Human urinary excretion kinetics of the antimycotic climbazole: Biomonitoring of two new metabolites after oral and dermal dosage.

Climbazole is an antimycotic compound used in cosmetic products as a preservative or as an active ingredient in anti-dandruff (AD) formulations. In this study we provide human toxicokinetic data on climbazole. Using our previously published analytical method, we investigated the urinary excretion of two climbazole metabolites, (OH)2-climbazole and cx-OH-climbazole, for 48h after oral ingestion (n = 5, 49-77µg/kg bw) and for 72h after dermal application of either a climbazole-containing rinse-off AD shampoo or a leave-on hair tonic (n = 2×3). In total, 23.9% (18.0-33.4%) of the oral dose were excreted as the two abovementioned metabolites over 48h. In one volunteer, who used an over-the-counter phytopharmaceutical, metabolite excretion was about three times lower and we found influences on diastereoselectivity of (OH)2-climbazole formation using a modified analytical method. After dermal application, urinary concentration maxima occurred considerably later than after oral intake. The two different dermal exposure scenarios also revealed a relevance of exposure duration and product formulation on the systemic availability of climbazole. Back-calculated oral-dose-equivalent intakes from the dermal exposures showed a maximum climbazole intake of 18.5µg/kg bw/d after hair tonic use, or 6.6µg/kg bw/d after AD shampoo application.

Antifungal climbazole alters androgenic pathways in mammalian cells.

Among antifungal agents used in pharmaceuticals and personal care products, the synthetic azole climbazole (CBZ; 1-(4-Chlorophenoxy)-1-(imidazol-1-yl)-3,3-dimethylbutan-2-one) acts on the fungus Malassezia. Despite concerns surrounding its effects on health, based on alterations to reproduction and steroidogenesis found in fish, little is known about its mechanism of action as an endocrine disrupting chemical (EDC) in mammalian cells. In this study, using OECD test guidelines, we investigated the effects of CBZ (i) in H295R cells, on the production of estradiol and testosterone, as well as intermediate metabolites in steroidogenesis pathway, and (ii) in HeLa9903 and AR-EcoScreen cell lines, on the transactivation of estrogen and androgen receptors. Our results are the first evidence in H295R cells, that CBZ treatment (from 0.3 µM) decreased secreted levels of testosterone and estradiol. This was associated with reduced 17α-hydroxypregnenolone and 17α-hydroxyprogesterone levels. The altered levels of these metabolites were associated with a decrease in cytochrome P450 17α-hydroxylase/17,20-lyase (Cyp17A1) activity without any effect on its protein level. CBZ was also found to exert antagonistic effects toward androgen and estrogen α receptors. These results give insights into the toxicological mechanism of action of CBZ. Many azoles share structural similarities; therefore, caution should be adopted due to their potential toxicity.

Human adenovirus type 3 restores pharmacologically inhibited exosomal cargo in lung carcinoma cells.

Introduction: Drug repurposing is fast growing and becoming an attractive approach for identifying novel targets, such as exosomes for cancer and antiviral therapy. Exosomes are a specialized class of extracellular vesicles that serve as functional mediators in intercellular communication and signaling that are important in normal physiological functions. A continuously growing body of evidence has established a correlation between the abnormal release of exosomes with various viral disease pathologies including cancer. Cells that are virus-infected release exosomes known to influence the process via the loading and transfer of viral components, such as miRNA, small (s) RNA, DNA, and proteins. Inhibition of exosome release may abate the spread and severity of viral infection, thus making exosomes an attractive target for antiviral therapies. We previously demonstrated the pharmacological inhibition of exosomes. Methods: Herein, we used a cell-based assay to determine the effect of Human adenovirus type 3 (HAdV3) on the exosome inhibition process by azole and Heparin derivatives. HAdV3-infected cells were treated with two concentrations of each inhibitor at different time points. Results: HAdV3 activities led to increased total sRNA, DNA, and exosome particle concentrations via particle tracking in the presence of Climbazole and Heparin relative to uninfected exosomes. In addition, there was an increased expression of classical markers such as ALG-2 interacting protein X (ALIX), and tetraspanin (CD63), (p < 0.05) and upregulated transcription factor interferon regulatory factor (IRF) 8 in the presence of HAdV3 after 24 hours (h) of treatment. Whereas higher concentrations of Climbazole and Heparin sodium salt were found to inhibit total exosome protein (p < 0.001) and exo-RNA (p < 0.01) content even in the presence of HAdV3 relative to infected exosomes only. Activities of HAdV3 in the presence of selected inhibitors resulted in the positive regulation of exosome related DNA damage/repair signaling proteins. Blocking exosome secretion partially obstructed viral entry. Immunological studies revealed that HAdV3 fiber protein levels in A549 cells were reduced at all concentrations of Climbazole and Heparin and both multiplicities of infections (p < 0.001). Discussion: Our findings suggest that while HAdV may bolster inhibited exosome content and release when modulating certain activities of the endosomal pathway mediators, HAdV entry might be constrained by the activities of these pharmacological agents.

Evaluation of novel cosmetic shampoo formulations against Malassezia species: Preliminary results of anti-dandruff shampoo formulations.

OBJECTIVES: Malassezia species are common, clinically relevant, and lipid-dependent yeasts of humans. They are also the leading causes of the dandruff problem of humans, and the azoles are used primarily in their topical and systemic treatment. Resistance to azoles is an emerging problem among Malassezia sp., which indicates the need of new drug assessments that will be effective against dandruff and limit the use of azoles and other agents in treatment. Among them, the efficacy of various combinations of piroctone olamine and climbazole against Malassezia sp. is highly important. Here, we assessed the efficacies of various piroctone olamine and climbazole formulations against Malassezia sp. in comparison with ketoconazole. METHODS: A total of nine formulations were included in the study, where each formulation was prepared from different concentrations of piroctone olamine and climbazole and both. All formulations contained the same ingredients as water, surfactants, hair conditioning agents, and preservatives. Malassezia furfur CBS1878, Malassezia globosa CBS7874, and Malassezia sympodialis CBS9570 were tested for antifungal susceptibility of each formulation by agar diffusion method. Sizes of the inhibition zones were compared with standard medical shampoo containing 2% ketoconazole, and the data were analyzed by Dunnett's multiple-comparison test. RESULTS: For all Malassezia sp. strains, climbazole 0.5% and piroctone olamine/climbazole (0.1%/0.1% and 0.1%/0.5%) combinations were found to have the same effect as the medical shampoo containing 2% ketoconazole. Piroctone olamine/climbazole 1.0%/0.1% formulation showed the same efficacy as 2% ketoconazole on M. furfur and M. sympodialis, while 0.1%/0.5% formulation to only M. furfur. For M. globosa, none of the formulations tested were as effective as ketoconazole. CONCLUSION: The species distribution of Malassezia sp. varies depending on the anatomical location on the host. According to the results of this study, climbazole and piroctone olamine combinations seem to be promising options against the dandruff problem with their high antifungal/anti dandruff efficacy.

Rational development of topical climbazole formulations.

Dandruff, or pityriasis capitis simplex, is a common scalp condition associated with excessive flaking and scaling of the epidermal tissue. Other features include irregular corneocyte turnover, irritation, itching and an impaired skin barrier function. Previously we reported the characterization of climbazole (CBZ), an antifungal agent used in the management of dandruff. Skin permeation of CBZ from neat solvents was also investigated. In the present work we evaluated CBZ permeation in human skin in vitro from more complex formulations that better represent products used by consumers. The various systems studied were composed of propylene glycol (PG), Transcutol®P (TC), octyl salicylate (OSal) and isopropyl alcohol (IPA). As well as measurement of skin uptake and penetration of CBZ, where possible, the skin retention and permeation of the various solvents was also determined. All vehicles promoted skin permeation of CBZ but no significant differences in amount permeated were evident between the binary vehicles (PG:TC, TC:OSal) and the ternary vehicle studied (PG:IPA:OSal). The binary vehicles generally promoted more skin uptake of CBZ compared with the neat solvents (PG, TC, OSal) studied previously. Permeation and skin extraction of CBZ from the PG:TC vehicles increased with increasing PG content; a similar trend was evident for the PG:IPA:OSal systems. New methods were developed and validated for measurement of PG, TC and OSal. Analysis of the individual solvents indicated that PG permeation was also independent of the amounts of other solvents in the binary or ternary systems. Consistent with previous findings higher proportions of TC permeated compared with PG for the PG:TC binary systems; TC also permeated the skin more rapidly than PG from these vehicles. For OSal, skin extraction was generally higher for TC:OSal compared with the PG:IPA:OSal vehicle. However, increasing the content of OSal did not appear to influence CBZ skin uptake nor permeation. Interestingly, the effects of the various PG:TC vehicles on CBZ skin delivery contrast with results we previous reported for the same systems for a different active. This confirms that with reference to skin permeation, formulation effects and/or skin penetration enhancement should be expected to vary and may not be predicted for specific vehicles.

An azole fungicide climbazole damages the gut-brain axis in the grass carp.

Azole antifungal climbazole has frequently been detected in aquatic environments and shows various effects in fish. However, the underlying mechanism of toxicity through the gut-brain axis of climbazole is unclear. Here, we investigated the effects of climbazole at environmental concentrations on the microbiota-intestine-brain axis in grass carp via histopathological observation, gene expression and biochemical analyses, and high-throughput sequencing of the 16 S rRNA. Results showed that exposure to 0.2 to 20 µg/L climbazole for 42 days significantly disrupted gut microbiota and caused brain neurotoxicity in grass carp. In this study, there was an alteration in the phylum and genus compositions in the gut microbiota following climbazole treatment, including reducing Fusobacteria (e.g., Cetobacterium) and increasing Actinobacteria (e.g., Nocardia). Climbazole disrupted intestinal microbial abundance, leading to increased levels of lipopolysaccharide and tumor necrosis factor-alpha in the gut, serum, and brain. They passed through the impaired intestinal barrier into the circulation and caused the destruction of the blood-brain barrier through the gut-brain axis, allowing them into the brain. In the brain, climbazole activated the nuclear factor kappaB pathway to increase inflammation, and suppressed the E2-related factor 2 pathway to produce oxidative damage, resulting in apoptosis, which promoted neuroinflammation and neuronal death. Besides, our results suggested that this neurotoxicity was caused by the breakdown of the microbiota-gut-brain axis, mediated by reduced concentrations of dopamine, short chain fatty acids, and intestinal microbial activity induced by climbazole.

Climbazole causes cell apoptosis and lipidosis in the liver of grass carp.

Climbazole, an azole, is widely used in personal care products, pharmaceuticals, and pesticides and is frequently detected in surface water. Climbazole has showed endocrine-disrupting effects. However, the effects of climbazole in fish are still largely unclear. In this study, grass carp (Ctenopharyngodon idella) and liver cell lines (L8824 cells) were treated with climbazole at concentrations ranging from 0.2 to 20 µg/L for 42 days in vivo and 24 h in vitro to evaluate the effects on the liver, respectively. Pathological, biochemical, and gene transcription and expression analyses were conducted to examine the hepatotoxicity. Our results showed that climbazole significantly decreased the hepatosomatic index, caused cell apoptosis in vivo and in vitro, and finally accumulated lipids in the liver. Beside, climbazole increased ROS levels, reduced Nrf2 and Keap1 mRNA and protein levels, and further decreased transcription of Nrf2-dependent downstream antioxidant enzyme genes, causing oxidative stress. Moreover, climbazole increased transcription and protein levels of apoptosis-related genes. Finally, climbazole damaged mitochondrial function and structure, disrupted liver lipid metabolism. Overall, climbazole caused hepatotoxicity, leading to a high ecological risk for aquatic organisms.

Does climbazole instigate a threat in the environment as persistent, mobile and toxic compound? Unveiling the occurrence and potential ecological risks of its phototransformation products in the water cycle.

Persistent, mobile, and toxic chemicals (PMT), such as the antimycotic climbazole-(CBZ), proliferate in water cycle and imperil drinking water quality, sparking off research about their environmental fate. Unlike the parent compound, its transformation products-(TPs) are scarcely investigated, much less as PMTs. To this end, phototransformation of CBZ was investigated. A novel suspect-screening workflow was developed and optimized by cross-comparing the results of the identified photo-TPs against literature data to create an enhanced HRMS-database for environmental investigations of CBZ/TPs in the water cycle. In total, 24 TPs were identified, 14 of which are reported for the first time. Isomerism, dechlorination, hydroxylation, and cleavage of the ether or C-N bond are suggested as the main transformation routes. A screening of CBZ/TPs was conducted in wastewater, leachates, surface, and groundwater, revealing a maximum concentration of 464.8 ng/L in groundwater. In silico and in vitro methods were used for toxicity assessment, indicating toxicity for CBZ and some TPs. Seemingly, CBZ is rightly considered as PMT, and a higher potential to occur in surface or groundwater than non-PM chemicals appears. Likewise, the occurrence of TPs due to PMT properties or emission patterns was evaluated.

Characterization of Climbazole-Bovine serum albumin interaction by experimental and in silico approaches.

Interactive association of an antifungal drug, climbazole (CBZ) with the carrier protein in bovine circulation, bovine serum albumin (BSA) was explored by fluorescence and absorption spectroscopy along with in silico techniques. The fluorescence and absorption spectral alterations of the protein upon addition of CBZ affirmed the complex foration between CBZ and BSA. The inverse temperature dependence behaviour of the KSV values as well as the hyperchromic result of the protein's absorption signals characterized CBZ-triggered quenching of BSA fluorescence as the static quenching. A weak binding affinity (Ka = 3.12-1.90-× 103 M-1) was reported towards the CBZ-BSA association process. Interpretation of thermodynamic data (entropy change = +14.68 J mol-1 K-1 and enthalpy change = -15.07 kJ mol-1) and in silico analyses anticipated that hydrophobic forces, van der Waals forces and hydrogen bonds were the key intermolecular forces in the complex stabilization. Inclusion of CBZ to BSA produced microenvironmental perturbations around Tyr and Trp residues, and also significantly defended temperature-induced destabilization of BSA. The binding locus of CBZ was detected in the proximity of Sudlow's sites I (subdomain IIA) and II (subdomain IIIA) of BSA, exhibiting greater preference towards site II, as revealed by competitive site-marker displacement investigations and in silico analysis. The stability of the CBZ-BSA complex was further validated by the molecular dynamics simulation assessments.

Biodegradation of fluoroquinolone antibiotics and the climbazole fungicide by Trichoderma species.

Filamentous fungi Trichoderma have been able to efficiently degrade fluoroquinolone antibiotics namely ciprofloxacin (CIP) and ofloxacin (OFL) as well as the fungicide climbazole (CLB) that are persistent in conventional activated sludge processes. All targeted compounds were biotransformed by whole cells of Trichoderma spp., exactly T. harzanium and T. asperellum, and biosorption played a limited role in their elimination. However, contrasting results were obtained with the two strains. T. asperellum was more efficient against CIP, with a 81% degradation rate in 13 days of incubation, while T. harzianum was more efficient against CLB, with a 91% degradation rate. While in the case of OFL, both strains showed same efficiency with degradation rate around 40%. Adding a cytochrome P450 enzyme inhibitor hardly resulted in the modification of degradation kinetics supporting the implication of extracellular enzymes in chemical biotransformation. Transformation products were identified by liquid chromatography-high resolution-mass spectrometry and transformation pathways were proposed. Biotransformation of selected compounds included hydroxylation, oxidation/reduction and N- and O-dealkylation reactions, similarly to those reported with white rot fungi. CIP underwent transformations at the piperazinyl ring through oxidation and conjugation reactions, while OFL mainly underwent hydroxylation processes and CLB carbonyl reduction into alcohol. Consequently, Trichoderma spp. likely possess a machinery of unspecific enzymes, which makes their application in removal of pharmaceutical and personal care products attractive.

Effects of antimicrobial exposure on detrital biofilm metabolism in urban and rural stream environments.

The occurrence of antimicrobials and other pharmaceuticals in streams is increasingly being reported, yet the impacts of these contaminants of emerging concern on aquatic ecosystems are relatively unknown. Bacteria and fungi are vital components of stream environments and, therefore, exposure to antimicrobials may have important consequences for ecosystem services, such as carbon cycling. The objective of this study was to investigate how two antimicrobials, ciprofloxacin and climbazole, impact detrital biofilm metabolism in urban and rural streams. To establish baseline conditions, the biological oxygen demand (BOD) of red maple (Acer rubrum) biofilms was measured in one urban and one rural stream. In mesocosm studies, the BOD of biofilms on single- and mixed-species leaf litter from the same sites was measured after exposure to 10 µg/L of the antimicrobials, both in combination and individually. The presence of ciprofloxacin and climbazole did not affect BOD compared to the controls at the urban site, although significant differences were identified for select treatments at the rural site. In addition, the BOD of mixed-leaf biofilms was not significantly different from that of single species litter after exposure. Overall, exposure to 10 µg/L of the antimicrobials did not significantly impact community-level carbon processing by the leaf biofilms, and leaf mixtures did not result in increased biofilm BOD compared to single species leaves. The outcomes of this work demonstrate a need for further research for the understanding the effects of antimicrobials on rural streams to prevent unintended consequences to ecological processes and biota from future development, leaking septic systems, and wastewater spills.

Dummy molecularly imprinted solid phase extraction of climbazole from environmental water samples.

Dummy molecularly imprinted polymer (DMIP) for climbazole (CBZ) was synthesized for the first time employing miconazole (MNZ) as the dummy template together with methacrylic acid (MAA) monomer, ethylene glycol dimethacrylate (EGDMA) cross-linker and acetonitrile (ACN) porogen. The selectivity and capacity of the prepared MNZ-DMIP was estimated for CBZ by high-performance liquid chromatography (HPLC) and equilibrium binding experiments. Imprinting factor (IF) with a value of 7.0 was achieved, much higher than the CBZ templated MIP (IF = 3.5). Heterogeneous binding sites were found in the MNZ-DMIP, the corresponding saturation capacity and dissociation constant for the high and low affinity binding sites were 6.761 µmol g-1 and 0.3027 mmol L-1, 43.60 µmol g-1 and 4.055 mmol L-1, respectively. High efficient method based on dummy molecularly imprinted solid phase extraction (DMISPE) coupled with HPLC was established for the selective enrichment of CBZ in river and tap water using MNZ-DMIP as sorbent. DMISPE conditions including sample loading pH/volume, selective washing and elution solvents were carefully optimized. The developed method showed good recoveries (82.3-96.2%) and repeatability (RSDs 0.6-4.9%, n = 5) for samples spiked at three different concentration levels (0.2, 1.0 and 5.0 µg L-1). The detection limit was determined as 0.012 µg L-1. The results demonstrated good potential of this method for sample pretreatment of CBZ in environmental water samples.

Degradation of climbazole by UV/chlorine process: Kinetics, transformation pathway and toxicity evaluation.

Climbazole is an antifungal agent widely used in household personal care products, and it was found persistent in chlorination disinfection process. Here we investigated the kinetics and mechanism of climbazole degradation by UV/chlorine process. The results showed that the UV/chlorine process dramatically enhanced degradation of climbazole when compared to the UV photolysis and chlorination alone. The neutral condition (pH 7) produced the highest reaction rate for the climbazole by UV/chlorine among the various pH conditions. Dissolved organic matter and inorganic ions in natural water showed moderate inhibition effects on the degradation of climbazole in the UV/chlorine process. Hydroxyl radical (OH and chlorine radical (Cl) were found to be the main reactive species in the degradation of climbazole, with the second-order rate constant of 1.24 × 1010 M-1 s-1 and 6.3 × 1010 M-1 s-1, respectively. In addition, the OH and Cl in the UV/chlorine at 100 µM accounted for 82.2% and 7.7% contributions to the removal of climbazole, respectively. Eleven of main transformation products of climbazole were identified in the UV/chlorine process. These oxidation products did not cause extra toxicity than climbazole itself. The findings from this study show that the combination of chlorination with UV photolysis could provide an effective approach for removal of climbazole during conventional disinfection process.

Topical delivery of climbazole to mammalian skin.

Dandruff is a common condition, affecting up to half the global population of immunocompetent adults at some time during their lives and it has been highly correlated with the over-expression of the fungus Malassezia spp. Climbazole (CBZ) is used as an antifungal and preservative agent in many marketed formulations for the treatment of dandruff. While the efficacy of CBZ in vitro and in vivo has previously been reported, limited information has been published about the uptake and deposition of CBZ in the skin. Hence, our aim was to investigate the skin permeation of CBZ as well as the influence of various solvents on CBZ skin delivery. Four solvents were selected for the permeability studies of CBZ, namely propylene glycol (PG), octyl salicylate (OSal), Transcutol® P (TC) and polyethylene glycol 200 (PEG). The criteria for selection were based on their wide use as excipients in commercial formulations, their potential to act as skin penetration enhancers and their favourable safety profiles. 1% (w/v) solutions of CBZ were applied under infinite and finite dose conditions using Franz type diffusion cells to human and porcine skin. In line with the topical use of CBZ as an antidandruff agent, comparatively low amounts of CBZ penetrated across the skin barrier (<1% of the applied dose of CBZ). Finite dose studies resulted in a higher extraction of CBZ from human skin compared with infinite dose studies (p < 0.05). CBZ was also taken up to a higher extent in porcine skin (>7-fold) compared with human skin (p < 0.05). Nevertheless, no statistical differences were observed in the amounts that permeated across the different membranes. These preliminary results confirm the potential of simple formulations of CBZ to target the outer layers of the epidermis. The PG and OSal formulations appear to be promising vehicles for CBZ in terms of overall skin extraction and penetration. Future work will expand the range of vehicles studied and explore the reasons underlying the retention of CBZ in the outer layers of the skin.

Removal, biotransformation and toxicity variations of climbazole by freshwater algae Scenedesmus obliquus.

Climbazole (CBZ) is an antibacterial and antifungal agent widely used in personal care products. In this study, we investigated the interactions between climbazole (CBZ) and freshwater microalgae Scenedesmus obliquus (S. obliquus). Dose-effect relationships between CBZ concentrations and growth inhibitions or chlorophyll a content were observed. After 12 days of incubation, the algae density and chlorophyll a content in 2 mg/L treatment group was 56.6% and 15.8% of those in the control group, respectively. Biotransformation was the predominant way to remove CBZ in the culture solution, whereas the contribution of bioaccumulation and bioadsorption were negligible. More than 88% of CBZ was removed by S. obliquus across all treatments after 12 days of incubation, and the biotransformation of CBZ followed the first order kinetic model with half-lives of approximately 4.5 days at different treatments. CBZ-alcohol (CBZ-OH) was the only biotransformation product identified in algal solution. Moreover, the toxicity of biotransformation products was much lower than its corresponding precursor compound (CBZ). The results of this study revealed that S. obliquus might have a great impact on the environmental fates of CBZ and could be further applied to remove organic pollutants in aquatic environment.

Retinyl propionate and climbazole combination demonstrates clinical improvement to the appearance of hyperpigmentation and deep wrinkling with minimal irritation.

OBJECTIVE: To evaluate a combination of retinyl propionate and climbazole (RPC) compared to 0.1% retinol for its efficacy, tolerance and ageing appearance. METHOD: Forty-five healthy Caucasian females, ages 40-70, with moderately photodamaged facial skin, were recruited for a 16-week randomized, double-blind, IRB-approved facial study. The efficacy of RPC treatment was compared to 0.1% retinol, in the same product base formulation, with twice daily, split-face product application. Changes in overall photodamage, fine lines and wrinkles, pigmentation and irritation were visually evaluated and measured by instrumentation. Subjective appraisal of efficacy was self-assessed from images where subjects were blinded to treatment and time point. Irritancy potential was also evaluated in a 5-day randomized, double-blind, IRB-approved patch study. RESULTS: Treatment with RPC resulted in significant (P < 0.05) improvement in ageing attributes compared to 0.1% retinol treatment, with minimal irritation. More than 50% of subjects showed improvement to deep wrinkles in the crow's feet area after 5 weeks of product application, and continued improvement to deep wrinkles was observed throughout the course of the study. Similarly, improvement was observed for the appearance of lines and wrinkles in the nasolabial fold (NLF) and for mottled hyperpigmentation. The results from subjective self-assessment confirmed in vivo clinical assessments. In a separate patch study, significantly less irritation was observed with the RPC product as compared to the 0.1% retinol control product. CONCLUSION: RPC delivered significant skin anti-ageing benefits comparable or greater than 0.1% retinol, with minimal irritation.

Ex-vivo measurement of scalp follicular infundibulum delivery of zinc pyrithione and climbazole from an anti-dandruff shampoo.

Efficient delivery of anti-dandruff (AD) actives into the scalp follicular infundibulum as well as onto the scalp surface is critical for the efficacy of AD shampoos. A method involving scalp cyanoacrylate (CA) biopsy sampling, a tailor made cutting device, ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis, scanning electron microscopy (SEM) measurement and Raman imaging has been developed for the measurement of delivery of zinc pyrithione (ZPT) and climbazole (CBZ) from an AD shampoo into the scalp follicular infundibulum. Scalp CA biopsy enables the sampling of ZPT and CBZ delivered into the scalp follicular infundibula as well as onto the scalp surface. Raman imaging of scalp CA biopsy samples allows the visualization of the spatial distribution of ZPT and CBZ deposited on the scalp. A tailor made cutting device enables the separation of the scalp follicular infundibulum sample (20µm below the scalp surface) from the scalp surface samples (including top 20µm of infundibula). UHPLC-MS/MS was used as a sensitive and specific methodology enabling the quantification of ZPT and CBZ without interference. Using this method, both ZPT and CBZ were successfully quantified and spacially visualized within the scalp follicular infundibulum, after scalp was washed with an AD shampoo.

Enantioselective reductive transformation of climbazole: A concept towards quantitative biodegradation assessment in anaerobic biological treatment processes.

An efficient chiral method-based using liquid chromatography-high resolution-mass spectrometry analytical method has been validated for the determination of climbazole (CBZ) enantiomers in wastewater and sludge with quantification limits below the 1 ng/L and 2 ng/g range, respectively. On the basis of this newly developed analytical method, the stereochemistry of CBZ was investigated over time in sludge biotic and sterile batch experiments under anoxic dark and light conditions and during wastewater biological treatment by subsurface flow constructed wetlands. CBZ stereoselective degradation was exclusively observed under biotic conditions, confirming the specificity of enantiomeric fraction variations to biodegradation processes. Abiotic CBZ enantiomerization was insignificant at circumneutral pH and CBZ was always biotransformed into CBZ-alcohol due to the specific and enantioselective reduction of the ketone function of CBZ into a secondary alcohol function. This transformation was almost quantitative and biodegradation gave good first order kinetic fit for both enantiomers. The possibility to apply the Rayleigh equation to enantioselective CBZ biodegradation processes was investigated. The results of enantiomeric enrichment allowed for a quantitative assessment of in situ biodegradation processes due to a good fit (R2 > 0.96) of the anoxic/anaerobic CBZ biodegradation to the Rayleigh dependency in all the biotic microcosms and was also applied in subsurface flow constructed wetlands. This work extended the concept of applying the Rayleigh equation towards quantitative biodegradation assessment of organic contaminants to enantioselective processes operating under anoxic/anaerobic conditions.

Climbazole boosts activity of retinoids in skin.

OBJECTIVE: To explore whether climbazole enhances retinoid-associated biological activities in vitro and in vivo. METHODS: Primary human dermal fibroblasts (HDFs) were treated from six to 48 h with either retinoids (retinol, retinyl propionate, retinyl palmitate) alone or in combination with climbazole, and then assessed for cellular retinoic acid-binding protein 2 (CRABP2) mRNA expression by RT-qPCR. Next, skin equivalent (SE) cultures were topically treated with retinol or retinyl propionate, with or without climbazole, and then measured for biological changes in retinoid biomarkers. Lastly, an IRB-approved clinical study was conducted on the outer forearm of 16 subjects to ascertain the effects of low (0.02%) or high (0.1%) levels of retinol, retinyl propionate (0.5%), climbazole (0.5%) or a combination of retinol (0.02%)/climbazole (0.5%). Indicators of retinoid activities were measured after 3 weeks. RESULTS: Treatment of HDFs with retinol or retinyl propionate was unaffected by climbazole but alone, resulted in a significantly (P < 0.01) higher sustained CRABP2 mRNA expression than those treated with retinyl palmitate or vehicle control. In SEs, climbazole combined with either retinol or retinyl propionate boosted retinoid related activity greater than the retinoid only, reflected by a dose-response, downregulation of loricrin (LOR) and induction of keratin 4 (KRT4) proteins. In vivo, retinol (0.1%) and retinyl propionate (0.5%) significantly increased most evaluated biomarkers, as expected. Low-dose retinol or climbazole alone did not increase these biomarkers; however, in combination, significant (P < 0.05) increases in retinoid and ageing biomarkers were detected. CONCLUSION: Climbazole boosted retinoid activity both in the SE model, after a combined topic treatment with either retinol or retinyl propionate, and in vivo, in combination with a low level of retinol. Based upon the evidence presented here, we suggest that the topical skin application of climbazole in combination with retinoids could deliver skin ageing benefits more than a less robust retinoid alone.

Inhibition of 11ß-hydroxysteroid dehydrogenase 2 by the fungicides itraconazole and posaconazole.

Impaired 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2)-dependent cortisol inactivation can lead to electrolyte dysbalance, hypertension and cardiometabolic disease. Furthermore, placental 11ß-HSD2 essentially protects the fetus from high maternal glucocorticoid levels, and its impaired function has been associated with altered fetal growth and a higher risk for cardio-metabolic diseases in later life. Despite its important role, 11ß-HSD2 is not included in current off-target screening approaches. To identify potential 11ß-HSD inhibitors among approved drugs, a pharmacophore model was used for virtual screening, followed by biological assessment of selected hits. This led to the identification of several azole fungicides as 11ß-HSD inhibitors, showing a significant structure-activity relationship between azole scaffold size, 11ß-HSD enzyme selectivity and inhibitory potency. A hydrophobic linker connecting the azole ring to the other, more polar end of the molecule was observed to be favorable for 11ß-HSD2 inhibition and selectivity over 11ß-HSD1. The most potent 11ß-HSD2 inhibition, using cell lysates expressing recombinant human 11ß-HSD2, was obtained for itraconazole (IC50 139±14nM), its active metabolite hydroxyitraconazole (IC50 223±31nM) and posaconazole (IC50 460±98nM). Interestingly, experiments with mouse and rat kidney homogenates showed considerably lower inhibitory activity of these compounds towards 11ß-HSD2, indicating important species-specific differences. Thus, 11ß-HSD2 inhibition by these compounds is likely to be overlooked in preclinical rodent studies. Inhibition of placental 11ß-HSD2 by these compounds, in addition to the known inhibition of cytochrome P450 enzymes and P-glycoprotein efflux transport, might contribute to elevated local cortisol levels, thereby affecting fetal programming.