Main Research Findings

1) Treatment with (-)-clausenamide was found to potentiate levels of synaptic transmission through the dentate gyrus in rats.

2) (-)-Clausenamide has been shown to improve memory impairments through a multi-targeted effect, making it a potential candidate for anti-dementia treatment.

 

Selected Data

1) This study performed by the research team of Xu et al investigated the effects and mechanisms of action of clausenamide enantiomers on synaptic transmission within the dentate gyrus of rats, specifically focusing on their nootropic potential. The experimental subjects were male Wistar rats, weighing between 180-220g. Surgical preparation for electrophysiological recordings was performed under deep urethane carbamate anesthesia. Rats were secured in a stereotaxic frame, a standard apparatus for precise head positioning. Sterile surgical conditions were maintained throughout the implantation of electrodes and cannulae. For precise targeting, a guide cannula, a monopolar recording electrode, and a bipolar stimulating electrode were implanted into specific brain regions of the right hemisphere [1].

The recording electrode was positioned in the hilar region of the right dorsal dentate gyrus (3.8 mm posterior to bregma, 2.5 mm lateral to midline, 3.0-3.5 mm from dura). The stimulating electrode was placed in the right lateral perforant path (7.5 mm posterior to bregma, 4.2 mm lateral to midline, 2.3 mm from dura), which projects to the dentate gyrus. Stainless steel electrodes, with an outer diameter of 0.1 mm, were insulated with Teflon except for their tips, ensuring localized electrical activity. Electrode placement was verified by adjusting their depth until the population spike amplitude (PSA) reached its maximum, a criterion for optimal positioning for granular cell population excitation. For experiments involving freely moving animals, the entire electrode/cannula assembly was securely fixed to the skull with dental acrylic, and rats were allowed a minimum of 7 days for recovery before behavioral testing commenced [1].

Electrophysiological recordings aimed to quantify synaptic transmission. A single current pulse of 0.15 ms duration was delivered to the perforant path every 30 seconds. The stimulation intensity was carefully adjusted to evoke 50% of the maximal PSA, a critical step to ensure that the recordings were within the dynamic range for detecting potentiation. PSA, reflecting the excitation level of the granular cell population, was measured as the voltage difference between specific points of the waveform. In anesthetized animals, synaptic responses were averaged over 10 consecutive records, and baseline measurements were established by averaging PSA over 6 time points collected within 30 minutes before drug administration or tetanus. For freely moving rats, daily PSAs were averaged over 30 minutes to represent the synaptic transmission level for that day, with a 3-day baseline established before drug administration. Long-term potentiation (LTP) was induced by a high-frequency stimulation (HFS) protocol consisting of 10 bursts of 5 pulses at a frequency of 200 Hz, with 200 ms interburst intervals. LTP was considered successfully induced if the PSA increased to over 130% of its baseline level and maintained this increase for more than 40 minutes.

To investigate the underlying mechanisms, the activities of calcineurin and calpain were measured biochemically. Immediately following electrophysiological recording, rats were decapitated. The brain cortex and hippocampus were dissected on ice and separated for calcineurin and calpain assays. Tissues were homogenized for enzyme activity analyses. Calcineurin activity, a calcium-dependent phosphatase, was determined by measuring inorganic phosphate release from O-phospho-DL-tyrosine. A 200 µl sample was incubated in a reaction solution composed of 25 mM MOPS, 1 mM MnCl2, and 6.6 mM O-phospho-DL-tyrosine, at pH 7.0, for 40 minutes. Absorbance was read at 660 nm after adding malachite green reagent. Calpain activity, a calcium-dependent protease, was measured using casein as a substrate. The reaction solution composed of 20 mg/ml casein, 50 mM Tris-HCl pH 7.4, 10 mM DTT, 0.1 mM EGTA, 0.65 mM CaCl2, and hippocampal homogenate was incubated for 30 minutes at 25°C, and absorbance was read at 595 nm after adding Coomassie brilliant blue G-250 dye. Protein content was determined by the Bradford method [1].

Chemicals used in the study included 16 clausenamide enantiomers, IMM, PUMC & CAMS, with a purity of ≥98.5%. For in vivo experiments in anesthetized animals, clausenamide was dissolved in DMSO and then diluted in 0.9% NaCl solution to desired concentrations. Corresponding DMSO in saline served as vehicle controls. Drugs were delivered via intracerebral ventricular (icv) microinjection (5 µl), with doses calculated to achieve specific brain concentrations. For experiments on freely moving rats, (-)-clausenamide and (+)-clausenamide were suspended in 0.5% Tween-80 and administered orally at a dose of 8 mg/kg [1].

2) The research presented by the research team of Chu et al delves into the synthesis, characterization, and pharmacological evaluation of clausenamide (clau) enantiomers, particularly focusing on (-)-clau, as a potential multi-target drug candidate for dementia. The foundational material, clausenamide, is a small molecule compound originally isolated from the traditional Chinese herbal medicine, Clausena lansium. The study’s approach begins with the intricate chemical synthesis of the various clau enantiomers. Given that the clau molecule possesses four chiral centers, predicting the existence of 16 enantiomers, the researchers had to employ sophisticated synthetic strategies to produce these compounds. Further advancements in production technology, including a newly developed catalytic asymmetric approach, ensured a sufficient supply of (-)-clau for clinical needs [2]

Following the chemical synthesis and characterization, the study employed a variety of in vitro and in vivo experimental models to assess the pharmacological effects of (-)-clau and, for comparative purposes, its enantiomer (+)-clau. For in vivo studies, ten different animal models of memory impairment were utilized. These included amyloid precursor protein (APP) mice, aged rats (24-27 months-old), rats with memory impairment induced by aggregated beta-amyloid (Aβ) injection, and models of cerebral ischemia. Additionally, memory impairment was induced chemically using various compounds or by inducing diabetes mellitus (DM). To evaluate cognitive function in these animal models, established behavioral tasks were primarily employed. The Passive Avoidance Task (PAT) was used to measure latency to enter a dark compartment (step-through) or to descend from a platform (step-down), as well as the number of errors. The Morris Water Maze (MWM) was used to assess spatial learning and memory by measuring escape latency and retention time in the target quadrant. The compounds were administered orally, typically at dosages ranging from 5-10 mg/kg for (-)-clau and 500 mg/kg for piracetam, a known anti-dementia agent used as a positive control.

The mechanistic investigations involved a combination of cellular and molecular techniques. To study the impact on intracellular calcium (Ca²⁺) concentrations, primary cultured neurons were used, and Ca²⁺ levels were measured at various concentrations of (-)-clau. The modulation of the cholinergic system was assessed by measuring choline acetyltransferase (ChAT) activity in frontal cortex neurons and in the neocortex, hippocampus, and striatum of adult mice following oral administration of (-)-clau. Acetylcholinesterase (ACE) activity was also analyzed to understand its inhibition by (-)-clau. Synaptic plasticity was evaluated through long-term potentiation (LTP) recordings in hippocampal slice preparations and in vivo in anesthetized or awake rats, utilizing electrical stimulation to induce LTP and pharmacological agents like nimodipine and R-2-amino-5-phosphonopentanoate to probe underlying mechanisms [2].

Confocal microscopy and y32P-ATP assays were used to examine CaMKII activity and phosphorylation states of signaling molecules like ERK and CREB. Synaptogenesis and structural plasticity were assessed through quantitative synapse analysis and measurement of cerebral cortex thickness and mossy fiber sprouting. For AD pathogenesis, the anti-apoptotic effects were studied in five models of apoptosis, including: ow potassium in cerebellar granule cells, growth factor deprivation in cortical neurons, 6-hydroxyldopamine in PC12 cells, ischemia/reperfusion in rats, and Aβ1-40 infusion/natural aging. The expression of pro-apoptotic genes, p53 and c-Myc, as well as mitochondrial functions of cytochrome C release, glutathione content, methane dicaroxylic aldehyde production, mitochondrial membrane potential, complex I and IV activity, were analyzed.

Tau hyperphosphorylation was investigated in streptozotocin-induced diabetic mice, with analyses of microtubule structure, tau phosphorylation at Ser, and activities of GSK-3, CDK-5, and PP1 using immunohistochemistry. Finally, pharmacokinetic analyses compared the tmax, Cmax, t1/2ß, AUC0-12 h, AUC0-∞, CL (or CL/F), and Va (or Va/F) for (-)-clau and its enantiomers following single intravenous or oral doses, along with studies on plasma protein binding and metabolic/excretory routes, and identification of metabolites, 7-hydroxyl-clau and 4-hydroxyl-clau. These comprehensive methods allowed for a detailed understanding of the multi-target effects and chirality-dependent properties of clausenamide [2].

 

Discussion

1) This study investigated the effects of 16 clausenamide enantiomers on basal synaptic transmission in the dentate gyrus of anesthetized rats, along with a detailed comparison of (-)-clausenamide and (+)-clausenamide, and the underlying mechanisms. The findings provided compelling evidence for the nootropic potential of (-)-clausenamide.

First, an initial screen of 16 clausenamide enantiomers, each at 2 µM, revealed that at least three enantiomers exhibited LTP-like effects on basal synaptic transmission in anesthetized rats: (+)-cis-neo-clausenamide, (-)-clausenamide, and (+)-epi-clausenamide. Specifically, at 15, 30, and 60 minutes after drug administration, these compounds significantly increased the population spike amplitude (PSA) above baseline. Among these, (-)-cis-clausenamide also showed a potentiating effect, although its significance was primarily observed at 30 and 60 minutes post-administration [1].

To further elucidate the chiral dependence and mechanisms, the study focused on a typical enantiomeric pair: (-)-clausenamide and (+)-clausenamide. In anesthetized animals, a single icv administration of (-)-clausenamide (2 µM) consistently and significantly increased PSA, inducing LTP-like effects. The PSA reached 131.8% ± 0.4% at 15 minutes, 138.5% ± 8.9% at 30 minutes, and a remarkable 158.1% ± 4.2% at 60 minutes post-administration, all significantly higher than baseline. In contrast, (+)-clausenamide showed no or very little effect on PSA, indicating a clear chiral specificity in its synaptic modulatory activity.

To exclude the confounding influence of anesthesia, the effects of (-)-clausenamide and (+)-clausenamide on synaptic transmission were also investigated in freely moving rats. Oral administration of 8 mg/kg (-)-clausenamide resulted in a significant increase in PSA by 128.6% ± 9.5%, starting after 3 days of treatment and reaching a maximal level of 163.2% ± 10.5% on the 5th day. This elevated PSA was sustained for up to 5 days after the termination of administration, demonstrating a long-lasting effect. Conversely, (+)-clausenamide administered orally had no significant effect on PSA in freely moving rats, further reinforcing the chiral specificity observed in anesthetized animals. These findings also indicated that (-)-clausenamide could effectively cross the blood-brain barrier, providing a strong biological basis for its potential as an orally administered cognitive enhancer [1].

The study also examined the interaction between (-)-clausenamide-induced synaptic potentiation and electrically induced LTP. When 2 µM (-)-clausenamide was injected 10 minutes prior to HFS, it enhanced HFS-induced LTP. In contrast, (+)-clausenamide had little to no effect on HFS-induced LTP, suggesting that (-)-clausenamide’s potentiation shares common mechanisms with electrical LTP induction. To investigate this further, the effects of (-)-clausenamide on synaptic transmission were examined in the presence of Nimodipine, an L-type voltage-dependent calcium channel blocker, and APV, an NMDA receptor blocker. Interestingly, Nimodipine completely inhibited the (-)-clausenamide-induced LTP, with PSA values at 30 and 60 minutes remaining at baseline levels of 98.4% ± 16.7% and 100.8% ± 9.8% respectively. This critical observation strongly suggested the involvement of voltage-dependent calcium channels (VDCCs) in the potentiating effect of (-)-clausenamide. Surprisingly, APV pretreatment did not affect (-)-clausenamide’s action on synaptic transmission, indicating that NMDARs are not primarily required for this specific potentiating effect. This contrasts with the typical understanding of LTP, where NMDARs are crucial.

Further mechanistic investigation focused on calcium-dependent signaling molecules. The activities of calcineurin and calpain, two key protein phosphatases and proteases involved in synaptic plasticity, were measured in the cortex and hippocampus. (-)-Clausenamide treatment significantly increased the activity of both calcineurin and calpain compared to control values. Crucially, the observed increases in calcineurin and calpain activities were completely blocked by Nimodipine, further supporting the central role of VDCCs in the (-)-clausenamide-mediated potentiation. The fact that (-)-clausenamide increased intracellular calcium concentration in cultured neurons in previous studies, combined with the current findings, solidifies the hypothesis that (-)-clausenamide exerts its effects by enhancing calcium influx through VDCCs, thereby activating downstream signaling pathways involving calcineurin and calpain, ultimately leading to LTP-like effects in the dentate gyrus, providing a pharmacological basis for understanding its nootropic mechanisms [1].

2) The findings reported by the research team of Chu et al highlight the efficacy and multi-target mechanisms of (-)-clausenamide in improving cognitive function and ameliorating key pathological features of neurodegenerative diseases, particularly Alzheimer’s disease (AD). A central result was the demonstration of the chirality-dependent nootropic effect of clau. Across ten different animal models of memory impairment, oral administration of (-)-clau consistently improved performance in cognitive tasks. Specifically, it decreased errors and prolonged latency in PAT and reduced escape latency while increasing retention time in the target quadrant of the MWM. In contrast, (+)-clau, the enantiomer, showed no detectable effects, unequivocally establishing (-)-clau as the eutomer and (+)-clau as the distomer in cognitive improvement. Furthermore, (-)-clau proved more potent than the established anti-dementia drug piracetam, requiring significantly lower dosages of 5-10 mg/kg vs. 500 mg/kg, to achieve similar benefits. These in vivo results, spanning diverse models of memory impairment induced by Aβ, aging, ischemia, and various chemicals, strongly support (-)-clau as an effective drug candidate for memory enhancement [2].

Mechanistically, the study unveiled several key actions of (-)-clau. A crucial finding was its ability to induce a mild, controlled elevation of intracellular Ca²⁺ concentrations in primary cultured neurons at physiological concentrations. This modulation was primarily achieved by opening voltage-dependent calcium channels (VDCCs), increasing Ca²⁺ influx, and activating the inositol triphosphate (IP3) signaling pathway to release Ca²⁺ from intracellular stores. This Ca²⁺ transient was shown to mediate neurotrophic factor-like effects, promoting neuronal survival, neurite outgrowth, and antagonizing neural apoptosis induced by growth factor deprivation. The cholinergic system, a key player in memory, was significantly modulated by (-)-clau. In vitro studies revealed that (-)-clau promoted ChAT activity in frontal cortex neurons, supported cholinergic neuron survival and neurite outgrowth, and increased ACh release from synaptosomes. In vivo, (-)-clau increased ChAT activity and ACh concentrations in the neocortex, hippocampus, and striatum across various rodent models, including those with cholinergic system damage, APP overexpression, or natural senescence. Importantly, (-)-clau reversibly inhibited ACE, though with lower potency than other ACE inhibitors, indicating a broader modulatory effect rather than just blocking hydrolysis [2].

Another significant result was the upregulation of synaptic plasticity, particularly LTP in the hippocampus. (-)-Clau enhanced basal synaptic transmission and induced VDCC-dependent LTP in hippocampal synaptic pathways, both in slice preparations and in vivo in rats. This LTP induction was blocked by nimodipine, a VDCC antagonist, but not by APV, an NMDA receptor blocker, confirming its VDCC-dependent nature. Furthermore, (-)-clau activated key signaling pathways involved in cognition, including the PLC/PKC-CREB pathway, leading to the expression of memory-related genes like Zif/268 and BDNF. It also activated CaM-dependent pathways, notably CaMKII-ERK-CREB signaling, which was rapidly phosphorylated in the hippocampus and cortex, influencing neuronal CREB phosphorylation and contributing to LTP induction. These findings collectively demonstrate that (-)-clau supports synaptic plasticity at both functional and structural levels, enhancing learning and memory.

Beyond its nootropic effects, (-)-clau exhibited significant anti-Alzheimer’s disease properties by inhibiting multiple etiological processes. It effectively inhibited Aβ-induced intracellular Ca²⁺ overload and apoptosis, with its anti-apoptotic effects observed across five different models of apoptosis. (-)-Clau significantly reduced apoptosis rates in a dose-dependent manner and inhibited the expression of pro-apoptotic genes like p53 and c-Myc. Moreover, it demonstrated mitochondrial protection by increasing glutathione content, decreasing methane dicaroxylic aldehyde production, reversing collapsed mitochondrial membrane potential, and attenuating complex I and IV activity. The study also found that (-)-clau effectively prevented tau hyperphosphorylation and neurodegeneration. In streptozotocin-induced diabetic mice, (-)-clau treatment ameliorated dementia symptoms, fragmentation and dissolution of axonal microtubules, and tau hyperphosphorylation at Ser. This effect was mediated by targeting key protein kinases and phosphatases: (-)-clau increased PKC activities, reduced CDK-5 activity, and improved protein phosphate 1 (PP1) activity, which is crucial for tau dephosphorylation [2].

Finally, the pharmacokinetic analysis revealed a chirality-dependent profile for clau enantiomers. (-)-Clau showed faster absorption, distribution, and elimination compared to its enantiomers, characterized by smaller tmax, Cmax, t1/2ß, AUC0-12h, AUC0-∞, and greater CL/F and Va/F values. This rapid pharmacokinetic profile was partly explained by its lower plasma protein binding fraction compared to (+)-clau. The metabolism of clau was also chirality-dependent, with (-)-clau and its metabolites primarily excreted via the gastrointestinal route, while (+)-clau predominantly used the biliary route. Importantly, the main metabolite of (-)-clau, 7-hydroxyl-clau, retained similar biological activities, suggesting that its rapid actions are supplemented by active metabolites. These comprehensive results firmly establish (-)-clau as a promising chiral drug candidate with multifaceted therapeutic potential for cognitive enhancement and AD treatment [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Xu L, Liu SL, Zhang JT. (-)-Clausenamide potentiates synaptic transmission in the dentate gyrus of rats. Chirality. 2005;17(5):239-244. doi:10.1002/chir.20150

[2] Chu S, Liu S, Duan W, et al. The anti-dementia drug candidate, (-)-clausenamide, improves memory impairment through its multi-target effect. Pharmacol Ther. 2016;162:179-187. doi:10.1016/j.pharmthera.2016.01.002

 

(-)-Clausenamide is sold for laboratory research use only. Terms of sale apply. Not for human consumption, nor medical, veterinary, or household uses. Please familiarize yourself with our Terms & Conditions prior to ordering.