10 research outputs found
Metabolic Activation and Major Protein Target of a 1-Benzyl-3-carboxyazetidine Sphingosine-1-phosphate-1 Receptor Agonist
1-{4-[(4-Phenyl-5-trifluoromethyl-2-thienyl)Âmethoxy]Âbenzyl}Âazetidine-3-carboxylic
acid (MRL-A) is a potent sphingosine-1-phosphate-1 receptor agonist,
with potential application as an immunosuppressant in organ transplantation
or for the treatment of autoimmune diseases. When administered orally
to rats, radiolabeled MRL-A was found to undergo metabolism to several
reactive intermediates, and in this study, we have investigated its
potential for protein modification in vivo and in vitro. MRL-A irreversibly
modified liver and kidney proteins in vivo, in a dose- and time-dependent
manner. The binding was found to occur selectively to microsomal and
mitochondrial subcellular fractions. Following a nonspecific proteolytic
digestion of liver and kidney proteins, a single major amino acid
adduct was observed. This adduct was characterized with LC/MS/UV and
NMR spectroscopy and was found to be the product of an unprecedented
metabolic activation of the azetidine moiety leading to the formation
of a ring-opened α,β-unsaturated imine conjugated to the
ε-amino group of a lysine residue. The formation of this adduct
was not inhibited when rats were pretreated with 1-aminobenzotriazole,
indicating that P450 enzymes were not involved in the metabolic activation
of MRL-A. Rather, our findings suggested that MRL-A underwent bioactivation
via a β-oxidation pathway. Several other minor adducts were
identified from protein hydrolysates and included lysine, serine,
and cysteine conjugates of MRL-A. These minor adducts were also detected
in microsomal incubations fortified with the cofactors for acyl-CoA
synthesis and in hepatocytes. Trypsin digestion of crude liver homogenates
from rats treated with radiolabeled MRL-A led to the identification
of a single radioactive peptide. Its sequence, determined by LC/MS
analysis, revealed that the target of the major reactive species of
MRL-A in vivo is Lys676 of long chain acyl-CoA synthetase-1 (ACSL1).
This lysine residue has been found to be critical for ACSL1 activity,
and its modification has the potential to lead to biological consequences
such as cardiac hypertrophy or thermogenesis dysregulation
Variability in P-Glycoprotein Inhibitory Potency (IC 50
A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC 50 determinations. Each laboratory followed its in-house protocol to determine in vitro IC50 values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells-Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLCPK1- MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC50 values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC50 values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC50 values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratiobased equation generally resulted in severalfold lower IC 50 values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC50 values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC 50 determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided. © 2013 by The American Society for Pharmacology
Variability in P-Glycoprotein Inhibitory Potency (IC50) Using Various in Vitro Experimental Systems: Implications for Universal Digoxin Drug- Drug Interaction Risk Assessment Decision Criterias
A P-glycoprotein (P-gp) IC(50) working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC(50) determinations. Each laboratory followed its in-house protocol to determine in vitro IC(50) values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells—Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK(1)-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC(50) values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC(50) values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC(50) values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC(50) values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC(50) values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC(50) determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided