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1.
J Neurooncol ; 168(2): 269-274, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38630388

ABSTRACT

PURPOSE: Diffuse midline gliomas (DMG) include all midline gliomas with a point mutation to the histone H3 gene resulting in the substitution of a lysine with a methionine (K27M). These tumors are classified as World Health Organization grade 4 with a mean survival between 9- and 19-months following diagnosis. There is currently no standard of care for DMG, and palliative radiation therapy has been proven to only extend survival by months. Our current study aims to report current treatment trends and predictors of the overall survival of DMG. METHODS: We searched the National Cancer Database for adult patients treated for DMG from 2016 to 2020. Patients were required to have been treated with primary radiation directed at the brain with or without concurrent chemotherapy. Univariable and multivariable Cox regressions were used to determine predictors of overall survival. RESULTS: Of the 131 patients meeting the inclusion criteria, 113 (86%) received radiation and chemotherapy. Based on multivariable Cox regression, significant predictors of survival were Charlson-Deyo comorbidity index and race. Patients with a Charlson-Deyo score of 1 had 2.72 times higher odds of mortality than those with a score of 0. Patients not identifying as White or Black had 2.67 times higher odds of mortality than those identifying as White. The median survival for all patients was 19 months. CONCLUSIONS: Despite being considered ineffective, chemotherapy is still administered in most adult patients diagnosed with DMG. Significant predictors of survival were Charlson-Deyo comorbidity index and race.


Subject(s)
Brain Neoplasms , Glioma , Humans , Male , Female , Adult , Brain Neoplasms/therapy , Brain Neoplasms/genetics , Brain Neoplasms/mortality , Brain Neoplasms/pathology , Glioma/therapy , Glioma/genetics , Glioma/mortality , Middle Aged , Survival Rate , Young Adult , Aged , Retrospective Studies , Combined Modality Therapy , Prognosis , United States/epidemiology , Databases, Factual , Follow-Up Studies
2.
Bioorg Med Chem Lett ; 31: 127624, 2021 01 01.
Article in English | MEDLINE | ID: mdl-33096162

ABSTRACT

Calcitonin gene-related peptide (CGRP) receptor antagonists have been shown clinically to be effective treatments for migraine. Zavegepant (BHV-3500, BMS-742413) is a high affinity antagonist of the CGRP receptor (hCGRP Ki = 0.023 nM) that has demonstrated efficacy in the acute treatment of migraine with intranasal delivery in a Phase 2/3 trial, despite showing low oral bioavailability in rats (FPO = 1.7%). Using zavegepant as a template, we sought to improve oral bioavailability through a series of azepinones which were designed in an attempt to reduce the number of rotatable bonds. These efforts led to the discovery of compound 21 which was able to mostly maintain high affinity binding (hCGRP Ki = 0.100 nM) and in vivo efficacy in the marmoset facial blood flow assay, while greatly improving oral bioavailability (rat FPO = 17%).


Subject(s)
Azepines/pharmacology , Calcitonin Gene-Related Peptide Receptor Antagonists/pharmacology , Indazoles/pharmacology , Receptors, Calcitonin Gene-Related Peptide/metabolism , Azepines/chemistry , Calcitonin Gene-Related Peptide Receptor Antagonists/chemistry , Dose-Response Relationship, Drug , Humans , Indazoles/chemistry , Molecular Structure , Structure-Activity Relationship
3.
Case Rep Neurol ; 11(3): 271-276, 2019.
Article in English | MEDLINE | ID: mdl-31607893

ABSTRACT

We present a 26-year-old female with HbSC disease who presented to the emergency department multiple times with pain and shortness of breath, eventually developing unresponsiveness and a brief episode of pulseless electrical activity. She was admitted to the intensive care unit with multisystem organ failure and found to have diffuse ischemic strokes. Infectious workup revealed disseminated anaplasmosis and babesiosis, which had likely caused sickle cell crisis, atypical hemolytic-uremic syndrome, and ischemic brain injury. She was started on eculizumab therapy as well as antimicrobial therapy with doxycycline, clindamycin, and atovaquone. The patient was given tracheostomy and a percutaneous feeding tube. Unfortunately, she did not have significant neurologic recovery after prolonged hospital stay and was discharged to a skilled nursing facility with significant neurologic burden.

4.
SLAS Discov ; 24(7): 738-744, 2019 08.
Article in English | MEDLINE | ID: mdl-31076010

ABSTRACT

The Caco-2 permeability assay is a well-accepted in vitro model to evaluate compounds' potential for oral absorption at early discovery. However, for many lipophilic compounds, no meaningful Caco-2 data could be generated due to their low solubility in assay buffer and/or poor recovery from the assay. In our previous study, we reported an organic catch approach to improve compound recovery. To further reduce compound loss and increase solubility in aqueous buffer, we explored the addition of bovine serum albumin (BSA). However, in contrast to the commonly used BSA level at 4%, a lower level of BSA was selected in an effort to minimize the potential risk of missing the identification of efflux substrates, and to avoid the extensive sample cleanup needed for 4% BSA. Through a systematic evaluation, it was found that 0.5% BSA was effective in enhancing compound solubility and reducing nonspecific binding, which allowed reliable assessment of the permeability and efflux potential for lipophilic compounds. Also, with an optimized sample handling process, no extra sample cleanup was required before liquid chromatography-mass spectrometry (LC-MS) analysis. The implementation of this assay has enabled accurate permeability assessment for compounds that had poor solubility and/or poor mass balance under the non-BSA assay conditions.


Subject(s)
Cell Membrane Permeability , Drug Discovery/methods , High-Throughput Screening Assays , Serum Albumin, Bovine/metabolism , Adsorption , Animals , Caco-2 Cells , Cattle , Cell Membrane Permeability/drug effects , Chromatography, Liquid , Humans , Mass Spectrometry , Solubility
5.
Bioorg Med Chem Lett ; 26(4): 1229-32, 2016 Feb 15.
Article in English | MEDLINE | ID: mdl-26832218

ABSTRACT

Calcitonin gene-related peptide (CGRP) is a potent neuropeptide implicated in the pathophysiology of migraine. In the course of seeking CGRP antagonists with improved oral bioavailability, metabolic stability, and pharmacokinetic properties, lower molecular weight, structurally simpler piperidine and piperazine analogs of BMS-694153 were prepared. Several were found to have nM binding affinity in vitro. The synthesis and SAR of these substituted piperidine and piperazine CGRP antagonists are discussed.


Subject(s)
Calcitonin Gene-Related Peptide/antagonists & inhibitors , Indazoles/chemistry , Piperazines/chemistry , Piperidines/chemistry , Quinazolinones/chemistry , Calcitonin Gene-Related Peptide/metabolism , Cell Line, Tumor , Cell Membrane Permeability/drug effects , Humans , Indazoles/chemical synthesis , Indazoles/pharmacology , Inhibitory Concentration 50 , Piperazine , Quinazolinones/chemical synthesis , Quinazolinones/pharmacology , Structure-Activity Relationship
6.
J Pharmacol Exp Ther ; 356(2): 293-304, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26582730

ABSTRACT

The muscarinic acetylcholine receptor subtype 1 (M1) receptors play an important role in cognition and memory, and are considered to be attractive targets for the development of novel medications to treat cognitive impairments seen in schizophrenia and Alzheimer's disease. Indeed, the M1 agonist xanomeline has been shown to produce beneficial cognitive effects in both Alzheimer's disease and schizophrenia patients. Unfortunately, the therapeutic utility of xanomeline was limited by cholinergic side effects (sweating, salivation, gastrointestinal distress), which are believed to result from nonselective activation of other muscarinic receptor subtypes such as M2 and M3. Therefore, drug discovery efforts targeting the M1 receptor have focused on the discovery of compounds with improved selectivity profiles. Recently, allosteric M1 receptor ligands have been described, which exhibit excellent selectivity for M1 over other muscarinic receptor subtypes. In the current study, the following three compounds with mixed agonist/positive allosteric modulator activities that are highly functionally selective for the M1 receptor were tested in rats, dogs, and cynomologous monkeys: (3-((1S,2S)-2-hydrocyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one; 1-((4-cyano-4-(pyridin-2-yl)piperidin-1-yl)methyl)-4-oxo-4H-quinolizine-3-carboxylic acid; and (R)-ethyl 3-(2-methylbenzamido)-[1,4'-bipiperidine]-1'-carboxylate). Despite their selectivity for the M1 receptor, all three compounds elicited cholinergic side effects such as salivation, diarrhea, and emesis. These effects could not be explained by activity at other muscarinic receptor subtypes, or by activity at other receptors tested. Together, these results suggest that activation of M1 receptors alone is sufficient to produce unwanted cholinergic side effects such as those seen with xanomeline. This has important implications for the development of M1 receptor-targeted therapeutics since it suggests that dose-limiting cholinergic side effects still reside in M1 receptor selective activators.


Subject(s)
Muscarinic Agonists/metabolism , Muscarinic Agonists/pharmacology , Receptor, Muscarinic M1/agonists , Receptor, Muscarinic M1/metabolism , Animals , CHO Cells , Cricetinae , Cricetulus , Dogs , Dose-Response Relationship, Drug , Humans , Macaca fascicularis , Male , Mice , Rats , Rats, Sprague-Dawley
7.
J Med Chem ; 58(10): 4220-9, 2015 May 28.
Article in English | MEDLINE | ID: mdl-25901762

ABSTRACT

Allosteric modulators of G protein-coupled receptors (GPCRs) have a number of potential advantages compared to agonists or antagonists that bind to the orthosteric site of the receptor. These include the potential for receptor selectivity, maintenance of the temporal and spatial fidelity of signaling in vivo, the ceiling effect of the allosteric cooperativity which may prevent overdose issues, and engendering bias by differentially modulating distinct signaling pathways. Here we describe the discovery, synthesis, and molecular pharmacology of δ-opioid receptor-selective positive allosteric modulators (δ PAMs). These δ PAMs increase the affinity and/or efficacy of the orthosteric agonists leu-enkephalin, SNC80 and TAN67, as measured by receptor binding, G protein activation, ß-arrestin recruitment, adenylyl cyclase inhibition, and extracellular signal-regulated kinases (ERK) activation. As such, these compounds are useful pharmacological tools to probe the molecular pharmacology of the δ receptor and to explore the therapeutic potential of δ PAMs in diseases such as chronic pain and depression.


Subject(s)
Receptors, Opioid, delta/metabolism , Structure-Activity Relationship , Animals , Arrestins/metabolism , Benzamides/pharmacology , Binding, Competitive , CHO Cells , Cell Membrane/drug effects , Cell Membrane/metabolism , Chemistry Techniques, Synthetic , Cricetulus , Drug Discovery , Drug Evaluation, Preclinical/methods , Enkephalin, Leucine/pharmacology , Enzyme Activation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Molecular Structure , Molecular Targeted Therapy , Piperazines/pharmacology , Protein Binding , Quinolines/pharmacology , beta-Arrestins
8.
Drug Metab Lett ; 8(2): 109-18, 2014.
Article in English | MEDLINE | ID: mdl-25429513

ABSTRACT

The study presented here identified and utilized a panel of solubility enhancing excipients to enable the generation of flux data in the Human colon carcinoma (Caco-2) system for compounds with poor solubility. Solubility enhancing excipients Dimethyl acetamide (DMA) 1 % v/v, polyethylene glycol (PEG) 400 1% v/v, povidone 1% w/v, poloxamer 188 2.5% w/v and bovine serum albumin (BSA) 4% w/v did not compromise Caco-2 monolayer integrity as assessed by trans-epithelial resistance measurement (TEER) and Lucifer yellow (LY) permeation. Further, these excipients did not affect P-glycoprotein (P-gp) mediated bidirectional transport of digoxin, permeabilities of high (propranolol) or low permeability (atenolol) compounds, and were found to be inert to Breast cancer resistant protein (BCRP) mediated transport of cladribine. This approach was validated further using poorly soluble tool compounds, atazanavir (poloxamer 188 2.5% w/v) and cyclosporine A (BSA 4% w/v) and also applied to new chemical entity (NCE) BMS-A in BSA 4% w/v, for which Caco-2 data could not be generated using the traditional methodology due to poor solubility (<1 µM) in conventional Hanks balanced salt solution (HBSS). Poloxamer 188 2.5% w/v increased solubility of atazanavir by >8 fold whereas BSA 4% w/v increased the solubility of cyclosporine A and BMS-A by >2-4 fold thereby enabling permeability as well as efflux liability estimation in the Caco-2 model with reasonable recovery values. To conclude, addition of excipients such as poloxamer 188 2.5% w/v and BSA 4% w/v to HBSS leads to a significant improvement in the solubility of the poorly soluble compounds resulting in enhanced recoveries without modulating transporter-mediated efflux, expanding the applicability of Caco-2 assays to poorly soluble compounds.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP-Binding Cassette Transporters/metabolism , Excipients/pharmacology , Neoplasm Proteins/metabolism , Pharmaceutical Preparations/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2 , Biological Transport/drug effects , Caco-2 Cells , Humans , Isoquinolines/metabolism , Permeability , Pharmaceutical Preparations/chemistry , Solubility
9.
J Biomol Screen ; 19(10): 1409-14, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25296658

ABSTRACT

Plastic labware is used in all processes of modern pharmaceutical research, including compound storage and biological assays. The use of these plastics has created vast increases in productivity and cost savings as experiments moved from glass test tubes and capillary pipettes to plastic microplates and multichannel liquid handlers. One consequence of the use of plastic labware, however, is the potential release of contaminants and their resultant effects on biological assays. We report herein the identification of biologically active substances released from a commonly used plastic microplate. The active contaminants were identified by gas chromatography-mass spectroscopy as dodecan-1-ol, dodecyl 3-(3-dodecoxy-3-oxopropyl)sulfanylpropanoate, and dodecanoic acid, and they were found to be selective monoamine oxidase-B inhibitors.


Subject(s)
Drug Evaluation, Preclinical/instrumentation , Monoamine Oxidase Inhibitors/pharmacology , Plastics/chemistry , 3-Mercaptopropionic Acid/analogs & derivatives , 3-Mercaptopropionic Acid/pharmacology , Dodecanol/chemistry , Dodecanol/pharmacology , Drug Evaluation, Preclinical/methods , Gas Chromatography-Mass Spectrometry , Lauric Acids/pharmacology , Monoamine Oxidase/metabolism , Plastics/pharmacology , Signal-To-Noise Ratio , Sulfides/pharmacology
10.
Pediatr Int ; 55(3): e42-5, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23782376

ABSTRACT

Bleeding from gastric varices due to splenic vein obstruction is extremely rare in children, but it can be catastrophic. Reported herein is the case of a teenager with splenic vein thrombosis and chronic decompensated liver disease from autoimmune hepatitis who presented with massive gastric variceal bleeding. Standard medical management did not control the bleeding. Due to decompensated liver disease and continuous active bleeding, emergency partial splenic artery embolization was preferred over splenectomy or a shunt procedure. Bleeding was successfully controlled by partial splenic artery embolization by decreasing the inflow of blood into the portal system. It is concluded that emergency partial splenic artery embolization is a safer alternative life-saving procedure to manage severe gastric variceal bleeding due to splenic vein obstruction in a patient with high surgical risk. To our knowledge, only one other patient with similar management has been reported in the pediatric age group.


Subject(s)
Embolization, Therapeutic , Emergencies , Esophageal and Gastric Varices/therapy , Gastrointestinal Hemorrhage/therapy , Adolescent , Angiography , Autoantibodies/blood , Autoimmune Diseases/complications , Autoimmune Diseases/diagnosis , Autoimmune Diseases/pathology , Endoscopy, Digestive System , Esophageal and Gastric Varices/diagnosis , Gastrointestinal Hemorrhage/diagnosis , Humans , Intensive Care Units, Pediatric , Intubation, Gastrointestinal , Liver/pathology , Liver Cirrhosis/complications , Liver Cirrhosis/diagnosis , Liver Cirrhosis/psychology , Male , Muscle, Smooth/immunology , Muscle, Smooth/pathology , Necrosis , Spleen/pathology , Splenic Artery , Splenic Infarction/diagnosis , Tomography, X-Ray Computed
11.
Drug Metab Dispos ; 41(4): 827-35, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23382458

ABSTRACT

The studies reported here were conducted to investigate the transport characteristics of apixaban (1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide) and to understand the impact of transporters on apixaban distribution and disposition. In human permeability glycoprotein (P-gp)- and breast cancer resistance protein (BCRP)-cDNA-transfected cell monolayers as well as Caco-2 cell monolayers, the apparent efflux ratio of basolateral-to-apical (PcB-A) versus apical-to-basolateral permeability (PcA-B) of apixaban was >10. The P-gp- and BCRP-facilitated transport of apixaban was concentration- and time-dependent and did not show saturation over a wide range of concentrations (1-100 µM). The efflux transport of apixaban was also demonstrated by the lower mucosal-to-serosal permeability than that of the serosal-to-mucosal direction in isolated rat jejunum segments. Apixaban did not inhibit digoxin transport in Caco-2 cells. Ketoconazole decreased the P-gp-mediated apixaban efflux in Caco-2 and the P-gp-cDNA-transfected cell monolayers, but did not affect the apixaban efflux to a meaningful extent in the BCRP-cDNA-transfected cell monolayers. Coincubation of a P-gp inhibitor (ketoconazole or cyclosporin A) and a BCRP inhibitor (Ko134) provided more complete inhibition of apixaban efflux in Caco-2 cells than separate inhibition by individual inhibitors. Naproxen inhibited apixaban efflux in Caco-2 cells but showed only a minimal effect on apixaban transport in the BCRP-transfected cells. Naproxen was the first nonsteroidal antiinflammatory drug that was demonstrated as a weak P-gp inhibitor. These results demonstrate that apixaban is a substrate for efflux transporters P-gp and BCRP, which can help explain its low brain penetration, and low fetal exposures and high milk excretion in rats.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP-Binding Cassette Transporters/metabolism , Fibrinolytic Agents/pharmacokinetics , Neoplasm Proteins/metabolism , Pyrazoles/pharmacokinetics , Pyridones/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/antagonists & inhibitors , Adenosine/analogs & derivatives , Adenosine/pharmacology , Animals , Biological Transport/drug effects , Caco-2 Cells/metabolism , Cell Line, Transformed , Cell Membrane Permeability/drug effects , Cyclosporine/pharmacology , Digoxin/pharmacokinetics , Diketopiperazines , Dose-Response Relationship, Drug , Drug Interactions , Heterocyclic Compounds, 4 or More Rings , Humans , Ketoconazole/pharmacology , Male , Naproxen/pharmacology , Neoplasm Proteins/antagonists & inhibitors , Rats
12.
J Pharm Sci ; 101(8): 2755-62, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22611052

ABSTRACT

The Caco-2 cell culture system is widely employed as an in vitro model for prediction of intestinal absorption of test compounds in early drug discovery. Poor recovery is a commonly encountered issue in Caco-2 assay, which can lead to difficulty in data interpretation and underestimation of the apparent permeability of affected compounds. In this study, we systematically investigated the potential sources of compound loss in our automated, high-throughput Caco-2 assay, sample storage, and analysis processes, and as a result found the nonspecific binding to various plastic surfaces to be the major cause of poor compound recovery. To minimize the nonspecific binding, we implemented a simple and practical approach in our assay automation by preloading collection plates with organic solvent containing internal standard prior to transferring incubations samples. The implementation of this new method has been shown to significantly increase recovery in many compounds previously identified as having poor recovery in the Caco-2 permeability assay. With improved recovery, permeability results were obtained for many compounds that were previously not detected in the basolateral samples. In addition to recovery improvement, this new approach also simplified sample preparation for liquid chromatography-tandem mass spectrometric analysis and therefore achieved time and cost savings for the bioanalyst.


Subject(s)
Cell Membrane Permeability , High-Throughput Screening Assays/methods , Tandem Mass Spectrometry/methods , Caco-2 Cells , Chromatography, Liquid/methods , Humans , Intestinal Absorption , Pharmacokinetics
13.
Bioanalysis ; 4(5): 581-93, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22409555

ABSTRACT

BACKGROUND: High-resolution MS (HRMS) has recently received a considerable interest in quantitative bioanalysis using full-scan acquisition mode. The benefits include complete elimination of compound-specific MS method development, and simultaneous collection of mass spectral data on both targeted and non-targeted components. One additional advantage that has not been widely discussed is its suitability for simultaneous quantitation of, theoretically, an unlimited number of compounds, which is not possible with selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. MATERIALS & METHODS: We took advantage of this unique bioanalytical capability of HRMS and developed a novel in vitro ADME workflow of cassette incubation of as many as 32 compounds, followed by quantitative bioanalysis using full-scan acquisition on an Orbitrap HRMS. The workflow was evaluated for a serum protein-binding assay and a parallel artificial membrane permeability (PAMPA) assay. RESULTS: The bioanalytical assay displayed acceptable sensitivity, selectivity and linearity for all compounds in the cassettes, and the biological results obtained using this approach were similar to those from discrete incubation and analysis, demonstrating the feasibility of the workflow. Additional benefits of this platform include a saving of analysis time due to the reduced sample numbers from the cassette approach, as well as cost saving due to the reduction in the required assay reagents. CONCLUSION: Cassette incubation with bioanalysis using HRMS is a feasible approach for high-throughput in vitro ADME assays evaluated in this study.


Subject(s)
Biological Assay/methods , Mass Spectrometry , Pharmaceutical Preparations/analysis , Pharmaceutical Preparations/chemistry
15.
Toxicol In Vitro ; 25(8): 1934-43, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21767630

ABSTRACT

Phospholipidosis (PLD) in preclinical species can lead to regulatory delays thereby creating incentives to screen for PLD during drug discovery. The objective of this work was to compare, optimize, and validate in vitro PLD assays in primary mouse macrophages and hepatocyte- (HepG2, HuH7) or macrophage-derived cells lines (I.13.35, RAW264.7) and to evaluate whether primary cells were better at predicting PLD. Assay precision, determined by a measure of signal to noise window (Z'), within assay variability, and day-to-day variability, using amiodarone, was generally acceptable for all cell types; however, precision limits for HepG2 and HuH7 were slightly below assay acceptance criteria. Up to 66 known PLD inducers and non-inducers were subsequently tested to validate the assays. The concordance for predicting PLD in primary macrophages, I-13.35, RAW264.7, HuH7, and HepG2 cells was 91%, 74%, 73%, 62%, and 62% respectively using a decision limit of EC50≤125 µM as a positive finding. Increasing the number of negative controls tested in RAW264.7 cells and changing the decision limit to ≥4-fold increase in PLD, improved the specificity and overall concordance to 88%. RAW264.7 cells were selected as the primary screen for predicting PLD, and together with the primary macrophages, were integrated into an overall testing paradigm proposed for use in PLD risk identification.


Subject(s)
Drug Evaluation, Preclinical/methods , Drug-Related Side Effects and Adverse Reactions , Hepatocytes/drug effects , Lipidoses/chemically induced , Macrophages, Peritoneal/drug effects , Phospholipids/metabolism , Animals , Cell Line , Cell Survival/drug effects , Fluorescent Dyes/metabolism , Hep G2 Cells , Hepatocytes/metabolism , Humans , Lipidoses/metabolism , Macrophages, Peritoneal/metabolism , Macrophages, Peritoneal/ultrastructure , Male , Mice , Mice, Inbred BALB C , Microscopy, Electron , Phosphatidylethanolamines/metabolism
16.
Rapid Commun Mass Spectrom ; 25(9): 1231-40, 2011 May 15.
Article in English | MEDLINE | ID: mdl-21488121

ABSTRACT

The evaluation of interactions between drug candidates and transporters such as P-glycoprotein (P-gp) has gained considerable interest in drug discovery and development. Inhibition of P-gp can be assessed by performing bi-directional permeability studies with in vitro P-gp-expressing cellular model systems such as Caco-2 (human colon carcinoma) cells, using digoxin as a substrate probe. Existing methodologies include either assaying (3)H-digoxin with liquid scintillation counting (LSC) detection or assaying non-labeled digoxin with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis at a speed of several minutes per sample. However, it is not feasible to achieve a throughput high enough using these approaches to sustain an early liability screen that generates more than a thousand samples on a daily basis. To address this challenge, we developed an ultrafast (9 s per sample) bioanalytical method for digoxin analysis using RapidFire™, an on-line solid-phase extraction (SPE) system, with MS/MS detection. A stable isotope labeled analog, d3-digoxin, was used as internal standard to minimize potential ionization matrix effect during the RF-MS/MS analysis. The RF-MS/MS method was more than 16 times faster than the LC-MS/MS method but demonstrated similar sensitivity, selectivity, reproducibility, linearity and robustness. P-gp inhibition results of multiple validation compounds obtained with this RF-MS/MS method were in agreement with those generated by both the LC-MS/MS method and the (3)H-radiolabel assay. This method has been successfully deployed to assess P-gp inhibition potential as an important early liability screen for drug-transporter interaction.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Chromatography, Liquid/methods , Digoxin/analysis , High-Throughput Screening Assays/methods , Tandem Mass Spectrometry/methods , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Caco-2 Cells , Cyclosporine/chemistry , Cyclosporine/pharmacology , Digoxin/chemistry , Digoxin/metabolism , Drug Discovery/methods , Drug Discovery/standards , Humans , Linear Models , Models, Biological , Reproducibility of Results , Sensitivity and Specificity , Solid Phase Extraction , Tritium
17.
Rapid Commun Mass Spectrom ; 24(24): 3593-601, 2010 Dec 30.
Article in English | MEDLINE | ID: mdl-21080511

ABSTRACT

Quantification of small molecules using liquid chromatography/tandem mass spectrometry (LC/MS/MS) on a triple quadrupole mass spectrometer has become a common practice in bioanalytical support of in vitro adsorption, distribution, metabolism and excretion (ADME) screening. The bioanalysis process involves primarily three indispensable steps: MS/MS optimization for a large number of new chemical compounds undergoing various screening assays in early drug discovery, high-throughput sample analysis with LC/MS/MS for those chemically diverse compounds using the optimized MS/MS conditions, and post-acquisition data review and reporting. To improve overall efficiency of ADME bioanalysis, an integrated system was proposed featuring an automated and unattended MS/MS optimization, a staggered parallel LC/MS/MS for high-throughput sample analysis, and a sophisticated software tool for LC/MS/MS raw data review as well as biological data calculation and reporting. The integrated platform has been used in bioanalytical support of a serum protein binding screening assay with high speed, high capacity, and good robustness. In this new platform, a unique sample dilution scheme was also introduced. With this dilution design, the total number of analytical samples was reduced; therefore, the total operation time was reduced and the overall throughput was further improved. The performance of the protein binding screening assay was monitored with two controls representing high and low binding properties and an acceptable inter-assay consistency was achieved. This platform has been successfully used for the determination of serum protein binding in multiple species for more than 4000 compounds.


Subject(s)
Blood Proteins/metabolism , Drug Discovery/methods , High-Throughput Screening Assays/methods , Animals , Blood Proteins/chemistry , Chromatography, Liquid , Dogs , Humans , Linear Models , Macaca fascicularis , Male , Mice , Pharmacokinetics , Protein Binding , Rats , Reproducibility of Results , Systems Integration , Tandem Mass Spectrometry
18.
Assay Drug Dev Technol ; 7(3): 294-303, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19530896

ABSTRACT

In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.


Subject(s)
Cytochrome P-450 CYP3A/biosynthesis , Drug Evaluation, Preclinical/methods , Cell Line, Tumor , Cryopreservation , Dose-Response Relationship, Drug , Efficiency , Enzyme Induction/drug effects , Humans , Luciferases/genetics , Luciferases/metabolism , Pregnane X Receptor , Receptors, Steroid/biosynthesis , Receptors, Steroid/genetics , Reference Standards , Reproducibility of Results , Structure-Activity Relationship , Transfection
19.
J Biomol Screen ; 14(5): 566-72, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19470712

ABSTRACT

Disposable plastic labware is ubiquitous in contemporary pharmaceutical research laboratories. Plastic labware is routinely used for chemical compound storage and during automated liquid-handling processes that support assay development, high-throughput screening, structure-activity determinations, and liability profiling. However, there is little information available in the literature on the contaminants released from plastic labware upon DMSO exposure and their resultant effects on specific biological assays. The authors report here the extraction, by simple DMSO washing, of a biologically active substance from one particular size of disposable plastic tips used in automated compound handling. The active contaminant was identified as erucamide ((Z)-docos-13-enamide), a long-chain mono-unsaturated fatty acid amide commonly used in plastics manufacturing, by gas chromatography/mass spectroscopy analysis of the DMSO-extracted material. Tip extracts prepared in DMSO, as well as a commercially obtained sample of erucamide, were active in a functional bioassay of a known G-protein-coupled fatty acid receptor. A sample of a different disposable tip product from the same vendor did not release detectable erucamide following solvent extraction, and DMSO extracts prepared from this product were inactive in the receptor functional assay. These results demonstrate that solvent-extractable contaminants from some plastic labware used in the contemporary pharmaceutical research and development (R&D) environment can be introduced into physical and biological assays during routine compound management liquid-handling processes. These contaminants may further possess biological activity and are therefore a potential source of assay-specific confounding artifacts.


Subject(s)
Automation , Drug Discovery/instrumentation , Equipment Contamination , Erucic Acids/chemistry , Plastics/chemistry , Animals , Cell Line , Dimethyl Sulfoxide/chemistry , Equipment Reuse , Humans , Solvents/chemistry
20.
J Biomol Screen ; 12(2): 248-54, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17259590

ABSTRACT

Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r(2) > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.


Subject(s)
Biological Assay/methods , Cryopreservation/methods , Pharmaceutical Preparations/metabolism , Receptors, Steroid/metabolism , Transcriptional Activation , Caco-2 Cells , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Membrane Permeability/drug effects , Drug-Related Side Effects and Adverse Reactions , Humans , Liver Neoplasms/pathology , Mifepristone/metabolism , Mifepristone/pharmacology , Pregnane X Receptor , Receptors, Steroid/agonists , Receptors, Steroid/antagonists & inhibitors , Reproducibility of Results , Rifampin/metabolism , Rifampin/pharmacology , Sulfinpyrazone/metabolism , Sulfinpyrazone/pharmacology , Transfection
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