Five µM ivermectin added one hour post infection (hpi) significantly inhibited the production of infectious EB () and chlamydial 16s rRNA accumulation (), as evaluated by harvesting infected HeLa cultures and infecting fresh HeLa monolayers followed by fluorescent microscopy to determine infectious units per mL (IFU/ml, ), or by RNA extraction of infected HeLa cultures followed by cDNA synthesis and qPCR for C. trachomatis 16s rRNA (). Treatment with 1 µM ivermectin at 1 hpi modestly decreased the size of inclusions, while inclusions in cells treated with 5 µM ivermectin had significantly reduced dimensions (). Treatment with 10 µM ivermectin completely inhibited inclusion development. Of note, 5 µM ivermectin did not reduce the number of cells in which inclusions were apparent, suggesting that the effect of ivermectin was not related to uptake of bacteria into host cells.
Ivermectin inhibits chlamydial infection of epithelial cells.
Ivermectin inhibits the development of chlamydial inclusions in epithelial cells.
To exclude the possibility that ivermectin may be inhibiting infection through an indirect cytotoxic effect on the host cells, we verified that treatment of HeLa cells with up to 10 mM ivermectin for 4 to 24 hours did not cause increased cell death () or lactate dehydrogenase (LDH) release ().
Ivermectin is not cytotoxic to epithelial cells.
For use in humans as an anti-helminthic, ivermectin is typically received orally at a dose of 150 µg/kg body weight, and peak plasma concentrations from such dosage reach around 60 nM 
. Ivermectin has also been used and well-tolerated at 500 µg/kg body weight via topical administration in veterinary applications 
, which could potentially be an attractive mode of use if it is found to be effective against intracellular bacterial pathogens which primarily infect readily accessible mucosal surfaces. Ivermectin targets glutamate-gated chloride channels in nerve and muscle cells and gamma-aminobutyric acid (GABA) related chloride channels of invertebrates 
, as well as mammalian GABA receptors 
Ivermectin has also been reported to interact with the purinergic receptor, P2X4
, which can be stimulated by low (micromolar) concentrations of ATP; and we have observed that stimulation of C. trachomatis
-infected epithelial cells with micromolar concentrations of ATP leads to chlamydial growth inhibition 
. However, addition of apyrase (2.5 U/ml) to cells immediately prior to ATP addition prevents ATP-mediated chlamydial growth inhibition (unpublished data), but does not modify the impact of ivermectin on chlamydial growth (1 or 2.5 U/ml apyrase, 5 µM ivermectin, N
3; data not shown). These results suggest that ivermectin does not inhibit infection through P2X4
We have here demonstrated that ivermectin inhibits C. trachomatis infection in epithelial cells. While the concentrations of ivermectin necessary for this inhibitory action in vitro are higher than what is achieved distal to absorption sites in current human therapy, topical application may allow therapeutic use of ivermectin against sexually-transmitted infection, or against eye infection with ocular strains of C. trachomatis. Additionally, as the target of this particular activity in human cells has yet to be identified, other avermectins or structurally modified avermectin molecules may have greater potency. As it seems that ivermectin mediates this response through interaction with a host cell target, potential efficacy against other obligate intracellular bacteria or parasites is worthy of exploration.