Challenges and opportunities for in vitro-in vivo extrapolation of aldehyde oxidase-mediated clearance: Towards a roadmap for quantitative translation

Underestimation of AO-mediated clearance by current in vitro assays leads to uncertainty in human dose projections, thereby reducing the likelihood of success in drug development. In the present studywe first evaluated the current drug development practices for AO substrates. Next, the overall predictive performance of in vitro-in vivo extrapolation (IVIVE) of unbound hepatic intrinsic clearance (CLint,u) and unbound hepatic intrinsic clearance by AO (CLint,u,AO) was assessed using a comprehensiveliterature database of in vitro (human cytosol/ S9/ hepatocytes) and in vivo (iv/oral) data collated for 22AO substrates (total of 100 datapoints from multiple studies). Correction for unbound fraction in the incubation (fuinc) was done by experimental data or in silico predictions. The fraction metabolized by AO (fmAO) determined via in vitro/in vivo approaches was found to be highly variable. The geometric mean fold errors (gmfe) for scaled CLint,u (mL/min/kg) were 10.4 for human hepatocytes, 5.6 for human liver cytosols, and 5.0 for human liver S9, respectively. Application of these gmfe’s as empirical scaling factors improved predictions (45-57% within 2-fold of observed) compared with no correction (11-27% within 2-fold), with the scaling factors qualified by leave-one-out cross-validation. A road map for quantitative translation was then proposed following a critical evaluation on the in vitro and clinical methodology to estimate in vivo fmAO. In conclusion, the study provides the most robust system-specific empirical scaling factors to-date as a pragmatic approach for the prediction of in vivo CLint,u,AO in the early stages of drug development

Group II Intron-Anchored Gene Deletion in Clostridium

Clostridium plays an important role in commercial and medical use, for which targeted gene deletion is difficult. We proposed an intron-anchored gene deletion approach for Clostridium, which combines the advantage of the group II intron “ClosTron” system and homologous recombination. In this approach, an intron carrying a fragment homologous to upstream or downstream of the target site was first inserted into the genome by retrotransposition, followed by homologous recombination, resulting in gene deletion. A functional unknown operon CAC1493–1494 located in the chromosome, and an operon ctfAB located in the megaplasmid of C. acetobutylicum DSM1731 were successfully deleted by using this approach, without leaving antibiotic marker in the genome. We therefore propose this approach can be used for targeted gene deletion in Clostridium. This approach might also be applicable for gene deletion in other bacterial species if group II intron retrotransposition system is established

LEARN: A multi-centre, cross-sectional evaluation of Urology teaching in UK medical schools

OBJECTIVE: To evaluate the status of UK undergraduate urology teaching against the British Association of Urological Surgeons (BAUS) Undergraduate Syllabus for Urology. Secondary objectives included evaluating the type and quantity of teaching provided, the reported performance rate of General Medical Council (GMC)-mandated urological procedures, and the proportion of undergraduates considering urology as a career. MATERIALS AND METHODS: LEARN was a national multicentre cross-sectional study. Year 2 to Year 5 medical students and FY1 doctors were invited to complete a survey between 3rd October and 20th December 2020, retrospectively assessing the urology teaching received to date. Results are reported according to the Checklist for Reporting Results of Internet E-Surveys (CHERRIES). RESULTS: 7,063/8,346 (84.6%) responses from all 39 UK medical schools were included; 1,127/7,063 (16.0%) were from Foundation Year (FY) 1 doctors, who reported that the most frequently taught topics in undergraduate training were on urinary tract infection (96.5%), acute kidney injury (95.9%) and haematuria (94.4%). The most infrequently taught topics were male urinary incontinence (59.4%), male infertility (52.4%) and erectile dysfunction (43.8%). Male and female catheterisation on patients as undergraduates was performed by 92.1% and 73.0% of FY1 doctors respectively, and 16.9% had considered a career in urology. Theory based teaching was mainly prevalent in the early years of medical school, with clinical skills teaching, and clinical placements in the later years of medical school. 20.1% of FY1 doctors reported no undergraduate clinical attachment in urology. CONCLUSION: LEARN is the largest ever evaluation of undergraduate urology teaching. In the UK, teaching seemed satisfactory as evaluated by the BAUS undergraduate syllabus. However, many students report having no clinical attachments in Urology and some newly qualified doctors report never having inserted a catheter, which is a GMC mandated requirement. We recommend a greater emphasis on undergraduate clinical exposure to urology and stricter adherence to GMC mandated procedures

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Establishing a physiologically-based pharmacokinetic framework for aldehyde oxidase and dual aldehyde oxidase-CYP substrates

Abstract Aldehyde oxidase (AO) contributes to the clearance of many approved and investigational small molecule drugs, which are often dual substrates of AO and drug‐metabolizing enzymes such as cytochrome P450s (CYPs). As such, the lack of established framework for quantitative translation of the clinical pharmacologic correlates of AO‐mediated clearance represents an unmet need. This study aimed to evaluate the utility of physiologically based pharmacokinetic (PBPK) modeling in the development of AO and dual AO‐CYP substrates. PBPK models were developed for capmatinib, idelalisib, lenvatinib, zaleplon, ziprasidone, and zoniporide, incorporating in vitro functional data from human liver subcellular fractions and human hepatocytes. Prediction of metabolic elimination with/without the additional empirical scaling factors (ESFs) was assessed. Clinical pharmacokinetics, human mass balance, and drug–drug interaction (DDI) studies with CYP3A4 modulators, where available, were used to refine/verify the models. Due to the lack of clinically significant AO‐DDIs with known AO inhibitors, the fraction metabolized by AO (fmAO) was verified indirectly. Clearance predictions were improved by using ESFs (GMFE ≤1.4‐fold versus up to fivefold with physiologically‐based scaling only). Observed fmi from mass balance studies were crucial for model verification/refinement, as illustrated by capmatinib, where the fmAO (40%) was otherwise underpredicted up to fourfold. Subsequently, independent DDI studies with ketoconazole, itraconazole, rifampicin, and carbamazepine verified the fmCYP3A4, with predicted ratios of the area under the concentration–time curve (AUCR) within 1.5‐fold of the observations. In conclusion, this study provides a novel PBPK‐based framework for predicting AO‐mediated pharmacokinetics and quantitative assessment of clinical DDI risks for dual AO‐CYP substrates within a totality‐of‐evidence approach

The first pilot project of the consortium for top-down proteomics: A status report

Pilot Project #1—the identification and characterization of human histone H4 proteoforms by top-down MS—is the first project launched by the Consortium for Top-Down Proteomics (CTDP) to refine and validate top-down MS. Within the initial results from seven participating laboratories, all reported the probability-based identification of human histone H4 (UniProt accession P62805) with expectation values ranging from 10(−13) to 10(−105). Regarding characterization, a total of 74 proteoforms were reported, with 21 done so unambiguously; one new PTM, K79ac, was identified. Inter-laboratory comparison reveals aspects of the results that are consistent, such as the localization of individual PTMs and binary combinations, while other aspects are more variable, such as the accurate characterization of low-abundance proteoforms harboring >2 PTMs. An open-access tool and discussion of proteoform scoring are included, along with a description of general challenges that lie ahead including improved proteoform separations prior to mass spectrometric analysis, better instrumentation performance, and software development