Tacrine

A study nurse conducted preliminary eligibility screening over the phone or via an online questionnaire. Participants had to be 60 years of age or older, with an exception that individuals between 55 and 59 years old were eligible if their own age was within 15 years of symptom onset of their youngest-affected first-degree relative. Participants’ family history of “AD-like dementia” was ascertained either by a compelling AD diagnosis from an experienced clinician or, if such a report was not available, by use of a structured questionnaire developed for the Cache County Study (Breitner, 1999 (link)). The questionnaire was intended to establish memory or concentration issues sufficiently severe to cause disability or loss of function, having an insidious onset and gradual progression (as opposed to typical consequences of a stroke or other sudden insult).
An on-site eligibility visit (visit label EL00) then included more specific questions on family history of AD dementia, medical and surgical history, pharmacological profile, lifestyle habits, as well as physical and neurological examinations, blood and urine sampling. The blood sample was used for genotyping (see section 3.1) only after an individual was declared eligible to the program. The CAIDE score (Cardiovascular Risk Factors, Aging, and Incidence of Dementia risk score) was derived using data collected at entry into the program (age, sex, education, systolic blood pressure, body mass index (BMI), cholesterol, physical activity and APOE ε4 status) (Kivipelto, 2006 (link)). Two cognitive screening instruments assessed integrity of cognition: the Montreal Cognitive Assessment (MoCA) and the Clinical Dementia Rating (CDR) Scale (Morris, 1993 , Nasreddine, 2005 (link)) including its brief cognitive test battery. When cognitive status was in doubt (MoCA typically ≤ 26/30 or CDR > 0), a complete evaluation (2.5 h of testing) was performed by a certified neuropsychologist. The aim of this assessment was to determine if the cognitive deficits detected by the screening tests fell within the range of mild cognitive impairment (MCI), did not meet MCI criteria or were simply circumstantial, see section ‘Management of cognitive decline’ for more details.
Subsequently, during the enrollment visit (visit label EN00), a ~ 30-minute Magnetic Resonance Imaging (MRI) session was acquired to rule out structural brain disease, while simultaneously ensuring participants’ familiarity with the MRI environment. Handedness was determined using the Edinburgh Handedness Inventory (Oldfield, 1971 (link)), and an electrocardiogram was performed. Enrollment also required further documentation of stable general health, availability of a study partner to provide information on daily functioning, and willingness to comply with study protocols (Table 1 for detailed inclusion/exclusion criteria). Specific INTREPAD clinical trial inclusion/exclusion criteria are in the publication describing results of the trial (Meyer, 2019 (link)). In brief, they were similar except for additional criteria related to gastrointestinal tract problems and specific contraindicated concomitant medication. Final determination of eligibility for PREVENT-AD program was made by clinical consensus between one or more study physicians, a research nurse, and a neuropsychologist. All consent procedures fulfilled modern requirements for human subjects’ protection, while avoiding excess participant burden. Consent forms were carefully crafted to use simple but comprehensive language (typically at an 8th grade reading level). Protocols, consent forms and study procedures were approved by McGill Institutional Review Board and/or Douglas Mental Health University Institute Research Ethics Board. Specific consent forms were presented prior to each experimental procedure.Table 1

Inclusion and Exclusion Criteria.

Inclusion criteria
→ Self-reported parental or multiple-sibling (2 or more*) history of Alzheimer-like dementia → Age 60 years or older (persons aged 55–59 years and < 15 years younger than their affected index relative were also eligible) → Minimum of 6 years of formal education → Study partner available to provide information on cognitive status → Sufficient fluency in spoken and written French and/or English → Ability and intention to participate in regular visits → Agreement for periodic donation of blood and urine samples → Agreement to participate in periodic multimodal assessments via MRI and LP for CSF collection (LP optional at first, then mandatory (in 2017) for participation) → Agreement to limit use of medicines as required by clinical trial protocols, if applicable → Provision of informed consent of the different protocols
Exclusion criteria
→ Cognitive disorders - Known or identified during eligibility assessments (MoCA and CDR or exhaustive neuropsychological evaluation when needed) → Use of acetyl-cholinesterase inhibitors including tacrine, donepezil, rivastigmine, galantamine → Use of memantine or other approved prescription cognitive enhancer → Use of vitamin E at>600 i.u. / day or aspirin at > 325 mg / day → Use of opiates (oxycodone, hydrocodone, tramadol, meperidine, hydromorphone) → Use of NSAIDs or regular use of systemic or inhalation corticosteroids → Clinically significant hypertension (accepted if controlled medically), anemia, significant liver or kidney disease → Concurrent use of warfarin, ticlopidine, clopidrogel, or similar anti-coagulant → Current plasma Creatinine > 1.5 mg/dl (132 mmol/l) → Current alcohol, barbiturate or benzodiazepine abuse/dependence

Inclusion and exclusion criteria for the PREVENT-AD observational cohort. INTREPAD trial inclusion/exclusion criteria are specified in the publication describing the results (18). *8 participants had only 1 sibling affected with AD-like dementia. Refer to ‘List_participants_family_history_1_sibling_v1.0.txt’ file. MRI: magnetic resonance imaging; LP: lumbar puncture; CSF: cerebrospinal fluid; MoCA: Montreal Cognitive Assessment; CDR: Clinical Dementia Rating; NSAID: non-steroidal anti-inflammatory drug.

Inhibitions of the activities of AChE, BChE, MAO-A, and MAO-B by the 15 compounds were investigated at an inhibitor concentration of 10 µM. IC₅₀ values were also determined. The reference reversible inhibitors of AChE and BChE, MAO-A, and MAO-B used were tacrine (or donepezil), toloxatone and lazabemide, respectively, and the reference irreversible inhibitors of MAO-A and MAO-B used were clorgyline and pargyline, respectively. Kinetic parameters, inhibition types, and K_i values of PJ5 (for BChE), PJ13 and PJ15 (for AChE) were determined as the methods previously described^{43 (link)}. Enzyme kinetics were investigated at five different substrate concentrations, that is, at 0, ~ 1/2 × IC₅₀, IC₅₀, and 2 × IC₅₀ for each inhibitor. The inhibition types and K_i values were determined using Lineweaver–Burk Plots and secondary plots.

All chemicals and drugs – DPPH, 6-hydroxy-2,5,7,8-tetramethylchromium-2-carboxylic acid (Trolox), ABTS, ferrozine, EDTA, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), acetylthiocholine iodide and butyrylthiocholine iodide, reduced glutathione, gallic acid, tacrine, pargyline, clorgyline, isatin, L-deprenyl were purchased from Sigma-Aldrich®, Argentina. TBA, trichloroacetic acid (TCA) and butylated hydroxytoluene (BHT) were purchased from Biopack® Productos Químicos, Argentina. Vitamin E was purchased from Casasco Laboratories (Argentina).
Murine mAChE and recombinant hBChE were kindly provided by Xavier Brazzolotto and Florian Nachon (IRBA, Brétigny-sur-Orge, France). Recombinant human microsomal hMAO (expressed in baculovirus-infected insect cells (BTI-TN-5B1-4 cells)), horseradish peroxidase (type II, lyophilized powder) and p-tyramine hydrochloride were obtained from Sigma Aldrich (Sigma Aldrich, St. Louis, MO, USA). 10-Acetyl-3,7-dihydroxyphenoxazine (Amplex Red) was synthesized according to [98 ]. All reagents and solvents used were analytical or HPLC grade.

Corina online (Molecular Networks and Altamira) was used to create three-dimensional structures of compounds, that were then prepared using Sybyl 8.0 (Tripos). Protonation states were inspected, hydrogen atoms were added, atom types were checked and Gesteiger-Marsili charges were assigned. All ligands were docked to acetylcholinesterase from 2CKM and to butyrylcholinesterase based on a 1P0I crystal structure using GoldSuite 5.1 (CCDC). Before docking, the proteins were prepared in the following way: all histidine residues were protonated at Nε, the hydrogen atoms were added, ligand and water molecules were removed; the binding site was defined as all amino acid residues within 10 Å from bis-(7)-tacrine for AChE, and 20 Å from the glycerol molecule for BuChE. A standard set of genetic algorithms with a population size of 100, number of operations 100 000, and clustering with a tolerance of 1 Å was applied. After docking process, 10 ligand poses, sorted by GoldScore (for AChE) and ChemScore (for BuChE) were obtained. The results were visualised by PyMOL 0.99rc6 (DeLano Scientific LLC).