Long-Term Outcomes in KEYNOTE-052: Phase II Study Investigating First-Line Pembrolizumab in Cisplatin-Ineligible Patients With Locally Advanced or Metastatic Urothelial Cancer.

PURPOSE: The phase II single-arm KEYNOTE-052 study evaluated the efficacy and safety of first-line pembrolizumab for patients with locally advanced or metastatic cisplatin-ineligible urothelial carcinoma (UC). PATIENTS AND METHODS: Three hundred seventy patients received pembrolizumab 200 mg intravenously every 3 weeks for up to 24 months. Positive tumor programmed death ligand 1 (PD-L1) expression was defined as combined positive score (CPS) ≥ 10. Response was assessed by independent central review every 9 weeks per RECIST v1.1. The primary end point was objective response rate (ORR). RESULTS: At data cutoff (September 26, 2018), the minimum follow-up was 2 years since the last patient enrolled. ORR was 28.6% (95% CI, 24.1% to 33.5%); 33 patients (8.9%) and 73 patients (19.7%) achieved complete and partial response, respectively. The median duration of response was 30.1 months (95% CI, 18.1 months to not reached [NR]); responses lasted ≥ 12 and ≥ 24 months in 67% and 52% of patients, respectively. Forty patients with complete or partial response completed 2 years of study treatment, and 32 had ongoing response at completion. Median overall survival (OS) was 11.3 months (95% CI, 9.7 to 13.1 months), and 12- and 24-month OS rates were 46.9% and 31.2%, respectively. In patients with CPS ≥ 10, ORR was 47.3% (95% CI, 37.7% to 57.0%) and median OS was 18.5 months (95% CI, 12.2 to 28.5 months). In patients with lymph node-only disease, ORR was 49.0% (95% CI, 34.8% to 63.4%), and median OS was 27.0 months (12.4 months to NR). There were no new safety signals. CONCLUSION: First-line pembrolizumab confers meaningful and durable clinical response in cisplatin-ineligible patients with advanced UC and is associated with prolonged OS, particularly with PD-L1 CPS ≥ 10 and lymph node-only disease.

A Machine Learning Approach to Identify a Circulating MicroRNA Signature for Alzheimer Disease.

BACKGROUND: Accurate diagnosis of Alzheimer disease (AD) involving less invasive molecular procedures and at reasonable cost is an unmet medical need. We identified a serum miRNA signature for AD that is less invasive than a measure in cerebrospinal fluid. METHODS: From the Oxford Project to Investigate Memory and Aging (OPTIMA) study, 96 serum samples were profiled by a multiplex (>500 analytes) microRNA (miRNA) reverse transcription quantitative PCR analysis, including 51 controls, 32 samples from patients with AD, and 13 samples from patients with mild cognitive impairment (MCI). Clinical diagnosis of a subset of AD and the controls was confirmed by postmortem (PM) histologic examination of brain tissue. In a machine learning approach, the AD and control samples were split 70:30 as the training and test cohorts. A multivariate random forest statistical analysis was applied to construct and test a miRNA signature for AD identification. In addition, the MCI participants were included in the test cohort to assess whether the signature can identify early AD patients. RESULTS: A 12-miRNA signature for AD identification was constructed in the training cohort, demonstrating 76.0% accuracy in the independent test cohort with 90.0% sensitivity and 66.7% specificity. The signature, however, was not able to identify MCI participants. With a subset of AD and control participants with PM-confirmed diagnosis status, a separate 12-miRNA signature was constructed. Although sample size was limited, the PM-confirmed signature demonstrated improved accuracy of 85.7%, largely owing to improved specificity of 80.0% with comparable sensitivity of 88.9%. CONCLUSION: Although additional and more diverse cohorts are needed for further clinical validation of the robustness, the miRNA signature appears to be a promising blood test to diagnose AD.

Discovery of MK-8719, a Potent O-GlcNAcase Inhibitor as a Potential Treatment for Tauopathies.

Inhibition of O-GlcNAcase (OGA) has emerged as a promising therapeutic approach to treat tau pathology in neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Beginning with carbohydrate-based lead molecules, we pursued an optimization strategy of reducing polar surface area to align the desired drug-like properties of potency, selectivity, high central nervous system (CNS) exposure, metabolic stability, favorable pharmacokinetics, and robust in vivo pharmacodynamic response. Herein, we describe the medicinal chemistry and pharmacological studies that led to the identification of (3aR,5S,6S,7R,7aR)-5-(difluoromethyl)-2-(ethylamino)-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]thiazole-6,7-diol 42 (MK-8719), a highly potent and selective OGA inhibitor with excellent CNS penetration that has been advanced to first-in-human phase I clinical trials.

Pembrolizumab alone or in combination with chemotherapy as first-line therapy for patients with advanced gastric or gastroesophageal junction adenocarcinoma: results from the phase II nonrandomized KEYNOTE-059 study.

BACKGROUND: The multicohort, phase II, nonrandomized KEYNOTE-059 study evaluated pembrolizumab ± chemotherapy in advanced gastric/gastroesophageal junction cancer. Results from cohorts 2 and 3, evaluating first-line therapy, are presented. METHODS: Patients ≥ 18 years old had previously untreated recurrent or metastatic gastric/gastroesophageal junction adenocarcinoma. Cohort 3 (monotherapy) had programmed death receptor 1 combined positive score ≥ 1. Cohort 2 (combination therapy) received pembrolizumab 200 mg on day 1, cisplatin 80 mg/m2 on day 1 (up to 6 cycles), and 5-fluorouracil 800 mg/m2 on days 1-5 of each 3-week cycle (or capecitabine 1000 mg/m2 twice daily in Japan). Primary end points were safety (combination therapy) and objective response rate per Response Evaluation Criteria in Solid Tumors version 1.1 by central review, and safety (monotherapy). RESULTS: In the combination therapy and monotherapy cohorts, 25 and 31 patients were enrolled; median follow-up was 13.8 months (range 1.8-24.1) and 17.5 months (range 1.7-20.7), respectively. In the combination therapy cohort, grade 3/4 treatment-related adverse events occurred in 19 patients (76.0%); none were fatal. In the monotherapy cohort, grade 3-5 treatment-related adverse events occurred in seven patients (22.6%); one death was attributed to a treatment-related adverse event (pneumonitis). The objective response rate was 60.0% [95% confidence interval (CI), 38.7-78.9] (combination therapy) and 25.8% (95% CI 11.9-44.6) (monotherapy). CONCLUSIONS: Pembrolizumab demonstrated antitumor activity and was well tolerated as monotherapy and in combination with chemotherapy in patients with previously untreated advanced gastric/gastroesophageal junction adenocarcinoma. CLINICAL TRIAL: ClinicalTrials.gov NCT02335411.

Clinical Utility of the Combined Positive Score for Programmed Death Ligand-1 Expression and the Approval of Pembrolizumab for Treatment of Gastric Cancer.

CONTEXT.­: Regulatory approval of pembrolizumab for treatment of gastric and gastroesophageal junction (G/GEJ) adenocarcinoma required a reproducible scoring method for use of programmed death ligand-1 (PD-L1) protein expression as a companion diagnostic to identify likely responders to therapy. OBJECTIVE.­: To develop an immunohistochemical scoring algorithm that includes PD-L1 expression for tumor and immune cells, that is, the combined positive score. DESIGN.­: Four previously treated tumor types in the KEYNOTE-012 and KEYNOTE-028 studies were analyzed descriptively with a version of the PD-L1 immunohistochemical 22C3 pharmDx assay labeled for investigational use only to determine the relative importance of PD-L1 expression in tumor versus immune cells as a biomarker for pembrolizumab response. A combined positive score was developed as a novel scoring method and was compared with the tumor proportion score in cohort 1 from the KEYNOTE-059 study (G/GEJ cancer). External reproducibility was assessed. RESULTS.­: Per combined positive score cutoff of 1 or more, the prevalence of PD-L1 expression in patients with G/GEJ cancer was 57.6% (148 of 257 patients), with reasonable enrichment of responses (odds ratio, 2.8). Per tumor proportion score cutoff of 1% or more, prevalence was 12.5% (32 of 257 patients), with minimal enrichment (odds ratio, 1.4). External reproducibility assessments demonstrated interpathologist overall agreement of 96.6% (591 of 612; 95% CI, 94.0%-98.7%) and intrapathologist overall agreement of 97.2% (595 of 612; 95% CI, 95.3%-98.9%). CONCLUSIONS.­: Combined positive score is a robust, reproducible PD-L1 scoring method that predicts response to pembrolizumab in patients with G/GEJ cancer. This novel scoring method supported US Food and Drug Administration approval of pembrolizumab as third-line therapy for G/GEJ cancer and has facilitated investigation in other indications.

Early intervention of tau pathology prevents behavioral changes in the rTg4510 mouse model of tauopathy.

Although tau pathology, behavioral deficits, and neuronal loss are observed in patients with tauopathies, the relationship between these endpoints has not been clearly established. Here we found that rTg4510 mice, which overexpress human mutant tau in the forebrain, develop progressive age-dependent increases in locomotor activity (LMA), which correlates with neurofibrillary tangle (NFT) pathology, hyperphosphorylated tau levels, and brain atrophy. To further clarify the relationship between these endpoints, we treated the rTg4510 mice with either doxycycline to reduce mutant tau expression or an O-GlcNAcase inhibitor Thiamet G, which has been shown to ameliorate tau pathology in animal models. We found that both doxycycline and Thiamet G treatments starting at 2 months of age prevented the progression of hyperactivity, slowed brain atrophy, and reduced brain hyperphosphorylated tau. In contrast, initiating doxycycline treatment at 4 months reduced neither brain hyperphosphorylated tau nor hyperactivity, further confirming the relationship between these measures. Collectively, our results demonstrate a unique behavioral phenotype in the rTg4510 mouse model of tauopathy that strongly correlates with disease progression, and that early interventions which reduce tau pathology ameliorate the progression of the locomotor dysfunction. These findings suggest that better understanding the relationship between locomotor deficits and tau pathology in the rTg4510 model may improve our understanding of the mechanisms underlying behavioral disturbances in patients with tauopathies.

Extending a Systems Model of the APP Pathway: Separation of ß- and γ-Secretase Sequential Cleavage Steps of APP.

The abnormal accumulation of amyloid-ß (Aß) in the brain parenchyma has been posited as a central event in the pathophysiology of Alzheimer's disease. Recently, we have proposed a systems pharmacology model of the amyloid precursor protein (APP) pathway, describing the Aß APP metabolite responses (Aß40, Aß42, sAPPα, and sAPPß) to ß-secretase 1 (BACE1) inhibition. In this investigation this model was challenged to describe Aß dynamics following γ-secretase (GS) inhibition. This led an extended systems pharmacology model, with separate descriptions to characterize the sequential cleavage steps of APP by BACE1 and GS, to describe the differences in Aß response to their respective inhibition. Following GS inhibition, a lower Aß40 formation rate constant was observed, compared with BACE1 inhibition. Both BACE1 and GS inhibition were predicted to lower Aß oligomer levels. Further model refinement and new data may be helpful to fully understand the difference in Aß dynamics following BACE1 versus GS inhibition.

First-line pembrolizumab in cisplatin-ineligible patients with locally advanced and unresectable or metastatic urothelial cancer (KEYNOTE-052): a multicentre, single-arm, phase 2 study.

BACKGROUND: More than half of all patients with advanced urothelial cancer cannot receive standard, first-line cisplatin-based chemotherapy because of renal dysfunction, poor performance status, or other comorbidities. We assessed the activity and safety of first-line pembrolizumab in cisplatin-ineligible patients with locally advanced and unresectable or metastatic urothelial cancer. METHODS: In this multicentre, single-arm, phase 2 study (KEYNOTE-052), cisplatin-ineligible patients with advanced urothelial cancer who had not been previously treated with systemic chemotherapy were recruited from 91 academic medical centres in 20 countries. Enrolled patients received intravenous pembrolizumab 200 mg every 3 weeks. The primary endpoint was objective response (the proportion of patients who achieved complete or partial response) in all patients and by PD-L1 expression status according to the Response Evaluation Criteria in Solid Tumors, version 1.1, as assessed by independent central review. PD-L1 expression was assessed in tumour and inflammatory cells from tumour biopsies provided at study entry. Activity and safety were analysed in all patients who received at least one dose of pembrolizumab (all-patients-treated population). This study is registered with ClinicalTrials.gov, number NCT02335424, and follow-up is ongoing. FINDINGS: Between Feb 24, 2015, and Aug 8, 2016, 374 patients were enrolled and 370 patients received at least one dose of pembrolizumab. 89 (24%, 95% CI 20-29) of 370 patients had a centrally assessed objective response, and as of Sept 1, 2016 (data cutoff), 74 (83%) of 89 responses were ongoing. Median follow-up was 5 months (IQR 3·0-8·6). A PD-L1-expression cutoff of 10% was associated with a higher frequency of response to pembrolizumab; 42 (38%, 95% CI 29-48) of 110 patients with a combined positive score of 10% or more had a centrally assessed objective response. The most common grade 3 or 4 treatment-related adverse events were fatigue (eight [2%] of 370 patients), alkaline phosphatase increase (five [1%]), colitis, and muscle weakness (both four [1%]). 36 (10%) of 370 patients had a serious treatment-related adverse event. 17 (5%) of 370 patients died from non-treatment-related adverse events associated with death, and one patient died from treatment-related adverse events (myositis in addition to grade 3 thyroiditis, grade 3 hepatitis, grade 3 pneumonia, and grade 4 myocarditis). INTERPRETATION: First-line pembrolizumab has antitumour activity and acceptable tolerability in cisplatin-ineligible patients with urothelial cancer, most of whom were elderly, had poor prognostic factors, or had serious comorbidities. In view of this result, pembrolizumab has become a new treatment option for patients who are cisplatin-ineligible or not suitable candidates for chemotherapy. Pembrolizumab in the first-line setting is being further assessed in the phase 3 KEYNOTE-361 trial (ClinicalTrials.gov, NCT02335424). FUNDING: Merck & Co.

A multinational study distinguishing Alzheimer's and healthy patients using cerebrospinal fluid tau/Aß42 cutoff with concordance to amyloid positron emission tomography imaging.

INTRODUCTION: Changes in cerebrospinal fluid (CSF) tau and amyloid ß (Aß)42 accompany development of Alzheimer's brain pathology. Robust tau and Aß42 immunoassays were developed to establish a tau/Aß42 cutoff distinguishing mild-to-moderate Alzheimer's disease (AD) subjects from healthy elderly control (HC) subjects. METHODS: A CSF tau/Aß42 cutoff criteria was chosen, which distinguished the groups and maximized concordance with amyloid PET. Performance was assessed using an independent validation cohort. RESULTS: A tau/Aß42 = 0.215 cutoff provided 94.8% sensitivity and 77.7% specificity. Concordance with PET visual reads was estimated at 86.9% in a ∼50% PET positive population. In the validation cohort, the cutoff demonstrated 78.4% sensitivity and 84.9% specificity to distinguish the AD and HC populations. DISCUSSION: A tau/Aß42 cutoff with acceptable sensitivity and specificity distinguished HC from mild-to-moderate AD subjects and maximized concordance to brain amyloidosis. The defined cutoff demonstrated that CSF analysis may be useful as a surrogate to imaging assessment of AD pathology.

Systems Pharmacology Analysis of the Amyloid Cascade after ß-Secretase Inhibition Enables the Identification of an Aß42 Oligomer Pool.

The deposition of amyloid-ß (Aß) oligomers in brain parenchyma has been implicated in the pathophysiology of Alzheimer's disease. Here we present a systems pharmacology model describing the changes in the amyloid precursor protein (APP) pathway after administration of three different doses (10, 30, and 125 mg/kg) of the ß-secretase 1 (BACE1) inhibitor MBi-5 in cisterna magna ported rhesus monkeys. The time course of the MBi-5 concentration in plasma and cerebrospinal fluid (CSF) was analyzed in conjunction with the effect on the concentrations of the APP metabolites Aß42, Aß40, soluble ß-amyloid precursor protein (sAPP) α, and sAPPß in CSF. The systems pharmacology model contained expressions to describe the production, elimination, and brain-to-CSF transport for the APP metabolites. Upon administration of MBi-5, a dose-dependent increase of the metabolite sAPPα and dose-dependent decreases of sAPPß and Aß were observed. Maximal inhibition of BACE1 was close to 100% and the IC50 value was 0.0256 µM (95% confidence interval, 0.0137-0.0375). A differential effect of BACE1 inhibition on Aß40 and Aß42 was observed, with the Aß40 response being larger than the Aß42 response. This enabled the identification of an Aß42 oligomer pool in the systems pharmacology model. These findings indicate that decreases in monomeric Aß responses resulting from BACE1 inhibition are partially compensated by dissociation of Aß oligomers and suggest that BACE1 inhibition may also reduce the putatively neurotoxic oligomer pool.

Pharmacological Inhibition of O-GlcNAcase Does Not Increase Sensitivity of Glucocorticoid Receptor-Mediated Transrepression.

Glucocorticoid signaling regulates target genes by multiple mechanisms, including the repression of transcriptional activities of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) though direct protein-protein interactions and subsequent O-GlcNAcylation of RNA polymerase II (pol II). Recent studies have shown that overexpression of O-linked ß-N-acetylglucosamine transferase (OGT), which adds an O-linked ß-N-acetylglucosamine (O-GlcNAc) group to the C-terminal domain of RNA pol II, increases the transrepression effects of glucocorticoids (GC). As O-GlcNAcase (OGA) is an enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, we hypothesized that the potentiation of GC effects following OGT overexpression could be similarly observed via the direct inhibition of OGA, inhibiting O-GlcNAc removal from pol II. Here we show that despite pharmacological evidence of target engagement by a selective small molecule inhibitor of OGA, there is no evidence for a sensitizing effect on glucocorticoid-mediated effects on TNF-α promoter activity, or gene expression generally, in human cells. Furthermore, inhibition of OGA did not potentiate glucocorticoid-induced apoptosis in several cancer cell lines. Thus, despite evidence for O-GlcNAc modification of RNA pol II in GR-mediated transrepression, our data indicate that pharmacological inhibition of OGA does not potentiate or enhance glucocorticoid-mediated transrepression.

An integrated multi-study analysis of intra-subject variability in cerebrospinal fluid amyloid-ß concentrations collected by lumbar puncture and indwelling lumbar catheter.

INTRODUCTION: Amyloid-ß (Aß) has been investigated as a diagnostic biomarker and therapeutic drug target. Recent studies found that cerebrospinal fluid (CSF) Aß fluctuates over time, including as a diurnal pattern, and increases in absolute concentration with serial collection. It is currently unknown what effect differences in CSF collection methodology have on Aß variability. In this study, we sought to determine the effect of different collection methodologies on the stability of CSF Aß concentrations over time. METHODS: Grouped analysis of CSF Aß levels from multiple industry and academic groups collected by either lumbar puncture (n=83) or indwelling lumbar catheter (n=178). Participants were either placebo or untreated subjects from clinical drug trials or observational studies. Participants had CSF collected by lumbar puncture or lumbar catheter for quantitation of Aß concentration by enzyme linked immunosorbent assay. Data from all sponsors was converted to percent of the mean for Aß40 and Aß42 for comparison. Repeated measures analysis of variance was performed to assess for factors affecting the linear rise of Aß concentrations over time. RESULTS: Analysis of studies collecting CSF via lumbar catheter revealed tremendous inter-subject variability of Aß40 and Aß42 as well as an Aß diurnal pattern in all of the sponsors' studies. In contrast, Aß concentrations from CSF samples collected at two time points by lumbar puncture showed no significant differences. Repeated measures analysis of variance found that only time and draw frequency were significantly associated with the slope of linear rise in Aß40 and Aß42 concentrations during the first 6 hours of collection. CONCLUSIONS: Based on our findings, we recommend minimizing the frequency of CSF draws in studies measuring Aß levels and keeping the frequency standardized between experimental groups. The Aß diurnal pattern was noted in all sponsors' studies and was not an artifact of study design. Averaging Aß concentrations at each time point is recommended to minimize the effect of individual variability. Indwelling lumbar catheters are an invaluable research tool for following changes in CSF Aß over 24-48 hours, but factors affecting Aß concentration such as linear rise and diurnal variation need to be accounted for in planning study designs.

Evaluation of cynomolgus monkeys for the identification of endogenous biomarkers for hepatic transporter inhibition and as a translatable model to predict pharmacokinetic interactions with statins in humans.

Inhibition of hepatic transporters such as organic anion transporting polypeptides (OATPs) 1B can cause drug-drug interactions (DDIs). Determining the impact of perpetrator drugs on the plasma exposure of endogenous substrates for OATP1B could be valuable to assess the risk for DDIs early in drug development. As OATP1B orthologs are well conserved between human and monkey, we assessed in cynomolgus monkeys the endogenous OATP1B substrates that are potentially suitable to assess DDI risk in humans. The effect of rifampin (RIF), a potent inhibitor for OATP1B, on plasma exposure of endogenous substrates of hepatic transporters was measured. From the 18 biomarkers tested, RIF (18 mg/kg, oral) caused significant elevation of plasma unconjugated and conjugated bilirubin, which may be attributed to inhibition of cOATP1B1 and cOATP1B3 based on in vitro to in vivo extrapolation analysis. To further evaluate whether cynomolgus monkeys are a suitable translational model to study OATP1B-mediated DDIs, we determined the inhibitory effect of RIF on in vitro transport and pharmacokinetics of rosuvastatin (RSV) and atorvastatin (ATV). RIF strongly inhibited the uptake of RSV and ATV by cOATP1B1 and cOATP1B3 in vitro. In agreement with clinical observations, RIF (18 mg/kg, oral) significantly decreased plasma clearance and increased the area under the plasma concentration curve (AUC) of intravenously administered RSV by 2.8- and 2.7-fold, and increased the AUC and maximum plasma concentration of orally administered RSV by 6- and 10.3-fold, respectively. In contrast to clinical findings, RIF did not significantly increase plasma exposure of either intravenous or orally administered ATV, indicating species differences in the rate-limiting elimination pathways.

Soluble BACE-1 Activity and sAßPPß Concentrations in Alzheimer's Disease and Age-Matched Healthy Control Cerebrospinal Fluid from the Alzheimer's Disease Neuroimaging Initiative-1 Baseline Cohort.

ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) plays an important role in the development of Alzheimer's disease (AD), freeing the amyloid-ß (Aß) N-terminus from the amyloid-ß protein precursor (AßPP), the first step in Aß formation. Increased BACE1 activity in AD brain or cerebrospinal fluid (CSF) has been reported. Other studies, however, found either no change or a decrease with AD diagnosis in either BACE1 activity or sAßPPß, the N-terminal secreted product of BACE1 (sBACE1) activity on AßPP. Here, sBACE1 enzymatic activity and secreted AßPPß (sAßPPß) were measured in Alzheimer's Disease Neuroimaging Initiative-1 (ADNI-1) baseline CSF samples and no statistically significant changes were found in either measure comparing healthy control, mild cognitively impaired, or AD individual samples. While CSF sBACE1 activity and sAßPPß demonstrated a moderate yet significant degree of correlation with each other, there was no correlation of either analyte to CSF Aß peptide ending at residue 42. Surprisingly, a stronger correlation was demonstrated between CSF sBACE1 activity and tau, which was comparable to that between CSF Aß42 and tau. Unlike for these latter two analytes, receiver-operator characteristic curves demonstrate that neither CSF sBACE1 activity nor sAßPPß concentrations can be used to differentiate between healthy elderly and AD individuals.

Cerebrospinal fluid biomarkers distinguish postmortem-confirmed Alzheimer's disease from other dementias and healthy controls in the OPTIMA cohort.

Cerebrospinal fluid (CSF) amyloid-ß (Aß) and tau have been studied as markers of Alzheimer's disease (AD). Combined Aß42 and t-tau distinguishes AD from healthy controls with a sensitivity and specificity (sens/spec) near 89% across studies. This study examined these markers in the homogeneous OPTIMA cohort, using extensive longitudinal follow up and postmortem evaluation to confirm clinicopathological status. Baseline CSF was analyzed from 227 participants with AD (97% autopsy-confirmed), mild cognitive impairment (MCI; 73% confirmed), other dementia syndrome (ODS; 100% confirmed), and controls (CTL; 27% confirmed, follow up approximately 9-13 years). Biomarker concentrations were analyzed using validated ELISAs. AD patients had lower CSF Aß42 and higher t-tau, p-tau, t-tau/Aß42, and t-tau/Aß40 compared to CTLs, with MCI intermediate. CTL and MCI participants who progressed to AD demonstrated more AD-like profiles. Aß40, sAßPPα, and sAßPPß were lower in AD compared to CTL. High-level discriminators of AD from CTL were t-tau/Aß40 (AUROC 0.986, sens/spec of 92%/94%), p-tau/Aß42 (AUROC 0.972, sens/spec of 94%/90%), and Aß42 (AUROC 0.941, sens/spec of 88%). For discriminating AD from ODS, p-tau/Aß42 demonstrated sens/spec of 88%/100% (95%/86% at the AD versus CTL cutoff) and Aß42 demonstrated sens/spec of 84%/100% (88%/100% at the AD versus CTL cutoff). In a well-characterized, homogeneous population, a single cutoff for baseline CSF Aß and tau markers can distinguish AD with a high level of sens/spec compared to other studies. It may be important to characterize sources of demographic and biological variability to support the effective use of CSF diagnostic assays in the broader AD population.

CNS amyloid-ß, soluble APP-α and -ß kinetics during BACE inhibition.

BACE, a ß-secretase, is an attractive potential disease-modifying therapeutic strategy for Alzheimer's disease (AD) as it results directly in the decrease of amyloid precursor protein (APP) processing through the ß-secretase pathway and a lowering of CNS amyloid-ß (Aß) levels. The interaction of the ß-secretase and α-secretase pathway-mediated processing of APP in the rhesus monkey (nonhuman primate; NHP) CNS is not understood. We hypothesized that CNS inhibition of BACE would result in decreased newly generated Aß and soluble APPß (sAPPß), with increased newly generated sAPPα. A stable isotope labeling kinetics experiment in NHPs was performed with a (13)C6-leucine infusion protocol to evaluate effects of BACE inhibition on CNS APP processing by measuring the kinetics of sAPPα, sAPPß, and Aß in CSF. Each NHP received a low, medium, or high dose of MBI-5 (BACE inhibitor) or vehicle in a four-way crossover design. CSF sAPPα, sAPPß, and Aß were measured by ELISA and newly incorporated label following immunoprecipitation and liquid chromatography-mass spectrometry. Concentrations, kinetics, and amount of newly generated APP fragments were calculated. sAPPß and sAPPα kinetics were similar, but both significantly slower than Aß. BACE inhibition resulted in decreased labeled sAPPß and Aß in CSF, without observable changes in labeled CSF sAPPα. ELISA concentrations of sAPPß and Aß both decreased and sAPPα increased. sAPPα increased by ELISA, with no difference by labeled sAPPα kinetics indicating increases in product may be due to APP shunting from the ß-secretase to the α-secretase pathway. These results provide a quantitative understanding of pharmacodynamic effects of BACE inhibition on NHP CNS, which can inform about target development.

Adenosine analogue inhibitors of S-adenosylhomocysteine hydrolase.

Elevated plasma homocysteine (Hcy) levels are an independent risk factor for the onset and progression of Alzheimer's disease. Reduction of Hcy to normal levels therefore presents a new approach for disease modification. Hcy is produced by the cytosolic enzyme S-adenosylhomocysteine hydrolase (AHCY), which converts S-adenosylhomocysteine (SAH) to Hcy and adenosine. Herein we describe the design and characterization of novel, substrate-based S-adenosylhomocysteine hydrolase inhibitors with low nanomolar potency in vitro and robust activity in vivo.

A sensitive aß oligomer assay discriminates Alzheimer's and aged control cerebrospinal fluid.

A hallmark of Alzheimer's disease (AD) brain is the amyloid ß (Aß) plaque, which is comprised of Aß peptides. Multiple lines of evidence suggest that Aß oligomers are more toxic than other peptide forms. We sought to develop a robust assay to quantify oligomers from CSF. Antibody 19.3 was compared in one-site and competitive ELISAs for oligomer binding specificity. A two-site ELISA for oligomers was developed using 19.3 coupled to a sensitive, bead-based fluorescent platform able to detect single photons of emitted light. The two-site ELISA was >2500× selective for Aß oligomers over Aß monomers with a limit of detection ∼ 0.09 pg/ml in human CSF. The lower limit of reliable quantification of the assay was 0.18 pg/ml and the antibody pairs recognized Aß multimers comprised of either synthetic standards, or endogenous oligomers isolated from confirmed human AD and healthy control brain. Using the assay, a significant 3- to 5-fold increase in Aß oligomers in human AD CSF compared with comparably aged controls was demonstrated. The increase was seen in three separate human cohorts, totaling 63 AD and 54 controls. CSF oligomers ranged between 0.1 and 10 pg/ml. Aß oligomer levels did not strongly associate with age or gender, but had an inverse correlation with MMSE score. The C statistic for the Aß oligomer ROC curve was 0.86, with 80% sensitivity and 88% specificity to detect AD, suggesting reasonable discriminatory power for the AD state and the potential for utility as a diagnostic marker.

Pyroglutamate-modified amyloid-ß protein demonstrates similar properties in an Alzheimer's disease familial mutant knock-in mouse and Alzheimer's disease brain.

BACKGROUND: N-terminally truncated, pyroglutamate-modified amyloid-ß (Aß) peptides are major constituents of amyloid deposits in Alzheimer's disease (AD). METHODS: Using a newly developed ELISA for Aß modified at glutamate 3 with a pyroglutamate (pE3Aß), brain pE3Aß was characterized in human AD in an AD mouse model harboring double knock-in amyloid precursor protein (APP)-KM670/671NL and presenilin 1 (PS1)-P264L (APP/PS1-dKI) mutations, and in a second mouse model with transgenic overexpression of human APP695 with APP-KM670/671NL (Tg2576). RESULTS: pE3Aß increased in the AD brain versus age-matched controls, with pE3Aß/total Aß at 45 and 10%, respectively. Compared to controls, the AD brain demonstrated 8.5-fold increased pE3Aß compared to non-pE3Aß species, which increased 2.7-fold. In the APP/PS1-dKI brain, pE3Aß/total Aß increased from 7% at 3 months to 16 and 19% at 15 and 19 months, respectively. In Tg2576, pE3Aß/total Aß was only 1.5% at 19 months, suggesting that APP/PS1-dKI, despite less total Aß compared to Tg2576 at comparable ages, more closely mimics AD brain pathology. CONCLUSION: This report supports a significant role for pE3Aß in AD pathogenesis by confirming that pE3Aß represents a large fraction of Aß within the AD brain. Compared to the age-matched control brain, pE3Aß increased to a greater extent compared to Aß species without this N-terminal modification. Further, the APP/PS1-dKI model more closely resembles the AD brain in this regard, compared to the Tg2576 model.

Characterization of plasma ß-secretase (BACE1) activity and soluble amyloid precursor proteins as potential biomarkers for Alzheimer's disease.

Reduction in cerebrospinal fluid (CSF) amyloid ß42 (Aß42) and elevation in total tau and phospho-thr181 tau consistently differentiate between Alzheimer's disease (AD) and age-matched control subjects. In contrast, CSF ß-site APP-cleaving enzyme activity (BACE1) and soluble amyloid precursor proteins α and ß (sAPPα and sAPPß) are without consistent patterns in AD subjects. Plasma sampling is much easier, with fewer side effects, and is readily applied in primary care centers, so we have developed and validated novel plasma BACE activity, sAPPß, and sAPPα assays and investigated their ability to distinguish AD from age-matched controls. Plasma BACE activity assay was sensitive and specific, with signal being immunodepleted with a specific BACE1 antibody and inhibited with a BACE1-specific inhibitor. Plasma sAPPß and sAPPα assays were specific, with signal diluting linearly, immunodepleted with specific antibodies, and at background levels in APP knockout mice. In rhesus monkeys, BACE1 but not γ-secretase inhibitor led to significant lowering of plasma sAPPß with concurrent elevation of plasma sAPPα. AD subjects showed a significant increase in plasma BACE1 activity, sAPPß, sAPPα, and Aß42 (P < 0.001) compared with age-matched controls. In conclusion, plasma BACE activity and sAPP endpoints provide novel investigative biomarkers for AD diagnosis and potential pharmacodynamic biomarkers for secretase inhibitor studies.