In vitro study of the molecular mechanisms responsible for the filaricide effect of macrocyclic lactones combined with doxycycline against Dirofilaria immitis.

Abstract

Macrocyclic lactones (MLs) are antihelminthic drugs that can prevent heartworm disease in dogs, caused by Dirofilaria immitis infections, during transmission season. They have been also shown to eliminate adult parasites after long-term administration, with a so called “slow-kill” effect. In addition, recent studies have established that antibiotic treatment of Dirofilaria immitis-infected dogs inhibit parasite embryogenesis, larval development, microfilarial production, and long-term survival of adults. This is due to the presence of an bacterial endosymbiont, Wolbachia, which is required for normal parasite development, fertility and survival. These bacteria, whose presence have been reported in most human filarial nematode parasites, due to their obligate nature has been a target for several drug discovery initiatives. Thus, several protocols combining of the antibiotic doxycycline with a ML have been tested. Previous studies have shown them to provide a more rapid adulticidal and microfilaricidal effect than either one of the two drugs alone. Unfortunately, female worms have been observed to have a higher tolerance to treatments compared to male worms. The actual reason behind the increase in efficacy of the doxycycline/ML combination therapy on D. immitis is still unknown. Therefore, to understand better the mechanism of action laying behind the observed synergistic effect of the two drugs when administered in combination, an in vitro study on adult worms of D. immitis, focused on the response to different treatments has been carried out. In particular, since ivermectin, one of the most common ML used for prevention HWD, has been reported to interact with membrane transporters, primarily ABC transporter, the present study was aimed at evaluating whether transporters may be involved in the synergic effect of the combination treatment. For this purpose, adult worms of D. immitis were collected and treated in vitro for either 24 or 48 hours with either doxycycline alone, ivermectin alone or the two drugs combination. Changes in modulation of seven transporter genes have been analyzed by relative quantitative real-time PCR and doxycycline residual concentration in the treatments media were measured by HPLC analysis. Results of the gene expression study following 24 hours of treatments reported a sex-dependent gene expression. In female worms, this was characterized by a down-regulation following single ivermectin administration, a dramatic up-regulation of all transporter genes following the single doxycycline treatment, with balanced effect when the two drugs were given together. In male, the expressions were different. In fact, while ivermectin alone induced a general up-regulation, the single doxycycline treatment induced a slight down-regulation in all genes. Similarly to what reported in female, when administered in combination a balanced effect was reported also in male worms. Although the sex-dependence was conserved also in the results obtained following the 48 hours of treatment, the actual gene expression trends within each sex change entirely. Indeed, while females’ gene expression of all transporters per all tested treatments returned to the control level, males’ genes expression profile resemble the trend observed in female after 24 hours of treatments. Furthermore, no definitive proof of the actual change in excretion of either doxycycline have been collected with HPLC analysis. These results correlate the synergic adulticide effect observed in previous in vivo studies with the actual change in modulation of the transporter genes, and they highlighted a sex-dependent transporter genes modulation, which could be connected to the different Wolbachia load found in the two sexes. In conclusion, despite these results, further studies are required to further elucidate whether the modulation of cellular efflux may be connected, also partially, to the adulticide effect of DOXY/ML combinations in heartworm-infected dogs.