Clustering molecular dynamics conformations of the CC'-loop of the PD-1 immuno-checkpoint receptor.

Molecular mechanisms within the checkpoint receptor PD-1 are essential for its activation by PD-L1 as well as for blocking such an activation via checkpoint inhibitors. We use molecular dynamics to scrutinize patterns of atomic motion in PD-1 without a ligand. Molecular dynamics is performed for the whole extracellular domain of PD-1, and the analysis focuses on its CC'-loop and some adjacent Cα-atoms. We extend previous work by applying common nearest neighbor clustering (Cnn) and compare the performance of this method with Daura clustering as well as UMAP dimension reduction and subsequent agglomerative linkage clustering. As compared to Daura clustering, we found Cnn less sensitive to cutoff selection and better able to return representative clusters for sets of different 3D atomic conformations. Interestingly, Cnn yields results quite similar to UMAP plus linkage clustering.

Flexible Risk Evidence Combination Rules in Breast Cancer Precision Therapy.

Evidence theory by Dempster-Shafer for determination of hormone receptor status in breast cancer samples was introduced in our previous paper. One major topic pointed out here is the link between pieces of evidence found from different origins. In this paper the challenge of selecting appropriate ways of fusing evidence, depending on the type and quality of data involved is addressed. A parameterized family of evidence combination rules, covering the full range of potential needs, from emphasizing discrepancies in the measurements to aspiring accordance, is covered. The consequences for real patient samples are shown by modeling different decision strategies.

Molecular dynamics identifies semi-rigid domains in the PD-1 checkpoint receptor bound to its natural ligand PD-L1.

Cells in danger of being erroneously attacked by leucocytes express PD-L1 on their surface. These cells activate PD-1 on attacking leucocytes and send them to death, thus curbing erroneous, autoimmune attack. Unfortunately, cancer cells exploit this mechanism: By expressing PD-L1, they guard themselves against leucocyte attack and thereby evade immune clearance. Checkpoint inhibitors are drugs which re-enable immune clearance of cancer cells by blocking the binding of PD-L1 to PD-1 receptors. It is therefore of utmost interest to investigate these binding mechanisms. We use three 600 ns all-atom molecular dynamics simulations to scrutinize molecular motions of PD-1 with its binding partner, the natural ligand PD-L1. Usually, atomic motion patterns are evaluated against whole molecules as a reference, disregarding that such a reference is a dynamic entity by itself, thus degrading stability of the reference. As a remedy, we identify semi-rigid domains, lending themselves as more stable and reliable reference frames against which even minute differences in molecular motion can be quantified precisely. We propose an unsupervised three-step procedure. In previous work of our group and others, minute differences in motion patterns proved decisive for differences in function. Here, several highly reliable frames of reference are established for future investigations based on molecular motion.

Conformational flexibility of a free and TCR-bound pMHC-I protein investigated by long-term molecular dynamics simulations.

BACKGROUND: Major histocompatibility complexes (MHCs) play a crucial role in the cell-mediated adaptive immune response as they present antigenic peptides (p) which are recognized by host T cells through a complex formation of the T cell receptor (TCR) with pMHC. In the present study, we report on changes in conformational flexibility within a pMHC molecule upon TCR binding by looking at molecular dynamics (MD) simulations of the free and the TCR-bound pMHC-I protein of the LC13-HLA-B*44:05-pEEYLQAFTY complex. RESULTS: We performed long-term MD simulations with a total simulation time of 8 µs, employing 10 independent 400 ns replicas for the free and the TCR-bound pMHC system. Upon TCR ligation, we observed a reduced dynamic flexibility in the central residues of the peptide and the MHC α1-helix, altered occurrences of hydrogen bonds between the peptide and the MHC, a reduced conformational entropy of the peptide-binding groove, as well as a decreased solvent accessible surface area. CONCLUSIONS: In summary, our results from 8 µs MD simulations indicate a restricted conformational space of the MHC peptide-binding groove upon TCR ligation and suggest a minimum simulation time of approximately 100 ns for biomolecules of comparable complexity to draw meaningful conclusions. Given the relatively long total simulation time, our results contribute to a more detailed view on conformational flexibility properties of the investigated free and TCR-bound pMHC-I system.

Decision Theory versus Conventional Statistics for Personalized Therapy of Breast Cancer.

Estrogen and progesterone receptors being present or not represents one of the most important biomarkers for therapy selection in breast cancer patients. Conventional measurement by immunohistochemistry (IHC) involves errors, and numerous attempts have been made to increase precision by additional information from gene expression. This raises the question of how to fuse information, in particular, if there is disagreement. It is the primary domain of Dempster-Shafer decision theory (DST) to deal with contradicting evidence on the same item (here: receptor status), obtained through different techniques. DST is widely used in technical settings, such as self-driving cars and aviation, and is also promising to deliver significant advantages in medicine. Using data from breast cancer patients already presented in previous work, we focus on comparing DST with classical statistics in this work, to pave the way for its application in medicine. First, we explain how DST not only considers probabilities (a single number per sample), but also incorporates uncertainty in a concept of 'evidence' (two numbers per sample). This allows for very powerful displays of patient data in so-called ternary plots, a novel and crucial advantage for medical interpretation. Results are obtained according to conventional statistics (ODDS) and, in parallel, according to DST. Agreement and differences are evaluated, and the particular merits of DST discussed. The presented application demonstrates how decision theory introduces new levels of confidence in diagnoses derived from medical data.

ABCB1 and ABCG2 Together Limit the Distribution of ABCB1/ABCG2 Substrates to the Human Retina and the ABCG2 Single Nucleotide Polymorphism Q141K (c.421C> A) May Lead to Increased Drug Exposure.

The widely expressed and poly-specific ABC transporters breast cancer resistance protein (ABCG2) and P-glycoprotein (ABCB1) are co-localized at the blood-brain barrier (BBB) and have shown to limit the brain distribution of several clinically used ABCB1/ABCG2 substrate drugs. It is currently not known to which extent these transporters, which are also expressed at the blood-retinal barrier (BRB), may limit drug distribution to the human eye and whether the ABCG2 reduced-function single-nucleotide polymorphism (SNP) Q141K (c.421C > A) has an impact on retinal drug distribution. Ten healthy male volunteers (five subjects with the c.421CC and c.421CA genotype, respectively) underwent two consecutive positron emission tomography (PET) scans after intravenous injection of the model ABCB1/ABCG2 substrate [11C]tariquidar. The second PET scan was performed with concurrent intravenous infusion of unlabelled tariquidar to inhibit ABCB1 in order to specifically reveal ABCG2 function.In response to ABCB1 inhibition with unlabelled tariquidar, ABCG2 c.421C > A genotype carriers showed significant increases (as compared to the baseline scan) in retinal radiotracer influx K 1 (+62 ± 57%, p = 0.043) and volume of distribution V T (+86 ± 131%, p = 0.043), but no significant changes were observed in subjects with the c.421C > C genotype. Our results provide the first evidence that ABCB1 and ABCG2 may together limit the distribution of systemically administered ABCB1/ABCG2 substrate drugs to the human retina. Functional redundancy between ABCB1 and ABCG2 appears to be compromised in carriers of the c.421C > A SNP who may therefore be more susceptible to transporter-mediated drug-drug interactions at the BRB than non-carriers.

Influence of ABC transporters on the excretion of ciprofloxacin assessed with PET imaging in mice.

Ciprofloxacin is a commonly prescribed fluoroquinolone antibiotic which is cleared by active tubular secretion and intestinal excretion. Ciprofloxacin is a known substrate of the ATP-binding cassette (ABC) transporters breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 4 (MRP4). In this work, we used positron emission tomography (PET) imaging to investigate the influence of BCRP, MRP4, MRP2 and P-glycoprotein (P-gp) on the excretion of [18F]ciprofloxacin in mice. Dynamic 90-min PET scans were performed after intravenous injection of [18F]ciprofloxacin in wild-type mice without and with pre-treatment with the broad-spectrum MRP inhibitor MK571. Moreover, [18F]ciprofloxacin PET scans were performed in Abcc4(-/-), Abcc2(-/-), Abcc4(-/-)Abcg2(-/-) and Abcb1a/b(-/-)Abcg2(-/-) mice. In addition to non-compartmental pharmacokinetic (PK) analysis, a novel three-compartment PK model was developed for a detailed assessment of the renal disposition of [18F]ciprofloxacin. In MK571 pre-treated mice, a significant increase in the blood exposure to [18F]ciprofloxacin was observed along with a significant reduction in the renal and intestinal clearances. PK modelling revealed a significant reduction in renal radioactivity uptake (CL1) and in the rate constants for transfer of radioactivity from the corticomedullary renal region into blood (k2) and urine (k3), respectively, after MK571 administration. No changes in the renal clearance or in the estimated kidney PK model parameters were observed in any of the studied knockout models, while a significant reduction in the intestinal clearance was observed in Abcc2(-/-) and Abcc4(-/-)Abcg2(-/-) mice. Our data failed to reveal a role of any of the studied ABC transporters in the tubular secretion of ciprofloxacin. This may indicate that ciprofloxacin is handled in the kidneys by more than one transporter family, most likely with a great degree of mutual functional redundancy. Our study highlights the potential of PET imaging for an assessment of transporter-mediated renal excretion of radiolabelled drugs.

Decision theory for precision therapy of breast cancer.

Correctly estimating the hormone receptor status for estrogen (ER) and progesterone (PGR) is crucial for precision therapy of breast cancer. It is known that conventional diagnostics (immunohistochemistry, IHC) yields a significant rate of wrongly diagnosed receptor status. Here we demonstrate how Dempster Shafer decision Theory (DST) enhances diagnostic precision by adding information from gene expression. We downloaded data of 3753 breast cancer patients from Gene Expression Omnibus. Information from IHC and gene expression was fused according to DST, and the clinical criterion for receptor positivity was re-modelled along DST. Receptor status predicted according to DST was compared with conventional assessment via IHC and gene-expression, and deviations were flagged as questionable. The survival of questionable cases turned out significantly worse (Kaplan Meier p < 1%) than for patients with receptor status confirmed by DST, indicating a substantial enhancement of diagnostic precision via DST. This study is not only relevant for precision medicine but also paves the way for introducing decision theory into OMICS data science.

Safety, tolerability, pharmacokinetics and pharmacodynamics of parenterally administered dutogliptin: A prospective dose-escalating trial.

AIMS: Animal studies suggest that inhibition of dipeptidyl peptidase 4 (DPP-IV) may improve heart function and survival after myocardial infarction by increasing cardiac myocytes' regenerative capacity. Parenterally administered dutogliptin may provide continuous strong DPP-IV inhibition to translate these results into humans. This trial investigated the safety and tolerability, as well as pharmacokinetics and pharmacodynamics, of parenterally administered dutogliptin after single and repeated doses. METHODS: In an open-label trial, volunteers received dutogliptin at increasing doses of 30-120 mg subcutaneously or 30 mg intravenously in the single-dose cohorts. Subjects in the multiple-dose cohort received 60, 90 or 120 mg dutogliptin subcutaneously once daily on 7 consecutive days. RESULTS: Forty healthy males were included in the trial. No related serious adverse events occurred. Mild local injection site reactions with no requirement for intervention comprised 147 of 153 (96%) related adverse events. Subcutaneous bioavailability was approximately 100%. Multiple injections at daily intervals did not lead to the accumulation of the study drug. The accumulation ratios based on AUC0-24h range from 0.90 to 1.03, supporting this argument. All subjects receiving ≥60 mg dutogliptin yielded a maximum DPP-IV inhibition >90%. The duration of DPP-IV inhibition over time increased in a dose-dependent manner and was highest in the 120-mg multiple-dosing cohort with a maximum AUEC0-24h of 342 h % (standard deviation: 73), translating into 86% DPP-IV inhibition 24 hours after dosing. CONCLUSION: Parenteral injection of dutogliptin was safe and subcutaneous bioavailability is excellent. DPP-IV inhibition increased dose dependently to >86% over 24 hours after multiple doses of 120 mg dutogliptin.

Assessment of brain delivery of a model ABCB1/ABCG2 substrate in patients with non-contrast-enhancing brain tumors with positron emission tomography.

BACKGROUND: P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) are two efflux transporters expressed at the blood-brain barrier which effectively restrict the brain distribution of the majority of currently known anticancer drugs. High-grade brain tumors often possess a disrupted blood-brain tumor barrier (BBTB) leading to enhanced accumulation of magnetic resonance imaging contrast agents, and possibly anticancer drugs, as compared to normal brain. In contrast to high-grade brain tumors, considerably less information is available with respect to BBTB integrity in lower grade brain tumors. MATERIALS AND METHODS: We performed positron emission tomography imaging with the radiolabeled ABCB1 inhibitor [11C]tariquidar, a prototypical ABCB1/ABCG2 substrate, in seven patients with non-contrast -enhancing brain tumors (WHO grades I-III). In addition, ABCB1 and ABCG2 levels were determined in surgically resected tumor tissue of four patients using quantitative targeted absolute proteomics. RESULTS: Brain distribution of [11C]tariquidar was found to be very low across the whole brain and not significantly different between tumor and tumor-free brain tissue. Only one patient showed a small area of enhanced [11C]tariquidar uptake within the brain tumor. ABCG2/ABCB1 ratios in surgically resected tumor tissue (1.4 ± 0.2) were comparable to previously reported ABCG2/ABCB1 ratios in isolated human micro-vessels (1.3), which suggested that no overexpression of ABCB1 or ABCG2 occurred in the investigated tumors. CONCLUSIONS: Our data suggest that the investigated brain tumors had an intact BBTB, which is impermeable to anticancer drugs, which are dual ABCB1/ABCG2 substrates. Therefore, effective drugs for antitumor treatment should have high passive permeability and lack ABCB1/ABCG2 substrate affinity. TRIAL REGISTRATION: European Union Drug Regulating Authorities Clinical Trials Database (EUDRACT), 2011-004189-13. Registered on 23 February 2012, https://www.clinicaltrialsregister.eu/ctr-search/search?query=2011-004189-13.

Intramolecular Domain Movements of Free and Bound pMHC and TCR Proteins: A Molecular Dynamics Simulation Study.

The interaction of antigenic peptides (p) and major histocompatibility complexes (pMHC) with T-cell receptors (TCR) is one of the most important steps during the immune response. Here we present a molecular dynamics simulation study of bound and unbound TCR and pMHC proteins of the LC13-HLA-B*44:05-pEEYLQAFTY complex to monitor differences in relative orientations and movements of domains between bound and unbound states of TCR-pMHC. We generated local coordinate systems for MHC α1- and MHC α2-helices and the variable T-cell receptor regions TCR Vα and TCR Vß and monitored changes in the distances and mutual orientations of these domains. In comparison to unbound states, we found decreased inter-domain movements in the simulations of bound states. Moreover, increased conformational flexibility was observed for the MHC α2-helix, the peptide, and for the complementary determining regions of the TCR in TCR-unbound states as compared to TCR-bound states.

Towards Improved Pharmacokinetic Models for the Analysis of Transporter-Mediated Hepatic Disposition of Drug Molecules with Positron Emission Tomography.

Positron emission tomography (PET) imaging with radiolabeled drugs holds great promise to assess the influence of membrane transporters on hepatobiliary clearance of drugs. To exploit the full potential of PET, quantitative pharmacokinetic models are required. In this study, we evaluated the suitability of different compartment models to describe the hepatic disposition of [11C]erlotinib as a small-molecule model drug which undergoes transporter-mediated hepatobiliary excretion. We analyzed two different, previously published data sets in healthy volunteers, in which a baseline [11C]erlotinib PET scan was followed by a second PET scan either after oral intake of unlabeled erlotinib (300 mg) or after intravenous infusion of the prototypical organic anion-transporting polypeptide inhibitor rifampicin (600 mg). We assessed a three-compartment (3C) and a four-compartment (4C) model, in which either a sampled arterial blood input function or a mathematically derived dual input function (DIF), which takes the contribution of the portal vein to the liver blood supply into account, was used. Both models provided acceptable fits of the observed PET data in the liver and extrahepatic bile duct and gall bladder. Changes in model outcome parameters between scans were consistent with the involvement of basolateral hepatocyte uptake and canalicular efflux transporters in the hepatobiliary clearance of [11C]erlotinib. Our results demonstrated that inclusion of a DIF did not lead to substantial improvements in model fits. The models developed in this work represent a step forward in applying PET as a tool to assess the impact of hepatic transporters on drug disposition and their involvement in drug-drug interactions.

The role of the nAChR subunits α5, ß2, and ß4 on synaptic transmission in the mouse superior cervical ganglion.

Our previous immunoprecipitation analysis of nicotinic acetylcholine receptors (nAChRs) in the mouse superior cervical ganglion (SCG) revealed that approximately 55%, 24%, and 21% of receptors are comprised of α3ß4, α3ß4α5, and α3ß4ß2 subunits, respectively. Moreover, mice lacking ß4 subunits do not express α5-containing receptors but still express a small number of α3ß2 receptors. Here, we investigated how synaptic transmission is affected in the SCG of α5ß4-KO and α5ß2-KO mice. Using an ex vivo SCG preparation, we stimulated the preganglionic cervical sympathetic trunk and measured compound action potentials (CAPs) in the postganglionic internal carotid nerve. We found that CAP amplitude was unaffected in α5ß4-KO and α5ß2-KO ganglia, whereas the stimulation threshold for eliciting CAPs was significantly higher in α5ß4-KO ganglia. Moreover, intracellular recordings in SCG neurons revealed no difference in EPSP amplitude. We also found that the ganglionic blocking agent hexamethonium was the most potent in α5ß4-KO ganglia (IC50 : 22.1 µmol/L), followed by α5ß2-KO (IC50 : 126.7 µmol/L) and WT ganglia (IC50 : 389.2 µmol/L). Based on these data, we estimated an IC50 of 568.6 µmol/L for a receptor population consisting solely of α3ß4α5 receptors; and we estimated that α3ß4α5 receptors comprise 72% of nAChRs expressed in the mouse SCG. Similarly, by measuring the effects of hexamethonium on ACh-induced currents in cultured SCG neurons, we found that α3ß4α5 receptors comprise 63% of nAChRs. Thus, in contrast to our results obtained using immunoprecipitation, these data indicate that the majority of receptors at the cell surface of SCG neurons consist of α3ß4α5.

Impact of P-Glycoprotein Function on the Brain Kinetics of the Weak Substrate 11C-Metoclopramide Assessed with PET Imaging in Humans.

PET with avid substrates of P-glycoprotein (ABCB1) provided evidence of the role of this efflux transporter in effectively restricting the brain penetration of its substrates across the human blood-brain barrier (BBB). This may not reflect the situation for weak ABCB1 substrates including several antidepressants, antiepileptic drugs, and neuroleptics, which exert central nervous system effects despite being transported by ABCB1. We performed PET with the weak ABCB1 substrate 11C-metoclopramide in humans to elucidate the impact of ABCB1 function on its brain kinetics. Methods: Ten healthy male subjects underwent 2 consecutive 11C-metoclopramide PET scans without and with ABCB1 inhibition using cyclosporine A (CsA). Pharmacokinetic modeling was performed to estimate the total volume of distribution (VT) and the influx (K1) and efflux (k2) rate constants between plasma and selected brain regions. Furthermore, 11C-metoclopramide washout from the brain was estimated by determining the elimination slope (kE,brain) of the brain time-activity curves. Results: In baseline scans, 11C-metoclopramide showed appreciable brain distribution (VT = 2.11 ± 0.33 mL/cm3). During CsA infusion, whole-brain gray matter VT and K1 were increased by 29% ± 17% and 9% ± 12%, respectively. K2 was decreased by 15% ± 5%, consistent with a decrease in kE,brain (-32% ± 18%). The impact of CsA on outcome parameters was significant and similar across brain regions except for the pituitary gland, which is not protected by the BBB. Conclusion: Our results show for the first time that ABCB1 does not solely account for the "barrier" property of the BBB but also acts as a detoxifying system to limit the overall brain exposure to its substrates at the human blood-brain interface.

A Proof-of-Concept Study to Inhibit ABCG2- and ABCB1-Mediated Efflux Transport at the Human Blood-Brain Barrier.

The adenosine triphosphate-binding cassette transporters P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) are 2 efflux transporters at the blood-brain barrier (BBB) that effectively restrict brain distribution of dual ABCB1/ABCG2 substrate drugs, such as tyrosine kinase inhibitors. Pharmacologic inhibition of ABCB1/ABCG2 may improve the efficacy of dual-substrate drugs for treatment of brain tumors, but no marketed ABCB1/ABCG2 inhibitors are currently available. In the present study, we examined the potential of supratherapeutic-dose oral erlotinib to inhibit ABCB1/ABCG2 activity at the human BBB. Methods: Healthy men underwent 2 consecutive PET scans with 11C-erlotinib: a baseline scan and a second scan either with concurrent intravenous infusion of the ABCB1 inhibitor tariquidar (3.75 mg/min, n = 5) or after oral intake of single ascending doses of erlotinib (300 mg, n = 7; 650 mg, n = 8; or 1,000 mg, n = 2). Results: Although tariquidar administration had no effect on 11C-erlotinib brain distribution, oral erlotinib led, at the 650-mg dose, to significant increases in volume of distribution (23% ± 13%, P = 0.008), influx rate constant of radioactivity from plasma into brain (58% ± 26%, P = 0.008), and area under the brain time-activity curve (78% ± 17%, P = 0.008), presumably because of combined partial saturation of ABCG2 and ABCB1 activity. Inclusion of further subjects into the 1,000-mg dose group was precluded by adverse skin events (rash). Conclusion: Supratherapeutic-dose erlotinib may be used to enhance brain delivery of ABCB1/ABCG2 substrate anticancer drugs, but its clinical applicability for continuous ABCB1/ABCG2 inhibition at the BBB may be limited by safety concerns.

Effect of Rifampicin on the Distribution of [11C]Erlotinib to the Liver, a Translational PET Study in Humans and in Mice.

Organic anion-transporting polypeptides (OATPs) mediate the uptake of various drugs from blood into the liver in the basolateral membrane of hepatocytes. Positron emission tomography (PET) is a potentially powerful tool to assess the activity of hepatic OATPs in vivo, but its utility critically depends on the availability of transporter-selective probe substrates. We have shown before that among the three OATPs expressed in hepatocytes (OATP1B1, OATP1B3, and OATP2B1), [11C]erlotinib is selectively transported by OATP2B1. In contrast to OATP1B1 and OATP1B3, OATP2B1 has not been thoroughly explored yet, and no specific probe substrates are currently available. To assess if the prototypical OATP inhibitor rifampicin can inhibit liver uptake of [11C]erlotinib in vivo, we performed [11C]erlotinib PET scans in six healthy volunteers without and with intravenous pretreatment with rifampicin (600 mg). In addition, FVB mice underwent [11C]erlotinib PET scans without and with concurrent intravenous infusion of high-dose rifampicin (100 mg/kg). Rifampicin caused a moderate reduction in the liver distribution of [11C]erlotinib in humans, while a more pronounced effect of rifampicin was observed in mice, in which rifampicin plasma concentrations were higher than in humans. In vitro uptake experiments in an OATP2B1-overexpressing cell line indicated that rifampicin inhibited OATP2B1 transport of [11C]erlotinib in a concentration-dependent manner with a half-maximum inhibitory concentration of 72.0 ± 1.4 µM. Our results suggest that rifampicin-inhibitable uptake transporter(s) contributed to the liver distribution of [11C]erlotinib in humans and mice and that [11C]erlotinib PET in combination with rifampicin may be used to measure the activity of this/these uptake transporter(s) in vivo. Furthermore, our data suggest that a standard clinical dose of rifampicin may exert in vivo a moderate inhibitory effect on hepatic OATP2B1.

A Prediction Method for P-glycoprotein-Mediated Drug-Drug Interactions at the Human Blood-Brain Barrier From Blood Concentration-Time Profiles, Validated With PET Data.

The purpose of this study was to establish physiologically based pharmacokinetic models to predict in humans the brain concentration-time profiles and P-glycoprotein (Pgp)-mediated brain drug-drug interactions between the model Pgp substrate (R)-[11C]verapamil (VPM), the model dual Pgp/breast cancer resistance protein (BCRP) substrate [11C]tariquidar (TQD), and the Pgp inhibitor tariquidar. The model predictions were validated with results from positron emission tomography studies in humans. Using these physiologically based pharmacokinetic models, the differences between predicted and observed areas under the concentration-time curves (AUC) of VPM and TQD in the brain were within a 1.2-fold and 2.5-fold range, respectively. Also, brain AUC increases of VPM and TQD after Pgp inhibitor administration were predicted with 2.5-fold accuracy when in vitro inhibition constant or half-maximum inhibitory concentration values of tariquidar were used. The predicted rank order of the magnitude of AUC increases reflected the results of the clinical positron emission tomography studies. Our results suggest that the established models can predict brain exposure from the respective blood concentration-time profiles and rank the magnitude of the Pgp-mediated brain drug-drug interaction potential for both Pgp and Pgp/BCRP substrates in humans.

Effect of P-glycoprotein inhibition at the blood-brain barrier on brain distribution of (R)-[11 C]verapamil in elderly vs. young subjects.

AIMS: The efflux transporter P-glycoprotein (ABCB1) acts at the blood-brain barrier (BBB) to restrict the distribution of many different drugs from blood to the brain. Previous data suggest an age-associated decrease in the expression and function of ABCB1 at the BBB. In the present study, we investigated the influence of age on the magnitude of an ABCB1-mediated drug-drug interaction (DDI) at the BBB. METHODS: We performed positron emission tomography scans using the model ABCB1 substrate (R)-[11 C]verapamil in five young [26 ± 1 years, (mean ± standard deviation)] and five elderly (68 ± 6 years) healthy male volunteers before and after intravenous administration of a low dose of the ABCB1 inhibitor tariquidar (3 mg kg-1 ). RESULTS: In baseline scans, the total distribution volume (VT ) of (R)-[11 C]verapamil in whole-brain grey matter was not significantly different between the elderly (VT  = 0.78 ± 0.15) and young (VT  = 0.79 ± 0.10) group. After partial (incomplete) ABCB1 inhibition, VT values were significantly higher (P = 0.040) in the elderly (VT  = 1.08 ± 0.15) than in the young (VT  = 0.80 ± 0.18) group. The percentage increase in (R)-[11 C]verapamil VT following partial ABCB1 inhibition was significantly greater (P = 0.032) in elderly (+40 ± 17%) than in young (+2 ± 17%) volunteers. Tariquidar plasma concentrations were not significantly different between the young (786 ± 178 nmol l-1 ) and elderly (1116 ± 347 nmol l-1 ) group. CONCLUSIONS: Our results provide the first direct evidence of an increased risk for ABCB1-mediated DDIs at the BBB in elderly persons, which may have important consequences for pharmacotherapy of the elderly.

Assessment of P-Glycoprotein Transport Activity at the Human Blood-Retina Barrier with (R)-11C-Verapamil PET.

P-glycoprotein (ABCB1) is expressed at the blood-retina barrier (BRB), where it may control distribution of drugs from blood to the retina and thereby influence drug efficacy and toxicity. Methods: We performed PET scans with the ABCB1 substrate (R)-11C-verapamil on 5 healthy male volunteers without and with concurrent infusion of the ABCB1 inhibitor tariquidar. We estimated the rate constants for radiotracer transfer across the BRB (K1, k2) and total retinal distribution volume VTResults: During ABCB1 inhibition, retinal VT and influx rate constant K1 were significantly, by 1.4 ± 0.5-fold and 1.5 ± 0.3-fold, increased compared with baseline. Retinal efflux rate constant k2 was significantly decreased by 2.8 ± 1.0-fold. Conclusion: We found a significant increase in (R)-11C-verapamil distribution to the retina during ABCB1 inhibition, which provides first in vivo evidence for ABCB1 transport activity at the human BRB. The increase in retinal distribution was approximately 2.5-fold less pronounced than previously reported for the blood-brain barrier.

Spatiotemporal multistage consensus clustering in molecular dynamics studies of large proteins.

The aim of this work is to find semi-rigid domains within large proteins as reference structures for fitting molecular dynamics trajectories. We propose an algorithm, multistage consensus clustering, MCC, based on minimum variation of distances between pairs of Cα-atoms as target function. The whole dataset (trajectory) is split into sub-segments. For a given sub-segment, spatial clustering is repeatedly started from different random seeds, and we adopt the specific spatial clustering with minimum target function: the process described so far is stage 1 of MCC. Then, in stage 2, the results of spatial clustering are consolidated, to arrive at domains stable over the whole dataset. We found that MCC is robust regarding the choice of parameters and yields relevant information on functional domains of the major histocompatibility complex (MHC) studied in this paper: the α-helices and ß-floor of the protein (MHC) proved to be most flexible and did not contribute to clusters of significant size. Three alleles of the MHC, each in complex with ABCD3 peptide and LC13 T-cell receptor (TCR), yielded different patterns of motion. Those alleles causing immunological allo-reactions showed distinct correlations of motion between parts of the peptide, the binding cleft and the complementary determining regions (CDR)-loops of the TCR. Multistage consensus clustering reflected functional differences between MHC alleles and yields a methodological basis to increase sensitivity of functional analyses of bio-molecules. Due to the generality of approach, MCC is prone to lend itself as a potent tool also for the analysis of other kinds of big data.