Abstract:  Fatty acid synthase (FASN), a multi-enzyme complex, is involved in lipid biosynthesis. FASN is over-expressed in different types of cancers and is being widely investigated for its role in cancer progression, diagnosis and therapy. Here, three inhibitors targeting different domains of FASN – cerulenin, triclosan and orlistat – were evaluated for their anti-proliferative efficacy in ocular cancer, retinoblastoma (RB) cells and their toxicity (if any) in normal cells. FASN inhibitors were tested in cultured retinoblastoma Y79 cells, normal fibroblast (3T3) and Müller glial (MIOM1) cells. Cell viability was determined by MTT-based assay, and IC₅₀ (50% inhibitory concentration) of the FASN inhibitors was calculated in neoplastic and non-neoplastic cells. The IC₅₀ after 48 and 96 hr of incubation with the three anti-FASN agents showed that cerulenin, triclosan and orlistat inhibited retinoblastoma cell proliferation in a dose- and time-dependent manner. The cancer cells exhibited differential dose- and time-dependent response/sensitivities to cerulenin, triclosan and orlistat. The 48-hr neoplastic IC₅₀ dosages were, however, not toxic to the normal cells. These findings were confirmed by phase-contrast microscopic assessment of cell morphology. Therapeutic index (TI) was calculated as a ratio of the IC₅₀ normal cells, to the IC₅₀ neoplastic cells. Relative to normal MIOM1 cells, TI was 9.18 for cerulenin, while 5.32 for triclosan and 1.72 for orlistat. The TI computed relative to 3T3 cells was 28.64, 7.10 and 2.58 for cerulenin, triclosan and orlistat, respectively. DNA fragmentation analysis suggests that FASN inhibitors induced apoptotic DNA damage in retinoblastoma cells. Thus, FASN inhibition can be an effective strategy in retinoblastoma therapy.

Abstract:  We evaluated the pharmacokinetics of clindamycin and the dose of clindamycin phosphate necessary to treat peritonitis after intraperitoneal administration of clindamycin phosphate to patients on continuous ambulatory peritoneal dialysis (CAPD). This was an open-label, prospective, single-dose study conducted at the two levels of institutional clinical care in South Korea. Twelve patients (six men and six women; all older than 25 years), mean CAPD duration of 38.2 months with various origins without peritonitis, received 600 mg clindamycin phosphate mixed with only the first 2-L dialysate (1.5% dextrose). The 1.5%, 1.5%, 2.5% and 1.5% dextrose dialysates were serially exchanged every 6 hr. If patients were non-anuric, 24-hr urine samples were also collected. Clindamycin phosphate was incompletely activated to clindamycin in the dialysate. The clindamycin concentration in the dialysate was greater than the effective concentration (5 μg/mL) at 6.87 μg/mL up to 6 hr. So, 600 mg clindamycin phosphate per every 6 hr dialysate is effective for treatment of peritonitis. It has been reported that the clindamycin concentrations in the dialysate may be higher in CAPD patients with peritonitis. Thus, we can expect that intraperitoneal administration of <600 mg clindamycin phosphate per every 6 hr dialysate could be maintained over 5 μg/mL in patients with peritonitis. The transfer of clindamycin was unidirectional from the dialysate to the plasma.

Abstract:  Cadmium is a heavy metal that is known to cause toxicity to cells and, at low concentrations, can initiate apoptosis. This study was undertaken with the aim of defining the role of phospholipase C (PLC) in mediating cadmium-induced apoptosis in human embryonic kidney (HEK 293) cells. We have shown that intracellular Ca²⁺ levels increased significantly in HEK 293 cells after 24-hr exposure to Cd. The activity of the calcium-dependent protease calpain rose by four times. The PLC–specific inhibitor, U73122, prevented the Cd-dependent increase in Ca²⁺ levels and also abolished Cd-dependent calpain and caspase 3 activation as well as Cd-dependent mitochondrial Bax accumulation. Inhibition of PLC also leads to an increased cell viability following exposure to Cd. Taken together, the results show that the PLC pathway is involved in mediating Cd-induced apoptosis in HEK 293 cells.

Abstract:  Previous studies on animals have revealed that garlic (Allium sativum) is effective in reducing blood and tissue lead concentrations. The aim of this study was to investigate therapeutic effects of garlic and compare it with d-penicillamine in patients with chronic lead poisoning. After coordination and obtaining informed consent, clinical examinations and blood lead concentration (BLC) of 117 workers at a car battery industry were investigated. BLC was determined by heated graphite atomization technique of an atomic absorption spectrometer. The workers were randomly assigned into two groups of garlic (1200 μg allicin, three times daily) and d-penicillamine (250 mg, three times daily) and treated for 4 weeks. BLC was determined again 10 days post-treatment. Clinical signs and symptoms of lead poisoning were also investigated and compared with the initial findings. Clinical improvement was significant in a number of clinical manifestations including irritability (p = 0.031), headache (p = 0.028), decreased deep tendon reflex (p = 0.019) and mean systolic blood pressure (0.021) after treatment with garlic, but not d-penicillamine. BLCs were reduced significantly (p = 0.002 and p = 0.025) from 426.32 ± 185.128 to 347.34 ± 121.056 μg/L and from 417.47 ± 192.54 to 315.76 ± 140.00 μg/L in the garlic and d-penicillamine groups, respectively, with no significant difference (p = 0.892) between the two groups. The frequency of side effects was significantly (p = 0.023) higher in d-penicillamine than in the garlic group. Thus, garlic seems safer clinically and as effective as d-penicillamine. Therefore, garlic can be recommended for the treatment of mild-to-moderate lead poisoning.

Abstract:  The frequency distribution of SNPs and haplotypes in the ABCB1, SLCO1B1 and SLCO1B3 genes varies largely among continental populations. This variation can lead to biases in pharmacogenetic studies conducted in admixed populations such as those from Brazil and other Latin American countries. The aim of this study was to evaluate the influence of self-reported colour, geographical origin and genomic ancestry on distributions of the ABCB1, SLCO1B1 and SLCO1B3 polymorphisms and derived haplotypes in admixed Brazilian populations. A total of 1039 healthy adults from the north, north-east, south-east and south of Brazil were recruited for this investigation. The c.388A>G (rs2306283), c.463C>A (rs11045819) and c.521T>C (rs4149056) SNPs in the SLCO1B1 gene and c.334T>G (rs4149117) and c.699G>A (rs7311358) SNPs in the SLCO1B3 gene were determined by Taqman 5′-nuclease assays. The ABCB1 c.1236C>T (rs1128503), c.2677G>T/A (rs2032582) and c.3435C>T (rs1045642) polymorphisms were genotyped using a previously described single-base extension/termination method. The results showed that genotype and haplotype distributions are highly variable among populations of the same self-reported colour and geographical region. However, genomic ancestry showed that these associations are better explained by a continuous variable. The influence of ancestry on the distribution of alleles and haplotype frequencies was more evident in variants with large differences in allele frequencies between European and African populations. Design and interpretation of pharmacogenetic studies using these transporter genes should include genomic controls to avoid spurious conclusions based on improper matching of study cohorts from Brazilian populations and other highly admixed populations.

Abstract:  The analgesic concentrations of oxycodone in acute post-operative pain management have not been established. Here, we have evaluated the minimum effective concentration (MEC) and the minimum effective analgesic concentration (MEAC) of oxycodone in pain after laparoscopic cholecystectomy (LCC) in 23 adult patients. The patients were provided with 0.1 mg/kg of oxycodone i.v. 10–15 min. before the end of surgery. After surgery, when the wound pain at rest was ≥3/10 and/or ≥5/10 during wound compression, a first blood sample was obtained (MEC). A second blood sample was obtained after titration with 2 mg i.v. of oxycodone to wound pain <3/10 at rest and <5/10 during wound compression (MEAC). A third blood sample was obtained at the recurrence of the wound pain (the second MEC), and the final blood sample when pain relief was obtained a second time (the second MEAC). At the first onset of pain (MEC), mean P-oxycodone was 21 ng/mL (95% CI 13–29 ng/mL). At the first pain relief (MEAC), P-oxycodone was 55 ng/mL (19–91 ng/mL). The second MEC was 34 ng/mL (11–57 ng/mL), and the second MEAC was 47 ng/mL (14–80 ng/mL). In conclusion, the estimated MEC, 20–35 ng/mL, and MEAC, 45–50 ng/mL, values of P-oxycodone in patients after LLC were significantly higher than those proposed previously. Early pain after LCC appeared to be a feasible method to estimate the analgesic efficacy of oxycodone in acute pain management.

Abstract:  Monoisoamyl DMSA (MiADMSA), a lipophilic chelating agent has emerged as a promising drug for the treatment of arsenic. The present study aimed at exploring the optimum dose and route of administration for achieving maximum arsenic elimination with minimal side effects. We also carried out a pharmacokinetic analysis of this drug to support arsenic chelation. Rats were exposed to arsenic (25 ppm) for 6 months and later received MiADMSA (50 or 100 mg/kg) orally and via i.p. route for 5 days. Oxidative stress parameters and arsenic levels in soft tissues, liver function test and histopathology of liver and kidney were performed. Plasma kinetic of MiADMSA (plasma-free drug and total drug) at 50 and 100 mg/kg p.o. was carried out. Arsenic exposure resulted in significant oxidative stress and hepatotoxicity. MiADMSA at 50 mg/kg dose administered orally provided about 45% and 75% protection against oxidative stress and in lowering body arsenic burden, respectively, against 25% and 40% via i.p. route. Pharmacokinetic analysis supported prolonged availability of the drug through oral administration. Collectively, these findings led us to conclude that oral administration of MiADMSA was more effective than intraperitoneal administration and that the minimum effective dose with least side effects was 50 mg/kg.

Abstract:  Excessive and sustained increases in oxidative stress and apoptosis have been implicated in the pathogenesis of many diseases. In this study, we demonstrated that 4-hydroxynonenal (4-HNE), a product of lipid peroxidation in a range of concentration (0.1–50 μM) showed cytotoxic effects on SH-SY5Y cell culture at a concentration >5 μM at 4 hr of exposure. 4-HNE dose dependently decreased cell viability and significantly promoted reactive oxygen species formation and enhanced oxidative stress as reflected in the increased level of lipid peroxidation and catalase activity and decreased glutathione peroxidase activity as well as glutathione levels. 4-HNE-induced oxidative stress was associated with increased transcriptional and translational expressions of Bax and p53 in SH-SY5Y cells. Mitochondrial-mediated apoptosis was confirmed by increased expression and activity of caspase-3. Our data demonstrate that 4-HNE induces neuronal cell death through abnormal expression of apoptotic markers (p53, Bax and caspase-3). Oxidative stress may be involved in the initial priming of SH-SY5Y cells to 4-HNE-induced cytotoxicity in vitro.

Abstract:  Imazalil, cypermethrin and carbendazim are detected in plants for human nutrition. To explore whether their combinations, applied orally in low doses, would induce changes in metabolic patterns and hepatotoxicity, a subchronic in vivo experiment was conducted. Doses of 10 mg/kg of imazalil (im) and cypermethrin (cy) and 20 mg/kg of carbendazim (car) and their combinations (im, 10 mg/kg + cy, 10 mg/kg; im, 10 mg/kg + car, 20 mg/kg; car, 20 mg/kg + im, 10 mg/kg) were given to Swiss mice daily over 28 days. After 24 hr from the last dose, the relationships of cytotoxicity biomarkers were analysed: serum lactate dehydrogenase, aspartate transaminase, alanine transferase, amylase, alkaline phosphatase, creatine kinase, creatinine and total proteins. Individual pesticides showed different toxic potential (cy > im > car) generally characterized by increase in enzyme activities. Histological analysis showed that cypermethrin, but not imazalil or carbendazim, alone can cause mild necrosis. Combinations generally caused decrease in the activity of enzymes, indicating liver damage. Low doses of carbendazim in combination with low doses of imazalil or cypermethrin caused very pronounced hepatic necrosis, more than any of the three individually applied pesticides or combination of imazalil and cypermethrin. In fruits and vegetables for human consumption, residues of these three pesticides and prolonged combined intake of low doses, which by themselves acutely would not cause any effect, may have similar hepatotoxic effects.

Abstract:  A number of drugs, like sibutramine, which are used clinically in weight control, act on serotonergic metabolism. However, their relation with zinc and free radical (FR) production in central nervous system remains unknown. This study aimed to evaluate the effect of sibutramine and zinc on FR production. Female Wistar rats (about 250 g) were used in this study. The animals received 400 μg/kg of zinc and 10 mg/kg of sibutramine intraperitoneally every 36 hr for 15 days. At the end of the study, the rats were killed and their brains used for the measurement of lipid peroxidation thiobarbituric acid-reactive substances (TBARS), reduced glutathione (GSH), hydrogen peroxide (H₂O₂), calcium and 5-hydroxyindole acetic acid (5-HIAA) levels, all by means of validated methods. Corporal weight and food consumption were found to be decreased in the zinc/sibutramine group. TBARS decreased in cortex, hemispheres and medulla oblongata. GSH decreased in cortex, hemispheres and cerebellum in the sibutramine group. Zinc given alone and in combination with sibutramine decreased H₂O₂ concentration in cortex, hemispheres and cerebellum but increased calcium and 5-HIAA concentration in all brain regions. Our results suggest that sibutramine and zinc are associated with weight loss, an effect that was more pronounced in the group treated with both drugs. Reduction in oxidative stress may be involved in these effects.

Abstract:  This study aimed to analyse medication errors committed by health care professionals leading to toxicological consultations at the Czech Toxicological Information Centre and to identify the categories of special concern for further interventions of health care quality experts.

Abstract:  The fact that there is a complex and bidirectional communication between the immune and nervous systems has been well demonstrated. Lipopolysaccharide (LPS), a component of gram-negative bacteria, is widely used to systematically stimulate the immune system and generate profound physiological and behavioural changes, also known as ‘sickness behaviour’ (e.g. anhedonia, lethargy, loss of appetite, anxiety, sleepiness). Different ethological tools have been used to analyse the behavioural modifications induced by LPS; however, many researchers analysed only individual tests, a single LPS dose or a unique ethological parameter, thus leading to disagreements regarding the data. In the present study, we investigated the effects of different doses of LPS (10, 50, 200 and 500 μg/kg, i.p.) in young male Wistar rats (weighing 180–200 g; 8–9 weeks old) on the ethological and spatiotemporal parameters of the elevated plus maze, light-dark box, elevated T maze, open-field tests and emission of ultrasound vocalizations. There was a dose-dependent increase in anxiety-like behaviours caused by LPS, forming an inverted U curve peaked at LPS 200 μg/kg dose. However, these anxiety-like behaviours were detected only by complementary ethological analysis (stretching, grooming, immobility responses and alarm calls), and these reactions seem to be a very sensitive tool in assessing the first signs of sickness behaviour. In summary, the present work clearly showed that there are resting and alertness reactions induced by opposite neuroimmune mechanisms (neuroimmune bias) that could lead to anxiety behaviours, suggesting that misunderstanding data could occur when only few ethological variables or single doses of LPS are analysed. Finally, it is hypothesized that this bias is an evolutionary tool that increases animals’ security while the body recovers from a systemic infection.

Abstract:  Connective tissue growth factor (CTGF) plays a pathogenic role in diabetic nephropathy (DN). Rosmarinic acid (RA) is a naturally occurring phenolic acid. This study was conducted to investigate the efficacy of RA on DN and to elucidate the potential mechanism. High glucose (HG)-stimulated cultured human renal proximal tubular epithelial cells (HK-2) analysed CTGF expression by western blotting, and it was investigated whether extracellular signal-regulated kinase (ERK) signalling pathway was involved. Using streptozotocin (STZ)-induced rat animal models, diabetic rats were randomized to receive intragastric (i.g.) doses of RA. Renal tissue, blood and urine samples were collected to determine biochemical index and analyse protein expression. In vitro study, RA reduced CTGF excretion in HG-induced HK-2 cells through the ERK signalling pathway. In an in vivo study, I.g. of RA 7.5 or 15 mg/kg significantly ameliorated renal function and increased body-weight. Meanwhile, RA reduced renal CTGF expression by immunohistochemical staining and reduced serum levels of CTGF. Besides, there were no significant differences in glycaemia levels between the RA groups compared with the STZ-treated group. Furthermore, RA ameliorated renal pathology. These results suggest that RA exerts an early renal protective role to DN. Inhibition of CTGF may be a potential target in DN therapy, which highlights the possibility of using RA in the treatment of DN.

Abstract:  Recent studies have demonstrated that nuclear factor-κB (NF-κB) and high-mobility group box 1 (HMGB1) are associated with the pathophysiology of sepsis. The present study was carried out to investigate the effects of protocatechuic aldehyde (PA) on an experimental model of sepsis induced by caecal ligation and puncture (CLP) in rats and to elucidate the potential mechanism in the cultured murine macrophage cell line, RAW264.7 cells. Treatment of RAW 264.7 cells with PA blocked TNF-α-induced NF-κB phosphorylation and decreased HMGB1 expression. Septic rats received doses of 50 mg of PA alone or plus Imipenem by intravenous bolus injection into the tail vein. The results showed that PA reduced serum levels of HMGB1 and triggering the receptor expressed on myeloid cells, it attenuated myeloperoxidase in the lung, liver and small intestine, while it up-regulated serum level of IL-10. Meanwhile, PA alone or plus Imipenem reduced CLP-induced lethality in septic rats. These data indicate that the anti-septic effect of PA is mediated by decreasing local and systemic levels of a wide spectrum of inflammatory mediators. The protective effects of PA might block the inflammatory cascades through HMGB1 and NF-κB signalling pathway. Our studies enhance the case for the use of PA in sepsis, and PA therefore seems promising in the treatment of sepsis in human beings.

Abstract:  Intravenous lipid emulsion has been used in the resuscitative treatment of intoxications caused by local anaesthetics and tricyclic antidepressants with seemingly beneficial results. We studied the effect of intravenous lipid emulsion on the plasma concentration of amitriptyline and haemodynamic recovery in a pig model of amitriptyline intoxication. Twenty pigs were anaesthetized (1% isoflurane in 21% O₂) and given amitriptyline 15 mg/kg intravenously for 15 min. In random fashion immediately thereafter, either 20% lipid emulsion (ClinOleic^®, Lipid group) or Ringer’s acetate (Control group) was infused for 30 min.; first 1.5 ml/kg for 1 min., followed by 0.25 ml/kg/min. for 29 min. The amitriptyline concentration in total and lipid-poor plasma and haemodynamic parameters were measured until 30 min. after the infusions. Lipid infusion prevented the decrease in plasma total amitriptyline concentration, resulting in a 90% higher (p < 0.001) total concentration and significantly (p = 0.014) lower free fraction of plasma amitriptyline in the Lipid group (1.1%) compared with the Control group (3.0%) at 30 min. Haemodynamic recovery from the intoxication as measured by heart rate, arterial pressure or cardiac output was similar in both groups. However, five pigs in the Lipid group and two pigs in the Control group died. In conclusion, a marked entrapment of amitriptyline by intravenous lipid emulsion was observed but this did not improve the pigs’ haemodynamic recovery from severe amitriptyline intoxication. Care should be exercised in the antidotal use of lipid emulsion until controlled human studies indicate its efficacy and safety.

Abstract:  There has been recent interest in combining antiplatelets, angiotensin-converting enzyme inhibitors (ACEIs) and statins in primary and secondary ischaemic stroke prevention. This observational study was performed to evaluate the impact of adding ACEIs and/or statins to antiplatelets on post-stroke in-hospital mortality. Ischaemic stroke patients attending a hospital in Malaysia over an 18-month period were evaluated. Patients were categorized according to their vital status at discharge. Data included demographic information, risk factors, clinical characteristics and previous medications with particular attention on antiplatelets, ACEIs and statins. In-hospital mortality was compared among patients who were not taking antiplatelets, ACEIs or statins before stroke onset versus those who were taking antiplatelets alone or in combination with either ACEIs, statins or both. Data analysis was performed using spss version 15. Overall, 637 patients met the study inclusion criteria. After controlling for the effects of confounders, adding ACEIs or statins to antiplatelets significantly decreased the incidence of death after stroke attack by 68% (p = 0.036) and 81% (p = 0.010), respectively, compared to patients on antiplatelets alone or none of these medications. Additionally, the addition of both ACEIs and statins to antiplatelet medication resulted in the highest reduction (by 94%) of the occurrence of death after stroke attack (p < 0.001). Our results suggest that adding ACEIs and/or statins to antiplatelets for patients at risk of developing stroke, either as a primary or as a secondary preventive regimen, was associated with a significant reduction in the incidence of mortality after ischaemic stroke than antiplatelets alone. These results might help reduce the rate of ischaemic stroke morbidity and mortality by enhancing the application of specific therapeutic and management strategies for patients at a high risk of acute stroke.

Abstract:  The hybrid created from the crossbreeding of European and African bees, known as the Africanised bee, has provided numerous advantages for current beekeeping. However, this new species exhibits undesirable behaviours, such as colony defence instinct and a propensity to attack en masse, which can result in serious accidents. To date, there is no effective treatment for cases of Africanised bee envenomation. One promising technique for developing an efficient antivenom is the use of phage display technology, which enables the production of human antibodies, thus avoiding the complications of serum therapy, such as anaphylaxis and serum sickness. The aim of this study was to produce human monoclonal single-chain Fv (scFv) antibody fragments capable of inhibiting the toxic effects of Africanised bee venom. We conducted four rounds of selection of antibodies against the venom and three rounds of selection of antibodies against purified melittin. Three clones were selected and tested by enzyme-linked immunosorbent assay to verify their specificity for melittin and phospholipase A2. Two clones (C5 and C12) were specific for melittin, and one (A7) was specific for phospholipase A2. In a kinetic haemolytic assay, these clones were evaluated individually and in pairs. The A7–C12 combination had the best synergistic effect and was chosen to be used in the assays of myotoxicity inhibition and lethality. The A7–C12 combination inhibited the in vivo myotoxic effect of the venom and increased the survival of treated animals.

Abstract:  The ability of three newly developed reversible inhibitors of acetylcholinesterase (AChE) (K298, K344 and K474) and currently available carbamate pyridostigmine to increase the resistance of mice against soman and the efficacy of antidotal treatment of soman-poisoned mice was compared. Neither pyridostigmine nor new reversible inhibitors of AChE were able to increase the LD₅₀ value of soman. Thus, the pharmacological pre-treatment with pyridostigmine or newly synthesized inhibitors of AChE was not able to protect mice against soman-induced lethal acute toxicity. The pharmacological pre-treatment with pyridostigmine alone or with K474 was able to slightly increase the efficacy of antidotal treatment (the oxime HI-6 in combination with atropine) of soman-poisoned mice, but the increase in the efficacy of antidotal treatment was not significant. The other newly developed reversible inhibitors of AChF (K298, K344) were completely ineffective. These findings demonstrate that pharmacological pre-treatment of soman-poisoned mice with tested reversible inhibitors of AChF is not promising.

Abstract:  The clinical benefit of implementing Bayesian approach for lithium drug monitoring was evaluated. Intervention group (N = 42) and historical control group (N = 55) patients were each divided into two groups: Dosage with immediate-release lithium carbonate or a sustained-release formulation, lithium citrate. Bayesian approach was performed in the intervention groups, and estimation of lithium steady-state trough concentration was obtained from non-steady-state blood sample, collected about 12 hr after the first lithium study dose. The estimate was compared with the actually measured steady-state concentration. In the control group, lithium monitoring was traditionally performed as steady-state blood sampling. Predicted and measured lithium concentrations were comparable. The desired lithium dose was reached significantly faster in the intervention group compared to control; 2.47 ± 2.22 days versus 9.96 ± 11.24 days (mean ± S.D.) (p = 0.0003). Bayesian approach was an advantage for the clinicians as a fast and safe aid to obtain the optimal lithium treatment dose.

Abstract:  Considerable evidence supports the presence of oxidative stress in cystic fibrosis (CF). The disease has long been associated with both increased production of reactive oxygen species and impaired antioxidant status, in particular during the chronic pulmonary infection with Pseudomonas aeruginosa, which is the main cause of morbidity and mortality in CF. Guinea pigs are unable to synthesize ascorbate (ASC) or vitamin C, a major antioxidant of the lung, and thus like human beings rely on its presence in the diet. On this basis, guinea pigs receiving ASC-deficient diet have been used as a model of oxidative stress. The aim of our study was to investigate the consequences of a 7-day biofilm-grown P. aeruginosa lung infection in 3-month-old guinea pigs receiving either ASC-sufficient or ASC-deficient diet for at least 2 months. The animals receiving ASC-deficient diet showed significantly higher mortality during infection and increased respiratory burst of peripheral polymorphonuclear neutrophils (PMNs) compared with the animals receiving ASC sufficient diet. The inflammatory response at the site of lung infection consisted of PMNs and mononuclear leucocytes (MN), and higher PMN/MN ratios were present in animals on ASC-deficient diet compared with animals on ASC sufficient diet. Measurements of the ASC levels in the lung were significantly decreased in infected compared with non-infected animals. Interestingly, the infection by itself decreased the antioxidant capacity of the plasma (measured as plasma oxidizability) more than the ASC-deficient diet, suggesting a high consumption of the antioxidants during infection. Our data show that poor antioxidant status exacerbates the outcome of biofilm-related infections.

Abstract:  The effect of the natural product 3,3′-diindolylmethane (DIM) on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in MG63 human osteosarcoma cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. DIM at concentrations of 40–80 μM induced a [Ca²⁺]_i rise in a concentration-dependent manner. The response was reduced partly by removing Ca²⁺. DIM-evoked Ca²⁺ entry was suppressed by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished DIM-induced [Ca²⁺]_i rise. Incubation with DIM also inhibited thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished DIM-induced [Ca²⁺]_i rise. At concentrations of 10–50 μM, DIM killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data implicate that DIM (20 and 40 μM) induced apoptosis in a concentration-dependent manner. In sum, in MG63 cells, DIM induced a [Ca²⁺]_i rise by evoking phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-sensitive store-operated Ca²⁺ channels. DIM caused cell death that may involve apoptosis.

Abstract:  Seaweeds have attracted special interest as good sources of sulphated polysaccharides (SP) for use in pharmaceutical industries and biotechnology. In this study, we evaluated the effects of SP from the red seaweed Gracilaria cornea (Gc-TSP) in nociceptive and inflammatory models. In mice, Gc-TSP (3, 9 or 27 mg/kg) significantly reduced nociceptive responses, as measured by the number of writhes, at all tested doses. In a formalin test, Gc-TSP significantly reduced licking time in both phases of the test at a dose of 27 mg/kg. In a hot-plate test, the antinociceptive effect was observed only in animals treated with 27 mg/kg of Gc-TSP, suggesting that the analgesic effect occurs through a central action mechanism at the highest dose. Gc-TSP (3, 9 or 27 mg/kg) caused only a slight reduction in neutrophil migration in the rat peritoneal cavity. However, lower doses of Gc-TSP (3 and 9 mg/kg) significantly inhibited paw oedema induced by carrageenan, especially at 3 hr after treatment. Reduction in oedema was confirmed by myeloperoxidase activity in the affected paw tissue. In addition, treatment (s.c.) of animals with different doses of Gc-TSP inhibited paw oedema induced by dextran within the first hour in all doses tested. After 14 consecutive days of intraperitoneal administration of Gc-TSP (9 mg/kg), we measured the wet weight of the liver, kidney, heart, spleen and thymus and performed biochemical, haematological and histopathological evaluations. No systemic damage was found. These results indicate that Gc-TSP possesses analgesic and anti-inflammatory effects and is a potentially important tool worthy of further study.

Abstract:  Prolonged consumption of ethanol produces prefrontal cortex (PFC) dysfunction in patients, and this has been demonstrated using structural, physiological and psychological measurements. We therefore wanted to develop an animal model of PFC dysfunction to study whether this state changes sensitivity for ethanol or other behavioural/motivational measures. Adolescent Wistar rats were first screened in the novel object recognition task to establish a pre-treatment baseline measure of locomotor activity, anxiety-like behaviour and PFC function. Animals were divided into four treatment groups [saline, 5 mg/kg phencyclidine (PCP), 2.5g/kg ethanol, ethanol + PCP] and injected i.p. for 5 days followed by a 2-day washout. On the 8th day, animals were allowed to explore a Y-maze for 10 min. and spontaneous alternations were recorded using the ANY-maze tracking system. PCP, a classic drug used to induce PFC dysfunction in animals, did not significantly reduce the % correct alternations relative to the 70% level achieved by the saline group. Ethanol and the combination of Ethanol + PCP, however, significantly reduced alternations to approximately 30%. The combined dose was not additive in terms of Y-maze impairment, and these animals had less total distance travelled and greater time immobile relative to the other groups. We therefore concluded that injection of 2.5 g/kg ethanol for 5 days in Wistar rats produces a more substantial, consistent and valid PFC dysfunction than 5 mg/kg PCP.

Abstract:  Up-regulation of vascular endothelial growth factor (VEGF) is important to hepatocyte regeneration in the late stages of acetaminophen (APAP) toxicity in the mouse. This study was conducted to examine the relationship of hypoxia-inducible factor 1α (HIF-1α) to VEGF and hepatocyte regeneration in APAP toxicity using an inhibitor of HIF-1α DNA-binding activity, echinomycin (EC). B6C3F1 male mice were treated with APAP (200 mg/kg IP), followed by EC (0.15 mg IP) and killed at 4 hr. Serum alanine aminotransferase (ALT), necrosis, hepatic glutathione (GSH) and APAP protein adducts were comparable in the APAP/EC and the APAP/veh mice at 4 hr. Additional studies showed that high dose EC (0.3 mg) reduced hepatic VEGF but also lowered hepatic GSH. Subsequent studies were performed using the 0.15-mg dose of EC. Although EC 0.15 mg had no effect on hepatic VEGF levels at 8 hr, by 24 hr VEGF levels were decreased by 40%. Toxicity (ALT and histopathology) was comparable in the APAP and APAP/EC groups at 24 and 48 hr. Proliferating cell nuclear antigen expression was reduced by both Western blot analysis and immunohistochemical staining in the APAP/EC mice at 48 hr. The data support the hypothesis that induction of HIF-1α, its binding to DNA and subsequent expression of VEGF are important factors in hepatocyte regeneration in APAP toxicity in the mouse.

Abstract:  The aim of the study was to evaluate the pentoxifylline administration on the foetal–placental circulation and neonatal outcome in women with threatened preterm labour. Pentoxifylline was given as a supplement to standard tocolytic therapy in a group of 43 patients (pentoxifylline group) as an intravenous infusion and oral supplementation in a total dosage of 800 mg/day. The drug was administered within 3 weeks after admission. No pentoxifylline was given in the control group (53 patients). Doppler velocimetry of pulsatility indices (PI) of the umbilical (UA) and middle cerebral (MCA) arteries as well as cerebro-placental ratio (CPR) were calculated. Also, the neonatal outcome was estimated in both groups. From the second week of therapy with pentoxifylline, the PI decreased in umbilical artery and increased in the MCA, whereas in the control group, there were no changes. The value of PIUA, evaluated after the third week of pentoxifylline administration, was statistically significantly lower when compared to data obtained on admission (mean: 0.99 ± 0.22 versus 0.82 ± 0.12; p = 0.016). Pentoxifylline significantly increased CPR values calculated after third week of drug administration, which were statistically significantly higher in the pentoxifylline group when compared with respective data in the control group (mean: 2.30 versus 1.61; p = 0.001). The risk of severe neonatal complications was significantly lower in the pentoxifylline group (p = 0.026). Pentoxifylline changed foetal–placental blood circulation in patients with threatened preterm labour and improved neonatal outcome.

Abstract:  Curcumin (diferuloylmethane), a phenolic compound obtained from the rhizome of Curcuma longa, has been found to inhibit cell proliferation in various human cancer cell lines, including non-small cell lung cancer (NSCLC). Thymidine phosphorylase (TP) is considered an attractive therapeutic target, because increased TP expression can suppress cancer cell death induced by DNA-damaging agents. Mitomycin C (MMC), a chemotherapeutic agent used to treat NSCLC, inhibits tumour growth through DNA cross-linking and breaking. Whether MMC can affect TP expression in NSCLC is unknown. Therefore, in this study, we suggested that curcumin enhances the effects of MMC-mediated cytotoxicity by decreasing TP expression and ERK1/2 activation. Exposure of human NSCLC cell lines H1975 and H1650 to curcumin decreased MMC-elicited phosphorylated MKK1/2-ERK1/2 protein levels. Moreover, curcumin significantly decreased MMC-induced TP protein levels by increasing TP mRNA and protein instability. Enhancement of ERK1/2 activation by constitutively active MKK1/2 (MKK1/2-CA) increased TP protein levels and cell viability in curcumin- and MMC-co-treated cells. In contrast, U0126, a MKK1/2 inhibitor, augmented the cytotoxic effect and the down-regulation of TP by curcumin and MMC. Specific inhibition of TP by siRNA significantly enhanced MMC-induced cell death and cell growth inhibition. Our results suggest that suppression of TP expression or administration of curcumin along with MMC may be a novel lung cancer therapeutic modality in the future.

Abstract:  Markers of dopamine D₁ receptor activation were determined to elucidate intracellular mechanisms associated with the combined effects of caffeine and 3,4 methylenedioxymethamphetamine (MDMA), reported previously to produce increased toxicity, when compared with either drug alone. Caffeine (10 mg/kg) and MDMA (15 mg/kg) were administered to male Sprague Dawley rats alone and in combination. One hour after drug administration, core body temperature and phosphorylation of the dopamine D₁-related intracellular markers, cAMP response element binding protein (CREB), the dopamine and c-AMP-regulated phosphoprotein of 32 kDa (DARPP-32) and expression of the immediate early gene and cellular activation marker c-fos were determined in the hypothalamus. Co-administration of caffeine with MDMA increased core body temperature when compared with MDMA or caffeine treatment alone. Pre-treatment with the dopamine D₁ receptor antagonist SCH 23390 (1 mg/kg, i.p.), 30 min. prior to caffeine and MDMA administration, produced a hypothermic response to MDMA that was unaffected by caffeine. Co-administration of caffeine with MDMA increased p-CREB, p-DARPP-32 and c-fos expression when compared with either treatment alone. Pre-treatment with SCH-23390 attenuated the changes in p-CREB, p-DARPP and c-fos. The results show an enhanced intracellular response when caffeine is combined with MDMA but not with either agent alone suggestive of synergistic intracellular actions convergent on a dopamine D₁ receptor signalling pathway. A dopamine-related synergy associated with the combined administration of caffeine and MDMA may have important use and safety implications for recreational drug users.

Abstract:  This study investigated the potential antinociceptive efficacy of a novel synthetic curcuminoid analogue, 2,6-bis-(4-hydroxy-3-methoxybenzylidene)cyclohexanone (BHMC), using chemical- and thermal-induced nociception test models in mice. BHMC (0.03, 0.1, 0.3 and 1.0 mg/kg) administered via intraperitoneal route (i.p.) produced significant dose-related inhibition in the acetic acid-induced abdominal constriction test in mice with an ID₅₀ of 0.15 (0.13–0.18) mg/kg. It was also demonstrated that BHMC produced significant inhibition in both neurogenic (first phase) and inflammatory phases (second phase) of the formalin-induced paw licking test with an ID₅₀ of 0.35 (0.27–0.46) mg/kg and 0.07 (0.06–0.08) mg/kg, respectively. Similarly, BHMC also exerted significant increase in the response latency period in the hot-plate test. Moreover, the antinociceptive effect of the BHMC in the formalin-induced paw licking test and the hot-plate test was antagonized by pre-treatment with the non-selective opioid receptor antagonist, naloxone. Together, these results indicate that the compound acts both centrally and peripherally. In addition, administration of BHMC exhibited significant inhibition of the neurogenic nociception induced by intraplantar injections of glutamate and capsaicin with ID₅₀ of 0.66 (0.41–1.07) mg/kg and 0.42 (0.38–0.51) mg/kg, respectively. Finally, it was also shown that BHMC-induced antinociception was devoid of toxic effects and its antinociceptive effect was associated with neither muscle relaxant nor sedative action. In conclusion, BHMC at all doses investigated did not cause any toxic and sedative effects and produced pronounced central and peripheral antinociceptive activities. The central antinociceptive activity of BHMC was possibly mediated through activation of the opioid system as well as inhibition of the glutamatergic system and TRPV1 receptors, while the peripheral antinociceptive activity was perhaps mediated through inhibition of various inflammatory mediators.

Abstract:  Transient receptor potential vanilloid-1 (TRPV1) has been implicated as a mediator of itch in allergic rhinitis. To address this possibility, we synthesized a TRPV1 blocker (SB-705498) for nasal administration in patients with seasonal allergic rhinitis. The pharmacological activity of SB-705498 was confirmed on human TRPV1-expressing HEK293 cells, using fluorometric calcium imaging, and in patients with allergic rhinitis subjected to nasal capsaicin challenges. The effect of SB-705498 was studied in patients with seasonal allergic rhinitis subjected to daily allergen challenges for 7 days, using a double-blind, placebo-controlled, randomized and cross-over design. SB-705498 was delivered by nasal lavage 2 min. before each allergen challenge. Primary end-point was total nasal symptom score on days 5–7. Nasal peak inspiratory flow (nPIF) and eosinophil cationic protein (ECP) content in nasal lavages were also monitored. Daily topical applications of SB-705498 at a concentration that inhibited capsaicin-induced nasal symptoms had no effect on total symptom score, nPIF and ECP levels in allergen-challenged patients with seasonal allergic rhinitis. The individual symptoms, nasal itch or sneezes, were also not affected. These findings may indicate that TRPV1 is not a key mediator of the symptoms in allergic rhinitis. However, additional studies, using drug formulations with a prolonged duration of action, should be conducted before TRPV1 is ruled out as a drug target in allergic rhinitis.

Abstract:  Radial artery frequently develops spasm and requires vasodilator therapy during coronary artery bypass graft surgery (CABG). Levosimendan was recently shown to oppose 5-hydroxytryptamine-induced contraction of radial artery (RA) grafts. The aim of the present study was to explore whether levosimendan retains its vasodilatory capacity following in vitro pre-incubation of RA segments with the inodilator. A possible cumulative effect of the drug in human platelets was also studied. Human isolated RA segments were pre-incubated in 0.16 μmol/L levosimendan containing solution or in 0.9% NaCl, Bretschneider, 5% albumin and a 5% human serum protein solution (Biseko) as controls for 45 min. Contractions were induced by three consecutive administrations of 5-hydroxytryptamine (0.31 μM) 45, 90 and 120 min. after exchanging the pre-incubation solutions with Krebs-Henseleit solution, uniformly. Receptor-independent contractions (KCl, 80 mmol/L), endothelium-dependent (acetylcholine, 1 μmol/L) and independent relaxations (papaverine, 100 μmol/L) were also investigated. Washed human platelets were pre-incubated with levosimendan (0.06 μmol/L) for 2 or 15 min. and aggregated with thrombin (0.1 IU/mL). Contractions of RA grafts induced by 5-hydroxytryptamine were significantly smaller 45 min. and 90 min. after the replacement of levosimendan with Krebs-Henseleit solution. Biseko solution also decreased the contraction of the graft at 45 min. Contractions did not change in time following the pre-incubations of radial arteries with 0.9% NaCl, Bretschneider and 5% albumin solutions. The grafts remained intact as assessed by their maximum contractions and endothelium-dependent and endothelium-independent relaxations at the end of the investigations. Platelets revealed larger anti-aggregatory effect to levosimendan following the enhancement of the incubation time. Results indicate that the antispasmodic and anti-aggregatory effects of levosimendan cumulate in the vascular tissue and in platelets. The storage of RA with the inodilator before implantation may help to prevent the intraoperative spasm of the graft and also thrombotic occlusion during CABG surgery.

Abstract:  The aim of this study was to investigate whether treatment with a ginger (Zingiber officinale) extract of high-fat diet (HFD)-fed rats suppresses Nuclear factor-kappa B (NF-κB)-driven hepatic inflammation and to subsequently explore the molecular mechanisms in vitro. Adult male Sprague-Dawley rats were treated with an ethanolic extract of Zingiber officinale (400 mg/kg) along with a HFD for 6 weeks. Hepatic cytokine mRNA levels, cytokine protein levels and NF-κB activation were measured by real-time PCR, Western blot and an NF-κB nuclear translocation assay, respectively. In vitro, cell culture studies were carried out in human hepatocyte (HuH-7) cells by treatment with Zingiber officinale (100 μg/mL) for 24 hr prior to interleukin-1β (IL-1β, 8 ng/mL)-induced inflammation. We showed that Zingiber officinale treatment decreased cytokine gene TNFα and IL-6 expression in HFD-fed rats, which was associated with suppression of NF-κB activation. In vitro, Zingiber officinale treatment decreased NF-κB-target inflammatory gene expression of IL-6, IL-8 and serum amyloid A1 (SAA1), while it suppressed NF-κB activity, IκBα degradation and IκB kinase (IKK) activity. In conclusion, Zingiber officinale suppressed markers of hepatic inflammation in HFD-fed rats, as demonstrated by decreased hepatic cytokine gene expression and decreased NF-κB activation. The study demonstrates that the anti-inflammatory effect of Zingiber officinale occurs at least in part through the NF-κB signalling pathway.

Abstract:  As a cancer chemotherapeutic agent, paclitaxel (Taxol^®) causes dose-related peripheral neuropathy in human beings. The mechanisms underlying this toxicity are currently unknown, and there are no validated treatments for its prevention or control. To assess whether lithium as a pre-treatment and at subtherapeutic dose could prevent the peripheral neuropathy produced by it, rats were treated with paclitaxel (2 mg/kg i.p. every other day for a total of 16 times) and/or lithium chloride (300 mg/l) via water supply. General toxicity and body-weight were measured regularly during the experiment. To evaluate the sensory and motor neuropathy hot-plate, open-field test and nerve conduction velocity were used. In rats treated with only paclitaxel, there was behavioural, electrophysiological and histological evidence of a mixed sensorimotor neuropathy after 16 injections. Lithium robustly reduced the rate of mortality and general toxicity. Paclitaxel-induced sensorimotor neuropathy was significantly improved as indicated by changes in hotplate latency, total distance moved and a significant increase in sciatic, sural and tail sensory or motor nerve conduction velocity. The same results were observed in histopathological examinations; however, dorsal root ganglion neurons did not significantly change in the paclitaxel-treated groups. These results suggest that lithium, at subtherapeutic doses, can prevent both motor and sensory components of paclitaxel neuropathy in rats. Thus, lithium at these doses, as an inexpensive and relatively safe salt, may be useful clinically in preventing the neuropathy induced by paclitaxel treatment.

Abstract:  UDP-glucuronosyltransferases (UGTs) are conjugation enzymes, which are regulated in a tissue-specific manner by endogenous and environmental factors. In this study, we focused on UGT1A isoforms broadly expressed in hepatic and extrahepatic tissues and examined the expression and inducibility of UGT1As (UGT1A1 and UGT1A3–1A10) in MCF-7 cells (human breast carcinoma cell line). Reverse transcription polymerase chain reaction (RT-PCR) analysis demonstrated that UGT1A1, UGT1A6 and UGT1A9 mRNAs as well as the mRNAs of transcriptional regulators (AhR, aryl hydrocarbon receptor; Arnt, AhR nuclear translocator; ERα, oestrogen receptor α; ERβ, oestrogen receptor β; and GR, glucocorticoid receptor) are expressed in MCF-7 cells. UGT1A6 mRNA level in MCF-7 cells was significantly increased to 1.9 times by β-naphthoflavone (BNF), whereas UGT1A1 and UGT1A9 mRNA levels were not affected by BNF. There were no significant changes in the mRNAs of UGT1A1, UGT1A6 and UGT1A9 in MCF-7 cells by treatment with phenobarbital (PB) and dexamethasone (DEX) in MCF-7 cells. The kinetics of 7-ethyl-10-hydroxycamptothecin (SN-38), 5-hydroxytryptamine (5-HT) and 4-methylumbelliferone (4-MU) glucuronidation by microsomes from control and BNF-treated MCF-7 cells fitted with the Michaelis–Menten model, and the V_max and CL_int values were significantly increased to 7.5–8.5 times and 5.9–10.4 times by BNF treatment, respectively. These findings suggest that BNF induces UGT1A6 in MCF-7 cells and that the increase may be mediated by AhR but not pregnane X receptor (PXR)/constitutive androstane receptor (CAR). The information gained in this study should help predict and assess the toxicity of environmental chemicals.

Abstract:  Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme of bile acid production in human beings, and organic anion-transporting polypeptide 1B1 (OATP1B1) may influence bile acid hepatic uptake and cholesterol and bile acid synthesis rate. Our purpose was to investigate the effects of gender and CYP7A1 and SLCO1B1 polymorphisms on the fasting plasma concentrations of bile acids, bile acid synthesis marker and total cholesterol in a Finnish population. Fasting plasma concentrations of 16 endogenous bile acids, their synthesis marker (7α-hydroxy-4-cholesten-3-one) and total cholesterol were measured in 243 samples from 143 healthy volunteers. The volunteers were genotyped for 6 haplotype-tagging single-nucleotide polymorphisms (SNPs) of CYP7A1 and two functionally relevant SNPs in SLCO1B1. The mean plasma concentrations of chenodeoxycholic acid, glycochenodeoxycholic acid, ursodeoxycholic acid and glycoursodeoxycholic acid were 61–111% higher in men than in women (P ≤ 0.001). Accordingly, the mean concentration of total bile acids was 51% higher in men than in women (P = 0.001). The CYP7A1 rs8192879 and rs1023652 SNPs were associated with deoxycholic acid and hyodeoxycholic acid concentrations, respectively, but the associations were not significant after correction for multiple testing. None of the six CYP7A1 SNPs was associated with the plasma concentrations of cholesterol or 7α-hydroxy-4-cholesten-3-one. SLCO1B1 genotype was associated with total plasma cholesterol concentration only, but the association was not significant after correction for multiple testing. In general, the gender contributes substantially more to variation in fasting plasma bile acid concentrations than CYP7A1 or SLCO1B1 polymorphism do. Common genetic variability in CYP7A1 is unlikely to play a significant role in cholesterol metabolism and bile acid homeostasis under normal physiological conditions.

Abstract:  The objective of this study is to evaluate the effects of bupropion as an add-on therapy to selective serotonin reuptake inhibitor (SSRI) on patients of major depressive disorder with partial response. This prospective, randomized, controlled and single-blind study was conducted in sixty patients suffering from major depressive disorder as per Diagnostic and Statistical Manual (DSM)-IV TR criteria, who were having Hamilton depression rating scale (HDRS) score ≥16 after 4 weeks of treatment with SSRIs. Group A received SSRI plus placebo and group B received SSRI plus bupropion. Evaluation was performed based on changes in HDRS score, Montgomery and Asberg depression rating scale (MADRS), Amritsar depressive inventory (ADI) and spontaneously reported adverse effects. There was a significant decrease in the HDRS, MADRS and ADI scores as compared to baseline in both groups. However, the mean decrease in depression score was more in group B than in group A. The percentage decrease of remitters was also significantly more in group B (60% as per HDRS score and 63% as per MADRS score), as compared to group A (24% as per HDRS score and 27% as per MADRS score) (p < 0.05), at the end of treatment. In conclusion, bupropion add-on can act as augmenting agent in patients of depression with partial response to SSRIs.

Abstract:  Abnormal feeding behaviours have long been linked to disruptions in brain dopaminergic activity. Dopamine is metabolized, amongst others, by catechol-O-methyltransferase (COMT). Normally, COMT only plays a subordinate role in dopamine metabolism. However, changes in COMT activity, especially in the prefrontal cortex, become more important during events that evoke dopamine release. The current study investigated the effect of acute COMT inhibition on feeding in Wistar rats and C57BL/6 mice using a selective, brain penetrating COMT inhibitor (OR-1139). Furthermore, the effect of a long-term lack of COMT on feeding behaviour was studied in COMT-deficient (COMT −/−) mice. Apart from following the gross feeding behaviour of fasted rats and mice, the first 4 hr of re-feeding were recorded with a video camera to allow a more detailed analysis of feeding microstructure. In the acute study, rats and mice received a single injection of OR-1139 (3, 10 or 30 mg/kg), just before the experiment. We found that rats and mice receiving OR-1139 had fewer very short meals but more long meals than the controls. Treated mice even ate more frequently than the controls, but other feeding parameters remained unchanged. Conversely, COMT −/− mice displayed an increased latency to initiate the first meal and spent less total time eating than wild-type mice. In conclusion, although decreased/lack of COMT activity did not robustly alter feeding behaviour of female rodents, we observed some alterations in the microstructure of feeding. However, these minor changes were highly dependent on the extent and fashion in which COMT was manipulated.

Abstract:  Recent investigations have postulated a link between oxidative stress and endoplasmic reticulum (ER) dysfunction in cerebral ischaemic/reperfusion (I/R) injury. Diabetes is common amongst elderly patients with stroke and has been postulated to aggravate brain I/R damage by triggering oxidative as well as ER stress. We investigated whether treatment with edaravone (1–10 mg/kg), a potent free radical scavenger protects against cerebral I/R injury in rats associated with comorbid type 2 diabetes. Diabetic rats exposed to 2-hr middle cerebral artery occlusion (MCAO) and 22 hr of reperfusion significantly had increased infarct, oedema volume and functional neurological deficits as compared to sham-operated rats. Also, the massive DNA fragmentation accompanied by significant increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) positive cells was noticed in the ipsilateral penumbral brain region of diabetic I/R rats. The effects of I/R injury were associated with significant up-regulation of 78 kDa-glucose-regulated protein (GRP78), CCAAT/enhancer binding protein homologous protein or growth arrest DNA damage-inducible gene 153 (CHOP/GADD153) and activation of caspase-12, markers of ER stress/apoptosis. Treatment with edaravone (3 and 10 mg/kg) significantly diminished the cerebral infarct, oedema volume and improved functional recovery of neurological deficits. In addition, edaravone treatment ameliorated the DNA fragmentation concomitantly with a significant decrease in induction of GRP78, CHOP/GADD153 immunoreactivity/expression and activation of caspase-12 in the ischaemic brain hemispheres. Overall, the present data indicate that edaravone offers good neuroprotection against diabetic stroke by interrupting the ER stress-mediated apoptotic pathways involving CHOP/GADD153 and caspase-12.

Abstract:  Acetaminophen has been used as an analgesic for more than a hundred years, but its mechanism of action has remained elusive. Recently, it has been shown that acetaminophen produces analgesia by the activation of the brain endocannabinoid receptor CB1 through its para-aminophenol (p-aminophenol) metabolite. The objective of this study was to determine whether p-aminophenol could be toxic for in vitro developing mouse cortical neurons as a first step in establishing a link between acetaminophen use and neuronal apoptosis. We exposed developing mouse cortical neurons to various concentrations of drugs for 24 hr in vitro. Acetaminophen itself was not toxic to developing mouse cortical neurons at therapeutic concentrations of 10–250 μg/ml. However, concentrations of p-aminophenol from 1 to 100 μg/ml produced significant (p < 0.05) loss of mouse cortical neuron viability at 24 hr compared to the controls. The naturally occurring endocannabinoid anandamide also caused similar 24-hr loss of cell viability in developing mouse cortical neurons at concentrations from 1 to 100 μg/ml, which indicates the mechanism of cell death could be through the cannabinoid receptors. The results of our experiments have shown a detrimental effect of the acetaminophen metabolite p-aminophenol on in vitro developing cortical neuron viability which could act through CB1 receptors of the endocannabinoid system. These results could be especially important in recommending an analgesic for children or individuals with traumatic brain injury who have developing cortical neurons.

Abstract:  The effects of the K_v7 channel modulators retigabine (opener) and XE991 (blocker) on rat bladder function were investigated ex vivo and in vivo to assess the potential of K_v7 openers for the treatment of overactive bladder. In organ bath studies, capsaicin-stimulated rat urinary bladder rings were exposed to retigabine and XE991 and the effect on tension and amplitude was evaluated. In anaesthetized rats, retigabine (0.01–1 mg/kg, i.v.) effects on bladder function, in which overactivity was induced by continuous infusion of 0.5% acetic acid, were assessed. The effect of retigabine (10 mg/kg, p.o.) on cystometric parameters was also measured in conscious rats with capsaicin-induced irritated bladders. Localization of K_v7 subunits within bladder tissue was analysed by RT-qPCR and western blotting. In organ bath studies, retigabine robustly reduced capsaicin-induced contractility of bladder rings and this effect was blocked by XE991 confirming the specificity of action via K_v7 channels. In anaesthetized rats with acetic acid–irritated bladders, retigabine markedly increased bladder capacity with no concomitant reduction in blood pressure. Retigabine also reduced bladder pressure and delayed voiding in conscious rats with capsaicin-irritated bladders. K_v7.1, K_v7.4 and K_v7.5 subunit mRNA transcripts were detected in rat bladder. Western blot analysis confirmed that K_v7.4 subunit protein was expressed in rat bladder. These results suggest that retigabine and other K_v7 channel positive modulators may have beneficial effects on bladder overactivity partly via activation of K_v7 channels expressed in bladder tissue.

Abstract:  Recombinant human parathyroid hormone [rhPTH(1–84)] represents a new class of anabolic agents for the treatment of osteoporosis. The present study was designed to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of rhPTH(1–84) after single- and multiple-dose subcutaneous administration in healthy Chinese volunteers. Six cohorts of 32 volunteers received a single dose of rhPTH(1–84) at 0.5–5.0 μg/kg, and two cohorts of 12 volunteers received 2.0 and 3.0 μg/kg of rhPTH(1–84) once daily for 7 consecutive days to assess its safety and tolerability. The results indicated that rhPTH(1–84) appeared to be safe and well tolerated. Additionally, pharmacokinetics of rhPTH(1–84) and its active N-terminal fragment rhPTH(1–34) were investigated after administration of single 1.0, 2.0 and 4.0 μg/kg doses of rhPTH(1–84) in 30 other volunteers and after multiple doses of 2.0 μg/kg once daily for 7 consecutive days. The pharmacokinetic parameters for rhPTH(1–84) and rhPTH(1–34) after subcutaneous administration of a single dose of 1.0, 2.0 and 4.0 μg/kg were as follows: C_max = (110.54 ± 59.18), (149.70 ± 50.61) and (372.52 ± 94.96) pg/mL; (53.93 ± 6.27), (61.12 ± 11.28) and (89.04 ± 7.08) pg/mL, respectively. AUC_0–10 = (268.87 ± 47.72), (538.93 ± 146.89) and (1364.11 ± 176.82) pg hr/mL; (197.20 ± 50.78), (207.15 ± 72.08) and (344.05 ± 77.06) pg hr/mL, respectively. t_1/2 = (2.34 ± 1.93), (2.58 ± 1.18) and (2.74 ± 1.31) hr; (3.37 ± 1.82), (4.39 ± 3.79), and (3.99 ± 1.85) hr, respectively. Plasma C_max and AUC values of rhPTH(1–84) and rhPTH(1–34) were found to be dose proportional. The pharmacokinetic parameters for rhPTH(1–84) and rhPTH(1–34) after administration of multiple doses of 2.0 μg/kg were as follows: C_{ss_max} = (164.96 ± 52.61) and (75.05 ± 7.31) pg/mL; C_{ss_min} = (6.99 ± 7.73) and (2.05 ± 2.82) pg/mL; AUC_ss = (567.26 ± 118.41) and (306.02 ± 77.55) pg hr/mL; t_1/2 = (1.81 ± 0.89) and (2.27 ± 1.11) hr; DF = (6.93 ± 2.64) and (6.00 ± 1.37), respectively. After multiple doses, the pharmacokinetic parameters for rhPTH(1–84) were consistent with those after single dose. However, the mean C_max and AUC_0–10 of rhPTH(1–34) after multiple dosing were significantly higher than the corresponding values obtained after single-dose administration. Serum total calcium and phosphate concentrations increased and decreased significantly at 4 hr post-dosing, respectively.

Abstract:  L-dopa is still the gold standard in the symptomatic treatment of Parkinson’s disease (PD), and thus, it is the most commonly used drug in the non-clinical assessment of new drug therapies to PD, including those intended to improve the effect of L-dopa. In unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rat model of PD, the results from L-dopa-induced rotation tests are often unpredictable. While repeated administration of L-dopa improves the rotation, the exact mechanisms underlying the extensive variability in rotation responses between rats and testing times are unclear. In the present study, we aimed to assess whether the route of administration (oral or intraperitoneal) or the form of L-dopa (base or methyl ester) is associated with the extensive variation in rotation responses to L-dopa in 6-OHDA rats. We also wanted to examine the dependence between L-dopa (base or methyl ester)-induced rotational behaviour and the extent of dopamine and dopa decarboxylase enzyme loss in the lesioned striatum. It was found that variation in plasma levels of L-dopa as well as the administration route explains a part of the variability in rotation. There were small but significant differences in striatal dopamine depletion (indicative of degree of lesion) between the groups, which may partially account for the various patterns in L-dopa-induced rotational behaviour. While apomorphine-induced rotation test is a useful tool for primary screening of the success of 6-OHDA lesion, it is not useful at predicting the rotational performance of 6-OHDA rats to L-dopa. The exact mechanisms and causes of the variability in the rotation responses to L-dopa in 6-OHDA rats still remain to be clarified.

Abstract:  The monoterpene (−)-borneol is present in essential oils of several medicinal plants. The aim of this study was to evaluate (−)-borneol effects on rat thoracic aorta artery rings. The cumulative addition of (−)-borneol (10⁻⁹–3 × 10⁻⁴ M) on a phenylephrine-induced pre-contraction (10⁻⁶ M) promoted a vasorelaxant effect in a concentration-dependent manner and independent of vascular endothelium. A similar effect was obtained on KCl-induced pre-contractions (80 mM). (−)-Borneol (10⁻⁵–3 × 10⁻⁴ M) inhibited contractions induced by cumulative addition of CaCl₂ (10⁻⁶–3 × 10⁻² M) in depolarizing medium without Ca²⁺ in a concentration-dependent manner. On S-(−) Bay K 8644-induced pre-contractions (10⁻⁷ M), (−)-borneol did not induce significant changes compared with KCl-induced pre-contractions. In a Ca²⁺-free medium, (−)-borneol (10⁻⁵, 10⁻⁴ or 10⁻³ M) interfered in calcium mobilization from phenylephrine (10⁻⁶ M)- or caffeine (20 mM)-sensitive intracellular stores. The involvement of K⁺ channels was evaluated by tetraethylammonium (3 mM), 4-aminopyridine (1 mM) and glibenclamide (10⁻⁵ M) pre-treatment, and (−)-borneol-induced vasorelaxation was markedly attenuated. Thus, this vasorelaxant effect can probably be attributed to calcium influx blockade through voltage-operated calcium channels (Ca_VL), calcium mobilization from intracellular stores and potassium channels activation.

Abstract:  Features of airway remodelling have been described using tissue obtained from fatal cases of asthma and bronchial biopsies from mildly, moderately and severely asthmatic patients. Epithelial detachment and smooth muscle mass enhancement are common features of asthmatic bronchial tissue. This study is the first to investigate the inhibitory effect of nonylphenol (NP) on human bronchial epithelial cell lines BEAS-2B and HBE135-E6E7 (HBE). The results show that NP inhibits bronchial epithelial proliferation via the Fas/Fas ligand apoptotic system. We also treated BEAS-2B and HBE with NP and harvested the condition medium (CM), which was then added to bronchial smooth muscle cells (BSMC). Cultures of BSMC with NP-BEAS-2B-CM and NP-HBE-CM increased BSMC proliferation and migration. Exposure of BEAS-2B and HBE to NP caused epithelial cells to produce inflammatory cytokines IL-6 and IL-8, which subsequently induced BSMC proliferation and migration. Depleting both IL-6 and IL-8 completely reversed the effect of NP-BEAS-2B-CM- and NP-HBE-CM-mediated BSMC proliferation and migration, suggesting that this effect is a synergistic influence of IL-6 and IL-8. This study is the first to demonstrate that NP not only induces bronchial epithelial apoptosis via the Fas-mediated pathway but also stimulates the bronchial epithelium to secrete IL-6 and IL-8, which cause bronchial smooth muscle proliferation and migration – major features in asthma remodelling.

Abstract:  The objective of this study was to evaluate the ability of schisandrin A (SchA) to inhibit the P450 enzyme CYP3A in vivo. Male Sprague–Dawley rats were intragastrically administered with varied doses of SchA (8 mg/kg or 16 mg/kg or 32 mg/kg) or 75 mg/kg ketoconazole for three consecutive days. Ketoconazole, a chemical inhibitor of CYP3A, was used as positive control. Subsequently, changes in hepatic microsome CYP3A activity and the pharmacokinetic profiles of midazolam (MDZ), a specific CYP3A substrate, were studied as indicators of rat hepatic microsomal activity of CYP3A. Differences in the plasma concentrations of MDZ and its related metabolites and the hepatic microsome concentrations of 1′-hydroxymidazolam were analysed by high-performance liquid chromatography. The current results provide direct and explicit evidence that SchA produced concentration-dependent inhibition of MDZ metabolite formation in rat liver microsomes (p < 0.01 or p < 0.001). Regular SchA consumption also caused concentration-dependent increase in C_max and area under the concentration–time curve (AUC_0–t and AUC_0–∞) of peroral MDZ (p < 0.05 or p < 0.01) compared to vehicle-treated rats, whereas those of its metabolites (1′-hydroxymidazolam) were reduced (p < 0.05 or p < 0.01). Analysis of the data suggests that changes in the pharmacokinetic profiles of peroral MDZ in the rat model were contributed mainly to SchA inhibition of CYP3A activity. These results suggest that SchA, as an inhibitor of CYP3A, possesses a clinically beneficial property of altering the disposition of drugs metabolized by CYP3A.

Abstract:  Vitamin K antagonists (VKA) are highly effective anticoagulants but their use is hampered by multiple interactions with food and medicine and a narrow therapeutic range. The large variation in dose requirements has led to the development of several dosing algorithms based on pharmacogenetic and clinical variables. In contrast, evidence about the influence of behavioural (i.e. diet and exercise) and socio-psychological factors is sparse. To investigate the impact of pharmacogenetic, clinical, behavioural and socio-psychological factors on maintenance dose of VKA. In a cross-sectional study, we interviewed 250 consecutive patients from an anticoagulant clinic and subsequently measured pharmacogenetic and anthropometric variables. Statistical analyses were carried out using linear regression and multivariable models with visualization features. In both types of analyses, the strongest determinants of VKA dose were polymorphisms in the VKORC1 and CYP2C9 genes and age. Half of the variation in VKA dose could be explained by a linear regression model including four variables, while a multivariable model with 20 pharmacogenetic and clinical variables explained 60%. A multivariable model including 94 predictor variables was not notably better regarding predictive performance, but visualization of this model offered information about the correlation structure between predictor variables. The strongest determinants of VKA dose are well-known pharmacogenetic variables and age. The variables describing health-related behaviour and socio-psychological factors are strongly inter-correlated and not useful in dosing algorithms.

Abstract:  The standard treatment for ovarian cancer in advanced stages is post-surgery treatment with taxane–platin chemotherapy. Despite an initial high response rate, most patients eventually relapse. The dose-limiting toxicities of paclitaxel are neutropenia and neuropathy, but the inter-individual variability is large. The aim of this prospective study was to investigate the impact of genetic variants in key drug metabolizing/transporter genes on toxicity and compliance. CYP2C8*3 and three ABCB1 polymorphisms were chosen for primary analysis, and a host of other candidate genes was explored in 92 prospectively recruited Scandinavian Caucasian women with primary ovarian cancer who were treated with paclitaxel and carboplatin. A single investigator assessed the clinical toxicity in 97% of the patients. Patients carrying variant alleles of ABCB1 C3435T experienced more pronounced neutrophil decrease (63%, 72% and 80% for 3435CC, CT and TT, respectively; p-value 0.03). A similar association was found for G2677T/A, p-value 0.02. For C1236T, there was a trend with p-value 0.06. No statistically significant correlations were found for paclitaxel compliance and sensory neuropathy in the primary analysis. Variants in the drug transporter ABCB1 gene are possibly associated with the neutrophil suppressing effect of paclitaxel in patients with ovarian cancer. This finding has implications for the understanding of bone marrow suppression and future tailored chemotherapy.