Pharmacokinetics of the acetylcholinesterase oxime reactivator, HI-6, in rhesus monkeys (Macaca mulatta): effect of atropine, diazepam, and methoxyflurane anesthesia

The pharmacokinetics of the bispyridinium oxime HI-6 (CAS reg. no. 34433-31-3; 1-(((4-aminocarbonyl)pyridinio)methoxy)methyl)-2-[hydroxy i mino)methyl)- pyridinium dichloride) was investigated in rhesus monkeys (Macaca mulatta). The effects of methoxyflurane anesthesia, administration of atropine with and without diazepam were determined on the serum half-life (t1/2), clearance rate (CL), and the volume of distribution (Vd) following intramuscular (IM) administration of HI-6 (30 mg kg-1). The control t1/2, CL and Vd of HI-offere 27 min, 8.6 ml min-1 kg-1 and 0.34 l kg-1, respectively. These parameters were unaffected by the co-administration of either atropine (0.5 mg kg-1, IM) or atropine and diazepam (0.5 mg kg-1, IM + 0.2 mg kg-1 IV, respectively). Methoxyflurane anesthesia resulted in a significant increase in the HI-6 t1/2 to 61 min concomitant with a decrease in the CL to 4.1 ml min-1 kg-1 with no change in the Vd. The increase in the t1/2 of HI-6 in methoxyflurane anesthetized monkeys is probably the result of a decrease in the clearance rate and, thus, excretion of HI-6 by the kidneys.     

Are hypodense eosinophils in children activated or immature?

In patients with allergic asthma and rhinitis high numbers of hypodense eosinophils (HE) have been demonstrated. In a previous study we reported that asthmatic and healthy children had more HE than their adult counterparts. We assumed that this might, in part, he due to the presence of immature eosinophils in children. To distinguish between immature and activated eosinophils, determination of eosinophil cationic protein (ECP) might be interesting as it is known that high serum levels of ECP are associated with increased activation of eosinophiis. In this study we determined (he levels of ECP in scrum in asthmatic and healthy children and adults trying to distinguish activated from immature eosinophils. We found that ECP levels were not increased in children (healthy and asthmatic) compared to adults (healthy and asthmatic). This supports the hypothesis that increased numbers of HE in childhood are, at least in part, immature eosinophils. Nevertheless, we could confirm that inflammation was present in children because soluble interleukin-2-receptor (slL-2R), a marker of lymphocyte activation, was higher in asthmatic children as compared to healthy children. IL-6, a marker of macrophage/monocyte activation, was not different in the different patient groups. We conclude that although signs of inflammation are present in childhood asthma, the increased numbers of HE in children are in part due to the presence of immature eosinophils.     

The in vivo microdialysis recovery of dopamine is altered independently of basal level by 6-hydroxydopamine lesions to the nucleus accumbens

The present study was designed to test the hypothesis that the active neurotransmitter processes of release and uptake affect the in vivo microdialysis recovery of dopamine (DA) in the nucleus accumbens (N ACC) of the rat. The in vivo recovery for DA was established for rats which had received either unilateral infusions of the neurotoxin 6-hydroxydopamine (6-OHDA, 8 μg) or vehicle (0.2 μg ascorbate). In the quantitative dialysis method used (point of no net flux method), DA is added to the perfusate at concentrations above and below the expected extracellular concentration (0, 5, 10 and 20 nM) and DA is measured in the dialysate from the brain to generate a series of points. A linear fit is performed, the slope of which is the in vivo recovery of the dialysis probe. The in vivo recovery of the 6-OHDA group was 30 ± 3% which was significantly lower (P < 0.002) than the in vivo recovery of the control group which was 60 ± 3% (mean ± SEM; n = 6/group). The zero intercept of this regression is the point of no net flux, which is the extracellular concentration of DA independent of the probe sampling characteristics. The extracellular DA concentration for the 6-OHDA group was 7.8 ± 1.1nM, which was not significantly different than the control group which was 6.9 ± 0.7nM. The tissue DOPAC/DA ratios of the 6-OHDA lesioned hemispheres were significantly higher than the contralateral hemispheres of the same animals (0.62 ± 0.1vs.0.27 ± 0.1; P < 0.02) while the DOPAC/DA ratios in the control group were not significantly different (0.24 ± 0.1vs.0.27 ± 0.1). The fractional DA efflux from the terminals in the 6-OHDA group was significantly higher than the fractional DA efflux of the control group (0.52 ± 0.08vs.0.03 ± 0.003; P < 0.0001), indicating that the remaining terminals have increased turnover of DA. Despite the increased turnover, however, the number of remaining release and uptake sites are not sufficient to maintain the high in vivo recovery observed in the control group.     

Repair of DNA lesion O 6-methylguanine in hepatocellular carcinogenesis

Evidence from both experimental carcinogenesis and studies in human cirrhotic liver suggest that defective repair of the promutagenic DNA base lesion, O ⁶-methylguanine, is a factor in the multistep process of hepatocellular carcinogenesis. Ubiquitous environmental alkylating agents such as N-nitroso compounds can produce O ⁶-methylguanine in cellular DNA. Unrepaired, O ⁶-methylguanine can lead to the formation of G ? A transition mutations, a known mechanism of human oncogene activation and tumour suppressor gene inactivation. Combined treatment of rodents with an agent producing O ⁶-methylguanine in DNA, and an agent promoting cell proliferation, leads to development of hepatic nodules and hepatocellular carcinoma (HCC), cell division, hence DNA replication, being required for the propagation of tumorigenic mutation(s) in hepatocyte DNA. The paramount importance of O ⁶-methylguanine in hepatocellular carcinogenesis is indicated by the observation that transgenic mice engineered to have increased hepatic levels of repair enzyme O ⁶-methylguanine-DNA methyltransferase (MGMT) are significantly less prone to hepatocellular carcinogenesis following alkylating agent treatment. Cirrhosis is a universal risk factor for development of human HCC, and a condition that is characterized by increased hepatocyte proliferation as a result of tissue regeneration. Levels of the human repairing enzyme for O ⁶-methylguanine were found to be significantly lower in cirrhotic liver than in normal tissue. In accord with findings from animal models, this suggested a mechanism in which persistence of O ⁶-methylguanine due to defective DNA repair by MGMT, together with increased hepatocyte proliferation, might lead to specific gene mutation(s) and hepatocellular carcinogenesis. Screening for the presence and persistence of O ⁶-methylguanine in human DNA presently involves formidable technical difficulty. Indications are that such limitations might be overcome by the use of an ultrasensitive method such as immuno-polymerase chain reaction (PCR). This approach should allow parallel measurement of DNA adduct and repair enzyme in routine liver biopsy samples. It might also enable investigation of O ⁶-methylguanine in human genes specifically associated with hepatocellular carcinogenesis. Given the wide variation in human MGMT levels observed between individuals, tissues, and cells, this technology should be adapted to permit the ultrasensitive localisation and measurement of adducts and repairing enzyme in liver biopsy tissue sections. Ability to ultrasensitively measure O ⁶-methylguanine, and its repair enzyme, should prove valuable in the risk assessment of cirrhotic patients for developing hepatocellular carcinoma. Received for publication on July 6, 1998; accepted on Aug. 12, 1998     

Are cytokines possible mediators of cancer cachexia?

The possible role of cytokines in the development of cancer cachexia was reviewed from the literature. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, IL-6, interferon (IFN)-gamma and leukemia inhibitory factor (LIF) can elicit many but not all host changes seen in cancer cachexia, including loss of appetite, loss of body weight, and the induction of acute-phase protein synthesis. However, these cytokines are not always demonstrated in the circulation of the cancer patients. The inability to detect circulating cytokines may be due to their low rate of production, their short half-life and rapid clearance from plasma, or their mode of action (autocrine or paracrine). Different cytokines are induced to stimulate the same response. This is very different from hormonal regulation, where a hormone acts on a cell directly through a specific receptor without depending on other mediators. Specific antibodies including anti-IFN-gamma, anti-TNF and anti-IL-6 antibodies, as well as the cyclooxygenase inhibitor indomethacin, have been used to reverse cancer cachexia. Overlapping physiologic activities make it unlikely that a single substance is the sole cause of cancer cachexia. It is hoped that further investigation on other cytokines and their possible relationships with hormones will help to clarify the mechanisms of cancer cachexia in the near future.This work was supported by a grant from the Japan-Sweden Foundation in 1991.     

Monoamine oxidase-B inhibition: a comparison of in vivo and ex vivo measures of reversible effects

Summary A behavioural test involving potentiation of the effects of an acute injection of -phenylethylamine (10 mg kg^–1 i.p.) was used to assess the time-course of type-B MAO inhibition after administration of (–)deprenyl (5 mg kg^–1 i.p.) and of MD 240928 (20 mg kg^–1 i.p.) respectively. Potentiation of the effects of -phenylethylamine was observed 1 h after injection of (–)deprenyl or MD 240928. This effect was still evident 120 h after administration of (–)deprenyl but not 24 h after administration of MD 240928. Comparisons of ex vivo estimates of MAO activity yielded a corresponding time-course for the recovery of this enzyme. The extent of MAO inhibition required for potentiation of the effects of -phenylethylamine was inferred from a comparison of the behavioural test results and the ex vivo MAO activity observed after (–)deprenyl administration. These comparisons indicate a significant underestimation of MD 240928-induced MAO inhibition using ex vivo measures. This underestimation is interpreted as evidence fordilution effects in the ex vivo assay of MAO inhibition. The potentiation of effects of -phenylethylamine under the present conditions is proposed as a useful and simple test for effects of reversible type-B MAO inhibitors.     

SNHG6靶向miR-101/TGFBR1促进AMI小鼠左心室心肌纤维化

目的探讨SNHG6对急性心肌梗死(AMI)小鼠左心室心肌的影响。 方法将30只雄性C57/BL6小鼠构建成AMI小鼠后随机均分为AMI组、AMI+SNHG6组、AMI+miR-101-3p组、AMI+SNHG6+miR-101-3p组、AMI+miR-101-3p+TGFBR1组,另设正常小鼠6只为假手术组。qRT-PCR检测AMI小鼠SNHG6、miR-101-3p表达水平。心脏彩超检测各组小鼠左室射血分数(LVEF);马松和天狼星红染色法以及免疫组化分析各组小鼠左心室心肌纤维化变化。将H9C2细胞株分为阴性对照组(转染空质粒)、SNHG6组(转染质粒SNHG6)、miR-101-3p组(转染质粒miR-101-3p)。Western blotting检测各组TGFBR1蛋白表达;采用双荧光素酶报告基因法预测并验证SNHG6/miR-101-3p/TGFBR1荧光素酶活性及调控机制。 结果AMI小鼠较假手术组SNHG6表达显著增加,miR-101-3p降低(P＜0.05)。与AMI组比较,AMI+SNHG6组小鼠LVEF降低,心肌纤维化程度加重(P＜0.05);AMI+miR-101-3p组LVEF升高,心肌纤维化程度减轻(P＜0.05)。AMI+SNHG6+miR-101-3p组较AMI+SNHG6组LVEF升高、心肌纤维化程度减轻(P＜0.05),而AMI+miR-101-3p+TGFBR1组较AMI+miR-101-3p组LVEF降低、心肌纤维化程度加重(P＜0.05)。双荧光素酶报告基因法验证显示,miR-101-3p组SNHG6、TGFBR1野生型质粒的荧光素酶活性较阴性对照组明显降低(P＜0.05)。 结论SNHG6抑制miR-101-3p上调TGFBR1加重AMI小鼠左心室心肌纤维化。     

Regional brain kinetics of 6-fluoro-(β-11C)-L-dopa and (β-11C)-L-dopa following COMT inhibition. A study in vivo using positron emission tomography

Summary The regional brain kinetics of (-11C)-L-dopa and 6-fluoro-(-11C)-L-dopa was measured in six Rhesus monkeys using positron emission tomography (PET). Radioactivity accumulated specifically in the striatal region and the increase in L-dopa-derived radioactivity utilization with time was calculated using surrounding brain as a reference area, this being devoid of dopaminergic activity. The rate constant for selective striatal utilization i.e. grossly decarboxylation was 0.0110 ± 0.0007 (S.D) and 0.0057 ± 0.0006 min1 for (-11C)-L-dopa and 6-fluoro-(-11C)-L-dopa, respectively. After pre-treatment of the monkeys with the peripherally and centrally active catecholamine-O-methyl transferase (COMT) inhibitor Ro 40-7592 10 mg/kg, the decarboxylation rate remained unchanged (0.0112 ± 0.0015 min-1) for (11C)-L-dopa, whereas an increase in rate was measured for 6-fluoro-(-11C)L-dopa (0.0092 ± 0.0015 min–1). Differences in the distribution of radiolabelled metabolites i.e. the corresponding O-methyl-L-dopa in the reference area is most probably the reason for the difference in calculated decarboxylation rate seen between the radiotracers. The higher decarboxylation rate measured for 6-fluoro-(-11C)-L-dopa after blockade of COMT shows that the radiolabelled metabolites i.e. 6-fluoro-O-methyl-(-11C)-L-dopa significantly contributes to background radioactivity.     

Gene therapy of malignant brain tumors: a rat glioma line bearing the herpes simplex virus type 1-thymidine kinase gene and wild type retrovirus kills other tumor cells.

Tumor cells infected with a retrovirus vector (VIK) containing the herpes simplex virus thymidine kinase (HSV-TK) gene can be selectively killed by treatment with nucleoside analogues, such as ganciclovir. To mediate delivery of the HSV-TK gene to "recipient" tumor cells, "donor" C6 rat glioma cells infected with the VIK vector (C6VIK) were superinfected with wild type Moloney murine leukemia virus (WT Mo-MLV). These modified donor cells (C6VIKWT) produced both wild type retrovirus and the VIK vector. In culture, C6VIKWT cells were 300-fold more sensitive to the toxicity of ganciclovir than were C6VIK cells, suggesting that the presence of wild type retrovirus contributed to the toxicity. Co-culture of C6VIKWT cells with the C6 subline, C6BAG, sensitized the latter to ganciclovir treatment. Nude mice inoculated subcutaneously with a mixture of C6VIKWT and C6BAG cells showed regression of subsequent tumors when treated with ganciclovir. The observations show that tumor cells modified in culture by infection with a retrovirus bearing the HSV-TK gene and wild type retrovirus are not only sensitive to ganciclovir, but can transfer this sensitivity to neighboring "naive" tumor cells in culture and in vivo.     

Involvement of adenylate cyclase and protein kinase C in the α2-adrenoceptor-mediated inhibition of noradrenaline and 5-hydroxytryptamine release in rat hypothalamic slices

Summary In superfused rat hypothalamic slices prelabelled with [³H]-noradrenaline, the ₂-adrenoceptor agonist UK 14304 inhibited in a concentration-dependent manner the electrically-evoked release of tritium. This inhibition was antagonized by the ₂-adrenoceptor blocking agent idazoxan, which by itself increased the electrically-evoked tritium overflow. Exposure to forskolin, an adenylate cyclase activator, increased the electrically-evoked release of [³H]-noradrenaline. In the presence of forskolin (1 mol/l), both the inhibitory effect of UK 14304 and the increasing effect of idazoxan on the electrically-evoked release of [³H]-noradrenaline were less pronounced than in the absence of the adenylate cyclase activator. Exposure to forskolin and to the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine shifted to the right the concentration-effect curve for UK 14304 in a similar manner as that observed in the presence of forskolin alone. Exposure to phorbol-12,13-dibutyrate (0.01–10 mol/l), a drug which activates protein kinase C, increased the electrically-evoked release of [³H]-noradrenaline. In the presence of phorbol-12,13-dibutyrate (0.1 and 1 mol/l), the concentration effect curve for UK 14304 on tritium overflow was significantly shifted to the right. The increasing effect of idazoxan on tritium overflow was significantly less pronounced in the presence of 1 mol/l phorbol-12,13-dibutyrate.In superfused rat hypothalamic slices prelabelled with [³H]-5-hydroxytryptamine, the ₂-adrenoceptor agonist UK 14304 significantly inhibited the electrically-evoked release of tritium. Exposure to forskolin increased in a concentration-dependent manner [³H]-5-hydroxytryptamine overflow, but did not modify the UK 14304-mediated inhibition. Exposure to 3-isobutyl-1-methylxanthine enhanced the electrically-evoked release of [³H]-5-hydroxytryptamine. In the presence of both forskolin (1 mol/l) and 3-isobutyl-l-methylxanthine (1 mmol/l), the concentration-response curve for UK 14304 was significantly shifted to the right. Exposure to phorbol-12,13-dibutyrate (0.01–10 mol/l) enhanced in a concentration-dependent manner the electrically-evoked overflow of [³H]-5-hydroxytryptamine. In the presence of phorbol-12,13-dibutyrate (0.1 and 1 mol/l), UK 14304 was significantly less potent to inhibit tritium release than in the absence of the protein kinase C activator.It is concluded that both cyclic AMP and phosphoinositide turnover are involved in the modulation of noradrenaline and 5-hydroxytryptamine release by presynaptic ₂-adrenoceptors in rat hypothalamic slices. However, these interactions do not represent definitive proof for a cause-effect relationship for the second messengers mediating the ₂-adrenoceptor induced inhibition of transmitter release either as autoreceptor or as heteroreceptor.Send offprint requests to S. Z. Langer at the above address