AB Toxins: A Paradigm Switch from Deadly to Desirable

To ensure their survival, a number of bacterial and plant species have evolved a common strategy to capture energy from other biological systems. Being imperfect pathogens, organisms synthesizing multi-subunit AB toxins are responsible for the mortality of millions of people and animals annually. Vaccination against these organisms and their toxins has proved rather ineffective in providing long-term protection from disease. In response to the debilitating effects of AB toxins on epithelial cells of the digestive mucosa, mechanisms underlying toxin immunomodulation of immune responses have become the focus of increasing experimentation. The results of these studies reveal that AB toxins may have a beneficial application as adjuvants for the enhancement of immune protection against infection and autoimmunity. Here, we examine similarities and differences in the structure and function of bacterial and plant AB toxins that underlie their toxicity and their exceptional properties as immunomodulators for stimulating immune responses against infectious disease and for immune suppression of organ-specific autoimmunity.     

Molecular Motions as a Drug Target: Mechanistic Simulations of Anthrax Toxin Edema Factor Function Led to the Discovery of Novel Allosteric Inhibitors

Edema Factor (EF) is a component of Bacillus anthracis toxin essential for virulence. Its adenylyl cyclase activity is induced by complexation with the ubiquitous eukaryotic cellular protein, calmodulin (CaM). EF and its complexes with CaM, nucleotides and/or ions, have been extensively characterized by X-ray crystallography. Those structural data allowed molecular simulations analysis of various aspects of EF action mechanism, including the delineation of EF and CaM domains through their association energetics, the impact of calcium binding on CaM, and the role of catalytic site ions. Furthermore, a transition path connecting the free inactive form to the CaM-complexed active form of EF was built to model the activation mechanism in an attempt to define an inhibition strategy. The cavities at the surface of EF were determined for each path intermediate to identify potential sites where the binding of a ligand could block activation. A non-catalytic cavity (allosteric) was found to shrink rapidly at early stages of the path and was chosen to perform virtual screening. Amongst 18 compounds selected in silico and tested in an enzymatic assay, 6 thiophen ureidoacid derivatives formed a new family of EF allosteric inhibitors with IC50 as low as 2 micromolars.     

Venoconstrictor responses to ergosine and ergosinine: Evidence for the isomerization of ergosinine

Ergosine and its D-isolysergic acid derivative ergosinine were investigated on canine saphenous veins both in vivo and in vitro. Following local i.v. infusion in vivo, about 5 times higher doses of ergosinine were necessary to produce the same venoconstrictor response as induced by ergosine. When administered orally, however, both ergot alkaloids were equi-effective. In vitro methiothepin, a 5-HT receptor blocker with high affinity for 5-HT₁ receptors, antagonized venoconstrictor responses to 5-HT and ergosine within the same concentration range, being significantly less potent when tested against norepinephrine. The reverse was true for the α₂-selective adrenoceptor blocker yohimbine, which was significantly more potent against norepinephrine and ergosine than against 5-HT, suggesting that ergosine has affinity to both 5-HT₁-like receptors and α₂-adrenoceptors. Concentration-response curves to norepinephrine were shifted to the right in a parallel fashion when ergosine or ergosinine were present in the organ baths, suggesting competitive antagonism. The blocking potency of ergosinine increased with increasing incubation times in Krebs-Henseleit solution becoming similar to that of ergosine when an incubation time of 2 hr was applied. It is suggested that the pharmacological activity of ergosinine is the consequence of an isomerization into its natural stereoisomer ergosine, which may occur both in vivo and in vitro.     

Predictions of in Vivo Plasma Concentrations from in Vitro Release Kinetics: Application to Doxepin Parenteral (I.M.) Suspensions in Lipophilic Vehicles in Dogs

A flow through dissolution system was applied to obtain biorelevant dissolution rates from controlled release systems for parenteral administration using the antidepressant doxepin as a model compound. Plasma concentrations were simulated using the disposition function of doxepin obtained from administration of an aqueous doxepin solution (Aponal^®) to beagle dogs. Input functions were obtained from in vitro dissolution experiments with three parenteral controlled release suspensions of doxepin hydrochloride (DHC1), doxepin pamoate (DP-1), and microspheres of doxepin hydrochloride in poly D,L-lactid-co-glycolid (MC-1) in isopropyl myristate. The predicted plasma concentrations were compared with experimentally obtained concentrations in vivo. Good correlations (r>0.88) between observed and predicted data were obtained for all formulations investigated. Similarly, in vivo release kinetics calculated by the Loo-Riegelman method were compared with release kinetics measured in vitro and showed good correlations (r>0.89). It is anticipated that the in vitro dissolution system permits assessment of the clinical relevance of observed variations in dissolution rates e.g. between batches of one formulation.     

Jejunal Permeability: A Comparison Between the Ussing Chamber Technique and the Single-Pass Perfusion in Humans

Pharmaceutical Research -     

New insights in the metabolism of oxybutynin: evidence of N-oxidation of propargylamine moiety and rearrangement to enaminoketone

1. Oxybutynin hydrochloride is an antimuscarinic agent prescribed to patients with an overactive bladder (OAB) and symptoms of urinary urge incontinence. Oxybutynin undergoes pre-systemic metabolism, and the N-desethyloxybutynin (Oxy-DE), is reported to have similar anticholinergic effects.

2. We revisited the oxidative metabolic fate of oxybutynin by liquid chromatography–tandem mass spectrometry analysis of incubations with rat and human liver fractions, and by measuring plasma and urine samples collected after oral administration of oxybutynin in rats. This investigation highlighted that not only N-deethylation but also N-oxidation participates in the clearance of oxybutynin after oral administration.

3. A new metabolic scheme for oxybutynin was delineated, identifying three distinct oxidative metabolic pathways: N-deethylation (Oxy-DE) followed by the oxidation of the secondary amine function to form the hydroxylamine (Oxy-HA), N-oxidation (Oxy-NO) followed by rearrangement of the tertiary propargylamine N-oxide moiety (Oxy-EK), and hydroxylation on the cyclohexyl ring.

4. The functional activity of Oxy-EK was investigated on the muscarinic receptors (M_1-3) demonstrating its lack of antimuscarinic activity.

5. Despite the presence of the α,β-unsaturated function, Oxy-EK does not react with glutathione indicating that in the clearance of oxybutynin no reactive and potentially toxic metabolites were formed.     

Ethionine toxicity in vitro: the correlation of data from rat hepatocyte suspensions and monolayers with in vivo observations

The hepato-steatogenic compound ethionine has been used to investigate the correlations between in␣vivo and in vitro toxicity data. The aim was to find a suitable model of toxicity in hepatocyte suspensions or monolayers in vitro, which could predict the known toxicity of ethionine in vivo and which could be implemented in screening compounds of unknown toxicity. Thus a variety of markers of cytotoxicity, metabolic competence and liver-specific functions were investigated in rat hepatocyte suspensions and monolayers and compared with in vivo data in the rat. The following markers were measured in the appropriate system: (1) Neutral red uptake; 3-(4,5 dimethyl)thiazol-2-yl,-2,5-diphenyl tetrazolium bromide (MTT) reduction; lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) leakage (cytotoxicity). (2) ATP levels, protein synthesis and glutathione (GSH) levels (metabolic competence). (3) Urea and triglyceride synthesis and β-oxidation (liver specific functions). Ethionine (0–30 mM) did not affect the markers of direct cytotoxicity, except neutral red uptake, which was reduced by 18 and 30 mM ethionine after 20 h in culture. ATP and GSH depletion occurred in hepatocyte suspensions at the highest concentrations of ethionine (20 and 30 mM) after 1 h. In monolayers, GSH levels were reduced after 4 h, but not 20 h. Urea synthesis was increased in hepatocyte suspensions from 1 to 3 h by 10–30 mM ethionine and reduced after 20 h in cultured hepatocytes (18–30 mM). Protein synthesis was reduced and β-oxidation was increased in ethionine-treated hepatocyte suspensions. Unfortunately, there was no measurable effect on triglyceride accumulation within cells (the major biochemical change in␣vivo) in either system. Ethionine treated hepatocytes in suspension showed the same rate of triglyceride synthesis and transportation out of cells as control cells. Thus, hepatocyte suspensions were able to mimic the early biochemical effects of ethionine in vivo (ATP and GSH depletion, inhibition of protein synthesis) and some effects on urea synthesis, but monolayer cultures appeared to be less sensitive to the toxicity of ethionine. However, neither in vitro system was able to model the effects of ethionine on the accumulation of triglycerides in vivo. Received: 16 June 1998 / Accepted: 29 June 1998     

In vitro and in vivo techniques to assess the performance of gastro-retentive drug delivery systems: a review

Background: Interest in gastro-retentive drug delivery systems (GRDDS) can be attributed to the desire for increased patient convenience (once daily dosing) and to increase the therapeutic index (reduced C_max, increased C_min). A range of evaluation techniques for GRDDS exist for in vitro and in vivo evaluation. Objective: The aim of the present review is to describe the methodologies used for in vitro and in vivo evaluations of GRDDS. Methods: The proposed critical parameters for floating and swelling types of GRDDS are discussed. Modifications in dissolution testing and improved biorelevant testing methods are also described. The in vivo techniques for measuring gastro-retentive performance are also summarised. Conclusion: The described methods can be used as assessment techniques for the evaluation and development of GRDDS. With these techniques, it is possible (with appropriate controls) to determine if a GRDDS provides hope for advantages of extended residence time in the stomach.     

Prediction of Drug Transport Through the Blood-Brain Barrier in Vivo: A Comparison Between Two in Vitro Cell Models

Purpose. Studies were conducted to evaluate whether the use of an in vitro model of the blood-brain barrier (BBB) resulted in more accurate predictions of the in vivo transport of compounds compared to the use of a human intestinal cell line (Caco-2). Methods. The in vitro BBB model employs bovine brain capillary endothelial cells co-cultured with primary rat astrocytes. The Caco-2 cells originate from a human colorectal carcinoma. The rat was used as experimental animal for the in vivo studies. Results. Strong correlations (r = 0.93-0.95) were found between the results generated by the in vitro model of the BBB and two different methodologies to measure the permeability across the BBB in vivo. In contrast, a poor correlation (r = 0.68) was obtained between Caco-2 cell data and in vivo BBB transport. A relatively poor correlation (r = 0.74) was also found between the two in vitro models. Conclusion. The present study illustrates the limitations of the Caco-2 model to predict BBB permeability of compounds in vivo. The results emphasize the fact that the BBB and the intestinal mucosa are two fundamentally different biologic barriers, and to be able to make accurate predictions about the in vivo CNS penetration of potential drug candidates, it is important that the in vitro model possesses the main characteristics of the in vivo BBB.     

Cardiac arrhythmia: in vivo screening in the zebrafish to overcome complexity in drug discovery

Importance of the field: Cardiac arrhythmias remain a major challenge for modern drug discovery. Clinical events are paroxysmal, often rare and may be asymptomatic until a highly morbid complication. Target selection is usually based on limited information and though highly specific agents are identified in screening, the final efficacy is often compromised by unanticipated systemic responses, a narrow therapeutic index and substantial toxicities.

Areas covered in this review: Our understanding of complexity of arrhythmogenesis has grown dramatically over the last 2 decades, and the range of potential disease mechanisms now includes pathways previously thought only tangentially involved in arrhythmia. This review surveys the literature on arrhythmia mechanisms from 1965 to the present day, outlines the complex biology underlying every rhythm disturbance, and highlights the problems for rational target identification. The rationale for in vivo screening is described and the utility of the zebrafish for this approach and for complementary work in functional genomics is discussed. Current limitations of the model in this setting and the need for careful validation in new disease areas are also described.

What the reader will gain: An overview of the complex mechanisms underlying most clinical arrhythmias, and insight into the limits of ion channel conductances as drug targets. An introduction to the zebrafish as a model organism, in particular for cardiovascular biology. Potential approaches to overcoming the hurdles to drug discovery including in vivo screening of zebrafish genetic disease models.

Take home message: In vivo screening in faithful disease models allows the effects of drugs on integrative physiology and disease biology to be captured during the screening process in a manner agnostic to potential drug target or targets. This systematic strategy bypasses current gaps in our understanding of disease biology, but emphasizes the importance of the rigor of the disease model.     

Difference in metabolic profile of potassium canrenoate and spironolactone in the rat: Mutagenic metabolites unique to potassium canrenoate

The metabolic fates of potassium canrenoate (PC) and spironolactone (SP) were compared for the rat in vivo and in vitro. Approximately 18% of an in vivo dose of SP was metabolized to canrenone (CAN) and related compounds in the rat. In vitro, 20–30% of SP was dethioacetylated to CAN and its metabolites by rat liver 9000 g supernatant (S9). Thus, the major route of SP metabolism is via pathways that retain the sulfur moiety in the molecule. PC was metabolized by rat hepatic S9 to 6, 7- and 6, 7-epoxy-CAN. The -epoxide was further metabolized to its 3- and 3-hydroxy derivatives as well as its glutathione (GSH) conjugate. Both 3- and 3-hydroxy-6, 7-epoxy-CAN were shown to be direct acting mutagens in the mouse lymphoma assay, whereas 6, 7- and 6, 7-epoxy-CAN were not. These mutagenic metabolites, their precursor epoxides and their GSH conjugates were not formed from SP under identical conditions. The above findings appear to be due to inhibition of metabolism of CAN formed from SP by SP and/or its S-containing metabolites, since the in vitro metabolism of PC by rat hepatic microsomes was appreciably reduced in the presence of SP. The hypothesized mechanism(s) for this inhibition is that SP and its S-containing metabolites specifically inhibit an isozyme of hepatic cytochrome P-450 or SP is a preferred substrate over PC/CAN for the metabolizing enzymes. Absence of the CAN epoxide pathway in the metabolism of SP provides a possible explanation for the observed differences in the toxicological profiles of the two compounds.     

Investigational drugs for the treatment of Zika virus infection: a preclinical and clinical update

ABSTRACT

Introduction: The Zika virus (ZIKV) infection results in severe neurological complications and has emerged as a threat to public health worldwide. No drugs or vaccines are available for use in the clinic and the need for novel and effective therapeutic agents is urgent.

Areas covered: This review describes the latest progress of antiviral development for the treatment of ZIKV infection; it primarily focuses on the literature describing 20 potential anti-ZIKV drugs/agents currently being tested in vivo or in clinical trials. The paper also discusses the need for novel ZIKV inhibitors and the critical issues for successful antiviral drug development.

Expert opinion: So far, 20 compounds have been tested in vivo and three in the clinical trials; progressing these compounds to the clinic is a challenge. Novel ZIKV inhibitors that target virus or host factors are urgently needed. Knowledge-driven drug repurposing, structure-based discovery, RNA interference, long noncoding RNAs, miRNAs, and peptide inhibitors may pave the way for the discovery of such novel agents.     

Nitrotyrosine attenuates the hemodynamic effects of adrenoceptor agonists in vivo: relevance to the pathophysiology of peroxynitrite

Peroxynitrite, which attenuates catecholamine-mediated hemodynamic responses in vivo, nitrates free tyrosine residues to form the specific product, 3-nitro- -tyrosine. The chemical structure of 3-nitro- -tyrosine is similar to that of the endogenous catecholamines. Therefore, 3-nitro- -tyrosine may interfere with catecholamine hemodynamic function in vivo. The hemodynamic responses produced by norepinephrine (1-4 μg/kg, i.v., n = 6), epinephrine (0.5-4 μg/kg, i.v., n = 7), phenylephrine (1-8 μg/kg, i.v., n = 5), and isoproterenol (100-400 ng/kg, i.v., n = 5) were attenuated, while the hemodynamic responses produced by arginine vasopressin (50-250 ng/kg; i.v., n = 5) were unaffected following the administration of 3-nitro- -tyrosine (2.5 μmol/kg, i.v.) in pentobarbital-anesthetized rats. These results demonstrate substantial and selective attenuation of the hemodynamic effects produced by - and β-adrenoceptor agonists, raising the possibility that 3-nitro- -tyrosine may play a role in the hemodynamic dysfunction associated with inflammatory conditions in which the formation of peroxynitrite is favored.     

龟肌肽、鳖肌肽和龟鳖粉体内抗甲型流感病毒作用的初步观察

目的：了解龟肌肽、鳖肌肽和龟鳖粉体内抗甲型流感病毒感染的效果。方法：用甲型流感病毒PR－8株（H3N2）建立NIH小鼠呼吸道病毒感染模型。感染小鼠尾静脉注射不同浓度的龟肌肽和鳖肌肽或用不同浓度的龟鳖粉灌胃。结果：尾静脉注射生理盐水的对照组小鼠感染甲型流感病毒后均死亡。注射高浓度龟肌肽（100mg/kg)、低浓度龟肌肽（50mg/kg)、高浓度鳖肌肽（100mg/kg)、低浓度鳖肌肽（50mg/kg)和病毒唑（10mg/kg)的小鼠感染该病毒后存活率分别为64．3％、42．9％、50．0％、50．0％和85．7％;口服高浓度龟鳖粉（6g/kg) 和低浓度龟鳖粉（3g/kg)的小鼠感染该病毒后的存活率分别为35．8％利28．6％。高浓度龟肌肽试验组小鼠的存活率接近病毒唑对照组（χ^2=1.71,P＞0．05）,其它各试验组小鼠的存活率均低于病毒唑对照组（χ^2=4.09-9.33,P＜0．05）。结论：龟肌肽、鳖肌肽和龟鳖粉具有一定的体内抗甲型流感病毒感染的作用,但以高浓度龟肥肽抗病毒作用最强。     

Patent Evaluation: A New Approach to the Production of Human Monoclonal Antibodies

Summary

Novelty: A technique is described for the immortalization of human B-cells producing either IgG, IgM or IgA, thereby creating cell lines producing natural, human monoclonal antibodies. This technique may have major implications in the use of antibodies in human therapy where a lack of antigenicity is required. It will circumvent and simplify the current methods of humanizing mouse monoclonals.

Biology: Human blood is depleted of T-cells by rosetting and the resulting B-cell population of lymphocytes is transformed with Epstein Barr virus in conjunction with at least one of the cytokines, Interleukin-4 or Interleukin-6 plus 8-mercaptoguanosine. The cells are cloned out by limiting dilution in microliter trays and tested for antigen specific antibody production. Antibody production increases with time. IgG is produced in the largest quantities.     

Quantitative prediction of in vivo inhibitory interactions involving glucuronidated drugs from in vitro data: the effect of fluconazole on zidovudine glucuronidation

AIMS: Using the fluconazole-zidovudine (AZT) interaction as a model, to determine whether inhibition of UDP-glucuronosyltransferase (UGT) catalysed drug metabolism in vivo could be predicted quantitatively from in vitro kinetic data generated in the presence and absence bovine serum albumin (BSA). METHODS: Kinetic constants for AZT glucuronidation were generated using human liver microsomes (HLM) and recombinant UGT2B7, the principal enzyme responsible for AZT glucuronidation, as the enzyme sources with and without fluconazole. K(i) values were used to estimate the decrease in AZT clearance in vivo. RESULTS: Addition of BSA (2%) to incubations decreased the K(m) values for AZT glucuronidation by 85-90% for the HLM (923 +/- 357 to 91 +/- 9 microm) and UGT2B7 (478-70 microm) catalysed reactions, with little effect on V(max). Fluconazole, which was shown to be a selective inhibitor of UGT2B7, competitively inhibited AZT glucuronidation by HLM and UGT2B7. Like the K(m), BSA caused an 87% reduction in the K(i) for fluconazole inhibition of AZT glucuronidation by HLM (1133 +/- 403 to 145 +/- 36 microm) and UGT2B7 (529 to 73 microm). K(i) values determined for fluconazole using HLM and UGT2B7 in the presence (but not absence) of BSA predicted an interaction in vivo. The predicted magnitude of the interaction ranged from 41% to 217% of the reported AUC increase in patients, depending on the value of the in vivo fluconazole concentration employed in calculations. CONCLUSIONS: K(i) values determined under certain experimental conditions may quantitatively predict inhibition of UGT catalysed drug glucuronidation in vivo.     

A dissolution rate apparatus for the prediction of initial drug absorption patterns in beagles: Tolbutamide tablets

An apparatus utilizing liquid turbulence to simulate hydrodynamic conditions generated by gastrointestinal peristalsis was designed to estimate drug release from solid oral dosage forms. This turbulence was achieved through special arrangement of a pipetting pump to a dissolution chamber. By adjusting the flow rate of the pump to deliver and withdraw a fixed volume of dissolution medium per minute, a correlation was developed between dissolution rates and absorption patterns in beagles of two commercial tolbutamide tablets, A and B, and a micronized tolbutamide suspension. On the basis of this relationship, it was possible to predict the initial absorption patterns of two misformulated tablets, C and D.