Polyoxins and the related nikkomycin compounds showed inhibition of chitin synthase [2
]. These proved to be useful tools to elucidate cellular mechanisms in fungal cell wall formation, maintenance, and susceptibility. Hector et al. observed that some of these compounds suppressed infections by Coccidioides posadasii
, particularly NikZ, which led to promising in vivo studies, including an INDA (Investigational New Drug Application) and human Phase I trials, now moving towards Phase II. Nikkomycin Z is fungicidal against endemic dimorphic fungi [3
In one of the more extensive reports of NikZ activity against a wide range of fungal species, Li and Rinaldi [26
] examined NikZ in vitro antifungal properties alone and in combination with fluconazole or itraconazole, testing against 110 isolates of some 24 species (checkerboard method). Selected results are summarized here in Table 1
. NikZ in combination showed synergistic activity against several important fungi and additive activity against others. The authors note a “marked synergism” between NikZ and ITRAconazole against A. fumigatus
and A. flavus
] (p. 1401).
This review will focus first on the mechanism of action and origins of NikZ, then review NikZ impact on Coccidioides since this is well studied, and then move to other endemic dimorphic fungi, and finally, opportunistic Candida and Aspergillus.
4.1. NikZ Mechanism of Action: Cell Wall
In medically important fungi, cell wall processes have been studied very closely, such as the different cellular responses to maintain cell wall integrity. Fungal cell walls include chitin, beta glucans, and cellulose as structural components surrounding the bilayer [27
]. The amounts of these structural components differ significantly between different species.
In a typical cross section, a chitin component is illustrated as a first shell just surrounding the lipid bilayer membrane, surrounded in turn by a second enveloping layer composed of various glucan components. Chitin synthase inhibitors such as NikZ are fatally disruptive to some fungi, particularly the spherule-endospore phase of C. immitis
] and presumably similar phases in H. capsulatum
spp., endemic dimorphic fungi susceptible to NikZ.
Zhang et al. [31
] detail the fungal mechanisms involved in chitin synthesis, including microvesicles, the chitosomes, for intracellular transport. Fungi contain various chitin synthases, which activate differently during the fungal life cycle, and this timing has an impact on drug design. Studies of chitin in yeast cell walls have identified three chitin synthases, CSI, CSII, and CSIII, considered the most specific and potent of chitin synthase inhibitors [32
]. Cabib et al. studied chitin synthase III activity using NikZ, a specific inhibitor of CSIII in a chs2
mutant to study morphological changes and viability in Saccharomyces cerevisiae
]. A 1983 study of Polyoxin D (related to NikZ) as an inhibitor of chitin synthesis in C. immitis
concluded that chitin is important to maintain the structural integrity of the spherule phase [2
Echinocandin drugs block synthesis or incorporation of beta glucans into fungal cell walls. A common fungal response to cell wall damage is production of excess chitin, typically reducing susceptibility to echinocandin drugs. Combining an echinocandin with a chitin inhibitor such as NikZ increases potency against C. albicans
and A. fumigatus
4.2. NikZ In Vivo—Early Studies in Coccidioides
Early observations that NikZ is fungicidal against dimorphic fungi prompted particular attention targeting Coccidioides
]. Other pathogens are discussed below.
Hector, working for a Bayer subsidiary, filed a patent in 1987 for nikkomycin X and Z to treat mammals infected with fungi with >10% by weight chitin in the cell wall in the parasitic phase [3
]. In vitro, immature spherules exposed to 0.1 or 0.4 mM NikZ or NikZ (50, 200 ng/mL) stopped fungal growth within 8 h. The low dose of 20 ng/mL stopped endosporulation but “a small percentage” of cells appeared only weakened.
The in vivo studies concluded that NikX and NikZ “appear to be potentially fungicidal.”
Challenging mice with 500 C. immitis
arthrospores intranasally and treating them orally with NikZ or NikX, 100 mg/kg TID (300 mpkd, mg/kg/day) for 9 days gave “negligible” fungal burden, “sterilized,” in the lung, liver, and spleen, as shown in Table 2
A. Increasing the challenge inoculation by a factor of 10 and lowering the dose to 75 mg/kg BID (150 mpkd) or TID (225 mpkd) showed significant efficacy. One infected group was not started on NikX therapy until after the first death, giving a late-intervention cohort of 8 from which 4 survived. Table 2
B. A 10K CFU challenge terminated at 4 days showed no fungi in the lungs of mice treated with NikZ at 150 mg/kg/day (75 BID). Table 2
In 1990, Hector et al. extended the evaluation of NikZ and NikX in murine models against coccidioidomycosis, histoplasmosis, and blastomycosis [34
]. Table 3
details coccidioidomycosis results. Other diseases are discussed later. Oral NikZ at 20 and 50 mg/kg BID for 10 days protected 100% of the animals against pulmonary coccidioidomycosis. NikX was significantly less effective in this model. Oral NikZ at 100 mg/kg/day (divided BID) reduced lung CFU by log10
5.98. Half of this dose QD reduced burden by log10
2.58, 80% of the QD dose (40 mg/kg/day) reduced burden by an additional log10
2.51 (5.09 less than untreated). It was found that “divided doses are more effective.”
A murine model of meningocerebral coccidioidomycosis showed that oral NikZ at 50 mg/kg BID (100 mpkd) for 21 days reached the CNS, with 60% survival to end of test at day 65 [34
]. Untreated animals survived ≤ 9 days. Systemic blastomycosis and histoplasmosis are discussed below.
Shubitz et al. [35
] reported the murine model results of NikZ dose levels and timing against C. posadasii
, modifying Hector et al.’s paradigm of fatal dose challenge by using instead a sub-acute lethal dose (500 arthrospores) and delaying treatment until the fungal burden was increased 10–100 fold, 120 h after intra-nasal infection. Drug administration was subcutaneous (10, 20, 40, and 80 mg/kg BID), for seven days. By the seventh day of treatment, significant fungal burden was prevented (5 of 6 subjects had no detectable CFUs).
When evaluating therapy duration and dose frequency, treating BID was found to be more effective than treating QD (21 days QD at 80 mpkd prevented fungal burden almost to the levels achieved in 7 days BID, 33% of the QD duration). Evaluating dose level and frequency, fungal burdens were similar after treating 7 days QD at 80 mpkd or BID at 20 mpkd (4× higher dose required with QD). Fungal CFU burden did not increase after therapy, which was evidence of a fungicidal impact, and extending therapy beyond 7 days did not improve the outcome. Fungal burdens evaluated 2 or 21 days after therapy were statistically indistinguishable, tested after 7 or 21 days QD therapy. Therefore, it was evident that a relatively brief therapy at a high enough dose and frequency can sharply suppress disease, a durable suppression consistent with a fungicidal effect. NikZ therapy completely prevented infection of the spleen, in contrast with 67% infection in the untreated group (no numbers reported), showing NikZ prevented dissemination. In a connected PK study, an s.c. dose of 40 mg/kg gave an AUC of 18.6 µg h/mL.
The authors concluded this suggested a potential human dose of 250–500 mg BID, 7.7–15.4 mg/kg/day (mouse equivalent 63–125 mg/kg/day).
Recently, a 2020 meeting abstract reported that NikZ was significantly effective in a model of murine disseminated coccidioidomycosis after IV inoculation with a fatal dose of C. immitis
Addressing the natural incidence of coccidioidomycosis, a small trial in dogs with natural disease showed significant benefit from NikZ therapy [37
]. Canine therapy generally follows human guidelines, with generally similar outcomes. For humans, IDSA guidelines suggest fluconazole for “3 to 6 months or longer, depending on the clinical response” [38
]. When treating dogs with oral NikZ BID for only 60 days, 78% improved (7 of the 9 dogs that completed the study), and 33% (3 of 9) showed “resolution or near resolution of symptoms” after oral dosing at 250 mg BID for 10 ± 5 kg and 500 mg BID for 22.5 ± 7.5 kg dogs, a rough human equivalent of 2000 mg/day. These dogs had naturally occurring, fairly well-established disease (1, 1.5, 3, 8, and mostly 12 weeks since diagnosis). The subjects presented with various comorbidities [37
In summary, NikZ is fungicidal against Coccidioides spp., useful at smaller doses if given more frequently (BID superior to QD), reaching maximum impact in these murine models by 7 days, with persistent benefit unchanged if measured at 2 or 21 days post therapy, all supportive of the interpretation that the effect is fungicidal against Coccidioides.