| 1. |
- Behere, Anish, 1993-
(författare)
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Ex‘PLA’ining the progression of pathological proteins in Alzheimer’s and Parkinson’s diseases : see(d)ing is believing
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most common forms of neurodegenerative disorders affecting approximately 50 million people worldwide. The underlying neuropathological processes leading to AD and PD share many similarities, i.e. aberrant protein aggregation of tau and alpha-synuclein (αSyn) in the brain. Monitoring tau and αSyn aggregation is challenging, due to morphological heterogeneity of the aggregating species and problems in preserving the antigen conformation ex vivo.In paper-I, we validated the usefulness of proximity ligation assay (PLA), a technique that enabled us to visualize previously undetected early αSyn pathology in the A30P-tg mouse model of PD. We observed an age-progressive increase in the levels of phosphorylated αSyn (pSynS129) and the compactness of aggregates in the brain. Although loss of dopaminergic neurons was not found, a subtle dysregulation of other catecholamines was recorded in the older mice.In paper-II, we revealed a wide distribution of pSynS129 aggregates in alpha-synucleinopathy-patient brains. By using a PLA setup with certain antibody pair combinations on brain sections, we observed unique staining patterns, which could not be visualized using regular immunohistochemistry (IHC). In A30P-tg mice, the morphological pattern of the PLA signals indicated an intracellular shift of pSynS129 from the periphery towards the neuronal soma.In Paper-III, we demonstrated that multiplex pTauS202,T205-pTauT231, singleplex pTauT231 and singleplex pSynS129 PLAs can recognize an extensive tau and αSyn pathology compared to regular IHC. We found that using our PLA approach we could differentiate between pTauS202,T205 and pTauT231 pathology in AD brains, whereas IHC could not. Similarly, in the PD brain, singleplex pSynS129 PLA detected novel structures, i.e. apparent thick intercellular tunnelling nanotubes and early aggregates; whereas pSynS129 IHC was limited to the detection of mature pathology. Lastly, we demonstrated that our multiplex PLA approach detected co-aggregates of pSynS129-pTau.In Paper-IV, in an αSyn seeding mouse model we observed pSynS129 immunoreactivity close to the striatal injection site one day post-injection (dpi). Intriguingly, this type of staining disappeared with the concurrent formation of peri-nuclear pSynS129 inclusions in specific brain regions after 14 dpi. In parallel, astrocytic activation prior to pSynS129 inclusion formation was observed.In conclusion, we have developed several novel PLAs that detect both tau and αSyn pathology with a higher ex vivo sensitivity and specificity than currently used immunostaining methods. This thesis work provides valuable insights that potentially could be used for the development of future biomarkers for tauopathies and synucleinopathies.
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| 2. |
- Faresjö Melander, Rebecca, 1990-
(författare)
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Factors influencing transferrin receptor-mediated brain delivery : Evaluating preclinical antibody-based proteins for PET imaging in Alzheimer’s disease
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibody-based proteins targeting amyloid-beta (Aβ) could be used as radioligands in positron emission tomography (PET) to study Alzheimer’s disease (AD) pathology in the living brain. The prospective advantages of antibody-based PET are to detect pathology earlier, with higher sensitivity, and to evaluate treatment effects of emerging immunotherapies against Aβ. However, antibodies and other proteins are too large to cross the blood-brain barrier (BBB). This can be circumvented by fusing antibodies with transferrin-receptor (TfR) binders that penetrate the BBB via receptor-mediated transcytosis. In this thesis, I evaluated different bispecific antibody-based proteins that bind both TfR and Aβ. The overall aim was to determine which factors are important for TfR-mediated brain delivery of these proteins and their use as PET radioligands. In paper I, we studied a large, high TfR-avidity antibody compared with a smaller antibody fragment fusion with lower TfR avidity. The small antibody had fast elimination from blood and was cleared from the brain earlier than the large antibody, thus providing better signal-to-noise ratio for brainPET. In paper II, antibody-like proteins (affibodies), even smaller than the previously studied antibody, had enhanced TfR-mediated brain delivery but had an imbalance in binding to TfR and Aβ. This resulted in poor pathology-related retention of 125I-radiolabeled affibodies. In paper III, we observed that aged mice had poorer brain delivery of the bispecific antibody, mAb3D6-scFv8D3, compared with young mice. Age was also related to increased blood cell binding of the bispecific antibody, and a lower dose resulted in higher relative delivery to the brain parenchyma. In paper IV, we evaluated single domain llama-based antibodies, VHHs, which bound both mouse and human TfR, and were characterized by rapid elimination from blood and brain. The VHHs were fused to an Aβ binding antibody fragment, scFv3D6, which enabled increased brain retention of the 125I-radiobeled antibodies in an AD mouse model, and, thus, provided high contrast to healthy controls.In conclusion, antibody format, size, mouse age, dose, and TfR binding were important factors influencing brain delivery and retention.
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| 3. |
- Meier, Silvio R., 1990-
(författare)
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Preclinical PET imaging and therapy of Alzheimer's disease
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The main histopathological hallmarks of Alzheimer’s disease are extracellular amyloid-beta (Aβ) plaques and intracellular neurofibrillary tangles, containing tau protein. Because of misfolded and aggregated proteins, activated microglia and astroglia react with a neuroinflammatory response, which may contribute to disease progression and severity. To date, there is no treatment available that stops the underlying mechanisms of the disease, but several new drug candidates entered clinical trials every year during the last decade. New treatments, aiming to clear Aβ from the brain parenchyma or to reduce Aβ production, are dependent on diagnostic tools to follow changes in brain Aβ pathology in vivo. The presence of brain amyloid, verified with positron emission tomography (PET), is a regularly used criterion for enrolling patients in clinical trials. However, current amyloid radioligands such as [11C]Pittsburgh Compound B ([11C]PiB) have some disadvantages, e.g. early saturation during disease progression and reduced binding to diffuse Aβ pathology. Currently available radioligands for imaging of neuroinflammation are also suboptimal. In this thesis, we investigated the potential of a brain-penetrating, bispecific Aβ antibody as a PET ligand to detect effects of treatment. In paper I and II, we demonstrated that this ligand can follow Aβ disease progression and that Aβ reduction due to treatment with a BACE-1 inhibitor can be quantified in a mouse model of AD. In paper II we also compared antibody-PET with [11C]PiB-PET and showed that the two ligands provided differing read-outs.In paper III we created and investigated an antibody-based radioligand against the triggering receptor expressed on myeloid cells 2. Compared to wild type mice, transgenic animals displayed higher total in vivo exposure, calculated as the area under the concentration curve based on PET at 24 h, 48 h and 72 h post injection. However, differences were not evident in single time point PET images.In paper IV we investigated brain delivery of a nanobody against GFAP with and without active transcytosis over the blood-brain barrier in vivo. Brain uptake with active transcytosis was two times higher. However, brain retention after 8 h, 24 h or 48 h did not differ between transgenic and wild type mice. In paper V we studied the potential of a hexavalent and bispecific antibody construct against soluble Aβ aggregates for PET or immunotherapy in vivo. Its brain retention increased with age when applied at tracer doses in genetically modified mice. However, when applied at therapeutic dose, it had no or very low impact on Aβ levels measured in brain homogenates.
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| 4. |
- Tobias, Gustavsson, 1985-
(författare)
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Brain distribution of a bispecific antibody targeting Aβ
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer’s disease (AD) is characterised by aberrant protein aggregation in the brain with subsequent synaptic loss, neuroinflammation, and brain atrophy that ultimately clinically manifests as cognitive impairment. Histopathological findings in AD are extracellular plaques of the protein amyloid-beta (Aβ), Aβ in blood vessels (CAA), and intracellular neurofibrillary tangles (NFT) of hyperphosphorylated tau. The FDA recently approved the antibody aducanumab for AD treatment, and several antibodies are now in clinical phase 3 trials, demonstrating that Aβ-directed immunotherapy is a viable treatment option in AD. In this thesis we evaluated the therapeutic Aβ antibodies 3D6 and RmAb158 in comparison with the bispecific RmAb158-scFv8D3, which penetrates the blood-brain barrier (BBB) by transferrin receptor mediated transcytosis. Emphasis lies in antibody brain uptake and intra brain distribution, in their use as potential treatment options in AD and how such treatment affects BBB integrity. Vascular disturbances are common side effects of anti-Aβ immunotherapy. However, we demonstrated that the BBB permeability of large molecules is unchanged following acute 3D6 treatment in an Aβ mouse model (paper I). Next, brain uptake and distribution of radioiodinated RmAb158 and its bispecific variant RmAb158-scFv8D3 were investigated with SPECT in an Aβ mouse model. Due to its active transport across BBB, RmAb158-scFv8D3 had a higher brain uptake than RmAb158, resulting in greater total brain exposure, and higher concentration at Aβ plaques (paper II). In paper III, we labelled RmAb158-scFv8D3 with the radiometal indium-111 (111In), using chelators CHX-A”-DTPA or DOTA, and SPECT was used to investigate brain retention and biodistribution. The 111In-labelled bispecific antibody entered the brain, and although brain retention was higher in Aβ mice, the wild type (wt) background was high. SPECT revealed high bone uptake of all tracers, and subsequent ex vivo measurement pinpointed retention to the bone marrow. With the knowledge gained from paper II, we addressed whether RmAb158-scFv8D3 would improve treatment efficacy in different treatment regimes. We also assessed the immunogenicity of different antibody constructs upon chronic administration (paper IV). As all tested bispecific antibody constructs elicited a humoral response, immune cell depletion was necessary before repeated antibody treatment. Overall, long-term treatment of RmAb158-scFv8D3 did lower total brain Aβ but compared with RmAb158, it did not improve treatment efficacy.In conclusion, acute anti-Aβ immunotherapy did not negatively affect BBB integrity, and bispecific antibodies displayed improved brain distribution and long-term accumulation at parenchymal Aβ. However, this did not translate into an added treatment effect in a chronic therapeutic setting.
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| 5. |
- Bonvicini, Gillian
(författare)
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Harnessing the molecular Trojan horse : Evaluating properties of preclinical Aβ immunoPET radioligands for optimized brain delivery via the transferrin receptor
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- With high specificity and selectivity to targets, antibodies are prime candidates for positron emission tomography (PET) radioligands. They do not passively cross the blood-brain barrier which has hindered their development for imaging intrabrain targets, like amyloid-β (Aβ) in Alzheimer’s disease. The molecular Trojan horse strategy with antibodies that bind to both the transferrin receptor (TfR) and an intrabrain target improves brain delivery of therapeutic antibodies. However, therapeutic antibodies are typically dosed substantially higher than antibody-based PET (immunoPET) radioligands.This thesis evaluated the effects of affinity, valency, and dose on the brain delivery of preclinical Aβ immunoPET radioligands via the TfR.Paper I investigated whether immunoPET with TfR-mediated brain delivery could image Aβ with similar sensitivity in rats as it has in mice. To our knowledge, this was the first time TfR-hijacking to deliver a radioligand to image Aβ was successfully demonstrated in rats; suggesting this strategy could eventually be translated to clinics.Affinity to TfR influences therapeutic delivery to the brain. In Paper II, we compared four Biacore setups and one on-cell assay for determining apparent affinities to the TfR. Absolute affinity determination was challenging since several assay conditions impacted the kinetic parameters. A directional TfR capture in Biacore may be optimal since it determined kinetic parameters while mimicking in vivo receptor conditions. Papers I and III investigated how antibody affinity affects brain delivery at tracer doses and indicated that stronger TfR affinity yielded higher brain delivery. The antibodies in Paper III lacked effector function. The resulting pharmacokinetic profiles in Aβ pathology-presenting mice indicated this may have improved target accumulation of the immunoPET radioligand.In Paper IV, we screened a novel library of monovalent and bivalent affinity variants of the anti-mouse TfR antibody, 8D3. A pair of monovalent and bivalent antibodies with an apparent affinity of 10 nM was identified and evaluated in vivo. Monovalent binding yielded higher brain uptake at a tracer dose but whether bivalent binding steered the antibody towards lysosomal degradation was unclear.In conclusion, monovalency, high affinity binding, and ablated effector function are likely beneficial properties for TfR-mediated brain delivery of an immunoPET radioligand at a tracer dose.
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| 6. |
- Xiong, Mengfei
(författare)
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Visualising neurodegeneration in the living brain : Preclinical evaluation of PET radioligands
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- With an ageing population, the number of people suffering from Alzheimer’s disease (AD) and Parkinson’s disease (PD) escalates yearly. Pathological hallmarks of AD and PD include aggregated proteins and synaptic dysfunction. Developing imaging probes targeting specific pathological hallmarks is highly valuable in aiding early diagnosis and treatment assessment.The thesis focused on evaluating positron emission tomography (PET) imaging probes that can visualise different pathological changes in preclinical models of neurodegeneration. Ligands targeting synaptic vesicle protein 2A (SV2A), alpha-synuclein (αSyn), and amyloid-beta (Aβ) are investigated.In paper I, we compared synaptic density in transgenic AD and PD mouse models to their wild-type age-matched controls using SV2A PET. In the hippocampus, lower synaptic density was found in the PD mice compared to the control. In paper II, we continued using SV2A PET and studied synaptic density in ageing mice. Synaptic density remained steady for most of the lifespan but slightly decreased in old age. In paper III, we developed and evaluated five antibody-based PET radioligands targeting αSyn aggregates. By conjugating anti-αSyn antibodies with the transferrin receptor (TfR) binder 8D3, we increased antibody brain entry significantly. These bispecific antibodies displayed high specificity and selectivity to αSyn aggregates. The most promising candidate successfully imaged brain-deposited αSyn but was unable to detect endogenously expressed αSyn in PD mouse models. In light of this, further investigation of antibody brain entry, distribution, and elimination is needed. Thus, in paper IV, we used microdialysis to compare the brain pharmacokinetics of a bispecific antibody targeting TfR and Aβ and its regular monospecific version that only binds to Aβ. The bispecific antibody showed distinct pharmacokinetics and entered the brain more efficiently than the regular antibody. Lastly, in paper V, we studied the impact of anti-Aβ antibody treatment on amyloid PET. AD mice were short-term treated with anti-Aβ antibody mAb158 and underwent [11C]PiB ex vivo autoradiography. We found a trend indicating that the treatment reduced the [11C]PiB signal despite no reduction in total Aβ levels.Our results contribute to an increased understanding of PET radioligands imaging neurodegeneration. Furthermore, it provides valuable information for designing and developing new PET radioligands.
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| 7. |
- Fang, Xiaotian T., 1990-
(författare)
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Preclinical PET imaging of Alzheimer's disease progression
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Amyloid PET imaging with [11C]PIB enabled detection of Aβ for the first time in vivo. However, [11C]PIB is a small molecule that binds only the insoluble Aβ plaque. Rather, the soluble Aβ aggregates are considered the cause of Alzheimer’s disease (AD). As such, a more sensitive and specific PET tracer is needed for tracking longitudinal AD pathology.Soluble Aβ aggregates likely interact with the metabotropic glutamate receptor 5 (mGluR5) to cause neurotoxic effects. However, with [11C]ABP688 PET we were unable to detect aberrant mGluR5 binding in AD mouse models, although we find elevated mGluR5 protein levels with immunoblotting.Antibodies are highly specific large molecules that can bind specifically to soluble Aβ aggregates, thus they can be a good marker for AD pathology. Unfortunately, due to their large size they cannot cross the blood-brain barrier (BBB). However, it is possible to shuttle antibodies into the brain by taking advantage of endogenous transporter systems on the BBB. By creating bispecific antibodies binding both to soluble Aβ aggregates and to the transferrin receptor (BBB target), we successfully transported the antibody into the brain and could visually detect soluble Aβ aggregates with PET.Recombinant expression further improved and optimized antibody design, creating smaller bispecific antibody-based constructs that had better pharmacokinetic properties allowing for earlier PET scanning (1 day instead of 3), and more sensitive signal.Lastly, using TCO-tetrazine click chemistry, we indirectly labeled our antibodies with fluorine-18, and could successfully perform PET already 11 h post-injection with a fluorine-18 labeled antibody.
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| 8. |
- Loryan, Irena, Associate Professor (Docent), 1977-, et al.
(författare)
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Brain Distribution of Drugs: : Pharmacokinetic Considerations
- 2020
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Ingår i: Handbook of Experimental Pharmacology. - Cham : Springer Berlin/Heidelberg.
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Bokkapitel (refereegranskat)abstract
- It is crucial to understand the basic principles of drug transport, from the site of delivery to the site of action within the CNS, in order to evaluate the possible utility of a new drug candidate for CNS action, or possible CNS side effects of non-CNS targeting drugs. This includes pharmacokinetic aspects of drug concentration-time profiles in plasma and brain, blood–brain barrier transport and drug distribution within the brain parenchyma as well as elimination processes from the brain. Knowledge of anatomical and physiological aspects connected with drug delivery is crucial in this context. The chapter is intended for professionals working in the field of CNS drug development and summarizes key pharmacokinetic principles and state-of-the-art experimental methodologies to assess brain drug disposition. Key parameters, describing the extent of unbound (free) drug across brain barriers, in particular blood–brain and blood–cerebrospinal fluid barriers, are presented along with their application in drug development. Special emphasis is given to brain intracellular pharmacokinetics and its role in evaluating target engagement. Fundamental neuropharmacokinetic differences between small molecular drugs and biologicals are discussed and critical knowledge gaps are outlined.
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| 9. |
- Rofo, Fadi, et al.
(författare)
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Wide-Ranging Effects on the Brain Proteome in a Transgenic Mouse Model of Alzheimer's Disease Following Treatment with a Brain-Targeting Somatostatin Peptide
- 2021
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Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 12:13, s. 2529-2541
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimer’s disease is the most common neurodegenerative disorder characterized by the pathological aggregation of amyloid-β (Aβ) peptide. A potential therapeutic intervention in Alzheimer’s disease is to enhance Aβ degradation by increasing the activity of Aβ-degrading enzymes, including neprilysin. The somatostatin (SST) peptide has been identified as an activator of neprilysin. Recently, we demonstrated the ability of a brain-penetrating SST peptide (SST-scFv8D3) to increase neprilysin activity and membrane-bound Aβ42 degradation in the hippocampus of mice overexpressing the Aβ-precursor protein with the Swedish mutation (APPswe). Using LC–MS, we further evaluated the anti-Alzheimer’s disease effects of SST-scFv8D3. Following a triple intravenous injection of SST-scFv8D3, the LC–MS analysis of the brain proteome revealed that the majority of downregulated proteins consisted of mitochondrial proteins regulating fatty acid oxidation, which are otherwise upregulated in APPswe mice compared to wild-type mice. Moreover, treatment with SST-scFv8D3 significantly increased hippocampal levels of synaptic proteins regulating cell membrane trafficking and neuronal development. Finally, hippocampal concentrations of growth-regulated α (KC/GRO) chemokine and degradation of neuropeptide-Y were elevated after SST-scFv8D3 treatment. In summary, our results demonstrate a multifaceted effect profile in regulating mitochondrial function and neurogenesis following treatment with SST-scFv8D3, further suggesting the development of Alzheimer’s disease therapies based on SST peptides.
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| 10. |
- Syvänen, Stina, 1978-
(författare)
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Blood-Brain Barrier Transport : Investigation of Active Efflux using Positron Emission Tomography and Modelling Studies
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis examines the transport of exogenous molecules across the blood-brain barrier (BBB), focusing on active efflux, using positron emission tomography (PET), computer simulation and modelling. P-glycoprotein (P-gp) inhibition was studied using [11C]verapamil and [11C]hydroxyurea was investigated as a new marker for active efflux transport. Simulations were carried out to explore the importance of the efflux transporter location in the BBB. Brain concentrations of [11C]verapamil, [11C]GR205171 and [18F]altanserin were compared in various laboratory animal species and in humans.A central aspect of the studies has been the novel combination of dynamic PET imaging of the brain pharmacokinetics of a labelled drug, administered through an exponential infusion scheme allowing time-resolved consequence analysis of P-gp inhibition, and mathematical modelling of the obtained data. The methods are applicable to drugs under development and can be used not only in rodents but also in higher species, potentially even in humans, to investigate the effects of P-gp or other transporters on drug uptake in the brain.The inhibition of P-gp by cyclosporin A (CsA) and the subsequent change in brain concentrations of [11C]verapamil occurred rapidly in the sense that [11C]verapamil uptake increased rapidly after CsA administration but also in the sense that the increased uptake was rapidly reversible. The P-gp inhibition was best described by an inhibitory indirect effect model in which CsA decreased the transport of [11C]verapamil out of the brain. The model indicated that approximately 90% of the transport of [11C]verapamil was P-gp-mediated. The low brain concentrations of [11C]hydroxyurea appeared to be a result of slow transport across the BBB rather than active efflux. This exemplifies why the extent and the rate of brain uptake should be approached as two separate phenomena. The brain-to-plasma concentration ratios for the three studied radiotracers differed about 10-fold be-tween species, with lower concentrations in rodents than in humans, monkeys and pigs. The increase in brain concentrations after P-gp inhibition was somewhat greater in rats than in the other species.The findings demonstrate a need to include the dynamics of efflux inhibition in the experimental design and stress the importance of the choice of species in preclinical studies of new drug candidates.
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