RESUMEN
Liquid-liquid phase separation (LLPS) is thought to be a main driving force in the formation of membraneless organelles. Examples of such organelles include the centrosome, central spindle, and stress granules. Recently, it has been shown that coiled-coil (CC) proteins, such as the centrosomal proteins pericentrin, spd-5, and centrosomin, might be capable of LLPS. CC domains have physical features that could make them the drivers of LLPS, but it is unknown if they play a direct role in the process. We developed a coarse-grained simulation framework for investigating the LLPS propensity of CC proteins, in which interactions that support LLPS arise solely from CC domains. We show, using this framework, that the physical features of CC domains are sufficient to drive LLPS of proteins. The framework is specifically designed to investigate how the number of CC domains, as well as the multimerization state of CC domains, can affect LLPS. We show that small model proteins with as few as two CC domains can phase separate. Increasing the number of CC domains up to four per protein can somewhat increase LLPS propensity. We demonstrate that trimer-forming and tetramer-forming CC domains have a dramatically higher LLPS propensity than dimer-forming coils, which shows that multimerization state has a greater effect on LLPS than the number of CC domains per protein. These data support the hypothesis of CC domains as drivers of protein LLPS, and have implications in future studies to identify the LLPS-driving regions of centrosomal and central spindle proteins.
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Proteínas Intrínsecamente Desordenadas , Proteínas Intrínsecamente Desordenadas/metabolismo , Separación de Fases , Dominios Proteicos , Orgánulos/metabolismoRESUMEN
The canine spontaneous cancer model is increasingly utilized to evaluate new combined cancer immunotherapy approaches. While the major leukocyte subsets and phenotypes are closely related in dogs and humans, the functionality of T cells and antigen presenting cells in the two species has not been previously compared in detail. Such information would be important in interpreting immune response data and evaluating the potential toxicities of new cancer immunotherapies in dogs. To address this question, we used in vitro assays to compare the transcriptomic, cytokine, and proliferative responses of activated canine and human T cells, and also compared responses in activated macrophages. Transcriptomic analysis following T cell activation revealed shared expression of 515 significantly upregulated genes and 360 significantly downregulated immune genes. Pathway analysis identified 33 immune pathways shared between canine and human activated T cells, along with 34 immune pathways that were unique to each species. Activated human T cells exhibited a marked Th1 bias, whereas canine T cells were transcriptionally less active overall. Despite similar proliferative responses to activation, canine T cells produced significantly less IFN-γ than human T cells. Moreover, canine macrophages were significantly more responsive to activation by IFN-γ than human macrophages, as reflected by co-stimulatory molecule expression and TNF-α production. Thus, these studies revealed overall broad similarity in responses to immune activation between dogs and humans, but also uncovered important key quantitative and qualitative differences, particularly with respect to T cell responses, that should be considered in designing and evaluating cancer immunotherapy studies in dogs.
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Citocinas , Neoplasias , Humanos , Perros , Animales , Citocinas/metabolismo , Linfocitos T/metabolismo , Activación de Linfocitos , Perfilación de la Expresión Génica , Neoplasias/genética , Neoplasias/terapia , Neoplasias/veterinariaRESUMEN
Membraneless liquid compartments based on phase-separating biopolymers have been observed in diverse cell types and attributed to weak multivalent interactions predominantly based on intrinsically disordered domains. The design of liquid-liquid phase separated (LLPS) condensates based on de novo designed tunable modules that interact in a well-understood, controllable manner could improve our understanding of this phenomenon and enable the introduction of new features. Here we report the construction of CC-LLPS in mammalian cells, based on designed coiled-coil (CC) dimer-forming modules, where the stability of CC pairs, their number, linkers, and sequential arrangement govern the transition between diffuse, liquid and immobile condensates and are corroborated by coarse-grained molecular simulations. Through modular design, we achieve multiple coexisting condensates, chemical regulation of LLPS, condensate fusion, formation from either one or two polypeptide components or LLPS regulation by a third polypeptide chain. These findings provide further insights into the principles underlying LLPS formation and a design platform for controlling biological processes.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Péptidos , Animales , Proteínas Intrínsecamente Desordenadas/metabolismo , Mamíferos/metabolismoRESUMEN
Liquid-liquid phase separation (LLPS) is thought to be a main driving force in the formation of membraneless organelles. Examples of such organelles include the centrosome, central spindle, and stress granules. Recently, it has been shown that coiled-coil (CC) proteins, such as the centrosomal proteins pericentrin, spd-5, and centrosomin, might be capable of LLPS. CC domains have physical features that could make them the drivers of LLPS, but it is unknown if they play a direct role in the process. We developed a coarse-grained simulation framework for investigating the LLPS propensity of CC proteins, in which interactions which support LLPS arise solely from CC domains. We show, using this framework, that the physical features of CC domains are sufficient to drive LLPS of proteins. The framework is specifically designed to investigate how the number of CC domains, as well as multimerization state of CC domains, can affect LLPS. We show that small model proteins with as few as two CC domains can phase separate. Increasing the number of CC domains up to four per protein can somewhat increase LLPS propensity. We demonstrate that trimer-forming and tetramer-forming CC domains have a dramatically higher LLPS propensity than dimer-forming coils, which shows that multimerization state has a greater effect on LLPS than the number of CC domains per protein. These data support the hypothesis of CC domains as drivers of protein LLPS, and has implications in future studies to identify the LLPS-driving regions of centrosomal and central spindle proteins.
RESUMEN
Vinblastine (VBL) is a vinca alkaloid-class cytotoxic chemotherapeutic that causes microtubule disruption and is typically used to treat hematologic malignancies. VBL is characterized by a narrow therapeutic index, with key dose-limiting toxicities being myelosuppression and neurotoxicity. Pharmacokinetics (PK) of VBL is primarily driven by ABCB1-mediated efflux and CYP3A4 metabolism, creating potential for drug-drug interaction. To characterize sources of variability in VBL PK, we developed a physiologically based pharmacokinetic (PBPK) model in Mdr1a/b(-/-) knockout and wild-type mice by incorporating key drivers of PK, including ABCB1 efflux, CYP3A4 metabolism, and tissue-specific tubulin binding, and scaled this model to accurately simulate VBL PK in humans and pet dogs. To investigate the capability of the model to capture interindividual variability in clinical data, virtual populations of humans and pet dogs were generated through Monte Carlo simulation of physiologic and biochemical parameters and compared to the clinical PK data. This model provides a foundation for predictive modeling of VBL PK. The base PBPK model can be further improved with supplemental experimental data identifying drug-drug interactions, ABCB1 polymorphisms and expression, and other sources of physiologic or metabolic variability.
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Antineoplásicos , Vinblastina , Humanos , Perros , Ratones , Animales , Vinblastina/farmacocinética , Citocromo P-450 CYP3A/genética , Antineoplásicos/farmacocinética , Interacciones Farmacológicas , Transporte BiológicoRESUMEN
Insulin has been commonly adopted as a peptide drug to treat diabetes as it facilitates the uptake of glucose from the blood. The development of oral insulin remains elusive over decades owing to its susceptibility to the enzymes in the gastrointestinal tract and poor permeability through the intestinal epithelium upon dimerization. Recent experimental studies have revealed that certain O-linked glycosylation patterns could enhance insulin's proteolytic stability and reduce its dimerization propensity, but understanding such phenomena at the molecular level is still difficult. To address this challenge, we proposed and tested several structural determinants that could potentially influence insulin's proteolytic stability and dimerization propensity. We used these metrics to assess the properties of interest from [Formula: see text] aggregate molecular dynamics of each of 12 targeted insulin glyco-variants from multiple wild-type crystal structures. We found that glycan-involved hydrogen bonds and glycan-dimer occlusion were useful metrics predicting the proteolytic stability and dimerization propensity of insulin, respectively, as was in part the solvent-accessible surface area of proteolytic sites. However, other plausible metrics were not generally predictive. This work helps better explain how O-linked glycosylation influences the proteolytic stability and monomeric propensity of insulin, illuminating a path towards rational molecular design of insulin glycoforms.
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Insulina , Simulación de Dinámica Molecular , Dimerización , Insulina/análogos & derivados , Insulina/química , PolisacáridosRESUMEN
Cannabidiol (CBD), the major non-psychoactive compound found in cannabis, is frequently used both as a nutraceutical and therapeutic. Despite anecdotal evidence as an anticancer agent, little is known about the effect CBD has on cancer cells. Given the intractability and poor prognoses of brain cancers in human and veterinary medicine, we sought to characterize the in vitro cytotoxicity of CBD on human and canine gliomas. Glioma cells treated with CBD showed a range of cytotoxicity from 4.9 to 8.2 µg/ml; canine cells appeared to be more sensitive than human. Treatment with >5 µg/ml CBD invariably produced large cytosolic vesicles. The mode of cell death was then interrogated using pharmacologic inhibitors. Inhibition of apoptosis was sufficient to rescue CBD-mediated cytotoxicity. Inhibition of RIPK3, a classical necroptosis kinase, also rescued cells from death and prevented the formation of the large cytosolic vesicles. Next, cellular mitochondrial activity in the presence of CBD was assessed and within 2 hours of treatment CBD reduced oxygen consumption in a dose dependent manner with almost complete ablation of activity at 10 µg/ml CBD. Fluorescent imaging with a mitochondrial-specific dye revealed that the large cytosolic vesicles were, in fact, swollen mitochondria. Lastly, calcium channels were pharmacologically inhibited and the effect on cell death was determined. Inhibition of mitochondrial channel VDAC1, but not the TRPV1 channel, rescued cells from CBD-mediated cytotoxicity. These results demonstrate the cytotoxic nature of CBD in human and canine glioma cells and suggest a mechanism of action involving dysregulation of calcium homeostasis and mitochondrial activity.
RESUMEN
BACKGROUND: Intra-articular (IA) antibiotic usage is prevalent in equine practice. However, recent emergence of antimicrobial resistance prompts re-evaluation of antibiotic selection, particularly when used prophylactically. Furthermore, many commonly used antibiotics exert direct cytotoxicity to equine cells, and appropriate IA doses have not been defined. OBJECTIVES: To screen antibiotics in vitro as an initial assessment of cytotoxicity against normal equine joint cells in monolayer culture and explant tissues. STUDY DESIGN: In vitro experimental study. METHODS: Chondrocytes and synovial cells were harvested from three horses and plated on 24-well plates (100 000 cells/wells in triplicate) for 48 hours prior to addition of antibiotics. Joint cells were exposed to antibiotics (n = 15) at various doses (25-0.39 mg/mL in complete DMEM media) for 24 hours and viability was assessed by trypan blue dye exclusion. The half maximal inhibitory concentration (IC50) was determined for each antibiotic. Cartilage explants were obtained from 3 horses, minced and exposed to antibiotics (n = 5) for 72 hours. Live/dead staining was performed, and fluorescence was visualised using Olympus IX83 spinning disk confocal microscope. Percentage of live vs dead cells was quantified. RESULTS: Antibiotics from different antimicrobial classes expressed dose-dependent but variable cytotoxicity to equine joint cells in vitro. Aminoglycosides and doxycycline had the lowest IC50 (most toxic). Ampicillin sulbactam, imipenem, tobramycin, ceftiofur sodium and amoxicillin had IC50 > 25 mg/mL for at least one cell line, representing potentially less cytotoxic alternatives. MAIN LIMITATIONS: Further studies are necessary to extrapolate these in vitro data results to the in vivo joint environment. CONCLUSIONS: Targeted IA antibiotic therapy would involve selection of the safest antibiotics (highest IC50) with efficacy based on bacterial culture/sensitivity. Antimicrobial selection and evidence-based dosing may minimise damage to native articular cartilage and synovial cells and development of antimicrobial resistance when IA antibiotics are used in equine practice.
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Cartílago Articular , Condrocitos , Animales , Antibacterianos/toxicidad , CaballosRESUMEN
Since the discovery of CHD1L in 2008, it has emerged as an oncogene implicated in the pathology and poor prognosis of a variety of cancers, including gastrointestinal cancers. However, a mechanistic understanding of CHD1L as a driver of colorectal cancer has been limited. Until now, there have been no reported inhibitors of CHD1L, also limiting its development as a molecular target. We sought to characterize the clinicopathologic link between CHD1L and colorectal cancer, determine the mechanism(s) by which CHD1L drives malignant colorectal cancer, and discover the first inhibitors with potential for novel treatments for colorectal cancer. The clinicopathologic characteristics associated with CHD1L expression were evaluated using microarray data from 585 patients with colorectal cancer. Further analysis of microarray data indicated that CHD1L may function through the Wnt/TCF pathway. Thus, we conducted knockdown and overexpression studies with CHD1L to determine its role in Wnt/TCF-driven epithelial-to-mesenchymal transition (EMT). We performed high-throughput screening (HTS) to identify the first CHD1L inhibitors. The mechanism of action, antitumor efficacy, and drug-like properties of lead CHD1L inhibitors were determined using biochemical assays, cell models, tumor organoids, patient-derived tumor organoids, and in vivo pharmacokinetics and pharmacodynamics. Lead CHD1L inhibitors display potent in vitro antitumor activity by reversing TCF-driven EMT. The best lead CHD1L inhibitor possesses drug-like properties in pharmacokinetic/pharmacodynamic mouse models. This work validates CHD1L as a druggable target and establishes a novel therapeutic strategy for the treatment of colorectal cancer.
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Adenocarcinoma/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , ADN Helicasas/antagonistas & inhibidores , Proteínas de Unión al ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/uso terapéutico , Proteínas de Neoplasias/antagonistas & inhibidores , Adenocarcinoma/mortalidad , Animales , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Apoptosis , Línea Celular Tumoral , Neoplasias Colorrectales/mortalidad , Daño del ADN , ADN Helicasas/genética , ADN Helicasas/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/toxicidad , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Ensayos Analíticos de Alto Rendimiento , Humanos , Estimación de Kaplan-Meier , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Organoides/efectos de los fármacos , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequeñas , Factores de Transcripción TCF/fisiología , Transcripción Genética/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genética , Vía de Señalización Wnt/fisiologíaRESUMEN
Metastasis is the major cause of mortality for patients with cancer, and dysregulation of developmental signaling pathways can significantly contribute to the metastatic process. The Sine oculis homeobox homolog 1 (SIX1)/eyes absent (EYA) transcriptional complex plays a critical role in the development of multiple organs and is typically downregulated after development is complete. In breast cancer, aberrant expression of SIX1 has been demonstrated to stimulate metastasis through activation of TGFß signaling and subsequent induction of epithelial-mesenchymal transition (EMT). In addition, SIX1 can induce metastasis via non-cell autonomous means, including activation of GLI-signaling in neighboring tumor cells and activation of VEGFC-induced lymphangiogenesis. Thus, targeting SIX1 would be expected to inhibit metastasis while conferring limited side effects. However, transcription factors are notoriously difficult to target, and thus novel approaches to inhibit their action must be taken. Here we identified a novel small molecule compound, NCGC00378430 (abbreviated as 8430), that reduces the SIX1/EYA2 interaction. 8430 partially reversed transcriptional and metabolic profiles mediated by SIX1 overexpression and reversed SIX1-induced TGFß signaling and EMT. 8430 was well tolerated when delivered to mice and significantly suppressed breast cancer-associated metastasis in vivo without significantly altering primary tumor growth. Thus, we have demonstrated for the first time that pharmacologic inhibition of the SIX1/EYA2 complex and associated phenotypes is sufficient to suppress breast cancer metastasis. SIGNIFICANCE: These findings identify and characterize a novel inhibitor of the SIX1/EYA2 complex that reverses EMT phenotypes suppressing breast cancer metastasis.
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Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de Homeodominio/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Proteína BRCA1/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Estimación de Kaplan-Meier , Células MCF-7 , Ratones , Metástasis de la Neoplasia/prevención & control , Proteínas Nucleares/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Tirosina Fosfatasas/metabolismo , RNA-Seq , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Metastasis is the cause of 90% of mortality in cancer patients. For metastatic colorectal cancer (mCRC), the standard-of-care drug therapies only palliate the symptoms but are ineffective, evidenced by a low survival rate of â¼11%. T-cell factor (TCF) transcription is a major driving force in CRC, and we have characterized it to be a master regulator of epithelial-mesenchymal transition (EMT). EMT transforms relatively benign epithelial tumor cells into quasi-mesenchymal or mesenchymal cells that possess cancer stem cell properties, promoting multidrug resistance and metastasis. We have identified topoisomerase IIα (TOP2A) as a DNA-binding factor required for TCF-transcription. Herein, we describe the design, synthesis, biological evaluation, and in vitro and in vivo pharmacokinetic analysis of TOP2A ATP-competitive inhibitors that prevent TCF-transcription and modulate or reverse EMT in mCRC. Unlike TOP2A poisons, ATP-competitive inhibitors do not damage DNA, potentially limiting adverse effects. This work demonstrates a new therapeutic strategy targeting TOP2A for the treatment of mCRC and potentially other types of cancers.
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Neoplasias Colorrectales/tratamiento farmacológico , Transición Epitelial-Mesenquimal/efectos de los fármacos , Factores de Transcripción TCF/genética , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacología , Adenosina Trifosfato/metabolismo , Animales , Unión Competitiva , Línea Celular Tumoral , Neoplasias Colorrectales/patología , ADN-Topoisomerasas de Tipo II/metabolismo , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Terapia Molecular Dirigida , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Relación Estructura-Actividad , Factores de Transcripción TCF/metabolismo , Inhibidores de Topoisomerasa II/farmacocinética , Transcripción GenéticaRESUMEN
Doxorubicin (DOX) area-under-the-curve (AUC) was calculated for 40 dogs with spontaneously occurring cancers using a previously validated limited-sampling approach. All dogs were administered a dose of 30 mg/m2 by intravenous infusion and serum samples were collected at 5, 45 and 60 minutes post-infusion. DOX and its major metabolite, doxorubicinol (doxol), were quantified in serum samples using high-performance liquid chromatography tandem-mass spectrometry. Wide interpatient variability was observed in the predicted DOX AUC with a coefficient of variation of 34%. A significant relationship was found between DOX AUC and absolute white blood cell count (P = 0.003), absolute neutrophil count (ANC; P = 0.002) and surviving fraction of neutrophils (P = 0.03) approximately 1 week after dosing (nadir). No changes in other hematologic parameters (red blood cells, platelets, lymphocytes, haemoglobin) were found to correlate with DOX AUC. The absolute dose (mg) and the dose per unit body weight (mg/kg) were not significantly correlated with nadir ANC. No relationships were found between maximum serum doxol concentration and myelosuppression. Baseline ANC was also significantly correlated to nadir ANC and a model was constructed using baseline ANC and DOX AUC that significantly described the nadir ANC. These findings demonstrate the important relationship between systemic DOX exposure and degree of neutropenia in dogs, and suggest a potential for individualized, pharmacokinetically-guided DOX dosing in dogs.
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Antibióticos Antineoplásicos/efectos adversos , Antibióticos Antineoplásicos/farmacocinética , Enfermedades de los Perros/tratamiento farmacológico , Doxorrubicina/efectos adversos , Doxorrubicina/farmacocinética , Neoplasias/veterinaria , Neutropenia/veterinaria , Animales , Antibióticos Antineoplásicos/sangre , Área Bajo la Curva , Cromatografía Líquida de Alta Presión/veterinaria , Colorado , Enfermedades de los Perros/sangre , Perros , Doxorrubicina/análogos & derivados , Doxorrubicina/sangre , Doxorrubicina/uso terapéutico , Femenino , Modelos Lineales , Masculino , Neoplasias/tratamiento farmacológico , Neutropenia/sangre , Neutropenia/inducido químicamente , Facultades de Medicina VeterinariaRESUMEN
Cyclophosphamide (CP), a prodrug that is enzymatically converted to the cytotoxic 4-hydroxycyclophosphamide (4OHCP) by hepatic enzymes, is commonly used in both human and veterinary medicine to treat cancers and modulate the immune system. We investigated the metabolism of CP in humans, dogs, cats, and mice using liver microsomes; apparent K M, V max, and intrinsic clearance (V max/K M) parameters were estimated. The interspecies and intraspecies variations in kinetics were vast. Dog microsomes were, on average, 55-fold more efficient than human microsomes, 2.8-fold more efficient than cat microsomes, and 1.2-fold more efficient than mouse microsomes at catalyzing CP bioactivation. These differences translated to cell-based systems. Breast cancer cells exposed to 4OHCP via CP bioactivation by microsomes resulted in a stratification of cytotoxicity that was dependent on the species of microsomes measured by IC50: dog (31.65 µM), mouse (44.95 µM), cat (272.6 µM), and human (1857 µM). The contributions of cytochrome P450s, specifically, CYP2B, CYP2C, and CYP3A, to CP bioactivation were examined: CYP3A inhibition resulted in no change in 4OHCP formation; CYP2B inhibition slightly reduced 4OHCP in humans, cats, and mice; and CYP2C inhibition drastically reduced 4OHCP formation in each species. Semiphysiologic modeling of CP metabolism using scaled metabolic parameters resulted in simulated data that closely matched published pharmacokinetic profiles, determined by noncompartmental analysis. The results highlight differential CP metabolism delineated by species and demonstrate the importance of metabolism on CP clearance.
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Antineoplásicos Alquilantes/farmacocinética , Ciclofosfamida/farmacocinética , Inmunosupresores/farmacocinética , Modelos Biológicos , Profármacos/farmacocinética , Animales , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/uso terapéutico , Enfermedades de los Gatos/tratamiento farmacológico , Enfermedades de los Gatos/inmunología , Gatos , Línea Celular Tumoral , Ciclofosfamida/metabolismo , Ciclofosfamida/uso terapéutico , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/farmacología , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/inmunología , Perros , Femenino , Humanos , Inmunosupresores/metabolismo , Inmunosupresores/uso terapéutico , Masculino , Ratones , Microsomas Hepáticos , Neoplasias/tratamiento farmacológico , Oxidación-Reducción/efectos de los fármacos , Profármacos/metabolismo , Profármacos/uso terapéuticoRESUMEN
Membrane transport proteins are fundamental components of blood-tissue barriers and affect the absorption, distribution and elimination, and interactions of many of the drugs commonly used in veterinary medicine. A quantitative, simultaneous measurement of these proteins across dog tissues is not currently available, nor is it possible with current immune-based assays such as western blot. In the present study, we aimed to develop a sensitive and specific liquid chromatography tandem-mass spectrometry (LC/MS/MS) based quantitation method that can simultaneously quantitate 14 ATP-binding cassette transporters. We applied this method to a panel of normal canine tissues and compared the LC/MS/MS results with relative messenger RNA (mRNA) abundance using quantitative real-time polymerase chain reaction (qRT-PCR). Our LC/MS/MS method is sensitive, with lower limits of quantitation ranging from 5 to 10 fmol/µg of protein. We were able to detect and/or quantitate each of the 14 transporters in at least one normal dog tissue. Relative protein and mRNA abundance within tissues did not demonstrate a significant correlation in all cases. The results presented here will provide for more accurate predictions of drug movement in dogs through incorporation into physiologically based pharmacokinetic (PBPK) models; the method described here has wide applicability to the quantitation of virtually any proteins of interest in biologic samples where validated canine antibodies do not exist.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Cromatografía Liquida/veterinaria , Perros/metabolismo , Espectrometría de Masas en Tándem/veterinaria , Transportadoras de Casetes de Unión a ATP/análisis , Transportadoras de Casetes de Unión a ATP/genética , Animales , Cromatografía Liquida/métodos , Péptidos/análisis , ARN Mensajero/análisis , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND: Liver disease (LD) prolongs mirtazapine half-life in humans, but it is unknown if this occurs in cats with LD and healthy cats. HYPOTHESIS/OBJECTIVES: To determine pharmacokinetics of administered orally mirtazapine in vivo and in vitro (liver microsomes) in cats with LD and healthy cats. ANIMALS: Eleven LD and 11 age-matched control cats. METHODS: Case-control study. Serum was obtained 1 and 4 hours (22 cats) and 24 hours (14 cats) after oral administration of 1.88 mg mirtazapine. Mirtazapine concentrations were measured by liquid chromatography with tandem mass spectrometry. Drug exposure and half-life were predicted using limited sampling modeling and estimated using noncompartmental methods. in vitro mirtazapine pharmacokinetics were assessed using liver microsomes from 3 LD cats and 4 cats without LD. RESULTS: There was a significant difference in time to maximum serum concentration between LD cats and control cats (median [range]: 4 [1-4] hours versus 1 [1-4] hours; P = .03). The calculated half-life of LD cats was significantly prolonged compared to controls (median [range]: 13.8 [7.9-61.4] hours versus 7.4 [6.7-9.1] hours; P < .002). Mirtazapine half-life was correlated with ALT (P = .002; r = .76), ALP (P < .0001; r = .89), and total bilirubin (P = .0008; r = .81). The rate of loss of mirtazapine was significantly different between microsomes of LD cats (-0.0022 min-1 , CI: -0.0050 to 0.00054 min-1 ) and cats without LD (0.01849 min-1 , CI: -0.025 to -0.012 min-1 ; P = .002). CONCLUSIONS AND CLINICAL IMPORTANCE: Cats with LD might require less frequent administration of mirtazapine than normal cats.