Intratumoral estrogen sulfotransferase induction contributes to the anti-breast cancer effects of the dithiocarbamate derivative TM208

Aim:

Sulfotransferase-catalyzed sulfation is the most important pathway for inactivating estrogens. Thus, activation of estrogen sulfotransferase (EST) may be an alternative approach for the treatment of estrogen-dependent breast cancer. In this study we investigated the involvement of EST in anti-breast cancer effects of the dithiocarbamate derivative TM208 in vitro and in vivo.

Methods:

The viability of human breast cancer MCF-7 cells was determined using a SBB assay. Nude mice bearing MCF-7 cells were orally administered TM208 (50 and 150 mg·kg⁻¹·d⁻¹) for 18 days. The xenograft tumors and uteri were collected. The mRNA expression of EST was examined with real-time PCR. EST protein was detected with Western blot, ELISA or immunohistochemical staining assays. A radioactive assay was used to measure the EST activity. Uterotropic bioassay was used to examine the uterine estrogen responses.

Results:

Treatment with TM208 (10, 15 and 20 μmol/L) concentration-dependently increased EST expression in MCF-7 cells in vitro. Co-treatment with triclosan, an inhibitor of sulfonation, abolished TM208-induced cytotoxicity in MCF-7 cells. TM208 exhibited an apparent anti-estrogenic property: it exerted more potent cytotoxicity in E2-treated MCF-7 cells. In the nude mice bearing MCF-7 cells, TM208 administration time-dependently increased the expression and activity of EST, and blocked the gradual increase of E2 concentration in the xenograft tumors. Furthermore, TM208 administration blocked the estrogens-stimulated uterine enlargement. Tamoxifen, a positive control drug, produced similar effects on the expression and activity of EST in vitro and in vivo.

Conclusion:

The induction of EST and reduction of estrogen concentration contribute to the anti-breast cancer action of TM208 and tamoxifen. TM208 may be developed as anticancer drug for the treatment of estrogen receptor-positive breast cancer.     

Triptolide inhibits human breast cancer MCF-7 cell growth via downregulation of the ERα-mediated signaling pathway

Aim:

To investigate the anticancer mechanisms of triptolide, a diterpenoid isolated from the plant Tripterygium wilfordii Hook F, against human breast cancer cells and the involvement of the estrogen receptor-α (ERα)-mediated signaling pathway in particular.

Methods:

Human breast cancer ERα-positive MCF-7 cells and ERα-negative MDA-MB-231 cells were tested. PrestoBlue assay was used to evaluate the cell viability. The levels of ERα mRNA and protein were detected with real-time PCR and immunoblotting, respectively. Mouse models of MCF-7 or MDA-MB-231 xenograft tumors were treated with triptolide (0.4 mg·kg⁻¹·d⁻¹, po) or a selective estrogen receptor modulator tamoxifen (mg·kg⁻¹·d⁻¹, po) for 3 weeks, and the tumor weight and volume were measured.

Results:

Triptolide (5–200 nmol/L) dose-dependently inhibited the viability of both MCF-7 and MDA-MB-231 cells, with a more potent inhibition on MCF-7 cells. Knockdown of ERα in MCF-7 cells by siRNA significantly attenuated the cytotoxicity of triptolide, whereas overexpression of ERα in MDA-MB-231 cells markedly enhanced the cytotoxicity. Triptolide dose-dependently decreased the expression of ERα in MCF-7 cells and MCF-7 xenograft tumors. Furthermore, treatment of MCF-7 cells with triptolide inhibited the phosphorylation of ERK1/2 in dose- and time-dependent manners. In the mice xenografted with MCF-7 cells, treatment with triptolide or tamoxifen resulted in significant reduction in the tumor weight and volume. Similar effects were not obtained in the mice xenografted with MDA-MB-231 cells.

Conclusion:

The anticancer activity of triptolide against ERα-positive human breast cancer is partially mediated by downregulation of the ERα-mediated signaling pathway.     

Pharmacokinetics of panaxatrol disuccinate sodium, a novel anti-cancer drug from Panax notoginseng, in healthy volunteers and patients with advanced solid tumors

Aim:

To evaluate single-dose and multiple-dose pharmacokinetics of panaxatrol disuccinate sodium in healthy volunteers and patients with advanced solid tumors.

Methods:

In the single-dose pharmacokinetic study, 27 healthy volunteers received panaxatrol disuccinate sodium in three doses (70, 100, and 140 mg·m⁻²). In the multiple-dose pharmacokinetic study, Panaxatrol disuccinate sodium was administered to 8 patients at 100 mg·m⁻² daily in a 30-day continuous intravenous injection. Determination of the panaxatrol disuccinate sodium plasma concentration was performed by an LC-MS method. The pharmacokinetic analysis system — Drug and Statistics (DAS) — was applied to assess plasma panaxatrol disuccinate sodium concentration-time data.

Results:

After a single intravenous dose of 70, 100, or 140 mg·m⁻² was administered to subjects, panaxatrol disuccinate sodium distributed broadly, and the plasma concentration of panaxatrol disuccinate sodium declined rapidly. No significant differences were observed in the main pharmacokinetic parameters among the three dosing groups, including AUC_0–t, MRT_0–t, VRT_0–t, t_1/2Z, CL_z/F, V_z/F, and C₀ (P>0.05). In the multiple-dose pharmacokinetic study, the mean steady-state peak concentration (C_max), trough concentration (C_min), average concentration (C_av), mean steady state AUC (AUC_ss) and the degree of fluctuation were 13.96±15.48 mg·L⁻¹, 0.18±0.29 mg·L⁻¹, 0.15±0.29 mg·L⁻¹, 3.58±6.94 mg·L⁻¹·h, and 148.00±117.18, respectively. At any given dose of panaxatrol disuccinate sodium, interindividual variability in the pharmacokinetic parameters was obvious.

Conclusion:

The effect of the dose level on single-dose pharmacokinetics of panaxatrol disuccinate sodium was not significant. No accumulation was observed with exposure to 100 mg·m⁻² panaxatrol disuccinate sodium in the 30-day continuous intravenous injection. All subjects were evaluated for tolerability throughout the study. Thus, the phase II dose of panaxatrol disuccinate sodium may be considered to be 100 mg·m⁻² for a 30-day continuous intravenous injection to treat patients with advanced solid tumors.     

AVE8134, a novel potent PPARα agonist, improves lipid profile and glucose metabolism in dyslipidemic mice and type 2 diabetic rats

Aim:

AVE8134 is a structurally novel potent PPARα agonist. The aim of this study is to investigate the efficacy of AVE8134 on lipid profile and glucose metabolism in dyslipidemic mice and type 2 diabetic rats.

Methods:

A cell based PPAR Gal4 transactivation assay was constructed for testing the activities of AVE8134 at 3 different PPAR isoforms in vitro. Transgenic human Apo A1 (hApo A1) mice and insulin-resistant ZDF rats were used to evaluate the effects of AVE8134 in vivo.

Results:

AVE8134 was a full PPARα dominated PPAR agonist (the values of EC₅₀ for human and rodent PPARα receptor were 0.01 and 0.3 μmol/L, respectively). AVE8134 was not active at PPARδ receptor. In female hApo A1 mice, AVE8134 (1–30 mg·kg⁻¹·d⁻¹, po for 12 d) dose-dependently lowered the plasma triglycerides, and increased the serum HDL-cholesterol, hApo A1 and mouse Apo E levels. In female ZDF rats, AVE8134 (3–30 mg·kg⁻¹·d⁻¹ for 2 weeks) improved insulin-sensitivity index. In pre-diabetic male ZDF rats (at the age of 7 weeks), AVE8134 (10 mg·kg⁻¹·d⁻¹ for 8 weeks) produced an anti-diabetic action comparable to rosiglitazone, without the PPARγ mediated adverse effects on body weight and heart weight. In male ZDF rats (at the age of 6 weeks), AVE8134 (20 mg·kg⁻¹·d⁻¹ for 12 weeks) increased mRNA levels of the target genes LPL and PDK4 about 20 fold in the liver, and there was no relevant effect with rosiglitazone.

Conclusion:

AVE8134 improves lipid profile and glucose metabolism in dyslipidemic mice and type 2 diabetic rats.     

Resveratrol dimers are novel sphingosine kinase 1 inhibitors and affect sphingosine kinase 1 expression and cancer cell growth and survival

BACKGROUND AND PURPOSE

Sphingosine kinase 1 catalyses formation of the bioactive lipid, sphingosine 1-phosphate, which protects cancer cells from apoptosis. Therefore, sphingosine kinase 1 is a novel target for intervention with anti-cancer agents. We have assessed the effect of the anti-cancer agent, resveratrol and its dimers (ampelopsin A and balanocarpol) on sphingosine kinase 1 activity and on survival of MCF-7 breast cancer cells.

EXPERIMENTAL APPROACH

Ampelopsin A and balanocarpol were purified from Hopea dryobalanoides and their effect on sphingosine kinase 1 activity and expression, [³H] thymidine incorporation, ERK-1/2 phosphorylation and PARP activity assessed in MCF-7 cells.

KEY RESULTS

Resveratrol, ampelopsin A and balanocarpol were novel inhibitors of sphingosine kinase 1 activity. Balanocarpol was a mixed inhibitor (with sphingosine) of sphingosine kinase 1 with a K_ic= 90 ± 10 µM and a K_iu of ∼500 µM. Balanocarpol and ampelopsin A also induced down-regulation of sphingosine kinase 1 expression and reduced DNA synthesis, while balanocarpol stimulated PARP cleavage in MCF-7 breast cancer cells. Resveratrol was a competitive inhibitor (with sphingosine) of sphingosine kinase 1 with a K_ic= 160 ± 40 µM, reduced sphingosine kinase 1 expression and induced PARP cleavage in MCF-7 cells.

CONCLUSIONS AND IMPLICATIONS

Each molecule of balanocarpol may bind at least two sphingosine kinase 1 catalytic molecules to reduce the activity of each simultaneously. These findings suggest that resveratrol, ampelopsin A and balanocarpol could perturb sphingosine kinase 1-mediated signalling and this might explain their activity against MCF-7 breast cancer cells.

LINKED ARTICLE

This article is commented on by Hergst and Yun, pp. 1603–1604 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01898.x     

Evodiamine improves congnitive abilities in SAMP8 and APP(swe)/PS1(ΔE9) transgenic mouse models of Alzheimer's disease

Aim:

To investigate the effect of evodiamine (a quinolone alkaloid from the fruit of Evodia rutaecarpa) on the progression of Alzheimer''s disease in SAMP8 and APP^swe/PS1^ΔE9 transgenic mouse models.

Methods:

The mice at age of 5 months were randomized into the model group, two evodiamine (50 mg·kg⁻¹·d⁻¹ and 100 mg·kg⁻¹·d⁻¹) groups and an Aricept (2 mg·kg⁻¹·d⁻¹) group. The littermates of no-transgenic mice and senescence accelerated mouse/resistance 1 mice (SAMR1) were used as controls. After 4 weeks of treatment, learning abilities and memory were assessed using Morris water-maze test, and glucose uptake by the brain was detected using positron emission tomography/computed tomography (PET/CT). Expression levels of IL-1β, IL-6, and TNF-α in brain tissues were detected using ELISA. Expression of COX-2 protein was determined using Western blot.

Results:

In Morris water-maze test, evodiamine (100 mg·kg⁻¹·d⁻¹) significantly alleviated the impairments of learning ability and memory. Evodiamine (100 mg·kg⁻¹·d⁻¹) also reversed the inhibition of glucose uptake due to development of Alzheimer''s disease traits in mice. Furthermore, the dose of evodiamine significantly decreased the expression of IL-1β, IL-6, TNF-α, and COX-2 that were involved in the inflammation due to Alzheimer''s disease.

Conclusion:

The results indicate that evodiamine (100 mg·kg⁻¹·d⁻¹) improves cognitive abilities in the transgenic models of Alzheimer''s disease.     

Low dose of propranolol down-modulates bone resorption by inhibiting inflammation and osteoclast differentiation

BACKGROUND AND PURPOSE

Bones are widely innervated, suggesting an important role for the sympathetic regulation of bone metabolism, although there are controversial studies. We investigated the effects of propranolol in a model of experimental periodontal disease.

EXPERIMENTAL APPROACH

Rats were assigned as follows: animals without ligature; ligated animals receiving vehicle and ligated animals receiving 0.1, 5 or 20 mg·kg⁻¹ propranolol. After 30 days, haemodynamic parameters were measured by cardiac catheterization. Gingival tissues were removed and assessed for IL-1β, TNF-α and cross-linked carboxyterminal telopeptides of type I collagen (CTX) by elisa, or intercellular adhesion molecule 1 (ICAM-1), receptor activator of NF-κ B ligand (RANKL) and osteoprotegerin (OPG) by Western blot analysis. Sections from the mandibles were evaluated for bone resorption. Also, we analysed the ability of propranolol to inhibit osteoclastogenesis in vitro.

RESULTS

Propranolol at 0.1 and 5 mg·kg⁻¹ reduced the bone resorption as well as ICAM-1 and RANKL expression. However, only 0.1 mg·kg⁻¹ reduced IL-1β, TNF-α and CTX levels as well as increased the expression of OPG, but did not alter any of the haemodynamic parameters. Propranolol also suppressed in vitro osteoclast differentiation and resorptive activity by inhibiting the nuclear factor of activated T cells (NFATc)1 pathway and the expression of tartrate-resistant acid phosphatase (TRAP), cathepsin K and MMP-9.

CONCLUSIONS AND IMPLICATIONS

Low doses of propranolol suppress bone resorption by inhibiting RANKL-mediated osteoclastogenesis as well as inflammatory markers without affecting haemodynamic parameters.     

Evidence for a differential opioidergic involvement in the analgesic effect of antidepressants: prediction for efficacy in animal models of neuropathic pain?

BACKGROUND AND PURPOSE

Antidepressants are one of the recommended treatments for neuropathic pain. However, their analgesic action remains unpredictable, and there are no selection criteria for clinical use. Better knowledge of their mechanism of action could help highlight differences underlying their unequal efficacy.

EXPERIMENTAL APPROACH

We compared the activity of a tricyclic antidepressant (clomipramine) with selective 5-HT and noradrenaline reuptake inhibitors (milnacipran and duloxetine) in streptozocin-induced diabetic and chronic constriction nerve injury-induced neuropathic rats, after repeated injections. We looked for an opioidergic mechanism in their action.

KEY RESULTS

Abolition of mechanical hyperalgesia was observed in mononeuropathic rats after five injections of clomipramine (5 mg·kg⁻¹, s.c.) and milnacipran (10 or 20 mg·kg⁻¹, i.p.) and in diabetic rats after clomipramine. An additional antinociceptive effect was obtained with five injections of duloxetine (3 mg·kg⁻¹, i.p.) in both models and milnacipran (10 mg·kg⁻¹, i.p.) in diabetic rats. These effects were observed with plasma antidepressant concentrations similar to those found in patients treated for neuropathic pain. Naloxone (1 mg·kg⁻¹, i.v.) only suppressed the anti-hyperalgesic effects of clomipramine in both models of pain and of milnacipran in the traumatic model.

CONCLUSIONS AND IMPLICATIONS

The opioid system appears to be involved in the mechanism of action of antidepressants that only have an anti-hyperalgesic effect but not in those that have a stronger (i.e. antinociceptive) effect. These differences between the antidepressants occurred whatever the aetiology of the neuropathy and, if confirmed in clinical trials, could be used to decide which antidepressant is administered to a patient with neuropathic pain.     

Primary afferents with TRPM8 and TRPA1 profiles target distinct subpopulations of rat superficial dorsal horn neurones

Background and purpose:

The transient receptor potential (TRP) channels, transient receptor potential melastatin-1 (TRPM8) and transient receptor potential ankyrin-1 (TRPA1), are expressed in subpopulations of sensory neurones and have been proposed to mediate innocuous and noxious cold sensation respectively. The aim of this study was to compare TRPM8 and TRPA1 modulation of glutamatergic afferent transmission within the spinal dorsal horn.

Experimental approach:

Whole cell patch clamp recordings were made from rat spinal cord slices in vitro to examine the effect of TRP agonists and temperature on glutamatergic excitatory postsynaptic currents (EPSCs).

Key results:

Icilin (3 or 100 µmol·L⁻¹), menthol (200 µmol·L⁻¹) and capsaicin (1 µmol·L⁻¹) reduced the amplitude of primary afferent evoked EPSCs in subpopulations of lamina I and II neurones. In a subpopulation of superficial neurones, innocuous cold (threshold 29°C), 3 µmol·L⁻¹ icilin (EC₅₀ 1.5 µmol·L⁻¹) and menthol (EC₅₀ 263 µmol·L⁻¹) increased the rate of spontaneous miniature EPSCs. In the majority of lamina I and II neurones, 100 µmol·L⁻¹ icilin (EC₅₀ 79 µmol·L⁻¹), allyl isothiocyanate (EC₅₀ 226 µmol·L⁻¹), cinnamaldehyde (EC₅₀ 38 µmol·L⁻¹) and capsaicin (1 µmol·L⁻¹) increased miniature EPSC rate. The response to 100 µmol·L⁻¹, but not 3 µmol·L⁻¹ icilin, was abolished by ruthenium red, while neither was affected by iodoresiniferatoxin. Responsiveness to 3 µmol·L⁻¹, but not to 100 µmol·L⁻¹ icilin, was highly predictive of innocuous cold responsiveness. Neurones responding to 3 µmol·L⁻¹ icilin and innocuous cold were located more superficially than those responding to 100 µmol·L⁻¹ icilin.

Conclusions and implications:

Activation of TRPM8 and TRPA1 presynaptically modulated glutamatergic transmission onto partially overlapping but distinct populations of superficial dorsal horn neurones. Spinal TRPM8 and TRPA1 channels may therefore provide therapeutic targets in cold hyperesthesia.     

Synergism of irbesartan and amlodipine on hemodynamic amelioration and organ protection in spontaneously hypertensive rats

Aim:

To investigate the synergism of low-doses of amlodipine and irbesartan on reduction of blood pressure variability (BPV), amelioration of baroreflex sensitivity (BRS) and organ protection in spontaneously hypertensive rats (SHR).

Methods:

The rats were administered amlodipine (1 mg·kg⁻¹·d⁻¹) alone, irbesartan (10 mg·kg⁻¹·d⁻¹) alone, or the combination of the two drugs for 4 months. The drugs were mixed into the rat chow. Blood pressure (BP) was continuously monitored in conscious animals. After the determination of BRS, the rats were killed for morphological evaluation of organ damages.

Results:

The combination of low-dose irbesartan and amlodipine had statistically significant synergism on reduction of BP and BPV, amelioration of BRS and organ protection in SHR. Multiple regression analysis showed that the decrease in left ventricular hypertrophy was associated with the decrease in systolic BPV (r=0.665, P<0.01); the decrease in aortic hypertrophy was associated with the increase in BRS (r=0.656, P<0.01); and the amelioration in renal lesion was associated with the increase in BRS (r=0.763, P<0.01) and the decrease in systolic BPV (r=0.706, P<0.01).

Conclusion:

Long-term treatment with a combination of low-doses of amlodipine and irbesartan showed significant synergism on reduction of BP and BPV, restoration of BRS and organ protection in SHR. Besides BP reduction, the enhancement of BRS and reduction of BPV might contribute to the organ protection.     

Self-augmentation of the lengthening of repolarization is related to the shape of the cardiac action potential: implications for reverse rate dependency

Background and purpose:

The aims of the present work were to study the mechanism of the reverse rate dependency of different interventions prolonging cardiac action potential duration (APD).

Experimental approach:

The reverse rate-dependent lengthening effect of APD-prolonging interventions and the possible involvement of I_Kr (rapid component of the delayed rectifier potassium current) and I_K1 (inward rectifier potassium current) were studied by using the standard microelectrode and the whole-cell patch-clamp techniques in dog multicellular ventricular preparations and in myocytes isolated from undiseased human and dog hearts.

Key results:

All applied drugs – dofetilide (1 µmol·L⁻¹), BaCl₂ (10 µmol·L⁻¹), BAY-K-8644 (1 µmol·L⁻¹), veratrine (1 µg·mL⁻¹) – lengthened APD in a reverse rate-dependent manner regardless of their mode of action, suggesting that reverse rate dependency may not represent a specific mechanism of APD prolongation. The E-4031-sensitive current (I_Kr) and the Ba²⁺-sensitive current (I_K1) were recorded during repolarizing voltage ramps having various steepness and also during action potential waveforms with progressively prolonged APD. Gradually delaying repolarization results in smaller magnitude of I_Kr and I_K1 currents at an isochronal phase of the pulses. This represents a positive feedback mechanism, which appears to contribute to the reverse rate-dependent prolongation of action potentials.

Conclusions and implications:

Action potential configuration may influence the reverse rate-dependent APD prolongation due to the intrinsic properties of I_Kr and I_K1 currents. Drugs lengthening repolarization by decreasing repolarizing outward, or increasing depolarizing inward, currents are expected to cause reverse rate-dependent APD lengthening with high probability, regardless of which current they modify.     

Pharmacokinetics and in vivo potency of soluble epoxide hydrolase inhibitors in cynomolgus monkeys

BACKGROUND AND PURPOSE

Soluble epoxide hydrolase inhibitors (sEHIs) possess anti-inflammatory, antiatherosclerotic, antihypertensive and analgesic properties. The pharmacokinetics (PK) and pharmacodynamics in terms of inhibitory potency of sEHIs were assessed in non-human primates (NHPs). Development of a sEHI for use in NHPs will facilitate investigations on the role of sEH in numerous chronic inflammatory conditions.

EXPERIMENTAL APPROACH

PK parameters of 11 sEHIs in cynomolgus monkeys were determined after oral dosing with 0.3 mg·kg⁻¹. Their physical properties and inhibitory potency in hepatic cytosol of cynomolgus monkeys were examined. Dose-dependent effects of the two inhibitors 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) and the related acetyl piperidine derivative, 1-trifluoromethoxyphenyl-3-(1-acetylpiperidin-4-yl) urea (TPAU), on natural blood eicosanoids, were determined.

KEY RESULTS

Among the inhibitors tested, TPPU and two 4-(cyclohexyloxy) benzoic acid urea sEHIs displayed high plasma concentrations (>10 × IC₅₀), when dosed orally at 0.3 mg·kg⁻¹. Although the 4-(cyclohexyloxy) benzoic acid ureas were more potent against monkey sEH than piperidyl ureas (TPAU and TPPU), the latter compounds showed higher plasma concentrations and more drug-like properties. The C_max increased with dose from 0.3 to 3 mg·kg⁻¹ for TPPU and from 0.1 to 3 mg·kg⁻¹ for TPAU, although it was not linear over this range of doses. As an indication of target engagement, ratios of linoleate epoxides to diols increased with TPPU administration.

CONCLUSION AND IMPLICATIONS

Our data indicate that TPPU is suitable for investigating sEH biology and the role of epoxide-containing lipids in modulating inflammatory diseases in NHPs.     

Preclinical characterization of WAY-211612: a dual 5-HT uptake inhibitor and 5-HT1A receptor antagonist and potential novel antidepressant

Background and purpose

As a combination of 5-HT selective reuptake inhibitor (SSRI) with 5-HT_1A receptor antagonism may yield a rapidly acting antidepressant, WAY-211612, a compound with both SSRI and 5-HT_1A receptor antagonist activities, was evaluated in preclinical models.

Experimental approach

Occupancy studies confirmed the mechanism of action of WAY-211612, while its in vivo profile was characterized in microdialysis and behavioural models.

Key results

WAY-211612 inhibited 5-HT reuptake (K_i = 1.5 nmol·L⁻¹; K_B = 17.7 nmol·L⁻¹) and exhibited full 5-HT_1A receptor antagonist activity (K_i = 1.2 nmol·L⁻¹; K_B = 6.3 nmol·L⁻¹; I_max 100% in adenyl cyclase assays; K_B = 19.8 nmol·L⁻¹; I_max 100% in GTPγS). WAY-211612 (3 and 30 mg·kg⁻¹, po) occupied 5-HT reuptake sites in rat prefrontal cortex (56.6% and 73.6% respectively) and hippocampus (52.2% and 78.5%), and 5-HT_1A receptors in the prefrontal cortex (6.7% and 44.7%), hippocampus (8.3% and 48.6%) and dorsal raphe (15% and 83%). Acute or chronic treatment with WAY-211612 (3–30 mg·kg⁻¹, po) raised levels of cortical 5-HT approximately twofold, as also observed with a combination of an SSRI (fluoxetine; 30 mg·kg⁻¹, s.c.) and a 5-HT_1A antagonist (WAY-100635; 0.3 mg·kg⁻¹, s.c). WAY-211612 (3.3–30 mg·kg⁻¹, s.c.) decreased aggressive behaviour in the resident-intruder model, while increasing the number of punished crossings (3–30 mg·kg⁻¹, i.p. and 10–56 mg·kg⁻¹, po) in the mouse four-plate model and decreased adjunctive drinking behaviour (56 mg·kg⁻¹, i.p.) in the rat scheduled-induced polydipsia model.

Conclusions and implications

These findings suggest that WAY-211612 may represent a novel antidepressant.     

Testosterone ameliorates streptozotocin-induced memory impairment in male rats

Aim： To study the effects of testosterone on streptozotocin （STZ）-induced memory impairment in male rats.
Methods： Adult male Wistar rats were intracerebroventricularly （icv） infused with STZ （750 μg） on d 1 and d 3, and a passive avoidance task was assessed 2 weeks after the first injection of STZ. Castration surgery was performed in another group of rats, and the passive avoidance task was assessed 4 weeks after the operation. Testosterone （1 mg·kg^-1·d^-1, sc）, the androgen receptor antagonist flutamide （10 mg·kg^-1·d^-1, ip）, the estrogen receptor antagonist tamoxifen （1 mg·kg^-1·d^-1, ip） or the aromatase inhibitor letrozole （4 mg·kg^-1·d^-1, ip） were administered for 6 d after the first injection of STZ.
Results： STZ administration and castration markedly decreased both STL1 （the short memory） and STL2 （the long memory） in passive avoidance tests. Testosterone replacement almost restored the STL1 and STL2 in castrated rats, and significantly prolonged the STL1 and STL2 in STZ-treated rats. Administration of flutamide, letrozole or tamoxifen significantly impaired the memory in intact rats, and significantly attenuated the testosterone replacement in improving STZ- and castration-induced memory impairment.
Conclusion： Testosterone administration ameliorates STZ- and castration-induced memory impairment in male Wistar rats.     

Baicalin attenuates air embolism-induced acute lung injury in rat isolated lungs

Background and purpose

Baicalin has been reported to have anti-inflammatory effects and protect against various tissue injuries. However, the effect of baicalin on air embolism-induced acute lung injury has not been tested yet.

Experimental approach

Acute lung injury was induced by infusion of air at a rate of 0.25 mL·min⁻¹ for 1 min into the pulmonary artery of rat isolated lungs. At the end of the experiment, samples were collected for assessment of lung injury, biochemical analysis and histology. Different doses of baicalin (1, 2 and 4 mg·kg⁻¹) were given into the perfusate before air infusion.

Key results

Air embolism elicited a significant increase in microvascular permeability (K_f), lung weight gain, wet/dry weight ratio, pulmonary artery pressure and protein concentration in the bronchoalveolar lavage fluid. Levels of the cytokines, tumour necrosis factor α and cytokine-induced neutrophil chemoattractant-1 in perfusate, and malondialdehyde levels and myeloperoxidase activities in lung tissue were also significantly increased. In addition, histological examination showed increased neutrophil infiltration in lung tissues. Furthermore, nuclear factor-κB activity and degradation of IκB-α were significantly increased in lungs. Pretreatment of the lungs with baicalin (4 mg·kg⁻¹) showed a statistically significant difference in all of the assessed parameters, except for alteration in the pulmonary artery pressure.

Conclusions and implications

Our study suggests that baicalin attenuated air embolism-induced acute lung injury and may be considered a useful adjunct drug therapy in this clinical condition.     

Yhhu3813 is a novel selective inhibitor of c-Met kinase that inhibits c-Met-dependent neoplastic phenotypes of human cancer cells

Aim： c-Met kinase deregulation is strongly associated with the formation, progression and dissemination of human cancers. In this study we identified Yhhu3813 as a small-molecule inhibitor of c-Met kinase and characterized its antitumor properties both in vitro and in vivo.
Methods： The activities of different kinases were measured using ELISA assays and signaling proteins in the cells were detected with Western blotting. Cell proliferation was assessed using SRB or MTT assay in twenty human cell lines and cell cycle distribution was determined with flow cytometry. Transwell-based assay was used to evaluate cell migration and invasion. Cell invasive growth was detected by a morphogenesis assay. c-Met overactivated human NSCLC cell line EBC-1 xenografts were used to evaluate the in vivo anti-tumor efficacy.

Results： Yhhu3813 potently inhibited c-Met kinase activity in vitro with an IC50 value of 2.4±0.3 nmol/L, 〉400-fold higher than that for a panel of 15 different tyrosine kinases, suggesting a high selectivity of Yhhu3813. The compound （20, 100 and 500 nmol/L） dose-dependently inhibited the phosphorylation of c-Met and its key downstream Akt and Erk signal cascades in multiple c-Met aberrant human cancer cell lines, regardless of the mechanistic complexity in c-Met activation across different cellular contexts. In 20 human cancer cell lines harboring different backgrounds of c-Met expression/activation, Yhhu3813 potently inhibited c-Met-driven cell proliferation via arresting cells at G1/S phase. Furthermore, Yhhu3813 substantially impaired c-Met-mediated cell migration, invasion, scattering, and invasive growth. Oral administration of EBC-1 xenograft mice with Yhhu3813 （50 or 100 mg·kg-1·d-1, qd, for 2 weeks） dose-dependently suppressed the tumor growth, which was correlated with a reduction in the intratumoral proliferation index and c-Met signaling.

Conclusion： Yhhu3813 is a potent selective inhibitor of c-Met that inhibits c-Met-dependent neoplastic phenotypes of human cancer cells in vitro and in vivo.     

Potassium 2-（1-hydroxypentyl）-benzoate promotes long-term potentiation in Aβ1-42-injected rats and APP/PS1 transgenic mice

Aim： Potassium 2-（1-hydroxypentyl）-benzoate （d/-PHPB） is a new drug candidate for ischemic stroke. The aim of this study was to investigate the effects of dI-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 Aβ1-42-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 dI-PHPB （100 mg·kg-1·d1, po） reversed the downregulation of p-GluN2B, but did not change GluN1 level in the hippocampus. Furthermore, chronic administration of d/-PHPB reversed the memory deficits in APP/PS1 transgenic mice. In the dentate region of normal rats, injection of dI-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 dI-PHPB （100 mg·kg-1·d-1, po） reversed LTP impairment in Aβ1-42 -injected normal rats and APP/PS1 transgenic mice. Conclusion： Chronic administration of d/-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.     

Chronic ethanol consumption up-regulates protein-tyrosine phosphatase-1B (PTP1B) expression in rat skeletal muscle

Aim:

To investigate the potential effects of chronic ethanol intake on protein-tyrosine phosphatase-1B (PTP1B) and the insulin receptor signaling pathway in rat skeletal muscle.

Methods:

Rats received ethanol treatment at a daily dose of 0 (control), 0.5 (group L), 2.5 (group M) or 5 g·kg⁻¹ (group H) via gastric gavage for 22 weeks. In vivo insulin sensitivity was measured using a hyperinsulinemic-euglycemic clamp. Expression of PTP1B in skeletal muscles was examined at both the mRNA (real-time PCR) and protein (Western blot) levels. PTP1B activity was assayed with a p-nitrophenol phosphate (PNPP) hydrolysis method. Changes of insulin signaling in skeletal muscle were analyzed with Western blotting.

Results:

The activity and expression of PTP1B were dose-dependently elevated 1.6 and 2.0 fold in the skeletal muscle by ethanol, resepctively, at the doses of 2.5 and 5 g·kg⁻¹·d⁻¹. Total IRβ and IRS-1, as well as their phosphorylated forms, were decreased by ethanol at the two higher doses. Moreover, chronic ethanol consumption resulted in a significant inhibition of the association between IRS-1 and the p85 subunit of phosphatidylinositol 3-kinase, inhibition of Akt phosphorylation and reduced levels of mitogen-activated protein kinase phosphorylation.

Conclusion:

Chronic ethanol intake at 2.5 and 5 g·kg⁻¹·d⁻¹ sufficient doses can down-regulate the expression of IRβ, P-IRβ, and IRS-1, as well as the phosphorylated forms of IRS-1 and Akt, in rat skeletal muscle, possibly through increased PTP1B activity.     

Metabolism of novel anti-HIV agent 3-cyanomethyl-4-methyl-DCK by human liver microsomes and recombinant CYP enzymes

Aim:

To investigate the metabolism of 3-cyanomethyl-4-methyl-DCK (CMDCK), a novel anti-HIV agent, by human liver microsomes (HLMs) and recombinant cytochrome P450 enzymes (CYPs).

Methods:

CMDCK was incubated with HLMs or a panel of recombinant cytochrome P450 enzymes including CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5. LC-ion trap mass spectrometry was used to separate and identify CMDCK metabolites. In the experiments with recombinant cytochrome P450 enzymes, specific chemical inhibitors combined with CYP antibodies were used to identify the CYP isoforms involved in CMDCK metabolism.

Results:

CMDCK was rapidly and extensively metabolized by HLMs. Its intrinsic hepatic clearance estimated from the in vitro data was 19.4 mL·min⁻¹·kg⁻¹, which was comparable to the mean human hepatic blood flow rate (20.7 mL·min⁻¹·kg⁻¹). The major metabolic pathway of CMDCK was oxidation, and a total of 14 metabolites were detected. CYP3A4 and 3A5 were found to be the principal CYP enzymes responsible for CMDCK metabolism.

Conclusion:

CMDCK was metabolized rapidly and extensively in human hepatic microsomes to form a number of oxidative metabolites. CYP3A4 and 3A5 were the predominant enzymes responsible for the oxidation of CMDCK.