Will non-steroid approaches to the treatment of inflammation replace our need for glucocorticoids?

Glucocorticoids are widely used as anti-inflammatory agents, however, their use is limited by side effects. A long-term goal of pharmaceutical research has been the development of an agent that will mimic the anti-inflammatory actions of glucocorticoids without the undesirable side effects. This article reviews the diverse pharmacological approaches used to identify likely glucocorticoid substitutes and compares successful and unsuccessful approaches. All anti-inflammatory drugs that demonstrate clinical utility share an ability to modulate lymphocyte function. Given that glucocorticoids are also potent inhibitors of lymphocyte function, this property may be essential for clinically useful anti-inflammatory activity.

Metalloprotease inhibitors as anti-inflammatory agents: an evolving target?

The metalloproteases (MMPs) are a family of enzymes that are important for tissue remodeling. These enzymes have been implicated in a number of pathologies, including cancer, arthritis, atherosclerosis and chronic obstructive pulmonary disease. Thus, inhibitors of MMPs may have utility in the therapy of inflammatory diseases, particularly in arthritis where current therapies do not halt the progression of the disease. Many compounds have been identified as inhibitors of MMPs, and some have progressed to the clinic. However, no compound developed as an MMP inhibitor has been licensed for clinical use thus far. This review discusses this therapeutic area and compares inhibitors of MMPs with other novel therapeutic approaches in the treatment of inflammatory disease. Inhibitors of MMPs may find utility in disorders not currently targeted, but where MMPs are involved in the pathology.

Pharmacological characterisation of cannabinoid receptors inhibiting interleukin 2 release from human peripheral blood mononuclear cells.

The effects of a range of cannabinoid receptor agonists and antagonists on phytohaemagglutinin-induced secretion of interleukin-2 from human peripheral blood mononuclear cells were investigated. The nonselective cannabinoid receptor agonist WIN55212-2 ((R)-(+)-[2,3-dihydro-5-methyl-3-[4-morpholinylmethyl]pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthyl) methanone mesylate) and the selective cannabinoid CB(2) receptor agonist JWH 015 ((2-methyl-1-propyl-1H-indol-3-yl)-1-napthalenylmethanone) inhibited phytohaemagglutinin (10 microg/ml)-induced release of interleukin-2 in a concentration-dependent manner (IC(1/2max), WIN55212-2=8.8 x 10(-7) M, 95% confidence limits (C.L.)=2.2 x 10(-7)-3.5 x 10(-6) M; JWH 015=1.8 x 10(-6) M, 95% C.L.=1.2 x 10(-6)-2.9 x 10(-6) M, n=5). The nonselective cannabinoid receptor agonists CP55,940 ((-)-3-[2-hydroxy-4-(1,1-dimethyl-hepthyl)-phenyl]4-[3-hydroxypropyl]cyclo-hexan-1-ol), Delta(9)-tetrahydrocannabinol and the selective cannabinoid CB(1) receptor agonist ACEA (arachidonoyl-2-chloroethylamide) had no significant (P>0.05) inhibitory effect on phytohaemagglutinin-induced release of interleukin-2. Dexamethasone significantly (P<0.05) inhibited phytohaemagglutinin-induced release of interleukin-2 in a concentration-dependent manner (IC(1/2max)=1.3 x 10(-8) M, 95% C.L.=1.4 x 10(-9)-3.2 x 10(-8) M). The cannabinoid CB(1) receptor antagonist SR141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride) (10(-6) M) did not antagonise the inhibitory effect of WIN55212-2 whereas the cannabinoid CB(2) receptor antagonist SR144528 (N-(1,S)-endo-1,3,3-trimethyl bicyclo(2,2,1)heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) antagonised the inhibitory effect of WIN55212-2 (pA(2)=6.3+/-0.1, n=5). In addition, CP55,940 (10(-6) M) and Delta(9)-tetrahydrocannabinol (10(-6) M) also antagonised the inhibitory effects of WIN55212-2 (pA(2)=6.1+/-0.1, n=5 and pA(2)=6.9+/-0.2, n=5). In summary, WIN55,212-2 and JWH 015 inhibited interleukin-2 release from human peripheral blood mononuclear cells via the cannabinoid CB(2) receptor. In contrast, CP55,940 and Delta(9)-tetrahydrocannabinol behaved as partial agonists/antagonists in these cells.

Inhibition of interleukin-8 release in the human colonic epithelial cell line HT-29 by cannabinoids.

We have investigated the effects of cannabinoid agonists and antagonists on tumour necrosis factor-alpha (TNF-alpha)-induced secretion of interleukin-8 from the colonic epithelial cell line, HT-29. The cannabinoid receptor agonists [(-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)-phenyl]4-[3-hydroxypropyl]cyclo-hexan-1-ol] (CP55,940); Delta-9-tetrahydrocannabinol; [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl) methyl] pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthyl) methanone mesylate] (WIN55,212-2) and 1-propyl-2-methyl-3-naphthoyl-indole (JWH 015) inhibited TNF-alpha induced release of interleukin-8 in a concentration-dependent manner. The less active enantiomer of WIN55212-2, [S(-)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthyl) methanone mesylate (WIN55212-3), and the cannabinoid CB(1) receptor agonist arachidonoyl-2-chloroethylamide (ACEA) had no significant effect on TNF-alpha-induced release of interleukin-8. The cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1,4-pyrazole-3-carboxamide hydrochloride (SR141716A; 10(-6) M) antagonised the inhibitory effect of CP55,940 (pA(2)=8.3+/-0.2, n=6) but did not antagonise the inhibitory effects of WIN55212-2 and JWH 015. The cannabinoid CB(2) receptor antagonist N-(1,S)-endo1,3,3-trimethylbicyclo(2,2,1)heptan-2-yl)-5(4-chloro-3-methyl-phenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528; 10(-6) M) antagonised the inhibitory effects of CP55,940 (pA(2)=8.2+/-0.8, n=6), WIN55212-2 (pA(2)=7.1+/-0.3, n=6) and JWH 015 (pA(2)=7.6+/-0.3, n=6), respectively. Western immunoblotting of HT-29 cell lysates revealed a protein with a size that is consistent with the presence of cannabinoid CB(2) receptors. We conclude that in HT-29 cells, TNF-alpha-induced interleukin-8 release is inhibited by cannabinoids through activation of cannabinoid CB(2) receptors.

Nicotinic acetylcholine receptor subunits and receptor activity in the epithelial cell line HT29.

In the present study we have used RT-PCR to investigate nicotinic acetylcholine receptor (nAChR) subunit expression, and studied the effect of nicotine on TNFalpha-induced cytokine (IL-8) release in the epithelial cell line HT29. RNA was extracted using a commercial kit and amplified by RT-PCR. RT-PCR products were separated by electrophoresis and visualised using ethidium bromide. IL-8 release was measured by ELISA from cells activated for 6 h with TNFalpha (50 ng ml(-1)) in the absence and presence of nicotine (10(-11)-10(-6) M). HT29 cells contained mRNA for beta1, alpha4, alpha5, and alpha7 nAChR subunits. Activation of HT29 cells increased IL-8 release from undetectable amounts to 3.92 +/- 0.51 ng ml(-1) (n = 5). Nicotine significantly inhibited TNFalpha-induced IL-8 release in a concentration related manner with peak inhibition occurring at 10(-7) M (2.39 +/- 0.78 ng ml(-1), n = 5). Our data suggests that, while HT29 cells express mRNA for nAChR subunits, the only nAChR subunits that could form functional receptors and inhibit IL-8 release are alpha7.

Induction of apoptosis with tobacco smoke and related products in A549 lung epithelial cells in vitro.

BACKGROUND: This study has investigated the ability of tobacco smoke, and ingredients of tobacco smoke, to induce apoptosis in the airway epithelial cell line A549. METHOD: A549 cells were treated with 80 microg/ml Tobacco smoke condensate (TSC), 10 mM Nicotine, 10 microM paraldehyde, 10 microM hydrogen peroxide, 1 microM Taxol (Paclitaxel), 100%, 50% and 25% cigarette smoke extract (CSE). Following 4-48 h incubation apoptosis was measured morphologically following staining of cells with DAPI. TUNEL staining was also used to assess DNA damage after 24 and 48 h incubation. In addition, loss of mitochondrial cytochrome C and activation of Bax-alpha, early events in the apoptotic process, were measured after 4 h of incubation. RESULTS: Incubation of A549 cells with vehicle, Taxol, TSC, nicotine, paraldehyde, hydrogen peroxide and CSE caused a time-dependent detachment of the cells from the flask between 6 and 48 h. DAPI staining revealed that the cells remaining adhered to the flask appeared healthy whereas some of those that had detached appeared to be either apoptotic or indeterminate. Treatment with Taxol, TSC, nicotine, paraldehyde, hydrogen peroxide and CSE caused a significant increase in the number of apoptotic cells. Similarly, treatment with Taxol, TSC, nicotine, hydrogen peroxide and CSE caused a significant increase in the number of apoptotic cells among the cells that had detached from the culture plate. After 4 h of incubation, Taxol, TSC, hydrogen peroxide and CSE caused a significant reduction in mitochondrial cytochrome C and an increase in cytosolic cytochrome C. At the same time point, hydrogen peroxide and CSE significantly increased the concentration of Bax-alpha in the mitochondria. CONCLUSION: Tobacco smoke initiates apoptosis in A549 airway epithelial cells as a result of mitochondrial damage and that this results in a cell detachment and full apoptosis. This effect appears to result from factors in tobacco smoke other than nicotine and may result from free radical activity. However, additional stable factors may also be involved since the free radical content of TSC is likely to be low.