RESUMEN
Oxidative stress is a reflection of the imbalance between the production of reactive oxygen species (ROS) and the scavenging capacity of the antioxidant system. Excessive ROS, generated from various endogenous oxidative biochemical enzymes, interferes with the normal function of liver-specific cells and presumably plays a role in the pathogenesis of liver fibrosis. Once exposed to harmful stimuli, Kupffer cells (KC) are the main effectors responsible for the generation of ROS, which consequently affect hepatic stellate cells (HSC) and hepatocytes. ROS-activated HSC undergo a phenotypic switch and deposit an excessive amount of extracellular matrix that alters the normal liver architecture and negatively affects liver function. Additionally, ROS stimulate necrosis and apoptosis of hepatocytes, which causes liver injury and leads to the progression of end-stage liver disease. In this review, we overview the role of ROS in liver fibrosis and discuss the promising therapeutic interventions related to oxidative stress. Most importantly, novel drugs that directly target the molecular pathways responsible for ROS generation, namely, mitochondrial dysfunction inhibitors, endoplasmic reticulum stress inhibitors, NADPH oxidase (NOX) inhibitors, and Toll-like receptor (TLR)-affecting agents, are reviewed in detail. In addition, challenges for targeting oxidative stress in the management of liver fibrosis are discussed.
Asunto(s)
Células Estrelladas Hepáticas/metabolismo , Hepatocitos/metabolismo , Cirrosis Hepática/terapia , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Mitocondrias/efectos de los fármacos , NADPH Oxidasas/antagonistas & inhibidores , Receptores Toll-Like/antagonistas & inhibidoresRESUMEN
Formaldehyde fixation is widely used for long-term maintenance of tissue. However, due to formaldehyde-induced crosslinks, fixed tissue proteins are difficult to extract, which hampers mass spectrometry (MS) proteomic analyses. Recent years have seen the use of different combinations of high temperature and solubilizing agents (usually derived from antigen retrieval techniques) to unravel formaldehyde-fixed paraffin-embedded tissue proteomes. However, to achieve protein extraction yields similar to those of fresh-frozen tissue, high-temperature heating is necessary. Such harsh extraction conditions can affect sensitive amino acids and post-translational modifications, resulting in the loss of important information, while still not resulting in protein yields comparable to those of fresh-frozen tissue. Herein, the objective is to evaluate cleavable protein crosslinkers as fixatives that allow tissue preservation and efficient protein extraction from fixed tissue for MS proteomics under mild conditions. With this goal in mind, disuccinimidyl tartrate (DST) and dithiobis(succinimidylpropionate) (DSP) are investigated as cleavable fixating reagents. These compounds crosslink proteins by reacting with amino groups, leading to amide bond formation, and can be cleaved with sodium metaperiodate (cis-diols, DST) or reducing agents (disulfide bonds, DSP), respectively. Results show that cleavable protein crosslinking with DST and DSP allows tissue fixation with morphology preservation comparable to that of formaldehyde. In addition, cleavage of DSP improves protein recovery from fixed tissue by a factor of 18 and increases the number of identified proteins by approximately 20 % under mild extraction conditions compared with those of formaldehyde-fixed paraffin-embedded tissue. A major advantage of DSP is the introduction of well-defined protein modifications that can be taken into account during database searching. In contrast to DSP fixation, DST fixation followed by cleavage with sodium metaperiodate, although effective, results in side reactions that prevent effective protein extraction and interfere with protein identification. Protein crosslinkers that can be cleaved under mild conditions and result in defined modifications, such as DSP, are thus viable alternatives to formaldehyde as tissue fixatives to facilitate protein analysis from paraffin-embedded, fixed tissue.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Fijadores/química , Proteoma/análisis , Succinimidas/química , Fijación del Tejido/métodos , Animales , Cromatografía Liquida , Hígado/citología , Ácido Peryódico/química , Proteoma/química , Proteómica/métodos , Ratas , Espectrometría de Masas en TándemRESUMEN
Cisplatin occupies a crucial role in the treatment of various malignant tumors. However, its efficacy and applicability are heavily restricted by severe systemic toxicities and drug resistance. Our study exploits the active targeting of supramolecular metallacages to enhance the activity of cisplatin in cancer cells while reducing its toxicity. Thus, Pd2L4 cages (L = ligand) have been conjugated to four integrin ligands with different binding affinity and selectivity. Cage formation and encapsulation of cisplatin was proven by NMR spectroscopy. Upon encapsulation, cisplatin showed increased cytotoxicity in vitro, in melanoma A375 cells overexpressing αvß3 integrins. Moreover, ex vivo studies in tissue slices indicated reduced toxicity toward healthy liver and kidney tissues for cage-encapsulated cisplatin. Analysis of metal content by ICP-MS demonstrated that the encapsulated drug is less accumulated in these organs compared to the "free" cisplatin.
Asunto(s)
Antineoplásicos/administración & dosificación , Cisplatino/administración & dosificación , Portadores de Fármacos/metabolismo , Integrina alfaVbeta3/metabolismo , Melanoma/tratamiento farmacológico , Estructuras Metalorgánicas/metabolismo , Paladio/metabolismo , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Cisplatino/farmacología , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Humanos , Ligandos , Masculino , Melanoma/metabolismo , Estructuras Metalorgánicas/química , Paladio/química , Ratas WistarRESUMEN
The vitamin D-deficient model, established in the C57BL/6 mouse after 8 weeks of feeding vitamin D-deficient diets in the absence or presence of added calcium, was found associated with elevated levels of plasma parathyroid hormone (PTH) and plasma and liver cholesterol, and a reduction in cholesterol 7α-hydroxylase (Cyp7a1, rate-limiting enzyme for cholesterol metabolism) and renal Oat3 mRNA/protein expression levels. However, there was no change in plasma calcium and phosphate levels. Appraisal of the liver revealed an up-regulation of mRNA expressions of the small heterodimer partner (Shp) and attenuation of Cyp7a1, which contributed to hypercholesterolemia in vitamin D-deficiency. When vitamin D-sufficient or D-deficient mice were further rendered hypercholesterolemic with 3 weeks of feeding the respective, high fat/high cholesterol (HF/HC) diets, treatment with 1α,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ], active vitamin D receptor (VDR) ligand, or vitamin D (cholecalciferol) to HF/HC vitamin D-deficient mice lowered the cholesterol back to baseline levels. Cholecalciferol treatment partially restored renal Oat3 mRNA/protein expression back to that of vitamin D-sufficient mice. When the protein expression of protein kinase C (PKC), a known, negative regulator of Oat3, was examined in murine kidney, no difference in PKC expression was observed for any of the diets with/without 1,25(OH)2 D3 /cholecalciferol treatment, inferring that VDR regulation of renal Oat3 did not involve PKC in mice. As expected, plasma calcium levels were not elevated by cholecalciferol treatment of vitamin D-deficient mice, while 1,25(OH)2 D3 treatment led to hypercalcemia. In conclusion, vitamin D-deficiency resulted in down-regulation of liver Cyp7a1 and renal Oat3, conditions that are alleviated upon replenishment of cholecalciferol.
Asunto(s)
Colesterol 7-alfa-Hidroxilasa/biosíntesis , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Riñón/metabolismo , Hígado/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/biosíntesis , Deficiencia de Vitamina D/enzimología , Deficiencia de Vitamina D/genética , Animales , Ácidos y Sales Biliares/metabolismo , Calcifediol/sangre , Calcio/sangre , Calcio/farmacología , Colecalciferol/farmacología , Colesterol/sangre , Colesterol/metabolismo , Colesterol 7-alfa-Hidroxilasa/sangre , Colesterol 7-alfa-Hidroxilasa/genética , Dieta/métodos , Vesícula Biliar/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Transportadores de Anión Orgánico Sodio-Independiente/genética , Hormona Paratiroidea/sangre , Vitamina D/análogos & derivados , Vitamina D/sangreRESUMEN
Hepatotoxicity is one of the major reasons for withdrawal of drugs from the market. Therefore, there is a need to screen new drugs for hepatotoxicity in humans at an earlier stage. The aim of this study was to validate human precision-cut liver slices (PCLS) as an ex vivo model to predict drug-induced cholestasis and identify the possible mechanisms of cholestasis-induced toxicity using gene expression profiles. Five hepatotoxicants, which are known to induce cholestasis (alpha-naphthyl isothiocyanate, chlorpromazine, cyclosporine, ethinyl estradiol and methyl testosterone) were used at concentrations inducing low (<30 %) and medium (30-50 %) toxicity, based on ATP content. Human PCLS were incubated with the drugs in the presence of a non-toxic concentration (60 µM) of a bile acid mixture (portal vein concentration and composition) as model for bile acid-induced cholestasis. Regulated genes include bile acid transporters and cholesterol transporters. Pathway analysis revealed that hepatic cholestasis was among the top ten regulated pathways, and signaling pathways such as farnesoid X receptor- and liver X receptor-mediated responses, which are known to play a role in cholestasis, were significantly affected by all cholestatic compounds. Other significantly affected pathways include unfolded protein response and protein ubiquitination implicating the role of endoplasmic reticulum stress. This study shows that human PCLS incubated in the presence of a physiological bile acid mixture correctly reflect the pathways affected in drug-induced cholestasis in the human liver. In the future, this human PCLS model can be used to identify cholestatic adverse drug reactions of new chemical entities.
Asunto(s)
Colestasis/inducido químicamente , Hígado/efectos de los fármacos , 1-Naftilisotiocianato/toxicidad , Anciano , Clorpromazina/efectos adversos , Colestasis/genética , Ciclosporina/efectos adversos , Relación Dosis-Respuesta a Droga , Etinilestradiol/efectos adversos , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , Metiltestosterona/efectos adversos , Persona de Mediana Edad , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transcriptoma/efectos de los fármacos , Adulto JovenRESUMEN
Human precision-cut liver slices (hPCLS) are a valuable ex vivo model that can be used in acute toxicity studies. However, a rapid decline in metabolic enzyme activity limits their use in studies that require a prolonged xenobiotic exposure. The aim of the study was to extend the viability and function of hPCLS to 5 days of incubation. hPCLS were incubated in two media developed for long-term culture of hepatocytes, RegeneMed®, and Cellartis®, and in the standard medium WME. Maintenance of phase I and II metabolism was studied both on gene expression as well as functional level using a mixture of CYP isoform-specific substrates. Albumin synthesis, morphological integrity, and glycogen storage was assessed, and gene expression was studied by transcriptomic analysis using microarrays with a focus on genes involved in drug metabolism, transport and toxicity. The data show that hPCLS retain their viability and functionality during 5 days of incubation in Cellartis® medium. Albumin synthesis as well as the activity and gene expression of phase I and II metabolic enzymes did not decline during 120-h incubation in Cellartis® medium, with CYP2C9 activity as the only exception. Glycogen storage and morphological integrity were maintained. Moreover, gene expression changes in hPCLS during incubation were limited and mostly related to cytoskeleton remodeling, fibrosis, and moderate oxidative stress. The expression of genes involved in drug transport, which is an important factor in determining the intracellular xenobiotic exposure, was also unchanged. Therefore, we conclude that hPCLS cultured in Cellartis® medium are a valuable human ex vivo model for toxicological and pharmacological studies that require prolonged xenobiotic exposure.
Asunto(s)
Enzimas/metabolismo , Hígado/metabolismo , Técnicas de Cultivo de Órganos/métodos , Adenosina Trifosfato/metabolismo , Albúminas/biosíntesis , Proteínas Portadoras/metabolismo , Medios de Cultivo , Fibrosis/genética , Regulación de la Expresión Génica , Humanos , Inactivación Metabólica , Hígado/efectos de los fármacos , Hígado/patología , Estrés Oxidativo/genética , Xenobióticos/metabolismo , Xenobióticos/farmacocinéticaRESUMEN
Drug-induced cholestasis (DIC) is one of the leading manifestations of drug-induced liver injury (DILI). As the underlying mechanisms for DIC are not fully known and specific and predictive biomarkers and pre-clinical models are lacking, the occurrence of DIC is often only reported when the drug has been approved for registration. Therefore, appropriate models that predict the cholestatic potential of drug candidates and/or provide insight into the mechanism of DIC are highly needed. We investigated the application of rat precision-cut liver slices (PCLS) to predict DIC, using several biomarkers of cholestasis: hepatocyte viability, intracellular accumulation of total as well as individual bile acids and changes in the expression of genes known to play a role in cholestasis. Rat PCLS exposed to the cholestatic drugs chlorpromazine, cyclosporine A and glibenclamide for 48 h in the presence of a 60 µM physiological bile acid (BA) mix reflected various changes associated with cholestasis, such as decrease in hepatocyte viability, accumulation and changes in the composition of BA and changes in the gene expression of Fxr, Bsep and Ntcp. The toxicity of the drugs was correlated with the accumulation of BA, and especially DCA and CDCA and their conjugates, but to a different extent for different drugs, indicating that BA toxicity is not the only cause for the toxicity of cholestatic drugs. Moreover, our study supports the use of several biomarkers to test drugs for DIC. In conclusion, our results indicate that PCLS may represent a physiological and valuable model to identify cholestatic drugs and provide insight into the mechanisms underlying DIC.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Hígado/efectos de los fármacos , Técnicas de Cultivo de Órganos/métodos , Pruebas de Toxicidad/métodos , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/genética , Animales , Ácidos y Sales Biliares/metabolismo , Colestasis/inducido químicamente , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Ratas Wistar , Simportadores/genéticaRESUMEN
Although intestinal P-glycoprotein (P-gp) has been extensively studied in vitro and in animals, its activity and the consequences of P-gp inhibition for drug disposition and toxicity in humans are still difficult to accurately extrapolate from these studies. Moreover, existing in vitro models do not take into consideration that the intestine is heterogeneous with respect to P-gp expression. Recently, we reported rat precision-cut intestinal slices (PCIS) as a physiological ex vivo model to study the regional gradient of P-gp activity and inhibition. Here we extended the application of PCIS to the human intestine. For this purpose rhodamine 123 (R123) accumulation in the presence or absence of the P-gp inhibitors verapamil, cyclosporine A, quinidine, ketoconazole, PSC833 and CP100356 was measured in PCIS of human duodenum, jejunum, ileum and colon. R123 accumulation in the presence of the P-gp inhibitors appeared to be most enhanced in the ileum compared to the other regions. Moreover, the regional differences in accumulation are in line with published differences in abundance of P-gp. The rank order of the potency of the P-gp inhibitors, reflected by their IC50 , was comparable to that in rat PCIS. However, the increase in accumulation of the P-gp substrate R123 by the inhibitors was larger in human ileum PCIS than in rat PCIS, indicating species difference in P-gp abundance. These data show that human PCIS are an appropriate ex vivo model to study the activity of intestinal P-gp and predict the inhibitory effect of drugs and of transporter-mediated drug-drug interactions in the human intestine. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Colon/efectos de los fármacos , Colon/metabolismo , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Moduladores del Transporte de Membrana/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adulto , Anciano , Transporte Biológico , Relación Dosis-Respuesta a Droga , Duodeno/efectos de los fármacos , Duodeno/metabolismo , Femenino , Colorantes Fluorescentes/metabolismo , Humanos , Íleon/efectos de los fármacos , Íleon/metabolismo , Técnicas In Vitro , Yeyuno/efectos de los fármacos , Yeyuno/metabolismo , Cinética , Masculino , Persona de Mediana Edad , Rodamina 123/metabolismoRESUMEN
P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A) are differentially expressed along the intestine and work coordinately to reduce the intracellular concentration of xenobiotics and the absorption of orally taken drugs. Drug-drug interactions (DDIs) based on P-gp/CYP3A interplay are of clinical importance and require preclinical investigation. We investigated the P-gp/Cyp3a interplay and related DDIs with different P-gp inhibitors in the various regions of the rat intestine ex vivo using precision-cut intestinal slices (PCIS) with quinidine (Qi), a dual substrate of P-gp and Cyp3a, as the probe. The results showed that P-gp efflux was the main factor limiting the intracellular Qi content at concentrations below 5µM, whereas both efflux and metabolism were saturated at [Qi] > 50µM. The selective P-gp inhibitors CP100356 [N-(3,4-dimethoxyphenethyl)-4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2[1H]-yl)-6,7-dimethoxyquinazolin-2-amine] and PSC833 [valspodar, 6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-l-valine-cyclosporin A] enhanced the Qi accumulation in slices in line with the different P-gp expression in the intestinal regions and, as a result, also enhanced metabolism in the jejunum and ileum. Dual inhibitors of both P-gp and Cyp3a (verapamil and ketoconazole) increased the concentration of Qi in the jejunum and ileum, but less 3-hydroxy-quinidine was produced due to inhibition of Cyp3a. The results indicate that the P-gp/Cyp3a interplay depends on the concentration of the drug and on the intestinal region under study. Furthermore, due to the P-gp/Cyp3a interplay, DDIs can lead to remarkable changes in the intracellular concentration of both the parent drug and the metabolite, which varies among the intestinal regions and depends on the selectivity of the inhibitors, with potentially important implications for disposition and toxicity of drugs and their metabolites.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Citocromo P-450 CYP3A/metabolismo , Interacciones Farmacológicas/fisiología , Íleon/metabolismo , Yeyuno/metabolismo , Preparaciones Farmacéuticas/metabolismo , Animales , Ciclosporinas/metabolismo , Absorción Intestinal , Cetoconazol/metabolismo , Masculino , Quinidina/metabolismo , Ratas , Ratas Wistar , Valina/metabolismo , Verapamilo/metabolismoRESUMEN
Human toxicity screening is an important stage in the development of safe drug candidates. Hepatotoxicity is one of the major reasons for the withdrawal of drugs from the market because the liver is the major organ involved in drug metabolism, and it can generate toxic metabolites. There is a need to screen molecules for drug-induced hepatotoxicity in humans at an earlier stage. Transcriptomics is a technique widely used to screen molecules for toxicity and to unravel toxicity mechanisms. To date, the majority of such studies were performed using animals or animal cells, with concomitant difficulty in interpretation due to species differences, or in human hepatoma cell lines or cultured hepatocytes, suffering from the lack of physiological expression of enzymes and transporters and lack of nonparenchymal cells. The aim of this study was to classify known hepatotoxicants on their phenotype of toxicity in humans using gene expression profiles ex vivo in human precision-cut liver slices (PCLS). Hepatotoxicants known to induce either necrosis (n = 5) or cholestasis (n = 5) were used at concentrations inducing low (<30%) and medium (30-50%) cytotoxicity, based on ATP content. Random forest and support vector machine algorithms were used to classify hepatotoxicants using a leave-one-compound-out cross-validation method. Optimized biomarker sets were compared to derive a consensus list of markers. Classification correctly predicted the toxicity phenotype with an accuracy of 70-80%. The classification is slightly better for the low than for the medium cytotoxicity. The consensus list of markers includes endoplasmic reticulum stress genes, such as C2ORF30, DNAJB9, DNAJC12, SRP72, TMED7, and UBA5, and a sodium/bile acid cotransporter (SLC10A7). This study shows that human PCLS are a useful model to predict the phenotype of drug-induced hepatotoxicity. Additional compounds should be included to confirm the consensus list of markers, which could then be used to develop a biomarker PCR-array for hepatotoxicity screening.
Asunto(s)
Colestasis/inducido químicamente , Perfilación de la Expresión Génica , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Necrosis/inducido químicamente , Toxicogenética , Acetaminofén/toxicidad , Anciano , Benzofuranos/toxicidad , Ácidos y Sales Biliares/toxicidad , Cloranfenicol/toxicidad , Clorpromazina/toxicidad , Colestasis/genética , Colchicina/toxicidad , Ciclosporina/toxicidad , Dietilnitrosamina/toxicidad , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Etinilestradiol/toxicidad , Femenino , Hepatocitos/metabolismo , Humanos , Masculino , Metiltestosterona/toxicidad , Persona de Mediana Edad , Necrosis/genética , Fenotipo , Adulto JovenRESUMEN
OBJECTIVES: To assess the ability of a previously developed hybrid physiology-based pharmacokinetic-pharmacodynamic (PBPKPD) model in rats to predict the dopamine D2 receptor occupancy (D2RO) in human striatum following administration of antipsychotic drugs. METHODS: A hybrid PBPKPD model, previously developed using information on plasma concentrations, brain exposure and D2RO in rats, was used as the basis for the prediction of D2RO in human. The rat pharmacokinetic and brain physiology parameters were substituted with human population pharmacokinetic parameters and human physiological information. To predict the passive transport across the human blood-brain barrier, apparent permeability values were scaled based on rat and human brain endothelial surface area. Active efflux clearance in brain was scaled from rat to human using both human brain endothelial surface area and MDR1 expression. Binding constants at the D2 receptor were scaled based on the differences between in vitro and in vivo systems of the same species. The predictive power of this physiology-based approach was determined by comparing the D2RO predictions with the observed human D2RO of six antipsychotics at clinically relevant doses. RESULTS: Predicted human D2RO was in good agreement with clinically observed D2RO for five antipsychotics. Models using in vitro information predicted human D2RO well for most of the compounds evaluated in this analysis. However, human D2RO was under-predicted for haloperidol. CONCLUSIONS: The rat hybrid PBPKPD model structure, integrated with in vitro information and human pharmacokinetic and physiological information, constitutes a scientific basis to predict the time course of D2RO in man.
Asunto(s)
Antipsicóticos/farmacología , Antipsicóticos/farmacocinética , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Receptores de Dopamina D2/metabolismo , Esquizofrenia/tratamiento farmacológico , Animales , Antipsicóticos/administración & dosificación , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Antagonistas de los Receptores de Dopamina D2/administración & dosificación , Antagonistas de los Receptores de Dopamina D2/farmacocinética , Antagonistas de los Receptores de Dopamina D2/farmacología , Humanos , Modelos Biológicos , Ratas , Esquizofrenia/metabolismoRESUMEN
The use of diclofenac (DCF), a nonsteroidal anti-inflammatory drug, is associated with a high prevalence of gastrointestinal side effects. In vivo studies in rodents suggested that reactive metabolites of DCF produced by the liver or the intestine might be responsible for this toxicity. In the present study, precision-cut intestinal slices (PCIS) prepared from the jejunum of 18 human donors were used as an ex vivo model to investigate whether DCF intestinal metabolites are responsible for its intestinal toxicity in man. PCIS were incubated with a concentration range of DCF (0-600 µM) up to 24 h. DCF (≥400 µM) caused direct toxicity to the intestine as demonstrated by ATP depletion, morphological damage, caspase 3 activation, and lactate dehydrogenase leakage. Three main metabolites produced by PCIS (4'-hydroxy DCF, 5-hydroxy DCF, and DCF acyl glucuronide) were detected by HPLC. Protein adducts were detected by immunohistochemical staining and showed correlation with the intestinal metabolites. DCF induced similar toxicity to each of the samples regardless of the variation in metabolism among them. Less metabolites were produced by slices incubated with 400 µM DCF than with 100 µM DCF. The addition of the metabolic inhibitors such as ketoconazole, cimetidine, or borneol decreased the metabolite formation but increased the toxicity. The results suggest that DCF can induce intestinal toxicity in human PCIS directly at therapeutically relevant concentrations, independent of the reactive metabolites 4'-OH DCF, 5-OH DCF, or diclofenac acylglucuronide produced by the liver or formed in the intestine.
Asunto(s)
Antiinflamatorios no Esteroideos/toxicidad , Diclofenaco/análogos & derivados , Glucurónidos/toxicidad , Yeyuno/efectos de los fármacos , Yeyuno/metabolismo , Adenosina Trifosfato/metabolismo , Adulto , Anciano , Antiinflamatorios no Esteroideos/metabolismo , Caspasa 3/metabolismo , Diclofenaco/metabolismo , Diclofenaco/toxicidad , Femenino , Glucurónidos/metabolismo , Humanos , Inmunohistoquímica , Técnicas In Vitro , Yeyuno/patología , L-Lactato Deshidrogenasa/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
Induction of fibrosis during prolonged culture of precision-cut liver slices (PCLS) was reported. In this study, the use of rat PCLS was investigated to further characterize the mechanism of early onset of fibrosis in this model and the effects of antifibrotic compounds. Rat PCLS were incubated for 48h, viability was assessed by ATP and gene expression of PDGF-B and TGF-ß1 and the fibrosis markers Hsp47, αSma and Pcol1A1 and collagen1 protein expressions were determined. The effects of the antifibrotic drugs imatinib, sorafenib and sunitinib, PDGF-pathway inhibitors, and perindopril, valproic acid, rosmarinic acid, tetrandrine and pirfenidone, TGFß-pathway inhibitors, were determined. After 48h of incubation, viability of the PCLS was maintained and gene expression of PDGF-B was increased while TGF-ß1 was not changed. Hsp47, αSma and Pcol1A1 gene expressions were significantly elevated in PCLS after 48h, which was further increased by PDGF-BB and TGF-ß1. The increased gene expression of fibrosis markers was inhibited by all three PDGF-inhibitors, while TGFß-inhibitors showed marginal effects. The protein expression of collagen 1 was inhibited by imatinib, perindopril, tetrandrine and pirfenidone. In conclusion, the increased gene expression of PDGF-B and the down-regulation of fibrosis markers by PDGF-pathway inhibitors, together with the absence of elevated TGF-ß1 gene expression and the limited effect of the TGFß-pathway inhibitors, indicated the predominance of the PDGF pathway in the early onset of fibrosis in PCLS. PCLS appear a useful model for research of the early onset of fibrosis and for testing of antifibrotic drugs acting on the PDGF pathway.
Asunto(s)
Cirrosis Hepática/tratamiento farmacológico , Hígado/efectos de los fármacos , Proteínas Proto-Oncogénicas c-sis/antagonistas & inhibidores , Animales , Becaplermina , Benzamidas/farmacología , Bencilisoquinolinas/farmacología , Cinamatos/farmacología , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Depsidos/farmacología , Regulación hacia Abajo , Expresión Génica , Proteínas del Choque Térmico HSP47/genética , Proteínas del Choque Térmico HSP47/metabolismo , Mesilato de Imatinib , Hígado/metabolismo , Masculino , Modelos Biológicos , Niacinamida/análogos & derivados , Niacinamida/farmacología , Técnicas de Cultivo de Órganos , Perindopril/farmacología , Compuestos de Fenilurea/farmacología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-sis/genética , Proteínas Proto-Oncogénicas c-sis/metabolismo , Piridonas/farmacología , Pirimidinas/farmacología , Ratas , Ratas Wistar , Sorafenib , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Ácido Valproico/farmacología , Ácido RosmarínicoRESUMEN
OBJECTIVES: Dopamine D2 receptor occupancy (D2RO) is the major determinant of efficacy and safety in schizophrenia drug therapy. Excessive D2RO (>80%) is known to cause catalepsy (CAT) in rats and extrapyramidal side effects (EPS) in human. The objective of this study was to use pharmacokinetic and pharmacodynamic modeling tools to relate CAT with D2RO in rats and to compare that with the relationship between D2RO and EPS in humans. METHODS: Severity of CAT was assessed in rats at hourly intervals over a period of 8 h after antipsychotic drug treatment. An indirect response model with and without Markov elements was used to explain the relationship of D2RO and CAT. RESULTS: Both models explained the CAT data well for olanzapine, paliperidone and risperidone. However, only the model with the Markov elements predicted the CAT severity well for clozapine and haloperidol. The relationship between CAT scores in rat and EPS scores in humans was implemented in a quantitative manner. Risk of EPS not exceeding 10% over placebo correlates with less than 86% D2RO and less than 30% probability of CAT events in rats. CONCLUSION: A quantitative relationship between rat CAT and human EPS was elucidated and may be used in drug discovery to predict the risk of EPS in humans from D2RO and CAT scores measured in rats.
Asunto(s)
Antipsicóticos , Catalepsia/metabolismo , Antagonistas de los Receptores de Dopamina D2 , Modelos Biológicos , Receptores de Dopamina D2/metabolismo , Animales , Antipsicóticos/efectos adversos , Antipsicóticos/farmacocinética , Antipsicóticos/farmacología , Benzodiazepinas/efectos adversos , Benzodiazepinas/farmacocinética , Benzodiazepinas/farmacología , Encéfalo/metabolismo , Catalepsia/etiología , Simulación por Computador , Antagonistas de los Receptores de Dopamina D2/efectos adversos , Antagonistas de los Receptores de Dopamina D2/farmacocinética , Antagonistas de los Receptores de Dopamina D2/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Isoxazoles/efectos adversos , Isoxazoles/farmacocinética , Isoxazoles/farmacología , Cadenas de Markov , Olanzapina , Palmitato de Paliperidona , Pirimidinas/efectos adversos , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Ratas , Risperidona/efectos adversos , Risperidona/farmacocinética , Risperidona/farmacología , Índice de Severidad de la EnfermedadRESUMEN
A new series of gold(I) N-heterocyclic carbene (NHC) complexes based on xanthine ligands have been synthesized and characterized by mass spectrometry, NMR, and X-ray diffraction. The compounds have been tested for their antiproliferative properties in human cancer cells and nontumorigenic cells in vitro, as well as for their toxicity in healthy tissues ex vivo. The bis-carbene complex [Au(caffein-2-ylidene)2][BF4] (complex 4) appeared to be selective for human ovarian cancer cell lines and poorly toxic in healthy organs. To gain preliminary insights into their actual mechanism of action, two biologically relevant in cellulo targets were studied, namely, DNA (more precisely a higher-order DNA structure termed G-quadruplex DNA that plays key roles in oncogenetic regulation) and a pivotal enzyme of the DNA damage response (DDR) machinery (poly-(adenosine diphosphate (ADP)-ribose) polymerase 1 (PARP-1), strongly involved in the cancer resistance mechanism). Our results indicate that complex 4 acts as an efficient and selective G-quadruplex ligand while being a modest PARP-1 inhibitor (i.e., poor DDR impairing agent) and thus provide preliminary insights into the molecular mechanism that underlies its antiproliferative behavior.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Cafeína/química , Oro/química , Metano/análogos & derivados , Animales , Antineoplásicos/síntesis química , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacología , Humanos , Concentración 50 Inhibidora , Ligandos , Metano/química , Estructura Molecular , Xantina/químicaRESUMEN
Drug-induced liver injury is one of the leading causes of drug withdrawal from the market. In this study, we investigated the applicability of protein profiling of the incubation medium of human, mouse and rat precision-cut liver slices (PCLS) exposed to liver injury-inducing drugs for biomarker identification, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. PCLS were incubated with acetaminophen (APAP), 3-acetamidophenol, diclofenac and lipopolysaccharide for 24-48 h. PCLS medium from all species treated with APAP demonstrated similar changes in protein profiles, as previously found in mouse urine after APAP-induced liver injury, including the same key proteins: superoxide dismutase 1, carbonic anhydrase 3 and calmodulin. Further analysis showed that the concentration of hepcidin, a hepatic iron-regulating hormone peptide, was reduced in PCLS medium after APAP treatment, resembling the decreased mouse plasma concentrations of hepcidin observed after APAP treatment. Interestingly, comparable results were obtained after 3-acetamidophenol incubation in rat and human, but not mouse PCLS. Incubation with diclofenac, but not with lipopolysaccharide, resulted in the same toxicity parameters as observed for APAP, albeit to a lesser extent. In conclusion, proteomics can be applied to identify potential translational biomarkers using the PCLS system.
Asunto(s)
Biomarcadores/orina , Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Perfilación de la Expresión Génica , Hígado/efectos de los fármacos , Proteómica , Acetaminofén/administración & dosificación , Acetaminofén/toxicidad , Animales , Calmodulina/metabolismo , Anhidrasa Carbónica III/metabolismo , Diclofenaco/administración & dosificación , Diclofenaco/toxicidad , Hepcidinas/metabolismo , Humanos , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/toxicidad , Hígado/metabolismo , Ratones , Ratas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Pruebas de ToxicidadRESUMEN
The inclusion of nanoparticles dispersed in a hydrophilic matrix is one of the formulation strategies to improve the bioavailability of orally administered Biopharmaceutics Classification System (BCS) class II and IV drugs by increasing their dissolution rate in the intestine. To confirm that the increased dissolution rate results in increased bioavailability, in vitro and in vivo animal experiments are performed, however, translation to the human situation is hazardous. In this study, we used a range of in vitro and ex vivo methods, including methods applying human tissue, to predict the in vivo oral bioavailability of a model BCS class II CB-1 antagonist, formulated as a nanoparticle solid dispersion. The enhanced dissolution rate from the nanoparticle formulation resulted in an increased metabolite formation in both rat and human precision-cut intestinal slices, suggesting increased uptake and intracellular drug concentration in the enterocytes. In Ussing chamber experiments with human tissue, both the metabolite formation and apical efflux of the metabolite were increased for the nanoparticulate solid dispersion compared with a physical mixture, in line with the results in intestinal slices. The pharmacokinetics of the different formulations was studied in rats in vivo. The nanoparticle formulation indeed improved the absorption of the cannabinoid receptor 1 (CB-1) antagonist and the delivery into the brain compared with the physical mixture. In conclusion, the combined approach provides a valuable set of tools to investigate the effects of formulation on the absorption of poorly soluble compounds in human intestine and may provide relevant information on the oral bioavailability in humans early in the development process.
Asunto(s)
Antagonistas de Receptores de Cannabinoides/administración & dosificación , Absorción Intestinal , Nanopartículas/administración & dosificación , Receptor Cannabinoide CB1/antagonistas & inhibidores , Animales , Encéfalo/metabolismo , Antagonistas de Receptores de Cannabinoides/química , Antagonistas de Receptores de Cannabinoides/farmacocinética , Química Farmacéutica , Humanos , Masculino , Ratas , Ratas Wistar , SolubilidadRESUMEN
Idiosyncratic drug-induced liver injury (IDILI) is a major problem during drug development and has caused drug withdrawal and black-box warnings. Because of the low concordance of the hepatotoxicity of drugs in animals and humans, robust screening methods using human tissue are needed to predict IDILI in humans. According to the inflammatory stress hypothesis, the effects of inflammation interact with the effects of a drug or its reactive metabolite, precipitating toxic reactions in the liver. As a follow-up to our recently published mouse precision-cut liver slices model, an ex vivo model involving human precision-cut liver slices (hPCLS), co-incubated for 24 h with IDILI-related drugs and lipopolysaccharide (LPS), was developed to study IDILI mechanisms related to inflammatory stress in humans and to detect potential biomarkers. LPS exacerbated the effects of ketoconazole and clozapine toxicity but not those of their non-IDILI-related comparators, voriconazole and olanzapine. However, the IDILI-related drugs diclofenac, carbamazepine, and troglitazone did not show synergistic toxicity with LPS after incubation for 24 h. Co-incubation of ketoconazole and clozapine with LPS decreased the levels of glutathione in hPCLS, but this was not seen for the other drugs. All drugs affected LPS-induced cytokine release, but interestingly, only ketoconazole and clozapine increased the level of LPS-induced TNF release. Decreased levels of glutathione and cysteine conjugates of clozapine were detected in IDILI-responding livers following cotreatment with LPS. In conclusion, we identified ketoconazole and clozapine as drugs that exhibited synergistic toxicity with LPS, while glutathione and TNF were found to be potential biomarkers for IDILI-inducing drugs mediated by inflammatory stress. hPCLS appear to be suitable for further unraveling the mechanisms of inflammatory stress-associated IDILI.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Clozapina/toxicidad , Cetoconazol/toxicidad , Adolescente , Adulto , Anciano , Femenino , Humanos , Técnicas In Vitro , Lipopolisacáridos/toxicidad , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
The aim of this study was to develop a pharmacokinetic-pharmacodynamic (PKPD) model that quantifies the efficacy of haloperidol, accounting for the placebo effect, the variability in exposure-response, and the dropouts. Subsequently, the developed model was utilized to characterize an effective dosing strategy for using haloperidol as a comparator drug in future antipsychotic drug trials. The time course of plasma haloperidol concentrations from 122 subjects and the Positive and Negative Syndrome Scale (PANSS) scores from 473 subjects were used in this analysis. A nonlinear mixed-effects modeling approach was utilized to describe the time course of PK and PANSS scores. Bootstrapping and simulation-based methods were used for the model evaluation. A 2-compartment model adequately described the haloperidol PK profiles. The Weibull and Emax models were able to describe the time course of the placebo and the drug effects, respectively. An exponential model was used to account for dropouts. Joint modeling of the PKPD model with dropout model indicated that the probability of patients dropping out is associated with the observed high PANSS score. The model evaluation results confirmed that the precision and accuracy of parameter estimates are acceptable. Based on the PKPD analysis, the recommended oral dose of haloperidol to achieve a 30% reduction in PANSS score from baseline is 5.6 mg/d, and the corresponding steady-state effective plasma haloperidol exposure is 2.7 ng/mL. In conclusion, the developed model describes the time course of PANSS scores adequately, and a recommendation of haloperidol dose was derived for future antipsychotic drug trials.
Asunto(s)
Antipsicóticos/uso terapéutico , Haloperidol/uso terapéutico , Modelos Biológicos , Esquizofrenia/tratamiento farmacológico , Adolescente , Adulto , Anciano , Antipsicóticos/farmacocinética , Femenino , Haloperidol/farmacocinética , Humanos , Masculino , Persona de Mediana Edad , Método de Montecarlo , Dinámicas no Lineales , Pacientes Desistentes del Tratamiento , Escalas de Valoración Psiquiátrica , Factores de Tiempo , Adulto JovenRESUMEN
1.The precision-cut intestinal slice (PCIS) technology is a relatively new addition to the battery of in vitro assays for evaluation of xenobiotic toxicity, metabolism, and transport. 2.The intestine is an important target for drug-induced toxicity due to its high exposure after oral administration. Therefore, the prediction of drug-induced intestinal side effects remains a significant safety issue in pharmaceutical development. Although animal experiments have been proven useful, species differences and the requirement for reduction of animal use warrant the development of in vitro methods which can apply human tissue. 3.The enterocytes lining the villi express high activities of enzymes and transporters involved in drug disposition. They vary highly in activities: along the length of the intestine and along the villi, gradients of expression levels of the enzymes and proteins exist, which necessitates an in vitro model that can reflect the different regions of the intestine. 4.In this chapter, the application of PCIS in studies on transport and toxicity of xenobiotics is reviewed. PCIS can be prepared from each region of the intestine and from various species in a similar manner, and the results published so far indicate that they represent a promising model to evaluate intestinal toxicity and transport.