Comparison of Neutralizing Antibody Assays for Receptor Binding and Enzyme Activity of the Enzyme Replacement Therapeutic Naglazyme® (Galsulfase)

Most patients receiving Naglazyme® (galsulfase, rhASB) enzyme replacement therapy for mucopolysaccharidosis type VI develop an antibody response. To evaluate the impact of this response, two in vitro neutralizing antibody (NAb) assays were developed based on the two steps of the mechanism of action. Neutralization of enzyme activity was detected by inhibition of rhASB cleavage of a fluorogenic substrate. Neutralization of receptor binding was detected by decreased binding of labeled rhASB to immobilized soluble receptor. For the enzyme activity NAb assay, serum pretreatment was required to isolate antibodies from interfering phosphate ions, with sensitivity of ≤5 μg/mL. The receptor binding NAb assay used a five-fold dilution, with sensitivity of ≤40 μg/mL. Cutpoints for percent inhibition were based on 95% confidence intervals from naïve sera. Clinical samples were similarly likely to be positive in both assays than positive for neutralization of only one step in the mechanism of action. The two NAb assays yielded complementary information about potential neutralization of rhASB. Relative estimated sensitivity between neutralization assays did not correlate with the number of positive clinical samples or patients. In vitro NAb assays based on a well-understood mechanism of action provide specific information about the NAb mechanism.     

Development and Implementation of an Advising Program’s Meet-and-Greet Session

Objective. To describe the implementation and perceptions of an advising program’s meet-and-greet session on student/faculty interactions.Design. Student advisees and faculty advisors attended a meet-and-greet program designed to facilitate introductions. Two online surveys evaluating program perceptions were electronically distributed to participants.Assessment. Twenty-eight advisors and 226 students attended; 17 faculty members and 42% (n=95) of students completed the survey. Advisors and advisees found the program valuable (100%, 85%) and recommended holding it again (100%, 93%), respectively. Most advisors agreed that the event improved success in meeting advisees while reducing time needed to schedule and meet with advisees. Students felt more comfortable contacting advisors after participating, with 83% agreeing it was more convenient than scheduling separate meeting times.Conclusion. An advising meet-and-greet program facilitated initial advisee/advisor meetings while reducing self-reported faculty time/resources. This activity could be implemented by other institutions seeking to promote student advising relationships.     

Initial Development and Psychometric Properties of a New Measure of Substance Use Disorder “Recovery Progression”: The Recovery Progression Measure (RPM)

There is a growing literature around substance use disorder treatment outcomes measures. Various constructs have been suggested as being appropriate for measuring recovery outcomes, including “recovery capital” and “treatment progression.” However, these previously proposed constructs do not measure changes in psychosocial functioning during the recovery process. Therefore, a new psychometric assessment, the “Recovery Progression Measure” (RPM), has been developed to measure this recovery oriented psychosocial change. Aims: The aims of this study were to evaluate the reliability and factor structure of the RPM via data collected from 2218 service users being treated for their substance dependence. Method: Data were collected from service users accessing the Breaking Free Online (BFO) substance use disorder treatment and recovery program, which has within its baseline assessment a 36-item psychometric measure previously developed by the authors to assess the six areas of functioning described in the RPM. Reliability analyses and exploratory factor analyses (EFA) were conducted to examine the underlying factor structure of the RPM measure. Results: Internal reliability of the RPM measure was found to be excellent (α > .70) with the overall assessment to have reliability α = .89, with item-total correlations revealing moderate–excellent reliability of individual items. EFA revealed the RPM to contain an underlying factor structure of eight components. Discussion: This study provides initial data to support the reliability of the RPM as a recovery measure. Further work is now underway to extend these findings, including convergent and predictive validity analyses.     

Clinical pharmacology and efficacy of rotigotine (Neupro® patch) in the treatment of restless leg syndrome

Introduction: Restless legs syndrome/Willis Ekbom disease (RLS/WED) is a sensorimotor disorder characterized by unpleasant sensations in the legs accompanied by an urge to move them, that typically occurs and tend to worsen in the evening/night or during period of inactivity. Standard medications for RLS/WED are dopamine agonists and calcium channel α-2-δ ligands. The clinical spectrum of RLS/WED is very broad, ranging from individuals suffering from the disease during limited periods up to those severely affected, with daily symptoms. In such cases a long-acting drug like rotigotine should be considered.

Areas covered: The clinical pharmacology and efficacy of rotigotine was examined to evaluate the evidence supporting its use in RLS/WED.

Expert opinion: The rotigotine transdermal patch provides constant delivery of the drug, maintaining a stable plasma concentration over 24 hours by means of a single daily application. Several randomized, double-blind, placebo-controlled trials have demonstrated the efficacy of rotigotine in improving moderate-to-severe RLS/WED symptoms. Rotigotine is generally well tolerated. The most common adverse effects were application-site reactions, dose-dependent, more frequently reported in the first period of treatment. Incidence of augmentation in RLS/WED patients treated with oral dopamine agonists is higher when compared with the use of transdermal rotigotine.     

Development of Infrared Imaging to Measure Thermogenesis in Cell Culture: Thermogenic Effects of Uncoupling Protein-2, Troglitazone,and β-Adrenoceptor Agonists

Purpose. Although the effects of thermogenic agents in cell culture can be measured by direct microcalorimetry, only a few samples can be analyzed over several hours. In this report, we describe a robust non-invasive technique to measure real-time thermogenesis of cells cultured in microtiter plates using infrared thermography. Methods. Yeast were transformed with uncoupling protein-2 (UCP2) or exposed to carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) or rotenone. Adipocytes were exposed to rotenone, FCCP, cycloheximide, troglitazone, or CL316243. Thermogenesis was measured using infrared thermography. Results. Thermogenesis increased after exposing yeast to the mitochondrial uncoupler, FCCP, or transforming the cells with UCP2. Further, thermogenesis in adipocytes was stimulated by CL316243, a ₃-adrenoceptor agonist being developed to treat obesity. The protein synthesis inhibitor, cycloheximide, did not inhibit CL316243-mediated thermogenesis. In contrast, the mitochondrial proton transport inhibitor, rotenone, inhibited thermogenesis in yeast and adipocytes. Similarly, the antidiabetic agent, troglitazone, suppressed thermogenesis in adipocytes. Although increased UCP synthesis resulted in increased thermogenesis in yeast, UCP expression did not correlate with thermogenesis in adipocytes. Conclusions. The results, taken together with the high resolution (0.002°C) and robustness (384-well format) of the approach, indicate infrared-imaging is a rapid and effective method for measuring thermogenesis in vitro.     

Development and Advanced Validation of an Optimized Method for the Quantitation of Aβ42 in Human Cerebrospinal Fluid

Cerebrospinal fluid (CSF) biomarkers have been extensively utilized in the diagnosis of Alzheimer’s disease (AD) and characterization of progression. One important CSF biomarker is the amyloid beta 42 (Aβ₄₂) peptide, a key player in AD pathogenesis. The INNOTEST® Aβ₄₂ ELISA kit has been widely used but an advanced level of method development and validation has not been reported. To support a clinical trial in AD, we successfully completed a Good Laboratory Practices (GLP)-level validation of the method to establish the parameters of precision, accuracy, parallelism, selectivity, specificity, and linearity of dilution of the assay in CSF matrix, as well as CSF storage stability. Several modifications were required to optimize the assay and ensure consistent results in a clinical-trial setting. These included the use of additional calibrators, an adjusted standard curve range, a minimum required dilution (MRD) of CSF by 6-fold to avoid matrix interference and mitigation of analyte adsorption to labware by the addition of Tween-20. The optimized method displayed a quantitative range of 375–4,500 pg/mL. The inter-assay precision was ≤12.1 % CV and the inter-assay relative accuracy was ≤10.9 % absolute bias, bringing the total error of the assay to ≤23 %. The intra-assay precision of the assay at the high validation standard and below was ≤5.5 % CV; this enables sensitive detection of biomarker changes across a therapeutic regime. The INNOTEST® Aβ₄₂ ELISA kit, modified as reported here, may be appropriate for many applications, including regulatory agency acceptable clinical diagnosis and pharmacodynamic assessment.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-012-9360-7) contains supplementary material, which is available to authorized users.Key words: AB42, Alzheimer, biomarker, enzyme-linked immunosorbent assay, method validation     

Total syntheses of schizandriside,saracoside and (±)-isolariciresinol with antioxidant activities

Lignans are widely distributed in plants and exhibit significant pharmacological effects, including anti-tumor and antioxidative activities. Here, we describe the total synthesis of schizandriside (1), a compound we previously isolated from Saraca asoca by monitoring antioxidative activity using the 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay. Starting from a tandem Michael-aldol reaction, the lignan skeleton was synthesized in 6 steps, including a cyclization step. To determine the stereochemistry of 1, we synthesized the natural product (±)-isolariciresinol (18) from alcohol 17. Comparison of the spectral data showed good agreement. Glycosylation was investigated using four different glycosyl donors. Only the Koenigs–Knorr condition using silver trifluoromethanesulfonate with 1,1,3,3-tetramethylurea provided the glycosylated product. Deprotection and purification using reverse-phase high-performance liquid chromatography gave schizandriside (1) and its diastereomer saracoside (2). Synthesized 1, 2 and 18 showed antioxidant activity with IC₅₀?=?34.4, 28.8, 53.0 μM, respectively.     

Subacute and subchronic toxicity of Avalon® mixture (bentazone+dicamba) to rats

Subacute and subchronic toxicity of the herbicide Avalon^®, a mixture of bentazone and dicamba, were tested on rats. Avalon^® was administered at dose levels of 250, 500 and 1000 mg/kg body weight/day for 28 and 90 days. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) activities were monitored together with biochemistry parameters. The results showed that the mixture caused increases in the activities of ALT, AST and ALP, elevated concentrations of sodium, albumin and albumin/globulin ratio in males. In females, ALT activity, cholesterol and phosphate levels were increased. The changes generally were dose related and, in most cases, females exhibited lower susceptibility than males. The effects of a mixture are, in the most cases, different from the effects of the individual substances. The effects of bentazone were not prevalent which would be expected taking the composition of the mixture into account.     

Development and validation of immunoassays to quantify the half-antibody exchange of an IgG4 antibody, natalizumab (Tysabri®) with endogenous IgG4

Natalizumab is a humanized IgG4 monoclonal antibody which binds human α4 integrin and is approved for treatment of multiple sclerosis and Crohn's disease. Assessment of the in vivo disposition of natalizumab presents a unique assay development challenge due to the ability of human IgG4 antibodies to undergo half-antibody exchange in vivo. Such exchange generates IgG4 molecules of mixed specificity comprising a natalizumab heavy-light chain pair coupled to an IgG4 heavy-light chain pair of unknown specificity. Since exchanged and non-exchanged species cannot be quantified independently using a single enzyme linked immunosorbent assay (ELISA), a novel quantitation strategy was developed employing two ELISAs: one measuring total natalizumab including both intact and exchanged molecules, and the second measuring only intact natalizumab. The presence and amount of exchanged natalizumab in serum is calculated by the difference in values obtained in the two assays. To evaluate assay performance, a control reagent was created from natalizumab and an irrelevant humanized monoclonal IgG4 antibody. Subsequent validation demonstrated that both assays are specific, accurate, and precise within the working ranges of the assays (1.5-10μg/mL for total and 0.5-12μg/mL for intact natalizumab assays). The mean accuracy, intra- and inter-assay precision for both assays were 82-113%, ≤9% and ≤20%, respectively. Additionally, the limits of detection of intact and exchanged natalizumab were established using statistical methods. The utility of the two-assay strategy was confirmed by analyzing samples from a pharmacokinetic study in rats using different variants of natalizumab administered along with another human IgG4 antibody as an exchange partner.     

“Fit-for-Purpose” Method Validation and Application of a Biomarker (C-terminal Telopeptides of Type 1 Collagen) in Denosumab Clinical Studies

Biomarkers are used to study drug effects, exposure–response relationships, and facilitate early decision making during development. Denosumab, a fully human monoclonal antibody against receptor activator of nuclear factor-κB ligand, profoundly inhibits bone resorption. C-terminal telopeptides of type I collagen (CTx), a bone resorption biomarker, provides early indications of denosumab effectiveness and informs protracted clinical outcomes (e.g., bone mineral density). Because of the dynamic relationship between denosumab and CTx, a precise and robust assay was desired. Thus, we adopted a fit-for-purpose approach to modify and validate a commercial CTx diagnostic kit to meet the intended applications of a quantitative pharmacodynamic biomarker for denosumab development. Seven standards were prepared to replace five calibrators provided in the kit. Three quality controls (QC) and two sample controls were used to characterize and monitor assay performance. Robotic workstations were used for standard and QC preparation and assay execution. Method validation experiments were conducted with rigor and procedures similar to those used for drug bioanalysis. The method demonstrated a linear range of 0.0490–2.34 ng/mL with four-parameter logistic regression. Inter-assay total error of validation samples in serum was ≤26.7%. Extensive tests were conducted on selectivity in sera from target populations, specificity, stability, parallelism, and dilutional linearity. Applications to samples from numerous clinical studies confirmed that the CTx method was reliable, robust, and fit for use as an early indicator of denosumab effectiveness. Refinement supported the confidence for use in pharmacokinetic/pharmacodynamic modeling, dose selections, correlation to clinical effects, and formulation bioequivalence work.     

Recommendations on the Development of a Bioanalytical Assay for 4β-Hydroxycholesterol,an Emerging Endogenous Biomarker of CYP3A Activity

The availability of reliable assays for measuring 4β-hydroxycholesterol (4β-HC), a CYP3A metabolite of cholesterol, is an important step in qualifying this endogenous moiety as a biomarker of CYP3A activity. Liquid and gas chromatographic methods with mass spectrometric detection have been developed with varying sensitivities, with or without derivatization. Care must be taken to chromatographically resolve 4β-HC from the multiple isobaric cholesterol oxidation products present in plasma, including 4α-hydroxycholesterol (4α-HC). Plasma concentrations of 4β-HC are low in humans (10–60 ng/ml), lower than many other cholesterol metabolites and far less than cholesterol itself. Stability of 4β-HC has been established for at least 12 months at ?20°C in plasma samples obtained with a typical clinical workflow. Oxidation of plasma cholesterol during storage produces both 4β-HC and 4α-HC, and 4α-HC may be used as assessment of sample quality. As 4β-HC concentrations over time in untreated individuals have low intra-individual variability, assay precision and reproducibility are the key assay attributes in assessing CYP3A4 induction, and potentially inhibition. Assessment of CYP3A4/5 activity with 4β-HC relies on the differences between pre- and post-dose concentrations, in which each subject acts as their own control. To reduce analytical variability, samples from a single subject should be analyzed together to facilitate interpretation of study results. As an endogenous biomarker, 4β-HC offers the opportunity for less invasive assessment of CYP3A induction potential of new drugs during clinical development.     

Investigation of the Mechanism of Therapeutic Protein-Drug Interaction Between Methotrexate and Golimumab,an Anti-TNFα Monoclonal Antibody

A prominent example of human therapeutic protein-drug interaction (TP-DI) is between methotrexate (MTX) and anti-TNFα mAbs. One plausible mechanism for this TP-DI is through the pharmacodynamic effect of MTX on immunogenicity. However, there is no definitive evidence to substantiate this mechanism, and other competing hypotheses, such as MTX suppressing FcγRI expression thereby affecting mAb PK, have also been proposed. In order to understand this mechanism, a cynomolgus monkey study was conducted using golimumab as a model compound. Golimumab elicited high incidences of immunogenicity in healthy cynomolgus monkeys. Concomitant dosing of MTX delayed the onset and reduced the magnitude of anti-drug antibody (ADA) formation. The impact of MTX on golimumab PK correlated with the ADA status. Prior to ADA formation, MTX has no discernable effect on golimumab PK. Additionally, no alteration in FcγRI expression was observed following MTX treatment. The impact of MTX on golimumab immunogenicity and PK has been observed in patients with rheumatoid arthritis, psoriatic arthritis (PsA), and ankylosing spondylitis. In a representative phase 3 study of golimumab in patients with PsA, patients not receiving concomitant MTX was reported to have ~?30% lower steady-state trough golimumab levels compared to those who received MTX. However, further analysis showed that PsA patients who were negative for ADA in both treatment groups had comparable trough levels of golimumab. Taken together, our results suggest that the mechanism of TP-DI between MTX and golimumab can mostly be attributed to the pharmacodynamic effect of MTX, i.e., the lowering of immunogenicity and immunogenicity-mediated clearance of mAbs.     

Effect of Roundup® (glyphosate formulation) in the energy metabolism and reproductive traits of Hyalella castroi (Crustacea, Amphipoda, Dogielinotidae)

Roundup^? (glyphosate formulation) is a nonselective and posts emergent herbicide used for controlling aquatic weeds and different concentrations are used in cultures around the world. The objective of this investigation was to examine the effects of Roundup^? (glyphosate formulation) on the biochemical composition, levels of lipoperoxidation, Na⁺/K⁺ATPase activity and reproductive traits in the Hyalella castroi. Amphipods were collected in summer 2009, in the southern Brazilian highlands. In the laboratory, the animals were kept in aquariums under controlled conditions for 7 days, and after this period they were exposed to 0.36, 0.52, 1.08 and 2.16 mg/l of glyphosate for 7 days. After the period of exposure, the animals were immediately frozen for determination of glycogen, proteins, lipids, triglycerides, cholesterol, levels of lipoperoxidation, and Na⁺/K⁺ATPase activity. During each day of the cultivation reproductive traits (number of reproductive pairs, ovigerous females and eggs in the marsupium) were observed. All concentrations of Roundup^? induced significant decreases in all biochemical parameters and Na⁺/K⁺ATPase activity, and significant increase in lipoperoxidation levels. Showing this form a potentially toxic effect at very low concentrations, this pattern of results can lead to significant changes in trophic structure of limnic environments because these amphipods are important links in food chain in these habitats.     

Development of a Virosomal RSV Vaccine Containing 3D-PHAD® Adjuvant: Formulation,Composition, and Long-Term Stability

Purpose

Characterization of virosomes, in late stage preclinical development as vaccines for Respiratory Syncytial Virus (RSV), with a membrane-incorporated synthetic monophosphoryl lipid A, 3D-PHAD® adjuvant.

Methods

Virosomes were initially formed by contacting a lipid film containing 3D-PHAD® with viral membranes solubilized with the short chain phospholipid DCPC, followed by dialysis, later by adding solubilized 3D-PHAD to viral membranes, or to preformed virosomes from DMSO.

Results

Virosomes formed from lipid films contained the membrane glycoproteins G and F, at similar F to G ratios but lower concentrations than in virus, and the added lipids, but only a fraction of the 3D-PHAD®. By single particle tracking (SPT), the virosome size distribution resembled that seen by cryo-electron microscopy, but dynamic light scattering showed much larger particles. These differences were caused by small virosome aggregates. Measured by SPT, virosomes were stable for 300 days. 3DPHAD ® incorporation in virosomes could be enhanced by providing the adjuvant from DCPC solubilized stock, but also by adding DMSO dissolved adjuvant to pre-formed virosomes. Virosomes with 0.1 mg/mg of 3D-PHAD®/viral protein from DMSO induced antibody titers similar to those by virosomes containing 0.2 mg/mg of DCPC-solubilized 3D-PHAD®.

Conclusions

Stable 3D-PHAD® adjuvanted RSV virosomes can be formulated.

     

Successful administration of BI 695501, an adalimumab biosimilar,using an autoinjector (AI): results from a Phase II open-label clinical study (VOLTAIRE®-RL)

Background: This study examined the patient handling experience and self-injection success of patients with rheumatoid arthritis (RA) administering BI 695501 using an AI.

Methods: This Phase II, 7-week, open-label, interventional study (NCT02636907) included adult patients with moderately to severely active RA not adequately controlled by DMARDs, with no experience of self-injecting with AI/pen. Patients self-injected BI 695501 via AI every 2 weeks in the AI Assessment Period (AAP). Training was given on first injection; AI handling events were recorded. Percentage of self-injection success was the primary end point. Patients could enter a 42-week pre-filled syringe (PFS) safety extension.

Results: The AAP was completed by 73/77 patients. In total, 216/218 (99.1%) self-injections on Days 15, 29, and 43, were successful. Nine (11.7%) patients had drug-related adverse events (AEs). Two patients reported four serious AEs (SAEs), none drug-related. Overall (in the AAP and PFS extension), 28 (36.4%) patients had drug-related AEs; nine patients had SAEs, one was considered drug-related. Five (6.5%) patients reported injection-site reactions in the AAP; 13 (18.1%) in the PFS extension.

Conclusions: After training, almost all patients were successfully able to self-administer BI 695501 using an AI. BI 695501 via AI (and via PFS in the extension) was well tolerated.

Clinical Trial Registration: NCT02636907     

Review of the Basic and Clinical Pharmacology of Sulfobutylether-β-Cydodextrin (SBECD)

Despite its use in commercially available drugs such as intravenous voriconazole, there is little known in the medical literature about the clinical pharmacology of the solubilizing agent, sulfobutylether-β-cyclodextrin (SBECD). This paper summarizes all known data on SBECD pharmacokinetics and safety. In animals, volume of distribution approximates extracellular water volume and clearance is determined by the glomerular filtration rate. SBECD does not have any apparent effects on cardiovascular or respiratory systems, nor on autonomic and somatic functions in animals. In 1- and 6-month studies in rats and dogs, the most noteworthy findings were renal tubular vacuolation and foamy macrophages in the liver and lungs. Mild toxicity in the kidney and liver as a consequence of vacuolation occurred in rats at the maximum dose of 3000 mg/kg, which is approximately 50-fold greater than the SBECD dose typically administered in man. Doses up to 1500 mg/kg produced no histopathological evidence of toxicity in dog kidneys. SBECD has also been studied in healthy volunteers and subjects with renal dysfunction. Whereas plasma SBECD levels accumulate in those with renal compromise, there were no deleterious effects on renal function. Nonetheless, serum creatinine levels should be monitored in subjects with renal compromise receiving multiple doses of SBECD.