RESUMEN
Microphysiological systems (MPS) are comprised of one or multiple cell types of human or animal origins that mimic the biochemical/electrical/mechanical responses and blood-tissue barrier properties of the cells observed within a complex organ. The goal of incorporating these in vitro systems is to expedite and advance the drug discovery and development paradigm with improved predictive and translational capabilities. Considering the industry need for improved efficiency and the broad challenges of model qualification and acceptance, the International Consortium for Innovation and Quality (IQ) founded an IQ MPS working group in 2014 and Affiliate in 2018. This group connects thought leaders and end users, provides a forum for crosspharma collaboration, and engages with regulators to qualify translationally relevant MPS models. To understand how pharmaceutical companies are using MPS, the IQ MPS Affiliate conducted two surveys in 2019, survey 1, and 2021, survey 2, which differed slightly in the scope of definition of the complex in vitro models under question. The surveys captured demographics, resourcing, rank order for organs of interest, compound modalities tested, and MPS organ-specific questions, including nonclinical species needs and cell types. The major focus of this manuscript is on results from survey 2, where we specifically highlight the context of use for MPS within safety, pharmacology, or absorption, disposition, metabolism, and excretion and discuss considerations for including MPS data in regulatory submissions. In summary, these data provide valuable insights for developers, regulators, and pharma, offering a view into current industry practices and future considerations while highlighting key challenges impacting MPS adoption. SIGNIFICANCE STATEMENT: The application of microphysiological systems (MPS) represents a growing area of interest in the drug discovery and development framework. This study surveyed 20+ pharma companies to understand resourcing, current areas of application, and the key challenges and barriers to internal MPS adoption. These results will provide regulators, tech providers, and pharma industry leaders a starting point to assess the current state of MPS applications along with key learnings to effectively realize the potential of MPS as an emerging technology.
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Industria Farmacéutica , Sistemas Microfisiológicos , Animales , Humanos , Descubrimiento de DrogasRESUMEN
The beta-site APP cleaving enzyme 1, known as BACE1, has been a widely pursued Alzheimer's disease drug target owing to its critical role in the production of amyloid-beta. We have previously reported the clinical development of LY2811376 and LY2886721. LY2811376 advanced to Phase I before development was terminated due to nonclinical retinal toxicity. LY2886721 advanced to Phase II, but development was halted due to abnormally elevated liver enzymes. Herein, we report the discovery and clinical development of LY3202626, a highly potent, CNS-penetrant, and low-dose BACE inhibitor, which successfully addressed these key development challenges.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Compuestos Heterocíclicos con 2 Anillos/farmacología , Inhibidores de Proteasas/farmacología , Pirazinas/farmacología , Pirroles/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Barrera Hematoencefálica/fisiología , Encéfalo/metabolismo , Cristalografía por Rayos X , Perros , Estabilidad de Medicamentos , Compuestos Heterocíclicos con 2 Anillos/síntesis química , Compuestos Heterocíclicos con 2 Anillos/farmacocinética , Humanos , Células de Riñón Canino Madin Darby , Masculino , Ratones , Microsomas Hepáticos/metabolismo , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacocinética , Unión Proteica , Pirazinas/síntesis química , Pirazinas/farmacocinética , Pirroles/síntesis química , Pirroles/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
BACKGROUND: The United States faces a national crisis involving opioid medications, where currently more than 130 people die every day. To combat this epidemic, a better understanding is needed of how opioids penetrate into the central nervous system (CNS) to facilitate pain relief and, potentially, result in addiction and/or misuse. Animal models, however, are a poor predictor of blood-brain barrier (BBB) transport and CNS drug penetration in humans, and many traditional 2D cell culture models of the BBB and neurovascular unit have inadequate barrier function and weak or inappropriate efflux transporter expression. Here, we sought to better understand opioid transport mechanisms using a simplified microfluidic neurovascular unit (NVU) model consisting of human brain microvascular endothelial cells (BMECs) co-cultured with astrocytes. METHODS: Human primary and induced pluripotent stem cell (iPSC)-derived BMECs were incorporated into a microfluidic NVU model with several technical improvements over our previous design. Passive barrier function was assessed by permeability of fluorescent dextrans with varying sizes, and P-glycoprotein function was assessed by rhodamine permeability in the presence or absence of inhibitors; quantification was performed with a fluorescent plate reader. Loperamide, morphine, and oxycodone permeability was assessed in the presence or absence of P-glycoprotein inhibitors and cortisol; quantification was performed with mass spectrometry. RESULTS: We first report technical and methodological optimizations to our previously described microfluidic model using primary human BMECs, which results in accelerated barrier formation, decreased variability, and reduced passive permeability relative to Transwell models. We then demonstrate proper transport and efflux of loperamide, morphine, and oxycodone in the microfluidic NVU containing BMECs derived from human iPSCs. We further demonstrate that cortisol can alter permeability of loperamide and morphine in a divergent manner. CONCLUSIONS: We reveal a novel role for the stress hormone cortisol in modulating the transport of opioids across the BBB, which could contribute to their abuse or overdose. Our updated BBB model represents a powerful tool available to researchers, clinicians, and drug manufacturers for understanding the mechanisms by which opioids access the CNS.
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Analgésicos Opioides/farmacocinética , Astrocitos/fisiología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/fisiología , Células Endoteliales/fisiología , Hidrocortisona/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Modelos Neurológicos , Astrocitos/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Microvasos/citologíaRESUMEN
The discovery of mammalian cardiac progenitor cells has suggested that the heart consists of not only terminally differentiated beating cardiomyocytes, but also a population of self-renewing stem cells with the potential to generate new cardiomyocytes (Anderson, D., Self, T., Mellor, I. R., Goh, G., Hill, S. J., and Denning, C. 2007. Transgenic enrichment of cardiomyocytes from human embryonic stem cells. Mol. Ther. 15, 2027-2036; Bearzi, C., Rota, M., Hosoda, T., Tillmanns, J., Nascimbene, A., De Angelis, A., Yasuzawa-Amano, S., Trofimova, I., Siggins, R. W., Lecapitaine, N., Cascapera, S., Beltrami, A. P., D'Alessandro, D. A., Zias, E., Quaini, F., Urbanek, K., Michler, R. E., Bolli, R., Kajstura, J., Leri, A., et al. 2007. Human cardiac stem cells. Proc. Natl. Acad. Sci. U.S.A. 104, 14068-14073; Wu, S. M., Chien, K. R., and Mummery, C. 2008. Origins and fates of cardiovascular progenitor cells. Cell 132, 537-543). A consequence of longevity is continual exposure to environmental and xenobiotic stresses, and recent literature suggests that hematopoietic stem cell pools tightly control cell health through upregulation of the integrated stress response and consequent cellular mechanisms such as apoptosis. However, whether or not this biological response is conserved in progenitor cells for later lineages of tissue-specific stem cells is not well understood. Using human-induced pluripotent stem cells (iPSC) of both cardiac progenitor and mature cardiomyocyte lineages, we found that the integrated stress response was upregulated in the iPSC cardiac progenitors leading to an increased sensitivity for apoptosis relative to the mature cardiomyocytes. Of interest, C/EBP homologous protein (CHOP) signaling plays a mechanistic role in the cell death phenotype observed in iPSC progenitors, by which depletion of CHOP prevents cell death following cellular stress by thapsigargin exposure. Our studies suggest that the integrated stress response plays a unique role in maintaining iPSC cardiac progenitor cellular integrity by removing unhealthy cells via apoptosis following environmental and xenobiotic stresses, thus preventing differentiation and self-renewal of damaged cells.
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Estrés del Retículo Endoplásmico/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Xenobióticos/toxicidad , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteína 9 Asociada a CRISPR/genética , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Linaje de la Célula , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Estrés del Retículo Endoplásmico/genética , Técnicas de Silenciamiento del Gen , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Factor de Transcripción CHOP/metabolismo , Transcriptoma/efectos de los fármacosRESUMEN
Injection site reactions (ISRs) are commonly encountered in the development of parenteral drugs, and severe ISRs can lead to preclinical and clinical dose limiting toxicities. Tools to assess the risk of clinical ISRs during drug development are not well established. We developed an in vitro ISR screen using L6 rat myotubes to assess compounds for irritation risk. Reference compounds that were either known to induce ISRs or were non-irritating in the clinical setting were used to validate this method. We evaluated three compounds, two with known clinical ISRs (mitoxantrone and doxorubicin) and one without clinical ISR (metoprolol), using a preclinical in vivo rat model and the L6 in vitro model at clinically relevant concentrations, and showed that the L6 assay is a better prognostic indicator for clinical ISR risk. We then utilized this assay during early preclinical development to guide optimization of structure activity relationship (SAR), selection of dose concentrations for pre-clinical in vivo experiments, and prioritization of alternative formulations to minimize ISR risk. Our studies indicate that the L6 assay is a better measure of clinical ISR risk than current in vivo preclinical models, and that it can help guide not only compound selection, but also selection of dose concentration and formulation.
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Doxorrubicina/toxicidad , Irritantes/toxicidad , Mitoxantrona/toxicidad , Fibras Musculares Esqueléticas/efectos de los fármacos , Pruebas de Irritación de la Piel/métodos , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Doxorrubicina/administración & dosificación , Femenino , Inyecciones , Irritantes/administración & dosificación , Mitoxantrona/administración & dosificación , Fibras Musculares Esqueléticas/patología , Ratas , Ratas Sprague-Dawley , Medición de RiesgoRESUMEN
Due to the complex physiology of the testes, in vitro models have been largely unsuccessful at modeling testicular toxicity in vivo. We conducted a pilot study to evaluate the utility of the Durand ex vivo rat seminiferous tubule culture model [1-3] that supports spermatogenesis through meiosis II, including the formation of round spermatids. We used this system to evaluate the toxicity of four known testicular toxicants: 1,3-dinitrobenzene (DNB), 2-methoxyacetic acid (MAA), bisphenol A (BPA), and lindane over 21 days of culture. This organotypic culture system demonstrated the ability to successfully model in vivo testicular toxicity (Sertoli cell toxicity and disruption of meiosis) for all four compounds. These findings support the application of this system to study molecules and evaluate mechanisms of testicular toxicity.
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Túbulos Seminíferos/efectos de los fármacos , Pruebas de Toxicidad/métodos , Acetatos/toxicidad , Animales , Compuestos de Bencidrilo/toxicidad , Células Cultivadas , Dinitrobencenos/toxicidad , Células Germinativas/efectos de los fármacos , Hexaclorociclohexano/toxicidad , Masculino , Fenoles/toxicidad , Proyectos Piloto , Ratas , Ratas Sprague-Dawley , Células de Sertoli/efectos de los fármacos , Técnicas de Cultivo de TejidosRESUMEN
Mdr1a-, Bcrp-, and Mrp2-knockout rats are a more practical species for absorption, distribution, metabolism, and excretion (ADME) studies than murine models and previously demonstrated expected alterations in the pharmacokinetics of various probe substrates. At present, gene expression and pathology changes were systematically studied in the small intestine, liver, kidney, and brain tissue from male SAGE Mdr1a, Bcrp, and Mrp2 knockout rats versus wild-type Sprague-Dawley controls. Gene expression data supported the relevant knockout genotype. As expected, Mrp2 knockout rats were hyperbilirubinemic and exhibited upregulation of hepatic Mrp3. Overall, few alterations were observed within 112 ADME-relevant genes. The two potentially most consequential changes were upregulation of intestinal carboxylesterase in Mdr1a knockouts and catechol-O-methyltransferase in all tissues of Bcrp knockout rats. Previously reported upregulation of hepatic Mdr1b P-glycoprotein in proprietary Wistar Mdr1a knockout rats was not observed in the SAGE counterpart investigated herein. Relative liver and kidney weights were 22-53% higher in all three knockouts, with microscopic increases in hepatocyte size in Mdr1a and Mrp2 knockout rats and glomerular size in Bcrp and Mrp2 knockouts. Increased relative weight of clearing organs is quantitatively consistent with reported increases in the clearance of drugs that are not substrates of the knocked-out transporter. Overall, SAGE knockout rats demonstrated modest compensatory changes, which do not preclude their general application to study transporter-mediated pharmacokinetics. However, until future studies elucidate the magnitude of functional change, caution is warranted in rare instances of extensive metabolism by catechol-O-methyltransferase in Bcrp knockouts and intestinal carboxylesterase in Mdr1a knockout rats, specifically for molecules with free catechol groups and esters subject to gut-wall hydrolysis.
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Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Técnicas de Inactivación de Genes , Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/deficiencia , Transportadoras de Casetes de Unión a ATP/genética , Animales , Técnicas de Inactivación de Genes/métodos , Hepatocitos/citología , Hepatocitos/metabolismo , Glomérulos Renales/citología , Glomérulos Renales/metabolismo , Hígado/citología , Hígado/metabolismo , Masculino , Transporte de Proteínas/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
A primary goal of lead optimization is to identify compounds with improved absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. A number of reports have linked computed molecular properties to desirable in vivo ADMET outcomes, but a significant limitation of these analyses is the failure to control statistically for possible covariates. We examine the relationship between molecular properties and in vitro surrogate assays vs in vivo properties within 173 chemical series from a database of 3773 compounds with rodent pharmacokinetic and toxicology data. This approach identifies the following pairs of surrogates as most predictive among those examined: rat primary hepatocyte (RPH) cytolethality/volume of distribution (V(d)) for in vivo toxicology outcomes, scaled microsome metabolism/calculated logP for in vivo unbound clearance, and calculated logD/kinetic aqueous solubility for thermodynamic solubility. The impact of common functional group substitutions is examined and provides insights for compound design.
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Descubrimiento de Drogas/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Preparaciones Farmacéuticas/metabolismo , Farmacocinética , Absorción , Animales , Disponibilidad Biológica , Femenino , Preparaciones Farmacéuticas/química , Ratas , Solubilidad , TermodinámicaAsunto(s)
Carcinógenos/toxicidad , Hidrazinas/toxicidad , Hígado/efectos de los fármacos , Xenobióticos/toxicidad , Alanina Transaminasa/antagonistas & inhibidores , Alanina Transaminasa/metabolismo , Animales , Aspartato Aminotransferasas/antagonistas & inhibidores , Aspartato Aminotransferasas/metabolismo , Carcinógenos/administración & dosificación , Relación Dosis-Respuesta a Droga , Hidrazinas/administración & dosificación , Hígado/enzimología , Masculino , Fosfato de Piridoxal/metabolismo , Ratas , Ratas Sprague-Dawley , Xenobióticos/administración & dosificaciónRESUMEN
In order to determine a threshold for nongenotoxic carcinogens, the traditional risk assessment approach has been to identify a mode of action (MOA) with a nonlinear dose-response. The dose-response for one or more key event(s) linked to the MOA for carcinogenicity allows a point of departure (POD) to be selected from the most sensitive effect dose or no-effect dose. However, this can be challenging because multiple MOAs and key events may exist for carcinogenicity and oftentimes extensive research is required to elucidate the MOA. In the present study, a microarray analysis was conducted to determine if a POD could be identified following short-term oral rat exposure with two nongenotoxic rodent carcinogens, fenofibrate and methapyrilene, using a benchmark dose analysis of genes aggregated in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) biological processes, which likely encompass key event(s) for carcinogenicity. The gene expression response for fenofibrate given to rats for 2days was consistent with its MOA and known key events linked to PPARα activation. The temporal response from daily dosing with methapyrilene demonstrated biological complexity with waves of pathways/biological processes occurring over 1, 3, and 7days; nonetheless, the benchmark dose values were consistent over time. When comparing the dose-response of toxicogenomic data to tumorigenesis or precursor events, the toxicogenomics POD was slightly below any effect level. Our results suggest that toxicogenomic analysis using short-term studies can be used to identify a threshold for nongenotoxic carcinogens based on evaluation of potential key event(s) which then can be used within a risk assessment framework.
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Carcinógenos/toxicidad , Fenofibrato/toxicidad , Metapirileno/análisis , Metapirileno/toxicidad , Neoplasias/inducido químicamente , Toxicogenética/métodos , Animales , Carcinógenos/administración & dosificación , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/genética , Relación Dosis-Respuesta a Droga , Femenino , Fenofibrato/administración & dosificación , Expresión Génica , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/genética , Metapirileno/administración & dosificación , Neoplasias/genética , Nivel sin Efectos Adversos Observados , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Medición de RiesgoRESUMEN
Drug safety remains a high profile issue at a time when the cost and time required to develop a new drug are at an all time high. Balancing risk against the expected clinical benefit is the primary purpose of preclinical and clinical testing. We offer an expanded view on the application of predictive strategies and technologies to early safety decisions and suggestions to narrow the focus for improving preclinical safety testing to the problems that contribute most to adverse drug reactions.
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Evaluación Preclínica de Medicamentos/tendencias , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Pruebas de Toxicidad/tendencias , Animales , Ensayos Clínicos como Asunto , Diseño de Fármacos , Humanos , Tecnología Farmacéutica/métodosRESUMEN
Marked species-specific responses to agonists of the peroxisome proliferator-activated alpha receptor (PPAR alpha) have been observed in rats and dogs, two species typically used to assess the potential human risk of pharmaceuticals in development. In this study, we used primary cultured rat and dog hepatocytes to investigate the underlying mechanisms of a novel PPAR alpha and -gamma coagonist, LY465608, relative to fenofibrate, a prototypical PPAR alpha agonist. As expected, rat hepatocytes incubated with these two agonists demonstrated an increase in peroxisome number as evaluated by electron microscopy, whereas the peroxisome number remained unchanged in dog hepatocytes. Biochemical analysis showed that rat hepatocytes responded to PPAR agonists with an induction of both peroxisomal and mitochondrial beta-oxidation (PBox and MBox) activities. Dog hepatocytes treated with both PPAR agonists, however, did not show increased PBox activity but did demonstrate increased MBox activity. Analysis of peroxisomal beta-oxidation gene expression markers by quantitative real-time PCR confirmed that PPAR agonists induced the peroxisomal enzymes, acyl-coenzyme A (CoA) oxidase (Acox), enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase (Ehhadh), and 3-ketoacyl-CoA thiolase (Acaa1) at the transcriptional level in rat hepatocytes, but not dog hepatocytes. Expression of mRNA for the mitochondrial beta-oxidation gene hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase (Hadhb), however, increased in both rat and dog hepatocytes, consistent with biochemical measurements of peroxisomal and mitochondrial beta-oxidation. Repeat-dose nonclinical safety studies of LY465608 revealed abnormities in mitochondrial morphology and evidence of single-cell necrosis following 30 days of dosing exclusively in dogs, but not in rats. Microarray analysis indicated that dog hepatocytes, but not rat hepatocytes, treated with LY465608 had an expression profile consistent with abnormalities in the regulation of cell renewal and death, oxidative stress, and mitochondrial bioenergetics, which may explain the canine-specific toxicity observed in vivo with this compound. This increased sensitivity to mitochondrial toxicity of canine hepatocytes relative to rat hepatocytes identified using gene expression was confirmed using the fluorescent indicator tetramethylrhodamine ethyl ester (TMRE) and flow cytometry. At doses of 0.1 microM LY465608, canine hepatocytes showed a greater shift in fluorescence indicative of mitochondrial damage than observed with rat hepatocytes treated at 10 microM. In summary, using rat and dog primary hepatocytes, we replicated the pharmacologic and toxicologic effects of LY465608 observed in vivo during preclinical development and propose an underlying mechanism for these species-specific effects.
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Hepatocitos/efectos de los fármacos , Compuestos Orgánicos/farmacología , PPAR alfa/agonistas , PPAR gamma/agonistas , Animales , Bovinos , Células Cultivadas , Perros , Femenino , Fenofibrato/farmacología , Fenofibrato/toxicidad , Citometría de Flujo/métodos , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , Hipolipemiantes/farmacología , Hipolipemiantes/toxicidad , Masculino , Microscopía Electrónica , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Compuestos Orgánicos/toxicidad , Oxidación-Reducción , Peroxisomas/efectos de los fármacos , Peroxisomas/metabolismo , Peroxisomas/ultraestructura , Ratas , Ratas Endogámicas F344 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la EspecieRESUMEN
Cadmium toxicity has been evaluated in a number of in vivo and in vitro toxicological studies. In vivo Cd toxicity exhibits sexual dimorphism with females being more susceptible to Cd uptake, accumulation, and toxicity in the liver. Research to date does not explain why females are more sensitive to Cd-induced hepatotoxicity. Recent studies demonstrate that progesterone sensitizes female F(344) rats and TRL-1215 cells to Cd toxicity, however the mode of action is still unclear. Approximately one half of the Cd entering the cytoplasm does so through receptor operated Ca(2+) channels. Progesterone treatment of human spermatozoa and Xenopus laevis oocytes causes a rapid influx of Ca(2+) suggesting a possible mechanism. Since hepatocytes have progesterone receptors on their cellular membrane and Ca(2+) influx into the cytoplasm occurs following progesterone treatment we evaluated the hypothesis that progesterone facilitates the uptake and accumulation of Cd via Ca(2+) channels, leading to enhanced toxicity. Primary isolated rat hepatocytes were treated with Cd, progesterone, and/or verapamil for 4 h and cytolethality was measured. Pretreatment with the Ca(2+) channel blocker verapamil increased the Cd concentration producing 50% lethality (LC(50)) by 2-fold, thus decreasing Cd cytolethality. In contrast, pretreatment with progesterone decreased the Cd LC(50) by 2-fold resulting in enhanced Cd cytolethality. Verapamil treatment reversed the progesterone enhanced Cd cytolethality. Verapamil and/or progesterone in the absence of Cd did not affect hepatocyte viability. Overall, the results of this study demonstrate that inhibition of progesterone-induced Ca(2+) influx with the Ca(2+) channel blocker verapamil, decreases Cd cytolethality in primary isolated rat hepatocytes. These findings indicate that progesterone activation of receptor-mediated Ca(2+) channels is involved in the sexually dimorphic hepatotoxicity seen following acute Cd exposure.