A white paper--consensus and recommendations of a global harmonization team on assessing the impact of immunogenicity on pharmacokinetic measurements.

The Global Bioanalysis Consortium (GBC) set up an international team to explore the impact of immunogenicity on pharmacokinetic (PK) assessments. The intent of this paper is to define the field and propose best practices when developing PK assays for biotherapeutics. We focus on the impact of anti-drug antibodies (ADA) on the performance of PK assay leading to the impact on the reported drug concentration and exposure. The manuscript describes strategies to assess whether the observed change in the drug concentration is due to the ADA impact on drug clearance rates or is a consequence of ADA interference in the bioanalytical method applied to measure drug concentration. This paper provides the bioanalytical scientist guidance for developing ADA-tolerant PK methods. It is essential that the data generated in the PK, ADA, pharmacodynamic and efficacy/toxicity evaluations are viewed together. Therefore, the extent for the investigation of the PK sensitivity to the presence of ADA should be driven by the project needs and risk based.

Pharmacokinetics and hematological effects of the PEGylated thrombopoietin peptide mimetic GW395058 in rats and monkeys after intravenous or subcutaneous administration.

GW395058, a potent PEGylated peptide human thrombopoietin receptor (HuTPOr) agonist in vitro, is being evaluated for the treatment of thrombocytopenia. GW395058 shares no sequence homology with TPO. In this report the pharmacokinetics and hematological effects of GW395058 in rats and monkeys are described. Doses eliciting thrombocytosis in rodents (2 or 10 microg/kg s.c.) produced insufficient plasma concentration data for pharmacokinetic parameter estimate calculations. At higher i.v. doses in rats (500, 1,000 or 2,000 microg/kg) serum t1/2 (half-life) values were >20 h, and the area under the concentration time curve increased proportionally with dose. In cynomolgus monkeys GW395058 plasma t1/2 values ranged from 37 to 68 h after s.c. or i.v. dosing, and similar values were observed in rhesus monkeys following s.c. dosing. Rat platelet counts increased following 2 (1.6-fold) or 10 microg/kg (fourfold) s.c. doses. Cynomolgus and rhesus monkey platelet counts did not change significantly at comparable s.c. doses, but did increase slightly (

Disposition of digoxin immune Fab in patients with kidney failure.

Digoxin and digoxin immune Fab, its antidote, are eliminated renally. However, the disposition of Fab in severe kidney disease is poorly described. Therefore, the disposition of Fab and its relationship to total and free digoxin were studied in five digoxin-toxic patients with end-stage renal disease (n = 4) or severe renal dysfunction (n = 1) with a mean (+/- SD) serum creatinine of 5.9 +/- 1.2 mg/dl (four patients were receiving long-term hemodialysis). Serum was drawn after a clinically neutralizing Fab dose (80 to 160 mg) every 12 to 24 hours for 204 to 327 hours. Fab concentrations were assessed by radioimmunoassay, whereas total digoxin concentrations were assessed with a modified radioimmunoassay or fluorescence polarization immunoassay. The concentration-time profile of Fab appeared to be similar to the concentration-time profile of total digoxin. The mean (+/- SD) half-lives of the alpha and beta disposition phases of Fab were 13 +/- 5 hours and 96 +/- 31 hours, respectively, which were similar to the alpha and beta parameter estimates of total digoxin (14 +/- 4 and 123 +/- 16 hours, respectively). Steady-state volume of distribution and systemic clearance of Fab were 0.29 +/- 0.11 L/kg and 0.057 +/- 0.022 ml/min/kg, respectively. Thus, in comparison to values reported in patients with normal renal function, the elimination of Fab and total digoxin are markedly delayed in patients with end-stage renal disease, which may necessitate prolonged clinical monitoring.

The pharmacokinetics and pharmacodynamics of the prostacyclin analog 15AU81 in the anesthetized beagle dog.

15AU81, a tricyclic benzindene analog of prostacyclin, is currently under preclinical evaluation as a potential treatment for congestive heart failure. The cardiovascular effects of 15AU81 were evaluated in anesthetized beagle dogs given 4-h infusions at rates of either 0.1, 0.3, 1.0, or 3.0 micrograms/kg/min. Plasma samples taken from these dogs prior to, during, and after the infusion, were analyzed for 15AU81 by a radioimmunoassay (RIA). This report integrates the vasodilatory effects and plasma concentration data from the 15AU81 infusion study. Pharmacokinetic analysis of mean data indicated a biphasic decay of 15AU81 in plasma, with an initial half-life of approximately 2 min, and a terminal half-life of approximately 20 min. Visual inspection of plots of drug effect and drug concentration against time indicated a close relationship between plasma concentration of 15AU81 and the onset of decreases in total peripheral resistance (TPR) and pulmonary vascular resistance (PVR). In general, the decreases in TPR and PVR induced by 15AU81 were maintained during infusion. Concentration-effect plots indicated some hysteresis in TPR vs plasma concentrations of 15AU81 after termination of the infusion; possible explanations for this hysteresis include the presence of saturating concentrations of 15AU81 at the effect site, with a delay in the clearance of 15AU81 from the effect site compared to its clearance from plasma, and/or the presence of active metabolites at the effect site. A fit of the TPR and PVR data to the Emax pharmacodynamic model predicted that the maximum decrease in TPR achievable with 15AU81 in anesthetized dogs was 66%, and that the concentration of 15AU81 producing 50% of the maximum effect (EC50) was 8.6 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioimmunoassay for the chemical stable prostacyclin analog, 15AU81: a preliminary pharmacokinetics study in the dog.

15AU81 is a chemically stable tricyclic benzindene analog of prostacyclin, with possible application to the treatment of congestive heart failure. Effective pharmacological doses in a dog model are in the microgram to sub-microgram/kg range, necessitating an analytical method of high sensitivity for determination of the drug in plasma. This report describes the development and validation of a radioimmunoassay for 15AU81, and its application to a pharmacokinetic study in the beagle dog. An antiserum elicited by immunization with a 15AU81-bovine thyroglobulin conjugate was employed, along with 3H-15AU81, in the radioimmunoassay. Analogs of 15AU81, as well as a glucuronide metabolite produced by a dog liver subcellular fraction in vitro, were used to demonstrate the specificity of the radioimmunoassay. Specificity was confirmed by comparative analysis by radioimmunoassay and by a quantitative GC/MS procedure of plasma samples from dogs dosed with 15AU81. The limit of quantitation in dog plasma was 1.6 ng/ml; accuracy and precision were both acceptable. The assay was applied to a study of the pharmacokinetics of 15AU81 in the beagle dog after intravenous or intratracheal administration of a single 20-micrograms/kg dose. Following intravenous dosing, 15AU81 was eliminated rapidly from plasma (t1/2, 2.8 min), while after intratracheal administration, clearance appeared to be somewhat slower and bioavailability was appreciable (mean, 46%), suggesting that this route of administration may be worthy of further evaluation.

Clinical and pharmacokinetic profiles of digoxin immune Fab in four patients with renal impairment.

Minimal pharmacokinetic data on digoxin immune Fab are currently available, especially in patients with impaired renal function. The serum concentration-time profiles of total digoxin, free digoxin, and digoxin immune Fab in four patients with moderate to severe renal impairment who received digoxin immune Fab are presented. The calculated elimination half-life of digoxin immune Fab was 25-73 hours. The calculated elimination half-life of total digoxin was 24-72 hours. Free digoxin concentrations rebounded to a peak of 1-2.9 ng/mL 44-97 hours after the administration of digoxin immune Fab. The areas under the curve for digoxin immune Fab were 213-1026 micrograms.h/mL, and total body clearances were 2.3-7.1 mL/min. The total digoxin concentrations peaked at 14-33 times the pre-Fab digoxin concentrations 5-30 hours after digoxin immune Fab administration. In comparing these data with data available from patients with normal renal function, the half-life of digoxin immune Fab and total digoxin was longer, the peak total digoxin concentration occurred later, the ratio of the peak total digoxin concentration to pre-Fab digoxin concentration was larger, and the rebound in free digoxin occurred later in patients with renal impairment. The Fab dose should not be reduced in patients with renal impairment; however, post-Fab monitoring should be extended to compensate for the prolonged half-life of Fab and later rebound of free digoxin.

Immunofluorometric assay for lamotrigine (Lamictal) in human plasma.

An immunofluorometric assay (IFA) has been developed for the potential antiepileptic agent, lamotrigine (Lamictal). The assay involves competition between lamotrigine free in solution and bound to a bovine thyroglobulin conjugate on the surface of microtiter strip wells for a limited amount of polyclonal lamotrigine antisera. The end-point of this reaction, which indicates the concentration of lamotrigine present in the solution under analysis, is detected by adding Eu(3+)-labelled anti-rabbit IgG, followed by an enhancement solution to produce a fluorescent product. Thus, the higher the concentration of lamotrigine in the sample, the less intense the fluorescence produced. The assay displays minor cross-reactivity (0.05%) by the major glucuronide metabolite (in humans) and moderate cross-reactivity (2.7%) by a minor N-oxide metabolite (in rats) of the parent drug. No interference from these sources in the analysis of plasma samples from clinical trials was demonstrated by comparative sample analysis by IFA and high-performance liquid chromatography. Intraassay accuracy and precision were excellent, greater than 90% and less than 5% coefficient of variation (CV), while interassay accuracy was greater than 95% and interassay precision (CV) was 8.8-17.0%. This assay is suitable for analysis of lamotrigine in plasma samples collected during clinical trials.

Treatment of digoxin intoxication in a renal failure patient with digoxin-specific antibody fragments and plasmapheresis.

A patient with renal failure due to myeloma kidney and coincident digitalis intoxication due to prescribed daily digoxin administration was treated with digoxin-specific antibody fragments and plasmapheresis. Rapid response to therapy was noted, removal of digoxin-antidigoxin antibody complexes was confirmed, and prevention of delayed rebound toxicity was documented. We suggest that this is the therapy of choice in similar individuals.

Pseudoephedrine and triprolidine in plasma and breast milk of nursing mothers.

Plasma and milk concentrations of pseudoephedrine and triprolidine were determined (by radioimmunoassay) in three lactating mothers over 12-48 h after ingestion of a combination medication containing 60 mg of pseudoephedrine hydrochloride and 2.5 mg of triprolidine hydrochloride monohydrate. Pseudoephedrine concentrations in milk were consistently higher than those in plasma. The total amount of drug in milk, as judged by areas under the respective curves (AUC), was two to three times greater than in plasma. Triprolidine concentrations in milk and plasma were more variable between subjects than those of pseudoephedrine. AUC values for milk and plasma were similar for one subject, while the plasma value exceeded that for milk in another woman. The fraction of the dose excreted in milk was estimated to be 0.4-0.7% for pseudoephedrine and 0.06-0.2% for triprolidine.