Rank Order Of Inhibitory Potency Research Articles

Mechanistic in vitro studies indicate that the clinical drug-drug interactions between protease inhibitors and rosuvastatin are driven by inhibition of intestinal BCRP and hepatic OATP1B1 with minimal contribution from OATP1B3, NTCP and OAT3.

Previous use of a mechanistic static model to accurately quantify the increased rosuvastatin exposure due to drug-drug interaction (DDI) with coadministered atazanavir underpredicted the magnitude of area under the plasma concentration-time curve ratio (AUCR) based on inhibition of breast cancer resistance protein (BCRP) and organic anion transporting polypeptide (OATP) 1B1. To reconcile the disconnect between predicted and clinical AUCR, atazanavir and other protease inhibitors (darunavir, lopinavir and ritonavir) were evaluated as inhibitors of BCRP, OATP1B1, OATP1B3, sodium taurocholate cotransporting polypeptide (NTCP) and organic anion transporter (OAT) 3. None of the drugs inhibited OAT3, nor did darunavir and ritonavir inhibit OATP1B3 or NTCP. All drugs inhibited BCRP-mediated estrone 3-sulfate transport or OATP1B1-mediated estradiol 17β-D-glucuronide transport with the same rank order of inhibitory potency (lopinavir>ritonavir>atazanavir>>darunavir) and mean IC50 values ranging from 15.5 ± 2.80 μM to 143 ± 14.7 μM or 0.220 ± 0.0655 μM to 9.53 ± 2.50 μM, respectively. Atazanavir and lopinavir also inhibited OATP1B3- or NTCP-mediated transport with a mean IC50 of 1.86 ± 0.500 μM or 65.6 ± 10.7 μM and 5.04 ± 0.0950 μM or 20.3 ± 2.13 μM, respectively. Following integration of a combined hepatic transport component into the previous mechanistic static model using the in vitro inhibitory kinetic parameters determined above for atazanavir, the newly predicted rosuvastatin AUCR reconciled with the clinically observed AUCR confirming additional minor involvement of OATP1B3 and NTCP inhibition in its DDI. The predictions for the other protease inhibitors confirmed inhibition of intestinal BCRP and hepatic OATP1B1 as the principal pathways involved in their clinical DDI with rosuvastatin.

PS-B03-6: COMPARISON OF SUPPRESSIVE EFFECTS ON CATECHOLAMINE SECRETION BETWEEN OLMESARTAN AND ENALAPRIL

Objective: The present study was designed to compare olmesartan, an angiotensin II receptor blocker (ARB) with enalapril, an angiotensin converting enzyme inhibitor (ACEI) in the suppressive effects on the secretion of catecholamines (CA) from the perfused model of the rat adrenal gland. Design and Methods: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: Both olmesartan (50 μM) and enalapril (50 μM) during perfusion into an adrenal vein for 90 min inhibited the CA secretory responses evoked by ACh (5.32 μM), DMPP (a selective neuronal nicotinic Nn receptor agonist, 100 μM), high K+ (a direct membrane-depolarizer, 56 μM), and McN-A-343 (a selective muscarinic M1 receptor agonist, 100 μM) in a time-dependent manner. Also, in the presence of enalapril or olmesartan, the secretory responses of CA evoked by veratridine (an activator of voltage-dependent Na+ channels, 100 μM), Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10 μM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 μM) were significantly reduced. Based on the same concentration (50 μM) of enalapril and olmesartan, for the CA release evoked by Ach, high K+, DMPP, McN-A-343, angiotensin II, veratridine, Bay-K-8644 and cyclopiazonic acid, the following rank order of inhibitory potency was obtained: olmesartan > enalapril. In the simultaneous presence of enalapril and olmesartan, Ach-evoked CA secretory responses were more strongly inhibited in comparison with that of enalapril- or olmesartan-treated alone. Conclusion: Collectively, these results demonstrate that both enalapril and olmesartan inhibit the CA secretory responses evoked by activation of both cholinergic and angiotensin II receptors stimulation as well as by direct membrane depolarization in the perfused model of the isolated rat adrenal medulla. When these two drugs were used in combination, their effects were enhanced, which may also be of clinical benefit. Based on concentration used in this study, it seems that the inhibitory effect of olmesartan on the CA secretion is more potent than that of enalapril.

Synergistic Effects of Adavosertib, WEE‐1 Inhibitor in Combination with Cytarabine in the Jurkat Leukemia Cell Model

Current treatment for leukemia focuses on DNA damaging treatments (DNAdt), including chemo (cytarabine, vincristine, doxorubicin, etc.) and/or radiotherapy. However, in both adult and pediatric patients with Acute Lymphoblastic Leukemia (ALL), value of these treatments are limited by development of chemoresistance. Recent studies evaluated inhibition of the G2‐M gatekeeper (Wee‐1 kinase) as a possible alternative treatment for other types of cancer, such as: breast, prostate, pancreatic, lung, leukemia, glioblastoma, among others. Adavosertib (AZD‐1775), a well‐documented Wee‐1 kinase inhibitor, has shown synergistic; chemo‐sensitization effects, and is currently undergoing clinical trials. Therefore, our working hypothesis is that: AZD‐1775 sensitizes Leukemia cell lines to DNA‐damaging drugs like cytarabine, resulting in a drug combination with significant synergistic effects. This study evaluates the cytotoxic and chemo‐sensitization effect of AZD‐1775 on an ALL T‐cell model (Jurkat). A broad range of drug combinations (high throughput 384 well plates format) in cell viability assays (96 hours; resazurin‐based) was used to determine cytotoxic effects of AZD‐1775 and cytarabine. Corresponding IC50 values were calculated using GraphPad Prism and the nature of the interactions (synergy, additivity, antagonism) determined with open access software CompuSyn, Combenefit, and SynergyFinder. The Combenefit freeware permitted fitting and analysis of the data to various synergy models (Loewe‐additivity, Bliss‐independence and Highest‐single Agent) while SynergyFinder allowed for an additional model (Zero Interaction Potency), and the classical model of CompuSyn allowed estimates of drug combination indexes (CI) and dose‐reduction indexes (DRI). The individual agents exhibited the following rank order of inhibitory potencies (IC50±SEM): cytarabine (160nM±6nM) ≥ AZD‐1775 (370nM±9nM). Drug combinations results demonstrate a strong and statistically significant synergistic effect with the highest synergistic interactions obtained with AZD‐1775 concentrations of 58nM to 270nM, and cytarabine concentrations of 8nM to 125nM. These synergistic interactions were observed independent on the model and software used for the analysis. Furthermore, the drug combination found to produce the highest synergistic effects in our resazurin‐based cytotoxic assay (AZD‐1775 = 97nM; Cytarabine = 63nM), was also used in a study using Jurkat, normal PBMCs, and activated T‐cells with cells analyzed for cell proliferation, viability and biochemical markers evaluated by Western Blot. At these low concentrations combination no synergistic effects were observed in normal PBMC cells; yet, our findings strongly demonstrate that AZD‐1775 can act as a chemosensitizer agent in ALL capable of potentiating the effect and susceptibility to cytarabine.

COMPARISON OF INHIBITORY EFFECTS EVOKED BY OLMESARTAN AND ENALAPRIL ON CATECHOLAMINE SECRETION

Objective: The present study was designed to compare enalapril, an angiotensin converting enzyme inhibitor (ACEI) with olmesartan an angiotensin II receptor blocker (ARB) in the inhibitory effects on the secretion of catecholamines (CA) from the perfused model of the rat adrenal gland. Design and method: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: Both enalapril (50 microM) and olmesartan (50 microM) during perfusion into an adrenal vein for 90 min inhibited the CA secretory responses evoked by ACh (5.32 microM), DMPP (a selective neuronal nicotinic Nn receptor agonist, 100 microM), high K+ (a direct membrane-depolarizer, 56 mM), and McN-A-343 (a selective muscarinic M1 receptor agonist, 100 microM) in a time-dependent manner. Also, in the presence of enalapril or olmesartan, the secretory responses of CA evoked by veratridine (an activator of voltage-dependent Na+ channels, 100 microM), Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10 microM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 microM) were significantly reduced. Based on the same concentration (50 microM) of enalapril and olmesartan, for the CA release evoked by Ach, high K+, DMPP, McN-A-343, angiotensin II, veratridine, Bay-K-8644 and cyclopiazonic acid, the following rank order of inhibitory potency was obtained: olmesartan> enalapril. In the simultaneous presence of enalapril and olmesartan, Ach-evoked CA secretory responses were more strongly inhibited in comparison with that of enalapril- or olmesartan-treated alone. Conclusions: Collectively, these results demonstrate that both enalapril and olmesartan inhibit the CA secretory responses evoked by activation of both cholinergic and angiotensin II receptors stimulation as well as by direct membrane depolarization in the perfused model of the isolated rat adrenal medulla. When these two drugs were used in combination, their effects were enhanced, which may also be of clinical benefit. Based on concentration used in this study, it seems that the inhibitory effect of olmesartan on the CA secretion is more potent than that of enalapril.

A Comprehensive Study of the Chemosensitizing Effects of AZD‐1775, WEE‐1 Inhibitor in Combination with DNA Damaging Treatments in the Jurkat Leukemia Cell Model

Current treatment for leukemia focuses on DNA damaging treatments (DNAdt), including chemo (cytarabine, vincristine, doxorubicin, etc.) and/or radiotherapy. However, Acute Lymphoblastic Leukemia (ALL) mechanisms of resistance limit the value of these treatments. Recent studies evaluated the G2‐M gatekeeper Wee1 inhibition as a possible alternative treatment for other types of cancer, such as: breast, prostate, pancreatic, lung, leukemia, glioblastoma, among others. AZD‐1775, a well‐documented Wee1 (G2‐M‐Gatekeeper) inhibitor, has shown chemosensitizing, synergistic effects, and currently is undergoing clinical trials. Therefore, our working hypothesis is that: AZD‐1775 sensitizes Leukemia cell lines to DNA‐damaging drugs, via a mitotic catastrophe process. This study evaluates the cytotoxic and chemo‐sensitization effect of AZD‐1775 on an ALL T‐cell model (Jurkat). Even more, this study innovates the understanding of AZD‐1775 by carefully crafting a panel of drugs with different mechanisms that induce DNA damage. Cell viability assays (resazurin‐based; high throughput 384 well plates) were done to determine the inhibitory effects of AZD‐1775 and a series of DNAdt drugs alone and in combinations. The corresponding IC50 value for all curves obtained were calculated using GraphPAD Prism (Vers.7). The nature of the interactions (synergy, additivity, antagonism) between AZD‐1775 and the different DNAdt drugs was determined with Combenefit and the CompuSyn Software. The Combenefit freeware permitted fitting and analysis of the data to various synergy models (Loewe‐additivity, Bliss‐independence and Highest‐single Agent); while, GraphPad Prism and CompuSyn allowed estimates of drug combination indexes (CI) and dose‐reduction indexes (DRI). The individual agents exhibited the following rank order of inhibitory potencies (IC50±SEM): cytarabine (160nM±6nM SEM) ≥ AZD‐1775 (370nM±9nM SEM) >> etoposide (2μM±60nM SEM) = cisplatin (3μM±127nM SEM) > 5′‐fluorouracil (34μM±2.7μM SEM). The results demonstrate a significant synergistic effect of AZD‐1775 with cytarabine, 5FU, cisplatin and etoposide. Conversely, most of these drugs were also capable of potentiating the effect of AZD‐1775 in a synergistic manner. In all models of synergy analysis, statistically significant synergistic interactions were confined to specific concentration ranges of drug‐combinations. For example, the most significant synergistic interactions were obtained with AZD‐1775 concentrations of 58nM to 270nM, and cytarabine concentrations of 8nM to 125nM, with the Bliss‐independence model. In conclusion, our findings strongly suggest that AZD‐1775 can be used as a chemosensitizing agent in ALL, capable of potentiating the effect and susceptibility to DNAdt; whilst, allowing the patient to require lower doses of these chemotherapies to decrease adverse effects.Support or Funding InformationPartially supported by NIGMS/INBRE award P20GM103475. The content is solely the responsibility of the author and does not necessarily represent the official views of any of the supporting agencies.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

O-Methylbulbocapnine and Dicentrine Suppress LPS-Induced Inflammatory Response by Blocking NF-κB and AP-1 Activation through Inhibiting MAPKs and Akt Signaling in RAW264.7 Macrophages.

The natural aporphine alkaloids including crebanine (CN), O-methylbulbocapnine (OMP), and dicentrine (DC), and protoberberine alkaloids, tetrahydropalmatine (THP) and N-methyl tetrahydropalmatine (NTHP), have been found in Stephania venosa. Previous reports demonstrated CN and THP exhibited anti-inflammatory properties. In this study, we investigated anti-inflammatory effect of CN analogs including OMP, DC, THP, and NTHP in RAW264.7 macrophages. The pre-treatment of macrophages with CN, OMP and DC suppressed lipopolysaccharide (LPS)-induced pro-inflammatory cytokines and mediators including interleukin-6 (IL-6), tumor necrosis factor alpha, prostaglandin E2 and nitric oxide, in which the rank-order of inhibitory potency was DC>CN≥OMP. Whereas, high dose THP (30-40 µg/mL) reduced LPS-induced IL-6 production in RAW264.7 cells but NTHP did not effect. Moreover, CN, OMP and DC inhibited the LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2. OMP and DC inhibited LPS-induced nuclear factor kappa B (NF-κB) activation by suppressing the phosphorylation of NF-κB at Ser536, but not the nucleus translocation and inhibitor of kappaB (IκB)-α degradation. In addition, OMP and DC also reduced the phosphorylation and nucleus translocation of activator protein-1 (AP-1). Furthermore, OMP and DC suppressed the LPS-activated myeloid differentiation factor 88 (MyD88), Akt and mitogen-activated protein kinases (MAPKs) signaling pathway, which were the upstream signaling regulators of AP-1 and NF-κB. Collectively, OMP and DC have an anti-inflammatory effect on RAW264.7 macrophages by the suppression of pro-inflammatory cytokines and mediators. The inhibitory property of OMP and DC is mediated by blockage the activation of MyD88, MAPKs, Akt, NF-κB and AP-1 signaling molecules.

PS 07-04 COMPARISON OF INHIBITORY EFFECTS BETWEEN GINSENOSIDE-Rb2 AND Rg2 ON ADRENAL CATECHOLAMINE RELEASE

Objective: The present study was attempted to compare ginsenoside-Rb2 (Rb2) one of panaxadiol saponins with Ginsenoside-Rg2 (Rg2), another panaxadiol saponin in the inhibitory effects on the secretion of catecholamines (CA) from the perfused model of the rat adrenal gland. Design and Method: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: Both Rb2 and Rg2 during perfusion into an adrenal vein for 90 min inhibited the CA release evoked by ACh, angiotensin II, DMPP, high K+, and McN-A-343 in a time-dependent manner. Also, in the presence of Rb2 or Rg2, the CA release evoked by veratridine (an activator of voltage-dependent Na+ channels), Bay-K-8644 (a voltage-dependent L-type Ca2+ channel activator), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor) were significantly reduced. Both two ginsenosides also greatly induced NO release from adrenal medulla. Based on the same concentration of Rb2 and Rg2, for the CA release evoked by ACh, high K+, DMPP, McN-A-343, Ang II, veratridine, Bay-K-8644 and cyclopiazonic acid, as well as for No release, the following rank order of inhibitory potency was obtained: Rg2> Rb2. In the simultaneous presence of fimasartan, an antagonist of angiotensin II receptors and Ginsenoside-Rg2, ACh- or angiotensin II-evoked CA secretion was more strongly inhibited compared with that of fimasartan- or Rg2-treated alone. Conclusions: Collectively, these results demonstrate that both Rb2 and Rg2 can inhibit the CA secretion evoked by activation of both cholinergic and AT1 receptors stimulation from the perfused rat adrenal medulla. When these two ginsenosides were used in combination with a different antihypertensive agent, antihypertensive effects were enhanced, which may also be of clinical benefit. Based on concentrations used in this study, the inhibitory effect of ginsenoside-Rg2 on the CA secretion seems to be more potent than that of ginsenoside-Rb2.

Rat precision-cut intestinal slices to study P-gp activity and the potency of its inhibitors ex vivo

Rat Precision-Cut Intestinal Slices (PCIS) were evaluated as ex vivo model to study the regional gradient of P-gp activity, and to investigate whether the rank order of inhibitory potency of P-gp inhibitors can be correctly reproduced in this model with more accurate IC50 values than with current in vitro models. PCIS were prepared from small intestine (duodenum, jejunum, ileum) and colon. Rhodamine 123 (R123) was used as P-gp substrate, while verapamil, cyclosporine A, quinidine, ketoconazole, PSC833 and CP100356 were employed as P-gp inhibitors. Increase in tissue accumulation of R123 in the presence of the inhibitors was considered as an indication of the inhibitory effect. The P-gp inhibitors increased the tissue accumulation of R123 in a concentration dependent manner. Fluorescence microscopy elucidated that this increase occurred predominantly in the enterocytes. The rank order of the corresponding IC50 values agreed well with reported values from cell lines expressing rat P-gp. The activity of and inhibitory effects on P-gp were significantly higher in ileum compared to the other regions. These data suggest that rat PCIS are a reliable ex vivo model to study the activity of intestinal P-gp and the inhibitory effect of drugs. PCIS have potential as ex vivo model for the prediction of transporter-mediated drug–drug interactions.

Pharmacological characterisation of α6β4⁎ nicotinic acetylcholine receptors assembled from three chimeric α6/α3 subunits in tsA201 cells

The distribution and physiological functions of the α6 subunit-containing (α6⁎) nicotinic acetylcholine receptors in the central nervous system make them interesting putative therapeutic targets in several disorders. However, investigations into the receptors have been complicated by their inefficient functional expression in vitro. In the present study we have characterized and compared the pharmacological properties displayed by α6β4 and α6β4β3 nicotinic acetylcholine receptors assembled in tsA201 cells from the classical α6/α3 chimera (C1) and two novel α6/α3 chimeras (C6F223L and C16F223L) identified in a recent study (Jensen et al., 2013). Whereas the Bmax values exhibited by [3H]epibatidine at wild-type α6β4, C1β4, C6F223Lβ4 and C16F223Lβ4 receptors differed substantially, the radioligand and seven orthosteric nicotinic agonists exhibited very similar KD and Ki values at the four receptors. In the FLIPR™ Membrane Potential Blue assay, the agonists exhibited the same rank order of potencies [(±)-epibatidine>sazetidine A>varenicline>(−)-cytisine~(S)-nicotine>acetylcholine>carbachol] at the C1β4, C1β4β3, C6F223Lβ4, C6F223Lβ4β3, C16F223Lβ4 and C16F223Lβ4β3 receptors, albeit the absolute EC50 values differed somewhat between the receptors. Furthermore, four reference antagonists displayed the same rank order of inhibitory potencies at the six receptors [α-conotoxin PIA>2,2,6,6-tetramethylpiperidin-4-yl heptanoate>mecamylamine>dihydro-β-erythroidine]. Although all interpretations based on these results should be made keeping the molecular modifications in the α6 surrogate subunits in mind, this study sheds light on the pharmacological properties of α6β4⁎ nicotinic acetylcholine receptors and demonstrates the applicability of the C6F223L and C16F223L chimeras for studies of these receptors.

Comparison of Inhibitory Effects between Enalapril and Losartan on Adrenal Catecholamine Secretion

❙ABSTRACT❙ Background: The present study was attempted to compare enalapril, an angiotensin-converting enzyme inhibitor with losartan an angiotensin II (Ang II) receptor blocker in the inhibitory effects on the secretion of catecholamines (CA) from the perfused model of the rat adrenal gland. Methods: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: Both enalapril and losartan during perfusion into an adrenal vein for 90 minutes inhibited the CA release evoked by acetylcholine (ACh), 1.1-dimethyl-4-phenyl piperazinium (DMPP, a selective Nn agonist), high K + (a direct membrane-depolarizer), 3-(m-chloro-phenyl-carbamoyl-oxy-2-butynyl- trimethyl ammonium (McN-A-343, a selective M1 agonist), and Ang II in a time-dependent manner. Also, in the presence of enalapril or losartan, the CA release evoked by veratridine (an activator of voltage-dependent Na + channels), 6-dimethyl-3-nitro-4-(2-trifluoromethyl phenyl)-pyridine-5-carboxylate (BAY-K-8644, an L-type Ca - 2+ channel activator), and cyclopiazonic acid (a cytoplasmic Ca 2+ -ATPase inhibitor) were significantly reduced. Based on the same concentration of enalapril and losartan, for the CA release evoked by ACh, high K + , DMPP, McN-A-343, Ang II, veratridine, BAY-K-8644, and cyclopiazonic acid, the following rank order of inhibitory potency was obtained: losartan > enalapril. In the simultaneous presence of enalapril and losartan, ACh-evoked CA secretion was more strongly inhibited compared with that of enalapril- or losartan-treated alone. Conclusions: Collectively, these results demonstrate that both enalapril and losartan inhibit the CA secretion evoked by activation of both cholinergic and Ang II type-1 receptors stimulation in the perfused rat adrenal medulla. When these two drugs were used in combination, their effects were enhanced, which may also be of clinical benefit. Based on concentration used in this study, the inhibitory effect of losartan on the CA secretion seems to be more potent than that of enalapril. (J Korean Soc Hypertens 2014;20(2):51-67)

Molecular, pharmacological, and signaling properties of octopamine receptors from honeybee (Apis mellifera) brain

G protein-coupled receptors are important regulators of cellular signaling processes. Within the large family of rhodopsin-like receptors, those binding to biogenic amines form a discrete subgroup. Activation of biogenic amine receptors leads to transient changes of intracellular Ca²⁺-([Ca²⁺](i)) or 3',5'-cyclic adenosine monophosphate ([cAMP](i)) concentrations. Both second messengers modulate cellular signaling processes and thereby contribute to long-lasting behavioral effects in an organism. In vivo pharmacology has helped to reveal the functional effects of different biogenic amines in honeybees. The phenolamine octopamine is an important modulator of behavior. Binding of octopamine to its receptors causes elevation of [Ca²⁺](i) or [cAMP](i). To date, only one honeybee octopamine receptor that induces Ca²⁺ signals has been molecularly and pharmacologically characterized. Here, we examined the pharmacological properties of four additional honeybee octopamine receptors. When heterologously expressed, all receptors induced cAMP production after binding to octopamine with EC₅₀(s) in the nanomolar range. Receptor activity was most efficiently blocked by mianserin, a substance with antidepressant activity in vertebrates. The rank order of inhibitory potency for potential receptor antagonists was very similar on all four honeybee receptors with mianserin >> cyproheptadine > metoclopramide > chlorpromazine > phentolamine. The subroot of octopamine receptors activating adenylyl cyclases is the largest that has so far been characterized in arthropods, and it should now be possible to unravel the contribution of individual receptors to the physiology and behavior of honeybees.

In Vitro and Ex Vivo Inhibition of Human Telomerase by Anti-HIV Nucleoside Reverse Transcriptase Inhibitors (NRTIs) but Not by Non-NRTIs

Telomerase is a specialized reverse transcriptase responsible for the de novo synthesis of telomeric DNA repeats. In addition to its established reverse transcriptase and terminal transferase activities, recent reports have revealed unexpected cellular activities of telomerase, including RNA-dependent RNA polymerization. This telomerase characteristic, distinct from other reverse transcriptases, indicates that clinically relevant reverse transcriptase inhibitors might have unexpected telomerase inhibition profiles. This is particularly important for the newer generation of RT inhibitors designed for anti-HIV therapy, which have reported higher safety margins than older agents. Using an in vitro primer extension assay, we tested the effects of clinically relevant HIV reverse transcriptase inhibitors on cellular telomerase activity. We observed that all commonly used nucleoside reverse transcriptase inhibitors (NRTIs), including zidovudine, stavudine, tenofovir, didanosine and abacavir, inhibit telomerase effectively in vitro. Truncated telomere synthesis was consistent with the expected mode of inhibition by all tested NRTIs. Through dose-response experiments, we established relative inhibitory potencies of NRTIs on in vitro telomerase activity as compared to the inhibitory potencies of the corresponding dideoxynucleotide triphosphates. In contrast to NRTIs, the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine and efavirenz did not inhibit the primer extension activity of telomerase, even at millimolar concentrations. Long-term, continuous treatment of human HT29 cells with select NRTIs resulted in an accelerated loss of telomere repeats. All tested NRTIs exhibited the same rank order of inhibitory potencies on telomerase and HIV RT, which, according to published data, were orders-of-magnitude more sensitive than other DNA polymerases, including the susceptible mitochondria-specific DNA polymerase gamma. We concluded that telomerase activity could be inhibited by common NRTIs, including currently recommended RTI agents tenofovir and abacavir, which warrants large-scale clinical and epidemiological investigation of the off-target effects of long-term highly active antiretroviral therapy (HAART) with these agents.

276 COMPARISON OF INHIBITORY EFFECTS BETWEEN LOSARTAN, OLMESARTAN AND FIMASARTAN ON CATECHOLAMINE RELEASE IN THE PERFUSED ADRENAL MEDULLA

Recently, we have found that both losartan (Noh et al. 2009) and olmesartan (Lim et al., 2010) inhibit the secretion of catecholamines (CA) from the perfused rat adrenal medulla. Therefore, the aim of the present study was to compare the inhibitory effects of three major ARBs (losartan, olmesartan and fimasartan) each other on the CA release from the isolated perfused model of the rat adrenal medulla. Losartan (LST, 15 μM), olmesartan (OST, 15 μM) and fimasartan (FST, 15 μM), perfused into an adrenal vein for 90 min, inhibited the CA secretory responses evoked by ACh (5.32 mM) and high K+ (56 mM, a direct membrane depolarizer), DMPP (100 μM, a selective neuronal nicotinic receptor agonist) and McN-A-343 (100 μM, a selective muscarinic M1 receptor agonist) in a time-dependent fashion, respectively. Also, in adrenal glands loaded with LST (15 μM), OST (15 μM) and FST (15 μM), the CA secretory responses evoked by Bay-K-8644 (10 μM, an activator of L-type Ca2+ channels), cyclopiazonic acid (10 μM, an inhibitor of cytoplasmic Ca2+-ATPase), and veratridine (100 μM, an activator of voltage-dependent Na+ channels) as well as by angiotensin ll (100 nM) were time-dependently reduced, respectively. Based on the same concentration (15 μM) of three ARBs, for the CA release evoked by Ach, high K+, DMPP, and McN-A-343, the following rank order of inhibitory potency was obtained: LST<OST<FST. Also, for the CA release evoked by Bay-K-8644, cyclopiazonic acid, and angiotensin ll, the following rank order of inhibitory potency was obtained: LST<OST<FST. However, for the CA release evoked by veratridine, the following rank order of inhibitory potency was obtained: LST<OST=FST. All three ARBs themselves did not affect basal CA output. Collectively, these experimental results suggest that all three ARBs inhibit the CA secretion evoked by cholinergic stimulation (both nicotininc and muscarinic receptors) as well as direct membrane depolarization from the perfused rat adrenal medulla. It seems that this inhibitory effect of all three ARBs may be mediated by blocking the influx of both Na+ and Ca2+ through their voltage-dependent ion channels into the adrenomedullary chromaffin cells as well as by inhibiting the Ca2+ release from its cytoplasmic calcium store. Based on the same concentration used in this study, it is also suggested that there is difference in potency of the inhibitory effects on the CA release between three ARBs, indicating that fimasartan seems to be more potent among three ARBs.

203 INFLUENCE OF SELF-FERMENTED PINE NEEDLE FRACTIONS ON CATECHOLAMINE RELEASE FROM THE RAT ADRENAL MEDULLA

Previously, we have found that self-fermented pine needle (Pinus densiflora) extract inhibits secretion of catecholamines (CA) from the perfused rat adrenal medulla (Shin & Lim, 2010), and also the contractile responses of isolated rat aortic strips induced by phenylephrine and high K+ (Yu et al., 2009). Therefore, the aim of the present study was to investigate the effects of several fractions obtained from methylene chloride (CH2Cl2) extract of self-fermented pine needle on the acetylcholine (ACh)-evoked CA release from the isolated perfused model of the rat adrenal medulla and to establish the mechanism of the most active fraction (Fr.)-induced inhibitory action on the CA release. We obtained 6 fractions from CH2Cl2 extract of self-fermented pine needle. For the ACh (5.32 mM)-evoked CA release, the following rank order of inhibitory potency was obtained: Fr.4>Fr.8-11>>Fr.3>Fr.6=Fr.7>Fr.1.2. Fr.4 (60 μg/mL) perfused into an adrenal vein for 90 min produced relatively time-dependent inhibition of the CA secretory responses to ACh (5.32 mM), high K+ (56 mM), DMPP (100 μM) and McN-A-343 (100 μM). Fr.4 itself did not affect basal CA secretion (data not shown). Also, in the presence of Fr.4 (60 μg/mL), the CA secretory responses to angiotensin II (AngII, 0.1 μM), veratridine (a selective Na+ channel activator, 10 μM), Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10 μM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 μM) were significantly reduced, respectively. In the simultaneous presence of Fr.4 (60 μg/mL) and L-NAME (an inhibitor of NO synthase, 30 μM), the inhibitory responses of Fr.4 on the CA secretion evoked by ACh, high K+, DMPP, McN-A-343, Bay-K-8644, cyclopiazonic acid, AngII and veratridine were considerably recovered to the extent of the corresponding control secretion compared with that of Fr.4-treatment alone. The level of NO released from adrenal medulla after the treatment of Fr.4 (60 μg/mL) was greatly elevated compared with the basal level. Taken together, these results demonstrate that Fr.4 inhibits the CA secretion from the isolated perfused rat adrenal medulla evoked by stimulation of cholinergic receptors as well as by direct membrane-depolarization. It seems that this inhibitory effect of Fr.4 is mediated by blocking the influx of Ca2+ and Na+ into the adrenomedullary chromaffin cells as well as by inhibition of Ca2+ release from the cytoplasmic calcium store, which is evoked at least partly through the increased NO production due to the activation of NO synthase. Based on these results, it is also thought that Fr. 4 isolated from CH2Cl2 extract of pine needle may contain beneficial antihypertensive components to prevent or treat hypertension.

Wide Spectrum of Inhibitory Effects of Sertraline on Cardiac Ion Channels

Sertraline is a commonly used antidepressant of the selective serotonin reuptake inhibitors (SSRIs) class. In these experiments, we have used the whole cell patch clamp technique to examine the effects of sertraline on the major cardiac ion channels expressed in HEK293 cells and the native voltage-gated Ca2+ channels in rat ventricular myocytes. According to the results, sertraline is a potent blocker of cardiac K+ channels, such as hERG, IKs and IK1. The rank order of inhibitory potency was hERG >IK1> IKs with IC50 values of 0.7, 10.5, and 15.2 µM, respectively. In addition to K+ channels, sertraline also inhibited INa and ICa, and the IC50 values are 6.1 and 2.6 µM, respectively. Modification of these ion channels by sertraline could induce changes of the cardiac action potential duration and QT interval, and might result in cardiac arrhythmia.

Pharmacological Effects of Imidafenacin (KRP-197/ONO-8025), a New Bladder Selective Anti-cholinergic Agent, in Rats

Imidafenacin (CAS 170105-16-5, KRP-197, ONO-8025) has been developed for the treatment of overactive bladder as a new anti-cholinergic with high affinities for muscarinic acetylcholine M3 and M1 receptors. The pharmacological profiles of imidafenacin on the urinary bladder function by determining carbamylcholine (CCh)-induced decrease in bladder capacity and distention-induced rhythmic bladder contraction in conscious rats were investigated. In addition, effects of imidafenacin on CCh-induced salivary secretion and performance in the Morris water maze task in rats were investigated to evaluate side effects, such as dry mouth and cognitive dysfunction in the central nervous system (CNS). Imidafenacin prevented the CCh-induced decrease in bladder capacity dose-dependently with an ID50 of 0.055 mg/kg. On the distention-induced rhythmic bladder contraction, imidafenacin, propiverine, tolterodine, oxybutynin and darifenacin showed inhibitory effects with ID30's of 0.17, 15, 3.0, 3.2 and 0.85 mg/kg, respectively. The rank order of inhibitory potency was: imidafenacin > darifenacin > tolterodine > or = oxybutynin > propiverine. Imidafenacin, propiverine, tolterodine, oxybutynin and darifenacin showed inhibitory effects on the CCh-stimulated salivary secretion with ID50's of 1.5, 14, 15, 4.4 and 1.2 mg/kg, respectively. The rank order of inhibitory potency was: darifenacin > or = imidafenacin > oxybutynin > propiverine > or = tolterodine. Imidafenacin at the doses of 1 and 10 mg/ kg did not affect the escape latencies in the Morris water maze task compared with those in vehicle controls. Oxybutynin at the dose of 100 mg/kg induced a significant increase in the escape latencies, but propiverine at the dose of 100 mg/kg did not induce significant changes. These results suggest that imidafenacin inhibits urinary bladder contraction to a greater extent than the salivary secretion (compared with the M3 receptor selective antagonist, darifenacin, and the non-selective antagonists, propiverine, tolterodine and oxybutynin) or the CNS functions, such as performance in the Morris water maze task (compared with oxybutynin). In conclusion, imidafenacin has organ selectivity for the bladder over the salivary gland, without influence on the central nervous system such as spatial learning and memory.

Extended kinase profile and properties of the protein kinase inhibitor nilotinib

As a drug used to treat imatinib-resistant and -intolerant, chronic and advanced phase chronic myelogenous leukaemia, nilotinib is well characterised as a potent inhibitor of the Abl tyrosine kinase activity of wild-type and imatinib-resistant mutant forms of BCR-Abl. Here we review the profile of nilotinib as a protein kinase inhibitor. Although an ATP-competitive inhibitor of Abl, nilotinib binds to a catalytically inactive conformation (DFG-out) of the activation loop. As a consequence of this, nilotinib exhibits time-dependent inhibition of Abl kinase in enzymatic assays, which can be extrapolated to other targets to explain differences between biochemical activity and cellular assays. Although these differences confound assessment of kinase selectivity, as assessed using a combination of protein binding and transphosphorylation assays, together with cellular autophosporylation and proliferation assays, well established kinase targets of nilotinib in rank order of inhibitory potency are DDR-1>DDR-2>BCR-Abl (Abl)>PDGFRα/β>KIT>CSF-1R. In addition nilotinib has now been found to bind to both MAPK11 (p38β) and MAPK12 (p38α), as well as with very high affinity to ZAK kinase. Although neither enzymatic nor cellular data are yet available to substantiate the drug as an inhibitor of ZAK phosphorylation, modeling predicts that it binds in an ATP-competitive fashion.

Extending matrix‐assisted laser desorption/ionization triple quadrupole mass spectrometry enzyme screening assays to targets with small molecule substrates

Mass spectrometry (MS)-based high-throughput screening (HTS) has tremendous potential as an alternative to current screening methods due to its speed, sensitivity, reproducibility and label-free readout. We recently reported that a new generation matrix-assisted laser desorption/ionization triple quadrupole (MALDI-QqQ) mass spectrometer is ideally suited for a variety of enzyme assays and screening protocols. However, all the targets measured to date had peptide substrates that were easily monitored by selected ion monitoring (SIM) without interference from the MALDI matrix. To further extend the application to enzymes with small molecule, non-peptide substrates, we evaluated this method for measuring enzyme activity and inhibition of acetylcholinesterase (AChE). Due to the potential of MALDI matrix interference, multiple reaction monitoring (MRM) was investigated for selective MS/MS transitions and to accurately measure the conversion of acetylcholine into choline. Importantly, ionization, detection and MRM transition efficiency differences between the substrate and product can be overcome by pre-balancing the MRM transitions during method development, thus allowing for a direct readout of the enzyme activity using the ratio of the substrate and product signals. Further validation of the assay showed accurate concentration-dependent inhibition measurements of AChE with several known inhibitors. Finally, a small library of 1008 drug-like compounds was screened at a single dose (10 microM) and the top 10 inhibitors from this primary screen were validated in a secondary screen to determine the rank order of inhibitory potency for each compound. Collectively, these data demonstrate that a MALDI-QqQMS-based readout platform is amenable to measuring small molecule substrates and products and offers significant advantages over current HTS methods in terms of speed, sensitivity, reproducibility and reagent costs.

Roles of Dopaminergic D1and D2Receptors in Catecholamine Release from the Rat Adrenal Medulla

The aim of the present study was designed to establish comparatively the inhibitory effects of D(1)-like and D(2)-like dopaminergic receptor agonists, SKF81297 and R(-)-TNPA on the release of catecholamines (CA) evoked by cholinergic stimulation and membrane depolarization from the isolated perfused model of the rat adrenal medulla. SKF81297 (30microM) and R-(-)-TNPA (30microM) perfused into an adrenal vein for 60 min, produced great inhibition in the CA secretory responses evoked by ACh (5.32x10(- 3) M), DMPP (10(-4) M), McN-A-343 (10(-4) M), high K(+) (5.6x10(-2) M), Bay-K-8644 (10microM), and cyclopiazonic acid (10microM), respectively. For the release of CA evoked by ACh, high K(+), DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid, the following rank order of inhibitory potency was obtained: SKF81297>R-(-)-TNPA. However, R(+)-SCH23390, a selectve D(1)-like dopaminergic receptor antagonist, and S(-)-raclopride, a selectve D(2)-like dopaminergic receptor antagonist, enhanced the CA secretory responses evoked by ACh, high K(+), DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid only for 0~4 min. The rank order for the enhancement of CA release evoked by high K(+), McN-A-343 and cyclopiazonic acid was R(+)-SCH23390>S(-)-raclopride. Also, the rank order for ACh, DMPP and Bay-K-8644 was S(-)-raclopride > R(+)-SCH23390. Taken together, these results demonstrate that both SKF81297 and R-(-)-TNPA inhibit the CA release evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors and the membrane depolarization from the isolated perfused rat adrenal gland without affecting the basal release, respectively, but both R(+)-SCH23390 and S(-)-raclopride facilitate the CA release evoked by them. It seems likely that the inhibitory effects of SKF81297 and R-(-)-TNPA are mediated by the activation of D(1)-like and D(2)-like dopaminergic receptors located on the rat adrenomedullary chromaffin cells, respectively, whereas the facilitatory effects of R(+)-SCH23390 and S(-)-raclopride are mediated by the blockade of D(1)-like and D(2)-like dopaminergic receptors, respectively: this action is possibly associated with extra- and intracellular calcium mobilization. Based on these results, it is thought that the presence of dopaminergic D(1) receptors may play an important role in regulation of the rat adrenomedullary CA secretion, in addition to well-known dopaminergic D(2) receptors.

Flavonoids and Vitamin E Reduce the Release of the Angiogenic Peptide Vascular Endothelial Growth Factor from Human Tumor Cells

Neoangiogenesis is required for tumor development and progression. Many solid tumors induce vascular proliferation by production of angiogenic factors, prominently vascular endothelial growth factor (VEGF). Because nutrition is a causative factor for tumor prevention and promotion, we determined whether secondary plant constituents, i.e., flavonoids, tocopherols, curcumin, and other substances regulate VEGF in human tumor cells in vitro. VEGF release (concurrent with synthesis) from MDA human breast cancer cells and, for comparison, U-343 and U-118 glioma cells was measured by ELISA. Of 21 compounds tested, 9 showed significant inhibitory activity at 0.1 micromol/L in MDA human breast cancer cells. The rank order of inhibitory potency was naringin > rutin > alpha-tocopheryl succinate (alpha-TOS) > lovastatin > apigenin > genistein > alpha-tocopherol >or= kaempferol > gamma-tocopherol; chrysin and curcumin were inactive except at a concentration of 100 micromol/L. Glioma cells were similarly sensitive, with U343 more than U118, especially for alpha-TOS and tocopherols. Among the tocopherol derivatives, alpha-TOS (0.1 micromol/L) was the most effective in reducing VEGF release. Overall, the glycosylated flavonoids (i.e., naringin, a constituent of citrus fruits, and rutin, a constituent of cranberries) induced the greatest response to treatment at the lowest concentration in MDA human breast cancer cells. Inhibition of VEGF release by flavonoids, tocopherols, and lovastatin in these models of neoplastic cells suggests a novel mechanism for mammary cancer prevention.