Induction of ABCG2/BCRP restricts the distribution of zidovudine to the fetal brain in rats.

Safety concerns for fetus development of zidovudine (AZT) administration as prophylaxis of vertical transmission of HIV persist. We evaluated the participation of the ATP-binding cassette efflux transporter ABCG2 in the penetration of AZT into the fetal brain and the relevance for drug safety. Oral daily doses of AZT (60mg/kg body weight) or its vehicle were administered between post gestational days 11 (E11) and 20 (E20) to Sprague-Dawley pregnant rats. At E21, animals received an intravenous bolus of 60mg AZT/kg body weight in the presence or absence of the ABCG2 inhibitor gefitinib (20mg/kg body weight, ip) and AZT in maternal plasma and fetal brain were measured by HPLC-UV. ABCG2 protein expression in placenta and fetal brain, as well as mitochondrial function and ultrastructure in fetal brain were also analyzed. In utero chronic exposure to AZT markedly induced ABCG2 expression in placenta and fetal brain whereas did not significantly alter mitochondrial functionality in the fetal brain. The area-under-the-concentration-time-curve of AZT significantly decreased in fetal brains isolated from AZT-exposed fetuses compared to control group, but this effect was abolished by ABCG2 inhibition. Our results suggest that the absence of mitochondrial toxicity in the fetal brain after chronic in utero administration of AZT could be attributed to its low accumulation in the tissue caused, at least in part, by ABCG2 overexpression. We propose that any interference with ABCG2 activity due to genetic, pathological or iatrogenic factors would increase the amount of AZT reaching the fetal brain, which could increase the risk of toxicity of this drug on the tissue.

Pharmacokinetic study of the variability of indinavir drug levels when boosted with ritonavir in HIV-infected children.

The aim of this work is to: (1) assess therapeutic drug monitoring of indinavir (IDV) during clinical routine practice in HIV-infected children, whose antiretroviral treatment includes IDV boosted with ritonavir (RTV), and (2) describe a possible relationship between IDV pharmacokinetics and MDR1 genotypes. In 21 ambulatory pediatric patients receiving IDV plus RTV, IDV plasma levels and MDR1 genotypes on exon 26 (C3435T) were determined. Nine of the 21 patients initially receiving 250 mg/m(2) IDV yielded trough levels below 0.10 microg/ml (median: 0.21, range: 0.04-1.31 microg/ml). When the dosage was increased to 400 mg/m(2) IDV plus 100 mg/m(2) RTV b.i.d., all, except 1 patient, achieved levels above 0.10 microg/ml. Pharmacokinetic analysis showed higher volume of distribution median values related to the C/C genotype in comparison with C/T or T/T genotypes for exon 26 (4.57 vs. 1.20 and 1.50 l/kg, respectively; p = 0.002). Although a higher median value of clearance was observed with the C/C genotype, the difference was not statistically significant (1.43 vs. 0.27 and 0.42 l/h, respectively; p = 0.052). These results may be explained by a reduced absorption of the drug, related with lower plasma IDV levels in patients carrying the C/C genotype in exon 26.

Indinavir-loaded pH-sensitive microparticles for taste masking: toward extemporaneous pediatric anti-HIV/AIDS liquid formulations with improved patient compliance.

The aim of this work was to develop indinavir pediatric anti-HIV/AIDS formulations enabling convenient dose adjustment, ease of oral administration, and improved organoleptic properties by means of the generation of drug-loaded microparticles made of a polymer that is insoluble under intake conditions and dissolves fast in the stomach in order to completely release the active agent. Indinavir-loaded microparticles made of a pH-dependent polymeric excipient soluble at pH < 5, Eudragit E100, were prepared using a double emulsion solvent diffusion technique and the in vitro release profiles characterized. Finally, taste masking properties were evaluated in blind randomized sensory experiments by ten healthy human volunteers. The use of a w/o/o emulsion system resulted in indinavir loads around 90%. Thermal analysis of the microparticles by differential scanning calorimetry revealed that indinavir appeared mainly dispersed at the molecular level. Concentrations of residual organic solvents as determined by gas chromatography were below the upper limits specified by the European Pharmacopeia for pharmaceutical oral formulations. Then, the behavior of drug-containing microparticles in aqueous media at different pH values was assessed. While they selectively dissolved in gastric-like medium, in tap water (intake conditions), the matrix remained almost unchanged and efficiently prevented drug dissolution. Finally, sensoring taste tests performed by volunteers indicated that systems with indinavir loads approximately 15% displayed acceptable taste. This work explored the production of indinavir-containing microparticles based on a common pharmaceutical excipient as a means for the improvement of medicines of drugs involved in the treatment of HIV/AIDS. For systems containing about 15% drug, taste studies confirmed the acceptability of the formulation. In pediatric regimes, this composition would require an acceptable amount of formulation (0.7-1.5 g).

[P-glycoprotein functional activity in peripheral blood lymphocytes in ulcerative colitis]. / Funcionalidad de la glicoproteína P linfocitaria en la colitis ulcerosa.

P-glycoprotein (P-gp), encoded by MDR-1, is a transmembrane efflux pump that has been involved in relevant clinical drug transport. It is expressed in lymphocytes, luminal epithelium of colon and other tissues with barrier function. MDR1 was proposed as a candidate gene for ulcerative colitis. The aim of the present work was to investigate the role of P-gp in therapeutic response of ulcerative colitis by studying its functionality in lymphocytes isolated from peripheral blood. Samples were taken from 27 patients with active colitis classified clinically in refractory (n = 16) and responders (n = 11) to treatment. Rhodamine 123 (a fluorescent P-glycoprotein substrate) efflux was studied by flow cytometry as absence and presence of an inhibitor (verapamil, 100 uM). Data were expressed evaluating the behaviour of two markers defined based on % of cells with maximum (M1)/minimum (M2) intracellular fluorescence, reflecting inactivity/activity of the pump. Results were compared with a group of healthy individuals (n = 68). Significant differences were observed in absence and presence of Verapamil inhibition, when comparing refractory vs. responders (p < 0.05) as well as refractory vs. healthy controls (p < 0.01). No differences were observed when comparing responders vs. controls (p > 0.05) (Kruskal-Wallis test and Dunn post-test). Rhodamine efflux assay was also performed in 12 patients who required therapeutic change; a significant diminish of rhodamine transport (p < 0.01) was observed without inhibitor when patients achieved clinical response. Finally, our results suggest a possible relevant role of P-gp in ulcerative colitis treatment response and a possible usefulness of P-gp functional assay in the early detection of individual therapeutic response.

Lymphocyte P-glycoprotein variability in healthy individuals.

The aim of the present work was to describe the distribution of lymphocyte P-glycoprotein activity on a population of healthy individuals, taking also into account sex and age. P-glycoprotein activity in lymphocytes was measured by the Rhodamine 123 efflux assay using flow cytometry, in the presence and absence of verapamil, a P-glycoprotein inhibitor. We obtained a range of P-glycoprotein activity from 1.04 to 3.79. The distribution of the activity in the population studied was better described by a bimodal model, according with the Kolmogorov-Smirnov test. The frequency adjusted to the following equation: F = 0.70 N (2.11; 0.43) + 0.30 N(3.29; 0.26), in which 0.70 and 0.30 represented the proportion of each group, and 0.43 and 0.26 were the standard deviations of the activity of each group, respectively. The study of the relationship between subjects' age and P-glycoprotein activity showed no statistical significance. When healthy volunteers were separated according to sex, similar distributions were observed, although for men an increase in proportion of higher P-glycoprotein function group was observed. The variability observed in the population studied was important, with some volunteers with very scarce activity and some with a fourfold higher activity. Characterization of P-glycoprotein functionality in the population represents a useful contribution to the beginning of pharmacological treatments that consider its effect on pharmacokinetics and pharmacodynamics of individualized patients.

Topotecan vitreous levels after periocular or intravenous delivery in rabbits: an alternative for retinoblastoma chemotherapy.

PURPOSE: To determine the extent and the mechanism by which topotecan, a candidate agent for the treatment of retinoblastoma, gains access to the vitreous when administered by periocular injection or intravenous infusion. METHODS: In vivo experiments were conducted in which albino rabbits received 1 mg topotecan by periocular injection (POI group; n = 30) or as a 30-minute intravenous infusion (IV group; n = 16). Plasma and vitreal topotecan concentrations were analyzed during the 10 hours after administration. A population pharmacokinetic model was fit to the data. Additionally, periocular injections were performed postmortem to study the effect of removing the blood vasculature barrier. RESULTS: Potentially active lactone topotecan levels were detected in the vitreous in the POI and IV groups. Both administration schedules induced high total topotecan plasma exposures because of absorption from the periocular depot, though plasma lactone area under the curve (AUC) was significantly higher in the IV group. Similar vitreal concentrations were found in treated and control eyes in the POI group. The transfer from the periocular compartment to the vitreous was negligible. The absence of drug levels in the control eye of the postmortem-injected rabbits confirmed the systemic delivery of topotecan. Local toxicity was not observed. CONCLUSIONS: As a consequence of a favored passage across the blood-retinal barrier, considerable topotecan vitreous levels were detected in a rabbit model after systemic or periocular administration. Transscleral entry in vivo was constrained by rapid clearance from the administration site.

Cytoskeleton of hippocampal neurons as a target for valproic acid in an experimental model of depression.

BACKGROUND: Atrophy of pyramidal hippocampal neurons and of the entire hippocampus has been reported in experimental models of depression and in depressive patients respectively. We investigated the efficacy of valproic acid (VPA) for reversing a depressive-like behaviour and a cytoskeletal alteration in the hippocampus, the loss of the light neurofilament subunit (NF-L). METHODS: Depressive-like behaviour was induced by inescapable stress. Animals were divided into four groups: two to assess the response to 21 days of treatment with 200 mg/kg (I.P.) of valproic acid, and two in which the treatment was interrupted and the effects of VPA were evaluated 90 days later. Depressive-like behaviour was evaluated by the quantification of escape movements in a swimming test. NF-L was quantified by immunohistochemistry in dentate gyrus and CA3 of hippocampus. RESULTS: VPA corrected the depressive-like behaviour and reversed the diminution of NF-L in the hippocampus. Ninety days after the end of the treatment, and in contrast to the results previously obtained with fluoxetine, no recurrence of the depressive-like behaviour was observed. CONCLUSIONS: Despite interruption of the treatment, a long-lasting effect of VPA was observed. A possible relationship between the effect on NF-L and the prevention of depressive-like behaviour recurrence could be suggested.

Chronic treatment with fluoxetine decreases seizure threshold in naïve but not in rats exposed to the learned helplessness paradigm: Correlation with the hippocampal glutamate release.

The proconvulsive effect of the new generation of antidepressants remains controversial. The authors investigated in naïve rats the effect of chronic treatment with fluoxetine (FLX) on the convulsive threshold and on two parameters of the hippocampal glutamatergic neurotransmission: the in vitro glutamate release and the binding of [3H] MK801 to NMDA receptors. While the acute treatment with FLX provoked no change either in seizure susceptibility or in the glutamate release, the chronic treatment decreased the convulsive threshold in coincidence with an increment in the in vitro glutamate release. No significant effects on the binding of [3H] MK801 to NMDA receptors were found to be attributable to the FLX treatment. We also assessed the effect of the chronic treatment with FLX on the seizure threshold in rats exposed to an experimental model of depression, the learned helplessness paradigm (LH). While a decrease in the K+-stimulated glutamate release was observed in non treated LH animals, when they were chronically injected with FLX, no changes in the epileptic susceptibility and no increments in the glutamate release were found. Our results indicate that chronic treatment with FLX decreases the epileptic threshold in naïve but not in LH rats and that this effect correlates with the levels of the hippocampal glutamate release.

Morphine withdrawal syndrome: involvement of the dopaminergic system in prepubertal male and female mice.

Morphine (MOR) withdrawal signs are more marked in males than in females. Considering that the influence of the dopaminergic system on these differences is unclear, we analyzed dopamine (DA) and dihydroxyphenylacetic-acid (DOPAC) brain levels during naloxone (NAL)-precipitated withdrawal as well as the involvement of D(1) and D(2) receptors in the expression of MOR withdrawal in either sex. Prepubertal Swiss-Webster mice received MOR (2 mg/kg, i.p.) twice daily for 9 days. On the tenth day, dependent animals received NAL (6 mg/kg, i.p.) after MOR and were sacrificed 30 min later. DA and DOPAC concentrations were determined in different brain areas using HPLC with electrochemical detection. Other pool of mice received either a D(1) (SCH 23390; 0.2 mg/kg, i.p.) or D(2) (raclopride; 0.3 mg/kg, i.p.) receptor antagonist before NAL and withdrawal signs were evaluated. DA and DOPAC levels only decreased in striatum and cortex of withdrawn males. Conversely, both DA receptor antagonists decreased the expression of MOR withdrawal signs in either sex. The neurochemical sex differences described here could partially explain the behavioral sex differences observed during MOR withdrawal. Additionally, SCH-23390 and raclopride effects suggest an important role of both DA receptors in the expression of MOR withdrawal in males and females.

Tetronic® 904-containing polymeric micelles overcome the overexpression of ABCG2 in the blood-brain barrier of rats and boost the penetration of the antiretroviral efavirenz into the CNS.

AIM: To assess the involvement of ABCG2 in the pharmacokinetics of efavirenz in the blood-brain barrier (BBB) and investigate a nanotechnology strategy to overcome its overexpression under a model of chronic oral administration. Materials & methods A model of chronic efavirenz (EFV) administration was established in male Sprague-Dawley rats treated with a daily oral dose over 5 days. Then, different treatments were conducted and drug concentrations in plasma and brain measured. RESULTS: Chronic treatment with oral EFV led to the overexpression of ABCG2 in the BBB that was reverted after a brief washout period. Moreover, gefitinib and the polymeric amphiphile Tetronic(®) 904 significantly inhibited the activity of the pump and potentiated the accumulation of EFV in CNS. The same effect was observed when the drug was administered within mixed micelles containing TetronicT904 as the main component. CONCLUSION: Tetronic 904-containing polymeric micelles overcame the overexpression of ABCG2 in the BBB caused by chronic administration of EFV then boosting its penetration into the CNS.

Tyrosine hydroxylase activity in the endocrine pancreas: changes induced by short-term dietary manipulation.

BACKGROUND: Tyrosine hydroxylase (TH) activity and its possible participation in the control of insulin secretion were studied in pancreatic islets of adult Wistar rats fed a standard commercial diet (SD) or carbohydrates alone (CHD) for one week. TH activity, norepinephrine (NE) content, and glucose-induced insulin secretion were assessed. Blood glucose and insulin levels were measured at the time of sacrifice. RESULTS: CHD rats had significantly higher blood glucose and lower insulin levels than SD rats (114.5 PlusMinus; 6.7 vs 80.7 PlusMinus; 7.25 mg/dl, p < 0.001; 20.25 PlusMinus; 2.45 vs 42.5 PlusMinus; 4.99 &mgr;U/ml, p < 0.01, respectively). Whereas TH activity was significantly higher in CHD isolated islets (600 PlusMinus; 60 vs 330 PlusMinus; 40 pmol/mg protein/h; p < 0.001), NE content was significantly lower (18 PlusMinus; 1 vs 31 PlusMinus; 5 pmol/mg protein), suggesting that TH activity would be inhibited by the end-products of catecholamines (CAs) biosynthetic pathway. A similar TH activity was found in control and solarectomized rats (330 PlusMinus; 40 vs 300 PlusMinus; 80 pmol/mg protein/h), suggesting an endogenous rather than a neural origin of TH activity. CHD islets released significantly less insulin in response to glucose than SD islets (7.4 PlusMinus; 0.9 vs 11.4 PlusMinus; 1.1 ng/islet/h; p < 0.02). CONCLUSIONS: TH activity is present in islet cells; dietary manipulation simultaneously induces an increase in this activity together with a decrease in glucose-induced insulin secretion in rat islets. TH activity - and the consequent endogenous CAs turnover - would participate in the paracrine control of insulin secretion.

Pharmacokinetic-pharmacodynamic modeling of the antinociceptive effect of baclofen in mice.

The aim of this study was the development of a pharmacokinetic-pharmacodynamic (PK/PD) model of the antinociceptive effect of baclofen in mice. We studied the dose response curve of the analgesic action of baclofen in mice by hot plate test. Baclofen produced a dose dependent antinociceptive effect with doses between 1-3 mg/kg administered intraperitoneally (i.p.) (ED50: 1.94 mg/kg of racemate) and this effect fits to a linear pharmacodynamic model. Blood and brain concentrations of (-)3H-baclofen were determined by Thin-Layer Chromatography (TLC) and counted in the scintillation-counter. The PK/PD models were analyzed with the PC-TOPFIT V.2.0 and the tests for distinguishing between models were several adjustment parameters as Akaike information criterion (AIC), Imbimbo criterion (Ip), standard deviation (SD) and the correlation coefficient (r2). Accordingly with these adjustment parameters, a 2 compartment open model was selected where plasma is the central compartment and brain is in the peripheral compartment. In this model, the effect is linked to the peripheral compartment. When the antinociceptive effect of baclofen was plotted against blood concentration, the resulting curve exhibited an anticlockwise hysteresis loop, but on the other hand, when the antinociceptive effect was plotted against the brain concentration, the hysteresis was collapsed. These results confirmed the selected model in our study, as the best adjustment was shown when the pharmacological response was linked to the peripheral compartment.

Enhanced intestinal permeability and oral bioavailability of enalapril maleate upon complexation with the cationic polymethacrylate Eudragit E100.

The low bioavailability of enalapril maleate associated to its instability in solid state motivated the development of a polyelectrolyte-drug complex between enalapril maleate and the cationic polymethacrylate Eudragit E100. The solid complexes were characterized by DSC-TG, FT-IR and X-ray diffraction. Their aqueous dispersions were evaluated for drug delivery in bicompartimental Franz cells and electrokinetic potentials. Stability in solid state was also evaluated using an HPLC-UV stability indicating method. Absorption of enalapril maleate was assessed thorough the rat everted gut sac model. In addition, urinary recovery after oral administration in rats was used as an indicator of systemic exposition. The solid materials are stable amorphous solids in which both moieties of enalapril maleate are ionically bonded to the polymer. Their aqueous dispersions exhibited controlled release over more than 7h in physiologic saline solution, being ionic exchange the fundamental mechanism that modified the extent and rate of drug release. Intestinal permeation of enalapril maleate was 1.7 times higher in the presence of the cationic polymer. This increase can be related with the capacity to adhere the mucosa due to the positive zeta potential of the complexes. As a consequence bioavailability was significantly improved (1.39 times) after oral administration of the complexes. In addition, no signs of chemical decomposition were observed after a 14months period. The results indicated that the products are new chemical entities that improve unfavorable properties of a useful drug.

Acetaminophen inhibits intestinal p-glycoprotein transport activity.

Repeated acetaminophen (AP) administration modulates intestinal P-glycoprotein (P-gp) expression. Whether AP can modulate P-gp activity in a short-term fashion is unknown. We investigated the acute effect of AP on rat intestinal P-gp activity in vivo and in vitro. In everted intestinal sacs, AP inhibited serosal-mucosal transport of rhodamine 123 (R123), a prototypical P-gp substrate. R123 efflux plotted against R123 concentration adjusted well to a sigmoidal curve. Vmax decreased 50% in the presence of AP, with no modification in EC50, or slope, ruling out the possibility of inhibition to be competitive. Inhibition by AP was absent at 0°C, consistent with interference of the active transport of R123 by AP. Additionally, AP showed no effect on normal localization of P-gp at the apical membrane of the enterocyte and neither affected paracellular permeability. Consistent with absence of a competitive inhibition, two further strategies strongly suggested that AP is not a P-gp substrate. First, serosal-mucosal transport of AP was not affected by the classical P-gp inhibitors verapamil or Psc 833. Second, AP accumulation was not different between P-gp knock-down and wild-type HepG2 cells. In vivo intestinal absorption of digoxin, another substrate of P-gp, was assessed in the presence or absence of AP (100 µM). Portal digoxin concentration was increased by 214%, in average, by AP, as compared with digoxin alone. In conclusion, AP inhibited P-gp activity, increasing intestinal absorption of digoxin, a prototypical substrate. These results suggest that therapeutic efficacy of P-gp substrates can be altered if coadministered with AP.

Efavirenz is a substrate and in turn modulates the expression of the efflux transporter ABCG2/BCRP in the gastrointestinal tract of the rat.

The oral bioavailability of the antiretroviral efavirenz (EFV) undergoes high inter and intra-individual variability, this fact supporting its therapeutic drug monitoring. Previously, it was demonstrated that the encapsulation of EFV within polymeric micelles increases the oral bioavailability of the drug. The breast cancer resistant protein (BCRP, ABCG2) is known to be inhibited by EFV in vitro. Since ABCG2 is profusely expressed in the gastrointestinal tract, the aim of the present work was to thoroughly investigate whether the intestinal permeability of EFV is modulated by ABCG2. The functional role of ABCG2 in mediating the transport of EFV at the intestinal level was consistent with the following findings: (a) an ABCG2 inhibitor, fumitremorgin C (5-10µM), significantly potentiated the mucosal-to-serosal permeation of the drug in everted gut sacs; (b) a five-day oral treatment with 20mg/kg EFV promotes the over-expression of ABCG2 in about 100%, this phenomenon being accompanied by a clear decline in the intestinal permeability of the antiretroviral and (c) the normalization of the ABCG2 expression within 24h after the last administration of EFV was coincident with the recovery of the ability of the drug to permeate through the small intestine wall. Interestingly, no interactions between EFV and P-glycoprotein (ABCB1) were apparent. Since the intestinal permeability of a drug could be associated with its in vivo absorbability, we suggest that the oral absorption of EFV is affected by modifications in the ABCG2 intestinal expression contributing to the intra-individual bioavailability variations.

Acetaminophen-induced stimulation of MDR1 expression and activity in rat intestine and in LS 174T human intestinal cell line.

The well-known analgesic and antipyretic drug N-acetyl-p-aminophenol (acetaminophen; APAP) has been previously reported to affect MDR1 expression in rat liver. In this study, we have investigated the effect of subtoxic doses of APAP on MDR1 expression and activity in rat intestine and human intestinal cells. Administration of APAP at increasing doses of 0.2, 0.3, and 0.6g/kg b.w., i.p. over three consecutive days, induced MDR1 expression in rat duodenum (+240%) and ileum (+160%) as detected by western blotting. This was accompanied by preserved localization of the protein at the surface of the villus, as detected by confocal immunofluorescence microscopy. MDR1 activity was increased by 50% in APAP treated rats, as evaluated by serosal to mucosal secretion of rhodamine 123 in everted intestinal sacs. Treatment with APAP also decreased by 65% the portal vein concentrations of digoxin found in anesthetized rats after intraduodenal administration of this drug, which is consistent with an APAP-induced increased efficacy of intestinal barrier for digoxin net absorption. Exposure of LS 174T human colon adenocarcinoma cells to subtoxic APAP concentration (5mM) induced an increase in MDR1 mRNA expression (+46%), which was accompanied with an enhanced ability (+78%) to reduce intracellular content of rhodamine 123. Taken together these data suggest the existence of APAP-induced stimulation of MDR1 transcription in the intestinal epithelium. These findings are of clinical relevance, as co-administration of APAP with other MDR1 substrates could indirectly inhibit the net intestinal absorption of these drugs, leading to changes in their pharmacokinetics and therapeutic efficacy.

Episcleral implants for topotecan delivery to the posterior segment of the eye.

Purpose. Intravenous or periocular topotecan has been proposed as new treatment modality for patients with advanced intraocular retinoblastoma, but systemic topotecan lactone exposure induced by both approaches may cause toxicity. The purpose of this study was to develop a topotecan-loaded ocular delivery system to minimize systemic exposure and achieve selective transscleral penetration. Methods. Biocompatible polymer implants containing low (0.3 mg) or high (2.3 mg) topotecan load were manufactured and characterized in vitro. Adrenaline (500 mug) was coloaded to induce local vasoconstriction in vivo in 2 of 4 animal groups. Implants were inserted into the episclera of rabbits, and topotecan (lactone and total) concentrations in ocular tissues and plasma were determined over a period of 48 hours. Results. In vitro, implants released 30% to 50% of the loaded drug within 48 hours and 45% to 70% by day 10. In vivo, topotecan lactone was highly accumulated in locally exposed ocular tissues (ranging from 10(5) to 10(6) ng/g in sclera and choroid and 10(2) to10(3) ng/g in retina) over 48 hours with all the formulations studied. Low vitreous topotecan lactone levels (approximately 5 ng/mL) were found in animals receiving concomitant local vasoconstriction and high load implants. Topotecan lactone concentrations in plasma and in contralateral eyes were minimal or undetectable as a marker of tissue selectivity of the proposed strategy. Conclusions. These studies may contribute to improving the efficacy and safety of chemotherapy treatments for retinoblastoma and may support the role of the local vasculature and tissues promoting drug clearance and local accumulation during transscleral drug delivery.

Relationship between P-glycoprotein activity measured in peripheral blood mononuclear cells and indinavir bioavailability in healthy volunteers.

Indinavir, a HIV-1 protease inhibitor, showed large inter-individual pharmacokinetic variability. It has been proposed as a substrate of P-glycoprotein (P-gp), an efflux transporter, that may contribute to limit indinavir bioavailability. A liquid formulation of indinavir was developed from indinavir capsules in order to study indinavir pharmacokinetics in healthy volunteers. Compartmental and noncompartmental analysis of indinavir plasma concentrations showed high inter-individual variability in terms of area under the curve (AUC) and maximal plasma concentration (C(max)). A significant negative association between AUC normalized to body weight (AUC x weight) and lymphocyte P-gp activity, using Rh123 efflux assay, was observed (p = 0.008; r = -0.75). AUC normalized to elimination rate constant (AUC x beta) also showed a significant negative relationship with lymphocyte P-gp activity (p = 0.03, r = -0.64). Apparent clearance (CL/[F x weight]) and volume of distribution (VD/[F x weight]) showed a positive correlation with P-gp activity. Conversely, elimination rate constant did not correlate with P-gp activity. Although there is not enough evidence of a correlation between lymphocitary and intestinal function of P-gp, our results suggest a relationship between a P-gp phenotype marker, Rh123 efflux assay in lymphocytes, and indinavir bioavailability.

Morphine withdrawal syndrome and its prevention with baclofen: Autoradiographic study of mu-opioid receptors in prepubertal male and female mice.

Although the expression of the morphine (MOR) withdrawal syndrome is more marked in male mice than in females, we have demonstrated that the GABAB agonist baclofen (BAC) is able to attenuate MOR withdrawal signs in either sex. In order to extend these previous observations, the aim of the present study was to evaluate the mu-opioid receptor labeling in various brain areas in mice of either sex, during MOR withdrawal and its prevention with BAC. Prepubertal Swiss-Webster mice were rendered dependent by intraperitonial (i.p.) injection of MOR (2 mg/kg) twice daily for 9 days. On the 10th day, dependent animals received naloxone (NAL; 6 mg/kg, i.p.) 60 min after MOR, and another pool of dependent mice received BAC (2 mg/kg, i.p.) previous to NAL. Thirty minutes after NAL, mice were sacrificed and autoradiography with [3H]-[D-Ala2, N-Me-Phe4, -glycol5] enkephalin (DAMGO) was carried out on mice brains at five different anatomical levels. Autoradiographic mapping showed a significant increase of mu-opioid receptor labeling during MOR withdrawal in nucleus accumbens core (NAcC), caudate putamen (CPu), mediodorsal thalamic nucleus (MDTh), basolateral and basomedial amygdala, and ventral tegmental area vs. respective control groups in male mice. In contrast, opiate receptor labeling was not significantly modified in any of the brain areas studied in withdrawn females. BAC reestablished mu-opioid receptor binding sites during MOR withdrawal only in NAcC of males, and a similar tendency was observed in CPu and MDTh, even when it was not statistically significant. The sexual dimorphism observed in the present study confirms previous reports indicating a greater sensitivity of males in response to MOR pharmacological properties. The present results suggest that the effect of BAC in preventing the expression of MOR withdrawal signs could be related with the ability of BAC to reestablish the mu-opioid receptor labeling in certain brain areas.

Chronic treatment with high doses of corticosterone decreases cytoskeletal proteins in the rat hippocampus.

Hypercortisolism is a common trait of Cushing's disease and depression. These two disorders also share hippocampal volume decrease and cognitive deficits. However, experimentally induced hypercortisolism induces neuronal atrophy, which has been proposed to be the phenomenon underlying the hippocampal shrinkage. We hypothesized that the above-mentioned atrophy is due to a deleterious effect of high concentrations of glucocorticoids on cytoskeletal proteins. One or two pellets (100 mg each) of corticosterone were subcutaneously implanted in adult rats. Twenty-one days later, light, medium and heavy subunits of intermediate neurofilaments (NFL, NFM and NFH) and the microtubule-associated protein 2 (MAP2) were quantified by immunohistochemistry in Ammon's horn and dentate gyrus. We also evaluated the in vitro glutamate release in hippocampal slices. Both doses of corticosterone induced a decrement of NFL, NFM and NFH in both hippocampal areas but only 200 mg decreased MAP2. This dose also diminished the potassium-stimulated glutamate release. All of these changes seemed not to be due to neuron loss, as no decrement in neuron-specific nuclear protein-positive cells was found. With the exception of NFL, the above-mentioned diminution was not observed in the globus pallidus, one of the brain regions with the lowest glucocorticoid receptor density. These results provide a subcellular insight into the trophic changes found in experimental models of hypercortisolism. The coincidence between decrements in MAP2 and glutamate release suggests possible links between high glucocorticoid levels, dendritic atrophy and the cognitive impairment reported in patients suffering from Cushing's disease and depression.