Measurement of cyclooxygenase inhibition in vivo: a study of two non-steroidal anti-inflammatory drugs in sheep

The anti-inflammatory effects of the non-steroidal anti-inflammatory drugs phenylbutazone (PBZ) and flunixin meglumine (FM) and the relationship between the effects and drug concentration in vivo were studied using a subcutaneous tissue-cage model in sheep. Intracaveal injection of carrageenan induced prostaglandin (PG) E2 production in tissue-cage exudate (maximal concentration, 101 nM) with significant increases in white blood cell (WBC) numbers, skin temperature over the inflamed cage and exudate leukotriene B4 (LTB4) concentration (P < 0.05). Intravenous PBZ, 4.4 mg kg-1 produced mild inhibition of exudate PGE2 generation (10%), but greater inhibition of serum TXB2 (75.3%). The IC50 for TXB2 was 36.0 microM. Phenylbutazone did not alter effects on skin temperature, WBC numbers or exudate LTB4 concentrations. Intravenous FM, 1.1 mg kg-1, significantly inhibited carrageenan-induced exudate PGE2 formation (Emax, 100%, IC50, < 0.4 nM) and serum TXB2 generation (Emax, 100%, IC50, 17 nM) for up to 32 h. Flunixin meglumine significantly inhibited the rise in skin temperature but had a limited effect on exudate WBC. Phenylbutazone and FM have distinct effects on carrageenan-induced cyclooxygenase (COX-2) and platelet COX (COX-1). Flunixin meglumine was a more potent COX inhibitor than PBZ and was more selective for the inducible form of COX in vivo.     

Constitutive cyclooxygenase (COX-1) and inducible cyclooxygenase (COX-2): rationale for selective inhibition and progress to date

While a great deal has been discovered concerning the potential physiological and pathological role of prostanoids, much is left to be determined. The widespread distribution of both COX-1 and COX-2 coupled with the capacity of most vascular beds, smooth muscle, as well as leukocytes to respond to prostanoids make drawing generalities difficult. The problems with the majority of currently used NSAIDs are clear and ulcerogenic liability is of obvious concern. Interestingly enough, the mechanism of that damage is still the subject of controversy as illustrated by the recent review and hypothesis of Somasundaram et al. In this treatise, the suggestion is made that the initial gastric damage is the result of uncoupling of oxidative phosphorylation which is independent but simultaneous with COX inhibition. At least two currently marketed NSAIDs have improved G.I. liability (nabumetone and etodolac) with efficacy equivalent to other more ulcerogenic NSAIDs. These drugs appear to have achieved that by a mechanism distinct from selective inhibition of COX-2. Whether or not selective COX-2 inhibitors will demonstrate an improved profile over these compounds remains to be shown. Unfortunately, clinical experience with nimsulide and CGP 28238 suggest that NSAID-like toxicity may still be an issue. The promise of selective COX-2 inhibitors remains largely untested. It is with great interest and expectation that the clinical evaluation of the more selective compounds of different structural types is awaited.     

Prescription of non-steroidal anti-inflammatory drugs

BACKGROUND AND PURPOSE: Tacrolimus, an immunosuppressant agent, has been shown to reduce tissue injury and leukocyte accumulation after transient ischemia. This study was designed to evaluate quantitatively the inhibitory effects of tacrolimus on leukocyte rolling and on subsequent leukocyte accumulation in vivo after transient retinal ischemia and the protective effects of tacrolimus on ischemia-induced neural damage. METHODS: Retinal ischemia was induced for 60 minutes in anesthetized pigmented rats by temporary ligation of the optic sheath. Tacrolimus was administered at 10 minutes after ischemic induction. At 4, 12, 24, and 48 hours after reperfusion, leukocyte behavior in the retinal microcirculation was evaluated in vivo with acridine orange digital fluorography. After 7 days of reperfusion, ischemia-induced retinal damage was evaluated histologically. RESULTS: Treatment with tacrolimus suppressed leukocyte rolling; the maximum number of rolling leukocytes was reduced by 60.1% at 12 hours after reperfusion (P<0.05). In tacrolimus-treated rats, the velocity of rolling leukocytes was significantly faster than in vehicle-treated rats (P<0.01). The subsequent leukocyte accumulation was reduced by 61.6% at 24 hours after reperfusion (P<0.01). Histological examination demonstrated the protective effect of tacrolimus on ischemia-induced retinal damage, which was more substantial in the inner retina (P<0.01). CONCLUSIONS: The present study demonstrated the inhibitory effect of tacrolimus on leukocyte rolling and on subsequent leukocyte accumulation and the therapeutic potency to neural injury after transient retinal ischemia.     

The classification of cyclooxygenase inhibitors

In summary, precise classification of COX inhibitors has important clinical implications for efficacy and toxicity. However, classification of these agents clinically is difficult because there are insufficient data to predict correlations between biochemical and pharmacologic properties and the clinical effect of a given agent. In any case, specific COX-2 inhibitors are expected to show antiinflammatory and analgesic activities equivalent to those of NSAID, as well as significant reductions in the incidence of the life threatening side effects (i.e., GI bleeding) associated with COX-1 inhibition. The advantages of preferential COX-2 inhibitors may be more subtle and therefore more difficult to verify in clinical trials.     

2-Iminopyrrolidines as potent and selective inhibitors of human inducible nitric oxide synthase

A series of substituted 2-iminopyrrolidines has been prepared and shown to be potent and selective inhibitors of the human inducible nitric oxide synthase (hiNOS) isoform versus the human endothelial nitric oxide synthase (heNOS) and the human neuronal nitric oxide synthase (hnNOS). Simple substitutions at the 3-, 4-, or 5-position afforded more potent analogues than the parent 2-iminopyrrolidine 1. The effect of ring substitutions on both potency and selectivity for the different NOS isoforms is described. Substitution at the 4- and 5-positions of the 2-iminopyrrolidine yielded both potent and selective inhibitors of hiNOS. In particular, (+)-cis-4-methyl-5-pentylpyrrolidin-2-imine, monohydrochloride (20), displayed potent inhibition of hiNOS (IC50 = 0.25 microM) and selectivities of 897 (heNOS IC50/hiNOS IC50) and 13 (hnNOS IC50/hiNOS IC50). Example 20 was shown to be an efficacious inhibitor of NO production in the mouse endotoxin assay. Furthermore, 20 displayed in vivo selectivity, versus heNOS isoform, by not elevating blood pressure at multiples of the effective dose in the mouse.     

Nizatidine in therapy and prevention of non-steroidal anti-rheumatic drug-induced ulcers in rheumatic patients

269 patients with various rheumatic disorders who had been treated with non-steroidal anti-inflammatory drugs (NSAID) for at least three weeks, were enrolled in this randomised double-blind multicenter trial. Entry criteria were both the presence of an ulcer in gastric and/or duodenal mucosa (> 3 mm and < 20 mm in diameter) as well as dyspeptic symptoms. The patients had been treated with 150 mg nizatidine nocte (n = 86), 2 x 150 mg/d (n = 93) and 2 x 300 mg/d (n = 90) nizatidine. All patients continued to take their original NSAID-medication. The three nizatidine-groups had been well matched with respect to important patient characteristics. After 8 weeks of treatment more than 90% of gastric and duodenal ulcers had been healed under all three nizatidine-dosages. There was a tendency for higher healing rates in case of gastric ulcers after 4 weeks following the higher dose of nizatidine. Erosions in stomach and duodenum as well as esophagitis had been improved to a similar degree with all nizatidine doses. The same holds with respect to improvement of clinical symptoms such as epigastric pain, heartburn etc. Consumption of additional antacids was similar in all three groups. In the subsequent prophylactic trial 237/221 patients had been followed for 3/6 months. 116/107 received in addition to their continued antirheumatic medication nizatidine 150 mg nocte and 121/114 patients 2 x 150 mg nizatidine daily. The cumulative relapse rates within 6 months averaged 5.5% in the low and 1.8% in the high dose group (n.s.).(ABSTRACT TRUNCATED AT 250 WORDS)     

Renal side-effects of non-steroidal antirheumatic drugs

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAID) are increasingly used for analgesia, as antirheumatics and to inhibit platelet aggregation. Renal side effects occur mainly in patients at risk, e.g. those with pre-existing renal insufficiency, or when used together with diuretics or a second NSAID. PATIENTS: In these patients, reversible impairment of renal function, disturbance of electrolyte homeostasis, edema and hypertension are quite common. Nephrotic syndrome with or without interstitial nephritis and renal failure is a rare complication of long-term NSAID therapy. Analgesic nephropathy may result from chronic NSAID use. These three renal complications are exemplified by case reports. CONCLUSIONS: Since side effects of NSAIDs are initially reversible, careful observation of patients can prevent chronic illness. Only rarely dialysis or treatment with glucocorticoids is indicated in patients with interstitial nephritis. Given the large number of patients taking NSAIDs, however, renal side effects are rare, and usually have no long-term consequences. Nevertheless, early detection of side effects is of importance for the prevention of long-term medical complications.     

Differential effects of inhibitors of cyclooxygenase (cyclooxygenase 1 and cyclooxygenase 2) in acute inflammation

The anti-inflammatory activity of drugs more selective for cyclooxgenase isoform inhibition (cyclooxygenase 1, cyclooxygenase 2), were compared in rat carrageenin-induced pleurisy. Suppression of inflammation by cyclooxygenase 2-selective inhibitors, NS-398 (N-[-2-cyclohexyloxy]-4-nitrophenyl methanesulphonamide) and nimesulide (4-nitro-2-phenoxy-methanesulfonanilide), and by piroxicam and aspirin, more selective for cyclooxygenase 1, was measured. Piroxicam and aspirin significantly inhibited inflammatory cell influx, exudate and prostaglandin E2 formation, 6 h after carrageenin injection. Cyclooxygenase 2 inhibitors had little effect on these parameters with NS-398 alone reducing prostaglandin E2 levels, but increasing levels of leukotriene B4. In contrast, at 3 h after carrageenin injection, cyclooxygenase 2 inhibitors significantly inhibited all inflammatory parameters however suppression with piroxicam and aspirin was greater, and more pronounced than at 6 h. NS-398 and nimesulide dosing did not reduce thromboxane B2 production from platelets isolated from rats with carrageenin-induced pleurisy, demonstrating that at the doses used, cyclooxygenase 2 inhibitors did not inhibit cyclooxygenase 1, as platelets contain only this isoform. Therefore, in the rat carrageenin-induced pleurisy, drugs more selective for the inhibition of cyclooxygenase 1 attenuate inflammation over a wider time frame than cyclooxygenase 2-selective drugs, suggesting a significant role for cyclooxygenase 1 in this model. Inhibition of cyclooxygenase 2 by NS-398 however, resulted in an increase in the potent chemoattractant leukotriene B4.     

Enhanced expression of inducible cyclooxygenase with age in murine macrophages

Macrophages (Mphi) from old mice produce more PGE2 than those from young mice, contributing to the dysregulation of the immune and inflammatory responses with age. This study was conducted to determine the mechanisms of the age-associated increase in Mphi PGE2 production. PGE2 production is influenced by the availability of the substrate arachidonic acid and by activity of the enzyme cyclooxygenase (Cox). We demonstrate that when the substrate is not the limiting factor, Mphi from old mice have significantly higher LPS-stimulated Cox activity than young mice, indicating that the age-associated increase in PGE2 production is due to increased enzyme activity and not to changes in substrate level. Cox activity is determined by the enzyme level and requires hydroperoxide for activation. Of the two Cox isoforms, Cox 1 is constitutively expressed in nearly all cells; whereas Cox 2 is induced by a wide range of ligands. Analysis of accumulated and de novo synthesis of constitutive Cox 1 and inducible Cox 2 proteins showed no age-related difference in Cox 1 protein levels, but Mphi from old mice had higher accumulated and newly synthesized LPS-stimulated Cox 2 protein levels than young mice. Furthermore, Mphi from old mice had higher LPS-stimulated levels of Cox 2 mRNA compared with those from young mice. Clearly, the age-associated increase in LPS-stimulated PGE2 production is due to increased Cox activity resulting from higher Cox 2 protein and mRNA expression. These findings have significant implications for age-associated immune and inflammatory dysregulation as well as the development of preventive and therapeutic strategies against them.     

New indole derivatives as potent and selective serotonin uptake inhibitors

A series of new indole derivatives (2-28) has been prepared in the search for novel 5-HT uptake inhibitors. These compounds were obtained by the condensation of N-(chloroalkyl) naphthalenesultam derivatives with the appropriate amine in presence of a base, at reflux of DMF or THF. The yields were moderate (12-56%), except for the piperazine derivative 20 (85%). The affinity of the compounds for uptake site and 5-HT2, alpha 1, and D2 receptors was measured. Some compounds were studied in vivo by their potentiating effect of 5-HTP-induced symptomatology. The most potent and selective (uptake, 5-HT2 versus alpha 1, D2 sites) compounds contain a 3-[(4-piperidinyl)methyl]indole moiety. 5-Fluoro-3-[(4-piperidinyl)methyl]indole itself (compound 1) displayed a high affinity for the uptake site but was devoided of in vivo activity. N-Methylation of this compound abolished the affinity. In contrast N-substitution by a two-carbon chain linked to a naphthalenesultam or related heterocycle led to compounds exhibiting high affinity for the uptake site. One of them, 1-[2-[4-((5-fluoro-1H-indol-3-yl)methyl-1- piperidinyl]ethyl]-5,6-dihydro-1H,4H-1,2,5-thiadiazolo[4,3,2- ij]quinoline 2,2-dioxide (compound 24), was found as active as fluoxetine in vivo.     

Pathomorphology of kidney lesions induced by non-steroidal anti-inflammatory drugs

Pathomorphological findings on nephrobiopsies from two groups of patients with NSAID-induced kidney lesions are provided. In the first group (9 patients) the disease developed in patients of different age after short-term use of NSAID and was clinically manifested by acute renal failure with extrarenal allergic symptoms (skin, rash, eosinophilia). Morphological features were acute tubulo-interstitial nephritis without glomerular lesions. In the second group (23 patients) the disease developed after longterm use of NSAID in older patients with a compromised renal blood circulation presenting clinically with nephrotic syndrome, and morphologically by acute tubulo-interstitial nephritis with minimal change (lipoid nephrosis).     

Quantitative analysis of non-steroidal anti-inflammatory drugs by capillary zone electrophoresis

The potential utility of capillary zone electrophoresis (CZE) for the separation and quantitative determination of some non-steroidal anti-inflammatory drugs (NSAIDs) was investigated. The influence of different parameters on migration times, peak symmetry, efficiency and resolution was studied; these parameters included the nature and concentration of the anionic and cationic components of the separation buffer. A buffer consisting of 75 mM glycine adjusted to pH 9.1 with triethanolamine was found to provide a very efficient and stable electrophoretic system for the CZE analysis of NSAIDs, giving RSD values of about 0.1 and 0.5% for the within-day reproducibility of migration times and peak areas, respectively at a concentration of 25 micrograms ml-1 (n = 5). Response was linear from 2-100 micrograms ml-1 for both sulindac and tiaprofenic acid, for which the LOQ values were 2.8 and 1.9 micrograms ml-1, respectively, using UV detection at 280 nm. Accuracy for each drug was 102-103%.     

Functional stability of hippocampal slices after treatment with cyclooxygenase inhibitors

The influence of cyclooxygenase inhibitors on functional stability of hippocampal slices, determined by electrophysiological criteria of recovery after slicing and long-term maintainence of population activity, was studied. Transient (3 min) treatment of slices during slicing with indomethacin (45 microM) or aspirin (0.5 mM) allowed registration of the population responses from the second minute. The activity reached 100% after 15 min incubation and could be registrated for 3 days under conditions of overnight hypothermia. The presence of the same drugs for the entire incubation period had the same effect. The present findings suggest that slicing is a crucial point for triggering of pathological events mediated by cyclooxygenase products and that blockade of cyclooxygenase provides for the further high longterm functional stability of brain slices.     

An update on eicosanoids and inhibitors of cyclooxygenase enzyme systems

There are 3 main enzymatic pathways for synthesis of eicosanoids from arachidonic acid, however, some compounds are also formed non-enzymatically. Among the enzymatic pathways, cyclooxygenase (COX) also known as prostaglandin synthase (PGHS), generates endoperoxides (PGG/H). These are converted into prostaglandins (PGs) and thromboxanes (TXs). The second pathway involves lipooxygenase (LOX) group of enzymes to provide hydroperoxyeicosatetraenoic acid (HpETEs) which in turn can be converted into leukotrienes (LTs), hepoxilins (HXs), trioxilins and lipoxins (LXs). The third pathway involves cytochrome P-450 which catalyses the formation of a number of monohydroxy fatty acids (hydroxyeicostetraenoic acids or HETEs) dihydroxy fatty acids (dihydroxyeicostetrienoic acids or DiHETrEs) and epoxyeicosatrienoic acids (EpETrEs: formerly called EETs). This system also provides leukotoxins. The non-enzymatic pathway leads to the formation of isoprostanes by free radical catalysed peroxidation of arachidonic acid. In addition, brain cells also convert arachidonic acid into arachidonylethanolamide (anandamide) which have the ability to bind to cannabinoid receptors. Most of these eicosanoids are either biologically active or are converted into metabolites which have biological activities. Cyclooxygenase is now known to exist in two separate isoforms which are called COX-1 and COX-2. While both isoforms catalyse the same reactions, the former is a constitutive enzyme and its activity is not markedly changed once the cell is fully grown. The later isoform is however inducible and its activity is several fold increased following the exposure of body cells to a number of stimuli and its contribution in the process of inflammation is now well documented. It is now believed that eicosanoids produced by COX-1 activity are essential for the physiological (house keeping) functions while those produced by COX-2 lead to various pathological changes in body tissues. Older nonsteroidal antiinflammatory drugs like aspirin and indomethacin are non selective inhibitors of COX activity and therefore, in addition to inhibiting COX-2 activity, inhibit the formation of eicosanoids by COX-1. The later are required for normal house keeping functions such as secretion of mucus for protection of gastrointestinal mucosa, maintenance of renal function and control of haemostasis. Use of older non-selective NSAIDs has been associated with a number of gastrointestinal, renal and other side effects. Recently drugs such as nimesulide and meloxicam with selective action on COX-2 have been discovered and introduced into medicine. Evidence available so far has indicated the low incidence of side effects with these drugs. While being useful for various arthritic and other conditions, it is unlikely that these drugs will replace aspirin for the cardiovascular disease.     

Augmented endothelium-dependent contraction to angiotensin II in the SHR aorta: role of an inducible cyclooxygenase metabolite

Several features of ion-channel-forming colicins have been illuminated by recent revelations: its four-domain structure, the mechanism and thermodynamics of binding to the gating loop of outer membrane porins, the mechanism of translocation, competition for the transperiplasmic excursion facilitated by the Tol or Ton transperiplasmic proteins, and the formation of a waisted, funnel-shaped transmembrane channel of well-characterized shape.     

General practitioners' perceptions of the tolerability of antidepressant drugs: a comparison of selective serotonin reuptake inhibitors and tricyclic antidepressants

OBJECTIVE: To examine inceptions and discontinuations of antidepressants in general practice. DESIGN: An observational study analysing data from an ongoing cross sectional postal survey. Every three months a representative sample of 250 doctors recorded prescribing activity for four weeks. This provided 4000 general practitioner weeks of recording per year. SETTING: A representative panel of general practitioners in England, Wales, and Scotland. SUBJECTS: Patients who began a new course of an antidepressant or had their treatment stopped or changed by the general practitioner between 1 July 1990 and 30 June 1995. MAIN OUTCOME MEASURES: Numbers of patients prescribed a new course of antidepressant; numbers discontinuing treatment; the ratio of antidepressant discontinuations to antidepressant inceptions; reasons for discontinuation; proportion of switches to another antidepressant. RESULTS: There were 13,619 inceptions and 3934 discontinuations of selective serotonin reuptake inhibitors and tricyclic antidepressants during the study. The number of newly prescribed courses of antidepressants increased by 116%, mostly due to an increase in prescribing of serotonin reuptake inhibitors. The ratio of total discontinuations to inceptions was significantly lower for serotonin reuptake inhibitors (22%) than for tricyclic antidepressants (33%). Differences persisted when controlled for age and sex of patients and severity of depression. However, there was more switching away from selective serotonin reuptake inhibitors when they failed (72%) than from tricyclic antidepressants (58%). CONCLUSIONS: Selective serotonin reuptake inhibitors are less likely than tricyclic antidepressants to be discontinued. A prospective study is needed in general practice to assess the implications of differences in discontinuation rates and switches on clinical and economic outcomes.