Combining an amyloid-beta (Aß) cleaving enzyme inhibitor with a γ-secretase modulator results in an additive reduction of Aß production.

A major hallmark of Alzheimer's disease (AD) is the deposition of amyloid-ß (Aß) peptides in amyloid plaques. Aß peptides are produced by sequential cleavage of the amyloid precursor protein by the ß amyloid cleaving enzyme (BACE) and the γ-secretase (γ-sec) complex. Pharmacological treatments that decrease brain levels of in particular the toxic Aß42 peptide are thought to be promising approaches for AD disease modification. Potent and selective BACE1 inhibitors as well as γ-sec modulators (GSMs) have been designed. Pharmacological intervention of secretase function is not without risks of either on- or off-target adverse effects. One way of improving the therapeutic window could be to combine treatment on multiple targets, using smaller individual doses and thereby minimizing adverse effect liability. We show that combined treatment of primary cortical neurons with a BACE1 inhibitor and a GSM gives an additive effect on Aß42 level change compared with the individual treatments. We extend this finding to C57BL/6 mice, where the combined treatment results in reduction of brain Aß42 levels reflecting the sum of the individual treatment efficacies. These results show that pharmacological targeting of two amyloid precursor protein processing steps is feasible without negatively interfering with the mechanism of action on individual targets. We conclude that targeting Aß production by combining a BACE inhibitor and a GSM could be a viable approach for therapeutic intervention in AD modification.

Alzheimer's disease: presenilin 2-sparing γ-secretase inhibition is a tolerable Aß peptide-lowering strategy.

γ-Secretase inhibition represents a major therapeutic strategy for lowering amyloid ß (Aß) peptide production in Alzheimer's disease (AD). Progress toward clinical use of γ-secretase inhibitors has, however, been hampered due to mechanism-based adverse events, primarily related to impairment of Notch signaling. The γ-secretase inhibitor MRK-560 represents an exception as it is largely tolerable in vivo despite displaying only a small selectivity between Aß production and Notch signaling in vitro. In exploring the molecular basis for the observed tolerability, we show that MRK-560 displays a strong preference for the presenilin 1 (PS1) over PS2 subclass of γ-secretases and is tolerable in wild-type mice but causes dose-dependent Notch-related side effect in PS2-deficient mice at drug exposure levels resulting in a substantial decrease in brain Aß levels. This demonstrates that PS2 plays an important role in mediating essential Notch signaling in several peripheral organs during pharmacological inhibition of PS1 and provide preclinical in vivo proof of concept for PS2-sparing inhibition as a novel, tolerable and efficacious γ-secretase targeting strategy for AD.

Second generation γ-secretase modulators exhibit different modulation of Notch ß and Aß production.

The γ-secretase complex is an appealing drug target when the therapeutic strategy is to alter amyloid-ß peptide (Aß) aggregation in Alzheimer disease. γ-Secretase is directly involved in Aß formation and determines the pathogenic potential of Aß by generating the aggregation-prone Aß42 peptide. Because γ-secretase mediates cleavage of many substrates involved in cell signaling, such as the Notch receptor, it is crucial to sustain these pathways while altering the Aß secretion. A way of avoiding interference with the physiological function of γ-secretase is to use γ-secretase modulators (GSMs) instead of inhibitors of the enzyme. GSMs modify the Aß formation from producing the amyloid-prone Aß42 variant to shorter and less amyloidogenic Aß species. The modes of action of GSMs are not fully understood, and even though the pharmacology of GSMs has been thoroughly studied regarding Aß generation, knowledge is lacking about their effects on other substrates, such as Notch. Here, using immunoprecipitation followed by MALDI-TOF MS analysis, we found that two novel, second generation GSMs modulate both Notch ß and Aß production. Moreover, by correlating S3-specific Val-1744 cleavage of Notch intracellular domain (Notch intracellular domain) to total Notch intracellular domain levels using immunocytochemistry, we also demonstrated that Notch intracellular domain is not modulated by the compounds. Interestingly, two well characterized, nonsteroidal anti-inflammatory drugs (nonsteroidal anti-inflammatory drug), R-flurbiprofen and sulindac sulfide, affect only Aß and not Notch ß formation, indicating that second generation GSMs and nonsteroidal anti-inflammatory drug-based GSMs have different modes of action regarding Notch processing.

First and second generation γ-secretase modulators (GSMs) modulate amyloid-ß (Aß) peptide production through different mechanisms.

γ-Secretase-mediated cleavage of amyloid precursor protein (APP) results in the production of Alzheimer disease-related amyloid-ß (Aß) peptides. The Aß42 peptide in particular plays a pivotal role in Alzheimer disease pathogenesis and represents a major drug target. Several γ-secretase modulators (GSMs), such as the nonsteroidal anti-inflammatory drugs (R)-flurbiprofen and sulindac sulfide, have been suggested to modulate the Alzheimer-related Aß production by targeting the APP. Here, we describe novel GSMs that are selective for Aß modulation and do not impair processing of Notch, EphB2, or EphA4. The GSMs modulate Aß both in cell and cell-free systems as well as lower amyloidogenic Aß42 levels in the mouse brain. Both radioligand binding and cellular cross-competition experiments reveal a competitive relationship between the AstraZeneca (AZ) GSMs and the established second generation GSM, E2012, but a noncompetitive interaction between AZ GSMs and the first generation GSMs (R)-flurbiprofen and sulindac sulfide. The binding of a (3)H-labeled AZ GSM analog does not co-localize with APP but overlaps anatomically with a γ-secretase targeting inhibitor in rodent brains. Combined, these data provide compelling evidence of a growing class of in vivo active GSMs, which are selective for Aß modulation and have a different mechanism of action compared with the original class of GSMs described.

Ziram causes dopaminergic cell damage by inhibiting E1 ligase of the proteasome.

The etiology of Parkinson disease (PD) is unclear but may involve environmental toxins such as pesticides leading to dysfunction of the ubiquitin proteasome system (UPS). Here, we measured the relative toxicity of ziram (a UPS inhibitor) and analogs to dopaminergic neurons and examined the mechanism of cell death. UPS (26 S) activity was measured in cell lines after exposure to ziram and related compounds. Dimethyl- and diethyldithiocarbamates including ziram were potent UPS inhibitors. Primary ventral mesencephalic cultures were exposed to ziram, and cell toxicity was assessed by staining for tyrosine hydroxylase (TH) and NeuN antigen. Ziram caused a preferential damage to TH+ neurons and elevated alpha-synuclein levels but did not increase aggregate formation. Mechanistically, ziram altered UPS function through interfering with the targeting of substrates by inhibiting ubiquitin E1 ligase. Sodium dimethyldithiocarbamate administered to mice for 2 weeks resulted in persistent motor deficits and a mild reduction in striatal TH staining but no nigral cell loss. These results demonstrate that ziram causes selective dopaminergic cell damage in vitro by inhibiting an important degradative pathway implicated in the etiology of PD. Chronic exposure to widely used dithiocarbamate fungicides may contribute to the development of PD, and elucidation of its mechanism would identify a new potential therapeutic target.

Monoacylglycerol lipase inhibition by organophosphorus compounds leads to elevation of brain 2-arachidonoylglycerol and the associated hypomotility in mice.

Three components of the cannabinoid system are sensitive to selected organophosphorus (OP) compounds: monoacylglycerol (MAG) lipase that hydrolyzes the major endogenous agonist 2-arachidonoylglycerol (2-AG); fatty acid amide hydrolase (FAAH) that cleaves the agonist anandamide present in smaller amounts; the CB1 receptor itself. This investigation considers which component of the cannabinoid system is the most likely contributor to OP-induced hypomotility in mice. Structure-activity studies by our laboratory and others rule against major involvement of a direct toxicant-CB1 receptor interaction for selected OPs. Attention was therefore focused on the OP sensitivities of MAG lipase and FAAH, assaying 19 structurally diverse OP chemicals (pesticides, their metabolites and designer compounds) for in vitro inhibition of both enzymes. Remarkably high potency and low selectivity is observed with three O-alkyl (C1, C2, C3) alkylphosphonofluoridates (C8, C12) (IC50 0.60-3.0 nM), five S-alkyl (C5, C7, C9) and alkyl (C10, C12) benzodioxaphosphorin oxides (IC50 0.15-5.7 nM) and one OP insecticide metabolite (chlorpyrifos oxon, IC50 34-40 nM). In ip-treated mice, the OPs at 1-30 mg/kg more potently inhibit brain FAAH than MAG lipase, but FAAH inhibition is not correlated with hypomotility. However, the alkylphosphonofluoridate-treated mice show dose-dependent increases in severity of hypomotility, inhibition of MAG lipase activity and elevation of 2-AG. Moderate to severe hypomotility is accompanied by 64 to 86% MAG lipase inhibition and about 6-fold elevation of brain 2-AG level. It therefore appears that OP-induced MAG lipase inhibition leads to elevated 2-AG and the associated hypomotility.

Blood acylpeptide hydrolase activity is a sensitive marker for exposure to some organophosphate toxicants.

Acylpeptide hydrolase (APH) unblocks N-acetyl peptides. It is a major serine hydrolase in rat blood, brain, and liver detected by derivatization with (3)H-diisopropyl fluorophosphate (DFP) or a biotinylated fluorophosphonate. Although APH does not appear to be a primary target of acute poisoning by organophosphorus (OP) compounds, the inhibitor specificity of this secondary target is largely unknown. This study fills the gap and emphasizes blood APH as a potential marker of OP exposure. The most potent in vitro inhibitors for human erythrocyte and mouse brain APH are DFP (IC(50) 11-17 nM), chlorpyrifos oxon (IC(50) 21-71 nM), dichlorvos (IC(50) 230-560 nM), naled (IC(50) 370-870 nM), and their analogs with modified alkyl substituents. (3)H-diisopropyl fluorophosphate is a potent inhibitor of mouse blood and brain APH in vivo (ED(50) 0.09-0.2 mg/kg and 0.02-0.03 mg/l for ip and vapor exposure, respectively). Mouse blood and brain APH and blood butyrylcholinesterase (BChE) are of similar sensitivity to DFP in vitro and in vivo (ip and vapor exposure), but APH inhibition is much more persistent in vivo (still >80% inhibition after 4 days). The inhibitory potency of OP pesticides in vivo in mice varies from APH selective (dichlorvos, naled, and trichlorfon), to APH and BChE selective (profenofos and tribufos), to ChE selective or nonselective (many commercial insecticides). Sarin administered ip at a lethal dose to guinea pigs inhibits blood acetylcholinesterase and BChE completely but erythrocyte APH only partially. Blood APH activity is therefore a sensitive marker for exposure to some but not all OP pesticides and chemical warfare agents.

Platelet-activating factor acetylhydrolase: selective inhibition by potent n-alkyl methylphosphonofluoridates.

Platelet-activating factor (PAF) is a potent endogenous phospholipid modulator of diverse biological activities, including inflammation and shock. PAF levels are primarily regulated by PAF acetylhydrolases (PAF-AHs). These enzymes are candidate secondary targets of organophosphorus (OP) pesticides and related toxicants. Previously known OP inhibitors of other serine hydrolases were tested with PAF-AH from mouse brain and testes of established functional importance compared with the structurally different human plasma enzyme. Several key OP pesticides and their oxon metabolites were very poor inhibitors of mouse brain and human plasma PAF-AH in vitro but moderately active for mouse brain and blood PAF-AH in vivo (e.g., tribufos defoliant and profenofos insecticide, presumably following oxidative bioactivation). OP compounds were then designed for maximum in vitro potency and selectivity for mouse brain PAF-AH vs. acetylcholinesterase (AChE). Lead compounds were found in a series of benzodioxaphosphorin 2-oxides. Ultrahigh potency and selectivity were achieved with n-alkyl methylphosphonofluoridates (long-chain sarin analogs): mouse brain and testes IC50 < or = 5 nM for C(8)-C(18) analogs and 0.1-0.6 nM for C(13) and C(14) compounds; human plasma IC50 < or = 2 nM for C(13)-C(18) analogs. AChE inhibitory potency decreased as chain length increased with maximum brain PAF-AH/AChE selectivity (>3000-fold) for C(13)-C(18) compounds. The toxicity of i.p.-administered PAF (LD50 ca. 0.5 mg/kg) was increased less than 2-fold by pretreatment with tribufos or the C(13)n-alkyl methylphosphonofluoridate. These studies with a mouse model indicate that PAF-AH is not a major secondary target of OP pesticide poisoning. The optimized PAF-AH inhibitors may facilitate investigations on other aspects of PAF metabolism and action.

Altered extracellular striatal in vivo biotransformation of the opioid neuropeptide dynorphin A(1-17) in the unilateral 6-OHDA rat model of Parkinson's disease.

The in vivo biotransformation of dynorphin A(1-17) (Dyn A) was studied in the striatum of hemiparkinsonian rats by using microdialysis in combination with nanoflow reversed-phase liquid chromatography/electrospray time-of-flight mass spectrometry. The microdialysis probes were implanted into both hemispheres of unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats. Dyn A (10 pmol microl(-1)) was infused through the probes at 0.4 microl min(-1) for 2 h. Samples were collected every 30 min and analyzed by mass spectrometry. The results showed for the first time that there was a difference in the Dyn A biotransformation when comparing the two corresponding sides of the brain. Dyn A metabolites 1-8, 1-16, 5-17, 10-17, 7-10 and 8-10 were detected in the dopamine-depleted striatum but not in the untreated striatum. Dyn A biotransformed fragments found in both hemispheres were N-terminal fragments 1-4, 1-5, 1-6, 1-11, 1-12 and 1-13, C-terminal fragments 2-17, 3-17, 4-17, 7-17 and 8-17 and internal fragments 2-5, 2-10, 2-11, 2-12, and 8-15. The relative levels of these fragments were lower in the dopamine-depleted striatum. The results imply that the extracellular in vivo processing of the dynorphin system is being disturbed in the 6-OHDA-lesion animal model of Parkinson's disease.

Motor effects of a dopamine stabilizer (GMC1111) in primate models of Parkinson and hemiparkinsonism.

The effects on motor behavior of a new potential dopamine stabilizer: 2-amino-6-(N,N-di-n-propylamino)thiazolo[4,5-f]indan (GMC1111) were investigated in common marmosets with 6-hydroxydopamine lesions within the median forebrain bundle (12 unilateral, 6 bilateral). GMC1111 was administered orally or subcutaneously (s.c.) to unilaterally 6-hydroxydopamine lesioned monkeys, either alone or together with s.c. injections of apomorphine (0.2 mg/kg) and the effect on rotational behavior was examined. GMC1111 (0.03-3.0 mg/kg) alone, orally or s.c., did not induce rotational behavior. When apomorphine and GMC1111 were injected simultaneously, rotations were nearly abolished in three monkeys with a baseline apomorphine-induced rotation rate below 13/min, whereas GMC1111 did not modify the rotations in three high-rotating animals (>17/min). Oral administration of GMC1111 (1.0 and 3.0 mg/kg) abolished the apomorphine-induced rotations in another six unilaterally dopamine-denervated monkeys, indicating a good oral bioavailability. A low dose of GMC1111 (0.3 mg/kg) administered s.c. to marmosets with bilateral nigrostriatal lesions produced a reduction of Parkinson symptoms of approximately the same degree as with levodopa/benserazide (15/3.75 mg/kg), while higher doses of GMC1111 were less effective. When levodopa/benserazide was administered together with various doses of GMC1111 (0.3-3.0 mg/kg), the levodopa-induced peak-dose dyskinesias were reduced with the highest dose of GMC1111 (3 mg/kg). Taken together, GMC1111 modifies dopaminergic activity in a normalizing direction. Parkinson symptoms, as well as levodopa-induced dyskinesias are both reduced. This suggests the arrival of another member of the new dopamine stabilizer family.

Tardive dyskinesia model in the common marmoset.

Thirteen adult common marmosets (Callithrix jacchus) were given once-monthly injections of haloperidol decanoate (5-15 mg/kg i.m.) for one year. Thereafter, drug-free and treatment periods alternated at 3-month intervals. After 2.5 to 14 months, 12 monkeys showed symptoms of tardive dyskinesia (TD), such as periocular and perioral twitchings, tongue protrusions, masticatory movements, and choreic movements in arms and legs. When TD symptoms were evident, the periodic treatment was interrupted and symptoms persisted for at least 5 months after the last haloperidol dose, worsened by injection of the anticholinergic drug biperiden. An injection of nondepot haloperidol (0.12 or 0.25 mg/kg) produced a reduction of TD symptoms. At the end of the study, nondepot haloperidol was injected once a week at two doses (0.12 and 0.25 mg/kg i.m.). A syndrome of excitation with peculiar behavior, interpreted as acute dystonia, was precipitated in all animals. The animals showed sustained retrocollis, climbing upside down, biting the perch, repetitive turnings, and frequent backward movements. The dystonic movements lasted approximately 6 hours and were reduced but not completely extinguished by biperiden (0.1 mg/kg). The TD syndrome registered in marmosets may provide a useful model for screening new antipsychotics for their propensity to induce TD.