Patient, Disease, and Drug-Related Risk Factors Associated with Phenytoin- Induced Cutaneous Adverse Drug Reactions in South Indian Epileptic Patients - A Prospective Case-Control Study.

BACKGROUND: Phenytoin is the most commonly reported aromatic Anti-Epileptic Drug (AED) to cause Cutaneous Adverse Drug Reactions (CADRs). Cutaneous adverse drug reactions may be immune or non-immune mediated. It has been observed that predisposition is multifactorial and that gene mutations alone cannot be the cause. OBJECTIVES: In this study, we investigated the patient, disease, and drug-related risk factors associated with phenytoin-induced cutaneous adverse drug reactions in South Indian epileptic patients. METHODS: This study was conducted as a single-center prospective case-control study over a period of 13 months. The Fisher's exact test and multivariate binary logistic regression analysis were used to test the association of single and multiple variables, respectively. RESULTS: This study comprised 26 patients with phenytoin-induced cutaneous adverse drug reactions (PHT-CARDs) and 32 phenytoin-tolerant controls with a mean age of 40.60±18.15 and 36.21±14.71 years, respectively. Among 26 phenytoin-induced cutaneous adverse drug reactions, 76.92% cases were mild-moderate reactions and 23.07% were severe. The onset latency period of these reactions ranged from 7-42 days. The multivariate analysis showed that multiple AEDs (OR =18.62, 95% CI 4.28-80.87, p=< .001) and comorbidities (OR= 5.98, 95% CI 1.33-26.78, p=.01) are risk factors for PHT-CADRs. PHT-SCARs were shown to be associated with previous allergy history (OR= 31, % CI 2.40-398.8, p=.008). CONCLUSION: The risk factors found to be associated with CARDs in South Indian Epileptic patients are multiple AEDs, comorbidities, and past allergic history. Therefore, physicians and other associated health care professionals should closely monitor the patients when phenytoin is employed.

The RING heterodimer BRCA1-BARD1 is a ubiquitin ligase inactivated by the platinum-based anticancer drugs.

The breast cancer susceptibility protein 1 (BRCA1) participates in the maintenance of cells genomic integrity through DNA repair, cell cycle checkpoint, protein ubiquitination, and transcriptional regulation. The N-terminus of BRCA1 contains a RING domain that preferentially forms a heterodimeric complex with BARD1. The BRCA1-BARD1 RING complex has an E3 ubiquitin ligase activity that plays an essential role in response to DNA damage. Preclinical and clinical studies have recently revealed that structural changes to the heterodimer result in alterations to the BRCA1-mediated DNA repair pathways in cancer cells, and lead to hypersensitivity to several chemotherapeutic agents. It is of interest to approach the BRCA1 RING domain as a potentially molecular target for platinum-based drugs for cancer therapy. A previous study has shown that the anticancer drug cisplatin formed intramolecular and intermolecular BRCA1 adducts in which His117 was the primary platinum-binding site, and conferred conformational changes and induced thermostability. Here, we have studied the functional consequence of the in vitro platination of the BRCA1 RING domain by a number of platinum complexes. The BRCA1 ubiquitin ligase activity was inhibited by transplatin > cisplatin > oxaliplatin > carboplatin in that order. The consequences of the binding of the platinum complexes on the reactivity of the BRCA1 were also discussed. The data raised the possibility of selectively targeting the BRCA1 DNA repair for cancer therapy.

Substitution of aspartic acid with glutamic acid at position 67 of the BRCA1 RING domain retains ubiquitin ligase activity and zinc(II) binding with a reduced transition temperature.

Breast cancer susceptibility protein 1 (BRCA1) participates in genomic integrity maintenance through DNA repair, cell cycle checkpoint, protein ubiquitination, and transcriptional regulation. The N-terminus of BRCA1 contains a RING domain which forms two Zn(2+) binding sites in an interleaved fashion. A number of deleterious BRCA1 missense mutations, which predispose an individual to a subset of hereditary breast and ovarian cancers, have been identified in the RING domain. Disruption of Zn(2+) binding sites and protein structure results in the inactivation of BRCA1 tumor suppression function. An unprecedented D67E BRCA1 mutation, identified in Thai familial breast cancer patients, is located in the vicinity of Zn(2+) binding site II, and its pathogenic significance remains elusive. The present study revealed that the D67E BRCA1 RING protein assumes a preformed structure in the absence of Zn(2+). The Zn(2+)-bound mutant protein was more folded, resulting in enhanced proteolytic resistance and dimerization. This indicated that the mutation retained Zn(2+) binding, and barely perturbed the native global structure of the BRCA1 RING domain. The complex between D67E BRCA1 and BARD1 RING domains exhibited a substantial ubiquitin ligase activity compared with a defective complex containing the C61G BRCA1 mutation. However, the D67E mutation was slightly less stable toward thermal denaturation. This implies that the D67E mutation might be a neutral or mild cancer-risk modifier of other defective mechanisms underlying BRCA1-mutation-related breast cancer.

Association of HLA-B*51:01, HLA-B*55:01, CYP2C9*3, and Phenytoin-Induced Cutaneous Adverse Drug Reactions in the South Indian Tamil Population.

Phenytoin (PHT) is one of the most commonly reported aromatic anti-epileptic drugs (AEDs) to cause cutaneous adverse reactions (CADRs), particularly severe cutaneous adverse reactions (SCARs). Although human leukocyte antigen (HLA)-B*15:02 is associated with PHT-induced Steven Johnson syndrome/toxic epidermal necrosis (SJS/TEN) in East Asians, the association is much weaker than it is reported for carbamazepine (CBZ). In this study, we investigated the association of pharmacogenetic variants of the HLA B gene and CYP2C9*3 with PHT-CADRs in South Indian epileptic patients. This prospective case-controlled study included 25 PHT-induced CADRs, 30 phenytoin-tolerant patients, and 463 (HLA-B) and 82 (CYP2C9*3) normal-controls from previous studies included for the case and normal-control comparison. Six SCARs cases and 19 mild-moderate reactions were observed among the 25 cases. Pooled data analysis was performed for the HLA B*51:01 and PHT-CADRs associations. The Fisher exact test and multivariate binary logistic regression analysis were used to identify the susceptible alleles associated with PHT-CADRs. Multivariate analysis showed that CYP2C9*3 was significantly associated with overall PHT-CADRs (OR = 12.00, 95% CI 2.759-84.87, p = 003). In subgroup analysis, CYP2C9*3 and HLA B*55:01 were found to be associated with PHT-SCARs (OR = 12.45, 95% CI 1.138-136.2, p = 0.003) and PHT-maculopapular exanthema (MPE) (OR = 4.041, 95% CI 1.125-15.67, p = 0.035), respectively. Pooled data analysis has confirmed the association between HLA B*51:01/PHT-SCARs (OR = 6.273, 95% CI 2.24-16.69, p = <0.001) and HLA B*51:01/PHT-overall CADRs (OR = 2.323, 95% CI 1.22-5.899, p = 0.037). In this study, neither the case nor the control groups had any patients with HLA B*15:02. The risk variables for PHT-SCARs, PHT-overall CADRs, and PHT-MPE were found to be HLA B*51:01, CYP2C9*3, and HLA B*55:01, respectively. These alleles were identified as the risk factors for the first time in the South Indian Tamil population for PHT-CADRs. Further investigation is warranted to establish the clinical relevance of these alleles in this population with larger sample size.

Cisplatin affects the conformation of apo form, not holo form, of BRCA1 RING finger domain and confers thermal stability.

The breast cancer suppressor protein 1 (BRCA1) has been shown to participate in genomic integrity maintenance. Preclinical and clinical studies have recently revealed that the inactivation of BRCA1 in cancer cells leads to chemosensitivity. Approaching the BRCA1 RING protein as a potentially molecular target for a platinum-based drug might be of interest in cancer therapy. In the present study, the in vitro platination of the BRCA1 RING protein by the anticancer drug cisplatin was observed. The protein contained a preformed structure in the apo form with structural changes and resistance to limited proteolysis after Zn2+ binding. SDS-PAGE and mass-spectrometric analyses revealed that cisplatin preferentially formed monofunctional and bifunctional BRCA1 adducts. Tandem mass spectrometry (MS/MS) of the 656.29(2+) ion indicated that the ion arose from [Pt(NH3)2(OH)]+ bound to the BRCA1 peptide (111)ENNSPEHLK(119). The product-ion spectrum revealed the Pt-binding site on His117. Circular dichroism showed that the apo form, not holo form, of BRCA1 underwent more folded structural rearrangement upon cisplatin binding. Cisplatin-bound protein exhibited an enhanced thermostability by 13 degrees , resulting from the favorably intermolecular cross-links driven by the free energy. Our findings demonstrated the first conformational and thermal evidences for a direct binding of cisplatin to the BRCA1 RING domain and could raise a possibility of selectively targeted treatment of cancer with less toxicity or improved response to conventional regimens.

Recognition properties and competitive assays of a dual dopamine/serotonin selective molecularly imprinted polymer.

A molecularly imprinted polymer (MIP) with dual dopamine/serotonin-like binding sites (DS-MIP) was synthesized for use as a receptor model of study the drug-interaction of biological mixed receptors at a molecular level. The polymer material was produced using methacrylic acid (MAA) and acrylamide (ACM) as functional monomers, N,N'-methylene bisacrylamide (MBAA) as cross-linker, methanol/water mixture (4:1, v/v) as porogen and a mixture of dopamine (D) and serotonin (S) as templates. The prepared DS-MIP exhibited the greatest rebinding of the template(s) in aqueous methanol solution with decreased recognition in acetonitrile, water and methanol solvent. The binding affinity and binding capacity of DS-MIP with S were found to be higher than those of DS-MIP with D. The selectivity profiles of DS-MIP suggest that the D binding site of DS-MIP has sufficient integrity to discriminate between species of non-optimal functional group orientation, whilst the S binding site of DS-MIP is less selective toward species having structural features and functional group orientations different from S. The ligand binding activities of a series of ergot derivatives (ergocryptine, ergocornine, ergocristine, ergonovine, agroclavine, pergolide and terguride) have been studied with the DS-MIP using a competitive ligand binding assay protocol. The binding affinities of DS-MIP were demonstrated in the micro- or submicro-molar range for a series of ergot derivatives, whereas the binding affinities were considerably greater to natural receptors derived from the rat hypothalamus. The DS-MIP afforded the same pattern of differentiation as the natural receptors, i.e. affinity for the clavines > lysergic acid derivatives > ergopeptines. The results suggest that the discrimination for the ergot derivatives by the dopamine and serotonin sites of DS-MIP is due to the structural features and functional orientation of the phenylethylamine and indolylethylamine entities at the binding sites, and the fidelity of the dopamine and serotonin imprinted cavities.

In vitro platination of human breast cancer suppressor gene1 (BRCA1) by the anticancer drug carboplatin.

Carboplatin is an anticancer drug for the treatment of cancers affecting various organs including ovary and testes. It essentially exerts its cytotoxicity against cancerous cells via covalent attachment of platinum atom to DNA, generating various platinum-DNA adducts. Platinum-DNA adducts inhibit biological processes essential for cellular viability. However, carboplatin interacts nonspecifically with DNA, resulting in damaging of normal cell DNA. Potential in vitro interaction of carboplatin with genes encoding tumor suppressor proteins such as human breast cancer suppressor gene 1(BRCA1) was herein investigated. The 696--bp fragment of the 3'-region of BRCA1 gene (nucleotide 4897--5592) was amplified by RT-PCR using mRNA templates isolated from human white blood cells. Retardation of the electrophoretic migration on agarose gel of drug-treated DNA, in the dose-response manner, was observed. Analysis by restriction digestion with PvuII and Eco O 109I suggested that the platination favorably occurred at the dGpG sequence of Eco O 109I-cleaved site. The semi-quantitative PCR-based assay was used to determine the lesion frequencies produced by carboplatin in the 696-bp fragment of the 3'-region of BRCA1 gene and in the 3,426-bp fragment of the BRCA1 exon 11 of human breast adenocarcinoma MCF-7 cells. A significant decrease in DNA amplification was observed at 400 microM of carboplatin with approximately 1--2 platinum atoms per BRCA1 fragment. Carboplatin caused slightly less damage at equimolar concentrations in cells than in cell-free BRCA1 fragment.

Temperature sensitive dopamine-imprinted (N,N-methylene-bis-acrylamide cross-linked) polymer and its potential application to the selective extraction of adrenergic drugs from urine.

A temperature sensitive dopamine-imprinted polymer was prepared in 80% aqueous methanol solution by free-radical cross-linking co-polymerisation of methacrylic acid and acrylamide at 60 degrees C in the presence of N,N-methylene-bis-acrylamide as the cross-linker and dopamine hydrochloride as template molecule. The resulting molecularly imprinted polymer (MIP) formed temperature responsive materials, which could be used for the selective separation of appropriate dopamine and adrenergic compounds from a liquid matrix at ambient temperatures. The thermoresponsive MIP exhibited a swelling-deswelling transition in 80% aqueous methanol solution at about 35 degrees C. The capacity of the thermoresponsive MIP to recognise the template molecule when present in aqueous methanol solution changed with temperature, with the highest selectivity found at 35 degrees C. Additionally, binding parameters obtained from Scatchard analyses indicate that increasing temperature resulted in an increased affinity and binding capacity of specific binding sites, but had less effect on non-selective binding sites. Subsequently, the thermoresponsive MIP was tested for its application as a sorbent material, utilisable in the selective solid-phase extraction (SPE) of dopamine and other adrenergic compounds (epinephrine, isoproterenol, salbutamol and serotonin) from urine samples. It was shown that the compounds that were structurally related to dopamine could be removed by elution, while dopamine and serotonin, the analytes of interest, remained strongly adsorbed to the adsorbent during SPE applications. The thermoresponsive MIP displayed different efficiency in clean-up and enrichments using the SPE protocol at different temperatures.

Interactions at the dimer interface influence the relative efficiencies for purine nucleotide synthesis and pyrophosphorolysis in a phosphoribosyltransferase.

Enzymes that salvage 6-oxopurines, including hypoxanthine phosphoribosyltransferases (HPRTs), are potential targets for drugs in the treatment of diseases caused by protozoan parasites. For this reason, a number of high-resolution X-ray crystal structures of the HPRTs from protozoa have been reported. Although these structures did not reveal why HPRTs need to form dimers for catalysis, they revealed the existence of potentially relevant interactions involving residues in a loop of amino acid residues adjacent to the dimer interface, but the contributions of these interactions to catalysis remained poorly understood. The loop, referred to as active-site loop I, contains an unusual non-proline cis-peptide and is composed of residues that are structurally analogous with Leu67, Lys68, and Gly69 in the human HPRT. Functional analyses of site-directed mutations (K68D, K68E, K68N, K68P, and K68R) in the HPRT from Trypanosoma cruzi, etiologic agent of Chagas' disease, show that the side-chain at position 68 can differentially influence the K(m) values for all four substrates as well as the k(cat) values for both IMP formation and pyrophosphorolysis. Also, the results for the K68P mutant are inconsistent with a cis-trans peptide isomerization-assisted catalytic mechanism. These data, together with the results of structural studies of the K68R mutant, reveal that the side-chain of residue 68 does not participate directly in reaction chemistry, but it strongly influences the relative efficiencies for IMP formation and pyrophosphorolysis, and the prevalence of lysine at position 68 in the HPRT of the majority of eukaryotes is consistent with there being a biological role for nucleotide pyrophosphorolysis.

Host Cell Reactivation and Transcriptional Activation of Carboplatin-Modified BRCA1.

The breast cancer susceptibility gene 1 (BRCA1) has been shown to maintain genomic stability through multiple functions in the regulation of DNA damage repair and transcription. Its translated BRCT (BRCA1 C-terminal domain) acts as a strong transcriptional activator. BRCA1 damaged by carboplatin treatment may lead to a loss of such functions. To address the possibility of the BRCA1 gene as a therapeutic target for carboplatin, we investigated the functional consequences of the 3'-terminal region of human BRCA1 following in vitro platination with carboplatin. A reduction in cellular BRCA1 repair of carboplatin-treated plasmid DNA, using a host cell reactivation assay, was dependent on the platination levels on the reporter gene. The transcriptional transactivation activity of the drug-modified BRCA1, assessed using a one-hybrid GAL4 transcriptional assay, was inversely proportional to the carboplatin doses. The data emphasized the potential of the BRCA1 gene to be a target for carboplatin treatment.

Development of a rubber elongation factor, surface-imprinted polymer-quartz crystal microbalance sensor, for quantitative determination of Hev b1 rubber latex allergens present in natural rubber latex products.

Molecularly imprinted polymers (MIPs) for screening to detect rubber latex allergens (Hev b1) in natural rubber based products were designed as artificial recognition polymeric materials coated onto a quartz crystal microbalance (QCM). The polymers were prepared using a stamp imprinting procedure after mixing optimum amounts of methacrylic acid-vinylpyrrolidone-dihydroxyethylene bisacrylamide and Hev b1 latex allergen proteins, obtained from rubber gloves. QCM measurements showed that the resulting polymer layers after removal of the proteins used in their preparation could incorporate structures and features down to nanometer scale of protein templates into the imprinted polymer much better than a non-specific control polymer under controlled sensor conditions and an optimized polymerization process. This selective polymer but not the non-selective polymer clearly distinguished between the latex allergen Hev b1 and proteins such as lysozyme, ovalbumin and bovine serum albumin, with a selectivity factor of from 2 to 4, and the response of the rubber elongation factors by an astonishing factor of 12. The imprinted cavities recognized specific binding sites and could distinguish among related hevein latex allergenic proteins isolated from fresh natural rubber latex; Hev b1, Hev b2, and Hev b3 with a selectivity factor of from 4 to 6. The different QCM measurements obtained presumably reflected slightly different conformations and affinities to the MIP binding sites. The sensor layers selectively adsorbed Hev b1 within minutes in amounts ranging from 10 to 1500 µg L⁻¹ and with a detection limit of 1 µg L⁻¹. This work has demonstrated that this new sensor provides a fast and reliable response to natural rubber latex protein, even after being extracted from the matrix of rubber gloves.

Cisplatin-damaged BRCA1 exhibits altered thermostability and transcriptional transactivation.

BRCA1 is a tumor suppressor gene. Its translated product has an important function in transcriptional activation and DNA repair pathways. Damaged BRCA1 due to cisplatin treatment may lead to loss of such functions. To address a potential drug target of BRCA1 for cisplatin treatment, we investigated the biophysical characterization and functional consequences of the 3'-terminal region of human BRCA1 after in vitro platination with cisplatin. To analyze the base/sequence specificity of cisplatin damage, the measurement for sensitivity of cisplatin-treated BRCA1 to restriction enzymes (EcoO109I and PvuII) and sequence gel analysis was conducted. The results suggested that the platination favorably occurred at the d(GpG) and the d(GpC) sites. An increase in drug concentrations resulted in increased interstrand crosslinks at the d(GpC) site. Cisplatin affected the transition temperature of the BRCA1 gene fragment in a biphasic fashion. DSC thermogram of DNA adducts was shifted to a lower transition temperature at lower cisplatin concentration. However, at higher drug concentration, the thermogram peaked at a slightly higher transition temperature with predominantly increased heat specific capacity. Reduction in cellular DNA repair of cisplatin-damaged plasmid DNA, using host cell reactivation assay, was a consequence of an increase in platination levels on the reporter gene. The GAL4-fused BRCA1 slightly enhanced the transcription of the reporter gene in the absence of GAL4 binding site. The transcriptional transactivation activity of cisplatin-modified BRCA1, when tested in "one-hybrid GAL4 transcriptional assay," was inversely proportional to cisplatin doses. Furthermore, the transcriptional transactivation activity was dramatically diminished in the presence of a second expression vector containing multiple cisplatin-damaged sites. The data provide the first evidence for direct interaction of cisplatin with BRCA1 and raise the possibility of BRCA1 as a therapeutic target for platinum drug-based chemotherapy.