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CONTEXT: Thyroid-stimulating hormone (or thyrotropin) receptor (TSHR) could be a selective target for small molecule ligands to treat thyroid cancer (TC). OBJECTIVE: We report a novel, orally efficacious ligand for TSHR that exhibits proliferation inhibitory activity against human TC in vitro and in vivo, and inhibition of metastasis in vivo. METHODS: A35 (NCATS-SM4420; NCGC00241808) was selected from a sublibrary of >200 TSHR ligands. Cell proliferation assays including BrdU incorporation and WST-1, along with molecular docking studies were done. In vivo activity of A35 was assessed in TC cell-derived xenograft (CDX) models with immunocompromised (NSG) mice. Formalin-fixed, paraffin-embedded sections of tumor and lung tissues were observed for the extent of cell death and metastasis. RESULTS: A35 was shown to stimulate cAMP production in some cell types by activating TSHR but not in TC cells, MDA-T32, and MDA-T85. A35 inhibited proliferation of MDA-T32 and MDA-T85 in vitro and in vivo, and pulmonary metastasis of MDA-T85F1 in mice. In vitro, A35 inhibition of proliferation was reduced by a selective TSHR antagonist. Inhibition of CDX tumor growth without decreases in mouse weights and liver function showed A35 to be efficacious without apparent toxicity. Lastly, A35 reduced levels of Ki67 in the tumors and metastatic markers in lung tissues. CONCLUSION: We conclude that A35 is a TSHR-selective inhibitor of TC cell proliferation and metastasis, and suggest that A35 may be a promising lead drug candidate for the treatment of differentiated TC in humans.
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Proliferación Celular , Receptores de Tirotropina , Neoplasias de la Tiroides , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Humanos , Ratones , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Proliferación Celular/efectos de los fármacos , Receptores de Tirotropina/antagonistas & inhibidores , Receptores de Tirotropina/metabolismo , Ligandos , Línea Celular Tumoral , Administración Oral , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos/administración & dosificación , Metástasis de la Neoplasia , Simulación del Acoplamiento Molecular , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , FemeninoRESUMEN
Efficiently circumventing the blood-brain barrier (BBB) poses a major hurdle in the development of drugs that target the central nervous system. Although there are several methods to determine BBB permeability of small molecules, the Parallel Artificial Membrane Permeability Assay (PAMPA) is one of the most common assays in drug discovery due to its robust and high-throughput nature. Drug discovery is a long and costly venture, thus, any advances to streamline this process are beneficial. In this study, â¼2,000 compounds from over 60 NCATS projects were screened in the PAMPA-BBB assay to develop a quantitative structure-activity relationship model to predict BBB permeability of small molecules. After analyzing both state-of-the-art and latest machine learning methods, we found that random forest based on RDKit descriptors as additional features provided the best training balanced accuracy (0.70 ± 0.015) and a message-passing variant of graph convolutional neural network that uses RDKit descriptors provided the highest balanced accuracy (0.72) on a prospective validation set. Finally, we correlated in vitro PAMPA-BBB data with in vivo brain permeation data in rodents to observe a categorical correlation of 77%, suggesting that models developed using data from PAMPA-BBB can forecast in vivo brain permeability. Given that majority of prior research has relied on in vitro or in vivo data for assessing BBB permeability, our model, developed using the largest PAMPA-BBB dataset to date, offers an orthogonal means to estimate BBB permeability of small molecules. We deposited a subset of our data into PubChem bioassay database (AID: 1845228) and deployed the best performing model on the NCATS Open Data ADME portal (https://opendata.ncats.nih.gov/adme/). These initiatives were undertaken with the aim of providing valuable resources for the drug discovery community.
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Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder that affects lysosome-related organelles, often leading to fatal pulmonary fibrosis (PF). The search for a treatment for HPS pulmonary fibrosis (HPSPF) is ongoing. S-MRI-1867, a dual cannabinoid receptor 1 (CB1R)/inducible nitric oxide synthase (iNOS) inhibitor, has shown great promise for the treatment of several fibrotic diseases, including HPSPF. In this study, we investigated the in vitro ADME characteristics of S-MRI-1867, as well as its pharmacokinetic (PK) properties in mice, rats, dogs, and monkeys. S-MRI-1867 showed low aqueous solubility (< 1 µg/mL), high plasma protein binding (>99%), and moderate to high metabolic stability. In its preclinical PK studies, S-MRI-1867 exhibited moderate to low plasma clearance (CLp) and high steady-state volume of distribution (Vdss) across all species. Despite the low solubility and P-gp efflux, S-MRI-1867 showed great permeability and metabolic stability leading to a moderate bioavailability (21-60%) across mouse, rat, dog, and monkey. Since the R form of MRI-1867 is CB1R-inactive, we investigated the potential conversion of S-MRI-1867 to R-MRI-1867 in mice and found that the chiral conversion was negligible. Furthermore, we developed and validated a PBPK model that adequately fits the PK profiles of S-MRI-1867 in mice, rats, dogs, and monkeys using various dosing regimens. We employed this PBPK model to simulate the human PK profiles of S-MRI-1867, enabling us to inform human dose selection and support the advancement of this promising drug candidate in the treatment of HPSPF.
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Síndrome de Hermanski-Pudlak , Fibrosis Pulmonar , Humanos , Ratas , Ratones , Animales , Perros , Fibrosis Pulmonar/diagnóstico por imagen , Fibrosis Pulmonar/tratamiento farmacológico , Síndrome de Hermanski-Pudlak/tratamiento farmacológico , Proyectos de InvestigaciónRESUMEN
Introduction: Niclosamide (Nc) is an FDA-approved anthelmintic drug that was recently identified in a drug repurposing screening to possess antiviral activity against SARS-CoV-2. However, due to the low solubility and permeability of Nc, its in vivo efficacy was limited by its poor oral absorption. Method: The current study evaluated a novel prodrug of Nc (PDN; NCATS-SM4705) in improving in vivo exposure of Nc and predicted pharmacokinetic profiles of PDN and Nc across different species. ADME properties of the prodrug were determined in humans, hamsters, and mice, while the pharmacokinetics (PK) of PDN were obtained in mice and hamsters. Concentrations of PDN and Nc in plasma and tissue homogenates were measured by UPLC-MS/MS. A physiologically based pharmacokinetic (PBPK) model was developed based on physicochemical properties, pharmacokinetic and tissue distribution data in mice, validated by the PK profiles in hamsters and applied to predict pharmacokinetic profiles in humans. Results: Following intravenous and oral administration of PDN in mice, the total plasma clearance (CLp) and volume of distribution at steady-state (Vdss) were 0.061-0.063 L/h and 0.28-0.31 L, respectively. PDN was converted to Nc in both liver and blood, improving the systemic exposure of Nc in mice and hamsters after oral administration. The PBPK model developed for PDN and in vivo formed Nc could adequately simulate plasma and tissue concentration-time profiles in mice and plasma profiles in hamsters. The predicted human CLp/F and Vdss/F after an oral dose were 2.1 L/h/kg and 15 L/kg for the prodrug respectively. The predicted Nc concentrations in human plasma and lung suggest that a TID dose of 300 mg PDN would provide Nc lung concentrations at 8- to 60-fold higher than in vitro IC50 against SARS-CoV-2 reported in cell assays. Conclusion: In conclusion, the novel prodrug PDN can be efficiently converted to Nc in vivo and improves the systemic exposure of Nc in mice after oral administration. The developed PBPK model adequately depicts the mouse and hamster pharmacokinetic and tissue distribution profiles and highlights its potential application in the prediction of human pharmacokinetic profiles.
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Preclinical pharmacokinetics (PK) and In Vitro ADME properties of GS-441524, a potential oral agent for the treatment of Covid-19, were studied. GS-441524 was stable in vitro in liver microsomes, cytosols, and hepatocytes of mice, rats, monkeys, dogs, and humans. The plasma free fractions of GS-441524 were 62-78% across all studied species. The in vitro transporter study results showed that GS-441524 was a substrate of MDR1, BCRP, CNT3, ENT1, and ENT2; but not a substrate of CNT1, CNT2, and ENT4. GS-441524 had a low to moderate plasma clearance (CLp), ranging from 4.1 mL/min/kg in dogs to 26 mL/min/kg in mice; the steady state volume distribution (Vdss) ranged from 0.9 L/kg in dogs to 2.4 L/kg in mice after IV administration. Urinary excretion appeared to be the major elimination process for GS-441524. Following oral administration, the oral bioavailability was 8.3% in monkeys, 33% in rats, 39% in mice, and 85% in dogs. The PK and ADME properties of GS-441524 support its further development as an oral drug candidate.
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(R,S)-Ketamine is rapidly metabolized to form a range of metabolites in vivo, including 12 unique hydroxynorketamines (HNKs) that are distinguished by a cyclohexyl ring hydroxylation at the 4, 5, or 6 position. While both (2R,6R)- and (2S,6S)-HNK readily penetrate the brain and exert rapid antidepressant-like actions in preclinical tests following peripheral administration, the pharmacokinetic profiles and pharmacodynamic actions of 10 other HNKs have not been examined. We assessed the pharmacokinetic profiles of all 12 HNKs in the plasma and brains of male and female mice and compared the relative potencies of four (2,6)-HNKs to induce antidepressant-relevant behavioral effects in the forced swim test in male mice. While all HNKs were readily brain-penetrable following intraperitoneal injection, there were robust differences in peak plasma and brain concentrations and exposures. Forced swim test immobility rank order of potency, from most to least potent, was (2R,6S)-, (2S,6R)-, (2R,6R)-, and (2S,6S)-HNK. We hypothesized that distinct structure-activity relationships and the resulting potency of each metabolite are linked to unique substitution patterns and resultant conformation of the six-membered cyclohexanone ring system. To explore this, we synthesized (5R)-methyl-(2R,6R)-HNK, which incorporates a methyl substitution on the cyclohexanone ring. (5R)-Methyl-(2R,6R)-HNK exhibited similar antidepressant-like potency to (2R,6S)-HNK. These results suggest that conformation of the cyclohexanone ring system in the (2,6)-HNKs is an important factor underlying potency and that additional engineering of this structural feature may improve the development of a new generation of HNKs. Such HNKs may represent novel drug candidates for the treatment of depression.
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Antidepresivos , Ketamina , Animales , Antidepresivos/uso terapéutico , Conducta Animal , Depresión/tratamiento farmacológico , Femenino , Ketamina/análogos & derivados , Ketamina/farmacología , Masculino , RatonesRESUMEN
Membrane permeability plays an important role in oral drug absorption. Caco-2 and Madin-Darby Canine Kidney (MDCK) cell culture systems have been widely used for assessing intestinal permeability. Since most drugs are absorbed passively, Parallel Artificial Membrane Permeability Assay (PAMPA) has gained popularity as a low-cost and high-throughput method in early drug discovery when compared to high-cost, labor intensive cell-based assays. At the National Center for Advancing Translational Sciences (NCATS), PAMPA pH 5 is employed as one of the Tier I absorption, distribution, metabolism, and elimination (ADME) assays. In this study, we have developed a quantitative structure activity relationship (QSAR) model using our â¼6500 compound PAMPA pH 5 permeability dataset. Along with ensemble decision tree-based methods such as Random Forest and eXtreme Gradient Boosting, we employed deep neural network and a graph convolutional neural network to model PAMPA pH 5 permeability. The classification models trained on a balanced training set provided accuracies ranging from 71% to 78% on the external set. Of the four classifiers, the graph convolutional neural network that directly operates on molecular graphs offered the best classification performance. Additionally, an â¼85% correlation was obtained between PAMPA pH 5 permeability and in vivo oral bioavailability in mice and rats. These results suggest that data from this assay (experimental or predicted) can be used to rank-order compounds for preclinical in vivo testing with a high degree of confidence, reducing cost and attrition as well as accelerating the drug discovery process. Additionally, experimental data for 486 compounds (PubChem AID: 1645871) and the best models have been made publicly available (https://opendata.ncats.nih.gov/adme/).
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Betametasona/farmacocinética , Dexametasona/farmacocinética , Ranitidina/farmacocinética , Verapamilo/farmacocinética , Administración Oral , Animales , Betametasona/administración & dosificación , Disponibilidad Biológica , Células CACO-2 , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Cultivadas , Dexametasona/administración & dosificación , Perros , Relación Dosis-Respuesta a Droga , Humanos , Concentración de Iones de Hidrógeno , Células de Riñón Canino Madin Darby , Ratones , Estructura Molecular , Redes Neurales de la Computación , Ranitidina/administración & dosificación , Ratas , Relación Estructura-Actividad , Verapamilo/administración & dosificaciónRESUMEN
The majority of FDA-approved HCV therapeutics target the viral replicative machinery. An automated high-throughput phenotypic screen identified several small molecules as potent inhibitors of hepatitis C virus replication. Here, we disclose the discovery and optimization of a 4-aminopiperidine (4AP) scaffold targeting the assembly stages of the HCV life cycle. The original screening hit (1) demonstrates efficacy in the HCVcc assay but does not show potency prior to or during viral replication. Colocalization and infectivity studies indicate that the 4AP chemotype inhibits the assembly and release of infectious HCV. Compound 1 acts synergistically with FDA-approved direct-acting antiviral compounds Telaprevir and Daclatasvir, as well as broad spectrum antivirals Ribavirin and cyclosporin A. Following an SAR campaign, several derivatives of the 4AP series have been identified with increased potency against HCV, reduced in vitro toxicity, as well as improved in vitro and in vivo ADME properties.
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Antivirales/farmacología , Descubrimiento de Drogas , Hepacivirus/efectos de los fármacos , Piperidinas/farmacología , Animales , Antivirales/síntesis química , Antivirales/química , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Piperidinas/síntesis química , Piperidinas/química , Ratas , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
Endoplasmic reticulum (ER) dysregulation is associated with pathologies including neurodegenerative, muscular, and diabetic conditions. Depletion of ER calcium can lead to the loss of resident proteins in a process termed exodosis. To identify compounds that attenuate the redistribution of ER proteins under pathological conditions, we performed a quantitative high-throughput screen using the Gaussia luciferase (GLuc)-secreted ER calcium modulated protein (SERCaMP) assay, which monitors secretion of ER-resident proteins triggered by calcium depletion. We identify several clinically used drugs, including bromocriptine, and further characterize them using assays to measure effects on ER calcium, ER stress, and ER exodosis. Bromocriptine elicits protective effects in cell-based models of exodosis as well as in vivo models of stroke and diabetes. Bromocriptine analogs with reduced dopamine receptor activity retain similar efficacy in stabilizing the ER proteome, indicating a non-canonical mechanism of action. This study describes a strategic approach to identify small-molecule drugs capable of improving ER proteostasis in human disease conditions.
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Retículo Endoplásmico/efectos de los fármacos , Proteoma/metabolismo , HumanosRESUMEN
Drug resistance is a constant threat to malaria control efforts making it important to maintain a good pipeline of new drug candidates. Of particular need are compounds that also block transmission by targeting sexual stage parasites. Mature sexual stages are relatively resistant to all currently used antimalarials except the 8-aminoquinolines that are not commonly used due to potential side effects. Here, we synthesized a new Torin 2 derivative, NCATS-SM3710 with increased aqueous solubility and specificity for Plasmodium and demonstrate potent in vivo activity against all P. berghei life cycle stages. NCATS-SM3710 also has low nanomolar EC50s against in vitro cultured asexual P. falciparum parasites (0.38 ± 0.04 nM) and late stage gametocytes (5.77 ± 1 nM). Two independent NCATS-SM3710/Torin 2 resistant P. falciparum parasite lines produced by growth in sublethal Torin 2 concentrations both had genetic changes in PF3D7_0509800, annotated as a phosphatidylinositol 4 kinase (Pfâ¯PI4KIIIß). One line had a point mutation in the putative active site (V1357G), and the other line had a duplication of a locus containing Pfâ¯PI4KIIIß. Both lines were also resistant to other Pfâ¯PI4K inhibitors. In addition NCATS-SM3710 inhibited purified Pfâ¯PI4KIIIß with an IC50 of 2.0 ± 0.30 nM. Together the results demonstrate that Pfâ¯PI4KIIIß is the target of Torin 2 and NCATS-SM3710 and provide new options for potent multistage drug development.
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Fluoxazolevir is an aryloxazole-based entry inhibitor of hepatitis C virus (HCV). We show that fluoxazolevir inhibits fusion of HCV with hepatic cells by binding HCV envelope protein 1 to prevent fusion. Nine of ten fluoxazolevir resistance-associated substitutions are in envelope protein 1, and four are in a putative fusion peptide. Pharmacokinetic studies in mice, rats and dogs revealed that fluoxazolevir localizes to the liver. A 4-week intraperitoneal regimen of fluoxazolevir in humanized chimeric mice infected with HCV genotypes 1b, 2a or 3 resulted in a 2-log reduction in viraemia, without evidence of drug resistance. In comparison, daclatasvir, an approved HCV drug, suppressed more than 3 log of viraemia but is associated with the emergence of resistance-associated substitutions in mice. Combination therapy using fluoxazolevir and daclatasvir cleared HCV genotypes 1b and 3 in mice. Fluoxazolevir combined with glecaprevir and pibrentasvir was also effective in clearing multidrug-resistant HCV replication in mice. Fluoxazolevir may be promising as the next generation of combination drug cocktails for HCV treatment.
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Antivirales/administración & dosificación , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Internalización del Virus/efectos de los fármacos , Animales , Carbamatos/administración & dosificación , Modelos Animales de Enfermedad , Perros , Quimioterapia Combinada , Genotipo , Hepacivirus/clasificación , Hepacivirus/genética , Hepacivirus/fisiología , Hepatitis C/virología , Humanos , Imidazoles/administración & dosificación , Masculino , Ratones , Pirrolidinas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Valina/administración & dosificación , Valina/análogos & derivados , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
The cell entry of SARS-CoV-2 has emerged as an attractive drug repurposing target for COVID-19. Here we combine genetics and chemical perturbation to demonstrate that ACE2-mediated entry of SARS-CoV and CoV-2 requires the cell surface heparan sulfate (HS) as an assisting cofactor: ablation of genes involved in HS biosynthesis or incubating cells with a HS mimetic both inhibit Spike-mediated viral entry. We show that heparin/HS binds to Spike directly, facilitates the attachment of viral particles to the cell surface to promote cell entry. We screened approved drugs and identified two classes of inhibitors that act via distinct mechanisms to target this entry pathway. Among the drugs characterized, Mitoxantrone is a potent HS inhibitor, while Sunitinib and BNTX disrupt the actin network to indirectly abrogate HS-assisted viral entry. We further show that drugs of the two classes can be combined to generate a synergized activity against SARS-CoV-2-induced cytopathic effect. Altogether, our study establishes HS as an attachment factor that assists SARS coronavirus cell entry, and reveals drugs capable of targeting this important step in the viral life cycle.
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PURPOSE: Metarrestin is a first-in-class pyrrolo-pyrimidine-derived small molecule targeting a marker of genome organization associated with metastasis and is currently in preclinical development as an anti-cancer agent. Here, we report the in vitro ADME characteristics and in vivo pharmacokinetic behavior of metarrestin. METHODS: Solubility, permeability, and efflux ratio as well as in vitro metabolism of metarrestin in hepatocytes, liver microsomes and S9 fractions, recombinant cytochrome P450 (CYP) enzymes, and potential for CYP inhibition were evaluated. Single dose pharmacokinetic profiles after intravenous and oral administration in mice, rat, dog, monkey, and mini-pig were obtained. Simple allometric scaling was applied to predict human pharmacokinetics. RESULTS: Metarrestin had an aqueous solubility of 150 µM at pH 7.4, high permeability in PAMPA and moderate efflux ratio in Caco-2 assays. The compound was metabolically stable in liver microsomes, S9 fractions, and hepatocytes from six species, including human. Metarrestin is a CYP3A4 substrate and, in mini-pigs, is also directly glucuronidated. Metarrestin did not show cytochrome P450 inhibitory activity. Plasma concentration-time profiles showed low to moderate clearance, ranging from 0.6 mL/min/kg in monkeys to 48 mL/min/kg in mice and moderate to high volume of distribution, ranging from 1.5 L/kg in monkeys to 17 L/kg in mice. Metarrestin has greater than 80% oral bioavailability in all species tested. The excretion of unchanged parent drug in urine was < 5% in dogs and < 1% in monkeys over collection periods of ≥ 144 h; in bile-duct cannulated rats, the excretion of unchanged drug was < 1% in urine and < 2% in bile over a collection period of 48 h. CONCLUSIONS: Metarrestin is a low clearance compound which has good bioavailability and large biodistribution after oral administration. Biotransformation appears to be the major elimination process for the parent drug. In vitro data suggest a low drug-drug interaction potential on CYP-mediated metabolism. Overall favorable ADME and PK properties support metarrestin's progression to clinical investigation.
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Sistema Enzimático del Citocromo P-450/química , Microsomas Hepáticos/metabolismo , Pirimidinas/administración & dosificación , Pirimidinas/farmacocinética , Pirroles/administración & dosificación , Pirroles/farmacocinética , Administración Oral , Animales , Biotransformación , Inhibidores Enzimáticos del Citocromo P-450/administración & dosificación , Inhibidores Enzimáticos del Citocromo P-450/farmacocinética , Sistema Enzimático del Citocromo P-450/metabolismo , Perros , Evaluación Preclínica de Medicamentos , Interacciones Farmacológicas , Femenino , Haplorrinos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Porcinos , Porcinos Enanos , Distribución TisularRESUMEN
A series of quinazolin-4-one based hydroxamic acids was rationally designed and synthesized as novel dual PI3K/HDAC inhibitors by incorporating an HDAC pharmacophore into a PI3K inhibitor (Idelalisib) via an optimized linker. Several of these dual inhibitors were highly potent (IC50 < 10 nM) and selective against PI3Kγ, δ and HDAC6 enzymes and exhibited good antiproliferative activity against multiple cancer cell lines. The lead compound 48c, induced necrosis in several mutant and FLT3-resistant AML cell lines and primary blasts from AML patients, while showing no cytotoxicity against normal PBMCs, NIH3T3, and HEK293 cells. Target engagement of PI3Kδ and HDAC6 by 48c was demonstrated in MV411 cells using the cellular thermal shift assay (CETSA). Compound 48c showed good pharmacokinetics properties in mice via intraperitoneal (ip) administration and provides a means to examine the biological effects of inhibiting these two important enzymes with a single molecule, either in vitro or in vivo.
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Diseño de Fármacos , Inhibidores de Histona Desacetilasas/síntesis química , Ácidos Hidroxámicos/síntesis química , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/síntesis química , Quinazolinonas/síntesis química , Animales , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos/métodos , Femenino , Células HEK293 , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Ratones , Ratones Endogámicos BALB C , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Estructura Terciaria de Proteína , Quinazolinonas/farmacología , RatasRESUMEN
The biosynthesis of sialic acid (Neu5Ac) leads to the intracellular production of cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac), the active sialic acid donor to nascent glycans (glycoproteins and glycolipids) in the Golgi. UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase myopathy is a rare autosomal recessive muscular disease characterized by progressive muscle weakness and atrophy. To quantify the intracellular levels of CMP-Neu5Ac as well as N-acetylmannosamine (ManNAc) and Neu5Ac in human leukocytes, we developed and validated robust liquid chromatography-tandem mass spectrometry methods. A fit-for-purpose approach was implemented for method validation. Hydrophilic interaction chromatography was used to retain three hydrophilic analytes. The human leukocyte pellets were lysed and extracted in a methanol-water mixture and the leukocyte extract was used for LC-MS/MS analysis. The lower limits of quantitation for ManNAc, Neu5Ac and CMP-Neu5Ac were 25.0, 25.0 and 10.0 ng/ml, respectively. These validated methods were applied to a clinical study.
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Cromatografía Liquida/métodos , Citidina Monofosfato/análogos & derivados , Leucocitos/química , Ácidos Siálicos/sangre , Espectrometría de Masas en Tándem/métodos , Citidina Monofosfato/sangre , Estabilidad de Medicamentos , Humanos , Límite de Detección , Modelos Lineales , Reproducibilidad de los ResultadosRESUMEN
Creatine transporter deficiency (CTD) is a metabolic disorder resulting in cognitive, motor, and behavioral deficits. Cyclocreatine (cCr), a creatine analog, has been explored as a therapeutic strategy for the treatment of CTD. We developed a rapid, selective, and accurate HILIC ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to simultaneously quantify the intracellular concentrations of cCr, creatine (Cr), creatine-d3 (Cr-d3), phosphocyclocreatine (pcCr), and phosphocreatine (pCr). Using HILIC-UPLC-MS/MS, we measured cCr and Cr-d3 uptake and their conversion to the phosphorylated forms in primary human control and CTD fibroblasts. Altogether, the data demonstrate that cCr enters cells and its dominant intracellular form is pcCr in both control and CTD patient cells. Therefore, cCr may replace creatine as a therapeutic strategy for the treatment of CTD.
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Encefalopatías Metabólicas Innatas/tratamiento farmacológico , Creatina/deficiencia , Creatinina/análogos & derivados , Fibroblastos/metabolismo , Imidazolidinas/metabolismo , Discapacidad Intelectual Ligada al Cromosoma X/tratamiento farmacológico , Fosfocreatina/análogos & derivados , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/deficiencia , Encefalopatías Metabólicas Innatas/metabolismo , Células Cultivadas , Cromatografía Líquida de Alta Presión/métodos , Creatina/metabolismo , Creatinina/farmacocinética , Creatinina/uso terapéutico , Humanos , Imidazolidinas/análisis , Discapacidad Intelectual Ligada al Cromosoma X/metabolismo , Fosfocreatina/análisis , Fosfocreatina/metabolismo , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/metabolismo , Cultivo Primario de Células , Espectrometría de Masas en Tándem/métodosRESUMEN
Small molecule Toll-like receptor-7 and -8 agonists (TLR-7/8a) can be used as vaccine adjuvants to induce CD8 T cell immunity but require formulations that prevent systemic toxicity and focus adjuvant activity in lymphoid tissues. Here, we covalently attached TLR-7/8a to polymers of varying composition, chain architecture and hydrodynamic behavior (â¼300 nm submicrometer particles, â¼10 nm micelles and â¼4 nm flexible random coils) and evaluated how these parameters of polymer-TLR-7/8a conjugates impact adjuvant activity in vivo. Attachment of TLR-7/8a to any of the polymer compositions resulted in a nearly 10-fold reduction in systemic cytokines (toxicity). Moreover, both lymph node cytokine production and the magnitude of CD8 T cells induced against protein antigen increased with increasing polymer-TLR-7/8a hydrodynamic radius, with the submicrometer particle inducing the highest magnitude responses. Notably, CD8 T cell responses induced by polymer-TLR-7/8a were dependent on CCR2+ monocytes and IL-12, whereas responses by a small molecule TLR-7/8a that unexpectedly persisted in vaccine-site draining lymph nodes (T1/2 = 15 h) had less dependence on monocytes and IL-12 but required Type I IFNs. This study shows how modular properties of synthetic adjuvants can be chemically programmed to alter immunity in vivo through distinct immunological mechanisms.
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Adyuvantes Inmunológicos/química , Linfocitos T CD8-positivos/efectos de los fármacos , Activación de Linfocitos , Micelas , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Animales , Linfocitos T CD8-positivos/inmunología , Línea Celular , Células Cultivadas , Citocinas/metabolismo , Femenino , Hidrodinámica , Ratones , Ratones Endogámicos C57BL , Unión ProteicaRESUMEN
BACKGROUND: (R,S)-ketamine has gained attention for its rapid-acting antidepressant actions in patients with treatment-resistant depression. However, widespread use of ketamine is limited by its side effects, abuse potential, and poor oral bioavailability. The ketamine metabolite, (2R,6R)-hydroxynorketamine, exerts rapid antidepressant effects, without ketamine's adverse effects and abuse potential, in rodents. METHODS: We evaluated the oral bioavailability of (2R,6R)-hydroxynorketamine in three species (mice, rats, and dogs) and also evaluated five candidate prodrug modifications for their capacity to enhance the oral bioavailability of (2R,6R)-hydroxynorketamine in mice. Oral administration of (2R,6R)-hydroxynorketamine was assessed for adverse behavioral effects and for antidepressant efficacy in the mouse forced-swim and learned helplessness tests. RESULTS: (2R,6R)-hydroxynorketamine had absolute bioavailability between 46-52% in mice, 42% in rats, and 58% in dogs. Compared to intraperitoneal injection in mice, the relative oral bioavailability of (2R,6R)-hydroxynorketamine was 62%, which was not improved by any of the candidate prodrugs tested. Following oral administration, (2R,6R)-hydroxynorketamine readily penetrated the brain, with brain to plasma ratios between 0.67-1.2 in mice and rats. Oral administration of (2R,6R)-hydroxynorketamine to mice did not alter locomotor activity or precipitate behaviors associated with discomfort, sickness, or stereotypy up to a dose of 450 mg/kg. Oral (2R,6R)-hydroxynorketamine reduced forced-swim test immobility time (15-150 mg/kg) and reversed learned helplessness (50-150 mg/kg) in mice. CONCLUSIONS: These results demonstrate that (2R,6R)-hydroxynorketamine has favorable oral bioavailability in three species and exhibits antidepressant efficacy following oral administration in mice.
Asunto(s)
Antidepresivos/administración & dosificación , Depresión/tratamiento farmacológico , Ketamina/análogos & derivados , Administración Oral , Animales , Antidepresivos/farmacocinética , Antidepresivos/farmacología , Conducta Animal/efectos de los fármacos , Disponibilidad Biológica , Modelos Animales de Enfermedad , Perros , Femenino , Ketamina/administración & dosificación , Ketamina/farmacocinética , Ketamina/farmacología , Locomoción/efectos de los fármacos , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Distribución TisularRESUMEN
A new series of quinazoline-based analogs as potent bromodomain-containing protein 4 (BRD4) inhibitors is described. The structure-activity relationships on 2- and 4-position of quinazoline ring, and the substitution at 6-position that mimic the acetylated lysine are discussed. A co-crystallized structure of 48 (CN750) with BRD4 (BD1) including key inhibitor-protein interactions is also highlighted. Together with preliminary rodent pharmacokinetic results, a new lead (65, CN427) is identified which is suitable for further lead optimization.
Asunto(s)
Proteínas Nucleares/antagonistas & inhibidores , Quinazolinas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Animales , Sitios de Unión , Proteínas de Ciclo Celular , Línea Celular Tumoral , Descubrimiento de Drogas , Humanos , Ratones , Microsomas Hepáticos/metabolismo , Estructura Molecular , Proteínas Nucleares/química , Quinazolinas/síntesis química , Quinazolinas/química , Quinazolinas/farmacocinética , Relación Estructura-Actividad , Factores de Transcripción/químicaRESUMEN
PURPOSE: Metarrestin is a first-in-class small molecule clinical candidate capable of disrupting the perinucleolar compartment, a subnuclear structure unique to metastatic cancer cells. This study aims to define the pharmacokinetic (PK) profile of metarrestin and the pharmacokinetic/pharmacodynamic relationship of metarrestin-regulated markers. METHODS: PK studies included the administration of single or multiple dose of metarrestin at 3, 10, or 25 mg/kg via intravenous (IV) injection, gavage (PO) or with chow to wild-type C57BL/6 mice and KPC mice bearing autochthonous pancreatic tumors. Metarrestin concentrations were analyzed by UPLC-MS/MS. Pharmacodynamic assays included mRNA expression profiling by RNA-seq and qRT-PCR for KPC mice. RESULTS: Metarrestin had a moderate plasma clearance of 48 mL/min/kg and a large volume of distribution of 17 L/kg at 3 mg/kg IV in C57BL/6 mice. The oral bioavailability after single-dose (SD) treatment was > 80%. In KPC mice treated with SD 25 mg/kg PO, plasma AUC0-∞ of 14400 ng h/mL, Cmax of 810 ng/mL and half-life (t1/2) of 8.5 h were observed. At 24 h after SD of 25 mg/kg PO, the intratumor concentration of metarrestin was high with a mean value of 6.2 µg/g tissue (or 13 µM), well above the cell-based IC50 of 0.4 µM. At multiple dose (MD) 25 mg/kg/day PO in KPC mice, mean tissue/plasma AUC0-24h ratio for tumor, spleen and liver was 37, 30 and 31, respectively. There was a good linear relationship of dosage to AUC0-24h and C24h. AUC0-24h MD to AUC0-24h SD ratios ranged from two for liver to five for tumor indicating additional accumulation in tumors. Dose-dependent normalization of FOXA1 and FOXO6 mRNA expression was observed in KPC tumors. CONCLUSIONS: Metarrestin is an effective therapeutic candidate with a favorable PK profile achieving excellent intratumor tissue levels in a disease with known poor drug delivery.