Orally Bioavailable Macrocyclic Peptide That Inhibits Binding of PCSK9 to the Low Density Lipoprotein Receptor.

BACKGROUND: Inhibition of PCSK9 (proprotein convertase subtilisin/kexin type 9)-low density lipoprotein receptor interaction with injectable monoclonal antibodies or small interfering RNA lowers plasma low density lipoprotein-cholesterol, but despite nearly 2 decades of effort, an oral inhibitor of PCSK9 is not available. Macrocyclic peptides represent a novel approach to target proteins traditionally considered intractable to small-molecule drug design. METHODS: Novel mRNA display screening technology was used to identify lead chemical matter, which was then optimized by applying structure-based drug design enabled by novel synthetic chemistry to identify macrocyclic peptide (MK-0616) with exquisite potency and selectivity for PCSK9. Following completion of nonclinical safety studies, MK-0616 was administered to healthy adult participants in a single rising-dose Phase 1 clinical trial designed to evaluate its safety, pharmacokinetics, and pharmacodynamics. In a multiple-dose trial in participants taking statins, MK-0616 was administered once daily for 14 days to characterize the safety, pharmacokinetics, and pharmacodynamics (change in low density lipoprotein cholesterol). RESULTS: MK-0616 displayed high affinity (Ki = 5pM) for PCSK9 in vitro and sufficient safety and oral bioavailability preclinically to enable advancement into the clinic. In Phase 1 clinical studies in healthy adults, single oral doses of MK-0616 were associated with >93% geometric mean reduction (95% CI, 84-103) of free, unbound plasma PCSK9; in participants on statin therapy, multiple-oral-dose regimens provided a maximum 61% geometric mean reduction (95% CI, 43-85) in low density lipoprotein cholesterol from baseline after 14 days of once-daily dosing of 20 mg MK-0616. CONCLUSIONS: This work validates the use of mRNA display technology for identification of novel oral therapeutic agents, exemplified by the identification of an oral PCSK9 inhibitor, which has the potential to be a highly effective cholesterol lowering therapy for patients in need.

Repeat serological testing for anti-citrullinated peptide antibody after commencement of therapy is not helpful in patients with seronegative rheumatoid arthritis.

AIM: To Investigate the prevalence of seroconversion to ACPA after commencement of triple disease-modifying anti-rheumatic drug (DMARD) treat-to-target therapy. BACKGROUND: Anti-citrullinated peptide antibody (ACPA) and rheumatoid factor (RF) define 'seropositive' rheumatoid arthritis (RA). Both predict unfavourable disease course, development of extra-articular features and treatment outcomes. We investigated the prevalence of seroconversion to ACPA after commencement of triple disease-modifying anti-rheumatic drug (DMARD) treat-to-target therapy. METHODS: DMARD-naïve patients with RA according to the 1987 American College of Rheumatology criteria and disease duration of <96 weeks were enrolled. RF and ACPA levels were recorded at baseline and sequentially during triple DMARD therapy. RESULTS: A total of 368 patients were followed for a median of 272 weeks. Of 154 patients seronegative for ACPA at recruitment, 10 (6.5%) seroconverted at some point. Nine of these were positive for RF at baseline and baseline RF titre was predictive of seroconversion. Four (2.6%) patients remained seropositive. No patients seroconverted from negative to positive for both RF and ACPA. Median time to seroconversion for ACPA was 29 months. CONCLUSION: Persistent seroconversion of ACPA from negative to positive after diagnosis in patients with RA is uncommon. ACPA and RF double negative patients are highly unlikely to ever develop ACPA positivity with a risk <1%. It is therefore unlikely to be helpful or cost effective to perform serial ACPA measurements in patients with seronegative RA after commencement of a treat-to-target strategy.

Factors associated with medication adherence in a longitudinal study of rheumatoid arthritis patients.

BACKGROUND: Identification of key determinants of medication adherence may assist with designing interventions to improve this important parameter. The aim of the study was to determine the rate and predictors of self-reported medication adherence in patients with rheumatoid arthritis (RA) over one-year follow-up. METHODS: Socio-demographic, disease, therapy and patient-related factors were obtained from a longitudinal observational cohort of RA patients between May 2014 and June 2016. Medication adherence was measured using self-reported Compliance Questionnaire for Rheumatology (CQR) at baseline, 6 and 12 months. Mixed-effects modelling was used to investigate the relationship between adherence and potential predictors. RESULT: Of 185 patients invited, 110 were included in the study. The median level of adherence was 71%-74% during the study period. Around 27%-30% of patients achieved > 80% adherence, while roughly one-fifth reported a CQR score within the lower quartile (CQR < 63%). After adjustment for potential confounders, increased age (ß = 0.19, P = 0.010), higher self-efficacy (ß = 0.89, P = 0.039) and higher medication necessity belief (ß = 1.12, P < 0.0001) were associated with better self-reported adherence. CONCLUSION: We found a moderate level of self-reported adherence over time and significant association with age, self-efficacy and medication necessity belief. The modifiable predictors of adherence found in this study can be used as a potential target for adherence-improving interventions.

Constraints on primary and secondary particulate carbon sources using chemical tracer and 14C methods during CalNex-Bakersfield.

The present study investigates primary and secondary sources of organic carbon for Bakersfield, CA, USA as part of the 2010 CalNex study. The method used here involves integrated sampling that is designed to allow for detailed and specific chemical analysis of particulate matter (PM) in the Bakersfield airshed. To achieve this objective, filter samples were taken during thirty-four 23-hr periods between 19 May and 26 June 2010 and analyzed for organic tracers by gas chromatography - mass spectrometry (GC-MS). Contributions to organic carbon (OC) were determined by two organic tracer-based techniques: primary OC by chemical mass balance and secondary OC by a mass fraction method. Radiocarbon (14C) measurements of the total organic carbon were also made to determine the split between the modern and fossil carbon and thereby constrain unknown sources of OC not accounted for by either tracer-based attribution technique. From the analysis, OC contributions from four primary sources and four secondary sources were determined, which comprised three sources of modern carbon and five sources of fossil carbon. The major primary sources of OC were from vegetative detritus (9.8%), diesel (2.3%), gasoline (<1.0%), and lubricating oil impacted motor vehicle exhaust (30%); measured secondary sources resulted from isoprene (1.5%), α-pinene (<1.0%), toluene (<1.0%), and naphthalene (<1.0%, as an upper limit) contributions. The average observed organic carbon (OC) was 6.42 ± 2.33 µgC m-3. The 14C derived apportionment indicated that modern and fossil components were nearly equivalent on average; however, the fossil contribution ranged from 32-66% over the five week campaign. With the fossil primary and secondary sources aggregated, only 25% of the fossil organic carbon could not be attributed. Whereas, nearly 80% of the modern carbon could not be attributed to primary and secondary sources accessible to this analysis, which included tracers of biomass burning, vegetative detritus and secondary biogenic carbon. The results of the current study contributes source-based evaluation of the carbonaceous aerosol at CalNex Bakersfield.

Adherence to combination DMARD therapy and treatment outcomes in rheumatoid arthritis: a longitudinal study of new and existing DMARD users.

Medication adherence is believed to be a major contributor to treatment outcomes yet studies quantifying this relationship as rare in rheumatoid arthritis (RA). To determine the association of adherence to DMARD therapy with treatment outcomes among new and existing DMARD users over 2 years. Relevant clinical parameters were obtained from a longitudinal cohort of RA patients, most of who were treated with combination therapy. Patients were classified as adherent if the proportion of days covered for each DMARD was ≥80%. Outcome measures were the change in the disease activity score in 28 joints (DAS28), simplified disease activity index (SDAI), modified health assessment questionnaires (mHAQ) and proportion of patients who achieved response criteria. An inverse propensity-score weighting method was used to estimate the association of adherence with each outcome. Of 194 patients invited, a total of 111 patients (new = 45 and existing = 66 DMARD users) met study eligibility. DMARD-naive patients demonstrated relatively higher rates of adherence compared to existing users. After controlling for confounding variables, adherence was significantly associated with reduction in DAS28 (ß = -1.5, 95% CI of ß = - 2.17 to -0.83, p < 0.0001), SDAI (ß = -9.44, 95% CI of ß = -15.53 to -3.35, p = 0.002) and mHAQ (ß = -0.269, 95% CI of ß, -0.462 to -0.077, p = 0.017) over 2 years among new patients and adherent patients were more likely to achieve most response criteria compared to non-adherent patients. Such associations were not replicated among existing DMARD users. Adherence to combination DMARD therapy was associated with improvements in disease activity and functional outcomes in the first 2 years of therapy.

Multiplexed Quantification of Proglucagon-Derived Peptides by Immunoaffinity Enrichment and Tandem Mass Spectrometry after a Meal Tolerance Test.

BACKGROUND: Proglucagon-derived peptides (PGDPs), which include glucagon-like peptide (GLP)-1, glucagon, and oxyntomodulin, are key regulators of glucose homeostasis and satiety. These peptide hormones are typically measured with immuno-based assays (e.g., ELISA, RIA), which often suffer from issues of selectivity. METHODS: We developed a multiplexed assay for measuring PGDPs including GLP-1 (7-36) amide, GLP-1 (9-36) amide, glucagon, and oxyntomodulin by mass spectrometry and used this assay to examine the effect of a meal tolerance test on circulating concentrations of these hormones. Participants fasted overnight and were either given a meal (n = 8) or continued to fast (n = 4), with multiple blood collections over the course of 3 h. Plasma samples were analyzed by microflow immunoaffinity (IA)-LC-MS/MS with an isotope dilution strategy. RESULTS: Assay performance characteristics were examined and established during analytical validation for all peptides. Intra- and interassay imprecision were found to be 2.2%-10.7% and 6.8%-22.5%, respectively. Spike recovery was >76%, and dilution linearity was established up to a 16-fold dilution. Immediately after the meal tolerance test, GLP-1 and oxyntomodulin concentrations increased and had an almost identical temporal relationship, and glucagon concentrations increased with a slight delay. CONCLUSIONS: IA-LC-MS/MS was used for the simultaneous and selective measurement of PGDPs. This work includes the first indication of the physiological concentrations and modulation of oxyntomodulin after a meal.

A Clustered Randomized Controlled Trial to Reduce Secondhand Smoke Exposure Among Nonsmoking Pregnant Women in Sichuan Province, China.

INTRODUCTION: Nonsmoking pregnant women in China have significant exposure to secondhand smoke (SHS). Few interventions have focused on pregnant women reducing their SHS exposure. METHODS: This clustered randomized controlled trial, conducted at eight hospitals in Sichuan, China, compared a prenatal health education intervention with usual clinical care as a control. The primary outcome was self-reported "no SHS exposure" before and 3 months after birth. The intervention consisted of three large group educational sessions, standardized clinician advice, brief monthly follow-up calls, and educational materials and resources. A random sample of participants was biochemically validated before birth with hair nicotine, a long-term biomarker of smoke exposure. RESULTS: Overall, 1181 participants were randomized to intervention (n = 526) and control (n = 655) groups. More participants in the intervention group than the control group reported no SHS exposure 3 months after birth (Total: 77.9% vs. 52.6%, P < .001; Home: 81.2% vs. 53.3%, P < .001). The intervention group also had greater changes in improved smoke-free homes and SHS knowledge and attitudes. Controlling for covariates, the intervention group was less likely to report SHS exposure than the control group (Total: OR = 0.47, 95% CI = 0.31 to 0.71; Home: OR = 0.33, 95% CI = 0.21 to 0.53), and this effect was sustained 3 months after birth. The adjusted log concentration of hair nicotine for the intervention group decreased by 0.28 log µg/g more than the control group. CONCLUSIONS: Our smoke-free health education intervention for nonsmoking pregnant women significantly reduced SHS exposure before and after birth. This intervention model can become part of a standard protocol for the care of pregnant women in hospital settings.

Fish oil in recent onset rheumatoid arthritis: a randomised, double-blind controlled trial within algorithm-based drug use.

BACKGROUND: The effects of fish oil (FO) in rheumatoid arthritis (RA) have not been examined in the context of contemporary treatment of early RA. This study examined the effects of high versus low dose FO in early RA employing a 'treat-to-target' protocol of combination disease-modifying anti-rheumatic drugs (DMARDs). METHODS: Patients with RA <12 months' duration and who were DMARD-naïve were enrolled and randomised 2:1 to FO at a high dose or low dose (for masking). These groups, designated FO and control, were given 5.5 or 0.4 g/day, respectively, of the omega-3 fats, eicosapentaenoic acid + docosahexaenoic acid. All patients received methotrexate (MTX), sulphasalazine and hydroxychloroquine, and DMARD doses were adjusted according to an algorithm taking disease activity and toxicity into account. DAS28-erythrocyte sedimentation rate, modified Health Assessment Questionnaire (mHAQ) and remission were assessed three monthly. The primary outcome measure was failure of triple DMARD therapy. RESULTS: In the FO group, failure of triple DMARD therapy was lower (HR=0.28 (95% CI 0.12 to 0.63; p=0.002) unadjusted and 0.24 (95% CI 0.10 to 0.54; p=0.0006) following adjustment for smoking history, shared epitope and baseline anti-cyclic citrullinated peptide. The rate of first American College of Rheumatology (ACR) remission was significantly greater in the FO compared with the control group (HRs=2.17 (95% CI 1.07 to 4.42; p=0.03) unadjusted and 2.09 (95% CI 1.02 to 4.30; p=0.04) adjusted). There were no differences between groups in MTX dose, DAS28 or mHAQ scores, or adverse events. CONCLUSIONS: FO was associated with benefits additional to those achieved by combination 'treat-to-target' DMARDs with similar MTX use. These included reduced triple DMARD failure and a higher rate of ACR remission.

Practical immunoaffinity-enrichment LC-MS for measuring protein kinetics of low-abundance proteins.

BACKGROUND: For a more complete understanding of pharmacodynamic, metabolic, and pathophysiologic effects, protein kinetics, such as production rate and fractional catabolic rate, can offer substantially more information than protein concentration alone. Kinetic experiments with stable isotope tracers typically require laborious sample preparation and are most often used for studying abundant proteins. Here we describe a practical methodology for measuring isotope enrichment into low-abundance proteins that uses an automated procedure and immunoaffinity enrichment (IA) with LC-MS. Low-abundance plasma proteins cholesteryl ester transfer protein (CETP) and proprotein convertase subtilisin/kexin type 9 (PCSK9) were studied as examples. METHODS: Human participants (n = 39) were infused with [(2)H(3)]leucine, and blood samples were collected at multiple time points. Sample preparation and analysis were automated and multiplexed to increase throughput. Proteins were concentrated from plasma by use of IA and digested with trypsin to yield proteotypic peptides that were analyzed by microflow chromatography-mass spectrometry to measure isotope enrichment. RESULTS: The IA procedure was optimized to provide the greatest signal intensity. Use of a gel-free method increased throughput while increasing the signal. The intra- and interassay CVs were <15% at all isotope enrichment levels studied. More than 1400 samples were analyzed in <3 weeks without the need for instrument stoppages or user interventions. CONCLUSIONS: The use of automated gel-free methods to multiplex the measurement of isotope enrichment was applied to the low-abundance proteins CETP and PCSK9.

Measurement of fractional synthetic rates of multiple protein analytes by triple quadrupole mass spectrometry.

BACKGROUND: Current approaches to measure protein turnover that use stable isotope-labeled tracers via GC-MS are limited to a small number of relatively abundant proteins. We developed a multiplexed liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM) assay to measure protein turnover and compared the fractional synthetic rates (FSRs) for 2 proteins, VLDL apolipoprotein B100 (VLDL apoB100) and HDL apoA-I, measured by both methods. We applied this technique to other proteins for which kinetics are not readily measured with GC-MS. METHODS: Subjects were given a primed-constant infusion of [5,5,5-D(3)]-leucine (D(3)-leucine) for 15 h with blood samples collected at selected time points. Apolipoproteins isolated by SDS-PAGE from lipoprotein fractions were analyzed by GC-MS or an LC-SRM assay designed to measure the M+3/M+0 ratio at >1% D(3)-leucine incorporation. We calculated the FSR for each apolipoprotein by curve fitting the tracer incorporation data from each subject. RESULTS: The LC-SRM method was linear over the range of tracer enrichment values tested and highly correlated with GC-MS (R(2) > 0.9). The FSRs determined from both methods were similar for HDL apoA-I and VLDL apoB100. We were able to apply the LC-SRM approach to determine the tracer enrichment of multiple proteins from a single sample as well as proteins isolated from plasma after immunoprecipitation. CONCLUSIONS: The LC-SRM method provides a new technique for measuring the enrichment of proteins labeled with stable isotopes. LC-SRM is amenable to a multiplexed format to provide a relatively rapid and inexpensive means to measure turnover of multiple proteins simultaneously.

Effectiveness and Success Factors of Bilateral Arm Training After Stroke: A Systematic Review and Meta-Analysis.

Bilateral arm training (BAT) presents as a promising approach in upper extremity (UE) rehabilitation after a stroke as it may facilitate neuroplasticity. However, the effectiveness of BAT is inconclusive, and no systematic reviews and meta-analyses have investigated the impact of different factors on the outcomes of BAT. This systematic review and meta-analysis aimed to (1) compare the effects of bilateral arm training (BAT) with unilateral arm training (UAT) and conventional therapy (CT) on the upper limb (UL) motor impairments and functional performance post-stroke, and (2) investigate the different contributing factors that may influence the success of BAT. A comprehensive literature search was performed in five databases. Randomized control trials (RCTs) that met inclusion criteria were selected and assessed for methodological qualities. Data relating to outcome measures, characteristics of participants (stroke chronicity and severity), and features of intervention (type of BAT and dose) were extracted for meta-analysis. With 25 RCTs meeting the inclusion criteria, BAT demonstrated significantly greater improvements in motor impairments as measured by Fugl-Meyer Assessment of Upper Extremity (FMA-UE) than CT (MD = 3.94, p = < 0.001), but not in functional performance as measured by the pooled outcomes of Action Research Arm Test (ARAT), Box and Block Test (BBT), and the time component of Motor Function Test (WMFT-time) (SMD = 0.28, p = 0.313). The superior motor impairment effects of BAT were associated with recruiting mildly impaired individuals in the chronic phase of stroke (MD = 6.71, p < 0.001), and applying a higher dose of intervention (MD = 6.52, p < 0.001). Subgroup analysis showed that bilateral functional task training (BFTT) improves both motor impairments (MD = 7.84, p < 0.001) and functional performance (SMD = 1.02, p = 0.049). No significant differences were detected between BAT and UAT for motor impairment (MD = -0.90, p = 0.681) or functional performance (SMD = -0.09, p = 0.457). Thus, our meta-analysis indicates that BAT may be more beneficial than CT in addressing post-stroke UL motor impairment, particularly in the chronic phase with mild UL paresis. The success of BAT may be dose-dependent, and higher doses of intervention may be required. BFTT appears to be a valuable form of BAT that could be integrated into stroke rehabilitation programs. BAT and UAT are generally equivalent in improving UL motor impairments and functional performance.

2021 White Paper on Recent Issues in Bioanalysis: Mass Spec of Proteins, Extracellular Vesicles, CRISPR, Chiral Assays, Oligos; Nanomedicines Bioanalysis; ICH M10 Section 7.1; Non-Liquid & Rare Matrices; Regulatory Inputs (Part 1A - Recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC & Part 1B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparabil ity & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.

Bilateral auricular lymphoplasmacytic lymphoma: barely mere coincidence.

We describe the first case of LPL simultaneously involving both auricles. Affected ears were the first manifestation of the disease that led to the diagnosis. The lack of appreciable systemic disease allowed sparing the patient from immunochemotherapy. Radiation therapy was used as a single modality and secured a stable remission. A putative pathogenesis of the paired auricular lymphoma is discussed and a literature review presented. While the role of genetic predisposition in our patient was uncertain, we postulate that symmetric ear lymphoma could have been caused by a combined effect of the homing of malignant lymphocytes whose localized growth was triggered by the hazardous environmental exposure.

Precision Medicine With Leflunomide: Consideration of the DHODH Haplotype and Plasma Teriflunomide Concentration and Modification of Outcomes in Patients With Rheumatoid Arthritis.

OBJECTIVE: Leflunomide is a commonly used disease-modifying drug in the treatment of rheumatoid arthritis (RA). Its effects are mediated via inhibition of dihydroorotate dehydrogenase (DHODH) by its active metabolite teriflunomide, and the pharmacokinetics of teriflunomide are highly variable. Our objective was to examine the association between the DHODH haplotype and plasma teriflunomide concentration with response to leflunomide in patients with RA where leflunomide was added to an existing disease-modifying drug regimen after failure to achieve an adequate response with conventional triple therapy. METHODS: Patients with RA who were taking, or were about to initiate, leflunomide were included. Participant characteristics, including the DHODH haplotype, were determined. Up to 5 plasma samples were collected after leflunomide was initiated for assays of total and free teriflunomide concentration. Disease activity was determined via the 28-joint Disease Activity Score (DAS28). The association between DAS28 scores and patient covariates was determined by linear mixed-effects modeling. RESULTS: A total of 67 patients were included in the study. The DAS28 score after initiation of leflunomide was associated with the baseline DAS28 score (ß = 0.70, P < 0.001) and was higher in those who carried the DHODH haplotype 2 (ß = 0.56. P = 0.01) and did not carry the shared epitope (ß = 0.56, P = 0.013). As total and free plasma teriflunomide concentration increased, the DAS28 score was significantly lower (P < 0.001 and P = 0.001, respectively). When considering threshold concentrations, teriflunomide concentrations >16 mg/liter were associated with a DAS28 score that was 0.33 lower, and when free teriflunomide concentration was >35 µg/liter, the DAS28 score was 0.32 lower. CONCLUSION: Teriflunomide concentration and carriage of the DHODH haplotype 2 are associated with response to leflunomide in patients with RA, and a total plasma teriflunomide concentration of at least 16 mg/liter is needed to maximize the likelihood of response.

2020 White Paper on Recent Issues in Bioanalysis: BMV of Hybrid Assays, Acoustic MS, HRMS, Data Integrity, Endogenous Compounds, Microsampling and Microbiome (Part 1 - Recommendations on Industry/Regulators Consensus on BMV of Biotherapeutics by LCMS, Advanced Application in Hybrid Assays, Regulatory Challenges in Mass Spec, Innovation in Small Molecules, Peptides and Oligos).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by Mass Spectrometry (hybrid assays, LCMS and HRMS) were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 1) Hybrid Assays, Innovation in Small Molecules, & Regulated Bioanalysis. Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation), Part 2B (Regulatory Input) and Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 5, and 6 (2021), respectively.

Application of an end-to-end biomarker discovery platform to identify target engagement markers in cerebrospinal fluid by high resolution differential mass spectrometry.

The rapid identification of protein biomarkers in biofluids is important to drug discovery and development. Here, we describe a general proteomic approach for the discovery and identification of proteins that exhibit a statistically significant difference in abundance in cerebrospinal fluid (CSF) before and after pharmacological intervention. This approach, differential mass spectrometry (dMS), is based on the analysis of full scan mass spectrometry data. The dMS workflow does not require complex mixing and pooling strategies, or isotope labeling techniques. Accordingly, clinical samples can be analyzed individually, allowing the use of longitudinal designs and within-subject data analysis in which each subject acts as its own control. As a proof of concept, we performed multifactorial dMS analyses on CSF samples drawn at 6 time points from n = 6 cisterna magna ported (CMP) rhesus monkeys treated with 2 potent gamma secretase inhibitors (GSI) or comparable vehicle in a 3-way crossover study that included a total of 108 individual CSF samples. Using analysis of variance and statistical filtering on the aligned and normalized LC-MS data sets, we detected 26 features that were significantly altered in CSF by drug treatment. Of those 26 features, which belong to 10 distinct isotopic distributions, 20 were identified by MS/MS as 7 peptides from CD99, a cell surface protein. Six features from the remaining 3 isotopic distributions were not identified. A subsequent analysis showed that the relative abundance of these 26 features showed the same temporal profile as the ELISA measured levels of CSF A beta 42 peptide, a known pharmacodynamic marker for gamma-secretase inhibition. These data demonstrate that dMS is a promising approach for the discovery, quantification, and identification of candidate target engagement biomarkers in CSF.

Is nurse-led telephone follow-up for post-operative obstructive sleep apnoea patients effective? A prospective observational study at a paediatric tertiary centre.

OBJECTIVES: To assess the effectiveness and patient satisfaction of a nurse-led telephone follow-up service in children following surgery for sleep disordered breathing (SDB)/obstructive sleep apnoea (OSA) in a tertiary paediatric hospital. DESIGN: Prospective observational uncontrolled study. PARTICIPANTS: Children under the age of 16 undergoing adenoid and/or tonsil surgery between June 2015 and June 2018 for SDB or OSA. Parents were contacted by telephone six weeks post-operatively by an ENT nurse specialist. The T-14 questionnaire was utilised to assess post-operative outcomes. Parents were subsequently asked to evaluate their experience of this nurse-led telephone consultation service between June 2016 and April 2017. RESULTS: 535 patients were included with an average post-operative T-14 score of 2.13 (95% CI 1.7-2.5). 430 patients were discharged following the nurse-led telephone consultation with a mean post-operative T-14 score 1.0 (95% CI 0.8-1.2). 105 patients were subsequently reviewed in clinic with an average T-14 score of 6.88 (95% CI 5.25-8.51). 36 (6.7%) patients had ongoing symptoms of SDB or OSA. 55 parents were invited to provide an evaluation of the nurse-led telephone FU clinic, which showed a 100% satisfaction rate with the service. CONCLUSION: A nurse-led telephone follow-up service is efficient and safe with high levels of parental satisfaction. It reduces unnecessary follow-up of uncomplicated patients whilst providing a robust safety net for those with ongoing problems.

Modifiable Lifestyle Factors Associated With Response to Treatment in Early Rheumatoid Arthritis.

OBJECTIVE: We aimed to evaluate the associations between response to algorithm-directed treat-to-target conventional synthetic disease-modifying antirheumatic drug therapy and potentially modifiable lifestyle factors, including dietary fish oil supplementation, body mass index (BMI), and smoking history in a rheumatoid arthritis (RA) inception cohort. METHODS: Patients with RA with a duration of less than 12 months were reviewed every 3 to 6 weeks to adjust therapy according to disease response. All patients received advice to take fish oil supplements, and omega-3 status was measured as plasma levels of eicosapentaenoic acid (EPA). Lifestyle factors and other variables potentially prognostic for 28-joint Disease Activity Score (DAS28) remission and DAS28 low disease activity (LDA) at the 12-month visit were included in multivariable logistic regression models. RESULTS: Of 300 participants, 57.7% reached DAS28 LDA, and 43.7% were in DAS28 remission at 1 year. Increase in plasma EPA was associated with an increase in the odds of being in LDA (adjusted odds ratio [OR] = 1.27; P < 0.0001) and remission (adjusted OR = 1.21; P < 0.001). There was some evidence that the effect of BMI on LDA might be modified by smoking history. An increase in BMI was associated with a decrease in the odds of being in LDA in current and former smokers but had no impact on LDA in patients who had never smoked. There were no meaningful associations between BMI or smoking history and remission. CONCLUSION: Omega-3 status, BMI, and smoking history are potential predictors of outcome in early RA. The possibility of an effect modification by smoking on the predictive value of BMI merits further investigation.

2019 White Paper on Recent Issues in Bioanalysis: Chromatographic Assays (Part 1 - Innovation in Small Molecules and Oligonucleotides & Mass Spectrometric Method Development Strategies for Large Molecule Bioanalysis).

The 2019 13th Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations on Innovation in Small Molecules and Oligonucleotides & Mass Spec Method Development Strategies for Large Molecules Bioanalysis. Part 2 (2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) and Part 3 (New Insights in Biomarkers Assays Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in drug discovery & development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and The Gene Therapy Bioanalytical Challenges) are published in volume 11 of Bioanalysis, issues 23 and 24 (2019), respectively.