BD Biosciences Discovery Labware

Woburn, MA, United States

BD Biosciences Discovery Labware

Woburn, MA, United States
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Elsby R.,Astrazeneca | Fox L.,BD Biosciences Discovery Labware | Stresser D.,BD Biosciences Discovery Labware | Layton M.,Astrazeneca | And 4 more authors.
European Journal of Pharmaceutical Sciences | Year: 2011

Methotrexate is the most commonly used drug to treat rheumatoid arthritis. Since clinical efficacy studies in rheumatoid arthritis are conducted on a background of methotrexate therapy, it is necessary to assess the potential of rheumatoid arthritis candidate drugs to perpetrate a drug-drug interaction (DDI) with methotrexate during development. Consequently, we need to identify the regulatory in vitro studies required to facilitate this assessment. We therefore reviewed the literature to ascertain the methotrexate disposition pathways implicated with known DDIs. Experiments were conducted to confirm that methotrexate was identified as a substrate for these pathways in our laboratory. The literature indicated active renal elimination (mediated by the human transporters OAT1, OAT3, MRP2 and BCRP) to be the principal pathway for methotrexate DDI risk. With the exception of MRP2, methotrexate was confirmed as a substrate of these transporters using oocyte and membrane vesicle test systems. A rheumatoid arthritis candidate drug (AZD9056) and sulfasalazine were subsequently assessed as inhibitors of OAT1, OAT3 and BCRP to determine their DDI potential towards methotrexate. AZD9056 was neither an inhibitor of OAT1 nor OAT3 and did not inhibit their transport of methotrexate. AZD9056 was an inhibitor of BCRP and weakly inhibited BCRP-mediated transport of methotrexate (IC50 = 92 μM). Sulfasalazine inhibited methotrexate transport mediated by all transporters studied (IC50 < 5 μM). Subsequent assessment of the in vitro data using [I]/IC50 ratios indicated that both AZD9056 and sulfasalazine were unlikely to cause a DDI with methotrexate in vivo. In conclusion, to support rheumatoid arthritis drug development it is proposed that regulatory in vitro studies for OAT1, OAT3 and BCRP inhibition be routinely conducted to assess the potential for a transporter-mediated DDI with methotrexate in vivo. © 2011 Elsevier B.V. All rights reserved.

Nandivada H.,University of Michigan | Nandivada H.,BD Biosciences Discovery Labware | Villa-Diaz L.G.,University of Michigan | O'Shea K.S.,University of Michigan | And 3 more authors.
Nature Protocols | Year: 2011

The culture of human embryonic stem (hES) cells in defined and xenogeneic-free conditions will contribute substantially to future biotechnological and medical applications. To achieve this goal, we developed the first fully defined synthetic polymer coating poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH) that sustains long-term growth of hES cells in different culture media. Here we describe a detailed protocol for the reproducible fabrication of PMEDSAH coating on tissue culture polystyrene dishes, and for the feeder-free culture of hES cells on PMEDSAH coating in defined culture medium. This culture system represents a key step toward the fully defined and xenogeneic-free culture of hES cells. © 2011 Nature America, Inc. All rights reserved.

Elsby R.,Astrazeneca | Smith V.,Astrazeneca | Fox L.,BD Biosciences Discovery Labware | Stresser D.,BD Biosciences Discovery Labware | And 3 more authors.
Xenobiotica | Year: 2011

Breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) can play a role in the absorption, distribution, metabolism, and excretion of drugs, impacting on the potential for drugdrug interactions. This study has characterized insect cell and mammalian cellderived ABC-transporterexpressing membrane vesicle test systems and validated methodologies for evaluation of candidate drugs as substrates or inhibitors of BCRP or MRP2. Concentration-dependent uptake of BCRP ([ 3H]oestrone 3-sulfate, [ 3H]methotrexate, [ 3H]rosuvastatin) and MRP2 ([ 3H]oestradiol 17β-glucuronide, [ 3H]pravastatin, carboxydichlorofluorescein) substrates, and inhibitory potencies (IC 50) of BCRP (sulfasalazine, novobiocin, fumitremorgin C) and MRP2 (benzbromarone, MK-571, terfenadine) inhibitors were determined. The apparent K m for probes [ 3H]oestrone 3-sulfate and [ 3H]oestradiol 17β-glucuronide was determined in insect cell vesicles to be 7.4 ± 1.7 and 105 ± 8.3 μM, respectively. All other substrates exhibited significant uptake ratios. Positive control inhibitors sulfasalazine and benzbromarone gave IC50 values of 0.74 ± 0.18 and 36 ± 6.1 μM, respectively. All other inhibitors exhibited concentration-dependent inhibition. There was no significant difference in parameters generated between test systems. On the basis of the validation results, acceptance criteria to identify substrates/inhibitors of BCRP and MRP2 were determined for insect cell vesicles. The approach builds on earlier validations to support drug registration and extends from those cell-based systems to encompass assay formats using membrane vesicles. © 2011 Informa UK, Ltd.

Zhang Y.,Pfizer | Li N.,Pfizer | Li N.,BD Biosciences Discovery Labware | Brown P.W.,Pfizer | And 2 more authors.
Rapid Communications in Mass Spectrometry | Year: 2011

P-Glycoprotein (P-gp/ABCB1) is expressed in membrane barriers to exclude pharmacological substrates from cells, and therefore influences the ADME/Tox properties and efficacy of therapeutics. In the present study, a liquid chromatography/tandem mass spectrometry (LC/MS/MS)-mediated targeted proteomics was developed to quantitate P-gp protein. With the aid of in silico predictive tools, a unique 9-mer tryptic peptide of P-gp protein was synthesized (with the stable isotope labeled (SIL) peptide as internal standard) and applied for quantitative LC/MS/MS method development. For LC/MS/MS quantification, the N-glycosylation of the peptide, polymorphism and transmembrane region was intended to be excluded during the peptide selection. The lower limit of quantification was established to be 0.025 nM with the linearity of the standard curve ranging to 20 nM of P-gp signature peptides in the matrix digested surrogate bovine serum albumin. The digestion efficiency, both the accuracy (relative error) and the precision (coefficient of variation) of the method, was verified by using the synthetic quantification peptide and the synthetic surrogate substrate peptide that mimics the sequence of tryptic peptide and associated flanking tryptic cleavage sites at the N- and C-terminals. By applying the method developed, the absolute amounts of human, dog and mouse P-gp (Mdr1a) were quantified in various biological samples. LC/MS/MS-mediated P-gp quantification was achieved as a highly sensitive, selective and reproducible assay and could be directly applicable to many current research needs related to P-gp. © 2011 John Wiley & Sons, Ltd.

U.S. FDA and EMEA guidance recommend that the preferred in vitro model for cytochrome P450 induction testing is human hepatocytes coupled with acceptable inducers as controls. However, there are surprisingly few published studies characterizing this model system for dose and time-dependence response to model inducing compounds. The concentration-dependent response and time-course for the induction of CYP1A2, CYP2B6 and CYP3A4 by inducing agents β-naphthoflavone, phenobarbital and rifampicin, respectively were examined in two or more donors using multiple end-points (mRNA, enzyme activity and Western blot analysis). Depending on the endpoint, exposure time for maximal response of CYP induction potential for the three enzymes ranged from 24 to 72 hours. Of the concentrations of BNF, PB and RIF tested, those which gave the maximal response were found to be 33 μM, > 2 mM and 10 μM, respectively.

PubMed | BD Biosciences Discovery Labware
Type: Journal Article | Journal: Drug discovery today. Technologies | Year: 2014

Over the past several years, drug companies have dedicated significant resources to address the biopharmaceutical properties bottleneck that is recognized as a major source of high attrition rates and longer development times for new drugs. Although it is well accepted that aqueous solubility is a crucial physicochemical parameter that directly influences the passive absorption and distribution of orally administered drugs, there is less agreement about the effective measurement and strategic use of solubility data, especially in the earlier stages of the drug development process.:

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