| 1. |
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| 2. |
- Fridén, Markus, et al.
(författare)
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Development of a High-Throughput Brain Slice Method for Studying Drug Distribution in the Central Nervous System
- 2009
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Ingår i: Drug Metabolism And Disposition. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0090-9556 .- 1521-009X. ; 37:6, s. 1226-1233
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Tidskriftsartikel (refereegranskat)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.
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| 3. |
- Fridén, Markus, 1978-
(författare)
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Development of Methods for Assessing Unbound Drug Exposure in the Brain : In vivo, in vitro and in silico
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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.
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| 4. |
- Fridén, Markus, et al.
(författare)
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Improved measurement of drug exposure in the brain using drug-specific correction for residual blood
- 2010
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Ingår i: Journal of Cerebral Blood Flow and Metabolism. - : SAGE Publications. - 0271-678X .- 1559-7016. ; 30:1, s. 150-161
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Tidskriftsartikel (refereegranskat)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.
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| 5. |
- Fridén, Markus, et al.
(författare)
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In vitro methods for estimating unbound drug concentrations in the brain interstitial and intracellular fluids
- 2007
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Ingår i: Drug Metabolism And Disposition. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0090-9556 .- 1521-009X. ; 35:9, s. 1711-1719
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Tidskriftsartikel (refereegranskat)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.
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| 6. |
- Fridén, Markus, et al.
(författare)
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Measurement of Unbound Drug Exposure in Brain : Modelling of pH Partitioning Explains Diverging Results between the Brain Slice and Brain Homogenate Methods
- 2011
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Ingår i: Drug Metabolism And Disposition. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0090-9556 .- 1521-009X. ; 39:3, s. 353-362
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Tidskriftsartikel (refereegranskat)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.
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| 7. |
- Fridén, Markus, et al.
(författare)
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Structure-brain exposure relationships in rat and human using a novel data set of unbound drug concentrations in brain interstitial and cerebrospinal fluids
- 2009
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 52:20, s. 6233-6243
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Tidskriftsartikel (refereegranskat)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.
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| 8. |
- Hammarlund-Udenaes, Margareta, et al.
(författare)
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Methodologies to assess brain drug delivery in lead optimization
- 2009
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Ingår i: Current Topics in Medicinal Chemistry. - : Bentham Science Publishers Ltd.. - 1568-0266 .- 1873-4294. ; 9:2, s. 148-162
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Forskningsöversikt (refereegranskat)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.
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| 9. |
- Henriksson, Gunnel, et al.
(författare)
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Humoral response to Clostridium difficile in inflammatory bowel disease, including correlation with immunomodulatory treatment
- 2019
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Ingår i: JGH Open. - : Wiley. - 2397-9070. ; 3:2, s. 154-158
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Tidskriftsartikel (refereegranskat)abstract
- Background and Aim: An abnormal immune response to intestinal bacteria has been observed in Crohn's disease (CD). Clostridium difficile infection incidence and severity are increased in CD, but reports on the humoral response have provided conflicting results. We aimed to shed light on the possible role of C. difficile in CD pathogenesis by paying attention to the influence of immunomodulatory treatment on the humoral response.Methods: A total of 71 consecutive outpatients with CD, 67 with ulcerative colitis (UC), and 121 healthy controls were analyzed for serum IgA and IgG to C. difficile toxins A and B.Results: IgA levels were similar in all study groups. IgG to toxin A was increased similarly in CD and UC (P = 0.02 for both). In contrast, IgG to toxin B was elevated only in CD patients not receiving disease-modifying anti-inflammatory bowel disease drugs (DMAID) (n = 16) (P = 0.0001), while the CD medication subgroup (n = 47) had a level similar to healthy controls. The UC results were not influenced by DMAID treatment.Conclusion: Our findings add support to the idea of a disturbed interaction between intestinal cells and the microbiota being part of the CD disease mechanism. An abnormal immune response to C. difficile toxin B may be a critical component of this interaction.
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| 10. |
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