Spectrum of cutaneous adverse reactions to aromatic antiepileptic drugs and human leukocyte antigen genotypes in Thai patients and meta-analysis.

Aromatic antiepileptic drugs (AEDs)-induced cutaneous adverse drug reactions (cADRs) add up to the limited use of the AEDs in the treatment and prevention of seizures. Human leukocyte antigen-B (HLA-B) alleles have been linked to AEDs-induced cADRs. We investigated the association between cADRs (including Stevens-Johnson syndrome; SJS/toxic epidermal necrolysis; TEN, drug reaction with eosinophilia and systemic symptoms; DRESS, and Maculopapular eruption; MPE) caused by AEDs (phenytoin, carbamazepine, lamotrigine, phenobarbital and oxcarbazepine) and HLA-B alleles in Thai population. Through the case-control study, 166 patients with AEDs-induced cADRs, 426 AEDs-tolerant patients (AEDs-tolerant controls), and 470 healthy subjects (Thai population) were collected. The HLA genotypes were detected using the polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) method. We also performed a meta-analysis with these data and other populations. The carrier rate of HLA-B*15:02 was significantly different between AEDs-induced cADRs group and AEDs-tolerant group (Odds ratio; OR 4.28, 95% Confidence interval; CI 2.64-6.95, p < 0.001), AEDs-induced cADRs group and Thai population (OR 2.15, 95%CI 1.41-3.29, p < 0.001). In meta-analysis showed the strong association HLA-B*15:02 with AEDs-induced cADRs (OR 4.77, 95%CI 1.79-12.73, p < 0.001). Furthermore, HLA-B*15:02 was associated with SJS/TEN induced by AEDs (OR 10.28, 95%CI 6.50-16.28, p < 0.001) Phenytoin (OR 4.12, 95%CI 1.77-9.59, p = 0.001) and carbamazepine (OR 137.69, 95%CI 50.97-371.98, p < 0.001). This study demonstrated that genetic association for AEDs-induced cADRs was phenotype-specific. A strong association between HLA-B*15:02 and AEDs-induced SJS/TEN was demonstrated with an OR of 10.79 (95%CI 5.50-21.16, p < 0.001) when compared with AEDs-tolerant group. On the other hand, the carrier rates of HLA-B*08:01, HLA-B*13:01, and HLA-B*56:02 were significantly higher in the DRESS group compared with the AEDs-tolerant group (p = 0.029, 0.007, and 0.017, respectively). The HLA-B*15:02 allele may represent a risk factor for AEDs-induced cADRs.

Association between HLA-B Alleles and Carbamazepine-Induced Maculopapular Exanthema and Severe Cutaneous Reactions in Thai Patients.

The HLA-B∗15:02 allele has been reported to have a strong association with carbamazepine-induced Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) in Thai patients. The HLA-B alleles associated with carbamazepine-induced maculopapular exanthema (MPE) and the drug reaction with eosinophilia and systemic symptoms (DRESS) among the Thai population have never been reported. The aim of the present study was to carry out an analysis of the involvement of HLA-B alleles in carbamazepine-induced cutaneous adverse drug reactions (cADRs) in the Thai population. A case-control study was performed by genotyping the HLA-B alleles of Thai carbamazepine-induced hypersensitivity reaction patients (17 MPE, 16 SJS/TEN, and 5 DRESS) and 271 carbamazepine-tolerant controls. We also recruited 470 healthy Thai candidate subjects who had not taken carbamazepine. HLA-B∗15:02 showed a significant association with carbamazepine-induced MPE (P = 0.0022, odds ratio (OR) (95% confidence interval [CI]) = 7.27 (2.04-25.97)) and carbamazepine-induced SJS/TEN (P = 4.46 × 10-13; OR (95% CI) = 70.91(19.67-255.65)) when compared with carbamazepine-tolerant controls. Carbamazepine-induced SJS/TEN also showed an association with HLA-B∗15:21 allele (P = 0.013; OR (95% CI) = 9.54 (1.61-56.57)) when compared with carbamazepine-tolerant controls. HLA-B∗58:01 allele was significantly related to carbamazepine-induced MPE (P = 0.007; OR (95% CI) = 4.73 (1.53-14.66)) and DRESS (P = 0.0315; OR (95% CI) = 7.55 (1.20-47.58)) when compared with carbamazepine-tolerant controls. These alleles may serve as markers to predict carbamazepine-induced cADRs in the Thai population.

Immunochromatographic determination of bacopaside I in biological samples.

We describe a novel immunochromatographic method for qualitative and quantitative analyses of bacopaside I, a bioactive constituent in Bacopa monnieri (L.) Wettst in biological samples. The assay was performed on polyethersulfone membrane using a polyclonal antibody raised against bacopaside I. The finalised method could quantitatively determine bacopaside I in the range of 31.3-1000.0ng and the detection and quantification limits were 1.0 and 31.3ng, respectively. The percentage recoveries of bacopaside I in blood and urine were nearly 100% indicating the accuracy of the extraction. The method was then applied for the determination of this compound in rat serum, urine and feces after an oral dose of 15mg/kg body weight. About 4% of the ingested dose of bacopaside I was detected in rat feces but none was detected in serum and urine which accorded with results from liquid chromatography tandem mass spectrometry. The accuracy, selectivity, sensitivity of the method are appropriate for in vivo pharmacokinetic studies.

Distribution and function of macrophage galactose-type C-type lectin 2 (MGL2/CD301b): efficient uptake and presentation of glycosylated antigens by dendritic cells.

Dendritic cells (DCs) express cell surface lectins that are potentially involved in the recognition, uptake, and presentation of glycosylated foreign substances. A unique calcium-type (C-type) lectin, the macrophage galactose (Gal)-type C-type lectin (MGL/CD301) expressed on DCs, is thought to participate in the recognition of molecules from both altered self and pathogens due to its monosaccharide specificity for Gal and N-acetylgalactosamine (GalNAc). Although mice have two MGL genes, Mgl1 and Mgl2, their distinct roles have not been previously explored. The present report characterizes the properties of MGL2 by examining its distribution and its role in antigen presentation by DCs. We generated an MGL2-specific monoclonal antibody and examined MGL2 expression in tissues by immunohistochemistry and in isolated cells by flow cytometry. The cells reactive with this antibody were shown to be a portion of MGL1-expressing cells, mostly conventional DCs. Internalization of soluble polyacrylamide polymers (PAA) with alpha-GalNAc residues (GalNAc-PAA) by bone marrow-derived DCs (BM-DCs) was mediated by MGL2, as revealed by a comparison of Mgl1(-/-) and Mgl2(-/-) BM-DCs with wild-type BM-DCs. Biotinylated GalNAc-PAA conjugated to streptavidin (SAv) was more efficiently presented to SAv-primed T cells by BM-DCs than beta-N-acetylglucosamine-PAA conjugated to SAv or SAv alone as shown by thymidine uptake and cytokine production. This is the first report that demonstrates the involvement of GalNAc residues in antigen uptake and presentation by DCs that lead to CD4(+) T cell activation.

Bioequivalence of generic lamotrigine 100-mg tablets in healthy Thai male volunteers: a randomized, single-dose, two-period, two-sequence crossover study.

BACKGROUND: Lamotrigine is an antiepileptic drug which has been used in the treatment of epilepsy and bipolar disorder. A search of the literature did not find previously published bioequivalence and pharmacokinetic evaluations of lamotrigine in healthy Thai male volunteers. OBJECTIVE: The aim of this study was to compare the pharmacokinetic parameters between 2 brands of lamotrigine in healthy Thai male volunteers. METHODS: A randomized, single-dose, 2-period, 2-sequence, crossover study design with a 2-week washout period was conducted in healthy Thai males. Subjects were randomized to receive either the test or reference formulation in the first period. All subjects were required to be nonsmokers and without a history of alcohol or drug abuse. Plasma samples were collected over a 120-hour period after 100-mg lamotrigine administration in each period. A validated high-performance liquid chromatography ultraviolet method was used to analyze lamotrigine concentration in plasma. Pharmacokinetic parameters were determined using a noncompartmental method. Bioequivalence between the test and reference products, as defined by the US Food and Drug Administration (FDA), is determined when the ratio for the 90% CIs of the difference in the means of the log-transformed AUC(0-t), AUC(0-infinity), and C(max) of the 2 products are within 0.80 and 1.25. Adverse events were determined by measuring vital signs after dosing. Subjects were also asked if they suffered from undesirable effects such as nausea, vomiting, dizziness, and headache. RESULTS: This bioequivalence study was performed in 24 healthy Thai males (mean [SD] age, 20.5 [1.3] years; range, 19-24 years; weight, 62.5 [7.4] kg; height, 172.8 [6.9] cm; body mass index, 20.9 [2.0] kg/m(2)). The mean (SD) C(max) and T(max) of the test formulation of lamotrigine were 1.7 (0.3) microg/mL and 1.2 (0.9) hours, respectively. The mean (SD) C(max) and T(max) of the reference formulation of lamotrigine were 1.7 (0.3) microg/mL and 1.4 (1.0) hours, respectively. The mean (SD) AUC(0-t) was 67.1 (13.2) microg/mL x h(-1) for the test product and 66.4 (14.6) microg/mL x h(-1) for the reference product. The mean (SD) AUC(0-infinity) was 74.9 (18.3) microg/mL x h(-1) for the test product and 74.3 (20.5) microg/mL x h(-1) for the reference product. The mean (SD) t((1/2)) values were 35.0 (7.6) hours for the test product and 34.7 (7.6) hours for the reference product. The mean test/reference ratios for AUC(0-t), AUC(0-infinity), and Cmax were 1.01, 1.01, and 1.05, respectively. The parametric 90% CIs for AUC(0-t), AUC(0-infinity), and Cmax were 0.98 to 1.05, 0.98 to 1.06, and 0.98 to 1.13, respectively. Following administration, dizziness or headache was reported in 2 subjects in the test group and 1 subject in the reference group. CONCLUSION: The results of this study suggest that the test product was bioequivalent to the reference product in these healthy Thai male subjects, based on the US FDA's regulatory definition.

Carbohydrate binding mechanism of the macrophage galactose-type C-type lectin 1 revealed by saturation transfer experiments.

Macrophage galactose-type C-type lectins 1 and 2 (MGL1/2) are expressed on the surfaces of macrophages and immature dendritic cells. Despite the high similarity between the primary sequences of MGL1 and MGL2, they display different ligand specificities. MGL1 shows high affinity for the LewisX trisaccharide, whereas MGL2 shows affinity for N-acetylgalactosamine. To elucidate the structural basis for the ligand specificities of the MGLs, we performed NMR analyses of the MGL1-LewisX complex. To identify the LewisX binding site on MGL1, a saturation transfer experiment for the MGL1-LewisX complex where sugar-CH/CH2-selective saturation was applied was carried out. To obtain sugar moiety-specific information on the interface between MGL1 and the LewisX trisaccharide, saturation transfer experiments where each of galactose-H5-, fucose-CH3-, and N-acetylglucosamine-CH3-selective saturations was applied to the MGL1-LewisX complex were performed. Based on these results, we present a LewisX binding mode on MGL1 where the galactose moiety is bound to the primary sugar binding site, including Asp-94, Trp-96, and Asp-118, and the fucose moiety interacts with the secondary sugar binding site, including Ala-89 and Thr-111. Ala-89 and Thr-111 in MGL1 are replaced with arginine and serine in MGL2, respectively. The hydrophobic environment formed by a small side chain of Ala-89 and a methyl group of Thr-111 is a requisite for the accommodation of the fucose moiety of the LewisX trisaccharide within the sugar binding site of MGL1.

The amino acids involved in the distinct carbohydrate specificities between macrophage galactose-type C-type lectins 1 and 2 (CD301a and b) of mice.

Binding specificities of mouse macrophage galactose-type C-type lectin 1 (MGL1/CD301a) and 2 (MGL2/CD301b) toward various oligosaccharides were compared by frontal affinity chromatography. MGL1 preferentially bound oligosaccharides containing Lewis(X) (Le(X)) trisaccharides among 111 oligosaccharides tested, whereas MGL2 preferentially bound globoside Gb4. The important amino acids for the preferential bindings were investigated by pair-wise site-directed mutagenesis at positions 61, 89, 97, 100, 110-113, 115, 124, and 125 in the soluble recombinant carbohydrate recognition domains (CRD) prepared in Escherichia coli and purified with galactose-Sepharose. Mutations of Val, Ala, Thr, and Phe at positions 61, 89, 111 and 125 on MGL1 CRD caused reductions in Le(X) binding. Mutations of MGL2 CRD at Leu, Arg, Arg, and Tyr at positions 61, 89, 115 and 125 were implicated in the preference for beta-GalNAc. Le(X) binding was observed with MGL2 mutants of Arg89Ala and Arg89Ala/Ser111Thr. MGL1 mutants of Ala89Arg and Ala89Arg/Pro115Arg showed beta-GalNAc bindings. Molecular modeling illustrated potential direct molecular interactions of Leu61, Arg89, and His109 in MGL2 CRD with GalNAc.