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Aims: Polyethylene glycol (PEG) is used in many applications including drug development. Due to exposure to environmental products, there is a high prevalence of preexisting anti-PEG antibodies in the global human population. The presence of anti-PEG antibodies is a concern for potentially reducing the efficacy of therapeutics after administration and represents a risk of safety events after exposure to PEGylated drug products. We developed and validated a creative and sensitive method for the detection of anti-PEG antibodies in human serum to support clinical programs for PEGylated drugs., Methods: In this method, biotin-PEG streptavidin beads were used to extract anti-PEG antibodies from human serum for analysis in an anti-PEG ELISA assay. The same serum sample was analyzed in an anti-drug antibody assay., Results: The anti-PEG antibody assay was validated with a screening cut point of 1.41 normalized signal, confirmatory cut point of 32.2% inhibition, sensitivity of 7.81 ng/mL and sufficient reproducibility, selectivity, and drug tolerance in accordance with the FDA 2019 Immunogenicity guidance., Conclusion: This method of removal of anti-PEG antibodies enables the use of a single sample to detect anti-drug and anti-PEG antibodies to support drug development programs.

Quality controls (QCs) are the primary indices of assay performance and an important tool in assay lifecycle management. Inclusion of QCs in the testing process allows for the detection of system errors and ongoing assessment of the reliability of the assay. Changes in the performance of QCs are indicative of changes in the assay behavior caused by unintended alterations to reagents or to the operating conditions. The focus of this publication is management of QC life cycle. A consensus view of the ligand binding assay (LBA) community on the best practices for factors that are critical to QC life cycle management including QC preparation, qualification, and trending is presented here.

The accuracy of reported sample results is contingent upon the quality of the assay calibration curve, and as such, calibration curves are critical components of ligand binding and other quantitative methods. Regulatory guidance and lead publications have defined many of the requirements for calibration curves which encompass design, acceptance criteria, and selection of a regression model. However, other important aspects such as preparation and editing guidelines have not been addressed by health authorities. The goal of this publication is to answer many of the commonly asked questions and to present a consensus and the shared views of members of the ligand binding assay (LBA) community on topics related to calibration curves with focus on providing recommendations for the preparation and editing of calibration curves.

This White Paper is focused on the technical aspects regarding quantifying pharmaceutically derived inorganic elements in biomatrices in support of GLP nonclinical and clinical studies using inductively coupled plasma (ICP) techniques. For decades ICP has been used in support of Environmental Protection Agency analyses and has more recently been applied for use in the pharmaceutical industry. Current bioanalytical method validation and sample analysis regulatory guidance applies to chromatographic platforms used for analysis of large- and small-molecule PK and TK assessments; however, it is not directly applicable to all aspects of various ICP techniques. Increasingly, quadrupole and high-resolution ICP-MS methods of analysis are being used to quantify inorganic elements contained in pharmaceutical compounds and biomatrices. Many elements occur endogenously in biomatrices, affecting quantification of blanks, standard curve samples, QC samples, and the selection of appropriate levels for the LLOQ.

Previously disclosed HIV (human immunodeficiency virus) attachment inhibitors, exemplified by BMS 806 (formally BMS378806, 1), are characterized by a substituted indole or azaindole ring linked to a benzoylpiperazine via a ketoamide or sulfonamide group. In the present report, we describe the discovery of a novel series of potent HIV entry inhibitors in which the indole or azaindole ring of previous inhibitors is replaced by a heterobiaryl group. Several of these analogues exhibited IC(50) values of less than 5 nM in a pseudotyped antiviral assay, and compound 13k was demonstrated to exhibit potency and selectivity similar to those of 1 against a panel of clinical viral isolates. Moreover, current structure-activity relationship studies of these novel biaryl gp120 inhibitors revealed that around the biaryl, a fine crevice might exist in the gp120 binding site. Taken in sum, these data reveal a hitherto unsuspected flexibility in the structure-activity relationships for these inhibitors and suggest new avenues for exploration and gp120 inhibitor design.

Background: Enfuvirtide is a potent inhibitor of systemic HIV-1 replication, but its penetration into the human central nervous system (CNS) has not been analysed. Here, we define cerebrospinal fluid (CSF) enfuvirtide pharmacokinetics and present a case illustrating the use of enfuvirtide as a probe to trace the origins of CSF HIV-1 quasispecies., Methods: Enfuvirtide CSF pharmacokinetics were assessed in 18 CSF and plasma sample pairs from four HIV-1-infected individuals. Enfuvirtide levels were measured by liquid chromatography tandem mass spectrometry using known standards and controls that included spiked CSF samples from untreated, HIV-negative individuals. A segment of the gp41 coding region encompassing the heptad repeat HR-1 and HR-2 domains was amplified from selected CSF and plasma samples and independent clones sequenced to assess resistance-associated mutations., Results: CSF and plasma samples obtained between 2 and 20 h after enfuvirtide injection showed plasma concentrations similar to previous reports (mean 3.687 SD +/- 1.828 mg/ml) with prolonged decay. By contrast, enfuvirtide in all CSF samples was below the assay detection limit of 0.025 mg/ml. In one individual, who developed a transient increase in CSF HIV-1 RNA, seven of seven CSF and plasma clones had identical enfuvirtide resistance-associated V38A mutations, suggesting that the CSF quasispecies derived from that of blood., Conclusions: Enfuvirtide penetration into CSF is negligible; thus, in clinical settings, where direct CNS drug exposure is crucial, this drug Is not likely to directly contribute to the local therapeutic effect. Enfuvirtide can be used as a tool to dissect the origin of the CNS virus.

The Third AAPS/FDA Bioanalytical Workshop, entitled "Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays" was held on May 1-3, 2006 in Arlington, VA. The format of this workshop consisted of presentations on bioanalytical topics, followed by discussion sessions where these topics could be debated, with the goal of reaching consensus, or identifying subjects where addition input or clarification was required. The discussion also addressed bioanalytical validation requirements of regulatory agencies, with the purpose of clarifying expectations for regulatory submissions. The proceedings from each day were reviewed and summarized in the evening sessions among the speakers and moderators of the day. The consensus summary was presented back to the workshop on the last day and was further debated. This communication represents the distillate of the workshop proceedings and provides the summary of consensus reached and also contains the validation topics where no consensus was reached.

Despite major advances in modern drug discovery and development, the number of new drug approvals has not kept pace with the increased cost of their development. Increasingly, innovative uses of biomarkers are employed in an attempt to speed new drugs to market. Still, widespread adoption of biomarkers is impeded by limited experience interpreting biomarker data and an unclear regulatory climate. Key differences preclude the direct application of existing validation paradigms for drug analysis to biomarker research. Following the AAPS 2003 Biomarker Workshop (J. W. Lee, R. S. Weiner, J. M. Sailstad, et al. Method validation and measurement of biomarkers in nonclinical and clinical samples in drug development. A conference report. Pharm Res 22:499-511, 2005), these and other critical issues were addressed. A practical, iterative, "fit-for-purpose" approach to biomarker method development and validation is proposed, keeping in mind the intended use of the data and the attendant regulatory requirements associated with that use. Sample analysis within this context of fit-for-purpose method development and validation are well suited for successful biomarker implementation, allowing increased use of biomarkers in drug development.

This study was performed to assess the feasibility of administering 1843U89, a potent, noncompetitive inhibitor of thymidylate synthase that does not require polyglutamation for activity, as a 2-min i.v. infusion daily for 5 days every 3 weeks, to determine whether folic acid supplementation ameliorates the toxic effects of 1843U89 and permits further dose escalation, and to recommend doses of 1843U89 administered without and with folic acid for further clinical evaluations. The study also sought to characterize the pharmacokinetic behavior of 1843U89 and to seek preliminary evidence of anticancer activity. Patients with advanced solid malignancies were treated with escalating doses of 1843U89 as a 2-min i.v. infusion daily for 5 days every 3 weeks. Initially, patients were treated in the absence of high-dose folic acid until dose-limiting toxicity was consistently noted. Next, patients were treated with escalating doses of 1843U89 preceded by 1000 mg of folic acid administered p.o. 30 min before each of the 5 daily doses of 1843U89. Patients (32) received 101 total courses of 1843U89 at doses ranging from 1 to 6 mg/m(2)/day with and without folic acid. At the 2 mg/m(2)/day dose level without folic acid, 2 of 7 new patients experienced dose-limiting toxicity, principally neutropenia, mucositis, and malaise in 3 of 11 courses. 1843U89 doses were further increased with folic acid to 6 mg/m(2)/day, but repetitive treatment was not feasible at this dose level because of an unacceptable high incidence of severe neutropenia and mucositis. Other toxicities included thrombocytopenia, rash, and fever. In contrast, repetitive treatment at the 5 mg/m(2)/day dose level was feasible. The pharmacokinetics of 1843U89 were neither dose dependent nor affected by folic acid. On day 1, clearance, terminal half-life, and steady-state volume of distribution values averaged 47.1 +/- 21.7 ml/min/m(2), 7.72 +/- 4.09 h, and 16.7 +/- 8.8 liter/m(2)/h, respectively. The results of the study indicate that the administration of 1843U89 as a 2-min infusion daily for 5 days every 3 weeks without and with folic acid is feasible at 1843U89 doses as high as 2 and 5 mg/m(2)/day, respectively. Because folic acid pretreatment results in no diminution of the antitumor activity of 1843U89 in preclinical studies and ameliorates the toxic effects of 1843U89 in both preclinical models and cancer patients, the therapeutic index of 1843U89 may be enhanced by folic acid pretreatment and, therefore, the development of 1843U89 with folic acid is warranted. However, the question of whether to administer 1843U89 at a dose of 2 mg/m(2)/day with folic acid, which is associated with negligible toxicity, or at its highest feasible dose with folic acid, 5 mg/m(2)/day, should be addressed in appropriately designed trials.

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 (

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.

Objective: To characterize the disposition of total and free serum digoxin following the administration of digoxin Fab antibody in patients with varying degrees of renal function., Design: Observational study of pharmacokinetics and pharmacodynamics., Setting: Critical care and telemetry units of two university-affiliated teaching institutions, Hartford Hospital and Henry Ford Hospital., Patients: Fourteen digoxin-intoxicated patients (baseline total digoxin > 3.2 nmol/mL) with mean (+/- SD) serum creatinine of 380.1 +/- 212.2 mumol/L who received digoxin Fab antibody therapy., Measurements: Serum was drawn every 12 to 24 hours for 80 to 327 hours after Fab administration. Total and free digoxin were assayed in serum by fluorescence polarization immunoassay or modified immunofluorometric assay., Results: Before Fab was administered, total digoxin ranged from 3.5 to 10.5 nmol/mL. After treatment with Fab, total digoxin increased rapidly to a mean (+/- SD) maximum of 51.8 +/- 22.7 nmol/mL and decreased to 7.2 +/- 4.7 nmol/mL at the last measurement. Total digoxin was eliminated in a two-phase fashion. The half-life of the initial phase of total digoxin decline was 11.6 +/- 4.1 hours, and the half-life of the second or terminal elimination phase was 118 +/- 57 hours. Free digoxin levels decreased rapidly following Fab therapy, to a mean nadir of 0.6 +/- 1.1 nmol/mL, but rebounded to a mean maximum free digoxin concentration of 1.7 +/- 1.3 nmol/mL in 77 +/- 46 hours. The time to maximum free digoxin rebound occurred later in patients with end-stage renal disease (n = 4) compared with other patients (127 +/- 40 hours compared with 55 +/- 28 hours)., Conclusion: Elimination of digoxin following Fab therapy is prolonged in digoxin-toxic patients with renal dysfunction. In addition, rebound of free digoxin is delayed in anephric patients. Monitoring free digoxin following the administration of Fab may be of value in selected patients to guide additional Fab dosing, confirm possible rebound toxicity, or guide the reinitiation of digoxin therapy.

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)

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.

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.

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.

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.