The anti-dementia drug candidate, (-)-clausenamide, improves memory impairment through its multi-target effect.

Multi-target drugs, such as the cocktail therapy used for treating AIDS, often show stronger efficacy than single-target drugs in treating complicated diseases. This review will focus on clausenamide (clau), a small molecule compound originally isolated from the traditional Chinese herbal medicine, Clausenalansium. The finding of four chiral centers in clau molecules predicted the presence of 16 clau enantiomers, including (-)-clau and (+)-clau. All of the predicted enantiomers have been successfully synthesized via innovative chemical approaches, and pharmacological studies have demonstrated (-)-clau as a eutomer and (+)-clau as a distomer in improving cognitive function in both normal physiological and pathological conditions. Mechanistically, the nootropic effect of (-)-clau is mediated by its multi-target actions, which include mild elevation of intracellular Ca(2+) concentrations, modulation of the cholinergic system, regulation of synaptic plasticity, and activation of cellular and molecular signaling pathways involved in learning and memory. Furthermore, (-)-clau suppresses the pathogenesis of Alzheimer's disease by inhibiting multiple etiological processes: (1) beta amyloid protein-induced intracellular Ca(2+) overload and apoptosis and (2) tau hyperphosphorylation and neurodegeneration. In conclusion, the nature of the multi-target actions of (-)-clau substantiates it as a promising chiral drug candidate for enhancing human cognition in normal conditions and treating memory impairment in neurodegenerative diseases.

Transport and uptake of clausenamide enantiomers in CYP3A4-transfected Caco-2 cells: An insight into the efflux-metabolism alliance.

The present study developed a CYP3A4-expressed Caco-2 monolayer model at which effects of the efflux-metabolism alliance on the transport and uptake of clausenamide (CLA) enantiomers as CYP3A4 substrates were investigated. The apparent permeability coefficients (Papp) of (-) and (+)CLA were higher in the absorptive direction than those in the secretory direction with efflux ratios (ER) of 0.709±0.411 and 0.867±0.250 (×10(-6)cm/s), respectively. Their bidirectional transports were significantly reduced by 75.6-87.5% after treatment with verapamil (a P-glycoprotein inhibitor) that increased the rate of metabolism by CYP3A4, whereas the CYP3A4 inhibitor ketoconazole treatment markedly enhanced the basolateral to apical flux of (-) and (+)CLA with ERs being 2.934±1.432 and 1.877±0.148(×10(-6)cm/s) respectively. These changes could be blocked by the duel CYP3A4/P-glycoprotein inhibitor cyclosporine A, consequently, Papp values for CLA enantiomers in both directions were significantly greater than those obtained by using verapamil or ketoconazole, and their ERs were similar to those following (-) or (+)-isomer treatment alone. Furthermore, the uptake of (-)CLA was more than that of (+)CLA in the transfected cells. Incubation with ketoconazole decreased the intracellular concentrations of the two enantiomers. This effect disappeared in the presence of a CYP3A4 inducer dexamethasone. These results indicated that CYP3A4 could influence P-gp efflux, transport and uptake of CLA enantiomers as CYP3A4 substrates and that a duel inhibition to CYP3A4/ P-glycoprotein could enhance their absorption and bioavailability, which provides new insight into the efflux-metabolism alliance and will benefit the clinical pharmacology of (-)CLA as a candidate drug for treatment of Alzheimer's disease.

Stereoselective Regulation of P-gp Activity by Clausenamide Enantiomers in Caco-2, KB/KBv and Brain Microvessel Endothelial Cells.

The (-)- and (+)-clausenamide (CLA) enantiomers have different pharmacokinetic effects in animals, but their association with putative stereoselective regulation of P-glycoprotein (P-gp) remains unclear. Using three cells expressing P-gp-Caco-2, KBv and rat brain microvessel endothelial cells(RBMEC), this study investigated the association of CLA enantiomers with P-gp. The results showed that the rhodamine 123 (Rh123) accumulation, an indicator of P-gp activity, in Caco-2, KBv and RBMECs was increased by (-)CLA (1 or 5 µmol/L) at 8.2%-28.5%, but reduced by (+)CLA at 11.7%-25.9%, showing stereoselectivity in their regulation of P-gp activity. Following co-treatment of these cells with each CLA enantiomer and verapamil as a P-gp inhibitor, the (+)-isomer clearly antagonized the inhibitory effects of verapamil on P-gp efflux, whereas the (-)-isomer had slightly synergistic or additive effects. When higher concentrations (5 or 10 µmol/L) of CLA enantiomers were added, the stimulatory effects of the (+)-isomer were converted into inhibitory ones, leading to an enhanced intracellular uptake of Rh123 by 24.5%-58.2%; but (-)-isomer kept its inhibition to P-gp activity, causing 30.0%-63.0% increase in the Rh123 uptake. The biphasic effects of (+)CLA were confirmed by CLA uptake in the Caco-2 cells. (+)CLA at 1 µmol/L had significantly lower intracellular uptake than (-)CLA with a ratio[(-)/(+)] of 2.593, which was decreased to 2.167 and 1.893 after CLA concentrations increased to 2.5 and 5 µmol/L. Besides, in the non-induced KB cells, (+)CLA(5 µmol/L) upregulated P-gp expression at 54.5% relative to vehicle control, and decreased Rh123 accumulation by 28.2%, while (-)CLA(5 µmol/L) downregulated P-gp expression at 15.9% and increased Rh123 accumulation by 18.0%. These results suggested that (-)CLA could be a P-gp inhibitor and (+)CLA could be a modulator with concentration-dependent biphasic effects on P-gp activity, which may result in drug-drug interactions when combined with other P-gp substrate drugs.

[Recent advances in pharmacological intervention for prediabetes].

Prediabetes is an abnormal condition between normal glucose metabolism and diabetes mellitus. Impaired glucose tolerance (IGT) is an indicator of high-risk state of prediabetes. Positive interventions of IGT, including life style changes and pharmacological intervention, can effectively postpone and reduce the development of prediabetes into type 2 diabetes mellitus, suggesting that IGT is a key point of diabetes prevention. Currently, pharmacological intervention for prediabetes is still at early stage. In this review, we summarizes recent clinical and preclinical studies on pharmacological intervention for prediabetes, and studies in the development of animal models with IGT and the application of new techniques. We also discuss the prospects of drugs for diabetes prevention, especially with the traditional Chinese medicine.

[Recent progress in vaccines against nicotine addiction].

Tobacco smoking is a global healthcare problem that poses a substantial and costly health burden. Nicotine is the major constituent responsible for the addiction to tobacco. Current strategies helping tobacco smokers have limited utility in increasing rates of smoking cessation, consequently indicating the need for alternative therapies. A novel therapeutic method is vaccination against nicotine. Nicotine vaccine can generate specific antibodies that can sequester nicotine from cigarette smoke in the blood, and prevent its access to the brain and minimize positive reinforcing effects, which may help smokers to stop smoking. The vaccine will have great potential for the treatment of nicotine addiction and for relapse prevention. Here we will review the current status of vaccines against nicotine addiction and discuss the problems associated with the development of nicotine vaccines.

Stereoselective excretion and first-pass metabolism of clausenamide enantiomers.

Stereoselective differences in pharmacokinetics between clausenamide (CLA) enantiomers have been found after intravenous and oral administration of each enantiomer to rats. The differences could be associated with excretion and first-pass metabolism of two enantiomers. The data from this study demonstrated that (-)CLA was mainly excreted in feces with 13.9% of dose, whereas (+)CLA in bile with 17.2%. A large portion of CLA enantiomers could be transformed into hydroxyl metabolites. In the in vitro metabolic system using rat liver microsomes, it was found that (-)CLA was cleared more than its antipode with peak height ratios [(+)/(-)] from 1.0 to 1.8 at the corresponding substrate concentrations from 0.25 to 2mM. Further study in rabbits showed that two enantiomers underwent an intermediate degree of first-pass metabolism. (-)CLA had lower concentrations and AUC0-8h in the portal vein and heart than those of (+)CLA with rates of hepatic extraction 64.7% for (-)-isomer and 50.8% for (+)-isomer, and intrinsic metabolic clearances of (-) and (+)CLA being 186.3 and 107.2 (l/h), respectively. The first-pass metabolism was involved in CYP3A enzymes in the gut and liver, and different levels of CYP3A1 expression induced by (-)CLA or (+)CLA. Immunohistochemical analyses revealed that (-)-isomer significantly increased the expression of CYP3A1, while (+)-isomer had no obvious effects on it. Taken together, the results provided new evidence that stereoselective pharmacokinetics of CLA enantiomers could be resulted from their stereoselective excretion, first-pass metabolism and induction to metabolizing enzymes, which might be important in understanding the clinic pharmacology of active eutomer, (-)CLA, for treatment of Alzheimer's disease.

The mineralocorticoid receptor-p38MAPK-NFκB or ERK-Sp1 signal pathways mediate aldosterone-stimulated inflammatory and profibrotic responses in rat vascular smooth muscle cells.

AIM: To explore the signalling pathways involved in aldosterone-induced inflammation and fibrosis in rat vascular smooth muscle cells (VSMCs). METHODS: Using Western blotting and real-time RT-PCR, we investigated the effects of aldosterone on the expression of cyclooxygenase-2 (Cox-2) and IL-6, two important proinflammatory factors, and TGFß1, a critical profibrotic factor, in VSMCs. RESULTS: Aldosterone treatment significantly increased the expression of Cox-2 and IL-6 and activation of p38MAPK and NF-κB. The expression of both Cox-2 and IL-6 could be blocked by the mineralocorticoid receptor (MR) antagonist spironolactone and the p38MAPK inhibitor SB203580. Also, the rapid phosphorylation of p38MAPK could be suppressed by SB203580 but not by spironolactone, implicating in nongenomic effects of aldosterone. Similar to SB203580 and spironolactone, the NF-κB inhibitor α-p-tosyl-L-lysine chloromethyl ketone (TLCK) markedly attenuated expression of Cox-2, indicating that MR, p38MAPK and NF-κB are associated with aldosterone-induced inflammatory responses. Furthermore, aldosterone enhanced expression of TGFß1 in rat VSMCs. This result may be related to activation of the MR/ERK-Sp1 signalling pathway because PD98059, an ERK1/2 inhibitor, significantly blocked the rapid phosphorylation of ERK1/2 and function of Sp1 and led to reduced expression of TGFß1. Spironolactone was also shown to significantly inhibit TGFß1 and Sp1 expression but not ERK1/2 phosphorylation. CONCLUSION: These results suggest that aldosterone-induced inflammatory responses and fibrotic responses may be mediated by the MR/p38MAPK-NF-κB pathways and the MR/ERK-Sp1 pathways in VSMCs, respectively.

Glycogen synthase kinase-3beta negatively regulates TGF-beta1 and Angiotensin II-mediated cellular activity through interaction with Smad3.

Glycogen synthase kinase-3beta (GSK3beta) is a major negative modulator of cardiac hypertrophy. Here we report that GSK3beta physically and functionally interacts with Smad3. The interaction between GSK3beta and Smad3 may participate in the negative regulation of transforming growth factor beta1 (TGF-beta1) and Angiotensin II-induced transcription and apoptosis. GSK3beta interacted directly with Smad3 to sequester it outside the nucleus and prevent its nuclear translocation. This resulted in the suppression of Smad3-mediated transcriptional activity and gene expression. GSK3beta counteracted the pro-apoptotic effect of Smad3 and attenuated Angiotensin II-induced apoptosis in cardiac myocytes. Furthermore, stimulation of these cells with TGF-beta1 and Angiotensin II led to the endogenous Smad3 disassociating from GSK3beta and inactivating GSK3beta by phosphorylation of its Ser9. These results uncovered a novel mechanism for the GSK3beta negative regulation of TGF-beta1/Smad3 and Angiotensin II/Smad3-mediated transcription and apoptosis by the identification of a crosstalk between GSK3beta and Smad3 signal pathway.

[Effects of (-), (+)-7-hydroxy-clausenamide on synaptic transmission in rat dentate gyrus in vivo].

AIM: To investigate effects of (-), (+)-7-hydroxy-clausenamide (7-OH-Clau) on basal synaptic transmission in the dentate gyrus of anesthetized rats. METHODS: Extracellular recording of the population spike (PS) of hippocampal dentate gyrus following application of low-frequency stimulation (1/30 Hz, 1.0 mA). RESULTS: Fifteen, thirty and sixty minutes after intracerebroventricular (icv) administration of an estimated final brain concentration of 2 x 10(-6) mol.L-1, (-)-7-OH-Clau caused over 30% increase relative to basal PS amplitude before administration, and 27%-41% increase relative to vehicle control (P < 0.05), while (+)-7-OH-Clau exhibited 18%-25% and 11%-20% decrease in the PS amplitude when compared with basal PS amplitude and vehicle control (P < 0.05), respectively. CONCLUSION: The basal synaptic transmission in the dentate gyrus of the rat hippocampus can be potentiated by (-)-7-OH-Clau and attenuated by (+)-7-OH-Clau, indicating that there was a stereoselective difference between the two enantiomers in the modulation of synaptic responses and plasticity.

[Enzyme kinetics of clausenamide enantiomers in rat liver microsomes].

AIM: To investigate the enzyme kinetics of (-)3S,4R,5R,6S-clausenamide[(-)-Clau] and (+)-3R,4S,5S,6R-clausenamide[(+)-Clau] catalyzed by rat liver microsomes and compare their stereoselective differences. METHODS: An in vitro metabolic system was built by using rat liver microsomes and NADPH-generating system. Clau and its metabolites were determined simultaneously by a reversed-phase high performance liquid chromatography. The kinetic parameters, K(m), Vmax, and metabolic rate, Vmax/K(m), were calculated by Eadie-Hofstee plot. RESULTS: In the metabolic system, (-)-Clau was found to be mainly metabolized to 7-hydroxy-, 5-hydoxy- and 4-hydroxy-Clau, and 7-hydroxylation was a preferential pathway which exhibited higher Vmax/K(m) value (0.135 microL.min-1.mg-1) than those of 5- and 4-hydroxylation (0.063 and 0.068 microL.min-1.mg-1, respectively). For (+)-Clau, it was mainly metabolized to 4-hydroxy-Clau, whereas 7-hydroxy- and 4-hydroxy-Clau were so small that they could not be detected systematically. 4-Hydroxylation of (+)-Clau showed highest Vmam/K(m) value (0.547 microL.min-1.mg-1) among all the metabolites tested, which was 8.0 times higher than that of 4-hydroxylation of its antipode. CONCLUSION: The data indicated that there were obvious substrate stereoselective differences in the hydroxylation metabolism of (+)- and (-)-Clau, which provided an explanation of the difference of pharmacokinetic characteristics of Clau enantiomers in rats.