Chitosan and hyaluronic acid nanoparticles as vehicles of epoetin beta for subconjunctival ocular delivery

Research Areas: Pharmacology & PharmacyNeuroprotection in glaucoma using epoetin beta (EPOβ) has yielded promising results. Our team has developed chitosan-hyaluronic acid nanoparticles (CS/HA) designed to carry EPOβ into the ocular globe, improving the drug’s mucoadhesion and retention time on the ocular surface to increase its bioavailability. In the present in vivo study, we explored the possibility of delivering EPOβ to the eye through subconjunctival administration of chitosan-hyaluronic acid-EPOβ (CS/HA-EPOβ) nanoparticles. Healthy Wistar Hannover rats (n = 21) were split into 7 groups and underwent complete ophthalmological examinations, including electroretinography and microhematocrit evaluations before and after the subconjunctival administrations. CS/HA-EPOβ nanoparticles were administered to the right eye (OD), and the contralateral eye (OS) served as control. At selected timepoints, animals from each group (n = 3) were euthanized, and both eyes were enucleated for histological evaluation (immunofluorescence and HE). No adverse ocular signs, no changes in the microhematocrits (≈45%), and no deviations in the electroretinographies in both photopic and scotopic exams were observed after the administrations (p < 0.05). Intraocular pressure remained in the physiological range during the assays (11–22 mmHg). EPOβ was detected in the retina by immunofluorescence 12 h after the subconjunctival administration and remained detectable until day 21. We concluded that CS/HA nanoparticles could efficiently deliver EPOβ into the retina, and this alternative was considered biologically safe. This nanoformulation could be a promising tool for treating retinopathies, namely optic nerve degeneration associated with glaucoma.info:eu-repo/semantics/publishedVersio

Epidemiological study of pesticide poisoning in domestic animals and wildlife in Portugal : 2014–2020

Research Areas: Veterinary SciencesABSTRACT - Nowadays the intentional poisoning of domestic and wild animals is a crime in the European Union (EU), but as in the past the poison is still used in rural areas of a number of European countries to kill animals that were considered harmful for human activities. From January 2014 up until October 2020, the Laboratory of Pharmacology and Toxicology of the Faculty of Veterinary Medicine (LFT-FMV) has done the analytical detection of poisoning substances in 503 samples of wildlife and domestic animals and pesticides residues were found in 239 of the samples analyzed. In this retrospective study, toxicology results from domestic species (dog, cat, sheep, cows, and horses), wildlife species (red foxes, birds of prey, lynx, and wild boar), and food baits, are presented. During this period the samples analyzed at the LFT-FMV, were received from all over the country. Analytical detections were performed via solvent extraction followed by thin layer chromatography. Molluscicides (47%, n = 109) and Carbamates (24%, n = 57) were found to be the first category of pesticides involved in intoxications, in both domestic and wild animals, followed by rodenticides (13%, n = 30)—in this group second and third generation, were the most represented; Strychnine is the third (11%, n = 26) even though this pesticide has been banned in Portugal since 1988 and in the European Union since 2006 and finally Organophosphates (5%, n = 11) in the small number. This study allowed to realize that a great number of positive samples involved banned pesticides (i.e., Aldicarb and Strychnine) but, at the same time, many positives cases were due to the exposure to commercially available products (i.e., Methiocarb and Anticoagulant rodenticides). Also, it’s possible to identify the areas where domestic species are the most affected (i.e., Setubal and Lisboa) and the areas where the wild animals are the mainly affected species (i.e., Faro, Castelo Branco, and Bragança).info:eu-repo/semantics/publishedVersio

Anacardium occidentale Bark as an Antidiabetic Agent

Anacardium occidentale L. is used throughout the world to treat type 2 diabetes. In Portugal, a traditional herbal preparation made with stem bark of this species (AoBTHP) has been used for more than 30 years to treat this pathology. The AoBTHP was standardized on total phenolic content, and its hypoglycemic activity was assessed using db/db mice (n = 26) for 92 days. Three doses (40.2, 71.5, and 127.0 mg/kg/day, per os) were tested, and glibenclamide (5 mg/kg/day) was used as positive control. During the study, glycemia was measured under non-fasting or fasting states. In sequence, thin-layer chromatography bioautographic assays were used for the detection of possible alpha- and beta-glucosidase inhibitors. A significant hypoglycemic effect in fasting glycemia in days 31 and 57 was observed with the three tested doses. The 71.5 mg/kg and 127.0 mg/kg AoBTHPs significantly reduced non-fasting glycemia on day 24. The highest dose showed the most significant hypoglycemic effect. Gallic acid was identified as the major alpha- and beta-glucosidase inhibitor. The 127 mg/kg/day AoBTHP dose showed a greater glucose-lowering effect than glibenclamide. For the first time, a standardized AoBTHP was tested using an in vivo diabetes model, and its usage was preclinically validated for type 2 diabetes treatment. The hypoglycemic activity of an AoBTHP can be related to the presence of alpha- and beta-glucosidase inhibitors, such as gallic acid, but other mechanisms can also be involved.info:eu-repo/semantics/publishedVersio

Evaluation of the humoral immune response induced by vaccination for canine distemper and parvovirus: a pilot study.

BACKGROUND: Canine Distemper Virus (CDV) and Canine Parvovirus (CPV) lead to infections with high mortality rates in dogs. These viruses affect unvaccinated dogs or dogs with incomplete vaccination protocols. Vaccination plays an important role in reducing death rates, preventing clinical cases and controlling the spread of virus However, the efficacy of vaccination might be affected by different factors including vaccine scheduling and the neutralization of the vaccine targets by maternal antibodies. In face of these factors, the main goals of this study are (i) to investigate the antibody responses of puppies undergoing different primary vaccination protocols against CPV and CDV and (ii) to estimate the time until seroreversion in adult dogs unvaccinated for at least 3 years. RESULTS: Antibody protection against CDV and CPV was evaluated in a total of 20 dogs: 5 puppies that initiated immunization at 6 weeks after birth (group A), 8 animals that started vaccination between 8 and 12 weeks of age (group B), and 7 adult dogs that have not been vaccinated for at least 3 years (group C). Blood samples were collected from each animal, with 3 to 4 weeks apart. Antibody responses were measured using indirect ELISA. In the second immunization point, no significant differences were found between the seroconversion of groups A and B for each viral infection (p = 0.81 and 0.20 for CDV and CPV, respectively). In the third immunization, there was evidence for a shorter time to achieve a protective titer against CPV in group B when compared to group A (p = 0.015). Similar evidence was not found for CDV (p-value = 0.41). In Group C, the average time until seroveversion was estimated at 2.86 years and 7.63 years for CDV and CPV, respectively. CONCLUSION: Vaccine response to CDV and CPV is specific in each individual. Effective immune protection in primary vaccination depends mainly on the initial titer of maternal antibodies acquired by the neonate. Other factors such as environmental exposure, immunization schedules and immune system activity influence the duration of immunity in adult dogs. The variability found reinforces the need to determine individual humoral immunity levels in order to assess vaccine efficacy

Rabbit derived VL single-domains as promising scaffolds to generate antibody–drug conjugates

© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.Antibody-drug conjugates (ADCs) are among the fastest-growing classes of therapeutics in oncology. Although ADCs are in the spotlight, they still present significant engineering challenges. Therefore, there is an urgent need to develop more stable and effective ADCs. Most rabbit light chains have an extra disulfide bridge, that links the variable and constant domains, between Cys80 and Cys171, which is not found in the human or mouse. Thus, to develop a new generation of ADCs, we explored the potential of rabbit-derived VL-single-domain antibody scaffolds (sdAbs) to selectively conjugate a payload to Cys80. Hence, a rabbit sdAb library directed towards canine non-Hodgkin lymphoma (cNHL) was subjected to in vitro and in vivo phage display. This allowed the identification of several highly specific VL-sdAbs, including C5, which specifically target cNHL cells in vitro and present promising in vivo tumor uptake. C5 was selected for SN-38 site-selective payload conjugation through its exposed free Cys80 to generate a stable and homogenous C5-DAB-SN-38. C5-DAB-SN-38 exhibited potent cytotoxicity activity against cNHL cells while inhibiting DNA-TopoI activity. Overall, our strategy validates a platform to develop a novel class of ADCs that combines the benefits of rabbit VL-sdAb scaffolds and the canine lymphoma model as a powerful framework for clinically translation of novel therapeutics for cancer.This work was supported by the Portuguese Funding Agency, Fundação para a Ciência e Tecnologia, FCT IP (SAICT/2017/32085, PTDC/QUI-OUT/3989/2021 and Ph.D. fellowship SFRH/BD/131468/2017 to ASA and SFRH/BD/90514/2012 to JD). CIISA has provided support through Project UIDB/00276/2020, funded by FCT and LA/P/0059/2020-AL4AnimalS. Research Institute for Medicines (iMed.ULisboa) acknowledges the financial support of Fundação para a Ciência e Tecnologia (Projects: PTDC/QUI-OUT/3989/2021; UIDB/04138/2020 and UIDP/04138/2020). The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project Nº 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC).info:eu-repo/semantics/publishedVersio

Topical Administration of a Nanoformulation of Chitosan-Hyaluronic Acid-Epoetin Beta in a Rat Model of Glaucoma

The present work investigates the effects of chitosan-hyaluronic acid-epoetin beta (CS/HA-EPOβ) nanoparticles after topical ocular administration in a rat glaucoma model. Wistar Hannover rats (n = 24) were submitted to a complete ophthalmological examination and electroretinography, followed by glaucoma induction in their right eye on day 1 of the study. Treatment group (T) received CS/HA-EPOβ nanocarriers (n = 12), while the control group (C) received only empty ones. Electroretinography was repeated on day 3 (n = 24) and before euthanasia on day 7 (n = 8), 14 (n = 8), and 21 (n = 8), followed by bilateral enucleation and histological assessment. The animals showed good tolerance to the nanoformulation. Maximum IOP values on the right eye occurred shortly after glaucoma induction (T = 62.6 ± 8.3 mmHg; C = 63.6 ± 7.9 mmHg). Animals from the treated group presented a tendency for faster recovery of retinal electrical activity (p > 0.05). EPOβ was detected on the retina of all treated eyes using immunofluorescence. Control animals presented with thinner retinas compared to the treated ones (p < 0.05). Therefore, topical ocular administration of CS/HA-EPOβ nanoparticles enabled EPOβ delivery to the retina of glaucomatous rats and promoted an earlier retinal recovery, confirming EPOβ’s neuroprotective effects. The encouraging results of this preclinical study pave the way for new strategies for topical ocular administration of neuroprotective compounds.This research was funded by the Fundação para a Ciência e a Tecnologia (FCT), Portugal (UID/DTP/04138/2019 and UIDB/04138/2020 to iMed.ULisboa, and is has also been funded by national funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., under the Project UIDB/00276/2020 for CIISA and Project LA/P/0059/2020 for AL4AnimalS; principal investigator grants CEECIND/03143/2017 (L. M. Gonçalves) and Beatriz Silva thanks to FCT for her fellowship SFRH/BD/130476/2017.info:eu-repo/semantics/publishedVersio

Colloidal nanosystems with mucoadhesive properties designed for ocular topical delivery

Over the last years, the scientific interest about topical ocular delivery targeting the posterior segment of the eye has been increasing. This is probably due to the fact that this is a non-invasive administration route, well tolerated by patients and with fewer local and systemic side effects. However, it is a challenging task due to the external ocular barriers, tear film clearance, blood flow in the conjunctiva and choriocapillaris and due to the blood-retinal barriers, amongst other features. An enhanced intraocular bioavailability of drugs can be achieved by either improving corneal permeability or by improving precorneal retention time. Regarding this last option, increasing residence time in the precorneal area can be achieved using mucoadhesive polymers such as xyloglucan, poly(acrylate), hyaluronic acid, chitosan, and carbomers. On the other hand, colloidal particles can interact with the ocular mucosa and enhance corneal and conjunctival permeability. These nanosystems are able to deliver a wide range of drugs, including macromolecules, providing stability and improving ocular bioavailability. New pharmaceutical approaches based on nanotechnology associated to bioadhesive compounds have emerged as strategies for a more efficient treatment of ocular diseases. Bearing this in mind, this review provides an overview of the current mucoadhesive colloidal nanosystems developed for ocular topical administration, focusing on their advantages and limitations.This work has been funded by national funds through FCT - Fundação para a Ciência e a Tecnologia, I.P., under the Project UIDB/00276/2020; FCT fellowship SFRH/BD/130476/2017.info:eu-repo/semantics/publishedVersio

Topical ocular delivery of nanoparticles with epoetin beta in Wistar Hannover rats

Topical instillation of drugs targeting the posterior ocular segment is an expanding area of research. Chitosan and hyaluronic acid have remarkable mucoadhesive properties and potentially enhance pre-corneal retention time after topical instillation. Bearing this in mind, we explored the possibility of delivering epoetin beta (EPOβ) to the posterior segment of the eye in a chitosan-hyaluronic acid (CS/HA-EPOβ) nanoparticulate system using the topical route of administration. Complete ophthalmological examinations, electroretinography and microhematocrit evaluations were performed in Wistar Hannover (WH) rats, before and after topical administration of nanoparticles. The right eye received CS/HA-EPOβ and the left eye received only empty nanocarriers (control). Animals were split into 6 groups and at designated timepoints, all animals from each group (n = 3) were euthanized and both eyes enucleated. Retinal morphology and EPOβ ocular distribution were assessed, respectively, through hematoxylin and eosin (HE) and immunofluorescence staining. After topical administration, no adverse ocular signs were noted and no significant changes either in microhematocrits nor in electroretinographies were detected. During the study, intraocular pressure (IOP) was always kept within physiological range bilaterally. No histological changes were detected in any of the ocular globes. Immunofluorescence enabled the identification of EPOβ in the retina 12 h after the administration, its presence still being detectable at day 21. In conclusion, CS/HA nanoparticles could efficiently deliver EPOβ to the retina of WH rats after topical instillation, being considered biologically safe. Topical administration of this nanoformulation could be a valuable tool for retinal neuroprotection, decreasing risks associated with more invasive routes of administration, being cost effective and also increasing long-term patients’ compliance.This research was funded by the Fundação para a Ciência e a Tecnologia (FCT), Portugal (UID/DTP/04138/2019 and UIDB/04138/2020 to iMed.ULisboa, and is has also been funded by national funds through FCT-Fundação para a Ciência e a Tecnologia, I.P., under the Project UIDB/00276/2020 for CIISA and the Project LA/P/0059/2020 for AL4AnimalS); principal investigator grants CEECIND/03143/2017 (L. M. Gonçalves) and Beatriz Silva thanks to FCT for her fellowship SFRH/BD/130476/2017.info:eu-repo/semantics/publishedVersio

Reaching for veterinary pharmacy services: an overlooked routine by community pharmacists?

Objectives Community pharmacists claim veterinary pharmacy as an integral part of their duties. There a paucity of studies investigating medication and health products’ dispensation for veterinary purposes. This study aimed to probe the Portuguese community pharmacists’ knowledge and behaviour towards veterinary pharmacy practice. Methods A cross-sectional exploratory survey, using an online questionnaire, was sent to a sample of academic-related community pharmacists in greater Lisbon, Portugal (N = 349). The questionnaire addressed participants’ specific education and training, counselling of products, and attitudes towards veterinary prescriptions. Logistic regression was used to estimated participants’ propensity for changing veterinary prescriptions independently. Everyday case scenarios were used to ascertain veterinary pharmacotherapy knowledge. Key Findings Participation reached 57.6% (N = 201), pointing to the theme’s relevance, with 83.6% females and a mean age of 36 years (SD =10.1). A large proportion (71.6%) declared to have received veterinary pharmacy education, although virtually all pharmacists (99.0%) perceived a skills deficit. The overall level of correct decisions regarding case scenarios reached 54%, although most professionals counsel pets’ treatments (85.6%). 18% of the sample declared to have changed a prescription without contacting the veterinarian. The propensity to independent medication changes was associated with being a male pharmacist, having one to two decades of professional experience, and working in community pharmacies with a permanent supply of prescribed veterinary pharmaceuticals. Conclusions Although recognizing limitations in veterinary pharmacy competencies, not all community pharmacists decide to minimize potential hazards to animals’ health as with human health. Present findings reinforce the urgency of further formal education and interprofessional collaboration.info:eu-repo/semantics/publishedVersio