Acyl glucuronide reactivity in perspective: biological consequences

The metabolic conjugation of exogenous and endogenous carboxylic acid substrates with endogenous glucuronic acid, mediated by the uridine diphosphoglucuronosyl transferase (UGT) superfamily of enzymes, leads to the formation of acyl glucuronide metabolites. Since the late 1970s, acyl glucuronides have been increasingly identified as reactive electrophilic metabolites, capable of undergoing three reactions: intramolecular rearrangement, hydrolysis, and intermolecular reactions with proteins leading to covalent drug-protein adducts. This essential dogma has been accepted for over a decade. The key question proposed by researchers, and now the pharmaceutical industry, is: does or can the covalent modification of endogenous proteins, mediated by reactive acyl glucuronide metabolites, lead to adverse drug reactions, perhaps idiosyncratic in nature? This review evaluates the evidence for acyl glucuronide-derived perturbation of homeostasis, particularly that which might result from the covalent modification of endogenous proteins and other macromolecules. Because of the availability of acyl glucuronides for test tube/in vitro experiments, there is now a substantial literature documenting their rearrangement, hydrolysis and covalent modification of proteins in vitro. It is certain from in vitro experiments that serum albumin, dipeptidyl peptidase IV, tubulin and UGTs are covalently modified by acyl glucuronides. However, these in vitro experiments have been specifically designed to amplify any interference with a biological process in order to find biological effects. The in vivo situation is not at all clear. Certainly it must be concluded that all humans taking carboxylate drugs that form reactive acyl glucuronides will form covalent drug-protein adducts, and it must also be concluded that this in itself is normally benign. However, there is enough in vivo evidence implicating acyl glucuronides, which, when backed up by in vivo circumstantial and documented in vitro evidence, supports the view that reactive acyl glucuronides may initiate toxicity/immune responses. In summary, though acyl glucuronide-derived covalent modification of endogenous macromolecules is well-defined, the work ahead needs to provide detailed links between such modification and its possible biological consequences. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

Arachidonic acid release from mammalian cells transfected with human groups IIA and X secreted phospholipase A(2) occurs predominantly during the secretory process and with the involvement of cytosolic phospholipase A(2)-alpha

Stable expression of human groups IIA and X secreted phospholipases A(2) (hGIIA and hGX) in CHO-K1 and HEK293 cells leads to serum- and interleukin-1beta-promoted arachidonate release. Using mutant CHO-K1 cell lines, it is shown that this arachidonate release does not require heparan sulfate proteoglycan- or glycosylphosphatidylinositol-anchored proteins. It is shown that the potent secreted phospholipase A(2) inhibitor Me-Indoxam is cell-impermeable. By use of Me-Indoxam and the cell-impermeable, secreted phospholipase A(2) trapping agent heparin, it is shown that hGIIA liberates free arachidonate prior to secretion from the cell. With hGX-transfected CHO-K1 cells, arachidonate release occurs before and after enzyme secretion, whereas all of the arachidonate release from HEK293 cells occurs prior to enzyme secretion. Immunocytochemical studies by confocal laser and electron microscopies show localization of hGIIA to the cell surface and Golgi compartment. Additional results show that the interleukin-1beta-dependent release of arachidonate is promoted by secreted phospholipase A(2) expression and is completely dependent on cytosolic (group IVA) phospholipase A(2). These results along with additional data resolve the paradox that efficient arachidonic acid release occurs with hGIIA-transfected cells, and yet exogenously added hGIIA is poorly able to liberate arachidonic acid from mammalian cells.

Unidirectional inversion of ibuprofen in Caco-2 cells: Developing a suitable model for presystemic chiral inversion study

Intestinal chiral inversion of ibuprofen is still lacking direct evidence. In a preliminary experiment, ibuprofen was found to undergo inversion in Caco-2 cells. This investigation was thus conducted to determine the characteristics and influence of some biochemical factors on the chiral inversion of ibuprofen in Caco-2 cells. The effects of substrate concentration (2.5-40 mu g/ml), cell density (0.5-2 x 10(6) cells/ well), content of serum (0-20%), coexistence of S ibuprofen (corresponding doses), sodium azide (10mm), exogenous Coenzyme A (CoA) (0.1 - 0.4 mm),. and palmitic acid (5-25 mu m) on inversion were examined. A stereoselective HPLC method based on the Chromasil-CHI-TBB column was developed for quantitative analysis of the drug in cell culture medium. The inversion ratio (F-i) and elimination rate constant were calculated as the indexes of inversion extent. Inversion of ibuprofen in Caeo-2 cells was found to be both dose and cell density dependent, indicating saturable characteristics. Addition of serum significantly inhibited the inversion, to an extent of 2.7 fold decrease at 20% content. Preexistence of S enantiomer exerted a significant inhibitory effect (p < 0.01 for all tests). Sodium azide decreased the inversion ratio from 0.43 to 0.32 (p < 0.01). Exogenous CoA and palmitic acid significantly promoted the inversion at all tested doses (p < 0.01 for all tests). This research provided strong evidence to the capacity and capability of intestinal chiral inversion. Although long incubation times up to 120 h were required, Caco-2 cells should be a suitable model for chiral inversion research of 2-APAs considering the human-resourced and well-defined characteristics from the present study.

Interactions among smoking, obesity, and symptoms of acid reflux in Barrett's esophagus

Background: Barrett's esophagus, a metaplastic precursor to esophageal adenocarcinoma, is becoming increasingly prevalent in many populations. Clinical studies suggest acid reflux causes Barrett's esophagus; however, no population-based estimates of risk have been reported, and the role of other health factors in modifying risk is unclear. Methods: We conducted a population-based case-control study in Brisbane, Australia. Cases were 167 patients with histologically confirmed Barrett's esophagus diagnosed between February and December 2003. Age-matched and sex-matched controls (n = 261) were randomly selected from a population register. Data on exposure to self-reported symptoms of acid reflux, smoking, obesity, and other factors were collected through self-completed questionnaires followed by telephone interview. Risks of Barrett's esophagus and Barrett's esophagus with dysplasia associated with these exposures were estimated by the odds ratio (OR) and 95% confidence interval (95% Cl), both crude and adjusted for other factors. Results: Self-reported weekly episodes of acid reflux were associated with greatly increased risks of Barrett's esophagus (adjusted OR, 29.7; 95% CI, 12.2-72.6) and Barrett's esophagus with dysplasia (OR, 59.7; 95% CI, 18.5-193). Smoking was also associated with risk of Barrett's esophagus. We found evidence of interactions between symptoms of acid reflux and smoking and obesity. Obese people with self-reported symptoms of acid reflux had markedly higher risks of Barrett's esophagus (OR, 34.4; 95% CI, 6.3-188) than people with reflux alone (OR, 9.3; 95% CI, 1.4-62.2) or obesity alone (OR, 0.7,95% CI, 0.2-2.4). Similarly, those reporting both acid reflux symptoms and smoking were at substantially higher risks of Barrett's esophagus (OR, 51.4; 95% CI, 14.1-188) than those reporting acid reflux or smoking alone. Conclusions: Although history of symptoms of acid reflux is the principle factor associated with Barrett's esophagus, risks are substantially increased by obesity and smoking.

Inhibitors of cyclo-oxygenase-2 and secretory phospholipase A2 preserve bone architecture following ovariectomy in adult rats

Epidemiological evidence and in vitro data suggest that COX-2 is a key regulator of accelerated remodeling. Accelerated states of osteoblast and osteoclast activity are regulated by prostaglandins in vitro, but experimental evidence for specific roles of cyclooxygenase-2 (COX-2) and secretory phospholipase A(2) (sPLA(2)) in activated states of remodeling in vivo is lacking. The aim of this study was to determine the effect of specific inhibitors of sPLA(2)-IIa and COX-2 on bone remodeling activated by estrogen deficiency in adult female rats. One hundred and twenty-four adult female Wistar rats were ovariectomized (OVX) or sham-operated. Rats commenced treatment 14 days after surgery with either vehicle, a COX-2 inhibitor (DFU at 0.02 mg/kg/day and 2.0 mg/kg/day) or a sPLA(2)-group-IIa inhibitor (KH064 at 0.4 mg/kg/day and 4.0 mg/kg/day). Treatment continued daily until rats were sacrificed at 70 days or 98 days post-OVX. The right tibiae were harvested, fixed and embedded in methylmethacrylate for structural histomorphometric bone analysis at the proximal tibial metaphysis. The specific COX-2 or sPLA(2) inhibitors prevented ovariectomy-induced (OVX-induced) decreases in trabecular connectivity (P < 0.05); suppressed the acceleration of bone resorption; and maintained bone turnover at SHAM levels following OVX in the rat. The sPLA2 inhibitor significantly suppressed increases in osteoclast surface induced by OVX (P < 0.05), while the effect of COX-2 inhibition was less marked. These findings demonstrate that inhibitors of COX-2 and sPLA(2)-IIa can effectively suppress OVX-induced bone loss in the adult rat by conserving trabecular bone mass and architecture through reduced bone remodeling and decreased resorptive activity. Moreover, we report an important role of sPLA(2)-IIa in osteoclastogenesis that may be independent of the COX-2 metabolic pathway in the OVX rat in vivo. (c) 2006 Elsevier Inc. All rights reserved.

Inhibition of COXs and 5-LOX and activation of PPARs by Australian Clematis species (Ranunculaceae)

The species of Clematis (Ranunculaceae) have been traditionally used for inflammatory conditions by indigenous Australians. We have previously reported that the ethanol extract of Clematis pickeringii inhibited COX-1. In this study, we examined the ethanol extracts and fractions of three Clematis species, Clematis pickeringii, Clematis glycinoides and Clematis microphylla, on cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX). We further examined the activating effects on the protein expression of peroxisome proliferator-activated receptor alpha (PPAR alpha) and gamma (PPAR-gamma) in HepG2 cells. The ethanol extracts of three Clematis species inhibited the activities of COX-1, COX-2 and 5-LOX in the different extents. The stem extract of Clematis pickeringii showed the highest inhibitory activities among the three species on COX-1, COX-2 and 5-LOX with the IC50 values of 73.5, 101.2 and 29.3 mu g/mL. One of its fractions also significantly elevated PPAR gamma expression by 173, 280 and 435% and PPAR gamma expression by 140, 228 and 296% at 4, 8 and 16 mu g/mL, respectively. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Regulation of bone biology by prostaglandin endoperoxide H synthases (PGHS): A rose by any other name...

It is well established that prostaglandins are essential mediators of bone resorption and formation. In the early 1990s, it was discovered that enzymatic reactions producing prostaglandins were regulated by two cyclooxygenase enzymes, one producing prostaglandins constitutively in tissues like the stomach, prostaglandin endoperoxide H synthase-1 (PGHS-1 or COX-1), and another induced by mitogens or inflammatory mediators (PGHS-2 or COX-2). This neat distinction has not been maintained because both enzymes act in different cell systems to provide physiological signaling, constitutively or by induction under certain conditions. For example, the regulation patterns of PGHS-1 and PGHS-2 are distinct, but the evidence shows that PGHS-2 functions constitutively in the skeleton. PGHS-2 hits quickly been established, therefore, as a key regulator of bone biology, capable of rapid and transient expression in bone cells, and mediating osteoclastogenesis, mechanotransduction, bone formation and fracture repair. The goal of this review is to Summarize the current state of our knowledge of PGHS regulation of bone metabolism and to identify some of the key unresolved challenges and questions that require further study. (c) 2006 Elsevier Ltd. All rights reserved.

Spectroscopic characterization of copper(II) binding to the immunosuppressive drug mycophenolic acid

Mycophenolic acid (MPA) is a drug that has found widespread use as an immunosuppressive agent which limits rejection of transplanted organs. Optimal use of this drug is hampered by gastrointestinal side effects which can range in severity. One mechanism by which MPA causes gastropathy may involve a direct interaction between the drug and gastric phospholipids. To combat this interaction we have investigated the potential of MPA to coordinate Cu(II), a metal which has been used to inhibit gastropathy associated with use of the NSAID indomethacin. Using a range of spectroscopic techniques we show that Cu(II) is coordinated to two MPA molecules via carboxylates and, at low pH, water ligands. The copper complex formed is stable in solution as assessed by mass spectrometry and H-1 NMR diffusion experiments. Competition studies with glycine and albumin indicate that the copper-MPA complex will release Cu(II) to amino acids and proteins thereby allowing free MPA to be transported to its site of action. Transfer to serum albumin proceeds via a Cu(MPA)(albumin) ternary complex. These results raise the possibility that copper complexes of MPA may be useful in a therapeutic situation.

Risk of serious NSAID-related gastrointestinal events during long-term exposure: a systematic review

Objective: Exposure to non-steroidal anti-inflammatory drugs (NSAIDs) is associated wit increased risk of serious gastrointestinal (GI) events compared with non-exposure. We investigated whether that risk is sustained over time. Data sources: Cochrane Controlled Trials Register (to 2002); MEDLINE, EMBASE, Derwent Drug File and Current Contents (1999-2002); manual searching of reviews (1999-2002). Study selection: From 479 search results reviewed and 221 articles retrieved, seven studies of patients exposed to prescription non-selective NSAIDs for more than 6 months and reporting time-dependent serious GI event rates were selected for quantitative data synthesis. These were stratified into two groups by study design. Data extraction: Incidence of GI events and number of patients at specific time points were extracted. Data synthesis: Meta-regression analyses were performed. Change in risk was evaluated by testing whether the slope of the regression line declined over time. Four randomised controlled trials (RCTs) provided evaluable data from five NSAID arms (aspirin, naproxen, two ibuprofen arms, and diclofenac). When the RCT data were combined, a small significant decline in annualised risk was seen: -0.005% (95% Cl, -0.008% to -0.001%) per month. Sensitivity analyses were conducted because there was disparity within the RCT data. The pooled estimate from three cohort studies showed no significant decline in annualised risk over periods up to 2 years: -0.003% (95% Cl, -0.008% to 0.003%) per month. Conclusions: Small decreases in risk over time were observed; these were of negligible clinical importance. For patients who need long-term (> 6 months) treatment, precautionary measures should be considered to reduce the net probability of serious GI events over the anticipated treatment duration. The effect of intermittent versus regular daily therapy on long-term risk needs further investigation.

A convergent solution-phase synthesis of the macrocycle Ac-Phe-[Orn-Pro-D-Cha-Trp-Arg], a potent new antiinflammatory drug

Relatively few cyclic peptides have reached the pharmaceutical marketplace during the past decade, most produced through fermentation rather than made synthetically. Generally, this class of compounds is synthesized for research purposes on milligram scales by solid-phase methods, but if the potential of macrocyclic peptidomimetics is to be realized, low-cost larger scale solution-phase syntheses need to be devised and optimized to provide sufficient quantities for preclinical, clinical, and commercial uses. Here, we describe a cheap, medium-scale, solution-phase synthesis of the first reported highly potent, selective, and orally active antagonist of the human C5a receptor. This compound, Ac-Phe[Orn-Pro-D-Cha-Trp-Arg], known as 3D53, is a macrocyclic peptidomimetic of the human plasma protein C5a and displays excellent antiinflammatory activity in numerous animal models of human disease. In a convergent approach, two tripeptide fragments Ac-Phe-Orn-(Boc)-Pro-OH and H-D-Cha-Trp(For)-Arg-OEt were first prepared by high-yielding solution-phase couplings using a mixed anhydride method before coupling them to give a linear hexapeptide which, after deprotection, was obtained in 38% overall yield from the commercially available amino acids. Cyclization in solution using BOP reagent gave the antagonist in 33% yield (13% overall) after HPLC purification. Significant features of the synthesis were that the Arg side chain was left unprotected throughout, the component Boe-D-Cha-OH was obtained very efficiently via hydrogenation Of D-Phe with PtO2 in TFA/water, the tripeptides were coupled at the Pro-Cha junction to minimize racemization via the oxazolone pathway, and the entire synthesis was carried out without purification of any intermediates. The target cyclic product was purified (>97%) by reversed-phase HPLC. This convergent synthesis with minimal use of protecting groups allowed batches of 50100 g to be prepared efficiently in high yield using standard laboratory equipment. This type of procedure should be useful for making even larger quantities of this and other macrocyclic peptidomimetic drugs.

D-Tyrosine as a chiral precusor to potent inhibitors of human nonpancreatic secretory phospholipase A(2) (IIa) with antiinflammatory activity

Few reported inhibitors of secretory phospholipase A(2) enzymes inhibit the IIa human isoform (hnpsPLA(2)-IIa) noncovalently at submicromolar concentrations. Herein, the simple chiral precursor D-tyrosine was derivastised to give a series of potent new inhibitors of hnpsPLA(2)-IIa. A 2.2-Angstrom crystal structure shows an inhibitor bound in the active site of the enzyme, chelated to a Ca2+ ion through carboxylate and amide oxygen atoms, H bonded through an amide NH group to His48, with multiple hydrophobic contacts and a T-shaped aromatic-group-His6 interaction. Antiinflammatory activity is also demonstrated for two compounds administered orally to rats.

Ion-trapping, microsomal binding, and unbound drug distribution in the hepatic retention of basic drugs

This study investigated the relative contribution of ion-trapping, microsomal binding, and distribution of unbound drug as determinants in the hepatic retention of basic drugs in the isolated perfused rat liver. The ionophore monensin was used to abolish the vesicular proton gradient and thus allow an estimation of ion-trapping by acidic hepatic vesicles of cationic drugs. In vitro microsomal studies were used to independently estimate microsomal binding and metabolism. Hepatic vesicular ion-trapping, intrinsic elimination clearance, permeability-surface area product, and intracellular binding were derived using a physiologically based pharmacokinetic model. Modeling showed that the ion-trapping was significantly lower after monensin treatment for atenolol and propranolol, but not for antipyrine. However, no changes induced by monensin treatment were observed in intrinsic clearance, permeability, or binding for the three model drugs. Monensin did not affect binding or metabolic activity in vitro for the drugs. The observed ion-trapping was similar to theoretical values estimated using the pHs and fractional volumes of the acidic vesicles and the pK(a) values of drugs. Lipophilicity and pK(a) determined hepatic drug retention: a drug with low pK(a) and low lipophilicity (e.g., antipyrine) distributes as unbound drug, a drug with high pK(a) and low lipophilicity (e.g., atenolol) by ion-trapping, and a drug with a high pK(a) and high lipophilicity (e.g., propranolol) is retained by ion-trapping and intracellular binding. In conclusion, monensin inhibits the ion-trapping of high pK(a) basic drugs, leading to a reduction in hepatic retention but with no effect on hepatic drug extraction.

Critical relationship between sodium valproate dose and human teratogenicity: results of the Australian register of anti-epileptic drugs in pregnancy

To compare the incidence of foetal malformations (FMs) in pregnant women with epilepsy treated with different anti-epileptic drugs (AED) and doses, and the influence of seizures, family and personal history, and environmental factors. A prospective, observational, community-based cohort study. Methods. A voluntary, Australia-wide, telephone-interview-based register prospectively enrolling three groups of pregnant women: taking AEDs for epilepsy; with epilepsy not taking AEDs; taking AEDs for a non-epileptic indication. Four hundred and fifty eligible women were enrolled over 40 months. Three hundred and ninety six pregnancies had been completed, with 7 sets of twins, for a total of 403 pregnancy outcomes. Results. 354 (87.8%) pregnancy outcomes resulted in a healthy live birth, 26 (6.5%) had a FM, 4 (1%) a death in utero, 1 (0.2%) a premature labour with stillbirth, 14 (3.5%) a spontaneous abortion and 4 lost to follow-up. The FM rate was greater in pregnancies exposed to sodium valproate (VPA) in the first trimester (116.0%) compared with those exposed to all other AEDs (16.0% vs. 2.4%, P < 0.01) or no AEDs (16.0% vs. 3.1 %, P < 0.01). The mean daily dose of VPA taken in pregnancy with FMs was significantly greater than in those without (11975 vs: 1128 mg, P < 0.01). The incidence of FM with VPA doses greater than or equal to 1100 mg was 30.2% vs. 3.2% with doses < 1100 mg (P < 0.01). Conclusions. There is a dose-effect relationship for FM and exposure to VPA during the first trimester of pregnancy, with higher doses of VPA associated with a significantly greater risk than with lower doses or with other AEDs. These results highlight the need to limit, where possible, the dose of VPA in pregnancy. (C) 2004 Elsevier Ltd. All rights reserved.