Search

Cognitive aging creates major individual and societal burden, motivating search for treatment and preventive care strategies. Behavioural interventions can improve cognitive performance in older age, but effects are small. Basic research has implicated dopaminergic signaling in plasticity. We investigated whether transient enhancement of dopaminergic neurotransmission via administration of L-dopa improves effects of cognitive training on performance.Sixty-three participants for this randomised, parallel-group, double-blind, placebo-controlled trial were recruited via newspaper advertisements. Inclusion criteria were: age of 65-75 years, Mini-Mental State Examination score >25, absence of serious medical conditions. Eligible subjects were randomly allocated to either receive 100/25mg L-dopa/benserazide (n=32) or placebo (n=31) prior to each of twenty cognitive training sessions administered during a four-week period. Participants and staff were blinded to group assignment. Primary outcomes were latent variables of spatial and verbal fluid intelligence.Compared to the placebo group, subjects receiving L-dopa improved less in spatial intelligence (−0.267 SDs; 95%CI [−0.498, −0.036]; p=0.024). Change in verbal intelligence did not significantly differ between the groups (−0.081 SDs, 95%CI [−0.242, 0.080]; p=0.323). Subjects receiving L-dopa also progressed slower through the training and the groups displayed differential volumetric changes in the midbrain. Adverse events occurred for 10 (31%) and 7 (23%) participants in the active and control groups, correspondingly. No statistically significant differences were found for the secondary cognitive outcomes.The results speak against early pharmacological interventions in older healthy adults to improve broader cognitive functions by targeting the dopaminergic system and provide no support for learning-enhancing properties of L-dopa supplements. The findings warrant closer investigation about the cognitive effects of early dopamine-replacement therapy in neurological disorders. This trial was preregistered at the European Clinical Trial Registry, EudraCT#2016-000891-54.SignificanceThe results put constraints on the hypothesis of a key role of the deteriorated dopaminergic system in age-related decline of learning abilities, and speak against early pharmacological interventions in older healthy adults to improve cognitive functions by targeting the dopaminergic system. Our findings also raise concerns about usefulness of novel L-dopa-containing supplements that claim to have neuroprotective and learning-enhancing properties, and present an urgent need to carefully investigate the cognitive outcomes of early pro-dopaminergic interventions in clinical populations often receiving substantially larger doses of L-dopa.

Background and Purpose There is a clear need for innovation in anti-tuberculosis drug development. The zebrafish larva is an attractive disease model in tuberculosis research. To translate pharmacological findings to higher vertebrates, including humans, the internal exposure of drugs needs to be quantified and linked to observed response.Experimental Approach In zebrafish studies, drugs are usually dissolved in the external water, posing a challenge to quantify internal exposure. We developed experimental methods to quantify internal exposure, including nanoscale blood sampling, and to quantify the bacterial burden, using automated fluorescence imaging analysis, with isoniazid as the test compound. We used pharmacokinetic-pharmacodynamic modelling to quantify the exposure-response relationship responsible for the antibiotic response. To translate isoniazid response to humans, quantitative exposure-response relationships in zebrafish were linked to simulated concentration-time profiles in humans, and two quantitative translational factors on sensitivity to isoniazid and stage of infection were included.Key Results Blood concentration was only 20% of the external drug concentration. The bacterial burden increased exponentially, and an isoniazid dose corresponding to 15 mg center dot L(-1)internal concentration (minimum inhibitory concentration) leads to bacteriostasis of the mycobacterial infection in the zebrafish. The concentration-effect relationship was quantified, and based on that relationship and the translational factors, the isoniazid response was translated to humans, which correlated well with observed data.Conclusions and Implications This proof of concept study confirmed the potential of zebrafish larvae as tuberculosis disease models in translational pharmacology and contributes to innovative anti-tuberculosis drug development, which is very clearly needed.

receptor activation was evaluated using quinpirole as a paradigm compound. ), as well as plasma concentrations of 13 hormones and neuropeptides, were measured. Experiments were performed at day 1 and repeated after 7-day s.c. drug administration. PK/PD modelling was applied to identify the in vivo concentration-effect relations and neuroendocrine dynamics. receptor expression levels on the pituitary hormone-releasing cells predicted the concentration-effect relationship differences. Baseline levels (ACTH, prolactin, TSH), hormone release (ACTH) and potency (TSH) changed with treatment duration. agonists in clinical practice. Further development towards quantitative systems pharmacology models will eventually facilitate mechanistic drug development. BACKGROUND AND PURPOSE EXPERIMENTAL APPROACH KEY RESULTS CONCLUSIONS AND IMPLICATIONS

Drug development targeting the central nervous system (CNS) is challenging due to poor predictability of drug concentrations in various CNS compartments. We developed a generic physiologically based pharmacokinetic (PBPK) model for prediction of drug concentrations in physiologically relevant CNS compartments. System-specific and drug-specific model parameters were derived from literature and in silico predictions. The model was validated using detailed concentration-time profiles from 10 drugs in rat plasma, brain extracellular fluid, 2 cerebrospinal fluid sites, and total brain tissue. These drugs, all small molecules, were selected to cover a wide range of physicochemical properties. The concentration-time profiles for these drugs were adequately predicted across the CNS compartments (symmetric mean absolute percentage error for the model prediction was

Purpose Predicting target site drug concentration in the brain is of key importance for the successful development of drugs acting on the central nervous system. We propose a generic mathematical model to describe the pharmacokinetics in brain compartments, and apply this model to predict human brain disposition. Methods A mathematical model consisting of several physiological brain compartments in the rat was developed using rich concentration-time profiles from nine structurally diverse drugs in plasma, brain extracellular fluid, and two cerebrospinal fluid compartments. The effect of active drug transporters was also accounted for. Subsequently, the model was translated to predict human concentration-time profiles for acetaminophen and morphine, by scaling or replacing system- and drug-specific parameters in the model. Results A common model structure was identified that adequately described the rat pharmacokinetic profiles for each of the nine drugs across brain compartments, with good precision of structural model parameters (relative standard error

A key challenge in the development of central nervous system drugs is the availability of drug target specific blood‐based biomarkers. As a new approach, we applied cluster‐based pharmacokinetic/pharmacodynamic (PK/PD) analysis in brain extracellular fluid (brainECF) and plasma simultaneously after 0, 0.17, and 0.86 mg/kg of the dopamine D2/3 agonist quinpirole (QP) in rats. We measured 76 biogenic amines in plasma and brainECF after single and 8‐day administration, to be analyzed by cluster‐based PK/PD analysis. Multiple concentration‐effect relations were observed with potencies ranging from 0.001–383 nM. Many biomarker responses seem to distribute over the blood‐brain barrier (BBB). Effects were observed for dopamine and glutamate signaling in brainECF, and branched‐chain amino acid metabolism and immune signaling in plasma. Altogether, we showed for the first time how cluster‐based PK/PD could describe a systems‐response across plasma and brain, thereby identifying potential blood‐based biomarkers. This concept is envisioned to provide an important connection between drug discovery and early drug development.

Background and Purpose Kv11.1 (hERG) channel blockade is an adverse effect of many drugs and lead compounds, associated with lethal cardiac arrhythmias. LUF7244 is a negative allosteric modulator/activator of Kv11.1 channels that inhibits early afterdepolarizations in vitro. We tested LUF7244 for antiarrhythmic efficacy and potential proarrhythmia in a dog model. Experimental Approach LUF7244 was tested in vitro for (a) increasing human IKv11.1 and canine IKr and (b) decreasing dofetilide‐induced action potential lengthening and early afterdepolarizations in cardiomyocytes derived from human induced pluripotent stem cells and canine isolated ventricular cardiomyocytes. In vivo, LUF7244 was given intravenously to anaesthetized dogs in sinus rhythm or with chronic atrioventricular block. Key Results LUF7244 (0.5–10 μM) concentration dependently increased IKv11.1 by inhibiting inactivation. In vitro, LUF7244 (10 μM) had no effects on IKIR2.1, INav1.5, ICa‐L, and IKs, doubled IKr, shortened human and canine action potential duration by approximately 50%, and inhibited dofetilide‐induced early afterdepolarizations. LUF7244 (2.5 mg·kg−1·15 min−1) in dogs with sinus rhythm was not proarrhythmic and shortened, non‐significantly, repolarization parameters (QTc: −6.8%). In dogs with chronic atrioventricular block, LUF7244 prevented dofetilide‐induced torsades de pointes arrhythmias in 5/7 animals without normalization of the QTc. Peak LUF7244 plasma levels were 1.75 ± 0.80 during sinus rhythm and 2.34 ± 1.57 μM after chronic atrioventricular block. Conclusions and Implications LUF7244 counteracted dofetilide‐induced early afterdepolarizations in vitro and torsades de pointes in vivo. Allosteric modulators/activators of Kv11.1 channels might neutralize adverse cardiac effects of existing drugs and newly developed compounds that display QTc lengthening.

Background and Purpose Because biological systems behave as networks, multi‐biomarker approaches increasingly replace single biomarker approaches in drug development. To improve the mechanistic insights into CNS drug effects, a plasma neuroendocrine fingerprint was identified using multi‐biomarker pharmacokinetic/pharmacodynamic (PK/PD) modelling. Short‐ and long‐term D2 receptor activation was evaluated using quinpirole as a paradigm compound. Experimental Approach Rats received 0, 0.17 or 0.86 mg·kg−1 of the D2 agonist quinpirole i.v. Quinpirole concentrations in plasma and brain extracellular fluid (brainECF), as well as plasma concentrations of 13 hormones and neuropeptides, were measured. Experiments were performed at day 1 and repeated after 7‐day s.c. drug administration. PK/PD modelling was applied to identify the in vivo concentration–effect relations and neuroendocrine dynamics. Key Results The quinpirole pharmacokinetics were adequately described by a two‐compartment model with an unbound brainECF‐to‐plasma concentration ratio of 5. The release of adenocorticotropic hormone (ACTH), growth hormone, prolactin and thyroid‐stimulating hormone (TSH) from the pituitary was influenced. Except for ACTH, D2 receptor expression levels on the pituitary hormone‐releasing cells predicted the concentration–effect relationship differences. Baseline levels (ACTH, prolactin, TSH), hormone release (ACTH) and potency (TSH) changed with treatment duration. Conclusions and Implications The integrated multi‐biomarker PK/PD approach revealed a fingerprint reflecting D2 receptor activation. This forms the conceptual basis for in vivo evaluation of on‐ and off‐target CNS drug effects. The effect of treatment duration is highly relevant given the long‐term use of D2 agonists in clinical practice. Further development towards quantitative systems pharmacology models will eventually facilitate mechanistic drug development.

Background: Cognitive aging creates major individual and societal burden, motivating search for treatment and preventive care strategies. Behavioural interventions can improve cognitive performance in older age, but effects are small. Basic research has implicated dopaminergic signaling in plasticity. We investigated whether transient enhancement of dopaminergic neurotransmission via administration of L-dopa improves effects of cognitive training on performance. Methods: Participants for this randomised, parallel-group, double-blind, placebo-controlled trial were recruited via newspaper advertisements. Inclusion criteria were: age of 65-75 years, Mini-Mental State Examination score >25, absence of serious medical conditions. Eligible subjects were randomly allocated to either receive 100/25mg L-dopa/benserazide or placebo prior to each of twenty cognitive training sessions administered during a four-week period. Participants and staff were masked to group assignment. Primary outcomes were latent variables of spatial and verbal fluid intelligence. This trial was preregistered at the European Clinical Trial Registry, EudraCT#2016-000891-54. Findings: Between January 1st and October 10th 2017, we screened 235 people from the population in Stockholm, Sweden and randomly assigned 63 eligible subjects to receive L-dopa (n=32) or placebo (n=31) during cognitive training. Compared to the placebo group, subjects receiving L-dopa improved less in spatial intelligence (-0·267 SDs; 95% CI [-0·498, -0·036]; p=0·024). Change in verbal intelligence did not significantly differ between the groups (-0·081 SDs, 95% CI [-0·242, 0·080]; p=0·323). Adverse events occurred for 10 subjects (31%) in the active and for 7 participants (23%) in the control groups. Interpretation: The results speak against early pharmacological interventions in older healthy adults to improve cognitive functions by targeting the dopaminergic system and provide no support for learning-enhancing properties of L-dopa supplements. The findings warrant closer investigation about the cognitive effects of early dopamine-replacement therapy in neurological disorders. Trial Registration Number: This trial was preregistered at the European Clinical Trial Registry, EudraCT#2016-000891-54. Funding: European Research Council (ERC Grant agreement #617280-REBOOT), Wallenberg Clinical Scholars, and Stiftelse Stockholms Sjukhem. Declaration of Interest: We declare no competing interests. Ethical Approval: The study protocol was approved by the regional ethics review board in Stockholm (2016-1897-31/1) and the Swedish medical product agency (20016000891-54). Participants provided written informed consent before enrolment.

The pharmacokinetics and pharmacodynamics of drugs are influenced by daily fluctuations in physiological processes. The aim of this study was to determine the effect of dosing time on the pharmacokinetics and brain distribution of morphine. To this end, 4 mg/kg morphine was administered intravenously to Wistar rats that were either pre-treated with vehicle or tariquidar and probenecid to inhibit processes involved in the active transport of morphine. Non-linear mixed effects modelling was used to describe the concentration-time profiles of morphine and its metabolite M3G in plasma and brain tissue. We found that the concentrations of morphine in the brain and of M3G in plasma depended on the time of day, which could be quantified by a 24-hour rhythm in the efflux of morphine from brain tissue back into the circulation, with the lowest efflux during the two light-dark phase transitions with a difference between peak and trough of 20%. The active processes involved in the clearance of morphine and its metabolite M3G from plasma also showed 24-hour variation with the highest value in the middle of the dark phase being 54% higher than the lowest value at the start of the light phase. Hence, time of day presents a considerable source of variation in the pharmacokinetics of morphine, which could be used to optimize the dosing strategy of morphine.

Background and PurposeKv11.1 (hERG) channel blockade is an adverse effect of many drugs and lead compounds, associated with lethal cardiac arrhythmias. LUF7244 is a negative allosteric modulator/activator of Kv11.1 channels that inhibits early afterdepolarizations in vitro. We tested LUF7244 for antiarrhythmic efficacy and potential proarrhythmia in a dog model.Experimental ApproachLUF7244 was tested in vitro for (a) increasing human IKv11.1 and canine IKr and (b) decreasing dofetilide‐induced action potential lengthening and early afterdepolarizations in cardiomyocytes derived from human induced pluripotent stem cells and canine isolated ventricular cardiomyocytes. In vivo, LUF7244 was given intravenously to anaesthetized dogs in sinus rhythm or with chronic atrioventricular block.Key ResultsLUF7244 (0.5–10 μM) concentration dependently increased IKv11.1 by inhibiting inactivation. In vitro, LUF7244 (10 μM) had no effects on IKIR2.1, INav1.5, ICa‐L, and IKs, doubled IKr, shortened human and canine action potential duration by approximately 50%, and inhibited dofetilide‐induced early afterdepolarizations. LUF7244 (2.5 mg·kg−1·15 min−1) in dogs with sinus rhythm was not proarrhythmic and shortened, non‐significantly, repolarization parameters (QTc: −6.8%). In dogs with chronic atrioventricular block, LUF7244 prevented dofetilide‐induced torsades de pointes arrhythmias in 5/7 animals without normalization of the QTc. Peak LUF7244 plasma levels were 1.75 ± 0.80 during sinus rhythm and 2.34 ± 1.57 μM after chronic atrioventricular block.Conclusions and ImplicationsLUF7244 counteracted dofetilide‐induced early afterdepolarizations in vitro and torsades de pointes in vivo. Allosteric modulators/activators of Kv11.1 channels might neutralize adverse cardiac effects of existing drugs and newly developed compounds that display QTc lengthening.

To understand the drivers in the biological system response to dopamine D2 receptor antagonists, a mechanistic semiphysiologically based (PB) pharmacokinetic-pharmacodymanic (PKPD) model was developed to describe prolactin responses to risperidone (RIS) and its active metabolite paliperidone (PAL). We performed a microdialysis study in rats to obtain detailed plasma, brain extracellular fluid (ECF), and cerebrospinal fluid (CSF) concentrations of PAL and RIS. To assess the impact of P-glycoprotein (P-gp) functioning on brain distribution, we performed experiments in the absence or presence of the P-gp inhibitor tariquidar (TQD). PK and PKPD modeling was performed by nonlinear mixed-effect modeling. Plasma, brain ECF, and CSF PK values of RIS and PAL were well described by a 12-compartmental semi-PBPK model, including metabolic conversion of RIS to PAL. P-gp efflux functionality was identified on brain ECF for RIS and PAL and on CSF only for PAL. In the PKPD analysis, the plasma drug concentrations were more relevant than brain ECF or CSF concentrations to explain the prolactin response; the estimated EC50 was in accordance with reports in the literature for both RIS and PAL. We conclude that for RIS and PAL, the plasma concentrations better explain the prolactin response than do brain ECF or CSF concentrations. This research shows that PKPD modeling is of high value to delineate the target site of drugs.

This study aimed to investigate the use of quinidine microdialysis to study potential changes in brain P-glycoprotein functionality after induction of status epilepticus (SE) by kainate. Rats were infused with 10 or 20 mg/kg quinidine over 30 min or 4 h. Plasma, brain extracellular fluid (brain ECF), and end-of-experiment total brain concentrations of quinidine were determined during 7 h after the start of the infusion. Effect of pretreatment with tariquidar (15 mg/kg, administered 30 min before the start of the quinidine infusion) on the brain distribution of quinidine was assessed. This approach was repeated in kainate-treated rats. Quinidine kinetics were analyzed with population modeling (NONMEM). The quinidine microdialysis assay clearly revealed differences in brain distribution upon changes in P-glycoprotein functionality by pre-administration of tariquidar, which resulted in a 7.2-fold increase in brain ECF and a 40-fold increase in total brain quinidine concentration. After kainate treatment alone, however, no difference in quinidine transport across the blood–brain barrier was found, but kainate-treated rats tended to have a lower total brain concentration but a higher brain ECF concentration of quinidine than saline-treated rats. This study did not provide evidence for the hypothesis that P-glycoprotein function at the blood–brain barrier is altered at 1 week after SE induction, but rather suggests that P-glycoprotein function might be altered at the brain parenchymal level.

Evidence suggests that the efflux transporter P-glycoprotein (P-gp) may play a facilitatory role in refractory epilepsy by limiting the brain access of antiepileptic drugs (AEDs). We have conducted a preliminary pharmacokinetic study of seven commonly used AEDs in mdr1a knockout mice, devoid of P-gp at the blood-brain barrier. A parallel group of matched wild-type mice served as controls. AEDs were administered by subcutaneous injection and serum and brain drug concentrations determined at 30, 60, and 240min post-dosing. The brain-serum concentration ratio for topiramate was higher in mdr1a(-/-) mice than in wild-type controls at all time points investigated. No consistent effects were observed with any other AED investigated. These findings suggest that topiramate may be a substrate for P-gp-mediated transport. Further studies employing a range of model systems are required to substantiate this observation and to address the potential role of drug transporters in refractory epilepsy.

Nearly all bodily processes exhibit circadian rhythmicity. As a consequence, the pharmacokinetic and pharmacodynamic properties of a drug may also vary with time of day. The objective of this study was to investigate diurnal variation in processes that regulate drug concentrations in the brain, focusing on P-glycoprotein (P-gp). This efflux transporter limits the distribution of many drugs in the brain. To this end, the exposure to the P-gp substrate quinidine was determined in the plasma and brain tissue after intravenous administration in rats at six different time points over the 24-h period. Our results indicate that time of administration significantly affects the exposure to quinidine in the brain. Upon inhibition of P-gp, exposure to quinidine in brain tissue is constant over the 24-h period. To gain more insight into processes regulating brain concentrations, we used intracerebral microdialysis to determine the concentration of quinidine in brain extracellular fluid (ECF) and cerebrospinal fluid (CSF) after intravenous administration at two different time points. The data were analyzed by physiologically based pharmacokinetic modeling using NONMEM. The model shows that the variation is due to higher activity of P-gp-mediated transport from the deep brain compartment to the plasma compartment during the active period. Furthermore, the analysis reveals that CSF flux is higher in the resting period compared to the active period. In conclusion, we show that the exposure to a P-gp substrate in the brain depends on time of administration, thereby providing a new strategy for drug targeting to the brain. Electronic supplementary material The online version of this article (doi:10.1208/s12248-014-9625-4) contains supplementary material, which is available to authorized users.

The antioxidant activity of flavonoids is believed to be caused by a combination of iron chelation and free radical scavenging activities. Several authors have attempted to separate the iron chelation and scavenging activity of flavonoids in order to study these processes individually. There are, however, several contradictions in the literature, and the outcome largely depends on the experimental conditions and the type of assay used. In order to investigate the contribution of iron chelation to the antioxidant activity of flavonoids, we determined the antioxidant activity of a group of flavonoids from several subclasses in an iron-independent (azobisamidinopropane, [ABAP]) lipid peroxidation (LPO) process and compared them with data from an iron-dependent (Fe2+/ascorbate) LPO process, which we determined earlier. These LPO data were compared with oxidation potentials, which were earlier found to have a good correlation with the scavenging activity of flavonoids. For most flavonoids, it was found that there was no difference between the LPO assays, indicating that iron chelation is either a constant factor among the flavonoids or is not significant in these types of assays. The IC50 values in the iron-independent LPO assay showed an excellent correlation with the oxidation potentials (Ep/2). Therefore, it can be concluded that for the majority of flavonoids tested, iron chelation does not play a role in the antioxidant activity in microsomal lipid peroxidation.

Children and adults with malignant diseases have a high risk of prevalence of the tumor in the central nervous system (CNS). As prophylaxis treatment methotrexate is often given. In order to monitor methotrexate exposure in the CNS, cerebrospinal fluid (CSF) concentrations are often measured. However, the question is in how far we can rely on CSF concentrations of methotrexate as appropriate surrogate for brain target site concentrations, especially under disease conditions.In this study, we have investigated the spatial distribution of unbound methotrexate in healthy rat brain by parallel microdialysis, with or without inhibition of Mrp/Oat/Oatp-mediated active transport processes by a co-administration of probenecid. Specifically, we have focused on the relationship between brain extracellular fluid (brainECF) and CSF concentrations. The data were used to develop a systems-based pharmacokinetic (SBPK) brain distribution model for methotrexate. This model was subsequently applied on literature data on methotrexate brain distribution in other healthy and diseased rats (brainECF), healthy dogs (CSF) and diseased children (CSF) and adults (brainECF and CSF).Important differences between brainECF and CSF kinetics were found, but we have found that inhibition of Mrp/Oat/Oatp-mediated active transport processes does not significantly influence the relationship between brainECF and CSF fluid methotrexate concentrations.It is concluded that in parallel obtained data on unbound brainECF, CSF and plasma concentrations, under dynamic conditions, combined with advanced mathematical modeling is a most valid approach to develop SBPK models that allow for revealing the mechanisms underlying the relationship between brainECF and CSF concentrations in health and disease.

Remoxipride is a selective dopamine D(2) receptor antagonist, and useful as a model compound in mechanism-based pharmacological investigations. To that end, studies in small animals with serial sampling over time are needed. For these small volume samples currently no suitable analytical methods are available. We propose analytical methods for the detection of low concentrations remoxipride in small sample volumes of plasma, brain homogenate, and brain microdialysate, using online solid phase extraction with liquid chromatography-tandem mass spectrometry. Method development, optimization and validation are described in terms of calibration curves, extraction yield, lower limit of quantification (LLOQ), precision, accuracy, inter-day- and intra-day variability. The 20 microl plasma samples showed an extraction yield of 76%, with a LLOQ of 0.5 ng/ml. For 0.6 ml brain homogenate samples the extraction yield was 45%, with a LLOQ of 1.8 ng/ml. The 20 microl brain microdialysate samples, without pre-treatment, had a LLOQ of 0.25 ng/ml. The precision and accuracy were well within the acceptable 15% range. Considering the small sample volumes, the high sensitivity and good reproducibility, the analytical methods are suitable for analyzing small sample volumes with low remoxipride concentrations.

The present study provides evidence that chronic intake of a high-fat diet induces a dramatic extravasation of immunoglobulins, indicating alterations in blood-brain barrier (BBB) functioning, in the brains of apolipoprotein E (apoE)-knockout mice, but not of C57Bl/6 control mice. Using sodium fluorescein as a marker for the permeability of the BBB, we found additional support for age-related disturbances of BBB function in apoE-knockout mice. Behavioral analysis of apoE-knockout mice compared with C57Bl/6 mice indicated that they were also less efficient in acquiring the spatial Morris water maze task. Furthermore, apoE-knockout mice are known to develop severe atherosclerosis, which is exacerbated with a high-fat diet. We therefore compared the apoE-knockout mice with the apoE3-Leiden transgenic mice, which are known to develop atherosclerosis. However, apoE3-Leiden mice that were kept on a high-fat, high-cholesterol diet and that developed atherosclerosis to an extent similar to the apoE-knockout mice, showed no signs of BBB disturbances. These results indicate for the first time that apoE plays an essential role in the maintenance of the integrity of the BBB during aging and that it protects the brain from neuropathology induced by a high-fat diet. We therefore hypothesize that the role of apoE in the maintenance of the integrity of the BBB may be the mechanism by which apoE affects the progression of neurodegeneration, as seen in Alzheimer's disease.

The role of mdr1a-encoded P-glycoprotein on transport of several fluoroquinolones across the blood-brain barrier was investigated. In vitro, P-glycoprotein substrates were selected by using a confluent monolayer of MDR1-LLC-PK1 cells. The inhibition of fluoroquinolones (100 microM) on transport of rhodamine-123 (1 microM) was compared with P-glycoprotein inhibitors verapamil (20 microM) and SDZ PSC 833 (2 microM). Subsequently, transport polarity of fluoroquinolones was studied. Sparfloxacin showed the strongest inhibition (26%) and a large polarity in transport, by P-glycoprotein activity. In vivo, using mdr1a (-/-) and wild-type mice, brain distribution of pefloxacin, norfloxacin, ciprofloxacin, fleroxacin and sparfloxacin was determined at 2, 4, and 6 h following intra-arterial infusion (50 nmol/min). Brain distribution of sparfloxacin was clearly higher in mdr1a (-/-) mice compared with wild-type mice. Sparfloxacin was infused (50 nmol/min) for 1, 2, 3 and 4 h in which intracerebral microdialysis was performed. At 4 h, in vivo recovery (dynamic-no-net-flux method) was 6.5+/-2.2 and 1.5+/-0.5%; brain(ECF) concentrations were 5.1+/-0.2 and 26+/-21 microM; and total brain concentrations were 7.2+/-0.3 and 23+/-0.3 microM in wild-type and mdr1a (-/-) mice, respectively. Plasma concentrations were similar (18.4+/-0.7 and 17.9+/-0.5 microM, respectively). In conclusion, sparfloxacin enters the brain poorly mainly because of P-glycoprotein activity at the blood-brain barrier.

Multidrug resistance protein 1 (MRP1) is a transporter protein that helps to protect normal cells and tumor cells against the influx of certain xenobiotics. We previously showed that Mrp1 protects against cytotoxic drugs at the testis-blood barrier, the oral epithelium, and the kidney urinary collecting duct tubules. Here, we generated Mrp1/Mdr1a/Mdr1b triple-knockout (TKO) mice, and used them together with Mdr1a/Mdr1b double-knockout (DKO) mice to study the contribution of Mrp1 to the tissue distribution and pharmacokinetics of etoposide. We observed increased toxicity in the TKO mice, which accumulated etoposide in brown adipose tissue, colon, salivary gland, heart, and the female urogenital system. Immunohistochemical staining revealed the presence of Mrp1 in the oviduct, uterus, salivary gland, and choroid plexus (CP) epithelium. To explore the transport function of Mrp1 in the CP epithelium, we used TKO and DKO mice cannulated for cerebrospinal fluid (CSF). We show here that the lack of Mrp1 protein causes etoposide levels to increase about 10-fold in the CSF after intravenous administration of the drug. Our results indicate that Mrp1 helps to limit tissue distribution of certain drugs and contributes to the blood-CSF drug-permeability barrier.

The cumulative dose-related cardiotoxicity of doxorubicin is believed to be caused by the production of oxygen- free radicals. 7-Monohydroxyethylrutoside (monoHER), a semisynthetic flavonoid and powerful antioxidant, was investigated with respect to the prevention of doxorubicin-induced cardiotoxicity in mice and to its influence on the antitumor activity of doxorubicin in vitro and in vivo. Non-tumor-bearing mice were equipped with a telemeter in the peritoneal cavity. They were given six weekly doses of 4 mg/kg doxorubicin i.v., alone or in combination with either 100 or 250 mg/kg monoHER i.p., 1 h prior to doxorubicin administration and for the following 4 days. Cardiotoxic effects were measured from electrocardiogram changes up to 2 weeks after treatment. Protection against cardiotoxicity was found to be dose dependent, with 53 and 75% protection, respectively, as calculated from the reduction in the increase in the ST interval. MonoHER and several other flavonoids with good antioxidant properties were tested for their antiproliferative effects in the absence or the presence of doxorubicin in A2780 and OVCAR-3 human ovarian cancer cells and MCF-7 human breast cancer cells in vitro. Some flavonoids were directly toxic at 50 and 100 microM, whereas others, including monoHER, did not influence the antiproliferative effects of doxorubicin at these concentrations. The influence of monoHER was further tested on the growth-inhibitory effect of 8 mg/kg doxorubicin i.v., given twice with an interval of 1 week in A2780 and OVCAR-3 cells that were grown as s.c. xenografts in nude mice. MonoHER, administered 1 h before doxorubicin in a dose schedule of 500 mg/kg i.p. 2 or 5 days per week, was not toxic and did not decrease the antitumor activity of doxorubicin. It can be concluded that monoHER showed a dose-dependent protection against chronic cardiotoxicity and did not influence the antitumor activity of doxorubicin in vitro or in vivo.