Anion exchange chromatography - Mass spectrometry for monitoring multiple quality attributes of erythropoietin biopharmaceuticals

Assessment of critical quality attributes of the biopharmaceutical erythropoietin (EPO) prior to product release requires the use of several analytical methods. We developed an MS-compatible anion exchange (AEX) method for monitoring multiple quality attributes of EPO biopharmaceuticals. AEX was performed using a stationary phase with quaternary ammonium functional groups and a pH gradient for elution. Baseline separation of charge variants and high-quality MS data were achieved using 30 mM ammonium formate pH 5.5 and 30 mM formic acid pH 2.5 as mobile phases. In a single experiment, assessment of critical quality attributes, such as charge heterogeneity, sialic acid content and number of N-ace-tyllactosamine units, was possible while providing additional information on other modifications such as O-acetylation and deamidation. In addition, good repeatability and robustness for the relative areas of the individual glycoforms and average number of Neu5Ac per EPO molecule were observed. The results were comparable to common pharmacopeia and standard methods with the advantage of requiring fewer analytical methods and less sample treatment saving time and costs. (C) 2020 The Authors. Published by Elsevier B.V.Proteomic

Functional selectivity of adenosine receptor ligands

Adenosine receptors are plasma membrane proteins that transduce an extracellular signal into the interior of the cell. Basically every mammalian cell expresses at least one of the four adenosine receptor subtypes. Recent insight in signal transduction cascades teaches us that the current classification of receptor ligands into agonists, antagonists, and inverse agonists relies very much on the experimental setup that was used. Upon activation of the receptors by the ubiquitous endogenous ligand adenosine they engage classical G protein-mediated pathways, resulting in production of second messengers and activation of kinases. Besides this well-described G protein-mediated signaling pathway, adenosine receptors activate scaffold proteins such as β-arrestins. Using innovative and sensitive experimental tools, it has been possible to detect ligands that preferentially stimulate the β-arrestin pathway over the G protein-mediated signal transduction route, or vice versa. This phenomenon is referred to as functional selectivity or biased signaling and implies that an antagonist for one pathway may be a full agonist for the other signaling route. Functional selectivity makes it necessary to redefine the functional properties of currently used adenosine receptor ligands and opens possibilities for new and more selective ligands. This review focuses on the current knowledge of functionally selective adenosine receptor ligands and on G protein-independent signaling of adenosine receptors through scaffold proteins

Mechanism-based pharmacokinetic-pharmacodynamic modeling of antilipolytic effects of adenosine A(1) receptor agonists in rats: prediction of tissue-dependent efficacy in

ABSTRACT We have developed a pharmacokinetic-pharmacodynamic strategy based on the operational model of agonism to obtain estimates of apparent affinity and efficacy of N 6 -cyclopentyladenosine (CPA) analogs for the adenosine A 1 receptor-mediated in vivo effect on heart rate in the rat. All analogs investigated produced a significant decrease of the heart rate after intravenous infusion. Individual concentration-effect curves were fitted to the operational model of agonism with the values of E max and n constrained to the intrinsic activity (273 bpm) and Hill slope (1.18), respectively, obtained with the agonist that displayed the highest intrinsic activity, 5Ј-deoxy-CPA. In all cases, the model converged and estimates of apparent affinity and efficacy were obtained for each agonist. Affinity estimates correlated well with pK i values for the adenosine A 1 receptor in rat brain homogenates. In addition, a highly significant correlation was found between the estimates of the in vivo efficacy parameter and the GTP shift (the ratio between K i in the presence and absence of GTP). In conclusion, the operational model of agonism can provide meaningful measures of agonist affinity and efficacy at adenosine A 1 receptors in vivo. The model should be of use in the development of partial adenosine A 1 receptor agonists. Adenosine is believed to exert its physiological effects through interactions with at least four receptor subtypes: the A 1 , A 2a , A 2b and A 3 receptors (see In the search for partial agonists, several series of adenosine derivatives have been synthesized leading to the identification of analogs of CPA for which the ratio between apparent affinity in the presence and absence of GTP for the adenosine A 1 receptor in radioligand binding studies is lower than for CP

High-performance liquid chromatography of the adenosine A1 agonist N6-cyclopentyladenosine and the A1 antagonist 8-cyclopentyltheophylline and its application in a pharmacokinetic study in rats

This report describes a rapid and sensitive analysis for the simultaneous detection of the adenosine A1 receptor ligands N6-cyclopentyladenosine (CPA) and 8-cyclopentyltheophylline (CPT) in rat blood. The method involved alkaline extraction of the compounds and internal standard N6-cyclohexyladenosine (CHA) with ethyl acetate, followed by isocratic reversed-phase high-performance liquid chromatography on a 3-microns MicroSphere C18 column with UV detection at 269 nm. The mobile phase consisted of a mixture of 10 mM acetate buffer (pH 4.0)-methanol-acetonitrile (56:40:4, v/v/v) with a flow-rate of 0.50 ml/min. The total run time was ca. 19 min. For CPA and CPT extraction yields were greater than 77 and 66% in the concentration range of 0.010-0.75 microgram/ml and 0.025-15 micrograms/ml, respectively, with intra- and inter-assay variations less than 9%. In 100 microliter blood samples the corresponding limits of detection were 3.3 and 6.2 ng/ml (signal-to-noise ratio = 3). CPA was found to be degraded in rat blood in vitro with a half-life of 24 min at 37 degrees C. The utility of the analytical method was established by analyzing blood samples from rats which had received an intravenous administration of 200 micrograms/kg CPA or 12 mg/kg CPT. Due to its rapidity and sensitivity this method is concluded to be particularly useful in pharmacokinetic studies with CPA and CP

Pharmacokinetic-pharmacodynamic relationship of the cardiovascular effects of adenosine A1 receptor agonist N6-cyclopentyladenosine in the rat

The purpose of the investigation was to develop a pharmacokinetic-pharmacodynamic model for the characterization of the cardiovascular effects of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) in individual normotensive rats. After the i.v. administration of 200 micrograms/kg (0.60 mumol/kg) of CPA, the time course of heart rate and arterial blood pressure was monitored in conjunction with serial blood sampling. Potential interference of the concentration-cardiovascular effect relationship by the development of acute tolerance or the formation of (inter)active metabolites was investigated by infusion of CPA with different rates and by determination of blood concentrations both by high-performance liquid chromatography and radioreceptor assay. In the individual rats the concentration-hemodynamic effect relationships were satisfactorily modeled according to the sigmoidal Emax pharmacodynamic model. For the negative chronotropic effect, the pharmacodynamic parameters proved to be independent of the infusion rate, indicating the absence of development of acute tolerance during the experiment. Potency (EC50) and intrinsic efficacy (Emax) were 2.7 +/- 0.5 ng/ml and -209 +/- 10 bpm, respectively (mean +/- S.E., n = 17). The concentrations of CPA as determined by the radioreceptor assay were identical to those determined by high-performance liquid chromatography, thereby excluding the formation of (inter)active metabolites. It is concluded that on the basis of this integrated pharmacokinetic-pharmacodynamic model, with the negative chronotropic effect as a pharmacodynamic endpoint, estimates of the potency and the intrinsic efficacy of adenosine A1 receptor agonists in vivo can be obtained after the administration of a single dos

Time course of action of three adenosine A1 receptor agonists with differing lipophilicity in rats: comparison of pharmacokinetic, haemodynamic and EEG effects

In this study we investigated the relationship between the pharmacokinetics and the cardiovascular and electroencephalogram (EEG) effects of three adenosine agonists with differing lipophilicity. Conscious normotensive rats received either 600 microg/kg N6-(p-sulphophenyl) adenosine (SPA), 200 microg/kg N6-cyclopentyladenosine (CPA) or 600 microg/kg 1-deaza-2-chloro-N6-cyclopentyladenosine (DCCA) in a 5-min intravenous infusion. Changes in haemodynamics and EEG were monitored in conjunction with arterial blood sampling to determine blood concentrations of the compounds. The three adenosine agonists showed large differences in pharmacokinetic properties, resulting in terminal half-lives of 66 +/- 10, 8.2 +/- 0.4 and 24 +/- 1 min (mean +/- SEM) for SPA, CPA, and DCCA respectively. SPA had a significantly lower blood clearance relative to CPA and DCCA, whereas DCCA had the largest volume of distribution and degree of plasma protein binding. The relationship between concentration and heart rate could be described adequately by the sigmoidal Emax model. For SPA, CPA, and DCCA the EC50 values based on free drug concentrations were 423 +/- 92, 1.8 +/- 0.4 and 9.5 +/- 1.1 nM respectively. These in vivo values correlated closely with the affinity of the compounds for the adenosine A1 receptor as determined in radioligand binding studies, with corresponding Ki values of 1410 +/- 220, 4.7 +/- 0.6 and 102 +/- 74 nM (mean +/- SEM) respectively. In the EEG, only CPA produced a small decrease in the amplitude of beta waves. This study demonstrates that the three adenosine analogues have large differences in pharmacokinetics, which complicates comparison of their cardiovascular and central responses simply on the basis of dose. The application of an integrated PK/PD approach permits estimates of potency and activity which are independent of underlying dose and pharmacokinetic