Biochemical and functional differences between eosinophils from animal species and man.

Eosinophils were isolated from peritoneal lavages of repeated horse serum-injected guinea pigs or rhesus monkeys and from peripheral blood of normal human donors. Eicosanoid metabolism and chemotaxis by these cells were studied by in vitro techniques. Upon calcium ionophore stimulation guinea pig eosinophils released thromboxane B2 (TXB2) and leukotriene B4 (LTB4) while monkey and human cells produced LTC4, LTB4, and 5-HETE. Guinea pig cells do not synthesize sulfidopeptide LTs, because they lack the specific LTA4 glutathione S-transferase. Guinea pig eosinophils exhibit maximal chemotactic responses to LTB4, zymosan activated plasma, and human recombinant C5a, while producing only a negligible response to platelet activating factor (PAF). Monkey and human cells responded maximally to PAF, but exhibit only a weak response to LTB4. These results suggest that the guinea pig eosinophils differ from monkey and human eosinophils in both the synthetic capacity and functional chemotaxis responses to lipid mediators.

Tröger's base. An alternate synthesis and a structural analog with thromboxane A2 synthetase inhibitory activity.

The synthesis of 2,8-dimethyl-6H,12H-5,11-methanodibenzo[b,f][1,5]diazocine (Tröger's base) from p-toluidine and of two Tröger's base analogs from other anilines by reaction with hexamethylenetetramine in trifluoroacetic acid is described. 2,3,6,7-Tetrahydro-9-methyl-2,6-di-p-tolyl-1H,5H-pyrimido[5,6,1-ij] quinazoline is formed as a secondary product in the reaction of p-toluidine and hexamethylenetetramine. One of the Tröger's base analogs, 2,8-bis(3'-pyridylmethyl)-6H,12H-5,11-methanodibenzo[b,f][1,5]d iazocine (5), is an effective inhibitor of the enzyme, thromboxane A2 (TxA2) synthase, with an ED50 of 30 ng/mL in a specified in vitro assay. Three analogs having substituents on the bridging methylene group of the bicyclic nucleus of the Tröger's base structure were prepared, but all were considerably less active than the aforementioned compound in the inhibition assay. The structures of these inhibitors of TxA2 synthase fall outside the classical structure-activity relationship that has been established for this class of enzyme inhibitors.

Thromboxane A2 synthase inhibitors. 5-(3-Pyridylmethyl)benzofuran-2-carboxylic acids.

The synthesis and screening of a series of 5-(3-pyridylmethyl)benzofuran-2-carboxylic acids as selective thromboxane A2 (TxA2) synthase inhibitors is outlined. The ability of these compounds to inhibit TxA2 biosynthesis was assayed using microsomal enzyme from human platelets. Substitution of the benzofuran ring caused small changes in potency; modification of the carboxylic acid group caused modest reductions in potency, and substitution of the pyridine ring resulted in large reductions of potency. 5-(3-Pyridylmethyl)benzofuran-2-carboxylic acid sodium salt (9b, sodium furegrelate) was chosen for further evaluation as a TxA2 synthase inhibitor.

Biological activity of leukotriene B4 analogs: inhibition of guinea pig eosinophil migration in vitro by the 2,6-disubstituted pyridine analogs U-75,302 and U-75,485.

A "late phase" antigen-induced bronchoalveolar eosinophilia has been demonstrated in ovalbumin sensitized guinea pigs (1,2). This in vivo response to antigen inhalation can be inhibited by a 2,6-disubstituted pyridine analog of LTB4, U-75,302(2) (3). In the present study, the mechanism of the drug action was studied by assessing the activity of U-75,302 and a second analog, U-75,485 to displace [3H]-leukotriene B4 binding at the guinea pig eosinophil membrane, as well as their action as chemoattractants or inhibitors of the directional migration of guinea pig eosinophils in vitro. Radioligand competition experiments demonstrated that both analogs interacted strongly with the high affinity LTB4 binding sites on guinea pig eosinophil membrane. Both analogs are powerful chemoattractants for guinea pig eosinophils since they induced directional migration of guinea pig eosinophils when administered alone. In addition, when the cells were treated with either analog and their chemotaxis response was measured in response to a natural chemoattractant, both U-75,302 and U-75,485 at concentrations of 0.1 to 100 microM dose dependently inhibited the LTB4 induced chemotaxis response. The EC50s obtained for U-75,302 and U-75,485 as inhibitors of LTB4 induced guinea pig eosinophil chemotaxis were estimated to be 11.5 +/- 5.5 microM and 5.4 +/- 2.5 microM respectively. Under the same conditions, they had no significant effect upon eosinophil migration induced by zymosan activated plasma at concentrations below 100 microM. We suggest that the inhibition of antigen-induced eosinophil infiltration in guinea pig airway in vivo by U-75,302 or U-75,485 may be a result of partial antagonism or desensitization at the LTB4 receptor level of guinea pig eosinophils.