RESUMEN
A series of macrocyclic PKCθ inhibitors based on a 1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one hinge binder has been studied. Different aromatic and heteroaromatic substituents have been explored in order to optimize potency, isoform selectivity as well as DMPK properties. The importance of the length of the macrocyclic linker has also been analyzed. In particular, it has been found that methyl substitutions on the linker can have a profound influence on both potency and metabolic stability. Several compounds showing very good profiles, suitable for in vivo testing, are disclosed.
RESUMEN
1. A novel method utilizing an internal standard in hepatocytes incubations has been developed and demonstrated to decrease the variability in the determination of intrinsic clearance (CLint) in this system. The reduced variability was shown to allow differentiation of lower elimination rate constants from noise. 2. The suggested method was able to compensate for a small but systematic error (0.5 µL/min/106 cells) caused by an evaporation of approximately 15% of the volume during the incubation time. 3. The approach was validated using six commercial drugs (ketoprofen, tolbutamide, phenacetin, etodolac and quinidine) which were metabolized by different pathways. 4. The suggested internal standard, MSC1815677, was extensively characterized and the acquired data suggest that it fulfills the requirements of an internal standard present during the incubation. The proposed internal standard was stable during the incubation and showed a low potential to inhibit drug metabolizing enzymes and transporters. With MSC1815677 we propose a novel simple, robust and cost-effective method to address the challenges in the estimation of low clearance in hepatocyte incubations.
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Hepatocitos/metabolismo , Supervivencia Celular , Humanos , Tasa de Depuración Metabólica , Estándares de ReferenciaRESUMEN
1. We compared direct scaling, regression model equation and the so-called "Poulin et al." methods to scale clearance (CL) from in vitro intrinsic clearance (CLint) measured in human hepatocytes using two sets of compounds. One reference set comprised of 20 compounds with known elimination pathways and one external evaluation set based on 17 compounds development in Merck (MS). 2. A 90% prospective confidence interval was calculated using the reference set. This interval was found relevant for the regression equation method. The three outliers identified were justified on the basis of their elimination mechanism. 3. The direct scaling method showed a systematic underestimation of clearance in both the reference and evaluation sets. The "Poulin et al." and the regression equation methods showed no obvious bias in either the reference or evaluation sets. 4. The regression model equation was slightly superior to the "Poulin et al." method in the reference set and showed a better absolute average fold error (AAFE) of value 1.3 compared to 1.6. A larger difference was observed in the evaluation set were the regression method and "Poulin et al." resulted in an AAFE of 1.7 and 2.6, respectively (removing the three compounds with known issues mentioned above). A similar pattern was observed for the correlation coefficient. Based on these data we suggest the regression equation method combined with a prospective confidence interval as the first choice for the extrapolation of human in vivo hepatic metabolic clearance from in vitro systems.
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Tasa de Depuración Metabólica , Fenómenos Farmacológicos , Hepatocitos/metabolismo , Humanos , Cinética , Hígado/metabolismo , Modelos Biológicos , Estudios Prospectivos , Análisis de RegresiónRESUMEN
Tetralysal® is a Galderma oral drug product (DP) marketed for the treatment of acne. Tetralysal® is sold in capsules containing either 150â¯mg or 300â¯mg of the drug substance. In the British Pharmacopoeia monograph for Lymecycline Capsules, the impurities already specified in the drug substance (A-G), visible in the European Pharmacopoeia ã1654ã, are also specified together with an unidentified impurity at RRT 1.6 (Impurity J). Based on both monographs Galderma has focused on characterizing most of specified and unspecified impurities to better understand the stability and degradation processes of the formulation. In this manuscript, through both formal synthesis, preparative LCMS and formal degradation studies, we are the first group to confirm the structural identities of 5 unidentified impurities (Impurity J (RRT 1.6), RRT 2.2, 2.4, 2.6 and 3.4), conditions which exacerbate the formation of all 5 impurities and response factors for RRT 2.2, 2.6 and 3.4.
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Contaminación de Medicamentos , Limeciclina , Cromatografía Líquida de Alta PresiónRESUMEN
Prediction of clearance in drug discovery currently relies on human primary hepatocytes, which can vary widely in drug-metabolizing enzyme activity. Potential alternative in vitro models include the HepaRG cell (from immortalized hepatoma cells), which in culture can express drug-metabolizing enzymes to an extent comparable to that of primary hepatocytes. Utility of the HepaRG cell will depend on robust performance, relative to that of primary hepatocytes, in routine high-throughput analysis. In this study, we compared intrinsic clearance (CL(int)) in the recently developed cryopreserved HepaRG cell system with CL(int) in human cryopreserved pooled hepatocytes and with CL(int) in vivo for 26 cytochrome P450 substrate drugs. There was quantitative agreement between CL(int) in HepaRG cells and human hepatocytes, which was linear throughout the range of CL(int) (1-2000 ml · min(-1) · kg(-1)) and not dependent on particular cytochrome P450 involvement. Prediction of CL(int) in HepaRG cells was on average within 2-fold of in vivo CL(int) (using the well stirred liver model), but average fold error was clearance-dependent with greater underprediction (up to at least 5-fold) for the more highly cleared drugs. Recent reporting of this phenomenon in human hepatocytes was therefore confirmed with the hepatocytes used in this study, and hence the HepaRG cell system appears to share an apparently general tendency of clearance-limited CL(int) in cell models. This study shows the cryopreserved HepaRG cell system to be quantitatively comparable to human hepatocytes for prediction of clearance of drug cytochrome P450 substrates and to represent a promising alternative in vitro tool.
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Criopreservación , Hepatocitos/metabolismo , Preparaciones Farmacéuticas/metabolismo , Línea Celular Tumoral , Criopreservación/métodos , Femenino , Hepatocitos/citología , Hepatocitos/patología , Humanos , Masculino , Tasa de Depuración Metabólica/fisiología , Valor Predictivo de las PruebasRESUMEN
Large multifunctional peptidase 7 (LMP7/ß5i/PSMB8) is a proteolytic subunit of the immunoproteasome, which is predominantly expressed in normal and malignant hematolymphoid cells, including multiple myeloma, and contributes to the degradation of ubiquitinated proteins. Described herein for the first time is the preclinical profile of M3258; an orally bioavailable, potent, reversible and highly selective LMP7 inhibitor. M3258 demonstrated strong antitumor efficacy in multiple myeloma xenograft models, including a novel model of the human bone niche of multiple myeloma. M3258 treatment led to a significant and prolonged suppression of tumor LMP7 activity and ubiquitinated protein turnover and the induction of apoptosis in multiple myeloma cells both in vitro and in vivo Furthermore, M3258 showed superior antitumor efficacy in selected multiple myeloma and mantle cell lymphoma xenograft models compared with the approved nonselective proteasome inhibitors bortezomib and ixazomib. The differentiated preclinical profile of M3258 supported the initiation of a phase I study in patients with multiple myeloma (NCT04075721).
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Ácidos Borónicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Furanos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Mieloma Múltiple/tratamiento farmacológico , Complejo de la Endopetidasa Proteasomal/química , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis , Compuestos de Boro/administración & dosificación , Bortezomib/administración & dosificación , Proliferación Celular , Femenino , Glicina/administración & dosificación , Glicina/análogos & derivados , Humanos , Ratones , Ratones Desnudos , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Proteolisis , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Proteasomes are broadly expressed key components of the ubiquitin-dependent protein degradation pathway containing catalytically active subunits (ß1, ß2, and ß5). LMP7 (ß5i) is a subunit of the immunoproteasome, an inducible isoform that is predominantly expressed in hematopoietic cells. Clinically effective pan-proteasome inhibitors for the treatment of multiple myeloma (MM) nonselectively target LMP7 and other subunits of the constitutive proteasome and immunoproteasome with comparable potency, which can limit the therapeutic applicability of these drugs. Here, we describe the discovery and structure-based hit optimization of novel amido boronic acids, which selectively inhibit LMP7 while sparing all other subunits. The exploitation of structural differences between the proteasome subunits culminated in the identification of the highly potent, exquisitely selective, and orally available LMP7 inhibitor 50 (M3258). Based on the strong antitumor activity observed with M3258 in MM models and a favorable preclinical data package, a phase I clinical trial was initiated in relapsed/refractory MM patients.
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Descubrimiento de Drogas , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Inhibidores de Proteasoma/síntesis química , Inhibidores de Proteasoma/química , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/metabolismo , Relación Estructura-ActividadRESUMEN
The alpha7 nicotinic acetylcholine receptor (nAChR) is a promising target for treatment of cognitive dysfunction associated with Alzheimer's disease and schizophrenia. Here, we report the pharmacological properties of 5-morpholin-4-yl-pentanoic acid (4-pyridin-3-yl-phenyl)-amide [SEN12333 (WAY-317538)], a novel selective agonist of alpha7 nAChR. SEN12333 shows high affinity for the rat alpha7 receptor expressed in GH4C1 cells (K(i) = 260 nM) and acts as full agonist in functional Ca(2+) flux studies (EC(50) = 1.6 microM). In whole-cell patch-clamp recordings, SEN12333 activated peak currents and maximal total charges similar to acetylcholine (EC(50) = 12 microM). The compound did not show agonist activity at other nicotinic receptors tested and acted as a weak antagonist at alpha3-containing receptors. SEN12333 treatment (3 mg/kg i.p.) improved episodic memory in a novel object recognition task in rats in conditions of spontaneous forgetting as well as cognitive disruptions induced via glutamatergic [5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate); MK-801] or cholinergic (scopolamine) mechanisms. This improvement was blocked by the alpha7-selective antagonist methyllycaconitine, indicating that it is mediated by alpha7 activation. SEN12333 also prevented a scopolamine-induced deficit in a passive avoidance task. In models targeting other cognitive domains, including attention and perceptual processing, SEN12333 normalized the apomorphine-induced deficit of prepulse inhibition. Neuroprotection of SEN12333 was demonstrated in quisqualate-lesioned animals in which treatment with SEN12333 (3 mg/kg/day i.p.) resulted in a significant protection of choline acetyltransferase-positive neurons in the lesioned hemisphere. Cumulatively, our results demonstrate that the novel alpha7 nAChR agonist SEN12333 has procognitive and neuroprotective properties, further demonstrating utility of alpha7 agonists for treatment of neurodegenerative and cognitive disorders.
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Trastornos del Conocimiento/tratamiento farmacológico , Morfolinas/uso terapéutico , Enfermedades Neurodegenerativas/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Agonistas Nicotínicos/uso terapéutico , Piridinas/uso terapéutico , Receptores Nicotínicos/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Calcio/metabolismo , Línea Celular , Cognición/efectos de los fármacos , Trastornos del Conocimiento/metabolismo , Humanos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Estructura Molecular , Morfolinas/química , Morfolinas/farmacocinética , Morfolinas/farmacología , Actividad Motora/efectos de los fármacos , Enfermedades Neurodegenerativas/metabolismo , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacocinética , Fármacos Neuroprotectores/farmacología , Agonistas Nicotínicos/química , Agonistas Nicotínicos/farmacocinética , Agonistas Nicotínicos/farmacología , Técnicas de Placa-Clamp , Unión Proteica , Piridinas/química , Piridinas/farmacocinética , Piridinas/farmacología , Ensayo de Unión Radioligante , Ratas , Ratas Long-Evans , Ratas Wistar , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Hepatic metabolic clearance is one of the most important factors driving the overall kinetics of chemicals including substances used in various product categories such as pesticides, biocides, pharmaceuticals, and cosmetics. A large number of in vitro systems from purified isozymes and subcellular organelles to hepatocytes in simple cultures and in complex scaffold setups are available for measuring hepatic metabolic clearance for different applications. However, there is currently no approach for systematically characterising and comparing these in vitro methods in terms of their design, applicability and performance. To address this, existing knowledge in the field of in vitro human hepatic metabolic clearance methods was gathered and analysed in order to establish a framework to systematically characterise methods based on a set of relevant components. An analogous framework would be also applicable for non-human in vitro systems. The components are associated with the biological test systems used (e.g. subcellular or cells), the in vitro method (e.g. number of cells, test item solubility), related analytical techniques, data interpretation methods (based on substrate depletion/metabolite formation), and performance assessments (precision and accuracy of clearance measurements). To facilitate the regulatory acceptance of this class of methods, it is intended that the framework provide the basis of harmonisation work within the OECD.
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Hígado/metabolismo , Humanos , Tasa de Depuración Metabólica , Modelos BiológicosRESUMEN
Wnt signaling pathway plays a critical role in numerous cellular processes, including tumor initiation, proliferation, invasion/infiltration, metastasis formation and resistance to chemotherapy. In a drug discovery project aimed at the identification of inhibitors of the canonical Wnt pathway, we selected a series of quinazoline 2,4-diones as starting point for the therapeutic treatment of glioblastoma multiforme. Despite of poor physico-chemical properties of hit compound 1, our medicinal chemistry effort allowed the discovery and characterization of lead compound 33 (SEN461), with improved ADME profile, good bioavailability and active in vitro and in vivo in glioblastoma, gastric and sarcoma tumors.
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Antineoplásicos/química , Antineoplásicos/farmacología , Quinazolinas/química , Quinazolinas/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Animales , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Concentración 50 Inhibidora , Masculino , Ratones , Quinazolinas/metabolismo , Quinazolinas/farmacocinética , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
To improve oral absorption of sanfetrinem, a broad-spectrum, beta-lactamase-stable antibiotic, two different ester prodrugs have been selected. Both prodrugs proved to be readily hydrolyzed after absorption before reaching the systemic circulation. The objective of this study was to evaluate the role of intestinal and liver metabolism in the conversion of the two prodrugs into the active compound. In vitro experiments were performed in different rat tissues involved in the absorption process. Moreover data obtained with in vitro experiments have been integrated with data obtained in vivo using a surgically prepared rat model which allows for the measurement of the amount of intact prodrug that overcomes the intestinal mucosa and its presence in the portal vein. Both prodrugs proved to be readily cleaved by jejunum and liver microsomes. The rates of ester hydrolysis with these two tissues were 10- to 30-fold higher than those calculated in intestinal juice at pH 7.4 and about 100-fold higher than in buffer at pH 5.5. These data suggest that both the intestinal wall and liver could play an important role in the conversion of the two prodrugs in active parent compound. In the in vivo experiment, relative to sanfetrinem levels, very low concentrations of intact esters were measured in the portal vein blood, indicating that the two prodrugs are nearly completely hydrolyzed to the active drug by the intestinal wall. In conclusion this study demonstrated that the intestinal epithelium plays a major role in the conversion of the two prodrugs into sanfetrinem. The liver, despite its high esterase activity seems to be only marginally involved.
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Antibacterianos/metabolismo , Mucosa Intestinal/metabolismo , Lactamas , Hígado/metabolismo , Profármacos/metabolismo , Administración Oral , Animales , Disponibilidad Biológica , Cromatografía Líquida de Alta Presión , Ésteres , Técnicas In Vitro , Yeyuno/metabolismo , Masculino , Modelos Animales , Especificidad de Órganos , Vena Porta , Ratas , Ratas Wistar , Venas CavasRESUMEN
Interendothelial cell tight junctions (TJs) proteins contribute to maintain the structural and functional integrity of the blood-brain barrier (BBB) and several efflux transporters regulate transport of compounds across BBB. A unique double compartment-model of the BBB, consisting of cerebral endothelial cells isolated from cryopreserved human glial tumors, alone and in the presence of human astroglial cells derived from the same tissue preparation was established. Endothelial cell viability and transendothelial electrical resistance (TEER) were measured in this model and three representative TJ proteins - occludin (OCLN), zonula occludens-1 (ZO-1) and claudin-5 (CLN-5) - as well as several drug efflux transporters - P-glycoprotein (P-gp), multidrug resistance protein-1 and 2 (MRP-1 and MRP-2), organic anion-transporting polypeptide-1 and 3 (oatp1 and oatp3) were analyzed at both the protein and gene transcript level. Functional activity of P-gp and MRP-1 was also assessed. Endothelial cell viability as well as TEER significantly increased in the presence of glial cells. A significant increase of expression of OCLN, ZO-1, and CLN-5 proteins as well as of several drug transporter proteins except oatp3 and MRP-1, was also found in the presence of glial cells. All the gene transcripts protein analyzed were found to be significantly increased in the presence of glial cells. A suitable functional activity of P-gp and MRP-1 was also found. These results demonstrate that this brain endothelium culture system mimics a physiologically relevant situation and may therefore provide a new tool for studying the effects of biological fluids such as serum and cerebrospinal fluid from patients with neurological disorders underlying a BBB alteration in disease pathogenesis.
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Underprediction of in vivo intrinsic clearance (CLint) from suspended human hepatocytes has recently been shown to be clearance-dependent although the mechanistic basis is currently unknown. One possible explanation is rate limiting transmembrane (passive) permeation into hepatocytes in vitro; evidence to support this has been minor to date, but there has not been a systematic exploration of the impact of passive permeability in vitro. To investigate the relationship between underprediction of in vivo CLint and potentially rate limiting permeability, permeability constants (Px, collated from published studies and determined experimentally in this study), using three alternative methodologies (parallel artificial membrane permeability assay (PAMPA), caco-2 permeability assay and calculated using an empirical model) were compared with CLint from suspended human hepatocytes for 65 drugs from a recently reported database of clearance predictions. Although there was an approximate correspondence between hepatocyte CLint and permeability measured by PAMPA (but not by caco-2 or modelling), prediction accuracy was not dependent on the relative permeability (measured as the ratio of CLint to permeability), indicating the absence of a general rate limitation by passive hepatocyte uptake on metabolic clearance. Further investigation into rate-dependent CLint in hepatocytes is required.
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Hepatocitos/metabolismo , Membranas Artificiales , Farmacocinética , Células CACO-2 , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , HumanosRESUMEN
A valuable strategy for the assessment of in vitro systems is proposed which involves a tiered approach consisting of four levels valuable for both selection of probe compounds and designing experiments for evaluation. At level 1, a Preliminary Assessment is used to triage novel systems based on existing information generated using the methods employed in the development of the system. There is no special requirement for specific probes or experimental conditions. At level 2, Metabolic and Transporter Competence and Cellular Integrity are investigated with a number of specific probes which are generally accepted as appropriate. The information obtained at this level (as with level 1) is largely qualitative in nature. At level 3, Quantitative Utility is established by kinetic studies conducted with specific probes under standard conditions of linearity with respect to time and protein concentration. It is essential that the latter be adhered to if subsequent scale up of the output metrics for uptake and clearance are to have appropriate (scalable) units. Finally level 4, Predictive Utility, is the most detailed level of evaluation involving several model compounds for which in vivo correlates are available. Model compounds have been collated that cover a wide range of metabolic clearance values, and it is important that comparisons are made with existing in vitro systems in order to show the added value of a novel approach including modelling and familiarity with in vivo investigations.
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Hígado/metabolismo , Modelos Biológicos , Farmacocinética , Animales , Humanos , Modelos Teóricos , Factores de TiempoRESUMEN
The functional in vitro study of the enantiomers of imidazolines 4-7 highlighted the role played by the nature of the ortho phenyl substituent in determining the preferred α(2C)-AR configuration. Indeed, the (S) enantiomers of 4-6 or (R) enantiomer of 7 behave as eutomers and activate this subtype as full agonists; the corresponding distomers are partial agonists. Because in clinical pain management with opioids α(2C)-AR agonists, devoid of the α(2A)-AR-mediated side effects, may represent an improvement over current therapies with clonidine like drugs, 4 and its enantiomers, showing α(2C)-agonism/α(2A)-antagonism, have been studied in vivo. The data suggest that partial α(2C)-activation is compatible with effective enhancement of morphine analgesia and reduction both of morphine tolerance acquisition and morphine dependence acquisition and expression. On the contrary, full α(2C)-activation appears advantageous in reducing morphine tolerance expression. Interestingly, the biological profile displayed by 4 (allyphenyline) and its eutomer (S)-(+)-4 has been found to be very unusual.
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Agonistas de Receptores Adrenérgicos alfa 2/síntesis química , Antagonistas de Receptores Adrenérgicos alfa 2/síntesis química , Compuestos Alílicos/síntesis química , Analgésicos/síntesis química , Imidazolinas/síntesis química , Dependencia de Morfina/prevención & control , Morfina/farmacología , Agonistas de Receptores Adrenérgicos alfa 2/química , Agonistas de Receptores Adrenérgicos alfa 2/farmacología , Antagonistas de Receptores Adrenérgicos alfa 2/química , Antagonistas de Receptores Adrenérgicos alfa 2/farmacología , Compuestos Alílicos/química , Compuestos Alílicos/farmacología , Analgésicos/química , Analgésicos/farmacología , Animales , Células CHO , Clonidina/farmacología , Cricetinae , Cricetulus , Agonismo Parcial de Drogas , Tolerancia a Medicamentos , Humanos , Imidazolinas/química , Imidazolinas/farmacología , Masculino , Ratones , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Alpha-7 nicotinic acetylcholine receptor (alpha7 nAChR) agonists are promising therapeutic candidates for the treatment of cognitive impairment. We report a series of novel, potent small molecule agonists (4-18) of the alpha7 nAChR deriving from our continuing efforts in the areas of Alzheimer's disease and schizophrenia. One of the compounds of the series containing a urea moiety (16) was further shown to be a selective agonist of the alpha7 nAChR with excellent in vitro and in vivo profiles, brain penetration, and oral bioavailability and demonstrated in vivo efficacy in multiple behavioral cognition models. Structural modifications leading to the improved selectivity profile and the biological evaluation of this series of compounds are discussed.