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Glutamate excitotoxicity via N-methyl-D-aspartate (NMDA) receptors is thought to be a factor involved in the loss of retinal neuronal cells, including retinal ganglion cells, in retinal diseases such as diabetic retinopathy and acute angle closure glaucoma. Herein we report the protective effect of systemic administration of ML233, an apelin receptor agonist, against retinal neuronal cell death induced by the intravitreal injection of NMDA into mice. Intraperitoneal administration of ML233 prevented the NMDA-induced reduction in the amplitude of scotopic threshold responses (STR), which mainly reflect the activity of the retinal ganglion cells. Immunohistochemical staining showed that ML233 inhibited the NMDA-induced loss of retinal ganglion cells and amacrine cells. In addition, ML233 suppressed the breakdown of spectrin αII, a neuronal cytoskeleton protein cleaved by calpain activation, in the retina after intravitreal injection of NMDA. Intraperitoneal administration of ML233 increased the phosphorylation of Akt, a potent anti-apoptotic protein in neurons, in the retina. Furthermore, oral administration of ML233 protected against the decrease in the STR amplitudes and the loss of retinal ganglion cells caused by NMDA. These results suggest that systemic administration of ML233 protected retinal neurons from NMDA receptor-mediated excitotoxicity and that drugs activating the apelin receptor may be a new candidate for preventing the progression of these retinal diseases.

Background and Objective: Little is known about acetaminophen (paracetamol) pharmacokinetics during pregnancy. The aim of this study was to develop a physiologically based pharmacokinetic (PBPK) model to predict acetaminophen pharmacokinetics throughout pregnancy., Methods: PBPK models for acetaminophen and its metabolites were developed in non-pregnant and pregnant women. Physiological and enzymatic changes in pregnant women expected to impact acetaminophen pharmacokinetics were considered. Models were evaluated using goodness-of-fit plots and by comparing predicted pharmacokinetic profiles with in vivo pharmacokinetic data. Predictions were performed to illustrate the average concentration at steady state (C ss,avg ) values, used as an indicator for efficacy, of acetaminophen achieved following administration of 1000 mg every 6 h. Furthermore, as a measurement of potential hepatotoxicity, the molar dose fraction of acetaminophen converted to N-acetyl-p-benzoquinone imine (NAPQI) was estimated., Results: PBPK models successfully predicted the pharmacokinetics of acetaminophen and its metabolites in non-pregnant and pregnant women. Predictions resulted in the lowest C ss,avg in the third trimester (median [interquartile range]: 4.5 [3.8-5.1] mg/L), while C ss,avg was 6.7 [5.9-7.4], 5.6 [4.7-6.3], and 4.9 [4.1-5.5] mg/L in non-pregnant, first trimester, and second trimester populations, respectively. Assuming a constant raised cytochrome P450 2E1 activity throughout pregnancy, the molar dose fraction of acetaminophen converted to NAPQI was highest during the first trimester (median [interquartile range]: 11.0% [9.1-13.4%]), followed by the second (9.0% [7.5-11.0%]) and third trimester (8.2% [6.8-10.1%]), compared with non-pregnant women (7.7% [6.4-9.4%])., Conclusion: Acetaminophen exposure is lower in pregnant than in non-pregnant women, and is related to pregnancy duration. Despite these findings, higher dose adjustments cannot be advised yet as it is unknown whether pregnancy affects the toxicodynamics of NAPQI. Information on glutathione abundance during pregnancy and NAPQI in vivo data are required to further refine the presented model.

Hypothesis: Because of its hydrophilic character the peptide drug Polymyxin B (PMB) cannot be incorporated in lipophilic nanocarrier systems such as self-emulsifying drug delivery systems (SEDDS) for oral administration. Due to the formation of imine conjugates between the primary amino groups of PMB and the carbonyl group of cinnamaldehyde, however, drug lipophilicity might be sufficiently raised for incorporation in SEDDS., Methods: Imine bonds were formed between the primary amino groups of PMB and the carbonyl group of cinnamaldehyde. PMB-cinnamaldehyde conjugate was characterized regarding degree of substitution, log P and release of PMB due to interaction with bovine serum albumin (BSA), SEDDS loading and cell viability., Results: 87.1% of primary amines formed imines with cinnamaldehyde. Log P was increased 69.183 - folds. BSA triggered release of PMB was 45.2%, 64.9% and 80.6% within 16 h. Log D SEDDS/Release medium of PMB-cinnamaldehyde conjugate was 3.4., Conclusion: According to these findings, the concept of imine bond formation with cinnamaldehyde can be considered as a novel concept for increasing lipophilicity of the hydrophilic antibiotic peptide PMB., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A novel structural class of iminopyridine derivative 1 was identified as a potent and selective human α1D adrenoceptor (α1D adrenergic receptor; α1D-AR) antagonist against α1A- and α1B-AR through screening of an in-house compound library. From initial structure-activity relationship studies, we found lead compound 9m with hERG K(+) channel liability. To develop analogues with reduced hERG K(+) channel inhibition, a combination of site-directed mutagenesis and docking studies was employed. Further optimization led to the discovery of (R)-9s and 9u, which showed antagonistic activity by a bladder strip test in rats with bladder outlet obstruction, as well as ameliorated cystitis-induced urinary frequency in rats. Ultimately, 9u was selected as a clinical candidate. This is the first study to show the utility of iminopyridine derivatives as selective α1D-AR antagonists and evaluate their effects in vivo.

Background: Glioblastoma is a malignant World Health Organization (WHO) grade IV glioma with a poor prognosis in humans. New therapeutics are desperately required. The nitrone OKN-007 (2,4-disulfophenyl-PBN) has demonstrated effective anti-glioma properties in several rodent models and is currently being used as a clinical investigational drug for recurrent gliomas. We assessed the regional effects of OKN-007 in the tumor necrotic core and non-necrotic tumor parenchyma., Methods: An F98 rat glioma model was evaluated using proton magnetic resonance spectroscopy ((1) H-MRS), diffusion-weighted imaging (DWI), morphological T2-weighted imaging (T2W) at 7 Tesla (30 cm-bore MRI), as well as immunohistochemistry and microarray assessments, at maximum tumor volumes (15-23 days following cell implantation in untreated (UT) tumors, and 18-35 days in OKN-007-treated tumors)., Results: (1) H-MRS data indicates that Lip0.9/Cho, Lip0.9/Cr, Lip1.3/Cho, and Lip1.3/Cr ratios are significantly decreased (all P < 0.05) in the OKN-007-treated group compared with UT F98 gliomas. The Cho/Cr ratio is also significantly decreased in the OKN-007-treated group compared with UT gliomas. In addition, the OKN-007-treated group demonstrates significantly lower ADC values in the necrotic tumor core and the nonnecrotic tumor parenchyma (both P < 0.05) compared with the UT group. There was also an increase in apoptosis following OKN-007 treatment (P < 0.01) compared with UT., Conclusion: OKN-007 reduces both necrosis and tumor cell proliferation, as well as seems to mediate multiple effects in different tumor regions (tumor necrotic core and nonnecrotic tumor parenchyma) in F98 gliomas, indicating the efficacy of OKN-007 as an anti-cancer agent and its potential clinical use., (© 2015 Wiley Periodicals, Inc.)

Cyclic imines (CIs) are a group of phytoplankton produced toxins related to shellfish food products, some of which are already present in UK and European waters. Their risk to shellfish consumers is poorly understood, as while no human intoxication has been definitively related to this group, their fast acting toxicity following intraperitoneal injection in mice has led to concern over their human health implications. A request was therefore made by UK food safety authorities to examine these toxins more closely to aid possible management strategies. Of the CI producers only the spirolide producer Alexandrium ostenfeldii is known to exist in UK waters at present but trends in climate change may lead to increased risk from other organisms/CI toxins currently present elsewhere in Europe and in similar environments worldwide. This paper reviews evidence concerning the prevalence of CIs and CI-producing phytoplankton, together with testing methodologies. Chemical, biological and biomolecular methods are reviewed, including recommendations for further work to enable effective testing. Although the focus here is on the UK, from a strategic standpoint many of the topics discussed will also be of interest in other parts of the world since new and emerging marine biotoxins are of global concern.

Free radicals are associated with glioma tumors. Here, we report on the ability of an anticancer nitrone compound, OKN-007 [Oklahoma Nitrone 007; a disulfonyl derivative of α-phenyl-tert-butyl nitrone (PBN)] to decrease free radical levels in F98 rat gliomas using combined molecular magnetic resonance imaging (mMRI) and immunospin-trapping (IST) methodologies. Free radicals are trapped with the spin-trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), to form DMPO macromolecule radical adducts, and then further tagged by immunospin trapping by an antibody against DMPO adducts. In this study, we combined mMRI with a biotin-Gd-DTPA-albumin-based contrast agent for signal detection with the specificity of an antibody for DMPO nitrone adducts (anti-DMPO probe), to detect in vivo free radicals in OKN-007-treated rat F98 gliomas. OKN-007 was found to significantly decrease (P < 0.05) free radical levels detected with an anti-DMPO probe in treated animals compared to untreated rats. Immunoelectron microscopy was used with gold-labeled antibiotin to detect the anti-DMPO probe within the plasma membrane of F98 tumor cells from rats administered anti-DMPO in vivo. OKN-007 was also found to decrease nuclear factor erythroid 2-related factor 2, inducible nitric oxide synthase, 3-nitrotyrosine, and malondialdehyde in ex vivo F98 glioma tissues via immunohistochemistry, as well as decrease 3-nitrotyrosine and malondialdehyde adducts in vitro in F98 cells via ELISA. The results indicate that OKN-007 effectively decreases free radicals associated with glioma tumor growth. Furthermore, this method can potentially be applied toward other types of cancers for the in vivo detection of macromolecular free radicals and the assessment of antioxidants., (Copyright © 2015. Published by Elsevier Inc.)

Integrin-targeted nanoparticles are promising for the delivery of small interfering RNA (siRNA) to tumor cells or tumor endothelium in cancer therapy aiming at silencing genes essential for tumor growth. However, during the process of optimizing and realizing their full potential, it is pertinent to gain a basic mechanistic understanding of the bottlenecks existing for nanoparticle-mediated intracellular delivery. We designed αvβ3 integrin-targeted nanoparticles by coupling arginine-glycine-aspartate (RGD) or RGD peptidomimetic (RGDp) ligands to the surface of poly(ethylene glycol) (PEG) grafted chitosan-poly(ethylene imine) hybrid nanoparticles. The amount of intracellular siRNA delivered by αvβ3-targeted versus non-targeted nanoparticles was quantified in the human non-small cell lung carcinoma cell line H1299 expressing enhanced green fluorescent protein (EGFP) using a stem-loop reverse transcription quantitative polymerase chain reaction (RT-qPCR) approach. Data demonstrated that the internalization of αvβ3-targeted nanoparticles was highly dependent on the surface concentration of the ligand. Above a certain threshold concentration, the use of targeted nanoparticles provided a two-fold increase in the number of siRNA copies/cell, subsequently resulting in as much as 90% silencing of EGFP at well-tolerated carrier concentrations. In contrast, non-targeted nanoparticles mediated low levels of gene silencing, despite relatively high intracellular siRNA concentrations, indicating that these nanoparticles might end up in late endosomes or lysosomes without releasing their cargo to the cell cytoplasm. Thus, the silencing efficiency of the chitosan-based nanoparticles is strongly dependent on the uptake and the intracellular trafficking in H1299 EGFP cells, which is critical information towards a more complete understanding of the delivery mechanism that can facilitate the future design of efficient siRNA delivery systems., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Polycationic polymers like poly(ethylene imine)s (PEIs) are extensively explored for the nonviral transfer of DNA or small RNAs (siRNAs). To enhance biocompatibility and alter pharmacokinetic properties, hyperbranched PEI was recently grafted with the nonligand oligosaccharides maltose or maltotriose at various degrees in a systematic study to yield (oligo-)maltose PEIs (OM-PEIs). In this paper, we investigate the in vivo biocompatibility and efficacy of a whole set of (OM-)PEIs and the corresponding (OM-)PEI-based DNA or siRNA complexes upon systemic (intravenous, i.v.) administration in mice. We determine the overall survival and animal welfare, hepatotoxicity, immune stimulation, erythrocyte aggregation, and the efficacy of DNA delivery in vivo. Higher-degree oligomaltose-grafting of PEI substantially decreases weight loss, abolishes lethality upon repeated treatment with the free polymers or with complexes, and abrogates hepatotoxicity, as determined by serum levels of liver enzymes. Immunostimulatory effects (TNF-α, IFN-γ) and erythrocyte aggregation are mainly observed upon treatment with partially maltotriose-grafted PEI or PEI-based complexes and are largely abolished upon higher-degree grafting. In vivo transfection experiments in mice bearing subcutaneous (s.c.) tumor xenografts reveal a strong dependence of reporter gene expression in a given organ on the mode of complex administration (i.v. vs intraperitoneal injection) and the OM-PEI architecture, with high-level maltose-grafted PEI (PEI-(2-Mal)) being most efficient for DNA delivery. We conclude that distinct differences between different patterns of maltose- or maltotriose-grafting are observed with regard to both biocompatibility and in vivo efficacy and identify optimal oligomaltose-PEIs for therapeutic applications.

In our previous work we have shown that the novel synthetic chromone derivative could effectively inhibit the Leishmania donovani replication in vitro and in vivo with less cytotoxicity on murine splenocytes. The aim of the present study is to explore the possible mechanism of anti-leishmanial effect of C-(6-methyl-4-oxo-4H-1-benzopyran-3-yl)-N-(p-tolyl) nitrone (designated as NP1) in vitro and in vivo in experimental visceral leishmaniasis caused by L. donovani. The cytotoxic effect of this derivative was studied in murine peritoneal macrophages by MTT method. NP1 at a dose of 17.06 μM showed 50% inhibition on L. donovani promastigotes but found less cytotoxic to the RAW 264.7 cells. Even the higher concentration of IC50 (up to four fold) did not exert much cytotoxic effect on RAW 264.7. Interestingly, NP1 at lower concentration (8.53 μM) could inhibit 50% of intracellular amastigotes in murine peritoneal macrophages. L. donovani is known to exert its pathogenic effects mainly by the suppression of NO generation and subversion of the cellular inflammatory responses in the macrophages. NP1 was found to induce a potent host-protective immune response by enhancing NO generation and iNOS2 expression at mRNA level and by up-regulating proinflammatory cytokines such as IL-12 and IFN-γ and limiting the expression of IL-10 in vivo. The NO dependent killing was further confirmed in iNOS(-/-) mice compared to wild type. In agreement with the fact, induced synthesis of IL-12 and IFN-γ and associated down-regulation of IL-10 by the treatment of NP1 clearly indicated the possibility of novel strategy of drug development against Leishmania infection., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Atopaxar is a reversible protease activated receptor (PAR)-1 thrombin receptor antagonist that interferes with platelet signaling. The effects of PAR-1 antagonists on biomarkers remain unknown. The primary objective was to assess the effects of atopaxar on biomarkers of inflammation and platelet activation. The LANCELOT-CAD trial randomized 720 subjects to atopaxar (50, 100, or 200 mg daily) or matching placebo for 24 weeks. Biomarkers were assessed at serial time points. A linear mixed model to account for repeated measures was used to evaluate the change in biomarker concentration from randomization across time to week 24. Least square means were determined from the linear mixed models. The concentration of sCD40L decreased on average over time by -553 (95 % CI -677, -429) ng/L in the combined atopaxar group versus -30.3 (-249 to 189) ng/L fall in the placebo arm (P < 0.001) and a dose-dependent trend was seen across treatment groups (P < 0.001 for trend). In contrast, Lp-PLA(2) mass rose on average over time by 12.6 (95 % CI 10.0, 15.3) ng/ml in the combined atopaxar group as compared with 2.6 (95 % CI -2.1, 7.3) ng/ml in the placebo arm (P < 0.001). Similarly, the concentration of IL-18 rose by 17.5 (95 % CI 12.4, 22.6) pg/ml in the atopaxar group versus a -1.2 (95 % CI -10.2, 7.8) pg/ml fall in the placebo group (P < 0.001). The effects of atopaxar on Lp-PLA(2) and IL-18 appeared to be dose-dependent (P < 0.001 for trend) and were observed in J-LANCELOT. Atopaxar did not have a significant effect on other inflammatory markers. In conclusion, atopaxar appeared to decrease sCD40L, but did not demonstrate an anti-inflammatory effect in patients with stable CAD. Although atopaxar increased the concentration of Lp-PLA(2) and IL-18, the clinical relevance of these findings remains unknown and warrants further investigation and validation.

Amphiphilic cationic block copolymers consisting of poly(ethylene glycol), poly(ε-caprolactone) and poly(ethylene imine) spontaneously assemble to nano-sized particulate carriers, which can be utilised for complexation of nucleic acids (small-interfering RNA), representing a multifunctional vector system, designed for drug and gene delivery. Apart from polymer design and charge ratio, a more homogeneous complexation could lead to a more uniform charge distribution, subsequently increasing colloidal stability, RNA protection and consequently transfection efficiency. Microfluidic mixing techniques, bringing cationic polymer and nucleic acid together at a constant ratio during the entire mixing process, have the potential for a gentler complexation. In the present study carriers were prepared by a solvent displacement technique. In a first step complex size for addition of RNA during (addition to the aqueous or the organic phase) or after (classical pipetting or microfluidic mixing) carrier assembly was determined by dynamic light scattering. Suitable N/P ratios have previously been selected by measuring size and ζ-potential as a function of N/P. Subsequently, for the most promising techniques (loading after assembly), colloidal stability, the ability to protect RNA as well as transfection efficiency in vitro were compared. Finally, parameters for the superior microfluidic mixing process were optimised with the help of a central composite design. Generally, gentler loading leads to more homogeneous complexes. Hence, possibly due to a more consistent surface coating, loading after carrier assembly resulted in less aggregation. In comparison to bulk mixing, microfluidic assembly exhibited smaller diameters (179±11 vs. 230±97nm), less heterogeneity (PDI=0.205±0.028 vs. 0.353±0.161), enhanced RNA protection (RNA recovery=30.6±1.0 vs. 15.4±1.4%) as well as increased transfection performance (34.8±1.5 vs. 24.5±2.2% knockdown). Therefore, microfluidic complexation represents a reproducible alternative for formulating gene delivery carriers with superior colloidal stability, RNA protection and transfection efficiency., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Atopaxar, also known as E 5555 is a novel reversible protease-activated receptor-1 (PAR-1) thrombin receptor antagonist. To date, Atopaxar has been investigated in phase II trials with focus on safety and tolerability in patients with acute coronary syndromes or stable coronary artery disease on top of standard antiplatelet therapy. Atopaxar was generally well tolerated, however a rise in liver enzymes and prolongation of the QTcF interval were observed. The data suggest, that atopaxar administration may promote some minor bleeding complications, but does not seem to significantly increase the risk of major bleeding. Although not powered for efficacy, the currently available data suggest potential benefits in patients at high risk for recurrent ischemic events on top of standard antiplatelet therapy. In conclusion, more studies (e.g. phase III) are needed to evaluate efficacy and safety of atopaxar.

Thrombin is a powerful agonist for a variety of cellular responses including platelet aggregation and vascular smooth muscle cell (SMC) proliferation. These actions are mediated by a thrombin receptor known as protease-activated receptor-1 (PAR-1). Recently we discovered that 1-(3-tert-butyl-4-methoxy-5-morpholinophenyl)-2-(5,6-diethoxy-7-fluoro-1-imino-1,3-dihydro-2H-isoindol-2-yl)ethanone hydrobromide (E5555, atopaxar) is a potent and selective thrombin receptor antagonist. This study characterized the pharmacological effects of E5555 on SMC proliferation in vitro and in a rat model of intimal thickening after balloon injury in vivo. E5555 selectively inhibited rat aortic SMC proliferation induced by thrombin and thrombin receptor-activating peptide (TRAP) with half maximal inhibitory concentration (IC(50)) values of 0.16 and 0.038 μM, respectively. E5555 did not inhibit rat SMC proliferation induced by basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) at concentrations up to 1μM. In addition, E5555 inhibited human aortic SMC proliferation induced by thrombin at concentrations of 0.3 and 3units/ml with IC(50) values of 0.028 and 0.079 μM, respectively, whereas it did not affect bFGF-induced proliferation at concentrations up to 1μM. Repeated oral administration of 30 mg/kg E5555 (once daily for 16 days) significantly reduced neointimal formation in the balloon-injured rat arterial model. These results suggested that a PAR-1 antagonist could be effective for treating restenosis following vascular intervention in addition to preventing thrombus formation. E5555 could thus have therapeutic potential for restenosis and chronic atherothrombotic disease., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Purpose: As the next step in the development of an intra-articular controlled release system to treat painful temporomandibular joint (TMJ) inflammation, we developed several biodegradable poly(DL-lactic-co-glycolic acid) (PLGA)-based microparticle (MP) formulations encapsulating a model anti-inflammatory small interfering RNA (siRNA) together with branched poly(ethylenimine) (PEI) as a transfecting agent. The effect of siRNA loading and N:P ratio on the release kinetics of siRNA-PEI polyplexes was determined, and the size and N:P ratio of the polyplexes released over time was characterized., Methods: Polyplex-loaded PLGA MPs were prepared using an established double emulsion technique. Increasing the pH of the release samples enabled siRNA-PEI dissociation and subsequent measurement of the release of each component over 28 days. Polyplex diameter was measured for all release samples and compared to freshly prepared siRNA-PEI under simulated physiologic conditions., Results: Systematic variation of siRNA loading and N:P ratio resulted in distinct siRNA and PEI release profiles. Polyplex diameter remained constant despite large variations in the relative amounts of siRNA and PEI. Excess PEI was sequestered through complexation with 500-1,000 nm diameter PLGA MP-derived particles, including small MPs and PLGA degradation products., Conclusions: These PLGA MP formulations show exciting potential as the first intra-articular TMJ controlled release system.

Background: Atopaxar (E5555) is a reversible protease-activated receptor-1 thrombin receptor antagonist that interferes with platelet signaling. The primary objective of the Lessons From Antagonizing the Cellular Effects of Thrombin-Acute Coronary Syndromes (LANCELOT—ACS) trial was to evaluate the safety and tolerability of atopaxar in patients with ACS., Methods and Results: Six hundred and three subjects were randomized within 72 hours of non-ST-elevation ACS to 1 of 3 doses of atopaxar (400-mg loading dose followed by 50, 100, or 200 mg daily) or matching placebo. The incidence of Clopidogrel in Unstable Angina to Prevent Recurrent Events (CURE) major or minor bleeding did not differ significantly between the combined atopaxar and placebo groups (3.08% versus 2.17%, respectively; P=0.63), and there was no dose-related trend (P=0.80). The incidence of CURE major bleeding was numerically higher in the atopaxar group compared with the placebo group (1.8% versus 0%; P=0.12). The incidence of cardiovascular death, myocardial infarction, stroke, or recurrent ischemia was similar between the atopaxar and placebo arms (8.03% versus 7.75%; P=0.93). The incidence of CV death, MI, or stroke was 5.63% in the placebo group and 3.25% in the combined atopaxar group (P=0.20). Dose-dependent trends for efficacy were not seen. Atopaxar significantly reduced ischemia on continuous ECG monitoring (Holter) at 48 hours compared with placebo (relative risk, 0.67; P=0.02). Transient dose-dependent transaminase elevation and relative QTc prolongation were observed with the highest doses of atopaxar., Conclusion: In patients after ACS, atopaxar significantly reduced early ischemia on Holter monitoring without a significant increase in major or minor bleeding. Larger trials are required to fully establish the efficacy and safety of atopaxar., Clinical Trial Registration: URL: http://www.ClinicalTrials.gov. Unique identifier: NCT00548587.

Background: Thrombin is a key mediator of platelet activation. Atopaxar is a reversible protease-activated receptor-1 antagonist that interferes with thrombin-mediated platelet effects. The phase II Lessons From Antagonizing the Cellular Effect of Thrombin-Coronary Artery Disease (LANCELOT-CAD) trial examined the safety and tolerability of prolonged therapy with atopaxar in subjects with CAD., Methods and Results: Subjects with a qualifying history were randomized in a double-blind fashion to 3 dosing regimens of atopaxar (50, 100, or 200 mg daily) or matching placebo for 24 weeks and followed up for an additional 4 weeks. The key safety end points were bleeding according to the Clopidogrel in Unstable Angina to Prevent Recurrent Events (CURE) and Thrombolysis in Myocardial Infarction (TIMI) classifications. Secondary objectives included platelet aggregation and major adverse cardiac events. Seven hundred and twenty subjects were randomized. Overall bleeding rates tended to be higher with atopaxar compared with placebo by CURE criteria (placebo, 0.6%; atopaxar, 3.9%; relative risk, 6.82, P=0.03; 50 mg, 3.9%; 100 mg, 1.7%; 200 mg, 5.9%; P for trend=0.01) and TIMI criteria (placebo, 6.8%; atopaxar, 10.3%; relative risk, 1.52, P=0.17; 50 mg, 9.9%; 100 mg, 8.1%; 200 mg, 12.9%; P for trend=0.07). There was no difference in major bleeding. Major adverse cardiac events were numerically lower in the atopaxar subjects. All atopaxar regimens achieved high levels of platelet inhibition. A transient elevation in liver transaminases and dose-dependent QTc prolongation without apparent complications were observed in higher-dose atopaxar treatment groups., Conclusions: In this dose-ranging study of patients with CAD, treatment with atopaxar resulted in platelet inhibition, more minor bleeding, and numerically but not statistically fewer ischemic events. Larger-scale trials are needed to determine whether these patterns translate into clinically meaningful effects., Clinical Trial Registration: URL: http://www.ClinicalTrials.gov. Unique identifier: NCT00312052.

Thrombin is a powerful agonist for platelets, the action of which is mediated by the thrombin receptor protease-activated receptor-1 (PAR-1). Recently, we discovered that E5555 (1-(3-tert-butyl-4-methoxy-5-morpholinophenyl)-2-(5,6-diethoxy-7-fluoro-1-imino-1,3-dihydro-2H-isoindol-2-yl) ethanone hydrobromide) is a potent thrombin receptor antagonist. We evaluated the anti-platelet and anti-thrombotic effects of E5555. E5555 inhibited the binding of a high-affinity thrombin receptor-activating peptide ([(3)H]haTRAP) to PAR-1 with a half maximal inhibitory concentration (IC(50)) value of 0.019μM. E5555 showed potent inhibitory effects on human platelet aggregation induced by thrombin and TRAP with IC(50) values of 0.064 and 0.031μM, respectively, but had no effect on platelet aggregation induced by either ADP or collagen. Similarly, E5555 showed potent and selective inhibitory effects on guinea pig platelet aggregation induced by thrombin and TRAP with IC(50) values of 0.13 and 0.097μM, respectively. The antithrombotic activity of E5555 in vivo was evaluated in a photochemically-induced thrombosis (PIT) model using guinea pigs. Oral administration of E5555 at 30 and 100mg/kg prolonged the time to occlusion by 1.8-fold and 2.4-fold, respectively, compared with controls. Furthermore, E5555 did not prolong bleeding time in guinea pigs at the highest tested dosage of 1000mg/kg. The drug interactions between E5555 and tissue plasminogen activator (tPA) were evaluated. Intravenous administration of 1mg/kg tPA significantly prolonged bleeding time, and its effects were not altered by the oral co-administration of 300mg/kg E5555. These results suggest that E5555 could be a therapeutic option for atherothrombotic disease., (Copyright © 2011 Elsevier B.V. All rights reserved.)

bPEI (polyethylenimine, 25 kDa, gold standard) is highly effective in transfection efficiency owing to its high buffering capacity, however, cytotoxicity limits its use in in vivo applications. We hypothesized that partial conversion of secondary amines in IPEI to tertiary amines, while preserving the overall number of amines, would result in improved buffering capacity, which may, in turn, improve transfection efficiency of the resulting nanoparticles with cell viability comparable to that of native IPEI. IPEI was crosslinked with BDE to obtain a series of IPEI nanoparticles (LPN-1 to LPN-8) which were obtained in approximately 80-85% yield. These particles were relatively non-toxic in vitro and in vivo. In vivo gene expression studies using LPN-5 in Balb/c mice through i.v. injection showed maximum expression of the reporter gene in the spleen. These results demonstrate the potential of these particles as efficient transfection reagents.

Aims: Two multicentre, randomized, double-blind, placebo-controlled Phase II studies assessed the safety and efficacy of the oral protease-activated receptor 1 (PAR-1) antagonist E5555 in addition to standard therapy in Japanese patients with acute coronary syndrome (ACS) or high-risk coronary artery disease (CAD)., Methods and Results: Patients with ACS (n = 241) or high-risk CAD (n = 263) received E5555 (50, 100, or 200 mg) or placebo once daily for 12 (ACS patients) or 24 weeks (CAD patients). The incidence of TIMI major, minor, and minimal bleeds requiring medical attention was similar in the placebo and combined E5555 (atopaxar) groups (ACS: 6.6% placebo vs. 5.0% E5555; CAD: 1.5% placebo vs. 1.5% E5555). There were no TIMI major bleeds and three CURE major bleeds (two with placebo; one with 100 mg E5555). There was a numerical increase in 'any' TIMI bleeding with the E5555 200 mg dose (ACS: 16.4% placebo vs. 23.0% E5555, P = 0.398; CAD: 4.5% placebo vs. 13.2% E5555, P = 0.081). The rate of major cardiovascular adverse events in the combined E5555 group was not different from placebo (ACS: 6.6% placebo vs. 5.0% E5555, P = 0.73; CAD: 4.5% placebo vs. 1.0% E5555, P = 0.066). There was a statistically significant dose-dependent increase in liver function abnormalities and QTcF with E5555. At trough dosing levels in both populations, mean inhibition of platelet aggregation was > 90% with 100 and 200 mg E5555, and 20-60% with 50 mg E5555., Conclusion: E5555 (50, 100, and 200 mg) did not increase clinically significant bleeding, although there was a higher rate of any TIMI bleeding with the highest two doses. All doses tested achieved a significant level of platelet inhibition. There was a significant dose-dependent increase in liver function abnormalities and QTcF. Although further study is needed, PAR-1 antagonism may have the potential to be a novel pathway for platelet inhibition to add on to the current standard of care therapy.

In non-viral gene delivery, the variance of transgenic expression stems from the low number of plasmids successfully transferred. Here, we experimentally determine Lipofectamine- and PEI-mediated exogenous gene expression distributions from single cell time-lapse analysis. Broad Poisson-like distributions of steady state expression are observed for both transfection agents, when used with synchronized cell lines. At the same time, co-transfection analysis with YFP- and CFP-coding plasmids shows that multiple plasmids are simultaneously expressed, suggesting that plasmids are delivered in correlated units (complexes). We present a mathematical model of transfection, where a stochastic, two-step process is assumed, with the first being the low-probability entry step of complexes into the nucleus, followed by the subsequent release and activation of a small number of plasmids from a delivered complex. This conceptually simple model consistently predicts the observed fraction of transfected cells, the cotransfection ratio and the expression level distribution. It yields the number of efficient plasmids per complex and elucidates the origin of the associated noise, consequently providing a platform for evaluating and improving non-viral vectors., ((c) 2009 Wiley Periodicals, Inc.)

In the present study, The PEI/DNA (PD) complexes was first prepared with positive surface charge under a suitable N/P ratio of 10. The redundant positive charge was partially and excessively shielded by a polysaccharide, hyaluronic acid (HA), in aqueous solution. The HA/PEI/DNA ternary complexes were characterized by assessing the zeta potential and size, then transferred to MDA-MB-435, MDA-MB-231, and MCF-7 cell lines with different amounts of HA-specific CD44 receptors on the surface. Consequently, The transfection efficiency of all the prepared complexes show a little increased to MCF-7 (low CD44 level) while a large increased to MDA-MB-231 and MDA-MB-435 cells (high CD44 level) with adding HA. Also, when HA:PEI charge ratio was 7.5%, the ternary complexes show the highest transfection efficiency. The prepared ternary complexes exhibited increased 2-13-fold fluorescence intensity and lower cell toxicity compared to the PD (N/P, 10). These results indicated that the positive HA/PEI/DNA ternary complexes (HA:PEI charge ratio, 7.5%) can target malignant breast cancer cells with high CD44 level and might be a promising candidate vector for gene therapy.

Using enhancers to improve the transfection efficiency of polyethylenimine (PEI) can circumvent the needs of chemical modifications as well as subsequent purification and characterization of the modified PEI. In this study, we found that incorporating trehalose into the transfection reagent could improve the transgene expression mediated by DNA-PEI complexes. Such enhancements were not observed when trehalose was replaced by other disaccharides. In an effort to explore the mechanisms, we examined how the timing of trehalose treatments and the durations of trehalose affected the percentages of cells expressing green fluorescent protein and the levels of intracellular ethidium monoazide labeled plasmid. Treatments with trehalose for 5-120 min prior to transfection could cause drops in transfection efficiency by 30-50%; such treatments, however, hardly affected the amounts of intracellular plasmid, indicating that the preexistence of intracellular trehalose could reduce transfection efficiency without lowering the endocytic activity. The transfection efficiency remained almost unchanged when the transfected cells were treated with trehalose after the removal of transfection reagents, indicating that trehalose had minimal effects on the machinery of protein synthesis. Despite the enhanced transgene expression, the presence of trehalose during transfection showed inhibitory effects on the internalization of DNA-PEI complexes. Additionally, the extent of enhancement in transgene expression strongly depended on the duration of trehalose. As the above observations suggested, only during the transfection process when complexes and trehalose coexisted, trehalose became an effective enhancer of transgene expression mediated by DNA-PEI complexes possibly by affecting the mechanisms of intracellular trafficking.

An effective vaccine strategy for HIV-1 will probably requires the induction and maintenance of both humoral and cellular immunity. We tested a new prime-boost approach of intranasal priming with 10 microg DNA plasmid in the PEI/DNA complexes and boosting with 10(7)PFU of replicative recombinant TianTan vaccinia virus (rTTV) expressing HIV-1 Gag in BALB/c mice. Intranasal priming with PEI/DNA complexes elicited strikingly stronger HIV-specific T-cell (p=0.0358) and IgA immune responses at mucosal sites of lung (p=0.0445) and vaginal tract (p=0.0469) than intranasal priming with naked DNA, though both are followed by the same rTTV boosting. Furthermore, an intramuscular boosting with rTTV could profoundly enhance both T-cell and antibody immune responses raised by intranasal priming. These results demonstrate that the combination of intranasal priming with PEI/DNA complexes and systemic boosting with rTTV is a preferable regimen for induction of both T-cell and humoral immune responses.

We have investigated the in vitro uptake, toxicity, phenotypic consequences and transfection efficiency of a stealth NGR/PEG/PDBA-coupled-SHA-PEI/pDNA targeting polyplex loaded with PLGA-PEG-PLGA tri-block copolymer in human monocyte-derived dendritic cells (DCs). Modification with PEG effectively shielded and reduced non-specific phagocytosis by immature DCs to approximately 20%. Coupling the NGR cell-specific peptide to the PEGylated polyplex (NGR/PEG/PDBA-SHA-PEI/pDNA) however resulted in specific and enhanced phagocytosis in DCs without any observable toxicity at the optimum concentration of 0.25% of the copolymer. DNase treatment had no effect on DNA integrity in the encapsulated polyplex. Confocal microscopy confirmed intracellular localization of the targeting NGR/PEG/PDBA-SHA-PEI/pDNA microparticles, resulting in more enhanced uptake of the radiolabeled plasmid DNA and approximately 5- and 10-fold increase over the control tri-block Pluronic F68 copolymer and the non-targeting polyplex, respectively. More importantly, phagocytosis of the targeting microparticles neither altered the functionality of immature DCs nor the phenotypic expression of DC-specific cell surface molecules, CD80, CD86, CD40 and CD54 (ICAM-1), suggesting that uptake of the targeting microparticles by themselves did not induce DC maturation. Taken together, these results suggest that PLGA-PEG-PLGA encapsulation of this stealth targeting polyplex has no negative effects on key properties of immature DCs and should pave the way for targeting DCs for vaccination purposes.

The positive charge on PEI was partially shielded by forming ionic nanocomposites with a polysaccharide, alginic acid, in aqueous solution, bypassing tedious chemical synthesis. The content of alginic acid was varied systematically to obtain a series of nanocomposites. The nanocomposites were first characterized by assessing the surface charge (zeta potential), size (DLS) and morphology (AFM) followed by evaluation for their DNA interaction ability, cytotoxicity and transfection efficiency on various cell lines. The transfection efficiency of PEI-alginate (6.26%) nanocomposites improved dramatically (2-16-fold over native PEI) in all the cell lines studied. However, a decrease in transfection efficiency was observed on deviating from this optimal concentration of alginic acid in nanocomposites. Cytotoxicity of PEI-alginate/DNA complexes was nearly abolished on increasing the concentration of alginic acid in nanocomposites. PEI-alginate (6.26%) nanocomposites also delivered SiRNAs efficiently into mammalian cells, resulting in 80% suppression of GFP expression. The cellular uptake and endosomal escape of PEI-alginate nanocomposites and PEI were found to follow a similar route when transfection was carried out in presence of chloroquine, bafilomycin A1, cytochalasin B and methyl-beta-cyclodextrin. The results demonstrate a versatile vector that can be used for efficient cytoplasmic delivery of a broad range of nucleic acids.

Aim: To investigate the properties of cationic nanoparticles composed of poly (ethyleneimine)-g-poly (methyl methacrylate) (PEI-PMMA) as gene delivery carriers and explore the mechanism of PEI-PMMA nanoparticles mediated gene transfer., Methods: PEI-PMMA nanoparticles were synthesized by free radical polymerization. The morphology of nanoparticles was observed by scanning electron microscopy. The particle size and zeta potential were measured by zeta sizer. The complex between pGL3 plasmid and nanoparticles was analyzed by gel electrophoresis; and PEI-PMMA nanoparticles mediated gene transfer into HeLa cells was observed by confocal laser scanning microscopy., Results: PEI-PMMA nanoparticles are spherical shape and monodispersity. The particle size and zeta potential are 172 nm and +50.3 mV, respectively. When pGL3 plasmid complexed with nanoparticles at N/P ratio of 5: 1 and 20: 1, the particle size of pGL3/nanoparticle complex are 133 and 139 nm and zeta potential is + 21.4 and + 33.7 mV, respectively. pGL3 plasmid complexed with nanoparticles completely at N/P ratio of 5: 1. PEI-PMMA nanoparticles can deliver pGL3 plasmid into HeLa cells by endocytosis and release pGL3 into the cytosol., Conclusion: PEI-PMMA nanoparticles effectively transferred DNA to target cells and it is a promising non-viral carrier for gene delivery.

The acute toxicity of the phycotoxin gymnodimine to female Swiss mice by intraperitoneal injection and by oral administration has been determined. Gymnodimine was highly toxic by injection, the LD50 being only 96 microg/kg. Animals either died within 10 min of injection or made a full recovery with no perceptible long-term effects. Gymnodimine was also toxic after oral administration by gavage (LD50 755 microg/kg), but was much less toxic when administered with food. No signs of toxicity were seen in mice voluntarily ingesting food containing gymnodimine at a level sufficient to give a dose of approximately 7500 microg/kg. Pre-treatment with physostigmine or neostigmine protected against injected gymnodimine, suggesting that the latter exerts its toxic effects via blockade of nicotinic receptors at the neuromuscular junction. The low toxicity of gymnodimine when ingested with food suggests that this compound is of low risk to humans, a conclusion that is consonant with anecdotal evidence for the absence of harmful effects in individuals consuming shellfish contaminated with gymnodimine.

Objective: To evaluate polypropylenimine dendrimers (generations 1-5: DAB 4, DAB 8, DAB 16, DAB 32, and DAB 64) as gene delivery systems., Methods: DNA binding was evaluated by measuring the reduced fluorescence of ethidium bromide, and molecular modelling of dendrimer-DNA complexes also was performed. Cell cytotoxicity was evaluated against the A431 cell line using the MTT assay. In vitro transfection was evaluated against the A431 cell line using the beta-galactosidase reporter gene and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulphate (DOTAP) served as a positive control., Results: Molecular modeling and experimental data revealed that DNA binding increased with dendrimer generation. Cell cytotoxicity was largely generation dependent, and cytotoxicity followed the trend DAB 64 > DAB 32 > DAB 16 > DOTAP > DAB 4 > DAB 8, whereas transfection efficacy followed the trend DAB 8 = DOTAP = DAB 16 > DAB 4 > DAB 32 = DAB 64., Conclusion: The generation 2 polypropylenimine dendrimer combines a sufficient level of DNA binding with a low level of cell cytoxicity to give it optimum in vitro gene transfer activity.

In functional pharmacological assays, talsaclidine has been described as a functionally preferential M1 agonist with full intrinsic activity, and less pronounced effects at M2- and M3 receptors. In accordance with this, cholinomimetic central activation measured in rabbits by EEG recordings occurred at a 10 fold lower dose than that inducing predominantly M3-mediated side effects. This pharmacological profile is also reflected in the clinical situation: Both in healthy volunteers and in Alzheimer patients--unlike after unspecific receptor stimulation through cholinesterase inhibitors--the mainly M3-mediated gastrointestinal effects (like nausea and vomiting) were not dose-limiting. Rather, sweating and hypersalivation, mediated through muscarinic receptors, occurred dose-dependently and were finally dose-limiting. In contrast to talsaclidine, sabcomeline had a less pronounced functional M1 selectivity in pharmacological assays. This was also shown in anaesthetized guinea pigs where sabcomeline alone induced bronchoconstriction, and in the rabbit EEG where central activation and cholinergic side effects occurred in the same dose range. Neither drug, however, showed convincing improvement of cognitive functions in patients with mild-to-moderate Alzheimer's disease. This asks for a reassessment of the muscarinic hypothesis for the treatment of this disease.

The effect of a new potential antiparkinsonian drug, N-(2-adamantyl)hexamethyleneimine hydrochloride (A-7), upon some behavioral parameters was studied in rats after subchronic 14-day treatment. Also studied was the effect of abrupt termination of a prolonged (22-day) drug administration. The subchronic treatment with A-7 at a dose of 10, 20, or 40 mg/kg did not produce significant changes in coordination, explorative behavior, muscle tone, and aggression level. At the same time, the 14-day treatment with A-7 at a dose of 10 mg/kg increased the pain threshold; A-7 at a dose of 40 mg/kg (but not 10 or 20 mg/kg) increased the spontaneous motor activity. Upon abrupt termination of the drug administration after a prolonged treatment sensitivity of the test animals with respect to the MFTP neurotoxin as compared to that observed in the non-drug control group.

Compound BP 2-94 is an orally available prodrug of the histamine H3-receptor agonist (R)-alpha-methylhistamine, which was found to produce higher plasma levels than the parent drug in humans. In the present study radioimmunoassay was carried out in dogs to investigate the generation of (R)-alpha-methylhistamine in vivo after intragastric administration of the prodrug. The effects of BP 2-94 on gastric acid secretion and on histamine, gastrin, and somatostatin release were also investigated. After intragastric administration of BP 2-94 (10 mg/kg), both the prodrug and (R)-alpha-methylhistamine were detected in plasma: plasma levels of (R)-alpha-methylhistamine decayed with a T1/2 of about 1 hr and displayed concentrations as high as 50-fold the EC50 of the drug at the H3 receptor for at least 2 hr. In conscious dogs provided with gastric fistula BP 2-94, administered at 10 and 30 mg/kg intragastrically, caused a dose-dependent inhibition (maximum reduction was about 80%) of the acid secretion stimulated by 2-deoxy-D-glucose, whereas (R)-alpha-methylhistamine (20 mg/kg, intragastrically) was ineffective. BP 2-94 (30 mg/kg, intragastrically) significantly reduced the acid secretion stimulated by bombesin, while leaving unaffected that induced by histamine. The increase in plasma gastrin levels induced by 2-deoxy-D-glucose, bombesin or a test meal was not significantly modified by BP 2-94 (30 mg/kg, intragastrically). In anesthetized dogs BP 2-94 (30 mg/kg, intragastrically) significantly reduced histamine release detected in the portal vein under bombesin infusion, whereas it did not modify gastrin and somatostatin plasma levels. These data indicate that BP 2-94 is a good prodrug of (R)-alpha-methylhistamine in the dog, causing an efficacious reduction of acid secretion induced by both 2-deoxy-D-glucose and bombesin. Moreover, the study of paracrine and hormonal mediators of acid secretion confirms that the main mechanism underlying inhibition of acid production induced by H3-receptor activation is the impairment of histamine release from gastric histaminocytes (possibly enterochromaffin-like cells).

1. Sabcomeline (SB-202026, 0.03 mg kg(-1), p.o.), a potent and functionally selective M1 receptor partial agonist, caused a statistically significant improvement in the performance of a visual object discrimination task by marmosets. No such improvement was seen after RS86 (0.1 mg kg(-1), p.o.). 2. Initial learning, which only required an association of object with reward and an appropriate response to be made, was not significantly affected. Reversal learning, which required both the extinction of the previously learned response and the acquisition of a new response strategy, was significantly improved after administration of sabcomeline (0.03 mg kg(-1), p.o.). 3. Sabcomeline (0.03 and 0.1 mg kg(-1), p.o.) had no significant effect on mean blood pressure measured for 2 h after administration in the conscious marmoset. 4. Sabcomeline (0.03 mg kg(-1), p.o.) caused none of the overt effects such as emesis or behaviours often seen after the administration of muscarinic agonists, e.g. face rubbing and licking. 5. This is the first study to demonstrate cognitive enhancement by a functionally selective M1 receptor partial agonist in a normal (i.e. non-cognitively impaired) non-human primate and this effect was seen at a dose which did not cause side effects. 6. Perseverative behaviour and deficient acquisition of new information are seen in patients with Alzheimer's disease (AD). Therefore the data suggest that sabcomeline might be of therapeutic benefit in the treatment of AD.

An attractive approach to the treatment of inflammatory conditions such as osteo- and rheumatoid arthritis, inflammatory bowel disease, and sepsis is through the selective inhibition of human inducible nitric oxide synthase (hiNOS) since localized excess nitric oxide (NO) release has been implicated in the pathology of these diseases. A series of monosubstituted iminohomopiperidinium salts possessing lipophilic functionality at ring positions 3, 5, 6, and 7 has been synthesized, and series members have demonstrated the ability to inhibit the hiNOS isoform with an IC50 as low as 160 nM (7). Compounds were found that selectively inhibit hiNOS over the human endothelial constitutive enzyme (heNOS) with a heNOS/hiNOS IC50 ratio in excess of 100 and as high as 314 (9). Potencies for inhibition of hiNOS and the human neuronal constitutive enzyme (hnNOS) are comparable. Substitution in the 3 and 7 positions provides compounds that exhibit the greatest degree of selectivity for hiNOS and hnNOS over heNOS. Submicromolar potencies for 6 and 7 in a mouse RAW cell assay demonstrated the ability of these compounds to inhibit iNOS in a cellular environment. These latter compounds were also found to be orally bioavailable and efficacious due to their ability to inhibit the increase in plasma nitrite/nitrate levels in a rat LPS model.

Nitrosoheptamethyleneimine (NHMI) was tested for carcinogenicity in Fischer-344 and Sprague-Dawley rats by intragastric administration and subcutaneous injection. Cumulative doses ranged from 5.5 to 1,200 mg/kg, and dosage schedules ranged from 40 serial administrations to one single injection. No difference in response was seen between sexes or strains of rats. Following the highest carcinogen doses by intragasritc administration, a high incidence of squamous-cell tumors occurred in the lung. The highest incidence of squamous-cell tumors occurred in the nasal cavity, trachea and esophagus. Following subcutaneous injection, tumors induced in the lungs were all alveologenic adenocarcinomas. The upper respiratory and gastrointestinal tracts were again the most commonly affected sites, with tumors similar to those of the first group. Intragastric administration was more effective in producing tumors than was subcutaneous injection, and administration of multiple small doses was more efficient than single large doses. The results demonstrated that the route and schedule of administration markedly influenced the tumor response to NHMI, both quantitatively and qualitatively.

RMI 12330 A, a compound of the lactamamide series, is a potent inhibitor of vasoactive intestinal polypeptide(VIP)- and prostaglandin (PG)-stimulated colonic secretion in the rat. This substance was tested on the adenylate cyclase system in human colonic mucosa. RMI 12330 A inhibited PGE2-, 16,16-dimethyl-PGE2- as well as VIP-sensitive adenylate cyclases in a dose-related manner. Half-maximal inhibition of hormone-stimulated enzyme activities occurred at a lactamimide concentration of about 0.15 mM. Lactamimide inhibition was non-competitive. Our results are compatible with the concept of RMI 12330 A acting as an inhibitor of colonic secretion via inhibition of hormone-sensitive adenylate cyclase. Since basal, NaF- and guanylyl-imidodiphosphate-stimulated enzyme activities were also affected by this compound, we may conclude that RMI 12330 A is a non-specific inhibitor of the human colonic adenylate cyclase system.