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[Endocrine and Diabetes] Pharmacokinetics and pharmacodynamics of LGD-3303, an orally available non-steroidal selective androgen receptor modulator (SARM)

2008-11-18

Selective androgen receptor modulators (SARMs) are a new class of molecules in development to treat a variety of diseases. SARMs maintain the beneficial effects of androgens, including increased muscle mass and bone density, while having reduced activity on unwanted side effects. The mechanisms responsible for the tissue selective activity of SARMs are not fully understood and the pharmacokinetic/pharmacodynamic (PK/PD) relationships are poorly described. Tissue specific compound distribution could potentially be a mechanism responsible for apparent tissue selectivity. We examined the PK/PD relationship of a novel SARM, LGD-3303, in a castrated rat model of androgen deficiency. LGD-3303 has potent activity on levator ani muscle but is a partial agonist on the preputial gland and ventral prostate. LGD-3303 never stimulated ventral prostate above intact levels in spite of increasing plasma concentrations of compound. Tissue selective activity was maintained when LGD-3303 was dosed orally or by continuous infusion, two routes of administration with markedly different time vs. exposure profiles. In spite of the greater muscle activity relative to prostate activity, local tissue concentrations of LGD-3303 were higher in the prostate than in the levator ani muscle. LGD-3303 has SARM properties that are independent of its pharmacokinetic profile suggesting that the principle mechanism for tissue selective activity is the result of altered molecular interactions at the level of the androgen receptor.

[Chemotherapy, Antibiotics, and Gene Therapy] New ifosfamide analogues designed for lower associated neurotoxicity and nephrotoxicity with modified alkylating kinetics leading to enhanced in vitro anticancer activity

2008-11-18

Ifosfamide is an useful prodrug with CYP450 metabolism associated to both antitumor activity and toxicities. Isophosphoramide mustard is the bisalkylating active metabolite and acrolein, the urotoxic one. Since acrolein toxicity is limited by co-administration of mesna, the incidence of urotoxicity has been lowered. Current evidence suggests that chloroacetaldehyde, a side-chain oxidation metabolite is responsible for neurotoxicity and nephrotoxicity. The aim of our research is to prevent chloroacetaldehyde formation using new enantioselective synthesized ifosfamide analogues i.e. C7,C9-dimethyl-ifosfamide. We hypothesise that reduced toxicogenic catabolism may induce less toxicity without change in the anticancer activity. Metabolites determination of the dimethyl-ifosfamide analogues was performed using liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) after in vitro biotransformation by drug-induced rat liver microsomes and human microsomes expressing the main CYP 3A4 and minor CYP2B6 enzymes. Both human and rat microsomes incubations produced the same N-dechloroalkylated and 4-hydroxylated metabolites. Then, a co-culture assay of 9L rat glioblastoma cells and rat microsomes was performed to evaluate their cytotoxicity. And finally, a mechanistic study using Phosphorus Nuclear Magnetic Resonance (³¹P-NMR) kinetics allowed estimating the alkylating activity of the modified mustards. The results showed that, analogues were still metabolized through the same N-deschloroalkylation pathway but they showed an increased activity. They were 4 to 6-fold more cytotoxic than ifosfamide on 9L and the generated dimethylated mustards were 28-fold faster alkylating agents than ifosfamide mustards. Among these new ifosfamide analogues, the 7S,9R enantiomer will be assessed for further in vivo investigations for its anticancer activity and its toxicological profile.

[Cardiovascular] Role of PKC{zeta} and Calcium Entry in KCl-Induced Vascular Smooth Muscle Calcium Sensitization and Feed Back Control of Cellular Calcium Levels

Ratz, P. H., Miner, A. S. — 2008-11-14

The degree of tonic force (F) maintenance induced in vascular smooth muscle (VSM) upon K⁺-depolarization (KCl) can be greatly reduced by inhibition of rhoA kinase (ROCK). We explored the possibility that a PKC isotype may also play a role in causing KCl-induced Ca²⁺ sensitization. In isometric rings of rabbit artery, the PKC inhibitors, Go 6983, GF-109203X and a cell permeable (myristoylated) pseudosubstrate inhibitor of PKC (PI_PKC), inhibited KCl-induced tonic F. Rather than inhibiting KCl-induced F, a myristoylated pseudosubstrate inhibitor of PKC/β that inhibited phorbol dibutyrate-induced F slightly potentiated KCl-induced tonic F. Whereas the ROCK inhibitor, H-1152, reduced basal phosphorylation of myosin light chain (MLC) phosphatase (MLCP) targeting subunit at threonine 853 (MYPT1-pT853), 3 µM and 10 µM GF-109203X inhibited only KCl-stimulated phosphorylation, not basal MYPT1-pT853. In fura-2-loaded tissues, GF-109203X and PI_PKC elevated basal [Ca²⁺]_i (Ca) and potentiated KCl-induced tonic increases in Ca while reducing KCl-induced tonic increases in F. Blockade by nifedipine of Ca²⁺ entry through voltage-operated Ca²⁺ channels (VOCCs) reduced KCl-induced Ca²⁺ sensitization and KCl-stimulated but not basal MYPT1-pT853. These data together support a model in which ROCK and PKC are constitutively active and function in "resting" muscle to regulate the basal levels of MYPT1-pT853 and Ca, respectively. In this model, KCl-induced increases in Ca activate PKC to feed forward and cause additional MYPT1p-T853 above that induced by constitutive ROCK, permitting Ca²⁺ sensitization and strong F maintenance. Active PKC also feeds back to attenuate the degree of KCl-induced increases in Ca.

[Cardiovascular] Increased Endothelial Nitric Oxide Synthase Expression Reduces Hypertension and Hyperinsulinemia in Fructose-treated Rats

2008-11-13

Endothelial dysfunction and decreased production of nitric oxide (NO) by endothelial NO synthase (eNOS) are implicated in the pathogenesis of hypertension and insulin resistance. As the potential influence of increased eNOS expression/activity on these parameters is unclear, the present study examined the effects of eNOS gene therapy on insulin resistance and blood pressure alterations in a fructose-induced hypertension model in rats. As predicted, two weeks of fructose consumption in the drinking water resulted in elevated systolic blood pressure and insulin resistance. These and other physiologic alterations were reversed within 2 weeks following a single intravenous injection of a vector containing the human eNOS cDNA (pcDNA3.1-eNOS), whereas injection of an empty vector (pcDNA3.1) was without effect. In support of the beneficial effects of pcDNA3.1-eNOS treatment being due to enhanced eNOS expression and activity, increased eNOS protein levels were documented in aorta, liver, kidney and heart of fructose-treated rats injected with pcDNA3.1-eNOS, and corresponding elevations in nitrite/nitrate and cGMP concentrations were observed in urine. Furthermore, pcDNA3.1-eNOS treatment prevented fructose-induced decreases in expression levels of IRS-1, the p110 catalytic subunit of phosphatidylinositol 3-kinase (PI3K), phosphorylated Akt, and phosphorylated AMP-activated protein kinases (AMPK) in liver, aorta and skeletal muscle. Cumulatively, the results of this study indicate that gene therapy with human eNOS decreased fructose-induced hypertension and insulin resistance in rats, and suggest potential signalling pathways that mediate these effects. These data highlight the potential utility of eNOS gene therapy in the treatment of hypertension and insulin resistance.

[Cellular and Molecular] Inhibition of LPS Stimulated COPD Macrophage Inflammatory Gene Expression by Dexamethasone and the p38 MAPK Inhibitor SB706504

2008-11-12

p38 MAPK signalling is known to be increased in COPD macrophages. We have studied the effects of the p38 MAPK inhibitor SB706504 and dexamethasone on COPD macrophage inflammatory gene expression, and protein secretion. We also studied the effects of combined SB706504 and dexamethasone treatment. LPS stimulated monocyte derived macrophages (MDMs) and alveolar macrophages (AM) were cultured with dexamethasone and /or SB706504. MDMs were used for gene array and protein studies, while TNF protein production was measured from AM. SB706504 caused transcriptional inhibition of a range of cytokines and chemokines in COPD MDMs. The use of SB706504 combined with dexamethasone caused greater suppression of gene expression (-8.90) compared to SB706504 alone (-2.04) or dexamethasone (-3.39). 23 genes were insensitive to the effects of both drugs, including IL-1β, IL-18 and CCL5. Also, the chromosome 4 chemokine cluster comprising CXCL1, CXCL2, CXCL3 and CXCL8 were all GC resistant. SB706504 significantly inhibited LPS stimulated TNF production from COPD and smoker AM, with near maximal suppression caused by combination treatment with dexamethasone. We conclude that SB706504 targets a subset of inflammatory macrophage genes, and when used with dexamethasone causes effective suppression of these genes. SB706504 and dexamethasone had no effect on the transcription of a subset of LPS regulated genes including IL-1β, IL-18 and CCL5, which are all known to be involved in the pathogenesis of COPD.

[Metabolism, Transport, and Pharmacogenomics] Functional characterization of multidrug and toxin extrusion protein 1 as a facilitative transporter for fluoroquinolones

Ohta, K.-y., Imamura, Y., Okudaira, N., Atsumi, R., Inoue, K., Yuasa, H. — 2008-11-12

Many fluoroquinolones are mainly eliminated by urinary excretion, in which tubular secretion by carrier-mediated transport systems has been suggested to be involved. In the present study, we examined the possibility that multidrug and toxin extrusion protein 1 (MATE1), which is abundantly expressed in the kidney, might be involved in that, using rat MATE1 (rMATE1) expressed in MDCKII cells. It was found that rMATE1 can transport fluoroquinolones such as ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, norfloxacin (NFX), pazufloxacin and tosufloxacin. Although rMATE1 has been known as an apical organic cation/H⁺ antiporter, detailed investigation of rMATE1-mediated uptake of NFX has revealed that it is not sensitive to intracellular acidification by treatments using NH₄Cl or nigericin, suggesting that transmembrane proton gradient is not involved in its transport as a driving force. However, it was dependent on extracellular pH, being greatest at pH 7.0 and smaller at both acidic and basic pH in agreement with the profile of zwitterionization of NFX. The basal-to-apical transcellular transport of NFX in rMATE1-expressing MDCKII cells was greater than that in mock cells and insensitive to acidification of the apical medium, demonstrating proton-gradient-independent functionality of rMATE1 in NFX efflux. Finally, rMATE1-mediated NFX uptake at pH 7.4 was saturable with the Michaelis constant of 55.3 µM and inhibited by cationic compounds, such as tetraethylammonium and cimetidine. These results suggest that rMATE1 mediates the transport of NFX by a facilitative manner. MATE1 may play a key role in the renal tubular secretion of fluoroquinolones.

[Metabolism, Transport, and Pharmacogenomics] Physiologically Based Pharmacokinetic Modeling to Predict Transporter-Mediated Clearance and Distribution of Pravastatin in Humans

Watanabe, T., Kusuhara, H., Maeda, K., Shitara, Y., Sugiyama, Y. — 2008-11-10

Hepatobiliary excretion mediated by transporters, OATP1B1 and MRP2, is the major elimination pathway of an HMG-Co A reductase inhibitor, pravastatin. The present study examined the effects of changes in the transporter activities on the systemic and liver exposure of pravastatin using a physiologically based pharmacokinetic model. Scaling factors, determined by comparing in vivo and in vitro parameters of pravastatin in rats for the hepatic uptake and canalicular efflux, were obtained. The simulated plasma and liver concentrations, and biliary excretion profiles were very close to the observed data in rats under linear and nonlinear conditions. In vitro parameters, determined in human cryopreserved hepatocytes and canalicular membrane vesicles, were extrapolated to in vivo parameters using the scaling factors obtained in rats. The simulated plasma concentrations of pravastatin were close to the reported values in humans. Sensitivity analyses showed that changes in the hepatic uptake ability altered the plasma concentration of pravastatin markedly, but had a minimal effect on the liver concentration, while changes in the ability of canalicular efflux altered the liver concentration of pravastatin markedly, but had a small effect on the plasma concentration. In conclusion, the model allows the prediction of the disposition of pravastatin in humans. The present study suggests that changes in the OATP1B1 activities may have a small and a large impact on the therapeutic efficacy and side effect (myopathy) of pravastatin, respectively, whereas those in the MRP2 activities may have opposite impacts (i.e., a large and a small impact on the therapeutic efficacy and side effect).

[Behavioral Pharmacology] Unconditioned behavioral effects of the powerful {kappa}-opioid hallucinogen, salvinorin A: Fast onset and entry into cerebrospinal fluid

Butelman, E. R., Prisinzano, T. E, Deng, H., Rus, S., Kreek, M. J. — 2008-11-10

Salvinorin A is the main active component of the widely available hallucinogenic plant, Salvia divinorum. Salvinorin A is a selective high efficacy -agonist in vitro, with some unique pharmacodynamic properties. Descriptive reports show that salvinorin A - containing products produce robust behavioral effects in humans. However, these effects have not been systematically characterized in human or non-human primates to date. The present studies therefore focused on the characterization of overt effects of salvinorin A, such as sedation (operationally defined as unresponsiveness to environmental stimuli) and postural relaxation, previously observed with centrally-penetrating -agonists in non-human primates. Salvinorin A was active in these endpoints (dose range: 0.01-0.1 mg/kg, i.v.) in non-human primates (n=3-5), similarly to the synthetic -agonist U69,593, used for comparison herein. Salvinorin A effects could be prevented by a clinically available opioid antagonist, nalmefene (0.1 mg/kg) at doses known to block -receptor mediated effects in non-human primates. When injected i.v., salvinorin A (0.032 mg/kg) could enter the CNS (as reflected in cisternal CSF) within 1 minute, and reach concentrations that are in the reported range of the affinity (K_i) of this ligand for brain -receptors. Consistent with this finding, specific translationally viable behavioral effects (e.g., facial relaxation and ptosis) could also be detected within 1-2 min of injection of salvinorin A. These are the first studies documenting rapid unconditioned effects of salvinorin A in a primate species, consistent with descriptive reports of rapid and robust effects of this powerful hallucinogen in humans.

[Chemotherapy, Antibiotics, and Gene Therapy] Carbendazim inhibits cancer cell proliferation by suppressing microtubule dynamics

Yenjerla, M., Cox, C., Wilson, L., Jordan, M. A. — 2008-11-10

Carbendazim (methyl 2-benzimidazolecarbamate) is widely used as a systemic fungicide in human food production and appears to act on fungal tubulin. However, it also inhibits proliferation of human cancer cells including drug- and multidrug-resistant and p53-deficient cell lines. Due to its promising preclinical antitumor activity, it has undergone Phase I clinical trials and is under further clinical development. While it weakly inhibits polymerization of brain microtubules and induces G2/M arrest in tumor cells, its mechanism of action in human cells has not been fully elucidated. We examined its mechanism of action in MCF7 human breast cancer cells and found that it inhibits proliferation (IC₅₀: 10 µM) and half-maximally arrests mitosis at a similar concentration (8 µM), in concert with suppression of microtubule dynamic instability without appreciable microtubule depolymerization. It induces mitotic spindle abnormalities and reduces the metaphase intercentromere distance of sister chromatids, indicating reduction of tension on kinetochores, thus leading to metaphase arrest. With microtubules assembled in vitro from pure tubulin, carbendazim also suppresses dynamic instability, reducing the dynamicity by 50% at 10 µM with only minimal (21%) reduction of polymer mass. Carbendazim binds to mammalian tubulin (K_d, 42.8 ± 4.0 µM). Unlike some benzimidazoles that bind to the colchicine-site in tubulin, carbendazim does not compete with colchicine nor does it compete with vinblastine for binding to brain tubulin. Thus carbendazim binds to an as yet unidentified site in tubulin and inhibits tumor cell proliferation by suppressing the growing and shortening phases of microtubule dynamic instability, thus inducing mitotic arrest.

[Gastrointestinal, Hepatic, Pulmonary, and Renal] Peroxisome-proliferator activated receptor-{alpha} contributes to the resolution of inflammation following renal ischemia/reperfusion injury

2008-11-07

This study was designed to elucidate the role of peroxisome-proliferator activated receptor (PPAR)- in the development of inflammation following ischemia/reperfusion injury of the kidney. We have evaluated the effects of ischemia/reperfusion on renal dysfunction, injury and inflammation in wild-type mice or mice in which the gene for PPAR- has been deleted (PPAR-^-/-), and then treated with the PPAR- agonist fenofibrate. Mice were subjected to bilateral renal ischemia (30 min) and reperfusion (24 h), and received fenofibrate (3 mg/kg, i.p.) prior to reperfusion. Plasma creatinine, urea and aspartate aminotransferase were all used as indicators of renal dysfunction and injury. Kidneys were used for histological and immunohistochemical analysis, and markers of inflammation. Fenofibrate significantly attenuated the degree of renal dysfunction, injury and inflammation caused by ischemia/reperfusion injury. The degree of renal dysfunction, injury and inflammation caused by ischemia/reperfusion was also significantly augmented in PPAR-^-/- mice when compared to their wild-type littermates. Interestingly, fenofibrate did not protect PPAR-^-/- mice against ischemia/reperfusion injury. We, therefore, propose that ligands of PPAR- may be useful in the treatment of renal ischemia/reperfusion injury, and that endogenous PPAR- limits the degree of renal dysfunction, injury and inflammation associated with ischemia/reperfusion injury.

[Cardiovascular] CGRP Receptor Antagonism Does Not Affect the Severity of Myocardial Ischemia during Atrial Pacing in Dogs with Coronary Artery Stenosis

Regan, C., Stump, G., Kane, S., Lynch, J. — 2008-11-07

CGRP is a sensory neuropeptide that also has potent vasodilator activity. There are conflicting preclinical reports regarding the effect of CGRP receptor antagonism in the setting of myocardial ischemia. The present studies were conducted in a canine model in which regional myocardial ischemia was reproducibly evoked by serial periods of atrial pacing (80 bpm above baseline rate) in the presence of a 40% stenosis of the left anterior descending (LAD) coronary artery. Ischemia severity was quantitated by changes in unipolar epicardial electrograms (EG) recorded in the area of ischemia. In validation studies, the calcium entry blocker diltiazem reduced ischemia severity (pre vs post-treatment: EG: 1.92±0.23 vs 0.54±0.24 mV, p<0.05) and tended to increase LAD flow (7.7±0.7 vs 9.4±1.4 ml/min, p=0.10), whereas the coronary constrictor serotonin increased ischemia severity (pre vs post-treatment: EG: 2.11±0.44 vs 4.90±1.46 mV, p<0.05) concomitant with a reduction in LAD flow (9.1±1.1 vs 5.4±1.5 ml/min, p<0.05). A 30 µg/kg/min i.v. infusion test dose of the CGRP receptor antagonist CGRP(_8-37) was validated by demonstrating complete block of the depressor effects of exogenous i.v. 0.03-0.3 µg/kg CGRP. This dose of CGRP(_8-37), administered either i.v. or i.a., had no effect on ischemia severity or paced LAD flow, indicating no intrinsic effect of CGRP receptor antagonism on the severity of acute myocardial ischemia. Likewise, the administration of a hemodynamically active dosing regimen of CGRP (0.03 µg/kg/min i.v.) had no effect on paced coronary flow or ischemia severity, suggesting no major role of CGRP in regulating ischemic blood flow.

[Inflammation, Immunopharmacology, and Asthma] Anti-Ccl2 Spiegelmer permits 75% dose reduction of cyclophosphamide to control diffuse proliferative lupus nephritis and pneumonitis in MRL-Fas(lpr) mice

2008-11-07

Cyclophosphamide (CYC) can control diffuse proliferative lupus nephritis (DPLN) by potent immunosuppression but remains associated with serious and life threatening complications. Drugs that specifically target mediators of DPLN may help to reduce CYC dose and side effects. Monocyte chemoattractant protein MCP-1/CCL2 mediates monocyte and T cell recruitment in DPLN and Ccl2-specific L-enantiomeric RNA Spiegelmer mNOX-E36 neutralizes the biological effects of murine Ccl2 in-vitro and in-vivo. We injected MRL^lpr/lpr mice with DPLN from 14 weeks of age with either vehicle, weekly 30 mg/kg CYC (full dose), monthly 30 mg/kg CYC (1/4 full dose), pegylated control Spiegelmer, pegylated anti-Ccl2 Spiegelmer (3/week), pegylated anti-Ccl2 Spiegelmer plus CYC 1/4 full dose and mycophenolate mofetil (MMF). At week 24, DPLN and autoimmune lung injury were virtually abolished with CYC full dose but not with CYC 1/4 full dose. The CYC 1/4 full dose/Spiegelmer combination was equipotent to CYC full dose on kidney and lung injury. CD3⁺CD4^-CD8^- and CD3⁺CD4⁺CD25⁺ T cells, serum IL-12p40 and TNF- levels which were all markedly affected by CYC full dose but not by CYC 1/4 full dose. No additive effects of anti-Ccl2 Spiegelmer were noted on bone marrow CFU-GM counts and 7/4 ^high monocyte counts, lymphoproliferation, spleen T cell depletion. In summary, anti-Ccl2 Spiegelmer permits 75% dose reduction of CYC for controling DPLN and pneumonitis in MRL-Fas(lpr) mice, sparing suppressive effects of full dose CYC on myelosuppression and T cell depletion. We propose anti-Ccl2 Spiegelmer therapy as novel strategy to reduce CYC toxicity in the treatment of severe lupus

[Gastrointestinal, Hepatic, Pulmonary, and Renal] Vitamin D3 Modulates the Expression of Bile Acid Regulatory Genes and Represses Inflammation in Bile Duct-Ligated Mice

2008-11-06

Vitamin D receptor (VDR), a nuclear receptor that regulates calcium homeostasis, has been found to function as a receptor for secondary bile acids. Since the in vivo role of VDR in bile acid metabolism remains unknown, we investigated the effect of VDR activation in a mouse model of cholestasis. We treated mice with 1-hydroxyvitamin D₃ [1(OH)D₃] after bile duct ligation (BDL) and examined mRNA expression and cytokine levels. 1(OH)D₃ treatment altered the expression of genes involved in bile acid synthesis and transport in the liver, kidney and intestine, but did not decrease bile acid levels in the plasma and liver of BDL mice. 1(OH)D₃ treatment suppressed mRNA expression of proinflammatory cytokines in the liver, and strongly decreased the plasma levels of proinflammatory cytokines in BDL mice. These findings indicate that 1(OH)D₃ regulates a network of bile acid metabolic genes and represses proinflammatory cytokine expression in BDL mice. VDR ligands have the potential to prevent the cholestasis-induced inflammatory response.

[Cardiovascular] Parstatin, the cleaved peptide on proteinase-activated receptor 1 activation, is a potent inhibitor of angiogenesis

2008-11-06

The proteolytic activation by thrombin of the proteinase-activated receptor 1 (PAR1) unveils the tethered peptide ligand and cleaves a 41 amino acids peptide. In this report we show that this peptide, which we have designated as "parstatin", is a potent inhibitor of angiogenesis. Synthesized parstatin suppressed both the basic angiogenesis and that stimulated by basic fibroblast growth factor and vascular endothelial growth factor in the chick embryo model in vivo and in the rat aortic rings assay. Parstatin also abrogated endothelial cell migration and capillary-like network formation on the Matrigel and fibrin angiogenesis models in vitro. Treatment of endothelial cells with parstatin resulted in inhibition of cell growth by inhibiting the phosphorylation of extracellular signal-regulated kinases in a specific and reversible fashion and by promoting cell cycle arrest and apoptosis through a mechanism involving activation of caspases. We have shown that parstatin acts as a cell-penetrating peptide, exerting its biological effects intracellularly. The uptake into cells and the inhibitory activity were dependent on parstatin hydrophobic region. These results support the notion that parstatin may represent an important negative regulator of angiogenesis with possible therapeutic applications.

[Cardiovascular] CHYMASE-DEPENDENT CONVERSION OF BIG ENDOTHELIN-1 IN THE MOUSE IN VIVO

Simard, E., Jin, D., Takai, S., Miyazaki, M., Brochu, I., D'Orleans-Juste, P. — 2008-11-05

The aim of this study was to identify the role of chymase in the conversion of exogenously administered big endothelin-1 in the mouse in vivo. Real time PCR analysis detected mRNA of mucosal mast cell chymase 4 and 5 as well as endothelin converting enzyme 1a and neutral endopeptidase 24.11 in pulmonary, cardiac and aorta homogenates derived from C57BL/6J mice with the later tissue expressing the highest levels of both chymase isoforms. Furthermore, hydrolysis of a fluorogenic peptide substrate, Suc-Leu-Leu-Val-Tyr-MCA, was sensitive to the chymase inhibitors Suc-Val-Pro-Phe^P (OPh)₂ (200 µM) and chymostatin (100 µM) in supernatants extracted from the same tissue homogenates. In anesthetized mice, big endothelin-1, endothelin-1 (1-31) and endothelin-1 triggered pressor responses (ED50s; 0.67, 0.89 and 0.16 nmol/kg) which were all reduced or potentiated by selective ET_A or ET_B receptor antagonists, respectively (BQ-123 or BQ-788; each at 1 mg/kg). The pressor responses to big endothelin-1 were significantly reduced by the neutral endopeptidase inhibitor thiorphan (1 mg/kg) or the endothelin converting enzyme inhibitor CGS 35066 (0.1 mg/kg). In contrast, the responses to endothelin-1 (1-31) were abolished by thiorphan, but unaffected by CGS 35066. In addition, Suc-Val-Pro-Phe^P (OPh)₂ (20-40 mg/kg) reduced by more than 60% the hemodynamic response to big endothelin-1 but not to endothelin-1 (1-31) and endothelin-1. Finally intravenous administration of big endothelin-1 induced Suc-Val-Pro-Phe^P (OPh)₂-sensitive increases in plasma immunoreactive levels of endothelin-1 (1-31) and endothelin-1. The present study suggests that chymase plays a pivotal role in the conversion and cardiovascular properties of big endothelin-1 in vivo.

[Neuropharmacology] Novel, potent and selective GABAC antagonists inhibit myopia development and facilitate learning and memory

2008-11-04

This study reports pharmacological and physiological effects of cis- and trans-(3-aminocyclopentanyl)butylphosphinic acid (cis- and trans-3-ACPBPA). These compounds are conformationally restricted analogs of the orally active GABA_B/C receptor antagonist, (3-aminopropyl)-n-butylphosphinic acid (CGP36742 or SGS742). Cis- (IC₅₀(1)=5.06µM and IC₅₀(2)=11.08µM; n=4) and trans-3-ACPMPA (IC₅₀(1)=72.58µM and IC₅₀(2)=189.7 µM; n=4) appear competitive at GABA_C receptors expressed in Xenopus oocytes, having no effect as agonists (1 mM) but exerting weak antagonist (1 mM) effects on human GABA_A and GABA_B receptors. Cis-3-ACPBPA was more potent and selective than the trans compound being over 100-times more potent at GABA_C than GABA_A or GABA_B receptors. Cis-3-ACPBPA was further evaluated on dissociated rat retinal bipolar cells and dose dependently inhibited the native GABA_C receptor (IC₅₀=47±4.5µM; n=6). When applied to the eye as intravitreal injections, cis- and trans-3-ACPBPA prevented experimental myopia development, and inhibited the associated vitreous chamber elongation, in a dose dependent manner in the chick model. Doses only 10-times greater than required to inhibit recombinant GABA_C receptors caused the anti-myopia effects. Using intraperitoneal administration, cis- (30 mg/kg) and trans-3-ACPBPA (100 mg/kg) enhanced learning and memory in male Wistar rats; compared to vehicle there was a significant reduction in time for rats to find the platform in the Morris Water Maze task (n=10; p<0.05). As the physiological effects of cis- and trans-3-ACPBPA are similar to those reported for CGP36742, the memory and refractive effects of CGP36742 may be due in part to its GABA_C activity.

[Toxicology] Acute toxicity of organophosphorus compounds in guinea pigs is sex and age dependent and cannot be solely accounted for by acetylcholinesterase inhibition

Fawcett, W. P., Aracava, Y., Adler, M., Pereira, E. F. R., Albuquerque, E. X. — 2008-11-04

This study was designed to test the hypothesis that the acute toxicity of the nerve agents VX, soman and sarin in guinea pigs is age and sex dependent and cannot be fully accounted for by the irreversible inhibition of acetylcholinesterase (AChE). The subcutaneous doses of nerve agents needed to decrease 24-h survival of guinea pigs by 50% (LD50s) were estimated by probit analysis. In all animal groups, the rank order of LD50s was sarin > soman > VX. The LD50 of soman was not influenced by sex or age of the animals. In contrast, the LD50s of VX and sarin were lower in adult male than in age-matched female or younger guinea pigs. A colorimetric assay was used to determine the concentrations of nerve agents that inhibit in vitro 50% of AChE activity (IC50s) in guinea pig brain extracts, plasma, red blood cell and whole blood. A positive correlation between LD50s and IC50s for AChE inhibition would support the hypothesis that AChE inhibition is a major determinant of the acute toxicity of the nerve agents. However, such a positive correlation was found only between LD50s and IC50s for AChE inhibition in brain extracts from neonatal and prepubertal guinea pigs. These results demonstrate for the first time that the lethal potencies of some nerve agents in guinea pigs are age and sex dependent. They also support the contention that mechanisms other than AChE inhibition contribute to the lethality of nerve agents.

[Cardiovascular] Angiotensin II-induced hypertension is associated with a selective inhibition of EDHF-mediated responses in the rat mesenteric artery

2008-11-04

Hypertension has been shown to be associated with impaired endothelium-derived hyperpolarizing factor (EDHF)-mediated arterial relaxation and hyperpolarization. Treatments of hypertensive rats with inhibitors of the renin-angiotensin system have been shown to restore both EDHF-mediated responses and the expression of connexins involved in the intercellular transfer of the hyperpolarization, in mesenteric arteries. The present study was designed to determine whether chronic treatment of rats with angiotensin II impairs EDHF-mediated responses and the expression of connexins in the mesenteric arterial wall. Male Wistar rats were treated with angiotensin II (0.4 mg/kg/day) during 21 days using osmotic mini-pumps. Arterial pressure was measured by tail-cuff plethysmography. Contractile responses and membrane potential were measured in isolated mesenteric arteries. The expression of the three connexins Cx37, Cx40 and Cx43 was quantified in segments of mesenteric arteries by immunohistochemistry and quantitative real time RT-PCR. Angiotensin II administration increased the mean systolic blood pressure. EDHF-mediated relaxation and hyperpolarization to acetylcholine and red wine polyphenols were significantly impaired in mesenteric arteries from angiotensin II-treated rats in comparison with control animals whereas NO-mediated relaxation was unaltered. The expression of connexins Cx37, Cx40 and Cx43 was significantly decreased in the mesenteric artery from angiotensin II-treated rats. These findings indicate that angiotensin II-induced hypertension is associated with a selective impairment of EDHF-mediated relaxation and hyperpolarization in the rat mesenteric artery. The inhibition of EDHF-mediated responses is due, at least partly, to a decreased expression of connexins Cx37, Cx40 and Cx43 in the arterial wall.

[Neuropharmacology] The neuroprotective effects of benzylideneacetophenone derivatives on excitotoxicity and inflammation via pJAK2/pSTAT3 and MAPK pathways

Jang, S., Jung, J.-C., Kim, D. H., Ryu, J. H., Lee, Y., Jung, M., Oh, S. — 2008-11-04

To search for new neuroprotective compounds, novel benzylideneacetophenone compounds (JC1-JC6) were synthesized, and their potential to prevent neurotoxicities were evaluated. All compounds (JC1-JC6) showed considerable effect on free radical scavenging, the inhibition of glutamate-induced neurotoxicity in cortical cells, and the suppression of lipopolysaccharide (LPS)-induced nitric oxide (NO) generation in microglia. (2E)-3-(4-Hydroxy-3-methoxyphenyl)phenylpro-2-en-l-one (JC3) exhibited the most potent neuroprotective effect in ischemia model using organotypic hippocampal culture (OHC) and middle cerebral artery occlusion (MCAO). Based on the above results, the mechanisms underlying the biological activity of JC3, which exhibited potent anti-excitotoxic and anti-inflammatory effects, were determined using cortical neurons and microglia. Compound JC3 exerted a neuroprotective effect on oxygen-glucose deprivation (OGD)- and hydrogen peroxide (H₂O₂)-induced cytotoxicity in cultured cortical cells. In addition, it suppressed the generation of NO, pro-inflammatory cytokines, and reactive oxygen species (ROS) in LPS-treated microglial cells. It also suppressed the activation of pJAK2/pSTAT3 and MAPK in activated microglia in cortex and striatum after 3 days of the MCAO in mice. These results demonstrated that JC3 might affect a set of intracellular signaling cascades, including the janus tyrosine kinase/signal transducers and activators of transcription (JAK/STAT) and mitogen-activated protein kinase (MAPK) pathways. This study suggests that benzylideneacetophenone derivative could be useful anti-neurotoxic agents.

[Neuropharmacology] Effect of Novel Negative Allosteric Modulators of Neuronal Nicotinic Receptors on Cells Expressing Native and Recombinant Nicotinic Receptors: Implications for Drug Discovery

2008-11-04

Allosteric modulation of nAChRs is considered to be one of the most promising approaches for drug design targeting nicotinic acetylcholine receptors (nAChRs). We have previously reported on the pharmacological activity of several compounds that appear to act non-competitively to inhibit the activation of 3β4* nAChRs. In the following studies the effects of 51 structurally-similar molecules on native and recombinant 3β4 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native 3β4* nAChR with IC₅₀ values ranging from 0.4 to 13.0 µM. Using cells expressing recombinant 3β4 nAChRs, these molecules inhibited calcium accumulation (a more direct assay to establish nAChR activity) with IC₅₀ values ranging from 0.7 to 38.2 µM. Radiolabeled nAChR binding studies to orthosteric sites showed no inhibitory activity on either native or recombinant nAChRs. Correlation analyses of the data from both functional assays suggested additional, non-nAChR activity of the molecules. To test this hypothesis, the effects of the drugs on neurosecretion stimulated through non-nAChR mechanisms were investigated; inhibitory effects ranged from no inhibition to 95% inhibition at concentrations of 10 µM. Correlation analyses of the functional data confirmed this hypothesis. Several of the molecules (24/51) increased agonist binding to native nAChRs, supporting allosteric interactions with nAChRs. Computational modeling and blind docking identified a binding site for our negative allosteric modulators near the orthosteric binding site of the receptor. In summary, these studies identify several molecules for potential development as negative allosteric modulators and document the importance of multiple screening assays for nAChR drug discovery.

[Neuropharmacology] A functional role for sodium dependent glucose transport across the blood-brain barrier during oxygen glucose deprivation

2008-11-03

In the current study we determined the functional significance of sodium dependent/independent glucose transporters at the neurovasculature during oxygen glucose deprivation (OGD). Confluent brain endothelial cells cocultured with astrocytes were exposed to varying degrees of in vitro stroke conditions. Glucose transporter 1 (GLUT1) and sodium glucose cotransporter (SGLT) activity were investigated by luminal membrane uptake and transport studies using [³H] D-glucose and also by [¹⁴C] alpha methyl D-glucopyranoside (AMG), a specific, nonmetabolized substrate of SGLT. In vivo middle cerebral artery occlusion (MCAO) experiments were tested to determine if blood-brain barrier (BBB) SGLT activity was induced during ischemia. Increases in luminal D-glucose and AMG uptake and transport were observed with in vitro stroke conditions. Specific inhibitor experiments suggest a combined role for both SGLT and GLUT1 at the BBB during OGD. A time dependent increase in the uptake of AMG was also seen in mice exposed to permanent focal ischemia and this increase was sensitive to SGLT inhibitor, phlorizin. Infarct and edema ratio during ischemia were significantly decreased by the inhibition of this transporter. These results show that both GLUT1 and SGLT play a role at the BBB in the blood-to-brain transport of glucose during ischemic conditions and inhibition of SGLT during stroke has the potential to improve stroke outcome. Pharmacological modulation of this novel BBB transporter could prove to be a brain vascular target in stroke.

[Gastrointestinal, Hepatic, Pulmonary, and Renal] PF-03716556, a novel, potent, and selective acid pump antagonist for the treatment of gastro-esophageal reflux disease

2008-11-03

Inhibition of H,K-ATPase is accepted as the most effective way of controlling gastric acid secretion. However, current acid suppressant therapy for gastro-esophageal reflux disease, using histamine H₂ receptor antagonists and proton pump inhibitors, does not fully meet the needs of all patients because of their mechanism of action. This study sought to characterize the in vitro and in vivo pharmacology of a novel acid pump antagonist, N-(2-Hydroxyethyl)-N,2-dimethyl-8-{[(4R)-5-methyl-3,4-dihydro-2H-chromen-4-yl]amino}imidazo[1,2-a]pyridine-6-carboxamide (PF-03716556), and to compare with other acid suppressants. Porcine, canine, and human recombinant gastric H,K-ATPase activities were measured by ion-leaky and ion-tight assay. The affinities for a range of receptors, ion channels, and enzymes were determined to analyze selectivity profile. Acid secretion in Ghosh-Schild rats and Heidenhain pouch dogs were measured by titrating perfusate and gastric juice samples. PF-03716556 demonstrated three-fold greater inhibitory activity than revaprazan, the only acid pump antagonist that has been available on the market, in ion-tight assay. The compound did not display any species differences, exhibiting highly selective profile including the canine kidney Na,K-ATPase. Kinetics experiments revealed that PF-03716556 has a competitive and reversible mode of action. More rapid onset of action than omeprazole and three-fold greater potency than revaprazan were observed in Ghosh-Schild rats and Heidenhain pouch dogs. PF-03716556, a novel acid pump antagonist, could improve upon or even replace current pharmacological treatment for GERD.

[Gastrointestinal, Hepatic, Pulmonary, and Renal] FXR Deficiency in Mice Leads to Increased Intestinal Epithelial Cell Proliferation and Tumor Development

2008-11-03

Increased dietary fat consumption is associated with colon cancer development. The exact mechanism by which fat induces colon cancer is not clear, however, increased bile-acid excretion in response to high-fat diet may promote colon carcinogenesis. The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily and bile acids are endogenous ligands of FXR. FXR is highly expressed in the intestine and liver where FXR is essential for maintaining bile-acid homeostasis. The role of FXR in intestine cancer development is not known. The current study evaluated the effects of FXR deficiency in mice on intestinal cell proliferation and cancer development. The results showed that FXR deficiency resulted in increased colon cell proliferation, which was accompanied by an up-regulation in the expression of genes involved in cell-cycle progression and inflammation, including cyclin D1 and IL-6. Most importantly, FXR deficiency led to an increase in the size of small intestine adenocarcinomas in adenomatous polypsosis coli mutant (APC^min) mice. Furthermore, following treatment with a colon carcinogen, azoxymethane (AOM), FXR deficiency increased the adenocarcinoma multiplicity and size in colon and rectum of C57BL/6 mice. Loss of FXR function also increased the intestinal lymphoid nodule numbers in the intestine. Taken together, the current study is the first to show that FXR deficiency promotes cell proliferation, inflammation and tumorigenesis in the intestine, suggesting that activation of FXR by non-bile-acid ligands may protect against intestinal carcinogenesis.

[Cardiovascular] Molecular pharmacology of human Cav3.2 T-type Ca2+ channels: Block by antihypertensives, antiarrhythmics, and their analogs

Perez-Reyes, E., Van Deusen, A. L., Vitko, I. — 2008-10-30

Antihypertensive drugs of the "calcium channel blocker" or "calcium antagonist" class have been used to establish the physiological role of L-type Ca²⁺ channels in vascular smooth muscle. In contrast, there has been limited progress on the pharmacology T-type Ca²⁺ channels. T-type channels play a role in cardiac pacemaking, aldosterone secretion, and renal hemodynamics, leading to the hypothesis that mixed T- and L-type blockers may have a therapeutic advantages over selective L-type blockers. The goal of this study was to identify compounds that block the Ca_v3.2 T-type channel with high affinity, focusing on two classes of compounds: phenylalkylamines (e.g. mibefradil) and dihydropyridines (e.g. efonidipine). Compounds were tested using a validated Ca²⁺ influx assay into a cell line expressing recombinant Ca_v3.2 channels. This study identified four clinically approved antihypertensive drugs (efonidipine, felodipine, isradipine, and nitrendipine) as potent T-channel blockers (IC₅₀ < 3 µM). In contrast, other widely prescribed dihydropyridines such as amlodipine and nifedipine were 10-fold less potent, making them a more appropriate choice in research studies on the role of L-type currents. In summary, the present results support the notion that many available antihypertensive drugs block a substantial fraction of T-current at therapeutically relevant concentrations, contributing to their mechanism of action.

[Endocrine and Diabetes] Regulation of plasma fructose and mortality in mice by the aldose reductase inhibitor lidorestat

2008-10-30

Aldose reductase (AR), an enzyme widely believed to be involved in the aberrant metabolism of glucose and development of diabetic complications, is expressed at low levels in the mouse. We studied whether expression of human AR (hAR), its inhibition with lidorestat, an AR inhibitor (ARI), and the presence of streptozotocin (STZ)-induced diabetes altered plasma fructose, mortality and/or vascular lesions in LDL receptor-deficient (Ldlr-/-) mice. Mice were made diabetic at 12 weeks of age with low dose STZ treatment. Four weeks later the diabetic animals (glucose >20mmol/l) were blindly assigned to a 0.15% cholesterol diet with or without ARI. After 4 and 6 weeks there were no significant differences in body weights or plasma cholesterol, triglyceride, and glucose levels between the groups. Diabetic Ldlr-/- mice receiving ARI had plasma fructose levels of 5.2 ± 2.3 µg/mL, placebo treated mice had plasma fructose levels of 12.08 ± 7.4 µg/mL, p< 0.01. Fructose metabolizing enzymes, fructose kinase and adolase B, were increased in hAR expressing mice. After 6 weeks, hAR/Ldlr-/- mice on the placebo-containing diet had greater mortality (31%, n = 9/26 vs 6%, n = 1/21, p<0.05). The mortality rate in the ARI treated group was similar to that in non-hAR-expressing mice. Therefore, diabetic hAR-expressing mice had increased fructose and greater mortality that was corrected by inclusion of lidorestat, an ARI, in the diet. If similar effects are found in humans, such treatment could improve clinical outcome in diabetic patients.

[Endocrine and Diabetes] Mechanism-Based Modeling of Nutritional and Leptin Influences on Growth in Normal and Type 2 Diabetic Rats

Landersdorfer, C. B., DuBois, D. C., Almon, R. R., Jusko, W. J. — 2008-10-29

Influences of genetic and nutritional factors on body weight, fat mass, and leptin production, and effects of leptin were assessed in normal (WKY) and diabetic (GK) rats by mechanism-based modeling. The study included 60 WKY and 60 GK rats: half received high-fat diet (HF), the others normal rat chow (N). Body weights and food consumption were measured twice weekly. Six rats per group were sacrificed at 4, 8, 12, 16, and 20 weeks. Abdominal fat was weighed and plasma leptin measured by ELISA. All data were co-modeled using NONMEM VI (FOCE-I). Weight gain was modeled as differences between energy intake and metabolic rate based on allometrically scaled lean body mass (LBM). The GK had higher metabolic rates (1.15 kcal/day/g LBM^0.75) than WKY-N (0.92) and WKY-HF (1.02) rats, and higher efficiency in transforming energy into body weight. Leptin effect was modeled as inhibition of food consumption. Total body fat was estimated from abdominal fat. Leptin production from fat was 4.7-fold higher for GK (3.03 ng/mL/day/g) than WKY (0.66 ng/mL/day/g). Leptin production rate from LBM was 0.53 ng/mL/day/g for all groups. The IC₅₀ for inhibition of food intake by leptin was about three-fold higher in GK vs. WKY, indicating leptin resistance for the effect on food consumption in GK. The GK had similar kcal intake, but lower body weights and fat mass than WKY, possibly due to higher metabolic rates. Our mechanism-based model explains intrinsic reasons for differences in growth, food intake and leptin concentrations among these two strains of rats.

[Endocrine and Diabetes] Chronic Treatment with the Dipeptidyl Peptidase-4 Inhibitor (R)-8-(3-Amino-piperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione (BI 1356) Increases Basal Glucagon-Like Peptide-1 and Improves Glycemic Control in Diabetic Rodent Models

Thomas, L., Tadayyon, M., Mark, M. — 2008-10-29

Antidiabetic effects of dipeptidyl peptidase (DPP)-4 inhibitors are exerted by potentiation of the biological activity of incretin hormones like glucagon-like peptide (GLP)-1. BI 1356 [proposed trade name ONDERO; (R)-8-(3-Amino-piperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione] is a novel competitive, selective, potent and long-acting DPP-4 inhibitor under clinical development for the treatment of type 2 diabetes. The effect of 1-2 months of chronic dosing of BI 1356 in two different animal models is investigated. The first is a primarily genetic model (Zucker Diabetic Fatty (ZDF) rats) and the second is a non-genetic model (mice with diabetes induced by a combination of high fat diet (HFD) and a low-dose streptozotocin (STZ)). BI 1356 was shown to lower HbA1c after multiple dosing in both models. The improvement of glycemic control achieved in disease models of different etiology suggests that BI 1356 would also be efficacious in treating a broad spectrum of type 2 diabetic patients. In addition, multiple dosing of BI 1356 leads to a sustained increase in basal levels of active GLP-1 in the systemic circulation with expected long-term benefits on pancreatic - and β-cells. The effects on HbA1c, as well as, GLP-1 were superior to the short-acting DPP-4 inhibitor vildagliptin, demonstrating the potential of BI 1356 as a once-daily treatment for type 2 diabetes at low therapeutic doses.

[Neuropharmacology] In vitro and in vivo pharmacological characterization of the neuropeptide S receptor antagonist [D-Cys(tBu)5]NPS

2008-10-29

Neuropeptide S (NPS) was identified as the endogenous ligand of an orphan receptor now referred to as NPSR. In the frame of a structure-activity study performed on NPS Gly⁵, the NPSR ligand [D-Cys(tBu)⁵]NPS was identified. [D-Cys(tBu)⁵]NPS up to 100 µM did not stimulate calcium mobilization in HEK293 cells stably expressing the mouse NPSR (HEK293_mNPSR), however, the peptide inhibited in a concentration dependent manner the stimulatory effects elicited by 10 and 100 nM NPS (pK_B 6.62). In Schild analysis experiments [D-Cys(tBu)⁵]NPS (0.1 - 100 µM) produced a concentration dependent and parallel rightward shift of the concentration response curve to NPS showing a pA₂ value of 6.44. 10 µM [D-Cys(tBu)⁵]NPS did not affect signalling at seven NPSR unrelated G-protein coupled receptors. In the mouse righting reflex (RR) recovery test, NPS given at 0.1 nmole intracerebroventricularly reduced the percent of animals losing the RR in response to diazepam 15 mg/kg and their sleeping time. [D-Cys(tBu)⁵]NPS (1-10 nmoles) was inactive per se but dose dependently antagonized the arousal-promoting action of NPS. Finally, NPSR-deficient mice were similarly sensitive to the hypnotic effects of diazepam as their wild-type littermates. However, the arousal promoting action of 1 nmole NPS could be detected in wild-type but not in mutant mice. In conclusion, [D-Cys(tBu)⁵]NPS behaves both in vitro and in vivo as a pure and selective NPSR antagonist, but with moderate potency. Moreover, using this tool together with receptor knockout mice studies we demonstrated that the arousal-promoting action of NPS is due to the selective activation of the NPSR protein.

[Neuropharmacology] DIFFERENTIAL PHARMACOLOGIES OF MECAMYLAMINE ENANTIOMERS: POSITIVE ALLOSTERIC MODULATION AND NON-COMPETITIVE INHIBITION

Fedorov, N. B., Benson, L. C., Graef, J., Lippiello, P. M., Bencherif, M. — 2008-10-28

(±) Mecamylamine is a racemic mixture of a widely used brain-permeant non-competitive inhibitor of muscle-type and neuronal nicotinic receptors (NNRs). The present studies evaluated whether the stereoisomers of this drug show different profiles for inhibition of the high-(HS) and low-sensitivity (LS) isoforms of the human (4β2) NNR subtype expressed in SH-EP1 cells. We found that at low concentrations (µM range) S-(+)-mecamylamine (TC-5214) was more effective than R-(-)-mecamylamine (TC-5213) in inhibiting the LS 4β2 NNRs. In addition we demonstrated that TC-5214 potentiated, and TC-5213 inhibited agonist-induced activation of HS 4β2 NNRs. The stereoselectivity of mecamylamine enantiomers at HS and LS 4β2 receptors demonstrates that TC-5214 is the preferred stereoisomer for selective activation of HS, while it is more effective in suppressing LS receptor function. This feature could be relevant to therapeutic applications where such a selective mechanism of action is required.

[Neuropharmacology] Deletion of the GluR5 subunit of kainate receptors affects the development of morphine tolerance

2008-10-28

Previous reports utilizing pharmacological antagonists implicate kainate receptor (KAR) activation in the development of morphine tolerance, dependence, conditioned place preference (CPP) and locomotor sensitization, but the role of GluR5-containing KAR in these effects remains unclear due to limited selectivity of the inhibitors employed. Therefore, we examined behavioral responses to systemic morphine in mice expressing a constitutive deletion of GluR5 (GluR5 KO). Unlike wildtype (WT) littermates, GluR5 KO mice do not develop tolerance following repeated morphine administration by subcutaneous (s.c.) injection or via s.c. pellet implantation. In contrast, GluR5 KO mice do not differ from WT with respect to thermal or mechanical nociceptive thresholds, acute morphine antinociception, morphine disposition in the central nervous system (CNS), morphine physical dependence as revealed by naloxone-precipitated withdrawal, or the development of place preference and locomotor hyper-responsiveness following chronic morphine administration. Surprisingly, the continuous s.c. infusion of the GluR2/GluR5-preferring antagonist LY293558 decreased the number of naloxone-precipitated jumps to a similar extent in WT and GluR5 KO mice. We observed opioid-induced hypersensitivity in both groups during morphine withdrawal as demonstrated by equivalent reductions in thermal and mechanical thresholds; however, this hypersensitivity was not evident during continuous systemic morphine infusion. Collectively, these data indicate that KAR containing the GluR5 subunit contribute to the development of morphine tolerance without affecting nociceptive thresholds, morphine analgesia or the disposition in CNS of morphine and its metabolite morphine-3-glucuronide. In addition, constitutive deletion of GluR5 does not alter the morphine-induced increase in locomotor activity or the acquisition of morphine reward as measured by a CPP paradigm.

[Cardiovascular] INCREASED OXIDANT ACTIVITY MEDIATES VASCULAR DYSFUNCTION IN VIBRATION INJURY

2008-10-27

Occupational exposure to hand-operated vibrating tools causes a spectrum of pathological changes in the vascular, neurological and musculoskeletal systems described as the Hand-Arm Vibration Syndrome (HAVS). Experiments were performed to determine the effects of acute vibration on the function of digital arteries. Rats paws were exposed to a vibrating platform (4 hrs, 125 Hz, constant acceleration of 49 m/s² root mean square), and digital artery function subsequently assessed in vitro using a pressure myograph system. Constriction to phenylephrine or 5-hydroxytryptamine was reduced in digital arteries from vibrated paws. However, after endothelium-denudation, constriction to the agonists was no longer impaired in vibrated arteries. Inhibition of NO synthase (NOS) with N^G-nitro-L-arginine methyl ester (L-NAME) increased constriction to phenylephrine or 5-hydroxytryptamine in vibrated but not control arteries, and abolished the vibration-induced depression in constrictor responses. However, NO activity, determined using the NO-sensitive probe 4-amino-5-methylamino-2', 7'-difluorofluorescein, was reduced in vibrated compared to control arteries. Concomitantly, endogenous levels of reactive oxygen species (ROS), determined using the ROS-sensitive probe 5-(and 6)-chloromethyl-2',7'-dichlorodihydro-fluorescein, were increased in vibrated compared to control arteries. The increased ROS levels were abolished by L-NAME or by catalase, which degrades extracellular H₂O₂. Catalase also increased constriction to phenylephrine or 5-hydroxytryptamine in vibrated but not control arteries, and abolished the vibration-induced depression in constrictor responses. The results suggest that acute vibration causes vascular dysfunction in digital arteries by increasing ROS levels, which is likely mediated by uncoupling of endothelial NOS. Therefore, therapeutic strategies to inhibit ROS or augment NO activity may be beneficial in HAVS.

[Behavioral Pharmacology] {alpha}1-Adrenoceptors mediate L-DOPA-induced activity in MPTP-lesioned macaques

Visanji, N., Johnston, T. H., Fox, S. H., Millan, M. J., Brotchie, J. M. — 2008-10-27

The mechanisms underlying L-DOPA's actions in Parkinson's disease (PD) remain to be fully elucidated. Noradrenaline formed from L-DOPA may stimulate ₁-adrenoceptors. We assess the involvement of ₁-adrenoceptors in actions of L-DOPA in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned macaques. In each animal, the minimal dose of L-DOPA required to alleviate parkinsonian symptoms was defined (12.5-25 mg/kg, p.o.). The effects of co-administration of the ₁-adrenoceptor antagonist prazosin on motor activity, parkinsonism and dyskinesia assessed. Anti-parkinsonian benefit was accompanied by mild dyskinesia. L-DOPA also elicited hyperactivity, i.e. activity greater than that seem in normal animals. Co-administration of prazosin (0.16-0.63mg/kg, p.o.) with L-DOPA did not significantly affect either its anti-parkinsonian actions or dyskinesia. However, prazosin significantly and dose-dependently attenuated L-DOPA-induced activity, reducing it to a level equivalent to that of normal animals. More specifically, during periods of pronounced L-DOPA-induced activity, prazosin attenuated the total, duration and rate of activity by 80%, 76% and 45%, respectively. These actions of prazosin were expressed in the absence of sedation. While activation of ₁-adrenoceptors plays no major role in the anti-parkinsonian and dyskinetic effects of L-DOPA per se, it does contribute to the induction of hyperactivity. ₁-adrenoceptors may be involved in pathological responses to L-DOPA treatment, including the dopamine dysregulation syndrome.

[Cardiovascular] Chronic cocaine-induced cardiac oxidative stress and MAPK activation: The role of Nox2 oxidase

2008-10-24

Chronic cocaine exposure is associated with severe cardiac complications but the mechanisms of cocaine cardiotoxicity remain unclear, and current therapies are unsatisfactory. We investigated the hypothesis of oxidative stress-mediated cardiotoxicity and the role of NADPH oxidase in this process in a mouse model of chronic escalating "binge" cocaine administration (mg/kg): d1-4 at 3x15mg; d5-8 at 3x20mg; d9-12 at 3x25mg and d13-14 at 3x30mg. Compared to vehicle controls, chronic "binge" cocaine administration significantly increased the cardiac NADPH-dependent superoxide production (1.96±0.4 fold) as detected by tiron (a superoxide scavenger)-inhibitable lucigenin-chemiluminescence and DHE fluorescence. Cocaine-induced reactive oxygen species (ROS) production was associated with significant increases (~2-fold) in the protein expressions of Nox2 (an isoform of NADPH oxidase) and its regulatory subunits: p22^phox, p67^phox, p47^phox, p40^phox and Rac1, and in p47^phox phosphorylation as detected by immunobloting (all P<0.03). Increased Nox2 activity was accompanied by the activation of ERK1/2, p38MAPK and JNK notably in the cardiomyocytes. Cell culture experiments revealed that cocaine-induced ROS production was primarily a directly action of cocaine on cardiac myocytes, which caused severe oxidative damage to myocytes and cell death as detected by TUNEL assay. These could be inhibited by inhibitors to PKC (bisindolymaleimide) or by depletion of Nox2 using siRNA. In conclusion, chronic cocaine administration directly causes severe myocardial oxidative stress through the activation of Nox2 oxidase. Increased ROS production contributes to MAPK activation and the subsequent myocyte damage. Inhibitors to NADPH oxidase or antioxidants may have therapeutic potential in the treatment of cocaine cardiotoxicity.

[Neuropharmacology] Modulation of sodium channel inactivation gating by a novel lactam: Implications for seizure suppression in chronic limbic epilepsy

2008-10-24

Epilepsy remains a devastating neurological disorder associated with recurrent, unprovoked, spontaneous epileptic seizures. Current treatments involve seizure suppression using antiepileptic drugs (AEDs); however, many patients remain refractory to current treatments or suffer serious side effects. In view of this continued need for more effective and safer AEDs, we have designed a novel compound, YWI92, based on a lactam structural class and evaluated its modulation of hNa_v1.2 currents and hippocampal neuron action potential firing. Furthermore, we have tested its AED activity using a chronic and acute rat seizure model. In a similar manner to lamotrigine (LTG), a clinically used AED, YWI92 exhibited tonic block of hNa_v1.2 channels and caused a hyperpolarizing shift in the steady-state inactivation curve when using a 30 second inactivating prepulse. YWI92 also delayed the time constants of channel repriming after a 30 second inactivating prepulse and exhibited use-dependent block at 20 Hz stimulation frequency. In membrane excitability experiments, YWI92 inhibited burst firing in CA1 neurons of animals with temporal lobe epilepsy (TLE) at concentrations that had little effect on CA1 neurons from control animals. These actions on neuronal activity translated into AED activity in the maximal electroshock (MES) acute seizure model (ED₅₀ 22.96 mg/kg) and importantly, in a chronic temporal lobe epilepsy model, where the mean number of seizures were reduced. Importantly, YWI92 exhibited no sedative/ataxic side effects at concentrations up to 500 mg/kg. In summary, greater affinity for inactivated Na channels, particularly after long depolarizing prepulses, may be important for both anticonvulsant activity and drug tolerability.

[Cardiovascular] PARTIAL A1 ADENOSINE RECEPTOR AGONIST REGULATES CARDIAC SUBSTRATE UTILIZATION IN INSULIN RESISTANT RATS IN VIVO

Shearer, J., Severson, D. L, Su, L., Belardinelli, L., Dhalla, A. — 2008-10-24

Reducing the availability and uptake of fatty acids is a plausible pharmaceutical target to ameliorate glucose intolerance and insulin resistance. CVT-3619 is a partial A₁ adenosine receptor agonist with anti-lipolytic properties. Aims of the present study were to examine the acute effects of CVT-3619 on whole body and cardiac glucose and fatty acid kinetics in vivo in normal and diet-induced insulin resistant rats. Male Sprague-Dawley rats were fed either a chow (CH) or high fat diet (HF) for 4wk. Catheters were then chronically implanted in the carotid artery and jugular vein for sampling and infusions respectively. Following 5d of recovery, fasted animals (10h) received either saline or CVT-3619 (0.4mg/kg bolus + 1mg/kg/hr). Indices of glucose and fatty acid utilization were obtained by the administration of 2-deoxy[¹⁴C]glucose and [9,10-³H]-(R)-2-bromopalmitate. HF feeding resulted in elevated, fasting insulin and FFA levels compared to CH. CVT-3619 caused a 64% and 86% reduction of FFA and insulin in HF (p<0.05) but less in (NS) CH diet fed animals. In HF diet fed rats, CVT-3619 increased whole body glucose clearance with no change in fatty acid kinetics. Similarly, analysis of cardiac tissue metabolism showed that CVT-3619 caused an increased glucose, but not fatty acid clearance in HF fed animals. Results show that the acute administration of CVT-3619 lowers circulating fatty acid levels leading to improved whole body and cardiac glucose clearance in a model of diet-induced insulin resistance. As such, CVT-3619 may be a treatment option for the restoration of substrate balance in the insulin resistant heart.

[Gastrointestinal, Hepatic, Pulmonary, and Renal] FXR-deficiency Induces Non-alcoholic Steatohepatitis in LDLr-knockout Mice Fed a High-fat Diet

Kong, B., Luyendyk, J. P, Tawfik, O., Guo, G. L — 2008-10-23

Non-alcoholic steatohepatitis (NASH) comprises dysregulation of lipid metabolism and inflammation. Identification of the various genetic and environmental susceptibility factors for NASH may provide novel treatments to limit inflammation and fibrosis in patients. This study utilized a mouse model of hypercholesterolemia, LDL receptor knockout (LDLr^-/-) mice fed a high-fat diet for 5 months, to test the hypothesis that farnesoid X receptor (FXR) deficiency contributed to NASH development. Either the high-fat diet or FXR deficiency increased serum ALT activity, whereas only FXR deficiency increased bile-acid and ALP levels. FXR deficiency and high-fat feeding increased serum cholesterol and triglycerides. Although high-fat led to macrosteatosis and hepatocyte ballooning in livers of mice regardless of genotype, no inflammatory infiltrate was observed in the livers of LDLr^-/- mice. In contrast, in the livers of LDLr^-/-/FXR^-/- mice foci of inflammatory cells were observed occasionally when fed the control diet and were greatly increased when fed the high-fat diet. Consistent with enhanced inflammatory cells, hepatic levels of TNF and ICAM-1 mRNA were increased by the high-fat diet in LDLr^-/-/FXR^-/- mice. In agreement with elevated levels of pro-collagen11 and TGF-β mRNA, type-1 collagen protein levels were increased in livers of LDLr^-/-/FXR^-/- mice fed high-fat diet. In conclusion, FXR deficiency induces pathologic manifestations required for NASH diagnosis in a mouse model of hypercholesterolemia, including macrosteatosis, hepatocyte ballooning and inflammation, which suggest a combination of FXR deficiency and high-fat diet is a risk factor for NASH development and activation of FXR may be a therapeutic intervention in the treatment of NASH.

[Gastrointestinal, Hepatic, Pulmonary, and Renal] Computational Strategies Unravel and Trace How Liver Disease Changes Hepatic Drug Disposition

Park, S., Ropella, G. E. P., Kim, S. H. K., Roberts, M. S., Hunt, C. A. — 2008-10-23

Liver disease changes the disposition properties of drugs, complicating drug therapy management. We present normal and "diseased" versions of an abstract, agent-oriented In Silico Liver (ISL), and validate their mechanisms against disposition data from perfused normal and diseased rat livers. Dynamic tracing features enabled spatiotemporal tracing of differences in dispositional events for diltiazem and sucrose across five levels, including interactions with representations of lobular microarchitectural features, cells, and intracellular factors that sequester and metabolize. Differences in attributes map to measures of histopathology. We measured disease caused differences in local, intralobular ISL effects, obtaining heretofore-unavailable views of how and where hepatic drug disposition may differ in normal and diseased rat livers from diltiazem's perspective. Exploration of disposition in less and more advanced stages of disease is feasible. The approach and technology represent an important step toward unraveling the complex changes from normal to disease states and their influences on drug disposition.

[Chemotherapy, Antibiotics, and Gene Therapy] Improved Pharmacokinetics and Efficacy of a Highly Stable Nanoliposomal Vinorelbine

2008-10-23

Effective liposomal formulations of vinorelbine have been elusive due to vinorelbine's hydrophobic structure and resulting difficulty in stabilizing the drug inside the nanocarrier. Triethylammonium salts of several polyanionic trapping agents were used initially to prepare minimally pegylated nanoliposomal vinorelbine formulations with a wide range of drug release rates. Sulfate, poly(phosphate), and sucroseoctasulfate were used to stabilize vinorelbine intraliposomally while in the circulation, with varying degrees of effectiveness. The release rate of vinorelbine from the liposomal carrier was affected by both the chemical nature of the trapping agent and the resulting drug-to-lipid ratio, with liposomes prepared using sucrose octasulfate displaying the longest half-life in the circulation (9.4 h) and in vivo retention in the nanoparticle (t_1/2=27.2 h). Efficacy was considerably improved in both a human colon carcinoma (HT29) and a murine (C26) colon carcinoma model when vinorelbine was stably encapsulated in liposomes using triethylammonium sucroseoctasulfate. Early difficulties in preparing highly pegylated formulations were later overcome by substituting a neutral distearoylglycerol anchor for the more commonly employed anionic distearoylphosphatidylethanolamine anchor. The new pegylated nanoliposomal vinorelbine displayed high encapsulation efficiency and in vivo drug retention, and was highly active against human breast and lung tumor xeongrafts. Acute toxicity of the drug in immunocompetent mice slightly decreased upon encapsulation in liposomes, with an MTD of 17.5 mg VRL/kg for free vinorelbine and 23.8 mg VRL/kg for nanoliposomal vinorelbine. Our results demonstrate that a highly active, stable, and long circulating liposomal vinorelbine can be prepared, and warrants further study in the treatment of cancer.

[Behavioral Pharmacology] Divergent Effects of Anandamide Transporter Inhibitors With Different Target Selectivity on Social Play Behavior in Adolescent Rats

Trezza, V., Vanderschuren, L. — 2008-10-23

The endocannabinoid system plays an important role in the modulation of affect, motivation and emotion. Social play behavior is a natural reinforcer in adolescent rats, and we have recently shown that interacting endocannabinoid, opioid and dopamine systems modulate social play. In the present study, we tested the hypothesis that, in contrast to administration of exogenous cannabinoid agonists, increasing local endocannabinoid signaling through anandamide transporter inhibition enhances social play. To this aim, we tested the effects of two anandamide transporter inhibitors with different target selectivity on social play behavior in adolescent rats. Interestingly, we found that the prototypical anandamide transporter inhibitor AM404 reduced social play, whereas its more selective analogue VDM11 enhanced it. The effects of AM404 were not mediated through its known pharmacological targets, since they were not blocked by the CB₁ cannabinoid receptor antagonist SR141716A, the CB₂ cannabinoid receptor antagonist SR144528, or by the TRPV1 vanilloid receptor antagonist capsazepine. In contrast, the increase in social play induced by VDM11 was dependent on cannabinoid, opioid and dopaminergic neurotransmission, since it was blocked by the CB₁ cannabinoid receptor antagonist SR141716A, the opioid receptor antagonist naloxone and the dopamine receptor antagonist alpha-flupenthixol. These findings support the notion that anandamide plays an important role in the modulation of social interaction in adolescent rats, and suggest that selective anandamide transporter inhibitors might be useful for the treatment of social dysfunctions. Furthermore, these results suggest that off-target effects may be responsible for some of the conflicting effects of anandamide transporter inhibitors on behavior.

[Metabolism, Transport, and Pharmacogenomics] Novel vasoconstrictor formulation to enhance intranasal targeting of neuropeptide therapeutics to the central nervous system

Dhuria, S. V., Hanson, L. R., Frey, W. H. — 2008-10-22

The intranasal route of drug administration is non-invasive, convenient, and rapidly targets therapeutics to the central nervous system (CNS) using olfactory and trigeminal neural pathways connecting the nasal passages to the brain. The purpose of this research was to enhance intranasal drug targeting to the CNS by incorporating a vasoconstrictor (phenylephrine, PHE) into nasal formulations containing therapeutic neuropeptides (hypocretin-1, HC, or the dipeptide L-Tyr-D-Arg, D-KTP). Concentrations in CNS tissues, peripheral tissues, and blood were determined at 30 minutes following intravenous or intranasal administration of ¹²⁵I-labeled neuropeptides with and without PHE. Compared to intranasal controls, inclusion of 1% PHE in nasal formulations significantly reduced absorption into the blood for HC (65% reduction) and D-KTP (56% reduction), while significantly increasing deposition into the olfactory epithelium by ~3-fold for both. 1% PHE significantly increased delivery to the olfactory bulbs for HC (2.1-fold) and D-KTP (3.0-fold), while significantly reducing concentrations in the trigeminal nerve for HC (65% reduction) and D-KTP (39% reduction) and in most remaining brain regions by ~50% for both. The dramatic reduction in blood concentrations with PHE contributed to brain-to-blood concentration ratios that were significantly increased for HC throughout the brain (1.6- to 6.8-fold) compared to intranasal controls. For D-KTP, 1% PHE significantly increased ratios only in the olfactory bulbs (5.3-fold). With a 5% PHE formulation, D-KTP ratios were significantly increased to additional brain areas (1.5- to 16-fold). Vasoconstrictor nasal formulations may have particular relevance for CNS therapeutics with adverse side effects where it would be advantageous to limit systemic exposure.

[Endocrine and Diabetes] Differential Effects of Diet-Induced Dyslipidemia and Hyperglycemia on Mesenteric Resistance Artery Structure and Function in Type 2 Diabetes

2008-10-21

Type 2 diabetes and dyslipidemia oftentimes present in combination. However, the relative roles of diabetes and diet-induced dyslipidemia in mediating changes in vascular structure, mechanics and function are poorly understood. Our hypothesis was that addition of a high-fat diet would exacerbate small artery remodeling, compliance and vascular dysfunction in Type 2 diabetes. Vascular remodeling indices (media to lumen ratio (M/L), collagen abundance and turnover and matrix metalloproteinase (MMP) dynamics), mechanical properties (vessel stiffness) and reactivity to pressure and vasoactive factors were measured in third order mesenteric arteries in control Wistar and Type 2 diabetic Goto-Kakizaki (GK) rats fed either a regular or high-fat diet. M/L ratios, total collagen and myogenic tone were increased in diabetes. Addition of high fat diet altered collagen patterns (mature vs new collagen) in favor of matrix accumulation. Addition of a high-fat diet caused increased constriction to endothelin-1(ET-1, 0.1-100nM), showed impaired vasorelaxation to both acetylcholine (ACh, 0.1nM - 1µM) and sodium nitroprusside (SNP, 0.1nM - 1µM) and increased cardiovascular risk factors in diabetes. These results suggest that moderate elevations in blood glucose as seen in our lean GK model of Type 2 diabetes promote resistance artery remodeling resulting in increased medial thickness whereas addition of high fat diet contribute to diabetic vascular disease predominantly by impairing vascular reactivity in the time frame used for this study. Although differential in their vascular effects, both hyperglycemia and diet-induced dyslipidemia need to be targeted for effective prevention and treatment of diabetic vascular disease.

[Neuropharmacology] Characterization of a Cannabinoid CB2 Receptor Selective Agonist, A-836339, in In Vitro Pharmacological assays and In Vivo Pain Models

2008-10-17

Studies demonstrating the anti-hyperalgesic and anti-allodynic effects of CB₂ receptor activation have been largely derived from the use of receptor selective ligands. Here we report the identification of A-836339 (2,2,3,3-tetramethyl-cyclopropanecarboxylic acid [3-(2-methoxy-ethyl)-4,5-dimethyl-3H-thiazol-(2Z)-ylidene]-amide), a potent and selective CB₂ agonist as characterized in in vitro pharmacological assays and in in vivo models of pain as well as CNS behavior models. In radioligand binding assays, A-836339 displays high affinities at CB₂ receptors and selectivity over CB₁ receptors in both human and rat. Similarly, A-836339 exhibits high potencies at CB₂ and selectivity over CB₁ receptors in recombinant FLIPR and cyclase functional assays. In Cerep assays, A-836339 exhibits a profile devoid of significant affinity at other GPCRs and ion channels. A-836339 was characterized extensively in various animal pain models. In the CFA model of inflammatory pain, A-836339 exhibits a potent CB₂ receptor mediated anti-hyperalgesic effect that is independent of CB₁ or µ-opioid receptors. A-836339 has also demonstrated efficacies in the CCI model of neuropathic pain, skin incision and Cap-SMH models. Further, no tolerance was developed in the CCI model after subchronic treatment with A-836339 for 5 days. In assessing CNS effects, A-836339 exhibited a CB₁ receptor mediated decrease of spontaneous locomotor activities at a higher dose, a finding consistent with the CNS activation pattern observed by phMRI. These data demonstrate that A-836339 is a useful tool for use of studying CB₂ receptor pharmacology and for investigation of the role of CB₂ receptor modulation for treatment of pain in pre-clinical animal models.

[Gastrointestinal, Hepatic, Pulmonary, and Renal] Therapeutic effect of lecithinized superoxide dismutase (PC-SOD) against colitis

2008-10-16

Ulcerative colitis (UC) involves intestinal mucosal damage induced by reactive oxygen species (ROS), in particular superoxide anion. Superoxide dismutase (SOD) catalyses dismutation of superoxide anion to hydrogen peroxide, which is subsequently detoxified by catalase. Lecithinized SOD (PC-SOD) is a new modified form of SOD which has overcome previous clinical limitations of SOD. In this study, we examined the action of PC-SOD using an animal model of UC, dextran sulfate sodium (DSS)-induced colitis. DSS-induced colitis was ameliorated by daily intervenous administration of PC-SOD. Unmodified SOD (U-SOD) produced a similar effect, but only at more than 30 times the concentration of PC-SOD. In vivo electron spin resonance analysis confirmed that the increase in the colonic level of ROS associated with development of colitis was suppressed by PC-SOD administration. The dose-response profile of PC-SOD was bell-shaped, but simultaneous administration of catalase restored the ameliorative effect at high doses of PC-SOD. Accumulation of hydrogen peroxide was observed with the administration of high doses of PC-SOD, an effect which was suppressed by the simultaneous administration of catalase. We also found that either a weekly intravenous administration or daily oral administration of PC-SOD conferred protection. These results suggest that PC-SOD achieves its ameliorative effect against colitis through decreasing the colonic level of ROS, and that its ineffectiveness at higher doses is due to the accumulation of hydrogen peroxide. Furthermore, we consider that intermittent or oral administration of PC-SOD can be applied clinically to improve the quality of life of UC patients.

[Cardiovascular] Opposite effects of two resveratrol tetramers, vitisin A and hopeaphenol on apoptosis of myocytes isolated from adult rat heart

2008-10-16

It has been reported that resveratrol from Vitis plants has various cardioprotective effects. Vitis plants also include various resveratrol tetramers. The aim of our study is to clarify the pharmacological properties of resveratrol tetramers. We isolated two resveratrol tetramers as major products of Vitis plants. One is vitisin A, a complex of two resveratrol dimers, (+)--viniferin and ampelopsin B, and the other is hopeaphenol composed of two moles of ampelopsin B. Unexpectedly, vitisin A (30-300 nM) dose-dependently facilitated swelling and depolarization of mitochondria, and cytochrome c release from mitochondria, that are indices of cardiomyocyte apoptosis. Furthermore, vitisin A induced apoptosis in the primary culture of adult rat ventricular myocytes. On the other hand, hopeaphenol (1-10 µM) dose-dependently inhibited Ca²⁺ (30 µM)-induced mitochondrial depolarization and cytochrome c release from mitochondria but had not affected mitochondrial swelling. Moreover, hopeaphenol inhibited vitisin A-induced apoptosis. In structural and functional studies, we further confirmed that vitisin B, one of the resveratrol tetramers having (+)--viniferin unit, induces mitochondrial swelling and cytochrome c release from mitochondria like as vitisin A, and that vitisifuran A, one of the resveratrol tetramers having ampelopsin B unit, inhibits Ca²⁺-induced cytochrome c release from mitochondria like as hopeaphenol. These results show that resveratrol tetramers have at least two opposite effects on cardiomyocytes: the one having (+)--viniferin unit induces cardiomyocyte apoptosis, and the other having ampelopsin B but not (+)--viniferin unit inhibits it.

[Cardiovascular] The endogenous brain constituent N-arachidonoyl L-serine is an activator of large conductance Ca2+-activated K+ channels

2008-10-15

The novel endocannabinoid-like lipid N-arachidonoyl L-serine (ARA-S) causes vasodilation through both endothelium-dependent and -independent mechanisms. We have analyzed the vasorelaxant effect of ARA-S in isolated vascular preparations and its effects on Ca²⁺-activated K⁺ currents in human embryonic kidney cells stably transfected with the -subunit of the human, large conductance Ca⁺-activated K⁺ (BK_Ca) channel (HEK293hSlo cells). ARA-S caused relaxation of rat isolated, intact and denuded, small mesenteric arteries pre-constricted with phenylephrine (pEC₅₀: 5.49 and 5.14, respectively), whereas it caused further contraction of vessels pre-constricted with KCl (pEC₅₀ 5.48 and 4.82, respectively). Vasorelaxation by ARA-S was inhibited by 100 nM iberiotoxin. In HEK293hSlo cells, ARA-S and its enantiomer N-arachidonoyl-D-serine enhanced the whole cell outward K⁺ current with similar potency (pEC₅₀: 5.63 and 5.32, respectively). The potentiation was not altered by β₁ subunit or mediated by ARA-S metabolites, stimulation of known cannabinoid receptors, G proteins, protein kinases or Ca²⁺-dependent processes; it was lost after patch excision or following membrane cholesterol depletion, but was restored after cholesterol reconstitution. BK_Ca currents were also enhanced by anandamide (pEC₅₀: 5.27) but inhibited by another endocannabinoid, virodhamine (pIC₅₀: 6.35), or by the synthetic cannabinoid O-1918 (pIC₅₀: 6.59), which blocks ARA-S-induced vasodilation. We conclude that (i) ARA-S directly activates BK_Ca channels. (ii) This interaction does not involve cannabinoid receptors or cytosolic factors but is dependent on the presence of membrane cholesterol. (iii) Direct BK_Ca channel activation likely contributes to the endothelium-independent component of ARA-S-induced mesenteric vasorelaxation. (iv) O-1918 is a BK_Ca channel inhibitor.