RESUMO
Perhexiline is a prophylactic antianginal agent developed in the 1970s. Although, therapeutically, it remained a success, the concerns of its severe adverse effects including hepatotoxicity caused the restricted use of the drug, and eventually its withdrawal from the market in multiple countries. In the clinical setting, cytochrome P450 (CYP) 2D6 is considered as a possible risk factor for the adverse effects of perhexiline. However, the role of CYP-mediated metabolism in the toxicity of perhexiline, particularly in the intact cells, remains unclear. Using our previously established HepG2 cell lines that individually express 14 CYPs (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7) and human liver microsomes, we identified that CYP2D6 plays a major role in the hydroxylation of perhexiline. We also determined that CYP1A2, 2C19, and 3A4 contribute to the metabolism of perhexiline. The toxic effect of perhexiline was reduced significantly in CYP2D6-overexpressing HepG2 cells, in comparison to the control cells. In contrast, overexpression of CYP1A2, 2C19, and 3A4 did not show a significant protective effect against the toxicity of perhexiline. Pre-incubation with quinidine, a well-recognized CYP2D6 inhibitor, significantly attenuated the protective effect in CYP2D6-overexpressing HepG2 cells. Furthermore, perhexiline-induced mitochondrial damage, apoptosis, and ER stress were also attenuated in CYP2D6-overexpressing HepG2 cells. These findings suggest that CYP2D6-mediated metabolism protects the cells from perhexiline-induced cytotoxicity and support the clinical observation that CYP2D6 poor metabolizers may have higher risk for perhexiline-induced hepatotoxicity.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Citocromo P-450 CYP1A2 , Humanos , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Perexilina/toxicidade , Perexilina/metabolismo , Inibidores do Citocromo P-450 CYP2D6 , Quinidina/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismoRESUMO
Perhexiline is an anti-anginal drug developed in the late 1960s. Despite its therapeutic success, it caused severe hepatoxicity in selective patients, which resulted in its withdrawal from the market. In the current study we explored the molecular mechanisms underlying the cytotoxicity of perhexiline. In primary human hepatocytes, HepaRG cells, and HepG2 cells, perhexiline induced cell death in a concentration- and time-dependent manner. Perhexiline treatment also caused a significant increase in caspase 3/7 activity at 2 h and 4 h. Pretreatment with specific caspase inhibitors suggested that both intrinsic and extrinsic apoptotic pathways contributed to perhexiline-induced cytotoxicity, which was confirmed by increased expression of TNF-α, cleavage of caspase 3 and 9 upon perhexiline treatment. Moreover, perhexiline caused mitochondrial dysfunction, demonstrated by the classic glucose-galactose assay at 4 h and 24 h. Results from JC-1 staining suggested perhexiline caused loss of mitochondrial potential. Blocking mitochondrial permeability transition pore using inhibitor bongkrekic acid attenuated the cytotoxicity of perhexiline. Western blotting analysis also showed decreased expression level of pro-survival proteins Bcl-2 and Mcl-1, and increased expression of pro-apoptotic protein Bad. Direct measurement of the activity of individual components of the mitochondrial respiratory complex demonstrated that perhexiline strongly inhibited Complex IV and Complex V and moderately inhibited Complex II and Complex II + III. Overall, our data demonstrated that both mitochondrial dysfunction and apoptosis underlies perhexiline-induced hepatotoxicity.
Assuntos
Fármacos Cardiovasculares/toxicidade , Mitocôndrias/efeitos dos fármacos , Perexilina/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
1. Perhexiline, a chiral anti-anginal agent, may be useful to develop new cardiovascular therapies, despite its potential hepatotoxicity. 2. This study compared Dark Agouti (DA) and Sprague-Dawley (SD) rats, as models of perhexiline's metabolism and hepatotoxicity in humans. Rats (n = 4/group) received vehicle or 200 mg/kg/d of racemic perhexiline maleate for 8 weeks. Plasma and liver samples were collected to determine concentrations of perhexiline and its metabolites, hepatic function and histology. 3. Median (range) plasma and liver perhexiline concentrations in SD rats were 0.09 (0.04-0.13) mg/L and 5.42 (0.92-8.22) ng/mg, respectively. In comparison, DA rats showed higher (p < 0.05) plasma 0.50 (0.16-1.13) mg/L and liver 24.5 (9.40-54.7) ng/mg perhexiline concentrations, respectively, 2.5- and 3.7-fold higher cis-OH-perhexiline concentrations, respectively (p < 0.05), and lower plasma metabolic ratio (0.89 versus 1.55, p < 0.05). In both strains, the (+):(-) enantiomer ratio was 2:1. Perhexiline increased plasma LDH concentrations in DA rats (p < 0.05), but had no effect on plasma biochemistry in SD rats. Liver histology revealed lower glycogen content in perhexiline-treated SD rats (p < 0.05), but no effects on lipid content in either strain. 4. DA rats appeared more similar to humans with respect to plasma perhexiline concentrations, metabolic ratio, enantioselective disposition and biochemical changes suggestive of perhexiline-induced toxicity.
Assuntos
Oxirredutases do Álcool/metabolismo , Fármacos Cardiovasculares/metabolismo , Fígado/efeitos dos fármacos , Modelos Animais , Perexilina/metabolismo , Ratos Sprague-Dawley , Animais , Fármacos Cardiovasculares/toxicidade , Família 2 do Citocromo P450 , Feminino , Fígado/metabolismo , Fígado/ultraestrutura , Perexilina/toxicidadeRESUMO
Proximal tubules of the kidneys are one of the most common targets of nephrotoxic drugs and chemicals. Screens to predict nephrotoxic potential of compounds with insights to mechanisms of toxicity facilitate lead optimization, guide structure-activity relationships, minimize risks of clinical nephrotoxicity and therefore are valuable in the process of drug discovery. We developed and characterized an in vitro assay multiplexed to measure several endpoints of cytotoxicity using HK-2 cells. Assays for lactate dehydrogenase, cellular caspase 3/7 activation, resazurin dye reduction and Hoechst 33342 DNA staining were multiplexed to maximize the ability to detect cell injury. Assays were performed after 5- or 24-h incubations to further enhance the sensitivity of detection of toxicity. Individual assays were optimized for cell density, assay linearity and assay performance under multiplexed conditions. Inducers of apoptosis (staurosporine) and necrosis (perhexiline) were used to validate the mechanistic aspects of cell death. Nephrotoxic compounds (5-fluorouracil, gentamicin, cisplatin, acetaminophen, para-aminophenol, potassium dichromate, ibuprofen, doxorubicin, cyclosporine, citrinin, puromycin) were used to determine the potential of this method to detect proximal tubule toxicity of compounds. Overall, this cost-effective multiplexed platform is more sensitive than a single endpoint assay, provides mechanistic cues of toxicity and is amenable for higher throughput screening.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Túbulos Renais Proximais/efeitos dos fármacos , Testes de Toxicidade/métodos , Apoptose/efeitos dos fármacos , Células Cultivadas , Análise Custo-Benefício , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Necrose/induzido quimicamente , Perexilina/toxicidade , Estaurosporina/toxicidade , Fatores de Tempo , Testes de Toxicidade/economiaRESUMO
Mallory-Denk bodies (MDB) are hepatocyte inclusions containing cytokeratin 8 (CK8) which can develop, along with other steatohepatitis lesions, in patients treated with amiodarone, perhexiline maleate or 4,4'-diethylaminoethoxyhexestrol. These drugs accumulate lipids, whose subsequent peroxidation liberates reactive by-products, like malondialdehyde (MDA). The formation of MDB has been previously reproduced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine or griseofulvin administration which cross-link CK8 by tissue transglutaminase, thus forming an entangled network, from which MDB progressively arise. The present study depicts the mechanisms initiating MDB formation by steatohepatitis-inducing drugs. Short incubation of hepatocytes with amiodarone (50 microM), 4,4'-diethylaminoethoxyhexestrol (50 microM) or perhexiline maleate (25 microM) increased the pool of CK8 monomers and increased cell calcium to activate Ca(++)-dependent transglutaminases which cross-linked the CK8 monomers into CK8-containing oligomers. The present study also provides the first evidence that MDA might directly participate in MDB formation, as this reactive agent cross-linked purified CK8 or albumin in vitro, disrupted the cytokeratin network of isolated hepatocytes, and bridged CK8 molecules. In conclusion, steatohepatitis-inducing drugs increase cell calcium and activate tissue transglutaminase, which cross-links CK8 to form a molecular scaffold, from which MDB might secondarily arise. Malondialdehyde also cross-links CK8, albeit through a different mechanism, and might also contribute to MDB formation.
Assuntos
Hepatócitos/efeitos dos fármacos , Corpos de Inclusão/efeitos dos fármacos , Queratina-8/efeitos dos fármacos , Malondialdeído/metabolismo , Amiodarona/toxicidade , Animais , Cálcio/metabolismo , Fígado Gorduroso/induzido quimicamente , Proteínas de Ligação ao GTP/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Hepatócitos/metabolismo , Hexestrol/análogos & derivados , Hexestrol/toxicidade , Corpos de Inclusão/metabolismo , Queratina-8/metabolismo , Masculino , Perexilina/análogos & derivados , Perexilina/toxicidade , Proteína 2 Glutamina gama-Glutamiltransferase , Proteínas , Ratos , Ratos Sprague-Dawley , Transglutaminases/efeitos dos fármacos , Transglutaminases/metabolismoRESUMO
Perhexiline is a prophylactic anti-anginal agent that ameliorates the metabolic basis for myocardial ischaemia and is increasingly used in the management of acute coronary syndromes. No intravenous preparation is available and usual oral loading regimens require 2-3 days to achieve therapeutic drug levels. Two patients presenting to hospital with single-dose over-dosage of perhexiline (6500 mg and 1000 mg, respectively) provided a basis for examining the safety of large single dosage of perhexiline and associated time-course of drug levels. Neither patient had previously taken perhexiline. Peak plasma perhexiline concentrations occurred within 12 h of ingestion and were 2.58 and 0.50 mg/L, respectively (therapeutic range 0.15-0.60 mg/L). The first patient developed transient nausea and vomiting; the second patient had no adverse effects. Subsequently, a series of 10 patients with acute coronary syndromes received an 800-mg loading dose. Peak concentrations occurred within 12 h postdose; the mean levels achieved were 0.40 +/- 0.16 mg/L (standard error of the mean). No serious adverse effects were seen. Two patients reported transient nausea or vomiting within 24 h of the loading dose. The utility of this rapid loading regimen for incremental suppression of myocardial ischaemia remains to be assessed.
Assuntos
Fármacos Cardiovasculares/administração & dosagem , Doença das Coronárias/tratamento farmacológico , Perexilina/administração & dosagem , Adulto , Fármacos Cardiovasculares/sangue , Fármacos Cardiovasculares/toxicidade , Esquema de Medicação , Overdose de Drogas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Perexilina/sangue , Perexilina/toxicidade , AutomedicaçãoRESUMO
Many cell models that are used to assess basic cytotoxicity show a good correlation with acute toxicity. However, their correlation with the toxicity seen following chronic in vivo exposure is less evident. The new human hepatoma cell line HBG BC2 possesses the capacity of being reversibly differentiated in vitro and of maintaining a relatively higher metabolic rate when in the differentiated state (3 weeks) as compared to HepG2 cells, and thus may allow the conduct of repeated toxicity testing on cells in culture. In order to evaluate the genetic background of HBG BC2 cells, the expression of selected genes was analyzed in untreated cultures and, in addition, the behavior of HBG BC2 cultures under conditions of repeated treatment was studied with acetaminophen as a test substance and coupled with the use of standard staining techniques to demonstrate toxicity. Results showed that cultures of HBG BC2 cells retained a capacity to undergo apoptosis and proliferation, allowing probable replacement of damaged cells in the culture monolayer. MTT reduction was used to evaluate the toxicity of acetaminophen, acetylsalicylic acid, perhexiline, and propranolol, after both single and repeated (3 times/week for 2 weeks) administration. Under the conditions of repeated treatment, cytotoxicity was observed at lower doses as compared to single administration. In addition, the lowest nontoxic doses were in the same range as plasma concentrations measured in humans under therapeutic use. Our results suggest that the new human hepatoma HBG BC2 cell line is of interest for the evaluation of cell toxicity under conditions of repeated administration.
Assuntos
Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Neoplasias Hepáticas/patologia , Testes de Toxicidade , Acetaminofen/toxicidade , Aspirina/toxicidade , Relação Dose-Resposta a Droga , Humanos , Perexilina/toxicidade , Propranolol/toxicidadeRESUMO
Toxic effects of a detriazinyl metabolite of almitrine (DTMA) were evaluated in rats and on cultured rat macrophages. In rats daily treated with DTMA for 16 weeks, spastic gaits with heel-lifting appeared, and lamellated and/or crystalloid bodies formed in sensory neurons, satellite cells, Schwann cells, and vascular endothelial cells of the dorsal root ganglia. The lysosomal lamellated bodies, which were not induced by almitrine, were produced also in cultured rat macrophages exposed to over 1 x 10(-5) M DTMA.
Assuntos
Almitrina/toxicidade , Fluorbenzenos/toxicidade , Gânglios Espinais/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Piperazinas/toxicidade , Medicamentos para o Sistema Respiratório/toxicidade , Administração Oral , Almitrina/administração & dosagem , Almitrina/química , Animais , Bloqueadores dos Canais de Cálcio/toxicidade , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/ultraestrutura , Feminino , Fluorbenzenos/administração & dosagem , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Lisossomos/efeitos dos fármacos , Lisossomos/ultraestrutura , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/ultraestrutura , Microscopia Eletrônica , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/ultraestrutura , Perexilina/toxicidade , Piperazinas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Medicamentos para o Sistema Respiratório/administração & dosagem , Medicamentos para o Sistema Respiratório/química , Células de Schwann/efeitos dos fármacos , Células de Schwann/ultraestrutura , Organismos Livres de Patógenos Específicos , Relação Estrutura-AtividadeRESUMO
Experience is reported with 41 patients taking perhexiline maleate for angina pectoris for periods of up to 70 months, while serum concentrations of the drug were monitored, and liver function tests and electromyographic tests were made before and during treatment. Severe side effects did not occur unless serum perhexiline levels were greater than 1.5 mg/L. The drug seems effective for prolonged dosage, and the monitoring of weight, liver function test results, and serum concentrations should prevent or reduce toxicity. A starting dose of 100 mg daily is recommended. The drug is not recommended for routine use in angina pectoris.
Assuntos
Angina Pectoris/tratamento farmacológico , Eletromiografia , Perexilina/análogos & derivados , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monitorização Fisiológica , Perexilina/administração & dosagem , Perexilina/sangue , Perexilina/toxicidadeRESUMO
The effects of perhexiline maleate on growth and drug sensitivity were studied in the P388 murine leukemia cell line and in an anthracycline-resistant subline (P388/ADR). At noninhibitory concentrations, perhexiline maleate markedly increased the sensitivity of P388/ADR cells to doxorubicin but did not have such an effect on anthracycline-sensitive cells. The effects of perhexiline maleate on P388/ADR cells were reversible. Perhexiline maleate also increased the accumulation of another anthracycline, daunorubicin, in P388/ADR cells but did not increase its accumulation in the anthracycline-sensitive cells. Perhexiline maleate did not affect the sensitivity of either cell line to methotrexate or to 6-mercaptopurine. However, its effects on the sensitivity and on drug accumulation of vinblastine, a drug to which P388/ADR cells are cross-resistant, were similar to those observed for the anthracyclines. Although perhexiline maleate has been reported to be a calcium antagonist in other systems, our data do not suggest that this mechanism is involved in its enhancement of the sensitivity of P388/ADR cells to doxorubicin. We suggest instead that this effect might be associated with alterations of cell lipid metabolism induced by perhexiline maleate.
Assuntos
Doxorrubicina/toxicidade , Leucemia P388/fisiopatologia , Leucemia Experimental/fisiopatologia , Perexilina/análogos & derivados , Animais , Transporte Biológico , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Daunorrubicina/metabolismo , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Cinética , Camundongos , Camundongos Endogâmicos , Perexilina/toxicidade , Vimblastina/toxicidadeRESUMO
Hepatic toxicity was observed in mice which had received Griseofulvin or Perhexilin Maleate over a period of several months. Treatment of griseofulvin alone gave rise to hepatitis with the presence of Mallory bodies (MB) whereas the same length of treatment with Perhexilin Maleate was associated with steatonecrosis with an absence of MB. When treatment was followed by a one month rest period hepatic lesions disappeared with no trace of sequelae. Cross-treatment studies showed that one week of Perhexiline Maleate was sufficient to induce MB in mice pretreated with Griseofulvin. Similarly, Griseofulvin administered to mice pretreated with Perhexilin Maleate gave rise to MB formation after one week as opposed to the usual two months incubation time (DENK et al.). The histological nature and mode of formation of these MB was identical to that encountered in acute alcoholic hepatitis. On addition, combined drug therapy employing Perhexilin Maleate suggests a particular hepatic toxicity in man in cases where the liver has become predisposed due to other therapeutic.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Griseofulvina/toxicidade , Perexilina/toxicidade , Piperidinas/toxicidade , Animais , Doença Hepática Induzida por Substâncias e Drogas/patologia , Fígado/patologia , Masculino , Camundongos , Perexilina/análogos & derivados , Fatores de TempoRESUMO
The effects of different doses of Pexid on cultured liver cells have been studied by ultrastructural and histoenzymological techniques. Two types of abnormal lysosomal inclusions were seen: clear matrix inclusions with either homogeneous or tiny lamellar structures, and dark matrix inclusions with clear vacuoles and either random or myelin-like lamellar structures. These patterns correspond to storage of triglycerides, phospholipids and gangliosides, either singly or together.
Assuntos
Fígado/efeitos dos fármacos , Perexilina/toxicidade , Piperidinas/toxicidade , Células Cultivadas , Gangliosídeos/análise , Glicogênio/metabolismo , Humanos , Corpos de Inclusão/ultraestrutura , Metabolismo dos Lipídeos , Fígado/enzimologia , Fígado/ultraestrutura , Microscopia Eletrônica , Perexilina/análogos & derivadosRESUMO
Perhexiline maleate is an anti-anginal drug believed to cause liver damage. The possibility that this effect could be potentiated by alcohol has been investigated. Rats fed both the drug and alcohol did not show more severe liver damage than those fed alcohol alone. Livers from rats fed perhexiline alone did not differ from controls. There is no evidence from this study that alcohol potentiates the effect of perhexiline in rat liver. These results may have a bearing in perhexiline-induced liver damage in humans, but clinical studies are required.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Etanol/toxicidade , Perexilina/toxicidade , Piperidinas/toxicidade , Animais , Sinergismo Farmacológico , Fígado/ultraestrutura , Hepatopatias/patologia , Masculino , Microscopia Eletrônica , RatosRESUMO
Perhexiline maleate therapy may produce a polyneuropathy as well as other side effects in man. The Schwann-cell origin of the neuropathy is demonstrated by morphologic studies. Ultrastructural studies of muscle and nerve biopsies showed the presence of intracytoplasmic inclusions, primarily in Schwann cells, muscle fibers, and endothelial cells. These inclusions were polymorphic and varied from one cell type to another. They were frequently associated with calcium deposits, and their structure suggested a lysosomal origin. It was possible experimentally to reproduce similar inclusions in mice. Furthermore, numerous tubular aggregates were observed in intoxicated mouse muscle fibers.