In vitro activity of amixicile against T. vaginalis from clinical isolates

Abstract

Trichomoniasis is a sexually transmitted infection in humans caused by the protozoan Trichomonas vaginalis, the leading causative agent of vaginitis in women and urethritis in men worldwide. Metronidazole is the standard treatment for trichomoniasis, with tinidazole as second-line. There are currently no FDA-approved non-nitroimidazole alternative treatments for resistant strains. This study compares the efficacy of a newly synthesized non-nitroimidazole oral drug, amixicile, to that of both metronidazole and the synthetic precursor of amixicile, nitazoxanide with in vitro sensitivity testing. One standard strain from ATCC and three patient-isolated strains of T. vaginalis were used to compare treatments under anaerobic conditions. The minimum inhibitory concentration for metronidazole, nitazoxanide, and amixicile were 12.5 μM, 100 μM, and 6.25 μM, respectively. These results suggest that amixicile may be highly active against T. vaginalis and warrants further investigation as a potential alternative to metronidazole in the treatment of trichomoniasis.

Trichomonas vaginalis is a flagellated anaerobic protozoan that infects the human urogenital tract. It causes trichomoniasis, a sexually transmitted infection (STI) presenting as vaginitis in women and urethritis in men (Centers for Disease Control and Prevention 2021). Trichomoniasis alone accounts for over 50% of curable STIs worldwide and is the most prevalent non-viral STI in the United States. 70–85% of cases are minimally symptomatic or asymptomatic. Untreated infection may last for several years and facilitate HIV transmission, gonorrhea and chlamydia co-infection, and risk of pregnancy complications including preterm delivery (Centers for Disease Control and Prevention 2021; Holmes 2008).

Oral metronidazole is the drug of choice against T. vaginalis, with tinidazole as second-line treatment. These are both derivatives of 5-nitroimidazole, the only class of antibiotics with known efficacy against and current FDA-approval to treat T. vaginalis infection (Centers for Disease Control and Prevention 2021). Metronidazole is activated by the oxidation steps of pyruvate metabolism in obligate anaerobe host cells, generating DNA-damaging toxic radicals (Weir and Le 2021; Sobel and Sobel 2015). Though metronidazole confers cure rates of over 90%, studies show that 17% of infected women experience recurrence within 3 months (Sobel and Sobel 2015; Agency 2015). Recurrence may be attributed to reinfection from an untreated sexual partner, or infection persistence due to treatment failure/resistance or noncompliance to therapy (Centers for Disease Control and Prevention 2021). Studies show a 4–10% rate of metronidazole resistance, most attributed to mutations that impair the activity of oxidation-reduction enzymes in T. vaginalis organisms needed for metronidazole activation (Centers for Disease Control and Prevention 2021; Graves et al. 2020). Identifying efficacious alternative therapies for T. vaginalis is urgently needed to treat resistant strains and prevent comorbidities associated with untreated trichomoniasis. The purpose of this study is to test the efficacy of amixicile, a systemic nitazoxanide-derived compound, against T. vaginalis in vitro.

We compared the efficacies of amixicile, metronidazole, and nitazoxamide against T. vaginalis by determining minimum inhibitory concentrations (MIC) in vitro. We used a standard T. vaginalis isolate from the American Type Culture Collection (ATCC) and three isolates cultured from de-identified vaginal specimens collected from patients at the Thomas Jefferson Health District Sexual Health Clinic whose wet mount microscopy showed at least one motile trichomonad. We cultured these isolates in LYI Entamoeba media solution containing human bovine serum, vitamins, concentrated penicillin/streptomycin, and tetracycline.

We prepared 0.1 M stock solutions of each drug candidate (amixicile, metronidazole, and nitazoxanide) in 10% dimethylsulfoxide (DMSO) and performed twofold serial dilutions with LYI media to achieve 6 drug concentrations ranging from 200 to 1.56 μM. We prepared 3 wells per dilution and included two controls (six total wells) containing only LYI solution and 10% DMSO. 100 μL of strain culture was added to each multi-well plate, giving approximately 1 × 10⁵ trichomonads per well. Each drug was tested against 9 samples of T. vaginalis – 3 samples of the ATCC strain and 2 samples of each of the 3 patient strains. These tests were conducted across 3 separate replicate studies. The cultures were incubated at 37°C with 5% CO₂ and samples were passaged every 48–72 hours. The plates were observed at 24 and 48 hours of incubation to determine the approximate percentage of living trichomonads and their motility. The most diluted concentration at which the majority of trichomonads were dead or immotile was recorded as the drug’s MIC.

Figure 1 depicts the susceptibilities of T. vaginalis to amixicile, metronidazole, and nitazoxanide in this study. As shown, the MICs of metronidazole are 12.5 μM against all 9 T. vaginalis samples, displaying consistency. The MICs of amixicile range from 1.56 to 12.5 μM, displaying lesser consistency, but with a lower median MIC (6.25 μM). Finally, the MICs of nitazoxanide are neither consistent nor low, ranging from 25 to 100 μM. Median MIC for metronidazole and amixicile are both significantly lower than that of nitazoxanide (p < 0.001), but the difference between metronidazole and amixicile is not statistically significant. While amixicile’s MICs varied, MICs as low as 1.56 μM suggest that T. vaginalis may be more susceptible to low concentrations of amixicile compared to metronidazole in vitro, demonstrating amixicile’s potential efficacy as an alternative therapy against T. vaginalis.

Figure 1.

Susceptibility of T. vaginalis to amixicile, metronidazole, and nitazoxanide

Amixicile is a novel antimicrobial derived from an FDA-approved drug, nitazoxanide, that is currently used in the treatment of Cryptosporidium and Giardia (Kennedy et al. 2016). Amixicile has shown efficacy in preclinical animal model studies against periodontal anaerobes, Clostridiodes difficile, and Helicobacter pylori (Gui et al. 2021; Kennedy et al. 2016). It has demonstrated high potency as well as low toxicity in animals, and has been shown to be non-inhibitory to various healthy bacteria and to support rapid repopulation of healthy mibrobiomes (Warren et al. 2012; Hoffman et al. 2014). Amixicile targets vitamin B1, a cofactor of the pyruvate ferredoxin oxidoreductase (PFOR) enzyme, a key player in the energy metabolism of anaerobic bacteria (Kennedy et al. 2016; Hoffman 2020). Because of the specificity of its target, amixicile may spare vaginal commensals that do not utilize the PFOR enzyme, reducing the risk of opportunistic infections such as Candida vaginitis from emerging post-treatment. Furthermore, amixicile, unlike nitazoxanide, functions as a systemic drug, making it likely to target organisms in the genitourinary tract. In pharmacokinetic studies in rats and mice, oral administration of amixicile yielded high plasma levels and undetectable level in feces in mice. Furthermore, plasma protein binding did not affect systemic bioavailability or therapeutic efficacy of amixicile against H. pylori, in a mouse model of infection (Hoffman et al. 2014). Recently, amixicile has also been shown to be effective as an alternative oral medication to treat periodontal disease in a non-human primate model, Macaca mulatta (Gui et al. 2021). There are currently no clinical reports of resistance to nitazoxanide, and research labs have been universally unsuccessful in developing mutant strains with resistance to nitazoxanide or amixicile (Hoffman 2020; Kennedy et al. 2016). Development of resistance to this antibiotic is unlikely, as mutations to the drug’s target would potentially lead to loss of function of the PFOR enzyme and would therefore be lethal for the pathogen (Hoffman 2020). Moreover, targeting a vitamin cofactor of an enzyme alone, makes mutation-based drug resistance unlikely (Kennedy et al. 2016).

The main limitation of this study is the small sample size precluding statistically significant difference between MICs of metronidazole and amixicile. However, MIC testing revealed T. vaginalis to be consistently more susceptible to the drug candidate, amixicile, at low concentrations as compared to both the current drug of choice, metronidazole, and amixicile’s synthetic precursor, nitazoxanide in vitro. Though this study did not test amixicile’s efficacy against metronidazole-resistant strains, these results raise the possibility that amixicile may be efficacious against T. vaginalis, a pathogen whose treatment options are currently limited to a single drug class, nitroimidazoles. This finding warrants larger studies to further investigate the activity of amixicile against antibiotic resistant strains as monotherapy or in combination with other agents, efficacy against T. vaginalis infection in an in vivo model, ability to preserve healthy genital microbiota, and potential to evade mutation-based resistance, all of which may make it a preferred drug candidate to the current standard therapy, metronidazole..