Molar Potency Research Articles

Bioactivity of 11 keto and hydroxy androgens in yeast and mammalian host cells

Recent studies have highlighted the potential role of 11oxygenated (keto or hydroxy) androgens in human reproductive function with 11keto androgens circulating at concentrations comparable with testosterone in women and children. However, the intrinsic androgenic bioactivities of 11 keto and hydroxy androgens are not fully characterized. We therefore investigated the full androgen dose-response curves using complementary in vitro yeast and mammalian (HEK293) host cell bioassays of 11 keto and hydroxy derivatives of the potent androgens, testosterone (T) and dihydrotestosterone (DHT), compared with their parent non-11 oxygenated steroids together with the pro-androgen precursor (androstenedione (A4)) and metabolites (androstanedione, androsterone). For potent androgens, the mammalian HEK293 host cell bioassay was 22–138 times more sensitive than the yeast host cell bioassay. In both androgen bioassays, 11keto derivatives displayed androgenic bioactivity but significantly lower molar potency than their parent non-keto steroids. By contrast, the 11hydroxy derivatives had minimal or no androgenic bioactivity. In both bioassays 5α-reduction increased androgenic potency. These findings confirm that that 11keto androgens may contribute directly to androgen status in women, children, and other conditions apart from healthy eugonadal men whereas 11hydroxy androgens have negligible androgenic potency although it cannot be excluded that they may be converted to more potent androgens in vivo.

Antimicrobial and Antifungal Activity of Rare Substituted 1,2,3-Thiaselenazoles and Corresponding Matched Pair 1,2,3-Dithiazoles.

We report our investigations into the underlying differences between 1,2,3-dithiazole and their ultra-rare counterpart, 1,2,3-thiaselenazole. This rare 1,2,3-thiaselenazole chemotype was afforded by sulfur extrusion and selenium insertion into the preconstructed 1,2,3-dithiazoles. We built a library of matched paired compounds to compare and contrast the two ring systems. This led to the development of both narrow and broad-spectrum antimicrobial compounds with sub-micro molar potency, limited to no toxicity and a further understanding of the transition state electronics through molecular simulations. We also identified the potent 4,5,6-trichlorocyclopenta[d][1,2,3]thiaselenazole 11a, for use against Candida albicans, Cryptococcus neoformans var. grubii, Staphylococcus aureus and Acinetobacter baumannii, all of which have limited clinical treatment options. The 1,2,3-thiaselenazole represents a new class of potential compounds for the treatment of a host of multi-resistant hospital derived infections.

Discovery of novel 3-(hydroxyalkoxy)-2-alkylchromen-4-one analogs as interleukin-5 inhibitors

A series of novel chromen-4-one analogs 9a-d and 10a-u was designed, synthesized and evaluated for their IL-5 inhibitory activity. Most of the chromen-4-one analogs showed strong inhibitory activity in low micro molar potency. Among them, 5-(cyclohexylmethoxy)-3-(3-hydroxypropoxy)-2-isopropyl-4H-chromen-4-one (10t, 90.0% inhibition at 30 μM, IC50 = 5.5 μM, CLogP = 4.76887) and 2-cyclohexyl-5-(cyclohexylmethoxy)-3-(3-hydroxypropoxy)-4H-chromen-4-one (10u, 95.5% inhibition at 30 μM, IC50 = 3.0 μM, CLogP = 5.96187) showed the best inhibition. The structure activity relationship reveals that the hydrophobic cyclohexylmethoxy group at the position 5 of the chromen-4-one ring A is preferable than at position 6 and the dual hydrogen bonding acceptor property on the chromen-4-one ring should be important for the inhibitory activity. In addition, the optimum length of the side chain at position 3 of chromen-4-one ring is critical for the donation of hydrogen to the binding site and the 3-hydroxypropoxy group showed the best activity. Moreover, the conformational restrictor (isopropyl, cyclohexyl group) at position 2 is much more favorable for the formation of effective conformer of side chain with hydrogen bonding donor property of these chromen-4-one analogs.

478 Pharmacological disruption of the Astrocytic Elevated Gene-1 (AEG1) in anticancer intervention: PB0412_3 (PB03) as a first-in-class AEG1 interacting agent

Background: AEG1 is highly expressed in most solid tumors conferring multidrug resistance, increased proliferation, angiogenesis, metastatic potential and poor outcome. AEG1 activates multiple signal transduction pathways such as PIK3/AKT, NFKB, MAPK, WNT and is a rational target for therapeutic intervention in cancer. Our previous data in advanced EGFR mutated and EGFR wild type lung cancer patients confirmed that AEG1 overexpression is indicative of poor outcome to targeted and pleiotropic therapy. In addition, AEG1 functionality is instrumental in the natural history of brain tumors. Material and Methods: We designed an in silico model based on inhibition of the NFkB (p65) binding domain of AEG1, identifying a possible interacting interface between the two proteins that could be modeled as a 3-dimensional structure. This interface was reduced and transformed into a 3-point pharmacophore for virtual screeningcomparing both molecular interaction fields and the pharmacophore. A total of 217,000 chemical entities (CEs) were tested in this model. Each CE was scored using the Hercules affinity ranking. The top sixty CEs amongst the high scores were selected, and thus 4 compounds were proposed for in vitro screening. Twenty analogs were then synthesized, and PB03, a small molecular weight polyheterocyclic compound, was selected as the CE lead. Results: PB03 displays antitumor activity at low mM and nM concentrations. PB03-induced growth inhibition appears to be independent of EGFR, KRAS, BRAF, PIK3CA and p53 mutational status, Her2 amplification, and BRCA1 and AEG1 mRNA expression. PB03 clusters in vitro molar potency (MP) of Glial (Gliobastoma, GBM) and Neural Crest (Neuroblastoma (NBM) and Melanoma) -derived tumors as compared with PB03 profile in other solid tumors and in non-tumoral cells (p < 0.0001). A lack of impact of MGMT methylation status on PB03 (MP) sensitivity has been observed. PB03 is between 500 and 1000 fold more potent than temozolomide and MTIC in GBM and NBM (p < 0.0001). The mean PB03 IC50s in human non-tumoral cell lines is 2.08mM versus 1.22mM in the solid tumor panel vs 0.40mM in the Glial and Neural Crest-derived panel vs 0.11mM in GBM+NBM. Preliminary data in PC9 shows downregulation of AEG1 mRNA expression upon exposure to PB03. Expansion of the Glial and Neural Crest-derived tumor panel, cell kinetics, drug-induced cell death and in vivo/PK-PD studies in in vivo models is ongoing. PB03 has a negative score when screened in the SCIfinder chemical data base confirming PB03 as a first-in-class CE. Conclusions: This is the first successful attempt at the pharmacological intervention against the AEG1 pathway. The observed clustering of Glial and Neural Crest-derived malignancies is unique and a tool for rational development.

Abstract 4601: Astrocytic elevated gene 1 (AEG1) a target for pharmacological anticancer intervention

Abstract AEG1 is highly expressed in solid tumors; hyperexpression confers multidrug resistance, increased tumor proliferation, angiogenesis, metastatic potential and poor outcome in most solid tumors. AEG1 activates multiple protumorogenic signal transduction pathways like PIK3/ALT, NFKB, MAMPK, WNT and is a rational target for therapeutic intervention in solid tumors.Also AEG1 functionality is instrumental in the natural history of brain tumors. Our data in NSCLCs confirm AEG1 overexpression as the main parameter (uni and multivariate analysis) indicative of outcome to targeted and cytotoxic therapy. We designed an in silico model based on inhibition of AEG1-p65 crosstalk, identifying a possible interacting interface between the 2 proteins that could be modeled as a three-dimensional structure. This interface was further reduced and transformed into a 3-point pharmacophore for virtual screening comparing both molecular interaction fields and the pharmacophore. A total of 217.000 chemical entities (CEs) were tested in this model giving a Hercules® affinity ranking. Sixty CEs with the highest Hercules® ranking were identified for a second (16 CEs) and third (5 CEs) selection to implement the first round of in vitro studies in our human solid tumor panel. Twenty analogs were generated to seek for a lead according to prospective selection criteria. PB0412-3 (PB3), a small molecule polyheterocyclic compound, was selected for further development. PB3 displays antitumor activity in the human solid tumor panel (median IC50 1.3 uM) and PB3-induced growth inhibition appears independent of EGFR, KRAS &amp; p53 mutational status, Her2 amplification, PIK3 mutations and BRCA1 expression;tumor cell lines bearing highest AEG1-mRNA expression levels were sensititive to PB3. Median IC50 in our glioblastoma (GBM) panel was 60 nM with no impact of MGMT methylation status on PB3's molar potency (MP). IC50s delta solid vs brain tumors were significant (p&amp;lt;.0001) representing a 25-fold increase in MP. Data from nitrosureas in the same GBM panel indicate PB3 is 250 times more potent than BCNU and temozolamide. Median IC50s in human non cancer lines is 2 uM. PB3 was re-screened in the in silico model; PB3 fully matches the AEG1 binder pharmacophore model and was screened in the Chemical Space web tool; results confirm PB3 as first-in-class CE. Functional studies are ongoing; preliminary data in PC9 NSCLC cell line demonstrate significant PB3-induced downregulation of AEG1 mRNA expression. Expansion of the CNS tumor panel, cell kinetics, drug-induced cell death &amp; in vivo/PK-PD studies in SC orthotopic models is ongoing. To the best of our knowledge, this is the first successful attempt at the Pharmacological intervention against the AEG1 pathway. A dual developmental plan for PB3 as local brain drug delivery and systemic therapy is proposed. Citation Format: Jose M. Jimeno, Gerardo Acosta, Miguel Ángel Molina, Nicky Karachaliou, Cristina Teixidó, Carlos Obiol, Oriol Villacañas, Jordi Bertran, María Sánchez Rouco, Ana Giménez Capitán, Santiago Ramón y Cajal, Miquel Taron, Rafael Rosell, Fernando Albericio. Astrocytic elevated gene 1 (AEG1) a target for pharmacological anticancer intervention. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4601. doi:10.1158/1538-7445.AM2014-4601

PQJS380

Acute lymphoblastic leukemia (ALL) is a malignant disorder of lymphoid progenitor cells that are committed to the B- or the T-cell lineage. The pathogenesis of ALL is heterogeneous and may be at least in part caused by genetic alterations. Although the modern sequencing technologies make it possible to rapidly discover novel genetic and epigenetic alterations and molecular targets for therapeutic intervention for ALL, conventional chemotherapy is still the most important therapeutic approach. Relapses and high morbidity and mortality remain major challenges particularly in adult patients with ALL. Therefore, development of novel chemotherapeutic agents remains in demand for ALL patients. In the course of seeking novel agents against ALL, we screened a library of small molecules and identified that PQJS380, a S-(E)-4-([7S,10S]-4-ethyl-7-isopropyl-2,5,8,12-tetraoxo-9-oxa-3,6,13,18-tetraaza-bicycle[13,2,1] octadec-1-en-10-yl)but-3-enyl octanethioate, showed potent anti-leukemia activity. PQJS380 inhibited the proliferation with IC50 values of 14.25 nM and 5 nM in REH and NALM-6 cells, respectively. PQJS380 had 10-fold higher molar potency than the front-line ALL drugs Ara-C and VP-16. The median IC50 value for leukemia blast cells from 17 patients with ALL was 52 nM. PQJS380 induced G1-phase arrest in REH cells, and S-phase in NALM-6 cells, respectively. Treatment of PQJS380 led to apoptosis in ALL cell lines (REH and NALM-6) and primary ALL cells. Our data supported that PQJS380 may be a promising lead compound for ALL treatment even though the precise targets remain to be elucidated.

Pharmacokinetic basis of the antiplatelet action of prasugrel

Prasugrel is the most recent development of thienopyridine-type antiplatelet drugs. Like the earlier-generation thienopyridines, i.e. ticlopidine and clopidogrel, prasugrel is also an inactive prodrug that requires metabolic processing in vivo to generate the active antiplatelet metabolite. The efficacy of this bioactivation is the key determinant for the pharmacodynamic potency of the compound, i.e. the irreversible blockade of the platelet P2Y12-ADP receptor. Prasugrel is rapidly absorbed from the gut. After oral administration of standard-loading doses of 60 mg, maximum plasma levels of the active metabolite are achieved within 1 h, effective, maximum inhibition of platelet aggregation at 1-2 h. Bioconversion of prasugrel into the active metabolite requires two metabolic steps that occur in sequence. The first is the generation of a thiolactone-intermediate, mainly by carboxyesterases-2 in the intestine, the second the cytochrome (CYP)-dependent conversion of the thiolactone into the active metabolite. This second step involves several cytochromes, most notably CYP3A4, CYP2C19, CYP2B6, and CYP2C9. The enzymatic generation of the active metabolite of prasugrel is much more effective than that of clopidogrel where only about 5% of oral clopidogrel is transformed into the active compound by two-step CYP-dependent procedures. About 70% of prasugrel metabolites are excreted in the urine and 30% in the feces. The molar potency of the respective active metabolites of prasugrel and clopidogrel is identical. Thus, the more rapid onset, higher potency and lower interindividual variability of antiplatelet effects of prasugrel as compared to clopidogrel in vivo are entirely because of its more efficient pharmacokinetics.

Angiogenic and Anti-Angiogenic Therapy for Gastrointestinal Ulcers: New Challenges for Rational Therapeutic Predictions and Drug Design

Gastrointestinal (GI) ulcers are essentially internal wounds that resist normal healing processes. Since their pathogenesis is poorly understood, and the etiologic (e.g., gastric acid, aspirin-like drugs, stress) and aggravating factors (e.g., H. pylori) are not well characterized, the remaining therapeutic option is to accelerate healing. Superficial mucosal lesions, i.e., erosions usually heal by epithelial regeneration and restitution, but when ulcers involve the muscularis propria, smooth muscle cells do not divide/regenerate. These deep lesions are filled by granulation tissue, i.e., angiogenesis followed by proliferation of connective tissue fibroblasts that deposit collagen over which adjacent surviving and dividing epithelial cells migrate to complete the healing. Our laboratory was the first to postulate that stimulation of angiogenesis alone might be sufficient to accelerate ulcer healing in the GI tract. Indeed, daily treatment of rats with bFGF, PDGF or VEGF markedly improved the healing of cysteamine-induced chronic duodenal ulcers, without any reduction in gastric acid secretion. These results were reproduced by a single dose of gene therapy by adenoviral vectors encoding PDGF or VEGF genes. The molar potency of angiogenic growth factors was 2-7 million times better than the antiulcerogenic effect of antisecretory H2 antagonists. Since histologically & pathologically gastroduodenal ulcers look similar to ulcers in the lower GI tract, we also predicted that the healing of experimental ulcerative colitis might be also improved by these angiogenic growth factors. Rectal enemas containing bFGF or PDGF indeed accelerated the healing of chemically induced ulcerative colitis in rats. VEGF, also known as VPF (vascular permeability factor), however, had no effect or slightly aggravated the colonic lesions. Injection of anti-VEGF neutralizing antibodies, however, counteracted the increased vascular permeability in the early stages of experimental ulcerative colitis and subsequently decreased the number of inflammatory cells in colonic ulcers in rats, resulting in significantly improved healing in the lower GI tract lesions. Thus, the three angiogenic growth factors tested exerted beneficial effect on gastroduodenal ulcers, and rectal enemas with bFGF or PDGF also accelerated the healing of experimental ulcerative colitis. Surprisingly, we achieved the latter effect with anti-VEGF antibodies, most likely because of the pro-inflammatory actions of VEGF in the pathogenesis of ulcerative colitis.

Insulin-releasing and metabolic effects of small molecule GLP-1 receptor agonist 6,7-dichloro-2-methylsulfonyl-3-N- tert-butylaminoquinoxaline

Much recent attention has focused on the GLP-1 receptor as a potential target for antidiabetic drugs. Enzyme resistant GLP-1 mimetics such as exenatide are now employed for the treatment of type 2 diabetes, but must be administered by injection. The present study has examined and compared the in vitro and in vivo metabolic actions of a small molecule GLP-1 receptor agonist 6,7-dichloro-2-methylsulfonyl-3-N- tert-butylaminoquinoxaline (DMB), with native GLP-1, exenatide and liraglutide. DMB significantly stimulated in vitro insulin secretion from BRIN-BD11 cells but with decreased molar potency compared to native GLP-1 or related mimetics. Administration of DMB in combination with glucose to mice significantly ( P < 0.05) decreased the overall glucose excursion compared to controls. Exenatide and liraglutide evoked similar ( P < 0.001) reductions of the overall glycaemic excursion, but were significantly ( P < 0.001 and P < 0.05; respectively) more effective than DMB. These observations were associated with prominently ( P < 0.05) enhanced glucose-mediated insulin release by exenatide and liraglutide, but not by DMB. Combined injection of DMB with either liraglutide or exenatide did not substantially improve glucose-lowering or insulin-releasing responses. However, administration of DMB in combination with exendin(9–39) did not impair its glucoregulatory actions. These results provide evidence to support the development and potential use of low molecular weight GLP-1 receptor agonists for the treatment of type 2 diabetes.

ORIGINAL RESEARCH—BASIC SCIENCE: Initial Characterization of Hydrogen Sulfide Effects in Female Sexual Function

ABSTRACT Introduction In our male animal models, hydrogen sulfide (H2S) displayed significant vasodilatory and smooth muscle relaxant effects suggestive of an endogenous physiological role in erectile process. Aim In this first exploratory study, we aimed to identify the existence and mechanism of H2S pathway in female sexual physiology. Methods Vaginal and clitoral cavernosal smooth muscle strips from New Zealand white rabbits (N = 12) were exposed to stable H2S donor, sodium hydrosulfide hydrate (NaHS.xH2O, 100 µM–1.6 mM), in isometric tension studies. The NaHS responses were repeated after incubations with (i) Nω-nitro-L-arginine (50 µM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µM) or cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2-amine (MDL 12,330A) (10 µM); and (ii) potassium chloride medium (high K+ 60 mM/low K+ 10 mM), tetraethylammonium (10 mM) or glibenclamide (100 µM). Relaxant effect of NaHS was also compared with those of nitroglycerine (0.18–78.2 µM) and sildenafil (0.084–25.3 µM). Additionally, samples (N = 16) were collected for estimations of plasma and tissue H2S and expression levels of cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) proteins. Main Outcome Measures In vitro evidences for H2S formation and its physiopharmacological effects. Results NaHS produced significant concentration-dependent relaxation of vaginal and cavernosal smooth muscles with inhibitions by combination of ODQ and MDL 12,330A (26.4%), Nω-nitro-L-arginine (22.2%), high K+ (15.1%) or glibenclamide (10.1%). Based on molar potency, NaHS was 18.3 and 6.3 times weaker than nitroglycerine and sildenafil, respectively. Quantitative assays indicated that plasma H2S level was 16.5 ± 2.58 µM, and H2S was synthesized in the clitoral and vaginal smooth muscles (1.8 and 3.9 nmol/mg soluble protein compared with 26.5 nmol/mg in the liver: positive control). Similarly, western blotting identified the protein expression bands of CSE (44.5 kDa) and CBS (63 kDa) in these genital tissue samples. Conclusion These pilot studies clearly indicate the smooth muscle relaxant effect of H2S in female genital tract, mediating through cyclic adenosine 3′:5′-monophosphate, nitric oxide-cyclic guanosine monophosphate and K+ATP channels. Taken together with biochemical and molecular evidences for endogenous formation, H2S pathway could be a contributing factor in female sexual responses.

Correlación entre la concentración molar del atracurio y su tiempo de comienzo o velocidad de acción

To study the correlation between the subparalyzing dose of atracurium and its onset time and speed of action. Six groups of patients received 0.36, 0.68, 0.98, 1.44, 1.77, or 2.06 times the median effective dose of atracurium. The maximal effect, onset time, molar potency (microM kg(-1)) and speed of action (relationship between time in seconds and percentage of maximum effect and between percentage of maximum effect and time) of each dose were measured. The values were converted to logarithms and square roots to test for correlations. The regression line provided the equation, coefficient of determination (R2), and statistical significance. The correlation between the logarithms of molar potency and onset time was poor and not statistically significant (R2 = 0.564, P = .138), whereas the correlations between the logarithms of molar potency and speed of action (seg/%) (s/maximum % effect, R2 = 0.952; and maximum % effect/s, R2 = 0.984) were strong and statistically significant (P < .0001) The regression formulas were able to predict the onset time. The results were similar when the values were converted to their square roots. The onset time of a neuromuscular blocker is linked to its effect and its permeability is linked to the size of its molecules or the molar potency, which is dose-dependent. In the case of atracurium, this correlation was poor and not significant. This finding coincides with those of other authors, who found that atracurium was distant from the regression line for equipotent quantities of different neuromuscular blockers. Substituting onset time with speed of action, however, made it possible to establish a strong and significant correlation. Our results suggest that the speed of action of atracurium depends on the size of its molecule.

Effects of big endothelin-1 in comparison with endothelin-1 on the microvascular blood flow velocity and diameter of rat mesentery in vivo

Synthetic big endothelin-1 (ET-1), a 39-residue precursor of ET-1, has been reported to elicit potent contractile action on helical strip specimens obtained from the porcine coronary artery, but its molar potency was found to be 140-fold lower than that of ET-1 [Saito, Y., Nakao, K., Mukoyama, M., Imura, H., 1990. Increased plasma endothelin level in patients with essential hypertension. N. Engl. J. Med. 322, 205]. It has been hypothesized that the increased rate of production and/or release of ET-1 from the vascular endothelium may contribute to the pathogenesis of hypertension. However, the effects of big ET-1 in comparison with ET-1 on the macrocirculation and microcirculation of the rat mesentery have not been well documented. Thus, our main purpose for this study was to examine the effects of both big ET-1 and ET-1 to clarify the role of phosphoramidon in inhibiting the conversion of big ET-1 to ET-1, by investigating the systemic blood pressure, microvascular blood flow velocity, and diameters of arterioles and venules of the rat mesentery. For this purpose, two groups of experiments were performed. In these experiments, the mesentery was arranged for in situ intravital microscopic observation under transillumination. In the first group of experiments, intravenous cumulative injections of big ET-1 or ET-1 were infused through a catheter inserted into the right jugular vein. Infusion of big ET-1 (1–8 nmol/kg) elicited a long-lasting significant pressor effect. Infusion of big ET-1 (1–2 nmol/kg) elicited a significant dose-dependent increase in the microvascular blood flow velocity both in arterioles (20–30 μm) and venules (30–40 μm). Microvascular diameters exhibited a slight but significant vasodilator effect. However, the infusion of big ET-1 (4–8 nmol/kg) elicited a dose-dependent significant decrease in the blood flow velocities, and diameters returned to control measurements. The administration of ET-1 (0.25–2 nmol/kg) induced a dose-dependent significant decrease in the blood flow velocity of arterioles and venules, and their diameters exhibited a vasoconstrictive effect more prominent in arterioles than in venules. In the second group of experiments, cumulative injections of phosphoramidon (30 mg/kg/10 min) were administered 10 min prior to the infusion of big ET-1. Phosphoramidon significantly suppressed the long-lasting significant pressor effect and significantly inhibited the dose-dependent increase and dose-dependent decrease in the microvascular blood flow velocity produced by big ET-1 in the rat mesenteric microcirculation. This study observed differences in the effects big ET-1 and ET-1 have on the rat mesenteric microcirculation and proposes a possible mechanism explaining these differences. Moreover, phosphoramidon markedly inhibited the conversion of big ET-1 to ET-1 in the rat mesenteric microcirculation, which may suggest an inhibition of the enzyme which converts big ET-1 to ET-1.

This Month in Anesthesiology

This Month in Anesthesiology

A Comparison of Median Effective Doses of Intrathecal Levobupivacaine and Ropivacaine for Labor Analgesia

The study was designed to determine and compare the median effective doses (MEDs) of intrathecal ropivacaine with levobupivacaine for labor analgesia. In this double-blind study, 100 parturients in early labor were randomized to receive either intrathecal ropivacaine or levobupivacaine. For each drug, the patients were assigned to receive one of the five doses studied, namely 1, 1.5, 2, 2.5, or 3 mg. Effective analgesia was defined as a pain score (0-100 visual analog scale) of less than 10 within 15 min of injection, lasting for 45 min or more after the induction of analgesia. MEDs were derived from probit analysis. The duration of analgesia rendered by the two drugs at 2.5 and 3 mg was also compared. The MED for levobupivacaine was 1.07 mg (95% confidence interval, 0.88-1.25 mg), and the MED for ropivacaine was 1.40 mg (95% confidence interval, 1.20-1.61 mg). Levobupivacaine was found to be 1.31 (95% confidence interval, 1.04-2.01) times more potent than ropivacaine. At doses of 2.5 mg or greater, there was no significant difference in duration of analgesia between levobupivacaine (median, 63.5 min; range, 46-123 min) and ropivacaine (median, 59.0 min; range, 47-93 min; P = 0.18). We detected no difference in the incidence of hypotension, nausea and vomiting, motor block, or abnormal fetal heart tracing between the two drugs. The MED of intrathecal ropivacaine for labor analgesia was significantly greater than levobupivacaine experimentally, but this significance was reduced when the comparison was based on molar potency. There was no difference in the duration of analgesia or adverse effects between the two drugs at higher doses (2.5 mg or greater).

S-nitroso proteome of Mycobacterium tuberculosis: Enzymes of intermediary metabolism and antioxidant defense.

The immune response to Mycobacterium tuberculosis (Mtb) includes expression of nitric oxide (NO) synthase (NOS)2, whose products can kill Mtb in vitro with a molar potency greater than that of many conventional antitubercular agents. However, the targets of reactive nitrogen intermediates (RNIs) in Mtb are unknown. One major action of RNIs is protein S-nitrosylation. Here, we describe, to our knowledge, the first proteomic analysis of S-nitrosylation in a whole organism after treating Mtb with bactericidal concentrations of RNIs. The 29 S-nitroso proteins identified are all enzymes, mostly serving intermediary metabolism, lipid metabolism, and/or antioxidant defense. Many are essential or implicated in virulence, including defense against RNIs. For each of two target enzymes tested, lipoamide dehydrogenase and mycobacterial proteasome ATPase, S-nitrosylation caused enzyme inhibition. Moreover, endogenously biotinylated proteins were driven into mixed disulfide complexes. Targeting of metabolic enzymes and antioxidant defenses by means of protein S-nitrosylation and mixed disulfide bonding may contribute to the antimycobacterial actions of RNIs.

Cytodifferentiation Enhances Erk Activation Induced by Endothelin-1 in Primary Cultured Astrocytes

We have previously demonstrated that endothelin-1 (ET-1)-induced extracellular signal-regulated kinase (Erk) activity via the ETB receptor (EDNRB) is mediated through two independent pathways, a protein kinase C-dependent pathway and a pertussis toxin (PTX)-sensitive pathway, in astrocytes. In this study, we showed that the molar potency of ET-1 to induce Erk activation was two orders of magnitude higher in dibutyryl cAMP (DBcAMP)-treated astrocytes than in quiescent astrocytes. This DBcAMP-enhanced molar potency of ET-1 in Erk activation was selectively inhibited by pretreatment of astrocytes with PTX. The expression level of EDNRB in astrocytes was markedly upregulated by DBcAMP-induced cytodifferentiation. However, this up-regulation was simply attributed to the high expression of low-affinity sites. The molar potency of ET-1 to induce both stimulation of inositol trisphosphate production and activation of protein kinase C in DBcAMP-treated astrocytes was similar to that in quiescent astrocytes. On the contrary, the molar potency of ET-1 to induce accumulation of Ras-GTP was two orders of magnitude higher in DBcAMP-treated astrocytes than in quiescent astrocytes, which was consistent with the case of ET-1-induced Erk activation. Moreover, the ET-1-induced Ras activation was PTX sensitive. These results suggest that cytodifferentiation selectively enhances the PTXsensitive Ras/Erk pathway induced by ET-1 in astrocytes, and that cytodifferentiation-induced EDNRB up-regulation might not contribute to this selective potentiation of ET-1 signaling.

The smooth muscle pharmacology of maximakinin, a receptor-selective, bradykinin-related nonadecapeptide from the venom of the Chinese toad, Bombina maxima

Structural homologues of vertebrate regulatory peptides found in defensive skin secretions of anuran amphibians often display enhanced bioactivity and receptor binding when compared with endogenous mammalian peptide ligands. Maximakinin, a novel N-terminally extended bradykinin (DLPKINRKGPRPPGFSPFR) from the skin venom of a Chinese toad ( Bombina maxima), displays such activity enhancement when compared with bradykinin but is additionally highly selective for mammalian arterial smooth muscle bradykinin receptors displaying a 50-fold increase in molar potency in this smooth muscle type. In contrast, a 100-fold decrease in molar potency was observed at bradykinin receptors in intestinal and uterine smooth muscle preparations. Maximakinin has thus evolved as a “smart” defensive weapon in the toad with receptor/tissue selective targeting. Natural selection of amphibian skin venom peptides for antipredator defence, through inter-species delivery by an exogenous secretory mode, produces subtle structural stabilisation modifications that can potentially provide new insights for the design of selectively targeted peptide therapeutics.

Host defense functions of proteolytically processed and parent (unprocessed) cathelicidins of rabbit granulocytes.

Members of the cathelicidin family are present in all mammals studied. Generally, these proteins contain a conserved N-terminal domain and a structurally and functionally divergent C-terminal region that expresses antibacterial or other activities when proteolytically released. Rabbit granulocytes produce CAP18, a cathelicidin that conforms to this structural and functional organization, and also 15-kDa protein isoforms (p15s) that share several key structural features with other cathelicidins but apparently do not undergo processing with release of an active peptide. To further define the importance of proteolysis in the antibacterial activities of these proteins, we have purified from granulocytes proCAP18, its C-terminal peptide (CAP18p), and two p15 isoforms to apparent homogeneity. Of these four polypeptides, only CAP18p was independently cytotoxic to encapsulated Escherichia coli (90% inhibitory concentration, approximately 600 nM) but it was approximately 50-fold less potent on a molar basis than the bactericidal/permeability-increasing protein (BPI). However, all four cathelicidin species, notably including proCAP18, exhibited antibacterial synergy with BPI, and the p15s also displayed synergy with CAP18p in the absence of BPI. Subnanomolar concentrations of proCAP18 blocked lipopolysaccharide-induced chemiluminescence of human leukocytes, showing a molar potency more than 100-fold greater than that of CAP18p ( approximately 20 nM) or BPI ( approximately 50 nM). Thus, while independent bactericidal activity of cathelicidins requires processing, other host-defense functions do not and are more potently expressed by the unprocessed protein than by the C-terminal peptide.

Immunophilins may limit calcineurin inhibition by cyclosporine and tacrolimus at high drug concentrations.

The immunosuppressive drugs cyclosporine (CsA) and tacrolimus (FK506 or FK) are qualitatively similar but differ in molar potency. Both drugs sterically inhibit the phosphatase activity of calcineurin (CN) but differ in molar potency. In our study we explored whether differential inhibition of CN explained the differences in molar potency of FK versus CsA. We compared their effects on NFATC2 dephosphorylation using Western analysis, interferon-gamma production using ELISA, and CN phosphatase activity using the CN assay in human peripheral blood leukocytes (PBL) and mouse spleen cell suspension. The FK concentration inhibiting 50% (IC50) of all three activities was approximately 0.2 microg/ml in human PBL, versus 5-20 microg/ml for CsA. Although inhibition of interferon-gamma secretion and NFATC2 dephosphorylation was complete, inhibition of CN phosphatase activity was incomplete with both drugs at saturation, particularly with FK. Inhibition of CN phosphatase activity was incomplete whether FK treatment was in vivo in mouse or in vitro in various human and mouse tissues, especially brain. Exogenous FKBP12 or CyPA increased CN phosphatase inhibition, suggesting that incomplete inhibition of CN phosphatase activity reflected limiting amounts of active immunophilin. These data contradict the prevailing assumption that immunophilins are abundant and not limiting for inhibition of CN by CsA or FK. Further, the observation that FK and CsA completely inhibit immune function without completely inhibiting CN suggests that the inhibition of immune function is not mediated by general CN inhibition but by inhibition of a subset of CN which is critical for lymphocyte activation.

Growth factors in ulcer healing: Lessons from recent studies

Growth factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and more recently vascular endothelial growth factor (VEGF) have been used extensively to heal experimental gastric, duodenal and colonic ulcers in animal models. Encouraging results have been reported in clinical trials with EGF and bFGF. Since our laboratory has been involved with the initial ulcer healing studies with bFGF, PDGF and VEGF, we summarize here the major lessons from these studies and from literature data. These conclusions relate to the role of: 1) gastrointestinal (GI) secretion; 2) epithelial versus vascular components of the healing; 3) efficacy in the upper and lower GI tract; 4) quality of ulcer healing; as well as 5) the endogenous origin; and 6) molar potency of growth factors. Namely, among these growth factors only EGF inhibits gastric acid and stimulates duodenal bicarbonate secretion, while chronic administration of bFGF slightly enhances gastric secretion and PDGF has no effect demonstrating that potent ulcer healing can be achieved without influencing acid base and mucus secretion. This might be related to the fact that these growth factors stimulate with varying potency virtually all the cellular elements needed for ulcer healing, e.g., epithelial cell proliferation and migration by EGF > bFGF > PDGF, fibroblast proliferation by bFGF > PDGF and angiogenesis by VEGF > bFGF >> PDGF >> EGF. Conceptually, the most interesting results were obtained recently with VEGF which is virtually specific for angiogenesis, illustrating that stimulation of vascular factors is sufficient for ulcer healing because epithelial cells apparently spontaneously proliferate and migrate over a dense granulation tissue to complete the healing process. Since these growth factors directly stimulate the cell components of ulcer healing, it is probably not surprising that they are active in both upper and lower GI tract lesions, produce good quality of ulcer healing in comparison with spontaneously healed duodenal ulcers which are hypovascular and muscle regeneration is not part of natural healing. Contrary to other antiulcer drugs, these growth factors are endogenously derived and play a role in the natural history of ulcer healing, and since these relatively large peptides (18–45 kDa) are active in ng quantities, their molar potency is 2–7 million times superior to cimetidine-like drugs. Thus growth factors are endogenously derived very potent antiulcer drugs which act independently of GI secretion, are active in upper and lower GI lesions, and since they stimulate virtually all the cells of the healing process, they produce an excellent quality of ulcer healing.