A3907, a systemic ASBT inhibitor, improves cholestasis in mice by multiorgan activity and shows translational relevance to humans.

BACKGROUND AND AIMS: Cholestasis is characterized by intrahepatic accumulation of bile constituents, including bile acids (BAs), which promote liver damage. The apical sodium-dependent BA transporter (ASBT) plays an important role in BA reabsorption and signaling in ileum, bile ducts, and kidneys. Our aim was to investigate the pharmacokinetics and pharmacological activity of A3907, an oral and systemically available ASBT inhibitor in experimental mouse models of cholestasis. In addition, the tolerability, pharmacokinetics, and pharmacodynamics of A3907 were examined in healthy humans. APPROACH AND RESULTS: A3907 was a potent and selective ASBT inhibitor in vitro. In rodents, orally administered A3907 distributed to the ASBT-expressing organs, that is, ileum, liver, and kidneys, and dose dependently increased fecal BA excretion. A3907 improved biochemical, histological, and molecular markers of liver and bile duct injury in Mdr2-/- mice and also had direct protective effects on rat cholangiocytes exposed to cytotoxic BA concentrations in vitro . In bile duct ligated mice, A3907 increased urinary BA elimination, reduced serum BA levels, and prevented body weight loss, while improving markers of liver injury. A3907 was well tolerated and demonstrated target engagement in healthy volunteers. Plasma exposure of A3907 in humans was within the range of systemic concentrations that achieved therapeutic efficacy in mouse. CONCLUSIONS: The systemic ASBT inhibitor A3907 improved experimental cholestatic disease by targeting ASBT function at the intestinal, liver, and kidney levels, resulting in marked clearance of circulating BAs and liver protection. A3907 is well tolerated in humans, supporting further clinical development for the treatment of cholestatic liver diseases.

Discriminative binding of tau PET tracers PI2620, MK6240 and RO948 in Alzheimer's disease, corticobasal degeneration and progressive supranuclear palsy brains.

Recent mechanistic and structural studies have challenged the classical tauopathy classification approach and revealed the complexity and heterogeneity of tau pathology in Alzheimer's disease (AD) and primary tauopathies such as corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP), progressing beyond distinct tau isoforms. In this multi-tau tracer study, we focused on the new second-generation tau PET tracers PI2620, MK6240 and RO948 to investigate this tau complexity in AD, CBD, and PSP brains using post-mortem radioligand binding studies and autoradiography of large and small frozen brain sections. Saturation binding studies indicated multiple binding sites for 3H-PI2620 in AD, CBD and PSP brains with different binding affinities (Kd ranging from 0.2 to 0.7 nM) and binding site densities (following the order: BmaxAD > BmaxCBD > BmaxPSP). Competitive binding studies complemented these findings, demonstrating the presence of two binding sites [super-high affinity (SHA): IC50(1) = 8.1 pM; and high affinity (HA): IC50(2) = 4.9 nM] in AD brains. Regional binding distribution studies showed that 3H-PI2620 could discriminate between AD (n = 6) and control cases (n = 9), especially in frontal cortex and temporal cortex tissue (p < 0.001) as well as in the hippocampal region (p = 0.02). 3H-PI2620, 3H-MK6240 and 3H-RO948 displayed similar binding behaviour in AD brains (in both homogenate competitive studies and one large frozen hemispherical brain section autoradiography studies) in terms of binding affinities, number of sites and regional patterns. Our small section autoradiography studies in the frontal cortex of CBD (n = 3) and PSP brains (n = 2) showed high specificity for 3H-PI2620 but not for 3H-MK6240 or 3H-RO948. Our findings clearly demonstrate different binding properties among the second-generation tau PET tracers, which may assist in further understanding of tau heterogeneity in AD versus non-AD tauopathies and suggests potential for development of pure selective 4R tau PET tracers.

Impact of elobixibat on serum and fecal bile acid levels and constipation symptoms in patients with chronic constipation.

BACKGROUND AND AIM: Elobixibat is a locally acting inhibitor of the ileal bile acid transporter. We compared bile acid metabolism between healthy subjects and patients with chronic constipation and assessed changes in the bile acid profile after elobixibat administration in the latter group. METHODS: Healthy subjects (n = 10) and patients with chronic constipation (n = 19) were assessed as inpatients for 7 days, during which they received meals containing ~60 g/day of fat. Patients with chronic constipation remained as inpatients for a further 7 days for once-daily elobixibat administration. Assessments included concentrations of fecal and serum bile acids, serum 7α-hydroxy-4-cholesten-3-one (C4) and fibroblast growth factor 19, and bowel movements and constipation symptoms. RESULTS: Fecal total and primary bile acids were significantly lower in patients with chronic constipation versus healthy subjects. Serum C4 and fibroblast growth factor 19 levels were comparable between groups. Elobixibat treatment increased fecal total and primary bile acids and decreased levels of fecal lithocholic acid and serum total as well as secondary bile acids in patients with chronic constipation. Bowel movements and other constipation-related symptoms were also improved by elobixibat to levels almost comparable with those of healthy subjects. CONCLUSIONS: Despite comparable C4 levels, patients with chronic constipation demonstrated decreased levels of fecal bile acids versus healthy subjects. Elobixibat treatment increased fecal bile acid excretion and reduced serum bile acid concentrations. The improvement of constipation after elobixibat treatment was associated with increased total bile acids, particularly primary bile acids.

Effects of odevixibat on pruritus and bile acids in children with cholestatic liver disease: Phase 2 study.

PURPOSE: Ileal bile acid transporter inhibition is a novel therapeutic concept for cholestatic pruritus and cholestatic liver disease progression. Odevixibat, a potent, selective, reversible ileal bile acid transporter inhibitor, decreases enteric bile acid reuptake with minimal systemic exposure. Oral odevixibat safety, tolerability, and efficacy in pediatric patients with cholestatic liver disease and pruritus were evaluated. PATIENTS AND METHODS: In this phase 2, open-label, multicenter study, children received 10‒200 µg/kg oral odevixibat daily for 4 weeks. Changes in serum bile acid levels (primary efficacy endpoint), pruritus, and sleep disturbance were explored. RESULTS: Twenty patients were enrolled (8 females; 1‒17 years; 4 re-entered at a different dose). Diagnoses included progressive familial intrahepatic cholestasis (n = 13; 3 re-entries), Alagille syndrome (n = 6), biliary atresia (n = 3), and other intrahepatic cholestasis causes (n = 2; 1 re-entry). Mean baseline serum bile acid levels were high (235 µmol/L; range, 26‒564) and were reduced in the majority (-123.1 µmol/L; range, -394 to 14.5, reflecting reductions of up to 98%). Patient-reported diary data documented improved pruritus (3 scales) and sleep. With 100 µg/kg, mean (SEM) decrease was 2.8 (1.1) points for pruritus (visual analogue itch scale 0-10) and 2.9 (0.9) points for sleep disturbance (Patient-Oriented Scoring Atopic Dermatitis scale 0-10). Reduced pruritus correlated significantly with reduced serum bile acids (P ≤ 0.007). Significant correlations were also observed between autotaxin levels and pruritus. All patients completed the study. No serious adverse events were treatment related; most adverse events, including increased transaminases, were transient. CONCLUSIONS: Orally administered odevixibat was well tolerated, reduced serum bile acids, and improved pruritus and sleep disturbance in children with cholestatic diseases.

Amyloid, tau, and astrocyte pathology in autosomal-dominant Alzheimer's disease variants: AßPParc and PSEN1DE9.

Autosomal-dominant Alzheimer's disease (ADAD) may be associated with atypical amyloid beta deposits in the brain. In vivo amyloid imaging using 11C-Pittsburgh compound B (PiB) tracer has shown differences in binding between brains from ADAD and sporadic Alzheimer's disease (sAD) patients. To gain further insight into the various pathological characteristics of these genetic variants, we performed large frozen hemisphere autoradiography and brain homogenate binding assays with 3H-PiB, 3H-MK6240-3H-THK5117, and 3H-deprenyl for detection of amyloid fibrils, tau depositions, and activated astrocytes, respectively, in two AßPParc mutation carriers, one PSEN1ΔE9 mutation carrier, and three sAD cases. The results were compared with Abeta 40, Abeta 42, AT8, and GFAP immunostaining, respectively, as well as with Congo red and Bielschowsky. PiB showed a very low binding in AßPParc. A high binding was observed in PSEN1ΔE9 and in sAD tissues but with different binding patterns. Comparable 3H-THK5117 and 3H-deprenyl brain homogenate binding was observed for AßPParc, PSEN1ΔE9, and sAD, respectively. Some differences were observed between 3H-MK6240 and 3H-THK5117 in ADAD. A positive correlation between 3H-deprenyl and 3H-THK5117 binding was observed in AßPParc, while no such correlation was found in PSEN1ΔE9 and sAD. Our study demonstrates differences in the properties of the amyloid plaques between two genetic variants of AD and sAD. Despite the lack of measurable amyloid fibrils by PiB in the AßPParc cases, high regional tau and astrocyte binding was observed. The lack of correlation between 3H-deprenyl and 3H-THK5117 binding in PSEN1ΔE9 and sAD in contrast of the positive correlation observed in the AßPParc cases suggest differences in the pathological cascade between variants of AD that warrant further exploration in vivo.

Systematic Review and Meta-analysis: Partial External Biliary Diversion in Progressive Familial Intrahepatic Cholestasis.

OBJECTIVES: We assessed available data on impact of partial external biliary diversion (PEBD) surgery on clinical outcomes in patients with progressive familial intrahepatic cholestasis (PFIC). METHODS: We performed a systematic literature review (PubMed) and meta-analysis to evaluate relationships between liver biochemistry parameters (serum bile acids, bilirubin, and alanine aminotransferase [ALT]) and early response (pruritus improvement) or long-term outcomes (need for liver transplant) in patients with PFIC who underwent PEBD. RESULTS: Searches identified 175 publications before September 2018; 16 met inclusion criteria. Receiver operating characteristic (ROC) analysis examined ability of liver biochemistry parameters to discriminate patients who demonstrated early and long-term response to PEBD from those who did not. Regarding pruritus improvement in 155 included patients in aggregate, 104 (67%) were responders, 14 (9%) had partial response, and 37 (24%) were nonresponders. In ROC analyses of individual patient data, post-PEBD serum concentration of bile acids, in particular, could discriminate responders from nonresponders for pruritus improvement (area under the curve, 0.99; P < 0.0001; n = 42); to a lesser extent, this was also true for bilirubin (0.87; P = 0.003; n = 31), whereas ALT could not discriminate responders from nonresponders for pruritus improvement (0.74; P = 0.06; n = 28). Reductions from pre-PEBD values in serum bile acid concentration (0.89; P = 0.0003; n = 32) and bilirubin (0.98; P = 0.002; n = 18) but not ALT (0.62; P = 0.46; n = 18) significantly discriminated decreased aggregate need for liver transplant. CONCLUSION: Changes in bile acids seem particularly useful in discriminating early and long-term post-PEBD outcomes and may be potential biomarkers of response to interruption of enterohepatic circulation in patients with PFIC.

Efficacy, long-term safety, and impact on quality of life of elobixibat in more severe constipation: Post hoc analyses of two phase 3 trials in Japan.

BACKGROUND: In two phase 3 trials, elobixibat, a locally acting ileal bile acid transporter inhibitor, resolved constipation and was well tolerated in Japanese patients with chronic constipation. We analyzed the efficacy, safety, and impact on quality of life (QOL) of elobixibat in patients with symptomatically more severe constipation in the two phase 3 trials. METHODS: This post hoc analysis of elobixibat treatment outcomes included data from a 2-week, randomized, placebo-controlled, phase 3 trial (10 mg/d), and a 52-week, open-label trial (5-15 mg/d) in subgroups with severe constipation defined as ≤2 spontaneous bowel movements (SBMs) and ≤3 Bristol Stool Form Scale score during the second week of the 2-week run-in period. We also analyzed the rates of abdominal pain, diarrhea, and QOL in subgroups according to sex, presence of constipation-predominant irritable bowel syndrome (IBS-C) and side effects. KEY RESULTS: In patients with severe constipation, there was significant improvement in the 10 mg elobixibat group compared to the placebo group in change in SBMs from baseline at week 1 (primary endpoint) of the 2-week trial. The differences between groups were reduced in patients with more severe constipation. Increasing the dose to 15 mg was effective for more severe constipation in improving the number of SBMs per week in the 52-week trial. Overall, elobixibat was well tolerated and improved QOL scores, irrespective of gender, presence of IBS-C or side effects. CONCLUSIONS & INFERENCES: Elobixibat is effective for symptomatically severe constipation, is well tolerated and improves QOL, irrespective of potentially confounding patient characteristics.

Tau PET imaging in neurodegenerative tauopathies-still a challenge.

The accumulation of pathological misfolded tau is a feature common to a collective of neurodegenerative disorders known as tauopathies, of which Alzheimer's disease (AD) is the most common. Related tauopathies include progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), Down's syndrome (DS), Parkinson's disease (PD), and dementia with Lewy bodies (DLB). Investigation of the role of tau pathology in the onset and progression of these disorders is now possible due the recent advent of tau-specific ligands for use with positron emission tomography (PET), including first- (e.g., [18F]THK5317, [18F]THK5351, [18F]AV1451, and [11C]PBB3) and second-generation compounds [namely [18F]MK-6240, [18F]RO-948 (previously referred to as [18F]RO69558948), [18F]PI-2620, [18F]GTP1, [18F]PM-PBB3, and [18F]JNJ64349311 ([18F]JNJ311) and its derivative [18F]JNJ-067)]. In this review we describe and discuss findings from in vitro and in vivo studies using both initial and new tau ligands, including their relation to biomarkers for amyloid-ß and neurodegeneration, and cognitive findings. Lastly, methodological considerations for the quantification of in vivo ligand binding are addressed, along with potential future applications of tau PET, including therapeutic trials.

Safety and efficacy of elobixibat for chronic constipation: results from a randomised, double-blind, placebo-controlled, phase 3 trial and an open-label, single-arm, phase 3 trial.

BACKGROUND: A subset of patients with constipation has reduced colonic bile acid concentrations, which are associated with slow colonic transit. In a previous study, elobixibat, a locally acting ileal bile acid transporter inhibitor, accelerated colonic transit in Japanese patients with functional constipation. In this study, we aimed to determine the efficacy of elobixibat for short-term treatment of chronic constipation, and safety, patient satisfaction, and quality of life with long-term treatment. METHODS: We did two phase 3 studies of patients aged 20-80 years in Japan with at least 6 months of chronic constipation, who satisfied Rome III criteria for functional constipation, including fewer than three spontaneous bowel movements per week. The first trial, including patients enrolled at 16 clinics, was a 2-week, randomised, double-blind, placebo-controlled study in which (after a 2-week run-in period) patients were randomly assigned (1:1) to either elobixibat 10 mg/day for 2 weeks or placebo. Randomisation was done with permuted block method (block size six) without stratification. Masking to treatment allocation was achieved with identical appearances of elobixibat and placebo, which were supplied in sealed, opaque containers. Group assignment was concealed from patients, investigators, and analysts. The second trial, including patients enrolled at 34 clinics or hospitals, was an open-label, 1-year study in which all patients received elobixibat; participants could titrate the dose to 5 mg/day or 15 mg/day, or maintain the 10 mg/day dose. In both studies, participants took the study drug as an oral tablet once per day before breakfast. The primary outcome of the 2-week randomised trial was the change from baseline (ie, last week of the 2-week run-in) in the frequency of spontaneous bowel movements during week 1 of treatment. The primary outcome of the 52-week open-label trial was safety (type, severity, and incidence of adverse drug reactions) at all times from treatment initiation. All efficacy analyses were based on the modified intention-to-treat (ITT) population without imputation for any missing data. Safety analyses included all patients who received at least one dose of study drug. These trials are registered with the Japan Pharmaceutical Information Center (numbers JapicCTI-153061 and JapicCTI-153062) and have been completed. FINDINGS: Between Nov 4, 2015, and June 11, 2016, we assigned 133 patients to treatment in the 2-week randomised trial: 70 to elobixibat (69 included in the modified ITT and safety populations) and 63 to placebo. The frequency of spontaneous bowel movements per week during week 1 of treatment was greater with elobixibat (least-squares mean 6·4, 95% CI 5·3-7·6) than with placebo (1·7, 1·2-2·2), p<0·0001). Between Oct 31, 2015, and March 15, 2017, we allocated 341 patients to 52 weeks of elobixibat (340 included in the modified ITT and safety populations). 163 (48%) patients in the 52-week trial had an adverse drug reaction, the most common of which were mild gastrointestinal disorders (in 135 [40%] patients). Inguinal hernia was reported in one patient with elobixibat in the 52-week study as a moderate adverse drug reaction. The most common adverse drug reactions in both trials were mild abdominal pain (13 [19%] patients with elobixibat and one [2%] with placebo in the 2-week randomised trial, and 82 [24%] patients in the 52-week trial) and diarrhoea (nine [13%] patients with elobixibat and none with placebo in the 2-week randomised trial and 50 [15%] in the 52-week trial). INTERPRETATION: Elobixibat resolved constipation in the short-term, and was well tolerated with both short-term and long-term treatment. The evidence supports the use of this novel approach to increase intracolonic concentrations of endogenous bile acid for the treatment of chronic constipation. FUNDING: EA Pharma and Mochida Pharmaceutical.

Comparative binding properties of the tau PET tracers THK5117, THK5351, PBB3, and T807 in postmortem Alzheimer brains.

BACKGROUND: The aim of this study was to compare the binding properties of several tau positron emission tomography tracers-THK5117, THK5351, T807 (also known as AV1451; flortaucipir), and PBB3-head to head in the same human brain tissue. METHODS: Binding assays were performed to compare the regional distribution of 3H-THK5117 and 3H-THK5351 in postmortem tissue from three Alzheimer's disease (AD) cases and three control subjects in frontal and temporal cortices as well as in the hippocampus. Competition binding assays between THK5351, THK5117, PBB3, and T807, as well as off-target binding of THK5117 and T807 toward monoamine oxidase B (MAO-B), were performed using binding assays in brain homogenates and autoradiography of three AD cases. RESULTS: Regional binding of 3H-THK5117 and 3H-THK5351 was similar, except in the temporal cortex, which showed higher 3H-THK5117 binding. Saturation studies demonstrated two binding sites for 3H-THK5351 (K d1 = 5.6 nM, Bmax = 76 pmol/g; K d2 = 1 nM, Bmax = 40 pmol/g). Competition studies in the hippocampus between 3H-THK5351 and unlabeled THK5351, THK5117, and T807 revealed super-high-affinity sites for all three tracers (THK5351 K i = 0.1 pM; THK5117 K i = 0.3 pM; T807 K i = 0.2 pM) and an additional high-affinity site (THK5351 K i = 16 nM; THK5117 K i = 20 nM; T807 K i = 78nM). 18F-T807, 11C-THK5351, and 11C-PBB3 autoradiography of large frozen sections from three AD brains showed similar regional binding for the three tracers, with lower binding intensity for 11C-PBB3. Unlabeled THK5351 and T807 displaced 11C-THK5351 to a similar extent and a lower extent, respectively, compared with 11C-PBB3. Competition with the MAO-B inhibitor 3H-L-deprenyl was observed for THK5117 and T807 in the hippocampus (THK5117 K i = 286 nM; T807 K i = 227 nM) and the putamen (THK5117 K i = 148 nM; T807 K i = 135 nM). 3H-THK5351 binding was displaced using autoradiography competition with unlabeled THK5351 and T807 in cortical areas by 70-80% and 60-77%, respectively, in the basal ganglia, whereas unlabeled deprenyl displaced 3H-THK5351 binding by 40% in the frontal cortex and 50% in the basal ganglia. CONCLUSIONS: THK5351, THK5117, and T807 seem to target similar binding sites, but with different affinities, whereas PBB3 seems to target its own binding site. Both THK5117 and T807 demonstrated off-target binding in the hippocampus and putamen with a ten times lower binding affinity to the MAO-B inhibitor deprenyl compared with 3H-THK5351.

Cortical laminar tau deposits and activated astrocytes in Alzheimer's disease visualised by 3H-THK5117 and 3H-deprenyl autoradiography.

Hyperphosphorylated tau protein deposits and, inflammatory processes are characteristic components of Alzheimer disease (AD) pathology. We here aimed to visualize in vitro the distribution of tau deposits and activated astrocytes across the cortical layers in autopsy AD brain tissue using the radiotracers 3H-THK5117 and 3H-deprenyl. 3H-THK5117 and 3H-deprenyl autoradiographies were carried out on frozen brain sections from three AD patients and one healthy control. 3H-THK5117 showed a distinct laminar cortical binding similar to 3H-deprenyl autoradiography, with an extensive binding in the superficial and deep layers of the temporal neocortices, whereas the middle frontal gyrus showed an even binding throughout the layers. Globally, eventhough some differences could be observed, AT8 (tau) and GFAP (astrocyte) immunostaining showed a laminar pattern comparable to their corresponding radiotracers within each AD case. Some variability was observed between the AD cases reflecting differences in disease phenotype. The similar laminar cortical brain distribution of tau deposits and activated astrocytes supports the hypothesis of a close pathological interconnection. The difference in regional binding patterns of 3H-THK5117 and AT8 antibody staining suggest additional tau binding sites detectable by 3H-THK5117.

Amyloid tracers binding sites in autosomal dominant and sporadic Alzheimer's disease.

INTRODUCTION: Amyloid imaging has been integrated into diagnostic criteria for Alzheimer's disease (AD). How amyloid tracers binding differ for different tracer structures and amyloid-ß aggregates in autosomal dominant AD (ADAD) and sporadic AD is unclear. METHODS: Binding properties of different amyloid tracers were examined in brain homogenates from six ADAD with APPswe, PS1 M146V, and PS1 EΔ9 mutations, 13 sporadic AD, and 14 control cases. RESULTS: 3H-PIB, 3H-florbetaben, 3H-AZD2184, and BTA-1 shared a high- and a varying low-affinity binding site in the frontal cortex of sporadic AD. AZD2184 detected another binding site (affinity 33 nM) in the frontal cortex of ADAD. The 3H-AZD2184 and 3H-PIB binding were significantly higher in the striatum of ADAD compared to sporadic AD and control. Polyphenol resveratrol showed strongest inhibition on 3H-AZD84 binding followed by 3H-florbetaben and minimal on 3H-PIB. DISCUSSION: This study implies amyloid tracers of different structures detect different sites on amyloid-ß fibrils or conformations.

Visualization of regional tau deposits using (3)H-THK5117 in Alzheimer brain tissue.

INTRODUCTION:  The accumulation of neurofibrillary tangles, composed of aggregated hyperphosphorylated tau protein, starts spreading early in specific regions in the course of Alzheimer's disease (AD), correlating with the progression of memory dysfunction. The non-invasive imaging of tau could therefore facilitate the early diagnosis of AD, differentiate it from other dementing disorders and allow evaluation of tau immunization therapy outcomes. In this study we characterized the in vitro binding properties of THK5117, a tentative radiotracer for positron emission tomography (PET) imaging of tau brain deposits. RESULTS: Saturation and competition binding studies of (3)H-THK5117 in post-mortem AD brain tissue showed the presence of multiple binding sites. THK5117 binding was significantly higher in hippocampal (p < 0.001) and temporal (p < 0.01) tissue homogenates in AD compared to controls. Autoradiography studies with (3)H-THK5117 was performed on large frozen brain sections from three AD cases who had been followed clinically and earlier undergone in vivo (18)F-FDG PET investigations. The three AD cases showed distinct differences in regional THK5117 binding that were also observed in tau immunohistopathology as well as in clinical presentation. A negative correlation between in vivo (18)F-FDG PET and in vitro (3)H-THK5117 autoradiography was observed in two of the three AD cases. CONCLUSIONS: This study supports that new tau PET tracers will provide further understanding on the role of tau pathology in the diversity of the clinical presentation in AD.

³H-deprenyl and ³H-PIB autoradiography show different laminar distributions of astroglia and fibrillar ß-amyloid in Alzheimer brain.

BACKGROUND: The pathological features in Alzheimer's disease (AD) brain include the accumulation and deposition of ß-amyloid (Aß), activation of astrocytes and microglia and disruption of cholinergic neurotransmission. Since the topographical characteristics of these different pathological processes in AD brain and how these relate to each other is not clear, this motivated further exploration using binding studies in postmortem brain with molecular imaging tracers. This information could aid the development of specific biomarkers to accurately chart disease progression. RESULTS: In vitro binding assays demonstrated increased [³H]-PIB (fibrillar Aß) and [³H]-PK11195 (activated microglia) binding in the frontal cortex (FC) and hippocampus (HIP), as well as increased binding of [³H]-L-deprenyl (activated astrocytes) in the HIP, but a decreased [³H]-nicotine (α4ß2 nicotinic acetylcholine receptor (nAChR)) binding in the FC of AD cases compared to age-matched controls. Quantitative autoradiography binding studies were also performed to investigate the regional laminar distributions of [³H]-L-deprenyl, [³H]-PIB as well as [¹²5I]-α-bungarotoxin (α7 nAChRs) and [³H]-nicotine in hemisphere brain of a typical AD case. A clear lamination pattern was observed with high [³H]-PIB binding in all layers and [³H]-deprenyl in superficial layers of the FC. In contrast, [³H]-PIB showed low binding to fibrillar Aß, but [³H]-deprenyl high binding to activated astrocytes throughout the HIP. The [³H]-PIB binding was also low and the [³H]-deprenyl binding high in all layers of the medial temporal gyrus and insular cortex in comparison to the frontal cortex. Low [³H]-nicotine binding was observed in all layers of the frontal cortex in comparison to layers in the medial temporal gyrus, insular cortex and hippocampus. Immunohistochemical detection in the AD case revealed abundant glial fibrillary acidic protein positive (GFAP+) reactive astrocytes and α7 nAChR expressing GFAP+ astrocytes both in the vicinity and surrounding Aß neuritic plaques in the FC and HIP. Although fewer Aß plaques were observed in the HIP, some hippocampal GFAP+ astrocytes contained Aß-positive (6 F/3D) granules within their somata. CONCLUSIONS: Astrocytosis shows a distinct regional pattern in AD brain compared to fibrillar Aß, suggesting that different types of astrocytes may be associated with the pathophysiological processes in AD.

Amyloid tracers detect multiple binding sites in Alzheimer's disease brain tissue.

Imaging fibrillar amyloid-ß deposition in the human brain in vivo by positron emission tomography has improved our understanding of the time course of amyloid-ß pathology in Alzheimer's disease. The most widely used amyloid-ß imaging tracer so far is (11)C-Pittsburgh compound B, a thioflavin derivative but other (11)C- and (18)F-labelled amyloid-ß tracers have been studied in patients with Alzheimer's disease and cognitively normal control subjects. However, it has not yet been established whether different amyloid tracers bind to identical sites on amyloid-ß fibrils, offering the same ability to detect the regional amyloid-ß burden in the brains. In this study, we characterized (3)H-Pittsburgh compound B binding in autopsied brain regions from 23 patients with Alzheimer's disease and 20 control subjects (aged 50 to 88 years). The binding properties of the amyloid tracers FDDNP, AV-45, AV-1 and BF-227 were also compared with those of (3)H-Pittsburgh compound B in the frontal cortices of patients with Alzheimer's disease. Saturation binding studies revealed the presence of high- and low-affinity (3)H-Pittsburgh compound B binding sites in the frontal cortex (K(d1): 3.5 ± 1.6 nM; K(d2): 133 ± 30 nM) and hippocampus (K(d1):5.6 ± 2.2 nM; K(d2): 181 ± 132 nM) of Alzheimer's disease brains. The relative proportion of high-affinity to low-affinity sites was 6:1 in the frontal cortex and 3:1 in the hippocampus. One control showed both high- and low-affinity (3)H-Pittsburgh compound B binding sites (K(d1): 1.6 nM; K(d2): 330 nM) in the cortex while the others only had a low-affinity site (K(d2): 191 ± 70 nM). (3)H-Pittsburgh compound B binding in Alzheimer's disease brains was higher in the frontal and parietal cortices than in the caudate nucleus and hippocampus, and negligible in the cerebellum. Competitive binding studies with (3)H-Pittsburgh compound B in the frontal cortices of Alzheimer's disease brains revealed high- and low-affinity binding sites for BTA-1 (Ki: 0.2 nM, 70 nM), florbetapir (1.8 nM, 53 nM) and florbetaben (1.0 nM, 65 nM). BF-227 displaced 83% of (3)H-Pittsburgh compound B binding, mainly at a low-affinity site (311 nM), whereas FDDNP only partly displaced (40%). We propose a multiple binding site model for the amyloid tracers (binding sites 1, 2 and 3), where AV-45 (florbetapir), AV-1 (florbetaben), and Pittsburgh compound B, all show nanomolar affinity for the high-affinity site (binding site 1), as visualized by positron emission tomography. BF-227 shows mainly binding to site 3 and FDDNP shows only some binding to site 2. Different amyloid tracers may provide new insight into the pathophysiological mechanisms in the progression of Alzheimer's disease.

Effect of muscarinic and nicotinic receptor antagonism on rat gastric motor activity.

BACKGROUND/AIMS: Our aim was to investigate whether muscarinic and nicotinic receptors mediate nitric oxide release during motor events in the rat stomach. METHODS: Isolated rat stomach volume changes were monitored in an organ bath setup with an intragastric balloon coupled to a barostat and studied in basal conditions and during electrical vagal stimulation (EVS). In conscious rats, the intragastric pressure (IGP) was measured during test meal infusion. RESULTS: In the presence of N(G)-nitro-L-arginine methyl ester (L-NAME; 0.1 mmol/l), EVS induced significant gastric contractions (mean +/- SEM = 0.27 +/- 0.04 ml; n = 6) that could be blocked by atropine (3 micromol/l) and hexamethonium (0.1 mmol/l). In the presence of atropine and/or hexamethonium, EVS-induced relaxations could not be blocked by L-NAME, while exogenous nitric oxide could still relax the stomach. In conscious rats, atropine (1 mg kg(-1)) initially decreased IGP, while during further distension it increased IGP. In the presence of L-NAME (30 mg kg(-1)) atropine consistently decreased IGP. L-NAME alone significantly increased IGP during the test meal infusion, but this effect was reduced in the presence of atropine. CONCLUSION: These findings indicate a role for nicotinic and muscarinic receptors in the vagal-stimulation-induced activation of nitrergic nerves in the rat stomach.

Bile acids: short and long term effects in the intestine.

Bile acids have secretory, motility and antimicrobial effects in the intestine. In patients with bile acid malabsorption the amount of primary bile acids in the colon is increased compared to healthy controls. Deoxycholic acid is affecting the intestinal smooth muscle activity. Chenodeoxycholic acid has the highest potency to affect intestinal secretion. Litocholic acid has little effect in the lumen of intestine compared to both deoxycholic acid and chenodeoxycholic acid. There is no firm evidence that clinically relevant concentrations of bile acids induce colon cancer. Alterations in bile acid metabolism may be involved in the pathophysiology of constipation.

Early integration of pharmacokinetic and dynamic reasoning is essential for optimal development of lead compounds: strategic considerations.

The aims of this report are firstly to raise awareness among kineticists and pharmacologists as to why pharmacokinetic-pharmacodynamic (PKPD) integration is essential for target validation (TV), optimizing development of lead compounds (lead generation [LG] and lead optimization [LO]) and scaling these to human. A related aim is to demonstrate strategic examples of PKPD collaborations that have improved the planning, execution and evaluation of experiments in primary and safety pharmacology. Examples include design of TV studies, design and data 'pruning' of PKPD studies in LO, analysis of data with marginal and substantial temporal (time) differences between exposure and response, design of safety pharmacology studies, assessment of safety margin and assessment of uncertainties in predictions of first dose in human.

A novel method for study of gastric mechanical functions in conscious mice.

A novel method has been developed for simultaneous study of gastric emptying, antral motility, and gastric muscle tone in conscious mice. Intragastric pressure was measured during infusion of an X-ray-opaque, viscous meal through a chronically implanted gastric fistula (0.25 ml/min). Compared with vehicle treatment, molsidomine (nitric oxide donor) and atropine (muscarinic receptor antagonist) treatment significantly reduced the area under the intragastric pressure curve (AUC) by 37 +/- 4% and 35 +/- 3%, respectively, (mean +/- S.E.M.) whereas N (G)-nitro-L-arginine methyl ester (L-NAME; nitric oxide synthase inhibitor) significantly increased the AUC by 20 +/- 3%. Atropine also significantly reduced the frequency and amplitude of stomach contraction-induced intragastric pressure waves while molsidomine only reduced the frequency. Gastric emptying, as assessed by X-ray imaging, was significantly delayed after L-NAME and atropine treatment. This methodology is the first to enable simultaneous assessment of gastric emptying, antral motility, and gastric tone in conscious mice and confirmed the important role of nitrergic and cholinergic innervation.