Effect of total flavonoids of Dracocephalum moldavica L. On neuroinflammation in Alzheimer's disease model amyloid-ß (Aß1-42)-peptide-induced astrocyte activation.

One of the main pathological features noted in Alzheimer's disease (AD) is the presence of plagues of aggregated ß-amyloid (Aß1-42)-peptides. Excess deposition of amyloid-ß oligomers (AßO) are known to promote neuroinflammation. Sequentially, following neuroinflammation astrocytes become activated with cellular characteristics to initiate activated astrocytes. The purpose of this study was to determine whether total flavonoids derived from Dracocephalum moldavica L. (TFDM) inhibited Aß1-42-induced damage attributed to activated C8-D1A astrocytes. Western blotting and ELISA were used to determine the expression of glial fibrillary acidic protein (GFAP), and complement C3 to establish the activation status of astrocytes following induction from exposure to Aß1-42. Data demonstrated that stimulation of C8-D1A astrocytes by treatment with 40 µM Aß1-42 for 24 hr produced significant elevation in protein expression and protein levels of acidic protein (GFAP) and complement C3 accompanied by increased expression and levels of inflammatory cytokines. Treatment with TFDM or the clinically employed drug donepezil in AD therapy reduced production of inflammatory cytokines, and toxicity initiated following activation of C8-D1A astrocytes following exposure to Aß1-42. Therefore, TFDM similar to donepezil inhibited inflammatory secretion in reactive astrocytes, suggesting that TFDM may be considered as a potential compound to be utilized in AD therapy.

Donepezil attenuates inflammation and apoptosis in ulcerative colitis via regulating LRP1/AMPK/NF-κB signaling.

This article focuses on the specific effects and mechanisms of donepezil (DNPZ) hydrochloride on inflammation and apoptosis in ulcerative colitis (UC). In vivo and in vitro models of UC were established using dextran sodium sulfate (DSS)-induced mice and NCM460 cells, respectively. Following oral administration of DNPZ, body weight, disease activity index (DAI) scores and colon lengths of mice were recorded. Histopathological damage was detected employing hematoxylin and eosin (H&E) staining. Inflammatory factors were tested using enzyme-linked immunosorbent assay and quantitative reverse transcription polymerase chain reaction, respectively. Apoptosis was estimated utilizing terminal deoxynucleotidyl transferase dUTP nick-end labeling and western blot. Low-density lipoprotein receptor-related protein 1 (LRP1)/AMP activated protein kinase (AMPK)/nuclear factor-κB (NF- κB) signaling proteins were detected utilizing western blot. NCM460 cell viability was assessed by cell counting kit (CCK)-8. We found that DNPZ partially restored body weight, reduced DAI scores and attenuated intestinal pathological damage in DSS-induced mice. Additionally, inflammatory factors decreased significantly after DNPZ treatment, accompanied by reduced apoptosis level. Phosphorylation (p)-AMPK increased and p-p65 decreased after DNPZ treatment, whereas LRP1 knockdown showed the opposite effect. Moreover, DNPZ treatment greatly restored NCM460 cell viability after DSS stimulation. DNPZ attenuated DSS-induced inflammation and apoptosis in NCM460 cells, which was reversed by LRP1 knockdown. In summary, DNPZ hydrochloride attenuates inflammation and apoptosis in UC via LRP1/AMPK/NF-κB signaling.

Combined Donepezil with Astaxanthin via Nanostructured Lipid Carriers Effective Delivery to Brain for Alzheimer's Disease in Rat Model.

Introduction: Donepezil (DPL), a specific acetylcholinesterase inhibitor, is used as a first-line treatment to improve cognitive deficits in Alzheimer's disease (AD) and it might have a disease modifying effect. Astaxanthin (AST) is a natural potent antioxidant with neuroprotective, anti-amyloidogenic, anti-apoptotic, and anti-inflammatory effects. This study aimed to prepare nanostructured lipid carriers (NLCs) co-loaded with donepezil and astaxanthin (DPL/AST-NLCs) and evaluate their in vivo efficacy in an AD-like rat model 30 days after daily intranasal administration. Methods: DPL/AST-NLCs were prepared using a hot high-shear homogenization technique, in vitro examined for their physicochemical parameters and in vivo evaluated. AD induction in rats was performed by aluminum chloride. The cortex and hippocampus were isolated from the brain of rats for biochemical testing and histopathological examination. Results: DPL/AST-NLCs showed z-average diameter 149.9 ± 3.21 nm, polydispersity index 0.224 ± 0.017, zeta potential -33.7 ± 4.71 mV, entrapment efficiency 81.25 ±1.98% (donepezil) and 93.85 ±1.75% (astaxanthin), in vitro sustained release of both donepezil and astaxanthin for 24 h, spherical morphology by transmission electron microscopy, and they were stable at 4-8 ± 2°C for six months. Differential scanning calorimetry revealed that donepezil and astaxanthin were molecularly dispersed in the NLC matrix in an amorphous state. The DPL/AST-NLC-treated rats showed significantly lower levels of nuclear factor-kappa B, malondialdehyde, ß-site amyloid precursor protein cleaving enzyme-1, caspase-3, amyloid beta (Aß1­42), and acetylcholinesterase, and significantly higher levels of glutathione and acetylcholine in the cortex and hippocampus than the AD-like untreated rats and that treated with donepezil-NLCs. DPL/AST-NLCs showed significantly higher anti-amyloidogenic, antioxidant, anti-acetylcholinesterase, anti-inflammatory, and anti-apoptotic effects, resulting in significant improvement in the cortical and hippocampal histopathology. Conclusion: Nose-to-brain delivery of DPL/AST-NLCs is a promising strategy for the management of AD.

Endophytic fungi: a potential source for drugs against central nervous system disorders.

Neuroprotection is one of the important protection methods against neuronal cells and tissue damage caused by neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, and multiple sclerosis. Various bioactive compounds produced by medicinal plants can potentially treat central nervous system (CNS) disorders. Apart from these resources, endophytes also produce diverse secondary metabolites capable of protecting the CNS. The bioactive compounds produced by endophytes play essential roles in enhancing the growth factors, antioxidant defence functions, diminishing neuroinflammatory, and apoptotic pathways. The efficacy of compounds produced by endophytic fungi was also evaluated by enzymes, cell lines, and in vivo models. Acetylcholine esterase (AChE) inhibition is frequently used to assess in vitro neuroprotective activity along with cytotoxicity-induced neuronal cell lines. Some of drugs, such as tacrine, donepezil, rivastigmine, galantamine, and other compounds, are generally used as reference standards. Furthermore, clinical trials are required to confirm the role of these natural compounds in neuroprotection efficacy and evaluate their safety profile. This review illustrates the production of various bioactive compounds produced by endophytic fungi and their role in preventing neurodegeneration.

Donepezil Reduces H2O2-Inflicted Oxidative Stress and Necroptosis in Cardiomyocytes.

OBJECTIVE: Necroptosis, as a form of regulated cell necrosis, could participate in myocardial oxidative damage. We investigated whether donepezil attenuates H2O2-induced oxidative stress injury and necroptosis in rat cardiomyocytes. METHODS: H9c2 cells were incubated with H2O2 (final concentration of 1 mM) and then intervened with donepezil at doses of 2.5 and 10 µM. Subsequently, the necroptosis inhibitor necrostatin-1 (Nec-1) was introduced to treat H9c2 cells. For cell function experiments, cell proliferation; the contents of creatine kinase (CK), lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA); the protein and mRNA levels of the necroptosis-related proteins receptor-interacting serine-threonine kinase 3 (RIP3) and mixed lineage kinase-like (MLKL); and calcium ion fluorescence intensity were detected using Cell Counting Kit-8, enzyme-linked immunosorbent assay (ELISA), Western blotting, quantitative reverse transcription polymerase chain reaction, and flow cytometry, respectively. RESULTS: Cell viability was conspicuously decreased; CK and LDH contents, RIP3 and MLKL expression levels, and MDA production were preeminently elevated; and the production of SOD, CAT, and GSH was prominently reduced under H2O2 stimulation, which were dose-dependently countered by donepezil intervention. Nec-1 decreased the cell necroptosis, oxidative stress, and calcium overload caused by H2O2. However, on the premise of donepezil intervention, the addition of Nec-1 failed to further improve the situation, suggesting that donepezil exerts cardioprotective effects partly by inhibiting RIP3 and MLKL levels. CONCLUSION: Donepezil reduced H2O2-inflicted oxidative stress and necroptosis in cardiomyocytes by suppressing RIP3 and MLKL levels and calcium ion overload.

Inhibition of Th17 cells by donepezil ameliorates experimental lung fibrosis and pulmonary hypertension.

Rationale: Pulmonary hypertension (PH) secondary to lung fibrosis belongs to WHO Group III, one of the most common subgroups of PH; however, it lacks effective treatment options. Cholinesterase inhibitor donepezil (DON) has been shown to effectively improve Group I PH. However, its effects on Group III PH are unknown. Methods: A lung fibrosis-induced PH mouse model was constructed using a single intratracheal instillation of bleomycin (BLM), after which DON was administered daily. Pulmonary artery and right ventricle (RV) remodeling were evaluated at the end of the study. Lung tissue in each group was analyzed using RNA sequencing, and the results were further verified with datasets from patients with PH. The mechanisms underlying DON-induced effects on PH were verified both in vivo and in vitro. Results: DON effectively improved pulmonary artery and RV remodeling in the BLM-induced mouse model. Transcriptomic profiles of lung tissue indicated that the expression of inflammatory and fibrotic genes was significantly changed in this process. In the animal model and patients with PH, T helper 17 lymphocytes (Th17) were the most common inflammatory cells infiltrating the lung tissue. DON significantly inhibited lung fibroblast activation; thus, preventing lung fibrosis and reducing the inflammatory response and Th17 cell infiltration in the BLM-induced lung tissue. In addition, Th17 cells could activate lung fibroblasts by secreting IL17A, and DON-mediated inhibition of Th17 cell differentiation was found to depend on the α7nAchR-JAK2-STAT3 pathway. Conclusion: DON can alleviate lung fibrosis and PH in an experimental mouse model. It inhibited pro-inflammatory Th17 cell differentiation, which is dependent on a cholinergic receptor pathway, thereby regulating fibroblast activation.

Pharmacokinetic/Pharmacodynamic Models of an Alzheimer's Drug, Donepezil, in Rats.

To investigate the relationship between the pharmacokinetics (PK) and pharmacodynamics (PD) of donepezil (Don), simultaneous examination of the PK of Don and the change in acetylcholine (ACh) in the cerebral hippocampus was analyzed using microdialysis in rats. Don plasma concentrations reached their maximum at the end of a 30-minute infusion. The maximum plasma concentrations (Cmaxs) of the major active metabolite, 6-O-desmethyl donepezil, were 9.38 and 13.3 ng/ml at 60 minutes after starting infusions at 1.25 and 2.5 mg/kg doses, respectively. The amount of ACh in the brain increased shortly after the start of the infusion and reached the maximum value at about 30 to 45 minutes, then decreased to the baseline with a slight delay from the transition of the Don concentration in plasma at a 2.5 mg/kg dose. However, the 1.25 mg/kg group showed little increase in ACh in the brain. The PK/PD models of Don, which were constructed using a general 2-compartment PK model with/without Michaelis-Menten metabolism and the suppressive effect of conversion of ACh to choline using an ordinary indirect response model, were able to effectively simulate Don's plasma and ACh profiles. The ACh profile in the cerebral hippocampus at a 1.25 mg/kg dose was effectively simulated using both constructed PK/PD models and parameters obtained at a 2.5 mg/kg dose by the PK/PD models and indicated that Don largely had no effect on ACh. When these models were used to simulate at 5 mg/kg, the Don PK were nearly linear, whereas the ACh transition had a different profile to lower doses. SIGNIFICANCE STATEMENT: Efficacy/safety of a drug and its pharmacokinetics (PK) are closely correlated. Therefore, it is important to understand the relationship between the drug's PK and its pharmacodynamics (PD). A quantitative procedure of achieving these goals is the PK/PD analysis. We constructed the PK/PD models of donepezil in rats. These models can predict the acetylcholine-time profiles from the PK. The modeling technique is a potential therapeutic application to predict the effect when changes in the PK are caused by pathological condition and co-administered drugs.

Neuroprotective effects of combined therapy with memantine, donepezil, and vitamin D in ovariectomized female mice subjected to dementia model.

Women older than 60 have a higher risk of dementia, aging-related cognitive decline, and Alzheimer's Disease (AD) than the rest of the population. The main reason is hormonal senescence after menopause, a period characterized by a decline in estrogen levels. Since the effectiveness of drugs currently approved for the treatment of AD is limited, it is necessary to seek the development of new therapeutic strategies. Vitamin D deficiency is prevalent in AD patients and individuals with dementia in general. The supplementation of this vitamin in dementia patients might be an interesting approach for increasing the effectiveness of pre-existing medications for dementia treatment. Thus, the present study aims to investigate the effect of vitamin D treatment associated with memantine and donepezil in female mice submitted to ovariectomy (OVX) for five months and subjected to a dementia animal model induced by intracerebroventricular injection of aggregated amyloid ßeta (Aß1-42). For this purpose, Balb/c mice were divided into five experimental groups, which received 17 days of combined therapy with vitamin D, donepezil, and memantine. Then, animals were subjected to behavioral tests. OVX groups exhibited reduced levels of estradiol (E2) in serum, which was not altered by the combined therapy. Higher levels of vitamin D3 were found in the OVX animals submitted to the triple-association treatment. Mice exposed to both OVX and the dementia animal model presented impairment in short and long-term spatial and habituation memories. Also, female mice exposed to Aß and OVX exhibited a reduction in brain-derived neurotrophic factor (BDNF) and interleukin-4 (IL-4) levels, and an increase in tumor necrose factor-α (TNFα) levels in the hippocampus. Besides, increased levels of IL-1ß in the hippocampus and cerebral cortex were observed, as well as a significant increase in immunoreactivity for glial fibrillary acidic protein (GFAP), an astrocytes marker, in the hippocampus. Notably, triple-association treatment reversed the effects of the exposition of mice to Aß and OVX in the long-term spatial and habituation memories impairment, as well as reversed changes in TNFα, IL-1ß, IL-4, and GFAP immunoreactivity levels in the hippocampus of treated animals. Our results indicate that the therapeutic association of vitamin D, memantine, and donepezil has beneficial effects on memory performance and attenuated the neuroinflammatory response in female mice subjected to OVX associated with a dementia animal model.

Forskolin, an Adenylcyclase/cAMP/CREB Signaling Activator Restoring Myelin-Associated Oligodendrocyte Destruction in Experimental Ethidium Bromide Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic neurodegenerative disease marked by oligodendrocyte loss, which results in central neuronal demyelination. AC/cAMP/CREB signaling dysregulation is involved in the progression of MS, including mitochondrial dysfunctions, reduction in nerve growth factors, neuronal inflammation, apoptosis, and white matter degeneration. Our previous research has shown that Forskolin (FSK), a naturally occurring direct adenylyl cyclase (AC)/cAMP/CREB activator, has neuroprotective potential to alleviate pathogenic factors linked with numerous neurological abnormalities. The current study intends to explore the neuroprotective potential of FSK at doses of 40 mg/kg and 60 mg/kg alone, as well as in combination with conventional medicines, such as Fingolimod (FNG), Donepezil (DON), Memantine (MEM), and Simvastatin (SIM) in EB-induced demyelinated experimental MS rats. Adult Wistar rats were divided into nine groups, and EB was infused stereotaxically in the rat brain's intracerebropeduncle (ICP) area. Chronic gliotoxin EB treatment results in demyelination as well as motor and cognitive dysfunctions. FSK, combined with standard medications, improves behavioral dysfunctions, such as neuromuscular and motor deficits and memory and cognitive abnormalities. Following pharmacological treatments improved remyelination by enhancing myelin basic protein and increasing AC, cAMP, and CREB levels in brain homogenates. Furthermore, FSK therapy restored brain mitochondrial-ETC complex enzymes and neurotransmitter levels while decreasing inflammatory cytokines and oxidative stress markers. The Luxol fast blue (LFB) stain results further indicate FSK's neuroprotective potential in preventing oligodendrocyte death. Therefore, the results of these studies contribute to a better understanding of the possible role that natural phytochemicals FSK could have in preventing motor neuron diseases, such as multiple sclerosis.

Effects of an acetylcholinesterase inhibitor and an N-methyl-D-aspartate receptor antagonist on inflammation and degeneration of the nucleus pulposus.

OBJECTIVE: The study aimed to examine the effects of two drugs, an acetylcholinesterase inhibitor (AChEI) and an N-methyl-D-aspartate receptor (NMDAR) antagonist, on degenerated annulus fibrosus (AF) and nucleus pulposus (NP) cells and the extracellular matrix (ECM) structure in vitro. PATIENTS AND METHODS: Tissue samples were obtained from patients with intervertebral disc herniation (four males and four females; classified as Pfirmann stage IV) and used to prepare cell cultures. Untreated cell culture samples served as the control group. Study group samples were treated with donepezil, memantine or a combination of the two drugs. Cell viability, toxicity and proliferation were evaluated in all groups. Western blotting was used to examine changes in protein expression of signal transducer and activator of transcription 3 (STAT3), phospho-STAT3 (ser727), hypoxia-inducible factor (HIF)-1 alpha (HIF-1α) and nucleotide-binding oligomerisation domain (NOD) leucine-rich repeat (LRR)-containing proteins (NLR) family pyrin domain containing 3 (NLRP3) inflammasome. The alpha significance value was < 0.05. RESULTS: Analysis of the microscopy and commercial kit results revealed that cell proliferation was suppressed, and no cell death was observed. The protein expression levels of NLRP3, STAT3, ser727 and HIF-1α were lower in the samples treated with donepezil and memantine at 72 h (p < 0.05). The protein expression levels of NLRP3, STAT3, ser727 and HIF-1α were higher in the samples treated with the combination of donepezil and memantine (p < 0.05). CONCLUSIONS: The combined administration of memantine a NMDAR antagonist which can prevent neurodegeneration and donepezil an AChEI used for pain relief increased the protein expression levels in the anabolic pathway. However, it did not reduce the protein expression levels in the catabolic pathway. Therefore, further studies are needed to provide extensive insight into whether it may be among the potential targets for the therapy of intervertebral disc (IVD) diseases.

[Galangin alleviates learning and memory impairments in APP/PS1 double- transgenic mice by regulating Akt/MEF2D/Beclin-1 signaling pathway].

The study aimed to investigate the effects of galangin on learning and memory impairments and Akt/MEF2 D/Beclin-1 signaling pathway in APP/PS1 double-transgenic mice. The mice in this experiment were divided into the normal group, model group, low-(25 mg·kg~(-1)), medium-(50 mg·kg~(-1)), and high-dose(100 mg·kg~(-1)) galangin groups, donepezil(3 mg·kg~(-1)) group, Akt inhibitor(25 mg·kg~(-1)) group, and autophagy inhibitor(30 mg·kg~(-1)) group, with ten in each group, and administered with the corresponding drugs for 30 successive days. On the 24 th day of medication, the water maze and dark avoidance tests were performed. The levels of p-tau, ß-amyloid peptide 1-42(Aß_(42)), acetylcholinesterase(AChE), ß-site amyloid precursor protein cleaving enzyme 1(BACE1), and amyloid precursor protein(APP) in hippocampus were detected by ELISA, the Beclin-1 mRNA expression by RT-PCR, the expression of Aß_(42) and glial fibrillary acidic protein(GFAP) by immunohistochemistry, and the expression of myocyte enhancer factor 2 D(MEF2 D) by immunofluorescence assay. The pathological changes in hippocampus were observed after HE staining, and the expression of Akt, MEF2 D, and Beclin-1 in hippocampus were assayed by Western blot. These results showed that compared with the normal group, the model group exhibited prolonged swimming time, increased number of errors and electric shocks, up-regulated p-tau, Aß_(42), APP, AChE, BACE1, GFAP, and Beclin-1, shortened incubation period, decreased p-Akt and MEF2 D, and obvious hippocampal injury. Compared with the model group, donepezil and galangin shortened the swimming time, reduced the number of errors and electric shocks, down-regulated the expression of p-tau, Aß_(42), APP, AChE, BACE1, GFAP, and Beclin-1, prolonged the incubation period, up-regulated p-Akt and MEF2 D, and improved the pathological changes in hippocampus. Compared with the autophagy inhibitor group, galangin prolonged the swimming time, elevated the number of errors and electric shocks, enhanced the expression of p-tau, Aß_(42), APP, AChE, BACE1, GFAP, and Beclin-1, shortened the incubation period, and diminished the expression of p-Akt and MEF2 D. In conclusion, galangin improves the learning and memory impairments and hippocampal neuron injury of APP/PS1 mice, which may be related to its regulation of Akt/MEF2 D/Beclin-1 signaling pathway.

Donepezil ameliorates oxygen-glucose deprivation/reoxygenation-induced brain microvascular endothelial cell dysfunction via the SIRT1/FOXO3a/NF-κB pathways.

Ischemic stroke is a disease in which brain tissue is damaged by a sudden rupture or blockage of a blood vessel in the brain that prevents blood from flowing to the brain. Extensive literature has demonstrated the neuroprotective effect of donepezil on brain injury, and this paper attempts to further reveal the effect of donepezil on brain microvascular endothelial cells dysfunction. Human brain microvascular endothelial cells (HBMECs) were treated with oxygen-glucose deprivation/reoxygenation (OGD/R) to induced brain microvascular endothelial cell dysfunction. The OGD/R-induced cell were added with different doses of donepezil with or without Sirtuin-1 (SIRT1) inhibitor EX527. Cell viability of HBMECs was examined by cell counting kit (CCK)-8 assay. OGD/R-treated cell migration was assessed by wound healing assay while angiogenesis in HBMECs was examined by tube formation assay and Western blot analysis. Endothelial cell dysfunction was assessed employing fluorescein isothiocyanate-dextran assay and Western blotting. SIRTI/FOXO3a/NF-kB signaling pathway-related protein expressions were detected using Western blotting. After pretreatment with SIRT1 inhibitor EX527, the above experiments were done again. Donepezil increased cell viability of OGD/R-induced HBMECs, promoted cell migration and angiogenesis, decreased cell permeability, and upregulated the expressions of tight junction proteins. In addition, donepezil regulated the expressions of SIRT1/FOXO3a/NF-κB signaling pathways. However, pretreatment with the SIRT1 inhibitor EX527 reversed the protective effect of donepezil on OGD/R-induced HBMECs. In summary, Donepezil ameliorates OGD/R-induced brain microvascular endothelial cell dysfunction via the SIRT1/FOXO3a/NF-κB pathways.

Association between human paraoxonase 2 protein and efficacy of acetylcholinesterase inhibiting drugs used against Alzheimer's disease.

Serum Paraoxonase 2 (PON2) level is a potential biomarker owing to its association with a number of pathophysiological conditions such as atherosclerosis and cardiovascular disease. Since cholinergic deficiency is closely linked with Alzheimer's disease (AD) progression, acetylcholinesterase inhibitors (AChEIs) are the treatment of choice for patients with AD. However, there is a heterogenous response to these drugs and mostly the subjects do not respond to the treatment. Gene polymorphism, the simultaneous occurrence of two or more discontinuous alleles in a population, could be one of the important factors for this. Hence, we hypothesized that PON2 and its polymorphic forms may be hydrolyzing the AChEIs differently, and thus, different patients respond differently. To investigate this, two AChEIs, donepezil hydrochloride (DHC) and pyridostigmine bromide (PB), were selected. Human PON2 wildtype gene and four mutants, two catalytic sites, and two polymorphic sites were cloned, recombinantly expressed, and purified for in vitro analysis. Enzyme activity and AChE activity were measured to quantitate the amount of DHC and PB hydrolyzed by the wildtype and the mutant proteins. Herein, PON2 esterase activity and AChE inhibitor efficiency were found to be inversely related. A significant difference in enzyme activity of the catalytic site mutants was observed as compared to the wildtype, and subsequent AChE activity showed that esterase activity of PON2 is responsible for the hydrolysis of DHC and PB. Interestingly, PON2 polymorphic site mutants showed increased esterase activity; therefore, this could be the reason for the ineffectiveness of the drugs. Thus, our data suggested that the esterase activity of PON2 was mainly responsible for the hydrolysis of AChEI, DHC, and PB, and that might be responsible for the variation in individual response to AChEI therapy.

Discovery of drug-like acetylcholinesterase inhibitors by rapid virtual screening of a 6.9 million compound database.

Cholinesterase inhibitors remain the mainstay of Alzheimer's disease treatment, and the search for new inhibitors with better efficacy and side effect profiles is ongoing. Virtual screening (VS) is a powerful technique for searching large compound databases for potential hits. This study used a sequential VS workflow combining ligand-based VS, molecular docking and physicochemical filtering to screen for central nervous system (CNS) drug-like acetylcholinesterase inhibitors (AChEIs) amongst the 6.9 million compounds of the CoCoCo database. Eleven in silico hits were initially selected, resulting in the discovery of an AChEI with a Ki of 3.2 µM. In vitro kinetics and in silico molecular dynamics experiments informed the selection of an additional seven analogues. This led to the discovery of two further AChEIs, with Ki values of 2.9 µM and 0.65 µM. All three compounds exhibited reversible, mixed inhibition of acetylcholinesterase. Importantly, the in silico physicochemical filter facilitated the discovery of CNS drug-like compounds, such that all three inhibitors displayed high in vitro blood-brain barrier model permeability.

Turning Donepezil into a Multi-Target-Directed Ligand through a Merging Strategy.

Thanks to the widespread use and safety profile of donepezil (1) in the treatment of Alzheimer's disease (AD), one of the most widely adopted multi-target-directed ligand (MTDL) design strategies is to modify its molecular structure by linking a second fragment carrying an additional AD-relevant biological property. Herein, supported by a proposed combination therapy of 1 and the quinone drug idebenone, we rationally designed novel 1-based MTDLs targeting Aß and oxidative pathways. By exploiting a bioisosteric replacement of the indanone core of 1 with a 1,4-naphthoquinone, we ended up with a series of highly merged derivatives, in principle devoid of the "physicochemical challenge" typical of large hybrid-based MTDLs. A preliminary investigation of their multi-target profile identified 9, which showed a potent and selective butyrylcholinesterase inhibitory activity, together with antioxidant and antiaggregating properties. In addition, it displayed a promising drug-like profile.

Cinnamoyl-N-Acylhydrazone-Donepezil Hybrids: Synthesis and Evaluation of Novel Multifunctional Ligands Against Neurodegenerative Diseases.

A new series of ten multifunctional Cinnamoyl-N-acylhydrazone-donepezil hybrids was synthesized and evaluated as multifunctional ligands against neurodegenerative diseases. The molecular hybridization approach was based on the combination of 1-benzyl-4-piperidine fragment from the anti-Alzheimer AChE inhibitor donepezil (1) and the cinnamoyl subunit from curcumin (2), a natural product with remarkable antioxidant, neuroprotective and anti-inflammatory properties, using a N-acylhydrazone fragment as a spacer subunit. Compounds 4a and 4d showed moderate inhibitory activity towards AChE with IC50 values of 13.04 and 9.1 µM, respectively. In addition, compound 4a and 4d showed a similar predicted binding mode to that observed for donepezil in the molecular docking studies. On the other hand, compounds 4a and 4c exhibited significant radical scavenging activity, showing the best effects on the DPPH test and also exhibited a significant protective neuronal cell viability exposed to t-BuOOH and against 6-OHDA insult to prevent the oxidative stress in Parkinson's disease. Similarly, compound 4c was capable to prevent the ROS formation, with indirect antioxidant activity increasing intracellular GSH levels and the ability to counteract the neurotoxicity induced by both OAß1-42 and 3-NP. In addition, ADMET in silico prediction indicated that both compounds 4a and 4c did not show relevant toxic effects. Due to their above-mentioned biological properties, compounds 4a and 4c could be explored as lead compounds in search of more effective and low toxic small molecules with multiple neuroprotective effects for neurodegenerative diseases.

Modulated delivery of donepezil using a combination of skin microporation and iontophoresis.

The present study investigated the transdermal delivery of donepezil hydrochloride across dermatomed porcine ear skin using passive and physical enhancement techniques. In vitro permeation studies were performed on Franz diffusion cells. Microneedles were fabricated in the lab using a polymeric blend of polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP). The fabricated microneedles were characterized using SEM. Effect of PVA-PVP microneedles and ablative laser (P.L.E.A.S.E) alone, and in combination with anodal iontophoresis on the delivery of donepezil hydrochloride was investigated. Scanning electron microscopy, histology, methylene blue staining, and confocal laser microscopy were used to characterize the microchannels created in the skin. Permeation of donepezil after passive delivery was found to be 26.87 ± 3.97 µg/sq.cm. Microneedles, laser, and iontophoresis significantly increased the permeation to 282.23 ± 8.28 µg/sq.cm, 1562 ± 231.8 µg/sq.cm and 623.4 ± 21.3 µg/sq.cm. Also, a significantly higher permeation was achieved with microneedles and laser in combination with iontophoresis (1000 ± 160.9 µg/sq.cm and 1700.4 ± 189.43 µg/sq.cm respectively). A sharp increase in flux was observed with a combination of skin microporation and iontophoresis, however, the same was not observed for iontophoretic delivery alone. Thus, flux can be successfully tailored with a combination of skin microporation and iontophoresis to suit patient needs.

Therapeutic Drug Monitoring of Rivastigmine and Donepezil Under Consideration of CYP2D6 Genotype-Dependent Metabolism of Donepezil.

BACKGROUND: The efficacy of acetylcholinesterase inhibitors (AChE-I) might depend on blood concentration. While rivastigmine metabolism is independent of the cytochrome P450 system, its isoenzymes, especially CYP2D6, metabolize donepezil. CYP2D6 polymorphisms can cause altered enzyme activity resulting in lower or higher than expected drug concentrations of donepezil. OBJECTIVE: We investigated correlations between clinical efficacy and serum concentrations of rivastigmine and donepezil under special consideration of CYP 2D6 genotype or gene dose-dependent metabolism of donepezil. METHODS: Serum concentrations of donepezil and rivastigmine were measured by liquid chromatography - tandem mass spectrometry (LC-MS/MS). Real-time quantitative polymerase chain reaction (PCR) and allele-specific PCR were performed to assess CYP2D6 genotype and gene dose. RESULTS: Patients treated with rivastigmine (n=28) or donepezil (n=48) were included in the study. Both gene dose and metabolism type significantly predicted the level of donepezil serum concentration (p=0.019 and p=0.013, respectively). In the rivastigmine group, changes of the word list delayed recall subtest before treatment and under stable medication were significantly associated with rivastigmine serum levels (ß=0.465; p=0.018). Drug serum concentrations were outside the recommended range in a substantial percentage of participants, which might have contributed to poor correlations between changes in cognitive measures and drug concentrations. Donepezil serum concentrations significantly depended on CYP2D6 gene dose. CONCLUSION: Testing AChE-I serum concentration should be considered in patients without clinical response to treatment or those with severe side effects. Patients with donepezil drug levels outside the recommended range might additionally profit from CYP2D6 genotyping or treatment with an AChE-I independent of CYP metabolism.

Acetylcholinesterase inhibition prevents alterations in cardiovascular autonomic control and gastric motility in L-NAME-induced hypertensive rats.

AIMS: Autonomic dysfunction in arterial hypertension affects cardiorespiratory control and gastric motility and has been characterized by increased sympathetic and reduced parasympathetic activity. In the present work we investigated the effects of anticholinesterase drugs [donepezil (DON) or pyridostigmine (PYR)] on cardiovascular, autonomic, and gastric parameters in L-NAME-induced hypertensive rats. MATERIALS AND METHODS: Daily oral gavage of L-NAME (70 mg/kg/day) was performed over 14 days in male Wistar rats (180-220 g), whereas daily oral gavage of DON or PYR (1.6 and 22 mg/kg/day, respectively) started 2 days after the L-NAME treatment initiation and lasted 12 days. The development of hypertension was verified by tail plethysmography technique. After the end of treatments, the animals were subjected to experimental protocols (6-12 animals per group; total number of animals used: 78). KEY FINDINGS: L-NAME hypertensive animals had no alterations in heart rate (HR) and intrinsic HR, but showed reduction in baroreflex sensitivity, parasympathetic tone, and gastric motility; and the sympathetic tone, chemoreflex sensitivity, and the LF (low frequency) band of systolic arterial pressure (SAP) variability were increased. DON or PYR attenuated the increase in mean arterial pressure (MAP) induced by L-NAME. Both anticholinesterase drugs were effective in preventing the decrease in baroreflex sensitivity, parasympathetic tone and gastric motility, and also prevented the increases in peripheral chemoreflex response and cardiac sympathetic tone. SIGNIFICANCE: Acetylcholinesterase inhibition with DON or PYR is a promising pharmacological approach to increase parasympathetic function, thus preventing the hypertension-induced alterations in the cardiovascular, gastrointestinal and autonomic systems.