Hyaluronate-functionalized hydroxyapatite nanoparticles laden with methotrexate and teriflunomide for the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA), an autoimmune inflammatory disorder is currently incurable. Methotrexate and Teriflunomide are routinely prescribed drugs but their uses are limited due to severe hepatotoxicity. Hyaluronic acid (HYA) is a targeting ligand for CD44 receptors overexpressed on inflamed macrophages. The present investigation aimed at design and fabrication of HYA coated hydroxyapatite nanoparticles (HA-NPs) loaded with Methotrexate (MTX) and Teriflunomide (TEF) (HAMT-NPs) to form HYA-HAMT-NPs for the treatment of RA. HYA-HAMT-NPs showed the nanoscale size of 274.9 ± 64 nm along with a zeta potential value of -26.80 ± 6.08 mV. FTIR spectra of HYA and HYA-HAMT-NPs proved the coating of HYA on HYA-HAMT-NPs. HYA-HAMT-NPs showed less cell viability compared to drugs on RAW 264.7 macrophage cells. A biodistribution study by gamma scintigraphy imaging further strengthened the results by revealing significantly higher (p<0.05) percentage radioactivity (76.76%) of HYA-HAMT-NPs in the synovial region. The results obtained by pharmacodynamic studies ensured the better efficacy of HYA-HAMT-NPs in preventing disease progression and promoting articular regeneration. Under hepatotoxicity evaluation, liver histopathology and liver enzyme assay revealed ~29% hepatotoxicity was reduced by HYA-HAMT-NPs when compared to conventional FOLITRAX-10 and AUBAGIO oral treatments. Overall, the results suggest that HYA-HAMT-NP is a promising delivery system to avoid drug-induced hepatotoxicity in RA.

Investigating dihydroorotate dehydrogenase inhibitor mediated mitochondrial dysfunction in hepatic in vitro models.

Inhibition of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzymatic step in de novo pyrimidine synthesis, has broad immunosuppressive effects in vivo and shows promise as a therapeutic target for the treatment of malignancies, viral infections and auto-immune diseases. Whilst there are numerous DHODH inhibitors under development, leflunomide and teriflunomide are the only FDA approved compounds on the market, each of which have been issued with black-box warnings for hepatotoxicity. Mitochondrial dysfunction is a putative mechanism by which teriflunomide and leflunomide elicit their hepatotoxic effects, however it is as yet unclear whether this is shared by other nascent DHODH inhibitors. The present study aimed to evaluate the propensity for DHODH inhibitors to mediate mitochondrial dysfunction in two hepatic in vitro models. Initial comparisons of cytotoxicity and ATP content in HepaRG® cells primed for oxidative metabolism, in tandem with mechanistic evaluations by extracellular flux analysis identified multifactorial toxicity and moderate indications of respiratory chain dysfunction or uncoupling. Further investigations using HepG2 cells, a hepatic line with limited capability for phase I xenobiotic metabolism, identified leflunomide and brequinar as positive mitochondrial toxicants. Taken together, biotransformation of some DHODH inhibitor species may play a role in mediating or masking hepatic mitochondrial liabilities.

Acute teriflunomide overdose with relatively mild symptoms: A case report.

WHAT IS KNOW AND OBJECTIVE: Teriflunomide is indicated for the treatment of adult patients with relapsing-remitting multiple sclerosis. CASE SUMMARY: We present a rare intoxication with a high dose (672 mg) of teriflunomide. According to its product label, the only known treatment is the administration of colestyramine and activated carbon (charcoal). No serious adverse events occurred during the time the patient was admitted (<24 h). No long-term overdose-related symptoms or complaints were reported. WHAT IS NEW AND CONCLUSION: The fact that after the acute overdose both adverse events and laboratory parameters were acceptable, prescribing colestyramine and activated carbon, as well as monitoring of laboratory parameters such as full blood count, liver and kidney values and QTc, seems sufficient during the early stage (<24 h after intake) of teriflunomide overdose.

Novel electrospun nanofibrous matrices prepared from poly(lactic acid)/poly(butylene adipate) blends for controlled release formulations of an anti-rheumatoid agent.

In the present work, a series of novel formulations consisting of poly(lactic acid)/poly(butylene adipate) (PLA/PBAd) electrospun blends was examined as controlled release matrices for Leflunomide's active metabolite, Teriflunomide (TFL). The mixtures were prepared using different ratios of PLA and PBAd in order to produce nanofibrous matrices with different characteristics. Miscibility studies of the blended polymeric fibers were performed through differential scanning calorimetry (DSC) and X-ray diffractometry (XRD). Hydrolytic degradation in the prepared fibers was evaluated at 37°C using a phosphate buffered saline solution. Different concentrations of (TFL) (5, 10, 15wt.%) were incorporated into nanofibers for examining the drug release behavior in simulated body fluids (SBF), at 37°C. The drug-loaded nanofibrous formulations were further characterized by Fourier Transform Infrared Spectroscopy (FTIR) spectroscopy, DSC and XRD. Gel permeation chromatography (GPC) analysis was used to evaluate the mechanism of TFL release. Artificial neural networks (ANN) and multi-linear-regression (MLR) models were used to evaluate the effect of % content of PBAd (X1) and TFL (X2) on an initial burst effect and a dissolution behavior. It was found that PLA/PBAd nanofibers have different diameters depending on the ratio of used polyesters and added drug. TFL was incorporated in an amorphous form inside the polymeric nanofibers. In vitro release studies reveal that a drug release behavior is correlated with the size of the nanofibers, drug loading and matrix degradation after a specific time. ANN dissolution modeling showed increased correlation efficacy compared to MLR.

Inhibition of hepatic cytochrome P450 enzymes and sodium/bile acid cotransporter exacerbates leflunomide-induced hepatotoxicity.

AIM: Leflunomide is an immunosuppressive agent marketed as a disease-modifying antirheumatic drug. But it causes severe side effects, including fatal hepatitis and liver failure. In this study we investigated the contributions of hepatic metabolism and transport of leflunomide and its major metabolite teriflunomide to leflunomide induced hepatotoxicity in vitro and in vivo. METHODS: The metabolism and toxicity of leflunomide and teriflunomide were evaluated in primary rat hepatocytes in vitro. Hepatic cytochrome P450 reductase null (HRN) mice were used to examine the PK profiling and hepatotoxicity of leflunomide in vivo. The expression and function of sodium/bile acid cotransporter (NTCP) were assessed in rat and human hepatocytes and NTCP-transfected HEK293 cells. After Male Sprague-Dawley (SD) rats were administered teriflunomide (1,6, 12 mg · kg(-1) · d(-1), ig) for 4 weeks, their blood samples were analyzed. RESULTS: A nonspecific CYPs inhibitor aminobenzotriazole (ABT, 1 mmol/L) decreased the IC50 value of leflunomide in rat hepatocytes from 409 to 216 µmol/L, whereas another nonspecific CYPs inhibitor proadifen (SKF, 30 µmol/L) increased the cellular accumulation of leflunomide to 3.68-fold at 4 h. After oral dosing (15 mg/kg), the plasma exposure (AUC0-t) of leflunomide increased to 3-fold in HRN mice compared with wild type mice. Administration of leflunomide (25 mg·kg(-1) · d(-1)) for 7 d significantly increased serum ALT and AST levels in HRN mice; when the dose was increased to 50 mg·kg(-1) · d(-1), all HRN mice died on d 6. Teriflunomide significantly decreased the expression of NTCP in human hepatocytes, as well as the function of NTCP in rat hepatocytes and NTCP-transfected HEK293 cells. Four-week administration of teriflunomide significantly increased serum total bilirubin and direct bilirubin levels in female rats, but not in male rats. CONCLUSION: Hepatic CYPs play a critical role in detoxification process of leflunomide, whereas the major metabolite teriflunomide suppresses the expression and function of NTCP, leading to potential cholestasis.

Toxicity of teriflunomide in aryl hydrocarbon receptor deficient mice.

The intracellular transcription factor aryl hydrocarbon receptor (AHR) is bound and activated by xenobiotics, thereby promoting their catabolism by inducing expression of cytochrome P450 oxidase (CYP) genes through binding xenobiotic response elements (XRE) in their promoter region. In addition, it is involved in several cellular pathways like cell proliferation, differentiation, regeneration, tumor invasiveness and immune responses. Several pharmaceutical compounds like benzimidazoles activate the AHR and induce their own metabolic degradation. Using newly generated XRE-reporter mice, which allow in vivo bioluminescence imaging of AHR activation, we show here that the AHR is activated in vivo by teriflunomide (TER), which has recently been approved for the treatment of multiple sclerosis. While we did not find any evidence that the AHR mediates the immunomodulatory effects of TER, AHR activation led to metabolism and detoxification of teriflunomide, most likely via CYP. Mice deficient for the AHR show higher blood levels of teriflunomide, suffer from enhanced thrombo- and leukopenia and elevated liver enzymes as well as from severe gastrointestinal ulcers and bleeding which are lethal after 8-11 days of treatment. Leukopenia, acute liver damage and diarrhea have also been described as common side effects in human trials with TER. These data suggest that the AHR is relevant for detoxification not only of environmental toxins but also of drugs in clinical use, with potential implications for the application of AHR-modifying therapies in conjunction to TER in humans. The XRE-reporter mouse is a useful novel tool for monitoring AHR activation using in vivo imaging.

Evidence supporting a role for dihydroorotate dehydrogenase, bioenergetics, and p53 in selective teriflunomide-induced apoptosis in transformed versus normal human keratinocytes.

We have demonstrated previously that the dihydroorotate dehydrogenase (DHODH) inhibitor teriflunomide (TFN) encourages apoptosis in transformed human keratinocytes. Here we sought to determine if this cytotoxic effect could be restricted to transformed keratinocytes relative to their normal human epidermal keratinocyte (NHEK) counterparts, and ascertain a potential mechanistic basis for the selectivity. The NHEK cells proliferated much slower than the premalignant HaCaT and malignant COLO 16 keratinocytes, and exogenous uridine added to the culture medium did not affect this growth. Similarly, DHODH expression and the bioenergetic characteristics of the normal cells were markedly dissimilar from those observed in the transformed cells indicating that de novo pyrimidine synthesis was involved with keratinocyte proliferation. Moreover, a short-term exposure to TFN caused a wild-type p53 response in the NHEK cells illustrating that pyrimidine metabolic stress could regulate this tumor suppressor protein in the normal cells. TFN-induced apoptosis occurred primarily in S phase HaCaT cells. This cell death was sensitive to uridine, an antioxidant, and a caspase inhibitor, and the suppression of Bcl-X(L) and the induction of Mn superoxide dismutase preceded it. These events suggested that mitochondrial/redox stress was involved with the cytotoxic effect of TFN. Conversely, a long-term exposure to TFN caused G(0)/G(1) arrest in the NHEK cells, which supported a cytoprotective role for p53 against TFN-induced apoptosis. Together, these results propose that TFN could be useful in the prevention or therapy of non-melanoma skin cancers and possibly other hyperproliferative keratinocytic diseases.

Fragrance material review on cinnamyl propionate.

A toxicologic and dermatologic review of cinnamyl propionate when used as a fragrance ingredient is presented.

Trans-4-aminocrotonic acid (TACA), a potent agonist of GABA(A) and GABA(C) receptors, shows a proconvulsant action in the electroconvulsive threshold test in mice.

In the present study, we evaluated TACA (a potent agonist of GABA(A) and GABA(C) receptors) in the electroconvulsive threshold test in mice. Surprisingly, TACA (at 15 and 25 mg/kg) significantly decreased the threshold. The highest ineffective dose of TACA was estimated as 10 mg/kg. The obtained results indicate that the drug enhancing GABAergic transmission may possess proconvulsant properties in the electroconvulsive test. Such effect was completely opposite to our primary assumption and expectance.

Subchronic oral toxicity study on the three flavouring substances: octan-3-ol, 2-methylcrotonic acid and oct-3-yl 2-methylcrotonate in Wistar rats.

Groups of 10 male and 10 female rats were administered 0, 25, 100 or 400 mg octan-3-ol/kg body weight per day, 77 mg 2-methylcrotonic acid/kg body weight per day or 163 mg oct-3-yl 2-methylcrotonate/kg body weight per day by gavage for 90 days. Relative liver weights of high-dose octan-3-ol males, and males and females dosed with oct-3-yl 2-methylcrotonate were significantly greater than those of the control. In male and female rats dosed with the highest level of octan-3-ol and in male rats dosed with 2-methylcrotonic acid, incidences of bile duct proliferation were increased. In the kidneys of males dosed with mid- and high level of octan-3-ol and oct-3-yl 2-methylcrotonate, tubular karyomegaly and desquamation of tubular epithelial cells were observed. Based on increased liver weight and microscopic evaluation of the liver and kidney, a no-observed-effect level (NOEL) of 25 mg/kg for octan-3-ol in rats was established. The histopathological evaluation of the liver of rats dosed with oct-3-yl 2-methylcrotonate revealed lesions corresponding to the lesions seen in rats dosed mid-dose with octan-3-ol. This observation is in accordance with the general assumption that oct-3-yl 2-methylcrotonate is completely hydrolysed to octan-3-ol and 2-methylcrotonic acid. However, when comparing the liver histopathology of oct-3-yl 2-methylcrotonate and 2-methylcrotonic acid and the kidney lesions of all three substances, conflicting results were seen and the present study does not allow the conclusion to be drawn that oct-3-yl 2-methylcrotonate and structurally-related esters are completely hydrolysed, at least under the conditions of the present study.

The relationship between mitochondrial activation and toxicity of some substituted carboxylic acids.

The activation of 4-bromocrotonic acid, 4-bromo-2-octenoic acid, valproic acid, and 3-methylglycidic acid by conversion to their CoA thioesters and the effects of these carboxylic acids on palmitoylcarnitine-supported respiration were studied with rat liver and rat heart mitochondria. 4-Bromocrotonic acid was activated by both liver and heart mitochondria, whereas 4-bromo-2-octenoic acid and valproic acid were only activated by liver mitochondria. 3-Methylglycidic acid was not a substrate of mitochondrial activation. All of the carboxylic acids that were activated also inhibited palmitoylcarnitine-supported respiration. 3-Methylglycidoyl-CoA was found to irreversibly inhibit 3-ketoacyl-CoA thiolase in a concentration-dependent and time-dependent manner. Together, these results lead to the conclusion that substituted medium-chain carboxylic acids, which enter mitochondria directly, may inhibit beta-oxidation as long as they are activated and perhaps further metabolized in the mitochondrial matrix to compounds that sequester CoA and/or inhibit beta-oxidation enzymes. Liver is more susceptible to inhibition by such xenobiotic carboxylic acids due to the broader substrate specificity of its mitochondrial medium-chain acyl-CoA synthetase (EC 6.2.1.2).

Some behavioral effects of prethcamide compared with those of its two components.

The effects of the two components of prethcamide (namely crotetamide and cropropamide) upon various behaviors in rats were compared with those of prethcamide itself to see if both were active or not and the kind of joint action shown when they were given in combination. Both crotetamide and cropropamide increase the motor activity of rats, reduce the rate of lever pressing in FR and VI food-reinforced schedules and increase the latency times in a multiple CRF-discrimination schedule. When given in combination the drugs show additive effects upon locomotor activity and FR or VI behaviors but they potentiate each other as regards the effects upon latency times in the multiple schedule. On the other hand, a clear antagonism between the two drugs has been found in the acute toxicity test.