Research Article17 September 2018Open Access Transparent process The BACE-1 inhibitor CNP520 for prevention trials in Alzheimer's disease Ulf Neumann Corresponding Author Ulf Neumann [email protected] orcid.org/0000-0001-6680-9177 Neuroscience, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Mike Ufer Mike Ufer Translational Medicine, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Laura H Jacobson Laura H Jacobson Neuroscience, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Marie-Laure Rouzade-Dominguez Marie-Laure Rouzade-Dominguez Translational Medicine, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Gunilla Huledal Gunilla Huledal PK Sciences, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Carine Kolly Carine Kolly Preclinical Safety, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Rainer M Lüönd Rainer M Lüönd Global Discovery Chemistry, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Rainer Machauer Rainer Machauer Global Discovery Chemistry, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Siem J Veenstra Siem J Veenstra Global Discovery Chemistry, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Konstanze Hurth Konstanze Hurth Global Discovery Chemistry, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Heinrich Rueeger Heinrich Rueeger Global Discovery Chemistry, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Marina Tintelnot-Blomley Marina Tintelnot-Blomley Global Discovery Chemistry, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Matthias Staufenbiel Matthias Staufenbiel Neuroscience, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Derya R Shimshek Derya R Shimshek Neuroscience, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Ludovic Perrot Ludovic Perrot Neuroscience, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Wilfried Frieauff Wilfried Frieauff Preclinical Safety, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Valerie Dubost Valerie Dubost Preclinical Safety, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Hilmar Schiller Hilmar Schiller PK Sciences, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Barbara Vogg Barbara Vogg PK Sciences, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Karen Beltz Karen Beltz PK Sciences, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Alexandre Avrameas Alexandre Avrameas Biomarker Discovery, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Sandrine Kretz Sandrine Kretz Biomarker Discovery, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Nicole Pezous Nicole Pezous Translational Medicine, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Jean-Michel Rondeau Jean-Michel Rondeau Chemical Biology and Therapeutics, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Nicolau Beckmann Nicolau Beckmann Musculoskeletal Diseases, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Andreas Hartmann Andreas Hartmann Preclinical Safety, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Stefan Vormfelde Stefan Vormfelde Translational Medicine, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Olivier J David Olivier J David Global Drug Development, Novartis, Basel, Switzerland Search for more papers by this author Bruno Galli Bruno Galli Global Drug Development, Novartis, Basel, Switzerland Search for more papers by this author Rita Ramos Rita Ramos Global Drug Development, Novartis, Basel, Switzerland Search for more papers by this author Ana Graf Ana Graf Global Drug Development, Novartis, Basel, Switzerland Search for more papers by this author Cristina Lopez Lopez Corresponding Author Cristina Lopez Lopez [email protected] orcid.org/0000-0003-4954-3754 Global Drug Development, Novartis, Basel, Switzerland Search for more papers by this author Ulf Neumann Corresponding Author Ulf Neumann [email protected] orcid.org/0000-0001-6680-9177 Neuroscience, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Mike Ufer Mike Ufer Translational Medicine, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Laura H Jacobson Laura H Jacobson Neuroscience, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Marie-Laure Rouzade-Dominguez Marie-Laure Rouzade-Dominguez Translational Medicine, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Gunilla Huledal Gunilla Huledal PK Sciences, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Carine Kolly Carine Kolly Preclinical Safety, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Rainer M Lüönd Rainer M Lüönd Global Discovery Chemistry, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Rainer Machauer Rainer Machauer Global Discovery Chemistry, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Siem J Veenstra Siem J Veenstra Global Discovery Chemistry, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Konstanze Hurth Konstanze Hurth Global Discovery Chemistry, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Heinrich Rueeger Heinrich Rueeger Global Discovery Chemistry, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Marina Tintelnot-Blomley Marina Tintelnot-Blomley Global Discovery Chemistry, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Matthias Staufenbiel Matthias Staufenbiel Neuroscience, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Derya R Shimshek Derya R Shimshek Neuroscience, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Ludovic Perrot Ludovic Perrot Neuroscience, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Wilfried Frieauff Wilfried Frieauff Preclinical Safety, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Valerie Dubost Valerie Dubost Preclinical Safety, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Hilmar Schiller Hilmar Schiller PK Sciences, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Barbara Vogg Barbara Vogg PK Sciences, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Karen Beltz Karen Beltz PK Sciences, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Alexandre Avrameas Alexandre Avrameas Biomarker Discovery, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Sandrine Kretz Sandrine Kretz Biomarker Discovery, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Nicole Pezous Nicole Pezous Translational Medicine, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Jean-Michel Rondeau Jean-Michel Rondeau Chemical Biology and Therapeutics, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Nicolau Beckmann Nicolau Beckmann Musculoskeletal Diseases, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Andreas Hartmann Andreas Hartmann Preclinical Safety, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Stefan Vormfelde Stefan Vormfelde Translational Medicine, Novartis Institute for BioMedical Research, Basel, Switzerland Search for more papers by this author Olivier J David Olivier J David Global Drug Development, Novartis, Basel, Switzerland Search for more papers by this author Bruno Galli Bruno Galli Global Drug Development, Novartis, Basel, Switzerland Search for more papers by this author Rita Ramos Rita Ramos Global Drug Development, Novartis, Basel, Switzerland Search for more papers by this author Ana Graf Ana Graf Global Drug Development, Novartis, Basel, Switzerland Search for more papers by this author Cristina Lopez Lopez Corresponding Author Cristina Lopez Lopez [email protected] orcid.org/0000-0003-4954-3754 Global Drug Development, Novartis, Basel, Switzerland Search for more papers by this author Author Information Ulf Neumann *,1, Mike Ufer2,10, Laura H Jacobson1,11, Marie-Laure Rouzade-Dominguez2, Gunilla Huledal3,12, Carine Kolly4, Rainer M Lüönd5, Rainer Machauer5, Siem J Veenstra5, Konstanze Hurth5, Heinrich Rueeger5, Marina Tintelnot-Blomley5, Matthias Staufenbiel1,13, Derya R Shimshek1, Ludovic Perrot1, Wilfried Frieauff4, Valerie Dubost4, Hilmar Schiller3, Barbara Vogg3, Karen Beltz3, Alexandre Avrameas6, Sandrine Kretz6, Nicole Pezous2, Jean-Michel Rondeau7, Nicolau Beckmann8, Andreas Hartmann4, Stefan Vormfelde2, Olivier J David9, Bruno Galli9, Rita Ramos9, Ana Graf9 and Cristina Lopez Lopez *,9 1Neuroscience, Novartis Institute for BioMedical Research, Basel, Switzerland 2Translational Medicine, Novartis Institute for BioMedical Research, Basel, Switzerland 3PK Sciences, Novartis Institute for BioMedical Research, Basel, Switzerland 4Preclinical Safety, Novartis Institute for BioMedical Research, Basel, Switzerland 5Global Discovery Chemistry, Novartis Institute for BioMedical Research, Basel, Switzerland 6Biomarker Discovery, Novartis Institute for BioMedical Research, Basel, Switzerland 7Chemical Biology and Therapeutics, Novartis Institute for BioMedical Research, Basel, Switzerland 8Musculoskeletal Diseases, Novartis Institute for BioMedical Research, Basel, Switzerland 9Global Drug Development, Novartis, Basel, Switzerland 10Present address: Idorsia Pharmaceuticals Ltd., Allschwil, Switzerland 11Present address: The Florey Institute of Neuroscience and Mental Health, Melbourne, Vic., Australia 12Present address: Swedish Orphan Biovitrum AB, Stockholm, Sweden 13Present address: Hertie Institute for Clinical Brain Research, Tübingen, Germany *Corresponding author. Tel: +41 79 845 6425; E-mail: [email protected] *Corresponding author. Tel: +41 61 324 0899; E-mail: [email protected] EMBO Mol Med (2018)10:e9316https://doi.org/10.15252/emmm.201809316 See also: JAD Zakaria & RJ Vassar (November 2018) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1) initiates the generation of amyloid-β (Aβ), and the amyloid cascade leading to amyloid plaque deposition, neurodegeneration, and dementia in Alzheimer's disease (AD). Clinical failures of anti-Aβ therapies in dementia stages suggest that treatment has to start in the early, asymptomatic disease states. The BACE-1 inhibitor CNP520 has a selectivity, pharmacodynamics, and distribution profile suitable for AD prevention studies. CNP520 reduced brain and cerebrospinal fluid (CSF) Aβ in rats and dogs, and Aβ plaque deposition in APP-transgenic mice. Animal toxicology studies of CNP520 demonstrated sufficient safety margins, with no signs of hair depigmentation, retina degeneration, liver toxicity, or cardiovascular effects. In healthy adults ≥ 60 years old, treatment with CNP520 was safe and well tolerated and resulted in robust and dose-dependent Aβ reduction in the cerebrospinal fluid. Thus, long-term, pivotal studies with CNP520 have been initiated in the Generation Program. Synopsis Alzheimer's disease (AD) is a chronic neurodegenerative disorder with increasing incidence in the aging societies, but without any disease-modifying treatment. Deposition of toxic forms of the protein Aβ in the brain is pathologic. Treatment with a BACE-1 inhibitor may prevent Aβ deposition. Recent BACE inhibitor clinical trials in patients at early or mild-to-moderate disease stage have failed, indicating that treatment needs to start earlier, before the onset of clinical symptoms. BACE inhibitor CNP520 was designed to meet the requirements of prevention treatment. CNP520 in preclinical models showed acute and chronic Aβ reduction, and a favorable safety profile. CNP520 is safe and well tolerated in humans, and dose-dependently reduced Aβ in cerebrospinal fluid. Prevention studies Generation I and II are underway in patients at enhanced risk to develop symptoms of AD. Introduction There is high unmet medical need for effective treatment of Alzheimer's disease (AD), one of the most prevalent and debilitating of neurological diseases. More than 6 million people in the United States suffer from dementia, and this number is predicted to rise to 15 million by 2060 (Brookmeyer et al, 2018). Currently available pharmacological therapies can treat only the symptoms of AD and have limited benefit. The pathophysiology of AD has been the subject of intense investigation in past decades, and a causal role for aggregated and deposited forms of amyloid-β (Aβ) is supported by a vast body of histopathological, genetic, and biomarker studies (Jonsson et al, 2012; Potter et al, 2013; Musiek & Holtzman, 2015; Scheltens et al, 2016). Consequently, investigational treatments targeting Aβ, such as anti-Aβ antibodies and inhibitors of beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1), are in advanced clinical development. However, several trials in early to moderate stages of dementia have failed to meet their primary endpoints or were stopped at interim analysis (Mullane & Williams, 2013; Hawkes, 2017; Egan et al, 2018; Honig et al, 2018). Recent evidence that Aβ deposition and measurable changes in brain biomarkers occur years before dementia symptoms appear suggests that in these trials, treatment was given too late during the course of the disease to be effective (Villemagne et al, 2011; Jack et al, 2013; Jones et al, 2016). The clinical development of drugs that target Aβ is therefore increasingly focused on treatment during the earlier stages of AD (the newly defined preclinical stage and during the stage of mild cognitive impairment) when the disease-modifying therapy targeting Aβ presumably will be of greatest benefit (Selkoe & Hardy, 2016). If successful, such a preventive treatment could have a profound clinical and public health impact if it is safe, well tolerated, and initiated early enough during the course of the disease to be effective. BACE-1 is a membrane-bound aspartyl protease required for the processing of the amyloid precursor protein (APP) to form the N-terminus of Aβ peptides and the protein-soluble APPβ (sAPPβ) (Vassar et al, 1999). Subsequent intramembrane proteolysis catalyzed by the γ-secretase complex releases amyloid-β peptides of 38–43 amino acids, which form the pathogenic oligomeric and fibrillary Aβ species (Haass, 2004; Masters & Selkoe, 2012). Inhibition of BACE-1 by low-molecular-weight compounds has emerged as a new concept for treatment of AD (Vassar et al, 2014; Eketjall et al, 2016; Kennedy et al, 2016; Timmers et al, 2016, 2017; Yan et al, 2016; Cebers et al, 2017) by preventing the generation and deposition of Aβ rather than just treating the dementia symptoms. However, limited selectivity for BACE-1 over cathepsin D (CatD), the off-target inhibition of which is a principal driver of ocular toxicity, and liver enzyme elevation have led to the termination of some early BACE-1 inhibitors in clinical trials (May et al, 2011, 2015; Zuhl et al, 2016). The termination of atabecestat (JNJ-54861911) in Phase III trials following liver enzyme elevation was recently announced. Although the small molecule BACE inhibitors, verubecestat and lanabecestat (recently stopped at interim analysis in Phase III trials), have a good selectivity for BACE-1 over CatD, they still exhibit undesirable characteristics: hair depigmentation in animal studies due to high BACE-2 inhibition and strong P-glycoprotein (P-gp)-mediated efflux that limits brain penetration (Cebers et al, 2016; Kennedy et al, 2016). We designed CNP520 specifically to avoid the above limitations and thus develop a BACE-1 inhibitor with a safety and tolerability profile suitable for long-term preventive treatment in AD. We are currently testing CNP520 in the Generation Program (Lopez Lopez et al, 2017), a pivotal program designed to assess efficacy and safety in an as yet cognitively unimpaired population at increased risk for developing clinical symptoms of AD based on their age and APOE4 genotype (NCT02565511 and NCT03131453). We herein describe the structure and the pharmacological, safety, and early clinical profiles of the BACE-1 inhibitor CNP520, with a special focus on the intended use of this compound for the prevention of AD. Results CNP520 structural and functional features are distinct from previous BACE-1 inhibitors We present here the structure of the BACE-1 inhibitor CNP520 for the first time (Fig 1A). It is the result of extensive structural optimization of the 3-amino-1,4-oxazine lead series of BACE-1 inhibitors and a detailed understanding of CNP520's binding to BACE-1 (Fig 1B). Our priorities were to develop a compound (i) with good brain penetration to minimize peripheral exposure and the associated risk of side effects, (ii) with sufficient selectivity over BACE-2 and cathepsins to avoid any clinically relevant interactions with these counter targets, and (iii) without any structural elements in the parent structure or main metabolites that would be mutagenic or genotoxic. Although a detailed description of the drug design process is beyond the scope here, we summarize below the key features of CNP520's structure. Figure 1. Design of CNP520 A. Molecular structure. B. X-ray structure of CNP520 bound to the BACE-1 active site (resolution: 1.35 Å), with binding pockets S1 (filled by P1) and S3 (filled by P3) and catalytic residues Asp32 and Asp228. C. Blood and brain tissue levels of CNP520 in the rat after a 30 μmol/kg (15.4 mg/kg) oral dose. Shown are unbound fraction (main graph) and total levels (insert) of CNP520 in the blood and brain, using values for rat plasma protein binding of 97.8% and unspecific binding to brain homogenate of 99%. Values are mean ± SD, with n = 5 per time point. D. Structure of the aminopyridine generated by metabolic cleavage of the amide bond in CNP520. Download figure Download PowerPoint The amino-oxazine head group mediates the key binding interactions to Asp32 and Asp228 in BACE-1 (Fig 1B, Appendix Fig S1, and Appendix Table S1), but, being the most polar part of the molecule, it also largely determines the permeation and distribution properties of CNP520. In a previous series of BACE-1 inhibitors, we discovered that weakly basic compounds show good passive membrane permeation (Lerchner et al, 2010). Consequently, the basicity of the cyclic amidine of CNP520 was fine-tuned to pKa = 7.2 by introduction of the electron-withdrawing trifluoromethyl group. Furthermore, we speculated that a bulky group at position 2 of the oxazine ring might reduce binding of CNP520 to P-gp, a major drug transporter located at the blood–brain barrier responsible for drug efflux. Net drug transport into the brain is determined by the balance between passive blood vessel membrane permeation and P-pg-mediated outward transport (Meredith et al, 2008). In vitro investigation of CNP520 transport properties in Madin–Darby canine kidney (MDCK) cells expressing human P-gp (Rueeger et al, 2011) proved our hypothesis: The rate for apical-to-basolateral transport of 5.6 × 10−6 cm/s indicated good passive membrane permeation, whereas the P-gp-driven basolateral-to-apical transport rate of 14.3 × 10−6 cm/s was not substantially higher. In agreement with the in vitro data, a distribution study in rats showed comparable unbound CNP520 concentrations in the brain and in blood (Fig 1C), confirming that significant efflux did not occur. Enzyme inhibition assays showed CNP520 to be a potent BACE-1 inhibitor that is selective for BACE-1 over other human pepsin-like aspartic proteases, including BACE-2 and CatD (Table 1). BACE-1 shares high structural similarity with the catalytic and ligand binding sites of these enzymes; however, we increased selectivity for BACE-1 by exploiting the size differences of remote substrate binding pockets, in particular the extended S3 pocket (Schechter & Berger, 1967). We discovered that a picolinic acid with para-trifluoromethyl substitution, pointing into the extended S3 pocket, provided threefold selectivity for BACE-1 over BACE-2 and 20,000-fold selectivity for BACE-1 over CatD, which translated to functional selectivity in vivo (see below). Finally, we identified the central fluoropyridyl moiety as a non-genotoxic building block that could replace earlier aniline-type substructures that generated aniline metabolites upon hydrolysis of the amide bond (Neumann et al, 2015). Thus, in vivo metabolism of CNP520 forms an ortho-aminopyridine-containing metabolite with no known safety flag (Fig 1D) and does not generate aniline with its associated genotoxicity risk (Smith, 2011). Table 1. In vitro potency of CNP520 Enzyme IC50 ± SEM (nM) Human BACE-1 11 ± 0.4 Mouse BACE-1 10 ± 0.3 Human BACE-2 30 ± 1.0 Human cathepsin D 205,000 ± 28,200 Human cathepsin E 66,400 ± 13,000 Porcine pepsin > 250,000 CHO cells (wild-type APP) 2.8 ± 0.2 CHO cells ("Swedish" APP) 44 ± 0.4 Recombinant catalytic domains were used for the human aspartyl proteases and for mouse BACE-1. IC50 values are means of three individual CNP520 batches, each measured in duplicate. IC50 values for the inhibition of Aβ40 release from APP-transfected CHO cells are means from six different CNP520 batches (each in triplicate, APP-wild-type cells) or three different batches (triplicates, APP-Swedish cells). CNP520 is a moderately lipophilic compound; the logD (partition coefficient octanol/buffer pH 6.8) of 3.5 is in the target range for an orally available and brain-penetrating drug. However, the non-specific binding of CNP520 to plasma proteins is relatively high (ranging from 95.9% in human to 98.9% in mouse); therefore, the unbound concentration of CNP520 in plasma is low, typically below 0.1 μM at pharmacologically active doses in animals and humans (see below). CNP520 has a benign non-clinical safety profile During our evaluation of the non-clinical safety of CNP520, we paid special attention to the physiological effects that have been ascribed to BACE-1 knockout (KO) mice, including demyelination/thinning of myelin sheets, muscle spindle and proprioception changes due to decreased processing of neuregulin-1, and retina atrophy via impairment of vascular endothelial growth factor-1 cleavage-induced choroidal neovascularization (Willem et al, 2006; Cai et al, 2012; Cheret et al, 2013). None of the effects listed above were observed in toxicology studies with CNP520. In long-term toxicology studies, dose-limiting non-specific central nervous system (CNS) effects (including impaired mobility and tremor) occurred in dogs at ≥ 30 mg/kg/day and in male rats (adverse effects only observed at doses ≥ 500 mg/kg/day). In female rats, we found delayed ovulation and slightly reduced fecundity and fertility indices (74 versus 90% in control animals) at ≥ 30 mg/kg/day, and focal skeletal muscle atrophy in gastrocnemius and quadriceps muscles without functional effects at 200 mg/kg/day. After CNP520 withdrawal, all changes either trended toward being, or were, fully reversible. The organs that were affected by drug toxicity in these animal studies either were not relevant to the clinical target population or the toxicities occurred at a dose substantially higher than the efficacious clinical dose. No morphological changes were identified in the CNS (brain, spinal cord, and central nerves), peripheral nervous system (peripheral nerves, spindle cells, and neuromuscular junctions), liver, endocrine pancreas, retina, or the skin of any species. We investigated the safety pharmacology profile of CNP520 in vitro and in vivo. In our assessment of CNP520 cardiovascular safety, we paid special attention to the human Ether-à-go-go-Related Gene (hERG) potassium channel in the heart, as it is an important potential off-target for aromatic compounds with a basic/amphiphilic structure (Kalyaanamoorthy & Barakat, 2017). Due to their basic cyclic amidine, combined with a rather hydrophobic P3 substituent, BACE-1 inhibitors are potential hERG ligands. In vitro, CNP520 inhibited hERG with an IC50 of 3.2 μM, 67-fold above the free Cmax of the highest clinical dose tested in a 3-month human study (0.048 μM at 85 mg). We observed no effect on heart rate, ECG morphology, or blood pressure in dogs at doses up to 200 mg/kg (free plasma Cmax = 0.23 μM) or in the repeat dose toxicology studies (2, 4, 13, and 39 weeks' duration). To further delineate the safety pharmacology profile of CNP520, we investigated its affinity for a panel of neurotransmitter receptors associated with drug abuse or dependences. Although a number of the receptors had binding affinities in the low micromolar range, the CNP520 concentrations used in these experiments were higher than the free drug concentration obtained in human plasma at the clinically relevant dose of 85 mg/day (Appendix Table S2). Thus, the safety margins are robust, and the potential for functional interactions between CNP520 and all of the receptors tested is low. In agreement with these results, neither rats nor dogs displayed clinical signs indicative of drug withdrawal (i.e., rearing, or altered locomotor activity, body temperature, food consumption, or body weight) after cessation of repeated dosing. Therefore, no potential for drug dependencies was identified from in vitro or in vivo toxicological studies. CNP520 has no relevant side effects due to inhibition of BACE-2 or cathepsin D CNP520 is highly BACE-1 specific, with the exception of BACE-2 (Table 1 and Appendix Table S2). To investigate whether CNP520 has any effects on melanin synthesis and hair pigmentation, we dosed C57/BL6 mice with CNP520 for 8 weeks; NB-360, which inhibits BACE-1 and BACE-2 equipotently and leads to hair depigmentation in mice because of reduced PMEL-17 processing (Shimshek et al, 2016), was used as positive control. No change in hair color was observed with CNP520 at doses that provided more than 90% Aβ40 reduction in the brain (Fig 2A). NB-360 in contrast caused obvious hair depigmentation in all mice, starting at 2–3 weeks. Neither did we observe any changes in other pigmented organs (hair follicle, choroid, and retina pigmented epithelium evaluated by histopathology). Figure 2. CNP520 treatment of male C57/BL6 mice for 8 weeks did not cause hair depigmentation A. Subjective assessment of hair depigmentation (score 0–5, with 0 = completely black and 5 = completely white) at two doses of either CNP520 or the BACE-1/-2 inhibitor NB-360 (Shimshek et al, 2016; mean ± SEM, n = 8/group), statistical comparisons were made for every scoring day, versus vehicle using Kruskal–Wallis with Dunn's post hoc test. B. Summary of doses, steady-state effects on Aβ40 in brain (% compared to vehicle-treated mice) and the ratios between total skin concentrations of CNP520 and NB-360 and their respective IC50 values for inhibition of BACE-2 in vitro. Download figure Download PowerPoint The complete absence of hair depigmentation in the CNP520 group was surprising because the threefold selectivity for BACE-1 over BACE-2 was not expected to be sufficient to cause such a striking difference. We speculated that both the concentration of CNP520 or NB-360 in the tissue in combination with their respective potencies for BACE-2 would also play a role in determining the level of inhibition of BACE-2 and thus whether depigmentation would occur. Therefore, we determined CNP520 and NB-360 total concentrations in the skin of tre
Immunization against Aβ has been shown to reduce amyloid accumulation and associated pathology in APP transgenic mouse models. Clinical trials with a vaccine comprising aggregated Aβ1–42 and the adjuvant QS21 were, however, stopped in phase 2 due to development of aseptic meningoencephalitis in 6% of the treated patients. Autopsy studies of two affected patients demonstrated a T–cell mediated autoimmune response presumably directed against Aβ. To avoid Aβ–specific T–cell responses we developed CAD106, an immunotherapeutic vaccine comprising the Aβ1–6 peptide covalently coupled to the Qβ virus–like particle (VLP). This N–terminal Aβ peptide is shorter than T–cell epitopes, is predicted not to induce T–cell responses and was shown to contain B– but not T–cell epitopes. T–cell help is provided by Qβ. The ordered and repetitive display of Aβ1–6 on the Qβ VLPs renders the peptide highly immunogenic, even in the absence of added adjuvant. CAD106 generated high Aβ antibody titers in mice, rabbits and monkeys. A 100% responder rate was observed in both young and old animals. CAD106–induced Aβ antibodies recognized the N–terminus of Aβ and selectively stained amyloid deposits in human and APP transgenic mouse brain sections. They also protected against toxicity of oligomeric or fibrillar Aβ in vitro. Immunization with CAD106 reproducibly prevented amyloid plaque accumulation in the brains of two different APP transgenic mouse models (APP24 and APP23) which develop mostly diffuse (Congo–Red negative) or mostly compact (Congo–Red positive) amyloid deposits, respectively. Furthermore, immunization of aged mice, showing advanced amyloid pathology already present in the brain, resulted in a reduction of further amyloid plaque accumulation. Neither adverse immune reactions nor increased incidence of microhemorrhages were observed in these studies. Importantly, immunization of different mouse strains with CAD106 did not induce Aβ–responsive T–cells even in the presence of additional adjuvant. The expected strong T–cell response to Qβ was however found. In contrast, Aβ1–42 immunization gave rise to Aβ–responsive T–cells. They could be re–stimulated in vitro with Aβ1–40/2 and Aβ6–20 but not with Aβ1–6, confirming the lack of a T–cell epitope on this peptide. CAD106 is currently being tested in a phase 1 study in humans.
Abstract To obtain stable derivatives of α‐unsubstituted pyrroles, the reaction of the test pyrrole 9 with a series of chalcones 14a – h were studied. Michael adducts 16b – h could be isolated. In order to synthesize coloured derivatives, the reaction of different pyrroles 9, 21, 23 , and 25 with diphenylpropynone 19 was investigated. In these cases, too, Michael ‐addition products were formed. The intense absorption band around 400 nm makes the identification of these derivatives easy.
We have developed inhibitors of glutathione reductase that improve on the inhibition of literature lead compounds by up to three orders of magnitude. Thus, analogues of Safranine O and menadione were found to be strong, reversible inhibitors of yeast glutathione reductase. Safranine O exhibited partial, uncompetitive inhibition with Ki and alpha values of 0.5 mM and 0.15, respectively. Thionine O was a partial (hyperbolic) uncompetitive inhibitor with Ki and alpha values of 0.4 microM and 0.15, respectively. LY83583 and 2-anilino-1,4-naphthoquinone also showed (hyperbolic) partial, uncompetitive inhibition with micromolar Ki values. For Nile Blue A a model for two-site binding with (parabolic) uncompetitive inhibition fitted the data with a Ki value of 11 microM and a kinetic cooperativity between the sites of 0.12, increased to 0.46 by preincubation of the enzyme and Nile Blue A in the presence of glutathione disulphide. Analysis of the effects of preincubation on the kinetics and cooperativity indicated the possibility of a slow conformational change in the homodimeric enzyme, the first such indication of kinetic cooperativity in the native enzyme to our knowledge. Further evidence of conformational changes for this enzyme came from studies of the effects of dimethyl sulphoxide which indicated that this co-solvent, which at low concentrations has no apparent effect on initial velocities under normal assay conditions, induced a slow conformational change in the enzyme. Thionine O, Nile Blue A and LY83583 were redox-cycling substrates producing superoxide ion, detectable by means of cytochrome c reduction, but leading to no loss of glutathione reductase activity, under aerobic or anaerobic conditions. The water-soluble Safranine analogues Methylene Blue, Methylene Green, Nile Blue A and Thionine O (5 mg/kg i.p. x 5) were effective antimalarial agents in vivo against P. berghei, but their effect was small and a higher dose (50 mg/kg i.p. x 1) was toxic in mice. Comparison was made with human glutathione reductase and its literature-reported interactions with several tricyclic inhibitors as studied by X-ray diffraction. It is possible that the conformational changes detected in the present study from alterations in detailed kinetic inhibition mechanisms may shed light on information transfer through the glutathione reductase molecule from the dimer interface ligand pocket to the active-site.
Abstract A synthetic route leading to bis‐heteroleptic cyclometalated complexes magnified image is described. The complexes [2‐(2′‐thienyl)pyridinato‐ N , C ‐ 3′ ]{2‐[3′‐(trimethylsilyl)2′‐thienyl]pyridinato‐ N, C 3′ }platinum(II) ([Pt(thpy) (TMS‐thpy)]; I ) and (l‐phenylpyrazolato‐ N 2 , C 2′ )[2‐(2′‐thienyl)pyridinato‐ N , C 3′ ]platinum ([Pt(Phpz)(thpy)]; II ) are characterized by UV/VIS, NMR, and mass spectroscopy. Thermal and photochemical oxidative addition reactions yield two out of the 10 possible pairs of enantiomers of octahedral Pt(IV) compounds.
Immunization against amyloid-β (Aβ) can reduce amyloid accumulation in vivo and is considered a potential therapeutic approach for Alzheimer9s disease. However, it has been associated with meningoencephalitis thought to be mediated by inflammatory T-cells. With the aim of producing an immunogenic vaccine without this side effect, we designed CAD106 comprising Aβ1–6 coupled to the virus-like particle Qβ. Immunization with this vaccine did not activate Aβ-specific T-cells. In APP transgenic mice, CAD106 induced efficacious Aβ antibody titers of different IgG subclasses mainly recognizing the Aβ3–6 epitope. CAD106 reduced brain amyloid accumulation in two APP transgenic mouse lines. Plaque number was a more sensitive readout than plaque area, followed by Aβ42 and Aβ40 levels. Studies with very strong overall amyloid reduction showed an increase in vascular Aβ, which atypically was nonfibrillar. The efficacy of Aβ immunotherapy depended on the Aβ levels and thus differed between animal models, brain regions, and stage of amyloid deposition. Therefore, animal studies may not quantitatively predict the effect in human Alzheimer9s disease. Our studies provided no evidence for increased microhemorrhages or inflammatory reactions in amyloid-containing brain. In rhesus monkeys, CAD106 induced a similar antibody response as in mice. The antibodies stained amyloid deposits on tissue sections of mouse and human brain but did not label cellular structures containing APP. They reacted with Aβ monomers and oligomers and blocked Aβ toxicity in cell culture. We conclude that CAD106 immunization is suited to interfere with Aβ aggregation and its downstream detrimental effects.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)