| 1. |
- 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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| 2. |
- Rofo, Fadi, et al.
(författare)
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Blood-brain barrier penetrating neprilysin degrades monomeric amyloid-beta in a mouse model of Alzheimer’s disease
- 2022
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Ingår i: Alzheimer's Research & Therapy. - : BioMed Central (BMC). - 1758-9193. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundAggregation of the amyloid-β (Aβ) peptide in the brain is one of the key pathological events in Alzheimer’s disease (AD). Reducing Aβ levels in the brain by enhancing its degradation is one possible strategy to develop new therapies for AD. Neprilysin (NEP) is a membrane-bound metallopeptidase and one of the major Aβ-degrading enzymes. The secreted soluble form of NEP (sNEP) has been previously suggested as a potential protein-therapy degrading Aβ in AD. However, similar to other large molecules, peripherally administered sNEP is unable to reach the brain due to the presence of the blood–brain barrier (BBB).MethodsTo provide transcytosis across the BBB, we recombinantly fused the TfR binding moiety (scFv8D3) to either sNEP or a previously described variant of NEP (muNEP) suggested to have higher degradation efficiency of Aβ compared to other NEP substrates, but not per se to degrade Aβ more efficiently. To provide long blood half-life, an Fc-based antibody fragment (scFc) was added to the designs, forming sNEP-scFc-scFv8D3 and muNEP-scFc-scFv8D3. The ability of the mentioned recombinant proteins to degrade Aβ was first evaluated in vitro using synthetic Aβ peptides followed by sandwich ELISA. For the in vivo studies, a single injection of 125-iodine-labelled sNEP-scFc-scFv8D3 and muNEP-scFc-scFv8D3 was intravenously administered to a tg-ArcSwe mouse model of AD, using scFc-scFv8D3 protein that lacks NEP as a negative control. Different ELISA setups were applied to quantify Aβ concentration of different conformations, both in brain tissues and blood samples.ResultsWhen tested in vitro, sNEP-scFc-scFv8D3 retained sNEP enzymatic activity in degrading Aβ and both constructs efficiently degraded arctic Aβ. When intravenously injected, sNEP-scFc-scFv8D3 demonstrated 20 times higher brain uptake compared to sNEP. Both scFv8D3-fused NEP proteins significantly reduced aggregated Aβ levels in the blood of tg-ArcSwe mice, a transgenic mouse model of AD, following a single intravenous injection. In the brain, monomeric and oligomeric Aβ were significantly reduced. Both scFv8D3-fused NEP proteins displayed a fast clearance from the brain.ConclusionA one-time injection of a BBB-penetrating NEP shows the potential to reduce, the likely most toxic, Aβ oligomers in the brain in addition to monomers. Also, Aβ aggregates in the blood were reduced.
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| 3. |
- Rofo, Fadi
(författare)
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Enhancing the therapeutic effect of biological drugs with protein engineering : Focusing on pre-clinical Alzheimer’s disease therapy
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Aggregation of the amyloid-β peptide (Aβ) is one of the main pathological hallmarks in Alzheimer’s disease (AD). The soluble Aβ aggregates (oligomers and protofibrils) have shown to be the most harmful species. Hence, targeting these aggregates can be of therapeutic potential.Protein therapy is one of the fastest growing fields in drug development with more than 100 FDA approved protein-drugs in the last decade. Despite that, protein-drugs (mainly antibodies) targeting Aβ displayed limited beneficial effects in AD clinical trials. This might be attributed to the presence of the blood-brain barrier (BBB) that hinders the entry of big molecules such as proteins into the brain. In paper I, we fused somatostatin peptide (SST) to the previously developed BBB transporter (scFv8D3). The new protein, SST-scFv8D3, exhibited a 120-times longer plasma half-life compared to SST, and reached the brain at high levels when intravenously administered. When tested in APPswe mouse model of AD, SST-scFv8D3 significantly enhanced neprilysin (NEP)-mediated degradation of hippocampal Aβ42 after only three injections. In paper II, treatment with SST-scFv8D3 displayed a wide-ranging effect on AD brain proteome. Mitochondrial and neuronal growth proteins were among the most altered protein-groups, where SST-scFv8D treatment shifted them towards wild-type levels.There is potential to increase the binding strength and selectivity of antibodies to small Aβ aggregates (oligomers), which are thought to be the most toxic Aβ species. In paper III, we developed a multivalent antibody format with additional binding sites having short distances between them. The new antibody format displayed a 40-fold reduction in the dissociation rate from Aβ protofibrils. Furthermore, the multivalent antibody could strongly bind small Aβ oligomers, which has been difficult to achieve with conventional IgG antibodies. In paper IV, we developed a bispecific version of the multivalent antibody capable of passing the BBB. A single intravenous injection of the new antibody format was enough to significantly clear soluble Aβ aggregates from the brain of tg-ArcSwe mice. In paper V, we developed recombinant proteins with NEP linked to an Fc-fragment to provide long half-life and to the above-mentioned BBB transporter. When applied at therapeutic doses, these proteins significantly degraded plasma Aβ, but displayed limited effects on brain Aβ concentration, probably due to their short retention times in the brain.In conclusion, we developed new protein-drugs with improved binding properties to Aβ, ability to cross the BBB, and therapeutic potential in pre-clinical mouse models of AD.
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| 4. |
- Gustafsson, Sofia, 1985-
(författare)
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Translational Aspects of Blood-Brain Barrier Transport and Brain Distribution of Drugs in Health and Disease
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A high unmet medical need in the area of CNS diseases coincides with high failure rates in CNS drug development. Efficient treatment of CNS disease is constrained by limited entrance of drugs into the brain owing to the blood-brain barrier (BBB), which separates brain from blood. Insufficient inter-species translation and lack of methods to evaluate therapeutic, unbound, drug concentrations in human brain also contribute to development failure. Further disease related changes in BBB properties and tissue composition raise a concern of altered drug neuropharmacokinetics (neuroPK) during disease. This calls for the evaluation of translational aspects of neuroPK parameters in health and disease, and exploration of strategies for neuroPK translations between rodents and humans.Positron emission tomography (PET) enables corresponding PK analysis in various species, although being restricted to measuring total, i.e. both unbound and nonspecifically bound, drug concentrations. However, the current work shows that PET can be used for the estimation of unbound, active, brain concentrations and for assessment of drug BBB transport, if compensation is made for intra-brain drug distribution and binding. Adapted PET designs could be applied in humans where rat estimates of drug intra-brain distribution may be used with reasonable accuracy for concentration conversions in healthy humans, but preferably not in Alzheimer’s disease (AD) patients. As shown in this thesis, a high variability in nonspecific drug tissue binding was observed in AD compared to rats and human controls that might lead to unacceptable bias of outcome values if used in PET. Furthermore, heterogeneity in drug tissue binding among brain regions in both rodents and humans was detected and must be considered in regional investigations of neuroPK. By the use of transgenic animal models of amyloid beta and alpha-synuclein pathology, the work further suggests that the BBB is able to uphold sufficient capacity for the transport of small molecular drugs and integrity towards large molecules despite the presence of hallmarks representative of neurodegenerative diseases.This thesis work provides insight into neurodegenerative disease impact on neuroPK and contributes with translational strategies for neuroPK evaluation from preclinical investigations to the clinic, aimed to aid drug development and optimal disease management.
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| 5. |
- Syvänen, Stina, et al.
(författare)
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PET Imaging in Preclinical Anti-Aβ Drug Development
- 2022
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Ingår i: Pharmaceutical research. - : Springer Nature. - 0724-8741 .- 1573-904X. ; 39:7, s. 1481-1496
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Forskningsöversikt (refereegranskat)abstract
- Positron emission tomography (PET), a medical imaging technique allowing for studies of the living human brain, has gained an important role in clinical trials of novel drugs against Alzheimer’s disease (AD). For example, PET data contributed to the conditional approval in 2021 of aducanumab, an antibody directed towards amyloid-beta (Aβ) aggregates, by showing a dose-dependent reduction in brain amyloid after treatment. In parallel to clinical studies, preclinical studies in animal models of Aβ pathology may also benefit from PET as a tool to detect target engagement and treatment effects of anti-Aβ drug candidates. PET is associated with a high level of translatability between species as similar, non-invasive protocols allow for longitudinal rather than cross-sectional studies and can be used both in a preclinical and clinical setting. This review focuses on the use of preclinical PET imaging in genetically modified animals that express human Aβ, and its present and potential future role in the development of drugs aimed at reducing brain Aβ levels as a therapeutic strategy to halt disease progression in AD.
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| 6. |
- Gustafsson, Sofia, et al.
(författare)
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Blood-Brain Barrier Integrity in a Mouse Model of Alzheimer’s Disease With or Without Acute 3D6 Immunotherapy
- 2018
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Ingår i: Neuropharmacology. - : Elsevier BV. - 0028-3908 .- 1873-7064. ; 143, s. 1-9
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Tidskriftsartikel (refereegranskat)abstract
- The blood-brain barrier (BBB) is suggested to be compromised in Alzheimer's disease (AD). The concomitant presence of vascular amyloid beta (AD) pathology, so called cerebral amyloid angiopathy (CAA), also predisposes impairment of vessel integrity. Additionally, immunotherapy against A beta may lead to further damage of the BBB. To what extent this affects the BBB passage of molecules is debated. The current study aimed to investigate BBB integrity to large molecules in transgenic mice displaying abundant A beta pathology and age matched wild type animals, with or without acute anti-A beta antibody treatment. Animals were administered a single i.v. injection of PBS or 3D6 (10 mg/kg), i.e. the murine version of the clinically investigated A beta antibody bapineuzumab, supplemented with [(125)]3D6. Three days post injections, a 4 kDa FITC and a 150 kDa Antonia Red dextran were administered i.v. to all animals. After termination, fluorescent detection in brain and serum was used for the calculation of dextran brain-to-blood concentration ratios. Further characterization of antibody fate and the presence of CAA were investigated using radioactivity measurements and Congo red staining. BBB passage of large molecules was equally low in wild type and transgenic mice, suggesting an intact BBB despite A beta pathology. Neither was the BBB integrity affected by acute 3D6 treatment. However, CAA was confirmed in the transgenes and local antibody accumulations were observed in the brain, indicating CAA-antibody interactions. The current study shows that independently of A beta pathology or acute 3D6 treatment, the BBB is intact, without extensive permeability to large molecules, including the 3D6 antibody.
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| 7. |
- Syvänen, Stina, et al.
(författare)
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Using PET Studies of P-gp Function to Elucidate Mechanisms Underlying the Disposition of Drugs
- 2010
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Ingår i: Current Topics in Medicinal Chemistry. - : Bentham Science Publishers Ltd.. - 1568-0266 .- 1873-4294. ; 10:17, s. 1799-1809
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Forskningsöversikt (refereegranskat)abstract
- This paper discusses the basic principles of drug/P-glycoprotein (P-gp) interaction, focusing on the methodology and design of positron emission tomography (PET) studies investigating P-gp function. The requirements of a good PET P-gp radiotracer are also evaluated. (R)-[C-11]verapamil is used as an example, as this drug is the most common tracer for P-gp studies, but [C-11]loperamide, [C-11]desmethyl-loperamide and other compounds are also mentioned. The article also discusses the various study designs that can be used for PET drug disposition studies, such as administration of the inhibitor before or after the radiolabeled drug (tracer) and the use of bolus injections or infusions. Concepts such as the unbound partition coefficient (K-p,K-uu) and the volume of distribution of unbound drug in brain (V-u,V-brain), which are not easily measured directly with PET, can be used to describe the impact of protein binding and non-specific binding on drug distribution in brain tissue. It is concluded that new imaging probes will be required if the role of PET in studies of the interactions of drugs with efflux transporters is to expand.
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| 8. |
- Bergström, Mats, et al.
(författare)
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Blood-brain barrier penetration of zolmitriptan--modelling of positron emission tomography data
- 2006
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Ingår i: Journal of Pharmacokinetics and Pharmacodynamics. - : Springer Science and Business Media LLC. - 1567-567X .- 1573-8744. ; 33:1, s. 75-91
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Tidskriftsartikel (refereegranskat)abstract
- Positron emission tomography (PET) with the drug radiolabelled allows a direct measurement of brain or other organ kinetics, information which can be essential in drug development. Usually, however, a PET-tracer is administered intravenously (i.v.), whereas the therapeutic drug is mostly given orally or by a different route to the PET-tracer. In such cases, a recalculation is needed to make the PET data representative for the alternative administration route. To investigate the blood-brain barrier penetration of a drug (zolmitriptan) using dynamic PET and by PK modelling quantify the brain concentration of the drug after the nasal administration of a therapeutic dose. [11C]Zolmitriptan at tracer dose was administered as a short i.v. infusion and the brain tissue and venous blood kinetics of [11C]zolmitriptan was measured by PET in 7 healthy volunteers. One PET study was performed before and one 30 min after the administration of 5 mg zolmitriptan as nasal spray. At each of the instances, the brain radioactivity concentration after subtraction of the vascular component was determined up to 90 min after administration and compared to venous plasma radioactivity concentration after correction for radiolabelled metabolites. Convolution methods were used to describe the relationship between arterial and venous tracer concentrations, respectively between brain and arterial tracer concentration. Finally, the impulse response functions derived from the PET studies were applied on plasma PK data to estimate the brain zolmitriptan concentration after a nasal administration of a therapeutic dose. The studies shows that the PET data on brain kinetics could well be described as the convolution of venous tracer kinetics with an impulse response including terms for arterial-to-venous plasma and arterial-to-brain impulse responses. Application of the PET derived impulse responses on the plasma PK from nasal administration demonstrated that brain PK of zolmitriptan increased with time, achieving about 0.5 mg/ml at 30 min and close to a maximum of 1.5 mg/ml after 2 hr. A significant brain concentration was observed already after 5 min. The data support the notation of a rapid brain availability of zolmitriptan after nasal administration.
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| 9. |
- De Lange, E. C. M., et al.
(författare)
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P-glycoprotein protein expression versus functionality at the blood-brain barrier using immunohistochemistry, microdialysis and mathematical modeling
- 2018
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Ingår i: European Journal of Pharmaceutical Sciences. - : Elsevier BV. - 0928-0987 .- 1879-0720. ; 124, s. 61-70
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Tidskriftsartikel (refereegranskat)abstract
- A proper understanding of P-gp mediated transport (functionality) at the blood-brain barrier (BBB) and beyond is needed to interpret, understand and predict pharmacokinetic (PK)- pharmacodynamic (PD) relationships of CNS drugs that are substrates of P-gp, especially since P-gp functionality may be different in different conditions. Often, P-gp expression is taken as a biomarker of transporter functionally. The aim of our study was to investigate whether brain capillary protein expression of P-gp is associated with changes in P-gp mediated drug efflux at the BBB. Status Epilepticus (SE) was induced by kainate in male rats. During 3-5 weeks post SE, hippocampal P-gp expression was determined using immunohistochemistry, while BBB P-gp functionally was assessed by microdialysis of quinidine, in absence and presence of the P-gp blocker tariquidar. The data were analyzed using Nonlinear Mixed Effect Modeling implemented in NONMEM. Following SE, changes in brain capillary P-gp expression were observed. However, no relation between BBB P-gp protein expression and BBB P-gp mediated drug efflux was found. This warrants a critical view on the interpretation of reported changes in BBB P-gp expression as a biomarker of BBB P-gp functionally.
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| 10. |
- Hammarlund-Udenaes, Margareta, et al.
(författare)
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On the rate and extent of drug delivery to the brain
- 2008
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Ingår i: Pharmaceutical research. - : Springer Science and Business Media LLC. - 0724-8741 .- 1573-904X. ; 25:8, s. 1737-1750
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Forskningsöversikt (refereegranskat)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.
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