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Intra- or extracellular aggregates of proteins are central pathogenic features in most neurodegenerative disorders. The accumulation of such proteins in diseased brains is believed to be the end-stage of a stepwise aggregation of misfolded monomers to insoluble cross-ß fibrils via a series of differently sized soluble oligomers/protofibrils. Several studies have shown how a-synuclein, amyloid-ß, tau and other amyloidogenic proteins can act as nucleating particles and thereby share properties with misfolded forms, or strains, of the prion protein. Although the roles of different protein assemblies in the respective aggregation cascades remain unclear, oligomers/protofibrils are considered key pathogenic species. Numerous observations have demonstrated their neurotoxic effects and a growing number of studies have indicated that they also possess seeding properties, enabling their propagation within cellular networks in the nervous system. The seeding behavior of oligomers differs between the proteins and is also affected by various factors, such as size, shape and epitope presentation. Here, we are providing an overview of the current state of knowledge with respect to the "prion-like" behavior of soluble oligomers for several of the amyloidogenic proteins involved in neurodegenerative diseases. In addition to providing new insight into pathogenic mechanisms, research in this field is leading to novel diagnostic and therapeutic opportunities for neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

An increasing number of studies argues that self-propagating protein conformations (i.e., prions) feature in the pathogenesis of several common neurodegenerative diseases. Mounting evidence contends that aggregates of the amyloid-β (Aβ) peptide become self-propagating in Alzheimer's disease (AD) patients. An important characteristic of prions is their ability to replicate distinct strains, the biological information for which is enciphered within different conformations of protein aggregates. To investigate whether distinct strains of Aβ prions can be discerned in AD patients, we performed transmission studies in susceptible transgenic mice using brain homogenates from sporadic or heritable (Arctic and Swedish) AD cases. Mice inoculated with the Arctic AD sample exhibited a pathology that could be distinguished from mice inoculated with the Swedish or sporadic AD samples, which was judged by differential accumulation of Aβ isoforms and the morphology of cerebrovascular Aβ deposition. Unlike Swedish AD- or sporadic AD-inoculated animals, Arctic AD-inoculated mice, like Arctic AD patients, displayed a prominent Aβ38-containing cerebral amyloid angiopathy. The divergent transmission behavior of the Arctic AD sample compared with the Swedish and sporadic AD samples was maintained during second passage in mice, showing that Aβ strains are serially transmissible. We conclude that at least two distinct strains of Aβ prions can be discerned in the brains of AD patients and that strain fidelity was preserved on serial passage in mice. Our results provide a potential explanation for the clinical and pathological heterogeneity observed in AD patients.

Background: Neuroinflammatory processes have been suggested to play a role in the pathophysiology of neurodegenerative diseases and post-hemorrhagic hydrocephalus, but have rarely been investigated in patients with idiopathic normal pressure hydrocephalus (iNPH). The aim of this study was to investigate whether levels of inflammatory proteins in CSF are different in iNPH compared to healthy controls and patients with selected neurodegenerative disorders, and whether any of these markers can aid in the differential diagnosis of iNPH. Methods: Lumbar CSF was collected from 172 patients from a single center and represented iNPH (n = 74), Alzheimer's disease (AD) (n = 21), mild cognitive impairment (MCI) due to AD (n = 21), stable MCI (n = 22), frontotemporal dementia (n = 13), and healthy controls (HC) (n = 21). Levels of 92 inflammatory proteins were analyzed using a proximity extension assay. As a first step, differences between iNPH and HC were investigated, and proteins that differed between iNPH and HC were then compared with those from the other groups. The linear regressions were adjusted for age, sex, and plate number. Results: Three proteins showed higher (MCP-1, p = 0.0013; CCL4, p = 0.0008; CCL11, p = 0.0022) and one lower (PD-L1, p = 0.0051) levels in patients with iNPH compared to HC. MCP-1 was then found to be higher in iNPH than in all other groups. CCL4 was higher in iNPH than in all other groups, except in MCI due to AD. PD-L1 was lower in iNPH compared to all other groups, except in stable MCI. Levels of CCL11 did not differ between iNPH and the differential diagnoses. In a model based on the four proteins mentioned above, the mean area under the receiver operating characteristic curve used to discriminate between iNPH and the other disorders was 0.91. Conclusions: The inflammatory cytokines MCP-1 and CCL4 are present at higher—and PD-L1 at lower—levels in iNPH than in the other investigated diagnoses. These three selected cytokines may have diagnostic potential in the work-up of patients with iNPH. [ABSTRACT FROM AUTHOR]

Background: Tools to identify individuals at preclinical stages of dementia disorders are needed to enable early interventions. Alterations in dual-task performance have been detected early in progressive neurodegenerative disorders. Hence, dual-task testing may have the potential to screen for cognitive impairment caused by neurodegeneration. Exploring correlations between dual-task performance and biomarkers of neurodegeneration is therefore of interest.Objective: To investigate correlations between Timed Up-and-Go dual-task (TUGdt) outcomes and Alzheimer's disease (AD) cerebrospinal fluid (CSF) biomarkers amyloid-β 42 (Aβ42), total tau (t-tau), and phosphorylated tau (p-tau).Methods: This cross-sectional cohort study included 90 participants (age range 49-84 years) undergoing memory assessment, who were subsequently diagnosed with AD, other dementia disorders, mild cognitive impairment, or subjective cognitive impairment. TUG combined with "Naming Animals" (TUGdt NA) and "Months Backwards" (TUGdt MB), respectively, were used to assess dual-task performance. The number of correct words and time taken to complete the tests were measured. The CSF biomarkers were analysed by ELISA. Spearman's rank correlation was used for analyses between TUGdt outcomes (TUGdt NA and TUGdt MB), and CSF biomarkers, adjusted for age, gender, and educational level.Results: The number of correct words, as well as the number of correct words/10 s during TUGdt NA correlated negatively to CSF t-tau and p-tau. No correlations were found between any time scores and CSF biomarkers.Conclusion: The correlations between TUGdt NA and t-tau and p-tau may indicate that neurodegeneration affects dual-task performance. Longitudinal studies are needed to further explore dual-task testing in screening for cognitive impairment due to neurodegeneration. [ABSTRACT FROM AUTHOR]

Abstract: Deposition of amyloid‐β (Aβ) is central to Alzheimer's disease (AD) pathogenesis and associated with progressive neurodegeneration in traumatic brain injury (TBI). We analyzed predisposing factors for Aβ deposition including monomeric Aβ40, Aβ42 and Aβ oligomers/protofibrils, Aβ species with pronounced neurotoxic properties, following human TBI. Highly selective ELISAs were used to analyze N‐terminally intact and truncated Aβ40 and Aβ42, as well as Aβ oligomers/protofibrils, in human brain tissue, surgically resected from severe TBI patients (n = 12; mean age 49.5 ± 19 years) due to life‐threatening brain swelling/hemorrhage within one week post‐injury. The TBI tissues were compared to post‐mortem AD brains (n = 5), to post‐mortem tissue of neurologically intact (NI) subjects (n = 4) and to cortical biopsies obtained at surgery for idiopathic normal pressure hydrocephalus patients (iNPH; n = 4). The levels of Aβ40 and Aβ42 were not elevated by TBI. The levels of Aβ oligomers/protofibrils in TBI were similar to those in the significantly older AD patients and increased compared to NI and iNPH controls (P < 0.05). Moreover, TBI patients carrying the AD risk genotype Apolipoprotein E epsilon3/4 (APOE ε3/4; n = 4) had increased levels of Aβ oligomers/protofibrils (P < 0.05) and of both N‐terminally intact and truncated Aβ42 (P < 0.05) compared to APOE ε3/4‐negative TBI patients (n = 8). Neuropathological analysis showed insoluble Aβ aggregates (commonly referred to as Aβ plaques) in three TBI patients, all of whom were APOE ε3/4 carriers. We conclude that soluble intermediary Aβ aggregates form rapidly after TBI, especially among APOE ε3/4 carriers. Further research is needed to determine whether these aggregates aggravate the clinical short‐ and long‐term outcome in TBI. [ABSTRACT FROM AUTHOR]

Background: Increased concentrations of cerebrospinal fluid (CSF) total tau (t-tau) and phosphorylated tau, as well as decreased amyloid-β 42 peptide, are biomarkers of Alzheimer's disease (AD) pathology, but few studies have shown an association with AD progression rate. We hypothesized that high CSF tau, as a marker of ongoing neurodegeneration, would predict a more aggressive course of AD, using time to nursing home placement (NHP) as the main outcome. Methods: Our sample inlcuded 234 patients with mild cognitive impairment (MCI) due to AD (n = 134) or mild to moderate AD (n = 100) who underwent lumbar puncture at a memory clinic and were followed for 2-11 years (median 4.9 years). Results: Individuals with CSF t-tau in the highest quartile (≥900 ng/L) had a higher ratio of NHP, both in the total cohort and in patients with MCI only (adjusted HR 2.17 [95 % CI 1.24-3.80]; HR 2.37 [95 % CI 1.10-5.09], respectively), than the lowest quartile. The association between high t-tau levels and future steep deterioration was confirmed in analyses with conversion to moderate dementia (HR 1.66; 95 % CI 1.08-2.56), rapid decline in Mini Mental State Examination score (≥4-point drop/12 months), and dying in severe dementia as outcomes. Conclusions: To our knowledge, this is the first study to show that high CSF t-tau levels predict early NHP and conversion to moderate dementia in an AD cohort. Selecting patients with high CSF t-tau, indicating more aggressive neurodegeneration and steeper decline, for AD immunotherapy trials might increase the possibility of showing contrast between active treatment and placebo. [ABSTRACT FROM AUTHOR]

Cystatin C is suggested to be involved in neurodegeneration and the development of Alzheimer's disease (AD) by binding to soluble amyloid-β (Aβ) peptides. Studies of cystatin C levels in cerebrospinal fluid (CSF) in relation to risk of AD are conflicting and relations between cystatin C, Aβ42, and tau levels in CSF in AD, mild cognitive impairment (MCI), and healthy controls are unknown. The objective of this study was to investigate cystatin C, Aβ42, and tau levels in CSF in AD, MCI, and controls. As a secondary aim, the relationships between cystatin C, Aβ42, and tau levels across disease groups were investigated. Cystatin C, Aβ42, total tau, and phosphorylated tau levels in CSF were analyzed by turbidimetry (cystatin C) and xMAP Luminex technology (Aβ and tau) in persons with AD (n=101), MCI (n=84), and healthy control subjects (n=28). Mean cystatin C levels were similar in cases of AD (5.6 μmol/L ± 1.7), MCI (5.4 μmol/L ± 1.48), and controls (5.6 μmol/L ± 1.6). However, CSF cystatin C levels were strongly and positively correlated with total tau and phosphorylated tau levels (r=0.61–0.81, p< 0.0001) and Aβ42 (r=0.35–0.65, p< 0.001) independent of age, gender, and APOE genotype. Mean CSF cystatin C levels did not differ between patients with AD and MCI and healthy controls. Interestingly, cystatin C levels were positively correlated with both tau and Aβ42 levels in CSF independent of age, gender, and APOE genotype. [ABSTRACT FROM AUTHOR]

Background/Aims: Alterations in gene dosage have recently been associated with neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease, and deletions of the progranulin (PGRN) locus were recently described in patients with frontotemporal lobar degeneration (FTLD). FTLD is a genetically complex neurodegenerative disorder with mutations in the PGRN and the microtubule-associated protein tau (MAPT) genes being the most common known causes of familial FTLD. In this study, we investigated 39 patients with FTLD, previously found negative for mutations in PGRN and MAPT, for copy number alterations of these 2 genes. Methods: Gene dosage analysis of PGRN and MAPT was performed using multiplex ligation-dependent probe amplification. Results: We did not identify any PGRN or MAPT gene dosage variations in the 39 FTLD patients investigated. Conclusion: We therefore conclude that alterations in gene copy number of PGRN and MAPT are not a cause of disease in this collection of FTLD patients. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Tau is a microtubule-associated protein that is important for establishing and maintaining neuronal morphology. In addition to its role in normal cells, tau protein is involved in many neurodegenerative diseases, e.g. Alzheimer’s disease (AD) and frontotemporal dementia, as the main component of intraneuronal aggregates. Alternative splicing of tau gene in the brain can give rise to at least six protein variants. A causative role of skewed tau exon 10 inclusion has been defined in frontotemporal dementia; however, no link was established between the aberrant splicing of tau and AD. Here, we applied a single-molecule-based technology, polymerase colony or polony, to simultaneously monitor tau splicing variant and haplotype profile in sporadic AD and normal brains. We found that the coordinated expression of tau exons 2 and 10 is altered in AD. Additional investigations of cis and trans mechanisms of this observation revealed a decreased protein expression of a known tau splicing factor, htra2-beta-1 in AD, thereby implicating a trans mechanism. Our results demonstrate that dysregulation of combinatorial splicing might serve as a signature for aging-related diseases, and the polony assay could be widely adapted for the study of other tauopathies. Furthermore, splicing-based therapeutics is an emerging area of drug development, and a well-defined and quantitative assay for monitoring single-gene transcriptome will be relevant for such development. [ABSTRACT FROM AUTHOR]

Aggregated forms of different proteins are common hallmarks for several neurodegenerative diseases, including Alzheimer's disease, and ligands that selectively detect specific protein aggregates are vital. Herein, we investigate the molecular requirements of thiophene‐vinyl‐benzothiazole based ligands to detect a specific type of Aβ deposits found in individuals with dominantly inherited Alzheimer's disease caused by the Arctic APP E693G mutation. The staining of these Aβ deposits was alternated when switching the terminal heterocyclic moiety attached to the thiophene‐vinyl‐benzothiazole scaffold. The most prevalent staining was observed for ligands having a terminal 3‐methyl‐1H‐indazole moiety or a terminal 1,2‐dimethoxybenzene moiety, verifying that specific molecular interactions between these ligands and the aggregates were necessary. The synthesis of additional thiophene‐vinyl‐benzothiazole ligands aided in pinpointing additional crucial chemical determinants, such as positioning of nitrogen atoms and methyl substituents, for achieving optimal staining of Aβ aggregates. When combining the optimized thiophene‐vinyl‐benzothiazole based ligands with a conventional ligand, CN‐PiB, distinct staining patterns were observed for sporadic Alzheimer's disease versus dominantly inherited Alzheimer's disease caused by the Arctic APP E693G mutation. Our findings provide chemical insights for developing novel ligands that allow for a more precise assignment of Aβ deposits, and might also aid in creating novel agents for clinical imaging of distinct Aβ aggregates in AD. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is characterized by a substantial loss of neurons and synapses throughout the brain. The exact mechanism behind the neurodegeneration is still unclear, but recent data suggests that spreading of amyloid-β (Aβ) pathology via extracellular vesicles (EVs) may contribute to disease progression. We have previously shown that an incomplete degradation of Aβ42 protofibrils by astrocytes results in the release of EVs containing neurotoxic Aβ. Here, we describe the cellular mechanisms behind EV-associated neurotoxicity in detail. EVs were isolated from untreated and Aβ42 protofibril exposed neuroglial co-cultures, consisting mainly of astrocytes. The EVs were added to cortical neurons for 2 or 4 days and the neurodegenerative processes were followed with immunocytochemistry, time-lapse imaging and transmission electron microscopy (TEM). Addition of EVs from Aβ42 protofibril exposed co-cultures resulted in synaptic loss, severe mitochondrial impairment and apoptosis. TEM analysis demonstrated that the EVs induced axonal swelling and vacuolization of the neuronal cell bodies. Interestingly, EV exposed neurons also displayed pathological lamellar bodies of cholesterol deposits in lysosomal compartments. Taken together, our data show that the secretion of EVs from Aβ exposed cells induces neuronal dysfunction in several ways, indicating a central role for EVs in the progression of Aβ-induced pathology. [ABSTRACT FROM AUTHOR]

Cellular assays reveal a decline in Aβ and tau pathological conformers in Alzheimer's disease brain samples. Puncta point the way: Amyloid plaques composed of Aβ peptides and neurofibrillary tangles composed of aberrant tau proteins are the key pathological hallmarks in the Alzheimer's disease (AD) brain. However, understanding which conformers of Aβ and tau play a pathological role at each step of AD pathogenesis has been difficult to elucidate. Aoyagi et al. have developed sensitive cellular assays that detect aberrant Aβ and tau in postmortem brain homogenates from patients with AD or other neurodegenerative diseases. Using fluorescent puncta as a readout, these assays now reveal that patients with AD who died at an older age have lower Aβ and tau pathological conformers than do patients who died at a younger age. The hallmarks of Alzheimer's disease (AD) are the accumulation of Aβ plaques and neurofibrillary tangles composed of hyperphosphorylated tau. We developed sensitive cellular assays using human embryonic kidney–293T cells to quantify intracellular self-propagating conformers of Aβ in brain samples from patients with AD or other neurodegenerative diseases. Postmortem brain tissue from patients with AD had measurable amounts of pathological Aβ conformers. Individuals over 80 years of age had the lowest amounts of prion-like Aβ and phosphorylated tau. Unexpectedly, the longevity-dependent decrease in self-propagating tau conformers occurred in spite of increasing amounts of total insoluble tau. When corrected for the abundance of insoluble tau, the ability of postmortem AD brain homogenates to induce misfolded tau in the cellular assays showed an exponential decrease with longevity, with a half-life of about one decade over the age range of 37 to 99 years. Thus, our findings demonstrate an inverse correlation between longevity in patients with AD and the abundance of pathological tau conformers. Our cellular assays can be applied to patient selection for clinical studies and the development of new drugs and diagnostics for AD. [ABSTRACT FROM AUTHOR]

Pathophysiological impairment of the neurovascular unit, including the integrity and dynamics of the blood-brain barrier (BBB), has been denoted both a cause and consequence of neurodegenerative diseases. Pathological impact on BBB drug delivery has also been debated. The aim of the present study was to investigate BBB drug transport, by determining the unbound brain-to-plasma concentration ratio (K p,uu,brain ), in aged AβPP-transgenic mice, α-synuclein transgenic mice, and wild type mice. Mice were dosed with a cassette of five compounds, including digoxin, levofloxacin (1 mg/kg, s.c.), paliperidone, oxycodone, and diazepam (0.25 mg/kg, s.c.). Brain and blood were collected at 0.5, 1, or 3 h after dosage. Drug concentrations were measured using LC-MS/MS. The total brain-to-plasma concentration ratio was calculated and equilibrium dialysis was used to determine the fraction of unbound drug in brain and plasma for all compounds. Together, these three measures were used to determine the K p,uu,brain value. Despite Aβ or α-synuclein pathology in the current animal models, no difference was observed in the extent of drug transport across the BBB compared to wild type animals for any of the compounds investigated. Hence, the present study shows that the concept of a leaking barrier within neurodegenerative conditions has to be interpreted with caution when estimating drug transport into the brain. The capability of the highly dynamic BBB to regulate brain drug exposure still seems to be intact despite the presence of pathology. [ABSTRACT FROM AUTHOR]

The protein alpha-synuclein (αSYN) plays a central role in synucleinopathies such as Parkinsons's disease (PD) and multiple system atrophy (MSA). Presently, there are no selective αSYN positron emission tomography (PET) radioligands that do not also show affinity to amyloid-beta (Aβ). We have previously shown that radiolabeled antibodies, engineered to enter the brain via the transferrin receptor (TfR), is a promising approach for PET imaging of intrabrain targets. In this study, we used this strategy to visualize αSYN in the living mouse brain. Five bispecific antibodies, binding to both the murine TfR and αSYN were generated and radiolabeled with iodine-125 or iodine-124. All bispecific antibodies bound to αSYN and mTfR before and after radiolabelling in an ELISA assay, and bound to brain sections prepared from αSYN overexpressing mice as well as human PD- and MSA subjects, but not control tissues in autoradiography. Brain concentrations of the bispecific antibodies were between 26 and 63 times higher than the unmodified IgG format 2 h post-injection, corresponding to about 1.5% of the injected dose per gram brain tissue. Additionally, intrastriatal αSYN fibrils were visualized with PET in an αSYN deposition mouse model with one of the bispecific antibodies, [124I]RmAbSynO2-scFv8D3. However, PET images acquired in αSYN transgenic mice with verified brain pathology injected with [124I]RmAbSynO2-scFv8D3 and [124I]RmAb48-scFv8D3 showed no increase in antibody retention compared to WT mice. Despite successful imaging of deposited extracellular αSYN using a brain-penetrating antibody-based radioligand with no cross-specificity towards Aβ, this proof-of-concept study demonstrates challenges in imaging intracellular αSYN inclusions present in synucleinopathies. • Five bispecific antibodies binding to both the transferrin receptor, to facilitate brain delivery, and alpha-synuclein, were generated and radiolabeled. • The radiolabeled bispecific antibodies displayed brain concentrations similar to what is seen for radioligands based on small molecules, and 26–63 times higher than unmodified antibodies. • Pathology was visualized on tissue sections prepared from transgenic mice expressing human alpha-synuclein, and on sections prepared from PD and MSA patients, but not on control tissue. • PET imaging visualized extracellular alpha-synuclein deposits in the living mouse brain but not intracellular alpha-synuclein inclusions in transgenic mice. [ABSTRACT FROM AUTHOR]

Abstract: Oxidative stress has been implicated in the etiology of neurodegenerative disorders with α-synuclein pathology. Lipid peroxidation products such as 4-oxo-2-nonenal (ONE) and 4-hydroxy-2-nonenal (HNE) can covalently modify and structurally alter proteins. Herein, we have characterized ONE- or HNE-induced α-synuclein oligomers. Our results demonstrate that both oligomers are rich in β-sheet structure and have a molecular weight of about 2000kDa. Atomic force microscopy analysis revealed that ONE-induced α-synuclein oligomers were relatively amorphous, with a diameter of 40–80nm and a height of 4–8nm. In contrast, the HNE-induced α-synuclein oligomers had a protofibril-like morphology with a width of 100–200nm and a height of 2–4nm. Furthermore, neither oligomer type polymerized into amyloid-like fibrils despite prolonged incubation. Although more SDS and urea stable, because of a higher degree of cross-linking, ONE-induced α-synuclein oligomers were less compact and more sensitive to proteinase K treatment. Finally, both ONE- and HNE-induced α-synuclein oligomers were cytotoxic when added exogenously to a neuroblastoma cell line, but HNE-induced α-synuclein oligomers were taken up by the cells to a significantly higher degree. Despite nearly identical chemical structures, ONE and HNE induce the formation of off-pathway α-synuclein oligomers with distinct biochemical, morphological, and functional properties. [ABSTRACT FROM AUTHOR]

■ Synaptic density is decreased in a mouse model of alpha-synucleinopathy. ■ Ageing appears to be associated with decreased synaptic density in wild type mouse brain. ■ Model-independent AUC-based brain-to-blood ratio can be used to evaluate [11C]UCB-J binding as an alternative to full pharmacokinetic modeling. The positron emission tomography (PET) radioligand [11C]UCB-J binds to synaptic vesicle protein 2A (SV2A) and is used to investigate synaptic density in the living brain. Clinical studies have indicated reduced [11C]UCB-J binding in Alzheimer's disease (AD) and Parkinson's disease (PD) brains compared to healthy controls. Still, it is unknown whether [11C]UCB-J PET can visualise synaptic loss in mouse models of these disorders. Such models are essential for understanding disease pathology and for evaluating the effects of novel disease-modifying drug candidates. In the present study, synaptic density in transgenic models of AD (ArcSwe) and PD (L61) was studied using [11C]UCB-J PET. Data were acquired during 60 min after injection, and time-activity curves (TACs) in different brain regions and the left ventricle of the heart were generated based on the dynamic PET images. The [11C]UCB-J brain concentrations were expressed as standardised uptake value (SUV) over time. The area under the SUV curve (AUC), the ratio of AUC in the brain to that in the heart (AUC brain/blood), and the volume of distribution (V T) obtained by kinetic modelling using the heart TAC as input were compared between transgenic and age-matched wild type (WT) mice. The L61 mice displayed 11–13% lower AUC brain/blood ratio and brain V T generated by kinetic modeling compared to the control WT mice. In general, also transgenic ArcSwe mice tended to show lower [11C]UCB-J brain exposure than age-matched WT controls, but variation within the different animal groups was high. Older WT mice (18–20 months) showed lower [11C]UCB-J brain exposure than younger WT mice (8–9 months). Together, these data imply that [11C]UCB-J PET reflects synaptic density in mouse models of neurodegeneration and that inter-subject variation is large. In addition, the study suggested that model-independent AUC brain/blood ratio can be used to evaluate [11C]UCB-J binding as an alternative to full pharmacokinetic modelling. [Display omitted] [ABSTRACT FROM AUTHOR]