Precision Medicine With Leflunomide: Consideration of the DHODH Haplotype and Plasma Teriflunomide Concentration and Modification of Outcomes in Patients With Rheumatoid Arthritis.

OBJECTIVE: Leflunomide is a commonly used disease-modifying drug in the treatment of rheumatoid arthritis (RA). Its effects are mediated via inhibition of dihydroorotate dehydrogenase (DHODH) by its active metabolite teriflunomide, and the pharmacokinetics of teriflunomide are highly variable. Our objective was to examine the association between the DHODH haplotype and plasma teriflunomide concentration with response to leflunomide in patients with RA where leflunomide was added to an existing disease-modifying drug regimen after failure to achieve an adequate response with conventional triple therapy. METHODS: Patients with RA who were taking, or were about to initiate, leflunomide were included. Participant characteristics, including the DHODH haplotype, were determined. Up to 5 plasma samples were collected after leflunomide was initiated for assays of total and free teriflunomide concentration. Disease activity was determined via the 28-joint Disease Activity Score (DAS28). The association between DAS28 scores and patient covariates was determined by linear mixed-effects modeling. RESULTS: A total of 67 patients were included in the study. The DAS28 score after initiation of leflunomide was associated with the baseline DAS28 score (ß = 0.70, P < 0.001) and was higher in those who carried the DHODH haplotype 2 (ß = 0.56. P = 0.01) and did not carry the shared epitope (ß = 0.56, P = 0.013). As total and free plasma teriflunomide concentration increased, the DAS28 score was significantly lower (P < 0.001 and P = 0.001, respectively). When considering threshold concentrations, teriflunomide concentrations >16 mg/liter were associated with a DAS28 score that was 0.33 lower, and when free teriflunomide concentration was >35 µg/liter, the DAS28 score was 0.32 lower. CONCLUSION: Teriflunomide concentration and carriage of the DHODH haplotype 2 are associated with response to leflunomide in patients with RA, and a total plasma teriflunomide concentration of at least 16 mg/liter is needed to maximize the likelihood of response.

Coated nanostructured lipid carriers targeting the joints - An effective and safe approach for the oral management of rheumatoid arthritis.

Oral treatment of rheumatoid arthritis (RA) with the immunomodulator, leflunomide (LEF), is associated with systemic side effects namely immunosuppression and hepatotoxicity. Herein, attempts to improve LEF therapeutic outcomes via nanostructured lipid carriers (NLCs) targeting inflamed rheumatic joints were executed. LEF-NLCs coated with either chondroitin sulphate (CHS) or chitosan (CS) were around 250 nm in size with negative or positive charge, respectively. Particle coating was evidenced by TEM and FTIR analysis. NLCs generally ensured sustained release profile up to 21 days, particularly extended in coated formulations. In vivo pharmacokinetic study of LEF suspension, uncoated NLCs, CHS- and CS-coated NLCs was carried out. Following oral administration in RA-induced rat model, joint diameter, paw inflammation, liver functions were measured, in addition to histological examination of liver, kidney and joints. Results revealed improved joint healing and reduced hepatotoxicity following treatment with nanoencapsulated LEF compared to LEF suspension, whereby CHS-NLCs ensued the highest Cmax, AUC and lowest TNF-α level. The dual potential of CHS to achieve active targeting to CD44-receptor and hence maximize LEF concentration at the target site in addition to its synergistic effect in joint healing endow promises for a competent oral nanosystem for targeted drug delivery to the joints.

Safety, Pharmacokinetics, and Pharmacogenetics of Single-Dose Teriflunomide Sodium and Leflunomide in Healthy Chinese Subjects.

BACKGROUND: Teriflunomide sodium, a novel derivative of leflunomide, was developed to treat systemic lupus erythematosus. OBJECTIVE: The objectives of this trial were to study the safety, pharmacokinetics, and pharmacogenetics of teriflunomide sodium in healthy Chinese subjects in order to support its accelerated development. METHODS: A clinical study was designed as a single-dose, randomized, parallel, open-label study. Healthy volunteers were randomly assigned to take teriflunomide sodium 10 mg or leflunomide 10 mg. Eligible healthy volunteers were monitored over a 98-day observation period. Blood and urine samples were collected and analyzed for teriflunomide and its metabolite concentrations, and ABCG2 and CYP2C9 genotypes were detected. The safety profile was also collected. RESULTS: All adverse events were mild in intensity, and all subjects completed this trial without any other treatment. After a single administration of teriflunomide sodium and leflunomide, teriflunomide maximal concentrations were 1.32 ± 0.341 mg/L and 0.718 ± 0.169 mg/L, and area under the concentration-time curve from time zero to infinity (AUC∞) was 423 ± 229 mg·h/L and 303 ± 159 mg·h/L, respectively. Overall, teriflunomide AUC∞ in ABCG2 34A/A mutants was 70.4% lower than in wild-type ABCG2 34G/G after administration of teriflunomide sodium. In addition, after administration of leflunomide, teriflunomide AUC∞ in ABCG2 34A/A mutants was 30.0% lower than in subjects carrying ABCG2 34G/G. CONCLUSIONS: Teriflunomide sodium was generally safe and well tolerated in healthy Chinese subjects. The relative bioavailability of teriflunomide between teriflunomide sodium and leflunomide after a single dose administration was approximately 150%. Additionally, ABCG2 34G>A was found to significantly affect teriflunomide pharmacokinetics, which suggested ABCG2 34G>A may be a significant influencing factor. CLINICAL TRIAL REGISTRATION: This study was registered at the China National Medical Products Administration ( http://www.nmpa.gov.cn ; registration number 2014L01935), and also at the China platform for registry and publicity of drug clinical trials ( http://www.chinadrugtrials.org.cn ; registration number CTR20150314).

LC-MS/MS Method for the Quantification of the Leflunomide Metabolite, Teriflunomide, in Human Serum/Plasma.

Leflunomide is a prodrug that is metabolized to the active metabolite, teriflunomide (A77 1726), to inhibit the enzyme dihydroorotate dehydrogenase and decrease the synthesis of pyrimidine nucleotides for DNA and RNA synthesis. Teriflunomide is primarily used for the treatment of rheumatoid arthritis and multiple sclerosis.A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated to quantify the drug teriflunomide over a concentration range of 5 ng/mL-200 µg/mL in serum or plasma. The calibration curve was divided into two separate overlapping regions of the analytical measurement range, with a high curve and a low curve range. Samples are first analyzed using the high-range calibration curve after a 100-fold dilution of the sample extract. Samples falling below the upper curve region are evaluated again without dilution and quantified, if possible, against the low curve calibration standards. This method can be used to support therapeutic drug monitoring of patients that are administered with leflunomide therapy.

Development of a simple HPLC-MS/MS method to simultaneously determine teriflunomide and its metabolite in human plasma and urine: Application to clinical pharmacokinetic study of teriflunomide sodium and leflunomide.

A simple high-performance liquid chromatography coupled with tandem mass spectrometry method was developed and fully validated to simultaneously determine teriflunomide (TER) and its metabolite 4-trifluoro-methylaniline oxanilic acid (4-TMOA) in human plasma and urine. Merely 50 µL plasma and 20 µL urine were employed in sample preparation using protein precipitation and direct dilution method, respectively. An Agilent Zorbax eclipse plus C18 column was selected to achieve rapid separation for TER and 4-TMOA within 3 min. Electrospray ionization under multiple reaction monitoring was used to monitor the ion transitions for TER (m/z 269.0 → 159.9), 4-TMOA (m/z 231.9 → 160.0), internal standard teriflunomide-d4 (m/z 273.0 → 164.0) and 2-amino-4-trifluoromethyl benzoic acid (m/z 203.8 → 120.1), operating in the negative ion mode. This method proved to have better accuracy and precision over concentration range of 10-5000 ng/mL in plasma as well as 10-10,000 ng/mL in urine. After a full validation, this method was successfully applied in a pharmacokinetic study of teriflunomide sodium and leflunomide in Chinese healthy volunteers.