Measure Of Potency Research Articles

Kinact/KI Value Determination for Penicillin-Binding Proteins in Live Cells.

Penicillin-binding proteins (PBPs) are an essential family of bacterial enzymes that are covalently inhibited by the β-lactam class of antibiotics. PBP inhibition disrupts peptidoglycan biosynthesis, which results in deficient growth and proliferation, and ultimately leads to lysis. IC50 values are often employed as descriptors of enzyme inhibition and inhibitor selectivity, but can be misleading in the study of time-dependent, covalent inhibitors. Due to this disconnect, the second-order rate constant, kinact/KI, is a more appropriate metric of covalent-inhibitor potency. Despite being the gold standard measurement of potency, kinact/KI values are typically obtained from in vitro assays, which limits assay throughput if investigating an enzyme family with multiple homologues (such as the PBPs). Therefore, we developed a whole-cell kinact/KI assay to define inhibitor potency for the PBPs in Streptococcus pneumoniae using the fluorescent, activity-based probe, Bocillin-FL. Our results align with in vitro kinact/KI data and show a comparable relationship to previously established IC50 values. These results support the validity of our in vivo kinact/KI method as a means of obtaining β-lactam potency for a suite of PBPs to enable structure-activity relationship studies.

Collaborative study on the cross-reactivity of two influenza B viral components in single radial immunodiffusion assay using quadrivalent influenza vaccines in Japan from 2015/16 to 2021–22 influenza season

A quadrivalent influenza vaccine (QIV) has been available in Japan since the 2015/2016 influenza season. Single radial immunodiffusion (SRID) assays are currently used worldwide to measure the hemagglutinin (HA) content of influenza vaccine components because they are simple, accurate, and the regulatory requirement, ensuring consistency in manufacture for the HA content. However, the cross-reactivity of antisera against the two lineages of the influenza B virus (IFVB) may cause inaccurate quantification of HA content in QIVs using the SRID assay. To examine cross-reactivity and develop an appropriate procedure for accurate measurement of vaccine potency, a collaborative study with four Japanese vaccine manufacturers was conducted to measure the HA contents of trivalent influenza vaccines (TIVs) and QIVs by SRID assay with a single and a mixture of reference antigens (refAgs) from each lineage of IFVB for seven influenza seasons from 2015/16 to 2021/22. The cross-reactivity of the two IFVB components in the SRID assay varied depending on the vaccine viruses. Our study demonstrated that it is useful to validate a suitable combination for each refAg and reference antiserum by selecting the combination showing similar HA contents between experimental TIV and QIV before lot release testing.

A human monoclonal antibody based immunoenzymetric assay to measure Fel d 1 concentrations in cat hair and pelt allergenic extracts.

In the United States, 19 allergen extracts of different specificities are standardized, which means that their potencies are determined in comparison to a US reference standard. For cat allergen extracts, potency is determined by measuring Fel d 1 content expressed in in Fel d 1 units, and with a unitage that correlates with skin test reactions (bioequivalent allergy units or BAU). Currently, Fel d 1 content is measured with a radial immunodiffusion (RID) assay that uses polyclonal sheep antisera to detect the allergenic protein by producing a white precipitin line in agar gel. However, the RID is considered cumbersome, and the polyclonal sera may qualitatively vary among animals and may recognize epitopes irrelevant to human allergic disease. In this report, we describe a quantitative two-site immunoenzymetric assay (IEMA) for Fel d 1 that uses immobilized capture and soluble biotin-labeled detection Fel d 1-specific human IgE monoclonal antibodies (mAb) that have been class-switched to IgG4. Together, they sandwich Fel d 1 molecules from extracts. Using purified natural Fel d 1 as a calibrator, the historically reported ∼4 micrograms Fel d 1/Fel d 1 unit assignment was directly measured in this mAb-based IEMA at 3.12 ± 0.24 micrograms of Fel d 1 per Fel d 1 unit. This IEMA appears to be equivalent to RID in the measurement of biological potencies of commercial cat hair and cat pelt extracts marketed in the United States.

Evaluating the Improvement of Blend Potency Measurements in the Feed Frame of a Rotary Tablet Press Using Combined NIR and Raman Spectroscopy.

This study investigated the added value of combining both near-infrared (NIR) and Raman spectroscopy into a single NIRaman Combi Fiber Probe for in-line blend potency determination in the feed frame of a rotary tablet press. A five-component platform formulation was used, containing acetylsalicylic acid as the Active Pharmaceutical Ingredient (API). Calibration models for the determination of 1 and 5%w/w label claim tablets were developed using NIR and Raman spectra of powder blends ranging from 0.75 to 1.25%w/w and 3.75 to 6.25%w/w API, respectively. Step-change experiments with deliberate 10% deviation steps from the label claims were performed, from which the collected spectra were used for model validation. For model development and validation, low-level data fusion was explored through concatenation of preprocessed NIR and Raman spectra. Mid-level data fusion was also evaluated, based on extracted features of the preprocessed data. Herewith, score vectors were extracted by transforming preprocessed spectra through Principal Component Analysis, followed by critical feature selection through Elastic Net Regression. Partial Least Squares regression was applied to regress singular, low-level or mid-level fused data versus blend potency. It could be concluded that irrespective of the data fusion technique, an increase in Step-Change Sensitivity (SCS) and decrease in Root Mean Squared Error (RMSE) was observed when predicting the 5%w/w step-change experiment. For the prediction of the 1%w/w step-change experiment, no added benefit with regard to SCS and RMSE was observed due to the addition of the noisy NIR spectra.

Automated ELISA for potency measurements of therapeutic antibodies and antibody fragments

Potency assays are essential for the development and quality control of biopharmaceutical drugs, but they are often a time limiting factor due to manual handling steps and consequently low analytical throughput. On the other hand, automation of potency assays can be challenging due to their complexity and the use of biological materials. ELISA (enzyme-linked immunosorbent assay) is widely used for potency determination and is a good candidate for automation as all ELISA types depend on the same basic steps: coating, blocking, sample incubation, detection, and signal measurement. Nevertheless, ELISA for relative potency measurements still require drug-specific development and assay validation thereby complicating automation efforts. To simplify potency testing by ELISA, we first developed a manual protocol generally applicable to different drugs and then adapted this protocol for automated measurements. We identified unexpected critical parameters which had to be adapted to transfer the manual ELISA to an automated liquid handling system and we demonstrated that gravimetric sample dilution is unnecessary with the automated protocol. Both manual and automated protocols were validated and compared using multiple biotherapeutics. The automated protocol showed similar or higher precision and accuracy when compared to the manual method.

Impacts of priming on distinct immunosuppressive mechanisms of mesenchymal stromal cells under translationally relevant conditions

BackgroundThe multimodal properties of mesenchymal stromal cells (MSCs), particularly their ability to modulate immune responses is of high interest in translational research. Pro-inflammatory, hypoxic, and 3D culture priming are promising and often used strategies to improve the immunosuppressive potency of MSCs, but the underlying mechanisms are not well understood. Therefore, the aims of this study were (i) to compare the effects of pro-inflammatory, hypoxic, and 3D culture priming on the in vitro immunosuppressive potential of MSCs, (ii) to assess if immunosuppressive priming effects are temporally preserved under standard and translationally relevant culture conditions, and (iii) to investigate if the three priming strategies engage the same immunosuppressive mechanisms.MethodsFunctional in vitro T cell suppressive potency measurements were conducted to assess the impact of pro-inflammatory, hypoxic, and 3D culture priming on the immunosuppressive potential of human bone marrow-derived MSCs. Primed MSCs were either cultured under standard cell culture conditions or translationally relevant culture conditions, and their transcriptomic adaptations were monitored over time. Next-generation sequencing was performed to assess if different priming strategies activate distinct immunosuppressive mechanisms.Results(i) Pro-inflammatory, hypoxic, and 3D culture priming induced profound transcriptomic changes in MSCs resulting in a significantly enhanced T cell suppressive potential of pro-inflammatory and 3D culture primed MSCs. (ii) Priming effects rapidly faded under standard cell culture conditions but were partially preserved under translationally relevant conditions. Interestingly, continuous 3D culture priming of MSCs maintained the immunosuppressive potency of MSCs. (iii) Next-generation sequencing revealed that priming strategy-specific differentially expressed genes are involved in the T cell suppressive capacity of MSCs, indicating that different priming strategies engage distinct immunosuppressive mechanisms.ConclusionPriming can be a useful approach to improve the immunosuppressive potency of MSCs. However, future studies involving primed MSCs should carefully consider the significant impact of translationally relevant conditions on the preservation of priming effects. Continuous 3D culture could act as a functionalized formulation, supporting the administration of MSC spheroids for a sustainably improved immunosuppressive potency.

Associations between hair-derived cannabinoid levels, self-reported use, and cannabis-related problems

RationaleAs cannabis potency and cannabis use are increasing in newly legalized markets, it is increasingly important to measure and examine the effects of cannabinoid exposure.ObjectivesThe current study aims to assess how hair-derived cannabinoid concentrations – offering insight into three-month cumulative exposure – are associated with common self-report measures of cannabis use and cannabis use-related problems.Methods74 near-daily dependent cannabis users self-reported their quantity of cannabis use, cannabis use-related problems, and estimated cannabis potency. Hair samples were provided to quantify Δ9-THC, CBD, and CBN using LC–MS/MS and THC-consumption was verified by analyzing THC-COOH in hair using GC–MS/MS.ResultsCannabinoids were detectable in 95.95% of the hair samples from individuals who tested positive on a urine screen for cannabis. Δ9-THC concentrations were positively associated with measures of self-reported potency (relative potency, potency category, and perceived ‘high’), but Δ9-THC, CBD, CBN concentrations and THC/CBD ratio were not associated with self-reported quantity of use. Self-reported potency, but not hair-derived concentrations, were associated with withdrawal and craving. Self-reported quantity of cannabis use, but not cannabinoid concentrations, were associated with cannabis use-related problems.ConclusionsThe use of hair-derived cannabinoid quantification is supported for detecting cannabis use in near-daily users, but the lack of associations between hair-derived cannabinoid concentrations and self-report measures of use does not support the use of hair analyses alone for quantification of cannabinoid exposure. Further research comparing hair-derived cannabinoid concentrations with other biological matrices (e.g. plasma) and self-report is necessary to further evaluate the validity of hair analyses for this purpose.

Fully automated high-throughput immuno-µPlaque assay for live-attenuated tetravalent dengue vaccine development.

Dengue fever has remained a continuing global medical threat that impacts half of the world's population. Developing a highly effective dengue vaccine, with live-attenuated tetravalent vaccines as leading candidates, remains essential in preventing this disease. For the development of live virus vaccines (LVVs), potency measurements play a vital role in quantifying the active components of vaccine drug substance as well as drug product during various stages of research, development, and post-licensure evaluations. Traditional plaque-based assays are one of the most common potency test methods, but they generally take up to weeks to complete. Less labor and time-intensive potency assays are thus called for to aid in the acceleration of vaccine development, especially for multivalent LVVs. Here, we introduce a fully automated, 96-well format µPlaque assay that has been optimized as a high-throughput tool to evaluate process and formulation development of a live-attenuated tetravalent dengue vaccine. To the best of our knowledge, this is the first report of a miniaturized viral plaque method for dengue with full automation via an integrated robotic system. Compared to the traditional manual plaque assay, this newly developed method substantially reduces testing time by approximately half and allows for the evaluation of over ten times more samples per run. The fully automated workflow, from cell culture to plaque counting, significantly minimizes analyst hands-on time and improves assay repeatability. The study presents a pioneering solution for the rapid measurement of LVV viral titers, offering promising prospects for advancing vaccine development through high-throughput analytics.

Aldehyde Dehydrogenase Enzyme in Relation to Cord Blood stem cells Potency

Abstract Introduction/Objective In umbilical cord blood transplantation (UCBT), the number of cluster of differentiation (CD)34+ cells and colony-forming units (CFUs) in the cord blood (CB) graft positively correlate with patient survival. Since aldehyde dehydrogenase (ALDH) activity is high in hematopoietic stem cells, the number of ALDH-bright (ALDHbr) cells was examined in comparison with the number of CD34+ cells and CFUs for the quality assessment of CB units, Aim and objectives; to correlate the aldehyde dehydrogenase (ALDH) enzyme assay with colony-forming unit–granulocyte/macrophage (CFU-GM) assay before cryopreservation of cord blood buffy coats (CBBC), in order to investigate the hypothesis that the ALDH assay might exclude apoptotic cells since it is based on enzyme activity. Methods/Case Report This study was carried out at Benha University Hospital on Fifty (50) samples of Human Umbilical Cord Blood which were processed for mononuclear cells (MNC) separation by density gradient centrifugation method using Ficoll-Paque media that layered on the diluted blood. In parallel, 1 million nucleated cells were sampled and prepared for flow cytometry for analysis of ALDH activity according to the manufacturer’s instructions. Results (if a Case Study enter NA) CD34+ ALDH+ (%) (Of MNC) showed significant positive correlation with ALDH++ (%) (Of CD34+) and numbers of total CFU/40000 cells. Also ALDH++ (%) (Of CD34+) showed significant positive correlation with numbers of total CFU/40000 cells. Conclusion The ALDH assay excludes nonviable and apoptotic cells, and therefore correlates well with CFU enumeration compared to the number of viable CD34+ cells. We propose that the ALDH assay might replace the CFU-GM method in CBU potency measurements.

Use of the TD50 99 % CI for single dose rodent carcinogenicity studies

The Tumorigenic dose 50 (TD50) is a widely used measure of carcinogenic potency representing the dosage at which 50 % of animals exposed to a compound will develop tumours. The popularity of the TD50 is in part due to the large amount of publicly available data. TD50s were calculated for a large number of compounds in the Carcinogenic Potency Database, which has since been extended in the freely available Lhasa Carcinogenicity database, containing TD50s from over 7500 studies and 1700 compounds. However, due to the age of these studies many are of low quality, often comprising only a single test dosage, therefore raising questions about the applicability of such TD50 sfor toxicological risk assessment. We investigate whether the lower 99 % confidence interval is sufficient to produce conservative TD50 estimates for these studies.

A synbiotic made of an autochthonous Enterococcus durans strain and microbial polysaccharides improves broiler chicken health

PurposeThe gastrointestinal tract (GI) of organically fed desi chickens fosters a microbiota that can exclude pathogens and be potentially probiotic for improving the gut health of commercial chickens. MethodsA panel of strains were selectively cultured from desi chicken intestine and screened for their antagonism towards specific pathogens. Treating the culture free supernatant (CFS) of such strains with NaOH and proteases gives a picture of the antibacterial compound's biochemistry and determining its minimum inhibitory concentration (MIC) provides a measure of potency. The most strongly antagonistic strains were tested in vitro for surviving the chicken GIT. Safety was examined using mucin degradation, gelatin liquefaction, hemolysis, antibiotic susceptibility and biogenic amine production. An in vivo trial was done on young male Ross chickens to evaluate the performance of a bespoke synbiotic formulation comprising of an Enterococcus durans strain (1x108 CFUg−1) with prebiotics Dextran or Levan (0.1% w/v) and with both and assessing its impact on broiler gut microbiota, body and carcass weight, histopathology of organ tissues, and biochemical markers. ResultsA panel of Enterococcus spp. (NPL1315-NPL1334) was obtained and the selected strains (NLP-1319, 20, 24, 27, 34) showed good bile, acid and phenol survival, and cell surface hydrophobicity but not autoaggregation and none of them were deemed unsafe. The synbiotic-fed chickens were significantly heavier, with lowered serum cholesterol, triglycerides and glucose levels and a lesser load of pathogens than the controls. ConclusionDesi chicken intestine can be a viable source of probiotics with good antagonistic potential capable of replacing antibiotics in poultry feed.

Unique allergen-specific human IgE monoclonal antibodies derived from patients with allergic disease.

Allergic reactions are mediated by human IgE antibodies that bind to and cross-link allergen molecules. The sites on allergens that are recognized by IgE antibodies have been difficult to investigate because of the paucity of IgE antibodies in a human serum. Here, we report the production of unique human IgE monoclonal antibodies to major inhaled allergens and food allergens that can be produced at scale in perpetuity. The IgE antibodies were derived from peripheral blood mononuclear cells of symptomatic allergic patients, mostly children aged 3-18 years, using hybridoma fusion technology. Total IgE and allergen-specific IgE was measured by ImmunoCAP. Their specificity was confirmed through ELISA and immunoblotting. Allergenic potency measurements were determined by ImmunoCAP inhibition. Biological activity was determined in vitro by comparing β-hexosaminidase release from a humanized rat basophilic cell line. Human IgE monoclonal antibodies (n = 33) were derived from 17 allergic patients with symptoms of allergic rhinitis, asthma, atopic dermatitis, food allergy, eosinophilic esophagitis, or red meat allergy. The antibodies were specific for five inhaled allergens, nine food allergens, and alpha-gal and had high levels of IgE (53,450-1,702,500 kU/L) with ratios of specific IgE to total IgE ranging from <0.01 to 1.39. Sigmoidal allergen binding curves were obtained through ELISA, with low limits of detection (<1 kU/L). Allergen specificity was confirmed through immunoblotting. Pairs of IgE monoclonal antibodies to Ara h 6 were identified that cross-linked after allergen stimulation and induced release of significant levels of β-hexosaminidase (35%-80%) from a humanized rat basophilic cell line. Human IgE monoclonal antibodies are unique antibody molecules with potential applications in allergy diagnosis, allergen standardization, and identification of allergenic epitopes for the development of allergy therapeutics. The IgE antibody probes will enable the unequivocal localization and validation of allergenic epitopes.

Electrostatics-Based Computational Design and Experimental Analysis of Buforin II Antimicrobial Peptide Variants with Increased DNA Affinities.

Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics in the treatment of bacterial infections in part due to their targeting of generic bacterial structures that make it more difficult to develop drug resistance. In this study, we introduce and implement a design workflow to develop more potent AMPs by improving their electrostatic interactions with DNA, which is a putative intracellular target. Using the existing membrane-translocating AMP buforin II (BF2) as a starting point, we use a computational workflow that integrates electrostatic charge optimization, continuum electrostatics, and molecular dynamics simulations to suggest peptide positions at which a neutral BF2 residue could be substituted with arginine to increase DNA-binding affinity either significantly or minimally, with the latter choice done to determine whether AMP binding affinity depends on charge distribution and not just overall monopole. Our analyses predicted that T1R and L8R BF2 variants would yield substantial and minimal increases in DNA-binding affinity, respectively. These predictions were validated with experimental peptide-DNA binding assays with additional computational analyses providing structural insights. Additionally, experimental measurements of antimicrobial potency showed that a design to increase DNA binding can also yield greater potency. As a whole, this study takes initial steps to support the idea that (i) a design strategy aimed to increase AMP binding affinity to DNA by focusing only on electrostatic interactions can improve AMP potency and (ii) the effect on DNA binding of increasing the overall peptide monopole via arginine substitution depends on the position of the substitution. More broadly, this design strategy is a novel way to increase the potency of other membrane-translocating AMPs that target nucleic acids.

Characterizing On-Chip Angiogenesis Induction in a Microphysiological System as a Functional Measure of Mesenchymal Stromal Cell Bioactivity.

Mesenchymal stromal cells (MSCs) continue to be proposed for clinical investigation to treat myriad diseases given their purported potential to stimulate endogenous regenerative processes, such as angiogenesis. However, MSC functional heterogeneity has hindered clinical success and still poses a substantial manufacturing challenge from a product quality control perspective. Here, a quantitative bioassay based on an enhanced-throughput is described, microphysiological system (MPS) to measure the specific bioactivity of MSCs to stimulate angiogenesis as a potential measure of MSC potency. Using this novel bioassay, MSCs derived from multiple donors at different passages are co-cultured with human umbilical vein endothelial cells and exhibit significant heterogeneity in angiogenic potency between donors and cell passage. Depending on donor source and cellular passage number, MSCs varied in their ability to stimulate tip cell dominant or stalk cell dominant phenotypes in angiogenic sprout morphology which correlated with expression levels of hepatocyte growth factor (HGF). These findings suggest that MSC angiogenic bioactivity may be considered as a possible potency attribute in MSC quality control strategies. Development of a reliable and functionally relevant potency assay for measuring clinically relevant potency attributes of MSCs will help to improve consistency in quality and thereby, accelerate clinical development of these cell-based products.

Design, synthesis and evaluation of novel 2-phenyl-3-(1H-pyrazol-4-yl)pyridine positive allosteric modulators for the M4 mAChR

Translation of muscarinic acetylcholine receptor (mAChR) agonists into clinically used therapeutic agents has been difficult due to their poor subtype selectivity. M4 mAChR subtype-selective positive allosteric modulators (PAMs) may provide better therapeutic outcomes, hence investigating their detailed pharmacological properties is crucial to advancing them into the clinic. Herein, we report the synthesis and comprehensive pharmacological evaluation of M4 mAChR PAMs structurally related to 1e, Me–C-c, [11C]MK-6884 and [18F]12. Our results show that small structural changes to the PAMs can result in pronounced differences to baseline, potency (pEC50) and maximum effect (Emax) measures in cAMP assays when compared to the endogenous ligand acetylcholine (ACh) without the addition of the PAMs. Eight selected PAMs were further assessed to determine their binding affinity and potential signalling bias profile between cAMP and β-arrestin 2 recruitment. These rigorous analyses resulted in the discovery of the novel PAMs, 6k and 6l, which exhibit improved allosteric properties compared to the lead compound, and probative in vivo exposure studies in mice confirmed that they maintain the ability to cross the blood-brain barrier, making them more suitable for future preclinical assessment.

Correlating physicochemical and biological properties to define critical quality attributes of a rAAV vaccine candidate

Recombinant adeno-associated viruses (rAAVs) are a preferred vector system in clinical gene transfer. A fundamental challenge to formulate and deliver rAAVs as stable and efficacious vaccines is to elucidate interrelationships between the vector's physicochemical properties and biological potency. To this end, we evaluated an rAAV-based coronavirus disease 2019 (COVID-19) vaccine candidate that encodes the Spike antigen (AC3) and is produced by a commercially viable process. First, state-of-the-art analytical techniques were employed to determine key structural attributes of AC3, including primary and higher-order structures, particle size, empty/full capsid ratios, aggregates, and multi-step thermal degradation pathway analysis. Next, several quantitative potency measures for AC3 were implemented, and data were correlated with the physicochemical analyses on thermally stressed and control samples. Results demonstrate links between decreasing AC3 physical stability profiles, invitro transduction efficiency in a cell-based assay, and, importantly, invivo immunogenicity in a mouse model. These findings are discussed in the general context of future development of rAAV-based vaccine candidates as well as specifically for the rAAV vaccine application under study.

Development and qualification of cell-based relative potency assay for a human respiratory syncytial virus (RSV) mRNA vaccine

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections worldwide. A safe and effective RSV vaccine has been an elusive goal but recent advances in vaccine technology have improved the likelihood that a vaccine for the prevention of RSV could be licensed in near future. We have developed an RSV vaccine V171 consisting of four lipids and messenger ribonucleic acid (mRNA) encoding an engineered form of the RSV F protein stabilized in its prefusion conformation. The lipids form lipid nanoparticles (LNP) with mRNA encapsulated during process, which protects the mRNA from degradation and enables the mRNA to be delivered into mammalian cells. Once inside the cells, the mRNA then can be translated into RSV F protein and elicit both humoral and cellular immune responses. Preclinical results and Phase I clinical trial results indicate that this mRNA vaccine targeting RSV F protein is a promising RSV vaccine approach and should be further evaluated in clinical trials. We have developed a cell-based relative potency assay to support the Phase II development of this vaccine. Test articles and a reference standard are tested with serial dilutions in a 96-well plate pre-seeded with Hep G2 cells. Cells were incubated for 16–18 h after transfection and then permeabilized and stained with a human monoclonal antibody specific to RSV F protein, followed by a fluorophore-conjugated secondary antibody. The plate is then analyzed for percentage of transfected cells and relative potency of the test article is calculated by comparing its EC50 to that of a reference standard. This assay takes advantage of the fact that due to the inherent variability in biological test systems an absolute measure of potency is more variable than a measure of activity relative to a standard. Targeting testing relative potency range 25–250 %, our assay showed an R2 close to 1 for linearity, relative bias of 1.05–5.41 %, and intermediate precision of 11.0 %. The assay has been used for testing of process development samples, formulation development samples, as well as drug product intermediate (DPI) and drug product (DP) in support of Phase II development of our RSV mRNA vaccine.

Specificity of the local lymph node assay (LLNA) for skin sensitisation

The local lymph node assay (LLNA) has provided a large dataset against which performance of non-animal approaches for prediction of skin sensitisation potential and potency can be assessed. However, a recent comparison of LLNA results with human data has argued that LLNA specificity is low, with many human non-sensitisers, particularly hydrophobic chemicals, being false positives. It has been suggested that such putative false positives result from hydrophobic chemicals causing cytotoxicity, which induces irritancy, in turn driving non-specific lymphocyte proliferation. This paper finds that the apparent reduced specificity of the LLNA largely reflects differences in definitions of the boundaries between weak skin sensitisers and non-sensitisers. A small number of LLNA false positives may be due to lymphocyte proliferation without skin sensitisation, but most alleged ‘false’ positives are in fact very weak sensitisers predictable from structure-activity considerations. The evidence does not support the hypothesis for hydrophobicity-induced false positives. Moreover, the mechanistic basis is untenable. Sound LLNA data, appropriately interpreted, remain a good measure of sensitisation potency, applicable across a wide hydrophilicity-hydrophobicity range. The standard data interpretation protocol enables detection of very low levels of sensitisation, irrespective of regulatory significance, but there is scope to interpret the data to give focus on regulatory significance.

High potency cannabis use, mental health symptoms and cannabis dependence: Triangulating the evidence

BackgroundCannabis potency (concentration of Δ-9-Tetrahydrocannabinol) has been associated with risks of adverse mental health outcomes and addiction but no studies have triangulated evidence from self-report and objective measures of cannabis potency. We hypothesised that users of high potency cannabis would have higher levels of (a) anxiety, (b) depression and (c) psychosis-like symptoms (d) cannabis dependence than users of lower potency cannabis. MethodsA cross-sectional study of 410 participants donated a sample of cannabis for analysis of THC concentration and reported their cannabis potency preference. These two exposure measures were investigated for their association with cannabis dependence, depression, anxiety, and psychosis-like symptoms in separate linear/logistic regression models. ResultsHigh potency cannabis preference was associated with a slight increased risk of cannabis dependence after adjusting for confounding, with the exception of cannabis use frequency (OR = 1.16, 95% CI 1.04–1.28). No association was found between THC concentration in cannabis and cannabis dependence. There was weak evidence of a small association between cannabis potency and depression and anxiety. There was no association between high potency cannabis preference or THC concentration in cannabis and psychosis-like symptoms. ConclusionsUsers of cannabis who preferred high potency types might be at increased risk of problematic cannabis use. This should be considered with caution as we were not able to triangulate these results with an objective measure of cannabis potency. More research is needed to understand the association between high potency cannabis use and depression and anxiety.

SIC50: Determining drug inhibitory concentrations using a vision transformer and an optimized Sobel operator

As a measure of cytotoxic potency, half-maximal inhibitory concentration (IC50) is the concentration at which a drug exerts half of its maximal inhibitory effect against target cells. It can be determined by various methods that require applying additional reagents or lysing the cells. Here, we describe a label-free Sobel-edge-based method, which we name SIC50, for the evaluation of IC50. SIC50 classifies preprocessed phase-contrast images with a state-of-the-art vision transformer and allows for the continuous assessment of IC50 in a faster and more cost-efficient manner. We have validated this method using four drugs and 1,536-well plates and also built a web application. We anticipate that this method will assist in the high-throughput screening ofchemical libraries (e.g., small-molecule drugs, small interfering RNA [siRNA], and microRNA and drug discovery).