RESUMO
Fifty two steroids and 9 Vitamin D analogs were docked into ten crystallographically-defined DNA dinucleotide sites and two human topoisomerase II ATP binding sites using two computational programs, Autodock and Surflex. It is shown that both steroids and Vitamin D analogs exhibit a propensity for non-covalent intercalative binding to DNA. A higher predicted binding affinity was found, however, for steroids and the ATP binding site of topoisomerase; in fact these drugs exhibited among the highest topo II binding observed in over 1370 docked drugs. These findings along with genotoxicity data from 26 additional steroids not subjected to docking analysis, support a mechanism wherein the long known, but poorly understood, clastogenicity of steroids may be attributable to inhibition of topoisomerase. A "proof of principle" experiment with dexamethasone demonstrated this to be the likely mechanism of clastogenicity of, at least, this steroid. The generality of this proposed mechanism of genotoxicity across the steroids and vitamin-D analogs is discussed.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , DNA/metabolismo , Esteroides/toxicidade , Vitamina D/toxicidade , Sítios de Ligação , Cristalografia/métodos , Humanos , Simulação de Acoplamento Molecular , Testes de Mutagenicidade , Software , Esteroides/metabolismo , Vitamina D/análogos & derivados , Vitamina D/metabolismoRESUMO
OBJECTIVE: To identify biomarkers that distinguish between active antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and remission in a manner superior or complementary to established markers of systemic inflammation. METHODS: Markers of vascular injury and angiogenesis were measured before and after treatment in a large clinical trial in AAV: 163 subjects enrolled in the Rituximab in ANCA-Associated Vasculitis trial were screened for the present study. Serum levels of E-selectin, intercellular adhesion molecule 3 matrix metalloproteinase protein 1 (MMP-1), MMP-3, MMP-9, P-selectin, thrombomodulin, and vascular endothelial growth factor were measured at study screening (time of active disease) and at month 6. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels had been measured at the time of the clinical visit. The primary outcome measure was the difference in marker level between screening and month 6 among patients whose disease was in remission (Birmingham Vasculitis Activity Score for Wegener's granulomatosis [BVAS/WG] score of 0) at month 6. RESULTS: All patients had severe active vasculitis at screening (mean ± SD BVAS/WG score 8.6 ± 3.2). Among the 123 patients whose disease was clinically in remission at month 6, levels of all markers except E-selectin showed significant declines. MMP-3 levels were also higher among the 23 patients with active disease at month 6 than among the 123 patients whose disease was in remission. MMP-3 levels correlated weakly with ESR and CRP levels. CONCLUSION: Many markers of vascular injury and angiogenesis are elevated in severe active AAV and decline with treatment, but MMP-3 appears to distinguish active AAV from remission better than the other markers studied. Further study of MMP-3 is warranted to determine its clinical utility in combination with conventional markers of inflammation and ANCA titers.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/sangue , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/fisiopatologia , Selectina E/sangue , Metaloproteinase 3 da Matriz/sangue , Neovascularização Patológica/sangue , Lesões do Sistema Vascular/sangue , Adulto , Idoso , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Anticorpos Monoclonais Murinos/uso terapêutico , Antirreumáticos/uso terapêutico , Biomarcadores/sangue , Sedimentação Sanguínea , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Diagnóstico Diferencial , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/fisiopatologia , Indução de Remissão , Rituximab , Lesões do Sistema Vascular/fisiopatologiaRESUMO
Treatment with drugs from multiple classes induces vascular injury with medial necrosis, hemorrhage, endothelial damage, and inflammation. Previous research has suggested early events might be occurring well in advance of the full lesions that appear forty-eight to seventy-two hours after dosing with SCH 351591, a PDE IV inhibitor. This study was performed to study early events in detail. Rats were dosed with 20 mg/kg of drug by gavage and sacrificed at times between fifteen and 240 minutes after dosing. Tissues were collected for histopathological analysis and gene expression studies. Serum was collected for biomarker analysis. The data from biomarker analysis showed a three-part response with an early phase that was maximal at fifteen to thirty minutes, a second phase from forty-five to 180 minutes, and the third phase that was starting to rise at four hours. The first phase included increases in lymphocytes, serum histamine, and serum nitrite. The second phase shows continued elevation of serum nitrite. The third phase was marked by an increase in serum GRO/CINC-1. At fifteen minutes, histopathology showed activation of mast cells, but not degranulation. Increases in endothelial activation and perivascular inflammatory cells were first apparent at thirty minutes and increased through 240 minutes.
Assuntos
Óxidos N-Cíclicos/toxicidade , Inflamação/induzido quimicamente , Inibidores da Fosfodiesterase 4/toxicidade , Quinolinas/toxicidade , Lesões do Sistema Vascular/induzido quimicamente , Animais , Biomarcadores/análise , Biomarcadores/sangue , Expressão Gênica/efeitos dos fármacos , Inflamação/patologia , Mesentério/irrigação sanguínea , Mesentério/patologia , Ratos , Ratos Sprague-Dawley , Tempo , Lesões do Sistema Vascular/sangue , Lesões do Sistema Vascular/patologiaRESUMO
BACKGROUND: The effects of histamine H1 antagonist chlorcyclizine on rat palate development were characterized following in utero exposure. METHODS: To identify the optimum dose for inducing cleft palate, pregnant rats were administered 30, 60, or 90 mg/kg chlorcyclizine on Gestation Days 11 to 14. Fetal palate gene expression was also assessed after 90 mg/kg chlorcyclizine at 8, 15 and 30 hours post-dose on Gestation Day 14 using microarray and qRT-PCR. RESULTS: Rats in the 60- and 90-mg/kg groups exhibited adverse clinical signs and body weight loss. Rats in the 90-mg/kg group also demonstrated increases in late resorptions and decreases in fetal weight. Effects in the low-dose group were limited to decreases in body weight gain. Fetal assessment on Gestation Day 21 revealed that findings were limited to the 60- and 90-mg/kg groups, and included cleft palate (80% of litters for both groups), high arched palate, small nose, micrognathia, high domed head, digits shortened/absent and small limb. The fetal incidence of cleft palate was higher at 90 mg/kg, thus this dose was selected to assess palate gene expression. The altered genes associated with chlorcyclizine-induced cleft palate included Wnt5a, Bmp2, Bmp4, Fgf10, Fgfr2, Msx1, and Insig1 but the magnitude of the change was relatively small (1.5- to 2-fold). CONCLUSIONS: Expression of several genes involved in palate, limb and digit development was altered in the fetal palate following in utero exposure to chlorcyclizine. The subtle perturbation and interplay of these genes may have profound effects on the dynamics of fetal palate development.
Assuntos
Fissura Palatina/induzido quimicamente , Embrião de Mamíferos/efeitos dos fármacos , Antagonistas dos Receptores Histamínicos H1/toxicidade , Palato/efeitos dos fármacos , Piperazinas/toxicidade , Animais , Biomarcadores/metabolismo , Fissura Palatina/genética , Fissura Palatina/patologia , Embrião de Mamíferos/anormalidades , Feminino , Reabsorção do Feto/induzido quimicamente , Peso Fetal/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Exposição Materna , Análise em Microsséries , Palato/anormalidades , Palato/metabolismo , Gravidez , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos , Aumento de PesoRESUMO
Drug resistant tumor "side-populations," enriched in cancer stem cells and identified by reduced accumulation of Hoechst 33342 under ABCG2-mediated efflux, may compromise therapeutic outcome. Side-population cells have predicted resistance to minor groove ligands, including the DNA topoisomerase I poison topotecan. We have used a stable Hoechst 33342-resistant murine L cell system (HoeR415) to study resistance patterns, removing the need for SP isolation before microarray analysis of gene expression and the tracking of cell cycle dynamics and cytotoxicity. The majority of HoeR415 cells displayed a side-population phenotype comparable with that of the side-population resident in the ABCG2 over-expressing A549 lung cancer cell line. Photo-crosslinking showed direct protection against minor groove ligand residence on DNA, driven by ABCG2-mediated efflux and not arising from any binding competition with endogenous polyamines. The covalent minor-groove binding properties of the drug FCE24517 (tallimustine) prevented resistance suggesting a mechanism for overcoming SP-related drug resistance. Hoechst 33342-resistant murine cells showed lower but significant crossresistance to topotecan, again attributable to enhanced ABCG2 expression, enabling cells to evade S-phase arrest. Hoechst 33342/TPT-resistant cells showed limited ancillary gene expression changes that could modify cellular capacity to cope with chronic stress including over-expression of Aldh1a1 and Mgst1, but under-expression of Plk2 and Nnt. There was no evidence to link the putative stem cell marker ALDH1A1 with any augmentation of the TPT resistance phenotype. The study has implications for the patterns of drug resistance arising during tumor repopulation and the basal resistance to minor groove-binding drugs of tumor side-populations.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Benzimidazóis/farmacologia , Citometria de Fluxo/métodos , Topotecan/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Linhagem Celular Tumoral , Separação Celular , Reagentes de Ligações Cruzadas/farmacologia , Distamicinas/farmacologia , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/farmacologia , Humanos , Ligantes , Camundongos , Compostos de Mostarda Nitrogenada/farmacologia , Poliaminas/químicaRESUMO
The 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase inhibitor, lovastatin (lova), has been reported to both sensitize to, and protect against, the toxic effects of the antitumor anthracycline doxorubicin (dox) in cellular and in vivo systems. The mechanism by which these effects occur has not yet been determined. In the present study, lova is shown to enhance the genotoxicity of dox in the V79 cell in vitro micronucleus assay and to do so, most likely, via noncovalent interaction with DNA adjacent to sites of dox binding. These studies confirm and extend the experimental evidence strongly suggesting the importance of noncovalent drug/DNA interactions in cellular responses to genotoxic stimuli.
Assuntos
Antibióticos Antineoplásicos/toxicidade , Doxorrubicina/toxicidade , Lovastatina/toxicidade , Mutagênicos/toxicidade , Animais , Linhagem Celular , Cricetinae , Cricetulus , DNA/efeitos dos fármacos , DNA/metabolismo , Sinergismo Farmacológico , Inibidores de Hidroximetilglutaril-CoA Redutases/toxicidade , Testes para MicronúcleosRESUMO
Drug-induced renal injury is a common finding in the early preclinical phase of drug development. But the specific genes responding to renal injury remain poorly defined. Identification of drug-induced gene changes is critical to provide insights into molecular mechanisms and detection of renal damage. To identify genes associated with the development of drug-induced nephrotoxicity, a literature survey was conducted and a panel of 48 genes was selected based on gene expression changes in multiple published studies. Male Sprague-Dawley rats were dosed daily for 1, 3 or 5 days to the known nephrotoxicants gentamicin, bacitracin, vancomycin and cisplatin, or the known hepatotoxicants ketoconazole, 1-naphthyl isothiocyanate and 4,4-diaminodiphenylmethane. Histopathological evaluation and clinical chemistry revealed renal proximal tubular necrosis in rats treated with the nephrotoxicants, but not from those treated with the hepatotoxicants. RNA was extracted from the kidney, and RT-PCR was performed to evaluate expression profiles of the selected genes. Among the genes examined, 24 genes are confirmed to be highly induced or repressed in rats treated with nephrotoxicants; further investigation identified that 5 of the 24 genes were also altered by hepatotoxicants. These data led to the identification of a set of genomic biomarker candidates whose expression in kidney is selectively regulated only by nephrotoxicants. Among those genes displaying the highest expression changes specifically in nephrotoxicant-treated rats were kidney injury molecule 1 (Kim1), lipocalin 2 (Lcn2), and osteopontin (Spp1). The establishment of such a genomic marker set offers a new tool in our ongoing quest to monitor nephrotoxicity.
Assuntos
Antibacterianos/toxicidade , Antineoplásicos/toxicidade , Marcadores Genéticos , Nefropatias/induzido quimicamente , Nefropatias/genética , Rim/efeitos dos fármacos , Animais , Bacitracina/toxicidade , Cisplatino/toxicidade , Expressão Gênica/genética , Perfilação da Expressão Gênica , Gentamicinas/toxicidade , Rim/metabolismo , Rim/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Toxicogenética , Vancomicina/toxicidadeRESUMO
Histopathological and immunohistochemical studies were conducted to characterize vascular injuries in rats treated with phosphodiesterase (PDE) IV inhibitors SCH 351591 or SCH 534385. Sprague-Dawley rats were administered PDE IV inhibitors by gavage at a range of doses and times. The two PDE IV inhibitors induced comparable levels of vascular injury, primarily in the mesentery and to a lesser extent in the pancreas, kidney, liver, small intestine, and stomach. Mesenteric vascular changes occurred as early as one hour, progressively developed over twenty-four to forty-eight hours, peaked at seventy-two hours, and gradually subsided from seven to nine days. The typical morphology of the vascular toxicity consisted of hemorrhage and necrosis of arterioles and arteries, microvascular injury, fibrin deposition, and perivascular inflammation of a variety of blood vessels. The incidence and severity of mesenteric vascular injury increased in a time- and dose-dependent manner in SCH 351591- or SCH 534385-treated rats. Mesenteric vascular injury was frequently associated with activation of mast cells (MC), endothelial cells (EC), and macrophages (MØ). Immunohistochemical studies showed increases in CD63 immunoreactivity of mesenteric MC and in nitrotyrosine immunoreactivity of mesenteric EC and MØ. The present study also provides a morphological and cellular basis for evaluating candidate biomarkers of drug-induced vascular injury.
Assuntos
Vasos Sanguíneos/efeitos dos fármacos , Óxidos N-Cíclicos/toxicidade , Inibidores da Fosfodiesterase 4 , Inibidores de Fosfodiesterase/toxicidade , Quinolinas/toxicidade , Doenças Vasculares/induzido quimicamente , Doenças Vasculares/patologia , Animais , Imuno-Histoquímica , Intestino Delgado/irrigação sanguínea , Intestino Delgado/patologia , Rim/irrigação sanguínea , Rim/patologia , Artérias Mesentéricas/patologia , Pâncreas/irrigação sanguínea , Pâncreas/patologia , Ratos , Ratos Sprague-Dawley , Estatísticas não Paramétricas , Estômago/irrigação sanguínea , Estômago/patologiaRESUMO
Drug-associated vascular injury can be caused by phosphodiesterase (PDE) IV inhibitors and drugs from several other classes. The pathogenesis is poorly understood, but it appears to include vascular and innate immunological components. This research was undertaken to identify changes in peripheral blood associated with vascular injury caused by PDE IV inhibitors. We evaluated twelve proteins, serum nitrite, and leukocyte populations in peripheral blood of rats treated with experimental PDE IV inhibitors. We found that these compounds produced histological microvascular injury in a dose- and time-dependent manner. Measurement of these serum proteins showed changes in eight of the twelve examined. Changes were seen in the levels of: tissue inhibitor of metalloproteinase-1, alpha1-acid glycoprotein, GRO/CINC-1, vascular endothelial growth factor, C-reactive protein, haptoglobin, thrombomodulin, and interleukin-6. No changes were seen in levels of tumor necrosis factor-alpha, hepatocyte growth factor, nerve growth factor, and granulocyte-monocyte colony stimulating factor. Serum levels of nitrite were also increased. Circulating granulocyte numbers were increased, and lymphocyte numbers were decreased. The changes in these parameters showed both a dose- and time-dependent association with histopathologic changes. These biomarkers could provide an additional tool for the nonclinical and clinical evaluation of investigational compounds.
Assuntos
Biomarcadores/sangue , Vasos Sanguíneos/efeitos dos fármacos , Óxidos N-Cíclicos/toxicidade , Inibidores da Fosfodiesterase 4 , Inibidores de Fosfodiesterase/toxicidade , Quinolinas/toxicidade , Doenças Vasculares/induzido quimicamente , Animais , Testes de Química Clínica , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Contagem de Leucócitos , Artérias Mesentéricas/patologia , Nitratos/sangue , Nitritos/sangue , Ratos , Ratos Sprague-Dawley , Doenças Vasculares/sangue , Doenças Vasculares/patologiaRESUMO
Non-genotoxic carcinogenicity of chemicals is currently routinely evaluated in 2-year rodent bioassays. Therefore, the development of early biomarkers for non-genotoxic carcinogenesis would result in substantial savings in time and expense. The current study investigates whether early changes in gene expression may be developed as markers for cancer. Animals were treated for 1 or 5 days with either non-genotoxic carcinogens (NGTCs) or non-carcinogens and gene expression was analyzed by quantitative PCR (qPCR). We tested two gene signatures previously reported to detect non-genotoxic carcinogens. Using one gene signature it was confirmed that 3/3 non-genotoxic carcinogens and 2/2 non-carcinogens are correctly identified with data from 1 or 5 days of dosing. In contrast an alternative signature correctly identified 0/3 and 2/3 non-genotoxic carcinogens at 1 and 5 days of treatment, respectively and 2/2 non-carcinogens at both time-points. Additionally, we evaluated a novel panel of putative biomarker genes, from the literature, many of which have roles in cell growth and division, including myc, cdc2 and mcm6. These genes were significantly induced by non-genotoxic carcinogens and not by non-carcinogens. Using the average fold-induction across this panel, 2/3 non-genotoxic carcinogens were detected at both 1 and 5 days. These data support the idea that acute changes in gene expression may provide biomarkers for non-genotoxic carcinogenesis but also highlight interesting differences in the sensitivities of distinct gene signatures.
Assuntos
Bioensaio/métodos , Biomarcadores/análise , Carcinógenos/toxicidade , Regulação Neoplásica da Expressão Gênica/genética , Valor Preditivo dos Testes , Animais , Testes de Carcinogenicidade , Relação Dose-Resposta a Droga , Masculino , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Medição de Risco , Fatores de TempoRESUMO
A large number of cationic amphiphilic drugs (CADs) are known to cause phospholipidosis (PLD) in vivo. In the present study, we have built upon our previous findings to further qualify the use of a fluorescently labeled phospholipid-based cell-culture assay to detect PLD-inducing drugs. In this paper, we demonstrate that 12 PLD-negative compounds and 11 drugs known to cause PLD in vivo are all correctly identified by using this assay. Interestingly, we found that in cells treated with certain CADs, the fluorescent phospholipid was sequestered in a very specific punctate pattern, which overlapped strongly with the staining pattern seen with a lysosomal marker protein. Our data also show that false positives can be generated with the fluorescence assay when compounds are used at concentrations that cause a >30% decrease in cell number in this assay. Confocal microscopy demonstrated that the staining pattern of fluorescent phospholipids in these cases may be differentiated from those of true positives by the fact that diffuse, rather than punctuate, fluorescence is observed. These studies confirm and expand our previous results showing that the fluorescent phospholipid assay is a highly sensitive, specific tool for detecting PLD-inducing drugs, if care is taken to rule out cytotoxicity-related artifact.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Corantes Fluorescentes/metabolismo , Lipidoses/induzido quimicamente , Técnicas de Sonda Molecular , Fosfolipídeos/metabolismo , Artefatos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Lipidoses/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Microscopia Confocal , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Assessment of cytochrome P450 (CYP) induction at the mRNA level in preclinical rodent studies has gained interest in recent years, but there are still concerns regarding correlations between the mRNA and the enzyme activity levels, especially in mice. The purpose of the present study was to systematically evaluate patterns of temporal changes of CYPs 1a1, 1a2, 2b10, 3a11, and 4a10 at mRNA, protein, and activity levels in order to determine to what extent mRNA levels could be used either qualitatively or quantitatively for the assessment of CYP enzyme induction. In this study, livers from male CD-1 mice treated daily with beta-naphthoflavone, phenobarbital, dexamethasone, clofibrate, and control vehicles were collected for RNA and microsomal analysis after 0.5, 1, 2, 4, and 8 days of daily dose. The results revealed a good correlation among mRNA, protein, and enzyme activity levels, with the best correlation at the time points between Days 2 and 8, suggesting that the appropriate time to monitor CYP mRNA may be beyond Day 2 of chemical treatments. Based on these results, we concluded that the mRNA approach is a useful tool to monitor CYP induction in mice, particularly when treatment duration is beyond 2 days.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Avaliação Pré-Clínica de Medicamentos/métodos , Fígado/efeitos dos fármacos , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Animais , Clofibrato/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Dexametasona/farmacologia , Indução Enzimática/efeitos dos fármacos , Estudos de Viabilidade , Isoenzimas , Fígado/enzimologia , Masculino , Camundongos , Fenobarbital/farmacologia , Reprodutibilidade dos Testes , Fatores de Tempo , beta-Naftoflavona/farmacologiaRESUMO
Phospholipidosis (PLD) is characterized by the excessive intracellular accumulation of phospholipids. It is well established that a large number of cationic amphiphilic drugs have the potential to induce PLD. In the present study, we describe two facile in vitro methods to determine the PLD-inducing potential of a molecule. The first approach is based on a recent study by (Sawada et al., 2005, Toxicol. Sci. 83, 282-292) in which 17 genes were identified as potential biomarkers of PLD in HepG2 cells. To confirm the utility of this gene panel, we treated HepG2 cells with PLD-positive and -negative compounds and then analyzed gene expression using real-time PCR. Our initial analysis, which used a single dose of each drug, correctly identified five of eight positive compounds and four of four negative compounds. We then increased the doses of the three false negatives (amiodarone, tamoxifen, and loratadine) and found that the changes in gene expression became large enough to correctly identify them as PLD-inducing drugs. Our results suggest that a range of concentrations should be used to increase the accuracy of prediction in this assay. Our second approach utilized a fluorescently labeled phospholipid (LipidTox) which was added to the media of growing HepG2 cells along with compounds positive and negative for PLD. Phospholipid accumulation was determined using confocal microscopy and, more quantitatively, using a 96-well plate assay and a fluorescent plate reader. Using an expanded set of compounds, we show that this assay correctly identified 100% of PLD-positive and -negative compounds. Dose-dependent increases in intracellular fluorescent phospholipid accumulation were observed. We found that this assay was less time consuming, more sensitive, and higher throughput than gene expression analysis. To our knowledge, this study represents the first validation of the use of LipidTox in identifying drugs that can induce PLD.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Corantes Fluorescentes/farmacologia , Expressão Gênica/efeitos dos fármacos , Lipidoses/induzido quimicamente , Fosfolipídeos/metabolismo , Testes de Toxicidade/métodos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Fluorescência , Perfilação da Expressão Gênica , Marcadores Genéticos , Humanos , Lipidoses/metabolismo , Microscopia Confocal , Preparações Farmacêuticas/classificação , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: To report a pediatric case of pegfilgrastim-induced hyperleukocytosis. CASE SUMMARY: A 3-year-old boy with medulloblastoma therapy presented with white blood cell (WBC) count 0.1 x 10(3)/microL and absolute neutrophil count (ANC) 0.014 x 10(3)/microL on day 27 following a course of induction chemotherapy. The patient received pegfilgrastim 200 microg/kg the following day. On his return 6 days later for the next planned course of chemotherapy, hyperleukocytosis was determined, with WBC 149 x 10(3)/microL and ANC 110 x 10(3)/microL ("neutrophil overshoot"). No sources of the elevated WBC count other than administration of pegfilgrastim (eg, steroids, antiepileptics, infection) were present. Chemotherapy was delayed until the WBC count had fallen to 35.2 x 10(3)/microL (ANC 28.9 x 10(3)/microL). No sequelae from this adverse effect occurred. DISCUSSION: Pegfilgrastim has unique saturable neutrophil receptor-mediated clearance, the ability for self-regulation. Due to this clearance mechanism, hyperleukocytosis associated with pegfilgrastim use is uncommon in adults and has not been previously reported in pediatrics. The pegfilgrastim dose in children is under investigation; however, 100-110 microg/kg has been effective and safe in this population. Use of the Naranjo probability scale suggested that pegfilgrastim was the probable cause of hyperleukocytosis in our patient. CONCLUSIONS: Pegfilgrastim 200 microg/kg, in excess of the 100 microg/kg dose used in limited pediatric clinical trials, appeared to exceed saturable neutrophil receptor-mediated clearance. The inability of this mechanism to self-regulate neutrophil counts in the normal range led to neutrophil overshoot. Routine pediatric use of the pegylated dosage form of G-CSF should await further published clinical trials to validate a safe and effective dose.
Assuntos
Fator Estimulador de Colônias de Granulócitos/efeitos adversos , Leucocitose/induzido quimicamente , Neutropenia/tratamento farmacológico , Antineoplásicos/uso terapêutico , Neoplasias Cerebelares/tratamento farmacológico , Pré-Escolar , Filgrastim , Humanos , Leucócitos/efeitos dos fármacos , Masculino , Meduloblastoma/tratamento farmacológico , Polietilenoglicóis , Proteínas RecombinantesRESUMO
Non-covalent genotoxic interaction between DNA and classical planar fused-ring intercalating agents, has been well understood for some time especially in the context of frameshift mutagenesis in bacterial systems. Recent evidence, however, suggests that a rather wide structural range of small non-fused ring molecules may also be capable of partial or complete DNA intercalation in mammalian cells. The present paper will review recent studies on the identification and characterization of such atypically-structured molecules utilizing both cell-based and three-dimensional computational analyses focusing principally on prediction and detection of these atypical molecules. Mechanistic aspects of genotoxicity of such non-covalent binding molecules, with emphasis on marketed pharmaceuticals, will also be discussed. A review and presentation of new data using catalytic DNA topo II inhibitors, confirms the notion that topoisomerase II poisoning arising via intercalation is the major mechanism of genotoxicity of these drugs.
Assuntos
DNA/química , DNA/efeitos dos fármacos , Substâncias Intercalantes/química , Substâncias Intercalantes/farmacologia , Animais , Linhagem Celular , Simulação por Computador , Cricetinae , Cricetulus , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ligantes , Modelos Moleculares , Estrutura Molecular , Conformação de Ácido Nucleico , Eletricidade Estática , Inibidores da Topoisomerase IIRESUMO
PURPOSE: The successful resumption of high-dose methotrexate in a 13-year-old boy with recurrent anaplastic large-cell lymphoma (ALCL) who suffered renal dysfunction after a 24-hour infusion of high-dose methotrexate and required treatment with carboxypeptidase G(2) (CPDG(2) ) is described. SUMMARY: A 13-year-old boy who had been diagnosed in 2001 with stage I ALCL was admitted to the hospital in February 2005 after he developed a smaller left axillary mass in the area of his original mass. Recurrent ALCL was diagnosed, and treatments were initiated based on branch K3 of the protocol published in the non-Hodgkin's lymphoma-Berlin-Frankfurt-Münster (NHL-BFM) trial 90. In the NHL-BFM 90 protocol, all AA and BB courses include high-dose methotrexate therapy, which consists of aggressive alkalinized hydration, methotrexate 5 g/m(2) given as an i.v. infusion over 24 hours, and leucovorin rescue. During course BB2, the boy's serum methotrexate values exceeded NHL-BFM goals at 36, 42, and 48 hours. Because the patient's elimination of methotrexate remained slow and his serum creatinine level remained above normal limits, CPDG(2) was obtained for the treatment of methotrexate toxicity. The patient tolerated the CPDG(2) without adverse effects, and the patient's serum methotrexate concentration decreased from 14.47 to 0.66 microM. The patient went on to complete six courses based on the protocol. High-dose methotrexate was resumed at 50% then 100% of the original dose. He is currently in remission on maintenance therapy. CONCLUSION: A 13-year-old boy with recurrent ALCL had methotrexate-induced nephrotoxicity following high-dose methotrexate. The resultant delayed methotrexate clearance required the standard therapies as well as use of investigational CPDG(2). High-dose methotrexate was successfully resumed.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/efeitos adversos , Metotrexato/administração & dosagem , Metotrexato/efeitos adversos , Insuficiência Renal/tratamento farmacológico , gama-Glutamil Hidrolase/uso terapêutico , Adolescente , Antimetabólitos Antineoplásicos/uso terapêutico , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Metotrexato/uso terapêutico , Recidiva Local de Neoplasia , Insuficiência Renal/induzido quimicamente , RetratamentoRESUMO
A range of genomics technologies are increasingly becoming integrated with existing scientific disciplines to broaden and strengthen existing capabilities and open new avenues of research in drug discovery and development. Examples of these new research fields are proteomics, pharmacogenomics, metabolomics and toxicogenomics. Here we review the application of toxicogenomics to improve the evaluation of drug safety, mechanism of action and toxicity in the drug discovery and development process.
Assuntos
Bases de Dados Genéticas , Toxicogenética , Desenho de Fármacos , Tratamento Farmacológico , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Perfilação da Expressão Gênica , HumanosRESUMO
We have previously noted that the Physicians' Desk Reference (PDR) contains over 80 instances in which a drug elicited a positive genotoxic response in one or more in vitro assays, despite having no obvious structural features predictive of covalent drug/DNA interactive potential or known mechanistic basis. Furthermore, in most cases, these drugs were "missed" by computational genotoxicity-predicting models such as DEREK, MCASE and TOPKAT. We have previously reported the application of a V79 cell-based model and a 3D DNA docking model for predicting non-covalent chemical/DNA interactions. Those studies suggested that molecules that are very widely structurally diverse may be capable of intercalating into DNA. To determine whether such non-covalent drug/DNA interactions might be involved in unexpected drug genotoxicity, we evaluated, using both models where possible, 56 marketed pharmaceuticals, 40 of which were reported as being clastogenic in in vitro cytogenetics assays (chromosome aberrations/mouse lymphoma assay). As seen before, the two approaches showed good concordance (62%) and 26 of the 40 (65%) drugs exhibiting in vitro clastogenicity were predicted as intercalators by one or both methods. This finding provides support for the hypothesis that non-covalent DNA interaction may be a common mechanism of clastogenicity for many drugs having no obvious structural alerts for covalent DNA interaction.
Assuntos
Substâncias Intercalantes/toxicidade , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Preparações Farmacêuticas/administração & dosagem , Animais , Linhagem Celular , Simulação por Computador , DNA/química , DNA/efeitos dos fármacos , DNA/genética , Ligação de Hidrogênio , Substâncias Intercalantes/administração & dosagem , Substâncias Intercalantes/química , Testes para Micronúcleos , Modelos Químicos , Estrutura Molecular , Preparações Farmacêuticas/química , Vigilância de Produtos Comercializados , Software , Eletricidade EstáticaRESUMO
In this report, results are presented from an international study of the in vitro micronucleus assay using Chinese hamster ovary cells. This study was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Test chemicals included mannitol, bleomycin, cytosine arabinoside, urethane and diethylstilboestrol. Mitomycin C was used as a positive control. Each chemical was evaluated in at least two laboratories following a variety of different protocols (short and long exposures, varying recovery times, with and without cytochalasin B) in order to help determine a standard protocol for routine testing in Chinese hamster ovary cells. Mannitol and urethane were negative, while bleomycin, cytosine arabinoside and diethylstilboestrol induced a dose dependent increase in micronucleated cells. In the presence of cytochalasin B, increases in micronuclei were observed in binucleated as well as mononucleated cells in cultures treated with bleomycin, cytosine arabinoside or diethylstilboestrol. Importantly, all three of these chemicals were detected in each of the different treatment/recovery regimens. No differences were seen in the sensitivity or accuracy of the responses in the presence of absence of cytochalasin B. Overall, these results demonstrate the suitability of Chinese hamster ovary cells for the in vitro micronucleus assay.
Assuntos
Testes para Micronúcleos/métodos , Mutagênicos/toxicidade , Aneugênicos/toxicidade , Animais , Bleomicina/toxicidade , Células CHO , Cricetinae , Citarabina/toxicidade , Citocalasina B , Dietilestilbestrol/toxicidade , Técnicas In Vitro , Cooperação Internacional , Manitol/toxicidade , Mitomicina , Uretana/toxicidadeRESUMO
Decades of mutagenesis and clastogenesis studies have yielded enough structure-activity-relationship (SAR) information to make feasible the construction of computational models for prediction of endpoints based on molecular structure and reactivity. Although there is cause for optimism that these approaches might someday reduce or eliminate the need for actual genotoxicity testing, we are in fact a long way from this. We provide an overview of the state of the art of such approaches, dissecting out how these models are suboptimal. It is clear that current programs still have limited predictive capabilities. We propose that one of the major contributing factors for the inherent lack of sensitivity (typically 50-60%) is inadequate coverage of non-covalent DNA interactions. Suboptimal specificity can be partly attributed to chemical space considerations with associated non-causal activity correlations.