Extensive depolarization and lack of recovery of leech Retzius neurons caused by 2,4 diaminobutyric acid.

In this paper we present, for the first time, a detailed account of electrophysiological effects of 2,4-diaminobutyric acid (2,4-DABA). 2,4-DABA is a neurotoxic non-protein amino acid produced by Cyanobacteria with a possible link to neurodegenerative disorders in animals and humans. Intracellular recordings were performed on Retzius nerve cells of the leech Haemopis sanguisuga using glass microelectrodes filled with 3 mol/L KCl. Our results show that 2,4-DABA is an excitatory amino acid, causing membrane depolarization in a concentration-dependent manner. The most prominent depolarizations of 39.63±2.22 mV and 47.05±4.33 mV, induced by 5×10-3 and 10-2 mol/L 2,4-DABA respectively, are several times larger than maximal depolarizations induced by either Glutamate, Aspartate, ß-N-methylamino-alanine (BMAA) or ß-N-oxalylamino-alanine (BOAA) on our model. These 2,4-DABA induced depolarizations evolve through two distinct stages, which is a novel phenomenon in electrical cell activity upon application of an excitatory amino acid, at least on our model. Involvement of two separate mechanisms, suggested by the two stage phenomenon, is discussed in the paper. We also provide evidence that 2,4-DABA induces irreversible functional disturbances in neurons in a concentration-dependent manner, since only half of the cells recovered normal electrical activity after application of 5×10-3 mol/L 2,4-DABA, and none recovered after application of 10-2 mol/L 2,4-DABA. Effects of both L-2,4-DABA and DL-2,4-DABA were tested and are not significantly different.

Patterns of antihypertensive medication use in kidney transplant recipients.

AIM: This study analyzed the prevalence of hypertension and the pattern of antihypertensive treatment before and after kidney transplantation. PATIENTS AND METHODS: The prevalence of hypertension and the class and daily dosage load of antihypertensive medications were analyzed in 116 patients before kidney transplantation and 1, 6, and 12 months after transplantation (67.2 % males, mean age 45.9 ± 11.4 years). Two patients died and eight had the allograft explanted, leaving 106 patients in the final analysis. Blood pressure (BP) was recorded on the day of transplantation and at every follow-up; it was considered uncontrolled at values > 130/80 mmHg. RESULTS: The prevalence of uncontrolled BP was significantly reduced after kidney transplantation (63.2 % before transplantation vs. 54.7, 41.5, and 25.5 % at the 1­, 6­, and 12-month follow-up, respectively, p < 0.001 for all). The number of prescribed antihypertensives did not change significantly during the follow-up (1.96 ± 1.03 before transplantation vs. 2.01 ± 0.88, 1.71 ± 0.78, and 1.73 ± 0.73 at the 1­, 6­, and 12-month follow-up, respectively, p > 0.05 for all). There was a significant decrease in antihypertensive drug load during the follow-up (1.08 ± 1.3 end of the study vs. 2.05 ± 2.32 before transplantation, p < 0.008). Before kidney transplantation, angiotensin-converting enzyme (ACE) inhibitors were most commonly prescribed (52.8 %), while after surgery ß­blockers gained prevalence (59.4-63.2 %). Mean arterial pressure decline correlated with an improvement of graft function. CONCLUSION: The prevalence of uncontrolled BP and the antihypertensive drug dosage load reduced significantly after kidney transplantation. ß­blockers were used more frequently than ACE inhibitors after kidney transplantation.

Acute Renal Failure - A Serious Complication in Patients After Kidney Transplantation.

Free radical-mediated injury releases proinflammatory cytokines and activates innate immunity. It has been suggested that the early innate response and the ischemic tissue damage play roles in the development of adaptive responses, which may lead to acute kidney rejection. Various durations of hypothermic kidney storage before transplantation add to ischemic tissue damage. The final stage of ischemic injury occurs during reperfusion that develops hours or days after the initial insult. Repair and regeneration processes occur together with cellular apoptosis, autophagy and necrosis and a favorable outcome is expected if regeneration prevails. Along the entire transplantation time course, there is a great demand for novel immune and nonimmune injury biomarkers. The use of these markers can be of great help in the monitoring of kidney injury in potential kidney donors, where acute kidney damage can be overlooked, in predicting acute transplant dysfunction during the early post-transplant periods, or in predicting chronic changes in long term followup. Numerous investigations have demonstrated that biomarkers that have the highest predictive value in acute kidney injury include NGAL, Cystatin C, KIM-1, IL-18, and L-FABP. Most investigations show that the ideal biomarker to fulfill all the needs in renal transplant has not been identified yet. Although, in many animal models, new biomarkers are emerging for predicting acute and chronic allograft damage, in human allograft analysis they are still not routinely accepted and renal biopsy still remains the gold standard.

Acute Renal Failure in Transplanted Kidneys.

Free radical-mediated injury releases proinflammatory cytokines and activates innate immunity. It has been suggested that the early innate response and the ischemic tissue damage play roles in the development of adaptive responses, which may lead to acute kidney rejection. Various durations of hypothermic kidney storage before transplantation add to ischemic tissue damage. The final stage of ischemic injury occurs during reperfusion that develops hours or days after the initial insult. Repair and regeneration processes occur together with cellular apoptosis, autophagy and necrosis and a favorable outcome is expected if regeneration prevails. Along the entire transplantation time course, there is a great demand for novel immune and nonimmune injury biomarkers. The use of these markers can be of great help in the monitoring of kidney injury in potential kidney donors, where acute kidney damage can be overlooked, in predicting acute transplant dysfunction during the early post-transplant periods, or in predicting chronic changes in long term followup. Numerous investigations have demonstrated that biomarkers that have the highest predictive value in acute kidney injury include NGAL, Cystatin C, KIM-1, IL-18, and L-FABP. Most investigations show that the ideal biomarker to fulfill all the needs in renal transplant has not been identified yet. Although, in many animal models, new biomarkers are emerging for predicting acute and chronic allograft damage, in human allograft analysis they are still not routinely accepted and renal biopsy still remains the gold standard.

An update on atrial fibrillation: focus on stroke risk reduction strategies.

Atrial fibrillation (AF) currently affects approximately 2% of the general adult population, and the number of patients suffering from AF constantly increases. Although the occurrence of AF rarely poses an immediate threat to patient's survival, the arrhythmia is associated with significant cardiovascular morbidity and mortality mostly resulting from ischemic stroke or systemic thromboembolism, or heart failure. Overall, patients with AF have a 5-fold greater risk of stroke compared to individuals in sinus rhythm, but individual stroke risk depends on the presence of various stroke risk factors, and optimal stroke prevention is essential for AF patients. Several major advances in AF-related stroke prevention have been achieved recently, including the refinements in stroke and bleeding risk assessment with an essential shift in the recognition of AF patients who should be offered oral anticoagulant (OAC) therapy, the advent of non-vitamin K antagonist oral anticoagulants (NOACs) which are increasingly used in the "real-world" setting, as well as the development of non-pharmacological means of thromboprophylaxis in AF patients (e.g., left atrial appendage [LAA] occluding devices). In this review article, we summarize these recent developments in stroke risk reduction strategies and discuss the main principles of decision-making regarding OAC therapy in AF patients.

The antinociceptive efficacy of morphine-ketamine-magnesium combination is influenced by the order of medication administration.

OBJECTIVE: Ketamine and magnesium, both N-methyl-D-aspartate (NMDA) receptor antagonists, enhance the antinociceptive effects of opioid analgesics in different animal models of pain, as well as in humans. This study aimed at evaluating whether magnesium sulphate added to morphine-ketamine combination produces a higher level of analgesia. MATERIALS AND METHODS: Analgesic activity was assessed by tail-immersion test in male Wistar rats (200-250 g). RESULTS: Magnesium sulphate (0.5-60 mg/kg, s.c.) and ketamine (5-30 mg/kg, i.p.) administered alone did not produce any effect. Magnesium sulphate (5 and 60 mg/kg) and ketamine (5 and 30 mg/kg) increased the antinociceptive effect of morphine (2.6 mg/kg, i.p.). Magnesium sulphate (5 mg/kg) increased the antinociceptive effect of the morphine (2.6 mg/kg)-ketamine (2.5 or 5 mg/kg) combination when magnesium sulphate was added to morphine after, and not before ketamine. It is also demonstrated that magnesium sulphate prolonged the duration of the antinociceptive effect of the morphine-ketamine combination. Low dose of morphine (2.6 mg/kg), ketamine (5 mg/kg) and magnesium sulfate (5 mg/kg) given together did not cause motor impairment that could be verified on a rotarod test. The antinociceptive effect of the triple combination was readily antagonized with naloxone (3 mg/kg, s.c.), a nonselective antagonist of opioid receptors, indicating that the effect is mediated via opioid receptors. CONCLUSIONS: This study revealed that the efficacy of the morphine-ketamine-magnesium sulphate combination in tail-immersion test in rats is influenced by the order of medication administration; a higher level of activity is demonstrated only when ketamine is added to morphine before magnesium sulphate.

A synergistic interaction between magnesium sulphate and ketamine on the inhibition of acute nociception in rats.

OBJECTIVE: Magnesium is an endogenous voltage-dependent NMDA receptor-channel blocker and ketamine is a non-competitive NMDA receptor antagonist. Magnesium may potentiate the effect of ketamine in analgesia and anaesthesia, but may also interact in an opposing manner. This study aimed at evaluating type of the interaction between magnesium sulphate and ketamine administered systemically in rats with an acute nociceptive pain (tail-immersion test). MATERIALS AND METHODS: Analgesic activity was assessed by tail-immersion test in male Wistar rats (200-250 g). The distal 5 cm of the tail was immersed in a warm water bath (55 ± 0.5°C) and the time for tail-withdrawal was measured as response latency. RESULTS: Magnesium sulphate (2.5-30 mg/kg, s.c.) and ketamine (2.5-30 mg/kg, i.p.) administered alone did not produce any effect. However, significant antinociception (synergistic interaction) was revealed at the following doses of ketamine: magnesium sulphate of 5:5 mg/kg, 2.5:5 mg/kg and 10:5 mg/kg. The effect was not dose-dependent, and a greater response was obtained when ketamine was administered before magnesium sulphate. CONCLUSIONS: This study revealed that (1) magnesium sulphate and ketamine given alone were not effective against acute nociceptive pain in rats, but (2) a combination of both drugs resulted in synergistically inhibited nociception, (3) which occurred only at selected low doses and proportions of the medications in a combination and (4) suggested the importance of the order of drug administration.

The analysis of transduction mechanisms associated with an acute action of homocysteine on isolated rat femoral artery.

The present study was undertaken in order to examine transduction mechanism involved in the single application of 100 µM homocysteine (Hcy) on isolated rat femoral artery (RFA) rings equilibrated on the basal tone; to establish if a single application of 100 µM Hcy alters contractile effect of phenylephrine (Phe), or oppositely the relaxant effect of acetylcholine (ACh) or bradykinin (BK) after 60-min-long incubation of 100 µM Hcy; and finally to identify morphological changes on the vascular wall after a 24-h-long incubation of 100 µM Hcy. Hcy produced contractile response of intact RFA, which was increased after endothelial denudation, while decreased by urapidil (an α1 receptor blocker), nifedipine (a voltage-gated L-type Ca++ channel blocker) or indomethacin (a cyclooxygenase inhibitor). The initial RFA contraction evoked by Phe was further increased by the single addition of Hcy, which was not the case when ouabain (an inhibitor of Na+/K+-ATPase) was preincubated. After 60-min-long incubation of Hcy relaxant actions of ACh and BK were unaltered, equieffective and equipotent. A 24-h-long incubation of RFA rings with Hcy produced an impairment of vascular endothelium, expressed as a minor or more pronounced interruption of endothelial cells.

Impact of age, weight and concomitant treatment on lamotrigine pharmacokinetics.

WHAT IS KNOWN AND OBJECTIVE: Lamotrigine metabolism may be substantially altered with concomitant administration of valproic acid and/or carbamazepine. Such alterations may require the adjustment of lamotrigine dose to ensure optimal treatment efficacy and safety. METHODS: The extent of lamotrigine interactions was investigated dependent on age, gender, weight and dose of concomitant carbamazepine and/or valproic acid in 65 patients with epilepsy. Lamotrigine plasma steady-state oral clearance (CLss/F) and area under the curve (AUCss) were calculated from the dose of drug, average steady-state concentration (Css) and interval of administration. Multiple regression analysis was used for the identification and quantification of factors that influenced lamotrigine pharmacokinetics. RESULTS AND DISCUSSION: Age and dose of carbamazepine and valproic acid had significant influence on lamotrigine CLss/F and AUCss. Carbamazepine was associated with a dose-dependent increase and valproic acid with a dose-dependent decrease of lamotrigine metabolism rate. The effect of carbamazepine was more pronounced. Younger patients were expected to metabolize lamotrigine more rapidly whereas overweight patients may be less susceptible to interactions. Gender had no influence on lamotrigine pharmacokinetics. WHAT IS NEW AND CONCLUSION: The efficacy and safety of lamotrigine may be altered by concomitant administration of carbamazepine and valproic acid. The models developed may be useful for estimating doses of lamotrigine for individual patients to minimize clinically significant interactions. Therapeutic monitoring is advisable when those drugs are used concomitantly.

Fentanyl analogs: structure-activity-relationship study.

Fentanyl is the prototype of the 4-anilidopiperidine class of synthetic opioid analgesics. This study was aimed to review the structure-activity-relationship (SAR) of fentanyl analogs substituted in the position 3, or 4 of the piperidine ring. Pharmacological results show that the groups in position 3 of the piperidine ring, which are larger than methyl, severely reduce the analgesic potency compared to fentanyl. It is likely that the steric factor alone (i.e. voluminosity of the group and cis/trans isomerism), rather than the polarity and/or chemical reactivity, plays a crucial role in the analgesic potency of this series. Although the duration of action, in general, does not depend on the stereochemistry, longer action of the most potent 3-alkyl fentanyl analogs such as cis-3-methyl- and cis-3-ethyl fentanyl, is more likely influenced by pharmacodynamic, rather than pharmacokinetic variables. Also, it is possible that the introduction of a functional group such as 3-carbomethoxy reduces the duration of action by altering pharmacokinetic properties. SAR findings obtained by evaluating the neurotoxic effects of fentanyl analogs substituted in the position 3 of the piperidine ring parallel the SAR findings on analgesia in regard to potency and duration of action. This might suggest that similar receptors are involved in producing both antinociceptive and neurotoxic effects of these drugs. It appears that both the potency and the duration of action in the series of fentanyl analogs substituted in position 4 of the piperidine ring is influenced only by the steric requirement and not by the chemical nature of the substituent.

Opioidergic mechanisms are not involved in the antihyperalgesic effects of carbamazepine and oxcarbazepine.

The mechanisms of the analgesic action of carbamazepine and oxcarbazepine, in particular the role of opioid receptors, have not been established precisely. The systemic effects of naloxone, an opioid receptor antagonist, on the antihyperalgesic effects of carbamazepine and oxcarbazepine were examined in the model of inflammatory hyperalgesia induced by the intraplantar (i.pl.) administration of concanavaline A (Con A, 0.8 mg/paw) into the rat hind paw. Naloxone (3 mg/kg; i.p.) did not alter the antihyperalgesic effects of either carbamazepine or oxcarbazepine. These results indicate that the opioid system of pain modulation does not play a significant role in the antihyperalgesic effects of carbamazepine and oxcarbazepine.

Role of alpha2-adrenoceptors in the local peripheral antinociception by carbamazepine in a rat model of inflammatory mechanical hyperalgesia.

The anticonvulsant carbamazepine was recently shown to possess local peripheral antinociceptive properties. In this study, we investigated whether alpha2-adrenergic receptors are involved in the local peripheral antihyperalgesic effects of carbamazepine and determined the type of interaction between carbamazepine and clonidine, an alpha2-adrenoceptor agonist. Intraplantar (i.pl.) coadministration of either carbamazepine (100-1000 nmol/paw) or clonidine (1.9-3.7 nmol/paw) with the proinflammatory compound concanavalin A (Con A; 0.8 mg/paw) caused a significant dose- and time-dependent reduction of the difference between the forces exerted by a rat's hind paws in a modified paw-pressure test. The coadministration of 260 and 520 nmol/paw (i.pl.) yohimbine, an alpha2-adrenoceptor antagonist, with carbamazepine, significantly depressed the local antihyperalgesic effect in a dose- and time-dependent manner whereas yohimbine by itself did not have any effect. The administration of a mixture of carbamazepine and clonidine at fixed dose fractions (1/4, 1/2 and 3/4) of ED50 caused a significant and dose-dependent reduction of Con A-induced hyperalgesia. Isobolographic analysis revealed an additive interaction. These results suggest that alpha2-adrenoceptors play a role in the local peripheral antihyperalgesic effects of carbamazepine and that local peripheral coadministration of carbamazepine with clonidine results in an additive antihyperalgesic effect.

Peripheral anti-hyperalgesia by oxcarbazepine: involvement of adenosine A1 receptors.

In this study we determined whether oxcarbazepine (OXC) could produce local peripheral antinociceptive effects in a rat model of inflammatory hyperalgesia, and whether adenosine receptors were involved. When coadministered with the pro-inflammatory compound concanavalin A, OXC (1000-3000 nmol/paw) caused a significant dose- and time-dependent anti-hyperalgesia. Caffeine (1000-1500 nmol/paw), a nonselective adenosine receptor antagonist, as well as 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (10-30 nmol/paw), a selective A1 receptor antagonist, coadministered with OXC, significantly depressed its anti-hyperalgesic effect. Drugs injected into the contralateral hind paw did not produce significant effects. These results indicate that OXC produces local peripheral anti-hyperalgesic effects, which is mediated via peripheral A1 receptors.

Comparison of liquid chromatography with fluorescence detection to liquid chromatography-mass spectrometry for the determination of fluoxetine and norfluoxetine in human plasma.

A comparison study on fluoxetine (FL) and norfluoxetine (NORFL) quantitation in human plasma was carried out between the recently developed liquid chromatographic method with fluorescence detection (LC-FLD) and an earlier established liquid chromatography-mass spectrometry (LC-MS) laboratory procedure. Comparative method evaluation was based on the analysis of plasma samples obtained from Parkinsonian patients receiving 20mg of FL per day. The LC-FLD method involves a two-step liquid extraction procedure without any derivatization, followed by direct chromatography on a Zorbax C8 reversed-phase column. The analytical results are discussed in terms of the method validation and the corresponding experimental protocol (r>/=0.998; CV<9%; LOQ 20 microg/l). There was good correlation between FL, as well as NORFL, plasma levels as determined by the LC-MS and LC-FLD techniques (r=0.9597, N=16 and r=0.9852, N=14 for FL and NORFL, respectively). The results confirm that direct FL/NORFL fluorimetric determination is acceptable for routine use in pharmacokinetic and clinical studies.

[Antibiotics in the prevention of the surgical site infection in orthopedic surgery]. / Antibiotici u prevenciji infekcije hirurske rane u ortopedskoj hirurgiji.

Surgical site infection is an actual problem of orthopaedic surgery. Despite considerable efforts that have been done during last several decades (e.g. improvements in surgical techniques, preoperative preparation of the surgical site, infection-control practice, use of preventive antibiotics) surgical site infection still affects about 0.5-2% of patients after closed fracture surgery or insertion of prosthetic devices. They are associated with substantial morbidity and mortality. The adherence to the principles of rationale preventive antibiotic therapy has an important role in the prevention of the surgical infection. In addition, it is well known that inappropriate use of antibiotic promote development of resistance, superinfections and increase the cost of the treatment. This paper focuses on the basic principles of rational use of antibiotics, i.e. appropriate selection of drug, dose, and duration of treatment in the prevention of surgical site infections in orthopaedic surgery.

The influence of L-arginine on heart rate and tissue oxygen extraction in haemorrhaged rabbits.

Several studies have already indicated some beneficial effects of L-arginine in haemorrhaged rats. The aim of our study was to assess whether intravenous bolus injection of L-arginine could improve some cardiovascular and metabolic parameters in anaesthetized haemorrhaged rabbits (intermittent bleeding; 40% of the estimated blood volume for 15 min). I.v. bolus injection of L-arginine ( 300 mg kg(-1)--L-Arg(300)) increased heart rate (app. 10%) and decreased venous haemoglobin saturation with oxygen (sO(2)) (app. 23%) 60 min after the cessation of bleeding, without changes in arterial pressure. D-arginine (300 mg kg(-1)i.v. bolus-D-Arg(300)) produced similar, but insignificant haemodynamic and metabolic changes. In addition, no difference was found between the effects of the L- and D-isomers. Accordingly, L-arginine produces beneficial effects on the heart rate and tissue oxygen extraction in haemorrhaged rabbits. However, such changes do not appear to be stereospecific.

Antinociceptive activity of the novel fentanyl analogue iso-carfentanil in rats.

A large number of fentanyl analogues have been synthesized so far, both to establish the structure-activity-relationship (SAR) and to find novel, clinically useful antinociceptive drugs. In this study, the newly synthesized fentanyl analogue 3-carbomethoxy fentanyl (iso-carfentanil) was compared to fentanyl for its antinociceptive activity (tail-immersion test) in rats. It was revealed that the introduction of a 3-carbomethoxy group in the piperidine ring of fentanyl skeleton reduced the potency and shortened the duration of action of the parent compound, i.e., fentanyl. The antinociceptive potency of 3-carbomethoxy fentanyl is influenced mainly by the steric factor (voluminosity of the carbomethoxy group and the cis/trans isomerism), while the chemical nature of the group is probably irrelevant. This is in agreement with SAR studies of other 3-substituted fentanyl analogues. In contrast to potency, the duration of action is not affected by cis/trans isomerism. It is assumed that the time course of action of 3-carbomethoxy fentanyl is influenced by the nature of the carbomethoxy group. Since the potency and the duration of action of this novel antinociceptive compound are interesting from the aspect of SAR studies and have potential promise for clinical application, 3-carbomethoxy fentanyl deserves to be extensively evaluated.

The synthesis and preliminary pharmacological evaluation of 4-methyl fentanyl.

The synthesis of 4-methyl fentanyl, a prototype of a novel class of fentanyl analogues has been effected in 5 steps, starting from N-ethoxycarbonyl-4-piperidone (approximately 20% overall yield). In the key step, N-phenylation of secondary aliphatic amide intermediare was achieved by a novel reaction, using diphenyliodonium chloride for the phenyl group transfer. Preliminary pharmacological results indicate that 4-methyl fentanyl is a super potent narcotic analgesic, about four times more potent than fentanyl.

Characterization of adenosine action in isolated rat renal artery. Possible role of adenosine A(2A) receptors.

Adenosine (0.1-300 microM) induced concentration- and endothelium-dependent relaxation of rat renal artery (RRA). N(G)-Nitro-L-arginine (L-NOARG, 10 microM) significantly reduced adenosine-elicited dilatation, but not the application of indomethacin (10 microM), ouabain (100 microM) or tetraethylammonium (TEA, 500 microM). In the presence of high concentration of K(+) (100 mM) or glibenclamide (1 microM), adenosine-evoked relaxation was almost abolished. 8-(3-Chlorostyril)caffeine (CSC, 0.3-3 microM), a selective A(2A)-antagonist, significantly reduced adenosine-evoked dilatation in a concentration-dependent manner (pA(2)=7.29). Conversely, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10 nM), an A(1)-antagonist, did not alter adenosine-induced relaxation. These results indicate that adenosine produces endothelium-dependent relaxation of isolated RRA. Dilatation evoked by adenosine is mediated by predominant releasing of endothelium-derived hiperpolarizing factor (EDHF) and also in one part of nitric oxide (NO) from endothelial cells. The obtained results also suggest that RRA response to adenosine is most likely initiated by activation of endothelial adenosine A(2A) receptors.