Advances in sheep analgesia

Sheep (Ovis aries) are widely used in experimental settings and breeding system, nevertheless pain treatment in this species seems to be overlooked. The first two studies described in this thesis were designed to evaluate administration of analgesics both in the experimental and clinical setting. The third study evaluated the pharmacokinetics and antinociceptive effects of tramadol and its metabolite O-desmethyltramadol (M1) in sheep in a preclinical model of pain. The first study consisted of a meta-analysis of the reported use of analgesics in sheep for experimental purposes. Studies involving experimental procedures in sheep carried out in selected years (2008-2011-2014) were identified using a search engine. A total of 75 papers were selected. The study showed that analgesic treatment was often not accurately reported. The second study consisted of an on-line questionnaire evaluating the current attitudes of Italian practitioners to assessment and treatment of pain in sheep. The questionnaire consisted of five sections regarding the demographic data, analgesic drugs and techniques used to treat pain, attitudes to pain relief and assessment of pain and the knowledge on the topic of sheep analgesia. Only a modest number of questionnaires were returned. The most commonly used drugs by sheep practitioners who replied to the questionnaire were non steroidal anti-inflammatory drugs and local anaesthetics. In the practitioners’ opinion the main reasons for analgesic drugs not to be administered to sheep was the lack of licensed drugs followed by costs, withholding times and regulations. The vast majority of practitioners were interested in improving their knowlwdge on sheep analgesia. The third study investigated the pharmacokinetic profile and antinociceptive effect of tramadol and M1 following intravenous administration in sheep. Six healthy adult sheep were administered 4 (T4) and 6 (T6) mg/kg of tramadol (T) and saline (SAL) over 2 minutes in a cross over design with a two weeks wash out period. At predetermined time points blood samples were collected, physiological parameters and mechanical nociceptive threshold (NMT) values were recorded. Tramadol and M1 fitted a two compartmental model and a non compartmental model respectively. Pharmacokinetic parameters were similar for T4 and T6. Tramadol and M1 plasma concentrations decreased rapidly. Physiological parameters were not statistically different between groups. No mechanical antinociceptive effects of tramadol were detected, as MNT values did not statistically differ between groups. In conclusion, these studies showed that there is great scope for improvement in pain assessment and treatment in sheep both in the research than clinical settings. Moreover more experimental and clinical studies regarding the pharmacokinetic and pharmacokinamic effects of analgesic drugs in sheep are advocated in order to improve their welfare

Pharmacokinetics and antinociceptive effects of tramadol and its metabolite O-desmethyltramadol following intravenous administration in sheep

Although sheep are widely used as an experimental model for various surgical procedures there is a paucity of data on the pharmacokinetics and efficacy of analgesic drugs in this species. The aims of this study were to investigate the pharmacokinetics of intravenously (IV) administered tramadol and its active metabolite O-desmethyltramadol (M1) and to assess the mechanical antinociceptive effects in sheep. In a prospective, randomized, blinded study, six healthy adult sheep were given 4 and 6\u2009mg/kg tramadol and saline IV in a cross-over design with a 2-week wash-out period. At predetermined time points blood samples were collected and physiological parameters and mechanical nociceptive threshold (MNT) values were recorded. The analytical determination of tramadol and M1 was performed using high performance liquid chromatography. Pharmacokinetic parameters fitted a two- and a non-compartmental model for tramadol and M1, respectively. Normally distributed data were analysed by a repeated mixed linear model. Plasma concentration vs. time profiles of tramadol and M1 were similar after the two doses. Tramadol and M1 plasma levels decreased rapidly in the systemic circulation, with both undetectable after 6\u2009h following drug administration. Physiological parameters did not differ between groups; MNT values were not statistically significant between groups at any time point. It was concluded that although tramadol and M1 concentrations in plasma were above the human minimum analgesic concentration after both treatments, no mechanical antinociceptive effects of tramadol were reported. Further studies are warranted to assess the analgesic efficacy of tramadol in sheep

Regional Formularies in Italy: current state and future perspectives

Regional Formularies in Italy: current state and future perspectives Regional Formularies (RF) are considered part of pharmaceutical policies implemented by regions to govern access of medicines to regional market. However, they have been actually challenged, because of their presumed impact on differences of patient's access across the regions. The paper aimed at investigating the current status of RF and Regional Therapeutic Committees (CTR) and at suggesting/recommending possible reforms. The current status was investigated through a questionnaire administered to the regional pharmaceutical departments. Recommendations were retrieved from a multi -stakeholder work group carried out on 30-31 March 2023, embedded into a Forum focused on the regional pharmaceutical policies. Nineteen out of twenty-one regions responded to the survey: 12 use RF, mainly managed by the CTR; the RF frequency of update and the time needed for drugs listing greatly vary across regions; pharmacists, specialists and general practitioners are always represented in CTR, whereas other healthcare professionals and experts are more rarely involved; in 3 regions the CTR does not publish any RF update; the CTR mainly rely, to take decisions, on the dimension of the target population, the cost of therapy compared to alternative treatments and the impact on pharmaceutical expenditure. The working group recommended to overcome the RFs, if they are merely considered a list of available drugs at regional level, focusing CTR activities to ensure market access and to govern the prescribing behaviour, and strengthening/anticipating the flow of information from the Italian Medicines Agency (AIFA) to the regions, to enable a more efficient approach to local access to drugs

Protective Efficacy of H9N2 Avian Influenza Vaccines Inactivated by Ionizing Radiation Methods Administered by the Parenteral or Mucosal Routes

H9N2 viruses have become, over the last 20 years, one of the most diffused poultry pathogens and have reached a level of endemicity in several countries. Attempts to control the spread and reduce the circulation of H9N2 have relied mainly on vaccination in endemic countries. However, the high level of adaptation to poultry, testified by low minimum infectious doses, replication to high titers, and high transmissibility, has severely hampered the results of vaccination campaigns. Commercially available vaccines have demonstrated high efficacy in protecting against clinical disease, but variable results have also been observed in reducing the level of replication and viral shedding in domestic poultry species. Antigenic drift and increased chances of zoonotic infections are the results of incomplete protection offered by the currently available vaccines, of which the vast majority are based on formalin-inactivated whole virus antigens. In our work, we evaluated experimental vaccines based on an H9N2 virus, inactivated by irradiation treatment, in reducing viral shedding upon different challenge doses and compared their efficacy with formalin-inactivated vaccines. Moreover, we evaluated mucosal delivery of inactivated antigens as an alternative route to subcutaneous and intramuscular vaccination. The results showed complete protection and prevention of replication in subcutaneously vaccinated Specific Pathogen Free White Leghorn chickens at low-to-intermediate challenge doses but a limited reduction of shedding at a high challenge dose. Mucosally vaccinated chickens showed a more variable response to experimental infection at all tested challenge doses and the main effect of vaccination attained the reduction of infected birds in the early phase of infection. Concerning mucosal vaccination, the irradiated vaccine was the only one affording complete protection from infection at the lowest challenge dose. Vaccine formulations based on H9N2 inactivated by irradiation demonstrated a potential for better performances than vaccines based on the formalin-inactivated antigen in terms of reduction of shedding and prevention of infection

Miconazole-like scaffold is a promising lead for Naegleria fowleri-specific CYP51 inhibitors

Developing drugs for brain infection by Naegleria fowleri is an unmet medical need. We used a combination of cheminformatics, target-, and phenotypic-based drug discovery methods to identify inhibitors that target an essential N. fowleri enzyme, sterol 14-demethylase (NfCYP51). A total of 124 compounds preselected in silico were tested against N. fowleri. Nine primary hits with EC50 ≤ 10 μM were phenotypically identified. Cocrystallization with NfCYP51 focused attention on one primary hit, miconazole-like compound 2a. The S-enantiomer of 2a produced a 1.74 Å cocrystal structure. A set of analogues was then synthesized and evaluated to confirm the superiority of the S-configuration over the R-configuration and the advantage of an ether linkage over an ester linkage. The two compounds, S-8b and S-9b, had an improved EC50 and KD compared to 2a. Importantly, both were readily taken up into the brain. The brain-to-plasma distribution coefficient of S-9b was 1.02 ± 0.12, suggesting further evaluation as a lead for primary amoebic meningoencephalitis

SARS-CoV-2 infection and replication in human gastric organoids

COVID-19 typically manifests as a respiratory illness, but several clinical reports have described gastrointestinal symptoms. This is particularly true in children in whom gastrointestinal symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. These observations raise the question of whether the virus can replicate within the stomach. Here we generate gastric organoids from fetal, pediatric, and adult biopsies as in vitro models of SARS-CoV-2 infection. To facilitate infection, we induce reverse polarity in the gastric organoids. We find that the pediatric and late fetal gastric organoids are susceptible to infection with SARS-CoV-2, while viral replication is significantly lower in undifferentiated organoids of early fetal and adult origin. We demonstrate that adult gastric organoids are more susceptible to infection following differentiation. We perform transcriptomic analysis to reveal a moderate innate antiviral response and a lack of differentially expressed genes belonging to the interferon family. Collectively, we show that the virus can efficiently infect the gastric epithelium, suggesting that the stomach might have an active role in fecal-oral SARS-CoV-2 transmission.Several clinical reports have described gastrointestinal symptoms for COVID-19, though whether the virus can replicate within the stomach remains unclear. Here the authors generate gastric organoids from human biopsies and show that the virus can efficiently infect gastric epithelium, suggesting that the stomach might have an active role in fecal-oral transmission

Advances in sheep analgesia

Sheep (Ovis aries) are widely used in experimental settings and breeding system, nevertheless pain treatment in this species seems to be overlooked. The first two studies described in this thesis were designed to evaluate administration of analgesics both in the experimental and clinical setting. The third study evaluated the pharmacokinetics and antinociceptive effects of tramadol and its metabolite O-desmethyltramadol (M1) in sheep in a preclinical model of pain. The first study consisted of a meta-analysis of the reported use of analgesics in sheep for experimental purposes. Studies involving experimental procedures in sheep carried out in selected years (2008-2011-2014) were identified using a search engine. A total of 75 papers were selected. The study showed that analgesic treatment was often not accurately reported. The second study consisted of an on-line questionnaire evaluating the current attitudes of Italian practitioners to assessment and treatment of pain in sheep. The questionnaire consisted of five sections regarding the demographic data, analgesic drugs and techniques used to treat pain, attitudes to pain relief and assessment of pain and the knowledge on the topic of sheep analgesia. Only a modest number of questionnaires were returned. The most commonly used drugs by sheep practitioners who replied to the questionnaire were non steroidal anti-inflammatory drugs and local anaesthetics. In the practitioners’ opinion the main reasons for analgesic drugs not to be administered to sheep was the lack of licensed drugs followed by costs, withholding times and regulations. The vast majority of practitioners were interested in improving their knowlwdge on sheep analgesia. The third study investigated the pharmacokinetic profile and antinociceptive effect of tramadol and M1 following intravenous administration in sheep. Six healthy adult sheep were administered 4 (T4) and 6 (T6) mg/kg of tramadol (T) and saline (SAL) over 2 minutes in a cross over design with a two weeks wash out period. At predetermined time points blood samples were collected, physiological parameters and mechanical nociceptive threshold (NMT) values were recorded. Tramadol and M1 fitted a two compartmental model and a non compartmental model respectively. Pharmacokinetic parameters were similar for T4 and T6. Tramadol and M1 plasma concentrations decreased rapidly. Physiological parameters were not statistically different between groups. No mechanical antinociceptive effects of tramadol were detected, as MNT values did not statistically differ between groups. In conclusion, these studies showed that there is great scope for improvement in pain assessment and treatment in sheep both in the research than clinical settings. Moreover more experimental and clinical studies regarding the pharmacokinetic and pharmacokinamic effects of analgesic drugs in sheep are advocated in order to improve their welfare.La pecora domestica (Ovis aries) è una delle specie animali più comunemente allevate ed utilizzate nella ricerca biomedica; nonostante questo l’analgesia in questo animale è stata a lungo trascurata. I primi due studi presentati in questa tesi sono stati elaborati allo scopo di valutare la somministrazione di analgesici nell’ambito clinico e sperimentale. Il terzo studio valuta la farmacocinetica e gli effetti antinocicettivi del tramadolo e del suo metabolita O desmethyltramadol (M1). Il primo studio consiste in una meta-analisi sull’ uso di farmaci analgesici riportato nelle pecore utilizzate a fini sperimentali. Studi riguardanti procedure sperimentali in pecore effettuati in anni selezionati (2008-2011-2014) sono stati identificati utilizzando un motore di ricerca. In totale, sono stati selezionati 75 articoli scientifici. Lo studio evidenzia mostra che la terapia antalgica spesso non viene accuratamente riportata. Il secondo studio consiste in un questionario on line redatto allo scopo di valutare l’attuale approccio dei veterinari italiani, che si occupano della specie ovina, alla valutazione ed al trattamento del dolore in questa specie. Il questionario era diviso in cinque sezioni riguardanti i dati demografici, l’uso di farmaci analgesici a tecniche utilizzate per apportare analgesia, e l’approccio utilizzato dai veterinari nella valutazione e trattamento del dolore nella specie ovina, ed, infine, la loro conoscenza riguardo tale argomento. Un numero limitato di veterinari ha completato il questionario. I farmaci più comunemente utilizzati dai veterinari che hanno risposto al questionario sono i farmaci antiinfiammatori non steroidei e gli anestetici locali. Secondo l’opinione dei veterinari, le ragioni principali per cui la terapia analgesica non viene effettuata nella specie ovina erano la mancanza di farmaci registrati, il loro costo, i tempi di sospensione e la regolamentazione riguardante il loro utilizzo. La maggior parte dei veterinari si dimostrava interessata a migliorare le proprie conoscenze riguardo l’analgesia nella specie ovina. Il terzo studio investiga la farmacocinetica e gli effetti antinocicettivi del tramadolo ed M1 dopo somministrazione endovenosa nelle pecore. Due dosi di tramadolo, 4 mg/kg (T4) e 6 mg/kg (T6), e soluzione salina (SAL) sono state somministrate in due minuti a sei pecore adulte e sane in uno studio randomizzato “in cieco” con un periodo di sospensione di due settimane. A tempi predeterminati, sono stati effettuati i prelievi di sangue per l’analisi farmacocinetica, e sono stati registrati i parametri fisiologici e i valori dopo stimolazione nocicettiva meccanica (MNT). Tramadolo ed M1 presentano rispettivamente una cinetica bi-compartimentale e non-compartimentale. I parametri farmacocinetici sono simili per le due dosi T4 e T6. Le concentrazioni plasmatiche di tramadolo ed M1 sono rapidamente diminuite. I parametri fisiologici non sono risultati statisticamente diversi tra i gruppi. Non sono stati evidenziati effetti antinociettivi del tramadolo; infatti i valori di MNT non sono risultati statisticamente diversi tra i gruppi. Concludendo, questi studi hanno dimostrato che ci sono ampi margini di miglioramento nella valutazione e trattamento del dolore nella specie ovina sia in ambito sperimentale sia clinico. Inoltre, sono necessari studi sperimentali e clinici riguardanti la farmacocinetica e farmacodinamica di farmaci analgesici nella specie ovina al fine di migliorarne il benessere

Practical use of opioids in cats: a state-of-the-art evidence-based review

Rationale Recent recognition of the need to improve pain management in cats has led to the investigation of the pharmacokinetics and efficacy of opioid analgesic drugs in this species. The results of these studies may be difficult to interpret because the effect of these drugs varies with dose, route of administration and the method used to assess them. As equipotency of different opioids is not known, it is hard to compare their effects. Animals do not verbalise the pain they feel and, in cats, it may be more difficult to recognise signs of pain in comparison with other species such as dogs. Aim This article reviews the use of opioid analgesics in cats. It must be remembered that not all drugs are licensed for use in cats, and that marketing authorisations vary between different countries. </jats:sec