Characterization of the In Vitro Inhibitory Potential of the Oligonucleotide Imetelstat on Human Cytochrome P450 Enzymes with Predictions of In Vivo Drug-Drug Interactions.

Imetelstat, a 13-base oligonucleotide (5'-TAGGGTTAGACAA-3'), is a potent, investigational telomerase inhibitor in clinical development for the treatment of hematologic myeloid malignancies. Modifications to imetelstat oligonucleotide chemistry include an N3'-P5' thio-phosphoramidate backbone linkage to improve biologic stability and the addition of a palmitoyl tail at the 5'-position to enhance cellular membrane permeability. Other oligonucleotides have been previously shown to have in vitro test-system-dependent outcomes when potent cytochrome P450 inhibition in human liver microsomes (HLM) is observed, but such inhibition is not observed in cryopreserved human hepatocytes (CHH). Outcomes in CHH are consistent with clinical reports in which no interactions were reported. In the present study, imetelstat was evaluated for in vitro inhibition of eight P450 enzymes, namely CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 in CHH (0.5 million cells/ml). Assays were performed using validated conditions, including short substrate times (10 minutes), and at the approximate substrate Km concentration. Imetelstat was found to have little to no inhibition of all P450 isoforms evaluated, with inhibitor concentration that causes 50% inhibition (IC50) values >100 µM. Maximum percent inhibition values for each P450 isoform at 100 µM imetelstat were <20% except for CYP2C8 activity, which was inhibited by 49%. Using a static mechanistic model, the predicted change in area under the curve of a victim drug coadministered with imetelstat was 1.04-fold, projecting no relevant clinical interaction. Overall, the results from this in vitro study suggest that clinical use of imetelstat is unlikely to affect the pharmacokinetics of concomitant therapies that undergo cytochrome P450-mediated metabolism.

Quantitative analysis of imetelstat in plasma with LC-MS/MS using solid-phase or hybridization extraction.

AIM: Imetelstat, a 13-mer oligonucleotide with a lipid tail is being evaluated for treating hematologic myeloid malignancies. This report describes the development of extraction and quantification methods for imetelstat. Methodology & results: Imetelstat was extracted using SPE (rat plasma) or by hybridization using a biotinylated capture probe (human plasma) and was quantified by LC-MS/MS. Calibration curves were established (0.1-50 µg/ml). Stability of imetelstat in plasma was demonstrated. Concentrations of imetelstat extracted using either of the methods and quantified with LC-MS/MS were comparable with a validated ELISA. CONCLUSION: Two extraction methods (solid phase and hybridization) were developed for quantifying imetelstat in plasma using LC-MS/MS. The hybridization extraction in combination with LC-MS/MS is a novel extraction approach.

Postmortem redistribution of fentanyl in the rabbit blood.

Postmortem redistribution of fentanyl in the rabbit was investigated after application of the 50-µg/h Durogesic pain patch. Patches were applied for 48 hours. Two cycles of patch administration were used before characterization of the postmortem redistribution. Fentanyl showed marked redistribution into the femoral and pulmonary veins of the rabbit through 48 hours after the animals were humanely killed and the pain patches removed. The plasma concentration of 2.34 ng/mL in the femoral blood before killing the animals increased 5.6-fold by 48 hours after patch removal to 13.2 ng/mL. This postmortem concentration is approximately 3-fold the C(max) determined during antemortem pharmacokinetic analysis, 4 ng/mL, which was achieved 24 hours after the application of the second 50-µg/h Durogesic pain patch. After blood sampling for 48 hours after animal termination with patch removal compared with sampling for 48 hours from animals not terminated and with patch removal, the exposure ratios in the terminated animals were approximately 30-fold, indicating that between the postmortem redistribution of fentanyl and the cessation of hepatic clearance of fentanyl in the rabbit, the postmortem redistribution of fentanyl leads to an elevated measures of postmortem blood concentrations relative to antemortem blood concentrations.

Nonclinical safety assessment of a synthetic peptide thrombopoietin agonist: effects on platelets, bone homeostasis, and immunogenicity and the implications for clinical safety monitoring of adverse bone effects.

RWJ-800088 is a novel, potent polyethylene glycol (PEG)-conjugated thrombopoietin (TPO) mimetic that increases platelet levels and protects against thrombocytopenia. A nonclinical safety program was customized for this peptide that takes into account its protein-like structure, synthetic chemical nature, agonist pharmacologic activity, and mode of administration. In repeat-dose toxicity studies, the salient findings were dose-related increases in circulating platelet counts, mean platelet volume, and megakaryocytes in the bone marrow with no antibody formation. Reversible myelofibrosis and hyperostosis were observed in rats, but not dogs, when the circulating platelet levels exceeded 3× those of vehicle controls. The bone effects were due to the exaggerated pharmacologic effect and excessive stimulation and elevation of megakaryocytes by TPO, which results in intramedullary proliferation of fibroblasts and mesenchymal cells followed by osseous metaplasia. These findings support the use of platelet elevations of >3× as a stopping criterion to prevent potential adverse bone-related effects in humans.

Mixed-effects modelling of the interspecies pharmacokinetic scaling of pegylated human erythropoietin.

The aim of this study was to develop a population pharmacokinetic model for interspecies allometric scaling of pegylated r-HuEPO (PEG-EPO) pharmacokinetics to man. A total of 927 serum concentrations from 193 rats, 6 rabbits, 34 monkeys, and 9 dogs obtained after a single dose of PEG-EPO, administered by the i.v. (dose range: 12.5-550 microg/kg) and s.c. (dose range: 12.5-500 microg/kg) routes, were pooled in this analysis. An open two-compartment model with first-order absorption and lag time (Tlag) and linear elimination from the central compartment was fitted to the data using the NONMEM V software. Body weight (WT) was used as a scaling factor and the effect of brain weight (BW), sex, and pregnancy status on the pharmacokinetic parameters was investigated. The final model was evaluated by means of a non-parametric bootstrap analysis and used to predict the PEG-EPO pharmacokinetic parameters in healthy male subjects. The systemic clearance (CL) in males was estimated to be 4.08WT1.030xBW-0.345 ml/h. In females, the CL was 90.7% of the CL in males. The volumes of the central (Vc) and the peripheral (Vp) compartment were characterized as 57.8WT0.959 ml, and 48.1WT1.150 ml, respectively. Intercompartmental flow was estimated at 2.32WT0.930 ml/h. Absorption rate constant (Ka) was estimated at 0.0538WT-0.149. The absolute s.c. bioavailability F was calculated at 52.5, 80.2, and 49.4% in rat, monkey, and dog, respectively. The interindividual variability in the population pharmacokinetic parameters was fairly low (<35%). Non-parametric bootstrap confirmed the accuracy of the NONMEM estimates. The mean model predicted pharmacokinetic parameters in healthy male subjects of 70 kg were estimated at: CL: 26.2 ml/h; Vc: 3.6l; Q: 286 l/h; Vp: 6.9l, and Ka: 0.031 h-1. The population pharmacokinetic model developed was appropriate to describe the time course of PEG-EPO serum concentrations and their variability in different species. The model predicted pharmacokinetics of PEG-EPO in humans suggest a less frequent dosing regimen relative to erythropoietin and darbepoetin, potentially leading to a simplification of anemia management.

Population pharmacokinetic analysis of pegylated human erythropoietin in rats.

The purpose of this study was to model the pharmacokinetics of the pegylated human erythropoietin (PEG-EPO) after single-dose administration in rats, and to evaluate the influence of weight, sex, and pregnancy status on the pharmacokinetic parameters. A total of 436 serum concentrations from 193 Sprague-Dawley rats were obtained from four pharmacokinetic/toxicokinetic studies, in which a single dose of PEG-EPO was administered by the intravenous (i.v.; dose range: 2.5 to 500 microg/kg) and subcutaneous (s.c.; dose range: 12.5 to 500 microg/kg) route. Pharmacokinetic analysis was performed using nonlinear mixed effect modeling (NONMEM V software) to determine the population mean of pharmacokinetic parameters and the variances of the interindividual random effects. The effect of weight, sex, and pregnancy status on the pharmacokinetic parameters was evaluated by forward inclusion and backward elimination process, using the likelihood ratio test. Nonparametric bootstrap analysis was employed as an internal model evaluation technique to qualify the model developed. An open two-compartment model with linear elimination from the central compartment, a first-order absorption with lag time characterized the serum concentration-time profiles of PEG-EPO after i.v. and s.c. administration. For a male rat of 0.24 kg, the average CL, Vc, Q, Vp, Ka, Tlag, and F was estimated to be 0.728 mL/h, 15.8 mL, 0.373 mL/h, 6.99 mL, 0.0618 h(-1), 3.13 h, and 48.8%, respectively. A twofold increase in weight corresponded with a 170 and 238% increase in CL and Vc, respectively. In female rats, Vp was reduced by 11%, whereas F was increased by 15%. No effect of pregnancy status on any of the parameters could be identified. The interindividual variability in CL, Vc, Vp, Ka, and F was estimated at 10.7, 14.7, 16.6, 11.0, and 13.6%, respectively. Nonparametric bootstrap analysis confirmed the accuracy and the precision of the NONMEM parameter estimates. A population pharmacokinetic approach was used to integrate the knowledge gathered from several pharmacokinetic/toxicokinetic studies in rats. The pharmacokinetics of PEG-EPO in the rat was successfully modeled using a two-compartmental model with a linear elimination from the central compartment and a first-order absorption process with lag time. Weight and sex, but not pregnancy status, were identified as covariates of interest during preclinical development. The population pharmacokinetic model developed will be further used for the purpose of interspecies scaling and PK/PD modeling.