Quantitative determination of MDR1 mRNA expression in peripheral blood lymphocytes: a possible role of genetic polymorphisms in the MDR1 gene.

BACKGROUND: P-glycoprotein is a transmembrane efflux pump that extrudes a wide variety of drugs, thereby reducing their intracellular access. In humans, P-glycoprotein is encoded by the MDR1 gene. Recently, several single nucleotide polymorphisms in the MDR1 gene were identified. Moreover, it was postulated that, in addition to the full-length P-glycoprotein, a 'mini' P-glycoprotein was also present in lymphocytes. MATERIALS AND METHODS: We investigated the effect of the genetic polymorphisms G2677T and C3435T in the MDR1 gene on MDR1 mRNA expression in FACS-sorted peripheral blood CD4+, CD8+, CD19+, and CD56+ cells. MDR1 mRNA expression was determined in 45 healthy individuals using a real-time quantitative RT-PCR. RESULTS: We detected the highest expression of MDR1 mRNA in CD56+ cells, followed by CD8+ > CD4+ > CD19+ cells. However, genetic polymorphisms of the MDR1 gene failed to affect (P > 0.05) MDR1 mRNA levels in the peripheral blood lymphocytes. Furthermore, the transcript levels for the MDR1 N-terminal half were almost two-fold lower than that of the MDR1 C-terminal half in all cell populations investigated (P < 0.0001). CONCLUSIONS: An almost two-fold difference in MDR1 C- and N-terminal half expressions supports the presence of mini-P-glycoprotein, an alternatively spliced form of the full-length molecule, in peripheral blood lymphocytes.

Differential effects of beta-carbolines and antidepressants on rat exploratory activity in the elevated zero-maze.

Present experiments were designed to compare the effects of antidepressants desipramine (10 and 20 mg/kg IP) and fluoxetine (5 and 10 mg/kg IP) with anxiogenic beta-carboline DMCM (0.5 and 1.0 mg/kg IP) in the elevated zero-maze test in rats. The second aim of this study was to assess the effects of pinoline (6-methoxy-1,2,3, 4-tetrahydro-beta-carboline) in the rat elevated zero-maze test in comparison with structurally unrelated beta-carboline DMCM and antidepressants. The time spent in the open part of the elevated zero-maze was not significantly affected by antidepressants, but was decreased by beta-carbolines pinoline and DMCM. The number of line crossings in the open parts and the number of head dips were also decreased more by beta-carbolines in comparison with antidepressants. Latency to enter the open part was statistically significantly increased only by DMCM. Measurement of locomotor activity in a separate experiment indicated that activity of the rats' time moving, distance traveled, and number of rearings were reduced by all four drugs studied. These results demonstrate that the effects of antidepressants in the elevated zero-maze test differ from the effects of the reference anxiogenic compound DMCM. The effects of pinoline and DMCM in the zero-maze test were similar, which suggests the involvement of mechanisms other than serotoninergic in the action of pinoline.

Digoxin: use pattern in Estonia and bioavailability of the local market leader.

BACKGROUND: In comparison with neighbouring Scandinavian countries, the use of digoxin in Estonia is high. The present study was carried out to determine the use pattern of digoxin in Estonia and bioavailability of the local market leader preparation in comparison with Lanoxin. METHOD: Drug use data were evaluated from the annual reports of wholesale companies. For the bioequivalence study, a single-blind cross-over randomised two-way single-dose comparative bioavailability study was performed using 14 healthy volunteers. Digoxin concentrations in serum samples and urine were measured by chemiluminescent competitive immunoassay. RESULTS: The use of digoxin in Estonia has increased by 35% during the period 1994-97. The steady market leader was the local generic drug. No statistically significant differences were found in any pharmacokinetic parameter between the generic preparation and Lanoxin. All parameters showed considerable variability. The total amount of drug excreted was the parameter with lowest inter- individual variation. CONCLUSION: The present study indicates that the generic digoxin preparation studied is bioequivalent to Lanoxin. The increasing use of digoxin in Estonia is not caused by low bioavailability of the local market leader but by therapeutic traditions.

Comparative bioavailability of three different preparations of rifampicin.

OBJECTIVE: To study the relative bioavailabilities of two generic rifampicin preparations with Rimactane. METHOD: Each of nineteen healthy volunteers received a single oral dose of 600 mg of rifampicin in an open cross-over randomised three-way single-dose design with a washout period of 7 days between each doses. Plasma concentrations of rifampicin were determined by HPLC. In vitro dissolution profiles of the same drugs were determined and compared with human bioavailability study results. RESULTS: No statistically significant difference was found in main bioavailability parameters between Rimactane and generic preparations studied. Both generic preparations also fulfilled the bioequivalence criteria based on the 90% confidence intervals. There was a good correlation between in vivo and in vitro results: faster dissolution time corresponded to the lower Tmax value; lower percentage of released compound to the lower AUC value. Significant intersubject variations were found in main bioavailability parameters; significant negative correlation was found between average AUC values and body weight of the volunteer. CONCLUSION: All three products were bioequivalent. Our results also suggest the suitability of one-compartmental model with lag time, first order input and first order output to describe the kinetics of rifampicin.

Long-term administration of MC4 receptor antagonist HS014 causes hyperphagia and obesity in rats.

In this study we investigated the long term effects of a potent and selective melanocortin 4 (MC4) receptor antagonist (HS014) on food intake, body weight, body composition and blood glucose levels in adult rats. HS014 was injected i.c.v. either by twice-daily injections (2 x 1 nmol) for 6 days or administered by continuous infusion with osmotic minipumps (0.16 nmol/h) for 2 weeks. The results show a considerable increase in food intake and body weight after both of the treatments without any signs of tachyphylaxis. After 2 weeks of treatment with osmotic pumps, the HS014-treated rats (average weight 425g) had 20% higher body weight than the controls rats (average 360 g). When i.c.v. injections were terminated, the body weight of the twice-daily HS014-treated rats returned to the levels of control group, whereas the rats treated with continuous infusion of HS014 remained hyperphagic and still gained weight. Blood glucose levels in the rats treated with HS014 infusion were significantly increased. Analysis of body composition in HS014-infused rats indicated that body weight was increased due to fat deposits. These data show for the first time that chronic administration of exogenous MC4 receptor antagonist causes hyperphagia and severe obesity in rats. These data also indicate that the melanocortic control of food intake is very robust and suggest that changes induced by such treatment overcome negative feedback signals.

Dose-dependent effects of noradrenergic denervation by DSP-4 treatment on forced swimming and beta-adrenoceptor binding in the rat.

DSP-4 is a neurotoxin highly selective for the noradrenergic nerve terminals originating from the locus coeruleus. Preliminary data suggested that its effect in a typical screening test for antidepressant drugs, the forced swimming test, is biphasic dependent on the dose. In the present study, DSP-4 was administered in four doses (5, 10, 30 and 50mg/kg) to male Wistar rats. Administration of the neurotoxin had a dose-dependent biphasic effect on immobility time in the forced swimming test 8 and 9 days later. Thus, DSP-4 at the dose of 10mg/kg increased immobility, but higher doses reduced this measure. The reduction of noradrenaline concentration in the frontal cortex and hippocampus was dose-dependent starting from the dose 10mg/kg. Cortical beta-adrenoceptor binding was increased by DSP-4 treatment at the doses 30mg/kg and 50mg/kg. These results suggest that the increase in immobility time in the forced swimming test is associated with presynaptic changes in noradrenaline availability, whereas the decrease in immobility observed after more complete denervation is associated with postsynaptic receptor supersensitivity.

Chronic mild unpredictable stress after noradrenergic denervation: attenuation of behavioural and biochemical effects of DSP-4 treatment.

Chronic mild unpredictable stress, which reduces rewarded behaviour in rats, is becoming increasingly popular as an animal model of depression. The effect of chronic mild stress (applied to animals housed five per cage for 15 days) on forced swimming and open field behaviour, and on beta-adrenoceptor binding was studied in naive rats and after the denervation of the locus coeruleus projections by DSP-4 (50 mg kg(-1)) treatment. In the forced swimming test, chronic mild stress reduced the immobility time on the second day of testing in both vehicle- and DSP-4-treated rats, indicating rather an antidepressant-like effect. This antidepressant-like effect of chronic mild stress in the forced swimming test was not present in individually housed rats which suggests that this paradigm is sensitive to housing conditions. Stress had no clear effect on the open field locomotion in naive animals (but caused a reduction in defecations), but completely blocked the DSP-4-induced decrease in the exploratory activity. As measured by 3H-dihydroalprenolol binding, DSP-4 treatment increased the beta-adrenoceptor affinity in the frontal cortex and the number of binding sites in the hippocampus and in the cerebral cortex (total-frontal cortex). Stress had no effect on the beta-adrenoceptor binding in the frontal cortex and cerebral cortex, but prevented the increase in affinity caused by DSP-4 treatment in the frontal cortex. In the hippocampus, chronic mild stress and DSP-4 treatment increased the number of beta-adrenoceptor binding sites. Neither chronic mild stress nor DSP-4 treatment had any effect on CCK(B) receptor binding in the cerebral cortex and striatum. These results show that chronic mild stress applied to group-housed rats can prevent the development of certain behavioural and biochemical changes caused by the denervation of the locus coeruleus projection areas.

Discovery of a novel superpotent and selective melanocortin-4 receptor antagonist (HS024): evaluation in vitro and in vivo.

Several novel cyclic MSH analogs were synthesized, and their binding properties were tested on cells transiently expressing the human melanocortin-1 (MC1), MC3, MC4, and MC5 receptors. We discovered a novel substance (HS024) that showed about 20-fold selectivity and very high affinity (Ki = 0.29 nM) for the MC4 receptor. HS024 (cyclic [AcCys3,Nle4,Arg5,D-Nal7,Cys-NH2(11)]alpha-MSH-(3-11)) has a 29-membered atom ring structure that includes an Arg in position 5. HS024 was found to antagonize an alphaMSH-induced cAMP response in cells expressing the human MC1, MC3, MC4, and MC5 receptor DNAs. HS024 also caused a dose-dependent increase in food intake, with a maximum response (4-fold increase) at a 1-nmol dose injected intracerebroventricularly in free feeding rats. We also tested SHU9119, a previously described nonselective MC receptor antagonist, and found HS024 and SHU9119 to have similar potencies for increasing food intake, although SHU9119 appeared to induce more serious side-effects. HS024 increased the food intake of free feeding rats to levels comparable to those in food-deprived rats, indicating that blockade of the MC4 receptor is a highly effective way to increase feeding. Moreover, we tested the effects of intracerebroventricular injections of HS024 in elevated plus-maze and open-field experiments on rats. In these tests, HS024 did not appear to affect emotionality or locomotor activity, suggesting that the MC4 receptor does not mediate the anxiogenic-like and locomotor effects related to the melanocortic peptides.

Selective antagonist for the melanocortin 4 receptor (HS014) increases food intake in free-feeding rats.

Recently, we discovered a cyclic analogue of MSH (melanocyte stimulating hormone), HS014, which is the first selective antagonist of the MC4 receptor. We have here studied the effects of this peptide on food intake in non-deprived male rats. Vehicle or five doses of HS014 (0.1-10 nmol) were administered ICV at midday. HS014 (0.33-3.3 nmol) significantly and in a dose-dependent manner increased food intake for the first 1 h. At 4 h after the injections, food intake was also significantly increased in rats treated with 1 and 3.3 nmol of HS014, whereas the lowest dose tested (0.1 nmol) was without effect. Cumulative food intake increased to 100% at 4 h after the injections. The highest dose of HS014 (10 nmol) induced sedation and inhibited feeding for first hour of testing. However, this dose also increased food consumption later. These data demonstrate that attenuation of central melanocortinergic tone with HS014 induces disinhibition of feeding and provides additional evidence for the hypothesis that activation of the MC4 receptor inhibits food intake. HS014 may be a useful tool for elucidating the role of the MC receptor subtypes in vivo. This is the first report demonstrating an increase in daytime food intake in free-feeding animals caused by a MC receptor active agent.

Comparison of the antioxidant activity of melatonin and pinoline in vitro.

Several recent experiments have shown that melatonin is an efficient antioxidant and free radical scavenger. In the present study the antioxidative effect of melatonin was compared with that of pinoline. Pinoline (6-methoxy-tetrahydro-beta-carboline) can be formed in the mammalian body under physiological conditions from 5-hydroxytryptamine or as a tricyclic metabolite of melatonin. Both melatonin and pinoline inhibited lipid peroxidation and showed comparable activity in a total antioxidant status test. Melatonin and pinoline concentration-dependently scavenged hydroxyl radicals with IC50 11.4+/-1.0 microM for melatonin and 62.3+/-3.8 microM for pinoline. These results support the importance of the indolic part of the molecule and the 5-methoxy group common to both compounds in terms of the ability of these molecules to quench the hydroxyl radicals. As pinoline has been shown to exert an antidepressant-like effect in behavioral experiments and has been reported to have a low toxicity, this compound should be further studied as a potential antidepressant with pronounced antioxidative effects. These results further support the importance of pineal gland in antioxidative protection.

Binding of pinoline on the 5-hydroxytryptamine transporter: competitive interaction with [3H] citalopram.

Pinoline (6-methoxy-1,2,3,4-tetrahydro-beta-carboline) is a naturally occurring compound in the mammalian body which inhibits 5-hydroxytryptamine (5-HT) uptake and exerts antidepressant-like behavioural effects in rats. The present study investigates the effects of pinoline on [3H]citalopram binding to the 5-HT transporter on rat brain. Our experiments revealed that pinoline inhibits [3H]citalopram binding with IC50 1255 +/- 167 nM and Ki 572 +/- 76 nM; Hill coefficient for inhibition was close to 1. In saturation experiments, pinoline co-incubated with [3H]citalopram, increased dose-dependently the Kd value but had no effect on the Bmax value of [3H]citalopram binding. Micromolar concentrations of pinoline did not have influence on the dissociation rate of specifically bound [3H]citalopram. Binding parameters of [3H]citalopram did not differ significantly in cerebral cortex and hippocampus of rats treated for 10 days with pinoline or vehicle. These results indicate that pinoline did not have any modulative influence on the activity of 5-HT transporter and it interacts competitively with citalopram on the substrate recognition site of the 5-HT transporter.

Different molecular forms of cholecystokinin and CCKB receptor binding in the rat brain after chronic antidepressant treatment.

Cholecystokinin (CCK) is a neuropeptide recently implicated in affective disorders. This study aimed at measuring the levels of different molecular forms of CCK and the binding characteristics of CCKB receptors in the rat brain after three weeks of treatment with four different antidepressants, imipramine, amitriptyline, desipramine, and citalopram (all at the dose of 10 mg/kg once per day i.p.). Chronic treatment with imipramine and desipramine had a significant immobility-reducing effect in the Porsolt's swim test. The effect of amitriptyline, albeit in the same direction, was not significant, and citalopram had no effect in this test. In the elevated plus-maze test of anxiety, all drugs tended to increase the number of open arm entries and the ratio open/total arm entries, but only the effects of imipramine were statistically significant. None of the treatments affected the total levels of CCK or the levels of CCK-8-sulphated, CCK-8-nonsulphated, CCK-5, or CCK-4 in the frontal cortex. There was no effect of the treatments on CCKB receptor binding in the frontal cortex, hippocampus, or striatum. Imipramine and amitriptyline, however, increased the affinity of CCKB receptor binding in the hypothalamus. Thus, no consistent effect of chronic antidepressant treatment on the CCK-ergic neurotransmission in the rats was found.

Behavioural effects of pinoline in the rat forced swimming, open field and elevated plus-maze tests.

Pinoline (6-methoxy-1,2,3,4-tetrahydro-beta-carboline) is a naturally occurring compound in the mammalian body which inhibits serotonin (5-hydroxytryptamine) uptake and monoamine oxidase-A activity. The present study was designed to assess potential antidepressant- or anxiolytic-like behavioural effects of pinoline in rat forced swimming, open field and elevated plus-maze tests. In the forced swimming test pinoline dose-dependently reduced the immobility time, starting from a dose of 8 mg kg-1. In the open field test pinoline reduced the total open field activity. This effect was significant at 20 mg kg-1, whereas the dose of 15 mg kg-1 only significantly reduced the number of rearings. In the plus-maze test pinoline decreased the total number of arm entries, the number of line crossings and time spent in the open part of the apparatus, but increased the number of approaches to the open part of the maze. The effects of pinoline were dose-dependent in all three behavioural tests used. Since antidepressant drugs reduce the immobility time in the forced swimming test but typically inhibit activity in open field and elevated plus-maze tests, the behavioural effects of pinoline resemble those of drugs with an antidepressant profile.

Autoradiographic localization of [3H]-pinoline binding sites in mouse tissues.

Pinoline (6-methoxy-1,2,3,4-tetrahydro-beta-carboline) has been found in similar concentrations to those of melatonin in various mammalian tissues. The present study investigates the subcellular distribution of in vivo administered [3H]-pinoline in mouse tissues using light and electron microscopic autoradiography. The antioxidative capacity of pinoline was determined in vitro and compared with that of the structurally similar pineal hormone melatonin. Autoradiograms revealed that [3H]-pinoline was present in all tissues studied 30 min after administration, but in most tissues binding was nonspecific. Adrenal glands showed the highest specific binding for [3H]-pinoline 0.5-1 h after administration of the labelled compound. In all tissues a large amount (approximately 40%) of radioactivity was located in nuclei of cells. Specific nuclear binding was the highest in adrenal glands and cerebral cortex. Both pinoline and melatonin potently inhibited lipid peroxidation. Therefore, we suggest that pinoline may act in cells and nuclei as an antioxidant. Direct genomic effects of pinoline cannot be excluded.