Marie A. Holahan

First Demonstration of Cerebrospinal Fluid and Plasma Aβ Lowering with Oral Administration of a β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Inhibitor in Nonhuman Primates

β-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic Aβ42 peptides in Alzheimers disease. Although previous mouse studies have shown brain Aβ lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of Aβ in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC50 ∼ 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain Aβ levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma Aβ levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPPβ, Aβ40, Aβ42, and plasma Aβ40 levels. CSF Aβ42 lowering showed an EC50 of ∼20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF Aβ lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

Acute gamma-secretase inhibition of nonhuman primate CNS shifts amyloid precursor protein (APP) metabolism from amyloid-beta production to alternative APP fragments without amyloid-beta rebound

The accumulation of amyloid β (Aβ) in Alzheimers disease is caused by an imbalance of production and clearance, which leads to increased soluble Aβ species and extracellular plaque formation in the brain. Multiple Aβ-lowering therapies are currently in development: an important goal is to characterize the molecular mechanisms of action and effects on physiological processing of Aβ, as well as other amyloid precursor protein (APP) metabolites, in models which approximate human Aβ physiology. To this end, we report the translation of the human in vivo stable-isotope-labeling kinetics (SILK) method to a rhesus monkey cisterna magna ported (CMP) nonhuman primate model, and use the model to test the mechanisms of action of a γ-secretase inhibitor (GSI). A major concern of inhibiting the enzymes which produce Aβ (β- and γ-secretase) is that precursors of Aβ may accumulate and cause a rapid increase in Aβ production when enzyme inhibition discontinues. In this study, the GSI MK-0752 was administered to conscious CMP rhesus monkeys in conjunction with in vivo stable-isotope-labeling, and dose-dependently reduced newly generated CNS Aβ. In contrast to systemic Aβ metabolism, CNS Aβ production was not increased after the GSI was cleared. These results indicate that most of the CNS APP was metabolized to products other than Aβ, including C-terminal truncated forms of Aβ: 1-14, 1-15 and 1-16; this demonstrates an alternative degradation pathway for CNS amyloid precursor protein during γ-secretase inhibition.

Application of an End-to-End Biomarker Discovery Platform to Identify Target Engagement Markers in Cerebrospinal Fluid by High Resolution Differential Mass Spectrometry

The rapid identification of protein biomarkers in biofluids is important to drug discovery and development. Here, we describe a general proteomic approach for the discovery and identification of proteins that exhibit a statistically significant difference in abundance in cerebrospinal fluid (CSF) before and after pharmacological intervention. This approach, differential mass spectrometry (dMS), is based on the analysis of full scan mass spectrometry data. The dMS workflow does not require complex mixing and pooling strategies, or isotope labeling techniques. Accordingly, clinical samples can be analyzed individually, allowing the use of longitudinal designs and within-subject data analysis in which each subject acts as its own control. As a proof of concept, we performed multifactorial dMS analyses on CSF samples drawn at 6 time points from n = 6 cisterna magna ported (CMP) rhesus monkeys treated with 2 potent gamma secretase inhibitors (GSI) or comparable vehicle in a 3-way crossover study that included a total of 108 individual CSF samples. Using analysis of variance and statistical filtering on the aligned and normalized LC-MS data sets, we detected 26 features that were significantly altered in CSF by drug treatment. Of those 26 features, which belong to 10 distinct isotopic distributions, 20 were identified by MS/MS as 7 peptides from CD99, a cell surface protein. Six features from the remaining 3 isotopic distributions were not identified. A subsequent analysis showed that the relative abundance of these 26 features showed the same temporal profile as the ELISA measured levels of CSF A beta 42 peptide, a known pharmacodynamic marker for gamma-secretase inhibition. These data demonstrate that dMS is a promising approach for the discovery, quantification, and identification of candidate target engagement biomarkers in CSF.

Systemic pan-AMPK activator MK-8722 improves glucose homeostasis but induces cardiac hypertrophy

Hitting a dozen enzymes with one drug The adenosine monophosphate-activated protein kinase (AMPK) controls cellular energy status. AMPK is activated when energy levels fall. This stimulates adenosine triphosphate (ATP)-generating pathways that promote glucose uptake and inhibits ATP-consuming pathways associated with glucose synthesis. In principle, these effects would be beneficial in metabolic diseases, including diabetes. Pharmacological activation of AMPK has been challenging, however, because in mammals, the enzyme exists as 12 distinct complexes. Myers et al. describe an orally available compound (MK-8722) that activates all 12 complexes (see the Perspective by Hardie). In animal models, MK-8722 ameliorated diabetes, but it also caused enlargement of the heart. MK-8722 may be a useful tool compound for laboratory research on AMPK function. Science, this issue p. 507; see also p. 455 In animals, a drug activating all 12 isoforms of the energy regulator AMPK benefits metabolism but may pose heart risks. 5′-Adenosine monophosphate–activated protein kinase (AMPK) is a master regulator of energy homeostasis in eukaryotes. Despite three decades of investigation, the biological roles of AMPK and its potential as a drug target remain incompletely understood, largely because of a lack of optimized pharmacological tools. We developed MK-8722, a potent, direct, allosteric activator of all 12 mammalian AMPK complexes. In rodents and rhesus monkeys, MK-8722–mediated AMPK activation in skeletal muscle induced robust, durable, insulin-independent glucose uptake and glycogen synthesis, with resultant improvements in glycemia and no evidence of hypoglycemia. These effects translated across species, including diabetic rhesus monkeys, but manifested with concomitant cardiac hypertrophy and increased cardiac glycogen without apparent functional sequelae.

Discovery of 6-(Fluoro-18F)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine ([18F]-MK-6240): A Positron Emission Tomography (PET) Imaging Agent for Quantification of Neurofibrillary Tangles (NFTs)

Neurofibrillary tangles (NFTs) made up of aggregated tau protein have been identified as the pathologic hallmark of several neurodegenerative diseases including Alzheimers disease. In vivo detection of NFTs using PET imaging represents a unique opportunity to develop a pharmacodynamic tool to accelerate the discovery of new disease modifying therapeutics targeting tau pathology. Herein, we present the discovery of 6-(fluoro-(18)F)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine, 6 ([(18)F]-MK-6240), as a novel PET tracer for detecting NFTs. 6 exhibits high specificity and selectivity for binding to NFTs, with suitable physicochemical properties and in vivo pharmacokinetics.

Demonstration of enhanced endogenous fibrinolysis in thrombin activatable fibrinolysis inhibitor-deficient mice

To investigate the importance of thrombin activatable fibrinolysis inhibitor (TAFI) in the stabilization of plasma clots, we have compared fibrinolysis in TAFI-deficient (KO) and wild-type (WT) littermate mice. TAFI-deficient mice were previously generated by targeted gene disruption. The level of TAFI activity generated in plasma from WT mice in the presence of added thrombin and thrombomodulin (activatable TAFI) is twice that of plasma from TAFI heterozygous mice (HET); no activatable TAFI is detected in TAFI KO plasma. In vitro, TAFI KO plasma clots lysed faster than WT plasma clots, and HET plasma clots lysed at an intermediate rate. The rate of clot lysis for KO mice is not changed in the presence of potato carboxypeptidase inhibitor, a specific inhibitor of TAFIa, whereas the WT and HET clot lysis rates are increased in the presence of potato carboxypeptidase inhibitor. C-terminal lysine residues are preserved on partially degraded clots from KO mice, but are absent from partially degraded WT clots. In vivo, in a batroxobin-induced pulmonary embolism model, KO mice displayed a lower retention of fibrin in the lungs than did WT mice. These results are the first demonstration of enhanced endogenous fibrinolysis in an in vivo model without the addition of exogenous thrombolytic.

Simple high-performance liquid chromatographic method to analyze serum creatinine has several advantages over the Jaffé picric acid reaction as demonstrated with a cimetidine dose response in rhesus monkeys.

A simple method for creatinine determination was developed using high-performance liquid chromatography (HPLC) to more accurately monitor serum creatinine levels in experimental animal models when compared to the Jaffé method. The new HPLC procedure will replace the traditional Jaffé method for rhesus monkey kidney function studies. We developed an isocratic method using a polymeric, hydrophilic, silica-based strong cation-exchange bed with a 5.0 mmol/l lithium acetate matrix, pH 4.9, which isolates creatinine with no detectable impurities as determined by three-dimensional ultraviolet-visible spectral analysis. Sample preparation includes deproteination with acetonitrile, evaporation, and resolubilization in mobile phase followed by quantitation with UV detection at 234 nm. Extraction efficiency across the measured range was 96 +/- 2%. From numerous extracted rhesus monkey creatinine curves (n=38) a slope of 251,100 +/- 756 (95% CI) and an intercept of 675.6 +/- 712.7 (95% CI) was calculated. Extraction efficiency and peak purity tests with human plasma were cross-compared with rhesus monkey serum producing equivalent results. An average of 120 samples can be run daily.

Nonpeptide GPIIb/IIIa inhibitors. 10. Centrally constrained alpha-sulfonamides are potent inhibitors of platelet aggregation

Abstract Potency enhancing features of two series of fibrinogen receptor antagonists were combined to give analogs with improved potency and oral activity. Antagonists containing either alkyl or aryl sulfonamides and a central isoindolinone structural constraint demonstrate high affinity for both activated and unactivated platelet receptors.

Nonpeptide glycoprotein IIb/IIIa inhibitors. 13. design and synthesis of an orally active pyrazolopiperazinone nonpeptide fibrinogen receptor antagonist

Abstract The synthesis and antiplatelet activity of a series of pyrazolopiperazinone nonpeptide fibrinogen receptor antagonists is reported. The sulfonamide analog 6 (L-734, 115), significantly inhibited ex vivo platelet aggregation 24 h after oral administration at doses of 1.0 and 2.0 mg/kg to dogs and rhesus monkeys, respectively.

Non-peptide fibrinogen receptor antagonists. 3. design and discovery of a centrally constrained inhibitor

Abstract Analysis of platelet aggregation inhibition results and rotational isomer preferences has provided an understanding of inhibitory potency for m-phthalic acid analogs 7–7. Constraint of the N-terminal amide led to compound 9, which is potent, selective, and orally active.