Preclinical pharmacology of robenacoxib: a novel selective inhibitor of cyclooxygenase-2.

This manuscript reports the results of preclinical studies in the rat with robenacoxib, a novel selective cyclooxygenase (COX)-2 inhibitor. Robenacoxib selectively inhibited COX-2 in vitro as evidenced from COX-1:COX-2 IC50 ratios of 27:1 in purified enzyme preparations and >967:1 in isolated cell assays. Binding to COX-1 was rapid and readily reversible (dissociation t(1/2) << 1 min), whilst COX-2 binding was slowly reversible (t(1/2) = 25 min). In vivo, robenacoxib inhibited PGE2 production (an index of COX-2 inhibition) in lipopolysaccharide (LPS)-stimulated air pouches (ID50 0.3 mg/kg) and for at least 24 h in zymosan-induced inflammatory exudate (at 2 mg/kg). Robenacoxib was COX-1 sparing, as it inhibited serum TxB2 synthesis ex vivo (an index of COX-1 inhibition) only at very high doses (100 mg/kg but not at 2-30 mg/kg). Robenacoxib inhibited carrageenan-induced paw oedema (ID50 0.40-0.48 mg/kg), LPS-induced fever (ID50 1.1 mg/kg) and Randall-Selitto pain (10 mg/kg). Robenacoxib was highly bound to plasma protein (99.9% at 50 ng/mL in vitro). After intravenous dosing, clearance was 2.4 mL/min/kg and volume of distribution at steady-state was 306 mL/kg. Robenacoxib was preferentially distributed into inflammatory exudate; the AUC for exudate was 2.9 times higher than for blood and the MRT in exudate (15.9 h) was three times longer than in blood (5.3 h). Robenacoxib produced significantly less gastric ulceration and intestinal permeability as compared with the reference nonsteroidal anti-inflammatory drug (NSAID), diclofenac, and did not inhibit PGE2 or 6-keto PGF(1alpha) concentrations in the stomach and ileum at 30 mg/kg. Robenacoxib also had no relevant effects on kidney function at 30 mg/kg. In summary, results of preclinical studies in rats studies suggest that robenacoxib has an attractive pharmacological profile for potential use in the intended target species, cats and dogs.

Characterization of CGS 8515 as a selective 5-lipoxygenase inhibitor using in vitro and in vivo models.

CGS 8515 inhibited 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 synthesis in guinea pig leukocytes (IC50 = 0.1 microM). The compound did not appreciably affect cyclooxygenase (sheep seminal vesicles), 12-lipoxygenase (human platelets), 15-lipoxygenase (human leukocytes) and thromboxane synthetase (human platelets) at concentrations up to 100 microM. CGS 8515 inhibited A23187-induced formation of leukotriene products in whole blood (IC50 values of 0.8 and 4 microM, respectively, for human and rat) and in isolated rat lung (IC50 less than 1 microM) in vitro. The selectivity of the compound as a 5-lipoxygenase inhibitor was confirmed in rat whole blood by the 20-70-fold separation of inhibitory effects on the formation of leukotriene from prostaglandin products. Ex vivo and in vivo studies with rats showed that CGS 8515, at an oral dose of 2-50 mg/kg, significantly inhibited A23187-induced production of leukotrienes in whole blood and in the lung. The effect persisted for at least 6 h in the ex vivo whole blood model. CGS 8515, at oral doses as low as 5 mg/kg, significantly suppressed exudate volume and leukocyte migration in the carrageenan-induced pleurisy and sponge models in the rat. Inhibitory effects of the compound on inflammatory responses and leukotriene production in leukocytes and target organs are important parameters suggestive of its therapeutic potential in asthma, psoriasis and inflammatory conditions.

Derivatives of 2-[[N-(Aminocarbonyl)-N-hydroxyamino]methyl]-1,4- benzodioxan as orally active 5-lipoxygenase inhibitors.

N-Hydroxyureas based on the 1,4-benzodioxan template were prepared from appropriately substituted 1,4-benzodioxan-2-methanols as the key intermediates and evaluated in the in vitro guinea pig polymorphonuclear leukocyte 5-lipoxygenase (5-LO) assay for their 5-LO inhibitory activity. Placement of a 7-phenoxy or 7-p-fluorophenoxy substituent resulted in a dramatic increase in in vitro potency. Selected compounds were subsequently assayed in an ex vivo dog model of LTB4 synthesis at a dose of 1.0 mg/kg. The 7-phenoxy derivatives 16 and 17 showed modest duration of action (DA) in this dog model. The 6-regioisomers 21 and 22 were less potent. Replacement of the 7-phenoxy group of 16 with the p-fluorophenoxy moiety enhanced the DA dramatically. Compound 18 (CGS 25667), which had an IC50 value of 100 nM in the in vitro guinea pig 5-LO assay, had a DA of 8.5 h (zileuton, DA = 8.5 h) at the oral dose of 1.0 mg/kg. Optical antipodes (24, 26) of 18 were independently synthesized in high (> 95%) enantiomeric purity from commercially available optically active glycidyl tosylates and evaluated. In the in vitro assay, the 2S-(-)-enantiomer (24, CGS 25997, IC50 = 85 nM) was found to be twice as active as the 2R-(+)-counterpart (26, CGS 25998, IC50 = 180 nM). In the ex vivo experiment, 24, which dose dependently inhibited plasma 5-LO activity, was shown to be significantly longer acting than 26, with a DA of 8.4 h when dosed orally at 1.0 mg/kg.

Substituted chromenes as potent, orally active 5-lipoxygenase inhibitors.

A series of chromene derivatives was synthesized and evaluated for their in vitro and ex vivo 5-lipoxygenase (5-LO) inhibitory activity. These compounds were prepared by condensation of appropriate salicyl aldehydes with alpha, beta-unsaturated carbonyl compounds, followed by transformation to the corresponding hydroxamic acids or N-hydroxyureas. Placement of phenoxy or p-fluorophenoxy substituents at the 6 position of the chromene ring led to a dramatic increase in the in vitro potency as demonstrated by the guinea pig PMN 5-LO assay. Chromene hydroxamic acids, in general, behaved poorly in the ex vivo dog model. On the other hand, replacement of the hydroxamic acid function with N-hydroxyurea yielded potent and long-lasting 5-LO inhibitors in the dog model. In most cases, the oral efficacy of the chromene N-hydroxyureas correlated very well with their in vitro activity. Compounds 43 (CGS 23885) and 55 (CGS 24891) are among the most potent inhibitors prepared, showing IC50 values of 48 and 51 nM, respectively. The values for the duration of action (DA) for compounds 43 and 55 are 21 and 20 h, respectively, following intravenous (i.v.) administration of 1.0 mg/kg. In the oral (po) experiments, 43 and 55 have DA's of 14 and 15 h, respectively, at a 1.0 mg/kg dose. In both iv and po experiments, 43 and 55 showed sustained maximal inhibition (> 95%) at earlier time points. The oral ED50 values of 43 and 55 in the ex vivo dog model are 0.23 and 0.23 mg/kg, respectively, at 6.0 h, and 2.37 and 1.63 mg/kg, respectively, at 24 h. Compound 43, which inhibits sheep seminal vesicle cyclooxygenase (CO) with an IC50 value of 36 microM, was shown to be a selective 5-lipoxygenase inhibitor in the ex vivo study. These compounds compare favorably with zileuton (A-64077) in all the parameters examined.

The effects of diclofenac sodium on arachidonic acid metabolism.

Evidence has been presented that inhibition by diclofenac sodium of the production of leukotrienes by cells participating in the inflammatory process is due to a decreased availability of intracellular arachidonic acid which results from enhanced uptake of the substrate into triglyceride pools. The diminished leukotriene production does not result from direct inhibition of 5-lipoxygenase or phospholipase A2. Reduced availability of arachidonic acid would also limit production of prostaglandins, although in this case manifestation is obscured by the potent inhibitory effect of diclofenac sodium on cyclooxygenase. This recently discovered action of diclofenac sodium, which has been characterized by studies on isolated leukocytes, appears to be operative in vivo. Consistent with this mechanism, and not explainable by classical cyclooxygenase inhibition, diclofenac sodium inhibited leukotriene production in whole blood from drug-treated animals and also suppressed leukocyte infiltration of subcutaneously implanted sponges. The latter effect contrasts with increased infiltration frequently obtained with other NSAIDs and thought to reflect enhanced production of leukotrienes. In conclusion, the findings suggest that patient acceptance or preference for diclofenac sodium is not merely subjective but has a logical scientific basis.

CGS 15435A, a thromboxane synthetase inhibitor with an extended duration of action: a comparison with dazoxiben.

CGS 15435A, a novel thromboxane (Tx) synthetase inhibitor (5-chloro-1-methyl-2-(3-pyridyl)-3-indolhexanoic acid HCl), had a selectivity for Tx synthetase 100,000-fold greater than that for cyclooxygenase, PGI2 synthetase and lipoxygenase enzymes. In conscious beagles, 1 h following a single 3 mg/kg p.o. dose, serum TxB2 was inhibited 95% by CGS 15435 and 82% by dazoxiben (DAZ). Unlike the short acting Tx synthetase inhibitor DAZ, CGS 15435A significantly inhibited TxB2 formation 4, 6, 12 and 24 h after dosing. Serum levels of 6-keto PGF1 alpha and PGE2 were significantly increased following the administration of either drug. CGS 15435A and DAZ were further examined in a model with known Tx involvement. Thrombotic sudden death, produced in anesthetized rabbits by injection of 0.75 mg/kg arachidonic acid (AA) i.v. resulted in a 45% fall in the platelet count and 0% survival. Pretreatment with DAZ (8.6 mumol/kg i.v.) at 0.25 or 2 h pre-AA resulted in 3 and 42% thrombocytopenia and 100 and 0% survival respectively. CGS 15435A (8.6 mumol/kg i.v.) prevented the increases in plasma TxB2 levels, thrombocytopenia and sudden death with pretreatment at 0.25 h (0% thrombocytopenia and 100% survival) or 24 h (11% thrombocytopenia and 83% survival) before AA. These data indicate that CGS 15435A is a potent and selective Tx synthetase inhibitor with a long duration of action, and suggest that the compound could be useful in chronic, non-symptomatic indications of Tx involvement.

Inhibition of LTB4 biosynthesis in situ by CGS 23885, a potent 5-lipoxygenase inhibitor, correlates with its pleural fluid concentrations in an experimentally induced rat pleurisy model.

An intrapleural injection of carrageenan in rats induced LTB4 and LTC4/D4/E4 biosynthesis, exudate formation, and cellular influx in the pleural cavity. An injection of calcium ionophore (A23187, 100 nmol) 16-18 h after carrageenan injection augmented leukotriene biosynthesis and exudate formation, but not cellular influx. The carrageenan-induced pleurisy model modifid by A23187 administration was used to study the oral effect of CGS 23885 (N-hydroxy-N-[(6-phenoxy-2H-1-benzopyran-3-yl)-methyl]urea), a potent 5-lipoxygenase (5-LO) inhibitor, on inflammatory parameters. CGS 23885 dose-dependently (1 to 30 mg/kg) inhibited the enhanced LTB4 and LTC4/D4/E4 (1 to 10 mg/kg) biosynthesis, but had no effect on enhanced exudate formation. An inhibitory effect of CGS 23885 of small magnitude on cellular influx due to carrageenan stimulation was seen at 30 mg/kg. The concentrations of CGS 23885 in the pleural fluid were dose-related, and a positive correlation (r2=0.989) between pleural fluid concentration of LTB4 and CGS 23885 was observed. The results confirm that CGS 23885 is a specific, orally active 5-LO inhibitor which can achieve concentrations in the pleural cavity sufficient to inhibit production of LTB4 and LTC4/D4/E4 in an ongoing inflammatory response.

Antimicrobial effectiveness of Locacorten-Vioform cream in Secondary infections of common dermatoses.

In a double-blind study involving 430 patients, Locacorten (0.02%)-Vioform (3%) cream was found to be highly effective in the treatment of dermatological conditions complicated by secondary bacterial involvement. Themicrobiological conversion and clinical improvement in patients treated with Locacorten-Vioform combinations were markedly greater than those achieved in the groups medicated with (1) Vioform 3%, (2) Locacorten 0.02%, and (3) placebo cream. Staphylococcus aureus was the most prevalent organism isolated from the skin lesions. All strains were sensitive to Vioform, as compared to 62% found to be resistant to other antimicrobials.

Antimicrobial susceptibility, growth kinetic and pathogenicity of thymidine-requiring Streptococcus species.

18 strains of thymidine-requiring streptococcal mutants (thy-) were isolated from urines of patients with urinary tract infection treated with trimethoprim-sulfameth-oxazole (TMP-SMZ) or trimethoprim-sulfadiazine (TMP-SDZ). All thy- mutants were catalase- and beta-lactamase-negative, grew in the presence of bile and esculin, and required from 0.3 to greater than 1280 micrograms/ml of thymidine for normal growth. The antibiotic susceptibility of wild-type and thy- mutants to 21 antimicrobial agents tested were comparable except to trimethoprim (TMP), TMP-SMZ and TMP-SDZ. Ampicillin, penicillin G, erythromycin and rifampin were among the most active compounds tested. Growth kinetic studies with a Streptococcus faecalis thy- mutant in a synthetic basal medium without thymidine resulted in a decrease of 2-3 logarithmic units in viable cells after 24 h of incubation. The addition of thymidine to this thymidine-deprived culture prevented the thymineless death of the cells. In vivo, this thy- mutant was less virulent than the wild-type strain in producing kidney infection in mice.

Synergistic activity of cefsulodin combined with cefoxitin and sulbactam against Bacteroides species.

The in-vitro activity of cefsulodin combined with sulbactam, cefoxitin or cefotaxime was investigated against 32 strains of beta-lactamase-producing Bacteroides species. Synergy of cefsulodin-sulbactam or cefsulodin-cefoxitin could be demonstrated against 30 of 32 and 32 of 32 strains tested at the concentrations readily achievable in serum. In the presence of 1 mg/l of sulbactam or cefoxitin, more than 90% of the Bacteroides isolated were inhibited by 32 mg/l of cefsulodin. The inhibitory activity of cefsulodin-sulbactam or cefsulodin-cefoxitin combinations was bactericidal against Bact. fragilis and Bact. vulgatus. In contrast, no synergistic inhibitory or bactericidal activities can be observed by the cefsoludin-cefotaxime combination. Both sulbactam and cefoxitin were potent inhibitors of beta-lactamases produced by Bact. fragilis and Bact. melaninogenicus suggesting that this inhibitory activity might be one of the factors contributing to the synergistic combinations.

Therapeutic efficacy and pharmacokinetic properties of rifampicin in a Bacteroides fragilis intra-abdominal abscess.

The efficacy of rifampicin in treating a Bacteroides fragilis infection was investigated and compared to clindamycin and metronidazole in an experimental model of intra-abdominal abscess in mice. Rifampicin, when given subcutaneously, showed activity superior to that of clindamycin in reducing the incidence of abscess formation as well as the number of Bacteroides organisms recovered from the abscess, and rifampicin was comparable in efficacy to metronidazole when given orally at the same dose level. The comparative pharmacokinetic properties of rifampicin and clindamycin demonstrated that the peak serum and abscess levels reached with rifampicin were significantly higher than those of clindamycin. The half-life of rifampicin in serum and in the abscess was longer than that of clindamycin.

Leukotriene biosynthesis inhibition by aryl and aroyl substituted naphthoquinones.

Naphthoquinones are known to inhibit 5-lipoxygenase. In particular, CGS 8515 (methyl 2-[(3,4-dihydro-3,4-dioxo-1-naphthalenyl)amino]benzoate) has been studied in detail. However this potent and selective 5-lipoxygenase inhibitor has a short duration of action and poor bioavailability. To improve the duration of action we have prepared a series of carbon substituted naphthoquinones. Several members of this series have demonstrated potent in vitro inhibition of 5-LO (IC50's less than 10(-6) M) and at doses of 1 mg/kg iv completely inhibited LTB4 production measured in ex vivo A23187-stimulated blood from dogs. The duration of action measured by the ex vivo assay was improved up to threefold over CGS 8515.

Efficacy of rifampicin in experimental Bacteroides fragilis and Pseudomonas aeruginosa mixed infections.

Experimental intraabdominal abscesses were produced in mice by intraperitoneal injection of Bacteroides fragilis and Pseudomonas aeruginosa. The therapeutic efficacy of rifampicin and cefsulodin alone, and in combination was investigated in this in-vivo experimental mixed intraabdominal abscess model. Treatment with rifampicin at 10, and 25 mg/kg or cefsulodin at 50, and 100 mg/kg singly or in combinations prevented mortality as compared to 68% mortality rate occurring in the untreated mice. Rifampicin, at 25 mg/kg dose, was very effective in preventing abscess formation and produced bacterial eradication. It prevented abscess formation in 80% of the mice and eradicated both Bacteroides and Pseudomonas in 100% and 75% of the abscesses of the mice. Cefsulodin failed to reduce the incidence of abscess formation, and to eradicate Bact. fragilis from the abscesses, although it significantly decreased Ps. aeruginosa in the abscesses. The combination of rifampicin at 10 mg/kg and cefsulodin at 100 mg/kg was more effective than either of the antibiotics alone and was as effective as rifampicin alone at 25 mg/kg levels. This combination was bactericidal against both organisms in the infected mice.

Characterization of CGS 8515 as a selective 5-lipoxygenase (5-LO) inhibitor.

1. CGS 8515 selectively inhibited 5-LO (IC50 = 0.1 microM) with negligible effect on CO, 12-LO, 15-LO and TxS at concentrations up to 100 microM. 2. CGS 8515 selectively inhibited A23187-induced formation of 5-LO products in rat and human whole blood with a 20-70 fold separation of effects over the formation of CO products. 3. Ex vivo and in vivo studies with rats showed that CGS 8515, at an oral dose of 2-50 mg/kg, significantly inhibited A23187-induced formation of LTs in whole blood and in the lung. The effect persisted for at least 6 h in the ex vivo blood model. 4. CGS 8515, at oral doses as low as 5 mg/kg, significantly suppressed exudate volume and leukocyte migration in the carrageenan-induced pleurisy and sponge models in the rat.

Hypolipidaemic activity of a novel acyclic ansamycin derivative. Synthesis and pharmacokinetic and pharmacodynamic studies in rodents.

Certain classes of the antibacterial agent rifamycin SV have recently been shown to possess marked hypolipidaemic activity. An acyclic oxazolylrifamycin has been prepared and its hypolipidaemic properties evaluated; it was found to be significantly more potent when administered orally to Wistar and Sprague-Dawley rats than other previously described rifamycin hypolipidaemics. The plasma decay rate of a bolus of intravenously administered 125I-LDL (low density lipoprotein) was significantly greater in treated rats than in rats receiving vehicle alone, compatible with a drug-induced increase in LDL catabolism. A bolus of radiolabelled drug was rapidly removed from the circulation by the liver, the presumed target organ. Compound 3 constitutes the first example of a new class of acyclic hypolipidaemic ansamycins.