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Anita Kovács, Nikolett Kis, Mária Budai-Szűcs, Attila Gácsi, Erzsébet Csányi, Ildikó Csóka, Szilvia BerkóInstitute of Pharmaceutical Technology and Regulatory Affairs, Faculty of Pharmacy, University of Szeged, Szeged 6720, HungaryCorrespondence: Szilvia BerkóUniversity of Szeged, Szeged 6720, Hungarytpdel 44/ Tel/Fax +36-62-545-573Email berkosz@pharm.u-szeged.huPurpose: The aim of our research work was to develop dermally applicable, lidocaine hydrochloride (LID-HCl)-containing semisolid in situ film-forming systems (FFSs) using the Quality by Design (QbD) approach to increase drug permeation into the skin.Methods: Silicones were used to improve the properties of formulations and to increase the permeation through the skin. The QbD approach was applied to ensure quality-based development. With initial risk assessment, the critical material attributes (CMAs) and the critical process parameters (CPPs) were identified to ensure the required critical quality attributes (CQAs).Results: During the initial risk assessment, four high-risk CQAs, namely in vitro drug release, in vitro drug permeation, drying properties, and mechanical properties, and three medium-risk CQAs, namely pH, viscosity, and film appearance were identified and investigated. Moreover, four high-risk CMAs were also considered during the formulation: permeation enhancing excipients, drying excipients, film-forming excipients, and emollients. During the experiments, LID-HCl influenced these critical parameters highly, thereby reducing the drying time. The formulation containing 25% silicone showed the best mechanical properties (49 mN skin adhesion, 20.3% film flexibility, 1.27 N film burst strength), which could predict better patient adherence. In addition, in vitro permeation studies showed that formulation containing 50% silicone has the fastest permeation rate. The flux of diffused API was 6.763 μg/cm2/h, which is much higher compared to the silicone-free formulation (1.5734 μg/cm2/h), and it can already be observed in the lower part of the dermis in 0.5 hour.Conclusion: Our results show that LID-HCl has great influence on the critical parameters of FFSs. The silicone content can improve the applicability of formulations and has a favorable effect on the permeation rate of LID-HCl into the skin.Keywords: in situ film-forming system, Quality by Design, formulation excipients, local anaesthesia

Szilvia Berkó,1 Stella Zsikó,1 Gábor Deák,2 Attila Gácsi,1 Anita Kovács,1 Mária Budai-Szűcs,1 László Pajor,2 Zoltán Bajory,2 Erzsébet Csányi1 1Institute of Pharmaceutical Technology and Regulatory Affairs, University of Szeged, Szeged, Hungary; 2Department of Urology, University of Szeged, Szeged, Hungary Purpose: Papaverine hydrochloride (PaHCl) is an old, well-known drug with spasmolytic activity but it has therapeutic effect in erectile dysfunction, too. As an intracavernous injection, it is not used in urologic clinics today because the side effects of the injection are pain, scarring or priapism. Our aim was to develop and test a topical semi-solid preparation containing PaHCl that would provide an alternative administration option by eliminating the undesirable side effects of the injection.Materials and methods: Lyotropic liquid crystal (LLC) systems were formulated as a semi-solid preparation with different concentrations of PaHCl. The characterization of the LLC structure was performed by polarization microscopy using a Leica image analyzer and rheological measurements. The drug diffusion and penetration tests were performed with in vitro synthetic membrane and an ex vivo human epidermis, using Franz diffusion cell to test the skin penetration of PaHCl. Human skin was investigated by Raman microscope to visualize the Active Pharmaceutical Ingredient (API) in different skin layers.Results: The results of diffusion and penetration showed reverse concentration dependency. The in vitro and ex vivo studies correlated with each other and the results of Raman microscopy. The LLC structure influenced the penetration results, the lower viscosity and lamellar structure increased penetration through the skin.Conclusion: Based on our results, a PaHCl containing topically used LLC formulation may be a suitable and effective alternative to the injectable formulation. Keywords: nanocarrier, dermal penetration, impotence, Raman microscopy

Despite numerous recent technological developments, conventional powder metallurgy (CPM) remains the most commonly applied technique for fabricating stainless-steel products. However, the compressibility of stainless-steel powders is still a critical issue. Martensitic stainless-steel powders (i.e., 17-4 PH) are significantly harder than austenitic stainless-steel powders (i.e., 316L and 1.4551). Herein, three different stainless-steel (DSS) powders (i.e., 17-4 PH, 316L, and 1.4551) were investigated using a high cold pressing pressure (i.e., 1.6 GPa) and a relatively low sintering temperature of 1200 °C during CPM. Dimensional and Archimedes density measurements, volume and radial shrinkage calculations, optical and scanning electron microscopy analyses, and compressive strength measurements were conducted to investigate the physical and mechanical properties. Using 1.6 GPa resulted in a 10 % and 6 % increase in the relative green density of the 17-4 PH and 316L samples, compared with the reported literature. For the first time, relative sintering densities of approximately 97 % and 99 % were obtained for 316L and 1.4551 at a sintering temperature of 1200 °C, respectively. Despite the insufficient sintering process, the relative density of the 17-4 PH sample increased by 2 % compared with the literature values reported for higher sintering temperatures. The shrinkage ratios were much lower than those reported in the literature. Furthermore, the present 316L samples achieved 90 % of the compressive yield strength observed in cast 316L, a 25 % increase compared with that previously reported in the literature. This study extends the CPM procedure for enhancing green densities, employing low sintering temperatures, and reducing the shrinkage values of stainless-steel powders with differing hardness.

Abstract The dog is a so far unique species to study interspecific communication and a promising evolutionary model for preverbal human communication. Recently cats were reported to show some similar skills to dogs. Here we directly compared both the testability and the success of companion dogs and cats in relying on human distal pointing gestures. Due to differences in their domestication, social and ecological background, and developmental processes, we expected better performance from dogs compared to cats. Using an object-choice task, cats made considerably fewer choices than dogs in the laboratory environment, and their tendency to make a choice declined during trials. They were slightly more testable at home, where their willingness to choose did not decrease over time. Dogs made more successful choices than cats, both at the group and individual level, irrespective of the type of the pointing gesture. Older cats were more successful. Despite the two species’ rather similar role nowadays as human companions, our results support previous findings suggesting that, compared to the dog, the cat is a less ideal model to study some human communicative abilities in a laboratory environment.

Abstract Dogs live in a complex social environment where they regularly interact with conspecific and heterospecific partners. Awake dogs are able to process a variety of information based on vocalisations emitted by dogs and humans. Whether dogs are also able to process such information while asleep, is unknown. In the current explorative study, we investigated in N = 13 family dogs, neural response to conspecific and human emotional vocalisations. Data were recorded while dogs were asleep, using a fully non-invasive event-related potential (ERP) paradigm. A species (between 250–450 and 600–800 ms after stimulus onset) and a species by valence interaction (between 550 to 650 ms after stimulus onset) effect was observed during drowsiness. A valence (750–850 ms after stimulus onset) and a species x valence interaction (between 200 to 300 ms and 450 to 650 ms after stimulus onset) effect was also observed during non-REM specific at the Cz electrode. Although further research is needed, these results not only suggest that dogs neurally differentiate between differently valenced con- and heterospecific vocalisations, but they also provide the first evidence of complex vocal processing during sleep in dogs. Assessment and detection of ERPs during sleep in dogs appear feasible.

Abstract We have investigated dogs’ (Canis familiaris) abilities in associating different sounds with appetitive stimuli of different incentive values. The association’s establishment was first tested on family dogs (n = 20) in a problem-solving behavioural paradigm (experiment 1), then in a problem-solving behavioural paradigm as well as an fMRI study on specially trained family dogs (n = 20) (experiment 2). The aim was to show behavioural and parallel neural effects of the association formed between the two sounds and two different associated appetitive stimuli. The latency of solving the problem was considered an indicator of the motivational state. In our first experiment, where only behaviour was studied, we found that dogs were quicker in solving a problem upon hearing the sound associated with food higher in reward value, suggesting that they have successfully associated the sounds with the corresponding food value. In our second experiment, this behaviour difference was not significant. In the fMRI study, the cerebral response to the two sounds was compared both before and after the associative training. Two bilateral regions of interest were explored: the caudate nucleus and the amygdala. After the associative training, the response in the caudate nucleus was higher to the sound related to a higher reward value food than to the sound related to a lower reward value food, which difference was not present before the associative training. We found an increase in the amygdala response to both sounds after the training. In a whole-brain representational similarity analysis, we found that cerebral patterns in the caudate nucleus to the two sounds were different only after the training. Moreover, we found a positive correlation between the dissimilarity index in the caudate nucleus for activation responses to the two sounds and the difference in latencies (i.e. high reward value associated sound condition latency—low reward value associated sound condition latency) to solve the behavioural task: the bigger the difference between the conditions in latency to solve the task, the greater the difference in the neural representation of the two sounds was. In summary, family dogs’ brain activation patterns reflected their expectations based on what they learned about the relationship between two sounds and their associated appetitive stimuli.

Abstract When addressing preverbal infants and family dogs, people tend to use specific speech styles. While recent studies suggest acoustic parallels between infant- and dog-directed speech, it is unclear whether dogs, like infants, show enhanced neural sensitivity to prosodic aspects of speech directed to them. Using functional magnetic resonance imaging on awake unrestrained dogs we identify two non-primary auditory regions, one that involve the ventralmost part of the left caudal Sylvian gyrus and the temporal pole and the other at the transition of the left caudal and rostral Sylvian gyrus, which respond more to naturalistic dog- and/or infant-directed speech than to adult-directed speech, especially when speak by female speakers. This activity increase is driven by sensitivity to fundamental frequency mean and variance resulting in positive modulatory effects of these acoustic parameters in both aforementioned non-primary auditory regions. These findings show that the dog auditory cortex, similarly to that of human infants, is sensitive to the acoustic properties of speech directed to non-speaking partners. This increased neuronal responsiveness to exaggerated prosody may be one reason why dogs outperform other animals when processing speech.

Family dogs exhibit neuropsychological deficits similar to attention-deficit/hyperactivity disorder (ADHD) symptoms in humans. Questionnaire methods have mostly been used to assess ADHD-like behaviours in dogs. In addition to our validated questionnaire (Dog ADHD and Functionality Rating Scale–DAFRS; 2024), we developed a simple behavioural test battery covering the ADHD symptom domains (i.e., inattention, hyperactivity, and impulsivity) in dogs. Our main aim was (i) to provide a final external validation step to the DAFRS by examining its associations with the test variables (N = 59); and (ii) to compare owner- and trainer-rated factor scores’ associations with the test variables (n = 38). We developed four tests covering the ADHD symptom domains: the attention test (inattention), the plush dog test (impulsivity), the leash test, and the sit test (hyperactivity). All four behavioural variables correlated with their respective questionnaire scores, i.e., the strongest for hyperactivity, and the least strong for inattention. Both owner- and trainer-rated scores (n = 38) correlated with the relevant test variables in an expected direction. Dogs’ training status was linked only to the sit test results. Test–retest analyses (n = 34) indicated moderate-to-excellent agreement across all behavioural variables. Our findings support the validity of our novel human-analogue questionnaire for dogs as the behavioural tests strongly correlate with the relevant questionnaire scores, indicating that the two constructs together can effectively assess inattention, hyperactivity, and impulsivity in dogs.

In their recent paper, Hansen Wheat et al. (Ecology and Evolution, 2022, 12, e9299) claimed that hand raised 23‐week‐old wolves showed the same attachment behaviour towards their handler in the Strange Situation Test (SST) (Determinants of infant behavior, 1969, 4, 111) as dogs. At first glance, their results seem to contradict previous findings that domestication caused a unique change in social‐affiliative behaviours in dogs (Animal Behaviour, 2005, 70, 1367). We argue that no persuading evidence was presented to claim that “wolves can show attachment behaviours towards humans comparable to those of dogs”. When dealing with a behaviour system (Child Development, 1977, 48, 1184), the subjects' behaviour must meet consistent criteria (Behavioural and Brain Science, 1978, 3, 417), and a few behavioural preferences should not be used to claim the presence of an attachment system, especially, if the experiment violates basic assumptions of the original test. We believe the intriguing scientific question is whether the dog‐owner relationship is qualitatively different from what could be observed in the wolf‐hand raiser relation. Assessing all available data, our answer is still yes; dogs are unique in this respect.

Simple Summary: Dogs use gazing behavior to communicate with humans. This study explored how family dogs differ in their gazing behavior in an uncertain situation and how these differences relate to personality, emotional comfort, and breed. We observed dogs responding to a novel stimulus—a remote-controlled toy car—and analyzed how often they looked at their owner or the experimenter in one minute. Based on this behavior, dogs were grouped into four clusters. Dogs that gazed 6–7 times at each human partner were rated as less aggressive than those that gazed only 2–3 times. They were also perceived as less lively than dogs that gazed 10–12 times at each partner. Additionally, dogs gazing more frequently at the owner than at the experimenter were reported to provide more emotional comfort to their owners, such as making them feel loved. Breed differences also emerged, with German shepherd dogs more likely to be low gazers compared to golden retrievers. Interestingly, a dog's age, sex, or willingness to approach the toy car did not affect their gazing behavior. These findings deepen our understanding of how dogs communicate with humans and the factors influencing this behavior, which can improve training, strengthen bonds, and enhance canine welfare. Gazing toward humans is fundamental in dog–human communication. This study aimed to characterize companion dogs based on their gazing behavior and to explore the potential links between gazing, personality (liveliness, confidence, aggressiveness, and attachment), emotional comfort provided to the owners, and breed. We observed 171 dogs in an ambiguous situation involving a moving stimulus, a remote-controlled toy car and gathered additional data with questionnaires. Our behavior analysis, based on gaze frequency directed at the owner and experimenter, identified four distinct clusters of dogs: low gazers, experimenter-focused gazers, owner-focused gazers, and frequent gazers. We found that experimenter-focused gazers exhibited lower levels of liveliness than frequent gazers and were reported to be less aggressive than low gazers and frequent gazers. Owner-focused gazers provided more emotional comfort to their owners than low gazers. Regarding breeds, 56.5% of German shepherd dogs were low gazers, compared to only 5.3% of golden retrievers. Age, sex, neutered status, and approaching the stimulus were not associated with cluster membership. These results highlight the complexity of dog–human communication, suggesting that personality, emotional factors, and breed shape how dogs seek guidance and support from humans or express anxiety. [ABSTRACT FROM AUTHOR]

Dogs’ ability to cooperate with humans is widely acknowledged, but the factors influencing their spontaneous cooperative tendencies are largely unknown. We investigated whether breed function, training experience, and owner-reported social motivation level contribute to spontaneous dog–owner cooperation. Family dogs (N = 100) of three breed groups (non-working dogs, cooperative/independent working breeds) with various training experiences were tested in an ‘out–of–reach’ task with their owners as their partners, who never directly asked for help during the test. We measured dogs’ behaviour along three main components of successful cooperation: paying attention, understanding the problem, and willingness to cooperate. Breed groups had no significant effect on dogs’ behaviour. No factor was associated with the behavioural variables related to not understanding the task. Dogs with high training levels and high social motivation showed more attention-related behaviours and were more likely to help the owner (training level and social motivation were not correlated with each other). Our results highlight the importance of training experience and social motivation in dogs’ attentiveness and spontaneous cooperativity. This also points to the need for careful sample balancing and experimental procedures that do not rely on specific trained skills.

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