Development of C1q Affinity Chromatography for the Study of C1q-IgG Interactions.

The classical complement system represents a central effector mechanism of Abs initiated by the binding of C1q to target bound IgG. Human C1q contains six heterotrimeric globular head groups that mediate IgG interaction, resulting in an avidity-driven binding event involving multiple IgG molecules binding a single C1q. Accordingly, surface bound IgG molecules are thought to assemble into noncovalent hexameric rings for optimal binding to the six-headed C1q. To study the C1q-Fc interaction of various Abs and screen for altered C1q binding mutants, we developed, to our knowledge, a novel HPLC-based method. Employing a single-chain form of C1q representing one C1q head group, our HPLC methodology was able to detect the interaction between the single-chain monomeric form of C1q and various ligands. We show that, despite a narrow window of specific binding owing to the low affinity of the monomeric C1q-IgG interaction, this approach clearly distinguished between IgG subclasses with established C1q binding properties. IgG3 displayed the strongest binding, followed by IgG1, with IgG2 and IgG4 showing the weakest binding. Fc mutants known to have increased C1q binding through oligomerization or enhanced C1q interaction showed greatly increased column retention, and IgG glycovariants displayed a consistent trend of increasing retention upon increasing galactosylation and sialylation. Furthermore, the column retention of IgG isotypes and glycovariants matches both the cell surface recruitment of C1q and complement-mediated cytotoxicity induced by each variant on an anti-CD20 Ab backbone. This methodology therefore provides a valuable tool for testing IgG Ab (glyco)variants for C1q binding, with clear relevance for therapeutic Ab development.

Mechanistic incorporation of FcRn binding in plasma and endosomes in a whole body PBPK model for large molecules.

Monoclonal antibodies, endogenous IgG, and serum albumin bind to FcRn in the endosome for salvaging and recycling after pinocytotic uptake, which prolongs their half-life. This mechanism has been broadly recognized and is incorporated in currently available PBPK models. Newer types of large molecules have been designed and developed, which also bind to FcRn in the plasma space for various mechanistic reasons. To incorporate FcRn binding affinity in PBPK models, binding in the plasma space and subsequent internalisation into the endosome needs to be explicitly represented. This study investigates the large molecules model in PK-Sim® and its applicability to molecules with FcRn binding affinity in plasma. With this purpose, simulations of biologicals with and without plasma binding to FcRn were performed with the large molecule model in PK-Sim®. Subsequently, this model was extended to ensure a more mechanistic description of the internalisation of FcRn and the FcRn-drug complexes. Finally, the newly developed model was used in simulations to explore the sensitivity for FcRn binding in the plasma space, and it was fitted to an in vivo dataset of wild-type IgG and FcRn inhibitor plasma concentrations in Tg32 mice. The extended model demonstrated a strongly increased sensitivity of the terminal half-life towards the plasma FcRn binding affinity and could successfully fit the in vivo dataset in Tg32 mice with meaningful parameter estimates.

Multi-Attribute Monitoring of Complex Erythropoetin Beta Glycosylation by GluC Liquid Chromatography-Mass Spectrometry Peptide Mapping.

Recombinant human erythropoetin (EPO) is an important biopharmaceutical mainly used for the treatment of anemia. It is highly heterogeneous because of common amino acid chemical degradations known to occur in protein therapeutics (e.g., oxidation and deamidation) and its complex glycosylation profile. Recently, multi-attribute monitoring (MAM), i.e., the quantification of multiple post-translational and chemical modifications in a single peptide mapping liquid chromatography-mass spectrometry (LC-MS)-based method, has received increased attention for the analysis of antibody-like biotherapeutic proteins. In this study, an MAM method for examination of residue-specific glycan profiles of EPO was established. The MAM method, by virtue of the increased sensitivity and selectivity provided with LC-MS, yielded additional site-specific information not afforded by the conventional quality control (QC) methods. Low abundant glycans as well as additional post-translational and chemical modifications could also be simultaneously detected by the MAM method. Our results demonstrate that desialylated N-oligosaccharides (DeNO) and N-acetylneuraminic acids (Neu5Ac) could be monitored by the developed MAM approach with data readout highly comparable to QC methods, while differences were observed for charge isoform distribution. In summary, the comparative data obtained demonstrate that MAM by LC-MS peptide mapping can, in principle, adequately replace selected QC methods and would add value to the in-process control and release testing strategy of EPO.

Pharmacokinetics, Safety, and Tolerability of Brivaracetam in Healthy Elderly Participants.

The pharmacokinetics, metabolism, safety, and tolerability of the antiseizure medication brivaracetam (BRV) were characterized in 16 healthy elderly participants (8 men/8 women) aged 65-78 years who received a single 200-mg oral dose of BRV on day 1, followed by 200 mg twice daily from day 3 until day 12. BRV and three metabolites were determined in plasma and urine. Adverse events, vital signs, electrocardiograms, laboratory tests, general and neurological examinations, and psychometric rating scales were recorded at regular intervals. No clinically relevant changes or abnormalities were detected. The adverse events were similar to those observed in pivotal trials. Rating scales indicated transiently increased sedation and decreased alertness. BRV pharmacokinetics and metabolism were unchanged relative to younger populations. Based on our observations in this healthy elderly population receiving oral BRV 200 mg twice daily (twice the maximum recommended dose), dose reductions are not warranted relative to other, younger populations. Further investigations may be necessary in frail elderly populations aged >80 years.

Not just what you did, but how: Children see distributors that count as more fair than distributors who don't.

To distribute resources in a fair way, identifying an appropriate outcome is not enough: We must also find a way to produce it. To solve this problem, young children spontaneously use number words and counting in fairness tasks. We hypothesized that children are also sensitive to other people's use of counting, as it reveals that the distributor was motivated to produce the outcome they believed was fair. Across four experiments, we show that U.S. children (N = 184 from the New Haven area; ages four to six; Approximately 58% White, 16% Black, 18% Hispanic, 4% Asian, and 4% other) believe that agents who count when distributing resources are more fair than agents who produce the same outcome without counting, even when both agents invest the same amount of effort. And vice versa, when the same two agents produce an unfair outcome, children now condemn the agent who counted. Our findings suggest that, from childhood, people understand that counting reflects a motivation to be precise and use this to evaluate other people's behavior in fairness contexts.

Caplacizumab Model-Based Dosing Recommendations in Pediatric Patients With Acquired Thrombotic Thrombocytopenic Purpura.

Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare and life-threatening autoimmune thrombotic microangiopathy. Caplacizumab, evaluated in phase II and III studies in adults, shortens the time to platelet count response and reduces aTTP exacerbations, has a favorable safety profile, and can potentially reduce refractoriness and mortality associated with aTTP. Since no children with aTTP were enrolled in these clinical trials, caplacizumab has been initially approved for use only in adult patients with aTTP (10 mg). Pediatric dosing recommendations were developed using model-based simulations. A semimechanistic pharmacokinetic/pharmacodynamic population model has been developed describing the interaction between caplacizumab and von Willebrand factor antigen (vWF:Ag) following intravenous and subcutaneous administration of caplacizumab in different adult populations, at various dose levels, using nonlinear mixed-effects modeling. Based on the allometrically scaled pharmacokinetic/pharmacodynamic model, different dosing regimens were simulated in 8000 children (aged 2-18 years). Simulated caplacizumab exposures and vWF:Ag levels across different age categories were compared to an adult reference group. A simulated daily dose of 5 mg in children weighing <40 kg and of 10 mg in children weighing ≥40 kg resulted in similar exposures and vWF:Ag suppression across age and weight groups. Despite the lack of pediatric clinical data, the results of this modeling and simulation analysis constituted the basis for the European extension of indication for caplacizumab (10 mg) to adolescents aged >12 years and with a body weight ≥40 kg. This represents a rare case in which regulatory authorities have deemed a modeling and simulation study robust enough to approve a variation of indication.

Giant left atrial myxoma - literature review and case presentation.

Primary cardiac tumors are an extremely rare pathology, representing only 5-10% of cardiac neoplasms, but among them, the most common are cardiac myxomas, that appear to originate from multipotent mesenchymal cells of the subendocardial and endocardial stroma. The incidence of cardiac myxomas is higher in females and they are usually diagnosed between the fourth and sixth decade of life. Most often, they are located in the left atrium, having the site of attachment at the level of the interatrial septum, especially at the level of the fossa ovalis and the adjacent limbus. Due to the increased risk of systemic embolization and intracardiac obstruction, cardiac myxomas have a definite indication for emergency surgical treatment. Cardiac myxomas are a very rare cause of transient ischemic attacks and stroke. We present the case of a 38-year-old patient who experienced four recurrent transient ischemic attacks and strokes. At the fourth cerebrovascular event, echocardiography was performed and it revealed a giant tumor located in the left atrium that was surgically removed. Pathological examination confirmed the diagnosis of cardiac myxoma. The postoperative evolution was favorable, both from a neurological and cardiac point of view. Although cardiac myxomas represent a rare cause of transient ischemic attacks and stroke, they must be considered as part of the assessment protocol for cerebrovascular events.

Physiologically based pharmacokinetics (PBPK).

Allometric scaling is widely used to predict human pharmacokinetic parameters from preclinical species, and many different approaches have been proposed over the years to improve its predictive performance. Nevertheless, prediction errors are commonly observed in the practical application of simple allometry, for example, in cases where the hepatic metabolic clearance is mainly determined by enzyme activities, which do not scale allometrically across species. Therefore, if good correlation was noted for some drugs, poor correlation was observed for others, highlighting the need for other conceptual approaches. Physiologically based pharmacokinetic (PBPK) models are now a well-established approach to conduct extrapolations across species and to generate simulations of pharmacokinetic profiles under various physiological conditions. While conventional pharmacokinetic models are defined by drug-related data themselves, PBPK models have richer information content and integrate information from various sources, including drug-dependent, physiological, and biological parameters as they vary in between species, subjects, or with age and disease state. Therefore, the biological and mechanistic bases of PBPK models allow the extrapolation of the kinetic behavior of drugs with regard to dose, route, and species. In addition, by providing a link between tissue concentrations and toxicological or pharmacological effects, PBPK modeling represents a framework for mechanistic pharmacokinetic-pharmacodynamic models.

Population pharmacokinetic and pharmacodynamic analysis in allergic diseases.

In this chapter, we introduce the concepts and methodologies of population analysis as applied to analyzing pharmacokinetic and pharmacodynamic data. One of the key determining characteristics of the population approach is that through it, one seeks not only to characterize deterministic trends in the data, but also to identify and estimate the magnitudes of the important sources of variability within the data. The first section of this chapter provides an introduction to the primary concepts of, and motivation for, population modeling by way of a hypothetical case study. Then, the various methodologies that have been employed throughout the history of population analysis are described in further detail. Of these, the most commonly employed today is nonlinear mixed-effects (NLME) modeling. Finally, notable examples of the application of population PK and PK/PD modeling to treatments for allergies and asthma are discussed. Population PK models have frequently been used to extrapolate exposures to special populations, such as pediatrics, as well as to optimize treatment regimens and trial designs for these populations. Population PK/PD models have most frequently been applied to analyzing and interpreting data from wheal and flare trials, but are also becoming increasingly important in the analysis of PD data from monoclonal antibodies.

From pharmacokinetics to therapeutics.

Whilst pharmacokinetics describe the relationship between dose levels and concentration-time profiles of a drug in the body and pharmacodynamics describe the concentration-response relationships, pharmacokinectics-pharmacodynamics(PK-PD) models link these two items providing a framework for modelling the time course of drug response. In this chapter, PK-PD models, describing the therapeutic effects of drugs used for the therapy of allergic diseases have been reviewed. Emphasis was given also to the description of the receptor occupancy, which is tightly related to the downstream clinical response. PK - PD models describing unwanted effects were also commented. An integrated use of these models allows choosing appropriate dosing regimens and providing an objective evaluation of the benefit/risk balance.

Malignant melanoma - the most severe skin cancer and neurological pathology.

OBJECTIVE: We report our clinical experience with malignant melanoma (MM) patients associated with neurological involvement. PATIENTS, MATERIALS AND METHODS: A database of patients admitted from 2014-2019 in the Ist Clinic of Neurology, Emergency County Hospital of Târgu Mures, Romania, was reviewed to identify patients with MM and neurological involvement. We assessed the demographic and clinical data regarding the neurological disorders and the primary tumor characteristics from the patient registries. Both histopathological and immunohistochemical analysis of the neoplasm was available for the entire cohort. RESULTS: We analyzed 13 982 patient files and 21 met all the inclusion and exclusion criteria. Brain metastases were found in 38.09% of the patients, spinal metastases in 9.52% of the patients, ischemic stroke by cancer-associated thrombosis in 42.85% of the patients and peripheral nervous system involvement in 19.04% of the patients. No statistically significant differences between the four categories of neurological disorders according to socio-demographic parameters, location of the primary tumor, existence of primary tumor ulceration, invasion of the lymph nodes or the presence and location of distant metastasis was found (p>0.05). Our presented patient is the first case of uveal melanoma with hemorrhagic brain metastasis before hepatic involvement. CONCLUSIONS: Neurological involvement in MM encompasses a myriad of variants and while the clinical setting varies from one patient to another, an underlining neoplasia should be evaluated in suspected patients.

Clinical pharmacology of caplacizumab for the treatment of patients with acquired thrombotic thrombocytopenic purpura.

Introduction: Caplacizumab is a humanized anti-von Willebrand Factor (vWF) Nanobody® for the treatment of acquired Thrombotic Thrombocytopenic Purpura (aTTP). Caplacizumab targets the A1-domain of vWF, inhibiting the interaction between vWF and platelets. Clinical studies conducted in aTTP patients confirmed the rapid and sustained complete suppression of the vWF activity using an initial intravenous dose of 10 mg, and a maintenance subcutaneous 10 mg daily dosing regimen, with corresponding favorable efficacy and safety profiles. Areas covered: The pharmacokinetics of caplacizumab are non-linear, characterized by a target-mediated disposition and the exposure is dependent upon drug and target concentration over time. The pharmacokinetics of caplacizumab are predictable when considering the turn-over of the circulating vWF and its modulation by the drug over time. Renal and hepatic impairment are not expected to influence the exposure to the drug, and no direct or indirect drug-drug pharmacokinetic interactions are anticipated based on the mechanism of action and the specificity of the pharmacodynamic effect of caplacizumab. Expert opinion: Caplacizumab prevents the interaction between vWF and platelets, offering a direct and rapid therapeutic intervention to stop microthrombosis. The combination of caplacizumab with plasma exchange and immunosuppression represents an important, potentially life-saving advance in the treatment of aTTP patients.

Retrospective population pharmacokinetic analysis of levetiracetam in children and adolescents with epilepsy: dosing recommendations.

OBJECTIVE: To characterize levetiracetam pharmacokinetics, identify significant covariate relationships and identify doses in children that achieve blood concentrations similar to those observed in adults. METHODS: Nonlinear mixed-effects modelling was used to analyse pooled data collected from 228 children with epilepsy aged 3 months to 18 years in five trials of adjunctive levetiracetam therapy. Simulations were used to identify dosing regimens achieving levetiracetam steady-state peak and trough plasma concentrations similar to those attained in adults receiving the recommended starting dose for adjunctive therapy (500 mg twice daily). The covariates considered for inclusion in the base model were age, bodyweight, gender, race, body surface area (BSA), body mass index (BMI), creatinine clearance (CL(CR)), levetiracetam dose, concomitant antiepileptic drug (AED) by category (neutral, enzyme inducer, inhibitor, combination of inducer and inhibitor), and benzodiazepines. RESULTS: A one-compartment model with first-order absorption and elimination best characterized the data. The following significant covariates were identified: (i) age on the absorption rate constant (k(a)); (ii) bodyweight, dose, CL(CR) and concomitant enzyme-inducing AED on plasma oral clearance (CL/F); and (iii) bodyweight on the apparent volume of distribution after oral administration (V(d)/F). The main explanatory covariates were age on k(a), bodyweight on CL/F and V(d)/F, and enzyme-inducing AED on CL/F, of which bodyweight was the most influential covariate. Dosing can be carried out with either 10 mg/kg of oral solution twice daily in children weighing <50 kg and a 500-mg tablet twice daily in those weighing >50 kg or, when patients favour a solid formulation, 10 mg/kg of oral solution twice daily in children weighing <20 kg, a 250-mg tablet twice daily in those weighing 20-40 kg, and a 500-mg tablet twice daily in those weighing >40 kg. All of these doses achieved steady-state peak and trough plasma concentrations similar to those observed in adults following the recommended starting dose for adjunctive therapy (500 mg twice daily). CONCLUSIONS: The most influential covariate of levetiracetam pharmacokinetics in children is bodyweight. A starting dose of levetiracetam 10 mg/kg twice daily ensures the same exposure in children as does 500 mg twice daily in adults.

Pharmacokinetics and metabolism of 14C-brivaracetam, a novel SV2A ligand, in healthy subjects.

This study was designed to investigate the human absorption, disposition, and mass balance of (14)C-brivaracetam, a novel high affinity SV2A ligand with potent anticonvulsant activity. Six healthy male subjects received a single p.o. dose of (14)C-brivaracetam (150 mg, 82 microCi, or 3.03 MBq). Serial blood and complete urine and feces were collected until 144 h postdose. Expired air samples were obtained until 24 h. Brivaracetam was rapidly absorbed, with C(max) of 4 mug/ml occurring within 1.5 h of dosing. Unchanged brivaracetam amounted to 90% of the total plasma radioactivity, suggesting a modest first-pass effect. Plasma protein binding of radioactivity was low (17.5%). Urinary excretion exceeded 90% after 2 days, and the final mass balance reached 96.8% of the radioactivity in urine and 0.7% in feces. Only 8.6% of the radioactive dose was recovered in urine as unchanged brivaracetam, the remainder being identified as non-cytochrome P450 (P450)- and P450-dependent biotransformation products resulting from hydrolysis of the amide moiety (M9, 34.2%), hydroxylation of the n-propyl side chain (M1b, 15.9%), and a combination of these two pathways leading to the hydroxy acid (M4b, 15.2%). Minor amounts of taurine and glucuronic acid conjugates and other oxidized derivatives were also identified. Brivaracetam is completely absorbed, is weakly bound to plasma proteins, extensively biotransformed through several metabolic pathways, and eliminated renally.

The pharmacokinetics, CNS pharmacodynamics and adverse event profile of brivaracetam after multiple increasing oral doses in healthy men.

AIMS: Brivaracetam is a novel synaptic vesicle protein 2A ligand that has shown potent activity in animal models of epilepsy. This study examined the pharmacokinetics, central nervous system pharmacodynamics and adverse event profile of multiple oral doses of brivaracetam in healthy male subjects. METHODS: Three successive panels of 12 healthy male subjects received double-blind brivaracetam 200, 400 or 800 mg day(-1) (all doses well above the expected therapeutic range) or placebo (9 : 3), in two divided doses, for 14 days. RESULTS: Brivaracetam was rapidly absorbed (t(max) approximately 2 h) and eliminated (t(1/2) 7-8 h). Volume of distribution was slightly lower than total body water. A small fraction of the dose (5-8%) was excreted unchanged in urine together with significant levels of metabolites, suggesting predominantly metabolic clearance. Based on 6-beta-hydroxycortisol/cortisol ratios in urine, there was no evidence of induction of CYP3A4 activity. Saliva and plasma brivaracetam levels were highly correlated. Adverse events were mostly mild to moderate, central nervous system-related and resolved within the first day of treatment. No clinically relevant changes were observed in laboratory tests, vital signs, physical examinations or ECGs. Pharmacodynamic tests showed dose-related sedation and decreased alertness that only persisted at 800 mg daily. CONCLUSIONS: Brivaracetam was well tolerated by healthy male volunteers at doses of 200-800 mg daily for 2 weeks, well above the expected clinically effective dose range. Brivaracetam had a favourable pharmacokinetic profile in this population, characterized by rapid absorption, volume of distribution limited to total body water, apparent single-compartment elimination and dose proportionality.

Population pharmacokinetics of levetiracetam in Japanese and Western adults.

OBJECTIVE: To assess the population pharmacokinetics of levetiracetam, a second-generation antiepileptic drug, in adult Japanese and Western populations. METHODS: Data were pooled from ten matched clinical trials conducted in Japan and in Europe and the USA, in which levetiracetam was administered orally to healthy subjects and subjects with epilepsy. Overall, 5408 plasma concentrations were available from 524 subjects in six clinical pharmacology studies and two confirmatory and two long-term safety studies of add-on treatment for partial epilepsy. A one-compartment open model with first-order absorption and elimination was fitted to the plasma concentrations using nonlinear mixed-effects modelling with first-order estimation. RESULTS: Ethnicity had no statistically significant effect on the pharmacokinetics of levetiracetam in the presence of the other covariates. Bodyweight, sex, creatinine clearance and concomitant intake of enzyme inducers or valproic acid had a statistically significant effect on apparent plasma clearance of levetiracetam. Bodyweight, disease and valproic acid had a statistically significant effect on the volume of distribution. Levetiracetam exposure (the area under the plasma concentration-time curve over the 12-hour dosing interval at steady state) was 12% higher in females than in males. Decreasing bodyweight from 70 kg to 40 kg was predicted to increase exposure by 16%, while halving creatinine clearance was predicted to increase exposure by 10%. Enzyme inducers reduced exposure by 8%, while valproic acid resulted in a 23% increase in exposure. The latter effect was assumed to arise from the known association between valproic acid and increased body fat, since levetiracetam is negligibly metabolised by cytochrome P450 enzymes. CONCLUSIONS: Population pharmacokinetic analysis points to the absence of ethnic differences in the pharmacokinetics of levetiracetam between Japanese and Western populations, other than those arising from bodyweight differences. Small, clinically non-relevant differences between individual demographic characteristics suggest that dose adjustment is usually not necessary.

Modeling and simulation of intravenous levetiracetam pharmacokinetic profiles in children to evaluate dose adaptation rules.

PURPOSE: To develop a pharmacokinetic model for intravenous levetiracetam in children, based on adult intravenous data and pediatric oral data. METHODS: Data from two adult Phase-I studies in which levetiracetam was given intravenously were utilized to develop the adult population pharmacokinetic two-compartment intravenous model. After model qualification, combination with an existing pediatric one-compartment oral population pharmacokinetic model enabled simulation of twice-daily intravenous infusions of levetiracetam in children. Median and 90% confidence intervals for C(trough), C(max) (end of infusion) and AUC(tau) were simulated for 2000 children and compared to the values observed in adults. RESULTS: The population pharmacokinetic two-compartment model successfully described intravenous levetiracetam pharmacokinetics in healthy adults. After combination with the oral pediatric population model, steady-state concentrations at the end of 15-, 30- and 60 min b.i.d. levetiracetam intravenous infusions in children were predicted to be 29-41, 17-24 and 6-13% higher than those observed after oral dosing of 30 mg/kg b.i.d. Concentrations returned to the range of oral exposures within 1h after the infusion peak. The combined model predicted that steady-state peak plasma concentrations and AUC(tau) in children receiving 30 mg/kg twice daily as 15 min intravenous infusions were within the range of predicted and observed C(max,ss) and AUC(tau )values of adults receiving 15 min intravenous infusions of 1500 mg levetiracetam. CONCLUSIONS: The simulations suggest that levetiracetam may be administered intravenously in children as 15 min infusions.

Applying mini-bore HPAEC-MS/MS for the characterization and quantification of Fc N-glycans from heterogeneously glycosylated IgGs.

High-performance anion-exchange chromatography (HPAEC) coupled to pulsed amperometric detection (PAD) is a highly sensitive method for the analysis of oligosaccharides without the need for prior derivatization. However, the method suffers from the lack of chemical information with peak assignments based on the retention times of authentic standards or known peaks of reference materials. Here we applied HPAEC coupled on-line with electrospray ion trap mass spectrometry (HPAEC-MS) using a prototype mini-bore (1mm I.D.) CarboPac PA200 column and challenged the analytical separation based method for the structural assignment of heterogeneous mixtures of N-glycans derived from immunoglobulin G from human plasma, glyco-engineered CHO cells, and Sp2/0 mouse myeloma cells. Compared to an analytical scale 3mm I.D. column, the mini-bore column demonstrated a superior performance with up to 8-fold improved limit of detection for specific N-glycans determined by PAD. Quantitative evaluation by extracted ion current chromatograms revealed detection limits in the 50-100 femtomole range using ion trap MS operated in positive ionization mode. In our hands HPAEC-MS/MS allowed the detection and quantification of even low abundant glycan species including biantennary complex-type, high mannose, hybrid and hybrid bisected structures. In comparison to the detection of N-glycans as lithiated or sodiated adducts, we obtained a 65-fold improved signal-to-noise ratio with protonated ions only. Relative quantitative evaluation by single ion current chromatograms was successfully applied and demonstrated an excellent performance with respect to selectivity in the relative quantification of heterogeneous samples of N-glycans compared to HPAEC-PAD and HILIC-UPLC of 2-AB labelled N-glycans.

Pharmacokinetic interaction of brivaracetam on carbamazepine in adult patients with epilepsy, with and without valproate co-administration.

OBJECTIVE: This Phase I, open-label, dose-escalation study investigated the effects of steady-state brivaracetam on the pharmacokinetics of carbamazepine in patients with epilepsy, with and without valproate co-administration. Valproate and brivaracetam inhibit epoxide hydrolase and increase carbamazepine epoxide levels. METHODS: Adult patients with epilepsy being chronically treated with carbamazepine alone (n=9) or with carbamazepine and valproate (n=9) received brivaracetam during successive 1-week periods at doses of 50mg, 100mg, 200mg, and 100mg twice daily (bid). Doses of carbamazepine and valproate must have been stable for at least 3 months. Trough plasma concentrations of carbamazepine, carbamazepine epoxide, and diol metabolites were determined on Days 1, 8, 15, 22, and 29, and at the end of study visit (ESV, 2-3weeks later). RESULTS: Eighteen patients with median (range) age of 45 (20-62) years and body weight of 74 (59-124) kg were enrolled and completed the study. In patients treated with carbamazepine alone, brivaracetam dose-dependently increased mean trough levels of carbamazepine epoxide from 1.38µg/mL on Day 1 pre-dose to 2.16µg/mL (+57%) on Day 8 (50mg bid), 2.72µg/mL (+97%) on Day 15 (100mg bid), 3.02µg/mL (+119%) on Day 22 (200mg bid), 2.67µg/mL (+94%) on Day 29 (100mg bid), and 1.22µg/mL (-12%) at ESV, respectively. In patients on carbamazepine and valproate, carbamazepine epoxide increased from 1.98µg/mL at baseline to 2.72µg/mL (+37%), 3.70µg/mL (+87%), 4.43µg/mL (+124%), 3.11µg/mL (+57%), and 1.94µg/mL (-2%), respectively. There was no trend for change in carbamazepine, carbamazepine diol or valproate levels. Brivaracetam levels increased linearly with dose. Brivaracetam was well tolerated. CONCLUSIONS: Carbamazepine epoxide plasma concentrations were approximately doubled by brivaracetam 100 or 200mg bid. Data are consistent with a dose-dependent and reversible inhibition of epoxide hydrolase by brivaracetam. Carbamazepine epoxide was approximately 0.7µg/mL higher in presence of valproate. There is no need to limit brivaracetam dosing when used concomitantly with carbamazepine.

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles--part 1: separation-based methods.

Immunoglobulin G (IgG) crystallizable fragment (Fc) glycosylation is crucial for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity, and for their pharmacokinetic and pharmacodynamics behavior. To monitor the Fc-glycosylation in bioprocess development, as well as product characterization and release analytics, reliable techniques for glycosylation analysis are needed. A wide range of analytical methods has found its way into these applications. In this study, a comprehensive comparison was performed of separation-based methods for Fc-glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods were compared for precision, accuracy, throughput and other features; special emphasis was placed on the detection of sialic acid-containing glycans. Seven, non-mass spectrometric methods were compared; the methods utilized liquid chromatography-based separation of fluorescent-labeled glycans, capillary electrophoresis-based separation of fluorescent-labeled glycans, or high-performance anion exchange chromatography with pulsed amperometric detection. Hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography of 2-aminobenzamide (2-AB)-labeled glycans was used as a reference method. All of the methods showed excellent precision and accuracy; some differences were observed, particularly with regard to the detection and quantitation of minor glycan species, such as sialylated glycans.