RÉSUMÉ
Oral delivery is the preferred route of administration and therefore good absorption after oral dosing is a prerequisite for a compound to be successful in the clinic. The prediction of oral bioavailability from in vitro permeability assays is thus a valuable tool during drug discovery and development. Caco-2 cell monolayers mimic the human intestinal epithelium in many aspects. These monolayers form tight junctions between cells and have been widely used as a model of human intestinal absorption. Caco-2 cells also express a variety of transporter proteins although the transformed nature of the cells results in unpredictable differentiation markers, transport properties and enzyme expression. Thus various modifications of the Caco-2 assay are used in laboratories across the globe. The purpose of this paper is to provide an overview of a time and resource saving 7-day Caco-2 assay protocol. We also discuss the impact of various experimental conditions on permeability measurements and its applications during lead optimization in early discovery and for clinical candidate characterization, specifically for prediction of absorption in human, at a later stage in drug development.
Sujet(s)
Dosage biologique , Absorption intestinale , Cellules Caco-2 , Découverte de médicament , Humains , Préparations pharmaceutiques/métabolismeRÉSUMÉ
Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of cancers and autoimmune diseases such as asthma, rheumatoid arthritis, and systemic lupus erythematous. We report the structure-guided optimization of pyridazine amide spleen tyrosine kinase inhibitors. Early representatives of this scaffold were highly potent and selective but mutagenic in an Ames assay. An approach that led to the successful identification of nonmutagenic examples, as well as further optimization to compounds with reduced cardiovascular liabilities is described. Select pharmacokinetic and in vivo efficacy data are presented.
Sujet(s)
Inhibiteurs de protéines kinases/synthèse chimique , Inhibiteurs de protéines kinases/pharmacologie , Protein-tyrosine kinases/antagonistes et inhibiteurs , Pyridazines/synthèse chimique , Pyridazines/pharmacologie , Rate/enzymologie , Amides/synthèse chimique , Amides/pharmacologie , Animaux , Biologie informatique , Simulation numérique , Conception de médicament , Canaux potassiques éther-à-go-go/effets des médicaments et des substances chimiques , Humains , Techniques in vitro , Souris , Microsomes du foie/métabolisme , Modèles moléculaires , Conformation moléculaire , Mutagenèse/effets des médicaments et des substances chimiques , Tests de mutagénicité , Inhibiteurs de protéines kinases/pharmacocinétique , Pyridazines/pharmacocinétique , Rats , Rate/effets des médicaments et des substances chimiques , Relation structure-activité , Diffraction des rayons XRÉSUMÉ
In the past few years, there have been many advances in the efforts to cure patients with hepatitis C virus (HCV). The ultimate goal of these efforts is to develop a combination therapy consisting of only direct-antiviral agents (DAAs). In this paper, we discuss our efforts that led to the identification of a bicyclic template with potent activity against the NS5B polymerase, a critical enzyme on the life cycle of HCV. In continuation of our exploration to improve the stilbene series, the 3,5,6,8-tetrasubstituted quinoline core was identified as replacement of the stilbene moiety. 6-Methoxy-2(1H)-pyridone was identified among several heterocyclic headgroups to have the best potency. Solubility of the template was improved by replacing a planar aryl linker with a saturated pyrrolidine. Profiling of the most promising compounds led to the identification of quinoline 41 (RG7109), which was selected for advancement to clinical development.
Sujet(s)
Antiviraux/pharmacologie , Antienzymes/pharmacologie , Hepacivirus/effets des médicaments et des substances chimiques , Quinoléines/pharmacologie , Sulfonamides/pharmacologie , Protéines virales non structurales/antagonistes et inhibiteurs , Animaux , Antiviraux/composition chimique , Antiviraux/pharmacocinétique , Chiens , Découverte de médicament , Antienzymes/composition chimique , Antienzymes/pharmacocinétique , Hepacivirus/enzymologie , Humains , Modèles moléculaires , Quinoléines/composition chimique , Quinoléines/pharmacocinétique , Rats , Sulfonamides/composition chimique , Sulfonamides/pharmacocinétiqueRÉSUMÉ
DYRK1B is a kinase over-expressed in certain cancer cells (including colon, ovarian, pancreatic, etc.). Recent publications have demonstrated inhibition of DYRK1B could be an attractive target for cancer therapy. From a data-mining effort, the team has discovered analogues of pyrido[2,3-d]pyrimidines as potent enantio-selective inhibitors of DYRK1B. Cells treated with a tool compound from this series showed the same cellular effects as down regulation of DYRK1B with siRNA. Such effects are consistent with the proposed mechanism of action. Progress of the SAR study is presented.
Sujet(s)
Inhibiteurs de protéines kinases/composition chimique , Inhibiteurs de protéines kinases/pharmacologie , Protein-Serine-Threonine Kinases/antagonistes et inhibiteurs , Protein-tyrosine kinases/antagonistes et inhibiteurs , Pyrimidines/composition chimique , Animaux , Sites de fixation , Cristallographie aux rayons X , Activation enzymatique/effets des médicaments et des substances chimiques , Période , Humains , Simulation de dynamique moléculaire , Inhibiteurs de protéines kinases/pharmacocinétique , Protein-Serine-Threonine Kinases/métabolisme , Structure tertiaire des protéines , Protein-tyrosine kinases/métabolisme , Pyrimidines/pharmacocinétique , Rats , Relation structure-activité ,RÉSUMÉ
The beagle dog is a widely used in vivo model to guide clinical formulation development and to explore the potential for food effects. However, the results in dogs are often not directly translatable to humans. Consequently, a physiologically based modeling strategy has been proposed, using the dog as a validation step to verify model assumptions before making predictions in humans. One current weakness in this strategy is the lack of validated tools to incorporate gut wall metabolism into the dog model. In this study, in vitro to in vivo extrapolation factors for CYP2B11 and CYP3A12 mediated metabolism were established based on tissue enzyme abundance data reported earlier. Thereafter, physiologically based modeling of intestinal absorption in beagle dog was conducted in GastroPlus using V(max) and K(m) determined in recombinant enzymes as inputs for metabolic turnover. The predicted fraction of absorbed dose escaping the gut wall metabolism (F(g)) of all five reference compounds studied (domperidone, felodipine, nitrendipine, quinidine, and sildenafil) were within a two-fold range of the value estimated from in vivo data at single dose levels. However, further in vivo studies and analysis of the dose-dependent pharmacokinetics of felodipine and nitrendipine showed that more work is required for robust forecasting of nonlinearities. In conclusion, this study demonstrates an approach for prediction of the gut wall extraction of CYP substrates in the beagle dog, thus enhancing the value of dog studies as a component in a strategy for the prediction of human pharmacokinetics.
Sujet(s)
Cytochrome P-450 enzyme system/métabolisme , Intestins/effets des médicaments et des substances chimiques , Foie/effets des médicaments et des substances chimiques , Administration par voie orale , Animaux , Biodisponibilité , Cellules Caco-2 , Cytochrome P-450 CYP2B1/composition chimique , Cytochrome P-450 enzyme system/composition chimique , Chiens , Dompéridone/pharmacocinétique , Félodipine/pharmacocinétique , Humains , Techniques in vitro , Cinétique , Nitrendipine/pharmacocinétique , Perméabilité , Pipérazines/pharmacocinétique , Purines/pharmacocinétique , Quinidine/pharmacocinétique , Citrate de sildénafil , Sulfones/pharmacocinétique , Distribution tissulaireRÉSUMÉ
We describe the discovery of several pyrrolopyrazines as potent and selective Syk inhibitors and the efforts that eventually led to the desired improvements in physicochemical properties and human whole blood potencies. Ultimately, our mouse model revealed unexpected toxicity that precluded us from further advancing this series.
Sujet(s)
Protéines et peptides de signalisation intracellulaire/antagonistes et inhibiteurs , Protein-tyrosine kinases/antagonistes et inhibiteurs , Pyrazines/synthèse chimique , Pyrroles/synthèse chimique , Animaux , Lymphocytes B/effets des médicaments et des substances chimiques , Lymphocytes B/enzymologie , Protéines du sang/métabolisme , Cristallographie aux rayons X , Humains , Souris , Microsomes du foie/métabolisme , Modèles moléculaires , Structure moléculaire , Liaison aux protéines , Pyrazines/pharmacologie , Pyrazines/toxicité , Pyrroles/pharmacologie , Pyrroles/toxicité , Relation structure-activité , Syk kinaseRÉSUMÉ
From a series of N-acyl 4-(3-pyridonyl)phenylalanine derivatives of 4, the trifluoromethyl derivative 28 was identified as a potent, dual acting alpha4 integrin antagonist with activity in primate models of allergic asthma. Investigation of a series of prodrug esters led to the discovery of the morpholinopropyl derivative 48 that demonstrated good intestinal fluid stability, solubility and permeability. Compound 48 gave high blood levels of 28 when dosed orally in cynomolgus monkeys. Surprisingly, hydrolysis of 48 was rapid in liver microsomes from the pharmacological species, mouse, rat and monkey, but slow in dog and human; in vivo studies also indicated there was prolonged exposure to unchanged prodrug in dogs.
Sujet(s)
Intégrine alpha4bêta1/antagonistes et inhibiteurs , Intégrines/antagonistes et inhibiteurs , Phénylalanine/analogues et dérivés , Animaux , Chiens , Esters/sang , Esters/pharmacologie , Humains , Souris , Phénylalanine/pharmacologie , Promédicaments/composition chimique , Promédicaments/pharmacologie , RatsRÉSUMÉ
N-Acyl 4-(5-pyrimidine-2,4-dionyl)phenylalanine derivatives of type 4 were designed to replace the 2,6-dichlorobenzoylamine portion of compound 1 in order to identify novel compounds with improved potency against α4-integrins. Several derivatives were identified as very potent dual-acting α4-integrin, α4ß1 and α4ß7 antagonists. Investigation of a limited number of prodrug esters led to the discovery of the ethyl ester prodrug 42, which demonstrated good intestinal fluid stability and good permeability. Despite low solubility, 42 gave acceptable blood levels of 30 when dosed orally in non-human primates. Additionally, 42 had an overall excellent profile and was selected for clinical trials. Investigation of N-acyl 4-(5-pyrimidine-2,4-dionyl)phenylalanine derivatives led to the discovery of several very potent dual-acting α4-integrin antagonists. Ethyl ester prodrug 42 advanced to human clinical trials based on the excellent intestinal fluid stability, good permeability and superior efficacy in non-human primates.
Sujet(s)
Intégrine alpha4bêta1/antagonistes et inhibiteurs , Intégrines/antagonistes et inhibiteurs , Phénylalanine/analogues et dérivés , Pyrimidines/pharmacologie , Animaux , Chiens , Esters/composition chimique , Esters/pharmacocinétique , Esters/pharmacologie , Humains , Macaca fascicularis , Souris , Phénylalanine/pharmacocinétique , Phénylalanine/pharmacologie , Promédicaments/composition chimique , Promédicaments/pharmacocinétique , Promédicaments/pharmacologie , Pyrimidines/composition chimique , Pyrimidines/pharmacocinétique , Rats , Relation structure-activitéRÉSUMÉ
The Janus kinases (JAKs) are involved in multiple signaling networks relevant to inflammatory diseases, and inhibition of one or more members of this class may modulate disease activity or progression. We optimized a new inhibitor scaffold, 3-amido-5-cyclopropylpyrrolopyrazines, to a potent example with reasonable kinome selectivity, including selectivity for JAK3 versus JAK1, and good biopharmaceutical properties. Evaluation of this analogue in cellular and in vivo models confirmed functional selectivity for modulation of a JAK3/JAK1-dependent IL-2 stimulated pathway over a JAK1/JAK2/Tyk2-dependent IL-6 stimulated pathway.
Sujet(s)
Anti-inflammatoires non stéroïdiens/synthèse chimique , Cyclopropanes/synthèse chimique , Janus kinase 1/antagonistes et inhibiteurs , Janus kinase 3/antagonistes et inhibiteurs , Pyrazines/synthèse chimique , Pyrroles/synthèse chimique , Administration par voie orale , Animaux , Anti-inflammatoires non stéroïdiens/pharmacocinétique , Anti-inflammatoires non stéroïdiens/pharmacologie , Cellules Caco-2 , Cristallographie aux rayons X , Cyclopropanes/pharmacocinétique , Cyclopropanes/pharmacologie , Techniques de knock-down de gènes , Tests de criblage à haut débit , Humains , Interleukine-2/physiologie , Janus kinase 1/génétique , Janus kinase 1/métabolisme , Janus kinase 3/génétique , Janus kinase 3/métabolisme , Souris , Modèles moléculaires , Pyrazines/pharmacocinétique , Pyrazines/pharmacologie , Pyrroles/pharmacocinétique , Pyrroles/pharmacologie , Petit ARN interférent/génétique , Rats , Récepteurs à l'interleukine-6/physiologie , Transduction du signal/effets des médicaments et des substances chimiques , Relation structure-activité , Lymphocytes T/effets des médicaments et des substances chimiques , Lymphocytes T/métabolismeRÉSUMÉ
3-[4-((1S,2S,3R,5S,7S)-5-Hydroxyadamantan-2-ylcarbamoyl)benzyl]-4-oxo-1-phenyl-1,4-dihydro-[1,8]naphthyridine-2-carboxylic acid methyl ester (4) was identified as a novel, druglike and selective quinolone pan JNK inhibitor. In this communication, some of the structure-activity relationship of the azaquinolone analogues leading to 4 is discussed. The focus is on how changes at the amide functionality affected the biochemical potency, cellular potency, metabolic properties, and solubility of this class of JNK inhibitors. Optimization of these properties led to the identification of the adamantyl analogue, 4. 4 achieved proof of mechanism in both rat and mouse TNF-α challenge models.
RÉSUMÉ
PURPOSE: This study describes the antiproliferative activity of the multikinase inhibitor R1530 in vitro and its antitumor and anti-angiogenic activity, pharmacokinetics, and tolerability in vivo. METHODS: The antiproliferative activity of R1530 was investigated in a range of human tumor, endothelial and fibroblast cell lines. Tolerability and antitumor activity were assessed in mice bearing a range of human tumor xenografts, and anti-angiogenic properties were established in the murine corneal pocket assay. R1530 pharmacokinetics in mice were established. RESULTS: R1530 strongly inhibited human tumor cell proliferation. Growth factor-driven proliferation of endothelial and fibroblast cells was also inhibited. Significant tumor growth inhibition was demonstrated in a lung cancer xenograft model with a range of once daily, weekly and twice-weekly doses of R1530 (3.125-50 mg/kg qd, 100 mg/kg qw, 100 mg/kg biw). Daily doses were most effective in the lung cancer model and also had significant growth inhibitory effects in models of colorectal, prostate, and breast tumors. Tumor regression occurred in all models treated with the maximum tolerated daily dose (50 mg/kg). The doses of 25 and 50 mg/kg qd resulted in biologically significant increased survival in all tested models. After oral administration in nude mice, R1530 showed good tissue penetration. Exposure was dose dependent up to 100 mg/kg with oral administration. CONCLUSIONS: R1530 has demonstrated activity against a range of tumor models in vitro and in vivo and is an effective inhibitor of angiogenesis. These findings support the approach of targeting multiple pathways in the search for potential agents with improved anticancer properties.
Sujet(s)
Antinéoplasiques/pharmacologie , Benzodiazépines/pharmacologie , Pyrazoles/pharmacologie , Animaux , Benzodiazépines/pharmacocinétique , Carcinome pulmonaire non à petites cellules/traitement médicamenteux , Lignée cellulaire tumorale , Prolifération cellulaire/effets des médicaments et des substances chimiques , Évaluation préclinique de médicament , Femelle , Humains , Tumeurs du poumon/traitement médicamenteux , Souris , Souris de lignée C57BL , Cellules NIH 3T3 , Pyrazoles/pharmacocinétique , Facteur de croissance endothéliale vasculaire de type A/antagonistes et inhibiteurs , Tests d'activité antitumorale sur modèle de xénogreffeRÉSUMÉ
Glucokinase (GK) is a glucose sensor that couples glucose metabolism to insulin release. The important role of GK in maintaining glucose homeostasis is illustrated in patients with GK mutations. In this publication, identification of the hit molecule 1 and its SAR development, which led to the discovery of potent allosteric GK activators 9a and 21a, is described. Compound 21a (RO0281675) was used to validate the clinical relevance of targeting GK to treat type 2 diabetes.
Sujet(s)
Diabète de type 2/traitement médicamenteux , Glucokinase/effets des médicaments et des substances chimiques , Hypoglycémiants/composition chimique , Sulfones/pharmacologie , Thiazoles/pharmacologie , Animaux , Glycémie , Lignée cellulaire , Cytotoxines , Relation dose-effet des médicaments , Découverte de médicament , Humains , Insuline , Mâle , Souris , Pharmacocinétique , Relation structure-activité , Sulfones/composition chimique , Sulfones/toxicité , Thiazoles/composition chimique , Thiazoles/toxicitéRÉSUMÉ
Targeting the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway represents a promising anticancer strategy. Recently, we have reported a novel class of potent and selective non-ATP-competitive MEK1/2 inhibitors with a unique structure and mechanism of action. RO5068760 is a representative of this class showing significant efficacy in a broad spectrum of tumors with aberrant mitogen-activated protein kinase pathway activation. To understand the relationship between systemic exposures and target (MEK1/2) inhibition as well as tumor growth inhibition, the current study presents a detailed in vivo characterization of efficacy, pharmacokinetics, and pharmacodynamics of RO5068760 in multiple xenograft tumor models. For inhibition of MEK1/2 as measured by the phosphorylated ERK levels, the estimated EC(50)s in plasma were 1.36 micromol/L (880 ng/mL) and 3.35 micromol/L (2168 ng/mL) in LOX melanoma and HT-29 colorectal cancer models, respectively. A similar EC(50) (1.41 micromol/L or 915 ng/mL) was observed in monkey peripheral blood lymphocytes. To achieve tumor growth inhibition (>or=90%), an average plasma drug concentration of 0.65 or 5.23 micromol/L was required in B-RafV600E or K-Ras mutant tumor models, respectively, which were remarkably similar to the IC(90) values (0.64 or 4.1 micromol/L) determined in vitro for cellular growth inhibition. With equivalent in vivo systemic exposures, RO5068760 showed superior efficacy in tumors harboring B-RafV600E mutation. The plasma concentration time profiles indicate that constant p-ERK suppression (>50%) may not be required for optimal efficacy, especially in highly responsive tumors. This study may facilitate future clinical trial design in using biochemical markers for early proof of mechanism and in selecting the right patients and optimal dose regimen.
Sujet(s)
Imidazolidines/pharmacologie , Imidazolidines/pharmacocinétique , MAP Kinase Kinase 1/antagonistes et inhibiteurs , MAP Kinase Kinase 2/antagonistes et inhibiteurs , Phénylbutyrates/pharmacologie , Phénylbutyrates/pharmacocinétique , Inhibiteurs de protéines kinases/pharmacologie , Inhibiteurs de protéines kinases/pharmacocinétique , Tests d'activité antitumorale sur modèle de xénogreffe , Animaux , Lignée cellulaire tumorale , Tests de criblage d'agents antitumoraux , Humains , Imidazolidines/sang , Imidazolidines/composition chimique , Macaca fascicularis , Souris , Souris nude , Phénylbutyrates/sang , Phénylbutyrates/composition chimique , Inhibiteurs de protéines kinases/sang , Inhibiteurs de protéines kinases/composition chimiqueRÉSUMÉ
RO4396686 is a small molecule KDR, FGFR, and PDGFR inhibitor with good pharmacokinetic properties in rodents. In a mouse corneal neovascularization assay, this compound inhibited VEGF-induced angiogenesis. Tested in a H460a xenograft tumor model this agent effected significant tumor growth inhibition at doses as low as 50mg/kg.
Sujet(s)
Inhibiteurs de l'angiogenèse/pharmacologie , Tumeurs expérimentales/vascularisation , Néovascularisation pathologique , Pyrimidines/pharmacologie , Inhibiteurs de l'angiogenèse/composition chimique , Animaux , Souris , Souris nude , Rats , Rat Wistar , Récepteur facteur croissance fibroblaste/antagonistes et inhibiteurs , Récepteurs aux facteurs de croissance dérivés des plaquettes/antagonistes et inhibiteurs , Facteur de croissance endothéliale vasculaire de type A/physiologie , Récepteur-2 au facteur croissance endothéliale vasculaire/antagonistes et inhibiteursRÉSUMÉ
(+/-)-1-(anti-3-Hydroxy-cyclopentyl)-3-(4-methoxy-phenyl)-7-phenylamino-3,4-dihydro-1H-pyrimido[4,5-d]pyrimidin-2-one (RO4383596) is a potent and selective inhibitor of the pro-angiogenic receptor tyrosine kinases KDR, FGFR, and PDGFR. This agent has an excellent pharmacokinetic profile and is highly efficacious in rodent models of angiogenesis upon oral administration.