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In order to further investigate the potential of rifamycins as antiinflammatory drugs, twenty-five semisynthetic rifamycins were tested at concentrations ranging from 10(-9) to 10(-5) M on in vitro human neutrophil functions such as locomotion, superoxide anion production, and degranulation, under different stimulatory conditions. They were also tested as antiproliferative agents on peripheral blood lymphocytes. The present semisynthetic derivatives are in general characterized by their carrying a hydrophilic substituent; they are rifamycin S or rifamycin SV derivatives carrying at C(3) either a carboxyalkyl side-chain or a glycosyl side-chain. Derivatives of the former group displayed inhibitory activities covering the whole range of activities tested, suggesting that the sum of these different effects could support their antiinflammatory activity in vivo. These derivatives, carrying a free carboxyl, are more water soluble than rifamycin SV at physiological pH, and may serve as antiinflammatory drugs for local administration, alternative to rifamycin SV, possibly giving higher efficacy and reduced side effects of pain and tissue swelling.

In our study we investigated the effect of rifamycin SV, rifamycin B, rifampicin and five semisynthetic derivatives on human neutrophil functions such as locomotion, superoxide production and degranulation stimulated by specific agonists. All compounds were tested at concentrations ranging from 10(-9) to 10(-5) M. Among the newly synthesized compounds the most active we found to be the derivatives carrying an acidic substituent at C3: these significantly lowered the superoxide generation induced by PMA throughout the entire concentration range, whereas rifamycin SV, rifamycin B and rifampicin were effective only at the highest concentrations. Moreover, chemotactic movement was significantly attenuated by derivative R4, rifamycin B and rifamycin SV at high doses; granule enzyme release was unaffected by all compounds.

Platelets activated with physiological agonists, such as thrombin, ADP, or collagen, released products able to modulate neutrophil functions. In particular, platelet supernatant contained an inhibitor of superoxide anion generation induced by phorbol ester and a chemotactic factor for human neutrophils. The proteolytic digestion of platelet supernatant completely abrogated chemotactic activity without interfering with the inhibitory effect, indicating the presence of different molecules involved in the modulation of different neutrophil functions. This was further confirmed by the pretreatment of platelets with aromatic benzamidine which abolished chemotactic activity, but did not affect superoxide inhibition of neutrophils. This report provides evidence for interaction of platelets and inflammatory cells, suggesting that platelets are able to induce accumulation of neutrophils and control their respiratory burst, which also has a critical role in tissue damaging in inflammation.

Two analogs of chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine were examined for their capacity to activate several functions of human neutrophils. The C-terminus methyl ester derivative of the chemotactic peptide was found to possess strong biological activity and was able to induce levels of chemotaxis, enzyme secretion, and superoxide generation comparable to those observed with the same concentrations of N-formyl-L-methionyl-L-leucyl-L-phenylalanine. The analog containing a tert-butyloxycarbonyl group at the N-terminus, as well as the C-terminal methyl ester, was completely devoid of activity towards neutrophils. From these results, it appears that the free carboxyl group is not necessary for biological function. In contrast, the substituent at the N-terminus may play a critical role in the induction of the cellular response.

Granulocyte superoxide production by different stimuli was studied in 14 patients suffering from rheumatoid arthritis, and in four cases defective O(2) generation was shown. The effect of two chemically related drugs, such as indomethacin and oxamethacin, was also evaluated, since we have previously investigated the action of antiinflammatory agents on cell locomotion. Indomethacin did not affect O(2) production, whereas oxamethacin reduced significantly superoxide generation in PMNs from all subjects tested. Moreover, the extent of the effect was dependent on the stimulant used, being larger when the activation of O(2) generating system was induced by opsonized zymosan.