RESUMO
Dual leucine zipper kinase (DLK, MAP3K12) was recently identified as an essential regulator of neuronal degeneration in multiple contexts. Here we describe the generation of potent and selective DLK inhibitors starting from a high-throughput screening hit. Using proposed hinge-binding interactions to infer a binding mode and specific design parameters to optimize for CNS druglike molecules, we came to focus on the di(pyridin-2-yl)amines because of their combination of desirable potency and good brain penetration following oral dosing. Our lead inhibitor GNE-3511 (26) displayed concentration-dependent protection of neurons from degeneration in vitro and demonstrated dose-dependent activity in two different animal models of disease. These results suggest that specific pharmacological inhibition of DLK may have therapeutic potential in multiple indications.
Assuntos
MAP Quinase Quinase Quinases/antagonistas & inibidores , Degeneração Neural/prevenção & controle , Doenças Neurodegenerativas/prevenção & controle , Inibidores de Proteínas Quinases/farmacologia , Animais , Modelos Animais de Doenças , Cães , Relação Dose-Resposta a Droga , Descoberta de Drogas , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Camundongos Endogâmicos C57BL , Modelos Químicos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , RatosRESUMO
BACKGROUND: NAD(+) is an endogenous analyte and is unstable during blood sample collection, both of which present obstacles for quantitation. Moreover, current procedures for NAD(+) sample collection require onsite treatment with strong acid to stabilize the NAD(+) in mouse blood cells. RESULTS: NAD(+) can be stabilized by addition of acid before the frozen mouse blood sample was thawed. A simple sample collection procedure was proposed to facilitate the analysis of NAD(+) in mouse blood and tissue samples. A LC-MS/MS method was developed for quantifying NAD(+) in mouse blood and various tissue samples. The described method was used to measure endogenous NAD(+) levels in mouse blood following oral administration of the nicotinamide phosphoribosyltransferase inhibitor GNE-617. CONCLUSION: This study presents a suitable assay and sample collection procedure for high throughput screening of NAD(+) samples in preclinical discovery studies.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , NAD/sangue , Animais , Coleta de Amostras Sanguíneas , CamundongosRESUMO
MEK, a kinase downstream of Ras and Raf oncogenes, constitutes a high priority target in oncology research. MEK small molecule inhibitors cause soft tissue mineralization in rats secondary to serum inorganic phosphorus (iP) elevation, but the molecular mechanism for this toxicity remains undetermined. We performed investigative studies with structurally distinct MEK inhibitors GEN-A and PD325901 (PD-901) in Sprague-Dawley rats. Our data support a mechanism that involves FGF-23 signal blockade in the rat kidney, causing transcriptional upregulation of 25-hydroxyvitamin D(3) 1-alpha-hydroxylase (Cyp27b1), the rate-limiting enzyme in vitamin D activation, and downregulation of 1,25-dihydroxyvitamin D(3) 24-hydroxylase (Cyp24a1), the enzyme that initiates the degradation of the active form of vitamin D. These transcriptional changes increase serum vitamin D levels, which in turn drive the increase in serum iP, leading to soft tissue mineralization in the rat.
Assuntos
Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Rim/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Fósforo/sangue , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Animais , Cálcio/sangue , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Homeostase/efeitos dos fármacos , Rim/enzimologia , Rim/metabolismo , Masculino , Estrutura Molecular , Hormônio Paratireóideo/sangue , Inibidores de Proteínas Quinases/química , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Espectrometria de Massas em Tandem , Vitamina D/sangueRESUMO
GNE-A (AR00451896; N-(4-(3-((3S,4R)-1-ethyl-3-fluoropiperidine-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-4-yloxy)-3-fluorophenyl)-2-(4-fluorophenyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide) is a potent, selective MET kinase inhibitor being developed as a potential drug for the treatment of human cancers. Plasma clearance was low in mice and dogs (15.8 and 2.44 mL/min/kg, respectively) and moderate in rats and monkeys (36.6 and 13.9 mL/min/kg, respectively). The volume of distribution ranged from 2.1 to 9.0 L/kg. The mean terminal elimination half-life ranged from 1.67 h in rats to 16.3 h in dogs. Oral bioavailability in rats, mice, monkeys, and dogs were 11.2%, 88.0%, 72.4%, and 55.8%, respectively. Allometric scaling predicted a clearance of 1.3-7.4 mL/min/kg and a volume of distribution of 4.8-11 L/kg in human. Plasma protein binding was high (96.7-99.0% bound). Blood-to-plasma concentration ratios (0.78-1.46) indicated that GNE-A did not preferentially distribute into red blood cells. Transporter studies in MDCKI-MDR1 and MDCKII-Bcrp1 cells suggested that GNE-A is likely a substrate for MDR1 and BCRP. Pharmacokinetic-pharmacodynamic modelling of tumour growth inhibition in MET-amplified EBC-1 human non-small cell lung carcinoma tumour xenograft mice projected oral doses of 5.6 and 13 mg/kg/day for 50% and 90% tumour growth inhibition, respectively. Overall, GNE-A exhibited favourable preclinical properties and projected human dose estimates.
Assuntos
Antineoplásicos/farmacocinética , Modelos Biológicos , Inibidores de Proteínas Quinases/farmacocinética , Pirazóis/farmacocinética , Piridazinas/farmacocinética , Absorção , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cães , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Haplorrinos , Humanos , Masculino , Camundongos , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Pirazóis/metabolismo , Pirazóis/farmacologia , Piridazinas/metabolismo , Piridazinas/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
A LC-MS/MS method has been developed to analyze tetranor PGE-M, the major urinary metabolite of PGE(2), that involves the acid-catalyzed dehydration of tetranor PGE-M and its deuterated (d(6)) analog followed by LC-MS/MS measurement of the dehydrated tetranor PGE-M product (tetranor PGA-M). We also report a method for quantification of creatinine in urine by LC-MS/MS to normalize tetranor PGE-M concentrations with that of urinary creatinine. These methods were used to study the effect of aspirin on urinary tetranor PGE-M levels in healthy male volunteers. Aspirin did not affect urinary creatinine concentrations but decreased urinary tetranor PGE-M concentrations by approximately 44%.
Assuntos
Cromatografia Líquida/métodos , Dinoprostona/metabolismo , Prostaglandinas/urina , Espectrometria de Massas em Tandem/métodos , Adulto , Aspirina/farmacologia , Creatinina/urina , Dinoprostona/urina , Estabilidade de Medicamentos , Humanos , Masculino , Reprodutibilidade dos TestesRESUMO
(3R)-4-(4-Chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl acetic acid (MK-0524) is a potent orally active human prostaglandin D(2) receptor 1 antagonist that is currently under development for the prevention of niacin-induced flushing. The major in vitro and in vivo metabolite of MK-0524 is the acyl glucuronic acid conjugate of the parent compound, M2. To compare metabolism of MK-0524 across preclinical species and humans, studies were undertaken to determine the in vitro kinetic parameters (K(m) and V(max)) for the glucuronidation of MK-0524 in Sprague-Dawley rat, beagle dog, cynomolgus monkey, and human liver microsomes, human intestinal microsomes, and in recombinant human UDP glucuronosyltransferases (UGT). A comparison of K(m) values indicated that UGT1A9 has the potential to catalyze the glucuronidation of MK-0524 in the liver, whereas UGT1A3 and UGT2B7 have the potential to catalyze the glucuronidation in the intestine. MK-0524 also was subject to phase I oxidative metabolism; however, the rate was significantly lower than that of glucuronidation. The rate of phase I metabolism was ranked as follows: rat approximately monkey > human intestine > dog > human liver with qualitatively similar metabolite profiles across species. In all the cases, the major metabolites were the monohydroxylated epimers (M1 and M4) and the keto-metabolite, M3. Use of inhibitory monoclonal antibodies and recombinant human cytochromes P450 suggested that CYP3A4 was the major isozyme involved in the oxidative metabolism of MK-0524, with a minor contribution from CYP2C9. The major metabolite in hepatocyte preparations was the acyl glucuronide, M2, with minor amounts of M1, M3, M4, and their corresponding glucuronides. Overall, the in vivo metabolism of MK-0524 is expected to proceed via glucuronidation, with minor contributions from oxidative pathways.
Assuntos
Hepatócitos/metabolismo , Indóis/metabolismo , Microssomos Hepáticos/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Animais , Cromatografia Líquida , Cães , Glucuronídeos/metabolismo , Humanos , Macaca mulatta , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , NADP/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Estrogens and selective estrogen receptor modulators (SERMs) are prescribed widely in the clinic to alleviate symptoms in postmenopausal women, and they are metabolized to reactive intermediates, which may elicit adverse effects. As part of our efforts to develop safer SERMs, in vitro covalent protein binding of (2S,3R)-(+)-3-(4-hydroxyphenyl)-2-[4-(2-piperidin-1-ylethoxy)phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (I) was evaluated. Radioactivity from [3H]I became covalently bound to proteins in a fashion that was both time- and NADPH-dependent in human liver microsomes and reached a value of 1106 pmol equiv/mg protein following a 45 min incubation. At least three pathways are involved in the bioactivation of I, namely, oxidative cleavage of the dihydrobenzoxathiin moiety to give a hydroquinone/para-benzoquinone redox couple, hydroxylation at position 5 or 7 of the benzoxathiin moiety leading to an o-quinone intermediate, and metabolism of the piperidine ring to give an iminium ion. The latter reactive intermediate was identified as its bis-cyano adduct when human liver microsomal incubations were performed in the presence of sodium cyanide. Structural modification of I, including a replacement of the piperidine with a pyrrolidine group, led to (2S,3R)-(+)-3-(3-hydroxyphenyl)-2-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (II), which did not form a reactive iminium ion. Following the incubation of II with human liver microsomes, covalent binding to proteins was reduced (461 pmol equiv/mg protein), the residual level of binding apparently due to the formation of a rearranged biphenyl quinone type metabolite. Studies with inhibitory antibodies and chemical inhibitors showed that P450 3A4 was the primary enzyme responsible for oxidative bioactivation of I and II in human liver microsomes. These studies thus demonstrated that gaining an understanding of bioactivation mechanisms may be exploited in terms of guiding structural modifications of drug candidates to minimize covalent protein binding and, hopefully, to lower the potential for drug-mediated adverse effects.
Assuntos
Sistema Enzimático do Citocromo P-450/fisiologia , Microssomos Hepáticos/metabolismo , Oxati-Inas/farmacocinética , Moduladores Seletivos de Receptor Estrogênico/farmacocinética , Benzoquinonas/metabolismo , Biotransformação , Citocromo P-450 CYP3A , Hepatócitos/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Ligação ProteicaRESUMO
[reaction: see text] During the course of drug metabolism studies, a major metabolite of compound 1 was detected in rhesus monkeys and assigned structure 4. The intriguing biotransformation of 1 leading to 4 was confirmed by a 19-step total synthesis starting from resorcinol (11), the key feature of which was the construction of the oxygen bridge utilizing a phenolic oxidation and trapping sequence. In addition, the synthesis of a related metabolite (5) is described.
Assuntos
Receptor alfa de Estrogênio/antagonistas & inibidores , Osteoporose/tratamento farmacológico , Oxati-Inas/síntese química , Oxati-Inas/farmacologia , Moduladores Seletivos de Receptor Estrogênico/síntese química , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Animais , Biotransformação , Macaca mulatta , Modelos Moleculares , Conformação Molecular , Oxirredução , Oxigênio/química , Resorcinóis/metabolismoRESUMO
The absorption, metabolism, and excretion of [14C]aprepitant, a potent and selective human substance P receptor antagonist for the treatment of chemotherapy-induced nausea and vomiting, was evaluated in rats and dogs. Aprepitant was metabolized extensively and no parent drug was detected in the urine of either species. The elimination of drug-related radioactivity, after i.v. or p.o. administration of [14C]aprepitant, was mainly via biliary excretion in rats and by way of both biliary and urinary excretion in dogs. Aprepitant was the major component in the plasma at the early time points (up to 8 h), and plasma metabolite profiles of aprepitant were qualitatively similar in rats and dogs. Several oxidative metabolites of aprepitant, derived from N-dealkylation, oxidation, and opening of the morpholine ring, were detected in the plasma. Glucuronidation represented an important pathway in the metabolism and excretion of aprepitant in rats and dogs. An acid-labile glucuronide of [14C]aprepitant accounted for approximately 18% of the oral dose in rat bile. The instability of this glucuronide, coupled with its presence in bile but absence in feces, suggested the potential for enterohepatic circulation of aprepitant via this conjugate. In dogs, the glucuronide of [14C]aprepitant, together with four glucuronides derived from phase I metabolites, were present as major metabolites in the bile, accounting collectively for approximately 14% of the radioactive dose over a 4- to 24-h period after i.v. dosing. Two very polar carboxylic acids, namely, 4-fluoro-alpha-hydroxybenzeneacetic acid and 4-fluoro-alpha-oxobenzeneacetic acid, were the predominant drug-related entities in rat and dog urine.
Assuntos
Antieméticos/farmacocinética , Morfolinas/farmacocinética , Antagonistas dos Receptores de Neurocinina-1 , Administração Oral , Animais , Antieméticos/sangue , Antieméticos/urina , Aprepitanto , Bile/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Cães , Fezes/química , Glucuronídeos/sangue , Glucuronídeos/urina , Injeções Intravenosas , Fígado/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Ácidos Mandélicos/sangue , Ácidos Mandélicos/urina , Espectrometria de Massas , Morfolinas/sangue , Morfolinas/urina , Fenilacetatos/sangue , Fenilacetatos/urina , Ratos , Ratos Sprague-Dawley , Especificidade da EspécieRESUMO
The pharmacokinetics, metabolism, and brain penetration of the neurokinin 1 (NK1) receptor antagonist (substance P receptor antagonist), aprepitant (MK-0869), were examined in ferrets. This species exhibits human-type NK1receptor pharmacology and is of proven value in the identification of clinically useful drugs for the treatment of chemotherapy-induced nausea and vomiting in humans. After a single p.o. dose of aprepitant at 1 or 2 mg/kg, plasma levels of the compound were between approximately 200 and 270 ng/ml, 24 h after dosing. In the brain cortex, concentrations of aprepitant reached between approximately 80 and 150 ng/g of tissue 24 h after dosing. The predominant radioactive component present in the plasma and the brain of ferrets at 24 or 48 h after a single oral dose of [14C]aprepitant at 3 mg/kg was the parent compound itself. The slow plasma clearance of aprepitant ( approximately 1.5 ml/min/kg) and its abundance in ferret brain were in accord with its efficacy in blocking the retching and vomiting at 24 and 48 h postdose when ferrets were challenged with the emetic anticancer drug, cisplatin. When aprepitant and some of its metabolites were assessed for their in vitro binding affinity to the human NK1receptor, aprepitant demonstrated the highest affinity. Collectively, these data suggested that aprepitant, rather than its metabolites, was responsible, primarily, for the antiemetic activity of this compound in the male ferret.