Conversion and pharmacokinetics profiles of a novel pro-drug of 3-n-butylphthalide, potassium 2-(1-hydroxypentyl)-benzoate, in rats and dogs.

Potassium 2-(1-hydroxypentyl)-benzoate (dl-PHPB) is a novel pro-drug of 3-n-butylphthalide (dl-NBP) that is used to treat ischemic stroke. Currently, dl-PHPB is in phase II-III clinical trials in China. In this study, we investigated the conversion and pharmacokinetics profiles of dl-PHPB in vitro and in vivo. The conversion of dl-PHPB to dl-NBP was pH- and calcium-dependent, and paraoxonase was identified as a major enzyme for the conversion in rat plasma. The pharmacokinetics, tissue distribution and excretion of dl-PHPB were studied and compared with equal-molar doses of dl-NBP in rats and dogs. The in vivo studies showed that dl-PHPB could be quickly and completely converted to dl-NBP. The plasma concentration-time course of converted dl-NBP after intravenous dl-PHPB administration was nearly the same as that after equal-molar dl-NBP. The Cmax and AUC of dl-NBP after oral dl-PHPB administration in rats and dogs were higher by 60% and 170%, respectively, than those after oral dl-NBP administration. Analysis of the tissue distribution of dl-PHPB revealed that converted dl-NBP was primarily distributed in fat, the brain and the stomach. In the brain, the levels of dl-NBP were relatively higher after dl-PHPB treatment by orally than after treatment with equal-molar dl-NBP. Approximately 3%-4% of dl-NBP was excreted within 72 h after dosing with dl-PHPB or dl-NBP, but no dl-PHPB was detected in urine or feces excrements. Our results demonstrate that the conversion of dl-PHPB is fast after oral or intravenous administration. Furthermore, the bioavailability of dl-PHPB was obviously better than that of dl-NBP.

The pathological roles of NDRG2 in Alzheimer's disease, a study using animal models and APPwt-overexpressed cells.

AIMS: To investigate the roles of N-myc downstream-regulated gene 2 (NDRG2) in the pathology of aging and neurodegenerative disease such as Alzheimer's disease (AD). RESULTS: In this study, we confirmed the upregulation of NDRG2 in the brains of aging and AD animal models. To explore the role of NDRG2 in the pathology of AD at molecular level, we conducted a cell-based assay of highly expressed wild-type human APP695 SK-N-SH cells (SK-N-SH APPwt). By silencing and overexpressing gene of NDRG2, we demonstrated that NDRG2-mediated increase in Aß1-42 was through the pathways of BACE1 and GGA3. NGRG2 improved tau phosphorylation via enhanced activity of CDK5 and decreased Pin1, but it was not affected by GSK3ß pathway. NDRG2 might also induce cell apoptosis through the extrinsic (caspase 8) apoptotic pathway by interaction with STAT3. CONCLUSION: Our study confirmed the upregulation of NDRG2 in AD animal models and demonstrated its important roles in AD pathology. NDRG2 might be a potential target for studying and treatment of AD.

Toxicokinetics and toxicity of potassium 2-(1-hydroxypentyl)-benzoate in beagle dogs.

Potassium 2-(1-hydroxypentyl)-benzoate (dl-PHPB) is a prodrug of 3-n-butylphthalide (dl-NBP) for treatment of cerebral ischemic stroke in China, which undergoes lactonization to form dl-NBP in plasma. And, the phase II-III clinical trial of dl-PHPB has been approved by China Food and Drug Administration (CFDA) in 2013. In this study, a toxicity and toxicokinetics evaluation of dl-PHPB was performed using beagle dogs at specially high-dose 108 mg/kg/day (65-fold higher than humans at MHRD) for 4 weeks by intravenous administration, with a 3-week recovery period. And the plasma concentrations of dl-PHPB along with its metabolite dl-NBP were determined by HPLC-UV method. The results showed that dl-PHPB was quickly metabolized into dl-NBP, and no significant accumulation was observed. A slight to moderate behavior-associated toxicity was revealed in the process of delivery; and recovered to normal at the end of administration. Changes in the blood hematological profiles included significantly increased NEUT levels and lower LYM% content. Meanwhile, a notable increase in TG content was also observed in the serum biochemical parameters at 4-week post-exposure. These findings were reversible during the recovery period. The information from these studies would be taken into consideration for the interpretation of toxicology findings and provide a reference for clinical safety assessment.

L-3-n-butylphthalide Rescues Hippocampal Synaptic Failure and Attenuates Neuropathology in Aged APP/PS1 Mouse Model of Alzheimer's Disease.

AIMS: Our previous studies showed that L-3-n-butylphthalide (L-NBP), an extract from seeds of Apium graveolens Linn (Chinese celery), improved cognitive ability in animal models of cerebral ischemia, vascular dementia, and Alzheimer's disease (AD). It is well known that cognitive deficit of AD is caused by synaptic dysfunction. In this study, we investigated the effect of L-NBP on hippocampal synaptic function in APP/PS1 AD transgenic mice and related mechanisms. METHODS: Eighteen-month-old APP/PS1 transgenic (Tg) mice were administrated 15 mg/kg L-NBP by oral gavage for 3 months. Synaptic morphology and the thickness of postsynaptic density (PSD) in hippocampal neurons were investigated by electron microscope. The dendritic spines, Aß plaques, and glial activation were detected by staining. The expressions of synapse-related proteins were observed by Western blotting. RESULTS: L-NBP treatment significantly increased the number of synapses and apical dendritic thorns and the thickness of PSD, increased the expression levels of synapse-associated proteins including PSD95, synaptophysin (SYN), ß-catenin, and GSK-3ß, and attenuated Aß plaques and neuroinflammatory responses in aged APP/PS1 Tg mice. CONCLUSION: L-NBP may restore synaptic and spine function in aged APP Tg mice through inhibiting Aß plaques deposition and neuroinflammatory response. Wnt/ß-catenin signaling pathway may be involved in L-NBP-related restoration of synaptic function.

L-3-n-Butylphthalide attenuates neuroinflammatory responses by downregulating JNK activation and upregulating Heme oxygenase-1 in lipopolysaccharide-treated mice.

Microglia activation-induced neuroinflammation contributes to neuronal damage in neurodegenerative diseases. Inhibition of microglia activation and reduction of major neurotoxic cytokines have been becoming a therapeutic strategy for neurodegenerative diseases. L-3-n-Butylphthalide (L-NBP) has shown the potent neuroprotective effects in stroke and Alzheimer's disease animal models. The present study investigated the immune modulatory effects of L-NBP on pro-inflammatory cytokines and microglia activation in brain tissue induced by systemic lipopolysaccharide (LPS) treatment in C57BL/6 mice. Our results showed that systemic LPS treatment induced microglia activation in the brain. L-NBP treatment significantly suppressed the expression of proinflammatory cytokines, such as tumor necrosis factor (TNFα), interlukin-1ß (IL-1ß), interlukin-6 (IL-6), and interlukin-10 (IL-10) in LPS-treated mice. At the meantime, L-NBP treatment decreased the morphological activation of microglia. In addition, the phosphorylation level of JNK MAP kinase-signaling pathway was also inhibited by L-NBP in LPS-treated mice. Furthermore, L-NBP upregulated the expression of heme oxygenase (HO)-1, a key element in the anti-inflammation and anti-oxidative stress. These results suggested that L-NBP might be a promising candidate in delaying and reversing the progress of neurodegenerative diseases by inhibiting microglia activation.

[Structure-activity relationship of gastrodin and parishins on learning and memory deficits induced by scopolamine].

Gastrodin, parishin and parishin C were purified from a water extract of GE (rhizome of Gastrodia elata, an herb medicine for treatment of neuronal disorders). In order to compare the pharmacological effects of gastrodin, parishin and parishin C on improving cognition deficits, we tested them in an animal model of cognition disorders induced by scopolamine and in a study of in vivo long-term potentiation (LTP) recordings. In the Morris water maze task, parishin C (15 and 50 mg·kg(-1), P<0.05) and parishin (150 mg·kg(-1), P<0.05), improved spatial learning and memory significantly. However, gastrodin showed no significant effects at the dose of 150 mg·kg(-1). In vivo LTP recordings showed that parishin C at 5,10 and 20 mg·kg(-1), parishin at 10, 30 and 100 mg·kg(-1) reversed the suppression of LTP by scopolamine in rats in a dose-dependent manner. However, gastrodin at 100 mg·kg(-1) showed only a modest effect. In summary, the action of parishin C in the improvement of dementia induced by scopolamine was more potent than parishin and gastrodin.

L-3-n-butylphthalide Promotes Neurogenesis and Neuroplasticity in Cerebral Ischemic Rats.

AIMS: This study investigated whether anticerebral ischemia new drug, l-3-n-butylphthalide (l-NBP), improved behavioral recovery and enhanced hippocampal neurogenesis after cerebral ischemia in rats. METHODS AND RESULTS: The middle cerebral artery of rats was blocked for 2 h. The daily oral administrations of 30 mg/kg l-NBP or vehicle were begun from the second day until the rats were sacrificed. L-NBP treatment markedly increased 5-bromo-2'-deoxyuridine (BrdU)-positive cells in the hippocampal dentate gyrus (DG) of injured hemisphere on day 28 after ischemia. The amount of newborn cells and newly mature neurons was also increased. The expressions of growth-associated protein-43 and synaptophysin were significantly elevated in l-NBP-treated rats. However, l-NBP markedly reduced the percentage of BrdU(+) /GFAP(+) cells. Additionally, the levels of catalytical subunit of protein kinase A (PKA), protein kinase B (Akt), and cAMP response element-binding protein (CREB) were significantly increased, and the activation of the signal transducer and activation of transcription 3 (STAT3) and the expressions of cleaved caspase-3 and Bax were obviously inhibited by l-NBP. Consequently, l-NBP attenuated the behavioral dysfunction. CONCLUSIONS: It first demonstrates that l-NBP may improve the behavioral outcome of cerebral ischemia by promoting neurogenesis and neuroplasticity. Activation of CREB and Akt and inhibition of STAT3 signaling might be involved in.

Potassium 2-(1-hydroxypentyl)-benzoate promotes long-term potentiation in Aß1-42-injected rats and APP/PS1 transgenic mice.

AIM: Potassium 2-(1-hydroxypentyl)-benzoate (dl-PHPB) is a new drug candidate for ischemic stroke. The aim of this study was to investigate the effects of dl-PHPB on memory deficits and long-term potentiation (LTP) impairment in animal models of Alzheimer's disease. METHODS: The expression of NMDA receptor subunits GluN1 and GluN2B in the hippocampus and cortex of APP/PS1 transgenic mice were detected using Western blot analysis. Memory deficits of the mice were evaluated with the passive avoidance test. LTP impairment was studied in the dentate region of Aß1-42-injected rats and APP/PS1 transgenic mice. RESULTS: APP/PS1 transgenic mice showed significantly lower levels of GluN1 and p-GluN2B in hippocampus, and chronic administration of dl-PHPB (100 mg · kg(-1) · d(-1), po) reversed the downregulation of p-GluN2B, but did not change GluN1 level in the hippocampus. Furthermore, chronic administration of dl-PHPB reversed the memory deficits in APP/PS1 transgenic mice. In the dentate region of normal rats, injection of dl-PHPB (100 µmol/L, icv) did not change the basal synaptic transmission, but significantly enhanced the high-frequency stimulation (HFS)-induced LTP, which was completely prevented by pre-injection of APV (150 µmol/L, icv). Chronic administration of dl-PHPB (100 mg · kg(-1) · d(-1), po) reversed LTP impairment in Aß1-42-injected normal rats and APP/PS1 transgenic mice. CONCLUSION: Chronic administration of dl-PHPB improves learning and memory and promotes LTP in the animal models of Alzheimer's disease, possibly via increasing p-GluN2B expression in the hippocampus.

[Targeted down-regulation of p53 gene expression by individual antisense RNA in vitro].

OBJECTIVE: To investigate the effect of specific blockage of mutant p53 gene by individualized antisense RNA in vitro. METHODS: Mutation status of p53 in human breast cancer cell lines was determined by immunocytochemical staining, PCR-SSCP and sequencing. Single strand antisense transcription system targeting specific p53 mutation site (mt-p53) was constructed, and corresponding antisense RNA was prepared. The hybridization of antisense RNA with its corresponding mt-p53 gene was confirmed by in-situ hybridization. Human breast cancer cells were transfected with antisense RNA by cationic liposome-mediated method. Time course of effects of antisense RNA was investigated by immunocytochemical staining and cell growth inhibiting assay. Expression of mt-p53 protein was examined by Western blot. Cell proliferation was evaluated by MTT assay and cell cycle distribution was determined by flow cytometry (FCM). Apoptosis was determined by TUNEL assay. RESULTS: Mutation of p53 exon 8 was found in MDA-MB-231 cells and antisense transcription system (pGEM3zf (+/-) p53exon8) was then constructed successfully. In transfected MDA-MB-231 cells, hybridization signals were observed in cytoplasm. Fourth-eight hours after transfection, the antisense RNA (ASp53exon8'RNA) had a significant retarding effect on p53 related proliferation inhibition, along with a decrease of p53 protein expression. CONCLUSIONS: ASp53exon8'RNA specifically blocks mt-p53 gene expression, resulting in an inhibition of MDA-MB-231 cell proliferation. Such an approach may be used as a therapeutic option against human malignancy.

[Investigation of discrepant proteins between two breast cancer cell lines with different metastatic abilities].

BACKGROUND & OBJECTIVE: Metastasis associated proteins play important roles in metastasis. This study was to investigate proteins which may be involved in breast cancer cell metastasis and further explore the potential mechanisms. METHODS: LM-MCF-7 and MCF-7, two breast cancer cell lines with different metastatic potentials, derived from the same parent cell line, were used in our study. Proteomics and Western blot were applied to identify differentially expressed proteins. Wound healing assay was performed to observe the effect of survivin gene on breast cancer cell migration by transfecting pcDNA3-Sur plasmid into MCF-7 cells. RESULTS: Eight differently expressed proteins, which were correlated with cell structures, cellular metabolism, apoptosis, protein enfold or interaction, were identified. Protein expression of nm23 and p27 was relatively higher in MCF-7 cells; while the expression of survivin, Bcl-2 and myosin light chain kinase was relatively higher in LM-MCF-7 cells. Increased migration ability was observed in MCF-7 cells which were transfected with pcDNA3-Sur. CONCLUSION: Metastasis associated proteins exist in breast cancer cell lines with different metastatic abilities. Survivin is closely related to the metastasis in breast cancer cells.

[Effect of 17beta-estradiol on Kv2.1 current and delayed rectifier potassium current in cultured rat hippocampal neurons].

AIM: To study the effects of 17beta-estradiol on Kv2.1 potassium channel current and delayed rectifier potassium current (IK) in cultured rat hippocampal neurons. METHODS: The effects of 17beta-estradiol on Kv2.1 channel current and IK in cultured rat hippocampal neurons were observed using the whole cell patch clamp techniques. RESULTS: 17beta-Estradiol was shown to reduce the amplitude of Kv2.1 current and IK in concentration-dependent manners. The IC50s of 17beta-estradiol blocking Kv2.1 and IK were 2.4 and 4.0 micromol x L(-1), respectively. 17beta-Estradiol (3 micromol x l(-1)) significantly shifted the steady-state activation and inactivation curves of Kv2.1 current to negative potentials. However, it only produced the shift of the steady-state activation curve of IK to the negative potential without effect on the steady-state inactivation of IK. CONCLUSION: 17beta-Estradiol inhibits Kv2.1 and IK of hippocampus at similar level. The inhibition of 17beta-estradiol on IK current may be partially via blocking Kv2.1 current.

[Chemopreventive effect of resveratrol to cancer].

BACKGROUND & OBJECTIVE: Carcinogenesis is a complex process and at least 3 stages, including initiation, promotion, and progression, have been proposed in the process of carcinogenesis. Resveratrol has attracted considerable attention due to its low toxicity and unique chemical structure. This study was designed to test chemopreventive effect of resveratrol to cancer using various animal models. METHODS: Ames assay and micronucleus formation assay were used to test the antimutagenic activities of resveratrol. Croton oil-induced enhancement of ornithine decarboxylase (ODC) activities of dorsal epidermis cells in mouse and mouse ear edema model were used to investigate the anti-promotion effect of resveratrol. In addition,7,12-dimenthylbenz[a]anthracene (DMBA)/croton oil-induced mouse skin tumor model was used to evaluate chemopreventive effect of resveratrol to cancer in vivo. RESULTS: In Ames test,100 microg/plate of resveratrol exhibited 42.2% of inhibition on the reversion of Salmonella typhimurium TA100 induced by methylmethansulfonate, and 200 microg/plate of resveratrol exhibited 91.8% of inhibition on the reversion induced by benzopyrene. Pretreatment of resveratrol prevented cyclophosphamide (CTX)-induced micronucleus formation of polychromatic erythrocytes of mice bone marrow in dose-dependent manner. Mice treated with 30 mg/kg of resveratrol for 6 days before croton oil exposure have palliative ear edema. Treatment of 180 mg/kg resveratrol for 3 days caused 69.3% decrease of ODC activities in croton oil-induced dorsal epidermis. It was shown that resveratrol could inhibit DMBA/croton oil-induced mouse skin papilloma, which includes prolonging the latent period of tumor occurrence, decreasing the incidence of papilloma, and reducing tumor number per mouse in dose-dependent manner. CONCLUSION: Resveratrol has the ability of anti-mutation and anti-promotion of cancer and merit further studies as a potential cancer chemopreventive agent.

[Inhibition of tacrine on delayed rectifier and transient outward potassium currents in cultured rat hippocampal neurons].

AIM: To study the effects of tacrine on IK and IA potassium current in primary cultured rat hippocampal neurons. METHODS: Whole cell patch clamp and primary rat hippocampal neuron cultures were used. RESULTS: Tacrine was shown to reduce the amplitude of IK and IA, in concentration-dependent manners. The IC50s at +40 mV for reduction of IK and IA were 23 and 52.6 mumol.L-1, respectively. Tacrine (30 mumol.L-1) shifted the steady state activation of IK and IA to negative potentials by 12 and 15 mV, respectively. The V1/2 of activation curves for IK current before and after the application of tacrine were (6.7 +/- 1.4) mV and (-5.4 +/- 1.3) mV, respectively. The k of activation curves for IK current was 13.4 + 1.3 and 12.5 + 1.4 without and with tacrine, respectively. The V1/2 of activation curve for IA current were (-9.9 +/- 2.6) mV and (-24 +/- 5) mV in the absence and presence of tacrine, respectively, and the k value was not changed. CONCLUSION: Tacrine inhibited IK and IA currents in rat hippocampal neurons and it is more potent for blocking IK.

Anti-invasion and anti-angiogenesis effect of taxol and camptothecin on melanoma cells.

Two highly invasive melanoma cell lines B16BL6 and B16F10 were used to investigate the anti-invasion and antiangiogenesis action of taxol and camptothecin (CPT). The adhesion of melanoma cells was tested by optical absorbance at 545 nm. The invasive activity of these cells was tested in a transwell chamber assay. The cell migration within a 3D collagen matrix was recorded with a time-lapse video recorder and analyzed by computer-assisted cell tracking. Gelatin zymography was used to study the metalloproteinase activity. The chicken chorioallantoic membrane (CAM) model was used to study the anti-angiogenesis effect of the two drugs. The results demonstrated that both taxol and CPT could inhibit the migration of B16F10 cells, and inhibit the adhesion of B16F10 to fibronectin and laminin. They can reduce the metalloproteinase secretion of HT1080 and exhibit the antiangiogenesis effect in the CAM model. Taxol showed a highly anti-invasion effect on B16BL6 cells while CPT did not exhibit such an effect.

Synthesis and cytotoxicity of 2alpha-amido docetaxel analogues.

Various 2-amido docetaxel analogues were prepared and evaluated for their cytotoxicities. Among them, m-methoxy and m-chlorobenzoylamido analogues were most active but not superior to docetaxel and paclitaxel, and D-seco analogues inactive. Change of 2-benzoate to 2-benzamide may not improve their activities to drug-resistant cell lines.