2,3��,4,4��,5��-Pentamethoxy-trans-stilbene, a resveratrol derivative, inhibits colitis-associated colorectal carcinogenesis in mice

Background and purpose:

Resveratrol, a naturally occurring polyphenolic antioxidant, has been shown to exhibit chemoprophylactic effects on cancer development. Previously, we reported that 2,3′,4,4′,5′-pentamethoxy-trans-stilbene (PMS), a methoxylated resveratrol derivative, exerted a highly potent anti-proliferative effect on human colon cancer cells as compared with its parent compound. In the present study, the chemopreventive effect of PMS was evaluated in a mouse model of colitis-associated colon carcinogenesis.

Experimental approach:

Seven-week-old Balb/c mice were injected i.p. with 10 mg·kg⁻¹ azoxymethane (AOM). After 1 week, 3% dextran sodium sulphate (DSS) was administered in the drinking water for 7 days followed by 14 days of tap water for recovery, and this cycle was repeated twice.

Key results:

Intragastric administration of PMS (25, 50 mg·kg⁻¹ body weight) for 16 weeks significantly reduced the multiplicity of colonic neoplasms by 15% and 35% (P < 0.01) respectively. Moreover, PMS at 50 mg·kg⁻¹ inhibited colon cancer cell proliferation and promoted apoptosis. Such changes were accompanied by reduction of Akt (protein kinase B) phosphorylation, inactivation of β-catenin and down-regulation of inducible nitric oxide synthase. In parallel, in vitro studies also demonstrated that PMS inhibited proliferation and induced apoptosis in the murine colon adenocarcinoma cell line Colon26 with concomitant inhibition of Akt phosphorylation and inactivation of β-catenin.

Conclusions and implications:

PMS effectively suppressed colon carcinogenesis in an AOM/DSS animal model and may merit further clinical investigation as a chemoprophylactic agent against colitis-associated colon cancer in humans.     

Piceatannol, a derivative of resveratrol, moderately slows INa inactivation and exerts antiarrhythmic action in ischaemia-reperfused rat hearts

Background and purpose:

Piceatannol is more potent than resveratrol in free radical scavenging in association with antiarrhythmic and cardioprotective activities in ischaemic-reperfused rat hearts. The present study aimed to investigate the antiarrhythmic efficacy and the underlying ionic mechanisms of piceatannol in rat hearts.

Experimental approach:

Action potentials and membrane currents were recorded by the whole-cell patch clamp techniques. Fluo-3 fluorimetry was used to measure cellular Ca²⁺ transients. Antiarrhythmic activity was examined from isolated Langendorff-perfused rat hearts.

Key results:

In rat ventricular cells, piceatannol (3–30 µmol·L⁻¹) prolonged the action potential durations (APDs) and decreased the maximal rate of upstroke (V_max) without altering Ca²⁺ transients. Piceatannol decreased peak I_Na and slowed I_Na inactivation, rather than induced a persistent non-inactivating current, which could be reverted by lidocaine. Resveratrol (100 µmol·L⁻¹) decreased peak I_Na without slowing I_Na inactivation. The inhibition of peak I_Na or V_max was associated with a negative shift of the voltage-dependent steady-state I_Na inactivation curve without altering the activation threshold. At the concentrations more than 30 µmol·L⁻¹, piceatannol could inhibit I_Ca,L, I_to, I_Kr, Ca²⁺ transients and Na⁺-Ca²⁺ exchange except I_K1. Piceatannol (1–10 µmol·L⁻¹) exerted antiarrhythmic activity in isolated rat hearts subjected to ischaemia-reperfusion injury.

Conclusions and implications:

The additional hydroxyl group on resveratrol makes piceatannol possessing more potent in I_Na inhibition and uniquely slowing I_Na inactivation, which may contribute to its antiarrhythmic actions at low concentrations less than 10 µmol·L⁻¹.     

Inhibition of EGFR autophosphorylation plays an important role in the anti-breast cancer efficacy of the dithiocarbamate derivative TM208

Aim： To investigate the effects of a novel dithiocarbamate derivative TM208 on human breast cancer cells as well as the pharmacoki- netic characteristics of TM208 in human breast cancer xenograft mice. Methods： Human breast cancer MCF-7 and MDA-MB-231 cells were treated with TM208 or a positive control drug tamoxifen. Cell pro- liferation was examined using SRB and colony formation assays. Cell apoptosis was analyzed with Annexin V-FITC/PI staining assay. Protein expression was examined with Western blot, ELISA and immunohistochemical analyses. MCF-7 breast cancer xenograft nude mice were orally administered TM208 （50 or 150 mg.k$1〈1-1） or tamoxifen （50 mg.kgl〈t-~） for 18 d. On d 19, the tumors were collected for analyses. Blood samples were collected from the mice treated with the high dose of TM208, and plasma concentrations of TM208 were measured using LC-MS/MS. Results： Treatment of MCF-7 and MDA-MB-231 cells with TM208 dose-dependently inhibited the cell proliferation and colony formation in vitro （the IC~o values were 36.38＋3.77 and 18.13＋0.76 pmol/L, respectively）. TM208 （20-150pmol/L） dose-dependently induced apoptosis of both the breast cancer cells in vitro. In MCF-7 breast cancer xenograft nude mice, TM208 administration dose-depend- ently reduced the tumor growth, but did not result in the accumulation of TM208 or weight loss. TM208 dose-dependently inhibited the phosphorylation of EGFR and ERK1/2 in both the breast cancer cells in vitro as well as in the MCF-7 xenograft tumor. Conclusion： Inhibition of EGFR autophosphorylation plays an important role in the anticancer effect of TM208 against human breast cancer.     

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.     

Telmisartan, ramipril and their combination improve endothelial function in different tissues in a murine model of cholesterol-induced atherosclerosis

BACKGROUND AND PURPOSE

Erectile dysfunction correlates with cardiovascular disease and its common risk factors due to the development of endothelial dysfunction. Positive effects on endothelial and erectile function have been described for substances inhibiting the renin-angiotensin-system. Here, we investigated in an atherosclerosis model, whether telmisartan (angiotensin receptor blocker) and ramipril (angiotensin converting enzyme inhibitor) are equivalent or the combination of both is superior in improving endothelial function in the aorta and the corpus cavernosum and in reducing atherosclerosis.

EXPERIMENTAL APPROACH

Wild-type (WT, C57/B6) and apolipoprotein-E-deficient (ApoE^−/−) mice were treated with a cholesterol-rich diet for 8 weeks. ApoE^−/− mice were supplemented with either telmisartan (20 mg·kg⁻¹·day⁻¹), ramipril (2.5 mg·kg⁻¹·day⁻¹) or the combination thereof.

KEY RESULTS

Systolic blood pressure significantly decreased in treatment groups (P < 0.001), with significantly smaller reduction under ramipril monotherapy (P < 0.05). Endothelial function (assessed by pharmacological stimulation of aortic rings and corpus cavernosum in organ bath chambers) was impaired in ApoE^−/− mice compared to WT animals, which was improved by all three treatments to a comparable extent (P < 0.05). Atherosclerotic lesion size in the ascending aorta and aortic sinus (P < 0.001), the amount of lipid peroxides in cavernosal and aortic tissue (P < 0.05) and free radical load (dihydroethidium-stain) (P < 0.05) were enhanced in untreated ApoE^−/− mice in comparison to WT animals and were significantly reduced by either treatment. In penile tissue, expression of eNOS could be restored by renin-angiotensin-aldosterone system blockade.

CONCLUSIONS AND IMPLICATIONS

Telmisartan and ramipril significantly improved endothelial function of aortic and cavernosal tissues in ApoE^−/− via reduction of oxidative stress. Combination of both agents does not enhance beneficial effects significantly.     

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.     

Quercetin potentiates insulin secretion and protects INS-1 pancreatic ��-cells against oxidative damage via the ERK1/2 pathway

BACKGROUND AND PURPOSE

Quercetin lowers plasma glucose, normalizes glucose tolerance tests and preserves pancreatic β-cell integrity in diabetic rats. However, its mechanism of action has never been explored in insulin-secreting β-cells. Using the INS-1 β-cell line, the effects of quercetin were determined on glucose- or glibenclamide-induced insulin secretion and on β-cell dysfunctions induced by hydrogen peroxide (H₂O₂). These effects were analysed along with the activation of the extracellular signal-regulated kinase (ERK)1/2 pathway. N-acetyl-L-cysteine (NAC) and resveratrol, two antioxidants also known to exhibit some anti-diabetic properties, were used for comparison.

EXPERIMENTAL APPROACH

Insulin release was quantified by the homogeneous time resolved fluorescence method and ERK1/2 activation tested by Western blot experiments. Cell viability was estimated by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) colorimetric assay.

KEY RESULTS

Quercetin (20 µmol·L⁻¹) potentiated both glucose (8.3 mmol·L⁻¹)- and glibenclamide (0.01 µmol·L⁻¹)-induced insulin secretion and ERK1/2 phosphorylation. The ERK1/2 (but not the protein kinase A) signalling pathway played a crucial role in the potentiation of glucose-induced insulin secretion by quercetin. In addition, quercetin (20 µmol·L⁻¹), protected β-cell function and viability against oxidative damage induced by 50 µmol·L⁻¹ H₂O₂ and induced a major phosphorylation of ERK1/2. In the same conditions, resveratrol or NAC were ineffective.

CONCLUSION AND IMPLICATIONS

Quercetin potentiated glucose and glibenclamide-induced insulin secretion and protected β-cells against oxidative damage. Our study suggested that ERK1/2 played a major role in those effects. The potential of quercetin in preventing β-cell dysfunction associated with diabetes deserves further investigation.     

Low dose of the liver X receptor agonist, AZ876, reduces atherosclerosis in APOE*3Leiden mice without affecting liver or plasma triglyceride levels

BACKGROUND AND PURPOSE

Liver X receptor (LXR) agonists are atheroprotective but often induce hypertriglyceridaemia and liver steatosis. We investigated the effect of a novel high-affinity LXR activator, AZ876, on plasma lipids, inflammation and atherosclerosis, and compared the effects with another LXR agonist, GW3965.

EXPERIMENTAL APPROACH

APOE*3Leiden mice were fed an atherogenic diet alone or supplemented with either AZ876 (5 or 20 µmol·kg⁻¹·day⁻¹) or GW3965 (17 µmol·kg⁻¹·day⁻¹) for 20 weeks. Total cholesterol and triglyceride levels were measured using commercial kits. Plasma cytokines were determined by using bead-based multiplex suspension array kits with the Luminex technology. Atherosclerosis was assessed histochemically and lesion composition was assessed by immunohistochemical methods.

KEY RESULTS

Low-dose AZ876 had no effect on plasma or liver lipids, whereas high-dose AZ876 increased plasma triglycerides (+110%) and reduced cholesterol (−16%) compared with controls. GW3965 increased plasma triglycerides (+70%). Low-dose AZ876 reduced lesion area (−47%); and high-dose AZ876 strongly decreased lesion area (−91%), lesion number (−59%) and severity. In either dose, AZ876 did not affect lesion composition. GW3965 reduced atherosclerosis and collagen content of lesions (−23%; P < 0.01). High-dose AZ876 and GW3965, but not low-dose AZ876, reduced inflammation as reflected by lower cytokine levels and vessel wall activation.

CONCLUSIONS AND IMPLICATIONS

We have identified a novel LXR agonist that when given in a low dose inhibits the progression of atherosclerosis without inducing anti-inflammatory effects, liver steatosis or hypertriglyceridaemia. Therefore, the primary protective action of a low-dose AZ876 is likely to be an increased reverse cholesterol transport.     

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.     

Spontaneous release of acetylcholine from autonomic nerves in the bladder

Background and purpose:

Bladder contractility is regulated by intrinsic myogenic mechanisms interacting with autonomic nerves. In this study, we have investigated the physiological role of spontaneous release of acetylcholine in guinea pig and rat bladders.

Experimental approach:

Conventional isotonic or pressure transducers were used to record contractile activity of guinea pig and rat bladders.

Key results:

Hyoscine (3 µmol·L⁻¹), but not tetrodotoxin (TTX, 1 µmol·L⁻¹), reduced basal tension, distension-evoked contractile activity and physostigmine (1 µmol·L⁻¹)-evoked contractions of the whole guinea pig bladder and muscle strips in vitro. ω-Conotoxin GVIA (0.3 µmol·L⁻¹) did not affect physostigmine-induced contractions when given either alone or in combination with ω-agatoxin IVA (0.1 µmol·L⁻¹) and SNX 482 (0.3 µmol·L⁻¹). After 5 days in organotypic culture, when extrinsic nerves had significantly degenerated, the ability of physostigmine to induce contractions was reduced in the dorso-medial strips, but not in lateral strips (which have around 15 times more intramural neurones). Most muscle strips from adult rats lacked intramural neurones. After 5 days in culture, physostigmine-induced or electrical field stimulation-induced contractions of the rat bladder strips were greatly reduced. In anaesthetized rats, topical application of physostigmine (5–500 nmol) on the bladder produced a TTX-resistant tonic contraction that was abolished by atropine (4.4 µmol·kg⁻¹ i.v.).

Conclusions and implications:

The data indicate that there is spontaneous TTX-resistant release of acetylcholine from autonomic cholinergic extrinsic and intrinsic nerves, which significantly affects bladder contractility. This release is resistant to blockade of N, P/Q and R type Ca²⁺ channels.British Journal of Pharmacology (2009) 157, 607–619; doi:10.1111/j.1476-5381.2009.00166.x; published online 3 April 2009     

Ketamine-induced ventricular structural, sympathetic and electrophysiological remodelling: pathological consequences and protective effects of metoprolol

BACKGROUND AND PURPOSE

Growing evidence suggests that long-term abuse of ketamine does harm the heart and increases the risk of sudden death. The present study was performed to explore the cardiotoxicity of ketamine and the protective effects of metoprolol.

EXPERIMENTAL APPROACH

Rats and rabbits were divided into control, ketamine, metoprolol alone and ketamine plus metoprolol groups. Ketamine (40 mg·kg⁻¹·day⁻¹, i.p.) and metoprolol (20 mg·kg⁻¹·day⁻¹, p.o.) were administered continuously for 12 weeks in rats and 8 weeks in rabbits. Cardiac function, electrophysiological disturbances, cardiac collagen, cardiomyocte apoptosis and the remodelling-related proteins were evaluated.

KEY RESULTS

Rabbits treated with ketamine showed decreased left ventricular ejection fraction, slowed ventricular conduction velocity and increased susceptibility to ventricular arrhythmia. Metoprolol prevented these pathophysiological alterations. In ketamine-treated rats, cardiac collagen volume fraction and apoptotic cell number were higher than those of control animals; these effects were prevented by co-administration of metoprolol. Consistently, the expressions of poly (ADP-ribose) polymerases-1, apoptosis-inducing factor and NF-κB-light-chain-enhancer of activated B cells were all increased after ketamine treatment and sharply reduced after metoprolol administration. Moreover, ketamine enhanced sympathetic sprouting, manifested as increased growth-associated protein 43 and tyrosine TH expression. These effects of ketamine were prevented by metoprolol.

CONCLUSIONS AND IMPLICATIONS

Chronic treatment with ketamine caused significant ventricular myocardial apoptosis, fibrosis and sympathetic sprouting, which altered the electrophysiological properties of the heart and increased its susceptibility to malignant arrhythmia that may lead to sudden cardiac death. Metoprolol prevented the cardiotoxicity of ketamine, indicating a promising new therapeutic strategy.     

Degarelix, a novel GnRH antagonist, causes minimal histamine release compared with cetrorelix, abarelix and ganirelix in an ex vivo model of human skin samples

AIMS

Early studies on gonadotrophin-releasing hormone (GnRH) antagonists pointed out histamine-mediated anaphylactic reactions as a potential adverse effect of these drug candidates. In this study we have compared the histamine-releasing potential of four approved and marketed antagonists, degarelix, cetrorelix, abarelix and ganirelix in an ex vivo model of human skin samples.

METHODS

Human skin samples were obtained during cosmetic plastic surgery and kept in oxygenated saline solution. The samples were incubated either without or at different concentrations of the antagonists (3, 30 or 300 µg ml⁻¹ for all, except for ganirelix 1, 10 or 100 µg ml⁻¹). The drug-induced effect was expressed as the increase relative to basal release. The histamine-releasing capacity of the skin was verified by a universal histamine releaser, compound 40/80.

RESULTS

Degarelix had no significant effect on basal histamine release in the 3 to 300 µg ml⁻¹ concentration range. The effect of ganirelix was moderate causing a nonsignificant increase of 81 ± 27% at the 100 µg ml⁻¹ concentration. At 30 and 300 µg ml⁻¹ concentrations abarelix (143 ± 29% and 362 ± 58%, respectively, P < 0.05) and cetrorelix (228 ± 111% and 279 ± 46%, respectively, P < 0.05) caused significantly increased histamine release.

CONCLUSIONS

In this ex vivo human skin model, degarelix displayed the lowest capacity to release histamine followed by ganirelix, abarelix and cetrorelix. These findings may provide indirect hints as to the relative likelihood of systemic anaphylactic reactions in clinical settings.     

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.     

Loss of multidrug and toxin extrusion 1 (MATE1) is associated with metformin-induced lactic acidosis

BACKGROUNDS AND PURPOSE

Lactic acidosis is a fatal adverse effect of metformin, but the risk factor remains unclear. Multidrug and toxin extrusion 1 (MATE1) is expressed in the luminal membrane of the kidney and liver. MATE1 was revealed to be responsible for the tubular and biliary secretion of metformin. Therefore, some MATE polymorphisms, that cause it to function abnormally, are hypothesized to induce lactic acidosis. The purpose of this study is to clarify the association between MATE dysfunction and metformin-induced lactic acidosis.

EXPERIMENTAL APPROACH

Blood lactate, pH and bicarbonate ion (HCO₃^-) levels were evaluated during continuous administration of 3 mg·mL⁻¹ metformin in drinking water using Mate1 knockout (−/−), heterozygous (+/−) and wild-type (+/+) mice. To determine the tissue accumulation of metformin, mice were given 400 mg·kg⁻¹ metformin orally. Furthermore, blood lactate data were obtained from diabetic patients given metformin.

KEY RESULTS

Seven days after metformin administration in drinking water, significantly higher blood lactate, lower pH and HCO₃^- levels were observed in Mate1^−/− mice, but not in Mate1^+/− mice. The blood lactate levels were not affected in patients with the heterozygous MATE variant (MATE1-L125F, MATE1-G64D, MATE2-K-G211V). Sixty minutes after metformin administration (400 mg·kg⁻¹, p.o.) the hepatic concentration of metformin was markedly higher in Mate1^−/− mice than in Mate1^+/+ mice.

CONCLUSION AND IMPLICATIONS

MATE1 dysfunction caused a marked elevation in the metformin concentration in the liver and led to lactic acidosis, suggesting that the homozygous MATE1 variant could be one of the risk factors for metformin-induced lactic acidosis.     

A cannabinoid receptor, sensitive to O-1918, is involved in the delayed hypotension induced by anandamide in anaesthetized rats

Background and purpose:

Intravenous injection of the endocannabinoid anandamide induces complex cardiovascular changes via cannabinoid CB₁, CB₂ and vanilloid TRPV1 receptors. Recently, evidence has been accumulating that in vitro, but not in vivo, anandamide relaxes blood vessels, via an as yet unidentified, non-CB₁ vascular cannabinoid receptor, sensitive to O-1918 (1,3-dimethoxy-5-2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-benzene). We here examined whether the anandamide-induced hypotension in urethane-anaesthetized rats was also mediated via a non-CB₁ vascular cannabinoid receptor.

Experimental approach:

Effects of two antagonists (O-1918 and cannabidiol) of the non-CB₁ vascular cannabinoid receptor on anandamide-induced changes in mean, systolic and diastolic blood pressure (MBP, SBP, DBP), mesenteric (MBF) and renal (RBF) blood flow and heart rate (HR) in urethane-anaesthetized rats was examined.

Key results:

In anaesthetized rats, anandamide (1.5–3 µmol·kg⁻¹) and its stable analogue methanandamide (0.5 µmol·kg⁻¹) caused a delayed and prolonged decrease in MBP, SBP, DBP, MBF and RBF by about 10–30% of the respective basal values without changing HR. In pithed rats, anandamide (3 µmol·kg⁻¹) decreased blood pressure by about 15–20% of the basal value without affecting HR, MBF and RBF. All vascular changes were reduced by about 30–70% by cannabidiol and O-1918 (3 µmol·kg⁻¹, each).

Conclusions and implications:

Non-CB₁ cannabinoid vascular receptors, sensitive to O-1918, contribute to the hypotensive effect of anandamide in anaesthetized rats. Activation of these receptors may be therapeutically important as the endocannabinoid system could be activated as a compensatory mechanism in various forms of hypertension.     

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.     

Ethyl pyruvate promotes spinal cord repair by ameliorating the glial microenvironment

BACKGROUND AND PURPOSE

Spinal cord injury (SCI) triggers a series of endogenous processes, including neuroinflammation and reactive astrogliosis, which may contribute to the failure of neural regeneration and functional recovery. In the present study, the effect of ethyl pyruvate on spinal cord repair was explored.

EXPERIMENTAL APPROACH

Functional assessment and histological analyses of astrogliosis, neuroinflammation, neuronal survival and axonal regeneration were performed to investigate the effects of ethyl pyruvate (0.086, 0.215, 0.431 or 0.646 mmol·kg⁻¹·day⁻¹) on spinal cord repair in a rat model of SCI. The effect of ethyl pyruvate (5, 10 or 15 mM) on astrocytic activation was also evaluated in an in vitro‘scratch-wound’ model.

KEY RESULTS

Functional assessment showed evident improvement of behavioural functions in the ethyl pyruvate-treated rats. Reactive astrogliosis was significantly inhibited in vivo, after injection of ethyl pyruvate (0.431 mmol·kg⁻¹day⁻¹), and in vitro‘scratch-wound’ model in the presence of 10 or 15 mM ethyl pyruvate. The difference between effective concentration in vitro and in vivo suggests that the inhibitory effect of ethyl pyruvate on astrogliosis in damaged spinal cord is indirect. In addition, ethyl pyruvate (0.431 mmol·kg⁻¹day⁻¹) attenuated SCI-induced neuroinflammation; it decreased the Iba-1-, ED-1- and CD11b-positive cells at the lesion site. Importantly, histological analyses showed a significantly greater number of surviving neurons and regenerative axons in the ethyl pyruvate-treated rats.

CONCLUSIONS AND IMPLICATIONS

Ethyl pyruvate was shown to inhibit astrogliosis and neuroinflammation, promote neuron survival and neural regeneration, and improve the functional recovery of spinal cord, indicating a potential neuroprotective effect of ethyl pyruvate against SCI.     

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.     

Moxonidine improves cardiac structure and performance in SHR through inhibition of cytokines, p38 MAPK and Akt

BACKGROUND AND PURPOSE

Regression of left ventricular hypertrophy by moxonidine, a centrally acting sympatholytic imidazoline compound, results from a sustained reduction of DNA synthesis and transient stimulation of DNA fragmentation. Because apoptosis of cardiomyocytes may lead to contractile dysfunction, we investigated in spontaneously hypertensive rats (SHR), time- and dose-dependent effects of in vivo moxonidine treatment on cardiac structure and function as well as on the inflammatory process and signalling proteins involved in cardiac cell survival/death.

EXPERIMENTAL APPROACH

12 week old SHR received moxonidine at 0, 100 and 400 µg·kg⁻¹·h⁻¹, s.c., for 1 and 4 weeks. Cardiac function was evaluated by echocardiography; plasma cytokines were measured by elisa and hearts were collected for histological assessment of fibrosis and measurement of cardiac proteins by Western blotting. Direct effects of moxonidine on cardiac cell death and underlying mechanisms were investigated in vitro by flow cytometry and Western blotting.

KEY RESULTS

After 4 weeks, the sub-hypotensive dose of moxonidine (100 µg) reduced heart rate and improved global cardiac performance, reduced collagen deposition, regressed left ventricular hypertrophy, inhibited Akt and p38 MAPK phosphorylation, and attenuated circulating and cardiac cytokines. The 400 µg dose resulted in similar effects but of a greater magnitude, associated with blood pressure reduction. In vitro, moxonidine inhibited norepinephrine-induced neonatal cardiomyocyte mortality but increased fibroblast mortality, through I₁-receptor activation and differential effects on downstream Akt and p38 MAPK.

CONCLUSIONS AND IMPLICATIONS

While the antihypertensive action of centrally acting imidazoline compounds is appreciated, new cardiac-selective I₁-receptor agonists may confer additional benefit.