Interaction of macrocyclic lactones with a Dirofilaria immitis P-glycoprotein

• Published in: International journal for parasitology 2016-09, Vol.46 (10), p.631-640
• Main Authors: Mani, Thangadurai, Bourguinat, Catherine, Keller, Kathy, Ashraf, Shoaib, Blagburn, Byron, Prichard, Roger K.
• Format: Article
• Language: English
• Subjects: Animals; Anthelmintic resistance; Anthelmintics - metabolism; Anthelmintics - therapeutic use; Anthelmintics - toxicity; ATP-Binding Cassette, Sub-Family B, Member 1 - genetics; ATP-Binding Cassette, Sub-Family B, Member 1 - metabolism; Blotting, Western - veterinary; Cloning, Molecular; Dirofilaria immitis; Dirofilaria immitis - chemistry; Dirofilaria immitis - drug effects; Dirofilaria immitis - genetics; Dirofilariasis - prevention & control; DNA, Complementary - metabolism; Dogs; Dose-Response Relationship, Drug; Gene Expression; Gene Expression Profiling - veterinary; Ivermectin; Lactones - metabolism; Lactones - therapeutic use; Lactones - toxicity; LLC-PK1 Cells; Macrocyclic lactones; Milbemycin oxime; Moxidectin; Nematoda; Real-Time Polymerase Chain Reaction - veterinary; Reverse Transcriptase Polymerase Chain Reaction - veterinary; RNA, Helminth - genetics; RNA, Helminth - isolation & purification; Selamectin; Swine; Transport assay
• ISSN: 0020-7519; 1879-0135
• DOI: 10.1016/j.ijpara.2016.04.004

[Display omitted] •Functional Dirofilaria immitis PGP-11 was stably expressed in mammalian cells.•Avermectins inhibited transport of fluorophores by Dim-PGP-11 in a saturable manner.•Milbemycins showed low, non-saturable inhibition of Dim-PGP-11 Rhodamine transport.•Dim-PGP-11 may have two to three drug binding sites. Dirofilaria immitis, a filarial nematode, causes dirofilariasis or heartworm disease in dogs, cats and wild canids. Effective prevention of the disease is mainly by the use of the macrocyclic lactone class of drugs as heartworm preventives, and no other class of drugs is effective for preventing infection. Macrocyclic lactones have been used for prevention of heartworm infection for more than 26years. However, prevention has been compromised by the development of resistance in recent years. The mechanism of macrocyclic lactone resistance in D. immitis has yet to be established. In other parasitic nematodes, P-glycoproteins (PGPs) have been implicated in macrocyclic lactone resistance. The presence of two polymorphic loci on D. immitis P-glycoprotein-11 (Dim-pgp-11) correlated with loss of efficacy of macrocyclic lactone anthelmintics, suggesting that PGPs may be involved in macrocyclic lactone resistance in D. immitis. We have identified the full length of Dim-Pgp-11 cDNA, expressed it in mammalian cells, and studied the functional activity of the expressed protein. We have characterised its interaction with the four macrocyclic lactone preventives, ivermectin, selamectin, moxidectin and milbemycin oxime, using the transport of different fluorescent substrates. The inhibitory effect of these macrocyclic lactones on the transport of two fluorophore probes, Rhodamine 123 and Hoechst 33342, by Dim-PGP-11 has been studied. The avermectins, ivermectin and selamectin, markedly inhibited Rhodamine 123 transport in a concentration-dependent and saturable manner, whereas the milbemycins, moxidectin and milbemycin oxime, were found to have different inhibition profiles with Rhodamine 123 transport. However, both avermectins and milbemycin preventives inhibited the transport of Hoechst 33342 by Dim-PGP-11 in a concentration-dependent and apparently saturable manner, although differences existed in terms of efficiency and potency of inhibition between the two sub-classes of macrocyclic lactones. We postulate that Dim-PGP-11 may have two to three drug binding sites, as with mammalian Pgp, including the ‘R’ site for Rhodamine 123 and the ‘H’ site for Hoech