RESUMEN
The (Z)-fluoro-olefin amide bioisosteric replacement is an effective tool for addressing various shortcomings of the parent amide. In an effort to fine tune ADME properties of BACE1 preclinical candidate AM-6494, a series of structurally distinct (Z)-fluoro-olefin containing analogs was developed that culminated in compound 15. Herein, we detail design considerations, synthetic challenges, structure activity relationship (SAR) studies, and in vivo properties of an advanced compound in this novel series of BACE1 inhibitors.
Asunto(s)
Alquenos/farmacología , Amidas/farmacología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Desarrollo de Medicamentos , Inhibidores Enzimáticos/farmacología , Hidrocarburos Fluorados/farmacología , Alquenos/síntesis química , Alquenos/química , Amidas/síntesis química , Amidas/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Células HEK293 , Humanos , Hidrocarburos Fluorados/síntesis química , Hidrocarburos Fluorados/química , Conformación Molecular , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
In vitro prediction of physiologically relevant transport of therapeutic molecules across the microcirculation represents an intriguing opportunity to predict efficacy in human populations. On-chip microvascular networks (MVNs) show physiologically relevant values of molecular permeability, yet like most systems, they lack an important contribution to transport: the ever-present fluid convection through the endothelium. Quantification of transport through the MVNs by current methods also requires confocal imaging and advanced analytical techniques, which can be a bottleneck in industry and academic laboratories. Here, it is shown that by recapitulating physiological transmural flow across the MVNs, the concentration of small and large molecule therapeutics can be directly sampled in the interstitial fluid and analyzed using standard analytical techniques. The magnitudes of transport measured in MVNs reveal trends with molecular size and type (protein versus nonprotein) that are expected in vivo, supporting the use of the MVNs platform as an in vitro tool to predict distribution of therapeutics in vivo.
Asunto(s)
Líquido Extracelular/fisiología , Microvasos/fisiología , Flujo Sanguíneo Regional/fisiología , Proteínas Sanguíneas/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Dispositivos Laboratorio en un Chip , Perfusión , Permeabilidad , Presión , Transporte de ProteínasRESUMEN
The identification of nonopioid alternatives to treat chronic pain has received a great deal of interest in recent years. Recently, the engineering of a series of Nav1.7 inhibitory peptide-antibody conjugates has been reported, and herein, the preclinical efforts to identify novel approaches to characterize the pharmacokinetic properties of the peptide conjugates are described. A cryopreserved plated mouse hepatocyte assay was designed to measure the depletion of the peptide-antibody conjugates from the media, with a correlation being observed between percentage remaining in the media and in vivo clearance (Pearson r = -0.5525). Physicochemical (charge and hydrophobicity), receptor-binding [neonatal Fc receptor (FcRn)], and in vivo pharmacokinetic data were generated and compared with the results from our in vitro hepatocyte assay, which was hypothesized to encompass all of the aforementioned properties. Correlations were observed among hydrophobicity; FcRn binding; depletion rates from the hepatocyte assay; and ultimately, in vivo clearance. Subsequent studies identified potential roles for the low-density lipoprotein and mannose/galactose receptors in the association of the Nav1.7 peptide conjugates with mouse hepatocytes, although in vivo studies suggested that FcRn was still the primary receptor involved in determining the pharmacokinetics of the peptide conjugates. Ultimately, the use of the cryopreserved hepatocyte assay along with FcRn binding and hydrophobic interaction chromatography provided an efficient and integrated approach to rapidly triage molecules for advancement while reducing the number of in vivo pharmacokinetic studies. SIGNIFICANCE STATEMENT: Although multiple in vitro and in silico tools are available in small-molecule drug discovery, pharmacokinetic characterization of protein therapeutics is still highly dependent upon the use of in vivo studies in preclinical species. The current work demonstrates the combined use of cryopreserved hepatocytes, hydrophobic interaction chromatography, and neonatal Fc receptor binding to characterize a series of Nav1.7 peptide-antibody conjugates prior to conducting in vivo studies, thus providing a means to rapidly evaluate novel protein therapeutic platforms while concomitantly reducing the number of in vivo studies conducted in preclinical species.
Asunto(s)
Dolor Crónico/tratamiento farmacológico , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoconjugados/farmacocinética , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Receptores Fc/metabolismo , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacocinética , Administración Intravenosa , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacocinética , Criopreservación , Evaluación Preclínica de Medicamentos/métodos , Hepatocitos , Antígenos de Histocompatibilidad Clase I/genética , Inmunoconjugados/administración & dosificación , Macaca fascicularis , Masculino , Tasa de Depuración Metabólica , Ratones , Ratones Noqueados , Péptidos/administración & dosificación , Péptidos/farmacocinética , Receptores Fc/genética , Distribución Tisular , Bloqueadores del Canal de Sodio Activado por Voltaje/administración & dosificaciónRESUMEN
The ß-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is one of the most hotly pursued targets for the treatment of Alzheimer's disease. We used a structure- and property-based drug design approach to identify 2-aminooxazoline 3-azaxanthenes as potent BACE1 inhibitors which significantly reduced CSF and brain Aß levels in a rat pharmacodynamic model. Compared to the initial lead 2, compound 28 exhibited reduced potential for QTc prolongation in a non-human primate cardiovascular safety model.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Xantenos/química , Xantenos/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Línea Celular , Células HEK293 , Humanos , Inhibidores de Proteasas/síntesis química , Ratas , Xantenos/síntesis químicaRESUMEN
ß-Secretase 1 (BACE1) represents an attractive target for the treatment of Alzheimer's disease. In the course of development of a novel small molecule BACE1 inhibitor (AMG-8718), retinal thinning was observed in a 1-month toxicity study in the rat. To further understand the lesion, an investigational study was conducted whereby rats were treated daily with AMG-8718 for 1 month followed by a 2-month treatment-free phase. The earliest detectable change in the retina was an increase in autofluorescent granules in the retinal pigment epithelium (RPE) on day 5; however, there were no treatment-related light microscopic changes observed in the neuroretina and no changes observed by fundus autofluorescence or routine ophthalmoscopic examination after 28 days of dosing. Following 2 months of recovery, there was significant retinal thinning attributed to loss of photoreceptor nuclei from the outer nuclear layer. Electroretinographic changes were observed as early as day 14, before any microscopic evidence of photoreceptor loss. BACE1 knockout rats were generated and found to have normal retinal morphology indicating that the retinal toxicity induced by AMG-8718 was likely off-target. These results suggest that AMG-8718 impairs phagolysosomal function in the rat RPE, which leads to photoreceptor dysfunction and ultimately loss of photoreceptors.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Benzopiranos/toxicidad , Inhibidores Enzimáticos/toxicidad , Piridinas/toxicidad , Retina/efectos de los fármacos , Enfermedades de la Retina/inducido químicamente , Compuestos de Espiro/toxicidad , Animales , Electrorretinografía , Masculino , Ratas , Ratas Sprague-Dawley , Retina/enzimología , Retina/patología , Enfermedades de la Retina/enzimología , Tomografía de Coherencia ÓpticaRESUMEN
1. A catheterized rat model was used to define the intestinal and hepatic components of oral bioavailability for an 11ß-HSD1 inhibitor, AMG 221. These data were integrated with standard in vivo metabolism studies to elucidate the components contributing to the oral disposition of a novel drug candidate. 2. Intestinal and hepatic extraction ratios of AMG 221 obtained using a five-catheter rat model were 0.56 and 0.32, respectively. Therefore, both intestinal and hepatic extraction contributed to the first-pass component of oral bioavailability. There was no evidence for significant gut extraction of systemically administered drug. 3. Mass balance data and in vivo metabolite characterization obtained after administration of [(14)C] AMG 221 to rat showed that AMG 221 was completely absorbed from the gut lumen following an oral dose, primarily excreted in urine and was almost completely metabolized prior to excretion. 4. Hepatic bioavailability (FH), measured in two animals at various time points after oral dose administration was somewhat variable but generally characterized by an initial reduction during the absorption phase followed by an increase during the elimination phase, consistent with hepatic distribution of AMG 221. 5. The five-catheter rat model afforded estimates of hepatic and intestinal contribution to oral bioavailability that were used with other data to define the preclinical ADME characteristics of a drug candidate.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Tiazoles/farmacocinética , Administración Oral , Animales , Disponibilidad Biológica , Radioisótopos de Carbono/metabolismo , Estructura Molecular , Ratas , Tiazoles/administración & dosificación , Tiazoles/química , Dispositivos de Acceso VascularRESUMEN
Desmoplasia in breast cancer leads to heterogeneity in physical properties of the tissue, resulting in disparities in drug delivery and treatment efficacy among patients, thus contributing to high disease mortality. Personalized in vitro breast cancer models hold great promise for high-throughput testing of therapeutic strategies to normalize the aberrant microenvironment in a patient-specific manner. Here, tumoroids assembled from breast cancer cell lines (MCF7, SKBR3, and MDA-MB-468) and patient-derived breast tumor cells (TCs) cultured in microphysiological systems including perfusable microvasculature reproduce key aspects of stromal and vascular dysfunction causing impaired drug delivery. Models containing SKBR3 and MDA-MB-468 tumoroids show higher stromal hyaluronic acid (HA) deposition, vascular permeability, interstitial fluid pressure (IFP), and degradation of vascular HA relative to models containing MCF7 tumoroids or models without tumoroids. Interleukin 8 (IL8) secretion is found responsible for vascular dysfunction and loss of vascular HA. Interventions targeting IL8 or stromal HA normalize vascular permeability, perfusion, and IFP, and ultimately enhance drug delivery and TC death in response to perfusion with trastuzumab and cetuximab. Similar responses are observed in patient-derived models. These microphysiological systems can thus be personalized by using patient-derived cells and can be applied to discover new molecular therapies for the normalization of the tumor microenvironment.
RESUMEN
A series of aminooxadiazoles was optimized for inhibition of Cdc7. Early lead isoquinoline 1 suffered from modest cell potency (cellular IC50=0.71 µM measuring pMCM2), low selectivity against structurally related kinases, and high IV clearance in rats (CL=18 L/h/kg). Extensive optimization resulted in azaindole 26 (Cdc7 IC50=1.1 nM, pMCM2 IC50=32 nM) that demonstrated robust lowering of pMCM2 in a mouse pharmacodynamic (PD) model when dosed orally. Modifications to improve the pharmacokinetic profile of this series were guided by trapping experiments with glutathione in rat hepatocytes.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Oxadiazoles/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Desnudos , Estructura Molecular , Oxadiazoles/síntesis química , Oxadiazoles/farmacología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
γ-Secretase modulators (GSMs) are potentially disease-modifying treatments for Alzheimer's disease. They selectively lower pathogenic Aß42 levels by shifting the enzyme cleavage sites without inhibiting γ-secretase activity, possibly avoiding known adverse effects observed with complete inhibition of the enzyme complex. A cell-based HTS effort identified the sulfonamide 1 as a GSM lead. Lead optimization studies identified compound 25 with improved cell potency, PKDM properties, and it lowered Aß42 levels in the cerebrospinal fluid (CSF) of Sprague-Dawley rats following oral administration. Further optimization of 25 to improve cellular potency is described.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Amidas/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Picolinas/farmacología , Enfermedad de Alzheimer/enzimología , Amidas/química , Animales , Células HEK293 , Humanos , Picolinas/química , Ratas , Ratas Sprague-DawleyRESUMEN
1. Extensive metabolism of a drug candidate can complicate the interpretation of comparative safety and efficacy data from humans and preclinical species. 2. The 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitor, AMG 221 underwent extensive oxidative metabolism to structurally similar but differentially active primary and secondary metabolites. Relative potency data from synthetic metabolites enabled a stepwise quantitative assessment of AMG 221 in vivo metabolism that compared relative exposure to metabolites in plasma across species and discerned which active metabolites to monitor in preclinical and clinical safety and efficacy studies. 3. Pooled plasma samples from AMG 221-dosed human subjects were profiled using high-resolution liquid chromatography-mass spectrometry (LC-MS) with a mass-defect-filter. The most abundant peak, M1 accounted for 47%-59% of peaks followed by AMG 221 at 27%-40%. Other metabolites were each less than 7%. Activity-normalized data indicated both M1 and AMG 221 should be monitored to assist pharmacokinetic-pharmacodynamic (PK-PD) modeling. 4. Rat and dog area under the plasma concentration time curve (AUC) exposures to M1 at preclinical no observable adverse effect level (NOAEL) doses were 2-42-fold higher than human, indicating M1 was not a disproportionate metabolite, as defined by International Committee on Harmonization (ICH) M3(R2) guidance. 5. Development decisions regarding active metabolite monitoring and potentially disproportionate metabolites in extensively metabolized drug candidates are enabled by metabolite synthesis and liquid chromatography high-resolution mass spectrometry (LC-HRMS)-based assessment of potency-normalized plasma metabolite AUCs.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , Tiazoles/farmacocinética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Animales , Área Bajo la Curva , Cromatografía Liquida , Perros , Humanos , Masculino , Microsomas Hepáticos/metabolismo , Ratas , Especificidad de la Especie , Espectrometría de Masas en TándemRESUMEN
Sequential proteolytic cleavage of the amyloid precursor protein (APP) by ß-site APP-cleaving enzyme 1 (BACE1) and the γ-secretase complex produces the amyloid-ß peptide (Aß), which is believed to play a critical role in the pathology of Alzheimer's disease (AD). The aspartyl protease BACE1 catalyzes the rate-limiting step in the production of Aß, and as such it is considered to be an important target for drug development in AD. The development of a BACE1 inhibitor therapeutic has proven to be difficult. The active site of BACE1 is relatively large. Consequently, to achieve sufficient potency, many BACE1 inhibitors have required unfavorable physicochemical properties such as high molecular weight and polar surface area that are detrimental to efficient passage across the blood-brain barrier. Using a rational drug design approach we have designed and developed a new series of hydroxyethylamine-based inhibitors of BACE1 capable of lowering Aß levels in the brains of rats after oral administration. Herein we describe the in vitro and in vivo characterization of two of these molecules and the overall relationship of compound properties [e.g., in vitro permeability, P-glycoprotein (P-gp) efflux, metabolic stability, and pharmacological potency] to the in vivo pharmacodynamic effect with more than 100 compounds across the chemical series. We demonstrate that high in vitro potency for BACE1 was not sufficient to provide central efficacy. A combination of potency, high permeability, low P-gp-mediated efflux, and low clearance was required for compounds to produce robust central Aß reduction after oral dosing.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Etilaminas/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Administración Oral , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/metabolismo , Proteínas Sanguíneas/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacocinética , Etilaminas/farmacocinética , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Técnicas In Vitro , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Unión Proteica , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes , Relación Estructura-ActividadRESUMEN
The blood-brain barrier (BBB) greatly restricts the entry of biological and engineered therapeutic molecules into the brain. Due to challenges in translating results from animal models to the clinic, relevant in vitro human BBB models are needed to assess pathophysiological molecular transport mechanisms and enable the design of targeted therapies for neurological disorders. This protocol describes an in vitro model of the human BBB self-assembled within microfluidic devices from stem-cell-derived or primary brain endothelial cells, and primary brain pericytes and astrocytes. This protocol requires 1.5 d for device fabrication, 7 d for device culture and up to 5 d for downstream imaging, protein and gene expression analyses. Methodologies to measure the permeability of any molecule in the BBB model, which take 30 min per device, are also included. Compared with standard 2D assays, the BBB model features relevant cellular organization and morphological characteristics, as well as values of molecular permeability within the range expected in vivo. These properties, coupled with a functional brain endothelial expression profile and the capability to easily test several repeats with low reagent consumption, make this BBB model highly suitable for widespread use in academic and industrial laboratories.
Asunto(s)
Barrera Hematoencefálica , Permeabilidad Capilar/fisiología , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Modelos Cardiovasculares , Astrocitos/citología , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/fisiología , Encéfalo/citología , Células Cultivadas , Células Endoteliales/citología , Humanos , Pericitos/citologíaRESUMEN
The novel biotransformation of an aminopyrrolidine to an aminopiperidine during the metabolism of 5-(4-chlorophenyl)-3-methyl-2-((2R)-2-(((1-methylethyl)amino)methyl)-1-pyrrolidinyl)-6-(4-pyridinyl)-4(3H)-pyrimidinone (AMG657417) was investigated using the NADPH-fortified S9 fraction from human liver. The major metabolite (M18) had a protonated molecule (MH(+) m/z 438) identical to that of AMG657417 except that it eluted earlier on a reverse-phase high-performance liquid chromatography. The structure of M18 had been identified as 5-(4-chlorophenyl)-3-methyl-2-((1-(1-methylethyl)-3-piperidinyl)amino)-6-(4-pyridinyl)-4(3H)-pyrimidinone (I) by liquid chromatography-mass spectrometry and proton NMR. M18 was not observed when AMG657417 was incubated with either microsomal or cytosolic fraction from human liver, suggesting the involvement of both microsomal and cytosolic enzymes in the biotransformation. The reaction mechanisms have been elucidated by trapping the intermediates formed during the biotransformation. An aldehyde intermediate was initially produced by hydroxylation and opening of the pyrrolidine ring of the parent molecule, followed by intramolecular Schiff-base formation between the exocyclic isopropylamine nitrogen and the aldehyde carbonyl to form a piperidinyl iminium ion. The iminium ion was then reduced to the piperidine product. The presence of the aldehyde intermediate was verified by the formation of semicarbazide conjugates in human liver microsomal, S9, and recombinant CYP3A4 incubations of AMG657417. The presence of the piperidinyl iminium ion intermediate was confirmed by the formation of cyanide conjugates in the incubations in human liver S9. Two cyanide conjugates with identical protonated molecule and product ion mass spectra were observed, indicating the likelihood of diastereomer formation. A chemical inhibition study in NADPH-fortified S9 fraction indicated that the oxidation of AMG657417 was catalyzed almost exclusively by CYP3A.
Asunto(s)
Citocromo P-450 CYP3A/metabolismo , Piperidinas/farmacocinética , Pirimidinonas/farmacocinética , Pirrolidinas/farmacocinética , Biotransformación , Cromatografía Líquida de Alta Presión/métodos , Humanos , Hidroxilación , Cetoconazol/farmacología , Espectroscopía de Resonancia Magnética/métodos , Espectrometría de Masas/métodos , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Piperidinas/metabolismo , Pirimidinonas/metabolismo , Pirrolidinas/metabolismo , Semicarbacidas/metabolismoRESUMEN
ß-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is an aspartyl protease that plays a key role in the production of amyloid ß (Aß) in the brain and has been extensively pursued as a target for the treatment of Alzheimer's disease (AD). BACE2, an aspartyl protease that is structurally related to BACE1, has been recently reported to be involved in melanosome maturation and pigmentation. Herein, we describe the development of a series of cyclopropylthiazines as potent and orally efficacious BACE1 inhibitors. Lead optimization led to the identification of 20, a molecule with biochemical IC50 BACE2/BACE1 ratio of 47. Administration of 20 resulted in no skin/fur color change in a 13-day mouse hypopigmentation study and demonstrated robust and sustained reduction of CSF and brain Aß40 levels in rat and monkey pharmacodynamic models. On the basis of a compelling data package, 20 (AM-6494) was advanced to preclinical development.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ciclopropanos/farmacología , Inhibidores Enzimáticos/farmacología , Tiazinas/farmacología , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/líquido cefalorraquídeo , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Ciclopropanos/química , Ciclopropanos/farmacocinética , Ciclopropanos/uso terapéutico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/uso terapéutico , Humanos , Masculino , Ratones , Modelos Moleculares , Fragmentos de Péptidos/líquido cefalorraquídeo , Fragmentos de Péptidos/metabolismo , Ratas Sprague-Dawley , Tiazinas/química , Tiazinas/farmacocinética , Tiazinas/uso terapéuticoRESUMEN
Motesanib diphosphate is a novel, investigational, highly selective oral inhibitor of the receptor tyrosine kinases vascular endothelial growth factor receptors 1, 2, and 3, the platelet-derived growth factor receptor, and the stem cell factor receptor (Kit). The in vitro metabolic profiles of [(14)C]motesanib were examined by using microsomes and hepatocytes from preclinical species and humans. Several oxidative metabolites were observed and characterized by tandem mass spectrometry, nuclear magnetic resonance spectroscopy, and coinjection with authentic standards. Cytochrome P450 (P450) 3A4 is the major isozyme involved in the oxidative biotransformation of motesanib, but the CYP2D6 and CYP1A isozymes also make minor contributions. In hepatocyte incubations, oxidative and conjugative pathways were observed for all species examined, and indoline N-glucuronidation was the dominant pathway. Three less common and novel phase II conjugates of the indoline nitrogen were detected in hepatocytes and in microsomes supplemented with specific cofactors, including N-carbamoyl glucuronide, N-glucose, and N-linked beta-N-acetylglucosamine. An N-glucuronide metabolite was the most frequently observed phase II conjugate in liver microsomes of all species, whereas the N-acetylglucosamine conjugate was observed only in monkey liver microsomes. Incubations with recombinant human UDP-glucuronosyltransferases (UGTs) and inhibition by the UGT1A4 and UGT1A1 substrates/inhibitors imipramine and bilirubin suggested that UGT1A4 is the major UGT isozyme catalyzing the N-glucuronidation of motesanib, with a minor contribution from UGT1A1. The in vitro metabolic profiles were similar between the human and preclinical species examined. All metabolites found in humans were also detected in other species.
Asunto(s)
Glucurónidos , Glucuronosiltransferasa/metabolismo , Microsomas Hepáticos/metabolismo , Administración Oral , Biotransformación , Sistema Enzimático del Citocromo P-450 , Glucurónidos/metabolismo , Hepatocitos/metabolismo , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Indoles/metabolismo , Espectroscopía de Resonancia Magnética , Redes y Vías Metabólicas , Metaboloma/efectos de los fármacosRESUMEN
Recent therapeutic success of large-molecule biologics has led to intense interest in assays to measure with precision their transport across the vascular endothelium and into the target tissue. Most current in vitro endothelial models show unrealistically large permeability coefficients due to a non-physiological paracellular transport. Thus, more advanced systems are required to better recapitulate and discern the important contribution of transcellular transport (transcytosis), particularly of pharmaceutically-relevant proteins. Here, a robust platform technology for the measurement of transport through a human endothelium is presented, which utilizes in vitro microvascular networks (MVNs). The self-assembled MVNs recapitulate the morphology and junctional complexity of in vivo capillaries, and express key endothelial vesicular transport proteins. This results in measured permeabilities to large molecules comparable to those observed in vivo, which are orders of magnitude lower than those measured in transwells. The permeability of albumin and immunoglobulin G (IgG), biopharmaceutically-relevant proteins, is shown to occur primarily via transcytosis, with passage of IgG regulated by the receptor FcRn. The physiological relevance of the MVNs make it a valuable tool to assess the distribution of biopharmaceuticals into tissues, and may be used to prioritize candidate molecules from this increasingly important class of therapeutics.
Asunto(s)
Permeabilidad Capilar/fisiología , Dispositivos Laboratorio en un Chip , Microcirculación/fisiología , Proteínas/metabolismo , Albúminas/metabolismo , Endotelio Vascular/metabolismo , Glicocálix/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Microvasos/fisiología , Perfusión , Receptores Fc/metabolismo , TranscitosisRESUMEN
Pim kinases are a family of constitutively active serine/threonine kinases that are partially redundant and regulate multiple pathways important for cell growth and survival. In human disease, high expression of the three Pim isoforms has been implicated in the progression of hematopoietic and solid tumor cancers, which suggests that Pim kinase inhibitors could provide patients with therapeutic benefit. Herein, we describe the structure-guided optimization of a series of quinazolinone-pyrrolodihydropyrrolone analogs leading to the identification of potent pan-Pim inhibitor 28 with improved potency, solubility, and drug-like properties. Compound 28 demonstrated on-target Pim activity in an in vivo pharmacodynamic assay with significant inhibition of BAD phosphorylation in KMS-12-BM multiple myeloma tumors for 16 h postdose. In a 2-week mouse xenograft model, daily dosing of compound 28 resulted in 33% tumor regression at 100 mg/kg.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Hematológicas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Pirroles/uso terapéutico , Quinazolinonas/uso terapéutico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Femenino , Humanos , Ratones SCID , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Pirroles/síntesis química , Pirroles/farmacocinética , Quinazolinonas/síntesis química , Quinazolinonas/farmacocinética , Relación Estructura-Actividad , Porcinos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) has attracted considerable attention during the past few years as a potential target for the treatment of diseases associated with metabolic syndrome. In our ongoing work on 11beta-HSD1 inhibitors, a series of new 2-amino-1,3-thiazol-4(5 H)-ones were explored. By inserting various cycloalkylamines at the 2-position and alkyl groups or spirocycloalkyl groups at the 5-position of the thiazolone, several potent 11beta-HSD1 inhibitors were identified. An X-ray cocrystal structure of human 11beta-HSD1 with compound 6d (Ki=28 nM) revealed a large lipophilic pocket accessible by substitution off the 2-position of the thiazolone. To increase potency, analogues were prepared with larger lipophilic groups at this position. One of these compounds, the 3-noradamantyl analogue 8b, was a potent inhibitor of human 11beta-HSD1 (Ki=3 nM) and also inhibited 11beta-HSD1 activity in lean C57Bl/6 mice when evaluated in an ex vivo adipose and liver cortisone to cortisol conversion assay.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , Adamantano/análogos & derivados , Hipoglucemiantes/síntesis química , Tiazoles/síntesis química , Triazoles/síntesis química , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/química , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Adamantano/síntesis química , Adamantano/farmacocinética , Adamantano/farmacología , Tejido Adiposo/metabolismo , Animales , Cortisona/metabolismo , Cristalografía por Rayos X , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Humanos , Hidrocortisona/metabolismo , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Ligandos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Estructura Molecular , Ratas , Estereoisomerismo , Relación Estructura-Actividad , Tiazoles/farmacocinética , Tiazoles/farmacología , Triazoles/farmacocinética , Triazoles/farmacologíaRESUMEN
In our continued efforts to search for potent and novel receptor tyrosine kinase (RTK) inhibitors as potential anticancer agents, we discovered, through a structure-based design, that 3-aminoindazole could serve as an efficient hinge-binding template for kinase inhibitors. By incorporating an N,N'-diaryl urea moiety at the C4-position of 3-aminodazole, a series of RTK inhibitors were generated, which potently inhibited the tyrosine kinase activity of the vascular endothelial growth factor receptor and the platelet-derived growth factor receptor families. A number of compounds with potent oral activity were identified by utilizing an estradiol-induced mouse uterine edema model and an HT1080 human fibrosarcoma xenograft tumor model. In particular, compound 17p (ABT-869) was found to possess favorable pharmacokinetic profiles across different species and display significant tumor growth inhibition in multiple preclinical animal models.
Asunto(s)
Inhibidores de la Angiogénesis/síntesis química , Indazoles/síntesis química , Compuestos de Fenilurea/síntesis química , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Adenosina Trifosfato/química , Administración Oral , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Animales , Sitios de Unión , Edema/inducido químicamente , Edema/patología , Estradiol , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Indazoles/química , Indazoles/farmacología , Masculino , Ratones , Modelos Moleculares , Células 3T3 NIH , Compuestos de Fenilurea/química , Compuestos de Fenilurea/farmacología , Fosforilación , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Relación Estructura-Actividad , Útero/efectos de los fármacos , Útero/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
As part of an ongoing effort at Amgen to develop a disease-modifying therapy for Alzheimer's disease, we have previously used the aminooxazoline xanthene (AOX) scaffold to generate potent and orally efficacious BACE1 inhibitors. While AOX-BACE1 inhibitors demonstrated acceptable cardiovascular safety margins, a retinal pathological finding in rat toxicological studies demanded further investigation. It has been widely postulated that such retinal toxicity might be related to off-target inhibition of Cathepsin D (CatD), a closely related aspartyl protease. We report the development of AOX-BACE1 inhibitors with improved selectivity against CatD by following a structure- and property-based approach. Our efforts culminated in the discovery of a picolinamide-substituted 3-aza-AOX-BACE1 inhibitor absent of retinal effects in an early screening rat toxicology study.