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1.	Backstrom, E., et al. (author) Possible Extraction of Drugs from Lung Tissue During Broncho-alveolar Lavage Suggest Uncertainty in the Procedure's Utility for Quantitative Assessment of Airway Drug Exposure 2022 In: Journal of Pharmaceutical Sciences. - : Elsevier. - 0022-3549 .- 1520-6017. ; 111:3, s. 852-858 Journal article (peer-reviewed)abstract Following inhaled dosing, broncho-alveolar lavage (BAL) is often used for sampling epithelial lining fluid (ELF) to determine drug concentration in the lungs. This study aimed to explore the technique's suitability. Urea is typically used to estimate the dilution factor between the BAL fluid and physiological ELF, since it readily permeates through all fluids in the body. As representatives of permeable small molecule drugs with high, medium and low tissue distribution properties, propranolol, diazepam, indomethacin and AZD4721 were infused intravenously to steady state to ensure equal unbound drug concentrations throughout the body. The results showed that propranolol had higher unbound concentrations in the ELF compared to the plasma whilst this was not the case for the other compounds. Experiments with different BAL volumes and repeated lavaging indicated that the amount of drug extracted is very sensitive to experimental procedure. In addition, the results show that the unbound concentrations in ELF compared to plasma differs dependent on molecule class and tissue distribution properties. Overall data suggests that lavaging can remove drug from lung tissue in addition to ELF and highlights significant uncertainty in the robustness of the procedure for determining ELF drug concentrations. (c) 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.
2.	Boger, Elin, et al. (author) A Mechanistic Lung-Disposition Model for Real-time Simulation of Pharmacokinetics and Pharmacodynamics for Pulmonary Drug Delivery 2014 In: In proceedings of the 7th Noordwijkerhout Symposium on Pharmacokinetics, Pharmacodynamics and Systems Pharmacology. Conference paper (other academic/artistic)
3.	Boger, Elin, et al. (author) A Novel In Vivo Receptor Occupancy Methodology for the Glucocorticoid Receptor : Toward An Improved Understanding of Lung Pharmacokinetic/Pharmacodynamic Relationships 2015 In: Journal of Pharmacology and Experimental Therapeutics. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0022-3565 .- 1521-0103. ; 353:2, s. 279-287 Journal article (peer-reviewed)abstract Investigation of pharmacokinetic/pharmacodynamic (PK/PD) relationships for inhaled drugs is challenging because of the limited possibilities of measuring tissue exposure and target engagement in the lung. The aim of this study was to develop a methodology for measuring receptor occupancy in vivo in the rat for the glucocorticoid receptor (GR) to allow more informative inhalation PK/PD studies. From AstraZeneca's chemical library of GR binders, compound 1 [N-(2-amino-2-oxo-ethyl)-3-[5-[(1R,2S)-2-(2,2-difluoropropanoylamino)-1-(2,3-dihydro-1,4-benzodioxin-6-yl) propoxy] indazol-1-yl]-N-methyl-benzamide] was identified to have properties that are useful as a tracer for GR in vitro. When given at an appropriate dose (30 nmol/kg) to rats, compound 1 functioned as a tracer in the lung and spleen in vivo using liquid chromatography-tandem mass spectrometry bioanalysis. The methodology was successfully used to show the dose-receptor occupancy relationship measured at 1.5 hours after intravenous administration of fluticasone propionate (20, 150, and 750 nmol/kg) as well as to characterize the time profile for receptor occupancy after a dose of 90 nmol/kg i.v. The dose giving 50% occupancy was estimated as 47 nmol/kg. The methodology is novel in terms of measuring occupancy strictly in vivo and by using an unlabeled tracer. This feature confers key advantages, including occupancy estimation not being influenced by drug particle dissolution or binding/dissociation taking place postmortem. In addition, the tracer may be labeled for use in positron emission tomography imaging, thus enabling occupancy estimation in humans as a translatable biomarker of target engagement.
4.	Boger, Elin, et al. (author) Physiologically Based Pharmacokinetic/Pharmacodynamic Modeling Accurately Predicts the Better Bronchodilatory Effect of Inhaled Versus Oral Salbutamol Dosage Forms 2018 In: Journal of Aerosol Medicine. - : MARY ANN LIEBERT, INC. - 1941-2711 .- 1941-2703. ; 31:0, s. 1-12 Journal article (peer-reviewed)abstract Background: Predicting local lung tissue pharmacodynamic (PD) responses of inhaled drugs is a longstanding challenge related to the lack of experimental techniques to determine local free drug concentrations. This has prompted the use of physiologically based pharmacokinetic (PBPK) modeling to potentially predict local concentration and response. A unique opportunity for PBPK model evaluation is provided by the clinical PD data for salbutamol, which in its inhaled dosage form (400g), produces a higher bronchodilatory effect than in its oral dosage form (2mg) despite lower drug concentrations in blood. The present study aimed at evaluating whether inhalation PBPK model predictions of free drug in tissue would be predictive of these observations.Methods: A PBPK model, including 24 airway generations, was parameterized to describe lung, plasma, and epithelial lining fluid concentrations of salbutamol administered intratracheally and intravenously to rats (100nmol/kg). Plasma and lung tissue concentrations of unbound (R)-salbutamol, the active enantiomer, were predicted with a humanized version of the model and related to effect in terms of forced expiratory volume in 1 second (FEV1).Results: In contrast to oral dosing, the model predicted inhalation to result in spatial heterogeneity in the target site concentrations (subepithelium) with higher free drug concentrations in the lung as compared with the plasma. FEV1 of inhaled salbutamol was accurately predicted from the PK/PD relationship derived from oral salbutamol and PBPK predictions of free concentration in airway tissue of high resistance (e.g., 6th generation).Conclusion: An inhalation PBPK-PD model was developed and shown predictive of local pharmacology of inhaled salbutamol, thus conceptually demonstrating the validity of PBPK model predictions of free drug concentrations in lung tissue. This achievement unlocks the power of inhalation PBPK modeling to interrogate local pharmacology and guide optimization and development of inhaled drugs and their formulations.
5.	Boger, E., et al. (author) Systems Pharmacology Approach for Prediction of Pulmonary and Systemic Pharmacokinetics and Receptor Occupancy of Inhaled Drugs 2016 In: CPT. - : Wiley. - 2163-8306. ; 5:4, s. 201-210 Journal article (peer-reviewed)abstract Pulmonary drug disposition after inhalation is complex involving mechanisms, such as regional drug deposition, dissolution, and mucociliary clearance. This study aimed to develop a systems pharmacology approach to mechanistically describe lung disposition in rats and thereby provide an integrated understanding of the system. When drug-and formulation-specific properties for the poorly soluble drug fluticasone propionate were fed into the model, it proved predictive of the pharmacokinetics and receptor occupancy after intravenous administration and nose-only inhalation. As the model clearly distinguishes among drug-specific, formulation-specific, and system-specific properties, it was possible to identify key determinants of pulmonary selectivity of receptor occupancy of inhaled drugs: slow particle dissolution and slow drug-receptor dissociation. Hence, it enables assessment of factors for lung targeting, including molecular properties, formulation, as well as the physiology of the animal species, thereby providing a general framework for rational drug design and facilitated translation of lung targeting from animal to man.
6.	Bäckström, Erica, et al. (author) Development of a Novel Lung Slice Methodology for Profiling of Inhaled Compounds 2016 In: Journal of Pharmaceutical Sciences. - : Elsevier BV. - 0022-3549 .- 1520-6017. ; 105:2, s. 838-845 Journal article (peer-reviewed)abstract The challenge of defining the concentration of unbound drug at the lung target site after inhalation limits the possibility to optimize target exposure by compound design. In this study, a novel rat lung slice methodology has been developed and applied to study drug uptake in lung tissue, and the mechanisms by which this occurs. Freshly prepared lung slices (500 μm) from drug-naive rats were incubated with drugs followed by determination of the unbound drug volume of distribution in lung (Vu,lung), as the total concentration of drug in slices divided by the buffer (unbound) concentration. Vu,lung determined for a set of inhaled drug compounds ranged from 2.21 mL/g for salbutamol to 2970 mL/g for dibasic compound A. Co-incubation with monensin, a modulator of lysosomal pH, resulted in inhibition of tissue uptake of basic propranolol to 13%, indicating extensive lysosomal trapping. Partitioning into cells was particularly high for the cation MPP+ and the dibasic compound A, likely because of the carrier-mediated transport and lysosomal trapping. The results show that different factors are important for tissue uptake and the presented method can be used for profiling of inhaled compounds, leading to a greater understanding of distribution and exposure of drug in the lung.
7.	Bäckström, Erica, et al. (author) Lung Retention by Lysosomal Trapping of Inhaled Drugs Can Be Predicted In Vitro With Lung Slices 2016 In: Journal of Pharmaceutical Sciences. - : Elsevier BV. - 0022-3549 .- 1520-6017. ; 105:11, s. 3432-3439 Journal article (peer-reviewed)abstract Modulating and optimizing the local pharmacokinetics of inhaled drugs by chemical design or formulation is challenged by the lack of predictive in vitro systems and in vivo techniques providing a detailed description of drug location in the lung. The present study investigated whether a new experimental setup of freshly prepared agarose-filled lung slices can be used to estimate lung retention in vitro, by comparing with in vivo lung retention after intratracheal instillation. Slices preloaded with inhaled beta-adrenergic compounds (salbutamol, formoterol, salmeterol, indacaterol or AZD3199) were incubated in a large volume of buffer (w/wo monensin to assess the role of lysosomal trapping), and the amount remaining in slices at different time points was determined with liquid chromatography-tandem mass spectrometry. The in vitro lung retention closely matched the in vivo lung retention (half-lives within 3-fold for 4/5 compounds), and monensin shortened the half-lives for all compounds. The results suggest that freshly prepared rat lungs slices can be used to predict lung retention and that slow kinetics of lysosomal trapping is a key mechanism by which retention in the lung and the effect duration of inhaled beta-adrenergic bronchodilators are prolonged.
8.	Bäckström, Erica, et al. (author) Uncovering the regional localization of inhaled salmeterol retention in the lung 2018 In: Drug Delivery. - : Taylor & Francis Group. - 1071-7544 .- 1521-0464. ; 25:1, s. 838-845 Journal article (peer-reviewed)abstract Treatment of respiratory disease with a drug delivered via inhalation is generally held as being beneficial as it provides direct access to the lung target site with a minimum systemic exposure. There is however only limited information of the regional localization of drug retention following inhalation. The aim of this study was to investigate the regional and histological localization of salmeterol retention in the lungs after inhalation and to compare it to systemic administration. Lung distribution of salmeterol delivered to rats via nebulization or intravenous (IV) injection was analyzed with high-resolution mass spectrometry imaging (MSI). Salmeterol was widely distributed in the entire section at 5 min after inhalation, by 15 min it was preferentially retained in bronchial tissue. Via a novel dual-isotope study, where salmeterol was delivered via inhalation and d(3)-salmeterol via IV to the same rat, could the effective gain in drug concentration associated with inhaled delivery relative to IV, expressed as a site-specific lung targeting factor, was 5-, 31-, and 45-fold for the alveolar region, bronchial sub-epithelium and epithelium, respectively. We anticipate that this MSI-based framework for quantifying regional and histological lung targeting by inhalation will accelerate discovery and development of local and more precise treatments of respiratory disease.
9.	Chen, Hongming, et al. (author) In silico prediction of unbound brain-to-plasma concentration ratio using machine learning algorithms 2011 In: Journal of Molecular Graphics and Modelling. - : Elsevier BV. - 1093-3263 .- 1873-4243. ; 29:8, s. 985-995 Journal article (peer-reviewed)abstract Distribution over the blood-brain barrier (BBB) is an important parameter to consider for compounds that will be synthesized in a drug discovery project. Drugs that aim at targets in the central nervous system (CNS) must pass the BBB. In contrast, drugs that act peripherally are often optimised to minimize the risk of CNS side effects by restricting their potential to reach the brain. Historically, most prediction methods have focused on the total compound distribution between the blood plasma and the brain. However, recently it has been proposed that the unbound brain-to-plasma concentration ratio (K(p,uu,brain)) is more relevant. In the current study, quantitative K(p,uu,brain) prediction models have been built on a set of 173 in-house compounds by using various machine learning algorithms. The best model was shown to be reasonably predictive for the test set of 73 compounds (R(2) = 0.58). When used for qualitative prediction the model shows an accuracy of 0.85 (Kappa = 0.68). An additional external test set containing 111 marketed CNS active drugs was also classified with the model and 89% of these drugs were correctly predicted as having high brain exposure.
10.	Colcloughl, Nicola, et al. (author) Building on the success of osimertinib : achieving CNS exposure in oncology drug discovery 2019 In: Drug Discovery Today. - : Elsevier BV. - 1359-6446 .- 1878-5832. ; 24:5, s. 1067-1073 Research review (peer-reviewed)abstract Due to the blood-brain barrier (BBB) limiting the exposure of therapeutics to the central nervous system (CNS), patients with brain malignancies are challenging to treat, typically have poor prognoses, and represent a significant unmet medical need. Preclinical data report osimertinib to have significant BBB penetration and emerging clinical data demonstrate encouraging activity against CNS malignancies. Here, we discuss the oncology drug candidates AZD3759 and AZD1390 as case examples of discovery projects designing in BBB penetrance. We demonstrate how these innovative kinase inhibitors were recognized as brain penetrant and outline our view of experimental approaches and strategies that can facilitate the discovery of new brain-penetrant therapies for the treatment of primary and secondary CNS malignancies as well as other CNS disorders.
11.	Ehrhardt, Carsten, et al. (author) Current Progress Toward a Better Understanding of Drug Disposition Within the Lungs : Summary Proceedings of the First Workshop on Drug Transporters in the Lungs 2017 In: Journal of Pharmaceutical Sciences. - : Elsevier BV. - 0022-3549 .- 1520-6017. ; 106:9, s. 2234-2244 Journal article (peer-reviewed)abstract The School of Pharmacy and Pharmaceutical Sciences at Trinity College Dublin hosted the "1st Workshop on Drug Transporters in the Lungs" in September 2016 to discuss the impact of transporters on pulmonary drug disposition and their roles as drug targets in lung disease. The workshop brought together about 30 scientists from academia and pharmaceutical industry from Europe and Japan and addressed the primary questions: What do we know today, and what do we need to know tomorrow about transporters in the lung? The 3 themes of the workshop were: (1) techniques to study drug transporter expression and actions in the lungs; (2) drug transporter effects on pulmonary pharmacokinetics-case studies; and (3) transporters as drug targets in lung disease. Some of the conclusions of the workshop were: suitable experimental models that allow studies of transporter effects are available; data from these models convincingly show a contribution of both uptake and efflux transporters on pulmonary drug disposition; the effects of transporters on drug lung PK is now better conceptualized; some transporters are associated with lung diseases. However, more work is needed to establish which of the available models best translate to the clinical situation.
12.	Ericsson, Therese, et al. (author) Benchmarking of Human Dose Prediction for Inhaled Medicines from Preclinical In Vivo Data 2017 In: Pharmaceutical research. - : SPRINGER/PLENUM PUBLISHERS. - 0724-8741 .- 1573-904X. ; 34:12, s. 2557-2567 Journal article (peer-reviewed)abstract A scientifically robust prediction of human dose is important in determining whether to progress a candidate drug into clinical development. A particular challenge for inhaled medicines is that unbound drug concentrations at the pharmacological target site cannot be easily measured or predicted. In the absence of such data, alternative empirical methods can be useful. This work is a post hoc analysis based on preclinical in vivo pharmacokinetic/pharmacodynamic (PK/PD) data with the aim to evaluate such approaches and provide guidance on clinically effective dose prediction for inhaled medicines. Five empirically based methodologies were applied on a diverse set of marketed inhaled therapeutics (inhaled corticosteroids and bronchodilators). The approaches include scaling of dose based on body weight or body surface area and variants of PK/PD approaches aiming to predict the therapeutic dose based on having efficacious concentrations of drug in the lung over the dosing interval. The most robust predictions of dose were made by body weight adjustment (90% within 3-fold) and by a specific PK/PD approach aiming for an average predicted 75% effect level during the dosing interval (80% within 3-fold). Scaling of dose based on body surface area consistently under predicted the therapeutic dose. Preclinical in vivo data and empirical scaling to man can be used as a baseline method for clinical dose predictions of inhaled medicines. The development of more sophisticated translational models utilizing free drug concentration and target engagement data is a desirable build.
13.	Fridén, Markus, et al. (author) Development of a High-Throughput Brain Slice Method for Studying Drug Distribution in the Central Nervous System 2009 In: Drug Metabolism And Disposition. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0090-9556 .- 1521-009X. ; 37:6, s. 1226-1233 Journal article (peer-reviewed)abstract New, more efficient methods of estimating unbound drug concentrations in the CNS combine the amount of drug in whole brain tissue samples measured by conventional methods with in vitro estimates of the unbound brain volume of distribution (Vu,brain). While the brain slice method is the most reliable in vitro method for measuring Vu,brain, it has not previously been adapted for the needs of drug discovery research. The aim of this study was to increase the throughput and optimize the experimental conditions of this method. Equilibrium of drug between the buffer and the brain slice within the 4-5 hours of incubation is a fundamental requirement. However, it is difficult to meet this requirement for many of the extensively binding, lipophilic compounds in drug discovery programmes. In this study, the dimensions of the incubation vessel and mode of stirring influenced the equilibration time, as did the amount of brain tissue per unit buffer volume. The use of casette experiments for investigating Vu,brain in a linear drug concentration range increased the throughput of the method. The Vu,brain for the model compounds ranged from mL*g brain(-1); the sources of variability are discussed. The optimized set-up of the brain slice method allows precise, robust estimation of Vu,brain for drugs with diverse properties, including highly lipophilic compounds. This is a critical step forward for the implementation of relevant measurements of CNS exposure in the drug discovery setting.
14.	Fridén, Markus, 1978- (author) Development of Methods for Assessing Unbound Drug Exposure in the Brain : In vivo, in vitro and in silico 2010 Doctoral thesis (other academic/artistic)abstract The blood-brain barrier is formed by tightly joined capillary cells with transporter proteins and acts as to regulate the brain concentration of nutrients as well as many drugs. When developing central nervous system drugs it is necessary to measure the unbound drug concentration in the brain, i.e. the unbound brain exposure. This is to ensure that the drug reaches the site of action. Furthermore, when designing new drugs it is extremely valuable to be able to predict brain exposure from a tentative drug structure. Established methods to measure total drug concentrations are of limited (if any) utility since the pharmacologically active, unbound, concentration is not obtained. The aim of the conducted research was to develop an efficient methodology to measure unbound drug in the brain and to generate a dataset for developing computational prediction models describing the relationship between drug structure and unbound brain exposure. First it was demonstrated that unbound brain exposure can be efficiently assessed using a combination of total drug concentrations in the brain and separate measurements of drug binding in the brain slices. The in vitro brain slice method was refined and made high-throughput. Improvements were also made to the in vivo measurements of total concentrations by introducing an appropriate correction for drug in residual blood. Modeling of a 43-drug dataset in the rat showed that unbound brain exposure is related to the drug hydrogen bonding potential and not to lipid solubility, which contrasts the common understanding. Further, the drug concentrations in cerebrospinal fluid approximated unbound concentrations in the brain (r2=0.80) and were also correlated with corresponding measurements in humans (r2=0.56). Therefore, rat-derived prediction models can be used when designing drugs for humans. This thesis work has provided drug industry and academia with efficient tools to obtain and to use relevant estimates of drug exposure in the brain for evaluating drugs candidates.
15.	Fridén, Markus, et al. (author) Identification of positron emission tomography (PET) tracer candidates by prediction of the target-bound fraction in the brain 2014 In: EJNMMI Research. - : Springer Science and Business Media LLC. - 2191-219X. ; 4, s. 1-8 Journal article (peer-reviewed)abstract Background: Development of tracers for imaging with positron emission tomography (PET) is often a time-consuming process associated with considerable attrition. In an effort to simplify this process, we herein propose a mechanistically integrated approach for the selection of tracer candidates based on in vitro measurements of ligand affinity (K-d), non-specific binding in brain tissue (V-u,V-brain), and target protein expression (B-max). Methods: A dataset of 35 functional and 12 non-functional central nervous system (CNS) PET tracers was compiled. Data was identified in literature for K-d and B-max, whereas a brain slice methodology was used to determine values for V-u,V-brain. A mathematical prediction model for the target-bound fraction of tracer in the brain (f(tb)) was derived and evaluated with respect to how well it predicts tracer functionality compared to traditional PET tracer candidate selection criteria. Results: The methodology correctly classified 31/35 functioning and 12/12 non-functioning tracers. This predictivity was superior to traditional classification criteria or combinations thereof. Conclusions: The presented CNS PET tracer identification approach is rapid and accurate and is expected to facilitate the development of novel PET tracers for the molecular imaging community.
16.	Fridén, Markus, et al. (author) Improved measurement of drug exposure in the brain using drug-specific correction for residual blood 2010 In: Journal of Cerebral Blood Flow and Metabolism. - : SAGE Publications. - 0271-678X .- 1559-7016. ; 30:1, s. 150-161 Journal article (peer-reviewed)abstract A major challenge associated with the determination of the unbound brain-to-plasma concentration ratio of a drug (K(p,uu,brain)), is the error associated with correction for the drug in various vascular spaces of the brain, i.e., in residual blood. The apparent brain vascular spaces of plasma water (V(water), 10.3 microL/g brain), plasma proteins (V(protein), 7.99 microL/g brain), and the volume of erythrocytes (V(er), 2.13 microL/g brain) were determined and incorporated into a novel, drug-specific correction model that took the drug-unbound fraction in the plasma (f(u,p)) into account. The correction model was successfully applied for the determination of K(p,uu,brain) for indomethacin, loperamide, and moxalactam, which had potential problems associated with correction. The influence on correction of the drug associated with erythrocytes was shown to be minimal. Therefore, it is proposed that correction for residual blood can be performed using an effective plasma space in the brain (V(eff)), which is calculated from the measured f(u,p) of the particular drug as well as from the estimates of V(water) and V(protein), which are provided in this study. Furthermore, the results highlight the value of determining K(p,uu,brain) with statistical precision to enable appropriate interpretation of brain exposure for drugs that appear to be restricted to the brain vascular spaces.
17.	Fridén, Markus, et al. (author) In vitro methods for estimating unbound drug concentrations in the brain interstitial and intracellular fluids 2007 In: Drug Metabolism And Disposition. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0090-9556 .- 1521-009X. ; 35:9, s. 1711-1719 Journal article (peer-reviewed)abstract Concentrations of unbound drug in the interstitial fluid of the brain are not rapidly measured in vivo. Therefore, measurement of total drug levels, i.e., the amount of drug per gram of brain, has been a common but unhelpful practice in drug discovery programs relating to central drug effects. This study was designed to evaluate in vitro techniques for faster estimation of unbound drug concentrations. The parameter that relates the total drug level and the unbound interstitial fluid concentration is the unbound volume of distribution in the brain (V(u,brain)). It was measured in vitro for 15 drugs using brain slice uptake and brain homogenate binding methods. The results were validated in vivo by comparison with V(u,brain) microdialysis results. The slice method results were within a 3-fold range of the in vivo results for all but one compound, suggesting that this method could be used in combination with total drug levels to estimate unbound interstitial fluid concentrations within reasonable limits. Although successful in 10 of 15 cases, the brain homogenate binding method failed to estimate the V(u,brain) of drugs that reside predominantly in the interstitial space or compounds that are accumulated intracellularly. Use of the simple methods described in this article will 1) allow quantification of active transport at the blood-brain barrier in vivo, 2) facilitate the establishment of a relationship between in vitro potency and in vivo activity for compounds acting on central nervous system targets, and 3) provide information on intracellular concentrations of unbound drug.
18.	Fridén, Markus, et al. (author) Measurement of Unbound Drug Exposure in Brain : Modelling of pH Partitioning Explains Diverging Results between the Brain Slice and Brain Homogenate Methods 2011 In: Drug Metabolism And Disposition. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0090-9556 .- 1521-009X. ; 39:3, s. 353-362 Journal article (peer-reviewed)abstract Currently used methodology for determining unbound drug exposure in brain combines measurement of the total drug concentration in the whole brain in vivo with estimation of brain tissue binding from one of two available in vitro methods: equilibrium dialysis of brain homogenate and the brain slice uptake method. This study of 56 compounds compares the fraction of unbound drug in brain (f(u,brain)), determined using the brain homogenate method, with the unbound volume of distribution in brain (V(u,brain)), determined using the brain slice method. Discrepancies were frequent and primarily related to drug pH partitioning, due to the preservation of cellular structures in the slice that are absent in the homogenate. A mathematical model for pH partitioning into acidic intracellular compartments was derived to predict the slice V(u,brain) from measurements of f(u,brain) and drug pKa. This model allowed prediction of V(u,brain) from f(u,brain) within a 2.2-fold error range for 95% of the drugs, as compared to a 4.5-fold error range using the brain homogenate f(u,brain) method alone. The greatest discrepancies between the methods occurred with compounds that are actively transported into brain cells, including gabapentin, metformin and prototypic organic cation transporter substrates. It is concluded that intra-brain drug distribution is governed by several diverse mechanisms in addition to non-specific binding and that the slice method is therefore more reliable than the homogenate method. Alternatively, predictions of V(u,brain) can be made from homogenate f(u,brain) using the presented pH partition model, although this model does not take into consideration possible active brain cell uptake.
19.	Fridén, Markus, et al. (author) Structure-brain exposure relationships in rat and human using a novel data set of unbound drug concentrations in brain interstitial and cerebrospinal fluids 2009 In: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 52:20, s. 6233-6243 Journal article (peer-reviewed)abstract New experimental methodologies were applied to measure the unbound brain-to-plasma concentration ratio (K(p,uu,brain)) and the unbound CSF-to-plasma concentration ratio (K(p,uu,CSF)) in rats for 43 structurally diverse drugs. The relationship between chemical structure and K(p,uu,brain) was dominated by hydrogen bonding. Contrary to popular understanding based on the total brain-to-plasma concentration ratio (logBB), lipophilicity was not a determinant of unbound brain exposure. Although changing the number of hydrogen bond acceptors is a useful design strategy for optimizing K(p,uu,brain), future improvement of in silico prediction models is dependent on the accommodation of active drug transport. The structure-brain exposure relationships found in the rat also hold for humans, since the rank order of the drugs was similar for human and rat K(p,uu,CSF). This cross-species comparison was supported by K(p,uu,CSF) being within 3-fold of K(p,uu,brain) in the rat for 33 of 39 drugs. It was, however, also observed that K(p,uu,CSF) overpredicts K(p,uu,brain) for highly effluxed drugs, indicating lower efflux capacity of the blood-cerebrospinal fluid barrier compared to the blood-brain barrier.
20.	Ganguly, Koustav, et al. (author) Computational modeling of lung deposition of inhaled particles in chronic obstructive pulmonary disease (COPD) patients : identification of gaps in knowledge and data 2019 In: Critical reviews in toxicology. - : TAYLOR & FRANCIS LTD. - 1040-8444 .- 1547-6898. ; 49:2, s. 160-173 Research review (peer-reviewed)abstract Computational modeling together with experimental data are essential to assess the risk for particulate matter mediated lung toxicity and to predict the efficacy, safety and fate of aerosolized drug molecules used in inhalation therapy. In silico models are widely used to understand the deposition, distribution, and clearance of inhaled particles and aerosols in the human lung. Exacerbations of chronic obstructive pulmonary disease (COPD) have been reported due to increased particulate matter related air pollution episodes. Considering the profound functional, anatomical and structural changes occurring in COPD lungs, the relevance of the existing in silico models for mimicking diseased lungs warrants reevaluation. Currently available computational modeling tools were developed for the healthy adult (male) lung. Here, we analyze the major alterations occurring in the airway structure, anatomy and pulmonary function in the COPD lung, as compared to the healthy lung. We also scrutinize the various physiological and particle characteristics that influence particle deposition, distribution and clearance in the lung. The aim of this review is to evaluate the availability of the fundamental knowledge and data required for modeling particle deposition in a COPD lung departing from the existing healthy lung models. The extent to which COPD pathophysiology may affect aerosol deposition depends on the relative contribution of several factors such as altered lung structure and function, bronchoconstriction, emphysema, loss of elastic recoil, altered breathing pattern and altered liquid volumes that warrant consideration while developing physiologically relevant in silico models.
21.	Hamm, Gregory R., et al. (author) Revealing the Regional Localization and Differential Lung Retention of Inhaled Compounds by Mass Spectrometry Imaging 2020 In: Journal of Aerosol Medicine. - : Mary Ann Liebert Inc. - 1941-2711 .- 1941-2703. ; 33:1, s. 43-53 Journal article (peer-reviewed)abstract Background: For the treatment of respiratory disease, inhaled drug delivery aims to provide direct access to pharmacological target sites while minimizing systemic exposure. Despite this long-held tenet of inhaled therapeutic advantage, there are limited data of regional drug localization in the lungs after inhalation. The aim of this study was to investigate the distribution and retention of different chemotypes typifying available inhaled drugs [slowly dissolving neutral fluticasone propionate (FP) and soluble bases salmeterol and salbutamol] using mass spectrometry imaging (MSI).Methods: Salmeterol, salbutamol, and FP were simultaneously delivered by inhaled nebulization to rats. In the same animals, salmeterol-d(3), salbutamol-d(3), and FP-d(3) were delivered by intravenous (IV) injection. Samples of lung tissue were obtained at 2- and 30-minute postdosing, and high-resolution MSI was used to study drug distribution and retention.Results: IV delivery resulted in homogeneous lung distribution for all molecules. In comparison, while inhalation also gave rise to drug presence in the entire lung, there were regional chemotype-dependent areas of higher abundance. At the 30-minute time point, inhaled salmeterol and salbutamol were preferentially retained in bronchiolar tissue, whereas FP was retained in all regions of the lungs.Conclusion: This study clearly demonstrates that inhaled small molecule chemotypes are differentially distributed in lung tissue after inhalation, and that high-resolution MSI can be applied to study these retention patterns.
22.	Hammarlund-Udenaes, Margareta, et al. (author) Methodologies to assess brain drug delivery in lead optimization 2009 In: Current Topics in Medicinal Chemistry. - : Bentham Science Publishers Ltd.. - 1568-0266 .- 1873-4294. ; 9:2, s. 148-162 Research review (peer-reviewed)abstract In the area of lead optimization for potential CNS-active drugs in medicinal chemistry, there is a great need for experimental methodologies that can generate data relevant to estimates of free (unbound) drug exposure within the CNS. The methods chosen have to be efficient and have to measure a pharmacologically relevant entity. The lack of methods for generating such data is probably linked with the lack of successful lead optimization strategies within CNS drug discovery. This article evaluates available methods for estimating drug delivery to the brain, and discusses the relevance of the methods from the perspective of CNS exposure to free drug. It is suggested that the extent of drug delivery is the most important investigative parameter, since permeability (rate of transfer) can vary within a relatively wide range and still allow effects within the CNS. Following this suggestion would shift the focus from the current way of thinking and could lead to the development of less lipophilic compounds than are currently being investigated. It is concluded that an extensive collection of quality data on brain drug delivery, transporter affinities and in vivo behavior is urgently required so as to be able to build relevant predictive in vitro and in silico models for the future. These models need to be much more focused on the asymmetry of active transport across the BBB than on permeability data.
23.	Hammarlund-Udenaes, Margareta, et al. (author) On the rate and extent of drug delivery to the brain 2008 In: Pharmaceutical research. - : Springer Science and Business Media LLC. - 0724-8741 .- 1573-904X. ; 25:8, s. 1737-1750 Research review (peer-reviewed)abstract To define and differentiate relevant aspects of blood-brain barrier transport and distribution in order to aid research methodology in brain drug delivery. Pharmacokinetic parameters relative to the rate and extent of brain drug delivery are described and illustrated with relevant data, with special emphasis on the unbound, pharmacologically active drug molecule. Drug delivery to the brain can be comprehensively described using three parameters: Kp,uu (concentration ratio of unbound drug in brain to blood), CLin (permeability clearance into the brain), and Vu,brain (intra-brain distribution). The permeability of the blood-brain barrier is less relevant to drug action within the CNS than the extent of drug delivery, as most drugs are administered on a continuous (repeated) basis. Kp,uu can differ between CNS-active drugs by a factor of up to 150-fold. This range is much smaller than that for log BB ratios (Kp), which can differ by up to at least 2,000-fold, or for BBB permeabilities, which span an even larger range (up to at least 20,000-fold difference). Methods that measure the three parameters Kp,uu, CLin, and Vu,brain can give clinically valuable estimates of brain drug delivery in early drug discovery programmes.
24.	Hu, Yang, et al. (author) Understanding the influence of nanocarrier-mediated brain delivery on therapeutic performance Other publication (other academic/artistic)
25.	Hu, Yang, 1989-, et al. (author) Understanding the Influence of Nanocarrier-Mediated Brain Delivery on Therapeutic Performance Through Pharmacokinetic-Pharmacodynamic Modeling. 2019 In: Journal of Pharmaceutical Sciences. - : Elsevier BV. - 0022-3549 .- 1520-6017. ; 108:10, s. 3425-3433 Journal article (peer-reviewed)abstract This study aimed at evaluating how encapsulation in a regular nanocarrier (NC) (providing extended circulation time) or in a brain-targeting NC (providing prolonged circulation time and increased brain uptake) may influence the therapeutic index compared with the unformulated drug and to explore the key parameters affecting therapeutic performance using a model-based approach. Pharmacokinetic (PK) models were built with chosen PK parameters. For a scenario where central effect depends on area under the unbound brain concentration curve and peripheral toxicity relates to peak unbound plasma concentration, dose-effect and drug-side effect curves were constructed, and the therapeutic index was evaluated. Regular NC improved the therapeutic index compared with the unformulated drug due to reduced peripheral toxicity, while brain-targeting NC enhanced the therapeutic index by lowering peripheral toxicity and increasing central effect. Decreasing drug release rate or systemic clearance of NC with drug still encapsulated could increase the therapeutic index. Also, a drug with shorter half-life would therapeutically benefit more from a NC encapsulation. This work provides insights into how a NC for brain delivery should be optimized to maximize the therapeutic performance and is helpful to predict if and to what extent a drug with certain PK properties would obtain therapeutic benefit from nanoencapsulation.
26.	Lindqvist, Annika, 1983-, et al. (author) Pharmacokinetic Considerations of Nanodelivery to the Brain : Using Modeling and Simulations to Predict Outcome of Liposomal Formulations 2016 In: European Journal of Pharmaceutical Sciences. - : Elsevier BV. - 0928-0987 .- 1879-0720. ; 92, s. 173-182 Journal article (peer-reviewed)abstract The use of nanocarriers is an intriguing solution to increase the brain delivery of novel therapeutics. The aim of this paper was to use pharmacokinetic analysis and simulations to identify key factors that determine the effective drug concentration-time profile at the target site in the brain. Model building and simulations were based on experimental data obtained from the administration of the opioid peptide DAMGO in glutathione tagged PEGylated liposomes to rats. Different pharmacokinetic models were investigated to explore the mechanisms of increased brain delivery. Concentration time profiles for a set of formulations with varying compound and carrier characteristics were simulated. By controlling the release rate from the liposome, the time profile and the extent of brain delivery can be regulated. The modeling did not support a mechanism of the liposomes passing the brain endothelial cell membrane in an intact form through endocytosis or transcytosis. The most likely process was found to be fusion of the liposome with the endothelial luminal membrane. The simulations revealed that low permeable compounds, independent on efflux, will gain the most from a nanocarrier formulation. The present model based approach is useful to explore and predict possibilities and limitations of carrier-based systems to the brain.
27.	Lindqvist, Annika, 1983- (author) Quantitative Aspects of Nanodelivery Across the Blood-Brain Barrier : Exemplified with the Opioid Peptide DAMGO 2015 Doctoral thesis (other academic/artistic)abstract The use of nanocarriers is an intriguing approach in the development of efficacious treatment for brain disorders. The aim of the conducted research was to evaluate and quantify the impact of a liposomal nanocarrier formulation on the brain drug delivery. A novel approach for investigating the blood-brain barrier transport of liposomal DAMGO is presented, including in vivo microdialysis in rat, a high quality LC-MS/MS bioanalytical method and pharmacokinetic model analysis of the data. Factors limiting the brain distribution of the free peptide DAMGO were also investigated. Microdialysis, in combination with plasma sampling, made it possible to separate the released drug from the encapsulated and to quantify the active substance in both blood and brain interstitial fluid over time.The opioid peptide DAMGO entered the brain to a limited extent, with a clearance out of the brain 13 times higher than the clearance into the brain. The brain to blood ratio of unbound drug was not affected when the efflux transporter inhibitors cyclosporine A and elacridar were co-administered with DAMGO. Nor was the transport affected in the in vitro Caco-2 assay using the same inhibitors. This indicates that DAMGO is not transported by P-glycoprotein (Pgp) or breast cancer resistant protein (Bcrp). The blood-brain barrier transport was significantly increased for DAMGO when formulated in liposomes, resulting in 2-3 fold higher brain to blood ratio of unbound DAMGO. The increased brain delivery was seen both for glutathione tagged PEGylated liposomes, as well as for PEGyalted liposomes without specific brain targeting. The improvement in brain delivery was observed only when DAMGO was encapsulated into the liposomes, thus excluding any effect of the liposomes themselves on the integrity of the blood-brain barrier. Modeling of the data provided additional mechanistic understanding of the brain uptake, showing that endocytosis or transcytosis of intact liposomes across the endothelial cell membranes were unlikely. A model describing fusion of the liposomes with the luminal membrane described the experimental data the best.In conclusion, the studies presented in this thesis all contribute to an increased understanding of how to evaluate and improve brain delivery of CNS active drugs and contribute with important insights to the nanocarrier field.
28.	Loryan, Irena, et al. (author) The brain slice method for studying drug distribution in the CNS 2013 In: Fluids and Barriers of the CNS. - : BioMed Central. - 2045-8118. ; 10, s. 6- Journal article (peer-reviewed)abstract The high-throughput brain slice method is a precise and robust technique for estimating the overall uptake of drugs into brain tissue through determination of the unbound volume of distribution in the brain (Vu,brain; ml·g brain-1). Vu,brain describes the relationship between the total drug concentration in the brain and the concentration of unbound drug in the brain interstitial fluid, regardless of blood–brain barrier function. The brain slice method is more physiologically based than the brain homogenate method with respect to the assessment of drug distribution in the brain because the cell-cell interactions, pH gradients and active transport systems are all conserved. The method provides information that is directly relevant to issues such as nonspecific binding to brain tissue, lysosomal trapping, and active uptake into the cells. For these reasons, the brain slice method is recommended for estimation of target-site pharmacokinetics in the early drug discovery process and fundamental pharmacological studies. This article provides a detailed protocol for the rat and mouse brain slice methods, with the aim of enabling simple, cost-effective profiling of compounds with diverse physicochemical properties. The procedure for assessing the viability of the brain slices after the 5 h incubation period is also described. The results are interpreted for a set of compounds covering a wide range of physicochemical properties and various pharmacological targets. Application of the method for evaluating the unbound intracellular-to-extracellular concentration ratio (Kp,uu,cell) and the unbound brain-to-plasma concentration ratio (Kp,uu,brain) is discussed.
29.	Loryan, Irena, Associate Professor (Docent), 1977-, et al. (author) Unbound Brain-to-Plasma Partition Coefficient, K-p,K-uu,K-brain-a Game Changing Parameter for CNS Drug Discovery and Development 2022 In: Pharmaceutical research. - : Springer Nature. - 0724-8741 .- 1573-904X. ; 39:7, s. 1321-1341 Journal article (peer-reviewed)abstract Purpose More than 15 years have passed since the first description of the unbound brain-to-plasma partition coefficient (K-p,K-uu,K-brain) by Prof. Margareta Hammarlund-Udenaes, which was enabled by advancements in experimental methodologies including cerebral microdialysis. Since then, growing knowledge and data continue to support the notion that the unbound (free) concentration of a drug at the site of action, such as the brain, is the driving force for pharmacological responses. Towards this end, K-p,K-uu,K-brain is the key parameter to obtain unbound brain concentrations from unbound plasma concentrations. Methods To understand the importance and impact of the K-p,K-uu,K-brain concept in contemporary drug discovery and development, a survey has been conducted amongst major pharmaceutical companies based in Europe and the USA. Here, we present the results from this survey which consisted of 47 questions addressing: 1) Background information of the companies, 2) Implementation, 3) Application areas, 4) Methodology, 5) Impact and 6) Future perspectives. Results and conclusions From the responses, it is clear that the majority of the companies (93%) has established a common understanding across disciplines of the concept and utility of K-p,K-uu,K-brain as compared to other parameters related to brain exposure. Adoption of the K-p,K-uu,K-brain concept has been mainly driven by individual scientists advocating its application in the various companies rather than by a top-down approach. Remarkably, 79% of all responders describe the portfolio impact of K-p,K-uu,K-brain implementation in their companies as 'game-changing'. Although most companies (74%) consider the current toolbox for K-p,K-uu,K-brain assessment and its validation satisfactory for drug discovery and early development, areas of improvement and future research to better understand human brain pharmacokinetics/pharmacodynamics translation have been identified.
30.	Nyman, Elin, et al. (author) Mechanisms of a sustained anti-inflammatory drug response in alveolar macrophages unraveled with mathematical modeling 2020 In: CPT. - : John Wiley & Sons. - 2163-8306. ; 9:12, s. 707-717 Journal article (peer-reviewed)abstract Both initiation and suppression of inflammation are hallmarks of the immune response. If not balanced, the inflammation may cause extensive tissue damage, which is associated with common diseases, e.g. asthma and atherosclerosis. Anti-inflammatory drugs come with side-effects which may be aggravated by high and fluctuating drug concentrations. To remedy this, an anti-inflammatory drug should have an appropriate pharmacokinetic half-life or better still: a sustained anti-inflammatory drug response. However, we still lack a quantitative mechanistic understanding of such sustained effects. Here, we study the anti-inflammatory response to a common glucocorticoid drug, Dexamethasone. We find a sustained response 22 hours after drug removal. With hypothesis testing using mathematical modeling, we unravel the underlying mechanism - a slow release of Dexamethasone from the receptor-drug complex. The developed model is in agreement with time-resolved training and testing data, and is used to simulate hypothetical treatment schemes. This work opens up for a more knowledge-driven drug development, to find sustained anti-inflammatory responses and fewer side effects.
31.	Sadiq, Muhammad Waqas, et al. (author) Lung pharmacokinetics of inhaled and systemic drugs : A clinical evaluation 2021 In: British Journal of Pharmacology. - : John Wiley & Sons. - 0007-1188 .- 1476-5381. ; 178:22, s. 4440-4451 Journal article (peer-reviewed)abstract Background and Purpose Human pharmacokinetic studies of lung-targeted drugs are typically limited to measurements of systemic plasma concentrations, which provide no direct information on lung target-site concentrations. We aimed to evaluate lung pharmacokinetics of commonly prescribed drugs by sampling different lung compartments after inhalation and oral administration. Experimental Approach Healthy volunteers received single, sequential doses of either inhaled salbutamol, salmeterol and fluticasone propionate (n = 12), or oral salbutamol and propranolol (n = 6). Each participant underwent bronchoscopies and gave breath samples for analysis of particles in exhaled air at two points after drug administration (1 and 6, 2 and 9, 3 and 12, or 4 and 18 h). Lung samples were taken via bronchosorption, bronchial brush, mucosal biopsy and bronchoalveolar lavage during each bronchoscopy. Blood samples were taken during the 24 h after administration. Pharmacokinetic profiles were generated by combining data from multiple individuals, covering all sample timings. Key Results Pharmacokinetic profiles were obtained for each drug in lung epithelial lining fluid, lung tissue and plasma. Inhalation of salbutamol resulted in approximately 100-fold higher concentrations in lung than in plasma. Salmeterol and fluticasone concentration ratios in lung versus plasma were higher still. Bronchosorption- and bronchoalveolar-lavage-generated profiles of inhaled drugs in epithelial lining fluid were comparable. For orally administered drugs, epithelial-lining-fluid concentrations were overestimated in bronchoalveolar-lavage-generated profiles. Conclusion and Implications Combining pharmacokinetic data derived from several individuals and techniques sampling different lung compartments enabled generation of pharmacokinetic profiles for evaluation of lung targeting after inhaled and oral drug delivery.
32.	Schou, Magnus, et al. (author) Pulmonary PET imaging confirms preferential lung target occupancy of an inhaled bronchodilator 2019 In: EJNMMI Research. - : Springer Science and Business Media LLC. - 2191-219X. ; 9 Journal article (peer-reviewed)abstract Background: Positron emission tomography (PET) is a non-invasive molecular imaging technique that traces the distribution of radiolabeled molecules in experimental animals and human subjects. We hypothesized that PET could be used to visualize the binding of the bronchodilator drug ipratropium to muscarinic receptors (MR) in the lungs of living non-human primates (NHP). The objectives of this study were two-fold: (i) to develop a methodology for quantitative imaging of muscarinic receptors in NHP lung and (ii) to estimate and compare ipratropium-induced MR occupancy following drug administration via intravenous injection and inhalation, respectively.Methods: A series of PET measurements (n=18) was performed after intravenous injection of the selective muscarinic radioligand C-11-VC-002 in NHP (n=5). The lungs and pituitary gland (both rich in MR) were kept in the field of view. Each PET measurement was followed by a PET measurement preceded by treatment with ipratropium (intravenous or inhaled).Results: Radioligand binding was quantified using the Logan graphical analysis method providing the total volume of distribution (V-T). Ipratropium reduced the V-T in the lung and pituitary in a dose-dependent fashion. At similar plasma ipratropium concentrations, administration by inhalation produced larger reductions in V-T for the lungs. The plasma-derived apparent affinity for ipratropium binding in the lung was one order of magnitude higher after inhalation (K-iih=1.01nM) than after intravenous infusion (K-iiv=10.84nM).Conclusion: Quantitative muscarinic receptor occupancy imaging by PET articulates and quantifies the therapeutic advantage of the inhaled route of delivery and provides a tool for future developments of improved inhaled drugs.
33.	Stridh, Sara, et al. (author) C-peptide normalizes glomerular filtration rate in hyperfiltrating conscious diabetic rats 2009 In: Oxygen transport to tissue xxx. - New York : Springer. - 9780387859972 - 0387859977 - 9780387859989 ; , s. 219-225 Book chapter (peer-reviewed)abstract Tubular electrolyte transport accounts for a major part of the oxygen consumed by the normal kidney. We have previously reported a close association between diabetes and increased oxygen usage, partly due to increased tubular electrolyte transport secondary to glomerular hyperfiltration during the early onset of diabetes. Several studies have shown that acute administration of C-peptide to diabetic rats with glomerular hyperfiltration results in normalized glomerular filtration rate (GFR). In this study, we validated a novel method for precise and repetitive GFR measurements in conscious rats and used C-peptide injection in diabetic rats for evaluation. First, GFR was determined in normoglycemic control rats before and after C-peptide administration. Thereafter, all rats were made diabetic by an i.v. streptozotocin injection. Fourteen days later, GFR was again determined before and after C-peptide administration. GFR was estimated from plasma clearance curves using a single bolus injection of FITC-inulin, followed by serial blood sampling over 155 min. FITC-inulin clearance was calculated using non-compartmental pharmacokinetic data analysis. Baseline GFR in normoglycemic controls was 2.10 +/- 0.18 ml/min, and was unaffected by C-peptide (2.23 +/- 0.14 ml/min). Diabetic rats had elevated GFR (3.06 +/- .034 ml/min), which was normalized by C-peptide (2.35 +/- 0.30 ml/min). In conclusion, the used method for estimation of GFR in conscious animals result in values that are in good agreement with those obtained from traditional GFR measurements on anaesthetized rats. However, multiple measurements from the same conscious subject can be obtained using this method. Furthermore, as previously shown on anaesthetized rats, C-peptide also normalizes GFR in hyperfiltrating conscious diabetic rats.
34.	Syvänen, Stina, et al. (author) Professor Margareta Hammarlund-Udenaes : a Modern and Visionary Mentor 2022 In: Pharmaceutical research. - : Springer Nature. - 0724-8741 .- 1573-904X. ; 39:7, s. 1291-1296 Journal article (other academic/artistic)
35.	Sällström, Johan, et al. (author) Simultaneous determination of renal plasma flow and glomerular filtration rate in conscious mice using dual bolus injection 2013 In: Journal of pharmacological and toxicological methods. - : Elsevier BV. - 1056-8719 .- 1873-488X. ; 67:3, s. 187-193 Journal article (peer-reviewed)abstract Introduction: The present report describes and evaluates a simple protocol for serial measurements of glomerular filtration rate (GFR) and renal plasma flow (RPF) in conscious mice. Methods: In conscious mice, a bolus of [H-3] methoxy-inulin and [C-14] para-amino-hippuric (PAH) was injected in the tail vein whereupon eight blood samples were taken during the following 75 min. Plasma concentrations were determined by liquid scintillation and clearances of the injected markers were calculated by non-compartmental pharmacokinetic data analysis of the plasma disappearance curves. In anaesthetized mice, the renal extraction ratio of PAH was determined by infusion of PAH and subsequent analysis of blood taken from the carotid artery and the renal vein. The acquired value (0.70 +/- 0.02) was used for all subsequent calculations of RPF. To evaluate the protocol, a crossover study was performed where either the vehicle or the angiotensin II AT1 receptor antagonist candesartan was given prior to the clearance measurements. Results: Baseline values of GFR and RPF were in line with those earlier reported in mice. Administration of candesartan increased RPF and reduced the filtration fraction, whereas GFR was unaltered. These changes are supported by earlier findings and demonstrate that GFR and RPF can be determined independently. Furthermore, modelling experiments demonstrated that acceptable results are obtained even if the number of blood samples is reduced to four which is a way to further simplify the procedure. Discussion: The method provides an effective way for repeated measurements of GFR and RPF in mice without potentially confounding effects of anaesthesia.
36.	Wieslander, Håkan, et al. (author) Deep learning and conformal prediction for hierarchical analysis of large-scale whole-slide tissue images 2021 In: IEEE journal of biomedical and health informatics. - : Institute of Electrical and Electronics Engineers (IEEE). - 2168-2194 .- 2168-2208. ; 25:2, s. 371-380 Journal article (peer-reviewed)abstract With the increasing amount of image data collected from biomedical experiments there is an urgent need for smarter and more effective analysis methods. Many scientific questions require analysis of image subregions related to some specific biology. Finding such regions of interest (ROIs) at low resolution and limiting the data subjected to final quantification at high resolution can reduce computational requirements and save time. In this paper we propose a three-step pipeline: First, bounding boxes for ROIs are located at low resolution. Next, ROIs are subjected to semantic segmentation into sub-regions at mid-resolution. We also estimate the confidence of the segmented sub-regions. Finally, quantitative measurements are extracted at high resolution. We use deep learning for the first two steps in the pipeline and conformal prediction for confidence assessment. We show that limiting final quantitative analysis to sub regions with high confidence reduces noise and increases separability of observed biological effects.

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