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The use of cannabis-based products for therapeutic purposes is a reality in the field of animal health. However, although cannabis is considered safe when appropriately used by human patients, cannabis-based products can pose a risk to companion animals such as dogs, depending on their composition or route of administration. Thus, this article discusses aspects of the safety and efficacy of different cannabis-based products in dogs' treatment through an integrative review. The review was systematically performed in Medline (via Pubmed

Ivermectin (IVM) is a drug widely used in veterinary and human medicine for the management of parasitic diseases. Its repositioning potential has been recently considered for the treatment of different diseases, such as cancer and viral infections. However, IVM faces some limitations to its formulations due to its low water solubility and bioavailability, along with reports of drug resistance. In this sense, novel technological approaches have been explored to optimize its formulations and/or to develop innovative medicines. Therefore, this review discusses the strategies proposed in the last decade to improve the safety and efficacy of IVM and to explore its novel therapeutic applications. Among these technologies, the use of micro/nano-drug delivery systems is the most used approach, followed by long-acting formulations. In general, the development of these novel formulations seems to run side by side in veterinary and human health, showing a shared interface between the two areas. Although the technologies proposed indicate a promising future in the development of innovative dosage forms containing IVM, its safety and therapeutic targets must be further evaluated. Overall, these approaches comprise tailoring drug delivery profiles, decreasing the risks of developing drug resistance, and supporting the application of IVM for reaching different therapeutic targets.

Serology assays are essential tools to mitigate the effect of COVID-19, help to identify previous SARS-CoV-2 infections or vaccination, and provide data for surveillance and epidemiologic studies. In this study, we report the production and purification process of the receptor-binding domain (RBD) of SARS-CoV-2 in HEK293 cells, which allowed the design, optimization, and validation of an indirect ELISA (iELISA) for the detection of human anti-RBD antibodies. To find the optimal conditions of this iELISA, a multivariate strategy was performed throughout design of experiments (DoE) and response surface methodology (RSM), one of the main tools of quality by design (QbD) approach. The adoption of this strategy helped to reduce the time and cost during the method development stage and to define an optimum condition within the analyzed design region. The assay was then validated, exhibiting a sensitivity of 94.24 (86.01-98.42%; 95% CI) and a specificity of 95.96% (89.98-98.89%; 95% CI). Besides, the degree of agreement between quality results assessed using kappa's value was 0.92. Hence, this iELISA represents a high-throughput technique, simple to perform, reliable, and feasible to be scaled up to satisfy the current demands. Since RBD is proposed as the coating antigen, the intended use of this iELISA is not only the detection of previous exposure to the virus, but also the possibility of detecting protective immunity. KEY POINTS: • RBD was produced in 1-L bioreactor and highly purified. • An iELISA assay was optimized applying QbD concepts. • The validation procedure demonstrated that this iELISA is accurate and precise.

We developed a fast, rabies virus-free, in vitro method, based on a blocking ELISA (bELISA), to detect and accurately quantify anti-rabies glycoprotein antibodies in serum of several animal species. In this method, purified rabies virus-like particles (VLPs) are used as antigen to coat the plates, while the presence of specific rabies immunoglobulins is revealed through blocking the recognition of these VLPs by a biotinylated monoclonal antibody. A quality by design approach was carried out in order to optimize the method performance, improving the sensitivity and, thereby, reducing the limit of detection of this assay. After the method validation, we confirmed that the bELISA method is able to detect a concentration of 0.06 IU/mL rabies immunoglobulins, titer lower than the 0.5 IU/mL cutoff value established as indication for correct vaccination. Further, we assessed the correlation between bELISA, the MNT, and the Platelia methods, confirming the accuracy of this new assay. On the other hand, precision was evaluated, obtaining acceptable repeatability and intermediate precision values, showing that this bELISA could be proposed as a potential alternative method, replacing the gold standard techniques in vaccination schemes and becoming a routine control technique within regional rabies surveillance programs.

The aim of this study was to assess the impact of BMI on perioperative outcomes in patients undergoing VATS lobectomy or segmentectomy. Data from 5088 patients undergoing VATS lobectomy or segmentectomy, included in the VATS Group Italian Registry, were collected. BMI (kg/m

Vaccination still represents the most efficient and inexpensive strategy in the control of hepatitis B virus (HBV) infection. However, about 10% of the population vaccinated with the current S yeast-derived vaccine fail to induce an adequate immune response. Our group has developed a new-generation hepatitis B vaccine candidate composed by the three surface proteins of the HBV. Here we describe the methods to develop and characterize a stable CHO-K1 recombinant cell line able to produce and secrete hepatitis B subviral envelope particles (HBV-SVPs) containing L and M glycoproteins in addition to S glycoprotein. In addition, Western blot and immunogold electron microscopy techniques to evaluate the size, morphology, and composition of the particles are explained. Finally, immunization protocols are described in order to study the immunogenicity of HBV-SVPs and the ability of the antibodies triggered by these particles to recognize the binding site of HBV with the hepatocyte.

The high degree of precision and control of 3D printing has given formulators the autonomy to engineer sophisticated and personalised medicines, starting a revolution in pharmaceutics. In addition, dosage forms with tailored drug release profile can be produced by changing some parameters of the 3D printing processes. Therefore, 3D printed medicines must be characterised in an orthogonal approach, to establish their physicochemical and biopharmaceutical features, and consequently to understand how these characteristics can be customised by changing the formulation and process parameters to ensure medicines' safety and efficacy. Given the recent regulation and commercialisation of 3D printed medicines, several methods and techniques have been transposed from official compendia; however, formulators must still make a critical assessment of their practical implications. A comprehensive review of the findings obtained by the characterisation of 3D printed oral dosage forms using traditional and advanced techniques is therefore presented here, to drive formulators towards a rational pharmaceutical development pathway. The characterisation methods have been classified in terms of their physicochemical or biopharmaceutical character. Interestingly, beyond the rise of modern characterisation techniques, the reassessment of data obtained by traditional methods has provided knowledge and a solid foundation to support the evolution of 3D printing techniques in pharmaceutics.

Chimeric virus-like particles are self-assembling structures composed of viral proteins that had been modified to incorporate sequences from different organisms, being able to trigger immune responses against the heterologous sequence. However, the identification of suitable sites for that purpose in the carrier protein is not an easy task. In this work, we describe the generation of rabies chimeric VLPs that expose a major antigenic site of foot-and-mouth disease virus (FMDV) by identifying suitable regions in rabies glycoprotein (RVG), as a proof of concept of a novel heterologous display platform for vaccine applications. To identify adequate sites for insertion of heterologous sequences without altering the correct folding of RVG, we identified regions that were evolutionally non-conserved in Lyssavirus glycoproteins and performed a structural analysis of those regions using a 3D model of RVG trimer that we generated. The heterologous sequence was inserted in three different sites within RVG sequence. In every case, it did not affect the correct folding of the protein and was surface exposed, being recognized by anti-FMDV antibodies in expressing cells as well as in the surface of VLPs. This work sets the base for the development of a heterologous antigen display platform based on rabies VLPs. Key points • Adequate regions for foreign epitope display in RVG were found. • G-H loop of FMDV was inserted in three regions of RVG. • The foreign epitope was detected by specific antibodies on fusion proteins. • G-H loop was detected on the surface of chimeric VLPs.

Rabies is an ancient fatal disease with no other available treatment than post-exposure vaccination, where the bite of infected animals, mainly dogs, is the leading cause of its transmission to human beings. In this context, global vaccination campaigns of companion animals, as well as wildlife reservoirs vaccination, are key factors to achieve the “Zero by 30” plan that pursues the eradication of dog-mediated human rabies by 2030. Rabies virus-neutralizing antibodies (VNAs) play an essential role in the disease protection, as it correlates with an adequate immune response and allows evaluating pre- or post-exposure prophylaxis efficacy. Hence, counting with reliable, accurate, and robust serological tests is of paramount importance. Currently, RFFIT and FAVN are the gold standard VNAs tests recommended by both the WHO and the OIE. Despite these methodologies are efficient and widely used, they present several drawbacks, as they are less easily to standardize and require the use of live rabies virus, containment facilities, and skilled professionals. Thus, in this review, we describe the state-of-the-art of alternative analytical methodologies currently available for rabies serology, with novel approaches based on pseudotyped recombinant viruses and emphasizing in the antigen binding methodologies that detect and quantify antibodies against the rabies glycoprotein. We discussed the wide range of assays that are interesting tools for a faster measurement of anti-rabies glycoprotein antibodies and, in some cases, less complex and more versatile than the gold standard methods. Finally, we discussed the key issues during the design and optimization steps of ELISA assays, highlighting the importance of validation and standardization procedures to improve rabies serology tests and, as a consequence, their results. Key points • An exhaustive revision of rabies serology testing was made. • No rabies serology assay can be thought as better than others for all intents and purposes. • The validation procedure guarantees reliable and consistent results among the globe.

Thoracoscopic lobectomy (VATS-L) for non-small-cell lung cancer (NSCLC) is a well-established option for early stage NSCLC, but the evidences are limited for octogenarians.The objectives of this multi-institutional study were to evaluate the post-operative outcomes of VATS-L in octogenarians and to estimate the post-operative quality of life (QoL) using a validated questionnaire (EuroQoL5D).Data from patients underwent VATS-L between 2014 and 2019 were analysed and divided into two groups: Group A (younger patients) and Group B (octogenarians). To define predictors for complications, univariate and multivariable logistic regression analysis were performed.7023 patients underwent VATS-L and 329 (4.6%) were octogenarians. 30-day and 90-day post-operative mortality were similar (0.95% vs 0.91%, p = 0.84 and 1.3% vs 1.2%, p = 0.58), whereas the percentage of patients who suffered from any complication (25.5% vs 31.9%, p = 0.012) and the complication rate (31.6% vs 45.2%, p=0.01) were higher for octogenarians. At discharge, the values of EuroQoL5D were worse in group B, but after one month these levels became similar. Age80 years had a significant influence on morbidity on both univariate and multivariable analyses (p = 0.025).VATS-L for NSCLC can be performed in selected octogenarians without increased risk of post-operative death, acceptable not-life-threatening complications and a moderate impact on QoL.

Equine chorionic gonadotropin (eCG) is a heterodimeric glycoprotein hormone produced by pregnant mares that has been used to improve reproductive performance in different domestic species. Several strategies to produce the hormone in a recombinant way have been reported; nevertheless, no approach has been able to produce a recombinant eCG (reCG) with significant in vivo bioactivity or in sufficient quantities for commercial purposes. For this reason, the only current product available on the market consists of partially purified preparations from serum of pregnant mares (PMSG). Herein, we describe a highly efficient process based on third-generation lentiviral vectors as delivery method for the production of reCG in suspension CHO-K1 cells, with productivities above 20 IU 106 cell−1.d−1 and 70% purification yields after one purification step. Importantly, reCG demonstrated biological activity in cattle, since around 30 μg of reCG were needed to exert the same biologic effect of 400 IU of PMSG in an ovulation synchronization protocol. The results obtained demonstrate that the developed strategy represents an attractive option for the production of reCG and constitutes an auspicious alternative for the replacement of animals as a source of PMSG.

Equine chorionic gonadotropin (eCG) is a heterodimeric glycoprotein hormone produced by pregnant mares that has been used to improve reproduction activity in different types of livestock. Several strategies to produce the hormone in a recombinant way have been reported; nevertheless, none approach has been able to produce a recombinant eCG (reCG) with significant in vivo bioactivity or in sufficient quantities for commercial purposes. For this reason, the only current product available on the market consists of partially purified preparations from serum of pregnant mares (PMSG). Herein, we describe a highly efficient process based on third-generation lentiviral vectors as delivery method for the production of reCG in suspension CHO-K1 cells, with productivities above 20 IU.106 cell-1.d-1 and 70% purification yields after one purification step. Importantly, reCG not only demonstrated biological activity in bovine cattle but also this bioactivity appeared to be higher than PMSG, since 140 IU of reCG were needed to exert the same biologic effect in an ovulation synchronization protocol compared to 400 IU of PMSG. The results obtained show that the developed strategy represents an attractive option to produce reCG and constitutes an auspicious alternative for the replacement of animals as a source of PMSG.

Ovotesticular Difference of Sex Development (OT DSD) is a rare condition characterized by histologic demonstration of ovarian and testicular tissue in the same individual. Descriptions in literature usually do not include long term follow-up data.The aim of this study is to describe clinical, biochemical and histological findings, as well as long term outcomes (including onset and progression of puberty) in patients with OT DSD.In a retrospective study of 31 patients, findings include predominantly male gender assignment at the time of referral (54.8%) and subsequent female gender of rearing (54.8%). The most frequent karyotype was 46,XX (58.1%). Ovotestis was the most frequent gonad (48.4%) Puberty could be evaluated in 20 patients, being spontaneous in 12 of them. Four patients with partial gonadectomy in infancy were able to enter female puberty spontaneously.It was observed that patients who preserved gonadal tissues were able to enter puberty spontaneously.

We carried out an investigation on rabies virus-like particles (RV-VLPs) expressed in HEK293 cells using serum free medium. These RV-VLPs were formulated with two different adjuvants in order to analyse the enhancement of the triggered immune response and its stability. In experiments in mice, RV-VLPs showed an enhanced humoral immune response when injected with adjuvant, in contrast to the obtained for the RV-VLPs without adjuvant addition. Besides, higher titers of neutralizing antibodies were induced when RV-VLPs were formulated with LipoSap® in comparison with the obtained with Alhydrogel®. At the same time, the positive effect of this adjuvant in vaccine’s potency and stability was demonstrated, showing that LipoSap® significantly increases the value obtained in NIH efficiency test for rabies vaccine, and proving that this value is maintained after 15 months storage at 4 °C. Further, we showed that RV-VLPs induces an immune response based on neutralizing antibodies when cat, dogs and bovines were vaccinated with only one dose of RV-VLPs. These results demonstrated that this vaccine candidate could be applied for the prevention of rabies in pets as well as for the control of paralytic rabies in cattle.

Substances with cytotoxic activity present in vaccines against the foot-and-mouth disease may interfere with methods used to detect residual live virus in the product or cause undesirable postvaccination reactions. This study describes a rapid in vitro test to detect cytotoxic activity in water-in-oil vaccines against foot-and-mouth disease using a commercial saponin as a cytotoxic agent and a solution of sheep’s red blood cells as substrate. Hemolytic and cytotoxic activity was analyzed using experimental and commercial vaccines prepared with and without saponin. The hemolytic and cytotoxic potential of preparations containing saponin was evident. In contrast, hemolytic and cytotoxic activities were not observed in vaccines without saponin in their composition. The method described here allows to easily detect if the vaccine under study has cytotoxic activity, making it possible to select the most appropriate method to process the sample to be used for the innocuity test. Additionally, due to undesirable effects that may be observed in animals receiving vaccines containing saponin in their formulation, the use of the rapid test described here allows to identify those vaccines with cytotoxic activity and to verify the presence of saponin on them, through the mass spectrometry method. RESUMO Substâncias com atividade citotóxica presentes em vacinas contra febre aftosa podem interferir com o método utilizado para a detecção de vírus ativo residual no produto ou causar reações pós-vacinais indesejáveis. O presente trabalho descreve uma prova rápida in vitro para detectar atividade citotóxica em vacinas oleosas contra febre aftosa utilizando uma saponina comercial como agente citotóxico e uma solução de hemácias de carneiro como substrato. Analisaram-se as atividades hemolítica e citotóxica utilizando-se vacinas experimentais e comerciais preparadas com e sem saponina. O potencial hemolítico e citotóxico dos preparados que continham saponina na sua formulação foi evidente. Em contraste, não se observou atividade hemolítica e citotóxica nas vacinas sem saponina. O método descrito permite conhecer rapidamente se a vacina em estudo apresenta atividade citotóxica e, dessa maneira, selecionar o método mais adequado para processar a amostra que será utilizada para investigar a presença de vírus ativo residual. Adicionalmente, devido aos efeitos indesejáveis que podem ser observados em animais que recebem vacinas que contêm saponina na sua formulação, o uso da prova rápida descrita permite selecionar aquelas vacinas com atividade citotóxica para posteriormente verificar a presença de saponina através de técnicas analíticas como a espectrometria de massas.

Validation of predictive risk models for prolonged air leak (PAL) is essential to understand if they can help to reduce its incidence and complications. This study aimed to evaluate both the clinical and statistical performances of 4 existing models. We selected 4 predictive PAL risk models based on their scientific relevance. We referred to these models as Chicago, Bordeaux, Leeds and Pittsburgh model, respectively, according to the affiliation place of the first author. These predicting risk models were retrospectively applied to patients recorded on the second edition of the Italian Video-Assisted Thoracoscopic Surgery Group registry. Predictions for each patient were calculated based on the logistic regression coefficient values provided in the original manuscripts. All models were tested for their overall performance, discrimination, and calibration. We recalibrated the original models with the re-estimation of the model intercept and slope. We used curve decision analysis to describe and compare the clinical effects of the studied risk models. Better statistical metrics characterize the models developed on larger populations (Chicago and Bordeaux models). However, no model has a valid benefit for threshold probability greater than 0.30. The Net benefit of the most performing model (Bordeaux model) at the threshold probability of 0.11 is 23 of 1000 patients, burdened by 333 false positive cases. One of 1000 is the Net benefit at the threshold probability of 0.3. The use of PAL scores based on preoperative predictive factors cannot be currently used in a clinical setting because of a high false positive rate and low positive predictive value.

A liquid chromatography (LC) method for the quantification of tretinoin (TTN) in different matrices (adhesive tape, cotton and porcine skin layers, stratum corneum, viable epidermis, and dermis) was validated and applied in in vitro porcine skin penetration/retention studies. This study proposes, for the first time, a method for assaying TTN in separated porcine skin layers (stratum corneum, viable epidermis, and dermis). The skin studies were carried out using tape stripping and cutaneous retention techniques. The procedures for the extraction of TTN from dermatological formulations (creams and gels) and biological and non-biological matrices used with the tape stripping and retention techniques were also evaluated. The LC method consisted of a mobile phase composed of a mixture of methanol, water, and glacial acetic acid (85:15:1, v/v); a C18 column used as the stationary phase; a flow rate of 1.0 mL min−1; an injection volume of 100 μL; and TTN detection at 342 nm. The method was linear in the range of 0.05–15.00 μg mL−1 (r = 0.9999), and it was precise and accurate. The limit of detection (LOD) and limit of quantification (LOQ) were 0.0165 μg mL−1 and 0.0495 μg mL−1, respectively. TTN was extracted from different matrices, showing good precision [relative standard deviation (RSD) of, Liquid chromatography; Tretinoin; Validation; Skin penetration and retention, Analytical chemistry; Chromatography; Pharmaceutical chemistry; Pharmaceutical science; Drug delivery

Introduction To explore baseline knowledge about avalanche guidelines and the Avalanche Victim Resuscitation Checklist (AVReCh) in Italy and the knowledge acquisition from a standardized lecture. Methods Standardized lecture material discussing AVReCh was presented during 8 mountain medicine courses from November 2014 to April 2016 in different regions of Italy. To determine the knowledge acquisition from the lecture, a pre- and postlecture survey was utilized. Results A total of 193 surveys were analyzed. More than 50% of the participants had never participated in lectures/courses on avalanche guidelines, and less than 50% of the participants knew about the AVReCh before the lecture. The correct temporal sequence of reportable information in the basic life support section of the AVReCh was selected by 40% of the participants before the lecture and by 75% after the lecture ( P P P Conclusions Health care providers and mountain rescue personnel are not widely aware of avalanche guidelines. The standardized lecture significantly improved knowledge of the principles of avalanche management related to core AVReCh elements. However, the effect that this knowledge acquisition has on avalanche victim survival or adherence to the AVReCh in the field is yet to be determined.

The application of nanocarrier aqueous suspensions as drug delivery systems offers an important challenge for formulators: their reduced physicochemical stability during storage. Here, a novel approach to produce dry redispersible nanocapsules by fluid bed granulation intended for oral administration is proposed. In addition, the influence of the cationic coating of nanoparticles on the properties of granules was evaluated. Lipid-core nanocapsules were prepared in aqueous dispersion with or without a cationic coating by chitosan to be used as the granulating liquid in the fluid bed granulation process. Granule properties, like volume-based mean particle size, yield, moisture content, morphological characteristics, flow properties, and redispersion behavior were evaluated. The granulation process had a yield close to 90%, affording granules with moisture content lower than 4% and good flow properties. Nanocapsules adhered to the surface of the primary particles which agglomerating to form the granules, but were released upon redispersion in water. Moreover, cationic coating of the nanocapsules improved the aqueous redispersion behavior of granules. In conclusion, this novel approach to produce granules as an intermediate or final dosage form by fluid bed granulation represents an innovative process to develop new oral nanomedicines. Furthermore, the cationic coating of nanocapsules has an important role on the aqueous redispersion properties of these granules.

Cognitive dysfunctions are the main symptoms associated with Alzheimer's disease (AD). In the last decade, the use of natural substances to prevent or alleviate neurological symptoms have attracted great interest. Curcumin, the main polyphenolic component of turmeric (Curcuma longa) rhizomes, has a variety of beneficial properties, including potent antioxidant and anti-inflammatory properties. Despite the therapeutic potential for neurodegenerative diseases including AD, the use of this natural compound as a drug in therapies has huge limitations due to its low water solubility, physicochemical instability, and poor bioavailability. Nanotechnology is an important tool to overcome these drawbacks. Here, the safety of curcumin and its nanoencapsulated form was evaluated using zebrafish embryos. We also investigated the potential protective effect of curcumin and nanoencapsulated curcumin (50 mg/kg, single injection, i.p.) against scopolamine-induced memory deficits in adult zebrafish. The embryotoxicity test revealed that the formulations in any dose tested did not cause embryo mortality or affect the hatching rate. Data showed that both curcumin and nanoencapsulated curcumin are effective in preventing scopolamine-induced cognitive impairments without altering the animals' locomotion. These findings suggest that these treatments are safe and could be useful for enhancing memory function in AD or other age-related disorders.

This study proposes a novel blend of drying adjuvants (lactose and polyvinylpyrrolidone) as an approach to produce dispersible powders containing nanocapsules for the development of skin delivery s...

Besides their clinical application, chronic misuse of opioids has often been associated to drug addiction due to their addictive properties, underlying neuroadaptations of AMPA glutamate-receptor-dependent synaptic plasticity. Topiramate (TPM), an AMPAR antagonist, has been used to treat psychostimulants addiction, despite its harmful effects on memory. This study aimed to evaluate the effects of a novel topiramate nanosystem on molecular changes related to morphine reinstatement. Rats were previously exposed to morphine in conditioned place preference (CPP) paradigm and treated with topiramate-chitosan nanoparticles (TPM-CS-NP) or non-encapsulated topiramate in solution (S-TPM) during CPP extinction; following memory performance evaluation, they were re-exposed to morphine reinstatement. While morphine-CPP extinction was comparable among all experimental groups, TPM-CS-NP treatment prevented morphine reinstatement, preserving memory performance, which was impaired by both morphine-conditioning and S-TPM treatment. In the NAc, morphine increased D1R, D2R, D3R, DAT, GluA1 and MOR immunoreactivity. It also increased D1R, DAT, GluA1 and MOR in the dorsal hippocampus. TPM-CS-NP treatment decreased D1R, D3R and GluA1 and increased DAT in the NAc, decreasing GluA1 and increasing D2 and DAT in the dorsal hippocampus. Taken together, we may infer that TPM-CS-NP treatment was able to prevent the morphine reinstatement without memory impairment. Therefore, TPM-CS-NP may be considered an innovative therapeutic tool due to its property to prevent opioid reinstatement because it acts modifying both dopaminergic and glutamatergic neurotransmission, which are commonly related to morphine addiction.