RESUMEN
SMARCA2 and SMARCA4 are the ATPases of the SWI/SNF chromatin remodeling complex, which play a significant role in regulating transcriptional activity and DNA repair in cells. SMARCA2 has become an appealing synthetic-lethal, therapeutic target in oncology, as mutational loss of SMARCA4 in many cancers leads to a functional dependency on residual SMARCA2 activity. Thus, for therapeutic development, an important step is understanding any potential safety target-associated liabilities of SMARCA2 inhibition. To best mimic a SMARCA2 therapeutic, a tamoxifen-inducible (TAMi) conditional knockout (cKO) rat was developed using CRISPR technology to understand the safety profile of Smarca2 genetic ablation in a model system that avoids potential juvenile and developmental phenotypes. As the rat is the prototypical rodent species utilized in toxicology studies, a comprehensive toxicological and pathological assessment was conducted in both heterozygote and homozygous knockout rats at timepoints up to 28 days, alongside relevant corresponding controls. To our knowledge, this represents the first TAMi cKO rat model utilized for safety assessment evaluations. No significant target-associated phenotypes were observed when Smarca2 was ablated in mature (11- to 15-week-old) rats; however subsequent induction of SMARCA4 was evident that could indicate potential compensatory activity. Similar to mouse models, rat CreERT2-transgene and TAMi toxicities were characterized to avoid confounding study interpretation. In summary, a lack of significant safety findings in Smarca2 cKO rats highlights the potential for therapeutics targeting selective SMARCA2 ATPase activity; such therapies are predicted to be tolerated in patients without eliciting significant on-target toxicities.
Asunto(s)
Neoplasias , Tamoxifeno , Ratones , Ratas , Animales , Tamoxifeno/toxicidad , Adenosina Trifosfatasas , MutaciónRESUMEN
The use of bile acids as functional biomarkers for hepatobiliary injury and disease has been proposed for decades, but the utility has been generally limited due to lack of sensitivity in diagnosis and assay availability. However, recent advances in liquid chromatography and mass spectrometry have allowed for highly sensitive profiling of individual bile acids across several different matrices. In the current work, a panel of 54 bile acids were quantified in plasma by high resolution mass spectrometry in the common species used for preclinical toxicity studies, including rat (both Wistar and Sprague-Dawley strains), Beagle dog, Cynomolgus macaque monkey, and New Zealand White rabbit. In each species, blood draws were collected across three days in such a way to derive overall interpretations of: 1) biological variability across species, 2) sex differences, 3) diurnal fluctuations in the bile acid pool (including over light/dark cycles), and 4) changes due to fed or fasting state. Various methods of normalization were applied to the dataset to overcome notable inter-individual variability in bile acid concentrations to allow for better data derivations and interpretation. As such, the current work elucidates not only key differences in the bile acid pool across species, but also informs best practices in protocol design and analytical methods for interpreting large sets of bile acid data. When taken together, these data facilitate better species translation and application of bile acids as biomarkers for hepatobiliary injury and disease.
Asunto(s)
Ácidos y Sales Biliares , Enfermedad Hepática Inducida por Sustancias y Drogas , Animales , Biomarcadores , Perros , Femenino , Macaca fascicularis , Masculino , Conejos , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
Polysorbate 80 (PS80) is commonly used in pre-clinical formulations. The dose threshold for cardiovascular (CV) changes and hypersensitivity reaction in the dog was assessed and compared to other species. PS80 was administered by intravenous (IV) bolus (.5, 1 mg/kg), IV infusion (.3, .5, 1, 3 mg/kg), subcutaneous (SC) injection (5, 10, 15 mg/kg) and oral gavage (10 mg/kg) to dogs with CV monitoring. Monkeys and minipigs received PS80 by IV infusion at 3 mg/kg. Plasma histamine concentration was measured following PS80 IV infusion and with diphenhydramine pre-treatment in dogs only. In dogs, PS80 was not associated with CV changes at doses up to 15 mg/kg SC and 10 mg/kg oral, but decreased blood pressure and increased heart rate with IV bolus at ≥ .5 mg/kg and IV infusion at ≥ 1.0 mg/kg and decreased body temperature with IV infusion at 3 mg/kg was observed. Transient edema and erythema were noted with all administration routes, in all three species including doses that were devoid of CV effects. In monkeys and minipigs, PS80 did not induce CV, cutaneous or histamine concentration changes. These results suggest that mild, transient skin changes occur following PS80 administration at doses that are not associated with CV effects in the dogs. In dogs, the cardiovascular effect threshold was <.5 mg/kg for IV bolus, .3 mg/kg for IV infusion, 15 mg/kg for SC injection, and 10 mg/kg for oral administration. Monkey and minipig were refractory to PS80-induced histamine release at 3 mg/kg by IV infusion over 15 minutes.
Asunto(s)
Anafilaxia , Polisorbatos , Anafilaxia/inducido químicamente , Animales , Perros , Histamina , Inyecciones Intravenosas , Polisorbatos/toxicidad , Porcinos , Porcinos EnanosRESUMEN
Systemic therapies targeting transforming growth factor beta (TGFß) or TGFßR1 kinase (ALK5) have been plagued by toxicities including cardiac valvulopathy and bone physeal dysplasia in animals, posing a significant challenge for clinical development in pulmonary indications. The current work aims to demonstrate that systemic ALK5-associated toxicities can be mitigated through localized lung delivery. Lung-selective (THRX-144644) and systemically bioavailable (galunisertib) ALK5 inhibitors were compared to determine whether lung selectivity is sufficient to maintain local tissue concentrations while mitigating systemic exposure and consequent pathway-related findings. Both molecules demonstrated potent ALK5 activity in rat precision cut lung slices (PCLS; p-SMAD3 half-maximal inhibitory concentration [IC50], 141 nM and 1070 nM for THRX-144644 and galunisertib, respectively). In 14-day repeat-dose studies in rats, dose-related cardiac valvulopathy was recapitulated with oral galunisertib at doses ≥150 mg/kg/day. In contrast, inhaled nebulized THRX-144644 did not cause similar systemic findings up to the maximally tolerated doses in rats or dogs (10 and 1.5 mg/kg/day, respectively). THRX-144644 lung-to-plasma ratios ranged from 100- to 1200-fold in rats and dogs across dose levels. THRX-144644 lung trough (24 h) concentrations in rats and dogs ranged from 3- to 17-fold above the PCLS IC50 across tolerated doses. At a dose level exceeding tolerability (60 mg/kg/day; 76-fold above PCLS IC50) minimal heart and bone changes were observed when systemic drug concentrations reached pharmacologic levels. In conclusion, the current preclinical work demonstrates that localized pulmonary delivery of an ALK5 inhibitor leads to favorable TGFß pathway pharmacodynamic inhibition in lung while minimizing key systemic toxicities.
Asunto(s)
Pulmón/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Administración Oral , Animales , Perros , Femenino , Pulmón/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Pirazoles/toxicidad , Quinolinas/toxicidad , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Nav1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 (1) and GDC-0310 (2), selective Nav1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound 3. This resulted in the discovery of GDC-0276 (1), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Nav1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 (2), which possesses improved Nav selectivity and pharmacokinetic profile over 1.
Asunto(s)
Azetidinas/farmacología , Benzamidas/farmacología , Descubrimiento de Drogas , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Sulfonamidas/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Animales , Azetidinas/química , Azetidinas/farmacocinética , Benzamidas/química , Benzamidas/farmacocinética , Células Cultivadas , Células HEK293 , Humanos , Piperidinas/química , Piperidinas/farmacocinética , Piperidinas/farmacología , Ratas Sprague-Dawley , Sulfonamidas/química , Sulfonamidas/farmacocinética , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacocinéticaRESUMEN
The integrative responses of the cardiovascular (CV) system are essential for maintaining blood flow to provide oxygenation, nutrients, and waste removal for the entire body. Progress has been made in independently developing simple in vitro models of two primary components of the CV system, namely the heart (using induced pluripotent stem-cell derived cardiomyocytes) and the vasculature (using endothelial cells and smooth muscle cells). These two in vitro biomimics are often described as immature and simplistic, and typically lack the structural complexity of native tissues. Despite these limitations, they have proven useful for specific "fit for purpose" applications, including early safety screening. More complex in vitro models offer the tantalizing prospect of greater refinement in risk assessments. To this end, efforts to physically link cardiac and vascular components to mimic a true CV microphysiological system (CVMPS) are ongoing, with the goal of providing a more holistic and integrated CV response model. The challenges of building and implementing CVMPS in future pharmacological safety studies are many, and include a) the need for more complex (and hence mature) cell types and tissues, b) the need for more realistic vasculature (within and across co-modeled tissues), and c) the need to meaningfully couple these two components to allow for integrated CV responses. Initial success will likely come with simple, bioengineered tissue models coupled with fluidics intended to mirror a vascular component. While the development of more complex integrated CVMPS models that are capable of differentiating safe compounds and providing mechanistic evaluations of CV liabilities may be feasible, adoption by pharma will ultimately hinge on model efficiency, experimental reproducibility, and added value above current strategies.
Asunto(s)
Células Endoteliales , Células Madre Pluripotentes Inducidas , Modelos Cardiovasculares , Miocitos Cardíacos , Reproducibilidad de los ResultadosRESUMEN
Drug-induced liver injury (DILI) continues to be a major cause of drug attrition and restrictive labeling. Given the importance of farnesoid X receptor (FXR) in bile acid homeostasis, drug-related FXR antagonism may be an important mechanism of DILI. However, a comprehensive assessment of this phenomenon broadly in the context of DILI is lacking. As such, we used an orthogonal approach comprising a FXR target gene assay in primary human hepatocytes and a commercially available FXR reporter assay to investigate the potential FXR antagonistic effects of an extensive test set of 159 compounds with and without association with clinical DILI. Data were omitted from analysis based on the presence of cytotoxicity to minimize false positive assay signals and other complications in data interpretation. Based on the experimental approaches employed and corresponding data, the prevalence of FXR antagonism was relatively low across this broad DILI test set, with 16-24% prevalence based on individual assay results or combined signals in both assays. Moreover, FXR antagonism was not highly predictive for identifying clinically relevant hepatotoxicants retrospectively, where FXR antagonist classification alone had minimal to moderate predictive value as represented by positive and negative likelihood ratios of 2.24-3.84 and 0.72-0.85, respectively. The predictivity did not increase significantly when considering only compounds with high clinical exposure (maximal or efficacious plasma exposures > 1.0 µM). In contrast, modest gains in predictive value of FXR antagonism were observed considering compounds that also inhibit bile salt export pump. In addition, we have identified novel FXR antagonistic effects of well-studied hepatotoxic drugs, including bosentan, tolcapone and ritonavir. In conclusion, this work represents a comprehensive evaluation of FXR antagonism in the context of DILI, including its overall predictivity and challenges associated with detecting this phenomenon in vitro.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Ácidos y Sales Biliares , Bioensayo , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Hepatocitos , Humanos , Estudios RetrospectivosRESUMEN
IRAK4 kinase activity transduces signaling from multiple IL-1Rs and TLRs to regulate cytokines and chemokines implicated in inflammatory diseases. As such, there is high interest in identifying selective IRAK4 inhibitors for the treatment of these disorders. We previously reported the discovery of potent and selective dihydrobenzofuran inhibitors of IRAK4. Subsequent studies, however, showed inconsistent inhibition in disease-relevant pharmacodynamic models. Herein, we describe application of a human whole blood assay to the discovery of a series of benzolactam IRAK4 inhibitors. We identified potent molecule 19 that achieves robust in vivo inhibition of cytokines relevant to human disease.
RESUMEN
Cyclic adenosine monophosphate-response element (CREB)-binding protein (CBP) and EP300E1A-binding protein (p300) are members of the bromodomain and extraterminal motif (BET) family. These highly homologous proteins have a key role in modulating transcription, including altering the status of chromatin or through interactions with or posttranslational modifications of transcription factors. As CBP and p300 have known roles for stimulating c-Myc oncogenic activity, a small-molecule inhibitor, GNE-781, was developed to selectively and potently inhibit the CBP/p300 bromodomains (BRDs). Genetic models have been challenging to develop due to embryonic lethality arising from germline homozygous mutations in either CBP or P300. Hence, the purpose of this study was to characterize the role of dual inhibition of these proteins in adult rats and dogs. Repeat dose toxicity studies were conducted, and toxicologic and pathologic end points were assessed. GNE-781 was generally tolerated; however, marked effects on thrombopoiesis occurred in both species. Evidence of inhibition of erythroid, granulocytic, and lymphoid cell differentiation was also present, as well as deleterious changes in gastrointestinal and reproductive tissues. These findings are consistent with many preclinical (and clinical) effects reported with BET inhibitors targeting BRD proteins; thus, the current study findings indicate a likely important role for CBP/p300 in stem cell differentiation.
Asunto(s)
Pirazoles/farmacología , Piridinas/farmacología , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Animales , Perros , Evaluación Preclínica de Medicamentos/métodos , Canales de Potasio Éter-A-Go-Go/efectos de los fármacos , Femenino , Humanos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
We report the unique pathogenesis and presentation of a rapidly progressive B-cell lymphoma in a 3-year-old female cynomolgus monkey on day 50 of a 13-week toxicity study. Clinical pathology evaluation revealed a marked leukocytosis with bicytopenia. A serum protein electrophoresis was consistent with monoclonal gammopathy. The architecture of the lymph node, spleen, and thymus were variably effaced by neoplastic cells, which also infiltrated other tissues. Immunohistochemistry of the affected tissues confirmed a predominant population of CD20+, CD79a+, CD3-, CD68-, and CD34-neoplastic cells. The full data best support a diagnosis of Stage V lymphoma. Nextgen sequencing and negative prestudy serology results suggested a recent infection by macaque lymphocryptovirus (mLCV) with a unique transcriptional profile comparable with a rarely observed direct LCV infection model. This infection model might be associated with a temporary lack of an LCV antigen-specific cytotoxic T-cell adaptive immune response. Consistent with the established mechanisms of LCV-related lymphoproliferation, MYC and BCL2L11 gene expression were increased and decreased, respectively. While there was no overt immunosuppression, immunophenotyping revealed the index animal had a relatively low NK cell count, which further decreased by >50% on day 24 of the study. In addition to the temporary lack of adaptive immunity, the low NK cell counts were suggestive of an impaired innate immunity to control the virally-transformed cells and the subsequent unchecked lymphoproliferation. To our knowledge, this is the first report of a Stage V lymphoma with a unique pathogenesis in an otherwise immunocompetent cynomolgus monkey.
Asunto(s)
Infecciones por Herpesviridae/veterinaria , Lymphocryptovirus/aislamiento & purificación , Linfoma de Células B/veterinaria , Enfermedades de los Monos/diagnóstico , Infecciones Tumorales por Virus/veterinaria , Animales , Femenino , Infecciones por Herpesviridae/diagnóstico , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Inmunofenotipificación/veterinaria , Lymphocryptovirus/genética , Linfoma de Células B/diagnóstico , Linfoma de Células B/patología , Linfoma de Células B/virología , Macaca fascicularis , Enfermedades de los Monos/patología , Enfermedades de los Monos/virología , Infecciones Tumorales por Virus/diagnóstico , Infecciones Tumorales por Virus/patología , Infecciones Tumorales por Virus/virologíaRESUMEN
Receptor interacting protein kinase 1 (RIP1) is a critical effector of inflammatory responses and cell death activation. Cell death pathways regulated by RIP1 include caspase-dependent apoptosis and caspase-independent necroptosis. The kinase activity of RIP1 has been associated with a number of inflammatory, neurodegenerative, and oncogenic diseases. In this study, we use the RIP1 kinase inhibitor GNE684 to demonstrate that RIP1 inhibition can effectively block skin inflammation and immune cell infiltrates in livers of Sharpin mutant (Cpdm; chronic proliferative dermatitis) mice in an interventional setting, after disease onset. On the other hand, genetic inactivation of RIP1 (RIP1 KD) or ablation of RIP3 (RIP3 KO) or MLKL (MLKL KO) did not affect testicular pathology of aging male mice. Likewise, infection with vaccinia virus or with mouse gammaherpesvirus MHV68 resulted in similar viral clearance in wild-type, RIP1 KD, and RIP3 KO mice. In summary, this study highlights the benefits of inhibiting RIP1 in skin inflammation, as opposed to its lack of relevance for testicular longevity and the response to certain viral infections.
Asunto(s)
Dermatitis/genética , Infecciones por Herpesviridae/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Piel/inmunología , Vaccinia/genética , Animales , Enfermedad Crónica , Dermatitis/inmunología , Dermatitis/patología , Dermatitis/virología , Modelos Animales de Enfermedad , Gammaherpesvirinae/inmunología , Gammaherpesvirinae/patogenicidad , Regulación de la Expresión Génica , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Inflamación , Hígado/inmunología , Hígado/patología , Hígado/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/deficiencia , Proteínas Quinasas/genética , Proteínas Quinasas/inmunología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Transducción de Señal , Piel/patología , Piel/virología , Testículo/inmunología , Testículo/patología , Testículo/virología , Vaccinia/inmunología , Vaccinia/patología , Vaccinia/virología , Virus Vaccinia/inmunología , Virus Vaccinia/patogenicidad , Replicación Viral/inmunologíaRESUMEN
A series of pyrazolopyrimidine inhibitors of IRAK4 were developed from a high-throughput screen (HTS). Modification of an HTS hit led to a series of bicyclic heterocycles with improved potency and kinase selectivity but lacking sufficient solubility to progress in vivo. Structure-based drug design, informed by cocrystal structures with the protein and small-molecule crystal structures, yielded a series of dihydrobenzofurans. This semisaturated bicycle provided superior druglike properties while maintaining excellent potency and selectivity. Improved physicochemical properties allowed for progression into in vivo experiments, where lead molecules exhibited low clearance and showed target-based inhibition of IRAK4 signaling in an inflammation-mediated PK/PD mouse model.
Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Aminoquinolinas/síntesis química , Aminoquinolinas/metabolismo , Aminoquinolinas/farmacología , Animales , Benzofuranos/síntesis química , Benzofuranos/metabolismo , Benzofuranos/farmacología , Dominio Catalítico , Femenino , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Ratones Endogámicos C57BL , Estructura Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Pirazoles/síntesis química , Pirazoles/metabolismo , Pirimidinas/síntesis química , Pirimidinas/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Use of genetically engineered rodents is often considered a valuable exercise to assess potential safety concerns associated with the inhibition of a target pathway. When there are potential immunomodulatory risks associated with the target, these genetically modified animals are often challenged with various pathogens in an acute setting to determine the risk to humans. However, the applicability of the results from infection models is seldom assessed when significant retrospective human data become available. Thus, the purpose of the current review is to compare the outcomes of infectious pathogen challenge in mice with genetic deficiencies in TNF-α, IL17, IL23, or Janus kinase pathways with infectious outcomes caused by inhibitors of these pathways in humans. In general, mouse infection challenge models had modest utility for hazard identification and were generally only able to predict overall trends in infection risk. These models did not demonstrate significant value in evaluating specific types of pathogens that are either prevalent (ie rhinoviruses) or of significant concern (ie herpes zoster). Similarly, outcomes in mouse models tended to overestimate the severity of infection risk in human patients. Thus, there is an emerging need for more human-relevant models that have better predictive value. Large meta-analyses of multiple clinical trials or post-marketing evaluations remains the gold-standard for characterizing the true infection risk to patients.
Asunto(s)
Susceptibilidad a Enfermedades/inmunología , Sistema Inmunológico/inmunología , Infecciones/inmunología , Modelos Animales , Animales , Animales Modificados Genéticamente , Humanos , Metaanálisis como Asunto , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Using structure- and ligand-based design principles, a novel series of piperidyl chromane arylsulfonamide Nav1.7 inhibitors was discovered. Early optimization focused on improvement of potency through refinement of the low energy ligand conformation and mitigation of high in vivo clearance. An in vitro hepatotoxicity hazard was identified and resolved through optimization of lipophilicity and lipophilic ligand efficiency to arrive at GNE-616 (24), a highly potent, metabolically stable, subtype selective inhibitor of Nav1.7. Compound 24 showed a robust PK/PD response in a Nav1.7-dependent mouse model, and site-directed mutagenesis was used to identify residues critical for the isoform selectivity profile of 24.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.7/química , Sulfonamidas/química , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Analgésicos/química , Analgésicos/metabolismo , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Sitios de Unión , Línea Celular , Supervivencia Celular/efectos de los fármacos , Dolor Crónico/tratamiento farmacológico , Dolor Crónico/patología , Perros , Semivida , Humanos , Ligandos , Masculino , Ratones , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Canal de Sodio Activado por Voltaje NAV1.7/genética , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Ratas , Relación Estructura-Actividad , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Bloqueadores del Canal de Sodio Activado por Voltaje/metabolismo , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/uso terapéuticoRESUMEN
Bile salt export pump (BSEP) inhibition has emerged as an important mechanism that may contribute to the initiation of human drug-induced liver injury (DILI). Proactive evaluation and understanding of BSEP inhibition is recommended in drug discovery and development to aid internal decision making on DILI risk. BSEP inhibition can be quantified using in vitro assays. When interpreting assay data, it is important to consider in vivo drug exposure. Currently, this can be undertaken most effectively by consideration of total plasma steady state drug concentrations (Css,plasma ). However, because total drug concentrations are not predictive of pharmacological effect, the relationship between total exposure and BSEP inhibition is not causal. Various follow-up studies can aid interpretation of in vitro BSEP inhibition data and may be undertaken on a case-by-case basis. BSEP inhibition is one of several mechanisms by which drugs may cause DILI, therefore, it should be considered alongside other mechanisms when evaluating possible DILI risk.
Asunto(s)
Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/antagonistas & inhibidores , Bilis/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Desarrollo de Medicamentos/métodos , Descubrimiento de Drogas/métodos , Hígado/efectos de los fármacos , Moduladores del Transporte de Membrana/toxicidad , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/química , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/genética , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/metabolismo , Animales , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Simulación por Computador , Diseño Asistido por Computadora , Diseño de Fármacos , Humanos , Técnicas In Vitro , Hígado/metabolismo , Moduladores del Transporte de Membrana/química , Modelos Biológicos , Conformación Proteica , Medición de Riesgo , Factores de Riesgo , Relación Estructura-ActividadRESUMEN
Drug-induced liver injury (DILI) has been the most frequent cause of post-marketing drug withdrawals in the last 50years. The multifactorial nature of events that precede severe liver injury in human patients is difficult to model in rodents due to a variety of confounding or contributing factors that include disease state, concurrent medications, and translational species differences. In retrospective analyses, a consistent risk factor for DILI has been the inhibition of the Bile Salt Export Pump (BSEP). One compound known for potent BSEP inhibition and severe DILI is troglitazone. The purpose of the current study is to determine if serum profiling of 19 individual bile acids by liquid chromatography-mass spectrometry (LC/MS) can detect perturbations in bile acid homeostasis in rats after acute intravenous (IV) administration of vehicle or 5, 25, or 50mg/kg troglitazone. Minimal serum transaminase elevations (approximately two-fold) were observed with no evidence of microscopic liver injury. However, marked changes in individual serum bile acids occurred, with dose-dependent increases in the majority of the bile acids profiled. When compared to predose baseline values, tauromuricholic acid and taurocholic acid had the most robust increase in serum levels and dynamic range, with a maximum fold increase from baseline of 34-fold and 29-fold, respectively. Peak bile acid increases occurred within 2hours (h) after dosing and returned to baseline values before 24h. In conclusion, serum bile acid profiling can potentially identify a mechanistic risk of clinical DILI that could be poorly detected by traditional toxicity endpoints.
Asunto(s)
Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/antagonistas & inhibidores , Ácidos y Sales Biliares/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Medición de Riesgo , Animales , Cromanos/toxicidad , Femenino , Masculino , Ratas , Ratas Sprague-Dawley , Tiazolidinedionas/toxicidad , TroglitazonaRESUMEN
The epigenetic regulator CBP/P300 presents a novel therapeutic target for oncology. Previously, we disclosed the development of potent and selective CBP bromodomain inhibitors by first identifying pharmacophores that bind the KAc region and then building into the LPF shelf. Herein, we report the "hybridization" of a variety of KAc-binding fragments with a tetrahydroquinoline scaffold that makes optimal interactions with the LPF shelf, imparting enhanced potency and selectivity to the hybridized ligand. To demonstrate the utility of our hybridization approach, two analogues containing unique Asn binders and the optimized tetrahydroquinoline moiety were rapidly optimized to yield single-digit nanomolar inhibitors of CBP with exquisite selectivity over BRD4(1) and the broader bromodomain family.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Animales , Asparagina/química , Asparagina/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular , Cristalografía por Rayos X , Femenino , Transferencia Resonante de Energía de Fluorescencia/métodos , Ratones Endogámicos , Simulación del Acoplamiento Molecular , Proteínas Nucleares/antagonistas & inhibidores , Dominios Proteicos , Pirazoles/química , Piridinas/química , Quinolinas/química , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción p300-CBP/química , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Inhibition of the bromodomain of the transcriptional regulator CBP/P300 is an especially interesting new therapeutic approach in oncology. We recently disclosed in vivo chemical tool 1 (GNE-272) for the bromodomain of CBP that was moderately potent and selective over BRD4(1). In pursuit of a more potent and selective CBP inhibitor, we used structure-based design. Constraining the aniline of 1 into a tetrahydroquinoline motif maintained potency and increased selectivity 2-fold. Structure-activity relationship studies coupled with further structure-based design targeting the LPF shelf, BC loop, and KAc regions allowed us to significantly increase potency and selectivity, resulting in the identification of non-CNS penetrant 19 (GNE-781, TR-FRET IC50 = 0.94 nM, BRET IC50 = 6.2 nM; BRD4(1) IC50 = 5100 nΜ) that maintained good in vivo PK properties in multiple species. Compound 19 displays antitumor activity in an AML tumor model and was also shown to decrease Foxp3 transcript levels in a dose dependent manner.
Asunto(s)
Antineoplásicos/farmacología , Proteína de Unión a CREB/antagonistas & inhibidores , Pirazoles/farmacología , Piridinas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Proteína de Unión a CREB/química , Perros , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Ratones , Dominios Proteicos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Pirazoles/síntesis química , Pirazoles/química , Pirazoles/farmacocinética , Piridinas/síntesis química , Piridinas/química , Piridinas/farmacocinética , ARN/genética , Ratas Sprague-Dawley , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure-activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 µM, BRET IC50 = 0.41 µM, BRD4(1) IC50 = 13 µM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.