Unplugging lateral fenestrations of NALCN reveals a hidden drug binding site within the pore region.

The sodium (Na+) leak channel (NALCN) is a member of the four-domain voltage-gated cation channel family that includes the prototypical voltage-gated sodium and calcium channels (NaVs and CaVs, respectively). Unlike NaVs and CaVs, which have four lateral fenestrations that serve as routes for lipophilic compounds to enter the central cavity to modulate channel function, NALCN has bulky residues (W311, L588, M1145, and Y1436) that block these openings. Structural data suggest that occluded fenestrations underlie the pharmacological resistance of NALCN, but functional evidence is lacking. To test this hypothesis, we unplugged the fenestrations of NALCN by substituting the four aforementioned residues with alanine (AAAA) and compared the effects of NaV, CaV, and NALCN blockers on both wild-type (WT) and AAAA channels. Most compounds behaved in a similar manner on both channels, but phenytoin and 2-aminoethoxydiphenyl borate (2-APB) elicited additional, distinct responses on AAAA channels. Further experiments using single alanine mutants revealed that phenytoin and 2-APB enter the inner cavity through distinct fenestrations, implying structural specificity to their modes of access. Using a combination of computational and functional approaches, we identified amino acid residues critical for 2-APB activity, supporting the existence of drug binding site(s) within the pore region. Intrigued by the activity of 2-APB and its analogues, we tested compounds containing the diphenylmethane/amine moiety on WT channels. We identified clinically used drugs that exhibited diverse activity, thus expanding the pharmacological toolbox for NALCN. While the low potencies of active compounds reiterate the pharmacological resistance of NALCN, our findings lay the foundation for rational drug design to develop NALCN modulators with refined properties.

Phenytoin promotes the proliferation of oligodendrocytes and enhances the expression of myelin basic protein in the corpus callosum of mice demyelinated by cuprizone.

Oligodendrocyte loss and myelin sheet destruction are crucial characteristics of demyelinating diseases. Phenytoin promotes the proliferation of endogenous neural precursor cells in the ventricular-subventricular zone in the postnatal brain that help restore the oligodendroglial population. This study aimed to evaluate whether phenytoin promotes myelin recovery of the corpus callosum of demyelinated adult mice. CD1 male mice were exposed to a demyelinating agent (0.2% cuprizone) for 8 weeks. We assembled two groups: the phenytoin-treated group and the control-vehicle group. The treated group received oral phenytoin (10 mg/kg) for 4 weeks. We quantified the number of Olig2 + and NG2 + oligodendrocyte precursor cells (OPCs), Rip + oligodendrocytes, the expression level of myelin basic protein (MBP), and the muscle strength and motor coordination. The oligodendroglial lineage (Olig2 + cells, NG2 + cells, and RIP + cells) significantly increases by the phenytoin administration when compared to the control-vehicle group. The phenytoin-treated group also showed an increased expression of MBP in the corpus callosum and better functional scores in the horizontal bar test. These findings suggest that phenytoin stimulates the proliferation of OPCs, re-establishes the oligodendroglial population, promotes myelin recovery in the corpus callosum, and improves motor coordination and muscle strength.

Frequency of drug combinations between enzyme-inducing first-generation antiepileptic drugs and inducible drugs in patients with epilepsy.

OBJECTIVE: The objective of the study was to systematically assess, through the analysis of administrative data, the frequency of combinations of first-generation enzyme-inducing (EI) antiepileptic drugs (AEDs) with drugs frequently prescribed in patients with epilepsy whose metabolism is induced by EIAEDs. METHODS: From the population of Tuscany (a region in Italy of about 3,750,000 habitants), patients who had been treated with at least one first-generation EIAEDs (carbamazepine, phenytoin, phenobarbital, and primidone) and had received prescriptions of an inducible non-AED (NON-AED) included in a prespecified list of 103 inducible drugs were identified. RESULTS: At the index date, 9221 patients with epilepsy were treated with at least one traditional EIAED, and there were 2538 drug combinations between EIAEDs and NON-AEDs, which may result in potentially serious clinical consequences, and 3317 combinations with NON-AEDs that have their metabolism consistently increased. CONCLUSIONS: Patients with epilepsy treated with traditional EIAEDs are at a very high risk of drug interactions.

Topical phenytoin nanostructured lipid carriers: design and development.

Phenytoin (PHT) is an antiepileptic drug that was reported to exhibit high wound healing activity. Nevertheless, its limited solubility, bioavailability, and inefficient distribution during topical administration limit its use. Therefore, this study aims to develop, characterize nanostructured lipid carriers (NLCs), and evaluate their potential in topical delivery of PHT to improve the drug entrapment efficiency and sustained release. The NLCs were prepared by hot homogenization followed by ultra sonication method using 23 factorial design. NLC formulations were characterized regarding their particle size (PS), zeta potential (ZP), entrapment efficiency percent (%EE), surface morphology, physicochemical stability, and in vitro release studies. The optimized NLC (F7) was further incorporated in 1%w/v carbopol gel and then characterized for appearance, pH, viscosity, stability, and in vitro drug release. The prepared NLCs were spherical in shape and possessed an average PS of 121.4-258.2 nm, ZP of (-15.4)-(-32.2) mV, and 55.24-88.80 %EE. Solid-state characterization revealed that the drug is dispersed in an amorphous state with hydrogen bond interaction between the drug and the NLC components. NLC formulations were found to be stable at 25 °C for six months. The stored F7-hydrogel showed insignificant changes in viscosity and drug content (p>.05) up to six months at 25 °C that pave a way for industrial fabrication of efficient PHT products. In vitro release studies showed a sustained release from NLC up to 48 h at pH 7.4 following non-Fickian Higuchi kinetics model. These promising findings encourage the potential use of phenytoin loaded lipid nanoparticles for future topical application.

Local anesthetic and antiepileptic drug access and binding to a bacterial voltage-gated sodium channel.

Voltage-gated sodium (Nav) channels are important targets in the treatment of a range of pathologies. Bacterial channels, for which crystal structures have been solved, exhibit modulation by local anesthetic and anti-epileptic agents, allowing molecular-level investigations into sodium channel-drug interactions. These structures reveal no basis for the "hinged lid"-based fast inactivation, seen in eukaryotic Nav channels. Thus, they enable examination of potential mechanisms of use- or state-dependent drug action based on activation gating, or slower pore-based inactivation processes. Multimicrosecond simulations of NavAb reveal high-affinity binding of benzocaine to F203 that is a surrogate for FS6, conserved in helix S6 of Domain IV of mammalian sodium channels, as well as low-affinity sites suggested to stabilize different states of the channel. Phenytoin exhibits a different binding distribution owing to preferential interactions at the membrane and water-protein interfaces. Two drug-access pathways into the pore are observed: via lateral fenestrations connecting to the membrane lipid phase, as well as via an aqueous pathway through the intracellular activation gate, despite being closed. These observations provide insight into drug modulation that will guide further developments of Nav inhibitors.

Application of Micropatterned Cocultured Hepatocytes to Evaluate the Inductive Potential and Degradation Rate of Major Xenobiotic Metabolizing Enzymes.

Long-term coculture models of hepatocytes are promising tools to study drug transport, clearance, and hepatoxicity. In this report we compare the basal expression of drug disposition genes and the inductive response of prototypical inducers (rifampin, phenobarbital, phenytoin) in hepatocyte two-dimensional monocultures and the long-term coculture model (HepatoPac). All the inducers used in the study increased the expression and activity of CYP3A4, CYP2B6 and CYP2C enzymes in the HepatoPac cultures. The coculture model showed a consistent and higher induction of CYP2C enzymes compared with the monocultures. The EC50 of rifampin for CYP3A4 and CYP2C9 was up to 10-fold lower in HepatoPac than the monocultures. The EC50 of rifampin calculated from the clinical drug interaction studies correlated well with the EC50 observed in the HepatoPac cultures. Owing to the long-term stability of the HepatoPac cultures, we were able to directly measure a half-life (t1/2) for both CYP3A4 and CYP2B6 using the depletion kinetics of mRNA and functional activity. The t1/2 for CYP3A4 mRNA was 26 hours and that for the functional protein was 49 hours. The t1/2 of CYP2B6 was 38 hours (mRNA) and 68 hours (activity), which is longer than CYP3A4 and shows the differential turnover of these two proteins. This is the first study to our knowledge to report the turnover rate of CYP2B6 in human hepatocytes. The data presented here demonstrate that the HepatoPac cultures have the potential to be used in long-term culture to mimic complex clinical scenarios.

Prediction of Drug-Drug Interactions Arising from CYP3A induction Using a Physiologically Based Dynamic Model.

Using physiologically based pharmacokinetic modeling, we predicted the magnitude of drug-drug interactions (DDIs) for studies with rifampicin and seven CYP3A4 probe substrates administered i.v. (10 studies) or orally (19 studies). The results showed a tendency to underpredict the DDI magnitude when the victim drug was administered orally. Possible sources of inaccuracy were investigated systematically to determine the most appropriate model refinement. When the maximal fold induction (Indmax) for rifampicin was increased (from 8 to 16) in both the liver and the gut, or when the Indmax was increased in the gut but not in liver, there was a decrease in bias and increased precision compared with the base model (Indmax = 8) [geometric mean fold error (GMFE) 2.12 vs. 1.48 and 1.77, respectively]. Induction parameters (mRNA and activity), determined for rifampicin, carbamazepine, phenytoin, and phenobarbital in hepatocytes from four donors, were then used to evaluate use of the refined rifampicin model for calibration. Calibration of mRNA and activity data for other inducers using the refined rifampicin model led to more accurate DDI predictions compared with the initial model (activity GMFE 1.49 vs. 1.68; mRNA GMFE 1.35 vs. 1.46), suggesting that robust in vivo reference values can be used to overcome interdonor and laboratory-to-laboratory variability. Use of uncalibrated data also performed well (GMFE 1.39 and 1.44 for activity and mRNA). As a result of experimental variability (i.e., in donors and protocols), it is prudent to fully characterize in vitro induction with prototypical inducers to give an understanding of how that particular system extrapolates to the in vivo situation when using an uncalibrated approach.

Concentration of antiepileptic drugs in persons with epilepsy: a comparative study in serum and saliva.

AIM OF THE STUDY: The monitoring of antiepileptic drugs (AEDs) in clinical setting is important for measuring the efficacy of drugs and their safety and in personalizing drug therapy. We investigated the levels of AED, carbamazepine (CBZ), phenytoin (PHT) and phenobarbital (PHB), to understand their association in saliva compared with those in serum during the therapy. MATERIALS AND METHODS: In this study, we performed a prospective study of 116 persons with epilepsy (PWE; mean age 26.90 ± 11.83 years). Serum and saliva samples were collected at trough levels from the patients, who were under the treatment of CBZ, PHT and PHB either alone or in combination of these drugs for at least three months. The drug levels were assessed by high-performance liquid chromatography. RESULTS AND CONCLUSIONS: The number of males (n = 88; 75.86%) was higher than females (n = 28; 24.14%) among the recruited patients. The intake of CBZ, PHT and PHB was observed in 49.14%, 68.10% and 38.79% of PWE, respectively. The levels of these AEDs showed a significant correlation (p < 0.05) between serum and saliva. Interestingly, the levels of mono-therapy or bi-therapy showed a significant association (p < 0.05) between serum and saliva, however, there was no significant association in case of poly-therapy. This is the first report in the Indian population on simultaneous estimation of the three commonly used AEDs, such as CBZ, PHT and PHB in serum and saliva implicating their associations, either in mono-therapy or bi-therapy in PWE.

Locating the route of entry and binding sites of benzocaine and phenytoin in a bacterial voltage gated sodium channel.

Sodium channel blockers are used to control electrical excitability in cells as a treatment for epileptic seizures and cardiac arrhythmia, and to provide short term control of pain. Development of the next generation of drugs that can selectively target one of the nine types of voltage-gated sodium channel expressed in the body requires a much better understanding of how current channel blockers work. Here we make use of the recently determined crystal structure of the bacterial voltage gated sodium channel NavAb in molecular dynamics simulations to elucidate the position at which the sodium channel blocking drugs benzocaine and phenytoin bind to the protein as well as to understand how these drugs find their way into resting channels. We show that both drugs have two likely binding sites in the pore characterised by nonspecific, hydrophobic interactions: one just above the activation gate, and one at the entrance to the the lateral lipid filled fenestrations. Three independent methods find the same sites and all suggest that binding to the activation gate is slightly more favourable than at the fenestration. Both drugs are found to be able to pass through the fenestrations into the lipid with only small energy barriers, suggesting that this can represent the long posited hydrophobic entrance route for neutral drugs. Our simulations highlight the importance of a number of residues in directing drugs into and through the fenestration, and in forming the drug binding sites.

A label-free untethered approach to single-molecule protein binding kinetics.

Single molecule approaches provide rich real-time dynamics of molecular interactions that are not accessible to ensemble measurements. Previous single molecule studies have relied on labeling and tethering, which alters the natural state of the protein. Here we use the double-nanohole (DNH) optical tweezer approach to measure protein binding kinetics at the single molecule level in a label-free, free-solution (untethered) way. The binding kinetics of human serum albumin (HSA) to tolbutamide and to phenytoin are in quantitative agreement with previous measurements, and our single-molecule approach reveals a biexponential behavior characteristic of a multistep process. The DNH optical tweezer is an inexpensive platform for studying the real-time binding kinetics of protein-small molecule interactions in a label-free, free-solution environment, which will be of interest to future studies including drug discovery.

A new dynamic in vitro modular capillaries-venules modular system: cerebrovascular physiology in a box.

BACKGROUND: The study of the cerebrovascular physiology is crucial to understand the pathogenesis of neurological disease and the pharmacokinetic of drugs. Appropriate models in vitro often fail to represent in vivo physiology. To address these issues we propose the use of a novel artificial vascular system that closely mimics capillary and venous segments of human cerebrovasculature while also allowing for an extensive control of the experimental variables and their manipulation. RESULTS: Using hollow fiber technology, we modified an existing dynamic artificial model of the blood-brain barrier (BBB) (DIV-capillary) to encompass the distal post-capillary (DIV-venules) segments of the brain circulatory system. This artificial brain vascular system is comprised of a BBB module serially connected to a venule segment. A pump generates a pulsatile flow with arterial pressure feeding the system. The perfusate of the capillary module achieves levels of shear stress, pressure, and flow rate comparable to what observed in situ. Endothelial cell exposure to flow and abluminal astrocytic stimuli allowed for the formation of a highly selective capillary BBB with a trans-endothelial electrical resistance (TEER; >700 ohm cm2) and sucrose permeability (< 1X10-u cm/sec) comparable to in vivo. The venule module, which attempted to reproduce features of the hemodynamic microenvironment of venules, was perfused by media resulting in shear stress and intraluminal pressure levels lower than those found in capillaries. Because of altered cellular and hemodynamic factors, venule segments present a less stringent vascular bed (TEER <250 Ohm cm2; Psucrose > 1X10-4 cm/sec) than that of the BBB. Abluminal human brain vascular smooth muscle cells were used to reproduce the venular abluminal cell composition. CONCLUSION: The unique characteristics afforded by the DIV-BBB in combination with a venule segment will realistically expand our ability to dissect and study the physiological and functional behavior of distinct segments of the human cerebrovascular network.

Revealing how phenytoin triggers liver damage and the potential protective effects of Balanites Aegyptiaca fruit extracts: Exploring Nrf2/MAPK/ Beclin-1 signaling pathways.

Phenytoin-induced liver injury (PHT ILII) is a serious condition that may necessitate discontinuation of the drug. This study investigates the mechanisms of PHT ILII and evaluates the protective effects of Balanites Aegyptiaca (BA) fruit extracts on the liver. We focus on the Nrf2/MAPK/NF-κB/Beclin-1 signaling pathways involved in oxidative stress and inflammation from drug-induced liver injury. Phytochemical analyses of BA fruit extracts (Bu-F and EA-F) are conducted. Molecular docking techniques explore the interaction between phenytoin (PHT) and the Nrf2/MAPK/NF-κB/Beclin-1 pathways. Thirty-six male rats are divided into Control, Bu-F, EA-F, PHT, Bu-F/PHT, and EA-F/PHT groups, and they are observed for 45 days. EA-F extract is rich in phenolics/flavonoids, while Bu-F extract mainly contains saponins.PHT ILII causes histological damage in liver tissues and affects Nrf-2, MAPK, TNF-α, IL-1ß, Mcp-1, Beclin-1, iNOS expression, and liver function markers (ALT, AST, ALP). However, EA-F/Bu-F extracts effectively improve the histological structure and significantly reduce biochemical/immunohistochemical parameters, restoring them to near-normal levels. EA-F extract is particularly effective.In conclusion, the Nrf2/MAPK /Beclin-1 pathways play a critical role in the development of PHT ILII. BA fruit extracts show promise as hepato-protective agents, with the EA-F extract demonstrating superior efficacy. These results lay the groundwork for new treatments for PHT ILII and drug-induced liver injuries.

Embryonic catalase protects against endogenous and phenytoin-enhanced DNA oxidation and embryopathies in acatalasemic and human catalase-expressing mice.

Oxidative stress and reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)), which is detoxified by catalase, are implicated in fetal death and birth defects. However, embryonic levels of catalase are only ∼ 5% of adult activity, and its protective role is not understood completely. Herein, we used mutant catalase-deficient mice [acatalasemic (aCat)] and transgenic mice expressing human catalase (hCat), which, respectively, exhibited 40-50% reductions and 2-fold elevations in the activities of embryonic and fetal brain catalase, to show that embryonic catalase protects the embryo from both physiological oxidative stress and the ROS-initiating antiepileptic drug phenytoin. Compared to wild-type (WT) catalase-normal controls, both untreated and phenytoin-exposed aCat mice exhibited a 30% increase in embryonic DNA oxidation and a >2-fold increase in embryopathies, both of which were completely blocked by protein therapy with exogenous catalase. Conversely, compared to WT controls, untreated and, to a lesser extent, phenytoin-exposed hCat mice were protected, with untreated hCat embryos exhibiting a 40% decrease in embryonic DNA oxidation and up to a 67% decrease in embryopathies. Embryonic catalase accordingly plays an important protective role, and both physiological and phenytoin-enhanced oxidative stress can be embryopathic.

Phenytoin Is Promoting the Differentiation of Dental Pulp Stem Cells into the Direction of Odontogenesis/Osteogenesis by Activating BMP4/Smad Pathway.

Background: The purpose of this study was the evaluation of the potential and mechanism of phenytoin to promote differentiation of human dental pulp stem cells (hDPSC) into odontoblasts/osteoblasts. Methods: Fourth-generation human hDPSC originating from healthy pulp of third molars was cultured in control as well as phenytoin-containing media (PHT) for 14 days. qPCR was applied to detect the expression of DSPP, DMP1, and ALP genes. Western blot analysis was used to confirm the findings. One-way analysis of variance (ANOVA) was used for statistical analysis (p < 0.05). Information about phenytoin was assessed from PubChem database, while targets of phenytoin were assessed from six databases. Drug targets were extracted based on the differentially expressed genes (‖logFC‖ ≥ 1, p < 0.05) in the experimental group (50 mg/L PHT, 14 days). GO BP and KEGG pathway enrichment analysis on the obtained drug targets was performed and the target protein functional network diagram was constructed. Results: A concentration below 200 mg/L PHT had no obvious toxicity to hDPSC. The expression of DSPP, DMP1, and ALP genes in the 50 mg/L PHT concentration group increased significantly. The WB experiment showed that the protein content of BMP4, Smad1/5/9, and p-Smad1/5 was significantly increased in 50 mg/L PHT in comparison with the NC group (the group without treatment of PHT) at 14 days. Conclusion: Phenytoin has the ability of promoting the differentiation of hDPSC into odontoblasts and osteoblasts. BMP4/Smad pathway, inducing odontogenic/osteogenic differentiation of hDPSC, appears a main process in this context.

The role of efflux transporters and metabolizing enzymes in brain and peripheral organs to explain drug-resistant epilepsy.

Drug-resistant epilepsy has been explained by different mechanisms. The most accepted one involves overexpression of multidrug transporters proteins at the blood brain barrier and brain metabolizing enzymes. This hypothesis is one of the main pharmacokinetic reasons that lead to the lack of response of some antiseizure drug substrates of these transporters and enzymes due to their limited entrance into the brain and limited stay at the sites of actions. Although uncontrolled seizures can be the cause of the overexpression, some antiseizure medications themselves can cause such overexpression leading to treatment failure and thus refractoriness. However, it has to be taken into account that the inductive effect of some drugs such as carbamazepine or phenytoin not only impacts on the brain but also on the rest of the body with different intensity, influencing the amount of drug available for the central nervous system. Such induction is not only local drug concentration but also time dependent. In the case of valproic acid, the deficient disposition of ammonia due to a malfunction of the urea cycle, which would have its origin in an intrinsic deficiency of L-carnitine levels in the patient or by its depletion caused by the action of this antiseizure drug, could lead to drug-resistant epilepsy. Many efforts have been made to change this situation. In order to name some, the administration of once-daily dosing of phenytoin or the coadministration of carnitine with valproic acid would be preferable to avoid iatrogenic refractoriness. Another could be the use of an adjuvant drug that down-regulates the expression of transporters. In this case, the use of cannabidiol with antiseizure properties itself and able to diminish the overexpression of these transporters in the brain could be a novel therapy in order to allow penetration of other antiseizure medications into the brain.

Assessment of porphyrogenicity of drugs and chemicals in selected hepatic cell culture models through a fluorescence-based screening assay.

Compounds that induce 5-aminolevulinic acid [ALA] synthase-1 and/or cytochromes P-450 may induce acute porphyric attacks in patients with the acute hepatic porphyrias [AHPs]. Currently, there is no simple, robust model used to assess and predict the porphyrogenicity of drugs and chemicals. Our aim was to develop a fluorescence-based in vitro assay for this purpose. We studied four different hepatic cell culture models: HepG2 cells, LMH cells, 3D HepG2 organoids, and 3D organoids of primary liver cells from people without known disease [normal human controls]. We took advantage of the fluorescent properties of protoporphyrin IX [PP], the last intermediate of the heme biosynthesis pathway, performing fluorescence spectrometry to measure the intensity of fluorescence emitted by these cells treated with selected compounds of importance to patients with AHPs. Among the four cell culture models, the LMH cells produced the highest fluorescence readings, suggesting that these cells retain more robust heme biosynthesis enzymes or that the other cell models may have lost their inducibility of ALA synthase-1 [ALAS-1]. Allyl isopropyl acetamide [AIA], a known potent porphyrogen and inducer of ALAS-1, was used as a positive control to help predict porphyrogenicity for tested compounds. Among the tested compounds (acetaminophen, acetylsalicylic acid, ß-estradiol, hydroxychloroquine sulfate, alpha-methyldopa, D (-) norgestrel, phenobarbital, phenytoin, sulfamethoxazole, sulfisoxazole, sodium valproate, and valsartan), concentrations greater than 0.314 mM for norgestrel, phenobarbital, phenytoin, and sodium valproate produced fluorescence readings higher than the reading produced by the positive AIA control. Porphyrin accumulation was also measured by HPLC to confirm the validity of the assay. We conclude that LMH cell cultures in multi-well plates are an inexpensive, robust, and simple system to predict the porphyrogenicity of existing or novel compounds that may exacerbate the AHPs.

Multi-biomarker approach to evaluate the neurotoxic effects of environmentally relevant concentrations of phenytoin on adult zebrafish Danio rerio.

Several studies have reported the presence of phenytoin (PHE) in wastewater treatment plant effluents, hospital effluents, surface water, and even drinking water. However, published studies on the toxic effects of PHE at environmentally relevant concentrations in aquatic organisms are scarce. The present study aimed to determine the effect of three environmentally relevant concentrations of PHE (25, 282, and 1500 ng L-1) on behavioral parameters using the novel tank test. Moreover, we also aimed to determine whether or not these concentrations of PHE may impair acetylcholinesterase (AChE) activity and oxidative status in the brain of Danio rerio adults. Behavioral responses suggested an anxiolytic effect in PHE-exposed organisms, mainly observed in organisms exposed to 1500 ng L-1, with a significant decrease in fish mobility and a significant increase in activity at the top of the tank. Besides the behavioral impairment, PHE-exposed fish also showed a significant increase in the levels of lipid peroxidation, hydroperoxides, and protein carbonyl content compared to the control group. Moreover, a significant increase in brain AChE levels was observed in fish exposed to 282 and 1500 ng L-1. The results obtained in the present study show that PHE triggers a harmful response in the brain of fish, which in turn generates fish have an anxiety-like behavior.

Potential herb-drug interaction risk of thymoquinone and phenytoin.

Thymoquinone is a main bioactive compound of Nigella sativa L. (N.sativa), which has been used for clinical studies in the treatment of seizures due to its beneficial neuroprotective activity and antiepileptic effects. It has been evidenced that thymoquinone may inhibit the activity of cytochrome P450 2C9 (CYP2C9). However, little is known about the effect of thymoquinone or N.sativa on the pharmacokinetic behavior of phenytoin, a second-line drug widely used in the management of status epilepticus. In this study, we systematically investigated the risk of the potential pharmacokinetic drug interaction between thymoquinone and phenytoin. The inhibitory effect of thymoquinone on phenytoin hydroxylation activity by CYP2C9 was determined using UPLC-MS/MS by measuring the formation rates for p-hydroxyphenytoin (p-HPPH). The potential for drug-interaction between thymoquinone and phenytoin was quantitatively predicted by using in vitro-in vivo extrapolation (IVIVE). Our data demonstrated that thymoquinone displayed effective inhibition against phenytoin hydroxylation activity. Enzyme kinetic studies showed that thymoquinone exerted a competitive inhibition against phenytoin hydroxylation with a Ki value of 4.45 ± 0.51 µM. The quantitative prediction from IVIVE suggested that the co-administration of thymoquinone (>18 mg/day) or thymoquinone-containing herbs (N.sativa > 1 g/day or N.sativa oil >1 g/day) might result in a clinically significant herb-drug interactions. Additional caution should be taken when thymoquinone or thymoquinone-containing herbs are co-administered with phenytoin, which may induce unexpected potential herb-drug interactions via the inhibition of CYP2C9.

Investigation of the effects of post-operative intraperitoneal, oral, and rectal phenytoin administration on colorectal anastomosis in rats.

BACKGROUND: Anastomotic leakage is the most feared complication after colonic anastomosis. The purpose of the study is to determine the effects of phenytoin applied by different application routes, on the healing process of colorectal anastomoses. METHODS: Wistar Albino rats were divided into Intraperitoneal Phenytoin Group, Oral Phenytoin Group (OAP), Rectal Phenytoin Group (RAP), and control groups. The molecular effect of phenytoin on the expression of vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-ß), fibroblast growth factor 2 (FGF2), and p53 genes was evaluated at mRNA and protein level. The effects of phenytoin on anastomotic bursting pressure analysis measured as well as pathohistological examinations. RESULTS: There are statistically significant increase in anastomotic bursting pressure values between control and application groups. Inflammatory cell infiltration of all groups increased in the intestinal anastomosis region compared to control. Collagen scores were found to be significantly higher in the OAP and RAP groups compared to the control group. mRNA of TGF-ß and FGF2 expression increased in all routes of phenytoin applications. CONCLUSION: Three different administration routes show considerably increase on the bursting pressure. Regarding the results of the expression of FGF2, TGF-ß, p53, and VEGF genes, there is a significant increase FGF2 and TGF-ß at mRNA and protein level in most administration routes.

Excretion of the principal urinary metabolites of phenytoin and absolute oral bioavailability determined by use of a stable isotope in patients with epilepsy.

BACKGROUND: The anticonvulsant properties of phenytoin (PHT) were discovered in 1938. Since then, it has been one of the most widely used antiepileptic drugs. It is slowly absorbed, extensively plasma protein-bound, exhibits a nonlinear, concentration-dependent pharmacokinetic profile, and has a narrow therapeutic range. METHODS: We determined PHT bioavailability during steady-state therapy by 1) measurement of the two principal deconjugated PHT urinary metabolites, 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) and 5-(3,4-dihydroxy-1,5-cyclohexadien-1-yl)-5-phenylhydantoin (DHD); and 2) direct determination of absolute bioavailability after simultaneous administration of an oral formulation and parenteral stable-labeled PHT (SL-PHT). Urinary metabolites were quantified by an isocratic HPLC-NI-APCI-MS method. The urinary dose recovery was calculated by dividing the molar recovery of the major PHT urinary metabolites by the molar dose received. RESULTS: Urinary metabolite recovery was surprisingly low, 35.4% ± 15.7% in younger patients (age 21-49 years old) and 32.9% ± 15.0% in patients aged 65 to 93 years. Absolute bioavailability was 86.4% ± 19.4% and 92.5% ± 25.2%, respectively. A weak, but significant, Spearman rank correlation was observed between urinary metabolite recovery and oral bioavailability (P = 0.00924, R = 0.166). CONCLUSION: This weak correlation may be the result of variability in urinary versus biliary-fecal excretion of p-HPPH glucuronide. This study demonstrates that daily PHT oral absorption exhibits wide interpatient variability, which may account for fluctuations in PHT concentration over time.