone eluted at 1.13 and 1.57 min, respectively, and were chromatographically resolved from the ion suppression and enhancement zones due to the biological matrix effect. Quantitation of the analytes was performed in electrospray negative ionization mode (ESI -ve) using multiple reaction monitoring (MRM) experiments. The weighted (1/x) calibration curve was quadratic over the plasma concentration range 1-2500 ng/mL with a correlation coefficient (r(2)) of 0.9966. The limit of quantitation (LOQ) of troglitazone in mouse plasma was lower than 1 ng/mL. The inter- and intra-day variations of the assay were lower than 12.1%; the overall accuracy ranged from 86.4-110.2% and recovery from spiked plasma was more than 60%. The developed method was successfully applied to determine troglitazone in mouse plasma after intraperitoneal administration.

Ultra-performance liquid chromatography/time-of-flight mass spectrometry based metabolomics of raw and steamed Panax notoginseng.

At present, metabolite profiling is of growing importance in herbal medicine fields such as breeding, formulation, quality control and clinical trials. This preliminary study indicated that ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOFMS)-based metabolomics allows direct detection of down-stream derivatives of metabolites, arising from the herbal formulation process. This analytical approach allows the discrimination and tentative authentication of unique biomarkers related to different herbal extracts using unsupervised multivariate principal component analysis (PCA). The tentative identification of biomarkers is complemented significantly by the accurate mass measurement of TOFMS and the high resolution and high retention time reproducibility rendered by UPLC. The application of this approach in herbal extract discrimination and ginsenoside biomarker discovery of raw and steamed Panax notoginseng (Burk.) F.H. Chen is demonstrated and discussed.

In vitro metabolism of leflunomide by mouse and human liver microsomes.

Leflunomide was found to be metabolized predominantly to A77-1726 and two novel hydroxylated metabolites, M1 and M2, in microsomes while A77-1726 was only biotransformed to M1. M1 and M2 were proposed to be the hydroxylated alpha-cyanoenol form of A77-1726 and the hydroxylated 5 methyl-isoxazole form of leflunomide, respectively.