Bleomycin (BLM) is a cytotoxic antibiotic used in veterinary oncology, primarily in electrochemotherapy (ECT), a local ablative therapy where electric pulses increase drug uptake in tumours. BLM is administered intravenously or intratumourally, with the standard intravenous dose for dogs being 15,000 IU/m², followed by electric pulses 8-10 minutes later. This protocol is derived from human oncology and lacks extensive pharmacological data in dogs. We studied BLM pharmacokinetics in 29 dogs with various tumours treated with intravenous BLM and ECT between 2017 and 2023. Samples were collected from serum of 15 dogs, serum and tumours of 8 dogs, and tumours of 6 dogs. The mean volume of distribution (Vd) of BLM was 224.5 ± 75.02 ml/kg, clearance (CL) was 7.04 ± 2.05 ml/kg/min, and area under the curve (AUC) was 65.87 ± 2.11 µg·min/l. The half-life (t₁/₂) of BLM in dogs was 22.03 ± 0.88 minutes. No significant difference was found in tumour BLM concentrations between 8 minutes post-administration and 2 minutes after pulse completion. These results support the recommended 8-28-minute window for applying electric pulses following intravenous BLM administration and may indicate no need for dose adjustments based on body weight or age.

The effects on the stress response, postanesthetic sedation, and altered behavior were evaluated following regional anesthesia and dental treatment in 40 dogs. Serum cortisol and blood glucose concentrations were measured following the administration of levobupivacaine (LBUP) 0.5% and dexmedetomidine (DEX) (0.5 µg/kg) or a placebo. The dogs were randomly assigned to 4 groups of 10 dogs each. All dogs received a regional nerve block using LBUP 0.5%. Group 1 (LBUP + DEX IV) also received DEX intravenously (IV); group 2 (LBUP + PLC IV) also received a placebo IV; group 3 (LBUP + DEX IO) also received DEX in one infraorbital (IO) block; and group 4 (LBUP + DEX IA) also received DEX in one inferior alveolar (IA) block. Serum cortisol and blood glucose concentrations were determined before the administration of oral blocks and at the end of the procedure. Sedation and behavior scores were assessed before premedication and hourly for 6 h after the end of anesthesia. Cortisol concentration did not change in any group at either evaluation time. The glucose concentration was higher ( P < .05) only in the LBUP + DEX IA group at the end of the procedure. The sedation score was higher until the end of the observation period only in the LBUP + DEX IV and LBUP + PLC IV groups. No change in behavior score was observed in any of the groups. The reduction of perioperative stress response in all groups was due to the use of LBUP and not DEX.

Astrocytes actively participate in neurotransmitter homeostasis by bidirectional communication with neuronal cells, a concept named the tripartite synapse, yet their role in dopamine (DA) homeostasis remains understudied. In the present study, we investigated the kinetic and molecular mechanisms of DA transport in cultured striatal astrocytes of adult rats. Kinetic uptake experiments were performed using radiolabeled [3H]-DA, whereas mRNA expression of the dopamine, norepinephrine, organic cation and plasma membrane monoamine transporters (DAT, NET, OCTs and PMAT) and DA receptors D1 and D2 was determined by qPCR. Additionally, astrocyte cultures were subjected to a 24 h treatment with the DA receptor agonist apomorphine, the DA receptor antagonist haloperidol and the DA precursor L-DOPA. [3H]-DA uptake exhibited temperature, concentration and sodium dependence, with potent inhibition by desipramine, nortriptyline and decynium-22, suggesting the involvement of multiple transporters. qPCR revealed prominent mRNA expression of the NET, the PMAT and OCT1, alongside lower levels of mRNA for OCT2, OCT3 and the DAT. Notably, apomorphine significantly altered NET, PMAT and D1 mRNA expression, while haloperidol and L-DOPA had a modest impact. Our findings demonstrate that striatal astrocytes aid in DA clearance by multiple transporters, which are influenced by dopaminergic drugs. Our study enhances the understanding of regional DA uptake, paving the way for targeted therapeutic interventions in dopaminergic disorders.

Astrocytes are crucial in the regulation of neurotransmitter homeostasis, and while their involvement in the dopamine (DA) tripartite synapse is acknowledged, it necessitates a more comprehensive investigation. In the present study, experiments were conducted on primary astrocyte cultures from the striatum and cortex of neonatal rats. The pharmacological intricacies of DA uptake, including dependence on time, temperature, and concentration, were investigated using radiolabelled [3H]-DA. The mRNA expression of transporters DAT, NET, PMAT, and OCTs was evaluated by qPCR. Notably, astrocytes from both brain regions exhibited prominent mRNA expression of NET and PMAT, with comparatively lower expression of DAT and OCTs. The inhibition of DA uptake by the DAT inhibitor, GBR12909, and NET inhibitors, desipramine and nortriptyline, impeded DA uptake in striatal astrocytes more than in cortical astrocytes. The mRNA expression of NET and PMAT was significantly upregulated in cortical astrocytes in response to the DA receptor agonist apomorphine, while only the mRNA expression of NET exhibited changes in striatal astrocytes. Haloperidol, a DA receptor antagonist, and L-DOPA, a DA precursor, did not induce significant alterations in transporter mRNA expression. These findings underscore the intricate and region-specific mechanisms governing DA uptake in astrocytes, emphasizing the need for continued exploration to unravel the nuanced dynamics of astrocytic involvement in the DA tripartite synapse.

Astrocytes, glial cells in the central nervous system, perform a multitude of homeostatic functions and are in constant bidirectional communication with neuronal cells, a concept named the tripartite synapse; however, their role in the dopamine homeostasis remains unexplored. The aim of this study was to clarify the pharmacological and molecular characteristics of dopamine transport in cultured cortical astrocytes of adult rats. In addition, we were interested in the expression of mRNA of dopamine transporters as well as dopamine receptors D1 and D2 and in the effect of dopaminergic drugs on the expression of these transporters and receptors. We have found that astrocytes possess both Na+-dependent and Na+-independent transporters. Uptake of radiolabelled dopamine was time-, temperature- and concentration-dependent and was inhibited by decynium-22, a plasma membrane monoamine transporter inhibitor, tricyclic antidepressants desipramine and nortriptyline, both inhibitors of the norepinephrine transporter. Results of transporter mRNA expression indicate that the main transporters involved in cortical astrocyte dopamine uptake are the norepinephrine transporter and plasma membrane monoamine transporter. Both dopamine receptor subtypes were identified in cortical astrocyte cultures. Twenty-four-hour treatment of astrocyte cultures with apomorphine, a D1/D2 agonist, induced upregulation of D1 receptor, norepinephrine transporter and plasma membrane monoamine transporter, whereas the latter was downregulated by haloperidol and L-DOPA. Astrocytes take up dopamine by multiple transporters and express dopamine receptors, which are sensitive to dopaminergic drugs. The findings of this study could open a promising area of research for the fine-tuning of existing therapeutic strategies.

This study investigated the cardiopulmonary effects and pharmacokinetics of dexmedetomidine (DEX) used as an adjunctive analgesic for regional anesthesia of the oral cavity with levobupivacaine in anesthetized dogs. Forty dogs were randomly assigned to four groups of 10 dogs. All dogs received levobupivacaine (4 blocks) with DEX IO (infraorbital block, n = 10) or IA (inferior alveolar block, n = 10) or placebo (PLC; n = 10) or DEX (n = 10) was injected intravenously (IV) after administration of levobupivacaine. The dose of DEX was always 0.5 µg/kg. Cardiopulmonary parameters were recorded, and blood was drawn for the quantification of DEX in plasma using LC-MS/MS. Heart rate was lower in all LB + DEX groups, while mean arterial pressure (MAP) was higher in the LB + DEX IV and LB + DEX IA groups compared to the LB + PLC IV group. Compared to DEX IV, IO and IA administration resulted in lower MAP up to 2 min after application. Absorption of DEX was faster at IO administration (Cmax and Tmax were 0.47 ± 0.08 ng/mL and 7.22 ± 1.28 min and 0.76 ± 0.09 ng/mL and 7.50 ± 1.63 min for the IO and IA block, respectively). The IA administration resulted in better bioavailability and faster elimination (t1/2 was 63.44 ± 24.15 min and 23.78 ± 3.78 min for the IO and IA block, respectively). Perineural administration of DEX may be preferable because of the less pronounced cardiovascular response compared to IV administration.

IntroductionData are lacking on the pharmacokinetic profile and safety of levobupivacaine (LB) used for regional anesthesia of the maxilla and mandibles in dogs.MethodsInfraorbital block (n = 10), inferior alveolar block (n = 10) or both infraorbital and inferior alveolar blocks (n = 10) were administered to dogs undergoing dental surgery under isoflurane anesthesia. The dose of LB was calculated as 0.11 ml/kg2/3 for the infraorbital block and 0.18 ml/kg2/3 for the inferior alveolar block. Blood samples were collected before and immediately after administration of the oral blocks, and 3, 4, 7, 12, 17, 32, 47, 62, 92, and 122 min thereafter. Quantification of LB in plasma was performed by LC-MS/MS.Results and discussionThe results are presented as median and interquartile range. In dogs in which all four quadrants of the oral cavity were desensitized with LB, the Cmax was 1,335 (1,030–1,929) ng/ml, the Tmax was 7 (4–9.5) min, and the AUC(0 → 120) was 57,976 (44,954–96,224) ng min/ml. Plasma concentrations of LB were several times lower than the reported toxic concentrations, and no signs of cardiovascular depression or neurotoxicity were observed in any of the dogs, suggesting that the occurrence of severe adverse effects after administration of LB at the doses used in this study is unlikely.

The plasma concentration profile of bleomycin in the distribution phase of patients younger than 65 years is needed to determine the suitable time interval for efficient application of electric pulses during electrochemotherapy. Additionally, bleomycin concentrations in the treated tumors for effective tumor response are not known. In this study, the pharmacokinetic profile of bleomycin in the distribution phase in 12 patients younger than 65 years was determined. In 17 patients, the intratumoral bleomycin concentration was determined before the application of electric pulses. In younger patients, the pharmacokinetics of intravenously injected bleomycin demonstrated a faster plasma clearance rate than that in patients older than 65 years. This outcome might indicate that the lowering of the standard bleomycin dose of 15,000 IU/m2 with intravenous bleomycin injection for electrochemotherapy is not recommended in younger patients. Based on the plasma concentration data gathered, a time interval for electrochemotherapy of 5–15 min after bleomycin injection was determined. The median bleomycin concentration in tumors 8 min after bleomycin injection, at the time of electroporation, was 170 ng/g. Based on collected data, the reduction of the bleomycin dose is not recommended in younger patients; however, a shortened time interval for application of electric pulses in electrochemotherapy to 5–15 min after intravenous bleomycin injection should be considered.

An experimental study on the effects of electroporation on pancreatic tissue was performed in pigs, and the fentanyl transdermal patch (FTP) was used postoperatively as part of multimodal pain management. Ingestion of an FTP, which resulted in fentanyl intoxication, was suspected 5 days after placement in one of the experimental pigs. The pig was first dysphoric, running in the stall, panting and vocalizing until it finally became depressed and it remained lying on the floor. Ingestion of an FTP was not observed but the fentanyl plasma concentration on the day of intoxication was 20.7 ng/ml, while at its peak after FTP administration it was only 0.492 ng/ml. The intoxication was successfully treated with a single intramuscular naloxone injection.

We used a combination of density functional theory (DFT) calculations and the implicit quantization of the acidic N-H and O-H bonds to assess the effect of deuteration on the binding of agonists (2-methylhistamine and 4-methylhistamine) and antagonists (cimetidine and famotidine) to the histamine H2 receptor. The results show that deuteration significantly increases the affinity for 4-methylhistamine and reduces it for 2-methylhistamine, while leaving it unchanged for both antagonists, which is found in excellent agreement with experiments. The revealed trends are interpreted in the light of the altered strength of the hydrogen bonding upon deuteration, known as the Ubbelohde effect, which affects ligand interactions with both active sites residues and solvent molecules preceding the binding, thus providing strong evidence for the relevance of hydrogen bonding for this process. In addition, computations further underline an important role of the Tyr250 residue for the binding. The obtained insight is relevant for the therapy in the context of (per)deuterated drugs that are expected to enter therapeutic practice in the near future, while this approach may contribute towards understanding receptor activation and its discrimination between agonists and antagonists.

OBJECTIVE:To investigate the pharmacokinetics of carprofen after a single intravenous (IV) dose and multiple oral doses administered to pigs undergoing electroporation of the pancreas. STUDY DESIGN:Prospective experimental study. ANIMALS:A group of eight female pigs weighing 31.74 ± 2.24 kg (mean ± standard deviation). METHODS:Carprofen 4 mg kg-1 was administered IV after placement of a central venous catheter during general anaesthesia with isoflurane. Blood samples were collected 30 seconds before and 5, 10, 20, 30 and 60 minutes and 2, 4, 6, 8, 12 and 24 hours after carprofen administration. Subsequently, the same dose of carprofen was administered orally, daily, for 6 consecutive days and blood collected at 36, 48, 60, 72, 96, 120, 144 and 168 hours after initial carprofen administration. Plasma was analysed using liquid chromatography with mass spectrometry. Standard pharmacokinetic parameters were calculated by compartmental analysis of plasma concentration-time curves. Data are presented as mean ± standard error. RESULTS:The initial plasma concentration of IV carprofen was estimated at 54.57 ± 3.92 μg mL-1 and decreased to 8.26 ± 1.07 μg mL-1 24 hours later. The plasma elimination curve showed a bi-exponential decline: a rapid distribution phase with a distribution half-life of 0.21 ± 0.03 hours and a slower elimination phase with an elimination half-life of 17.31 ± 3.78 hours. The calculated pharmacokinetic parameters were as follows: the area under the plasma concentration-time curve was 357.3 ± 16.73 μg mL-1 hour, volume of distribution was 0.28 ± 0.07 L kg-1 and plasma clearance rate was 0.19 ± 0.009 mL minute-1 kg-1. The plasma concentration of carprofen, administered orally from days 2 to 7, varied from 9.03 ± 1.87 to 11.49 ± 2.15 μg mL-1. CONCLUSIONS AND CLINICAL RELEVANCE:Carprofen can be regarded as a long-acting non-steroidal anti-inflammatory drug in pigs.

In this presentation we report a combined experimental and computational study concerning the effects of deuteration on the binding of histamine and other histaminergic ligands to 3H-tiotidine-labeled histamine H2 receptor in neonatal rat astrocytes. Binding affinities were measured by displacing radiolabeled tiotidine from H2 receptor binding sites present on cultured neonatal rat astrocytes. Quantum-chemical calculations were performed by employing the empirical quantization of nuclear motion within a cluster model of the receptor binding site extracted from the homology model of the entire H2 receptor. After incubation of the receptor in deuterium oxide (D2O), the hydrogen atoms, bound to electronegative elements, were replaced with deuterium atoms, which lead to a change in inter- and intramolecular bond lengths which lead to a change in the affinity of a agonists to histamine H2 receptor. Experiments clearly demonstrate that deuteration affects the binding by increasing the affinity for histamine and 4-methylhistamine and reducing the affinity for 2-methylhistamine, while basically leaving it unchanged for antagonists cimetidine and famotidine. Ab initio quantum-chemical calculations on the cluster system extracted from the homology H2 model along with the implicit quantization of the acidic N-H and O-H bonds demonstrate that these changes in the binding can be rationalized by the altered strength of the hydrogen bonding upon deuteration known as the Ubbelohde effect.

Pre-clinical and clinical data indicate differences in the responses of melanoma and carcinoma tumours to electrochemotherapy. The purpose of this study was to investigate the origin of this difference, whether it is due to the intrinsic difference in tumour cell susceptibility to the chemotherapeutic, or due to the tumour micro-environment. For this purpose, we performed a pre-clinical study in B16F1 melanoma and TS/A carcinoma tumours in mice, in which the antitumour effectiveness of electrochemotherapy with bleomycin, the intrinsic sensitivity of tumour cells in vitro, the pharmacokinetics of bleomycin in plasma and tumours, and the vascularization of tumours in vivo were evaluated. The results of the treatment show that carcinoma was significantly more responsive to electrochemotherapy than melanoma. This effect cannot be ascribed to the intrinsic sensitivity of these cells, as melanoma cells were more sensitive than carcinoma cells in vitro. The difference in responses could be ascribed to differences in the pharmacokinetics of bleomycin; at the time of electroporation in carcinomas, more bleomycin was accumulated. This effect could be due to differences in tumour vascularization, as carcinoma tumours had numerous well-distributed, small blood vessels, while melanomas were less vascularized, exhibiting predominantly larger vessels. In conclusion, this study provides evidence on the importance of the tumour micro-environment, particularly the tumour vasculature, in the responses of the tumours to bleomycin electrochemotherapy. Vasculature is important for the pharmacokinetics of bleomycin, influencing drug accumulation and drug distribution in tumours, and might be used as a predictive factor for the tumour response to electrochemotherapy.

In the few years, a rapid and continuous increase has been observed in the availability of new psychoactive substances, which in the last two years, is no longer exponential. In order to evade legal restrictions, they are commonly mislabelled as "not for human consumption" and easily obtained on the Internet or sold by street resellers and specialized stores (Univerzitetni kliniZni center Ljubljana, 2016). These new psychoactive substances are commonly referred to as new designer drugs. Such substances are mainly used by young people at private parties and clubs, but there are also cases of people over the age of fifty using them as well (Cadet-Tairou, 2016). These drugs can be chemically classified as synthetic cannabinoids, synthetic cathinones, ketamine, phenethylamines, piperazines, synthetic opioids, synthetic benzodiazepines, plant-based substances, and rarer aminoidanes, phencyclidine and tryptamines, to name but a few. Their consumption must be considered in cases of differential diagnosis of individuals for whom we suspect poisoning of this kind occurred, and these patients must be asked about the possible consumption of designer drugs.

BackgroundIn the present study, effectiveness of electrochemotherapy was compared in patients with nonmelanoma skin cancer (NMSC) of the head and neck using standard and reduced doses of bleomycin. MethodsTwenty-eight patients older than 65 years were prospectively treated with electrochemotherapy for nonmelanoma head and neck skin cancer. In the experimental group (n=12 patients; 24 lesions), reduced bleomycin doses (10000 IU/m(2)) were used. In the control group (n=16 patients; 28 lesions), the standard bleomycin doses (15000 IU/m(2)) were used. Tumor responses and side effects were monitored. These two groups were pair matched for the characteristics of patients (age, gender) and their tumors (diameter, histology type, recurrent lesions). ResultsComplete tumor response at 2 months post-electrochemotherapy with the reduced bleomycin dose was 100% and with the standard bleomycin dose it was 96%. No statistically significant difference regarding skin toxicity was observed between the 2 groups (P=.20). Skin toxicity of grade 3 or less was recorded only in the control group (7% of treated lesions). ConclusionPresented results show the comparable antitumor effectiveness of electrochemotherapy when using standard or reduced bleomycin dose in elderly patients with nonmelanoma head and neck skin cancer.

Differential scanning calorimetry provides unique signatures of blood plasma samples. Plasma samples from diseased individuals yield specific thermograms, which differ from each other and from plasma samples of healthy individuals. Thermograms from individuals suffering from chronic lymphocytic leukemia, multiple myeloma and acute myeloid leukemia were measured with DSC. To obtain additional information about thermal behaviour of plasma proteins immunoaffinity chromatography was introduced. An immunoextraction of HSA using a chromatographic column with immobilized anti-HSA was carried out in order to enrich less abundant plasma proteins, which could provide a further insight into disease development. Efficiency of HSA depletion and protein composition of fractionated plasma was validated by SDS-PAGE.

Clonidine has been shown effective in reducing sympathetic hyperactivity in neonatal abstinence syndrome (NAS). The aim of this study was to analyze clinical and laboratory characteristics of a group of newborns treated with clonidine for NAS due to maternal drug addiction and due to withdrawal from opioid analgesic therapy. Only one full-term newborn presented with metabolic acidosis and hyperkalemia; in others no clinical or laboratory adverse effects were detected. This report emphasizes the importance of alertness to potential adverse effects of clonidine therapy, and discusses possible pathophysiological aspects of hyperkalemia and metabolic acidosis during treatment for NAS.

Purpose With the aim to determine effective therapeutic window of electrochemotherapy, we analyzed bleomycin pharmacokinetic parameters in elderly patients.Methods In prospective clinical study in the treatment of tumors with electrochemotherapy, blood samples of patients older than 65 years were collected after the bolus intravenous injection of bleomycin (15,000 IU/m(2)). In serum samples, quantitative analysis was performed with liquid chromatography coupled to high-resolution mass spectrometry. Based on the data, the pharmacokinetic parameters of bleomycin elimination were determined.Results Pharmacokinetic analysis of the data revealed a monophasic serum clearance curve, which demonstrates slow elimination of bleomycin, being less than 500 ml/min and a half-time of 30 min.Conclusions Slow monophasic elimination of bleomycin from serum in elderly patients implies on the longer therapeutic window, from 8 to up to 40 min or even longer post-bleomycin injection for electrochemotherapy. However, prolonged therapeutic bleomycin serum concentrations may also affect the possible adverse effects, such as lung fibrosis and extensive necrosis of tumors due to the uptake of toxic bleomycin concentrations into the tumors. This may imply on lowering of bleomycin dosage, in particular in the elderly patients.