EULAR points to consider for therapeutic drug monitoring of biopharmaceuticals in inflammatory rheumatic and musculoskeletal diseases.

OBJECTIVE: To develop EULAR points-to-consider for therapeutic drug monitoring (TDM) of biopharmaceuticals in inflammatory rheumatic and musculoskeletal diseases (RMDs). METHODS: The points-to-consider were developed in accordance with EULAR standardised operation procedures by a multidisciplinary task force from eight European countries, based on a systematic literature review and expert consensus. Level of evidence and strength of the points-to-consider were determined, and mean levels of agreement among the task force were calculated using a 10-point rating scale. RESULTS: Six overarching principles and 13 points-to-consider were formulated. The level of agreement among the task force for the overarching principles and points-to-consider ranged from 8.4 to 9.9.The overarching principles define TDM and its subtypes, and reinforce the underlying pharmacokinetic/pharmacodynamic principles, which are relevant to all biopharmaceutical classes. The points-to-consider highlight the clinical utility of the measurement and interpretation of biopharmaceutical blood concentrations and antidrug antibodies in specific clinical scenarios, including factors that influence these parameters. In general, proactive use of TDM is not recommended but reactive TDM could be considered in certain clinical situations. An important factor limiting wider adoption of TDM is the lack of both high quality trials addressing effectiveness and safety of TDM and robust economic evaluation in patients with RMDs. Future research should focus on providing this evidence, as well as on further understanding of pharmacokinetic and pharmacodynamic characteristics of biopharmaceuticals. CONCLUSION: These points-to-consider are evidence-based and consensus-based statements for the use of TDM of biopharmaceuticals in inflammatory RMDs, addressing the clinical utility of TDM.

Structure of pyrazole derivatives impact their affinity, stoichiometry, and cooperative interactions for CYP2E1 complexes.

CYP2E1 plays a critical role in detoxification and carcinogenic activation of drugs, pollutants, and dietary compounds; however, these metabolic processes can involve poorly characterized cooperative interactions that compromise the ability to understand and predict CYP2E1 metabolism. Herein, we employed an array of ten azoles with an emphasis on pyrazoles to establish the selectivity of catalytic and cooperative CYP2E1 sites through binding and catalytic studies. Spectral binding studies for monocyclic azoles suggested two binding events, while bicyclic azoles suggested one. Pyrazole had moderate affinity toward the CYP2E1 catalytic site that improved when a methyl group was introduced at either position 3 or 4. The presence of methyl groups simultaneously at positions 3 and 5 blocked binding, and a phenyl group at position 3 did not improve binding affinity. In contrast, pyrazole fusion to a benzene or cyclohexane ring greatly increased affinity. The consequences of these binding events on CYP2E1 catalysis were studied through inhibition studies with 4-nitrophenol, a substrate known to bind both sites. Most pyrazoles shared a common mixed cooperative inhibition mechanism in which pyrazole binding rescued CYP2E1 from substrate inhibition. Overall, inhibitor affinities toward the CYP2E1 catalytic site were similar to those reported in binding studies, and the same trend was observed for binding at the cooperative site. Taken together, these studies identified key structural determinants in the affinity and stoichiometry of azole interactions with CYP2E1 and consequences on catalysis that further advance an understanding of the relationship between structure and function for this enzyme.

CT-P13 subcutaneous infliximab in gastroenterology and rheumatology.

The drug infliximab has been a key milestone in the treatment of inflammatory conditions such as Crohn's disease, ulcerative colitis, rheumatoid arthritis and the seronegative spondyloarthritides. Biosimilar drugs followed the originator, further improving access and diversity of therapy choice. Subcutaneous infliximab (CT-P13) holds potential for greater patient flexibility by self administration, reducing travel and hospital attendance for infusion, particularly relevant at a time of pandemic. We highlight the pharmacodynamic and pharmacokinetic basis of the subcutaneous device, clinical trials in rheumatology and gastroenterology and consider the safety and cost implications. Real-world switching data is required to confirm the efficacy data from clinical trials given the reduction in dosing flexibility compared with intravenous therapy.

Can training in musculoskeletal examination skills be effectively delivered by undergraduate students as part of the standard curriculum?

OBJECTIVES: There is a need to improve competence of musculoskeletal system (MSS) examination in medical students and junior doctors. Peer-assisted learning (PAL) is a technique whereby students learn from and with each other. This study aimed to determine whether PAL can be integrated into standard undergraduate medical curricula to improve MSS examination using the gait, arms, legs, spine (GALS) screening tool. METHODS: Fifty final-year students (trainers) were trained using GALS for MSS examination while attending a standard clinical medical attachment at Glasgow Royal Infirmary. These students delivered GALS training to a further 159 students (trainees). Pre/post-confidence questionnaire (100-mm visual analogue scale) and written feedback were obtained. Final Objective Structured Clinical Examination (OSCE) scores from an MSS station were compared with a control group of 229 students randomized to other hospitals for the standard MSS training. RESULTS: Analysis of completed trainer questionnaires (30/50) showed increased confidence in all parts of GALS after training [<47 (19) cf. >88 (12); P < 0.005]. Similarly, confidence in trainees (136/159) who answered the questionnaire increased [<43 (19) cf. >85 (15); P < 0.005]. Written comments highlighted that students would recommend PAL. OSCE results showed 84% (192/229) of students in the control group passed the MSS station, with 87% (139/159) of trainees (P = 0.3) and 100% (50/50) of trainers (P < 0.01). CONCLUSIONS: MSS examination skills are improved by integrating PAL into the undergraduate medical curriculum, with student confidence being increased, and higher OSCE scores.

The potential value of blood monitoring of biologic drugs used in the treatment of rheumatoid arthritis.

The advent of biological therapies has been a major therapeutic advance in rheumatology. Many patients now enjoy improved quality of life through better disease control. The number of therapies continues to grow both within drug class (including biosimilar drugs) and via new mechanisms. For the first time, nonbiological drugs such as small-molecule inhibitors (Janus kinase inhibitors) have shown clinical equivalence. However, clinical unmet need remains with up to a third of patients commenced on a biologic therapy having minimal or no response: (a) Generally, the first biologic used secures the best response, with likelihood of remission falling thereafter with successive therapies; (b) the success of strategy trials using biological therapies can be difficult to replicate in clinical practice due to a combination of patient factors and service limitations. Accordingly, ensuring optimization of initial treatment is an important consideration before switching to alternatives. Therapeutic drug monitoring (TDM) is the measurement of serum levels of a biologic drug with the aim of improving patient care. It is usually combined with detection of any antidrug antibodies that could neutralize the effect of the therapy. This technology has the potential to be a form of 'personalized medicine' by individualizing therapy, in particular, dosing and likelihood of sustained treatment response. It requires a clear relationship between drug dose, blood concentration and therapeutic effect. This paper will outline the technology behind TDM and unpack what we can learn from our colleagues in gastroenterology, where the adoption of TDM is at a more advanced stage than in rheumatology. It will explore and set out a number of clinical scenarios where rheumatologists might find TDM helpful in day-to-day practice. Finally, an outline is given of international developments, including regulatory body appraisals and guideline development.

Using Students' Concept-building Tendencies to Better Characterize Average-Performing Student Learning and Problem-Solving Approaches in General Chemistry.

We previously reported that students' concept-building approaches, identified a priori using a cognitive psychology laboratory task, extend to learning complex science, technology, engineering, and mathematics topics. This prior study examined student performance in both general and organic chemistry at a select research institution, after accounting for preparation. We found that abstraction learners (defined cognitively as learning the theory underlying related examples) performed higher on course exams than exemplar learners (defined cognitively as learning by memorizing examples). In the present paper, we further examined this initial finding by studying a general chemistry course using a different pedagogical approach (process-oriented guided-inquiry learning) at an institution focused on health science majors, and then extended our studies via think-aloud interviews to probe the effect concept-building approaches have on problem-solving behaviors of average exam performance students. From interviews with students in the average-achieving group, using problems at three transfer levels, we found that: 1) abstraction learners outperformed exemplar learners at all problem levels; 2) abstraction learners relied on understanding and exemplar learners dominantly relied on an algorithm without understanding at all problem levels; and 3) both concept-building-approach students had weaknesses in their metacognitive monitoring accuracy skills, specifically their postperformance confidence level in their solution accuracy.

Raynaud's of the Tongue.

We describe a 40-year-old lady who presents with a painful white tongue on exposure to the cold. The differential diagnosis and investigations are outlined with a discussion about this unusual presentation of a common problem. LEARNING POINTS: Raynaud's can affect areas of the body besides the digits.Lingual Raynaud's should be considered in patients with transient tongue pain.Alternative diagnoses should be sought if ulceration or permanent symptoms develop.

Neurosyphilis Presenting with Papillitis.

Syphilis is one of the oldest described infectious diseases in the world and is caused by the spirochete bacterium Treponema pallidum [1]. Although now a rare disease, incidence is increasing with the number of diagnoses of the disease rising in England from 1688 to 2713 between 2003 and 2012 (a 61% increase)[2]. Major outbreaks of syphilis have been documented in London, Manchester, Dublin, and Brighton particularly among men who have sex with men (MSM)[3]. Diagnosis remains difficult on account of multi-system symptoms, duration of the condition, and social stigma. LEARNING POINTS: Normal CSF pressure with indistinct optic disc margins suggests papillitis, not papilloedemaNeurosyphilis can cause various ocular symptoms and is a cause of unexplained high ESRWhen treating with penicillin, the physician should be aware of a potential Jarish-Herzheimer reaction.

Structural basis for cooperative binding of azoles to CYP2E1 as interpreted through guided molecular dynamics simulations.

CYP2E1 metabolizes a wide array of small, hydrophobic molecules, resulting in their detoxification or activation into carcinogens through Michaelis-Menten as well as cooperative mechanisms. Nevertheless, the molecular determinants for CYP2E1 specificity and metabolic efficiency toward these compounds are still unknown. Herein, we employed computational docking studies coupled to molecular dynamics simulations to provide a critical perspective for understanding a structural basis for cooperativity observed for an array of azoles from our previous binding and catalytic studies (Hartman et al., 2014). The resulting 28 CYP2E1 complexes in this study revealed a common passageway for azoles that included a hydrophobic steric barrier causing a pause in movement toward the active site. The entrance to the active site acted like a second sieve to restrict access to the inner chamber. Collectively, these interactions impacted the final orientation of azoles reaching the active site and hence could explain differences in their biochemical properties observed in our previous studies, such as the consequences of methylation at position 5 of the azole ring. The association of a second azole demonstrated significant differences in interactions stabilizing the bound complex than observed for the first binding event. Intermolecular interactions occurred between the two azoles as well as CYP2E1 residue side chains and backbone and involved both hydrophobic contacts and hydrogen bonds. The relative importance of these interactions depended on the structure of the respective azoles indicating the absence of specific defining criteria for binding unlike the well-characterized dominant role of hydrophobicity in active site binding. Consequently, the structure activity relationships described here and elsewhere are necessary to more accurately identify factors impacting the observation and significance of cooperativity in CYP2E1 binding and catalysis toward drugs, dietary compounds, and pollutants.

Off-label use of rituximab in systemic lupus erythematosus: a systematic review.

Considerable interest in the efficacy of rituximab (a monoclonal CD20 antibody) in patients with systemic lupus erythematosus (SLE) has been generated due to its unique mode of action, culminating in a series of randomized and open trials, and case reports. However, this use is off-license and two significant RCTs have reported negative findings, reopening the debate on clinical benefit. This review of the available data suggests that rituximab induces B-cell depletion in 95% of patients, and a significant reduction in disease activity is achieved with a relatively good safety profile in patients with SLE.

CYP2E1 substrate inhibition. Mechanistic interpretation through an effector site for monocyclic compounds.

In this study we offer a mechanistic interpretation of the previously known but unexplained substrate inhibition observed for CYP2E1. At low substrate concentrations, p-nitrophenol (pNP) was rapidly turned over (47 min(-1)) with relatively low K(m) (24 microM); nevertheless, at concentrations of >100 microM, the rate of pNP oxidation gradually decreased as a second molecule bound to CYP2E1 through an effector site (K(ss) = 260 microm), which inhibited activity at the catalytic site. 4-Methylpyrazole (4MP) was a potent inhibitor for both sites through a mixed inhibition mechanism. The K(i) for the catalytic site was 2.0 microM. Although we were unable to discriminate whether an EIS or ESI complex formed, the respective inhibition constants were far lower than K(ss). Bicyclic indazole (IND) inhibited catalysis through a single CYP2E1 site (K(i) = 0.12 microM). Similarly, 4MP and IND yielded type II binding spectra that reflected the association of either two 4MP or one IND molecule(s) to CYP2E1, respectively. Based on computational docking studies with a homology model for CYP2E1, the two sites for monocyclic molecules, pNP and 4MP, exist within a narrow channel connecting the active site to the surface of the enzyme. Because of the presence of the heme iron, one site supports catalysis, whereas the other more distal effector site binds molecules that can influence the binding orientation and egress of molecules for the catalytic site. Although IND did not bind these sites simultaneously, the presence of IND at the catalytic site blocked binding at the effector site.

Major histocompatibility complex class II (DR) antigen and costimulatory molecules on in vitro and in vivo activated human polymorphonuclear neutrophils.

We have previously shown that normal human peripheral blood polymorphonuclear neutrophils (PMNs) contain cytoplasmic 'stores' of three key molecules normally associated with antigen presentation and T-cell costimulation, i.e. major histocompatibility complex class II (DR) antigen, CD80 (B7-1) and CD86 (B7-2). These cytoplasmic molecules were found to translocate to the cell surface within a few minutes following cross-linking (X-L) of Mac-1: an early neutrophil activation signal. In this study we have compared X-L of Mac -1 in parallel with four other well documented in vitro neutrophil activators: phorbol myristate acetate, N-formyl methionyl leucyl phenylalanine, lipopolysaccharide, and phagocytosis of immunoglobulin G-Latex particles. In addition, we have used paired samples of neutrophils obtained from peripheral blood (as a control) and synovial fluid from patients with rheumatoid arthritis as a source of in vivo activated cells. With the exception of phagocytosis, all activators resulted in the rapid (within 30 min) generation of two populations of activated neutrophils (designated P1 and P2) based on flow-cytometry measurements of size, granularity and phenotype. Significant up-regulation of DR and costimulatory molecules was observed, predominantly on P2 cells, with all activators except phagocytosis. CD80 and CD86 were noted to respond to the various activation signals in a different pattern suggesting that their intracellular granule location may be different. Dual-staining confocal laser microscopy studies showed that CD80 is largely confined to secretory vesicles (SVs) while CD86 appears to have a much wider distribution being found in SVs and within secondary (specific) and primary (azurophilic) granules. Increased surface expression of these antigens was also observed on P2 synovial fluid neutrophils appearing as large heterogeneous clusters on the cell surface when visualized by confocal laser microscopy.

Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb.

Increased interest in using zebrafish as a model organism has led to a resurgence of fin regeneration studies. This has allowed for the identification of a large number of gene families, including signaling molecules and transcription factors, which are expressed during regeneration. However, in cases where no specific inhibitor is available for the gene product of interest, determination of a functional role for these genes has been difficult. Here we demonstrate that in vivo electroporation of morpholino oligonucleotides is a feasible approach for protein knock-down during fin regeneration. Morpholino oligonucleotides against fgfr1 and msxb were utilized and knock-down of both proteins resulted in reduced fin outgrowth. Importantly, Fgfr1 knock-down phenocopied outgrowth inhibition obtained with an Fgfr1 inhibitor. Furthermore, this method provided direct evidence for a functional role for msxb in caudal fin regeneration. Finally, knock-down of Fgfr1, but not Msxb, affected the blastemal expression of msxc, suggesting this technique can be used to determine epistasis in genetic pathways affecting regeneration. Thus, this convenient reverse genetic approach allows researchers to quickly (1) assess the function of genes known to be expressed during fin regeneration, (2) screen genes for functional relevance during fin regeneration, and (3) assign genes to the molecular pathways underlying fin regeneration.

Cre-mediated site-specific recombination in zebrafish embryos.

Cre-mediated site-specific recombination has become an invaluable tool for manipulation of the murine genome. The ability to conditionally activate gene expression or to generate chromosomal alterations with this same tool would greatly enhance zebrafish genetics. This study demonstrates that the HSP70 promoter can be used to inducibly control expression of an enhanced green fluorescent protein (EGFP) -Cre fusion protein. The EGFP-Cre fusion protein is capable of promoting recombination between lox sites in injected plasmids or in stably inherited transgenes as early as 2 hr post-heat shock induction. Finally, the levels of Cre expression achieved in a transgenic fish line carrying the HSP70-EGFP-cre transgene are compatible with viability and both male and female transgenic fish are fertile subsequent to induction of EGFP-Cre expression. Hence, our data suggests that Cre-mediated recombination is a viable means of manipulating gene expression in zebrafish.

Investigation of the kinetics and order of tyrosine phosphorylation in the T-cell receptor zeta chain by the protein tyrosine kinase Lck.

We report experiments to investigate the role of the physiologically relevant protein tyrosine kinase Lck in the ordered phosphorylation of the T-cell receptor zeta chain. Six synthetic peptides were designed based on the sequences of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the zeta chain. Preliminary 1H-NMR studies of recombinant zeta chain suggested that it is essentially unstructured and therefore that peptide mimics would serve as useful models for investigating individual ITAM tyrosines. Phosphorylation kinetics were determined for each tyrosine by assaying the transfer of 32P by recombinant Lck on to each of the peptides. The rates of phosphorylation were found to depend on the location of the tyrosine, leading to the proposal that Lck phosphorylates the six zeta chain ITAM tyrosines in the order 1N (first) > 3N > 3C > 2N > 1C > 2C (last) as a result of differences in the amino-acid sequence surrounding each tyrosine. This proposal was then tested on cytosolic, recombinant T-cell receptor zeta chain. After in vitro phosphorylation by Lck, the partially phosphorylated zeta chain was digested with trypsin. Separation and identification of the zeta chain fragments using LC-MS showed, as predicted by the peptide phosphorylation studies, that tyrosine 1N is indeed the first to be phosphorylated by Lck. We conclude that differences in the amino-acid context of the six zeta chain ITAM tyrosines affect the efficiency of their phosphorylation by the kinase Lck, which probably contributes to the distinct patterns of phosphorylation observed in vivo.

A vascular endothelial growth factor receptor-2 kinase inhibitor potentiates the activity of the conventional chemotherapeutic agents paclitaxel and doxorubicin in tumor xenograft models.

Inhibition of angiogenesis may have wide use in the treatment of cancer; however, this approach alone will not cause tumor regression but may only slow the growth of solid tumors. The clinical potential of antiangiogenic agents may be increased by combining them with conventional chemotherapeutics. 4-[4-(1-Amino-1-methylethyl)phenyl]-2-[4-(2-morpholin-4-yl-ethyl)phenylamino]pyrimidine-5-carbonitrile (JNJ-17029259) represents a novel structural class of 5-cyanopyrimidines that are orally available, selective, nanomolar inhibitors of the vascular endothelial growth factor receptor-2 (VEGF-R2) and other tyrosine kinases involved in angiogenesis, such as platelet-derived growth factor receptor, fibroblast growth factor receptor, VEGF-R1, and VEGF-R3, but have little activity on other kinase families. At nanomolar levels, JNJ-17029259 blocks VEGF-stimulated mitogen-activated protein kinase signaling, proliferation/migration, and VEGF-R2 phosphorylation in human endothelial cells; inhibits the formation of vascular sprouting in the rat aortic ring model of angiogenesis; and interferes with the development of new veins and arteries in the chorioallantoic membrane assay. At higher concentrations of 1 to 3 microM, this compound shows antiproliferative activity on cells that may contribute to its antitumor effects. JNJ-17029259 delays the growth of a wide range of human tumor xenografts in nude mice when administered orally as single-agent therapy. Histological examination revealed that the tumors have evidence of reduced vascularity after treatment. In addition, JNJ-17029259 enhances the effects of the conventional chemotherapeutic drugs doxorubicin and paclitaxel in xenograft models when administered orally in combination therapy. An orally available angiogenesis inhibitor that can be used in conjunction with standard chemotherapeutic agents to augment their activity may have therapeutic benefit in stopping the progression of cancer and preventing metastasis.