Potent and Selective BACE-1 Peptide Inhibitors Lower Brain Aß Levels Mediated by Brain Shuttle Transport.

Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidß (Aß) antibodies and secretase inhibitors. However, the blood-brain barrier (BBB) limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS) technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aß levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aß. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance.

Sembragiline: A Novel, Selective Monoamine Oxidase Type B Inhibitor for the Treatment of Alzheimer's Disease.

Monoamine oxidase B (MAO-B) has been implicated in the pathogenesis of Alzheimer's disease (AD) and other neurodegenerative disorders. Increased MAO-B expression in astroglia has been observed adjacent to amyloid plaques in AD patient brains. This phenomenon is hypothesized to lead to increased production of hydrogen peroxide and reactive oxygen species (ROS), thereby contributing to AD pathology. Therefore, reduction of ROS-induced oxidative stress via inhibition of MAO-B activity may delay the progression of the disease. In the present study we report the pharmacological properties of sembragiline, a novel selective MAO-B inhibitor specifically developed for the treatment of AD, and on its effect on ROS-mediated neuronal injury and astrogliosis in MAO-B transgenic animals. Sembragiline showed potent and long-lasting MAO-B-selective inhibition and did not inhibit MAO-A at doses where full inhibition of MAO-B was observed. Such selectivity should translate into a favorable clinical safety profile. Indeed, sembragiline neither induced the serotonin syndrome when administered together with the serotonin precursor l-5-hydroxytryptophan in combination with antidepressants such as fluoxetine, nor potentiated the pressor effect of tyramine. Additionally, in experiments using a transgenic animal model conditionally overexpressing MAO-B in astroglia, sembragiline protected against neuronal loss and reduced both ROS formation and reactive astrogliosis. Taken together, these findings warrant further investigation of the potential therapeutic benefit of MAO-B inhibitors in patients with AD and other neurologic disorders.

Region-specific permeability of the blood-brain barrier upon pericyte loss.

The blood-brain barrier (BBB) regulates differing needs of the various brain regions by controlling transport of blood-borne components from the neurovascular circulation into the brain parenchyma. The mechanisms underlying region-specific transport across the BBB are not completely understood. Previous work showed that pericytes are key regulators of BBB function. Here we investigated whether pericytes influence BBB permeability in a region-specific manner by analysing the regional permeability of the BBB in the pdgf-b ret/ret mouse model of pericyte depletion. We show that BBB permeability is heterogeneous in pdgf-b ret/ret mice, being significantly higher in the cortex, striatum and hippocampus compared to the interbrain and midbrain. However, we show that this regional heterogeneity in BBB permeability is not explained by local differences in pericyte coverage. Region-specific differences in permeability were not associated with disruption of tight junctions but may result from changes in transcytosis across brain endothelial cells. Our data show that certain brain regions are able to maintain low BBB permeability despite substantial pericyte loss and suggest that additional, locally-acting mechanisms may contribute to control of transport.

Trafficking of Endogenous Immunoglobulins by Endothelial Cells at the Blood-Brain Barrier.

The Blood-Brain Barrier (BBB) restricts access of large molecules to the brain. The low endocytic activity of brain endothelial cells (BECs) is believed to limit delivery of immunoglobulins (IgG) to the brain parenchyma. Here, we report that endogenous mouse IgG are localized within intracellular vesicles at steady state in BECs in vivo. Using high-resolution quantitative microscopy, we found a fraction of endocytosed IgG in lysosomes. We observed that loss of pericytes (key components of the BBB) in pdgf-b(ret/ret) mice affects the intracellular distribution of endogenous mouse IgG in BECs. In these mice, endogenous IgG was not detected within lysosomes but instead accumulate at the basement membrane and brain parenchyma. Such IgG accumulation could be due to reduced lysosomal clearance and increased sorting to the abluminal membrane of BECs. Our results suggest that, in addition to low uptake from circulation, IgG lysosomal degradation may be a downstream mechanism by which BECs further restrict IgG access to the brain.

Cargo Delivery into the Brain by in vivo identified Transport Peptides.

The blood-brain barrier and the blood-cerebrospinal fluid barrier prevent access of biotherapeutics to their targets in the central nervous system and therefore prohibit the effective treatment of neurological disorders. In an attempt to discover novel brain transport vectors in vivo, we injected a T7 phage peptide library and continuously collected blood and cerebrospinal fluid (CSF) using a cisterna magna cannulated conscious rat model. Specific phage clones were highly enriched in the CSF after four rounds of selection. Validation of individual peptide candidates showed CSF enrichments of greater than 1000-fold. The biological activity of peptide-mediated delivery to the brain was confirmed using a BACE1 peptide inhibitor linked to an identified novel transport peptide which led to a 40% reduction of Amyloid-ß in CSF. These results indicate that the peptides identified by the in vivo phage selection approach could be useful transporters for systemically administrated large molecules into the brain with therapeutic benefits.

Increased brain penetration and potency of a therapeutic antibody using a monovalent molecular shuttle.

Although biotherapeutics have vast potential for treating brain disorders, their use has been limited due to low exposure across the blood-brain barrier (BBB). We report that by manipulating the binding mode of an antibody fragment to the transferrin receptor (TfR), we have developed a Brain Shuttle module, which can be engineered into a standard therapeutic antibody for successful BBB transcytosis. Brain Shuttle version of an anti-Aß antibody, which uses a monovalent binding mode to the TfR, increases ß-Amyloid target engagement in a mouse model of Alzheimer's disease by 55-fold compared to the parent antibody. We provide in vitro and in vivo evidence that the monovalent binding mode facilitates transcellular transport, whereas a bivalent binding mode leads to lysosome sorting. Enhanced target engagement of the Brain Shuttle module translates into a significant improvement in amyloid reduction. These findings have major implications for the development of biologics-based treatment of brain disorders.

Gantenerumab: a novel human anti-Aß antibody demonstrates sustained cerebral amyloid-ß binding and elicits cell-mediated removal of human amyloid-ß.

The amyloid-ß lowering capacity of anti-Aß antibodies has been demonstrated in transgenic models of Alzheimer's disease (AD) and in AD patients. While the mechanism of immunotherapeutic amyloid-ß removal is controversial, antibody-mediated sequestration of peripheral Aß versus microglial phagocytic activity and disassembly of cerebral amyloid (or a combination thereof) has been proposed. For successful Aß immunotherapy, we hypothesized that high affinity antibody binding to amyloid-ß plaques and recruitment of brain effector cells is required for most efficient amyloid clearance. Here we report the generation of a novel fully human anti-Aß antibody, gantenerumab, optimized in vitro for binding with sub-nanomolar affinity to a conformational epitope expressed on amyloid-ß fibrils using HuCAL(®) phage display technologies. In peptide maps, both N-terminal and central portions of Aß were recognized by gantenerumab. Remarkably, a novel orientation of N-terminal Aß bound to the complementarity determining regions was identified by x-ray analysis of a gantenerumab Fab-Aß(1-11) complex. In functional assays gantenerumab induced cellular phagocytosis of human amyloid-ß deposits in AD brain slices when co-cultured with primary human macrophages and neutralized oligomeric Aß42-mediated inhibitory effects on long-term potentiation in rat brain. In APP751(swedish)xPS2(N141I) transgenic mice, gantenerumab showed sustained binding to cerebral amyloid-ß and, upon chronic treatment, significantly reduced small amyloid-ß plaques by recruiting microglia and prevented new plaque formation. Unlike other Aß antibodies, gantenerumab did not alter plasma Aß suggesting undisturbed systemic clearance of soluble Aß. These studies demonstrated that gantenerumab preferentially interacts with aggregated Aß in the brain and lowers amyloid-ß by eliciting effector cell-mediated clearance.

Mechanism of amyloid removal in patients with Alzheimer disease treated with gantenerumab.

BACKGROUND: Gantenerumab is a fully human anti-Aß monoclonal antibody in clinical development for the treatment of Alzheimer disease (AD). OBJECTIVES: To investigate whether treatment with gantenerumab leads to a measurable reduction in the level of Aß amyloid in the brain and to elucidate the mechanism of amyloid reduction. DESIGN: A multicenter, randomized, double-blind, placebo-controlled, ascending-dose positron emission tomographic study. Additionally, ex vivo studies of human brain slices from an independent sample of patients who had AD were performed. SETTING: Three university medical centers. PATIENTS: Patients with mild-to-moderate AD. INTERVENTION: Two consecutive cohorts of patients received 2 to 7 infusions of intravenous gantenerumab (60 or 200 mg) or placebo every 4 weeks. Brain slices from patients who had AD were coincubated with gantenerumab at increasing concentrations and with human microglial cells. MAIN OUTCOME MEASURES: Percent change in the ratio of regional carbon 11-labeled Pittsburgh Compound B retention in vivo and semiquantitative assessment of gantenerumab-induced phagocytosis ex vivo. RESULTS: Sixteen patients with end-of-treatment positron emission tomographic scans were included in the analysis. The mean (95% CI) percent change from baseline difference relative to placebo (n = 4) in cortical brain amyloid level was -15.6% (95% CI, -42.7 to 11.6) for the 60-mg group (n = 6) and -35.7% (95% CI, -63.5 to -7.9) for the 200-mg group (n = 6). Two patients in the 200-mg group showed transient and focal areas of inflammation or vasogenic edema on magnetic resonance imaging scans at sites with the highest level of amyloid reduction. Gantenerumab induced phagocytosis of human amyloid in a dose-dependent manner ex vivo. CONCLUSION: Gantenerumab treatment resulted in a dose-dependent reduction in brain amyloid level, possibly through an effector cell-mediated mechanism of action.

Separation of fast from slow anabolism by site-specific PEGylation of insulin-like growth factor I (IGF-I).

Insulin-like growth factor I (IGF-I) has important anabolic and homeostatic functions in tissues like skeletal muscle, and a decline in circulating levels is linked with catabolic conditions. Whereas IGF-I therapies for musculoskeletal disorders have been postulated, dosing issues and disruptions of the homeostasis have so far precluded clinical application. We have developed a novel IGF-I variant by site-specific addition of polyethylene glycol (PEG) to lysine 68 (PEG-IGF-I). In vitro, this modification decreased the affinity for the IGF-I and insulin receptors, presumably through decreased association rates, and slowed down the association to IGF-I-binding proteins, selectively limiting fast but maintaining sustained anabolic activity. Desirable in vivo effects of PEG-IGF-I included increased half-life and recruitment of IGF-binding proteins, thereby reducing risk of hypoglycemia. PEG-IGF-I was equipotent to IGF-I in ameliorating contraction-induced muscle injury in vivo without affecting muscle metabolism as IGF-I did. The data provide an important step in understanding the differences of IGF-I and insulin receptor contribution to the in vivo activity of IGF-I. In addition, PEG-IGF-I presents an innovative concept for IGF-I therapy in diseases with indicated muscle dysfunction.

In vitro pharmacological selectivity profile of oseltamivir prodrug (Tamiflu) and active metabolite.

Neuropsychiatric adverse events have been reported in influenza patients with and without exposure to oseltamivir (Tamiflu), triggering speculation as to whether oseltamivir may be interacting with any human receptors and contributing to such neuropsychiatric events. In this study, the in vitro selectivity profile of oseltamivir prodrug and active metabolite was investigated. Both compounds lacked clinically relevant pharmacological activities on human, rodent and primate neuraminidases and on a panel of 155 other molecular targets, including those relevant for mood, cognition and behavior. Neuropsychiatric adverse events observed in influenza patients are likely a phenomenon caused by the infection rather than by oseltamivir.

Thermodynamic studies on the interaction of antibodies with beta-amyloid peptide.

Antibodies against beta-amyloid peptides (Abetas) are considered an important therapeutic opportunity in Alzheimer's disease. Despite the vast interest in Abeta no thermodynamic data on the interaction of antibodies with Abeta are available as yet. In the present study we use isothermal titration calorimetry (ITC) and surface plasmon resonance to provide a quantitative thermodynamic analysis of the interaction between soluble monomeric Abeta(1-40) and mouse monoclonal antibodies (mAb). Using four different antibodies directed against the N-terminal, middle, and C-terminal Abeta epitopes, we measured the thermodynamic parameters for the binding to Abeta. Each antibody species was found to have two independent and equal binding sites for Abeta with binding constants in the range of 10(7) to 10(8) M(-1). The binding reaction was essentially enthalpy driven with a reaction enthalpy of DeltaH(0)(Abeta) approximately -19 to -8 kcal/mol, indicating the formation of tight complexes. The loss in conformational freedom was supported by negative values for the reaction entropy DeltaS(0)(Abeta). We also measured the heat capacity change of the 1mAb:2Abeta reaction. DeltaC(0)(p, abeta) was large and negative but could not be explained exclusively by the hydrophobic effect. The free energy of binding was found to be linearly correlated with the size of the epitope.

Disturbed cross talk between insulin-like growth factor I and AMP-activated protein kinase as a possible cause of vascular dysfunction in the amyloid precursor protein/presenilin 2 mouse model of Alzheimer's disease.

Cerebrovascular dysfunction appears to be involved in Alzheimer's disease (AD). In double mutant amyloid precursor protein/presenilin 2 (APP/PS2) mice, a transgenic model of AD, vessel homeostasis is disturbed. These mice have elevated levels of vascular endothelial growth factor (VEGF) and increased brain endothelial cell division but abnormally low brain vessel density. Examination of the potential involvement of insulin-like growth factor I (IGF-I) in these alterations revealed that treatment with IGF-I, a potent vessel growth promoter in the brain that ameliorates cognitive dysfunction in APP/PS2 mice, counteracted vascular dysfunction as follows: VEGF levels and endothelial cell proliferation were reduced, whereas vascular density was normalized. Notably, abnormally elevated brain IGF-I receptor levels in APP/PS2 mice were also normalized by IGF-I treatment. Analysis of possible processes involved in these alterations indicated that AMP-activated protein kinase (AMPK), a cell energy sensor that intervenes in angiogenic signaling and interacts with IGF-I, was also abnormally activated in APP/PS2 brains. Examination of the consequences of AMPK activation on cultured brain endothelial cells revealed increased VEGF levels together with enhanced endothelial cell proliferation and metabolism. Although these effects were also independently elicited by IGF-I, when both IGF-I and AMPK pathways were simultaneously activated on brain endothelial cells, VEGF production and endothelial cell proliferation ceased while cells remained metabolically activated (glucose use, peroxide production, and mitochondrial activity were elevated) and became more resistant to oxidative stress. Therefore, high IGF-I receptor and phosphoAMPK levels in APP/PS2 brains may reflect imbalanced IGF-I and AMPK angiogenic cross talk that could underlie vascular dysfunction in this model of AD.

A 13 kDa carboxy-terminal fragment of ApoE stabilizes Abeta hexamers.

The pathological role of ApoE4 in Alzheimer's disease (AD) is not fully elucidated yet but there is strong evidence that ApoE is involved in Abeta deposition, which is an early hallmark of AD neuropathology. Overexpression of ApoE in neuroblastoma cells (Neuro2a) leads to the generation of an intracellular 13 kDa carboxy-terminal fragment of ApoE comparable to fragments seen in brains of AD patients. ApoE4 generates more of this fragment than ApoE2 and E3 suggesting a potential pathological role of these fragments in Alzheimer's disease. Analysis of this intracellular ApoE4 fragment by protease digest followed by MALDI-TOF mass spectrometry showed the proteolytic cleavage site close to residue 187 of ApoE. We have engineered and expressed the corresponding ApoE fragments in vitro. The recombinant 13 kDa carboxy-terminal fragment inhibited fibril formation of Abeta; this contrasts with the full-length ApoE and the corresponding amino-terminal ApoE fragment. Moreover, we show that the 13 kDa carboxy-terminal fragment of ApoE stabilizes the formation of Abeta hexamers. Complexes of Abeta with the 13 kDa carboxy-terminal ApoE fragment show toxicity in PC12 cells comparable to Abeta fibrils. These data suggest that cleavage of ApoE, leading to the generation of this fragment, contributes to the pathogenic effect of ApoE4 in AD.

Altered metabolic profile in the frontal cortex of PS2APP transgenic mice, monitored throughout their life span.

The transgenic mouse line PS2APP (PS2N141I x APP(swe)) develops an age-related cognitive decline associated with severe amyloidosis, mimicking the pathophysiologic processes in Alzheimer disease (AD). In the quest for biomarkers to monitor, noninvasively, the progression of the disease, we used magnetic resonance imaging and 1H-spectroscopy to characterize PS2APP mice throughout their life span. Morphometric measurements revealed only small size differences to controls. The metabolic profile, however, showed clear indicators of hypometabolism with age in the PS2APP mice: both N-acetyl-aspartate and glutamate were significantly reduced in the older animals. These spectroscopic measures in vivo correlated well with the plaque load in the frontal cortex. A diagnostic test, based on these measures, reached 92% sensitivity and 82% specificity at age 20 months. These noninvasive biomarkers can be exploited in preclinical pharmaceutical research to cope with the high variability in transgenic animal models and to enhance the power of drug efficacy studies.

PS2APP transgenic mice, coexpressing hPS2mut and hAPPswe, show age-related cognitive deficits associated with discrete brain amyloid deposition and inflammation.

Transgenic mice, expressing mutant beta-amyloid precursor proteins (betaAPPs), have lead to a better understanding of the pathophysiological processes in Alzheimer's disease (AD). In many of these models, however, the temporal development of cognitive decline and the relationship to Abeta deposition and inflammation are unclear. We now report a novel transgenic mouse line, PS2APP (PS2N141I x APPswe), which develops a severe cerebral amyloidosis in discrete brain regions, and present a cross-sectional analysis of these mice at 4, 8, 12, and 16 months of age. Each age cohort was investigated for changes in behavior, electrophysiology of synapse efficacy, ELISA-determined Abeta load, histopathology, and in immunoelectron microscopy. Cognitive deficits were first observed at 8 months when Abeta deposits and inflammation were restricted to discrete brain regions, namely the subiculum and frontolateral (motor and orbital) cortex. As early as 5 months, electron microscopy revealed the presence, in these regions, of pre-plaque, immunogold-labeled extracellular fibrillar Abeta. At the same age, increased levels of insoluble Abeta were detected by ELISA, with Abeta1-40 levels exceeding those of Abeta1-42. Further cognitive decline occurred in an age-related manner, and this was accompanied by the spread of amyloidosis to ultimately affect not only neo- and limbic cortices, but also thalamic and pontine nuclei. Dentate gyrus post-tetanic potentiation was significantly attenuated at 17 months, and there were also significant differences in paired-pulse parameters. This systematic cross-sectional study of the behavioral and pathological changes in the PS2APP mouse indicates that it develops age-related cognitive decline associated with severe amyloidosis and inflammation in discrete brain regions and therefore is suitable for testing a range of potential symptomatic and disease-modifying therapies for AD.

Role of caspase-3 activation in cerebral ischemia-induced neurodegeneration in adult and neonatal brain.

These studies have addressed the role of caspase-3 activation in neuronal death after cerebral ischemia in different animal models. The authors were unable to show activation of procaspase-3 measured as an induction of DEVDase (Asp-Glu-Val-Asp) activity after focal or transient forebrain ischemia in rats. DEVDase activity could not be induced in the cytosolic fraction of the brain tissue obtained from these animals by exogenous cytochrome c/dATP and Ca2+. However, the addition of granzyme B to these cytosolic fractions resulted in a significant activation of DEVDase, confirming that the conditions were permissive to analyze proteolytic cleavage of the DEVD-AMC (7-amino-4-methyl-coumarin) substrate. Consistent with these findings, zVal-Ala-Asp-fluoromethylketone administered after focal ischemia did not have a neuroprotective effect. In contrast to these findings, a large increase in DEVDase activity was detected in a model of hypoxic-ischemia in postnatal-day-7 rats. Furthermore, in postnatal-day-7 animals treated with MK-801, in which it has been suggested that excessive apoptosis is induced, the authors were unable to detect activation of DEVDase activity but were able to induce it in vitro by the addition of cytochrome c/dATP and Ca2+ to the cytosolic fraction. Analysis of cytochrome c distribution did not provide definitive evidence for selective cytochrome c release in the permanent focal ischemia model, whereas in the transient model a small but consistent amount of cytochrome c was found in the cytosolic fraction. However, in both models the majority of cytochrome c remained associated with the mitochondrial fraction. In conclusion, the authors were unable to substantiate a role of mitochondrially derived cytochrome c and procaspase-3 activation in ischemia-induced cell death in adult brain, but did see a clear induction of caspase-3 in neonatal hypoxia.