D. A. Hartman

Etodolac selectively inhibits human prostaglandin G/H synthase 2 (PGHS-2) versus human PGHS-1

The isozymes of prostaglandin G/H synthase (PGHS) are shown to be differentially inhibited in vitro by currently marketed nonsteroidal anti-inflammatory drugs (NSAIDs) using microsomal rhPGHS-1 and rhPGHS-2. Comparison of selectivity ratios (IC50 rhPGHS-1/IC50 rhPGHS-2) demonstrated a 10-fold selectivity of etodolac (Lodine) for rhPGHS-2, whereas the other NSAIDs evaluated demonstrated no preference or a slight preference for inhibition of rhPGHS-1. In vitro enzyme results were supported by a human whole blood assay where etodolac also demonstrated a 10-fold selectivity for inhibition of PGHS-2 mediated TxB2 production. Taken together, these data may be key to explaining the clinically observed gastrointestinal safety of etodolac versus other marketed NSAIDs.

The effects of antiinflammatory and antiallergic drugs on cytokine release after stimulation of human whole blood by lipopolysaccharide and zymosan A

IL-1β, IL-8, IL-6 and TNFα, derived from infiltrating leukocytes, are important mediators of inflammation in arthritic and allergic diseases. Heparinized human whole blood was evaluated as a model to study the effects of various classes of antiinflammatory drugs on cytokine release/biosynthesis from leukocytes. Whole blood was stimulated with zymosan A (1.5 mg/ml) or LPS (5 µg/ml) for 4h to induce cytokine release. Dexamethasone was the most potent inhibitor of TNFα, IL-1β, IL-6 and IL-8 release from LPS stimulated blood leukocytes (IC50s of 0.19, 0.11 µM, 0.16 and 0.07 respectively). In LPS stimulated blood, SKF-86002, a 5-lipoxygenase/cytooxygenasae inhibitor, and rolipram, a PDE IV inhibitor, also inhibited the release of TNFα (IC50s of 33 and 11µM, respectively), IL-1β (IC50s of 11 and 30µM, respectively), IL-6 (IC50s of 56 and > 30, respectively) and IL-8 (IC50s of 6.7 and 15, respectively), whereas isoproterenol (1 µM) inhibited significantly only TNFα release. Nonsteroidal antiinflammatory drugs, 5-lipoxygenase inhibitors and immuno-suppressive drugs were inactive at 30 µM against LPS and zymosan A stimulation of cytokine release. Using zymosan A as the stimulus, only SKF-86002 (30 µM) showed significant inhibition of IL-1β (−59%). This 4h human blood assay has the potential to identify novel inhibitors and sites of actions (e.g. transcription, post-transcriptional and secretion) of new antiinflammatory drugs.

Inhibition of endotoxin-induced hypothermia and serum TNF-α levels in CD-1 mice by various pharmacological agents

Intraperitoneal injection of lipopolysaccharide (LPS) was used to elicit a sublethal, shock-like condition in mice. LPS, 2.5 mg/kg i.p., induced hypothermia, elevated serum TNF-α levels and lethality over a 48 h period in male CD-1 mice. The 5-lipoxygenase (LO) inhibitors, WY-50,295 tromethamine and zileuton (100 mg/kg p.o), significantly inhibited hypothermia at 4, 24 and 48 h after LPS. Interestingly, whereas cyclooxygenase (CO) inhibitors (ibuprofen, etodolac, naproxen and tenidap) at 40–80 mg/kg p.o. stimulated hypothermia at 4 h, they significantly reduced the later stages of hypothermia at 24–48 h. Rolipram (PDE-IV inhibitor) and dexamethasone significantly reduced hypothermia at 4–24 h and 1–24 h, respectively. All the anti-inflammatory agents significantly reduced elevated TNF-α levels at ∼70 min post-LPS, except for ibuprofen. In conclusion, these anti-inflammatory standards indicate that LPS-induced shock involves multiple lipid mediators (PGs, LTs and possibly PAF) and secondary cytokine generation. This sublethal model of LPS-induced shock represents a sensitive model for estimating the efficacy of potential drug candidates for the treatment of endotoxic shock.

The effects of anti-inflammatory and antiallergic drugs on the release of IL-1β and TNF-α in the human whole blood assay

Heparinized human whole blood was evaluated as a model to study the effects of various classes of anti-inflammatory drugs on IL-1β and TNF-α release from leukocytes. Human whole blood was stimulated with zymosan (1.5 mg/ml) or LPS (5 μg/ml) to induce significant cytokine release. As previously reported, the 5-lipoxygenase/cyclooxygenase (5-LO/CO) inhibitor, SKF86002 (30 μM), significantly inhibited both IL-1β and TNF-α release using either stimulus. In contrast, the cyclooxygenase (CO) inhibitors (naproxen and ibuprofen) and the lipoxygenase (5-LO) inhibitors (zileuton, L-663536 and BWA4C) did not effect IL-1β or TNF-α release/biosynthesis. Isoproterenol (β-agonist), rolipram (a PDE-IV inhibitor), and IBMX (a nonselective PDE inhibitor), significantly inhibited TNF-α but not IL-1β in the LPS model while having no effect in the zymosan model. This human whole blood assay is a unique and rapid method which can be used to identify novel inhibitors of IL-1β and TNF-α release/biosynthesis.

Sirolimus (rapamycin, Rapamune) and combination therapy with cyclosporin A in the rat developing adjuvant arthritis model: correlation with blood levels and the effects of different oral formulations.

Abstract.Objective and Design: To determine whole blood levels of sirolimus, a macrolide antibiotic in the rat developing adjuvant arthritis (AA) model after dosing orally with two different vehicles, and whether combinational doses of sirolimus and cyclosporin A (CsA) produced additive or synergistic inhibitory effects in this model.¶Material: Male Lewis rats (150-180g).¶Treatment: Arthritis was induced by the injection (0.5 mg/rat) of heat-killed Mycobacterium butyricum suspended in light mineral oil. Drugs were administered orally either in fine suspension (0.5% Tween 80) or in emulsion (phosal 50 PG in 1% Tween 80) at doses of 0.1 to 5 mg/kg in a 7 day, MWF or daily regimen.¶Method: Paw volumes (ml) were measured by automated mercury plethysmograph and sirolimus concentrations in whole blood were quantitated by liquid chromatography/mass spectroscopy.¶Results: At 72 h (7 days after adjuvant) after receiving the third oral dose (4.5 mg/kg p.o.), the phosal vehicle resulted in higher sirolimus blood levels (2.5 ng/ml) than in Tween 80 (1.6 ng/ml). After the rats received the last oral dose on day 14, (7 total doses of sirolimus at 4.5 mg/kg) the sirolimus blood levels (2 h after the last dose) were about 2 times higher for the phosal dosed rats (9.8 ng/ml) compared to Tween 80 dosed rats (4.6 ng/ml). Even 24 h after the last dose, sirolimus blood levels were still elevated in the phosal dosed rats (0.8 ng/ml) relative to 0.5% Tween 80 dosed rats (0.5 ng/ml). At day 16 in the rat developing model, sirolimus, when given in phosal vehicle, produced an ED50 of 0.28 mg/kg (i.e. inhibition of uninjected paw edema) that was about 5.5 times lower than using 0.5% Tween 80 as the suspending agent (ED50 = 1.6 mg/kg). When combining sirolimus and CsA using precalculated doses for producing an additive effect in this adjuvant model, an additive inhibitory effect on uninjected paw edema was observed at equal combinational doses of 0.5 and 2 mg/kg, respectively.¶Conclusions: The phosal vehicle used in administering sirolimus increases the absorption and whole blood levels in the rat and the elevated blood levels correlated positively with the therapeutic effect in the rat developing AA model. In addition, combination therapy using sirolimus and CsA produced an additive effect in rat developing AA.

Analgesic activities of PEM-420, the active eutomer of pemedolac

PEM-420, the active isomer of pemedolac, inhibited the writhing responses induced by phenylbenzoquinone (PBQ), acetic acid, and acetylcholine in mice with ED50s of 0.80, 0.92, and 0.075 mg/kg p.o., respectively. In the rat acetic acid writhing assay, PEM-420 exhibited an ED50 value of 8.4 mg/kg p.o. In the Randall-Selitto test, PEM-420 raised the pain threshold of the yeast-injected paw (ED50-0.55 mg/kg p.o.). Like other NSAIDs, PEM-420 inhibited the PBQ-induced production of PGI2 and PGE2 in the mouse peritoneal cavity, with ED50 values of 0.5 and 1.2 mg/kg p.o., respectively. It had weak ulcerogenic liability in rats (acute UD50=99 mg/kg p.o. in fasted rats; subacute UD50=74 mg/kg/day for 4 days in fed rats). The data indicate that PEM-420 is a potent and safe peripheral analgesic.

Cellular and Topical in vivo Inflammatory Murine Models in the Evaluation of Inhibitors of Phospholipase A2

Several novel inhibitors of human synovial fluid phospholipase A2 (HSF-PLA2) were evaluated in cellular models of inflammatory mediator release (murine macrophage and human neutrophil) and topical in vivo inflammatory skin models in mice to ascertain the scope of effects which might be observed for PLA2 inhibitors. Potent inhibition of HSF-PLA2 in vitro can be observed with compounds such as scalaradial and ellagic acid, which both have IC50 values of 0.02 microM (using autoclaved [3H]-arachidonic-acid (AA)-labelled Escherichia coli membranes as substrate). Luffariellolide, a manoalide analog, and aristolochic acid are less potent (IC50 = 5 and 46 microM, respectively) in this assay. An interesting observation is that ellagic acid in cellular assays does not inhibit macrophage eicosanoid production and only 30% inhibition of PAF biosynthesis can be obtained at 50 microM in the human neutrophil. Possibly due to its irreversible mechanism of action, scalaradial retained its potent activity in both the macrophage (IC50 for PGE2 production = 0.05 microM) and neutrophil assays (IC50 for PAF biosynthesis = 1 microM). Aristolochic acid is active in these cellular assays (macrophage IC50 = 2.5 microM and neutrophil IC50 = 100 microM), but is consistently less active than either scalaradial or luffariellolide. The relative potencies of these compounds were determined in several murine in vivo inflammatory models such as oxazolone contact hypersensitivity, AA-induced ear edema and phorbol ester (PMA)-induced ear edema. In the mouse model of oxazolone contact hypersensitivity, these PLA2 inhibitors have little effect (< or = 30% inhibition at 400 micrograms/ear) with scalaradial and luffariellolide being less effective than either aristolochic or ellagic acid. PMA-induced ear edema was effectively inhibited by scalaradial, luffariellolide and aristolochic acid (ED50 = 70, 50 and 50 micrograms/ear, respectively) whereas ellagic acid was less effective (ED50 = 230 micrograms/ear). In AA-induced ear edema, these PLA2 inhibitors had minimal effects, as would be expected for compounds which inhibit PLA2. These results, especially those of ellagic acid, suggest that caution should be taken in the extrapolation of potency against a purified human extracellular type PLA2 to the scope of activities these compounds might have in the cellular and in vivo models. The consistency of scalaradial and luffariellolide may be inherent to their irreversible mechanism of action, which is a factor to be accounted for in the extrapolation of enzyme data to cellular and in vivo models.

Kidney dysfunction in the arthritic rat

Kidney function and histopathology were investigated in the adjuvant-induced arthritic rat. Rats injected with Mycobacterium butyricum exhibited symptoms of arthritis (i.e. paw edema and loss of body weight) by day 9 which worsened and included systemic manifestations of the disease on days 16 and 30. Definite signs of kidney dysfunction were observed by day 16 which included elevated urine output and plasma creatinine values and decreased creatinine clearance. By day 30, these parameters were similar to the values obtained from normal rats; however, kidney weights from arthritic rats than those from normal rats. Histopathologic abnormalities observed in the kidneys of arthritic rats on day 30 included tubular lesions consisting of focal basophilia, edema, granular deposits and basement membrane thickening. Changes in the glomerulus included granular deposits with focal glomerulopathy. These findings are the first to clearly demonstrate kidney dysfunction and histopathologic alterations associated with the early expression of the adjuvant-induced disease process in the rat. Our observations in the rat suggest that renal dysfunction in man can be mediated by the inflammatory disease process and is not solely a drug treatment-induced side effect.

Pharmacological characterization of WAY-121,520 : a potent anti-inflammatory indomethacin-based inhibitor of 5-lipoxygenase (5-LO)/phospholipase A2 (PLA2)

WAY-121,520 inhibited human synovial fluid PLA2 (HSF-PLA2) (IC50=4 μM) using arachidonic acid-labeledE. coli as substrate. Further biochemical characterization of WAY-121,520 demonstrated potent inhibition of 5-lipoxygenase (5-LO) activity in the murine macrophage (LTC4, IC50=4nM) and rat PMN (LTB4, IC50=10 nM) and an ability to antagonize LTD4 binding to isolated guinea-pig trachea (pKB=6.0).In vivo anti-inflammatory activity was noted in murine TPA-induced (ED50=91 μg/ear) and arachidonic acid-induced (66% inhibition at 400 μg/ear) ear edema and in leukotriene-dependent antigen-induced bronchoconstriction in the guinea pig (73% inhibition at 50 mg/kg, p.o.). WAY-121,520 represents a novel series of indomethacin-based inhibitors of PLA2 with anti-inflammatory activity resulting from a combination of biochemical activities (inhibition of 5-LO and PLA2 and LTD4 antagonism). This agent may provide added therapeutic efficacy over more selective inhibitors.

Pulmonary pharmacology of WAY-126299A: A dual-acting 5-lipoxygenase inhibitor and leukotriene D4 antagonist

Leukotrienes have been implicated in the pathogenesis of asthma [1]. For this reason we previously sought a compound which could simultaneously act as a 5lipoxygenase (5-LO) inhibitor and a leukotriene D 4 (LTD4) receptor antagonist. WY50295 tromethamine (WY-50295T) exhibited this unique dual mechanism of action in vitro and in vivo, including the inhibition of leukotriene production in human neutrophils, rat whole blood and guinea-pig lung fragments [2, 3]. However, WY-50295T was recently found to be inactive in human whole blood, presumably due to a high degree of plasma protein binding. Therefore, we subsequently sought to identify a compound which exhibited dual 5-LO inhibitory and LTD4 antagonist activity, with oral potency and efficacy equivalent to WY-50295T in an animal model of asthma, which was also active in human whole blood. To our knowledge there are no compounds reported in the literature which exhibit 5-LO inhibitory activity in human whole blood and leukotriene antagonist activity. The present study compares WAY-126299A ((S)-6-(2-Benzothiazolylmethoxy)-N-hydroxy-.alpha., Ndimethyl-2-naphthalene acetamide sodium salt) to WY50295T and zileuton (a reference 5-LO inhibitor in development for the treatment of asthma [4-6]).