Transporters involved in adult rat cortical astrocyte dopamine uptake: Kinetics, expression and pharmacological modulation.

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.

Kinetic Properties and Pharmacological Modulation of High- and Low-Affinity Dopamine Transport in Striatal Astrocytes of Adult Rats.

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.

Cortical and Striatal Astrocytes of Neonatal Rats Display Distinct Molecular and Pharmacological Characteristics of Dopamine Uptake.

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.

Pharmacokinetics of carprofen in anaesthetized pigs: a preliminary study.

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.

Use of Differential Scanning Calorimetry and Immunoaffinity Chromatography to Identify Disease Induced Changes in Human Blood Plasma Proteome.

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.

Histamine and astrocyte function.

Astrocytes support the brain through numerous functional interactions in health and disease. The recent advances in our knowledge of astrocyte involvement in various neurological disorders raised up several questions about their role and functioning in the central nervous system. From the evidence discussed in this review, we show that histamine importantly influences the main astrocytic activities such as ion homeostasis, energy metabolism, neurotransmitter clearance, neurotrophic activity and immune response. These processes are mediated through at least three histamine receptor subtypes, H1, H2 and H3, expressed on the astrocyte surface. Thus, we recognize histamine as an important player in the modulation of astrocytic functions that deserves further considerations in exploring involvement of astrocytes in neurological disorders.

Serum Cortisol and Blood Glucose Concentrations in Anesthetized Dogs Administered Levobupivacaine and Low-Dose Dexmedetomidine for Regional Anesthesia of the Oral Cavity.

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.

Regional characteristics of histamine uptake into neonatal rat astrocytes.

Histaminergic signalling constitutes an attractive target for treatment of neuropsychiatric disorders. One obstacle to developing new pharmacological options has been failure to identify putative specific histamine transporter responsible for histamine clearance. Although high-affinity histamine uptake was detected in neonatal cortical astrocytes, its existence in other brain regions remains largely unexplored. We investigated whether cerebellar and striatal astrocytes participate in histamine clearance and evaluated the role of organic cation transporters (OCT) in astroglial histamine transport. Kinetic and pharmacological characteristics of histamine transport were determined in cultured astrocytes derived from neonatal rat cerebellum, striatum and cerebral cortex. As well as astrocytes of cortical origin, cultured striatal and cerebellar astrocytes displayed temperature-sensitive, high-affinity histamine uptake. Exposure to ouabain or Na(+)-free medium, supplemented with choline chloride markedly depressed histamine transport in cortical astrocytes. Conversely, histamine uptake in striatal and cortical astrocytes was ouabain-resistant and was only partially diminished during incubation in the absence of Na(+). Also, histamine uptake remained unaltered upon exposure to OCT inhibitor corticosterone, although OCTs were expressed in cultured astrocytes. Finally, histamine transport in cerebellar and striatal astrocytes was not sensitive to antidepressants. Despite common characteristics, cerebellar astrocytes had lower affinity, but markedly higher transport capacity for histamine compared to striatal astrocytes. Collectively, we provide evidence to suggest that cerebellar, striatal as well as cortical astrocytes possess saturable histamine uptake systems, which are not operated by OCTs. In addition, our data indicate that Na(+)-independent histamine carrier predominates in cerebellar and striatal astrocytes, whereas Na(+)-dependent transporter underlies histamine uptake in cortical astrocytes. Our findings implicate a role for histamine transporters in regulation of extracellular histamine concentration in cerebellum and striatum. Inhibition of histamine uptake might represent a viable option to modulate histaminergic neurotransmission.

The pharmacokinetics of levobupivacaine 0.5% after infraorbital or inferior alveolar block in anesthetized dogs.

Introduction: Data are lacking on the pharmacokinetic profile and safety of levobupivacaine (LB) used for regional anesthesia of the maxilla and mandibles in dogs. Methods: Infraorbital 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 discussion: The results are presented as median and interquartile range. In dogs in which all four quadrants of the oral cavity were desensitized with LB, the C max was 1,335 (1,030-1,929) ng/ml, the T max 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.

Cardiopulmonary Effects and Pharmacokinetics of Dexmedetomidine Used as an Adjunctive Analgesic to Regional Anesthesia of the Oral Cavity with Levobupivacaine in Dogs.

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.

Bleomycin Concentration in Patients' Plasma and Tumors after Electrochemotherapy. A Study from InspECT Group.

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.

Relevance of Hydrogen Bonds for the Histamine H2 Receptor-Ligand Interactions: A Lesson from Deuteration.

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.

Case Report: Intoxication in a Pig (Sus Scrofa Domesticus) After Transdermal Fentanyl Patch Ingestion.

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.

Controlled systemic release of interleukin-12 after gene electrotransfer to muscle for cancer gene therapy alone or in combination with ionizing radiation in murine sarcomas.

BACKGROUND: The present study aimed to evaluate the antitumor effectiveness of systemic interleukin (IL)-12 gene therapy in murine sarcoma models, and to evaluate its interaction with the irradiation of tumors and metastases. To avoid toxic side-effects of IL-12 gene therapy, the objective was to achieve the controlled release of IL-12 after intramuscular gene electrotransfer. METHODS: Gene electrotransfer of the plasmid pORF-mIL12 was performed into the tibialis cranialis in A/J and C57BL/6 mice. Systemic release of the IL-12 was monitored in the serum of mice after carrying out two sets of intramuscular IL-12 gene electrotransfer of two different doses of plasmid DNA. The antitumor effectiveness of IL-12 gene electrotransfer alone or in combination with local tumor or lung irradiation with X-rays, was evaluated on subcutaneous SA-1 and LPB tumors, as well as on lung metastases. RESULTS: A synergistic antitumor effect of intramuscular gene electrotransfer combined with local tumor irradiation was observed as a result of the systemic distribution of IL-12. The gene electrotransfer resulted in up to 28% of complete responses of tumors. In combination with local tumor irradiation, the curability was increased by up to 100%. The same effect was observed for lung metastases, where a potentiating factor of 1.3-fold was determined. The amount of circulating IL-12 was controlled by the number of repeats of gene electrotransfer and by the amount of the injected plasmid. CONCLUSIONS: The present study demonstrates the feasibility of treatment by IL-12 gene electrotransfer combined with local tumor or lung metastases irradiation on sarcoma tumors for translation into the clinical setting.

Suppression of membrane microvesiculation--a possible anticoagulant and anti-tumor progression effect of heparin.

Heparins (unfractionated and low molecular weight (LMWH) heparins) primarily used as anticoagulants, were found to be effective also in slowing down the development of some types of cancer. On the other hand, the number of microvesicles in the peripheral blood originating from the budding of cell membranes (mostly platelets) is increased in hypercoagulabile states as well as in cancer, indicating a possible common underlying mechanism. It was hypothesized that by mediating an attractive interaction between phospholipid membranes heparin suppresses microvesiculation and thereby acts as an anticoagulant and anti-tumor agent. In this work, the effect of LMWH nadroparin on phospholipid membranes was tested in vitro in a system of giant phospholipid vesicles (GPVs) created by electroformation and observed under the phase contrast microscope. Plasma of different blood donors containing different concentrations of nadroparin was added to the suspension of GPVs to induce adhesion between GPVs. The attractive interaction between membranes was assessed by measuring the average effective angle of contact between the adhered GPVs. It was found in healthy donors, in a donor with gastrointestinal cancer and in a donor with rheumatoid arthritis that adding therapeutic doses of nadroparin to the plasma samples enhanced adhesion of phospholipid membranes in a dose and time-dependent manner while nadroparin alone had no effect within the therapeutic concentration range. The results are in favor of the hypothesis that suppression of microvesiculation underlies both, the anticoagulant and the anti-tumor progression effect of heparin.

Prevention of microvesiculation by adhesion of buds to the mother cell membrane--a possible anticoagulant effect of healthy donor plasma.

Microvesicles (MVs) found in peripheral blood are derived from the budding of cell membranes and are associated with a higher risk of thrombosis. Recently, a hypothesis has been suggested that certain plasma proteins could suppress microvesiculation by mediating adhesion of the buds to the mother cell membrane. In a pilot study, we have tested this hypothesis by considering the relation between the amount of MVs in peripheral blood and the ability of plasma to induce adhesion between giant phospholipid vesicles (GPVs). MVs were isolated from human plasma and counted by flow cytometry. The adhesion between GPVs was measured by assessing the average angle of contact between the adhered vesicles. It was found that greater ability of plasma to induce adhesion relates to smaller concentration of MVs in plasma. The ratio between the concentration of MVs and the concentration of platelets proved the most efficient parameter to predict the propensity of the membrane to shed vesicles. Our results indicate that a stronger attractive interaction between GPVs mediated by plasma is associated with a smaller amount of MVs per platelets. Plasma that mediates stronger attractive interaction between GPVs might potentially be associated with a smaller risk of thrombosis.

Number of microvesicles in peripheral blood and ability of plasma to induce adhesion between phospholipid membranes in 19 patients with gastrointestinal diseases.

It was recently shown that the plasma protein-mediated attractive interaction between phospholipid membranes could in the budding process cause adhesion of the bud to the mother membrane [J. Urbanija, N. Tomsic, M. Lokar, A. Ambrozic, S. Cucnik, M. Kanduser, B. Rozman, A. Iglic, V. Kralj-Iglic, Coalescence of phospholipid membranes as a possible origin of anticoagulant effect of serum proteins, Chem. Phys. Lipids 150 (2007) 49-57]. Since in the in vivo conditions the budding of cell membranes leads to the release of microvesicles into the circulation, a hypothesis was put forward that the ability of plasma to cause adhesion between membranes supresses the microvesiculation process. In the present work, this hypothesis was tested in a population of 19 patients with gastrointestinal diseases. The number of microvesicles in peripheral blood of patients was determined by flow cytometry while the ability of plasma to cause adhesion between membranes was determined by adding patient's plasma to the suspension of giant phospholipid vesicles created by electroformation method, and measuring the average effective angle of contact between the adhered vesicles. Statistically significant negative correlations between the number of microvesicles and the average effective angle of contact (Pearson coefficient -0.50, p=0.031) and between the number of microvesicles per number of platelets and the average effective angle of contact (Pearson coefficient -0.64, p=0.003) were found, which is in favor of the above hypothesis. Patients with gastrointestinal cancer had larger number of microvesicles (difference 140%, statistical significance 0.033) and smaller average effective angle of contact (difference 20%, statistical significance 0.013) compared to patients with other gastrointestinal diseases.

Efficiency of electrochemotherapy with reduced bleomycin dose in the treatment of nonmelanoma head and neck skin cancer: Preliminary results.

BACKGROUND: In 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. METHODS: Twenty-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 (10 000 IU/m2 ) were used. In the control group (n = 16 patients; 28 lesions), the standard bleomycin doses (15 000 IU/m2 ) 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). RESULTS: Complete 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). CONCLUSION: Presented 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.

Vascularization of the tumours affects the pharmacokinetics of bleomycin and the effectiveness of electrochemotherapy.

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.

Bleomycin pharmacokinetics of bolus bleomycin dose in elderly cancer patients treated with electrochemotherapy.

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.