Diethylcarbamazine activity against Brugia malayi microfilariae is dependent on inducible nitric-oxide synthase and the cyclooxygenase pathway

Abstract

Background: Diethylcarbamazine (DEC) has been used for many years in the treatment of human lymphatic filariasis. Its mode of action is not well understood, but it is known to interact with the arachidonic acid pathway. Here we have investigated the contribution of the nitric oxide and cyclooxygenase (COX) pathways to the activity of DEC against B. malayi microfilariae in mice.

Methods: B. malayi microfilariae were injected intravenously into mice and parasitaemia was measured 24 hours later. DEC was then administered to BALB/c mice with and without pre-treatment with indomethacin or dexamethasone and the parasitaemia monitored. To investigate a role for inducible nitric oxide in DEC's activity, DEC and ivermectin were administered to microfilaraemic iNOS-/- mice and their background strain (129/SV). Western blot analysis was used to determine any effect of DEC on the production of COX and inducible nitric-oxide synthase (iNOS) proteins.

Results: DEC administered alone to BALB/c mice resulted in a rapid and profound reduction in circulating microfilariae within five minutes of treatment. Microfilarial levels began to recover after 24 hours and returned to near pre-treatment levels two weeks later, suggesting that the sequestration of microfilariae occurs independently of parasite killing. Pre-treatment of animals with dexamethasone or indomethacin reduced DEC's efficacy by almost 90% or 56%, respectively, supporting a role for the arachidonic acid and cyclooxygenase pathways in vivo. Furthermore, experiments showed that treatment with DEC results in a reduction in the amount of COX-1 protein in peritoneal exudate cells. Additionally, in iNOS-/- mice infected with B. malayi microfilariae, DEC showed no activity, whereas the efficacy of another antifilarial drug, ivermectin, was unaffected.

Conclusion: These results confirm the important role of the arachidonic acid metabolic pathway in DEC's mechanism of action in vivo and show that in addition to its effects on the 5-lipoxygenase pathway, it targets the cyclooxygenase pathway and COX-1. Moreover, we show for the first time that inducible nitric oxide is essential for the rapid sequestration of microfilariae by DEC.

Figures

Figure 1

DEC causes rapid sequestration of B. malayi microfilariae in BALB/c mice. BALB/c mice intravenously injected with B. malayi microfilariae were dosed orally with 100 mg/kg DEC and microfilaraemia monitored from 5 to 60 minutes post treatment, then at 24 hours and two weeks.

Figure 2

Indomethacin or dexamethasone pre-treatment reduces efficacy of DEC in BALB/c mice infected with B. malayi microfilariae. Indomethacin (10 mg/kg), dexamethasone (3 mg/kg) or vehicle was administered 30 minutes before oral dosing with DEC (100 mg/kg). Symbols are means of three mice for the DEC plus dexamethasone group (triangles), seven mice for the DEC plus indomethacin group (white circles) and four mice for the DEC-only group (black circles). Significantly different results from the DEC-only group are denoted by * (P < 0.017), ** (P = 0.001) or *** (P = 0.000).

Figure 3

DEC is ineffective against B. malayi microfilariae in the absence of iNOS. Efficacy of (a) a single, oral dose of DEC (100 mg/kg) or (b) a single, i.p. dose of ivermectin phosphate (1 mg/kg) in 129/SV and iNOS-/- mice infected with B. malayi microfilariae. Black symbols represent 129/SV mice, white symbols iNOS-/-. Squares indicate DEC administration, triangles ivermectin administration and circles untreated controls. Symbols represent mean results from at least three or four mice, except in the case of those treated with ivermectin (two animals) from two combined experiments which were representative of a further repeat. Significantly different microfilaraemias between 129/SV and iNOS-/- mice after DEC administration are denoted by * (P = 0.001) or ** (P = 0.000).

Figure 4

Western blot detection of COX-1 protein from peritoneal exudate cells. COX-1 protein was detected in 129/SV and iNOS-/- peritoneal exudate cells thirty minutes after i.p. injection of endotoxin-free water (control) or DEC (10 mg/kg). Proteins (10 μg) were separated on a 7.5% denaturing SDS polyacrylamide gel, transferred to PVDF membrane, incubated with rabbit anti-mouse COX-1, then goat anti-rabbit IgG-horse radish peroxidase conjugate and detected by chemiluminescence.