RESUMO
Drugs with prolonged on-target residence times often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here we made progress toward this elusive goal by targeting a noncatalytic cysteine in Bruton's tyrosine kinase (BTK) with reversible covalent inhibitors. Using an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, we identified potent and selective BTK inhibitors that demonstrated biochemical residence times spanning from minutes to 7 d. An inverted cyanoacrylamide with prolonged residence time in vivo remained bound to BTK for more than 18 h after clearance from the circulation. The inverted cyanoacrylamide strategy was further used to discover fibroblast growth factor receptor (FGFR) kinase inhibitors with residence times of several days, demonstrating the generalizability of the approach. Targeting of noncatalytic cysteines with inverted cyanoacrylamides may serve as a broadly applicable platform that facilitates 'residence time by design', the ability to modulate and improve the duration of target engagement in vivo.
Assuntos
Acrilamidas/farmacocinética , Linfócitos B/efeitos dos fármacos , Cianoacrilatos/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Tirosina Quinases/antagonistas & inibidores , Acrilamidas/síntese química , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/enzimologia , Linfócitos B/patologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Cianoacrilatos/síntese química , Dasatinibe , Feminino , Expressão Gênica , Humanos , Ligantes , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/síntese química , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Pirimidinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera , Relação Estrutura-Atividade , Especificidade por Substrato , Tiazóis/farmacocinética , Fatores de TempoRESUMO
Bruton's tyrosine kinase (BTK), expressed in B cells and cells of innate immunity, including microglia, is an essential signaling element downstream of the B-cell receptor and Fc-receptors. Tolebrutinib (PRN2246, SAR442168) is a potent BTK inhibitor that covalently binds the kinase, resulting in durable inhibition with the potential to target inflammation in the periphery and central nervous system (CNS). Tolebrutinib crosses the blood-brain barrier and potently inhibits BTK in microglial cells isolated from the CNS. A first-in-human randomized, double-blind, placebo-controlled study of tolebrutinib was conducted. The trial design consisted of five single ascending dose arms with oral administration of a single dose of 5, 15, 30, 60, and 120 mg (n = 6 per arm, n = 2 placebo), five multiple ascending dose arms with oral administration of 7.5, 15, 30, 60, and 90 mg (n = 8 per arm, n = 2 placebo) over 10 days, and one arm (n = 4) in which cerebral spinal fluid (CSF) exposure was measured 2 h after a single 120 mg dose. Tolebrutinib was well-tolerated in the study and all treatment-related treatment emergent adverse events were mild. Tolebrutinib was rapidly absorbed following oral administration with a rapid half-life of ~ 2 h. Peripheral BTK occupancy was assessed at various timepoints by an enzyme-linked immunosorbent assay-based readout using an irreversible probe. Assessments demonstrated extensive and prolonged peripheral BTK occupancy at steady-state with once daily doses as low as 7.5 mg. Further, CSF exposure was demonstrated 2 h after administration at 120 mg.
Assuntos
Inibidores de Proteínas Quinases , Tirosina Quinase da Agamaglobulinemia , Relação Dose-Resposta a Droga , Método Duplo-Cego , Meia-Vida , Humanos , Inibidores de Proteínas Quinases/efeitos adversosRESUMO
An increasing number of cancers are known to harbor mutations, translocations, or amplifications in the fibroblast growth factor receptor (FGFR) family of kinases. The FGFR inhibitors evaluated in clinical trials to date have shown promise at treating these cancers. Here, we describe PRN1371, an irreversible covalent inhibitor of FGFR1-4 targeting a cysteine within the kinase active site. PRN1371 demonstrated strong FGFR potency and excellent kinome-wide selectivity in a number of biochemical and cellular assays, including in various cancer cell lines exhibiting FGFR alterations. Furthermore, PRN1371 maintained FGFR inhibition in vivo, not only when circulating drug levels were high but also after the drug had been cleared from circulation, indicating the possibility of sustained FGFR inhibition in the clinic without the need for continuous drug exposure. Durable tumor regression was also obtained in multiple tumor xenografts and patient-derived tumor xenograft models and was sustained even using an intermittent dosing strategy that provided drug holidays. PRN1371 is currently under clinical investigation for treatment of patients with solid tumors. Mol Cancer Ther; 16(12); 2668-76. ©2017 AACR.
Assuntos
Piridonas/uso terapêutico , Pirimidinas/uso terapêutico , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Camundongos , Piridonas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aberrant signaling of the FGF/FGFR pathway occurs frequently in cancers and is an oncogenic driver in many solid tumors. Clinical validation of FGFR as a therapeutic target has been demonstrated in bladder, liver, lung, breast, and gastric cancers. Our goal was to develop an irreversible covalent inhibitor of FGFR1-4 for use in oncology indications. An irreversible covalent binding mechanism imparts many desirable pharmacological benefits including high potency, selectivity, and prolonged target inhibition. Herein we report the structure-based design, medicinal chemistry optimization, and unique ADME assays of our irreversible covalent drug discovery program which culminated in the discovery of compound 34 (PRN1371), a highly selective and potent FGFR1-4 inhibitor.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Piridonas/farmacologia , Pirimidinas/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Cães , Desenho de Fármacos , Estabilidade de Medicamentos , Feminino , Humanos , Absorção Intestinal , Macaca fascicularis , Masculino , Piridonas/administração & dosagem , Piridonas/síntese química , Piridonas/farmacocinética , Pirimidinas/administração & dosagem , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Ratos Sprague-Dawley , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Solubilidade , Relação Estrutura-AtividadeRESUMO
Amino carbamate adduct formation from the amino group of an aminoglycoside and carbon dioxide has been postulated as a mechanism for reducing nephrotoxicity in the aminoglycoside class compounds. In this study, sisomicin was used as a model compound for amino carbamate analysis. A high pH based reversed-phase high performance liquid chromatography (RP-HPLC) method is used to separate the amino carbamate from sisomicin. The carbamate is stable as the breakdown is inhibited at high pH and any reactive carbon dioxide is removed as the carbonate. The amino carbamate was quantified and the molar fraction of amine as the carbamate of sisomicin was obtained from the HPLC peak areas. The equilibrium constant of carbamate formation, Kc, was determined to be 3.3 × 10(-6) and it was used to predict the fraction of carbamate over the pH range in a typical biological systems. Based on these results, the fraction of amino carbamate at physiological pH values is less than 13%, and the postulated mechanism for nephrotoxicity protection is not valid. The same methodology is applicable for other aminoglycosides.
Assuntos
Carbamatos/química , Sisomicina/química , Aminoglicosídeos/química , Dióxido de Carbono/química , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Concentração de Íons de HidrogênioRESUMO
Novel derivatives of N-decylaminoethylvancomycin (2), containing appended hydrophilic groups were synthesized and their antibacterial activity and ADME properties were evaluated. The compounds were prepared by reacting amines with the C-terminus (C-) of 2 using PyBOP mediated amide formation, or with the resorcinol-like (R-) position of 2 using a Mannich aminomethylation reaction. These analogs retained the antibacterial activity of 2 against methicillin-resistant staphylococci and vancomycin-resistant enterococci. Compounds with a negatively charged auxiliary group also exhibited improved ADME properties relative to 2. In particular, R-phosphonomethylaminomethyl derivative 21 displayed good in vitro antibacterial activity, high urinary recovery and low distribution to liver and kidney tissues. Based on these results, 21 was advanced into development as TD-6424, and is currently in human clinical trials. The generic name telavancin has recently been approved for compound 21.
Assuntos
Aminoglicosídeos/farmacocinética , Antibacterianos/farmacologia , Vancomicina/análogos & derivados , Vancomicina/farmacocinética , Aminoglicosídeos/química , Animais , Fenômenos Químicos , Físico-Química , Cromatografia Líquida de Alta Pressão , Feminino , Bactérias Gram-Positivas/efeitos dos fármacos , Indicadores e Reagentes , Injeções Intravenosas , Lipoglicopeptídeos , Testes de Sensibilidade Microbiana , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Ultravioleta , Teicoplanina/farmacologia , Distribuição TecidualRESUMO
A reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed for the aminoglycoside (AG) plazomicin (ACHN-490). This method employed a high pH mobile phase (pH>11) with a gradient of 0.25 M ammonium hydroxide in water and acetonitrile, an XBridge C(18) column and UV detection at 210 nm. Although the molar UV absorption of plazomicin is weak, the high pH conditions of this method allow for higher loadings, which compensates for the inherent low UV sensitivity. Under these high pH conditions, impurities and degradants were base line separated from plazomicin. The mobile phases used for this method allowed for on-line mass detection for the impurities and degradants. The RP-HPLC method has been validated in terms of specificity, linearity and range, accuracy, and precision. The analytical method met specificity requirements of a homogenous peak with no interferences from the blank or from the known impurities in plazomicin. The linearity of the method for the plazomicin impurity determination was excellent, with a coefficient of determination (r(2)) of 0.9993, over the freebase (FB) concentration range of 0.0025-3.0 mg/mL. The method is capable of detecting impurities down to 0.1% of the peak area of plazomicin. A single point standard at a concentration of 1.0 mg/mL FB was validated over the range of 50-150% for quantitation of the freebase content (the assay) in bulk drug substance. The mean recoveries of FB are in the range 98.6-102.0% with a mean RSD (relative standard deviation) <1.0%. The study also examined the method precision for purity, impurities and the assay with two instruments on two different days. The method showed adequate accuracy and precision for the intended use. This high pH method was successfully used to determine the impurity and measure the drug content in the final plazomicin drug substance. In addition, the method with an on-line mass spectrometry detector has been used to characterize the structures of the impurities in plazomicin.
Assuntos
Antibacterianos/análise , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Sisomicina/análogos & derivados , Antibacterianos/química , Contaminação de Medicamentos , Concentração de Íons de Hidrogênio , Limite de Detecção , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Sisomicina/análise , Sisomicina/químicaRESUMO
The emergence and spread of multidrug-resistant gram-positive bacteria represent a serious clinical problem. Telavancin is a novel lipoglycopeptide antibiotic that possesses rapid in vitro bactericidal activity against a broad spectrum of clinically relevant gram-positive pathogens. Here we demonstrate that telavancin's antibacterial activity derives from at least two mechanisms. As observed with vancomycin, telavancin inhibited late-stage peptidoglycan biosynthesis in a substrate-dependent fashion and bound the cell wall, as it did the lipid II surrogate tripeptide N,N'-diacetyl-L-lysinyl-D-alanyl-D-alanine, with high affinity. Telavancin also perturbed bacterial cell membrane potential and permeability. In methicillin-resistant Staphylococcus aureus, telavancin caused rapid, concentration-dependent depolarization of the plasma membrane, increases in permeability, and leakage of cellular ATP and K(+). The timing of these changes correlated with rapid , concentration-dependent loss of bacterial viability, suggesting that the early bactericidal activity of telavancin results from dissipation of cell membrane potential and an increase in membrane permeability. Binding and cell fractionation studies provided direct evidence for an interaction of telavancin with the bacterial cell membrane; stronger binding interactions were observed with the bacterial cell wall and cell membrane relative to vancomycin. We suggest that this multifunctional mechanism of action confers advantageous antibacterial properties.
Assuntos
Aminoglicosídeos/farmacologia , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Parede Celular/metabolismo , Lipoglicopeptídeos , Resistência a Meticilina , Peptidoglicano/biossínteseRESUMO
The movement towards a 96-well format has greatly increased productivity and throughput in bioanalytical laboratories. Improvements in automated sample preparation and analytical methods have further contributed to increased productivity. We have focused on sample collection and transfer to the bioanalyst and have found improvements to the current available methods. The problem of manual transfers and plasma clotting issues can be overcome with the use of microtainers. Specifically, for illustrative purposes, three proprietary Theravance compounds were tested for stability, non-specific binding, and electrospray ion suppression in microtainers. There were no issues with stability, non-specific binding or ion suppression for the above compounds even after leaving plasma samples in the microtainers over long periods of time. The microtainers are robot-compatible and the resulting plasma can be transferred without clotting issues. To date, all in-house compounds successfully analyzed and tested using the microtainers have mass ranges between 200 and 1800 Da, pK(a) ranges between 3.8 and 10.3, and logD ranges between -1.7 and 4.2. Once samples are transferred into 96-well plates, flexibility in preparation and analysis is available. Together with automated sample preparation and the use of liquid chromatography/tandem mass spectrometry (LC/MS/MS) as an analytical tool, the use of microtainers as sample collection tubes and for sample storage saved considerable time, cost and effort in both of our pharmacokinetic (PK) and bioanalytical groups. This in turn has led to an increased efficiency and overall throughput in support of our drug discovery effort.