Repeated restraint stress alters sensitivity to the social consequences of ethanol in adolescent and adult rats

Human adolescents consume alcohol largely to enhance social interactions. Adolescent, but not adult rats likewise exhibit ethanol-induced social facilitation under low-stress circumstances. Since the relationship between stress and ethanol sensitivity across ontogeny still has yet to be well explored, the present study sought to characterize possible age-associated differences in the influence of stressor exposure on ethanol-induced changes in social behavior in adolescent [postnatal days (P) 30-36] and adult (P65-71) male and female Sprague-Dawley rats. Animals were repeatedly restrained (90 min/day) for 5 days, followed by examination of ethanol-induced (0, 0.25, 0.5, 0.75, or 1.0 g/kg) alterations in social behaviors on the last day. Results revealed typical age-related differences in sensitivity to ethanol among controls, with adolescents being uniquely sensitive to low-dose ethanol stimulation of social investigation and play fighting, but less sensitive than adults to the social suppression emerging at higher doses. At both ages, stressor exposure decreased sensitivity to social inhibitory effects of ethanol, while augmenting expression of ethanol's social facilitatory effects. Ethanol also attenuated the stress-related suppression of social motivation at both ages. These results suggest that repeated stressor exposure diminishes age-related differences in the social consequences of ethanol, with stress enhancing ethanol-induced social facilitation across age.     

Increases in anxiety-like behavior induced by acute stress are reversed by ethanol in adolescent but not adult rats

Repeated exposure to stressors has been found to increase anxiety-like behavior in laboratory rodents, with the social anxiety induced by repeated restraint being extremely sensitive to anxiolytic effects of ethanol in both adolescent and adult rats. No studies, however, have compared social anxiogenic effects of acute stress or the capacity of ethanol to reverse this anxiety in adolescent and adult animals. Therefore, the present study was designed to investigate whether adolescent [postnatal day (P35)] Sprague-Dawley rats differ from their adult counterparts (P70) in the impact of acute restraint stress on social anxiety and in their sensitivity to the social anxiolytic effects of ethanol. Animals were restrained for 90 min, followed by examination of stress- and ethanol-induced (0, 0.25, 0.5, 0.75, and 1 g/kg) alterations in social behavior using a modified social interaction test in a familiar environment. Acute restraint stress increased anxiety, as indexed by reduced levels of social investigation at both ages, and decreased social preference among adolescents. These increases in anxiety were dramatically reversed among adolescents by acute ethanol. No anxiolytic-like effects of ethanol emerged following restraint stress in adults. The social suppression seen in response to higher doses of ethanol was reversed by restraint stress in animals of both ages. To the extent that these data are applicable to humans, the results of the present study provide some experimental evidence that stressful life events may increase the attractiveness of alcohol as an anxiolytic agent for adolescents.     

Dasotraline as a selective cytochrome 2B6 inhibitor for reaction phenotyping

Reaction phenotyping using human liver microsomes or hepatocytes with chemical inhibitors is one of the most commonly applied methods to assess the fraction metabolized (f_m) of drug candidates by enzymes. The f_m information is critical to understanding the risk of victim drug–drug interactions in the clinic. Inhibitor selectivity is essential in order to generate reliable data and irreversible inhibitors are often preferred over reversible inhibitors to minimize the impact of inhibitor depletion. Although many selective cytochrome P450 (CYP) inhibitors are available, the identification of selective CYP2B6 inhibitors has been challenging due to cross inhibition to the other enzymes. In this study, dasotraline was evaluated as a selective inactivator of CYP2B6 under reaction phenotyping conditions with human hepatocytes. The results show that dasotraline is a very selective inactivator for CYP2B6 with minimal inhibition to other enzymes. A concentration of 0.1 μM dasotraline is recommended for reaction phenotyping with a hepatocyte cell density of 0.5 million cells/ml or 0.5 μM for 2 million cells/ml, when using a 15 minute preincubation, as well as the protocol of inactivator removal before the addition of substrates.     

A 5-HT1A agonist and a 5-HT2c antagonist reduce social interaction deficit induced by multiple ethanol withdrawals in rats

Rationale. Repeated withdrawals from chronic forced ethanol exposure sensitize animals to withdrawal-induced deficits in social interaction behavior. The deficits in social interaction behavior following withdrawal from continuous ethanol exposure can be reduced following acute treatments with 5-HT_2C antagonists or 5-HT_1A agonists. Objectives. The present study investigated whether prior treatment with these serotonergic agents during early withdrawals in rats subjected to repeated withdrawals from ethanol exposure would ameliorate the social interaction deficits observed following the final withdrawal. Methods. Sprague-Dawley rats were exposed to three cycles of 5 days forced ethanol (7%, w/v), with 2 days of control diet after the first and second cycles. Drugs were administered IP 4 h after removal of ethanol on the first and second cycles but not the third in one group and 4.5 h after removal of ethanol on the third cycle in another. The social interaction test was performed 5 h after removal of ethanol on the third cycle. Drugs tested included SB-242084, a 5-HT_2C antagonist; buspirone, a 5-HT_1A partial agonist; WAY-100635, a 5-HT_1A antagonist; ketanserin, a 5-HT_2A antagonist; ritanserin, a mixed 5-HT_2A/2C antagonist; and Ro-601075, a 5-HT_2C agonist. Results. Both SB-242084 and buspirone reduced ethanol withdrawal-induced deficits in social interaction when given either acutely 30 min before the test or at 4 h after withdrawal from the first and second cycles. WAY-100635 and ketanserin were completely ineffective regardless of mode of treatment. In contrast, the 5-HT_2C agonist, Ro-601075, accentuated the withdrawal-induced deficit in social interaction behavior in rats exposed to either 4.5 or 7% ethanol diet. Conclusions. These results support the utility of 5-HT_1A agonists and 5-HT_2C antagonists in reducing anxiety-like behavior induced by ethanol withdrawal and reducing the adaptive changes associated with repeated withdrawals. Electronic Publication     

Pharmacokinetics and safety of JTE-522, a novel selective cyclooxygenase-2 inhibitor,in healthy male volunteers

AIMS: The pharmacokinetics and safety profile of JTE-522, 4-(4-cyclohexyl-2 methyloxazol-5-yl)-2-fluorobenzensulphonamide, a novel selective cyclooxygenase-2 inhibitor were investigated in healthy male volunteers. METHODS: Initially, as a pilot study, five groups of two subjects were given oral doses of 3-100 mg of JTE-522. After safety assessment, subjects were given 150 and 200 mg of JTE-522. The effect of food-intake on the pharmacokinetics of JTE-522 at a dose of 150 mg was examined. In the multiple-dose study, subjects were given 150 mg of JTE-522 once a day for 7 days. Concentrations of unchanged JTE-522 in plasma, blood and urine were determined by high performance liquid chromatography (h.p.l.c.). Concentrations of metabolites were estimated with h.p.l.c. chromatograms and calibration curves for quantification of unchanged JTE-522. RESULTS: In the course of this study, no serious abnormality attributable to the test drug was observed, suggesting that JTE-522 was well tolerated in healthy subjects. In a single-dose study, the concentrations of JTE-522 in blood were much higher than the corresponding concentrations in plasma. JTE-522 was readily distributed to blood cells and percentage distribution into blood cells was more than 99.0%. However, the values of Cmax in blood at doses of 100, 150, 200 mg JTE-522 were 15241, 20445 +/- 3918 (16333-24556), 20965 +/- 3260 (17544-24386) ng ml-1, respectively. These findings suggest that JTE-522 has a high affinity for blood cells and the distribution into blood cells is limited at the higher doses of over 100 mg. In a multiple dose study, pharmacokinetic parameters including t1/2 and AUC after the fourth administration were comparable with that of the seventh administration. Thus, these findings suggest the absence of accumulation on the multiple-dosing of JTE-522. CONCLUSIONS: These results indicate that JTE-522 has an acceptable pharmacokinetic profile for clinical use without any serious adverse events as we verified in healthy young male volunteers.     

L-745,337: A selective inhibitor of cyclooxygenase-2 elicits antinociception but not gastric ulceration in rats

L-745,337 [5-methanesulphonamido-6-(2,4-difluorothiophenyl)-1-indanone] a selective cyclooxygenase-2 inhibitor reversed hyperalgesia induced by carrageenan in rats without causing gastric ulceration at doses 100 times those causing antinociception. In contrast, piroxicam and indomethacin produced ulcerations at antinociceptive doses. These findings demonstrate that L-745,337 possesses antinociceptive activity but has a reduced liability for gastric ulceration.     

A protease inhibitor against acute stress-induced visceral hypersensitivity and paracellular permeability in rats

In the present study, we investigated the effects of camostat mesilate (CM), a synthetic protease inhibitor, on visceral sensitivity and paracellular permeability induced by the acute restraint stress. We also explored the possible mechanisms underlying these effects. The acute restraint stress was induced by wrapping the fore shoulders, upper forelimbs and thoracic trunk of Sprague–Dawley rats for 2 h. Either CM (30, 100 and 300 mg/kg) or saline was intragastrically administrated to the rats 30 min before the acute restraint stress. Visceral perception was quantified as visceral motor response with an electromyography in a subset of rats. Paracellular permeability was determined in another subset of rats. We found that the visceral sensitivity and paracellular permeability were significantly reduced in the CM-treated rats. Moreover, the fecal protease activity was decreased in the CM-treated rats. The ZO-1 protein expression was markedly reduced by the stress treatment, but this decrease was suppressed by CM administration. Our data indicated that CM could efficiently inhibit visceral sensitivity and paracellular permeability induced by the acute restraint stress in rats. Therefore, CM might be an effective drug for the treatment of irritable bowel syndrome.     

环氧合酶-2抑制剂对大鼠机械通气所致肺损伤的影响

目的:探讨环氧合酶－2抑制剂对大鼠机械通气所致肺损伤的影响.方法:健康成年雄性SD大鼠30只,随机分为3组(n=10):常规潮气量通气组(TV组,潮气量8 mL/kg)、大潮气量通气组(HV组,潮气量40 mL/kg)、大潮气量通气＋环氧合酶-2选择性抑制剂组(HV+NS398组).HV+NS398组于机械通气前30 min腹腔注射NS398 8 mg/kg.于机械通气4h时处死大鼠,Western Blot法检测肺组织中水通道蛋白1(AQP1)表达;检测支气管肺泡灌洗液(BALF)中肿瘤坏死因子(TNF-α)、白介素6(IL-6)、血栓素B2(TXB2)含量及总蛋白浓度,计算肺组织湿/干重比(W/D).结果:与TV组相比,HV组AQP1表达降低(P＜0.05),BALF中TNF-α、IL-6、TXB2和总蛋白浓度增加,肺组织W/D升高(P＜0.05);与HV组相比,HV+NS398组AQP1表达升高,BALF中TNF-α、IL-6、TXB2和总蛋白浓度降低(P＜0.05),肺组织W/D降低(P＜0.05).结论:环氧合酶-2抑制剂可能通过上调AQP1的表达及减轻炎性反应程度,而缓解大鼠机械通气所致肺损伤.     

Antiviral effect of a selective COX-2 inhibitor on H5N1 infection in vitro

A selective cyclooxygenase-2 (COX-2) inhibitor has been previously shown to suppress the hyper-induced pro-inflammatory responses in H5N1 infected primary human cells. Here, we demonstrate that COX-2 inhibitors suppress H5N1 virus replication in human macrophages suggesting that H5N1 virus replication (more so than seasonal H1N1 virus) is dependent on activation of COX-2 dependent signaling pathways in host cells. COX-2 and its downstream signaling pathways deserve detailed investigation as a novel therapeutic target for treatment of H5N1 disease.     

γ1- and γ2-melanocyte stimulating hormones induce central anxiogenic effects and potentiate ethanol withdrawal responses in the elevated plus-maze test in mice

Little is known about the endogenous functions of γ1- and γ2-melanocyte stimulating hormones (γ1- and γ2-MSH). Although γ-MSHs bind to melanocortin receptor subtypes 3 and 4, we have previously shown that these peptides also influence non-melanocortinergic processes, such as dopaminergic and GABAergic. The aim of this study was to determine the effects of γ1- and γ2-MSH (at doses 0.3, 1 and 2 nmol/mouse/5 μl) on the anxiety levels in mice in elevated plus maze. Three experimental paradigms were performed to assess the effects of peptides on: a) ethanol withdrawal; b) acute ethanol-induced anxiolytic action; c) peptides per se.We used ethanol as the model substance, since its action involves either dopaminergic/GABAergic or melanocortinergic processes. γ-MSHs were administered intracisternally in mice and behavioural responses were assessed in the elevated plus maze test. This study provides the first demonstration of an anxiogenic effect of γ1- and γ2-MSH, their synergistic/additive effect on ethanol withdrawal-induced anxiety behaviour, and an antagonism of peptides involved in the anxiolytic action of ethanol. Furthermore, results suggest that γ-MSHs belong to an anxiogenic peptide family that may play an important role in anxiety disorders as well as in the development of alcohol dependence and/or alcohol withdrawal-induced behaviours.     

Depression-like deficits in rats improved by subchronic modafinil

Rationale  Attentional and sensorimotor gating deficits in human depression are observed as residual symptoms irrespective of antidepressant treatment. Clinical studies point to a benefit of modafinil in depression. No data are available on modafinil effects in depression-like animal models. Objectives  We investigated effects of modafinil on attention and sensorimotor gating after subchronic treatment during a restraint stress protocol inducing depression-like changes in rats. Materials and methods  Effects of modafinil were investigated (a) acutely in the forced swim test (FST) 1 h after administration of drug or placebo and (b) in a further experiment on cognition-related behaviour in rats after induction of depression-like changes using a restraint stress protocol for 15 days. Beginning from day 10, one restrained (R) and one non-restrained (NR) group were treated with modafinil (R-M and NR-M groups) and two groups with placebo (R-P and NR-P groups). At the end of protocol, behavioural testing was performed under conditions of nearly drug-free plasma. Depression-like behaviour was examined in the FST. Selective attention and sensorimotor gating were investigated as social novelty discrimination (SND) and prepulse inhibition (PPI) of acoustic startle response. Results  Restraint led to reduced body weight, decreased mobility in the FST and impaired cognitive capabilities in the SND and the PPI. Subchronic modafinil treatment reversed restraint-induced deficits in the FST, the SND and PPI, whereas it was without effect on body weight. Conclusions  The improvement of impaired attentional and information-processing functions under depression-like conditions suggests a benefit of modafinil in treatment of cognitive residual symptoms in affective disorders.     

Influence of simultaneous inhibition of cyclooxygenase-2 and inducible nitric oxide synthase in experimental colitis in rats

The inflamed mucosa in ulcerative colitis produces high amount of prostaglandin (PG) and nitric oxide (NO) through inducible enzymes: cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), respectively, implicating them as potential anti-inflammatory drug targets. COX-2 or iNOS-related treatments in different models of colitis have yielded ambiguous results ranging from exacerbation of disease to abolition of inflammation. iNOS and COX-2 induction is blocked by potent anti-inflammatory glucocorticoids, however, serious side effects including relapses limit their usefulness in colitis for long time. Simultaneous inhibition of iNOS and COX-2 was investigated in the current study in 2, 4, 6 trinitrobenzene sulphonic acid (TNBS)-induced colitis in rats. Treatment group received rofecoxib, aminoguanidine hydrochloride or their combination at different doses at 48, 36, 24, 12 and 1 h prior to induction of colitis and 12 h later. Colonic myeloperoxidase (MPO), COX-2, nitrate and nitrite, tumor necrosis factor-α (TNF-α) and lipid peroxidation were maximally reduced by combination of 10 mg/kg rofecoxib and 30 mg/kg of aminoguanidine hydrochloride in TNBS-induced colitis in rats. However, maximum increase in SOD and catalase was noted by this combination. Rats treated with rofecoxib, aminoguanidine hydrochloride and their combinations reduced the inflammation, acute colonic damage produced by TNBS as verified by macroscopic changes in colon. Combination of rofecoxib (10 mg/kg) and aminoguanidine hydrochloride (30 mg/kg) has maximal protective effect on colonic injury induced by TNBS enema which is probably, via mechanism of local inhibition of iNOS and COX-2 activity in colonic mucosa and support the idea that simultaneous inhibition of iNOS and COX-2 inhibitors have a promising potential in the treatment of colitis.     

Gender difference in the pharmacokinetic interaction between oral warfarin and oxolamine in rats: inhibition of CYP2B1 by oxolamine in male rats

The possible reason for the significantly greater AUC of oral warfarin with oral oxolamine in male Sprague-Dawley rats was evaluated. After oral administration of warfarin at a dose of 2 mg/kg to male rats with oxolamine at doses of 10 and 50 mg/kg, the AUC values of warfarin were significantly greater than the controls (254 and 330 versus 180 microg h/ml). However, the AUC values of warfarin were not affected by oxolamine in female rats. This could be due to inhibition of CYP2B1, 2C11 and 3A2 by oxolamine in male rats, since warfarin was metabolized via CYP1A1, 2B1, 2C6, 2C11 and 3A2 in rats and CYP2B1 is male dominant, and CYP2C11 and 3A2 are male specific. Therefore, phenytoin, torasemide and clarithromycin (mainly metabolized via CYP2B1/2, 2C11 and 3A2 in rats, respectively) were administered intravenously to male rats with or without oral oxolamine. After oral oxolamine at doses of 10 and 50 mg/kg, the AUC of phenytoin was significantly greater (1280 and 1640 versus 938 microg min/ml), however, the AUC values of torasemide and clarithromycin were independent of oxolamine. The above data suggest that the significantly greater AUC of oral warfarin with oral oxolamine could be due to inhibition of CYP2B1/2 by oxolamine in male rats.     

Suppression of alcohol and saccharin preference in rats by a novel Ca2+ channel inhibitor,Goe 5438

The effect of the novel 1,4-dihydronaphthyridine Ca²⁺ channel inhibitor Goe 5438 (CI-951) on voluntary ethanol consumption was examined in selectively bred alcohol-preferring (P) rats in a free choice two bottle preference test versus water. Intraperitoneally injected Goe 5438 dose-dependently (5, 10 or 20 µmol/kg, twice daily) inhibited ethanol and increased water intake over the 24 h period (injection day). The drug decreased ethanol preference, originally above 90%, by 6%, 19% and 45% at respective doses, on the injection day. That inhibitory effect of the highest dose of Goe 5438 on ethanol preference remained significant also on days 2 and 3 after injections (–51% and –18%, respectively). Goe 5438, in the highest dose, also tended to decrease granulated chow consumption during the injection day only. To further test whether the inhibition of ethanol preference is secondary to decrease in reinforcing properties of ethanol and not due to interference with satiety mechanisms, we compared the effect of two higher doses (10 and 20 µmol/kg, intraperitoneally, twice daily) of Goe 5438 on spontaneous preference for a non-caloric 0.04% saccharin solution in Sprague-Dawley rats. We observed a dose-dependent suppression of preference (by 44% and 58%, respectively) during the injection day, but not the subsequent 24 h period. However, Goe 5438 also significantly alleviated food pellet intake on the injection day. In conclusion, Goe 5438 produces potent and long-lasting inhibition of voluntary ethanol consumption, which may be secondary to attenuation of reinforcing properties of ethanol. Additionally, this particular Ca²⁺ channel inhibitor appears to have mild anorectic properties which may be conducive to acute suppression of alcohol intake.     

CYP2C9抑制剂对艾瑞昔布大鼠体内药动学的影响

目的：考察CYP2C9抑制剂胺碘酮对艾瑞昔布在大鼠体内药动学的影响。方法： 40只健康雄性SD大鼠随机分为2组（n=20）,实验组连续7 d灌胃胺碘酮灌胃液（40 mg·kg^-1,qd）,对照组灌胃等量空白灌胃液。2组均于第8天单次灌胃艾瑞昔布灌胃液20 mg·kg^-1,按确定时间点取血,LC-MS/MS法测定艾瑞昔布血药浓度,DAS 2.1.1软件拟合药时曲线并计算药动学参数,SPSS 13.0软件进行统计学分析。结果：实验组和对照组的主要药动学参数如下：AUC_{0-24 h}分别为（1 814.8±693.4） ng·h·mL^-1和（1 125.1±457.6） ng·h·mL^-1;AUC_0-∞分别为（2 091.6±887.1） ng·h·mL^-1和（1 331.3±592.6） ng·h·mL^-1;t_1/2分别为（7.8±4.5） h和（7.4±3.8） h;t_max分别为（1.7±0.6） h和（1.46±0.60） h;CL分别为（0.01±0.01） L·h^-1·kg^-1和（0.02±0.01） L·h^-1·kg^-1;V分别为（0.11±0.05） L·kg^-1和（0.17±0.07） L·kg^-1;C_max分别为（268.2±115.7） ng·mL^-1和（162.2±53.0） ng·mL^-1。与对照组相比,实验组大鼠的AUC_{0-24 h}、AUC_0-∞、C_max显著增大（P<0.05）,V、CL显著减小（P<0.05）,其他参数差异无统计学意义（P>0.05）。结论： CYP2C9抑制剂（胺碘酮）对艾瑞昔布在大鼠体内的药动学产生影响。     

A literature review of the patent publications on venetoclax – a selective Bcl-2 inhibitor: discovering the therapeutic potential of a novel chemotherapeutic agent

Introduction: Studies presented in patents show that a novel chemotherapeutic agent, venetoclax, might be useful in additional therapeutic indications. Venetoclax is approved in America for the treatment of patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). Venetoclax selectively inhibits the B-cell lymphoma-2 (Bcl-2) protein, an anti-apoptotic protein that can be overexpressed in most B-cell lymphoid malignancies.

Areas covered: This is a review of all the patents granted until November 2018, with venetoclax in the examples or claim section of the patent document. The patents include the synthesis, polymorphism, formulations, in vitro and in vivo efficacy as well as the therapeutic application of venetoclax.

Expert opinion: The approved indications for treatment with venetoclax are limited but expanding rapidly. Studies suggest that venetoclax might be useful in several other therapeutic indications, mostly other hematological malignancies. Numerous studies use venetoclax in combinations with other therapeutic agents. Such combinational treatment shows promising results in additional indications as well as drug-resistant cancers. Venetoclax is an interesting new therapeutic involved in a variety of clinical research. Patent applications in recent years even include venetoclax in somewhat exotic fields such as type 1 diabetes, asthma, and Zika virus treatment.     

Comparative effects of the dual metallopeptidase inhibitor,MDL 100,240 and of enalaprilat on regional and on cardiac haemodynamics in conscious,hypertensive, transgenic ((mRen-2)27) rats

1. Heterozygous, male, hypertensive, transgenic ((mRen-2)27) rats (350–450 g) were instrumented for the measurement of regional or cardiac haemodynamics (n = 16, in both groups). Animals were given continuous i.v. infusions of the angiotensin-converting enzyme inhibitor, enalaprilat, or the dual metallopeptidase inhibitor, MDL 100,240 (both at 3 mg kg⁻¹, 3 mg kg⁻¹ h⁻¹; n = 8 for regional and cardiac haemodynamics), for 32 h. Twenty four hours after the onset of infusion of enalaprilat or MDL 100,240, the bradykinin (B₂)-receptor antagonist, Hoe 140 (1 mg kg⁻¹, i.v.), was given and measurements were continued for a further 8 h, to assess any possible involvement of bradykinin.
2. Over the first 8 h of infusion, both enalaprilat and MDL 100,240 had significant antihypertensive effects, accompanied by similar regional vasodilatations. However, the blood pressure lowering effect of MDL 100,240 (−54 ± 9 mmHg) was greater than that of enalaprilat (−38 ± 4 mmHg), because the former caused a significantly greater reduction in cardiac index.
3. Between 8–24 h after the onset of infusion, there was a reduction in the effect of enalaprilat on blood pressure, because cardiac index rose, with no further increase in total peripheral conductance. In contrast, the antihypertensive effect of MDL 100,240 persisted, in spite of a recovery in cardiac index, because there was further vasodilatation, particularly in the mesenteric and hindquarters vascular beds.
4. There were no apparent haemodynamic changes associated with the injection of Hoe 140, and over the following 8 h, the difference between the haemodynamic effects of enalaprilat and MDL 100,240 persisted; there was little evidence of suppression of the effects of either drug.
5. These results are more consistent with the antihypertensive effects of enalaprilat or MDL 100,240 in transgenic ((mRen-2)27) rats being due to suppression of angiotensin II production, than due to inhibition of bradykinin degradation. The additional effects of MDL 100,240 may be accounted for by inhibition of the degradation of natriuretic peptides reducing cardiac output, initially, and decreasing vascular tone, subsequently. Alternatively, the additional increase in vascular conductance following treatment with MDL 100,240 may represent an autoregulatory response to the reduced pressure.

     

A high throughput in vitro mrp2 assay to predict in vivo biliary excretion

1. Prediction of biliary excretion is a challenge for drug discovery scientists due to the lack of in vitro assays. This study explores the possibility of establishing a simple assay to predict in vivo biliary excretion via the mrp2 transport system.

2. In vitro mrp2 activity was determined by measuring the ATP-dependent uptake of 5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF) in canalicular plasma membrane vesicles (cLPM) from rat livers. The CDCF uptake was time- and concentration-dependent (K_m of 2.2?±?0.3 µM and V_max of 115?±?26 pmol/mg/min) and strongly inhibited by the mrp2 inhibitors, benzbromarone, MK-571, and cyclosporine A, with IC₅₀ values ≤ 1.1 µM.

3. Low inhibition of CDCF uptake by taurocholate (BSEP inhibitor; 57 µM) and digoxin (P-gp inhibitor; 101 µM) demonstrated assay specificity towards mrp2.

4. A highly significant correlation (r²?=?0.959) between the in vitro IC₅₀ values from the described mrp2 assay and in vivo biliary excretion in rats was observed using 10 literature compounds.

5. This study demonstrated, for the first time, that a high throughput assay could be established with the capability of predicting biliary excretion in the rat using CDCF as a substrate.

     

The anxiogenic-like effect of caffeine in two experimental procedures measuring anxiety in the mouse is not shared by selective A2A adenosine receptor antagonists

Rationale: The elevated plus-maze and the light/dark box are two established anxiety tests in rodents, which are useful to screen putative anxiogenic effects of drugs. Objective: Caffeine is well known to promote anxious behaviour in humans and animal models, but the precise site of action of the drug is still a matter of debate. The present study investigated whether the anxiogenic effects of caffeine observed in mice depend on the blockade of A_2A receptor. First, the effects induced by the non-selective drug caffeine were compared with those elicited by two selective A_2A receptor antagonists over a wide range of doses in the same experimental conditions. The effects of A_2A or A₁ adenosine receptor agonists and of a selective A₁ adenosine receptor antagonist were also investigated. Second, wild-type and A_2A receptor knockout mice offered another approach to delineate the role played by A_2A receptor in caffeine’s anxiogenic effects. Methods: Mice were exposed to the elevated plus-maze or to the light/dark box for 5 min after acute or chronic administration of tested drugs. Results: Caffeine acutely administered (50 or 100 mg/kg IP) induced anxiety-like effects in both procedures. Its chronic administration (50 mg/kg IP twice daily) for 1 week or consumption in the drinking water (0.3 g/l) for 8 days or 2 months were also anxiogenic in the plus-maze test. The A_2A receptor antagonists ZM241385 (up to 60 mg/kg IP) and SCH58261 (up to 10 mg/kg IP) were devoid of acute effects in both tests. One week administration of ZM241385 (30 mg/kg IP) or SCH58261 (3 mg/kg IP) had no effects in the plus-maze test. An antagonist (DPCPX) and an agonist (CPA) at A₁ receptors had no acute effects on anxiety-related indices, whereas an A_2A receptor agonist (CGS 21680) displayed non-specific motor effects in the plus-maze test. Acute administration of caffeine (50 mg/kg IP) induced no clear-cut anxiety-like effects in the plus-maze test in A_2A receptor knockout mice that exhibited higher basal anxiety levels than wild-type mice. Chronic administration (50 mg/kg IP twice daily) for 1 week elicited less anxiety-like behaviour in A_2A receptor knockout than in wild-type mice. Conclusions: Adaptative mechanisms following mutation in A_2A receptors or their long-term blockade after chronic ingestion of caffeine may be responsible for increase proneness to anxiety. However, the short-term anxiety-like effect of caffeine in mice might not be related solely to the blockade of adenosine A_2A receptors, since it is not shared by A_2A selective antagonists. Received: 21 March 1999 / Final version: 24 June 1999     

Effect of 5-HT1A receptor agonists in two models of anxiety after dorsal raphe injection

The purpose of the present study was two-fold. Firstly, to present a more comprehensive analysis of the disinhibitory effects of 5-HT_1A receptor agonists after discrete dorsal raphe (DRN) injections (Higgins et al. 1988). Secondly, the effects of the 5-HT_1B receptor agonist CGS12066B and the 5-HT_1B/1C agonist mCPP were examined following injection into this nucleus. The increases in social interaction (SI) induced by intra-raphe injections of 8-OH DPAT (0.02–1 μg), buspirone (0.04–0.2 μg), ipsapirone (0.2 μg) and gepirone (0.2–1 μg) under a high light unfamiliar paradigm (HLU) were typically due to increased bout frequency, duration and a higher incidence of sniff, follow, allogroom behaviour. These increases were qualitatively similar to those seen in control animals tested under low light/familiar (LLF) conditions, thus supporting the belief that the drug-induced increases in SI reflected decreases in anxiety. Furthermore, at doses effective under the HLU condition, 8-OH DPAT, buspirone and gepirone failed to modify SI under conditions of minimal suppression (LLF paradigm). At doses which significantly increased punished responding in a water-lick conflict test 8-OH DPAT, ipsapirone and gepirone tended to also increase unpunished rates of drinking. However, in drug untreated rats, prior habituation to the test apparatus also increased unpunished drinking, suggesting some neophobia-induced suppression. At a comparatively high dose, the 5-HT_1B agonist CGS12066B (2.5 μg), but not the putative 5-HT_1B/1c agonist mCPP (0.5–12.5 μg), increased SI under the HLU condition. Considered along-side the other compounds described in this report, the relative potency of CGS12066B may be reflective of a 5-HT_1A receptor interaction. Together, these data support the proposal that the DRN is an important site through wich 5-HT_1A receptor agonists express their anxiolytic actions.