Impact of Heterotropic Allosteric Modulation on the Time-Dependent Inhibition of Cytochrome P450 3A4.

The current study was designed to investigate the influence of allosteric effectors on the metabolism of the prototypical cytochrome P450 (CYP) 3A4 substrate midazolam (MDZ), and on the determination in vitro time-dependent inhibition (TDI) of CYP3A4 using human liver microsomes (HLM). As the concentration of midazolam increased to 250 µM in HLMs, homotropic cooperativity resulted in a decrease in the 1'-hydroxymidazolam to 4-hydroxymidazolam ratio to a maximum of 1.1. The presence of varying concentrations of testosterone, progesterone (PGS), or carbamazepine (CBZ) in HLMs with MDZ could recapitulate the effect of homotropic cooperativity such that the formation rates of the 1'hydroxymidazolam and 4-hydroxymidazolam were equal even at low concentrations of MDZ. The presence of PGS (10 or 100 µM) and CBZ (100 or 1000 µM) in in vitro TDI determination of four known CYP3A4 time-dependent inactivators (clarithromycin, troleandomycin, mibefradil, raloxifene) simultaneously decreased potency and inactivation rate constant, resulting in fold changes in inactivation efficiency on average of 1.6-fold and 13-fold for the low and high concentrations of allosteric modulator tested, respectively. The formation of a metabolic-intermediate complex (MIC) for clarithromycin and troleandomycin decreased in the presence of the allosteric modulators in a concentration-dependent manner, reaching a new steady state formation that could not be overcome with increased incubation time. Maximum reduction of the MIC formed by clarithromycin was up to ∼91%, while troleandomycin MIC decreased up to ∼31%. These findings suggest that the absence of endogenous allosteric modulators may contribute to the poor translation of HLM-based drug-drug interaction predictions. SIGNIFICANCE STATEMENT: The reported overprediction of in vitro human liver microsome time-dependent inhibition of CYP3A4 and observed drug interactions in vivo remains an issue in drug development. We provide characterization of allosteric modulators on the CYP3A4 metabolism of the prototypical substrate midazolam, demonstrating the ability of the modulators to recapitulate the homotropic cooperativity of midazolam. Furthermore, we demonstrate that allosteric heterotropic cooperativity of CYP3A4 can impact the time-dependent inhibition kinetics of known mechanisms-based inhibitors, providing a potential mechanism to explain the overprediction.

Numerical analysis of time-dependent inhibition kinetics: comparison between rat liver microsomes and rat hepatocyte data for mechanistic model fitting.

Time-dependent inhibition (TDI) may confound drug interaction predictions. Recently, models were generated for an array of TDI kinetic schemes using numerical analysis of microsomal assays. Additionally, a distinct terminal inactivation step was identified for certain mechanism based inhibitors (MBI) following reversible metabolite intermediate complex (MIC) formation. Longer hepatocyte incubations potentially allow analysis of slow TDI and terminal inactivation. In the experiments presented here, we compared the quality of TDI parameterization by numerical analysis between hepatocyte and microsomal data. Rat liver microsomes (RLM), suspended rat hepatocytes (SRH) and sandwich-cultured rat hepatocytes (SCRH) were incubated with the prototypical CYP3A MBI troleandomycin and the substrate midazolam. Data from RLM provided a better model fit as compared to SRH. Increased CYP3A expression after dexamethasone (DEX) induction improved the fit for RLM and SRH. A novel sequential kinetic scheme, defining inhibitor metabolite production prior to MIC formation, improved the fit compared to direct MIC formation. Furthermore, terminal inactivation rate constants were parameterized for RLM and SRH samples with DEX-induced CYP3A. The low expression of CYP3A and experimental error in SCRH resulted in poor data for model fitting. Overall, RLM generated data better suited for elucidation of TDI mechanisms by numerical analysis.

Thyroid-Associated Orbitopathy: Evaluating Microstructural Changes of Extraocular Muscles and Optic Nerves Using Readout-Segmented Echo-Planar Imaging-Based Diffusion Tensor Imaging

OBJECTIVE: We aimed to investigate the ability of readout-segmented echo-planar imaging (rs-EPI)-based diffusion tensor imaging (DTI) in assessing the microstructural change of extraocular muscles (EOMs) and optic nerves in patients with thyroid-associated orbitopathy (TAO) as well as in evaluating disease activity.MATERIALS AND METHODS: We enrolled 35 TAO patients and 22 healthy controls (HCs) who underwent pre-treatment rs-EPI-based DTI. Mean, axial, and radial diffusivity (MD, AD, and RD) and fractional anisotropy (FA) of the medial and lateral EOMs and optic nerve for each orbit were calculated and compared between TAO and HC groups and between active and inactive TAO groups. Factors such as age, sex, disease duration, mediation, and smoking history between groups were also compared. Logistic regression analysis was used to evaluate the predictive value of significant variables for disease activity.RESULTS: Disease duration was significantly shorter in active TAOs than in inactive ones (p < 0.001). TAO patients showed significantly lower FA and higher MD, AD, and RD than HCs for both medial and lateral EOMs (p < 0.001), but not the AD value of lateral EOMs (p = 0.619). Active patients had significantly higher FA, MD, and AD than inactive patients for medial EOMs (p < 0.005), whereas only FA differed significantly in the lateral EOMs (p = 0.018). The MD, AD, and RD of optic nerves were significantly lower in TAO patients than HCs (p < 0.05), except for FA (p = 0.129). Multivariate analysis showed that the MD of medial EOMs and disease duration were significant predictors for disease activity. The combination of these two parameters showed optimal diagnostic efficiency for disease activity (area under the curve, 0.855; sensitivity, 68.4%; specificity, 96.9%).CONCLUSION: rs-EPI-based DTI is promising in assessing microstructural changes of EOMs and optic nerves and can help to indicate the disease activity of TAO, especially through the MD of medial EOMs.

Study on the inhibitory effect of furafylline and troleandomycin in the 7-methoxyresorufin-O-demethylase and nifedipine oxidase activities in hepatic microsomes from four poultry species using high-performance liquid chromatography coupled with fluorescence and ultraviolet detection.

The present study reports the in vitro studies with furafylline and troleandomycin (TAO) as specific inhibitors of activities 7-methoxyresorufin-O-demethylase (MROD) and nifedipine oxidase, catalyzed by cytochrome P450 1 A2 (CYP1 A2) and 3A4 human enzymes, respectively, in hepatic microsomes of quail, duck, turkey and chicken. The results suggest that in chicken and quail the MROD activity is carried out by orthologs CYP1 A4 and 1 A5, meanwhile in duck and turkey by a CYP1 A5 ortholog. The nifedipine oxidase activity is carried out by orthologs of the CYP3A family in the four bird species. The use of furafylline and TAO significantly decreased these activities (P < 0.05) and suggested that the biotransformation of resorufin methyl ether (RME) may be related to more than one avian ortholog.

Evaluation of Strategies for the Assessment of Drug-Drug Interactions Involving Cytochrome P450 Enzymes.

BACKGROUND AND OBJECTIVES: Drug-drug interactions (DDIs) can occur when one drug alters the metabolism of another drug. Drug metabolism mediated by cytochrome P450 enzymes (CYPs) is responsible for the majority of metabolism of known drugs and inhibition of CYP enzymes is a well-known cause of DDIs. In the current study, the use of various human liver microsomes (HLM)-based methods to determine occurrence of CYP-mediated metabolism-dependent inhibition (MDI) and possible follow-up studies were evaluated. METHODS: Human CYP inhibition was studied using the following methodologies: direct inhibition and (non-diluted) IC50-shift assays, a ferricyanide-based reversibility assay, a spectrophotometric metabolic intermediate complex (MIC) assay, and recording of reduced carbon monoxide (CO)-difference spectra. HLM incubations in the presence and absence of NADPH and glutathione (GSH) were performed to study the possible formation of CYP-dependent GSH adducts. HLM incubations with the radiolabeled inhibitors mifepristone and paroxetine were performed to study CYP-mediated covalent binding. RESULTS: Dihydralazine and furafylline displayed irreversible MDI of CYP1A2. Paroxetine displayed both quasi-irreversible and irreversible MDI of CYP2D6, formation of CYP-dependent GSH adducts was observed, while CYP-mediated covalent binding occurred which was decreased in the presence of GSH. Mifepristone displayed irreversible MDI of CYP3A4, formation of CYP-dependent GSH adducts was observed, while CYP-mediated covalent binding occurred which was decreased in the presence of GSH. Troleandomycin and verapamil displayed quasi-irreversible MDI of CYP3A4; MIC formation was observed, while no formation of CYP-dependent GSH adducts occurred. CONCLUSIONS: This study gives a representative overview of current methodologies that can be used to study CYP inhibition. The here presented strategy can be applied as a tool during risk evaluation of CYP-mediated DDIs.

Metabolism of megestrol acetate in vitro and the role of oxidative metabolites.

1. There is limited knowledge regarding the metabolism of megestrol acetate (MA), as it was approved by FDA in 1971, prior to the availability of modern tools for identifying specific drug-metabolizing enzymes. We determined the cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) that metabolize MA, identified oxidative metabolites and determined pharmacologic activity at the progesterone, androgen and glucocorticoid receptors (PR, AR and GR, respectively). 2. Oxidative metabolites were produced using human liver microsomes (HLMs), and isolated for mass spectral (MS) and nuclear magnetic resonance (NMR) analyses. We screened recombinant P450s using MA at 62 µM (HLM Km for metabolite 1; M1) and 28 µM (HLM Km for metabolite 2; M2). UGT isoforms were simultaneously incubated with UDPGA, nicotinamide adenine dinucleotide phosphate (NADPH), CYP3A4 and MA. Metabolites were evaluated for pharmacologic activity on the PR, AR and GR. CYP3A4 and CYP3A5 are responsible for oxidative metabolism of 62 µM MA. 3. At 28 µM substrate concentration, CYP3A4 was the only contributing enzyme. Mass spectral and NMR data suggest metabolism of MA to two alcohols. After oxidation, MA is converted into two secondary glucuronides by UGT2B17 among other UGTs. MA, M1 and M2 had significant pharmacologic activity on the PR while only MA showed activity on the AR and GR.

Comparison of Intraocular Pressure via Goldmann-applanation Tonometry and TonoPen in Thyroid-associated Ophthalmopathy Accompanying Restrictive Strabismus

PURPOSE: To compare the intraocular pressure (IOP) measured using Goldmann-applanation tonometry (GAT) and Tonopen® tonometry and to evaluate the factors influencing the measurement difference in patients with thyroid-associated ophthalmopathy (TAO)-related restrictive strabismus. METHODS: In 50 eyes of 50 patients who were diagnosed with TAO, IOP measurements were taken using both GAT and a Tonopen® and were subsequently compared between the devices. Factors influencing the measurement difference between the devices were determined, including the restriction of eyeball movement, eyeball deviation, exophthalmometry, central corneal thickness, refractive errors, and blood thyroid hormone levels. RESULTS: In the TAO patients, the GAT-measured IOP was higher than for Tonopen® (16.1 ± 4.7 vs. 13.8 ± 4.5 mmHg, respectively, p < 0.001). As the restriction of vertical eyeball movement increased, the IOP difference between the devices also increased (p = 0.037). The absolute IOP difference between the devices was positively correlated with restrictions in vertical eyeball movement (p = 0.027), degree of vertical strabismus (p = 0.021), and central corneal thickness (p ≤ 0.031). CONCLUSIONS: In patients with TAO accompanying vertical eyeball movement restriction, potential errors in IOP measurements should be considered between the different IOP-measuring devices.

Choroidal Thickness in Thyroid-associated Ophthalmopathy between Normal Tension Glaucoma Using Optical Coherence Tomography

PURPOSE: To compare the macular choroidal thickness in patients with thyroid-associated ophthalmopathy (TAO) with those with normal tension glaucoma (NTG). METHODS: A total of 70 normal eyes, 74 eyes with TAO and 60 eyes with NTG were enrolled in this study. All patients underwent spectral-domain optical coherence tomography (SD-OCT) (Cirrus HD-OCT, Carl Zeiss Meditec Inc., Dublin, CA, USA). Macular choroidal thickness was assessed using enhanced depth imaging. The average macular choroidal thickness was defined as the average value of three measurements: at the fovea and at the points located 1.5 mm in the nasal and temporal directions from the fovea. Generalized estimating equations were used to uncover factors affecting the average macular choroidal thickness. RESULTS: The average, superior and inferior quadrant retinal nerve fiber layer thicknesses were significantly thinner in the NTG group compared with the TAO and control groups (p < 0.001). The average macular choroidal thickness of the TAO group, NTG group and controls was 281.01 ± 60.06 µm, 241.66 ± 55.00 µm and 252.07 ± 55.05 µm, respectively, which were significantly different (p = 0.013). The subfoveal, nasal and temporal side choroidal thicknesses were significantly thinner in the NTG group compared with the TAO group (p = 0.014, 0.012 and 0.034, respectively). Subjects with TAO were associated with a thicker average macular choroidal thickness compared with the NTG group after adjusting for age, sex, spherical equivalent and intraocular pressure (β = 32.61, p = 0.017). CONCLUSIONS: Macular choroidal thickness was significantly thicker in patients with TAO compared with those with NTG. Further evaluation is required to determine if a thick choroid in subjects with TAO has any role in glaucomatous optic neuropathy.

Identification and Functional Characterization of ST3GAL5 and ST8SIA1 Variants in Patients with Thyroid-Associated Ophthalmopathy

PURPOSE: This study was conducted to identify and to functionally characterize genetic variants in ST3GAL5 and ST8SIA1 in Korean patients with thyroid-associated ophthalmopathy (TAO). MATERIALS AND METHODS: Genetic analyses were conducted using DNA samples from TAO patients (n=50) and healthy subjects (n=48) to identify TAO-specific genetic variants of ST3GAL5 or ST8SIA1. The effect of each genetic variant on the transcription or expression of these genes was examined. Additionally, correlations between functional haplotypes of ST3GAL5 or ST8SIA1 and clinical characteristics of the patients were investigated. RESULTS: Six promoter variants and one nonsynonymous variant of ST3GAL5 were identified, and four major promoter haplotypes were assembled. Additionally, three promoter variants and two major haplotypes of ST8SIA1 were identified. All ST3GAL5 and ST8SIA1 variants identified in TAO patients were also found in healthy controls. Promoter activity was significantly decreased in three promoter haplotypes of ST3GAL5 and increased in one promoter haplotype of ST8SIA1. Transcription factors activating protein-1, NKX3.1, and specificity protein 1 were revealed as having roles in transcriptional regulation of these haplotypes. The nonsynonymous variant of ST3GAL5, H104R, did not alter the expression of ST3GAL5. While no differences in clinical characteristics were detected in patients possessing the functional promoter haplotypes of ST3GAL5, exophthalmic values were significantly lower in patients with the ST8SIA1 haplotype, which showed a significant increase in promoter activity. CONCLUSION: These results from genotype-phenotype analysis might suggest a possible link between the ST8SIA1 functional promoter haplotype and the clinical severity of TAO. However, further studies with larger sample sizes are warranted.

Clinical Features of Dry Eye in Thyroid-Associated Ophthalmopathy According to Disease Activity

PURPOSE: To evaluate the clinical features of dry eye in thyroid-associated ophthalmopathy (TAO) according to disease activity and analyze the related factors. METHODS: This study included 157 patients diagnosed with TAO and dry eye between March 2009 and March 2015. According to the clinical activity score (CAS), TAO patients were divided into inactive (CAS < 3) and active (CAS ≥ 3) groups. Clinical features included age, sex, visual acuity, proptosis, palpebral fissure width, orbital computed tomography (CT) findings, thyroid hormones, and ocular surface parameters including tear film break-up time (TFBUT), Schirmer tests, keratoepitheliopathy scores, and ocular surface disease index (OSDI) were obtained and compared between the groups. In addition, correlations between clinical features and ocular surface parameters were analyzed in both groups. RESULTS: In the inactive and active TAO groups, CAS was 1.24 ± 0.69 and 4.23 ± 1.13, respectively (p = 0.001). Thyrotropin-binding inhibitory immunoglobulin was significantly higher in the active TAO group than in the inactive TAO group (p = 0.048). On orbital CT, extraocular muscle hypertrophy was more common in the active TAO group than the inactive TAO group (p = 0.020). No significant difference was found in age, sex, visual acuity, free T4, and thyroid-stimulating hormone between the two groups. During analysis of the tear film and ocular surface parameters, TFBUT (p = 0.006) was shorter and OSDI score (p = 0.028) was higher in the active TAO group than the inactive TAO group. TFBUT was negatively correlated with proptosis (r = -0.432, p = 0.001; r = -0.308, p = 0.032) and palpebral fissure width (r = -0.367 p = 0.012; r = -0.312, p = 0.031) in both groups. OSDI was positively correlated with proptosis in the active TAO group (r = 0.301, p = 0.033). CONCLUSIONS: TFBUT was shorter and OSDI score higher in dry eye patients with active TAO than in patients with inactive TAO. The TFBUT was negatively correlated with proptosis and palpebral fissure width in both groups.

Clinical Features of Dry Eye in Thyroid-Associated Ophthalmopathy According to Disease Activity

PURPOSE: To evaluate the clinical features of dry eye in thyroid-associated ophthalmopathy (TAO) according to disease activity and analyze the related factors. METHODS: This study included 157 patients diagnosed with TAO and dry eye between March 2009 and March 2015. According to the clinical activity score (CAS), TAO patients were divided into inactive (CAS < 3) and active (CAS ≥ 3) groups. Clinical features included age, sex, visual acuity, proptosis, palpebral fissure width, orbital computed tomography (CT) findings, thyroid hormones, and ocular surface parameters including tear film break-up time (TFBUT), Schirmer tests, keratoepitheliopathy scores, and ocular surface disease index (OSDI) were obtained and compared between the groups. In addition, correlations between clinical features and ocular surface parameters were analyzed in both groups. RESULTS: In the inactive and active TAO groups, CAS was 1.24 ± 0.69 and 4.23 ± 1.13, respectively (p = 0.001). Thyrotropin-binding inhibitory immunoglobulin was significantly higher in the active TAO group than in the inactive TAO group (p = 0.048). On orbital CT, extraocular muscle hypertrophy was more common in the active TAO group than the inactive TAO group (p = 0.020). No significant difference was found in age, sex, visual acuity, free T4, and thyroid-stimulating hormone between the two groups. During analysis of the tear film and ocular surface parameters, TFBUT (p = 0.006) was shorter and OSDI score (p = 0.028) was higher in the active TAO group than the inactive TAO group. TFBUT was negatively correlated with proptosis (r = -0.432, p = 0.001; r = -0.308, p = 0.032) and palpebral fissure width (r = -0.367 p = 0.012; r = -0.312, p = 0.031) in both groups. OSDI was positively correlated with proptosis in the active TAO group (r = 0.301, p = 0.033). CONCLUSIONS: TFBUT was shorter and OSDI score higher in dry eye patients with active TAO than in patients with inactive TAO. The TFBUT was negatively correlated with proptosis and palpebral fissure width in both groups.

Surgical Outcomes of Balanced Deep Lateral and Medial Orbital Wall Decompression in Korean Population: Clinical and Computed Tomography-based Analysis

PURPOSE: To evaluate the clinical outcomes of balanced deep lateral and medial orbital wall decompression and to estimate surgical effects using computed tomography (CT) images in Korean patients with thyroid-associated ophthalmopathy (TAO). METHODS: Retrospective chart review was conducted in TAO patients with exophthalmos who underwent balanced deep lateral and medial orbital wall decompression. Exophthalmos was measured preoperatively and postoperatively at 1 and 3 months. Postoperative complications were evaluated in all study periods. In addition, decompressed bone volume was estimated using CT images. Thereafter, decompression volume in each decompressed orbital wall was analyzed to evaluate the surgical effect and predictability. RESULTS: Twenty-four patients (48 orbits) with an average age of 34.08 ± 7.03 years were evaluated. The mean preoperative and postoperative exophthalmos at 1 and 3 months was 18.91 ± 1.43, 15.10 ± 1.53, and 14.91 ± 1.49 mm, respectively. Bony decompression volume was 0.80 ± 0.29 cm3 at the medial wall and 0.68 ± 0.23 cm3 at the deep lateral wall. Postoperative complications included strabismus (one patient, 2.08%), upper eyelid fold change (four patients, 8.33%), and dysesthesia (four patients, 8.33%). Postsurgical exophthalmos reduction was more highly correlated with the deep lateral wall than the medial wall. CONCLUSIONS: In TAO patients with exophthalmos, balanced deep lateral and medial orbital wall decompression is a good surgical method with a low-risk of complications. In addition, deep lateral wall decompression has higher surgical predictability than medial wall decompression, as seen with CT analysis.

Change in Quality of Life after Orbital Decompression Surgery in Patients with Dysthyroid Ophthalmopathy

PURPOSE: To evaluate the effect of orbital decompression surgery on quality of life in thyroid-associated ophthalmopathy (TAO) patients. METHODS: From August 2014 to December 2015, 80 patients diagnosed with TAO at our clinic were retrospectively analyzed. The patients were divided into 2 groups: 30 patients who underwent orbital decompression surgery and 50 patients who did not receive surgery. The Korean version of the Grave's ophthalmopathy specific quality of life (GO-QoL) questionnaire was completed by all patients. We compared questionnaire scores between groups and analyzed demographic and clinical factors affecting change in GO-QoL. RESULTS: The patients who underwent orbital decompression had lower mean GO-QoL score for appearance in comparison with patients without orbital decompression (p < 0.001). The mean GO-QoL score for appearance was increased from 28.8 ± 17.1 to 51.5 ± 18.8 after orbital decompression (p = 0.024). The mean GO-QoL score for visual function was not different between the patients who did not receive surgery and the orbital decompression group. The mean GO-QoL score for visual function was not changed after orbital decompression. There was a significant relation between postoperative proptosis degree and change in GO-QoL score for appearance after orbital decompression. CONCLUSIONS: GO-QoL score for appearance was significantly improved after orbital decompression surgery, and psychological interventions should be considered to enhance the quality of life outcomes.

The Change of Lacrimal Gland Volume in Korean Patients with Thyroid-associated Ophthalmopathy

PURPOSE: To describe the change of lacrimal gland volumes in Korean patients with thyroid-associated ophthalmopathy (TAO) via computed tomography (CT). METHODS: A retrospective review of CT images from 217 TAO patients and 135 control subjects was performed. The TAO patients were diagnosed between May 2005 and May 2014 and had a CT performed on initial presentation (330 orbital CT scans). These images were compared with 270 orbital CT scans from the control group, obtained between May 2013 and May 2014. An open source DICOM viewer was used to calculate the volume of the lacrimal gland. RESULTS: The mean volume of the lacrimal gland in TAO patients was 0.816 cm³ in the right orbit (standard deviation [SD], 0.048) and 0.811 cm3 in the left orbit (SD, 0.051), with no significant difference between right and left (p = 0.192). However, significant differences were observed between TAO patients and healthy individuals (p < 0.001). There was no significant difference between mean lacrimal gland volumes of males (0.812 cm³; SD, 0.037) and females (0.816 cm³; SD, 0.029) (p = 0.513). There was a negative correlation between gland volume and age in TAO patients (Pearson r = -0.479, p = 0.00). The subjective tearing (right: r = 0.244, p = 0.018; left: r = 0.226, p = 0.024), corneal superficial punctate keratopathy (right: r = 0.192, p = 0.040; left: r = 0.206, p = 0.036), and exophthalmometry (right: r = 0.182, p = 0.032; left: r = 0.180, p = 0.046) correlated with lacrimal gland volume. CONCLUSIONS: This study is the first to use CT images to calculate the lacrimal gland volume of Korean TAO patients. In TAO patients, the lacrimal gland volume was notably increased compared to control subjects. The lacrimal gland volume decreased with age, but there was no difference between gender and no difference between left and right. The lacrimal gland volume correlated with subjective tearing, corneal superficial punctate keratopathy and exophthalmometry.

Carbamazepine-Induced Liver Injury Requires CYP3A-Mediated Metabolism and Glutathione Depletion in Rats.

Carbamazepine (CBZ) is widely used as an antiepileptic agent and causes rare but severe liver injury in humans. It has been generally recognized that reactive metabolites formed via the metabolic activation reaction contribute to the onset of liver injuries by several drugs. However, the role of CBZ metabolism in the development of liver injury is not fully understood. In this study, we developed a novel rat model of CBZ-induced liver injury and attempted to elucidate the associated mechanisms by focusing on the metabolism of CBZ. The repeated administration of CBZ for 5 days in combination with l-buthionine sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor, resulted in increases in the plasma alanine aminotransferase (ALT) levels and centrilobular necrosis in the liver that were observed in various degrees. The CBZ and 2-hydroxy-CBZ concentrations in the plasma after the last CBZ administration were lower in the rats with high plasma ALT levels compared with those with normal plasma ALT levels, showing the possibility that the further metabolism of CBZ and/or 2-hydroxy-CBZ is associated with the liver injury. Although a single administration of CBZ did not affect the plasma ALT levels, even when cotreated with BSO, pretreatment with dexamethasone, a CYP3A inducer, increased the plasma ALT levels. In addition, the rats cotreated with troleandomycin or ketoconazole, CYP3A inhibitors, suppressed the increased plasma ALT levels. In conclusion, reactive metabolite(s) of CBZ produced by CYP3A under the GSH-depleted condition might be involved in the development of liver injury in rats.

Comparison of Patient Characteristics in Thyroid-Associated Ophthalmopathy with and without Strabismus

PURPOSE: To determine immunochemical and clinical differences in thyroid-associated ophthalmopathy (TAO) patients with restrictive strabismus and without strabismus. METHODS: A retrospective chart review of 15 TAO patients with strabismus (25 eyes) and 24 TAO patients without strabismus (39 eyes) who presented to the Ophthalmology Clinic between August 2011 and December 2013 was performed. Visual acuity, intraocular pressure (IOP), Hertel exophthalmometry, soft tissue score, and enlargement of extraocular muscles on computed tomography (CT) were obtained and compared in each group. Thyroid related autoantibody (thyroid-stimulating hormone receptor antibody, TRAb; thyroid peroxidase antibody, TPOAb; anti-thyroglobulin antibody, TgAb) titers and positive rates were obtained at the time of diagnosis or before treatment and analyzed. RESULTS: The gender and smoking proportion were not significantly different between the 2 groups. The mean age of TAO patients with strabismus was 52.53 years and of TAO patients without strabismus 40.33 years (p = 0.004). The differences in visual acuity and IOP between the 2 groups were not significant. Hertel exophthalmometry showed less proptotis in the TAO with strabismus group than the TAO without strabismus group (16.84 mm versus 18.67 mm). The soft tissue score was not significantly different. The extraocular muscle enlargement rate of TAO with strabismus was significantly higher than in TAO without strabismus group. In the TAO with strabismus group, TRAb level was higher than in the TAO without strabismus group (p = 0.021). CONCLUSIONS: The TAO with strabismus group was older and had higher positive rate, level of TRAb, and extraocular muscle enlargement rate on CT than the TAO without strabismus group. Furthermore, proptosis was less definite in the TAO with strabismus group.

Demethylation of neferine in human liver microsomes and formation of quinone methide metabolites mediated by CYP3A4 accentuates its cytotoxicity.

Neferine is a bisbenzylisoquinoline alkaloid isolated from the seed embryos of Nelumbonucifera Gaertn (Lotus) with various potent pharmacological effects. Recently, neferine has attracted attention for its anti-tumor activities. Our study explored its metabolism and cytotoxicity mechanism. Approaches using chemical inhibitors and recombinant human enzymes to characterize the involved enzymes and kinetic studies indicated that the demethylation of neferine by cytochrome P450 (CYP) 2D6 and CYP3A4 fitted a biphasic kinetic profile. Glutathione (GSH) was used as a trapping agent to identify reactive metabolites of neferine, and four novel GSH conjugates were detected with [M+H](+) ions at m/z 902.4, 916.2, 916.1, and 930.4. Based on its structure containing para-methylene phenol and results from a product ion scan, GSH tends to conjugate with C9' after undergoing oxidative metabolism to form the binding site predominated by CYP3A4. Furthermore, the addition of recombinant human GSTA1, GSTT1, and GSTP1 had little effect on the production of the GSH conjugates. In a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay, combined with the GSH modulators l-buthionine sulfoximine or N-acetyl-l-cysteine, neferine treatment of MDCK-hCYP3A4 and HepG2 cells revealed that CYP3A4 expression and cellular GSH content could cause an EC50 shift. Metabolic activation mediated by CYP3A4 and GSH depletion significantly enhanced neferine-induced cytotoxicity.

The Clinical Features of Ocular Myasthenia Gravis in Thyroid-Associated Ophthalmopathy

PURPOSE: There are some challenges to accurate diagnosis of ocular myasthenia gravis (MG) in thyroid-associated ophthalmopathy (TAO) patients because the clinical features of these diseases are similar. The aim of this study was to discuss the clinical features and treatment options that may help differentiate these 2 diseases. METHODS: We performed a retrospective analysis using the medical records of patients who visited our clinic and were diagnosed with ocular MG and TAO, from January 2002 to December 2012. The diagnosis of Ocular MG was made on the basis of clinical symptoms and signs with laboratory evaluation, including assays for antithyroid and antiacetylcholine receptor (AchRAb) antibodies, and the Ice, neostigmine, and electromyography tests. RESULTS: Of the 9 ocular MG patients with associated ophthalmopathy, 5 were male and 4 were female. The mean age was 36 +/- 16.0 years and the follow-up period was 45.6 +/- 36.6 months. Graves' disease (8 patients) was predominant and all patients showed abnormal thyroid function. Atypical symptoms and/or mild clinical features were predominant in ocular MG patients with TAO. Positive test results were obtained as follows: Neostigmine test 33.3%, electromyography 44.4%, ice test 77.8% and anti-AchR titer test 77.8%. Thyroid function test results were abnormal in all patients. In 3 patients who were first diagnosed with TAO, symptoms remained persistent despite steroid therapy then improved dramatically by administration of an anti-acetylcholinesterase agent. These patients were diagnosed with ocular MG in conjunction with TAO. CONCLUSIONS: Patients with thyroid disease who show atypical features, symptomatic changes with fatigue, odd appearing ptosis, and who, do not exhibit good response to treatment of TAO need to be examined for ocular MG with additional tests and treatment.

Drug-drug interactions and metabolism in cytochrome P450 2C knockout mice: application to troleandomycin and midazolam.

Drug-drug interactions (DDIs) may cause serious drug toxicity and delay development of candidate drugs. Screening using human liver microsomes and hepatocytes can help predict DDIs but do not always provide the degree of certainty required for confident progression of a candidate drug. Thus a suitable in vivo test system could be of great value. Here a Cyp2c knockout (KO) mouse was investigated for studying DDIs using midazolam (MDZ) a standard human CYP3A4 substrate and troleandomycin (TAO) a potent human CYP3A4 inhibitor. Pharmacokinetics (PK) and biotransformation of MDZ were investigated following dosing to Cyp2c KO and wild type mice before and after TAO treatment. The noteworthy differences in the metabolism of MDZ in Cyp2c KO compared to wild type mice confirms the important role that Cyp2c enzymes play in the murine metabolism of MDZ in vivo. The impact of Cyp3a inhibition produced a further increase in circulating MDZ concentrations in all individuals from both strains of mice though the impact of the elimination of the Cyp2c pathway in the KO mice on the AUC was less than perhaps expected. We have shown that TAO produces an increase in the MDZ concentration and a reduction in the 1'hydroxymidazolam/midazolam formation ratio but the expected difference in the magnitude of this effect between the wild type and the Cyp2c KO mice was not seen. The magnitude of the TAO effect was also smaller than is reported in humans. Hence further work is required before this animal model could be used to predict clinical interactions.

Green tea extract affects the cytochrome P450 3A activity and pharmacokinetics of simvastatin in rats.

Effects of green tea extract (GTE) on the activity of cytochrome P450 (CYP) enzymes and pharmacokinetics of simvastatin (SIM) were investigated in rats. Inhibitory effects of GTE on CYP3A activity were investigated in rat hepatic microsomes (RHM) using midazolam (MDZ) 1'-hydroxylation as a probe reaction. SD female rats received a single oral dose of GTE (400 mg/kg) or troleandomycin (TAO, a CYP3A selective inhibitor, 500 mg/kg), followed 30 min later by SIM (20 mg/kg). Plasma concentrations of SIM and its active metabolite, simvastatin acid, were determined up to 6 h after the SIM administration using LC/MS/MS. In RHM, GTE inhibited MDZ 1'-hydroxylation with IC50 and K(i)(app) values of 12.5 and 18.8 µg/mL, respectively, in a noncompetitive manner. Area under plasma concentration-time curves for SIM in the GTE and TAO groups were increased by 3.4- and 10.2-fold, respectively, compared with the control. The maximum concentrations of SIM were higher in the GTE (3.3-fold) and TAO (9.5-fold) groups. GTE alters the pharmacokinetics of SIM, probably by inhibiting intestinal CYP3A.