Development of a leakage impact assessment for patients with a stoma, who may be impacted by leakage.

For people living with a stoma leakage is unpredictable. Despite advances in stoma products, leakage can lead to soiling and this, along with worrying about leakage, can significantly affect patients' everyday lives and impact their quality of life. It is also associated with excessive product use and increased healthcare resources. Leakage therefore remains a major unmet need for many people living with a stoma. To address this, Coloplast Ltd in collaboration with the authors and a broader group of stoma care nurses have worked together to develop a first version of the Leakage Impact Assessment. This assessment is intended to identify patients who struggle with leakage and leakage worry, and who might benefit from the reassurance that a new digital leakage notification system, Heylo™, can provide. This article reviews the evidence for leakage and its impact on people living with a stoma and outlines the development process for the assessment.

Determining the feasibility of calculating pancreatic cancer risk scores for people with new-onset diabetes in primary care (DEFEND PRIME): study protocol.

INTRODUCTION: Worldwide, pancreatic cancer has a poor prognosis. Early diagnosis may improve survival by enabling curative treatment. Statistical and machine learning diagnostic prediction models using risk factors such as patient demographics and blood tests are being developed for clinical use to improve early diagnosis. One example is the Enriching New-onset Diabetes for Pancreatic Cancer (ENDPAC) model, which employs patients' age, blood glucose and weight changes to provide pancreatic cancer risk scores. These values are routinely collected in primary care in the UK. Primary care's central role in cancer diagnosis makes it an ideal setting to implement ENDPAC but it has yet to be used in clinical settings. This study aims to determine the feasibility of applying ENDPAC to data held by UK primary care practices. METHODS AND ANALYSIS: This will be a multicentre observational study with a cohort design, determining the feasibility of applying ENDPAC in UK primary care. We will develop software to search, extract and process anonymised data from 20 primary care providers' electronic patient record management systems on participants aged 50+ years, with a glycated haemoglobin (HbA1c) test result of ≥48 mmol/mol (6.5%) and no previous abnormal HbA1c results. Software to calculate ENDPAC scores will be developed, and descriptive statistics used to summarise the cohort's demographics and assess data quality. Findings will inform the development of a future UK clinical trial to test ENDPAC's effectiveness for the early detection of pancreatic cancer. ETHICS AND DISSEMINATION: This project has been reviewed by the University of Surrey University Ethics Committee and received a favourable ethical opinion (FHMS 22-23151 EGA). Study findings will be presented at scientific meetings and published in international peer-reviewed journals. Participating primary care practices, clinical leads and policy makers will be provided with summaries of the findings.

Assessing the potential of biopesticides to control the cabbage stem flea beetle Psylliodes chrysocephala.

BACKGROUND: Cabbage stem flea beetle (CSFB) is an economically important pest of oilseed rape crops in Europe that was effectively controlled by neonicotinoid insecticide seed treatments until they were banned by the European Union in 2013. Since then, CSFB has been a difficult pest to control effectively, in part due to many populations having developed resistance to pyrethroids, the only authorized insecticides used to control this pest in many countries. Alternative solutions are therefore necessary, such as biopesticides. We tested an entomopathogenic fungus, three entomopathogenic bacteria isolates, two fatty acids and azadirachtin against CSFB adults under laboratory conditions. We also tested the efficacy of the pyrethroid insecticide lambda-cyhalothrin. RESULTS: Fatty acids were effective, with up to 100% CSFB mortality after 24 h. The entomopathogenic fungus Beauveria bassiana resulted in up to 56% mortality 14 days after treatment. Entomopathogenic bacteria formulations and azadirachtin were not effective (<50% and <40% mortality, respectively). Results from a bioassay using lambda-cyhalothrin indicated that the CSFB used in this study were resistant to this insecticide. CONCLUSION: Entomopathogenic fungi and fatty acids could potentially be used to control CSFB as part of an integrated pest management programme. This study is the first to investigate the efficacy of different biopesticides to control CSFB under laboratory conditions. As such, these biopesticides require further testing to optimise the formulation and application methods, and to assess the impact on nontarget organisms. Finally, efficacy under field conditions must be determined to understand the influence of environmental variables. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Potential of Entomopathogenic Nematodes to Control the Cabbage Stem Flea Beetle Psylliodes chrysocephala.

Cabbage stem flea beetle (CSFB) is an important pest of oilseed rape that was controlled by neonicotinoid seed treatments until they were banned for this use in 2013. Since then, CSFB has been a difficult pest to control, partly due to widespread resistance to pyrethroid insecticides. Alternate solutions are necessary. Here, four entomopathogenic nematode (EPN) species were tested against CSFB adults under laboratory conditions. In addition, a bioassay was completed to test for EPN compatibility with a range of adjuvants (glycerin, xanthan gum and flame retardant) to protect EPNs from UV radiation and desiccation. Results show that EPNs have the potential to control CSFB adults under laboratory conditions. Heterorhabditis bacteriophora caused 75% CSFB mortality at a concentration of 4000 nematodes/mL after six days, Steinernema feltiae caused 80% CSFB mortality when applied at a concentration of 40,000 nematodes/mL after two days, Steinernema carpocapsae caused 85% mortality at a concentration of 10,000 nematodes/mL after six days, and Steinernema kraussei caused no more than 70% CSFB mortality overall compared to the water control, which led to 23% mortality. Steinernema feltiae and H. bacteriophora survival was 100% when exposed to adjuvants, except S. feltiae with glycerin and H. bacteriophora with flame retardant. Further research to evaluate the efficacy of EPN and adjuvants under field conditions is necessary.

BMI and HbA1c are metabolic markers for pancreatic cancer: Matched case-control study using a UK primary care database.

BACKGROUND: Weight loss, hyperglycaemia and diabetes are known features of pancreatic cancer. We quantified the timing and the amount of changes in body mass index (BMI) and glycated haemoglobin (HbA1c), and their association with pancreatic cancer from five years before diagnosis. METHODS: A matched case-control study was undertaken within 590 primary care practices in England, United Kingdom. 8,777 patients diagnosed with pancreatic cancer (cases) between 1st January 2007 and 31st August 2020 were matched to 34,979 controls by age, gender and diabetes. Longitudinal trends in BMI and HbA1c were visualised. Odds ratios adjusted for demographic and lifestyle factors (aOR) and 95% confidence intervals (CI) were calculated with conditional logistic regression. Subgroup analyses were undertaken according to the diabetes status. RESULTS: Changes in BMI and HbA1c observed for cases on longitudinal plots started one and two years (respectively) before diagnosis. In the year before diagnosis, a 1 kg/m2 decrease in BMI between cases and controls was associated with aOR for pancreatic cancer of 1.05 (95% CI 1.05 to 1.06), and a 1 mmol/mol increase in HbA1c was associated with aOR of 1.06 (1.06 to 1.07). ORs remained statistically significant (p < 0.001) for 2 years before pancreatic cancer diagnosis for BMI and 3 years for HbA1c. Subgroup analysis revealed that the decrease in BMI was associated with a higher pancreatic cancer risk for people with diabetes than for people without (aORs 1.08, 1.06 to 1.09 versus 1.04, 1.03 to 1.05), but the increase in HbA1c was associated with a higher risk for people without diabetes than for people with diabetes (aORs 1.09, 1.07 to 1.11 versus 1.04, 1.03 to 1.04). CONCLUSIONS: The statistically significant changes in weight and glycaemic control started three years before pancreatic cancer diagnosis but varied according to the diabetes status. The information from this study could be used to detect pancreatic cancer earlier than is currently achieved. However, regular BMI and HbA1c measurements are required to facilitate future research and implementation in clinical practice.

Cytochrome P450 168A1 from Pseudomonas aeruginosa is involved in the hydroxylation of biologically relevant fatty acids.

The cytochrome P450 CYP168A1 from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli followed by purification and characterization of function. CYP168A1 is a fatty acid hydroxylase that hydroxylates saturated fatty acids, including myristic (0.30 min-1), palmitic (1.61 min-1) and stearic acids (1.24 min-1), at both the ω-1- and ω-2-positions. However, CYP168A1 only hydroxylates unsaturated fatty acids, including palmitoleic (0.38 min-1), oleic (1.28 min-1) and linoleic acids (0.35 min-1), at the ω-1-position. CYP168A1 exhibited a catalytic preference for palmitic, oleic and stearic acids as substrates in keeping with the phosphatidylcholine-rich environment deep in the lung that is colonized by P. aeruginosa.

Patients with gastrointestinal irritability after TGN1412-induced cytokine storm displayed selective expansion of gut-homing αß and γδT cells.

Following infusion of the anti-CD28 superagonist monoclonal antibody TGN1412, three of six previously healthy, young male recipients developed gastrointestinal irritability associated with increased expression of 'gut-homing' integrin ß7 on peripheral blood αßT cells. This subset of patients with intestinal symptoms also displayed a striking and persistent expansion of putative Vδ2+ γδT cells in the circulation which declined over a 2-year period following drug infusion, concordant with subsiding gut symptoms. These data demonstrate that TGN1412-induced gastrointestinal symptoms were associated with dysregulation of the 'gut-homing' pool of blood αß and γδT cells, induced directly by the antibody and/or arising from the subsequent cytokine storm.

Immune reconstitution and clinical recovery following anti-CD28 antibody (TGN1412)-induced cytokine storm.

Cytokine storm can result from cancer immunotherapy or certain infections, including COVID-19. Though short-term immune-related adverse events are routinely described, longer-term immune consequences and sequential immune monitoring are not as well defined. In 2006, six healthy volunteers received TGN1412, a CD28 superagonist antibody, in a first-in-man clinical trial and suffered from cytokine storm. After the initial cytokine release, antibody effect-specific immune monitoring started on Day + 10 and consisted mainly of evaluation of dendritic cell and T-cell subsets and 15 serum cytokines at 21 time-points over 2 years. All patients developed problems with concentration and memory; three patients were diagnosed with mild-to-moderate depression. Mild neutropenia and autoantibody production was observed intermittently. One patient suffered from peripheral dry gangrene, required amputations, and had persistent Raynaud's phenomenon. Gastrointestinal irritability was noted in three patients and coincided with elevated γδT-cells. One had pruritus associated with elevated IgE levels, also found in three other asymptomatic patients. Dendritic cells, initially undetectable, rose to normal within a month. Naïve CD8+ T-cells were maintained at high levels, whereas naïve CD4+ and memory CD4+ and CD8+ T-cells started high but declined over 2 years. T-regulatory cells cycled circannually and were normal in number. Cytokine dysregulation was especially noted in one patient with systemic symptoms. Over a 2-year follow-up, cognitive deficits were observed in all patients following TGN1412 infusion. Some also had signs or symptoms of psychological, mucosal or immune dysregulation. These observations may discern immunopathology, treatment targets, and long-term monitoring strategies for other patients undergoing immunotherapy or with cytokine storm.

Isavuconazole and voriconazole inhibition of sterol 14α-demethylases (CYP51) from Aspergillus fumigatus and Homo sapiens.

Here we report the first evaluation of isavuconazole inhibition of Aspergillus fumigatus CYP51 and thus sterol biosynthesis in the fungus. Voriconazole and isavuconazole both bound tightly to recombinant A. fumigatus CYP51 isoenzymes A and B (AfCYP51A and AfCYP51B) isolated in Escherichia coli membranes. CYP51 reconstitution assays confirmed that AfCYP51A and AfCYP51B as well as three AfCYP51A mutants known to confer azole resistance (G54W, L98H and M220K) were strongly inhibited by both triazoles. Voriconazole bound relatively weakly to purified Homo sapiens CYP51 (HsCYP51), unlike isavuconazole that bound tightly. However, isavuconazole was a relatively poor inhibitor of HsCYP51 activity, with an IC50 value (half-maximal inhibitory concentration) of 25 µM, which was 55- to 120-fold greater than those observed for the A. fumigatus CYP51 enzymes, albeit not as poor an inhibitor of HsCYP51 as voriconazole with an IC50 value of 112 µM. Sterol analysis of triazole-treated A. fumigatus Af293 cells confirmed that isavuconazole and voriconazole both inhibited cellular CYP51 activity with the accumulation of 14-methylated sterol substrates and depletion of ergosterol levels. Isavuconazole elicited a stronger perturbation of the sterol composition in A. fumigatus Af293 than voriconazole at 0.0125 µg/mL, indicating increased potency. However, complementation studies in Saccharomyces cerevisiae using strains containing AfCYP51A and AfCYP51B showed isavuconazole to be equally as effective at inhibiting CYP51 activity as voriconazole. These in vitro studies suggest that isavuconazole is an effective alternative to voriconazole as an antifungal agent against the target CYP51 in A. fumigatus.

On the occurrence of cytochrome P450 in viruses.

Genes encoding cytochrome P450 (CYP; P450) enzymes occur widely in the Archaea, Bacteria, and Eukarya, where they play important roles in metabolism of endogenous regulatory molecules and exogenous chemicals. We now report that genes for multiple and unique P450s occur commonly in giant viruses in the Mimiviridae, Pandoraviridae, and other families in the proposed order Megavirales. P450 genes were also identified in a herpesvirus (Ranid herpesvirus 3) and a phage (Mycobacterium phage Adler). The Adler phage P450 was classified as CYP102L1, and the crystal structure of the open form was solved at 2.5 Å. Genes encoding known redox partners for P450s (cytochrome P450 reductase, ferredoxin and ferredoxin reductase, and flavodoxin and flavodoxin reductase) were not found in any viral genome so far described, implying that host redox partners may drive viral P450 activities. Giant virus P450 proteins share no more than 25% identity with the P450 gene products we identified in Acanthamoeba castellanii, an amoeba host for many giant viruses. Thus, the origin of the unique P450 genes in giant viruses remains unknown. If giant virus P450 genes were acquired from a host, we suggest it could have been from an as yet unknown and possibly ancient host. These studies expand the horizon in the evolution and diversity of the enormously important P450 superfamily. Determining the origin and function of P450s in giant viruses may help to discern the origin of the giant viruses themselves.

The Evolution of Azole Resistance in Candida albicans Sterol 14α-Demethylase (CYP51) through Incremental Amino Acid Substitutions.

Recombinant Candida albicans CYP51 (CaCYP51) proteins containing 23 single and 5 double amino acid substitutions found in clinical strains and the wild-type enzyme were expressed in Escherichia coli and purified by Ni2+-nitrilotriacetic acid agarose chromatography. Catalytic tolerance to azole antifungals was assessed by determination of the concentration causing 50% enzyme inhibition (IC50) using CYP51 reconstitution assays. The greatest increase in the IC50 compared to that of the wild-type enzyme was observed with the five double substitutions Y132F+K143R (15.3-fold), Y132H+K143R (22.1-fold), Y132F+F145L (10.1-fold), G307S+G450E (13-fold), and D278N+G464S (3.3-fold). The single substitutions K143R, D278N, S279F, S405F, G448E, and G450E conferred at least 2-fold increases in the fluconazole IC50, and the Y132F, F145L, Y257H, Y447H, V456I, G464S, R467K, and I471T substitutions conferred increased residual CYP51 activity at high fluconazole concentrations. In vitro testing of select CaCYP51 mutations in C. albicans showed that the Y132F, Y132H, K143R, F145L, S405F, G448E, G450E, G464S, Y132F+K143R, Y132F+F145L, and D278N+G464S substitutions conferred at least a 2-fold increase in the fluconazole MIC. The catalytic tolerance of the purified proteins to voriconazole, itraconazole, and posaconazole was far lower and limited to increased residual activities at high triazole concentrations for certain mutations rather than large increases in IC50 values. Itraconazole was the most effective at inhibiting CaCYP51. However, when tested against CaCYP51 mutant strains, posaconazole seemed to be the most resistant to changes in MIC as a result of CYP51 mutation compared to itraconazole, voriconazole, or fluconazole.

Adaption to glucose limitation is modulated by the pleotropic regulator CcpA, independent of selection pressure strength.

BACKGROUND: A central theme in (micro)biology is understanding the molecular basis of fitness i.e. which strategies are successful under which conditions; how do organisms implement such strategies at the molecular level; and which constraints shape the trade-offs between alternative strategies. Highly standardized microbial laboratory evolution experiments are ideally suited to approach these questions. For example, prolonged chemostats provide a constant environment in which the growth rate can be set, and the adaptive process of the organism to such environment can be subsequently characterized. RESULTS: We performed parallel laboratory evolution of Lactococcus lactis in chemostats varying the quantitative value of the selective pressure by imposing two different growth rates. A mutation in one specific amino acid residue of the global transcriptional regulator of carbon metabolism, CcpA, was selected in all of the evolution experiments performed. We subsequently showed that this mutation confers predictable fitness improvements at other glucose-limited growth rates as well. In silico protein structural analysis of wild type and evolved CcpA, as well as biochemical and phenotypic assays, provided the underpinning molecular mechanisms that resulted in the specific reprogramming favored in constant environments. CONCLUSION: This study provides a comprehensive understanding of a case of microbial evolution and hints at the wide dynamic range that a single fitness-enhancing mutation may display. It demonstrates how the modulation of a pleiotropic regulator can be used by cells to improve one trait while simultaneously work around other limiting constraints, by fine-tuning the expression of a wide range of cellular processes.

Manufacturing and characterization of long-acting levonorgestrel intrauterine systems.

Mirena® is long-acting (5 years) contraceptive intrauterine device. It is composed of a hollow cylindrical drug reservoir (containing Levonorgestrel and polydimethylsiloxane), which is covered with a release rate controlling silicone membrane. This structure presents a manufacturing challenge and to date, there have been no literature reports on the manufacturing, product design and quality evaluation of these hollow cylindrical intrauterine devices. It is vital to develop a reproducible and robust manufacturing process for these long-acting intrauterine devices or systems to obtain an understanding the in vitro and in vivo performance of such drug-device combinations. In this study, a twin-syringe method with a customized mold was developed to manufacture hollow cylindrical polydimethylsiloxane (PDMS)-based levonorgestrel intrauterine systems (LNG-IUSs). Different mold materials, curing temperatures and times were screened to fabricate PDMS-drug reservoirs with good quality characteristics (easy demolding, good appearance and appropriate physicochemical characteristics). The prepared PDMS-drug reservoirs were covered with the release rate controlling membrane to fabricate the LNG-IUSs. Physicochemical characterization (drug content and content uniformity, powder X-Ray diffraction (PXRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and Fourier-transform infrared spectroscopy (FTIR) of the PDMS-drug reservoirs with different drug loadings (10%, 25% and 50% w/w) was conducted. Real-time in vitro drug release testing of LNG-IUSs with different drug loading was performed in normal saline (0.9% w/v NaCl) at 37 °C using a water bath shaker rotating at 100 rpm. The prepared PDMS-drug reservoirs demonstrated good and reproducible quality characteristics including appearance (smooth surfaces), targeted drug loading and good drug content uniformity in the PDMS matrix. The PXRD showed that the crystallinity of the API was maintained inside the PDMS matrix. DSC, TGA and FTIR confirmed the structure of the drug and the PDMS, indicating no interaction between the drug and the PDMS matrix in the prepared LNG-IUSs. Real-time in vitro drug release from the LNG-IUSs with different drug loadings showed zero-order release kinetics, and the drug release rate (based on daily release percentage) was inversely proportional to the drug loading.

Functional importance for developmental regulation of sterol biosynthesis in Acanthamoeba castellanii.

The sterol metabolome of Acanthamoeba castellanii (Ac) yielded 25 sterols. Substrate screening of cloned AcCYP51 revealed obtusifoliol as the natural substrate which converts to ∆8,14-sterol (<95%). The combination of [2H3-methyl]methionine incubation to intact cultures showing C28-ergosterol incorporates 2-2H atoms and C29-7-dehydroporiferasterol incorporates 5 2H-atoms, the natural distribution of sterols, CYP51 and previously published sterol methyltransferase (SMT) data indicate separate ∆24(28)- and ∆25(27)-olefin pathways to C28- and C29-sterol products from the protosterol cycloartenol. In cell-based culture, we observed a marked change in sterol compositions during the growth and encystment phases monitored microscopically and by trypan blue staining; trophozoites possess C28/C29-∆5,7-sterols, viable encysted cells (mature cyst) possess mostly C29-∆5-sterol and non-viable encysted cells possess C28/C29-∆5,7-sterols that turnover variably from stress to 6-methyl aromatic sterols associated with changed membrane fluidity affording lysis. An incompatible fit of steroidal aromatics in membranes was confirmed using the yeast sterol auxotroph GL7. Only viable cysts, including those treated with inhibitor, can excyst into trophozoites. 25-Azacycloartanol or voriconazole that target SMT and CYP51, respectively, are potent enzyme inhibitors in the nanomolar range against the cloned enzymes and amoeba cells. At minimum amoebicidal concentration of inhibitor amoeboid cells rapidly convert to encysted cells unable to excyst. The correlation between stage-specific sterol compositions and the physiological effects of ergosterol biosynthesis inhibitors suggests that amoeba fitness is controlled mainly by developmentally-regulated changes in the phytosterol B-ring; paired interference in the ∆5,7-sterol biosynthesis (to ∆5,7) - metabolism (to ∆5 or 6-methyl aromatic) congruence during cell proliferation and encystment could be a source of therapeutic intervention for Acanthamoeba infections.

Co-production of 11α-hydroxyprogesterone and ethanol using recombinant yeast expressing fungal steroid hydroxylases.

BACKGROUND: Bioethanol production from sustainable sources of biomass that limit effect on food production are needed and in a biorefinery approach co-products are desirable, obtained from both the plant material and from the microbial biomass. Fungal biotransformation of steroids was among the first industrial biotransformations allowing corticosteroid production. In this work, the potential of yeast to produce intermediates needed in corticosteroid production is demonstrated at laboratory scale following bioethanol production from perennial ryegrass juice. RESULTS: Genes encoding the 11α-steroid hydroxylase enzymes from Aspergillus ochraceus (11α-SHAoch) and Rhizopus oryzae (CYP509C12) transformed into Saccharomyces cerevisiae for heterologous constitutive expression in p425TEF. Both recombinant yeasts (AH22:p11α-SHAoch and AH22:p509C12) exhibited efficient progesterone bioconversion (on glucose minimal medial containing 300 µM progesterone) producing either 11α-hydroxyprogesterone as the sole metabolite (AH22:p11α-SHAoch) or a 7:1 mixture of 11α-hydroxyprogesterone and 6ß-hydroxyprogesterone (AH22:p509C12). Ethanol yields for AH22:p11α-SHAoch and AH22:p509C12 were comparable resulting in ≥75% conversion of glucose to alcohol. Co-production of bioethanol together with efficient production of the 11-OH intermediate for corticosteroid manufacture was then demonstrated using perennial ryegrass juice. Integration of the 11α-SHAoch gene into the yeast genome (AH22:11α-SHAoch+K) resulted in a 36% reduction in yield of 11α-hydroxyprogesterone to 174 µmol/L using 300 µM progesterone. However, increasing progesterone concentration to 955 µM and optimizing growth conditions increased 11α-hydroxyprogesterone production to 592 µmol/L product formed. CONCLUSIONS: The progesterone 11α-steroid hydroxylases from A. ochraceus and R. oryzae, both monooxygenase enzymes of the cytochrome P450 superfamily, have been functionally expressed in S. cerevisiae. It appears that these activities in fungi are not associated with a conserved family of cytochromes P450. The activity of the A. ochraceous enzyme was important as the specificity of the biotransformation yielded just the 11-OH product needed for corticosteroid production. The data presented demonstrate how recombinant yeast could find application in rural biorefinery processes where co-production of value-added products (11α-hydroxyprogesterone and ethanol) from novel feedstocks is an emergent and attractive possibility.

The Tetrazole VT-1161 Is a Potent Inhibitor of Trichophyton rubrum through Its Inhibition of T. rubrum CYP51.

Prior to characterization of antifungal inhibitors that target CYP51, Trichophyton rubrum CYP51 was expressed in Escherichia coli, purified, and characterized. T. rubrum CYP51 bound lanosterol, obtusifoliol, and eburicol with similar affinities (dissociation constant [Kd ] values, 22.7, 20.3, and 20.9 µM, respectively) but displayed substrate specificity, insofar as only eburicol was demethylated in CYP51 reconstitution assays (turnover number, 1.55 min-1; Km value, 2 µM). The investigational agent VT-1161 bound tightly to T. rubrum CYP51 (Kd = 242 nM) with an affinity similar to that of clotrimazole, fluconazole, ketoconazole, and voriconazole (Kd values, 179, 173, 312, and 304 nM, respectively) and with an affinity lower than that of itraconazole (Kd = 53 nM). Determinations of 50% inhibitory concentrations (IC50s) using 0.5 µM CYP51 showed that VT-1161 was a tight-binding inhibitor of T. rubrum CYP51 activity, yielding an IC50 of 0.14 µM, whereas itraconazole, fluconazole, and ketoconazole had IC50s of 0.26, 0.4, and 0.6 µM, respectively. When the activity of VT-1161 was tested against 34 clinical isolates, VT-1161 was a potent inhibitor of T. rubrum growth, with MIC50, MIC90, and geometric mean MIC values of ≤0.03, 0.06, and 0.033 µg ml-1, respectively. With its selectivity versus human CYP51 and drug-metabolizing cytochrome P450s having already been established, VT-1161 should prove to be safe and effective in combating T. rubrum infections in patients.

Azole Antifungal Sensitivity of Sterol 14α-Demethylase (CYP51) and CYP5218 from Malassezia globosa.

Malassezia globosa cytochromes P450 CYP51 and CYP5218 are sterol 14α-demethylase (the target of azole antifungals) and a putative fatty acid metabolism protein (and a potential azole drug target), respectively. Lanosterol, eburicol and obtusifoliol bound to CYP51 with Kd values of 32, 23 and 28 µM, respectively, catalyzing sterol 14α-demethylation with respective turnover numbers of 1.7 min(-1), 5.6 min(-1) and 3.4 min(-1). CYP5218 bound a range of fatty acids with linoleic acid binding strongest (Kd 36 µM), although no metabolism could be detected in reconstitution assays or role in growth on lipids. Clotrimazole, fluconazole, itraconazole, ketoconazole, voriconazole and ketaminazole bound tightly to CYP51 (Kd ≤ 2 to 11 nM). In contrast, fluconazole did not bind to CYP5218, voriconazole and ketaminazole bound weakly (Kd ~107 and ~12 µM), whereas ketoconazole, clotrimazole and itraconazole bound strongest to CYP5218 (Kd ~1.6, 0.5 and 0.4 µM) indicating CYP5218 to be only a secondary target of azole antifungals. IC50 determinations confirmed M. globosa CYP51 was strongly inhibited by azole antifungals (0.15 to 0.35 µM). MIC100 studies showed itraconazole should be considered as an alternative to ketoconazole given the potency and safety profiles and the CYP51 assay system can be used in structure-activity studies in drug development.

l-DOPA as a small molecule surrogate to promote angiogenesis and prevent dexamethasone-induced ischemia.

The foreign body response to implantable biosensors has been successfully countered through the use of corticosteroids, such as dexamethasone. However, while controlling inflammation, dexamethasone also decreases angiogenesis, which may lead to delayed analyte readings. The concurrent application of VEGF with dexamethasone increases angiogenesis, but VEGF has physical stability issues and is not cost-effective. The use of l-DOPA, a small molecule drug shown to up-regulate VEGF in the Parkinsonian brain, can potentially resolve these issues by substituting for VEGF. In this work, l-DOPA was used for the first time as a pro-angiogenic agent to counteract dexamethasone-induced ischemia. Angiogenesis was modeled using the CAM assay and changes in blood vessel formation were recorded with both manual and digital techniques. As expected, dexamethasone reduced blood vessel formation in the CAM. Application of l-DOPA, on the other hand, increased blood vessel formation when dexamethasone and l-DOPA were administered simultaneously. This novel finding suggests the utility of l-DOPA in the field of implantable medical devices, such as biosensors, as well as tissue engineering applications where both a vascularized tissue environment and control of tissue response is desired.

The Investigational Drug VT-1129 Is a Highly Potent Inhibitor of Cryptococcus Species CYP51 but Only Weakly Inhibits the Human Enzyme.

Cryptococcosis is a life-threatening disease often associated with HIV infection. Three Cryptococcus species CYP51 enzymes were purified and catalyzed the 14α-demethylation of lanosterol, eburicol, and obtusifoliol. The investigational agent VT-1129 bound tightly to all three CYP51 proteins (dissociation constant [Kd] range, 14 to 25 nM) with affinities similar to those of fluconazole, voriconazole, itraconazole, clotrimazole, and ketoconazole (Kd range, 4 to 52 nM), whereas VT-1129 bound weakly to human CYP51 (Kd, 4.53 µM). VT-1129 was as effective as conventional triazole antifungal drugs at inhibiting cryptococcal CYP51 activity (50% inhibitory concentration [IC50] range, 0.14 to 0.20 µM), while it only weakly inhibited human CYP51 activity (IC50, ∼600 µM). Furthermore, VT-1129 weakly inhibited human CYP2C9, CYP2C19, and CYP3A4, suggesting a low drug-drug interaction potential. Finally, the cellular mode of action for VT-1129 was confirmed to be CYP51 inhibition, resulting in the depletion of ergosterol and ergosta-7-enol and the accumulation of eburicol, obtusifolione, and lanosterol/obtusifoliol in the cell membranes.

In Vitro Biochemical Study of CYP51-Mediated Azole Resistance in Aspergillus fumigatus.

The incidence of triazole-resistant Aspergillus infections is increasing worldwide, often mediated through mutations in the CYP51A amino acid sequence. New classes of azole-based drugs are required to combat the increasing resistance to existing triazole therapeutics. In this study, a CYP51 reconstitution assay is described consisting of eburicol, purified recombinant Aspergillus fumigatus CPR1 (AfCPR1), and Escherichia coli membrane suspensions containing recombinant A. fumigatus CYP51 proteins, allowing in vitro screening of azole antifungals. Azole-CYP51 studies determining the 50% inhibitory concentration (IC50) showed that A. fumigatus CYP51B (Af51B IC50, 0.50 µM) was 34-fold more susceptible to inhibition by fluconazole than A. fumigatus CYP51A (Af51A IC50, 17 µM) and that Af51A and Af51B were equally susceptible to inhibition by voriconazole, itraconazole, and posaconazole (IC50s of 0.16 to 0.38 µM). Af51A-G54W and Af51A-M220K enzymes were 11- and 15-fold less susceptible to inhibition by itraconazole and 30- and 8-fold less susceptible to inhibition by posaconazole than wild-type Af51A, confirming the azole-resistant phenotype of these two Af51A mutations. Susceptibility to voriconazole of Af51A-G54W and Af51A-M220K was only marginally lower than that of wild-type Af51A. Susceptibility of Af51A-L98H to inhibition by voriconazole, itraconazole, and posaconazole was only marginally lower (less than 2-fold) than that of wild-type Af51A. However, Af51A-L98H retained 5 to 8% residual activity in the presence of 32 µM triazole, which could confer azole resistance in A. fumigatus strains that harbor the Af51A-L98H mutation. The AfCPR1/Af51 assay system demonstrated the biochemical basis for the increased azole resistance of A. fumigatus strains harboring G54W, L98H, and M220K Af51A point mutations.