Glioblastoma multiforme is the most aggressive primary brain tumor in adults, which displays extremely poor prognosis. Protease-activated receptor 2 (PAR2) and its downstream effector SerpinB3 are overexpressed in aggressive glioblastomas. In this study we evaluated the antitumor activity of 1-piperidine propionic acid (1-PPA), an allosteric PAR2 inhibitor, in in vitro preclinical models of glioblastoma. PAR2 and SerpinB3 were analyzed at the transcriptional and protein level in glioblastoma cell lines and primary cultures. These were treated with 1-PPA alone or in association with temozolomide (TMZ) and the effects evaluated by Incucyte® technology. Pharmacokinetics and tissue distribution of 1-PPA were assessed in mice by LC-MS/MS. 1-PPA significantly reduced glioma cell proliferation, migration, and invasion, thus promoting apoptotic cell death, in a concentration-dependent manner. The combined treatment with TMZ led to a concentration-dependent decrease in cell proliferation (12-20%) compared to TMZ alone. Molecularly, 1-PPA downregulated PAR2 and SerpinB3 expression. Pharmacokinetic studies in healthy mice showed that 1-PPA is systemically bioavailable and distributes to several organs, including the brain. These data indicate that 1-PPA shows brain exposure and capability to affect different hallmarks of aggressiveness in glioblastoma cells, including hyperproliferation and invasion, supporting its further development as a novel therapeutic strategy in these tumors.

IntroductionWithin an integrative medicine approach that links veterinary and human health, red algae extracts are gaining attention as promising sources of natural bioactive compounds with potential anticancer properties. This study investigates the selectivity of Plocamium cartilagineum extract (PCE) against canine cancer cell lines and its transcriptional effects in C2 canine mastocytoma cells.MethodsInitial cytotoxicity was assessed in C2 canine mastocytoma cells using three assays: Alamar Blue (AB), Neutral Red Uptake (NRU), and Sulforhodamine B (SRB). Selectivity was then investigated using the AB assay in two canine cancer cell lines, C2 and CLBL-1, and two non-cancer canine cell lines, Cf2Th and MDCK. Following the selectivity assessment, the transcriptional responses induced by PCE in C2 cells were investigated by RNA sequencing (RNA-seq) and validated by quantitative PCR (qPCR).ResultsPCE exhibited cytotoxic activity in C2 cells, with IC50 values ranging from 42.15 to 77.75 µg/mL across the three assays. In the selectivity assessment, IC50 values ranged from 10.69 µg/mL in CLBL-1 cancer cells to 41.46 µg/mL in Cf2Th non-cancer cells. PCE showed preferential cytotoxicity toward cancer cells, with a selectivity index (SI) reaching 3.9 when comparing CLBL-1 cancer cells with Cf2Th non-cancer cells. RNA-seq and qPCR analyses revealed that PCE induced broad transcriptional changes in C2 cells, including the upregulation of CDKN1A and the downregulation of genes involved in cell-cycle regulation, such as CCNB2, CDC25, and PLK1. Functional enrichment analyses further identified biological processes associated with cell-cycle regulation, DNA repair-related pathways, cellular stress responses, and metabolism.ConclusionPCE exhibited selective cytotoxic activity toward canine cancer cell lines and induced broad transcriptional alterations in C2 mastocytoma cells. These findings support further investigation of red algae as potential anticancer candidates.

Psilocybin, the phosphorylated prodrug of psilocin, holds therapeutic promise across a range of neuropsychiatric conditions, yet its clinical utility is constrained by acute psychoactive effects. Here, we report the rational design, synthesis, and evaluation of a focused library of fluorinated reversible N-alkyl carbamate derivatives of psilocin aimed at reducing acute psilocin exposure and thereby limiting hallucinogenic-like effects. Carbamate bond stability was systematically modulated by varying the number and positioning of fluorine atoms on the alkyl promoiety. The resulting compounds exhibited finely tuned hydrolysis under physiological conditions. A selected lead compound (4e) showed favorable oral bioavailability and efficient brain penetration while undergoing partial bioconversion to psilocin. Notably, 4e displayed intrinsic serotonergic activity at 5-HT2A and 5-HT2C receptors but induced attenuated psychotropic effects relative to psilocybin. Overall, these findings highlight fluorinated carbamate chemistry as a versatile platform to control psilocin exposure and serotonergic signaling, rather than the development of a classical pharmacologically inert prodrug.

Introduction: A genetic algorithm (GA)-based approach was designed to predict drug-drug interactions (DDIs) triggered by cytochrome P450 3A (CYP3A) inhibition or induction in horses. Methods: Area under the concentration-time curve ratios (AUCRs), obtained from published in vivo DDI studies in horses, were used to compute the following parameters: (1) the contribution ratio (CR), i.e., the fraction of the substrate dose metabolized via the CYP3A pathway, and (2) the interacting drug's inhibitory potency or inducing efficacy (IR or IC, respectively). Results: AUCRs for 9 substrates, 12 inhibitors, and 1 inducer of equine CYP3A were predicted and validated with the developed method. More than 96% of predictions fell within the commonly accepted range of 50-200% of observed values. Conclusions: The proposed GA-based method may be a useful tool to estimate possible clinically relevant DDIs when co-administration of a CYP3A substrate and a CYP3A-interacting drug is anticipated.

IntroductionPasteurella multocida is a key bacterial agent involved in most respiratory disorders in rabbits. The objective of this study was to evaluate the risk of selecting Pasteurella multocida strains resistant to enrofloxacin (ENRO) in food producing rabbits treated with ENRO via drinking water, according to the standard husbandry practices. Indeed, despite the EU community guidelines recommend a prudent use of antibiotics and promote new strategies to prevent bacterial diseases, antimicrobial therapy remains the primary approach for pasteurellosis management in rabbits. Therefore, the potential risk of selecting resistant bacteria in food-producing animals requires identifying optimized dosage regimens to minimize resistance emergence and to extend the useful lifetime of the drug.MethodsIn this study, we isolated Pasteurella multocida strains from bacterial colonies sampled in nasal swabs collected from 6 healthy rabbits and 12 rabbits suffering respiratory disorders. Animals were sourced from industrial farms and were randomly selected to investigate the inter-individual variability in antimicrobial exposure associated with treatment via drinking water. Sick rabbits underwent an approved ENRO treatment (10 mg/kg for 5 days) administered via drinking water, following standard husbandry practices. We investigated the minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC), and the mutant prevention concentration (MPC) of ENRO against bacterial strains in healthy rabbits and in sick rabbits before and after treatment. We recorded plasma drug concentrations of treated animals, and we applied the mutant selection window (MSW) approach to each subject. Finally, we calculated the PK/PD indices for concentration-dependent antimicrobials to assess ENRO’s clinical efficacy and it’s potential for promoting resistance using published pharmacokinetic (PK) parameters and maximum drug plasma concentrations recorded in this study.ResultsHere we showed that treatment with ENRO improved clinical signs in rabbits with pasteurellosis but failed to completely eradicate the pathogen, consistent with previous studies. MPC-based analysis showed acquired resistance and potential ENRO-induced shift to a lesser sensitivity in the P. multocida population. Moreover, MSW analysis revealed that 45% of treated rabbits exhibited potential for drug resistance selection.ConclusionThese findings suggest that the current ENRO dosing regimen for pasteurellosis in rabbits is inadequate and may contribute to resistance development.

CYP1B1 is a key enzyme involved in xenobiotic and endogenous metabolism, yet its physiological role in bovine liver homeostasis remains unclear. In this study, we generated a CYP1B1 knockout (CYP1B1KO) bovine hepatocyte-like cell line to indirectly investigate its role in liver function. Transcriptomic analysis revealed alterations in immune regulation, epithelial barrier integrity, and detoxification pathways, with concurrent compensatory CYP1A1 upregulation. Beyond its physiological role, CYP1B1 was found to actively participate in Aflatoxin B1 (AFB1) metabolism, a mycotoxin posing significant health risks to humans and livestock. Molecular docking suggested that CYP1B1 facilitates the conversion of AFB1 into AFM1 and AFBO. In agreement with these predictions, CYP1B1KO cells exposed to AFB1 showed reduced AFM1 production and decreased cytotoxicity. Further transcriptomic analysis indicated that CYP1B1KO cells exhibited mitigated oxidative stress and inflammatory responses, along with downregulation of CYP3A74, a key enzyme in AFB1 bioactivation. This suggests that CYP1B1 KO reduces AFB1 toxicity by directly limiting AFB1 bioactivation and indirectly modulating the broader hepatic CYP network, further limiting the formation of toxic intermediates. These findings provide novel insights into CYP1B1’s function in bovine hepatocytes, highlighting its dual role in maintaining liver homeostasis and mediating AFB1 metabolism. The observed interplay between CYP1B1, CYP1A1, and CYP3A74 underscores the complexity of AFB1 biotransformation and warrants further investigation into the coordinated regulation of xenobiotic metabolism in cattle.

Vatinoxan, a peripheral alpha-2 antagonist, mitigates the cardiovascular effects of medetomidine but may cause hypotension during general anesthesia and alter the plasma concentrations of co-administered sedatives and analgesics. This study compares the effect of premedication with or without vatinoxan co-administered with medetomidine and methadone on sedation quality, cardiovascular and respiratory variables, and methadone plasma concentration in anesthetized dogs. Twenty healthy dogs undergoing elective ovariectomy were premedicated with methadone (0.2 mg/kg) and medetomidine (0.15 mg/m2), with vatinoxan (3 mg/m2) in group MV or without vatinoxan in group M. Sedation was assessed using a 21-point scale. Anesthesia was induced 20 min after and maintained with isoflurane. Cardiovascular and respiratory variables were recorded 15 min post-premedication, at traction on both ovarian pedicles, 90 min after premedication and at skin suture. Methadone plasma concentration was measured at 15, 90, and 240 min post-premedication. Sedation scores were significantly higher in group MV at 10 min post-premedication (p = 0.014). Only in group M, heart rate decreased significantly after premedication (p < 0.001). Vatinoxan administration did not affect intraoperative cardiovascular variables during nociceptive stimulation or in its absence. Hypotension was not observed in any dog. Time to recover from anesthesia and postoperative analgesia were not different between groups. Plasma methadone concentrations in group MV were not statistically different from group M, though slightly higher at 15  min post-premedication. For elective ovariectomy, adding vatinoxan to medetomidine and methadone provides rapid sedation, stable cardiovascular function during anesthesia, and smooth recovery causeing non-relevant variations in methadone plasma concentrations.

Within the “One Health, One Medicine” and comparative oncology paradigms, algal extracts have attracted attention, containing natural compounds (NCs) with biological activities, including anti-cancer properties. To characterize the biological effects of a Sphaerococcus coronopifolius extract (SCE), two canine mastocytoma and two normal cell lines were used. After a preliminary screening of three algal extracts, SCE cytotoxicity was measured using Alamar Blue, Sulforhodamine B, and Neutral Red Uptake assays. After assessing the selectivity versus tumor cells and its chemical characterization, SCE mechanisms of action were investigated using RNA-seq, quantitative PCR, flow cytometry and immunoblotting approaches. SCE showed an IC50 comprised between 25 and 35 μg/mL in tumor cell lines, but it also affected normal ones (selectivity index < 2.0). RNA-seq and flow cytometry revealed that SCE negatively affected cell cycle and mevalonate pathway in tumor cells. Additional flow cytometry and immunoblotting investigations suggested a concentration- and time-dependent pro-apoptotic effect of SCE and DNA damage events. In conclusion, SCE demonstrated promising anti-cancer activity in mastocytoma cell lines by targeting the mevalonate pathway, arresting the cell cycle, and inducing apoptosis and DNA damage. Furthermore, the results presented here reinforce the idea that NCs may be promising candidates in comparative anti-cancer chemotherapy.

A genetic algorithm (GA)-based framework was developed to predict drug-drug interactions (DDIs) caused by cytochrome P450 3A (CYP3A) inhibition or induction in dogs and cats. Area under the plasma concentration-time curve (AUC) ratios, obtained from published in vivo DDI studies, were used to calculate the following parameters: (a) the contribution ratio (CR), which represents the fraction of the dose of the victim drug metabolized via CYP3A, and (b) the inhibitory potency (inhibition ratio; IR) or inducing potency (IC) of the perpetrator drug. AUC ratios of 3 substrates, 4 inhibitors and 1 inducer of CYP3A in cats, and the AUC ratios of 10 substrates, 12 inhibitors and 3 inducers of CYP3A in dogs were successfully predicted and validated by the developed methodology within 50-200 % of observed values. This approach could represent a useful resource to predict the extent of DDIs in clinical scenarios requiring the simultaneous administration of a CYP3A substrate drug with a CYP3A perpetrator.

The cytochrome P450 1A (CYP1A) subfamily of xenobiotic metabolizing enzymes (XMEs) consists of two different isoforms, namely CYP1A1 and CYP1A2, which are highly conserved among species. These two isoenzymes are involved in the biotransformation of many endogenous compounds as well as in the bioactivation of several xenobiotics into carcinogenic derivatives, thereby increasing the risk of tumour development. Cattle (Bos taurus) are one of the most important food-producing animal species, being a significant source of nutrition worldwide. Despite daily exposure to xenobiotics, data on the contribution of CYP1A to bovine hepatic metabolism are still scarce. The CRISPR/Cas9-mediated knockout (KO) is a useful method for generating in vivo and in vitro models for studying xenobiotic biotransformations. In this study, we applied the ribonucleoprotein (RNP)-complex approach to successfully obtain the KO of CYP1A1 in a bovine foetal hepatocyte cell line (BFH12). After clonal expansion and selection, CYP1A1 excision was confirmed at the DNA, mRNA and protein level. Therefore, RNA-seq analysis revealed significant transcriptomic changes associated with cell cycle regulation, proliferation, and detoxification processes as well as on iron, lipid and mitochondrial homeostasis. Altogether, this study successfully generates a new bovine CYP1A1 KO in vitro model, representing a valuable resource for xenobiotic metabolism studies in this important farm animal species.

In human, the cytochrome P450 3A (CYP3A) subfamily of drug-metabolizing enzymes (DMEs) is responsible for a significant number of phase I reactions, with the CYP3A4 isoform superintending the hepatic and intestinal metabolism of diverse endobiotic and xenobiotic compounds. The CYP3A4-dependent bioactivation of chemicals may result in hepatotoxicity and trigger carcinogenesis. In cattle, four CYP3A genes (CYP3A74, CYP3A76, CYP3A28 and CYP3A24) have been identified. Despite cattle being daily exposed to xenobiotics (e.g., mycotoxins, food additives, drugs and pesticides), the existing knowledge about the contribution of CYP3A in bovine hepatic metabolism is still incomplete. Nowadays, CRISPR/Cas9 mediated knockout (KO) is a valuable method to generate in vivo and in vitro models for studying the metabolism of xenobiotics. In the present study, we successfully performed CRISPR/Cas9-mediated KO of bovine CYP3A74, human CYP3A4-like, in a bovine foetal hepatocyte cell line (BFH12). After clonal expansion and selection, CYP3A74 ablation was confirmed at the DNA, mRNA, and protein level. The subsequent characterization of the CYP3A74 KO clone highlighted significant transcriptomic changes (RNA-sequencing) associated with the regulation of cell cycle and proliferation, immune and inflammatory response, as well as metabolic processes. Overall, this study successfully developed a new CYP3A74 KO in vitro model by using CRISPR/Cas9 technology, which represents a novel resource for xenobiotic metabolism studies in cattle. Furthermore, the transcriptomic analysis suggests a key role of CYP3A74 in bovine hepatocyte cell cycle regulation and metabolic homeostasis

OBJECTIVE:To investigate pharmacokinetics (PK) of fentanyl administered by target-controlled infusion (TCI), and to develop a PK model optimized by covariates for TCI in anaesthetized dogs.STUDY DESIGN:Prospective clinical study.ANIMALS:A group of 20 client-owned dogs with spinal pain undergoing anaesthesia for magnetic resonance imaging.METHODS:Fentanyl was administered as an infusion to 20 anaesthetized dogs using a TCI system incorporating a previously described fentanyl two-compartment PK. Arterial blood samples were collected at specific time points during the infusion and over 60 minutes post-infusion for measurement of fentanyl plasma concentrations. The predictive performance of the Sano PK model was assessed by comparing predicted and measured plasma concentrations. A population PK analysis was then performed using a nonlinear mixed-effect modelling approach, allowing inter- and intra-individual variability estimation. Finally, a quantitative stepwise evaluation of the influence of various covariates such as weight, body condition score, size, size-related age, sex and type of premedication on the PK model was considered.RESULTS:Overall predictive performance of the Sano PK set of variables was not clinically acceptable in anaesthetized dogs. Fentanyl PK was best described by a three-compartment model. Weight and sex were found to affect the volume of distribution of the central compartment. Addition of these two covariate/variable associations resulted in a reduction of the objective function value (OFV) from -340.18 to -448.34, and of the median population weighted residual and the median population absolute weighted residual from 16.1% and 38.6% to 3.9% and 20.3%, respectively. Fentanyl infusions at measured concentrations up to 5.4 ng mL-1 in sevoflurane-anaesthetized dogs resulted in stable anaesthesia and smooth recoveries without complications.CONCLUSIONS AND CLINICAL RELEVANCE:A population three-compartment PK model for fentanyl TCI in anaesthetized dogs was developed. Weight and sex have been detected and incorporated as significant covariates.

The use of antimicrobials in agricultural, veterinary and medical practice exerts selective pressure on environmental microbiota, promoting the emergence and spread of antimicrobial resistance (AMR), a global concern for the One Health Initiative Task Force (OHITF). Honeybees have been studied as bioindicators of AMR in the environment, but little is known about beehive products like honey and pollen. The aim of this study was to assess the prevalence of AMR genes (ARGs) in beehive products and investigated their origins. Specifically, possible associations between ARGs, microbiota and other characteristics of different honey and pollen samples, including country of origin, flower type, type of commercial distribution and environmental factors, such as land use, weather and composition of the environment surrounding the beehives were investigated. We found that beehive products harboured ARGs conferring resistance to β-lactams, macrolides, (fluoro)quinolones and polymyxins. Most samples possessed resistance to multiple antimicrobial classes, with honey and pollen showing similar ARG profiles. Even if Lactobacillus and Acinetobacter genera were common in the microbial communities of both honey and pollen, Bacillus, Clostridium, and Bombella defined honey microbiota, while Pseudomonas and Vibrio were enriched in pollen. ErmB and blaTEM-1 co-occurred with Lactobacillus and Fructobacillus, while positive associations between β-lactams and macrolides and anthropogenic environments (i.e. industrial and commercial areas and non-irrigated arable lands) were found. Altogether, our findings suggest that ARGs in honey and pollen might originate from the honeybee foraging environment, and that the beehive products can be used as bioindicators of the AMR environmental contamination.

In cattle, phenobarbital (PB) upregulates target drug-metabolizing enzyme (DME) mRNA levels. However, few data about PB's post-transcriptional effects are actually available. This work provides the first, and an almost complete, characterization of PB-dependent changes in DME catalytic activities in bovine liver using common probe substrates and confirmatory immunoblotting investigations. As expected, PB increased the total cytochrome P450 (CYP) content and the extent of metyrapone binding; moreover, an augmentation of protein amounts and related enzyme activities was observed for known PB targets such as CYP2B, 2C, and 3A, but also CYP2E1. However, contradictory results were obtained for CYP1A, while a decreased catalytic activity was observed for flavin-containing monooxygenases 1 and 3. The barbiturate had no effect on the chosen hydrolytic and conjugative DMEs. For the first time, we also measured the 26S proteasome activity, and the increase observed in PB-treated cattle would suggest this post-translational event might contribute to cattle DME regulation. Overall, this study increased the knowledge of cattle hepatic drug metabolism, and further confirmed the presence of species differences in DME expression and activity between cattle, humans, and rodents. This reinforced the need for an extensive characterization and understanding of comparative molecular mechanisms involved in expression, regulation, and function of DMEs.

This work represents a step in the identification of hazards associated with the consumption of Italian Rhododendron honey, which could contain grayanotoxins, which can be found in plant belonging to genera of the Ericaceae family. An LC-MS/MS method was developed and validated to quantify Grayanotoxin I (GTX I) in 125 Italian Rhododendron honeys collected from 2017 to 2019. Sensitivity and linearity were demonstrated in the range 10-1000 mu g kg (-1), and a LOQ value of 10 mu g kg(-1) was obtained. Absolute recovery ranged from 88.2% to 99.3% and no matrix effect was measured. GTX I was found in the 30.4% of the samples, and the concentrations ranged from 12.3 to 103.5 mu g kg(-1). Multivariate statistical approaches were applied to investigate the relationship between the presence/absence of GTX I and different honey features (e.g. environmental factors, quality aspects). The main factors affecting the presence of GTX I resulted the environment, province, altitude and the purity of botanical origin. The mean GTX I level detected in the 'positive' Italian Rhododendron honeys was 119-668 times lower than GTX content found in mad honeys, which caused human intoxication, so the probability of an acute toxic syndrome, in this scenario, is remote.

A validated method in liquid chromatography tandem mass spectrometry was applied to assess the contamination profile of 17 pyrrolizidine alkaloids and their N-oxide metabolites (PAs/PANOs) in 775 honey samples of different origins, including from local beekeepers (579, LB), large retail and online stores (Italian (LRI) and foreign (LRF) products). Fifty-five percent of the honey samples contained the 17 monitored compounds at below the limit of quantification. The LB samples were characterised by a total mean concentration of PAs/PANOs at 6.7 and 3.3 times lower than those of the LRF and LRI samples, respectively. The PA echimidine characterised mainly honey samples from the large retail stores (75.8 % and 72.5 % of LRI- and LRF-contaminated samples, respectively). The LB samples showed a senecionine-type contamination profile, providing beekeepers clues to the plants responsible for contamination. The results highlighted that the consumption of the local honey samples analysed does not represent a double risk for human health compared to the consumption of products from large retail stores. Careful attention must be paid to honey imported from areas where lycopsamine-type and senecionine-type PAs/PANOs-producing plants are invasive. A machine learning approach could predict the origin of the sample given its contaminant profile at an accuracy of 0.74, which is useful for detecting fraud in the declared honey origin.