RESUMEN
Herein we disclose SAR studies that led to a series of isoindoline ureas which we recently reported were first-in-class, non-substrate nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. Modification of the isoindoline and/or the terminal functionality of screening hit 5 provided inhibitors such as 52 and 58 with nanomolar antiproliferative activity and preclinical pharmacokinetics properties which enabled potent antitumor activity when dosed orally in mouse xenograft models. X-ray crystal structures of two inhibitors bound in the NAMPT active-site are discussed.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Citocinas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Urea/análogos & derivados , Urea/farmacología , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Citocinas/química , Citocinas/metabolismo , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/uso terapéutico , Humanos , Isoindoles/química , Isoindoles/farmacocinética , Isoindoles/farmacología , Isoindoles/uso terapéutico , Ratones , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Nicotinamida Fosforribosiltransferasa/química , Nicotinamida Fosforribosiltransferasa/metabolismo , Relación Estructura-Actividad , Urea/farmacocinética , Urea/uso terapéuticoRESUMEN
Izencitinib (TD-1473), an oral, gut-selective pan-Janus kinase (JAK) inhibitor under investigation for treatment of inflammatory bowel diseases, was designed for optimal efficacy in the gastrointestinal tract while minimizing systemic exposures and JAK-related safety findings. The nonclinical safety of izencitinib was evaluated in rat and dog repeat-dose and rat and rabbit reproductive and developmental toxicity studies. Systemic exposures were compared with JAK inhibitory potency to determine effects at or above pharmacologic plasma concentrations (≥1× plasma average plasma concentration [Cave]:JAK 50% inhibitory concentration [IC50] ratio). In rats and dogs, 1000 and 30 mg/kg/day izencitinib, respectively, produced minimal systemic findings (ie, red/white cell changes) and low systemic concentrations (approximately 1× plasma Cave:JAK IC50 ratio) with an 8× nonclinical:clinical systemic area under the curve (AUC) margin compared with exposures at the highest clinically tested dose (300 mg, quaque die, once daily, phase 1 study in healthy volunteers). In dogs, it was possible to attain sufficient systemic exposures to result in immunosuppression characteristic of systemic JAK inhibition, but at high AUC margins (43×) compared with systemic exposures observed at the highest tested dose in humans. No adverse findings were observed in the gastrointestinal tract or systemic tissues. Izencitinib did not affect male or female fertility. Izencitinib did not affect embryonic development in rats and rabbits as commonly reported with systemic JAK inhibition, consistent with low maternal systemic concentrations (2-6× plasma Cave:JAK IC50 ratio, 10-33× nonclinical:clinical AUC margin) and negligible fetal exposures. In conclusion, the izencitinib gut-selective approach resulted in minimal systemic findings in nonclinical species at pharmacologic, clinically relevant systemic exposures, highlighting the impact of organ-selectivity in reducing systemic safety findings.
Asunto(s)
Quinasas Janus , Naftiridinas , Nitrilos , Administración Oral , Animales , Perros , Desarrollo Embrionario/efectos de los fármacos , Femenino , Voluntarios Sanos , Humanos , Enfermedades Inflamatorias del Intestino , Quinasas Janus/antagonistas & inhibidores , Masculino , Naftiridinas/farmacología , Naftiridinas/toxicidad , Nitrilos/farmacología , Nitrilos/toxicidad , Embarazo , Conejos , Ratas , Reproducción/efectos de los fármacos , Pruebas de ToxicidadRESUMEN
Probody therapeutics (Pb-Txs) are conditionally activated antibody-drug conjugates (ADCs) designed to remain inactive until proteolytically activated in the tumor microenvironment, enabling safer targeting of antigens expressed in both tumor and normal tissue. Previous attempts to target CD71, a highly expressed tumor antigen, have failed to establish an acceptable therapeutic window due to widespread normal tissue expression. This study evaluated whether a probody-drug conjugate targeting CD71 can demonstrate a favorable efficacy and tolerability profile in preclinical studies for the treatment of cancer. CX-2029, a Pb-Tx conjugated to maleimido-caproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E, was developed as a novel cancer therapeutic targeting CD71. Preclinical studies were performed to evaluate the efficacy and safety of this anti-CD71 PDC in patient-derived xenograft (PDX) mouse models and cynomolgus monkeys, respectively. CD71 expression was detected at high levels by IHC across a broad range of tumor and normal tissues. In vitro, the masked Pb-Tx form of the anti-CD71 PDC displayed a >50-fold reduced affinity for binding to CD71 on cells compared with protease-activated, unmasked anti-CD71 PDC. Potent in vivo tumor growth inhibition (stasis or regression) was observed in >80% of PDX models (28/34) at 3 or 6 mg/kg. Anti-CD71 PDC remained mostly masked (>80%) in circulation throughout dosing in cynomolgus monkeys at 2, 6, and 12 mg/kg and displayed a 10-fold improvement in tolerability compared with an anti-CD71 ADC, which was lethal. Preclinically, anti-CD71 PDC exhibits a highly efficacious and acceptable safety profile that demonstrates the utility of the Pb-Tx platform to target CD71, an otherwise undruggable target. These data support further clinical development of the anti-CD71 PDC CX-2029 as a novel cancer therapeutic.
Asunto(s)
Antineoplásicos , Inmunoconjugados , Neoplasias , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Plomo , Macaca fascicularis/metabolismo , Ratones , Neoplasias/tratamiento farmacológico , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A series of studies were conducted to explore the inductive potential of different fibric acid derivatives on the two alternative metabolic activation pathways of 2-phenylpropionic acid (2-PPA) (a model substrate for profen drugs), namely acyl-CoA formation and acyl glucuronidation, in vivo in rats, and to evaluate whether such treatment could potentially modulate the covalent binding of profens to hepatic protein. After administration of a single dose of 2-PPA (130 mg/kg) to rats pretreated with equimolar doses of clofibric acid (160 mg/kg/day), fenofibrate (260 mg/kg/day), or gemfibrozil (180 mg/kg/day) for 7 days, rat livers were collected and analyzed for covalent binding and hepatic levels of the two reactive metabolites over a 2-h period. Results showed that the three fibrates exhibited very different effects on the hepatic levels of 2-PPA-S-acyl CoA (2-PPA-CoA) in vivo, even though all three significantly increased acyl-CoA synthetase activity in vitro in liver homogenate. Treatment with clofibric acid markedly increased the hepatic exposure of 2-PPA-CoA by 2.9-fold and led to a 25% increase (p < 0.05) in covalent binding of 2-PPA to liver protein. In contrast, significant decreases of the hepatic levels of 2-PPA acyl glucuronide and/or 2-PPA-CoA by fenofibrate and gemfibrozil significantly lowered the covalent binding of 2-PPA to hepatic protein. Together, these results suggest that fibrates exhibit markedly different abilities to alter the extent of covalent binding of 2-PPA to hepatic protein by differentially modulating the hepatic exposure of the two reactive metabolites of 2-PPA, namely 2-PPA-CoA thioester and acyl glucuronide.
Asunto(s)
Ácido Clofíbrico/farmacocinética , Fenilpropionatos/metabolismo , Animales , Biotransformación/efectos de los fármacos , Biotransformación/fisiología , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Cancer cells are highly reliant on NAD+-dependent processes, including glucose metabolism, calcium signaling, DNA repair, and regulation of gene expression. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ salvage from nicotinamide, has been investigated as a target for anticancer therapy. Known NAMPT inhibitors with potent cell activity are composed of a nitrogen-containing aromatic group, which is phosphoribosylated by the enzyme. Here, we identified two novel types of NAM-competitive NAMPT inhibitors, only one of which contains a modifiable, aromatic nitrogen that could be a phosphoribosyl acceptor. Both types of compound effectively deplete cellular NAD+, and subsequently ATP, and produce cell death when NAMPT is inhibited in cultured cells for more than 48 hours. Careful characterization of the kinetics of NAMPT inhibition in vivo allowed us to optimize dosing to produce sufficient NAD+ depletion over time that resulted in efficacy in an HCT116 xenograft model. Our data demonstrate that direct phosphoribosylation of competitive inhibitors by the NAMPT enzyme is not required for potent in vitro cellular activity or in vivo antitumor efficacy. Mol Cancer Ther; 16(7); 1236-45. ©2017 AACR.
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Citocinas/antagonistas & inhibidores , Inhibidores Enzimáticos/administración & dosificación , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Animales , Señalización del Calcio/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Citocinas/genética , Reparación del ADN/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Humanos , Ratones , NAD/metabolismo , Nicotinamida Fosforribosiltransferasa/genética , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Proteína Portadora de Folato Reducido/genética , Animales , Antimetabolitos Antineoplásicos/efectos adversos , Antimetabolitos Antineoplásicos/sangre , Antimetabolitos Antineoplásicos/metabolismo , Cricetinae , Cricetulus , Humanos , Metotrexato/efectos adversos , Metotrexato/sangre , Metotrexato/metabolismo , Ratones , FarmacogenéticaRESUMEN
A high throughput, semi-automated clearance screening assay in hepatocytes was developed allowing a scientist to generate data for 96 compounds in one week. The 384-well format assay utilizes a Thermo Multidrop Combi and an optimized LC-MS/MS method. The previously reported LCMS/ MS method reduced the analytical run time by 3-fold, down to 1.2 min injection-to-injection. The Multidrop was able to deliver hepatocytes to 384-well plates with minimal viability loss. Comparison of results from the new 384-well and historical 24-well assays yielded a correlation of 0.95. In addition, results obtained for 25 marketed drugs with various metabolism pathways had a correlation of 0.75 when compared with literature values. Precision was maintained in the new format as 8 compounds tested in ≥39 independent experiments had coefficients of variation ≤21%. The ability to predict in vivo clearances using the new stability assay format was also investigated using 22 marketed drugs and 26 AbbVie compounds. Correction of intrinsic clearance values with binding to hepatocytes (in vitro data) and plasma (in vivo data) resulted in a higher in vitro to in vivo correlation when comparing 22 marketed compounds in human (0.80 vs 0.35) and 26 AbbVie Discovery compounds in rat (0.56 vs 0.17), demonstrating the importance of correcting for binding in clearance studies. This newly developed high throughput, semi-automated clearance assay allows for rapid screening of Discovery compounds to enable Structure Activity Relationship (SAR) analysis based on high quality hepatocyte stability data in sufficient quantity and quality to drive the next round of compound synthesis.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Drogas en Investigación/farmacocinética , Hepatocitos/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Animales , Automatización , Cromatografía Liquida , Evaluación Preclínica de Medicamentos/instrumentación , Hepatocitos/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Espectrometría de Masas en TándemRESUMEN
Systemic disposition of nucleosides and nucleoside analogs is dependent on renal handling of these compounds. There are five known, functionally characterized nucleoside transporters with varying substrate specificities for nucleosides: concentrative nucleoside transporters (CNT1-CNT3; Solute Carrier (SLC) 28A1-28A3), which mediate the intracellular flux of nucleosides, and equilibrative nucleoside transporters (ENT1-ENT2; SLC29A1-SLC29A2), which mediate bi-directional facilitated diffusion of nucleosides. All five of these transporters are expressed in the kidney. Concentrative nucleoside transporters primarily localize to the apical membrane of renal epithelial cells while equilibrative nucleoside transporters primarily localize to the basolateral membrane. These transporters work in concert to mediate reabsorptive flux of naturally occurring nucleosides and nucleoside analogs. In addition, equilibrative transporters also participate in secretory flux of some nucleoside analogs. Nucleoside transporters also serve in the targeting of nucleoside analog therapies to renal tumors. This review examines the role that these transporters play in renal disposition of nucleosides and nucleoside analogs in both systemic and kidney-specific therapies.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Riñón/metabolismo , Proteínas de Transporte de Nucleósidos/metabolismo , Nucleósidos/metabolismo , Animales , Humanos , Riñón/efectos de los fármacos , Proteínas de Transporte de Nucleósidos/química , Proteínas de Transporte de Nucleósidos/genética , Nucleósidos/administración & dosificación , Nucleósidos/químicaRESUMEN
Members of the concentrative nucleoside transporter (CNT) family (SLC28) mediate the transport of naturally-occurring nucleosides, and nucleoside analog drugs across the plasma membrane of epithelial cells. Each of the three CNT family members has a distinct specificity for naturally occurring nucleosides, and residues that contribute to the specificity of each transporter have been identified. In contrast, the molecular determinants of specificity for synthetic nucleoside analogs are not known. In this study, we take advantage of the large species difference that exists between human and rat CNT2 (hCNT2 and rCNT2) in their ability to transport the nucleoside analog drug cladribine, 2CdA, (rCNT2 > > > hCNT2) to identify the critical domains and amino acid residues that contribute to the observed difference in specificity between CNT2 orthologs. Using chimeric proteins of human and rat CNT2, we determined that the C-terminal half of CNT2 contained the determinants of 2CdA selectivity. We replaced key residues in the C terminus of hCNT2 with the equivalent residue in rCNT2. One residue in the C-terminal portion of CNT2 was found to significantly contribute to 2CdA selectivity: hCNT2-S354A. This mutant caused an increase of 5-6-fold over hCNT2. The 2-chloro pharmacophore, rather than the 2'-deoxyribose was responsible for the reduced 2CdA uptake by hCNT2. Our data are consistent with a model in which an increased capability for hydrogen bonding in critical amino acids that reside in the C terminus of rCNT2 contributes to its enhanced selectivity for 2CdA.
Asunto(s)
Proteínas de Transporte de Membrana , Nucleósidos , Proteínas Recombinantes de Fusión , Secuencia de Aminoácidos , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Cladribina/química , Cladribina/metabolismo , Humanos , Enlace de Hidrógeno , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Nucleósidos/química , Nucleósidos/metabolismo , Oocitos/fisiología , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , Xenopus laevisRESUMEN
Although considerable progress has been made toward characterizing human DNA sequence variation, there remains a deficiency in information on human phenotypic variation at the single-gene level. We systematically analyzed the function of all protein-altering variants of eleven membrane transporters in heterologous expression systems. Coding-region variants were identified by screening DNA from a large sample (n = 247-276) of ethnically diverse subjects. In total, we functionally analyzed 88 protein-altering variants. Fourteen percent of the polymorphic variants (defined as variants with allele frequencies > or =1% in at least one major ethnic group) had no activity or significantly reduced function. Decreased function variants had significantly lower allele frequencies and were more likely to alter evolutionarily conserved amino acid residues. However, variants at evolutionarily conserved positions with approximately normal activity in cellular assays were also at significantly lower allele frequencies, suggesting that some variants with apparently normal activity in biochemical assays may influence occult functions or quantitative degrees of function that are important in human fitness but not measured in these assays. For example, eight (14%) of the 58 variants for which we had measured the transport of at least two substrates showed substrate-specific defects in transport. These variants and the reduced function variants provide plausible candidates for disease susceptibility or variation in clinical drug response.
Asunto(s)
Frecuencia de los Genes/genética , Genómica , Proteínas de Transporte de Membrana/genética , Polimorfismo Genético/genética , Transporte Biológico/genética , Etnicidad , Expresión Génica/genética , Genómica/métodos , HumanosRESUMEN
Methotrexate (MTX) is used in patients with malignant and autoimmune diseases. This drug is primarily excreted unchanged in the urine, and its net excretion occurs via active secretory and reabsorptive processes. We characterized the interaction of MTX with human organic-anion transporting polypeptide transporter (OATP) 1A2, which is expressed in tissues important for MTX disposition and toxicity, such as the intestine, kidney, liver, and endothelial cells of the blood-brain barrier. In Xenopus laevis oocytes expressing OATP1A2, the uptake of the model substrate, estrone-3-sulfate (ES), was enhanced 30-fold compared with uninjected oocytes. MTX uptake in oocytes expressing OATP1A2 was saturable (Km = 457 +/- 118 microM; Vmax = 17.5 +/- 4.9 pmol/oocyte/60 min) and sensitive to extracellular pH. That is, acidic pHs stimulated MTX uptake by as much as 7-fold. Seven novel protein-altering variants were identified in 270 ethnically diverse DNA samples. Four protein-altering variants in OATP1A2 exhibited altered transport of ES and/or MTX. The common variant, protein reference sequence (p.) Ile13Thr, was hyperfunctional for ES and MTX and showed a 2-fold increase in the V(max) for ES. The common variant, p. Glu172Asp, exhibited reduced maximal transport capacity for ES and MTX. p. Arg168Cys was hypofunctional, and p. Asn277DEL was nonfunctional. Because of its expression on the apical membrane of the distal tubule and in tissues relevant to MTX disposition and toxicity, these findings suggest that OATP1A2 may play a role in active tubular reabsorption of MTX and in MTX-induced toxicities. Furthermore, genetic variation in OATP1A2 may contribute to variation in MTX disposition and response.