Atherosclerotic Plaque Epigenetic Age Acceleration Predicts a Poor Prognosis and Is Associated With Endothelial-to-Mesenchymal Transition in Humans.

BACKGROUND: Epigenetic age estimators (clocks) are predictive of human mortality risk. However, it is not yet known whether the epigenetic age of atherosclerotic plaques is predictive for the risk of cardiovascular events. METHODS: Whole-genome DNA methylation of human carotid atherosclerotic plaques (n=485) and of blood (n=93) from the Athero-Express endarterectomy cohort was used to calculate epigenetic age acceleration (EAA). EAA was linked to clinical characteristics, plaque histology, and future cardiovascular events (n=136). We studied whole-genome DNA methylation and bulk and single-cell transcriptomics to uncover molecular mechanisms of plaque EAA. We experimentally confirmed our in silico findings using in vitro experiments in primary human coronary endothelial cells. RESULTS: Male and female patients with severe atherosclerosis had a median chronological age of 69 years. The median epigenetic age was 65 years in females (median EAA, -2.2 [interquartile range, -4.3 to 2.2] years) and 68 years in males (median EAA, -0.3 [interquartile range, -2.9 to 3.8] years). Patients with diabetes and a high body mass index had higher plaque EAA. Increased EAA of plaque predicted future events in a 3-year follow-up in a Cox regression model (univariate hazard ratio, 1.7; P=0.0034) and adjusted multivariate model (hazard ratio, 1.56; P=0.02). Plaque EAA predicted outcome independent of blood EAA (hazard ratio, 1.3; P=0.018) and of plaque hemorrhage (hazard ratio, 1.7; P=0.02). Single-cell RNA sequencing in plaque samples from 46 patients in the same cohort revealed smooth muscle and endothelial cells as important cell types in plaque EAA. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally confirmed by TGFß-triggered endothelial-to-mesenchymal transition inducing rapid epigenetic aging in coronary endothelial cells. CONCLUSIONS: Plaque EAA is a strong and independent marker of poor outcome in patients with severe atherosclerosis. Plaque EAA was linked to mesenchymal endothelial and smooth muscle cells. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally validated. Epigenetic aging mechanisms may provide new targets for treatments that reduce atherosclerosis complications.

Female Gene Networks Are Expressed in Myofibroblast-Like Smooth Muscle Cells in Vulnerable Atherosclerotic Plaques.

BACKGROUND: Women presenting with coronary artery disease more often present with fibrous atherosclerotic plaques, which are currently understudied. Phenotypically modulated smooth muscle cells (SMCs) contribute to atherosclerosis in women. How these phenotypically modulated SMCs shape female versus male plaques is unknown. METHODS: Gene regulatory networks were created using RNAseq gene expression data from human carotid atherosclerotic plaques. The networks were prioritized based on sex bias, relevance for smooth muscle biology, and coronary artery disease genetic enrichment. Network expression was linked to histologically determined plaque phenotypes. In addition, their expression in plaque cell types was studied at single-cell resolution using single-cell RNAseq. Finally, their relevance for disease progression was studied in female and male Apoe-/- mice fed a Western diet for 18 and 30 weeks. RESULTS: Here, we identify multiple sex-stratified gene regulatory networks from human carotid atherosclerotic plaques. Prioritization of the female networks identified 2 main SMC gene regulatory networks in late-stage atherosclerosis. Single-cell RNA sequencing mapped these female networks to 2 SMC phenotypes: a phenotypically modulated myofibroblast-like SMC network and a contractile SMC network. The myofibroblast-like network was mostly expressed in plaques that were vulnerable in women. Finally, the mice ortholog of key driver gene MFGE8 (milk fat globule EGF and factor V/VIII domain containing) showed retained expression in advanced plaques from female mice but was downregulated in male mice during atherosclerosis progression. CONCLUSIONS: Female atherosclerosis is characterized by gene regulatory networks that are active in fibrous vulnerable plaques rich in myofibroblast-like SMCs.

Female gene networks are expressed in myofibroblast-like smooth muscle cells in vulnerable atherosclerotic plaques.

Women presenting with coronary artery disease (CAD) more often present with fibrous atherosclerotic plaques, which are currently understudied. Phenotypically modulated smooth muscle cells (SMCs) contribute to atherosclerosis in women. How these phenotypically modulated SMCs shape female versus male plaques is unknown. Here, we show sex-stratified gene regulatory networks (GRNs) from human carotid atherosclerotic tissue. Prioritization of these networks identified two main SMC GRNs in late-stage atherosclerosis. Single-cell RNA-sequencing mapped these GRNs to two SMC phenotypes: a phenotypically modulated myofibroblast-like SMC network and a contractile SMC network. The myofibroblast-like GRN was mostly expressed in plaques that were vulnerable in females. Finally, mice orthologs of the female myofibroblast-like genes showed retained expression in advanced plaques from female mice but were downregulated in male mice during atherosclerosis progression. Female atherosclerosis is driven by GRNs that promote a fibrous vulnerable plaque rich in myofibroblast-like SMCs.

Altered protein expression of membrane transporters in isolated cerebral microvessels and brain cortex of a rat Alzheimer's disease model.

There is growing evidence that membrane transporters expressed at the blood-brain barrier (BBB) and brain parenchymal cells play an important role in Alzheimer's disease (AD) development and progression. However, quantitative information about changes in transporter protein expression at neurovascular unit cells in AD is limited. Here, we studied the changes in the absolute protein expression of five ATP-binding cassette (ABC) and thirteen solute carrier (SLC) transporters in the isolated brain microvessels and brain cortical tissue of TgF344-AD rats compared to age-matched wild-type (WT) animals using liquid chromatography tandem mass spectrometry based quantitative targeted absolute proteomic analysis. Moreover, sex-specific alterations in transporter expression in the brain cortical tissue of this model were examined. Protein expressions of Abcg2, Abcc1 and FATP1 (encoded by Slc27a1) in the isolated brain microvessels of TgF344-AD rats were 3.1-, 2.0-, 4.3-fold higher compared to WT controls, respectively (p < 0.05). Abcc1 and 4F2hc (encoded by Slc3a2) protein expression was significantly up-regulated in the brain cortical tissue of male TgF344-AD rats compared to male WT rats (p < 0.05). The study provides novel information for the elucidation of molecular mechanisms underlying AD and valuable knowledge about the optimal use of the TgF344-AD rat model in AD drug development and drug delivery research.

Transcriptomic-based clustering of human atherosclerotic plaques identifies subgroups with different underlying biology and clinical presentation.

Histopathological studies have revealed key processes of atherosclerotic plaque thrombosis. However, the diversity and complexity of lesion types highlight the need for improved sub-phenotyping. Here we analyze the gene expression profiles of 654 advanced human carotid plaques. The unsupervised, transcriptome-driven clustering revealed five dominant plaque types. These plaque phenotypes were associated with clinical presentation and showed differences in cellular compositions. Validation in coronary segments showed that the molecular signature of these plaques was linked to coronary ischemia. One of the plaque types with the most severe clinical symptoms pointed to both inflammatory and fibrotic cell lineages. Further, we did a preliminary analysis of potential circulating biomarkers that mark the different plaques phenotypes. In conclusion, the definition of the plaque at risk for a thrombotic event can be fine-tuned by in-depth transcriptomic-based phenotyping. These differential plaque phenotypes prove clinically relevant for both carotid and coronary artery plaques and point to distinct underlying biology of symptomatic lesions.

The changing landscape of the vulnerable plaque: a call for fine-tuning of preclinical models.

For decades, the pathological definition of the vulnerable plaque led to invaluable insights into the mechanisms that underlie myocardial infarction and stroke. Beyond plaque rupture, other mechanisms, such as erosion, may elicit thrombotic events underlining the complexity and diversity of the atherosclerotic disease. Novel insights, based on single-cell transcriptomics and other "omics" methods, provide tremendous opportunities in the ongoing search for cell-specific determinants that will fine-tune the description of the thrombosis prone lesion. It coincides with an increasing awareness that knowledge on lesion characteristics, cell plasticity and clinical presentation of ischemic cardiovascular events have shifted over the past decades. This shift correlates with an observed changes of cell composition towards phenotypical stabilizing of human plaques. These stabilization features and mechanisms are directly mediated by the cells present in plaques and can be mimicked in vitro via primary plaque cells derived from human atherosclerotic tissues. In addition, the rapidly evolving of sequencing technologies identify many candidate genes and molecular mechanisms that may influence the risk of developing an atherosclerotic thrombotic event - which bring the next challenge in sharp focus: how to translate these cell-specific insights into tangible functional and translational discoveries?

Sex-dependent gene co-expression in the human body.

Many pathophysiological mechanisms in human health and disease are dependent on sex. Systems biology approaches are successfully used to decipher human disease etiology, yet the effect of sex on gene network biology is mostly unknown. To address this, we used RNA-sequencing data of over 700 individuals spanning 24 tissues from the Genotype-Tissue Expression project to generate a whole-body gene co-expression map and quantified the sex differences per tissue. We found that of the 13,787 genes analyzed in 24 tissues, 29.5% of the gene co-expression is influenced by sex. For example, skeletal muscle was predominantly enriched with genes co-expressed stronger in males, whereas thyroid primarily contained genes co-expressed stronger in females. This was accompanied by consistent sex differences in pathway enrichment, including hypoxia, epithelial-to-mesenchymal transition, and inflammation over the human body. Furthermore, multi-organ analyses revealed consistent sex-dependent gene co-expression over numerous tissues which was accompanied by enrichment of transcription factor binding motifs in the promoters of these genes. Finally, we show that many sex-biased genes are associated with sex-biased diseases, such as autoimmunity and cancer, and more often the target of FDA-approved drugs than non-sexbiased genes. Our study suggests that sex affects biological gene networks by differences in gene co-expression and that attention to the effect of sex on biological responses to medical drugs is warranted.

Plasma Testosterone Levels and Atherosclerotic Plaque Gene Expression in Men With Advanced Atherosclerosis.

Aims: Low plasma testosterone levels have been shown to predict worse outcome in men with severe atherosclerotic disease. We hypothesized that a low plasma testosterone level affects disease risk through changes in gene expression in atherosclerotic plaques. Therefore, we studied plasma testosterone levels in relation to gene expression levels in atherosclerotic plaque tissue of men with advanced atherosclerotic disease. Methods: Plasma testosterone levels were measured in 203 men undergoing carotid endarterectomy. The corresponding atherosclerotic plaque tissue was used for RNA sequencing. First, we assessed how often the androgen receptor gene was expressed in the plaque. Second, correlations between plasma testosterone levels and pre-selected testosterone-sensitive genes were assessed. Finally, differences within the RNA expression profile of the plaque as a whole, characterized into gene regulatory networks and at individual gene level were assessed in relation to testosterone levels. Results: Testosterone plasma levels were low with a median of 11.6 nmol/L (IQR: 8.6-13.8). RNA-seq of the plaque resulted in reliable expression data for 18,850 genes to be analyzed. Within the RNA seq data, the androgen-receptor gene was expressed in 189 out of 203 (93%) atherosclerotic plaques of men undergoing carotid endarterectomy. The androgen receptor gene expression was not associated with testosterone plasma levels. There were no significant differences in gene expression of atherosclerotic plaques between different endogenous testosterone levels. This remained true for known testosterone-sensitive genes, the complete transcriptomic profile, male-specific gene co-expression modules as well as for individual genes. Conclusion: In men with severe atherosclerotic disease the androgen receptor is highly expressed in plaque tissue. However, plasma testosterone levels were neither associated with pre-selected testosterone sensitive genes, gene expression profiles nor gene regulatory networks in late-stage atherosclerotic plaques. The effect of testosterone on gene expression of the late-stage atherosclerotic plaque appears limited, suggesting that alternate mechanisms explain its effect on clinical outcomes.

Sex-Stratified Gene Regulatory Networks Reveal Female Key Driver Genes of Atherosclerosis Involved in Smooth Muscle Cell Phenotype Switching.

BACKGROUND: Although sex differences in coronary artery disease are widely accepted with women developing more stable atherosclerosis than men, the underlying pathobiology of such differences remains largely unknown. In coronary artery disease, recent integrative systems biological studies have inferred gene regulatory networks (GRNs). Within these GRNs, key driver genes have shown great promise but have thus far been unidentified in women. METHODS: We generated sex-specific GRNs of the atherosclerotic arterial wall in 160 women and age-matched men in the STARNET study (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task). We integrated the female GRNs with single-cell RNA-sequencing data of the human atherosclerotic plaque and single-cell RNA sequencing of advanced atherosclerotic lesions from wild type and Klf4 knockout atherosclerotic smooth muscle cell (SMC) lineage-tracing mice. RESULTS: By comparing sex-specific GRNs, we observed clear sex differences in network activity within the atherosclerotic tissues. Genes more active in women were associated with mesenchymal cells and endothelial cells, whereas genes more active in men were associated with the immune system. We determined that key drivers of GRNs active in female coronary artery disease were predominantly found in (SMCs by single-cell sequencing of the human atherosclerotic plaques, and higher expressed in female plaque SMCs, as well. To study the functions of these female SMC key drivers in atherosclerosis, we examined single-cell RNA sequencing of advanced atherosclerotic lesions from wild type and Klf4 knockout atherosclerotic SMC lineage-tracing mice. The female key drivers were found to be expressed by phenotypically modulated SMCs and affected by Klf4, suggesting that sex differences in atherosclerosis involve phenotypic switching of plaque SMCs. CONCLUSIONS: Our systems approach provides novel insights into molecular mechanisms that underlie sex differences in atherosclerosis. To discover sex-specific therapeutic targets for atherosclerosis, an increased emphasis on sex-stratified approaches in the analysis of multi-omics data sets is warranted.

Intrinsic transcriptomic sex differences in human endothelial cells at birth and in adults are associated with coronary artery disease targets.

Sex differences in endothelial cell (EC) biology may reflect intrinsic differences driven by chromosomes or sex steroid exposure and gender differences accumulated over life. We analysed EC gene expression data from boy-girl twins at birth and in non-twin adults to detect sex differences at different stages of life, and show that 14-25% of the EC transcriptome is sex-biased. By combining data from both stages of life, we identified sex differences that are present at birth and maintained throughout life, and those that are acquired over life. Promisingly, we found that genes that present with an acquired sex difference in ECs are more likely to be targets of sex steroids. Annotating both gene sets with data from multiple genome-wide association studies (GWAS) revealed that genes with an intrinsic sex difference in ECs are enriched for coronary artery disease GWAS hits. This study underscores the need for treating sex as a biological variable.

Platelet RNA modules point to coronary calcification in asymptomatic women with former preeclampsia.

BACKGROUND AND AIMS: Women who develop preeclampsia during pregnancy are at a higher risk for developing cardiovascular disease. As platelets are affected by preeclampsia, we set out to identify whether platelets carry information in their transcriptome on cardiovascular risk in women with former preeclampsia. METHODS: Platelets were isolated from asymptomatic women with previous preeclampsia, who underwent screening with coronary computed tomography angiography. Platelet RNA was isolated and used to construct gene networks using an unbiased approach. Platelet gene modules assembled from the network were related to risk factors and clinical traits of these women, including coronary artery calcium scores (CACS). RESULTS: We found multiple gene modules which correlated with CACS (correlation coefficients: 0.44 to 0.59, p = 0.05 to 0.007). The genes from two clinically relevant modules were expressed at a higher level in the group with calcifications (p = 3.9 × 10-10 and 0.02) and enriched for platelet-related gene-sets such as platelet activation. The first of these modules was also enriched (ppermutation = 0.0546) for genes mapped to known coronary artery disease susceptibility loci. Additional unbiased network analyses in platelet RNA of patients with overt cardiovascular disease underlined the importance of the identified modules for disease by high preservation. (p = 1.6 × 10-9 to 1.7 × 10-47). CONCLUSIONS: We found platelet RNA modules that correlated with CACS in asymptomatic women with previous preeclampsia. Whether or not platelets directly contribute to this disease trajectory, or reflect the underlying plaque substrate remains to be determined, but enrichment for coronary artery disease susceptibility genes emphasizes the importance for the disease.

Photoacoustic imaging of gold nanorods in the brain delivered via microbubble-assisted focused ultrasound: a tool for in vivo molecular neuroimaging.

The protective barriers of the CNS present challenges during the treatment and monitoring of diseases. In particular, the blood brain barrier is a major hindrance to the delivery of imaging contrast agents and therapeutics to the brain. In this work, we use gas microbubble-assisted focused ultrasound to transiently open the blood brain barrier and locally deliver silica coated gold nanorods across the barrier. This particular nanoagent possesses a strong optical absorption which enables in vivo and ex vivo visualization of the delivered particles using ultrasound-guided photoacoustic imaging. The results of these studies demonstrate the potential of ultrasound-guided photoacoustics to image contrast agents delivered via microbubble-assisted focused ultrasound for longitudinal diagnostic imaging and for therapeutic monitoring of neurological diseases.

Blood-Based Biomarkers of Quinpirole Pharmacology: Cluster-Based PK/PD and Metabolomics to Unravel the Underlying Dynamics in Rat Plasma and Brain.

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.

Fingerprints of CNS drug effects: a plasma neuroendocrine reflection of D2 receptor activation using multi-biomarker pharmacokinetic/pharmacodynamic modelling.

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.

Sex differences in cardiovascular epigenetics-a systematic review.

BACKGROUND: Differences in cardiovascular diseases are evident in men and women throughout life and are mainly attributed to the presence of sex hormones and chromosomes. Epigenetic mechanisms drive the regulation of the biological processes that may lead to CVD and are possibly influenced by sex. In order to gain an overview of the status quo on sex differences in cardiovascular epigenetics, we performed a systematic review. MATERIALS AND METHODS: A systematic search was performed on PubMed and Embase for studies mentioning cardiovascular disease, epigenetics, and anything related to sex differences. The search returned 3071 publications to be screened. Primary included publications focused on cardiovascular and epigenetics research. Subsequently, papers were assessed for including both sexes in their studies and checked for appropriate sex stratification of results. RESULTS: Two independent screeners identified 75 papers in the proper domains that had included both sexes. Only 17% (13 papers out of 75) of these publications stratified some of their data according to sex. All remaining papers focused on DNA methylation solely as an epigenetic mechanism. Of the excluded papers that included only one sex, 86% (24 out 28) studied males, while 14% (4 out of 28) studied females. CONCLUSION: Our overview indicates that the majority of studies into cardiovascular epigenetics do not show their data stratified by sex, despite the well-known sex differences in CVD. All included and sex-stratified papers focus on DNA methylation, indicating that a lot of ground is still to gain regarding other epigenetic mechanisms, like chromatin architecture, and histone modifications. More attention to sex in epigenetic studies is warranted as such integration will advance our understanding of cardiovascular disease mechanisms in men and women.

Laser-activated perfluorocarbon nanodroplets: a new tool for blood brain barrier opening.

A major obstacle in the monitoring and treatment of neurological diseases is the blood brain barrier (BBB), a semipermeable barrier that prevents the delivery of many therapeutics and imaging contrast agents to the brain. In this work, we explored the possibility of laser-activated perfluorocarbon nanodroplets (PFCnDs) to open the BBB and deliver agents to the brain tissue. Specifically, near infrared (NIR) dye-loaded PFCnDs comprised of a perfluorocarbon (PFC) core with a boiling point above physiological temperature were repeatedly vaporized and recondensed from liquid droplet to gas bubble under pulsed laser excitation. As a result, this pulse-to-pulse repeated behavior enabled the recurring interaction of PFCnDs with the endothelial lining of the BBB, allowing for a BBB opening and extravasation of dye into the brain tissue. The blood brain barrier opening and delivery of agents to tissue was confirmed on the macro and the molecular level by evaluating Evans Blue staining, ultrasound-guided photoacoustic (USPA) imaging, and histological tissue analysis. The demonstrated PFCnD-assisted pulsed laser method for BBB opening, therefore, represents a tool that has the potential to enable non-invasive, cost-effective, and efficient image-guided delivery of contrast and therapeutic agents to the brain.

Development of a population pharmacokinetic model to predict brain distribution and dopamine D2 receptor occupancy of raclopride in non-anesthetized rat.

BACKGROUND: Raclopride is a selective antagonist of the dopamine D2 receptor. It is one of the most frequently used in vivo D2 tracers (at low doses) for assessing drug-induced receptor occupancy (RO) in animals and humans. It is also commonly used as a pharmacological blocker (at high doses) to occupy the available D2 receptors and antagonize the action of dopamine or drugs on D2 in preclinical studies. The aims of this study were to comprehensively evaluate its pharmacokinetic (PK) profiles in different brain compartments and to establish a PK-RO model that could predict the brain distribution and RO of raclopride in the freely moving rat using a LC-MS based approach. METHODS: Rats (n=24) received a 10-min IV infusion of non-radiolabeled raclopride (1.61µmol/kg, i.e. 0.56mg/kg). Plasma and the brain tissues of striatum (with high density of D2 receptors) and cerebellum (with negligible amount of D2 receptors) were collected. Additional microdialysis experiments were performed in some rats (n=7) to measure the free drug concentration in the extracellular fluid of the striatum and cerebellum. Raclopride concentrations in all samples were analyzed by LC-MS. A population PK-RO model was constructed in NONMEM to describe the concentration-time profiles in the unbound plasma, brain extracellular fluid and brain tissue compartments and to estimate the RO based on raclopride-D2 receptor binding kinetics. RESULTS: In plasma raclopride showed a rapid distribution phase followed by a slower elimination phase. The striatum tissue concentrations were consistently higher than that of cerebellum tissue throughout the whole experimental period (10-h) due to higher non-specific tissue binding and D2 receptor binding in the striatum. Model-based simulations accurately predicted the literature data on rat plasma PK, brain tissue PK and D2 RO at different time points after intravenous or subcutaneous administration of raclopride at tracer dose (RO <10%), sub-pharmacological dose (RO 10%-30%) and pharmacological dose (RO >30%). CONCLUSION: For the first time a predictive model that could describe the quantitative in vivo relationship between dose, PK and D2 RO of raclopride in non-anesthetized rat was established. The PK-RO model could facilitate the selection of optimal dose and dosing time when raclopride is used as tracer or as pharmacological blocker in various rat studies. The LC-MS based approach, which doses and quantifies a non-radiolabeled tracer, could be useful in evaluating the systemic disposition and brain kinetics of tracers.

Predicting Drug Concentration-Time Profiles in Multiple CNS Compartments Using a Comprehensive Physiologically-Based Pharmacokinetic Model.

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 <91%). In conclusion, the developed PBPK model can be used to predict temporal concentration profiles of drugs in multiple relevant CNS compartments, which we consider valuable information for efficient CNS drug development.

Diurnal variation in the pharmacokinetics and brain distribution of morphine and its major metabolite.

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, 4mg/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.

Target-Site Investigation for the Plasma Prolactin Response: Mechanism-Based Pharmacokinetic-Pharmacodynamic Analysis of Risperidone and Paliperidone in the Rat.

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.