Alexander Flohr

Generation of Aβ38 and Aβ42 Is Independently and Differentially Affected by Familial Alzheimer Disease-associated Presenilin Mutations and γ-Secretase Modulation

Alzheimer disease amyloid β-peptide (Aβ) is generated via proteolytic processing of the β-amyloid precursor protein by β- and γ-secretase. γ-Secretase can be blocked by selective inhibitors but can also be modulated by a subset of non-steroidal anti-inflammatory drugs, including sulindac sulfide. These drugs selectively reduce the generation of the aggregation-prone 42-amino acid Aβ42 and concomitantly increase the levels of the rather benign Aβ38. Here we show that Aβ42 and Aβ38 generation occur independently from each other. The amount of Aβ42 produced by cells expressing 10 different familial Alzheimer disease (FAD)-associated mutations in presenilin (PS) 1, the catalytic subunit of γ-secretase, appeared to correlate with the respective age of onset in patients. However, Aβ38 levels did not show a negative correlation with the age of onset. Modulation of γ-secretase activity by sulindac sulfide reduced Aβ42 in the case of wild type PS1 and two FAD-associated PS1 mutations (M146L and A285V). The remaining eight PS1 FAD mutants showed either no reduction of Aβ42 or only rather subtle effects. Strikingly, even the mutations that showed no effect on Aβ42 levels allowed a robust increase of Aβ38 upon treatment with sulindac sulfide. Similar observations were made for fenofibrate, a compound known to increase Aβ42 and to decrease Aβ38. For mutants that predominantly produce Aβ42, the ability of fenofibrate to further increase Aβ42 levels became diminished, whereas Aβ38 levels were altered to varying extents for all mutants analyzed. Thus, we conclude that Aβ38 and Aβ42 production do not depend on each other. Using an independent non-steroidal anti-inflammatory drug derivative, we obtained similar results for PS1 as well as for PS2. These in vitro results were confirmed by in vivo experiments in transgenic mice expressing the PS2 N141I FAD mutant. Our findings therefore have strong implications on the selection of transgenic mouse models used for screening of the Aβ42-lowering capacity of γ-secretase modulators. Furthermore, human patients with certain PS mutations may not respond to γ-secretase modulators.

Preclinical Profile of a Potent γ-Secretase Inhibitor Targeting Notch Signaling with In vivo Efficacy and Pharmacodynamic Properties

Notch signaling is an area of great interest in oncology. RO4929097 is a potent and selective inhibitor of gamma-secretase, producing inhibitory activity of Notch signaling in tumor cells. The RO4929097 IC50 in cell-free and cellular assays is in the low nanomolar range with >100-fold selectivity with respect to 75 other proteins of various types (receptors, ion channels, and enzymes). RO4929097 inhibits Notch processing in tumor cells as measured by the reduction of intracellular Notch expression by Western blot. This leads to reduced expression of the Notch transcriptional target gene Hes1. RO4929097 does not block tumor cell proliferation or induce apoptosis but instead produces a less transformed, flattened, slower-growing phenotype. RO4929097 is active following oral dosing. Antitumor activity was shown in 7 of 8 xenografts tested on an intermittent or daily schedule in the absence of body weight loss or Notch-related toxicities. Importantly, efficacy is maintained after dosing is terminated. Angiogenesis reverse transcription-PCR array data show reduced expression of several key angiogenic genes. In addition, comparative microarray analysis suggests tumor cell differentiation as an additional mode of action. These preclinical results support evaluation of RO4929097 in clinical studies using an intermittent dosing schedule. A multicenter phase I dose escalation study in oncology is under way.

Aminothiazoles as γ-secretase modulators.

We herein report the discovery of a new γ-secretase modulator class with an aminothiazole core starting from a HTS hit (3). Synthesis and SAR of this series are discussed. These novel compounds demonstrate moderate to good in vitro potency in inhibiting amyloid beta (Aβ) peptide production. Overall γ-secretase is not inhibited but the formation of the aggregating, toxic Aβ42 peptide is shifted to smaller non-aggregating Aβ peptides. Compound 15 reduced brain Aβ42 in vivo in APPSwe transgenic mice at 30mg/kg p.o.

The dynamics of Aβ distribution after γ-secretase inhibitor treatment, as determined by experimental and modelling approaches in a wild type rat.

Inhibition of the enzyme(s) that produce the Amyloid beta (Aβ) peptide, namely BACE and γ-secretase, is considered an attractive target for Alzheimer’s disease therapy. However, the optimal pharmacokinetic–pharmacodynamic modelling method to describe the changes in Aβ levels after drug treatment is unclear. In this study, turnover models were employed to describe Aβ levels following treatment with the γ-secretase inhibitor RO5036450, in the wild type rat. Initially, Aβ level changes in the brain, cerebral spinal fluid (CSF) and plasma were modeled as separate biological compartments, which allowed the estimation of a compound IC50 and Aβ turnover. While the data were well described, the model did not take into consideration that the CSF pool of Aβ most likely originates from the brain via the CSF drainage pathway. Therefore, a separate model was carried out, with the assumption that CSF Aβ levels originated from the brain. The optimal model that described the data involved two brain Aβ 40 sub-compartments, one with a rapid turnover, from which CSF Aβ 40 is derived, and a second quasi-static pool of ~20%. Importantly, the estimated in vivo brain IC50 was in a good range of the in vitro IC50 (ratio, 1.4). In conclusion, the PK/PD models presented here are well suited for describing the temporal changes in Aβ levels that occur after treatment with an Aβ lowering drug, and identifying physiological parameters.

4-Substituted-7-N-alkyl-N-acetyl 2-aminobenzothiazole amides: Drug-like and non-xanthine based A2B adenosine receptor antagonists

7-N-Acetamide-4-methoxy-2-aminobenzothiazole 4-fluorobenzamide (compound 1) was chosen as a drug-like and non-xanthine based starting point for the discovery of A(2B) receptor antagonists because of its slight selectivity against A(1) and A(2A) receptors and modest A(2B) potency. SAR exploration of compound 1 described herein included modifications to the 7-N-acetamide group, substitution of the 4-methoxy group by halogens as well as replacement of the p-flouro-benzamide side chain. This work culminated in the identification of compound 37 with excellent A(2B) potency, modest selectivity versus A(2A) and A(1) receptors, and good rodent PK properties.

Morphologic and Functional Effects of Gamma Secretase Inhibition on Splenic Marginal Zone B Cells

The γ-secretase complex is a promising target in Alzheimers disease because of its role in the amyloidogenic processing of β-amyloid precursor protein. This enzyme also catalyzes the cleavage of Notch receptor, resulting in the nuclear translocation of intracellular Notch where it modulates gene transcription. Notch signaling is essential in cell fate decisions during embryogenesis, neuronal differentiation, hematopoiesis, and development of T and B cells, including splenic marginal zone (MZ) B cells. This B cell compartment participates in the early phases of the immune response to blood-borne bacteria and viruses. Chronic treatment with the oral γ-secretase inhibitor RO4929097 resulted in dose-dependent decreased cellularity (atrophy) of the MZ of rats and mice. Significant decreases in relative MZ B-cell numbers of RO4929097-treated animals were confirmed by flow cytometry. Numbers of MZ B cells reverted to normal after a sufficient RO4929097-free recovery period. Functional characterization of the immune response in relation to RO4929097-related MZ B cell decrease was assessed in mice vaccinated with inactivated vesicular stomatitis virus (VSV). Compared with the immunosuppressant cyclosporin A, RO4929097 caused only mild and reversible delayed early neutralizing IgM and IgG responses to VSV. Thus, the functional consequence of MZ B cell decrease on host defense is comparatively mild.