Lipoic acid blocks seizures induced by pilocarpine via increases in δ-aminolevulinic dehydratase and Na, K-ATPase activity in rat brain

In the present study we investigated the effects of lipoic acid (LA) on δ-aminolevulinic dehydratase (δ-ALA-D) and Na⁺, K⁺-ATPase activities in rat brain after seizures induction by pilocarpine. Wistar rats were treated with 0.9% saline (i.p., control group), lipoic acid (10 mg/kg, i.p., LA group), pilocarpine (400 mg/kg, i.p., pilocarpine group), or the combination of LA (10 mg/kg, i.p.) with pilocarpine (400 mg/kg, i.p.), 30 min before administration of LA (LA plus pilocarpine group). After the treatments all groups were observed for 1 h. The enzyme activities (δ-ALA-D and Na⁺, K⁺-ATPase) were measured using spectrophotometric methods, and the results were compared with that obtained from saline and pilocarpine-treated animals. Neuroprotective effects of LA against seizures were evaluated based on those enzyme activities. The pilocarpine group showed a reduction in δ-ALA-D and Na⁺, K⁺-ATPase activities after seizures. In turn, LA plus pilocarpine abolished the appearance of seizures and reversed the decreased in δ-ALA-D and Na⁺, K⁺-ATPase activities produced by seizures, when compared to the pilocarpine seizing group. The results from the present study demonstrate that preadministration of LA abolished seizure episodes induced by pilocarpine in rat, probably by increasing δ-ALA-D and Na⁺, K⁺-ATPase activities in rat brain during seizures.     

Lead concentration in plasma as a biomarker of exposure and risk,and modification of toxicity by δ-aminolevulinic acid dehydratase gene polymorphism

Blood lead concentration (B-Pb), the main biomarker of lead exposure and risk, is curvi-linearily related to exposure. We assessed plasma lead (P-Pb) as a marker for both lead exposure and toxic effects. We examined claims that δ-aminolevulinic acid dehydratase genotype (ALAD) can modify lead toxicity. In 290 lead-exposed and 91 unexposed Chinese workers, we determined P-Pb, B-Pb, urinary lead (U-Pb), ALAD polymorphism (rs1800435, ALAD1/2; TaqMan assay), and also toxic effects on heme synthesis (blood zinc protoporphyrin and hemoglobin, urinary δ-aminolevulic acid), on the kidneys (urinary albumin, β₂-microglobulin and N-acetyl-β-d-glucosaminidase) and on the peripheral nervous system (sensory and motor conduction velocities).     

Effect of stanozolol on δ-aminolaevulinic acid synthase and hepatic monooxygenase activity in man and rat

Summary Stanozolol is an anabolic steroid which is used in the treatment of aplastic anaemia and has been recently advocated for the prophylaxis of vascular thrombosis. Similar steroid substances stimulate the activity of -aminolaevulinic acid synthase (ALA S), the rate limiting enzyme of haem biosynthesis, in rat hepatocytes and chick embryo liver cell cultures and activate acute hepatic porphyria. In the present study stanozolol (10 mg daily for 14 days) has been shown to increase significantly leucocyte ALA S activity in 9 healthy male subjects. There was a concomitant rise in urinary ALA and total porphyrin excretion but no change in antipyrine kinetics or urinary 6 B hydroxycortisol excretion. In a complementary study in male Sprague Dawley rats, stanozolol administered intraperitoneally, produced a dose-dependent increase in hepatic ALA S activity without changing hepatic cytochrome P 450 content. Stanozolol has been clearly shown to elevate ALA S activity, probably directly, and, thereby, porphyrin production without affecting hepatic monooxygenase activity. This porphyrinogenic effect may be relevant to the successful treatment of aplastic anaemia with anabolic steroids. Leucocyte ALA S activity may provide a human system for the study of drug porphyrinogenicity in vivo.     

Renal reabsorption and secretion of σ-aminolevulinic acid in the ratA

Renal mechanismsfor handling σ-aminolevulinic acid (ALA) were investigated in control and lead-exposed rats infused with ALA at a range of rates which produced plasma ALA as high as 2380 ng/mL. The presence of both reabsorption and secretion mechanisms was demonstrated. Reabsorption is the more efficient of the two mechanisms at low plasma ALA concentrations, and masks secretion in this range. Net secretion is observed at very high plasma ALA concentrations. Exposure to up to 2000 μg lead/mL of drinking water for 12 to 18 months had no measurable effect on net ALA excretion at a given plasma ALA level.     

Involvement of δ-opioid receptors in the effects of morphine on locomotor activity and the mesolimbic dopaminergic system in mice

Naltrindole (NTI) and naltriben (NTB), a benzofuran derivative of NTI, were recently synthesized as highly selective -opioid receptor antagonists. Both NTI and NTB failed to suppress the antinociceptive effect induced by morphine. In contrast, both NTI and NTB significantly suppressed the morphine-induced hyperlocomotion and increase in turnover of dopamine (DA) in the mouse limbic forebrain. These results suggest that -opioid receptors play, at least in part, a role in the morphine-induced hyperlocomotion and excitation of mesolimbic DA systems, but not antinociception.     

Relationship between lead concentration in blood and the activities of erythrocyte pyrimidine 5′-nucleotidase and delta-aminolevulinate dehydratase in the mice exposed to lead

The relationship between the activities of both pyrimidine 5-nucleotidase (Py5N) and delta-aminolevulinate dehydratase (ALA-D) in erythrocytes and the concentration of lead in blood was investigated in the mice which were given ad libitum a drinking water containing lead of 10 to 500 ppm, for 30 days.From these results, it was demonstrated that the erythrocyte PySN activity is inhibited by lead and its activity level is negatively correlated with the concentration of lead in blood (r=–0.78). In addition, it was suggested that the erythrocyte Py5N activity is a better indicator in the exposure to relatively high lead concentration while the ALA-D is a more sensitive indicator for evaluating the lead exposure of low and moderate levels.     

The effects of ethanol,estrogen, and hexachlorobenzene on the activities of hepatic δ-aminolevulinate synthetase, δ-aminolevulinate dehydratase,and uroporphyrinogen decarboxylase in male rats

To determine if clinically observed disorders in heme biosynthetic enzymes, known as sporadic porphyria cutanea tarda (PCT), could be reproduced in experimental animals, male Fischer rats were treated with ethanol, estrogen and hexachlorobenzene (HCB). A series of heme biosynthetic enzymes were assayed. In the rats given free access to 8% ethanol-drinking water for 15 weeks, -aminolevulinate (ALA) dehydratase was significantly reduced in erythrocytes. In the liver, ALA synthetase and uroporphyrinogen (UROgen) decarboxylase activities remained unchanged. In bone marrow cells, these activities did not change markedly. In the rats treated with estrogen (1 mg estrioltripropionate /rat/week, IM), no body weight gain was observed during the treatment for 15 weeks and urinary ALA excretion increased to 1.7 fold over normal level. In the liver, a significant increase was observed in the activity of ALA dehydratase, but other enzymes remained within the normal level. In bone marrow cells and erythrocytes, ALA dehydratase was also increased. ALA synthetase increased only in bone marrow cells to 2.1 times higher than the control level. In rats fed 0.3% HCB-diet for 8 weeks, urinary excretion of ALA, coproporphyrin and uroporphyrin increased to 2.4, 3.3 and 3.8 times higher than the controls, respectively. In the liver, an increase was observed in ALA synthetase, while a decrease was observed in ALA dehydratase and UROgen decarboxylase. In bone marrow cells and erythrocytes, ALA dehydratase was reduced and activities of other enzymes did not show any changes.These results indicate that alcohol, estrogen and HCB do not produce phenomena similar to those observed clinically in PCT.     

Effect of lead exposure on some biological indices related to porphyrin metabolism and the activity of erythrocyte pyrimidine 5′-nucleotidase in the mice

The urinary excretion of -aminolevulinic acid (ALA) and coproporphyrin (CP) isomers (I and III) was investigated in mice exposed to lead in drinking water (200 and 500 ppm) for 14 or 30 days. Furthermore, the inhibition of pyrimidine 5-nucleotidase (P5N) and -aminolevulinic acid dehydratase (ALAD) was studied in the same mice. In the lead-exposed mice, urinary excretion of ALA was significantly elevated (12-times control), while that of CP remained unchanged. This discrepancy on the urinary excretion may be of interest. The P5N activity in both erythrocytes and bone marrow cells was significantly inhibited by exposure to lead. However, the degree of inhibition was greater in the erythrocytes (45% inhibition) than in the bone marrow cells (25% inhibition). The inhibition of ALAD was much higher in erythrocytes (90% inhibition) than in liver (20–40% inhibition). The increase in erythrocyte protoporphyrin and the decrease in both hemoglobin and hematocrit were not observed in mice exposed to lead under the above conditions.     

Effects of some GABA-mimetic drugs on the antinociceptive activity of morphine and β-endorphin in rats

Summary Intracerebroventricular administration of muscimol, a potent GABA-receptor agonist, counteracted the antinociceptive effect of morphine or -endorphin in rats as measured by the tail flick method. Muscimol's activity was reversed by bicuculline. Isoguvacine, another GABA agonist, as well as nipecotic acid and guvacine, two inhibitors of neuronal and glial uptake of GABA, also antagonized morphine's antinociceptive effect. A role of the central GABA-ergic system in mediating opiate antinociception is proposed.This study was supported by C.N.R. grant no. CT 77.01401.04     

Assessment of the impact of CYP3A polymorphisms on the formation of α-hydroxytamoxifen and N-desmethyltamoxifen in human liver microsomes

Tamoxifen, an antiestrogen used in the prevention and treatment of breast cancer, is extensively metabolized by cytochrome P450 enzymes. Its biotransformation to α-hydroxytamoxifen (α-OHT), which may be genotoxic, and to N-desmethyltamoxifen (N-DMT), which is partially hydroxylated to 4-hydroxy-N-DMT (endoxifen), a potent antiestrogen, is mediated by CYP3A enzymes. However, the potential contribution of CYP3A5 and the impact of its low-expression variants on the formation of these metabolites are not clear. Therefore, we assessed the contributions of CYP3A4 and CYP3A5 and examined the impact of CYP3A5 genotypes on the formation of α-OHT and N-DMT, by using recombinant CYP3A4 and CYP3A5 and human liver microsomes (HLM) genotyped for CYP3A5 variants. We observed that the catalytic efficiency [intrinsic clearance (CL(int))] for α-OHT formation with recombinant CYP3A4 was 5-fold higher than that with recombinant CYP3A5 (0.81 versus 0.16 nl · min?1 · pmol cytochrome P450?1). There was no significant difference in CL(int) values between the three CYP3A5-genotyped HLM (*1/*1, *1/*3, and *3/*3). For N-DMT formation, the CL(int) with recombinant CYP3A4 was only 1.7-fold higher, relative to that with recombinant CYP3A5. In addition, the CL(int) for N-DMT formation by HLM with CYP3A5*3/*3 alleles was approximately 3-fold lower than that for HLM expressing CYP3A5*1/*1. Regression analyses of tamoxifen metabolism with respect to testosterone 6β-hydroxylation facilitated assessment of CYP3A5 contributions to the formation of the two metabolites. The CYP3A5 contributions to α-OHT formation were negligible, whereas the contributions to N-DMT formation ranged from 51 to 61%. Our findings suggest that polymorphic CYP3A5 expression may affect the formation of N-DMT but not that of α-OHT.     

The effects of dopamine D1 and D2 receptor antagonists on the rewarding effects of δ1 and δ2 opioid receptor agonists in mice

The effects of the dopamin D₁ antagonist SCH23390 and the D₂ antagonist sulpiride on the rewarding effects of opioid receptor agonists were examined in mice. Both [d-Pen², Pen⁵]enkephalin (DPDPE, 1–15 nmol, ICV), a selective ₁ opioid receptor agonist, and [d-Ala²]deltorphin II (DELT, 0.5–5 nmol, ICV), a selective ₂ opioid receptor agonist, produced a dose-dependent place preference in mice. The DPDPE (15 nmol, ICV)-induced place preference was abolished by BNTX (0.5 mg/kg, SC), a ₁ opioid receptor antagonist, but not by NTB (0.5 mg/kg, SC), a ₂ opioid receptor antagonist. In contrast, the DELT (5 nmol, ICV)-induced place preference was antagonized by NTB, but not BNTX. Pretreatment with SCH23390 (3 µg/kg, SC) abolished the DPDPE-induced place preference, but not affect the DELT-induced place preference. Moreover, pretreatment with sulpiride (40 mg/kg, SC) did not modify the place preference induced by DPDPE or DELT. In the present study, we found that the activation of both central ₁ and ₂ opioid receptors produced rewarding effects. Furthermore, these results suggest that the rewarding effects of ₁ opioid receptor agonist may be produced through activation of the central dopaminergic system, especially dopamine D₁ receptors, whereas the rewarding effects of ₂ opioid receptor agonists may be produced by some other mechanism(s).     

The inhibitory effects of cholalic acid and hyodeoxycholalic acid on the expression of TNFα and IL-1β after cerebral ischemia in rats

Previous studies have shown that Qing Kai Ling, a traditional Chinese medicine, was able to effectively prevent the inflammation from cerebral ischemia (Chen et al., 2002). The cholalic acid and hyodeoxycholalic acid (cholalic acid mixture) was major active components in Qing Kai Ling. To study the effects of cholalic acid mixture on the damage cascade of cerebral ischemia, rat model of focal cerebral ischemia was established by permanent occlusion of left middle cerebral artery. We found that the administration of cholalic acid mixture could reduce the ischemic infarct size after 24 h of ischemia, and cholalic acid mixture could be detected in cerebrospinal fluid after 2h of administration. We also found that the concentrations of tumor necrosis factor-alpha and interlukin-1beta in rat brain were significantly lower when compared to the untreated animals after 12 h and 24 h of ischemia. The concentrations of von Willebrand factor and neuron specific enolase in the plasma were remarkably decreased in cholalic acid mixture treated animals than in the untreated ones after 12h of ischemia. Our results suggested that cholalic acid mixture is able to decrease the expression of inflammation factors including tumor necrosis factor-alpha and interlukin-1beta after focal cerebral ischemia. These two authors contribute equally to the paper.     

Action of cannabidiol on the anxiety and other effects produced by δ9-THC in normal subjects

The object of the experiment was to verify whether cannabidiol (CBD) reduces the anxiety provoked by ⁹-TCH in normal volunteers, and whether this effect occurs by a general block of the action of ⁹-TCH or by a specific anxiolytic effect. Appropriate measurements and scales were utilized and the eight volunteers received, the following treatments in a double-blind procedure: 0.5 mg/kg ⁹-TCH, 1 mg/kg CBD, a mixture containing 0.5 mg/kg ⁹-TCH and 1 mg/kg CBD and placebo and diazepam (10 mg) as controls. Each volunteer received the treatments in a different sequence. It was verified that CBD blocks the anxiety provoked by ⁹-TCH, however this effect also extended to marihuanalike effects and to other subjective alterations induced by ⁹-TCH. This antagonism does not appear to be caused by a general block of ⁹-TCH effects, since no change was detected in the pulse-rate measurements. Several further effects were observed typical of CBD and of an opposite nature to those of ⁹-TCH.These results suggest that the effects of CBD, as opposed to those of ⁹-TCH, might be involved in the antagonism of effects between the two cannabinoids.With a fellowship from Capesfrom CNP_q     

Effects of An-Shen-Bu-Nao Syrup on the inducible nitric-oxide synthase activity and melatonin in brain of sleep deprivation rats

Objective： To study the effects of An-Shen-Bu-Nao Syrup （AS） on the inducible nitricoxide synthase （iNOS） activity and melatonin content in brain of sleep deprivation （SD） rats. Methods： Rats were divided into groups of control, SD model, SD＋AS （0.02ml/kg）, and SD＋ AS （0.06ml/kg） . The rats were fed with AS for 2 weeks and were subjected to sleep deprivation for 4 days, and then the iNOS activity in the front cortex was analyzed, and melatonin in the pineal gland was analyzed with HPLC. Results： Compared with the control rats, SD rats had the significantly elevated iNOS activity in the front cortex. Rats treated with AS （0. 06ml/kg） showed significantly decreased iNOS activity than SD rats.     

Mechanism-based pharmacokinetic–pharmacodynamic modeling of salvianolic acid A effects on plasma xanthine oxidase activity and uric acid levels in acute myocardial infarction rats

1.?Salvianolic acid A (SalA) was found to attenuate plasma uric acid (UA) concentration and xanthine oxidase (XO) activity in acute myocardial infraction (AMI) rats, which was characterized with developed mechanism-based pharmacokinetic–pharmacodynamic (PK-PD) model.

2.?AMI was induced in rats by coronary artery ligation. Surviving AMI rats received a single intravenous dose of 5?mg/kg of SalA and normal saline. The plasma SalA concentrations were determined by HPLC-MS/MS method. The plasma UA concentrations were determined by HPLC method and plasma XO activity were measured spectrophotometrically. An integrated mathematical model characterized the relationship between plasma UA and SalA.

3.?Pharmacokinetics was described using two-compartment model for SalA with linear metabolic process. In post-AMI rats, XO activity and UA concentrations were increased, while SalA dosing palliated this increase. These effects were well captured by using two series of transduction models, simulating the delay of inhibition on XO driven by SalA and UA elevation resulted from the multiple factors, respectively.

4.?The effect was well described by the developed PK-PD model, indicating that SalA can exert cardiovascular protective effects by decreasing elevated plasma UA levels induced by AMI.     

Anticancer activity and concurrent analysis of ursolic acid, β-sitosterol and lupeol in three different Hibiscus species (aerial parts) by validated HPTLC method

The genus Hibiscus contains about 275 species of flowering plants widely grown in the tropics and sub-tropics. The available literature revealed that several Hibiscus species exhibited excellent anticancer activity against several cancer cells like lung, breast, and liver. This motivated the authors to explore the anticancer property of other Hibiscus species (Hibiscus calyphyllus, H. deflersii and H. micranthus) along with development of a validated HPTLC method for the concurrent analysis of three anticancer biomarkers (ursolic acid, β-sitosterol and lupeol) in different Hibiscus species. The anticancer activity of various fractions (petroleum ether, toluene, dichloromethane, ethyl acetate and n-butanol) of all the Hibiscus species (aerial parts) were evaluated in vitro against HepG2 and MCF-7 cell lines using MTT assay. The HPTLC analysis was carried out using chloroform and methanol as mobile phase (97:3; v/v) on 20?×?10 cm glass-backed silica gel 60F254 plates and analyzed different phytoconstituents present in all fractions at λ?=?575?nm wavelength. Of the tested fractions of H. calyphyllus, H. deflersii and H. micranthus, HdP (H. deflersii petroleum ether fraction) exhibited the most potent cytotoxic effect on HepG2 and MCF-7 (IC50: 14.4 and 11.1?μg/mL, respectively) cell lines. Using the developed HPTLC method a compact and intense peak of ursolic acid, β-sitosterol and lupeol were obtained at R_f?=?0.22, 0.39 and 0.51, respectively. The LOD/LOQ (ng) for ursolic acid, β-sitosterol and lupeol were found as 42.30/128.20, 13.20/40.01 and 31.57/95.68, respectively in the linearity range 100–1200?ng/spot. The obtained result showed maximum presence of ursolic acid, β-sitosterol and lupeol (5.50, 11.85 and 7.47?μg/mg, respectively) in HdP which also supported its strong anticancer effect. Our data suggest that H. deflersii petroleum ether fraction (HdP) can be further subjected to the isolation of active cytotoxic phytoconstituents and establishment of their mechanism of action. The maiden developed HPTLC method for concurrent analysis of anticancer biomarkers may be further employed in the in process quality control of herbal formulation containing the said biomarkers.     

Effects of formulation and route of administration on the systemic availability of Ex-RAD®, a new radioprotectant, in preclinical species

ON 01210.Na (Ex‐RAD^®) is a novel small molecule under development by Onconova Therapeutics, Inc. as a radiation protection agent. The purpose of this investigation was to evaluate the effect of various formulation approaches on the systemic exposure of ON 01210.Na. In vitro experiments were used to characterize the plasma binding and metabolic stability of ON 01210.Na using hepatocytes from several animal species (mouse, rat, rabbit, dog, monkey and human). In vivo studies were performed in rats, rabbits, dogs and monkeys, and involved several routes of administration (intravenous, subcutaneous, oral). Plasma protein binding was high across species (>83%), and the rate of ON 01210.Na metabolism was highest in rat and mouse hepatocytes. After intravenous administration, ON 01210.Na demonstrated biphasic elimination from the plasma. Systemic exposure parameters (C_max, AUC) were dose‐proportional up to 100 mg/kg. Following subcutaneous dosing, ON 01210.Na showed relatively low bioavailability upon administration of the suspension formulation. Developing a solution formulation significantly increased the bioavailability of the drug. This solution formulation demonstrated significant oral bioavailability in rabbit (70%) and monkey (30%). The findings from these preclinical studies provide an overview of the systemic disposition of ON 01210.Na, aiding in the development of optimal formulations and routes of administration for pivotal animal efficacy and clinical safety studies. A solution formulation of ON 01210.Na for s.c. administration is being developed, in addition to an oral dosage form for potential use of the compound as a radioprotectant and a radiation‐mitigating agent in wider military and civilian populations. Copyright © 2011 John Wiley & Sons, Ltd.