Gino Villetti

Conformation-sensitive Antibodies against Alzheimer Amyloid-β by Immunization with a Thioredoxin-constrained B-cell Epitope Peptide

Immunotherapy against the amyloid-β (Aβ) peptide is a valuable potential treatment for Alzheimer disease (AD). An ideal antigen should be soluble and nontoxic, avoid the C-terminally located T-cell epitope of Aβ, and yet be capable of eliciting antibodies that recognize Aβ fibrils and neurotoxic Aβ oligomers but not the physiological monomeric species of Aβ. We have described here the construction and immunological characterization of a recombinant antigen with these features obtained by tandem multimerization of the immunodominant B-cell epitope peptide Aβ1-15 (Aβ15) within the active site loop of bacterial thioredoxin (Trx). Chimeric Trx(Aβ15)n polypeptides bearing one, four, or eight copies of Aβ15 were constructed and injected into mice in combination with alum, an adjuvant approved for human use. All three polypeptides were found to be immunogenic, yet eliciting antibodies with distinct recognition specificities. The anti-Trx(Aβ15)4 antibody, in particular, recognized Aβ42 fibrils and oligomers but not monomers and exhibited the same kind of conformational selectivity against transthyretin, an amyloidogenic protein unrelated in sequence to Aβ. We have also demonstrated that anti-Trx(Aβ15)4, which binds to human AD plaques, markedly reduces Aβ pathology in transgenic AD mice. The data indicate that a conformational epitope shared by oligomers and fibrils can be mimicked by a thioredoxin-constrained Aβ fragment repeat and identify Trx(Aβ15)4 as a promising new tool for AD immunotherapy.

Unbalanced oxidant-induced DNA damage and repair in COPD: a link towards lung cancer

Background Chronic obstructive pulmonary disease (COPD) is characterised by oxidative stress and increased risk of lung carcinoma. Oxidative stress causes DNA damage which can be repaired by DNA-dependent protein kinase complex. Objectives To investigate DNA damage/repair balance and DNA-dependent protein kinase complex in COPD lung and in an animal model of smoking-induced lung damage and to evaluate the effects of oxidative stress on Ku expression and function in human bronchial epithelial cells. Methods Protein expression was quantified using immunohistochemistry and/or western blotting. DNA damage/repair was measured using colorimetric assays. Results 8-OH-dG, a marker of oxidant-induced DNA damage, was statistically significantly increased in the peripheral lung of smokers (with and without COPD) compared with non-smokers, while the number of apurinic/apyrimidinic (AP) sites (DNA damage and repair) was increased in smokers compared with non-smokers (p=0.0012) and patients with COPD (p<0.0148). Nuclear expression of Ku86, but not of DNA-PKcs, phospho-DNA-PKcs, Ku70 or γ-H2AFX, was reduced in bronchiolar epithelial cells from patients with COPD compared with normal smokers and non-smokers (p<0.039). Loss of Ku86 expression was also observed in a smoking mouse model (p<0.012) and prevented by antioxidants. Oxidants reduced (p<0.0112) Ku86 expression in human bronchial epithelial cells and Ku86 knock down modified AP sites in response to oxidative stress. Conclusions Ineffective DNA repair rather than strand breakage per se accounts for the reduced AP sites observed in COPD and this is correlated with a selective decrease of the expression of Ku86 in the bronchiolar epithelium. DNA damage/repair imbalance may contribute to increased risk of lung carcinoma in COPD.

1-(3′,4′-Dichloro-2-fluoro[1,1′-biphenyl]-4-yl)-cyclopropanecarboxylic Acid (CHF5074), a Novel γ-Secretase Modulator, Reduces Brain β-Amyloid Pathology in a Transgenic Mouse Model of Alzheimer's Disease without Causing Peripheral Toxicity

Some nonsteroidal anti-inflammatory drugs has been shown to allosterically modulate the activity of γ-secretase, the enzymatic complex responsible for the formation of β-amyloid (Aβ). 1-(3′,4′-Dichloro-2-fluoro[1,1′-biphenyl]-4-yl)-cyclopropanecarboxylic acid (CHF5074) is a new γ-secretase modulator, devoid of anticyclooxygenase (COX) and Notch-interfering activities in vitro. We evaluated the effects of chronic CHF5074 treatment on brain Aβ pathology in Tg2576 transgenic mice. Twenty-eight animals of 9.5 to 10.5 months of age received CHF5074-medicated diet (375 ppm) or standard diet for 17 weeks. Compared with controls, CHF5074 treatment significantly reduced the area occupied by plaques and the number of plaques in cortex (–52.2 ± 5.6%, p = 0.0003 and –48.9 ± 6.6%, p = 0.0004, respectively) and hippocampus (–76.7 ± 6.4%, p = 0.004 and –66.2 ± 10.3%, p = 0.037, respectively). Biochemical analysis confirmed the histopathological measures, with CHF5074-treated animals showing reduced total brain Aβ40 (–49.2 ± 9.2%, p = 0.017) and Aβ42 (–43.5 ± 9.7%, p = 0.027) levels. In a human neuroglioma cell line expressing Swedish mutated form of amyloid precursor protein (H4swe), CHF5074 reduced Aβ42 and Aβ40 secretion, with an IC50 of 3.6 and 18.4 μM, respectively, values consistent with those measured in the brain of the CHF5074-treated Tg2576 mice (6.4 ± 0.4 μM). At 5 μM, no effects were observed on Notch intracellular cleavage in human embryonic kidney 293swe cells. CHF5074 was well tolerated by Tg2576 mice. No abnormal findings were observed upon histopathological examination of the gastrointestinal tract, indicating the absence of COX-related toxicity. Semiquantitative histochemical evaluation of goblet cells in the ileum of vehicle- and CHF5074-treated animals yielded similar results, suggesting no effects on Notch pathway. CHF5074 is therefore a promising therapeutic agent for Alzheimers disease.

CHF5074, a novel γ-secretase modulator, attenuates brain β-amyloid pathology and learning deficit in a mouse model of Alzheimer's disease

Background and purpose:  We evaluated the effects of 1‐(3′,4′‐dichloro‐2‐fluoro[1,1′‐biphenyl]‐4‐yl)‐cyclopropanecarboxylic acid (CHF5074), a new γ‐secretase modulator, on brain β‐amyloid pathology and spatial memory in transgenic mice expressing the Swedish and London mutations of human amyloid precursor protein (hAPP).

Acetaminophen, via its reactive metabolite N-acetyl-p-benzo-quinoneimine and transient receptor potential ankyrin-1 stimulation, causes neurogenic inflammation in the airways and other tissues in rodents

Acetaminophen [N-acetyl-p-aminophenol (APAP)] is the most common antipyretic/analgesic medicine worldwide. If APAP is overdosed, its metabolite, N-acetyl-p-benzo-quinoneimine (NAPQI), causes liver damage. However, epidemiological evidence has associated previous use of therapeutic APAP doses with the risk of chronic obstructive pulmonary disease (COPD) and asthma. The transient receptor potential ankyrin-1 (TRPA1) channel is expressed by peptidergic primary sensory neurons. Because NAPQI, like other TRPA1 activators, is an electrophilic molecule, we hypothesized that APAP, via NAPQI, stimulates TRPA1, thus causing airway neurogenic inflammation. NAPQI selectively excites human recombinant and native (neuroblastoma cells) TRPA1. TRPA1 activation by NAPQI releases proinflammatory neuropeptides (substance P and calcitonin gene-related peptide) from sensory nerve terminals in rodent airways, thereby causing neurogenic edema and neutrophilia. Single or repeated administration of therapeutic (15-60 mg/kg) APAP doses to mice produces detectable levels of NAPQI in the lung, and increases neutrophil numbers, myeloperoxidase activity, and cytokine and chemokine levels in the airways or skin. Inflammatory responses evoked by NAPQI and APAP are abated by TRPA1 antagonism or are absent in TRPA1-deficient mice. This novel pathway, distinguished from the tissue-damaging effect of NAPQI, may contribute to the risk of COPD and asthma associated with therapeutic APAP use.

CHF5074, a Novel γ-Secretase Modulator, Restores Hippocampal Neurogenesis Potential and Reverses Contextual Memory Deficit in a Transgenic Mouse Model of Alzheimer's Disease

The effects of compounds interfering with gamma-secretase, the enzymatic complex responsible of the formation of the amyloid-beta (Abeta) peptide from amyloid-beta protein precursor (AbetaPP), on plaque deposition in transgenic mouse models of Alzheimers disease are known but scanty data are available on the effects of these drugs on brain plasticity. We evaluated the effects of long-term treatment with CHF5074, a new gamma-secretase modulator, on hippocampal neurogenesis, cortical synaptophysin levels, and contextual memory in transgenic mice carrying the double Swedish mutation of AbetaPP (Tg2576). Six-month old Tg2576 mice were treated with CHF5074 (375 ppm in the diet) up to 15 months of age. Age-matched control transgenic and wild-type mice received standard diet. Compared to wild-type animals, transgenic controls showed a significant decrease in the number of doublecortin-positive neuroblasts in dentate gyrus, synaptophysin intensity in the cortex, freezing to context in the contextual fear conditioning test. Compared to transgenic controls, CHF5074 treatment of Tg2576 mice resulted in a significant attenuation of the neurogenesis impairment in hippocampus (p=0.036), normalization of synaptophysin levels in cortex (p< 0.001), attenuation of plaque burden in the cortex (p=0.033), increases astroglial reaction around plaques (p=0.001), and attenuation of activated microglia (p=0.040). These effects were associated to a complete reversal of contextual memory deficit (p=0.006). Contextual memory significantly correlated with synaptophysin immunoreactivity in the cortex (r=0.548, p=0.0038).

The γ-secretase modulator CHF5074 restores memory and hippocampal synaptic plasticity in plaque-free Tg2576 mice.

Abnormal amyloid-β (Aβ) production and deposition is believed to represent one of the main causes of Alzheimers disease (AD). γ-Secretase is the enzymatic complex responsible for Aβ generation from its precursor protein. Inhibition or modulation of γ-secretase represents an attractive therapeutic approach. CHF5074 is a new γ-secretase modulator that has been shown to inhibit brain plaque deposition and to attenuate memory deficit in adult AD transgenic mice after chronic treatment. To date, it is not known whether the positive behavioral effects of this compound also occur in young transgenic mice without plaque deposition. Here, we evaluated the effects of acute and subchronic treatment with CHF5074 on contextual and recognition memory and on hippocampal synaptic plasticity in plaque-free Tg2576 mice. We found that at 5 months of age, contextual memory impairment was significantly attenuated after acute subcutaneous administration of 30 mg/kg CHF5074. At 6 months of age, recognition memory impairment was fully reversed after a 4-week oral treatment in the diet (≈60 mg/kg/day). These cognitive effects were associated with a reversal of long-term potentiation (LTP) impairment in the hippocampus. A significant reduction in brain intraneuronal AβPP/Aβ levels and hyperphosphorylated tau, but no change in soluble or oligomeric Aβ levels was detected in Tg2576 mice showing functional recovery following CHF5074 treatment. We conclude that the beneficial effects of CHF5074 treatment in young transgenic mice occurred at a stage that precedes plaque formation and were associated with a reduction in intraneuronal AβPP/Aβ and hyperphosphorylated tau.

Pharmacological assessment of the duration of action of glycopyrrolate vs tiotropium and ipratropium in guinea-pig and human airways

1 Our study was aimed at investigating the duration of the bronchodilator action of the antimuscarinc drug glycopyrrolate compared to tiotropium and ipratropium. 2 In the guinea‐pig isolated trachea, the time (t1/2) necessary for a contractile response to carbachol (0.3 μM) to return to 50% recovery after washout of the antagonist was studied. The offset of the antagonist effect of glycopyrrolate, tiotropium and ipratropium (10 nM each) was t1/2=4.0±0.5, >4.5 and 0.5±0.1 h, respectively. At 4.5 h from the washout of the antagonist, the recovery of the response to carbachol was 50±8, 10±4 and 70±7%, respectively. 3 In the human isolated bronchus, the offset of the bronchodilator effects of glycopyrrolate (3 nM), tiotropium (1 nM) and ipratropium (10 nM) was t1/2=3.7±0.2; >6 and 3.0±0.2 h, respectively. At 6.0 h from the washout of the antagonist, the recovery of the response to carbachol (1 μM) was 101±10, 27±3 and 110±10%, respectively. 4 In anaesthetized guinea‐pigs, acetylcholine‐induced bronchoconstriction was markedly reduced by intratracheal instillation of glycopyrrolate (3 nmol kg−1; 88.1±4% inhibition), tiotropium (1.3 nmol kg−1; 86.2±5% inhibition) or ipratropium (1.45 nmol kg−1; 88.1±10% inhibition). These inhibitory effects assessed 3 or 24 h after antagonist administration were reduced to 69.9±5 and 29.7±6%; 28.3±5 and 14.2±5% for glycopyrrolate and ipratropium, respectively, whereas they remained stable (83.5±4; 70.6±6) for tiotropium. The residual inhibitory effect of glycopyrrolate was also assessed at 16 h from administration, and proved to be as low as that found at 24 h (31.2±10 vs 29.7±6%, respectively). 5 In conclusion, glycopyrrolate‐induced bronchodilation has a longer duration than that of ipratropium, but less than that of tiotropium. The efficacy of a possible glycopyrrolate‐based therapy for asthma or chronic obstructive pulmonary disease given once‐a‐day is not guaranteed by the present investigation.

4-Aminopyridine derivatives with antiamnesic activity

Acetylcholine (Ach) enhancement, useful in the treatment of Alzheimers disease (AD), may be obtained by means of ion channel modulators such as 4-aminopyridine (4-AP). 4-AP is also the central ring of tacrine, the first drug approved for the treatment of AD. The synthesis and pharmacological activity of three 4-AP derivatives, prepared with the aim of improving their antiamnesic activity, is here described. In two of these compounds 4-AP is connected to 4-aminobutyric acid (GABA), whereas in the third it is connected to 2-indolinone, i.e., the skeleton of linopirdine, another Ach enhancing agent. The new compounds showed potent antiamnesic activity in comparison with piracetam.

Preclinical evaluation of CHF3381 as a novel antiepileptic agent

CHF3381 [n-(2-indanyl)-glycinamide hydrochloride] has been selected on the basis of a screening program as the compound displaying the highest anticonvulsant activity in the maximal electroshock seizure (MES) test and the best therapeutic index with reference to the rotarod test in mice and rats. In this study, the antiepileptic activity and the behavioural toxicity of CHF3381 were characterised in multiple model systems. CHF3381 effectively prevented MES-induced convulsions when administered i.p. (ED50, 24 mg/kg and 7.5 mg/kg) or p.o. (ED50, 21 mg/kg and 21 mg/kg) in both mice and rats, respectively. The time course of oral anti-MES activity in the rat was related to the brain concentration profile of unchanged CHF3381. Interestingly, the brain drug levels were about 4-5 times higher than in plasma. CHF3381 was very effective in mice against picrotoxin-, and i.c.v. N-methyl-D-aspartate (NMDA)-induced hind limb tonic extension (ED50 Approximately/=10 mg/kg), but was a weaker antagonist of 4-amynopyridine- and bicuculline-induced tonic seizures (ED50 approximately/=100 mg/kg), and ineffective against pentylentetrazole- and picrotoxin-induced clonic seizures. CHF3381 antagonised the behavioural effects and lethality of i.p. administered NMDA (ED50 = 57 mg/kg p.o.), indicating that the compound may act as a functional NMDA antagonist. In keeping with this idea, CHF3381 weakly displaced [(3)H]-TCP from binding to NMDA receptor channels (Ki, 8.8 microM). In the rat amygdala kindling model, CHF3381 was more efficient against kindling development than against kindled seizures (minimally active dose = 80 vs. 120 mg/kg i.p). Furthermore, it significantly increased the seizure threshold in kindled rats at relatively low doses (40 mg/kg i.p.). In contrast with MK-801-induced hyperactivity, CHF3381 moderately reduced the spontaneous locomotor activity in mice at anticonvulsant doses. Toxic effects on motor performance (rotarod test) were found at high doses only (TD50 approximately/= 300 mg/kg p.o., congruent with 100 mg/kg i.p. in both mice and rats). Furthermore, CHF3381 did not impair passive avoidance and Morris water maze responding in the therapeutic range of doses. Finally, the development of tolerance after repeated doses was negligible. These data indicate that CHF3381 exerts anticonvulsant and antiepileptogenic effects in various seizure models and possesses good therapeutic window, with scarce propensity to cause neurological side-effects.