Deborah Dhuyvetter

BACE1 inhibition induces a specific cerebrospinal fluid β-amyloid pattern that identifies drug effects in the central nervous system.

BACE1 is a key enzyme for amyloid-β (Aβ) production, and an attractive therapeutic target in Alzheimers disease (AD). Here we report that BACE1 inhibitors have distinct effects on neuronal Aβ metabolism, inducing a unique pattern of secreted Aβ peptides, analyzed in cell media from amyloid precursor protein (APP) transfected cells and in cerebrospinal fluid (CSF) from dogs by immunoprecipitation-mass spectrometry, using several different BACE1 inhibitors. Besides the expected reductions in Aβ1-40 and Aβ1-42, treatment also changed the relative levels of several other Aβ isoforms. In particular Aβ1-34 decreased, while Aβ5-40 increased, and these changes were more sensitive to BACE1 inhibition than the changes in Aβ1-40 and Aβ1-42. The effects on Aβ5-40 indicate the presence of a BACE1 independent pathway of APP degradation. The described CSF Aβ pattern may be used as a pharmacodynamic fingerprint to detect biochemical effects of BACE1-therapies in clinical trials, which might accelerate development of novel therapies.

A translational assessment of preclinical versus clinical tools for the measurement of cardiac contractility: Comparison of LV dP/dtmax with echocardiography in telemetry implanted beagle dogs

INTRODUCTION Regarding evaluation of drug-induced changes in left ventricular contractility in safety pharmacology there is still a gap in knowledge between preclinically and clinically used measurements. METHODS As a step towards translation of preclinical to clinical outcomes, this study in telemetered dogs was initiated to compare indexes of contractility, such as LV dP/dt(max) (contractility measured as the maximum raise of pressure in the left ventricle) and LV dP/dt(max)/P (contractility measured as the maximum raise of pressure in the left ventricle, corrected for pressure) (telemetry; both commonly preclinically used) and EF (ejection fraction) and FS (fractional shortening) (echocardiography; both commonly clinically used). Different inotropic states were induced by minoxidil, milrinone, isoprenaline, clonidine, atenolol and verapamil. RESULTS Both techniques demonstrated reproducible changes in contractility which showed a clear linear association. A change in LV dP/dt(max) of 1000 mmHg/s (in the range of 2500 to 7500 mmHg/s; in healthy dogs) corresponded with a change in ejection fraction of approximately 7% and a fractional shortening of approximately 6%. A change of 10/s LV dP/dt(max)/P (in the range of 35 to 85/s; in healthy dogs) corresponded with a change in ejection fraction of approximately 7% and a fractional shortening of 7%. DISCUSSION The correlation found in this study could potentially enable a better--translational--assessment of the clinical relevance of changes in contractility indices measured with telemetry devices in preclinical safety studies.

Novel Pyrimidine Toll-like Receptor 7 and 8 Dual Agonists to Treat Hepatitis B Virus

Toll-like receptor (TLR) 7 and 8 agonists can potentially be used in the treatment of viral infections and are particularly promising for chronic hepatitis B virus (HBV) infection. An internal screening effort identified a pyrimidine Toll-like receptor 7 and 8 dual agonist. This provided a novel alternative over the previously reported adenine and pteridone type of agonists. Structure-activity relationship, lead optimization, in silico docking, pharmacokinetics, and demonstration of ex vivo and in vivo cytokine production of the lead compound are presented.

Young to Middle-Aged Dogs with High Amyloid-β Levels in Cerebrospinal Fluid are Impaired on Learning in Standard Cognition tests.

Understanding differences in Alzheimer’s disease biomarkers before the pathology becomes evident can contribute to an improved understanding of disease pathogenesis and treatment. A decrease in amyloid-β (Aβ)42 in cerebrospinal fluid (CSF) is suggested to be a biomarker for Aβ deposition in brain. However, the relevance of CSF Aβ levels prior to deposition is not entirely known. Dogs are similar to man with respect to amyloid-β protein precursor (AβPP)-processing, age-related amyloid plaque deposition, and cognitive dysfunction. In the current study, we evaluated the relation between CSF Aβ42 levels and cognitive performance in young to middle-aged dogs (1.5–7 years old). Additionally, CSF sAβPPα and sAβPPβ were measured to evaluate AβPP processing, and CSF cytokines were measured to determine the immune status of the brain. We identified two groups of dogs showing consistently low or high CSF Aβ42 levels. Based on prior studies, it was assumed that at this age no cerebral amyloid plaques were likely to be present. The cognitive performance was evaluated in standard cognition tests. Low or high Aβ concentrations coincided with low or high sAβPPα, sAβPPβ, and CXCL-1 levels, respectively. Dogs with high Aβ concentrations showed significant learning impairments on delayed non-match to position (DNMP), object discrimination, and reversal learning compared to dogs with low Aβ concentrations. Our data support the hypothesis that high levels of CSF Aβ in dogs coincide with lower cognitive performance prior to amyloid deposition. Further experiments are needed to investigate this link, as well as the relevance with respect to Alzheimer’s disease pathology progression.

Discovery of Potent and Centrally Active 6-Substituted 5-Fluoro-1,3-dihydro-oxazine β-Secretase (BACE1) Inhibitors via Active Conformation Stabilization

β-Secretase (BACE1) has an essential role in the production of amyloid β peptides that accumulate in patients with Alzheimers disease (AD). Thus, inhibition of BACE1 is considered to be a disease-modifying approach for the treatment of AD. Our hit-to-lead efforts led to a cellular potent 1,3-dihydro-oxazine 6, which however inhibited hERG and showed high P-gp efflux. The close analogue of 5-fluoro-oxazine 8 reduced P-gp efflux; further introduction of electron withdrawing groups at the 6-position improved potency and also mitigated P-gp efflux and hERG inhibition. Changing to a pyrazine followed by optimization of substituents on both the oxazine and the pyrazine culminated in 24 with robust Aβ reduction in vivo at low doses as well as reduced CYP2D6 inhibition. On the basis of the X-ray analysis and the QM calculation of given dihydro-oxazines, we reasoned that the substituents at the 6-position as well as the 5-fluorine on the oxazine would stabilize a bioactive conformation to increase potency.

Identification and Optimization of Pyrrolo[3,2- d ]pyrimidine Toll-like Receptor 7 (TLR7) Selective Agonists for the Treatment of Hepatitis B

Pyrrolo[3,2-d]pyrimidines were identified as a new series of potent and selective TLR7 agonists. Compounds were optimized for their activity and selectivity over TLR8. This presents an advantage over recently described scaffolds that have residual TLR8 activity, which may be detrimental to the tolerability of the candidate drug. Oral administration of the lead compound 54 effectively induced a transient interferon stimulated gene (ISG) response in mice and cynomolgus monkeys. We aimed for a high first pass effect, limiting cytokine induction systemically, and demonstrated the potential for the immunotherapy of viral hepatitis.

The performance of Aβ5-X isoforms as novel pharmacodynamic markers of BACE1 inhibition

progressors respectively) (P<0.05). Therewere no differences in composite vascular score, dementia severity (CMMSE and CDR-SOB score), age-related white matter (ARWMC) total score or treatment differences. No differences were noted in APOE status in 6 available data. We found statistically significant increase in GZMB, GZMH, TGFBR3, KLRD1, KIR2DL3 and KIR2DL4 expression (P <0.05) for fast AD-progressors (compared to slow progressors) consistently across 3 time points. Conclusions: Our initial results demonstrate relationship between mRNA levels and fast progression in early AD subjects. This has potential therapeutic implications for the independent role of inflammation and apoptosis in influencing AD progression and we await further confirmation with larger sample analyses.

Optimization of 1,4-Oxazine β-Secretase 1 (BACE1) Inhibitors Toward a Clinical Candidate

In previous studies, the introduction of electron withdrawing groups to 1,4-oxazine BACE1 inhibitors reduced the p Ka of the amidine group, resulting in compound 2 that showed excellent in vivo efficacy, lowering Aβ levels in brain and CSF. However, a suboptimal cardiovascular safety margin, based on QTc prolongation, prevented further progression. Further optimization resulted in the replacement of the 2-fluoro substituent by a CF3-group, which reduced hERG inhibition. This has led to compound 3, with an improved cardiovascular safety margin and sufficiently safe in GLP toxicity studies to progress into clinical trials.

Rational Design of Novel 1,3-Oxazine Based β-Secretase (BACE1) Inhibitors: Incorporation of a Double Bond To Reduce P-gp Efflux Leading to Robust Aβ Reduction in the Brain

Accumulation of Aβ peptides is a hallmark of Alzheimers disease (AD) and is considered a causal factor in the pathogenesis of AD. β-Secretase (BACE1) is a key enzyme responsible for producing Aβ peptides, and thus agents that inhibit BACE1 should be beneficial for disease-modifying treatment of AD. Here we describe the discovery and optimization of novel oxazine-based BACE1 inhibitors by lowering amidine basicity with the incorporation of a double bond to improve brain penetration. Starting from a 1,3-dihydrooxazine lead 6 identified by a hit-to-lead SAR following HTS, we adopted a p Ka lowering strategy to reduce the P-gp efflux and the high hERG potential leading to the discovery of 15 that produced significant Aβ reduction with long duration in pharmacodynamic models and exhibited wide safety margins in cardiovascular safety models. This compound improved the brain-to-plasma ratio relative to 6 by reducing P-gp recognition, which was demonstrated by a P-gp knockout mouse model.

2,4-Diaminoquinazolines as Dual Toll Like Receptor (TLR) 7/8 Modulators for the Treatment of Hepatitis B Virus

A novel series of 2,4-diaminoquinazolines was identified as potent dual Toll-like receptor (TLR) 7 and 8 agonists with reduced off-target activity. The stereochemistry of the amino alcohol was found to influence the TLR7/8 selectivity with the ( R) isomer resulting in selective TLR8 agonism. Lead optimization toward a dual agonist afforded ( S)-3-((2-amino-8-fluoroquinazolin-4-yl)amino)hexanol 31 as a potent analog, being structurally different from previously described dual agonists ( McGowan J. Med. Chem. 2016 , 59 , 7936 ). Pharmacokinetic and pharmacodynamic (PK/PD) studies revealed the desired high first pass profile aimed at limiting systemic cytokine activation. In vivo pharmacodynamic studies with lead compound 31 demonstrated production of cytokines consistent with TLR7/8 activation in mice and cynomolgus monkeys and ex vivo inhibition of hepatitis B virus (HBV).