Leukotriene B4 and oral cancer.

The quantitation of leukotriene B4 (LTB4) in induced squamous cell carcinoma (SCC) of the Syrian hamster cheek pouch and histologically proven human oral SCC was investigated by a combination of reverse phase-high performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA). Healthy tissue obtained from these same patients and animals treated with vehicle alone were used as controls. From both animal and human studies our results show a 10 to 30 fold increase in the levels of LTB4 found in tumour compared to control tissue. Furthermore, this dihydroxy acid was not detected in the mucosal tissue of normal subjects undergoing routine surgery. Since LTB4 is a potent inflammatory mediator and modulator of immune responses, its presence at biologically active concentrations in human squamous cell carcinoma suggests a possible role in the pathogenesis of head and neck cancer.

L-648,051, a novel cysteinyl-leukotriene antagonist is active by the inhaled route in man.

1. We have studied some of the pharmacological properties of inhaled L-648,051 which has been shown to be a selective cysteinyl-leukotriene (LT) antagonist in vitro and in vivo in various animal models. 2. The effects of three different doses (1.6, 6.0 and 12.0 mg) on the bronchoconstriction induced by inhaled LTD4 have been investigated in normal male subjects in a series of double-blind, placebo controlled studies. Furthermore, the specificity of the drug has been investigated by challenging subjects with histamine after pre-inhalation of 12.0 mg L-648,051. 3. At all doses L-648,051 partially blocked the bronchoconstriction induced by LTD4 inhalation in a dose related manner. At a dose of 12.0 mg, L-648,051 decreased the maximum fall in specific airways conductance (sGaw) (placebo, 49% vs L-648,051, 21%, P less than 0.01) and shortened the time to recovery from LTD4-induced bronchoconstriction (placebo, 41 min vs L-648,051, 19 min, P less than 0.01). 4. There was no evidence of partial agonist activity, and no effect on histamine-induced bronchospasm. Inhaled L-648,051 at all doses was well tolerated. 5. We conclude that LT antagonism is possible by the inhaled route in man. Inhaled L-648,051 is an active and selective LT-antagonist in man which is well tolerated and may prove to be a useful drug for assessing the role of leukotrienes in asthma and other lung diseases.

The biotransformation in vitro of cysteinyl leukotrienes in blood of normal and asthmatic subjects.

The metabolism of exogenous leukotriene C4 (LTC4), LTD4 and LTE4 (10(-8) M) was studied in vitro in blood of normal and asthmatic subjects for up to 2 hr by reverse-phase high performance liquid chromatography. In whole blood, incubation of LTC4 (T1/2 = 11.5 min) resulted in the formation of LTD4 and LTE4 whose biosynthesis was inhibited by serine borate (30 mM). Similar experiments performed with LTD4 (T1/2 = 5 min) produced a single metabolite (LTE4) which was inhibited by L-cysteine (10 mM). On the other hand, LTE4 represented a highly stable product in our in vitro system. The bioconversion of LTC4 or LTD4 was slower in plasma but this effect appeared more pronounced for the cysteinylglycinyl derivative. The bioconversion of LTD4 in whole blood or plasma was almost twice as rapid as LTC4. Experiments performed with asthmatic blood showed no significant difference in the survival of LTC4. These results suggest that blood may play a role in regulating the bioavailability of cysteinyl-containing LTs which could be of relevance to their excretion in man.

The metabolism "in vitro" of leukotriene B4 in blood of normal subjects and asthmatic patients.

The metabolism of exogenous leukotriene B4 (LTB4) was investigated in venous blood obtained from normal and asthmatic subjects. Using specific radioimmunoassay (RIA) and reverse-phase high performance liquid chromatography (RP-HPLC) techniques we have demonstrated that LTB4 is relatively stable during a 2 hr incubation period at 37 degrees C in our system in vitro. Nevertheless, chromatographic analysis revealed the presence of two products which had retention times identical to 20-hydroxy LTB4 (20-0H LTB4) and 20-carboxy LTB4 (20-C00H LTB4) in which the dicarboxylic derivative was the main metabolite present after 15 min incubation. The amount of LTB4 and its w-oxidation products observed after a 2 hr incubation period was 73% and 24% respectively. There was no basal release of LTB4 from blood. The appearance of these oxidative products was totally suppressed at 4 degrees C and with incubations performed with either venous plasma or Hartmann's control. No significant difference was observed in substrate metabolism between normal and asthmatic subjects. Our results demonstrate that LTB4 is slowly degraded in human whole blood through a cellular dependent process of w-oxidation which may be an important pathway for regulating the availability of this potent biologically active substance.

Lipid mediators in cystic fibrosis and chronic obstructive pulmonary disease.

In order to investigate the possible role of arachidonic acid metabolites as lipid mediators in cystic fibrosis and chronic obstructive pulmonary disease (COPD), sputum from patients with cystic fibrosis, chronic bronchitis, or bronchiectasis was analyzed for various eicosanoids using a combination of radioimmunoassay and bioassay. Leukotriene (LT) B4, cysteinyl-containing LTs, and prostaglandins (PGs) E2, F2 alpha, 6-oxo-PGF1 alpha, and thromboxane B2 were found in all sputum samples. Saliva, which can contaminate sputum, contained low concentrations of prostanoids but not LTs. Inflammatory cells, including polymorphonuclear leukocytes (PMNs) are present in sputum. Because LTB4 generated by these cells is chemotactic for PMNs, it is suggested that this dihydroxy acid contributes to the inflammation of cystic fibrosis and other diseases characterized by airway obstruction. The source of the cysteinyl-containing LTs is less clear; these LTs may constrict respiratory smooth muscle and/or stimulate mucus formation.

The detection of 5-lipoxygenase and cyclo-oxygenase products in sputum of patients with chronic bronchitis and bronchiectasis.

Leukotrienes (LTs) and prostanoids (Ps) were detected in sputum of patients with chronic bronchitis and/or bronchiectasis (CB/B) using selective superfusion bioassay and radioimmunoassay (RIA) techniques. Analysis of sputum extracts showed a 4-fold increase in the level of LTB4 compared to the cysteinyl-containing LTs (LTC4/LTD4). The measurement of cyclo-oxygenase products (COPs) indicated relatively greater amounts of the vasodilator prostaglandin E2 (PGE2) and prostacyclin (PGI2) compared to the vasoconstrictor prostaglandin F2 alpha (PGF2 alpha) and thromboxane A2 (TxA2) agents (70:30% of total COPs respectively). The presence of eicosanoids (LTs and Ps) in sputum of patients with CB/B suggest that these biologically active substances may act as mediators of bronchoconstriction and inflammation in these diseases.

Detection of sputum eicosanoids in cystic fibrosis and in normal saliva by bioassay and radioimmunoassay.

We have measured arachidonic acid (AA) metabolites, leukotrienes (LTs) and prostanoids (Ps), in sputum of patients with cystic fibrosis (CF) and in normal saliva using bioassay and radioimmunoassay (RIA). Almost three times as much LTB4 is present in CF extracts compared with slow reacting substances (SRSs). Leukotrienes were not detected in normal saliva. In CF sputum there is a three-fold increase in the level of the vasodilator prostanoid prostaglandin E2 (PGE2) and the stable metabolite of prostacyclin, 6-oxo PGF1 alpha compared with the vasoconstrictor prostaglandin F2 alpha (PGF2 alpha) and thromboxane B2 (TxB2), a hydrolysis product of thromboxane A2. Experiments with BW755c (25 micrograms ml-1, n = 3) indicated that the majority of this activity was not produced during the extraction procedure. The detection of LTs and Ps in sputum of CF patients shows that these substances are present at biologically active concentrations and may contribute to the pathophysiology of this disease.

Inactivation of prostaglandin E2 by rat isolated lung during the oestrous cycle.

The metabolism of prostaglandin E2 (PGE2) in lungs from female rats was measured during the stages of the oestrous cycle. In isolated lungs perfused through the pulmonary circulation, only 7-20% of PGE2 escaped metabolism, as measured by bioassay and radioimmunoassay. Within these limits, survival was highest at pro-oestrus compared with metoestrus and dioestrus. Uptake of PGE2 from the pulmonary vasculature, assessed by measuring the efflux of radioactivity derived from [14C]PGE2 injected into the pulmonary circulation of the isolated lung, did not show cycle-related variations. Assay of [14C]PGE2 metabolism by tissue homogenates prepared from lungs taken at different stages of the oestrous cycle showed a significant decrease in enzyme activity at pro-oestrus compared with dioestrus. It is concluded that PGE2 metabolism in isolated rat lung is affected by the oestrous cycle and that the increased PGE2 survival at pro-oestrus may be more readily explained by changes in enzyme activity than by changes in uptake of substrate.

Altered prostaglandin synthesis in isolated lungs of rats with streptozotocin-induced diabetes.

Metabolism of exogenous and endogenous arachidonic acid (AA) to several cyclo-oxygenase products has been studied by bioassay, radioimmunoassay and radiochemical assay in isolated lungs from rats made diabetic with streptozotocin and compared with that occurring in lungs from untreated rats. From exogenous AA, more PGE2, PGF2 alpha and PGD2 were formed in diabetic lungs than in control lungs, although amounts of PGI2 and TxA2 formed did not differ between the two sets of lungs. From endogenous AA, the synthesis of PGI2 in diabetic lungs was nearly half that in controls lungs. Such a deficiency in the unstimulated synthesis of a potent anti-aggregatory substance would contribute to the hyperaggregable state observed in diabetes.

Effects of the oestrous cycle on the metabolism of arachidonic acid in rat isolated lung.

1. The metabolism of exogenous arachidonic acid perfused through the pulmonary circulation was investigated in lungs taken from rats at different stages of the oestrous cycle. 2. Following perfusion with [14C]arachidonic acid there was more radioactivity associated with cyclo-oxygenase products in general at pro-oestrus than at any other stage of the cycle. 3. Production of 6-oxo-prostaglandin F1 alpha and hence of prostacyclin (PGI2) was also highest at pro-oestrus. 4. Production of thromboxane B2 was highest at pro-oestrus although it was never greater than PGI2 production at any stage. 5. Radioactivity retained in lung tissue was mostly present in phospholipid and free fatty acid fractions with the distribution at pro-oestrus being different from the other stages. 6. Following perfusion with [14C]oleic acid (which is not a substrate for cyclooxygenase), variations in the distribution of label in radioactivity in lung were also observed. However, these were not related to the stages of the oestrous cycle in the same way as those associated with arachidonic acid. 7. We conclude that both pathways of arachidonic acid metabolism in lung--oxidation via cyclo-oxygenase and incorporation into phospholipid - are affected by the progress of the oestrous cycle. 8. Altered arachidonate metabolism appeared to be associated chiefly with pro-oestrus and may be linked to those hormones involved in this stage of the oestrous cycle.