Novel antifungals and treatment approaches to tackle resistance and improve outcomes of invasive fungal disease

SUMMARY

Fungal infections are on the rise, driven by a growing population at risk and climate change. Currently available antifungals include only five classes, and their utility and efficacy in antifungal treatment are limited by one or more of innate or acquired resistance in some fungi, poor penetration into “sequestered” sites, and agent-specific side effect which require frequent patient reassessment and monitoring. Agents with novel mechanisms, favorable pharmacokinetic (PK) profiles including good oral bioavailability, and fungicidal mechanism(s) are urgently needed. Here, we provide a comprehensive review of novel antifungal agents, with both improved known mechanisms of actions and new antifungal classes, currently in clinical development for treating invasive yeast, mold (filamentous fungi), Pneumocystis jirovecii infections, and dimorphic fungi (endemic mycoses). We further focus on inhaled antifungals and the role of immunotherapy in tackling fungal infections, and the specific PK/pharmacodynamic profiles, tissue distributions as well as drug-drug interactions of novel antifungals. Finally, we review antifungal resistance mechanisms, the role of use of antifungal pesticides in agriculture as drivers of drug resistance, and detail detection methods for antifungal resistance.

INTRODUCTION

Fungi are ubiquitous organisms capable of survival in a vast variety of conditions. The number of different fungal species has been estimated at around 12 million (1). Over 200 fungal species are frequently identified as causes of infection in humans with an estimated annual incidence of 6.5 million invasive fungal infections and 3.8 million deaths, of which about 2.5 million (68%; range 35–90) were estimated directly attributable (2). With climate change, some fungal pathogens develop thermotolerance (3), resulting in the emergence of new and sometimes highly resistant fungal species as human pathogens (4). Use of antifungals in the environment has become essential for food security on a warming planet, but several antifungals used as environmental fungicides are similar to those used for treating humans (5, 6) and can thereby drive antifungal resistance in human pathogenic environmental fungi.

Fungal infections range from superficial involvement of the hair, nails, and skin, to invasive disease with deep-seated organ disease or with high-associated morbidity and mortality (7). Invasive fungal infections occur primarily in the immunocompromised population and more frequently in males (8), and certain races and ethnicities (9); social determinants of health and health disparities are highly contributory to patient risk (10). Additionally, the expanding immunosuppressed population presents a growing challenge with novel risk factors and longer durations of risk documented (11–13). Patient mortality remains high, with all-cause mortality in cases of invasive candidiasis >40%, while mortality rates in mold infections are similar (14–17). In cases caused by antifungal-resistant yeasts or molds, mortality increases further and exceeds 80% in some series (18).

The last two decades have seen significant advancements in fungal diagnostics and treatment (19–23). Attempts to improve patient outcomes are multifaceted. Patient and provider education are essential in attempts to decrease infections and aid in earlier disease identification to provide rapid diagnosis. Novel diagnostic strategies have brought forth preemptive therapeutic approaches, yet the limitations of currently available diagnostics still require the frequent use of antifungal prophylaxis or empirical treatment.

Currently available antifungals include only five classes: the polyenes [amphotericin B (AmB) formulations], the azoles (triazoles and tetrazoles), anti-metabolites (flucytosine), the echinocandins and other glucan synthase inhibitors, and the allylamines (terbinafine) that are infrequently used for treating invasive fungal diseases (IFDs) (24, 25). The use of each of these agents requires a detailed and nuanced understanding of their spectrum of activity, pharmacokinetics (PK), and toxicity. Innate or acquired resistance in some fungi limits antifungal utility in their treatment, poor penetration into “sequestered” sites limits efficacy (e.g., eye or central nervous system), and agent-specific side effects and drug interaction profiles require frequent patient reassessment and monitoring to alleviate tolerability concerns.

With climate change, some fungal pathogens develop thermotolerance (3), resulting in the emergence of new and sometimes highly resistant fungal species as human pathogens (4). Use of antifungals in the environment has become essential for food security on a warming planet, but several antifungals used as environmental fungicides are similar to those used for treating humans (5, 6) and can thereby drive antifungal resistance in human pathogenic environmental fungi. The extensive use of these limited systemic antifungal drugs in the clinic, on the other hand, has exacerbated the problem by driving antifungal-resistant fungal pathogens. Exemplary are the newly emerged drug-resistant fungal pathogens (e.g., multidrug-resistant Candida auris, fluconazole-resistant C. parapsilosis and Candida tropicalis, triazole-resistant Aspergillus fumigatus), which pose significant challenges to clinicians by causing fungal outbreaks and devastating infections and have thereby further heightened interest in novel agents for the treatment of priority fungal pathogens (4, 26–28). Resistance is also increasing in other fungal pathogens causing superficial fungal infections, including Trichophyton indotineae and Trichophyton rubrum (29). Additionally, there are no effective therapies currently available for some fungal infections (e.g., Lomentospora prolificans) (30) and no oral antifungal options for many more including a significant proportion of invasive Candida infections (31). These limitations have prompted the search for novel antifungal agents and for biologics tackling host susceptibility to circumvent the myriad of problems with currently available therapies. A substantial unmet need exists, and agents with novel mechanisms, favorable PK profile, and fungicidal mechanisms are urgently needed.

USE OF NOVEL ANTIFUNGALS IN AGRICULTURE: RISK VS BENEFIT

Fungal infections pose a major threat to plant health, as exemplified by the decimation of U.S. chestnut trees by Cryphonectria parasitica and the near-total destruction of banana monoculture by Fusarium oxysporum f. sp. cubense (32–34). It is estimated that fungi are responsible for up to 80% of all plant diseases, and every year, fungal plant diseases cause at least 125 million tons of loss of rice, soybean, wheat, corn, and potato, a quantity large enough to feed at least 596 million people (32, 35). The damage caused by fungi costs global agriculture $100–$200 billion per year (36). As such, fungicides have substantial economic importance in agriculture and are important for the protection of the global food supply (37). Fungicides can have both agricultural uses (e.g., crops, turf, or ornamentals) and non-agricultural uses (e.g., wood and paint preservation) (38). Resistance of plant pathogens to fungicides has been recognized as a problem since as early as the 1960s, and resistance to many of the currently available classes of pesticides has been documented. Limited options exist for effective fungal disease management (39, 40), highlighting a need for strategies to prevent the emergence of resistance among plant pathogens as well as for novel mechanisms of action for fungicides.

Of note, certain fungicides share mechanisms of action with antifungal drugs that are critically important to human and animal medicine, a fact that has risen concern among clinicians and public health officials about the development of cross-resistance and associated harms to human and animal health (Fig. 1) (41, 42). Of particular concern is the azole antifungal class, which acts by blocking synthesis of ergosterol, a key component of fungal cell membranes (43). Azole fungicides have become an essential component of agriculture with an estimated world marked value share of 20%–25% of all fungicides (38). Triazole fungicides, whose use in the United States has increased fourfold over the course of a decade (44), can select for Aspergillus fumigatus strains harboring unique genetic mutations conferring resistance to medical azoles (e.g., itra- and voriconazole), as shown first in the Netherlands (28, 45–51).

Fig 1.

Climate change and antifungal use and resistance in agriculture and humans.

Available evidence suggests that the use of these fungicides has led to severe azole-resistant A. fumigatus infections worldwide, including in the United States and particularly in Europe (51, 52). As new fungicides and uses are proposed, the potential exists for certain antifungal agents to select for pathogenic fungi that are resistant to medically important antimicrobial drugs, including both U.S. Food and Drug Administration (FDA)-approved drugs and those still undergoing late-stage clinical trials (e.g., olorofim and manogepix) (42).

Ipflufenoquin, a fungicide authorized in 2022 in the United States for use on pome fruits and almonds (53), and quinofumelin, another fungicide which is currently in development, share their mechanism of action with olorofim, an orally available antifungal agent that is currently undergoing phase III clinical evaluation. This has raised concern regarding risk of induction of cross-resistance (42), a concern that is further exacerbated as this cross-resistance can occur in vitro after ipflufenoquin exposure (54). Both ipflufenoquin and olorofim interfere with de novo pyrimidine biosynthesis via inhibition of the enzyme dihydroorotate dehydrogenase (DHODH) (42, 55, 56). The first-in-class (orotomide) antifungal drug, olorofim, shows promising in vitro and in vivo activity against difficult-to-treat mold infections such as those caused by azole-resistant A. fumigatus, Scedosporium species, and Lomentospora prolificans, as well as infections caused by thermally dimorphic fungi, including species resistant to azoles and amphotericin B (57). Notably, in vitro activity of olorofim and ipflufenoquin against Aspergillus species was found to be similar (42, 54). Furthermore, it was shown that mutants resistant to olorofim were also resistant to ipflufenoquin and that this cross-resistance was due to non-synonymous mutations in the gene encoding DHODH (the pyrE gene); some isolates containing resistance-conferring mutations to olorofim did not demonstrate a fitness cost when drug stress was absent (54, 58). Similarly, Aspergillus isolates that develop resistance to ipflufenoquin may also be resistant to olorofim (54).

Fosmanogepix is another first-in-class antifungal drug currently undergoing clinical trials and showing promise for difficult-to-treat infections (59). It kills yeasts by inhibiting glycosylphosphatidylinositol-anchored wall transfer protein (Gwt1), a protein that mediates cross-linking of cell wall mannoproteins to β-1,6-glucan and is necessary for biofilm and germ tube formation (60). This drug is particularly important clinically because of its activity again multidrug-resistant C. auris, an urgent public health threat with rapidly increasing spread throughout the United States (27). Broad-spectrum antifungal activity of manogepix (the active moiety of the prodrug fosmanogepix) has also been demonstrated against other important pathogenic yeasts and molds, including against resistant strains (61–63). Of concern, pyridine antifungal pesticides that also target Gwt1 are also in development, namely the antifungal pesticide aminopyrifen (64). Data to assess the risk of cross-resistance between aminopyrifen and manogepix are currently lacking but are needed to assess the potential risk to human health posed by use of the antifungal pesticide.

The first risk assessment on the impact of environmental usage of triazoles on the development and spread of resistance to medical triazoles in Aspergillus species by the European Centre for Disease Prevention and Control was published in 2013. Currently, to begin to address this issue, the U.S. government, through a U.S. government interagency process including the Environmental Protection Agency, the U.S. Department of Health and Human Services, and the U.S. Department of Agriculture, has begun the development of a proposed framework aiming to assess the risk to the effectiveness of human and animal drugs posed by certain antibacterial or antifungal pesticides (65), with a recent precedent of outlawing environmental agents due to their impact on human health (66).

Overall, the issue of cross-resistance between antifungal pesticide use in the environment and clinical antifungal drugs is a One Health problem requiring multi-sector collaboration among clinicians, veterinarians, agronomists, regulatory agencies, and other groups to evaluate and mitigate risks to human health posed by antifungal pesticide-induced cross-resistance (41, 67). The importance of novel fungicides for the protection of the food supply and economy must be balanced with consideration for the emergence of antifungal-resistant infections affecting human health, with the ultimate goal of preserving the use of antifungal compounds in humans, animals, and plants.

NEW AND HIGHLY RESISTANT FUNGI AND OUTBREAK EMERGENCE

Mechanisms of action of currently available antifungal drugs

Among the currently available antifungal agents, the triazoles (fluconazole, itraconazole, voriconazole, posaconazole, and isavuconazole) impede the production of one of the major membrane components, ergosterol, through binding to lanosterol 14-α demethylase (the product of the gene ERG11) resulting in the buildup of toxic precursor methyl-sterols. Echinocandins, such as micafungin, caspofungin, and anidulafungin, exert their action by inhibiting the production of a major cell wall carbohydrate, 1,3-β-D-glucan (BDG), through binding to the catalytic subunit of BDG synthase, the product of the FKS gene. Polyenes, such as AmB, kill fungi primarily by forming extramembranous sponge-like aggregates that extract ergosterol from lipid bilayers (68, 69), followed by osmotic disturbance and cell death (70–73). Flucytosine is another antifungal drug that can be converted to certain metabolites, which prevent RNA/DNA synthesis (74). Finally, terbinafine non-competitively inhibits squalene epoxidase and is believed to exert its growth inhibitory activity by disruption of ergosterol biosynthesis and accumulation of squalene (75).

Antifungal resistance and tolerance

Antifungal susceptibility is a critical characteristic of fungal organisms. Exposure to environmental antifungals or xenobiotics may result in the development of antifungal tolerance or resistance. This process may develop from single-point mutations within critical pathways resulting in changes to target site affinity. Overexpression of target site(s), efflux pumps, and other mechanisms has also been described and plays a significant role in antifungal susceptibility to antifungal agents (72).

A potential link between triazole tolerance and therapeutic failure has been suggested using in vivo murine and Galleria mellonella larvae models (76, 77), and will need to be further explored as these findings were to some extent also confirmed in a mice model, where trailing growth of C. tropicalis isolates in fluconazole, which likely represents the same phenomenon as tolerance (78) was associated with worse treatment response to fluconazole (79). Accordingly, understanding the cellular machinery conveying antifungal resistance is important and allows for the evaluation of novel drug targets to minimize the burden of resistance and extend the lifespan of the limited armamentarium of systemic antifungal drugs.

Antifungal resistance is defined as growth in the presence of inhibitory/fungicidal concentration above the clinical breakpoints of antifungal drugs, which can be measured in vitro by standard antifungal susceptibility testing protocols (80, 81). Susceptibility results are reported as minimum inhibitory concentrations (MICs) or minimum effective concentrations (MECs) (77).

In contrast, antifungal tolerance is a transient phenomenon, which refers to the slow growth or survival of a subpopulation of fungal cells in a clonal population without known stable genetic mutations (77). The term “tolerance” can vary depending on the activity of a given antifungal drug. For instance, in Candida species, “azole tolerance ” has been described as slow growth of a sub-population (10%–85%) in the presence of supra-MIC of fungistatic azoles, whereas “echinocandin tolerance ” has been described as survival of a very small sub-population (<0.1%) in the face of fungicidal concentrations of echinocandins (77, 82). Azole tolerance can be measured either through antifungal gradient strips or disk-diffusion assays, which use fraction of growth in the inhibition zone, or potentially by adopting modifications in the broth microdilution antifungal susceptibility testing protocol, also known as supra-MIC growth after 48 hours of incubation (77). On the other hand, measuring echinocandin tolerance requires extensive and time-consuming colony-forming unit (CFU) counting at different time points following exposure to echinocandins (or other fungicidal drugs), which are characterized by biphasic or monophasic killing curves (Fig. S1) (82). Of note, voriconazole exerts fungicidal activity against A. fumigatus, and voriconazole persistence is a newly introduced concept in medical mycology (83). The clinical implication of this concept has been exemplified by observation of therapeutic failure in invasive aspergillosis (IA) patients infected with susceptible isolates despite reaching therapeutic levels of correct azole drugs (84).

Remarkably, the antifungal-tolerant cells can give rise to progeny displaying the same MIC and tolerance level as the wild-type-susceptible parental strain (77, 82). Therefore, mechanisms underpinning antifungal tolerance are thought to be physiological/epigenetic. Such mechanisms encompass an extensive array of factors ranging from pre-existing stochastic metabolic variation in a clonal population, proteins exerting chaperon activities (Hsp90), genomic plasticity (aneuploidy and copy number variations), core stress responses, and epimutation (RNAi and heterochromatin-evoked tolerance) to name a few (Fig. 2) (71, 72, 77, 85, 86).

Fig 2.

Mechanisms underlying antifungal tolerance. Myriad of mechanisms contributes to antifungal tolerance, including genomic plasticity resulting in gene dosage alteration (such as Erg11 and efflux pump upregulation), chaperon activities to restore the protein structure and function of proteins (such as HSP90), mitochondrial loss (such as petite C. glabrata/N. glabratus isolates), and epimutation-mediated gene silencing (such as heterochromatin formation and RNAi-evoked target silencing). Note that core stress responses are not shown here.

Of note, current lines of research have unraveled that genetically, clonal microbial populations possess distinct metabolic states with majority and minority of rapidly and slowly growing cells, respectively. Such intrinsic metabolic differences are thought to allow the effective establishment of a community in a given niche by rapid replication of the major subpopulation at the time of abundance in the absence of stress. In contrast, the minority subpopulation could better persist in unfavorable environmental conditions (exposure to antifungal drugs, toxic compounds excreted from other competing microbes, etc.), which can rebuild the same community upon stress removal and, therefore, prevent the extinction of microbial communities (87, 88). Indeed, the lower metabolic rate could result in lesser production of drug targets, which provides a potent survival advantage upon exposure to fungicidal agents (89). This intrinsic, stochastic metabolic/physiologic difference is similar to echinocandin tolerance in C. glabrata/Nakaseomyces glabratus (82, 90). Furthermore, mitochondrial loss represents a severe metabolic adjustment employed by C. glabrata/N. glabratus to effectively cope with stresses imposed by host innate immune cells and antifungal drugs (89, 91, 92). Alarmingly, new studies have unraveled an unexpected prevalence of petite C. glabrata/N. glabratus isolates in clinical samples, which can potentially confer therapeutic failure (89, 92, 93).

Genomic plasticity has been identified as one of the most prevalent adaptive responses employed by C. albicans to azole drugs and adaptation to host (94–97). For instance, amplifying the entire left arm of chromosome 5, where ERG11 and TAC1 reside, may result in gene dosage alteration and enhance azole tolerance (94, 96). Similarly, high copy number variations, especially genes involved in azole resistance and core-stress response, feature another genome plasticity potentially underlying azole tolerance (97). Similarly, it has been shown that Cryptococcus neoformans can also adapt to high concentrations of fluconazole by doubling chromosome 1, where the ERG11, the drug target, and AFR1, a major azole efflux pump, reside (98). Interestingly, Cryptococcus isolates recovered from patients suffering from cryptococcal meningitis treated with fluconazole also show chromosome 1 aneuploidy and, subsequently, increase the azole tolerance subpopulation (99). Such plastic genomic changes can revert to the wild-type level without azoles (97). Recent studies have also identified copy number variations among fluconazole-resistant C. parapsilosis isolates (100). The involvement of copy number variation during exposure to fungicidal echinocandins in Candida species remains to be investigated. Still, it is plausible that suppression of the drug target expression coincided with mounting compensatory cell wall integrity pathways, such as overexpression of genes synthesizing chitin, which may provide a more robust survival response.

Core stress responses refer to a cascade of subcellular molecular pathways triggered by various environmental cues. These provide a survival advantage to extensive stresses, including cell wall and membrane integrity pathways (71, 72). Notably, activation of these pathways in response to environmental cues other than antifungals can also confer cross-tolerance to antifungal drugs. Strikingly, C. glabrata/N. glabratus internalization by macrophages or exposure to hydrogen peroxide and incubation in depleted media elicits effective responses conferring cross-tolerance to echinocandins (82).

Gene silencing via RNAi and DNA methylation represents an additional layer of protective responses (85, 86). RNAi-mediated gene silencing utilizes the combined action of various effector proteins guided by RNA molecules, which specifically degrade the mature mRNA of the drug target without exerting any impact on the DNA level (85). Of note, RNAi machinery is not present in some major fungal pathogens, such as C. glabrata/N. glabratus (101), and despite their identification in some other fungal pathogens, such as C. albicans (102) and Cryptococcus deneoformans (103), its association with drug resistance/tolerance and pathogenicity has been poorly characterized in such species. Heterochromatin-mediated gene silencing, such as methylation of lysine 9 on histone 3 (H3K9 me2), limits the accessibility of RNA polymerase machinery and thereby inhibits the transcription of an extensive number of genes (86). Therefore, the resultant epigenetic landscape changes confer cross-protection to numerous unrelated stresses, placing it as an ideal swift response to stresses (86). Finally, stresses conferred by antifungal exposure generate denatured proteins, whose buildup could be lethal to fungal cells. Therefore, fungal species are equipped with an array of chaperon molecules, such as HSP90, to restore the structure and, thereby, the function of client proteins, including calcineurin- and mitogen-activated protein kinases (73). Given that stresses generally lower metabolic activity and that chaperons require ATP, transcription inhibition is an energy-saving mechanism that could alleviate denatured proteins’ overwhelming buildup.

Interestingly, mechanisms underlying antifungal tolerance are analogous to innate immunity, where universal responses primarily interact with the assault and finally shape the trajectory of the secondary response, which results in stable genetic changes designed to counteract the impact of a given assault (resistance). Moreover, antifungal resistance typically imparts fitness-cost, such as virulence attenuation of (echinocandin resistant) C. albicans isolates (104) and sensitivity of fluconazole-resistant C. lusitaniae (105) and C. glabrata/N. glabratus isolates to oxidative stress (106, 107). Accordingly, antifungal tolerance provides a rapid response, enabling fungal cells to survive in the face of challenge while minimizing relative fitness cost.

Major mechanisms underlying antifungal resistance

Antifungal resistance can be either adaptive and emerge following exposure to antifungal drugs, such as azole and echinocandin resistance in major Candida species (with the exception of C. krusei/Pichia kudriavzevii) or intrinsic, which refers to pre-existing mechanisms counteracting the drug activity, such as fluconazole resistance in C. krusei/P. kudriavzevii (108) and Aspergillus fumigatus (109), and high echinocandin MICs in C. parapsilosis (110). This section will mainly discuss adaptive mechanisms of resistance applicable to a broad range of fungal species.

Drug resistance in fungal species can be categorized into three major categories, namely, (i) overexpression of efflux pumps, which actively pump antifungal compounds out of the cell (relevant for azole resistance), (ii) overexpression of drug target to compensate for the occupied antifungal target (relevant for azole resistance), and (iii) drug target alteration or silencing, which modulates the affinity of the target to the drug (relevant for azole and echinocandin resistance). Efflux pumps belong to two main groups, namely, major facilitator superfamily (such as MDRs) and ATP-binding cassette (such as CDRs), which pump azoles outside the cell in an ATP-independent and ATP-dependent manner, respectively (Fig. 3) (70–73). Typically, these efflux pumps are expressed through binding of specific transcription factors to their promoter (known as drug resistance binding element), such as MRR1 and TAC1 binding to MDR and CDR promoters, respectively. In fact, acquisition of gain-of-function mutations renders these transcription factors hyperactive, their localization to nucleus, followed by overexpression of efflux pumps. The prevalence of efflux pump overexpression mediated by gain-of-function mutation is a prevalent mechanism among diverse fungal species. Similarly, drug target overexpression is orchestrated by acquisition of gain of function in its pertinent transcription factors, such as UPC2 in Candida species. The efflux pump and drug target overexpression are general mechanisms exerted to counteract the azole drugs (70–73).

Fig 3.

Mechanisms generally governing antifungal resistance. Azole resistance involves multiple mechanisms, including the overexpression of efflux pumps (CDR1 and MDR1) due to hyperactive Tac1 and Mrr1, and the overexpression of the drug target using hyperactive UPC2. The drug target mutation is universal for both azole and echinocandin resistance. Mechanisms underpinning AmB resistance have not been shown.

Drug target alteration through acquisition of mutations is one of the most prevalent mechanisms underlying both azole and echinocandin resistance. For instance, certain mutations in ERG11/CYP51A have been reported to confer azole resistance among a wide range of fungal species (70–73). Similarly, echinocandin resistance is mainly driven by acquisition of mutation in a short stretch of the catalytic subunit of drug target, hotspot of FKS genes between both Candida and Aspergillus species. It should be noted that echinocandin resistance in C. glabrata/N. glabratus is mediated by acquisition of mutations in hotspot 1/hotspot 2 of either FKS1 or FKS2 (111). Importantly, new lines of studies have identified echinocandin-resistant isolates carrying mutation outside of the hotspot regions (112) as well as presumably echinocandin-susceptible isolates carrying mutation in hotspot region conferring therapeutic failure in real-life situations (113). Interestingly, although thought to be naturally echinocandin resistant, emerging studies from multiple countries have identified echinocandin R C. parapsilosis isolates harboring mutations in the hotspot region of the FKS1, highlighting that the naturally occurring mutation (P660A) is not sufficient to confer echinocandin R in vivo and in real life (26, 114–116). Secondly, application of clustered regularly interspaced palindromic repeats (CRISPR)-Cas9 technology has unraveled that acquisition/pre-existing certain amino acid changes located beyond the hotspot 1 and hotspot 2 of FKS1 in C. parapsilosis do not confer echinocandin resistance rather they confer echinocandin tolerance. Interestingly, isolates carrying some of these mutations also facilitated the emergence of bona fide echinocandin-resistant C. parapsilosis isolates carrying clinically known mutations in FKS1 following exposure to echinocandins (117).

Unlike azoles and echinocandins, polyenes (such as AmB) do not have a protein drug target, and the mechanisms underpinning AmB resistance are found to be diverse (118). Since AmB exerts its fungicidal activity through creating sponge-like (119) aggregates and ergosterol extraction and reactive oxygen species production (120, 121), it is plausible to assume that AmB-resistant isolates potentially lower the ergosterol content in the plasma membrane by inhibiting major genes involved in ergosterol biosynthesis pathway and/or reducing reactive oxygen species buildup either through overexpression of reactive oxygen species detoxifying genes or inhibiting mitochondrial activity. Some Candida species, such as C. haemulonii species complex, are known to have intrinsically high MICs against AmB, which has been attributed to concomitant distorted mitochondrial activities, altered redox homeostasis, and lower ergosterol on the membrane (121). Interestingly, it has been shown that clinical isolates of C. haemulonii displayed poor growth rate on alternative carbon sources, used low oxygen, and reduced mitochondrial membrane potential, which all indicated altered metabolic status and shifting toward the fermentative metabolic state. Given that mitochondria are one of the major sources of reactive oxygen species inside the cell (122) and contributes in ergosterol biosynthesis pathway (123), the lower mitochondrial activity of C. haemulonii isolates could lower the membrane ergosterol content in the basal state and a lower reactive oxygen species production following AmB exposure, which can provide an evolutionary protection against AmB. Moreover, authors identified that C. haemulonii isolates had significantly higher levels of catalase and superoxide dismutase, which were attributed to a lower reactive oxygen species levels following exposure to AmB and oxidative stress-inducing agents (121). Adaptive AmB resistance has been rarely identified, which has been mainly attributed to acquisition of de novo mutations in ERG2, ERG3, ERG6, and ERG11 among clinical isolates of C. lusitaniae and C. auris (124–128), with some polymorphisms conferring AmB and azole resistance simultaneously. Finally, a study recently suggested the involvement of post-translation modification in AmB-resistant C. krusei/P. kudriavzevii isolates, whose role is yet to be proven (129).

Fungal outbreaks

Drug-resistant fungal pathogens able to cause outbreaks pose a serious threat to healthcare settings, when compared to Candida species causing endogenous infections, such as C. albicans. Such infections are thought to be fueled by in- and outdoor environmental reservoirs feeding the outbreaks and have the potential to complicate treatment strategies by causing infections in antifungal-naïve patients, which may not only prolong the hospitalization duration and, therefore, the pertinent costs but also potentially increase the likelihood of mortality (26). During the last two decades, we have witnessed an increasing number of such pathogens, including but not limited to C. parapsilosis, C. auris, C. tropicalis, and species belonging to dematiaceous fungi and Mucorales as well as Aspergillus spp. Additionally, there has been an increasing number dermatophytes causing difficult-to-treat superficial fungal infections (130), which will be briefly discussed in this section.

Outbreaks due to fluconazole-resistant C. parapsilosis and C. auris isolates have been increasingly identified in multiple countries (26). Although initial selective pressure exerted by azole drugs has caused the emergence of fluconazole-resistant C. parapsilosis isolates, azole use is not the main driver of the outbreak severity as such fluconazole-resistant isolates may have adapted to environmental conditions (26). This hypothesis is supported by recurrence of fluconazole-resistant C. parapsilosis outbreaks even after application of extensive environmental decontamination and infection control strategies (131). Unlike C. parapsilosis, the simultaneous emergence of C. auris worldwide has been associated with multiple factors, including climate change and a better adaptation to human host body conditions as well as an increasing azole use in the clinic just to name a few (132). Outbreaks caused by fluconazole-resistant C. tropicalis, on the other hand, have been primarily concentrated in Southeast Asian countries, including Taiwan (133), China (134), and Thailand (135). The emergence of multidrug-resistant C. parapsilosis (114, 115, 136) and C. auris isolates in clinical settings is particularly challenging (137). Alarmingly, an increasing number of multidrug-resistant C. parapsilosis isolates in large Turkish hospitals were reported (114, 136). A recent whole-genome sequencing analysis has noted the emergence of novel multidrug-resistant C. parapsilosis isolates and, using multidimensional experimental approaches, has shown that multidrug-resistant C. parapsilosis isolates persistently causing outbreaks do not suffer fitness cost ex vivo and in vivo and produce prominent biofilm levels compared to wild-type-susceptible counterparts. Importantly, the biofilm produced by such multidrug-resistant C. parapsilosis isolates also has comparable tolerance to bleach and hydrogen peroxide relative to susceptible counterparts (138). As such, outbreak scenarios can select for drug-resistant isolates displaying remarkable adaptation to host and environmental stresses. Accordingly, minimization and/or eradication of outbreaks are of paramount importance to prevent the emergence of superbugs not only resistant to antifungal drugs but also host immune system (138).

Aspergillosis outbreaks have frequently been described over the decades and mostly associated with construction work in hospitals (139, 140). Although rare, outbreaks caused by species within the Fusarium genus, especially F. solani, can be life threatening as these species are inherently resistant to echinocandins, have elevated MICs to AmB and triazole drugs, and disseminated infections caused by these species are associated with extremely high mortality rates (50%–70%) (6, 141–144). Particularly concerning have been the recent fatal fusariosis outbreaks in New Mexico potentially linked to epidural and spinal anesthesia with the mortality rate of 72% (6); for that outbreak caused by multiresistant Fusarium spp., therapeutic use of manogepix, a novel antifungal that is currently under clinical development, has proven beneficial (61).

Mucormycosis is a devastating diagnosis, given the high mortality rates associated with these pathogens (40%–90%) (145), limited activity of some azole drugs (except for isavuconazole and posaconazole) (146), and disfigurement impacting the life quality of survivors (145, 147). Notably, outbreaks caused by mucormycosis have frequently been described following natural disasters or in soldiers with war injuries (148, 149), but also hospital-associated outbreaks associated with contaminated wooden spatula and bed linen have been described (150, 151). Mucormycosis has made headlines during the COVID-19 pandemic where India was one of the countries severely hit by COVID-19-associated mucormycosis (147, 152).

Dematiaceous fungi, also known as melanized/brown-pigmented fungi, are environmentally widespread and mostly reside in soil. Species within this group of pathogenic fungus are extremely heterogenous and accordingly cause a wide range of infections in humans, including superficial, pneumonia, and disseminated infections just to name a few. In fact, dematiaceous fungi are among the most common fungal pathogens causing allergic sinusitis and brain abscesses, and antifungal therapy in conjunction with surgical treatment is required for a better outcome since such infections are refractory to sole antifungal treatment. Unlike other fungal pathogens, these species can cause infection in immunocompetent patients. Similar to aforementioned fungal pathogens, infections due to dematiaceous fungi are on the rise (153–156), and several outbreaks have been reported so far, which have been mainly due to contaminated drugs, such as methylprednisolone (157, 158) injections and injectable steroids (156).

In parallel with fungal pathogens causing systemic infections, there has been an upsurge in the number of outbreaks caused by Trichophyton species, especially T. rubrum and T. indotineae (159, 160). Although initially rarely reported in some countries, there has been a notable increase in the prevalence of terbinafine-resistant T. indotineae in India from 2018 onward (159). Further concerning has been the emergence of clonal multidrug-resistant T. indotineae isolates in India, given the high genetic relatedness of the isolates, and that 36% of these isolates were resistant to terbinafine and 39.6% to griseofulvin and fluconazole (161). In fact, retrospective analysis of case series caused by T. mentagrophytes has shown the suboptimal efficacy of topical treatments, associated in part with the misuse of topical formulations containing both antifungals and corticosteroids, and effective treatment requires using oral therapeutic options, including itraconazole and voriconazole, whereas fluconazole was shown to be ineffective (160). Recently, an outbreak by T. mentagrophytes has been described among men who have sex with men in France and some other countries (130).

NOVEL ANTIFUNGALS FOR YEAST INFECTIONS

An overview of the clinical trials timeline and approval status for novel antifungals is displayed in Fig. 4.

Fig 4.

Clinical trials timeline and approval status for novel antifungals.

Rezafungin

Rezafungin (Rezzayo; formerly CD101) is a second-generation echinocandin developed for the treatment of invasive candidiasis, including candidemia, and for use as single-agent prophylaxis against IFDs in hematopoietic stem cell transplant (HSCT) recipients (162). Following a pivotal phase III clinical study, rezafungin gained FDA approval in the United States in March 2023 for treating invasive candidiasis in individuals aged ≥18 years in whom there are limited or no alternative treatment options (163, 164). In Europe, it was approved for invasive candidiasis in December 2023 (165, 166).

Rezafungin is a lipopeptide echinocandin and inhibits the formation of BDG, an integral component of the fungal cell wall, through inhibition of BDG synthase (167–170). It has a cyclic depsipeptide core and an N-linked acyl lipid side chain, the latter of which is responsible for antifungal activity. It was designed specifically to improve the chemical stability and tissue penetration relative to first-generation echinocandins (anidulafungin, caspofungin, and micafungin) (167, 169). As a structural analog of anidulafungin, it shares the same side chain and a similar core but with the C5 ornithine residue (anidulafungin) replaced with a choline aminal ether (rezafungin). This has resulted in substantive reduction of its degradation [<2% degradation after 9 months storage at 40°C (170), a prolonged half-life of the drug >130 hours or >5 times the half-life of other echinocandins] in humans with attainment of target drug levels with once-weekly dosing (167, 171–173). Rezafungin is chemically stable with a relative lack of reactive intermediates and is stable in mammalian hepatocytes (172). This stability allowed the development of formulations for topical or subcutaneous application, but to date, these have not been realized due to insufficient efficacy. Rezafungin is currently administered only by the intravenous route.

The abundant in vitro antifungal susceptibility data on rezafungin are mostly derived from surveillance studies using Clinical and Laboratory Standards Institute (CLSI) protocols (documents A3 and M27 4th ed. for yeasts) (80) and European Committee on Antimicrobial Susceptibility Testing (EUCAST; EDef. 7.4.1) methods (174–176). Differences in rezafungin MICs have been noted between CLSI and EUCAST protocols where MICs have varied by up to three twofold dilutions (177–180) which is triggered by methodological differences between CLSI and EUCAST. Rezafungin has potent in vitro activity against a broad range of yeast isolates. Of >1,600 contemporary clinical isolates tested in the SENTRY Antimicrobial Surveillance Program (2014–2021) (177, 180, 181), rezafungin MIC₉₀ values were 0.03 mg/L for C. krusei/P. kudriavzevii, 0.06 mg/L for C. albicans, C. tropicalis, and C. glabrata/N. glabratus, 0.12 mg/L for C. dubliniensis, and 2 mg/L for C. parapsilosis (180). The majority of C. albicans isolates can be expected to be inhibited by drug concentrations of ≤0.125 mg/L (177, 178). Table S1 summarizes MIC₅₀ and MIC₉₀ values for Candida species.

Generally, rezafungin MIC₅₀ and MIC₉₀ values are comparable to those of anidulafungin, caspofungin, and micafungin when determined by the CLSI method, although small differences have been found. For instance, for C. albicans and C. tropicalis, rezafungin MIC₅₀ values were higher than those for anidulafungin by about twofold but lower than those for caspofungin (~0.4–0.6-fold; Table S1). Rezafungin exhibited greater potency than caspofungin and micafungin against C. krusei/P. kudriavzevii, while caspofungin was more potent than rezafungin when C. parapsilosis was tested (rezafungin MIC₅₀/caspofungin MIC₅₀ ratio: 2.58). The intrinsic reduced susceptibility phenotype was seen with C. parapsilosis and C. guilliermondii/Meyerozyma guilliermondii compared with the phenotype of C. albicans (MIC₅₀ geometric means of 1.12 vs 0.023 mg/L; Table S1). Against C. auris, rezafungin has good activity, with slightly greater activity (182) than caspofungin and micafungin, although MIC₅₀s were higher than those for anidulafungin (Table S1). Most C. auris isolates can be expected to have MICs of 0.25 mg/L. Rezafungin MIC₉₀ values were 0.5 mg/L for Saccharomyces cerevisiae (179). For Candida species, there were no MIC increases over time, and an extended post-antibiotic effect against C. albicans, C. parapsilosis, and C. glabrata/N. glabratus is known similar to that of micafungin (183). Comparable results are found for EUCAST testing, although the MICs for rezafungin are much lower due to the technical differences between the assays (mainly with/without tween supplementation of the medium).

For Candida isolates harboring FKS hot spot mutations which may lead to echinocandin resistance, rezafungin exhibits the same potency as the other echinocandins with higher MICs than those of wild-type isolates without FKS mutations (167, 177, 184). The highest MIC differences between FKS mutants and wild-type strains were seen for C. auris FKS mutants carrying the S639P amino acid substitution (185). Mechanisms of resistance or tolerance to rezafungin are similar to the other echinocandins and are discussed in the general section on antifungal resistance. The potential to develop in vitro rezafungin resistance is low for common Candida species (186). The median frequency of spontaneous mutations conferring reduced susceptibility to rezafungin ranged from 1.35 times 10⁻⁸ to 3.86 times 10⁻⁹ . Serial passage showed ≅20 passages were required for resistance development (186).

Of interest, susceptibility testing of Candida isolates from patients with vulvovaginal candidiasis (VVC) using CLSI methods performed at pH 4 to mimic vaginal pH showed rezafungin had good activity against Candida with MICs (177, 185, 187). Notably, MICs (including for C. parapsilosis) were less than the anticipated intravaginal rezafungin concentration (>100 mg/L) that would be reached after topical administration (187). Moreover, when MIC values were studied in a vagina-simulative medium at pH 4.2 for topical rezafungin, the drug retained fungicidal anti-candidal activity (188).

Using provisional CLSI-approved “susceptible-only” breakpoints (189), susceptibility rates were 98.3% for C. glabrata/N. glabratus, 99.6% for C. parapsilosis, and 100% for other tested species (180). Suggested wild-type upper limits to distinguish wild-type and non-wild-type isolates are 0.06–0.125 mg/L for C. albicans, 0.126–0.25 mg/L for C. glabrata/N. glabratus and C. krusei/P. kudriavzevii, 0.25 mg/L for C. tropicalis, 0.5 mg/L for C. auris, and 4 mg/L for C. parapsilosis (178, 184). Finally, rezafungin is active against early and mature Candida biofilms. Rezafungin concentrations of 0.25–1.0 mg/L both reduced biofilm thickness and prevented development of mature biofilms (190). In time-kill studies of C. auris, rezafungin concentrations of ≥1 to ≥8 mg/L using RPMI-1640 medium with 50% human serum exhibited killing in the first 8–12 hours at clinically achievable trough concentrations (191), but regrowth occurred after 24 hours.

In murine models of invasive candidiasis, it has been established too that rezafungin exhibits concentration-dependent fungicidal activity with similar/better efficacy than the other echinocandins against Candida species including azole-resistant strains (192), C. dubliniensis and C. auris (172, 182, 193). Notably, the area under the curve (AUC)/MIC ratio to achieve efficacy endpoints was lower for rezafungin compared with other echinocandins (194). Rezafungin was more potent than AmB and micafungin against C. auris in reducing fungal burden in kidneys of immunosuppressed mice (rezafungin 20 mg/kg administered on alternate days, AmB 0.3 mg/kg, and micafungin 5 mg/kg given daily) (195). In mice with intra-abdominal candidiasis, rezafungin reached higher hepatic concentrations compared with micafungin, with improved survival (196).

Rezafungin also demonstrated benefits in immunosuppressed mice with C. albicans infection when administered prophylactically (197). Reduction of Candida CFU was greater with increasing drug concentrations (5, 10, or 20 mg/kg) and when the drug was given closer to the time of Candida infection (day −1, –3, or −5). For mice given 20 mg/kg of drug with one exception, C. albicans was cleared. At lower dose, there was little bioburden reduction when rezafungin was administered on day −5, but when given on day −3 or −1, the 10 mg/kg groups had negative culture, while the 5 mg/kg groups showed significant reductions in CFU burden (197).

Past and ongoing clinical trials for treating yeast infections

Finding in phase I studies on the improved PK/pharmacodynamic (PD) properties of rezafungin supported phase II/III clinical trials for the treatment of invasive candidiasis that have since been completed (Table S2).

The safety and efficacy of rezafungin for the treatment of candidemia and invasive candidiasis in humans has been studied in two double-blind, double-dummy randomized controlled trials compared against caspofungin (Table S2) (198, 199). The STRIVE study (NCT02734862) was a two-part phase II study of rezafungin at two different dosing regimens (400 mg once weekly, and 400 mg in week 1 followed by 200 mg once weekly) against a standard caspofungin regimen (70 mg intravenous once, followed by 50 mg daily). Efficacy was assessed in the modified intention-to-treat (mITT) population, which comprised 76, 46, and 61 patients in the high-dose, and low-dose rezafungin regimens, and caspofungin treatment groups, respectively. Overall cure rates at day 14 were comparable between the three treatment arms (60.5% vs 76.1% vs 67.2%, respectively). All-cause 30-day mortality rates and clinical cure at day 14 are given in Table S2. While not powered to detect efficacy differences between the treatment groups, the highest cure rates and lowest mortality were observed with the low-dose rezafungin regimen (198). Endophthalmitis, osteomyelitis, and other end-organ disease were excluded. Furthermore, the study found a 5-day reduction of intensive care unit (ICU) length of stay in the rezafungin arm (198).

The results from the STRIVE study informed the selection of the dosing regimen of rezafungin 400 mg for the first week followed by 200 mg weekly as the most effective dosing regimen for the phase III randomized double-blind ReSTORE study (identifier NCT03667690) (199). This study enrolled 199 adults with invasive candidiasis and/or candidemia (≅70% patients had only candidemia) with 93 and 94 patients in the rezafungin and caspofungin arms, respectively. In the mITT population, rezafungin met non-inferiority criteria compared with caspofungin with global cure rates at day 14 of 59.1% vs 60.6%, respectively. The corresponding 30-day all-cause mortality rates were 23.6% and 21.2%, respectively. Global cure comprised investigator-assessed clinical care, radiological cure, and mycological eradication (199). Similar results were seen in patients with candidemia alone and in those with end-organ invasive candidiasis.

Although not powered for statistical analysis, one important finding of the ReSTORE trial was that rezafungin efficacy (vs caspofungin) was seen early in the course of treatment as shown by the proportion of patients with negative blood cultures at 24 and 48 hours (54% vs 46% and 74% and 64%, respectively) and also rates of global cure at day 5 (56% vs 52%); global cure and mycological eradication at day 14 were independent of Candida species or baseline in vitro (200). An integrated analysis of the ReSTORE and STRIVE trials has further supported the efficacy for utility of rezafungin in invasive candidiasis (201). Collectively, in the mITT population analysis (n = 139 for rezafungin, 155 for caspofungin), the 30-day all-cause mortality was 18.7% for rezafungin and 19.4% for caspofungin (difference −1.5; 95% CI – 10.7 to 7.7). Day 5 mycological eradication rates in patients with positive blood cultures were 75.5% and 54.9%, respectively (difference 19.2; 95% CI 3.0–35.5) (201).

Safety

Two phase I randomized double-blind dose-escalation studies were performed to determine the safety of rezafungin (173). In the single-dose study (NCT02516904), 50, 100, 200, and 400 mg doses were given to sequential cohorts of patients (total n = 32). In the multiple dose study (NCT02551549) (n = 24), doses given were 100 mg weekly for 2 weeks, 200 mg weekly for 2 weeks, and 400 mg weekly for 3 weeks. There were no serious or severe adverse events or withdrawals from the study due to an adverse event or deaths in either study. Adverse events were all transient and resolved. Mild transient infusion reactions such as flushing, nausea, and chest tightness were seen with the third 400 mg dose in the multiple-dose study (173). No clinically meaningful laboratory abnormalities were seen. In a phase I, single-center, randomized, double-blind trial in healthy adults using single therapeutic and supratherapeutic doses of 1,400 mg, rezafungin did not prolong QT interval and had no apparent effect on repolarization or QRS duration (202).

In the ReSTORE study, treatment-emergent adverse events occurred in 91% of 98 patients in the rezafungin group vs 85% of 98 patients who received caspofungin—the most common treatment-emergent adverse events were pyrexia (14% in the rezafungin group vs 5% in the caspofungin group), hypokalemia (13% vs 9%), pneumonia (10% vs 3%), septic shock (10% vs 9%), anemia (each 9%), and hypomagnesemia (7% vs 3%). Serious AEs occurred in 56% and 53% of patients in the respective groups, and treatment-related adverse events occurred in 16% and 9%. Treatment-related serious AEs were reported in two rezafungin recipients (one infusion-related reaction and one urticaria) and three caspofungin recipients [raised transaminase indices and anaphylactic shock (199)].

Combined with data from the STRIVE trial, the most common adverse events in 151 patients receiving intravenous rezafungin 400 mg loading dose followed by 200 mg once weekly (incidence ≥5%) included hypokalemia, pyrexia, diarrhea, anemia, vomiting, and hypomagnesemia. Precautions to alert recipients/prescribers of rezafungin included infusion-related reactions, photosensitivity, and hepatic adverse reactions (164).

A phase III randomized double-blind trial is currently ongoing. This trial was designed to compare rezafungin (400 mg loading dose followed by 200 mg once weekly for a total of 13 weeks) vs a standard antimicrobial regimen (posaconazole or fluconazole plus trimethoprim-sulfamethoxazole, also for 13 weeks) in the prevention of IFD in allogeneic HSCT recipients (ReSPECT identifier NCT04368559; Table S2) (169). Adults receiving a matched HSCT who have acute myeloid leukemia (AML), other acute leukemia, and who have adequate renal and liver function will be included (168). In addition, a phase I trial (NCT05534529) is evaluating the PKs, safety, and tolerability of rezafungin in children.

A number of studies on rezafungin were discontinued. The safety and efficacy of a rezafungin 3% gel and rezafungin 6% ointment for treating moderate-to-severe acute vulvovaginal candidiasis were evaluated in the phase II RADIANT study (identifier NCT02733432) (Table S2) in comparison with oral fluconazole 150 mg daily. Although both formulations were safe and well tolerated, clinical and mycological cure rates at days 7, 14, and 29 were inferior to those of fluconazole and the trial discontinued (203, 204). Future studies should consider formulation modification with either increased or prolonged vaginal exposures to the active medication or the use of higher doses.

Studies on combination therapy including the new antifungals for treating yeast infections

First-generation echinocandins are well studied in combination with other antifungals against Candida species both in in vitro and in vivo animal models (205). However, there are no published results for rezafungin.

Echinocandin-azole interactions have ranged from synergistic to antagonistic effects with (205) results dependent on the Candida species and antifungal agent studied (206). Other studies have evaluated AmB-echinocandin combinations; synergistic results were observed in immunosuppressed mice using liposomal AmB (LAmB) combined with caspofungin where there was complete clearance of infection (207). In vitro studies have also been performed with echinocandin/AmB treatments against Candida biofilms on venous catheters (208) where combined anidulafungin/AmB at lower concentrations produced a significant increase in antifungal activity compared with the individual agent alone. Whether these observations can be extended to rezafungin is not yet known. There have been no reports of in vitro antagonism of rezafungin with commonly used antifungals (164).

Perspective on the future use to treat yeast infections

The promise of rezafungin lies in its prolonged half-life, favorable safety profile, and its “front-loaded” drug exposure that may limit the development of resistance. The fact that it can be given once weekly also has implications for reducing healthcare costs.

Projecting the future, a large part of rezafungin’s role would be in the targeted therapy of invasive candidiasis. Potential areas for use include those scenarios where prolonged therapy is required [e.g., in intra-abdominal candidiasis, for those requiring daily echinocandin therapy to ease patient healthcare burden, and those with resistant or refractory infection that may benefit from the unique PK parameters of rezafungin (e.g., endocarditis)] (209). The high front-loaded exposure and prolonged half-life may potentially allow for earlier hospital discharge. Such extended outpatient access with completion of therapy via once-weekly infusion may particularly benefit those with non-sanctuary sites of invasive candidiasis and even for candidemia. Until more data are available, rezafungin may initially be reserved for those indications where it will provide additional value. A recent analysis of the ECMM Candida III study has outlined that 16% of patients with candidemia required prolonged hospitalization, and another 4% received outpatient parenteral antifungal therapy (31), indicating that one out of five patients with candidemia may benefit from rezafungin right away.

A recent pharmacoeconomic study compared real-world costs of illness for patients with invasive candidiasis in a single hospital and modeled potential cost savings of rezafungin therapy based on the 5-day reduction of ICU length of stay observed (210). These findings are intriguing, and additional studies should be performed to fully evaluate potential cost savings with treatment. The ongoing phase III prophylaxis trial in HSCT recipients will provide valuable data in this population. A reduction in daily pill burden, given the anti-Candida, Pneumocystis, and Aspergillus activity of rezafungin, may be particularly attractive in this patient group. Other populations to study include liver transplant recipients or those with abdominal surgery with anastomotic leak. Finally, data are required in invasive candidiasis forms, such as endophthalmitis, and sanctuary-site infections.

Notwithstanding the potential niche areas of rezafungin above, further studies addressing alternate options in patients known to be allergic to the echinocandins are needed. One would expect that alternate antifungal treatment would be instituted for all echinocandin-allergic patients depending on IFD. The theoretical risk of in vivo development of resistance (FKS mutant selection) resulting from prolonged exposure to the drug with subtherapeutic levels for at least part of the treatment period is a valid concern. To our knowledge, there have been no signals thus far, either in vitro or clinical studies, to indicate clinical impact.

Ibrexafungerp

Ibrexafungerp (Brexafemme; formerly SCY-078) is a novel oral glucan synthase inhibitor representing the first agent of the triterpenoid antifungals (SCY-247 is now in early stage development as the second agent in this class and has been FDA approved for treatment of VVC) (211). Ibrexafungerp inhibits the biosynthesis of BDG in the fungal cell wall in a similar fashion to echinocandins and provides fungicidal activity against Candida spp. and fungistatic activity on Aspergillus spp., including azole-resistant strains (212), with only partial overlap in of BDG sites between ibrexafungerp and echinocandins, and there is limited cross-resistance between both antifungals (213).

Ibrexafungerp has in vitro activity against several Candida spp. (e.g., C. albicans, C. glabrata/N. glabratus, C. krusei/P. kudriavzevii, C. tropicalis), including C. auris (214–216) and C. glabrata/N. glabratus isolates, with FKS mutations causing echinocandin resistance. However, some Candida FKS mutants also have shown reduced susceptibility to ibrexafungerp (213), indicating that the antifungal activity of ibrexafungerp against echinocandin-resistant Candida spp. cannot uniformly be predicted (217). In a mouse model, ibrexafungerp showed good activity against fluconazole-resistant C. auris infections with the improvement of survival and reduction of kidney fungal burden (216). Ibrexafungerp is active against Aspergillus (e.g., A. fumigatus, A. niger, A. terreus), Cladosporium, and Alternaria but notably has no activity against the Mucorales nor Fusarium. In vitro investigation of ibrexafungerp activity against Mucorales in combination with AmB or posaconazole led to a significant reduction in fungal growth compared to untreated controls or ibrexafungerp alone (218).

Past and ongoing clinical trials for treating yeast infections

Ibrexafungerp was FDA approved for treatment of VVC in June 2021 (219). In one study performed in the United States (VANISH 303), patients with VVC were randomly assigned 2:1 to receive ibrexafungerp (300 mg orally twice for 1 day) or placebo. The primary endpoint was the percentage of patients with clinical cure defined as complete resolution of VVC signs and symptoms at test of cure (11 ± 3 days). Secondary endpoints included clinical improvement at test of cure (vulvovaginal signs and symptoms ≤1), symptom resolution at follow-up (25 ± 4 days), percentage of patients with mycological eradication, and overall success (clinical cure and mycological eradication). Results reported from 188 patients receiving ibrexafungerp and 98 receiving placebo showed that patients treated with ibrexafungerp had higher rates of clinical cure (50.5% vs 28.6%), clinical improvement (64.4% vs 36.7%), mycological eradication (49.5% vs 19.4%), complete resolution of symptoms at day 25 (59.6% vs 44.9%), and overall success (36% vs 12.6%). Ibrexafungerp was well tolerated, and the adverse events were mild in severity and primarily gastrointestinal (220).

In another study with comparable design performed in the United States and Bulgaria (VANISH 306; an extension of the VANISH 303 study), patients suffering from VVC received ibrexafungerp (n = 298) or placebo (n = 151). Ibrexafungerp again showed statistically better outcomes compared to placebo for primary and secondary endpoints (221). Another study compared two different ibrexafungerp dosages (1,250 mg loading dose, followed by 750 mg once for 2 days; or 1,250 mg loading dose followed by 750 mg once for 4 days) and fluconazole (150 mg once for 1 day) in patients with moderate-to-severe VVC (96 in total, 32 in each group). The rate of clinical cure at day 24 after randomization was 78.1% for ibrexafungerp (both dosages combined) and 65.6% for fluconazole, and mycological eradication was 70.3% and 68%, respectively (222).

A dose-finding study investigating oral ibrexafungerp vs fluconazole in subjects with acute VVC (DOVE) recruited patients in five different ibrexafungerp dosage groups or fluconazole 150 mg once. Based on gastrointestinal side effects with larger doses without corresponding increase of efficacy and patient convenience, ibrexafungerp 300 mg twice for 1 day was selected for further VVC studies, and presentation of results was focused on this dose. There were no differences between 27 ibrexafungerp- and 24 fluconazole-treated patients (clinical cure 51.9% vs 58.3% at day 25) (223). Ibrexafungerp-treated patients rarely required rescue medication compared to fluconazole (3.7% vs 29.2%). Ibrexafungerp (300 mg twice for 1 day) was associated with 46.7% treatment-related adverse events (25% in fluconazole-treated patients), which were self-limited (generally 1-day duration) and mostly mild-to-moderate gastrointestinal events (223).

Final data from an already completed study evaluating the efficacy and safety of ibrexafungerp in patients with fungal diseases refractory or intolerant to standard antifungal treatment (FURI, NCT03059992) are currently under review with publication forthcoming. Several interim analyses from this multicenter study, however, have been presented at various conferences (224–226). In one of the most recent presentations of interim FURI data, ibrexafungerp was prescribed to 113 patients in total, of which 49.5% had invasive candidiasis/candidemia, 28.3% had mucocutaneous candidiasis, 12.4% had VVC, and 9.7% had aspergillosis. Complete or partial response or clinical improvement in VVC (signs and symptoms ≤1) was achieved in 58.4% of patients, stable disease in 23.9% of patients, and 11.5% had progression (including two VVC patients). There was one death due to underlying disease and six indeterminate outcomes (224, 225). Overall, ibrexafungerp demonstrated potent activity against most fungal isolates from FURI patients, including isolates that demonstrated resistance against fluconazole, micafungin, or voriconazole (226). A subgroup of 17 patients receiving ibrexafungerp in the FURI study had intra-abdominal candidiasis. Most patients were enrolled based on refractoriness to current treatment (82%), and 71% of those patients had previously failed echinocandin therapy. Non-Candida albicans isolates were predominant (67%), with C. glabrata/N. glabratus the most common organism. Complete or partial response was 53%, stable disease 41%, and progression of disease 6%. There was one death not attributed to the fungal disease (227).

Seven patients with Candida urinary tract infections and recruited in FURI (two patients) and CARES (study to evaluate the efficacy and safety of oral ibrexafungerp in patients with candidiasis caused by C. auris) (five patients) received a loading dose of oral ibrexafungerp 750 mg twice a day for the first 2 days followed by oral ibrexafungerp 750 mg once daily. All patients showed a favorable response to ibrexafungerp with 86% complete response and partial response in one patient (one C. auris case) (228).

Recently, a study evaluating the efficacy and safety of oral ibrexafungerp as step-down therapy following intravenous echinocandin for the treatment of invasive candidiasis was started (MARIO, NCT05178862) (229). In this study, patients receiving intravenous echinocandin therapy and qualifying for oral step-down will be randomized to either fluconazole or ibrexafungerp in case of fluconazole-susceptible isolates. Patients with fluconazole-resistant isolates will be switched to either ibrexafungerp or best available treatments.

Studies on combination therapy including the new antifungals for treating yeast infections

While data on ibrexafungerp combination therapy for treating yeast infections are very limited, some data exist for combinations against molds. Investigations of ibrexafungerp with triazoles against Aspergillus spp. in a murine model of infection showed enhanced activity of ibrexafungerp in combination with azoles (isavuconazole), resulting in prolonged survival, decreased pulmonary injury, reduced residual fungal burden, and lower fungal biomarker (galactomannan, BDG) levels in comparison to monotherapy (230, 231). Murine models have also shown synergy with AmB for treating mucormycosis (212, 218, 232). Clinical experience with ibrexafungerp combination remains very limited; in one report, two cases were treated with ibrexafungerp/LAmB combination for Candida krusei/P. kudriavzevii arthritis (limited benefit of the combination) and refractory invasive pulmonary aspergillosis (IPA; successful combination treatment), respectively (233).

Perspective on the future use to treat yeast infections

Ibrexafungerp has already been approved for treatment of VVC in June 2021 but has the potential for other indications due to its broad spectrum of activity, including against Candida isolates resistant to other antifungals, resistance in other fungal species, oral availability, limited contraindications, low potential of drug-drug interactions, and high tissue penetration. Potential indications include oral treatment of invasive candidiasis/candidemia or completion of treatment following intravenous echinocandins, C. auris infections, and azole-resistant mold infections (alone or in combination with AmB lipid formulations) as well as prophylaxis against Candida and Aspergillus spp. in high-risk patients.

Encochleated amphotericin B

Encochleated AmB (CAmB, formerly MAT2203) is a novel formulation of AmB in which the antifungal is trapped in a nanocochleate structure consisting of a multilayer composed of lipid bilayers and calcium ions (234). The cochleated structure provides protection against degradation in the gastrointestinal tract allowing oral administration and targeted intracellular delivery of AmB into macrophages and reticuloendothelial cells. The active substance of the nanocrystal formulation is amphotericin and as such binds to ergosterol within the fungal cell membrane which causes membrane disruption. When the cochleate form is taken up by phagocytic cells, a calcium concentration gradient between the cochleate and cytoplasm is created, upon which the cochleate opens and the AmB molecules are released (234). This unique mechanism may serve to reduce toxicity, given the reduction in systemic exposure.

Two pathways can be envisioned for CAmB cochleates mechanism of action—Pathway 1 (Path-1) is the macrophage pathway; both activated and inactivated macrophages take up cochleates and target the fungal cell. Pathway 2 or Path-2 involves uptake by the fungal/yeast cells of rhodamine-labeled particle rapidly in a cell culture or biofilm. Growing yeasts, like Candida, will most likely absorb the nanoparticles at the place where there is a cell division, just before the cell wall forms over the cell membrane.

CAmB was shown to cause a similar improvement in survival and reduction in tissue fungal burden as intraperitoneal administration of AmB deoxycholate in a mouse model of systemic candidiasis (235). In a murine model of cryptococcal meningoencephalitis, CAmB was found to have efficacy equivalent to AmB deoxycholate in combination with flucytosine and was superior to oral fluconazole. Transport of fluorescent CAmB particles to the brain could be demonstrated in treated mice. Negligible toxicity was noted in additional toxicity studies consisting of a 28-day treatment schedule (236).

Past and ongoing clinical trials for treating yeast infections

Safety, tolerability, and efficacy of CAmB were evaluated in a phase II open-label dose-escalation study including four patients (three had STAT3 dominant-negative disease) with chronic mucocutaneous candidiasis who were intolerant or resistant to azole antifungal drugs (NCT02629419). Administration of CAmB resulted in clinical efficacy in both oropharyngeal candidiasis and esophageal candidiasis by 2 weeks of treatment with 200 or 400  mg twice-daily dosing. No signs of renal, hepatic, or hematologic toxicity were noted throughout the study period, including throughout the extension phase of up to 60 months (237).

In a phase II multicenter, randomized study (NCT02971007) of CAmB 200 and 400 mg for 5 days compared to a single dose of fluconazole (150 mg) for moderate-to-severe VVC including 137 patients, overall response was lower with CAmB as compared to fluconazole, and no serious adverse events occurred (238).

Two sequential trials were conducted to study CAmB in the setting of cryptococcal meningitis (EnACT trial, NCT04031833) (234, 239). In the phase II trial, 80 Ugandan adults with HIV and cryptococcal meningitis were randomized to oral CAmB + flucytosine with (n = 40) and without (n = 40) 2 intravenous loading doses and 41 control participants receiving intravenous AmB (22/41 liposomal AmB and 19/41 AmB deoxycholate) with flucytosine (239). Survival was 85% in the intravenous AmB control group as well as for the all-oral CAmB group and 90% in the oral group with two intravenous loading doses. Grade 3 and 4 laboratory adverse events occurred less frequently in the CAmB groups. Similar 2-week cerebrospinal fluid sterility was noted with CAmB combined with flucytosine, but there was more variability in Cryptococcus cerebrospinal fluid clearance in the all-oral regimen (239). Patients with cerebrospinal fluid pleocytosis had twofold higher mean rates of fungal clearance, but sample sizes preclude definitive conclusions.

Studies on combination therapy including the new antifungals for treating yeast infections

As mentioned above, CAmB is studied in combination with flucytosine for the treatment of cryptococcal meningitis. No specific studies on synergism and antagonism with other antifungals have been performed on CAmB specifically, and those properties are very likely reflective of what has been found for other polyenes, with synergism particularly with fosmanogepix (68, 240–243).

Perspective on the future use to treat yeast infections

While further research into the exact mode of action is required, CAmB is highly promising for the treatment of cryptococcal meningitis in combination with flucytosine, especially in low- and middle-income settings where the disease burden is the highest (244). Outpatient therapy may become an option with this oral drug as electrolyte monitoring and supplementation appear potentially unnecessary based on early clinical trial results. Advantages include the oral administration with no requirement for intravenous access and stability of the drug at room temperature. Major disadvantages include the inconvenient dosing scheme, requiring six times per day dosing. Future studies are also needed on the potential impact of CAmB on the gut mycobiome, as the oral formulation may have a different impact than intravenous formulations of AmB.

Oteseconazole

Oteseconazole (Vivjoa; formerly VT-1161, SHR8008) is a member of a next generation of azoles, i.e., tetrazoles designed to have greater specificity for the fungal Cyp51 enzyme (lanosterol 14α-demethylase) by replacing the triazole iron-binding group with a tetrazole that has been developed and FDA approved for treatment of VVC. This high specificity (and thereby reduced affinity with human CYP) is associated with reduced clinically significant drug-drug interactions and adverse events. Oteseconazole is available for oral administration at different regimens either alone or in combination with fluconazole (245).

Oteseconazole is active against Candida species, including C. krusei/P. kudriavzevii and fluconazole- and echinocandin-resistant C. glabrata/N. glabratus (MIC₅₀ and MIC₉₀ were 0.25 and 0.5 mg/L for 50 C. krusei/P. kudriavzevii and 0.12 and 1 mg/L for 34 C. glabrata/N. glabratus isolates tested) as well as against Cryptococcus species (246, 247). Overall, the in vitro activity of oteseconazole against C. glabrata/N. glabratus is comparable to those of itraconazole, voriconazole, and posaconazole, and decreased susceptibility to oteseconazole in C. glabrata/N. glabratus is driven by many known resistance mechanisms, and cross-resistance is thus expected (248). Activity was tested against 1,910 clinical isolates (87% C. albicans) from phase III clinical studies in patients with (recurrent vulvovaginal candidiasis) RVVC (249). The MIC₅₀ and MIC₉₀ values of oteseconazole for all isolates were 0.002 and 0.06 mg/L, which were significantly lower than for fluconazole (0.25 and 8 mg/L, respectively). Oteseconazole was also shown to be active against C. albicans biofilms as well as dual-species biofilms (250). No studies were found evaluating the activity of oteseconazole to C. auris.

The administration of a single oral oteseconazole dose (5 mg/kg) in a murine model of vaginal candidiasis revealed high oral bioavailability (73%) and high vaginal tissue levels (251). Doses of 7.5 mg/kg/day (about 3.5 times the steady state clinical exposure seen in patients treated for RVVC) administered to pregnant rats during organogenesis until lactation resulted in offspring with ocular abnormalities. There was no embryo-fetal toxicity or malformations following administration of oteseconazole at 40 mg/kg/day (about 10 times maximum human exposure for RVVC) during organogenesis in pregnant rats (252).

Past and ongoing clinical trials for treating yeast infections

After demonstrating safety and efficacy in phase II studies (NCT02267382, NCT01891331), three large phase III trials, referred to as VIOLET (NCT03562156, NCT03561701) and ultra VIOLET (NCT03840616), were initiated to investigate oteseconazole for treatment of RVVC (253). VIOLET comprised two global, phase III, multicenter, randomized, double-blind, placebo-controlled trials (CL-011 and CL-012). All participants (N = 863) were first treated with oral fluconazole (150 mg, days 1, 4, and 7). Patients with resolved symptoms (N = 656) were assigned to receive either maintenance therapy with oral oteseconazole (150 mg daily during 1 week followed by 150 mg weekly during 11 weeks) or placebo. The average percentage of participants with one or more RVVC episodes through week 48 was 6.7% (CL-011) and 3.9% (CL-012) in the oteseconazole groups vs 42.8% and 39.4% in the corresponding placebo groups. No adverse fetal abnormalities were observed during the eight pregnancies that occurred; 85% of 71 participants who enrolled for an extension study completed 96 weeks without experiencing a VVC episode, with an average time to recurrence of 92 weeks (254).

Oteseconazole was shown to be safe and efficacious in the treatment and prevention of RVVC and was non-inferior to fluconazole for the treatment of acute VVC in the ultraviolet trial (NCT03840616). Only 1 of 580 women treated during phase III clinical trials had to discontinue treatment (due to allergic dermatitis). The most frequently reported adverse reaction was headache (7.4%) and nausea (3.6%).

Most treatment-related adverse events were mild to moderate (255). Based on these results, oteseconazole was approved by the U.S. Food and Drug Administration in July 2022 for the treatment of RVVC in women who are not of childbearing potential.

An important limitation of the currently completed studies is the comparison with placebo instead of fluconazole for the maintenance phase. This is due to regulatory restrictions, as fluconazole is not approved to treat RVVC in the U.S. In a currently ongoing phase, three randomized, double-blind, multicenter study efficacy and safety of oteseconazole will be compared to fluconazole in patients with RVVC (NCT05074602).

Studies on combination therapy including the new antifungals for treating yeast infections

To the best of our knowledge, there are no data available on combination therapy of oteseconazole with other antifungal drugs.

Perspective on the future use to treat yeast infections

Despite the exceptionally low recurrence rates reported in patients with RVVC, the future of oteseconazole remains uncertain. Oteseconazole is not a treatment option for a substantial proportion of women due to the contraindication for females of reproductive potential. Due to the very long half-life, patients are expected to be exposed to the drug for 690 days (five times the t_1/2) after treatment, which precludes adequate mitigation of the embryo-fetal toxicity risk in women of reproductive potential. This may favor ibrexafungerp in the future for the treatment of women with RVVC. The results of a head-to-head comparison with fluconazole for the prevention of RVVC will be important for the future of oteseconazole. The long half-life may be better leveraged in the treatment of cryptococcal infections or molds/endemic fungi.

Manogepix/fosmanogepix

Fosmanogepix (formerly APX001) is a first-in-class intravenous and oral antifungal. Fosmanogepix is a prodrug that is rapidly and completely metabolized by systemic phosphatases to the active moiety manogepix (formerly APX001A). This novel agent interferes with cell wall synthesis by targeting fungal glycosylphosphatidylinositol-anchored cell wall transfer protein causing pleotropic effect with resultant loss of cell viability (21). Manogepix displays wide activity against Candida spp., with the exception of C. krusei/P. kudriavzevii and C. kefyr/Kluyveromyces marxianus. In an in vitro study, combination therapy with anidulafungin and fosmanogepix showed heightened efficacy against both drug-susceptible and drug-resistant strains of C. auris (256). Interestingly, as calcineurin is required for virulence for many fungal pathogens (257), there is growing evidence that calcineurin inhibitors can enhance the fungicidal potential of fosmanogepix through enhanced exposure to immunostimulatory glucans (258).

Past and ongoing clinical trials for treating yeast infections

Safety

Two phase II studies have evaluated the safety of fosmanogepix in patients with Candida infections. In the first phase II, multicenter, open-label, non-comparative, single-arm study evaluating fosmanogepix in non-neutropenic patients with candidemia (NCT03604705), participants received intravenous fosmanogepix 1,000 mg twice daily on day 1 followed by 600 mg intravenously once daily, with optional switch to 700 mg orally once daily after day 4. Although 20/21 (95%) of participants experienced an adverse event, these were thought to be related to candidemia or other underlying conditions and not fosmanogepix. The most common treatment-related adverse events were diarrhea, vomiting, peripheral edema, and pleural effusion. Overall fosmanogepix was well tolerated with no serious adverse events or treatment discontinuations in all 21 participants who were enrolled in the study (259).

Another multicenter, open-label, single-arm phase II study evaluated fosmanogepix for the treatment of candidemia/invasive candidiasis caused by C. auris in adults with limited antifungal treatment options (NCT04148287). The treatment regimen was intravenous fosmanogepix 1,000 mg twice daily over a 3-hour infusion on day 1, followed by 600 mg intravenously daily on day 2 and day 3, followed by 600 mg intravenously daily over a 3-hour infusion or 800 mg orally daily from days 4 to 42. Of nine participants, seven completed the treatment course. Two of those participants experienced serious adverse events, including cardiac arrest, multiple organ dysfunction, pneumonia, and hypotension. All experienced at least one treatment-related adverse event, including constipation, fever, diarrhea, and nausea (260). Safety and tolerability of manogepix are discussed further in the section describing manogepix for treatment of mold infections.

Efficacy

In the aforementioned phase II study evaluating fosmanogepix for the treatment of candidemia (NCT03604705), participants received fosmanogepix for up to 14 days. Of 21 enrolled participants, 1 participant did not have confirmed candidemia within 96 hours of the start of treatment and was excluded from analysis. The primary efficacy endpoint was treatment success, which was defined as clearance of Candida from blood cultures without any additional antifungal treatment and survival at day 42. The primary endpoint was observed in 16/20 (80%) of participants in the mITT population. Negative blood cultures occurred after a mean of 2.4 days after fosmanogepix initiation. Efficacy occurred in isolates resistant to AmB and anidulafungin. Overall, 85% of participants were alive at day 30, and the three deaths were not thought to be related to fosmanogepix (259).

In the phase II study evaluating fosmanogepix for the treatment of patients with candidemia/invasive candidiasis caused by C. auris (NCT04148287), the primary efficacy endpoint was clearance of C. auris from the bloodstream. In the case of a deep-seated infection, at least one negative culture from the affected site or clinical and radiological improvement without any additional antifungal treatment and survival at day 42 were required. Of nine participants, eight (89%) met the primary endpoint. The median time to first negative blood culture was 6 days. One participant was deceased at day 30 (260).

A phase I study of fosmanogepix in persons with hepatic dysfunction (NCT05582187) is currently recruiting participants. Goal enrollment is 18 participants. Another planned study in people with candidemia and/or invasive candidiasis (NCT05421858) has not yet started enrollment. Goal enrollment is 450 participants, with an estimated start date in mid-2024 and completion date of 15 July 2027.

Studies on combination therapy including the new antifungals for treating yeast infections

Synergistic effects have been demonstrated in vitro when fosmanogepix was used with anidulafungin against C. auris (256) as well as synergism with calcineurin inhibitors (258).

Perspective on the future use to treat yeast infections

Fosmanogepix may be used widely for antifungal prophylaxis and empirical treatment of fungal infections. Specifically for yeast, the lack of activity against C. krusei/P. kudriavzevii may pose a challenge for treatment of Candida infections when isolate identification is not available. However, the broad activity against other Candida spp., including multiresistant isolates, may create a niche for fosmanogepix to be utilized for invasive candidiasis and candidemia caused by Candida spp. other than C. krusei/P. kudriavzevii. It also has a broad spectrum of activity against non-Candida yeast, including difficult-to-treat yeast such as Trichosporon asahii, Exophiala dermatitidis, and Malassezia furfur.

NOVEL ANTIFUNGALS FOR PNEUMOCYSTIS INFECTIONS

Pneumocystis jirovecii pneumonia (PJP) is a severe and potentially life-threatening fungal infection. The vast majority of PJP infections occur in immunocompromised patients (261, 262). Trimethoprim-sulfamethoxazole (TMP-SMX) has been the gold-standard antimicrobial for PJP prophylaxis and treatment for decades (263, 264) and has proven high effectivity for pneumocystosis (265, 266). Nevertheless, the mortality rate of PJP, particularly in the HIV-negative patient population, is still high (267–269), and TMP-SMX-associated toxicities, including rash, fever, nephrotoxicity, bone marrow suppression, electrolyte disorders, and hepatotoxicity, require a change of treatment in up to 40% of patients (270–272), and resistance mechanisms to atovaquone have also been described (273). New PJP management strategies, including new antifungals and/or antifungal combination therapy, may overcome some of the limitations with current available antimicrobials and may improve the overall survival (274).

Rezafungin

Echinocandins have been proposed as an alternative treatment option for PJP (275) as they inhibit BDG synthases and, consequently, BDG production. However, BDG is only present in Pneumocystis asci (=cyst form) and not in the trophic form that, however, dominates in acute infection by approximately 10:1 over the asci (276, 277). Caspofungin, anidulafungin, and micafungin have demonstrated good in vitro activity against various Pneumocystis species (including P. jirovecii) with some inter-class variability (278, 279). The newly developed long-lasting echinocandin rezafungin may also be an effective antifungal in the prevention of P. jirovecii biofilm formation and reduction of viability of mature P. jirovecii biofilms (280).

Efficacy of rezafungin has been investigated in animal models for prevention and treatment of Pneumocystis pneumonia. In dexamethasoneimmunosuppressed mice infected with P. murina, rezafungin signifcantly reduced the trophic form and asci burden in contrast to control mice, with exception only of mice receiving low-dose rezafungin (0.2 mg/kg) where no significant reduction of fungal burden was observed (197, 281). A rezafungin dose of 20 mg/kg (the human equivalent in mice) was comparable to TMP-SMX in the clearance of the trophic form as well as P. murina asci (197, 281). In both groups, no asci or trophic forms could be observed microscopically following prophylaxis dosing experiments (197). Rezafungin prophylaxis for at least 4 weeks also prevented the re-activation of P. murina infection 6 weeks after discontinuation of rezafungin, despite ongoing immunosuppression (281). This finding is interesting, as other echinocandins effectively reduce the number of BDG-containing asci forms but not non-BDG-containing trophic forms, and infections re-activated after dicontinuation of prophylaxis (276). The high efficacy of rezafungin may be based on the long-acting pharmacokinetic and the fact that P. murina, as well as other Pneumocystis spp., may be highly depended on sexual reproduction resulting in formation of BDG-containing asci.

Treatment with rezafungin in an established P. murina pneumonia mouse model was also associated with a significant reduction of P. murina asci and trophic forms compared to the untreated control group but did not show survival differences (280).

In animal models, rezafungin seems to perform at least as well as the current gold standard for pneumocystosis prophylaxis and treatment (TMP-SMX), warranting further evaluation in clinical trials.

Past and ongoing clinical trials for treating Pneumocystis infections

In the ReSPECT trial (NCT04368559), rezafungin is currently under evaluation for the prevention of IFDs (including PJP) in allogeneic stem cell transplant recipients. In this randomized, double-blinded trial, subjects will receive either standard-of-care antimicrobial prophylaxis (fluconazole or posaconazole plus TMP-SMX) or rezafungin for a total duration of 13 weeks. The trial is currently enrolling subjects, and primary study completion is expected for August 2024.

In a randomized phase II trial, rezafungin is under evaluation as primary therapy in combination with TMP-SMX in HIV-positive adults with mild, moderate, or severe PJP vs TMP-SMX monotherapy (NCT05835479). A combination approach for treamtent of PJP may be very promising as the combination of an echinocandin plus TMP-SMX not only covers all stages of the Pneumocystis spp. life cycle but may also allow for dose reduction of TMP-SMX and therefore reduce TMP-SMX-associated toxicity and improve tolerability (274).

Studies on combination therapy including the new antifungals for treating Pneumocystis infections

To the best of our knowledge no data are available for rezafungin combination in PJP.

Perspective on the future use to treat Pneumocystis infections

In conclusion, BDG inhibition with new antifungals may be a promising future tool for PJP prevention and treatment. BDG synthase inhibitors are generally well tolerated with few adverse events. Most data exist for rezafungin; however, interventional clinical trials comparing rezafungin to TMP-SMX have just recently been launched, and results will be expected for the next years.

Ibrexafungerp

As no in vitro culture model for Pneumocystis has been established, in vitro data for other BDG synthase inhibitors like the first-in-class triterpenoid antifungal ibrexafungerp are lacking, but the mode of action indicates activity against Pneumocystis species and was therefore further studied in animal models.

In a dexamethasone-immunosuppressed mouse model, ibrexafungerp prophylaxis: 7.5, 15, and 30 mg/kg BID significantly reduced P. murina asci and trophic forms vs the control group, and in the group of mice that received 30 mg/kg BID, ibrexafungerp performed similar to TMP-SMX (282). With the 30 mg/kg BID dose, no more asci could be observed in mice after 6 weeks of prophylaxis (283).

For the treatment model, ibrexafungerp at doses of 15 and 30 mg/kg BID performed significantly better than TMP-SMX in reducing asci burden at day 7. In addition, ibrexafungerp at both doses reduced the fungal load of P. murina (asci and nuclei) at day 14 and day 21.

While animal models showed activity of ibrexafungerp in murine Pneumocystis jirovecii treatment and prophylaxis studies (283), to the best of our knowledge, no clinical evaluation of ibrexafungerp against PJP has been performed or is currently planned; therefore, the role of ibrexafungerp in Pneumocystis jirovecii prophylaxis or treatment is yet to be defined.

NOVEL ANTIFUNGALS FOR MOLD INFECTIONS

Olorofim

Olorofim (formerly F901318) is the first of the new orotomide class of antifungal agents and acts by reversibly inhibiting the DHODH enzyme that plays a crucial role in the biosynthesis of pyrimidine (284). Inhibition of DHODH disrupts the synthesis of uridine-50-monophosphate and uridine-50-triphosphate, which are vital for generating various cell wall components, cytosine, thymine, and uracil, and are essential for cell cycle regulation. Exposure of susceptible fungi to olorofim leads to cell cycle arrest and eventual cell lysis (284–286). It is active in vitro against Aspergillus spp., Scedosporium spp., Lomentosporium spp. (287–289), certain other rare molds (290, 291), dermatophytes (292), and dimorphic fungi including Coccidioides immitis (293) but has no activity against agents of mucormycosis, Candida spp., and Cryptococcus spp. (57). Of note, an antagonistic action of antifungal triazoles on the action of olorofim has been demonstrated in vitro with yet unknown implications for efficacy in vivo (294).

Olorofim has shown promising activity against mold infections in animal models. In a study of mice with neutropenia and chronic granulomatous disease who were infected with Aspergillus spp., olorofim-treated mice had lower fungal burden and improved survival at 10 days compared to the control group (295). In neutropenic mice infected with Scedosporium spp. and L. prolificans, fungal burdens and survival at day 10 were significantly lower in the olorofim-treated group compared to the control group (296).

Past and ongoing clinical trials for treating mold infections

Safety

Four clinical studies report data on safety and tolerability of olorofim. In a phase I study (NCT02142153) of 40 healthy male volunteers receiving a single intravenous infusion of olorofim or placebo, there were no serious adverse events. Paresthesia occurred in one participant receiving 0.75 mg/kg of olorofim and headache in another participant receiving 1.5 mg/kg of olorofim. Epistaxis occurred in two participants receiving olorofim (0.25 and 4 mg/kg) and one participant receiving placebo; eczema occurred in one participant receiving 3 mg/kg of olorofim (297).

In another phase I study of eight healthy male volunteers, olorofim given intravenously over a 5-day period, infusion site reactions and dizziness were the most commonly reported adverse events. Infusion site pain or phlebitis occurred in 44% and 39%, respectively, of individuals receiving olorofim compared to 17% of participants experiencing these side effects who received placebo. Dizziness was reported in 67% of individuals receiving olorofim compared to 17% who received placebo, and the intravenous formulation, as a consequence, has not been further developed (298). In a third phase I multiple-dose pharmacokinetic study of 10 healthy volunteers, intravenous olorofim was well tolerated, without serious adverse events. Mild adverse events included an increase in alanine transaminase (ALT) (2/10), nausea and diarrhea (1/10), and dizziness (1/10) (299).

In a single ascending dose study of olorofim in tablet form given to 40 healthy male volunteers, no serious adverse events were reported. Non-serious adverse events occurred slightly more frequently in participants taking olorofim compared to placebo. The occurrences were as follows: extremity pain in 0/30 participants in the olorofim group vs 1/10 in the placebo group, paresthesia and headache each occurred in 1/30 in the olorofim group vs 0/10 in the placebo group, epistaxis in 2/30 in the olorofim group vs 0/10 in the placebo group, and eczema in 1 out of 30 in the olorofim group vs 0/10 in the placebo group (297).

For study 32 (FORMULA, NCT03583164), a single-arm phase IIb study where olorofim was used for probable or proven IMIs in patients with limited or no treatment options, results from 202 enrolled patients were recently presented and published as an abstract (300). Olorofim was overall well tolerated, with a median duration of 84 days of treatment in the main study phase and up to 988 days in the extended protocol. The main adverse event, by FDA also classified as an adverse event of special interest, was elevation of hepatic transaminases, which occurred and judged at least possibly related to olorofim in 9.9% of subjects. This elevation of transaminases occurred typically within the first 12 weeks of dosing was usually managed successfully by olorofim dose reduction or pausing but required olorofim discontinuation in 2.5% of subjects enrolled (300). Diarrhea, nausea, and vomiting possibly suggestive of mild gastrointestinal upset/intolerance were noted in 9.9% of subjects enrolled (300).

Efficacy

There are currently no published clinical trial data on the clinical efficacy of olorofim. What is known is restricted to studies of published case reports and abstracts presented at conferences.

In the FORMULA study, patients with infections due to L. prolificans, Scedosporium spp., Aspergillus spp., Coccidioides spp., and other invasive molds with resistance to commercially available antifungal drugs or for patients refractory to traditional therapy were enrolled. Goal enrollment of this study was 200 participants. Interim analysis of outcomes of the first 100 patients was presented at ID week 2022, showing day 100 all-cause mortality in 17/53 (32%) of patients with IA and limited or no treatment options (301). Final results of 202 enrolled patients were recently presented at TIMM 2023 (300). Among patients with IA with limited or no treatment options, Data-Review-Committee-adjudicated partial or complete response was observed in 35/101 (34.7%) at day 42 and 34/101 (33.7%) at day 84, with all-cause mortality of 26/101 (25.7%) at day 84. For Lomentospora prolificans, partial or complete response was observed in 11/26 patients (42.3%) at days 42 and 84, with all-cause mortality of 11.5% (3/26). Partial or complete response was also observed in patients with Scedosporium spp. infections (8/22, 36.4% at day 42 and 5/22, 22.7% at day 84) with all-cause mortality observed in 9.1% (2/22) at days 42 and 84, and in patients with Scopulariopsis spp. infections (5/6, 83.3% at days 42 and 84 with no mortality) (300).

Other efficacy data are limited to case reports. In one case study, a 56-year-old Australian woman with acute T-cell lymphoblastic leukemia (ALL) was administered a combination of cyclophosphamide, vincristine, doxorubicin, and dexamethasone (Hyper-CVAD) and developed disseminated L. prolificans infection with fungemia, endophthalmitis, and osteomyelitis. She did not respond to dual antifungal therapy with voriconazole plus terbinafine and after 11 months from the onset of infection was started on olorofim monotherapy (loading dose of 180 mg followed by 60 mg twice daily followed by 90 mg twice daily). After 6 months, she developed radiologic improvement with decreased uptake on positron emission tomography scan and clinically improved with weight gain and stabilization of vision. She ultimately received a year of olorofim without any adverse effects (302).

In another case report, a 49-year-old Australian woman developed disseminated L. prolificans infection following a right breast implant, which spread to her soft tissue, ribs, and sternum. The implant was removed and infection site debrided, and she was treated with multiple antifungal agents, including voriconazole plus terbinafine, miltefosine, posaconazole, and anidulafungin, yet her infection persisted. She was administered olorofim (60 mg twice daily followed by 90 mg twice daily followed by 120 mg twice daily) for a total of 322 days. This regimen was well tolerated, and eventually, the surgical site healed (303).

OASIS, a phase III study evaluating olorofim vs treatment with LAmB followed by standard of care in patients with IA is currently ongoing (NCT05101187). Goal enrollment is 225 participants with study completion estimated to occur in March 2025.

Studies on combination therapy including the new antifungals for treating mold infections

To the best of our knowledge, there are no published results outlining synergism with other antifungal classes, while one study showed antagonism between olorofim and azoles (294). In a subanalysis of study 32 presented recently at TIMM 2023, 29/100 patients received olorofim with an azole (most often fluconazole; n = 13). Most of those 29 patients had IA (n = 16), while two of each had IFI due to L. prolificans and Scopulariopsis spp. and one due to Scedosporium spp. Similar efficacy outcomes were observed at day 42 in patients receiving olorofim with an azole in combination (41.4%; 12/29) vs those receiving olorofim monotherapy (45.0%; 32/71), with also similar outcomes at day 84 and regarding all-cause mortality rates (304).

These results indicate there is no meaningful reduction of clinical efficacy from a treatment combination of olorofim with azole drug class and no evidence of any clinical antagonism, while future studies are needed that focus specifically on combination with mold-active azoles to evaluate a potential benefit.

Perspective on the future use to treat mold infections

Olorofim will likely play an important role in the treatment of drug-resistant mold infections, including azole-resistant aspergillosis, the treatment of L. prolificans, which is typically resistant to most or all antifungal agents (30, 305, 306), and the treatment of Scedosporium spp. infection.

Given the favorable safety profile and fewer drug-drug interactions, olorofim will likely be used widely as oral step-down or salvage treatment for IA, while lack of a parenteral formulation may pose challenges to use olorofim as a first-line agent for IA, particularly in ICU settings and other settings where oral treatment is not an option. While some studies have indicated that the bioavailability when given via nasogastric tube was 87% of the oral bioavailability, there will likely still be hesitancy for utilization of the drug for first-line treatment of IA when other options are available. In contrast, olorofim will likely emerge as first-line treatment option for infections caused by Lomentospora prolificans but also Scedosporium spp., Scopulariopsis spp., and some Fusarium spp. that are resistant to currently available antifungals.

Manogepix/fosmanogepix

Manogepix displays wide activity against molds. In a mouse model, combination treatment with fosmanogepix plus LAmB was superior to monotherapy with either fosmanogepix or LAmB for the treatment of invasive pulmonary aspergillosis, invasive mucormycosis, and invasive fusariosis (242).

Past and ongoing clinical trials for treating mold infections

Safety

The safety and tolerability of fosmanogepix have been evaluated in four phase I clinical trials. The first study (NCT02956499) was a randomized, double-blind, placebo-controlled, single ascending dose, and multiple ascending dose-escalation study. Six cohorts of eight healthy volunteers received a single 3-hour infusion of 10, 30, 100, 200, 275, or 350 mg of fosmanogepix or placebo. Four cohorts of eight healthy volunteers received 50, 150, 300, or 600 mg daily over a 14-day period. Fosmanogepix was well tolerated at all doses with no serious adverse events. Headache was the most commonly reported adverse event with other adverse events being mild, transient, and requiring no treatment (307).

In a second phase I randomized, double-blind, placebo-controlled study (NCT02957929), healthy subjects were randomized to receive placebo or a single intravenous dose of 200 mg infused over 3 hours followed by a single oral tablet dose of 100, 300, and 500 mg separated by a 14-day washout period. In addition, participants were given a dose of 400 mg under both fed and fasting states. In this study, fosmanogepix was well tolerated without any serious adverse events observed. As with the first phase I study, adverse events were mild, transient, and resolved without treatment (308).

A third phase Ib open-label, multicenter study evaluated intravenous and oral fosmanogepix in patients with acute myeloid leukemia and neutropenia (NCT03333005). Of 21 adults with AML undergoing remission induction chemotherapy, 10 patients received intravenous fosmanogepix 600 mg every 24 hours, and 11 received oral fosmanogepix 500 mg every 24 hours, both over 14 days with 28 days of follow-up. This was in conjunction with remission induction chemotherapy with sequential high-dose cytarabine and mitoxantrone (S-HAM) or 7 + 3 regime plus posaconazole IFD prophylaxis. No serious adverse events were reported. The most frequent adverse events were mild and included nausea/vomiting, increase in ALT level, and delirium. One patient experienced fosmanogepix-related hypertension. Overall, fosmanogepix was safe and well tolerated (309).

Lastly, a fourth phase I study (a drug-drug interaction study of CYP3A4 inhibition and pan-CYP induction on APX001) (NCT04166669) is completed, but results have not been published yet.

A phase II open-label, multicenter study evaluated fosmanogepix for the treatment of invasive mold infections caused by Aspergillus spp. or other rare molds (NCT04240886). This study was terminated to prioritize a randomized comparative phase III trial with the same indication. The dosing regimen was identical to the previously mentioned phase II study (NCT04148287). Of 21 participants enrolled, 11 completed the study. All participants experienced at least one treatment-related adverse event, and 13/21 (62%) experienced serious adverse events (310).

Efficacy

At the time of study termination, in the phase II study evaluating fosmanogepix for the treatment of invasive mold infections caused by Aspergillus spp. or other rare molds (NCT04240886), 8/20 (40%) achieved treatment success by day 42, including 4/20 (20%) achieving complete response and 4/20 (20%) partial response. Sixty percent achieved treatment failure, including 2/20 (10%) with a stable response, 6/20 (30%) progression, and 4/20 (20%) death at day 84 (310).

Fosmanogepix has also been successfully utilized for treating cases with CNS fusariosis occurring as part of the recent outbreaks in Durango and Matamoros, Mexico, where it became the treatment standard (6, 311).

After fosmanogepix has been acquired by Basilea, phase III studies for mold infections, potentially for the salvage setting and in combination with LAmB, are currently in the planning stages.

Studies on combination therapy including the new antifungals for treating mold infections

Fosmanogepix has been shown to exhibit synergistic effects with AmB in a mouse model, leading to reduced lung and brain fungal burden as well as increased survival rates in invasive pulmonary aspergillosis and mucormycosis (242, 312). The combination of fosmanogepix with LAmB may therefore be a very promising treatment option.

Perspective on the future use to treat mold infections

Given its broad spectrum of activity, fosmanogepix will likely play an important role in the treatment of drug-resistant mold infections, including from Fusarium spp., Scedosporium spp., and L. prolificans. Fosmanogepix will also likely emerge as salvage treatment option (potentially in combination with LAmB) and potential first-line option for treating IA, particularly if there is concern for Aspergillus/Mucorales co-infection (24, 313). In combination with LAmB, fosmanogepix may also be utilized for treatment of mucormycosis (313). Availability as oral and parenteral formulation as well as its CNS penetration may allow utilization of the drug for long-term treatment of complicated mold infections.

BAL2062

BAL2062 (formerly GR-2397, VL-2397, ASP2397) is a first-in-class antifungal, siderophore-like molecule that resembles ferrichrome; however, it is differentiated by three amino acid changes and an aluminum rather than iron chelate (63). BAL2062 is transported into fungal cells via the Sit1 transporter, which is not found in humans, leading to fungal specificity. BAL2062 shows fungicidal activity against Aspergillus spp., including azole-resistant strains, activity against Fusarium solanii, and C. glabrata/N. glabratus. Safety has been demonstrated in a phase I first-in-human, randomized, double-blind, placebo-controlled dose-escalation study (NCT02956499), where no serious adverse events occurred, and the most common drug-related TEAEs were infusion site reactions (314). The safety profile, consistent PK, and lack of drug accumulation support further development of BAL2062 in patients with invasive aspergillosis.

NOVEL ANTIFUNGALS FOR ENDEMIC FUNGAL INFECTIONS

The global burden of the endemic mycoses (blastomycosis, coccidioidomycosis, histoplasmosis, paracoccidioidomycosis, sporotrichosis, and talaromycosis) continues to rise yearly and remains a significant cause of patient morbidity and mortality worldwide (315). These infections differ substantially in their geographic regions of endemicity, disease manifestations, and the approach to diagnosis and treatment. The presenting manifestations of infection may be acute, with severe life-threatening disease readily apparent, while others have subclinical disease that may reactivate at a later time if immunosuppression occurs (316). The treatment course thus varies substantially and ranges from no therapy (subclinical disease in the immunocompetent patient) to years or lifelong antifungal treatment (315).

Patients requiring long courses of antifungal therapy frequently exhibit drug-related adverse effects, and these may be cumulative. The nephrotoxicity, acid-base, and electrolyte disturbance of currently available AmB formulations are well known and obviate attempts for long-term therapy (317). The triazole class is frequently prescribed to those requiring long courses of treatment. Fluconazole has poor efficacy in the treatment of several of the endemic mycoses (315) and, when used in the treatment of coccidioidomycosis, is a frequent cause of xerosis, cheilitis, alopecia, and malaise, and self-discontinuation is not uncommon (318, 319). Itraconazole is limited by food and gastric pH effects for adequate absorption and may cause gastrointestinal disturbance, peripheral edema, or heart failure due to the negative inotropic effects (320, 321). Voriconazole is a significant photosensitizer (322), and rash is frequent in addition to hepatotoxicity or hyperfluorosis (323), and therapeutic drug monitoring is required (324). Posaconazole may cause drug-induced hypertension (325), and little data regarding isavuconazole efficacy in the treatment of endemics have thus far been presented (326). Novel antifungal agents are therefore urgently needed in this patient group.

Fosmanogepix

Fosmanogepix exerts pleiotropic effects within the host, and in vitro testing may not fully predict the spectrum of in vivo activity (327). Recent work evaluating the minimal effective concentration (MEC) causing abnormal hyphal growth (short abundant branching) in a broth microdilution assay found low values of tested isolates against the endemic mycoses (<0.008 µg/mL). Evaluation in a murine model of coccidioidomycosis demonstrated efficacy in both organ burden and survival studies (328). Survival in mice was significantly longer in the fosmanogepix-treated group compared to those receiving fluconazole.

Past and ongoing clinical trials for treating endemic mycoses

Human data in the treatment of the endemic mycoses are limited, although successful treatment of refractory disseminated coccidioidomycosis has been observed (GRT personal communication). Data regarding in vitro or in vivo effectiveness in other endemic mycoses are not yet available.

Perspective on the future use to treat endemic mycoses

The growing burden of the endemic mycoses globally will continue to see patients refractory, resistant, or intolerant to currently available agents. The efficacy of fosmanogepix and the potential for therapeutic CNS/cerebrospinal fluid drug concentrations mandate evaluation in these difficult-to-treat infections. Fosmanogepix has good activity against some of the commonly encountered dimorphic fungi, including Blastomyces, Coccidioides, and Histoplasma spp., and the potential for therapeutic CNS/cerebrospinal fluid drug concentrations, as well as its oral bioavailability mandates evaluation in these difficult-to-treat infections.

Olorofim

The activity of olorofim against the pezizomycotina includes the endemic mycoses. Low in vitro MICs have been observed for each of the endemics with MIC₉₀ values <0.015 µg/mL. In a murine model of coccidioidomycosis of the central nervous system, olorofim was highly active against Coccidioides spp., with decreased fungal burdens and eradication of the organism in some mice, with improved survival at day 30 in mice treated with olorofim compared to controls (293).

Past and ongoing clinical trials for treating endemic mycoses

A phase II trial of patients refractory, resistant, or intolerant to currently available antifungal agents has included patients with endemic mycoses (coccidioidomycosis = 41, sporotrichosis = 1, histoplasmosis = 2). Efficacy was demonstrated in a significant number of these patients lacking alternative antifungal options (300). While, due to the nature of disease, no mycological eradication was achieved in 41 patients with extrapulmonary coccidioidomycosis, clinical benefit (symptoms/signs) was observed in 31 patients (75.6%) achieving complete or partial clinical response at days 42 and 30 (73.2%) and at day 84 (300).

Olorofim was utilized in a 45-year-old man in the United States with insulin-dependent diabetes mellitus who developed CNS coccidioidomycosis infection which did not respond to antifungal treatment with fluconazole monotherapy followed by voriconazole monotherapy, itraconazole monotherapy, LAmB plus posaconazole, and posaconazole plus micafungin. He continued to clinically deteriorate and, after 8 months, was switched to posaconazole (300 mg daily) plus olorofim (120 mg twice daily). After 3 months, he clinically improved with normalization of his cerebrospinal fluid indices with negative cerebrospinal fluid coccidioidomycosis serology, and his serum Coccidioides complement fixation (CF) titer had decreased from 1:128 to 1:64. After 5 months of therapy, his Coccidioides CF titer had decreased to 1:32, and he was tolerating both antifungal agents well (329).

Perspective on the future use to treat endemic mycoses

The rapid fungicidal effects of olorofim make this an attractive choice, and future trials focused on shortening the duration of antifungals and potentially curative courses have significant potential. In particular, olorofim may have an important niche in the treatment of disseminated coccidioidomycoses, in which indefinite antifungal treatment is the mainstay due to difficulty in eradicating Coccidioides from the central nervous system, and intolerance to the commonly used azoles is common.

Oteseconazole

In vitro testing against the endemic mycoses has shown MICs for Blastomyces dermatitidis 1.0 µg/mL, Coccidioides of 1.5 µg/mL, and Histoplasma 0.43 µg/ml. Oral administration of VT-1161 in a murine model of coccidioidomycosis demonstrated oteseconazole-reduced spherule burden in the lungs by 100- to 1,000-fold, leading to improved mouse survival (330). While VT-1161 had similar effectiveness as fluconazole in vivo, it is hoped the better selectivity of this tetrazole antifungal will decrease potential host toxicity.

SUBA-itraconazole

A new formulation of itraconazole, super bioavailability itraconazole (SUBA-itra), has been developed and contains a solid dispersion of itraconazole in a pH-dependent polymeric matrix to enhance absorption (331). Phase I studies in healthy volunteers have shown SUBA-itra has little food or acid effect on bioavailability, a significant advance over the absorption concerns commonly encountered with conventional itraconazole formulations (332, 333) . SUBA-itra was approved by the U.S. FDA in December of 2018.

An open-label randomized comparative trial has been recently published comparing conventional itraconazole to SUBA-itra in the treatment of endemic mycoses (N = 88: Histoplasma N = 51, Blastomyces N = 18, Coccidioides N = 13, Sporothrix N = 6). Primary outcomes were response at day 42 and day 180. On day 42, clinical success was observed in SUBA-itra (69%) and conventional itraconazole (67%); and on day 180, clinical success was seen in SUBA-itra (60%) and conventional itraconazole-itra (65%). There was less drug variability in the SUBA-itra-treated group with similar overall drug concentrations between groups. Fewer adverse events and serious adverse events were seen in the SUBA-itra-treated group as well (334).

Encochleated amphotericin B

The broad-spectrum activity of AmB formulations is well described. AmB is recommended in the treatment of severe disease with each of the endemic mycoses. Following clinical improvement, patients undergo “step-down” to an oral triazole antifungal. The potential availability of an oral AmB formulation may allow for longer induction courses of AmB. The reduction in toxicity seen in a phase III trial of cryptococcal meningitis (239) may allow for longer courses of this fungicidal agent and reduce the need for intravenous therapy and/or accompanying hospitalizations. This agent also allows for patients intolerant or with toxicity to currently available agents an alternative therapeutic option.

Ibrexafungerp

In vitro testing of ibrexafungerp has shown low MICs against Coccidioides range <0.125–0.25 µg/mL. A phase III open-label study (FURI: NCT03059992) investigated the efficacy of ibrexafungerp in patients intolerant or refractory to standard-of-care antifungals. This study focused on invasive candidiasis and aspergillosis, although blastomycosis, coccidioidomycosis, and histoplasmosis patients were allowed (335). Detailed analysis, including the response characteristics of those with the endemic mycoses, is eagerly awaited.

NOVEL INHALED ANTFIFUNGALS

Inhaled antifungals can potentially overcome some commonly encountered problems of systemic antifungal drugs. By delivering high concentrations of the drug directly to the lungs, one can reach adequate concentrations at the site of infection and minimize systemic exposure and consequent drug interactions and toxicities (336). To date, existing intravenous formulations of antifungal agents (mainly AmB) are used off-label to be administered by nebulization. In recent years, increasing efforts have been made both to develop novel drugs specifically designed for inhalation delivery (opelconazole) (337) and to optimize existing antifungal compounds for administration by dry powder inhalation (itraconazole and voriconazole) (338).

Opelconazole

Opelconazole is the newest member of the triazole family and has specifically been designed for inhalation delivery. Like other triazoles, opelconazole inhibits lanosterol 14α-demethylase causing disruption of the fungal membrane integrity and displays potent activity against Aspergillus and Candida spp. (339). The drug has good physicochemical properties for inhalation: the distal airways are efficiently reached, and slow dissociation rates lead to slow absorption from lung tissue into the systemic circulation (340). In preclinical models, the antifungal effects against A. fumigatus increase on repeat dosing and when combined with systemic antifungal compounds (337).

Past and ongoing clinical trials

Opelconazole has successfully completed a phase I clinical trial: the compound was safe and well tolerated by both healthy subjects and subjects with mild asthma. The study also confirmed very slow systemic absorption from the lung (341). Moreover, a recent case report showed successful usage of opelconazole as salvage therapy for fungal bronchial anastomotic infection after lung transplantation (342). A phase II study of opelconazole prophylaxis vs antifungal standard of care for lung transplant recipients finished enrollment, and data are expected in the first quarter of 2024 (343). Furthermore, a phase III double-blind placebo-controlled trial is currently assessing safety and efficacy of opelconazole in combination with systemic antifungal drugs for the treatment of refractory invasive pulmonary aspergillosis, with enrollment ongoing (344).

Studies on combination therapy including inhaled antifungals for treating fungal infections

In an in vitro human alveolus bilayer model and in the lungs of neutropenic immunocompromised mice topical opelconazole with systemic voriconazole or posaconazole in subtherapeutic doses resulted in a synergistic interaction with potency improved over either compound as a monotherapy against both azole-susceptible and -resistant Aspergillus fumigatus invasion (345). Combination of opelconazole with systemic antifungals is also evaluated in the ongoing phase III study on salvage treatment of invasive aspergillosis (344).

Perspective on the future use to treat fungal infections

While both preclinical and early clinical results appear very promising, the recently completed phase II and currently ongoing phase III study will inform on the efficacy of opelconazole as antifungal prophylaxis in lung transplant patients and as add-on treatment of refractory invasive pulmonary aspergillosis. Opelconazole might also be beneficial in patients with allergic bronchopulmonary aspergillosis (ABPA) or cystic fibrosis (CF) with Aspergillus sensitization; however, phase II trials in these populations were terminated early during the COVID-19 pandemic (346, 347).

Inhaled formulations of existing triazoles

Off-label use of parenteral formulations of voriconazole and itraconazole is not suitable for delivery by inhalation; although they efficiently reach the distal zones of the lung (348, 349), they are rapidly absorbed in the systemic circulation (336, 350). Regarding inhaled posaconazole, clinical experience is very limited (351). However, multiple pharmaceutical companies are currently developing advanced formulations of either itraconazole (PUR1900 by Pulmatrix Inc.) or voriconazole (TFF-Vori by TFF Pharmaceuticals Inc. and ZP-059 by Zambon SpA) as dry powder inhalation systems, showing promising results in animal models (352, 353).

Past and ongoing clinical trials

PUR1900 has successfully completed a phase I clinical trial in both healthy and asthmatic subjects. The trial showed that after 2 weeks, PUR1900 inhalation at doses between 10 and 35 mg resulted in 106- to 400-fold lower plasma concentrations compared to reported steady-state plasma concentrations of oral itraconazole 200 mg (354). Subsequently, a phase II clinical trial was started in patients with ABPA (355), which was unfortunately stopped during the COVID-19 pandemic. Considering inhaled voriconazole, both TFF-Vori and ZP-059 have completed phase I clinical trials in healthy and asthmatic patients (356–358). Results of these phase I trials have not been published. TFF-Vori is currently available in the clinic via an active expanded access protocol providing TFF-Vori as add-on therapy for patients with pulmonary aspergillosis or other voriconazole-sensitive pulmonary fungal infections, who have limited or no other treatment options or who have had unfavorable response to adequate standard-of-care antifungal therapy, including voriconazole (359).

Perspective on the future use to treat fungal infections

While the recent phase I and II clinical trials first need to confirm safety and tolerance of these new formulations of established antifungal compounds, their future applications could be very broad. They could not only be used as antifungal prophylaxis in high-risk patients but also serve as add-on treatment of fungal tracheobronchitis and invasive pulmonary fungal infections in combination with systemic antifungal drugs. Moreover, administration by DPI forms a very attractive approach to treat ambulant patients with ABPA, CF, and CPA (chronic pulmonary aspergillosis). We may certainly expect future clinical trials in all these different patient populations.

Inhaled amphotericin B

AmB formulations are suitable compounds for delivery by nebulization, displaying long residence times in the lungs and minimal systemic absorption (360). Various formulations of AmB are currently used for nebulization, including AmB deoxycholate (D-AmB), LAmB, and AmB lipid complex (ABLC) (361). In terms of efficacy, comparative studies show no significant differences between D-AmB and lipid formulations nebulized AmB (either LAmB or ABLC) (362–364). However, D-AmB is associated with slightly higher treatment-related adverse events (cough, dyspnea, and nausea) and discontinuation, although not reaching statistical significance in a meta-analysis (365). To the authors’ knowledge, no clinical trials have compared the efficacy and safety profiles of inhaled lipid AmB formulations (ABLC vs LAmB).

Tolerance of inhaled lipid compounds of AmB is good with only mild and transient side effects (mostly cough, nausea, and bad taste), and nephrotoxicity is absent (366). Bronchospasms are rare and manageable with concomitant administration of bronchodilators. To limit bystander exposure of patient- or drug-derived aerosols during nebulization, caregivers should use personal protective equipment (face mask, gloves) and expiratory filters on nebulizers and ventilators (367, 368). Most evidence for inhaled AmB is generated in lung transplant recipients and hematology patients with prolonged neutropenia. It can be used in both prophylactic and curative setting: Table S3 gives an overview of current applications according to recent guidelines. In a curative setting, current guidelines (ISHLT, IDSA, AST‐IDCOP) position nebulized AmB compounds as adjunctive therapy in addition to systemic antifungals for treatment of invasive tracheobronchial aspergillosis associated with anastomotic endobronchial ischemia in lung transplant recipients (369–371). These recommendations are based on low-quality evidence derived from case series (372–375) and case reports (376). The role of inhaled AmB in curative treatment of IFD in other at-risk populations has never been elucidated. In a preventive setting, more data are available on the efficacy of nebulized AmB in both high-risk hematology patients and lung transplant recipients.

In prolonged neutropenic patients with underlying hematological disorders, two recent meta-analyses of randomized studies comparing prophylactic inhaled AmB vs placebo showed that inhaled AmB is effective in reducing IFD (377), without any clear effect on mortality (378). Furthermore, several non-randomized studies in hematology patients with historical control groups show lower IFD incidence rates while on nebulized AmB prophylaxis, with or without systemic antifungal prophylaxis (366, 379–381). Current guidelines (IDSA, ESCMID-ECMM-ERS, ECIL) therefore consider lipid formulations of nebulized AmB prophylaxis (in combination with systemic fluconazole) to be used in second intention when systemic posaconazole prophylaxis is contra-indicated or unavailable (370, 382, 383). Considering lung transplant recipients, there are no randomized studies on inhaled antifungal prophylaxis. However, several prospective and retrospective cohorts show a significant decrease in IPA incidence in comparison with historical controls without antifungal prophylaxis (338, 384–386). This leads to guidelines recommending nebulized AmB lipid formulations to be used as IPA prophylaxis post-lung transplantation. Several protocols exist considering the exact formulation, dosing, and treatment duration (369, 370, 382). Considering viral-associated pulmonary aspergillosis, to date only a few retrospective studies show reduced IPA incidences among mechanically ventilated COVID-19 patients on nebulized AmB prophylaxis (387–389). Prospective confirmation of these findings in randomized trials is lacking and, to our knowledge, not planned.

Studies on combination therapy including inhaled antifungals for treating fungal infections

A few clinical trials are currently investigating nebulized AmB formulations outside lung transplant recipients and hematology patients. As such, it is currently being investigated in a phase III randomized placebo-controlled trial as add-on therapy to oral itraconazole for the treatment of CPA (390). Furthermore, an ongoing phase I trial is evaluating its safety and tolerability in patients with CF (391). Other patient populations, such as those with other causes of bronchiectasis, are also of interest and would potentially benefit from inhalation therapy.

Perspective on the future use to treat fungal infections

While the (off-label) administration of antifungal agents via inhalation has been used for decades, only in recent years, antifungal compounds specifically designed for inhalation therapy have reached clinical trials. Characteristics of these compounds are efficient drug delivery to the distal airways, longer lung retention times, and low systemic exposure resulting in minimal systemic side effects and drug interactions. Both preclinical and early clinical results look promising, and inhaled antifungal drugs will potentially reshape the future of antifungal treatment strategies. While the exact role in prophylaxis and treatment of IFD is still a question that remains to be answered, we may expect their wide implementation into the clinic in the coming years, improving care for patients with acute invasive fungal pulmonary infections, ABPA, CF, and CPA. Despite its promise, studies are needed to confirm efficacy of prophylaxis, as lessons learned from inhaled pentamidine in the early AIDS epidemic can also be recalled: the unequal distribution of inhaled drug saw Pneumocystis recur in the upper lobes, and extra-pulmonary infection was rarely observed suggesting inhalational delivery may still allow for fungal inhalation and “escape” with dissemination.

INDICATIONS AND SITUATIONS IN WHICH NOVEL ANTIFUNGALS SHOULD BE CONSIDERED

A number of new antifungals are currently in late-stage clinical development. These include new agents of currently available antifungal classes, with preferable PK, safety, and efficacy profile that allows some of them to overcome existing antifungal resistance. Rezafungin is the first oral/iv echinocandin with a long half-life, allowing for once-weekly administration, with the added benefit of a front load mechanism, which has been recently approved in the U.S. and in Europe for treating invasive Candida infections, but trials are also investigating rezafungin for treating PJP. The once-weekly application will be a major advantage for long-term treatment of complicated Candida infections and also for treatment of resistant Candida infections, while future studies will show whether long subtherapeutic levels may also dive echinocandin resistance in colonizing isolates (392).

Opelconazole is the first formulation of a triazole designed for inhaled therapy and is currently evaluated in a phase III superiority study for salvage treatment of pulmonary aspergillosis. While not systemically active, the inhaled formulation allows for high drug concentrations in the lung without systemic drug-drug interactions or toxicity. Oteseconazole is currently available and is useful in cases of recurrent vulvovaginal yeast infections. The long half-life of oteseconazole, while preventing use in women of childbearing potential, could make it potentially attractive in some other patients due to the infrequency of dosing required and may have a role in the treatment of endemic mycoses. SUBA-itraconazole is attractive due to the reduction in adverse events compared to conventional itraconazole and the reduced food and gastric pH requirements easing the burden to patients for compliance.

CAmB represents a polyene with oral bioavailability and better safety profile that could open the door for oral polyene treatment of Cryptococcus, Candida, Aspergillus, and Mucorales infections. The fact that the drug has to be given six times a day may make it a less convenient alternative, however.

In contrast, ibrexafungerp, fosmanogepix, and olorofim are the first agents of new classes of antifungals with novel mechanisms of action offering new options and improvements in terms of efficacy and safety. Ibrexafungerp represents an oral echinocandin-like drug that will allow for oral step-down treatment of candidemia and invasive candidiasis when fluconazole is not an option. For multidrug-resistant Candida spp., including C. auris, ibrexafungerp will be a viable option. In addition, the drug will revolutionize treatment of recurrent and resistant vulvovaginal candidiasis and represent an option for long-term combination treatment (with an azole) for invasive aspergillosis. Fosmanogepix and olorofim are both broadly active against Aspergillus spp. Their spectrum of activity includes cryptic species, and a limited risk of cross-resistance is expected with currently used antifungals. Fosmanogepix and olorofim also show strong activity against other rare molds, particularly Lomentospora spp., Scedosporium spp., and Scopulariopsis spp. as well as endemic mycoses such as coccidioidomycosis, where these agents have the potential to become the primary treatment options. Fosmanogepix also shows activity against some Mucorales and some Fusarium spp., and broadly against Candida spp., including highly resistant Candida spp. such as C. auris, but not C. krusei; in contrast, olorofim lacks activity against yeast, Mucorales, and most Fusarium spp. Synergism with liposomal AmB, if confirmed, may open the door for fosmanogepix to be used in combination for treatment of aspergillosis, mucormycosis, fusariosis, among others.

In summary, the wide spectrum of activity of these new antifungals includes the classical yeasts and filamentous fungi (except for olorofim that is not active against yeasts) but also some rare molds and yeasts, endemic mycosis particularly coccidioidomycosis and/or pneumocystosis (Table 1 and Fig. 5). In addition to approved indications or possible indications under evaluation through phase II and III clinical trials, we want to emphasize on various situations in which these novel antifungals should be considered because of their spectrum and/or their formulation. These proposals summarized in Table 1 are authors’ own opinions that do not necessarily rely only on results from ongoing clinical trials.

TABLE 1.

Indications and situations in which novel antifungals should be considered^a

Drug family	Target	Spectrum of activity	Indications under evaluation (phase II and III clinical trials) or approved indications (noted)	Useful for additional indications (authors’ own opinion)
Olorofim Orotomide	Dihydroorotate dehydrogenase inhibition	Molds: Aspergillus spp., Lomentospora prolificans, Scedosporium spp., Rasamsonia spp., Paecilomyces variotii, Scopulariopsis spp. Endemic yeasts: Coccidioïdes immitis, Histoplasma capsulatum, Blastomyces dermatitidis, Madurella mycetomatis, Talaromyces marneffei	Invasive aspergillosis Refractory filamentous IFI such as Lomentospora prolificans and Scedosporium spp. infections	Coccidioïdomycosis Talaromycosis Other endemic fungi Chronic/allergic forms of aspergillosis
Ibrexafungerp Triterpenoid	1-3-β-D-glucan synthase inhibition	Yeasts: Candida spp. Molds: Aspergillus spp., Alternaria alternata, Cladosporium spp. Endemic yeasts: Coccidioïdes immitis, Histoplasma capsulatum, Blastomyces dermatitidis, Fonsecaea pedrosoï Pneumocystis jirovecii	Vulvovaginal candidiasis and recurrences (US FDA approved in June 2021) Invasive candidiasis including C. auris Invasive aspergillosis Refractory IFI Coccidioïdomycosis	Oral/esophagal candidiasis Cryptococcosis Pneumocystosis Prophylaxis of IFI (including pneumocystosis)
Fosmanogepix Gepix mannoprotein synthase	Gwtl inositol acyltransferase inhibition	Yeasts: Candida spp. except C. krusei/P. kudriavzevii, Cryptococcus spp., Trichosporon asahi, Exophiala dermatitidis, Malassezia furfur Molds: Aspergillus spp., Fusarium spp., Scedosporium spp., Lomentospora prolificans, Rasamsonia spp., Cladosporium spp., Paecilomyces variotii, Purpureocillium lilacinum, Scopulariopsis spp. Endemic yeasts: Coccidioides immitis, Histoplasma capsulatum, Blastomyces dermatitidis, Fonsecaea pedrosoi, Phialophora verrucosa	Invasive candidiasis including C. auris Invasive aspergillosis and rare mold infections (scedosporiosis, fusariosis, mucormycosis)	Cryptococcosis Coccidioidomycosis Lomentosporosis Endemic mycoses such as coccidioidomycosis Fusariosis and mucromycosis (in combination with liposomal amphotericin B)
Rezafungin Echinocandin	13-β-D-glucan synthase inhibition	Yeasts: Candida spp. Molds: Aspergillus spp. Pneumocystis jirovecii	Invasive candidiasis, prophylaxis of IFI in SCT recipients	Invasive candidiasis after discharge from hospital Joint bone candidiasis, endocarditis and other long-term Candida infections Intraabdominal candidiasis Pneumocystosis
Opelconazole Triazole	14-α-Demethylase inhibition	Yeasts: Candida albicans, C. auris, C. glabrata/N. glabratus, C. krusei/P. kudriavzevii, Cryptococcus spp. Molds: A. fumigatus (except azole resistant), A. flavus, A. terreus, Rhizopus	Salvage treatment for invasive aspergillosis in combination with SOC	Inhaled prophylaxis of pulmonary aspergillosis Inhaled therapy for invasive aspergillosis in non-neutropenic patients Inhaled therapy for chronic and allergic broncho-pulmonary aspergillosis

^a Approved indications and possible indications under evaluation through phase II and III clinical trials are outlined, as are additional situations in which these novel antifungals could be considered because of their spectrum and/or their formulation. These proposals are authors’ own opinions that do not necessarily rely only on results from ongoing clinical trials.

Fig 5.

Indications for novel antifungal drug classes and situations where they might be used off-label.

IMMUNOTHERAPY

Cellular immunotherapy

Conventional antifungal therapy presents challenges due to the limited number of drug classes, associated toxicity, drug-drug interactions, and the escalating global spread of antifungal resistance. To address these limitations, alternative treatment modalities involving immunomodulatory drugs and genetically modified immune cells have been proposed (Fig. 6).

Fig 6.

Promising immunotherapies for invasive fungal disease. Figure showing a selection of components of the antifungal host response, here arbitrarily shown in the setting of invasive aspergillosis. Potential immunotherapeutic agents (divided in cellular, molecular, or vaccination strategies) are depicted, showing their main method of action in the antifungal host response. CAR, chimeric antigen receptor; DC, dendritic cell. Figure created with BioRender.com.

In recent decades, different cellular immunotherapies aiming to restore and augment antifungal immunity have emerged (393). Examples include adoptive T-cell transfer, chimeric antigen receptor (CAR) T-cells, ex vivo pulsed dendritic cells (DCs), and antifungal-loaded leukocytes (394–397). Despite promising preclinical data, only a few of these strategies have transitioned to clinical studies, primarily because of the costs, scalability challenges, workload, and significant regulatory hurdles. For this reason, manufacturing techniques such as closed-system bioreactors have been developed to enhance the reproducibility of the cellular products and reduce costs (398).

The adoptive transfer of DCs pulsed with fungal antigens to boost antifungal immunity represents one classical example of cellular immunotherapy to treat invasive fungal infections. The stimulation of DCs with conidia or RNA from A. fumigatus was found to activate antigen-specific, interferon (IFN)-γ-producing T-cells in vitro and to confer resistance in mouse models otherwise susceptible to aspergillosis (394). More recently, myeloid DCs pulsed with selected fungal proteins were found to upregulate maturation markers, which was reflected in an increased production of proinflammatory cytokines and improved antigen presentation leading to stronger proliferation and IFN-γ secretion from autologous CD4+ and CD8+ T cells (399). Moreover, supernatants of pulsed myeloid DCs also significantly enhanced the oxidative burst in neutrophils after co-culture with the fungus.

The direct infusion of fungal-specific T-cells has also been proposed as a possible approach to treat invasive fungal infections. By using different clinical-grade manufacturing processes, the generated specific T-cells against A. fumigatus were predominantly CD4+ T cells with a central-memory phenotype, while CD8+ T cells mainly showed an effector-memory phenotype with efficient cytotoxic activity (400). Another protocol using selection of fungus-specific T-cells based on CD137 or CD154 expression also showed promise in generating cells with cross-reactivity to different fungal proteins (401).

In addition to DCs, cellular therapy involving other innate cells, such as granulocyte transfusions, has shown promise in treating invasive fungal infections in neutropenic patients (402). However, the transfusion of mature neutrophils has resulted in limited clinical benefit, with a potential benefit noted only in those receiving the highest quantity of neutrophils (403), likely owing to insufficient numbers and the very short lifespan of these donor cells. Furthermore, these cells may also display selective defects in antifungal immunity, as granulocyte colony-stimulating factor (G-CSF)/dexamethasone-mobilized neutrophils have an impaired capacity to kill C. albicans yeast, but not hyphae, as a consequence of an altered neutrophil granular content (404).

Conditionally immortalized murine neutrophil progenitors capable of continuous expansion have recently emerged as a potential alternative allowing the generation of unlimited number of homogenous granulocyte-macrophage progenitors (405). These cells were demonstrated to differentiate into mature neutrophils in vivo and to respond adequately to inflammatory stimuli. Their therapeutic benefit was further confirmed by the improved survival in experimental models of candidemia and pulmonary aspergillosis following the transfusion with conditionally immortalized progenitors (405). Importantly, granulocytes offer the possibility of ex vivo loading with elevated concentrations of antifungals and improve their local delivery (397). The incorporation of the lipophilic antifungal posaconazole in neutrophil-like HL60 cells was found to result in contact-dependent transfer of posaconazole to hyphae and decreased fungal viability in vitro and a neutropenic mouse model of invasive pulmonary aspergillosis (397).

The advent of cellular therapy, particularly chimeric antigen receptor T-cell technology, has revolutionized the treatment of hematological malignancies (406). CAR T-cells are engineered to target specific antigens present in affected cell types, enabling targeted elimination. Notably, T-cells engineered with the pattern recognition receptor dectin-1, targeting BDG, exhibited efficacy in inhibiting fungal growth in vitro and in animal models of aspergillosis (395). Another innovative approach involves T-cells engineered with a CAR that recognizes a conserved protein antigen in the cell wall of A. fumigatus hyphae (407). T-cells expressing this CAR, particularly CD8+ T cells, displayed direct antifungal effects in vivo, as demonstrated by the increased release of perforin and granzyme B and cytokine production that activated macrophages to potentiate the overall antifungal response. However, challenges such as the development of cytokine release syndrome or alloreactivity with the host are significant drawbacks of CAR T-cell therapy (408). Strategies to minimize adverse effects of cellular immunotherapy include incorporating safety switches into engineered cells, enabling their controlled destruction if adverse effects occur (409). Furthermore, advanced monitoring techniques such as cytokine profiling and imaging studies can be implemented to swiftly detect and manage potential side effects (410).

An alternative approach involves the use of natural killer (NK) cells for CAR engineering, aiming to mitigate potentially fatal side effects like cytokine storms, alloreactivity, or neurotoxicity. CAR NK cells, e.g., by using the NK92 cell line, offer a universal “off-the-shelf product” that is readily available, is very well characterized, and has good proliferation capacity and ease of transduction (411).

Current research is exploring new frontiers in cellular immunotherapy, including the use of genome editing technologies such as clustered regularly interspaced palindromic repeats/Cas9 to enhance the specificity and efficacy of engineered cells. For example, deletion of the inhibitory co-receptor cytotoxic T-lymphocyte-associated protein 4 was demonstrated to improve the proliferation and anti-tumor efficacy of CAR T-cells (412). Additionally, the identification of novel fungal targets and the development of more sophisticated engineering techniques hold promise for the future. Emerging studies are also investigating the potential of utilizing immune cells other than T-cells and NK cells, paving the way for a more diverse range of therapeutic options.

Molecular immunotherapy

Immunotherapeutic strategies can rely on non-cellular approaches as well. Increasing insights into the antifungal immune response by soluble mediators have provided several interesting targets that could be used when treating patients with fungal disease. We can broadly divide humoral strategies into two classes: humoral components that opsonize fungal structures, leading to improved recognition and clearance by the immune system, and molecular components that directly boost immune cell functions.

Soluble pattern recognition molecules and antibodies

Targets in the first class consist of innate soluble pattern recognition molecules (PRMs) and immunoglobulins. Some examples of PRMs, all studied in preclinical research, are mannose-binding lectin in the setting of candidiasis (413) and the long pentraxin-3 or surfactant protein D in aspergillosis (414–416). Antibodies directed against fungal epitopes have been studied for several types of fungal diseases, especially for cryptococcosis, candidiasis, and aspergillosis (417–421). Both PRMs and antibodies have been proven beneficial in preventing or treating fungal diseases in animal models. However, only a few antibodies, such as antibody 18B7 (production stopped due to lack of industrial support) for cryptococcosis and antibody efungumab [Mycograb; production stopped due to product safety and quality control (QC) issues] for candidiasis and cryptococcal meningitis, have been studied to a very limited extent in human patients (422–425).

Cytokines and checkpoint inhibitors

The second class consists of (recombinant) cytokines and checkpoint inhibitors that boost the functionality of the antifungal immune response. A major advantage of this class is the clinical availability of several of these drugs. However, the main disadvantage is the non-specificity of this approach, which may result in more adverse effects compared to narrow-spectrum immunotherapy, such as antifungal antibodies. Cryptococcosis, candidiasis, and aspergillosis are the main fungal diseases for which cytokines, mainly recombinant interferon-gamma (rIFN-γ) and colony-stimulating factors (CSFs), have been studied.

rIFN-γ may boost phagocytic function, leading to improved fungal clearance, and decreased IFN-γ signaling has been implied in the pathophysiology of invasive disease by several fungal species (426–432). It is recommended as prophylaxis for aspergillosis in patients with chronic granulomatous disease (433), and it has shown potential as treatment in a retrospective study in patients with CPA (434), for which a randomized feasibility study is currently being organized ongoing. For aspergillosis and candidiasis, however, studies are limited to case reports and series, and therefore high-quality evidence for its benefit is still lacking (435–438). More insight into Candida infections is expected soon, as a randomized controlled trial with rIFN-γ as adjunctive therapy in patients with candidemia is ongoing (NCT04979052).

Regarding colony-stimulating factors, which may boost myeloid cell myelopoiesis and function, mainly G-CSF and granulocyte-macrophage CSF (GM-CSF) have been studied in fungal disease. Both drugs are FDA approved. CSFs are typically administered prophylactically when neutropenia is expected (e.g., after allogeneic HSCT). Observational data regarding benefit of G-CSF in fungal infections are scarce (439–442). GM-CSF has a more pleiotropic effect and has been studied more extensively in fungal disease. It was shown to lead to lower 600-day IFD-related mortality in recipients of allogeneic HSCT compared to G-CSF (443). Moreover, a recent large case series reported potential benefit of GM-CSF in patients with hematological malignancies and refractory IFD, even when there is no baseline neutropenia (444).

Checkpoint inhibitors have recently emerged as potential adjunctive immunomodulatory therapy in IFD, countering immune cell exhaustion (445). Mainly in candidiasis, mucormycosis, and aspergillosis, these agents are increasingly investigated in preclinical models (446–451), with beneficial responses observed in several case reports, often in combination with IFN-γ (452–455). However, more (pre)clinical work is required to define the place of these agents in the antifungal armamentarium.

Vaccination

Vaccines could form the most cost-effective and efficient way to reduce fungal disease burden (425). Until recently, the often severely immunocompromised status of patients at risk for IFD formed a significant hurdle for vaccine development, as a functioning, adaptive immune system is a prerequisite to ensure an effective vaccine response. Nevertheless, several potential vaccines for prevention of invasive disease caused by Aspergillus, Cryptococcus, Candida, Coccidioides, and other fungal species are currently being studied in preclinical models (456–461). More research is urgently needed in this field that could ultimately redefine the prophylactic management of IFD.

DRUG-DRUG INTERACTIONS AND OTHER PK/PD OF NOVEL ANTIFUNGALS

With the emergence of new antifungal agents and their integration into treatment regimens, understanding their interactions with other medications has become paramount for ensuring patient safety and optimal therapeutic outcomes. The complexity of these interactions is illustrated by the experience with the triazole drugs. These drugs are known for their high potential to inhibit patient cytochrome P450 enzymes, leading to interactions with drugs metabolized through the same pathway. The introduction of novel antifungal drugs has expanded the treatment landscape but also presents unfamiliar drug-drug interaction risks: lower risk (fosmanogepix, olorofim, ibrexafungerp, oteseconazole) or unlikely (opelconazole, rezafungin, CAmB). However, novel oncologic and immunologic therapeutic agents may increase potential drug-drug interactions and should be carefully reviewed. The significance of recognizing and managing the interaction potential of new drugs cannot be overstated. Failure to address potential interactions may compromise treatment efficacy or lead to adverse effects, compromising patient well-being.

Medications may act as either an inducer/inhibitor or the substrate in drug-drug interactions. In some bidirectional drug interactions, a single drug may play both roles, acting as both an inducer/inhibitor and a substrate. Inhibition can involve direct (competitive) or indirect (non-competitive) blocking of specific metabolizing enzymes, leading to effects that can be reversible or irreversible.

Substrates fall into categories of sensitivity: sensitive or moderately sensitive. Sensitive index substrates refer to drugs that exhibit a fivefold or greater increase in the area under the concentration-time curve (AUC) when affected by potent inhibitors of a particular metabolic pathway. Moderately sensitive substrates, on the other hand, demonstrate an AUC increase ranging between two and less than five times. Inhibitor drugs are categorized based on the extent to which they elevate the AUC of sensitive index substrates within a specific metabolic pathway: 5 times or more (classified as strong), between 2 and less than 5 times (considered moderate), and between 1.25 and less than 2 times [referred to as weak by the United States Food and Drug Administration or mild by the European Medicines Agency (EMA)].

The evolving landscape of antifungal drugs underscores the importance of understanding their interactions with concomitant medications. Early data are promising with regard to their drug-drug interaction potential, but more data, and specifically real-world data, are urgently needed so substantiate these initial reports.

Previously described drug-drug interactions of novel antifungals are summarized in Table 2, while ongoing studies about drug-drug interactions of novel antifungal compounds are displayed in Table 3.

TABLE 2.

Drug-drug interactions of novel antifungals

	Substrate	Inhibitor	Inducer	Transporter
Ibrexafungerp	Substrate 3A4	Strong inhibition of CYP2C8 and moderate inhibition of CYP3A4		P-gp, BCRP, BSEP, MRP2, and OATP1B3 inhibitor.	(462)
Fosmanogepix		Weak inhibitor of CYP2C9 and CYP2D6	Weak inducer of CYP2B6; a moderate inducer of CYP1A2, CYP2C19, and CYP3A4	Ibrexafungerp may be an inhibitor of MDR1, OATP1B3, and weakly BSEP	(463, 464)
Olorofim	Substrate 3A4	Weak inhibitor of 3A4 and 2D6	Weak inducer 1A2 and 2B6
Rezafungin	Not a substrate, inducer, or inhibitor				(465)
Oteseconazole		Inhibitor of CYP3A4/5	Inducer of CYP3A4/5	Inhibitor of BCRP	(466)
Opelconazole	Substrate for CYP3A4 and CYP2C9a	No inhibition expected at clinical steady-state concentrations	No induction expected at clinical steady-state concentrations	As the expected clinical steady-state systemic Cmax for opelconazole is ≤0.01 µM (≤6830 pg/mL), it is considered improbable that inhibitory interactions with the substrates of uptake and efflux transporters will occur	Source: data on file, provided by Sponsor
Encochleated amphotericin B

^a Expected to be of little clinical relevance in patients with pulmonary aspergillosis, given the delivery of the drug directly to the respiratory system via inhalation and the low systemic concentration of opelconazole.

TABLE 3.

Ongoing studies about drug-drug interactions of novel antifungal compounds

Antifungal drug (pharmacologic effect)	ClinicalTrials.gov ID (status)	Design	Concomitant drug (pharmacologic effect)	Measurement
Ibrexafungerp (substrate of Cyp3A4, inhibitor of Cyp2C8)	NCT04092751 (completed)	Phase I Healthy subjects	Pravastatin (no major CYP450 interactions)	Pravastatin exposure
	NCT04092725 (completed)	Phase I Healthy subjects	Dabigatran (no major CYP450 interactions)	Dabigatran exposure
	NCT03672292 (completed)	Phase II Patients with IPA	Voriconazole (Cyp3A4 substrate and inhibitor)	Ibrexafungerp and voriconazole exposure
	Not registered	Phase I Healthy subjects	Rosiglitazone (Cyp2C8 substrate)	Rosiglitazone exposure
Olorofim (substrate of Cyp3A4, weak inhibitor of Cyp3A4)	NCT04171739 (completed)	Phase I Healthy subjects	Itraconazole (Cyp3A4 substrate and inhibitor) Rifampicin (pan-CYP inducer)	Olorofim exposure
	NCT03095547 (withdrawn)	Phase I Healthy subjects	Posaconazole (Cyp3A4 inhibitor) Pantoprazole (proton pump inhibitor, substrate of Cyp3A4) Tacrolimus and cyclosporine A (Cyp3A4 substrates)	Olorofim and posaconazole exposure Olorofim and pantoprazole exposure Olorofim, tacrolimus, and cyclosporine A exposure
	NCT02730442 (completed)	Phase I Healthy subjects	Fluconazole (Cyp3A4 substrate and inhibitor)	Olorofim exposure
	NCT02680808 (completed)	Phase I Healthy subjects	Midazolam (substrate of Cyp3A4)	Midazolam exposure
	NCT03036046 (withdrawn)	Phase II Patients with AML and chemotherapy	Fluconazole or posaconazole (Cyp3A4 substrates and inhibitors)	Olorofim exposure
Fosmanogepix (inhibitor of Cyp3A4)	NCT04166669 (completed)	Phase I Healthy subjects	Itraconazole (Cyp3A4 substrate and inhibitor) Rifampicin (pan-CYP inducer)	Fosmanogepix exposure
	NCT02957929 (completed)	Phase I Healthy subjects	Different CYP450 cytochrome substrates	Fosmanogepix exposure
Opelconazole	DOH-27-122022-5196a	Phase I Healthy subjects	Midazolam Caffeine	Area under the plasma concentration vs time curve, from time 0 to t, where t is the time of the last quantifiable concentration [AUC(0–t)] Maximum observed plasma concentration (Cmax)

^a South African National Clinical Trials Registry.

Key characteristics of selected novel antifungal compounds are displayed in Table 4, while tissue penetration of novel antifungal compounds is summarized in Fig. 7.

TABLE 4.

Key characteristics of selected novel antifungal agents

	Olorofim (OLO)	Ibrexafungerp (IBX)	Fosmanogepix (FMG)	Oteseconazole (OTC)	Rezafungin (REZ)	Opelconazole (OPL)
Formulation	Film coated 30 mg	Capsule 250 mg (IV under development)	FMG is a prodrug tablet 100 and 200 mg coated IV (20 mg/mL)	Capsule 150 mg	200 mg solid (cake or powder) for intravenous infusion	Suspension for oral inhalation 14.8 mg
Typical dose	150 mg BID loading dose 90 mg BID maintenance	750 mg BID loading 750 mg QD maintenance for invasive disease Lower dose of 300 mg BID for VVC/RVVC	1,000 mg intravenous BID (3-hour infusion) for 1 day, then 600 mg IV QD for at least 2 days, followed by either 600 mg IV QD or 700 mg PO QD.	600 mg on day 1 followed by 450 on day 2 then beginning on day 14 150 mg once weekly (indication RVVC)	400 mg loading once weekly by intravenous infusion, followed by a 200 mg dose once weekly thereafter	14.8 mg BID
Dose linearity	Yes	Yes from 10 to 1,600 mg after single dose	Yes; 10–350 mg SAD and 50–600 mg MAD; till 1,000 mg MAD	Yes, 20–320 mg	Yes, both SAD from 50 to 400 and MAD of 100–400 mg showed dose proportional increases with only minor accumulation at 400 mg dose.	Yes, SAD 1.4–28 mga
Variability	Moderate	Moderate	Moderate	Moderate	Moderate	Moderateb
Half-life	Resurgence	20 hours	~2 days	138 days	152 hours	Approx. 6.5 days, rate limited absorption from lung into the plasmaa
Bioavailability	~68%	~36%	~90%	>99.5%	Not applicable	Oral bioavailability of the swallowed fraction <0.7%
Food effect	About 12% increase in AUC when taken with food	Yes, food increases AUC by 40%	No food effect		Not applicable	Not relevant
Protein binding	99.8%	99.7%	>90%			96%

^a However, the significantly higher concentrations of drug in the lung relative to plasma overcome the potential impact of any variability in drug concentrations in the target tissue.

^b Systemic variability is typical of inhaled medicines, at target clinical dosing.

Fig 7.

Tissue penetration of new antifungals. CAmB is not displayed: while tissue presentation is thought to mirror that of other AmB formulations, there are no animal data or human data supporting this assumption in the public domain.

Fosmanogepix pharmacokinetics/pharmacodynamics

Fosmanogepix (APX001A) is a prodrug that is available as tablets as well as an intravenous formulation. The active moiety of fosmanogepix, manogepix results in pleiotropic effects on the fungal cell and, ultimately, severe growth defects and lethality (60). In phase II trials in patients with candidemia, it was given at a dose of 1,000 mg intravenous twice a day (3-hour infusion) for 1 day followed by either 600 mg intravenous once daily or 700 mg PO once daily and overall well tolerated and effective (59, 259, 467).

Preclinical pharmacokinetic/pharmacodynamic studies were conducted in murine models of disseminated candidiasis and of invasive pulmonary aspergillosis. In both situations, the free fraction area under the plasma concentration-time curve over MIC (fAUC/MIC) best correlated with efficacy (62, 468–470). In a rabbit model of hematogenous Candida meningoencephalitis, tissue/plasma concentration ratios of manogepix in meninges and brain tissue were approximately 1:1, which correlated with a significant decline of C. albicans in tissue vs control (471).

Once in the blood, fosmanogepix is rapidly converted by phosphatases into the active moiety, manogepix. In healthy adult volunteers, fosmanogepix showed linear- and dose-proportional pharmacokinetics of manogepix in either formulation and high oral bioavailability of over 90% independent of gastric filling (367). The median time to maximum concentration of drug in serum (T_max) was 3.0 hours for oral regimens and concordant with the 3-hour infusion time. Across doses, manogepix’s volume of distribution (VD) is approximately 2 L/kg, and systemic clearance (CL) is between 20 and 30 mL/h/kg with a half-life ranging from 53 to 73 hours after multiple dosages. Manogepix is extensively bound to plasma proteins (>90%) and, while its exact pattern of metabolism is not fully elucidated, excreted predominantly via bile and feces (367, 371). Of note, no fundamental differences in pharmacokinetics have been observed in immunocompromised neutropenic patients with leukemia (309).

Tissue- and organ-site penetration were investigated using the distribution, absorption, and elimination patterns of radioactivity of carbon 14-fosmanogepix in rats and monkeys (472). Radioactivity swiftly and extensively dispersed across various tissues following both administration routes in both species. In rats, organs with the highest radioactivity included bile, abdominal fat, reproductive fat, subcutaneous fat, and liver. Radioactivity was also present in tissues linked to invasive fungal infections, such as the lung, brain, and eye. Among monkeys, the highest maximum values were observed in bile, urine, uveal tract, bone marrow, abdominal fat, liver, and kidney cortex. Similar to rats, radioactivity was also detected in lung, brain, and eye tissues. Consequently, manogepix shows extensive distribution in major tissues in both rats and monkeys and is primarily eliminated through biliary/fecal excretion (371).

DDI

No relevant drug-drug interactions have been reported to date for fosmanogepix. Two phase I studies have been completed to assess potential interactions with drugs metabolized by the cytochrome P450 enzymes (NCT04166669 and NCT02957929). Their results are not yet available. No increases in manogepix levels were observed with coadministration of posaconazole in patients with leukemia (309).

Ibrexafungerp pharmacokinetics/pharmacodynamics

Ibrexafungerp (MK-3118; SCY-078) is available for oral administration and FDA approved in form of 150 mg tablets as Brexafemme at a dose of 300 mg twice a day for 1 day for treatment of vulvovaginal candidiasis (219). For invasive fungal infections in clinical trials, the targeted exposure is reached at a dose of 750 mg QD with a 750 mg twice daily loading dose on day 1 and day 2 (381).

Ibrexafungerp exhibits concentration- and time-dependent fungicidal activity against Candida spp. (473); correspondingly, the fAUC/MIC best correlated with efficacy in murine models of invasive candidiasis (474–476). Against Aspergillus spp., antifungal activity in vitro was fungistatic (477). In a murine neutropenic model of disseminated aspergillosis, ibrexafungerp was effective in prolonging survival and reducing fungal tissue burden (478), and combination with an antifungal triazole showed promising efficacy in all endpoints in the neutropenic rabbit model of invasive pulmonary aspergillosis (231).

In humans, ibrexafungerp showed linear pharmacokinetics over the investigated dosage range after single- and multiple dosing, and maximum plasma concentrations are reached after 4 and 6 hours, respectively. Oral bioavailability is approximately 35%, and intake with a high fat meal increases the oral bioavailability by around 40% which is not considered clinically significant. Ibrexafungerp is highly protein bound (greater than 99%), predominantly to albumin, and the mean steady-state VD is approximately 600 L. The compound is eliminated predominantly via metabolism and biliary excretion with an elimination half-life of around 20 hours. Ibrexafungerp undergoes hydroxylation by CYP3A4, followed by glucuronidation and sulfation of a hydroxylated inactive metabolite. In healthy volunteers, 90% of an oral dose of radioactive drug (51% unchanged) was recovered in feces, and 1% was recovered in urine. Published information on appropriate dosing in renal and hepatic impairment is limited at this stage. In the label, it has been stated that the pharmacokinetics of ibrexafungerp were not altered in subjects with mild (child-pugh class A) to moderate (child-pugh class B) hepatic impairment when the total AUC estimates were compared to healthy subjects, while the impact of severe hepatic impairment (child-pugh class C) on the pharmacokinetics of ibrexafungerp is unknown (219). Therefore, no dosage adjustment is recommended in patients with mild or moderate hepatic impairment (219). In preclinical reproduction studies, on exposure approximately five times the RHD based on AUC comparison, ibrexafungerp was associated with fetal malformations, and its use is contraindicated in pregnancy (219, 476, 479–481).

Of note, tissue distribution studies by quantitative whole-body autoradiography, mass balance, and elimination after single intravenous and oral doses of ibrexafungerp in rats demonstrated wide and excellent distribution in sites commonly associated with invasive fungal infection, including the lung, liver, kidney, spleen, and skin, with tissue:plasma ratios between 11 and 75. Penetration into brain tissue in the uninfected animals, however, was minor with a tissue:plasma ratio of 0.1 (479, 482).

DDI

Ibrexafungerp is a substrate of CYP3A4 and P-gp. In vitro, ibrexafungerp is an inhibitor of CYP2C8, CYP3A4, P-gp transporter, and OATP1B3 transporter. Ibrexafungerp is not an inducer of CYP3A4 (219, 479).

Caution is advised with strong CYP3A4 and P-gp inhibitors: Ketoconazole increases ibrexafungerp AUC to approximately sixfold. Diltiazem, a moderate CYP3A4 inhibitor, increases the AUC of ibrexafungerp by 2.5-fold, and the proton pump inhibitor pantoprazole decreased ibrexafungerp AUC by approximately 25%; the significance of these changes in exposure at ibrexafungerp doses used for treatment of invasive infections is unclear at present (219).

The effects of ibrexafungerp on substrates of CYP2C8, CYP3A4, P-gp, and OATP1B3 transporters were assessed using loading doses of the compound of 1,250–1,500 mg for 2 days followed by 750 mg once daily for 3–7 days. Ibrexafungerp did not increase the AUC of rosiglitazone, a moderate-sensitive CYP2C8 substrate (483). Ibrexafungerp resulted in 1.4-fold increase in the AUC of the sensitive CYP3A4 and P-gp substrate tacrolimus (484) and a 1.4-fold increase in the AUC of the P-gp substrate dabigatran (485). Finally, ibrexafungerp resulted in a 2.8-fold increase in the AUC of the OATP1B3 transporter substrate pravastatin (219). An evaluation of the pharmacokinetics and safety in combination with voriconazole for the treatment of invasive pulmonary aspergillosis (NCT03672292) is under way.

No clinically relevant effect was observed on the corrected QT interval, heart rate, PR, or QRS intervals at plasma ibrexafungerp concentrations of up to 4,000 ng/mL (479).

Olorofim pharmacokinetics/pharmacodynamics

Olorofim (F901318) is available as oral formulation in the form of film-coated tablets. It is administered at a dose of 90 mg BID with a loading dose of 150 mg BID on day 1 (486).

Antifungal efficacy of olorofim in vivo has been demonstrated in animal models of invasive pulmonary aspergillosis (295, 487, 488), systemic scedosporiosis, and lomentosporiosis (296), and in central nervous system coccidioidomycosis (293). Live-cell imaging experiments with A. fumigatus revealed time-dependent pharmacodynamics of olorofim in vitro; while olorofim initially had a fungistatic effect by inhibiting germination and growth, prolonged exposure (>24 hours) was fungicidal through hyphal swelling followed by cell lysis (285). Corresponding to these in vitro observations, dose-fractionating studies in neutropenic murine and rabbit models of invasive pulmonary aspergillosis using serum galactomannan as pharmacodynamic endpoint revealed a time-dependent antifungal efficacy with the ratio of the minimum total plasma concentration/MIC (C_min/MIC) as the pharmacodynamic index that best linked drug exposure with observed effect (487, 488) .

In humans, olorofim has linear pharmacokinetics over the therapeutic dose range. It is well absorbed with an oral bioavailability of 67% with first pass metabolism likely accounting the portion of drug that does not reach the systemic circulation. Intake with food decreases its exposure by about 12%, which is not considered clinically relevant (489). Protein binding is over 99%, and the volume of distribution is approximately 3 L/kg, indicating extensive tissue distribution. Olorofim is predominantly cleared by complex oxidative metabolism with <0.5% of a dose eliminated in urine and bile as intact drug and up to 14 metabolites detected in human plasma. Clearance is approximately 110 mL/h/kg with a terminal elimination half- life of 24–30 hours; use of loading dose shortens the time required to attain steady state. Olorofim plasma levels undergo some kind of resurgence with multiple peaks observed in most subjects that are temporally related to meal intake. These secondary peaks are considered to be due to food-triggered release of olorofim from a depot site and not a result of enterohepatic recycling. Dose adaptation in subjects with mild-to-moderate hepatic impairment or severe renal impairment is not needed (490, 491).

In tissue distribution studies following a single intravenous infusion of (14C)-olorofim to albino rats, radioactivity was greatest in liver, kidney, adrenal gland, and abdominal fat with tissue:plasma ratios of >3; lowest concentrations were observed in the eye and at the bone surface. Radioactive concentrations in all other tissues, where measurable, were generally similar to or up to threefold greater than plasma. Of note, levels of radioactivity in brain were up to 1.6-fold greater than those observed in plasma, and at the earlier timepoints, where the majority of plasma radiolabel is unchanged parent drug, penetration of drug-related material into the brain is likely to predominantly comprise intact olorofim (492).

DDI

Olorofim is both a substrate and a weak inhibitor of CYP3A4. Fluconazole (a moderate dual inhibitor of CYP2C9 and CYP3A4) and itraconazole (a strong CYP3A4 inhibitor) increase the exposure to olorofim by 2- to 2.5-fold as measured by the AUC. When coadministered with rifampicin, a strong CYP3A4 inducer, exposure to olorofim was reduced by 74%. Olorofim is a weak CYP3A4 inhibitor and increases the exposure of midazolam (a pure CYP3A4 substrate) by 1.5-fold. Data from in vitro studies and physiologically based pharmacokinetic modeling suggest that olorofim may be a weak CYP2D6 inhibitor and a weak CYP1A2 and 2B6 inducer; the clinical relevance of these findings is not known. Results of the respective studies (NCT04171739; NCT03095547; NCT02730442; NCT02680808; NCT03036046) have not yet been published but have been presented as abstracts and are provided in the investigator brochure of olorofim (245).

Opelconazole pharmacokinetics/pharmacodynamics

Opelconazole (PC945) is a drug specifically designed be delivered to and retained in the lung and lower airways. It is a suspension for oral inhalation with a particle size distribution of approximately 1–4 µm and is given at a typical dose of 14.8 mg twice daily by a 10-minute nebulization via an off-the-shelf nebulizer (337).

In preclinical studies in temporarily neutropenic mice infected with A. fumigatus intranasally, intranasal administration of opelconazole once daily (339) was effective as treatment and prophylaxis (345, 493), and the combination of intranasal treatment with systemic administration of approved triazoles demonstrated synergistic effects in an in vitro human alveolus bilayer model and in the lungs of infected mice (345). Concentrations of opelconazole in mice were below the limit of detection in plasma but readily measured in lung extracts. Concentrations were higher after extended prophylaxis suggesting accumulation in whole lung after repeat dosing. Whereas concentrations were low in BAL supernatant, high levels of opelconazole were measured in BAL cell pellets, and concentrations in BAL cells showed a statistically significant correlation with antifungal efficacy (494).

In humans, the plasma pharmacokinetics of opelconazole were assessed following single and repeat inhaled doses for 7 days in healthy subjects. Geometric mean C_max was 0.322 ng/mL at 4–5 hours (median t_max) after a single inhalation of 14 mg. Following repeat once-daily inhalation, day 7 C_max was 0.951 ng/mL 45 minutes after dosing. Increases in C_max and AUC0-24h were approximately dose proportional over the dose range studied (14–28 mg). No data have yet been published as to the compartmental intrapulmonary disposition of the compound (341).

Since only very small amounts of the compound reach the systemic circulation, inhalation of opelconazole is not likely to cause any relevant drug-drug interactions despite in vitro affinities for the CYP enzyme system (341), and there is little potential for clinically relevant changes in subjects with renal or hepatic impairment.

Oteseconazole pharmacokinetics/pharmacodynamics

Oteseconazole has potent activity in vitro against Candida spp., including fluconazole-resistant isolates (246–248) and has demonstrated efficacy in animal models of vulvovaginal candidiasis and oropharyngeal candidiasis (251, 495) in vulvovaginal candidiasis, and plasma concentrations of free drug were predictive of efficacy when in excess of the in vitro MIC (251). Although exposure-response relationships and the time course of pharmacodynamic response are unknown for oteseconazole (245), based on the mechanism of action, it is more than likely that the pharmacokinetic and pharmacodynamic relationships are similar to the other azoles and are concentration- and time dependent.

In humans, oteseconazole showed linear pharmacokinetics of a dose range of 20 mg to 320 mg. The estimated oral bioavailability based on human mass balance studies is around 75%. Food, specifically a high fat meal increases the exposure by 1.36 compared to a fasted intake. After absorption, oteseconazole is widely distributed over the body with an estimated volume of distribution of 432 L. Protein binding in plasma exceeds 99.5%. The compound does not undergo significant metabolism: Following administration of C14-oteseconazole, the majority of the dose (82%) was recovered in excreta; 56% was recovered in feces and 26% in urine. The median terminal half-life is very long at 138 days, and the compound is primarily excreted by biliary excretion and to a lesser extent renally in unchanged form. No tissue concentration data are available except for comparable concentrations in vaginal tissue relative to plasma (245).

No dose adjustment of oteseconazole is needed in patients with mild hepatic impairment (child-pugh A) and in patients with mild-to-moderate renal impairment. No data exist for patients with moderate or severe hepatic impairment or severe and end-stage renal disease (245).

DDI

Oteseconazole increased the AUC0-24h of rosuvastatin, a BCRP substrate, by 114%. No clinically significant differences in the pharmacokinetics of the following drugs were observed when co-administered with oteseconazole: midazolam, ethinyl estradiol, and norethindrone (CYP3A4 substrates), or digoxin (P-gp substrate) (245).

Rezafungin pharmacokinetics/pharmacodynamics

Rezafungin (CD101) is available as intravenous formulation and approved by both FDA and EMA for treatment of candidemia and invasive candidiasis in adults. It is administered once weekly by intravenous infusion, with an initial 400 mg loading dose followed by a 200 mg dose once weekly thereafter.

Rezafungin is a member of the class of antifungal echinocandins. Similar to other members of this class, it targets the fungal enzyme BDG synthase and inhibits the formation of BDG, an essential component of the fungal cell wall involved in cell wall integrity and cell division (169). It is active in vitro predominantly against Candida and Aspergillus spp. but also has useful activity against Pneumocystis jirovecii (178, 180, 496, 497).

In vitro, rezafungin exhibits concentration- and time-dependent fungicidal activity against most Candida isolates including C. auris (188, 191) and showed sustained growth inhibition following drug removal with equivalent or longer post-antifungal effect values than the comparator micafungin against all tested Candida spp. (183). In neutropenic animals, rezafungin demonstrated efficacy in treatment and prophylaxis of invasive candidiasis and aspergillosis (192, 197) and prophylactic efficacy against Pneumocystis jirovecii (197). In the neutropenic mouse-disseminated candidiasis model, the total and free-drug (f) AUC/MIC was a robust predictor of efficacy (R2 ≥0.72) for all Candida spp. tested (182, 193, 498). These animal studies showed that fAUC/MIC targets were likely to be exceeded for >90% of isolates with the studied human doses, supporting the use of once-weekly rezafungin regimens in patients with candidemia and invasive candidiasis (498). Subsequent population pharmacokinetic modeling using data from five phase I, one phase II, and one phase III study and target attainment simulations for treatment of candidemia and invasive candidiasis confirmed rezafungin coverage, at the clinical dose, for the majority of the MIC distributions in most cases at multiple dilutions above the MIC₉₀ values for five of the six Candida spp. tested. Similar to other echinocandins, rezafungin achieved stasis for C. parapsilosis only in non-clinical models despite good clinical responses (499).

In healthy human volunteers, at single (50, 100, 200, and 400 mg) or multiple once-weekly (100, 200, and 400 mg) doses, administered by intravenous infusion over 1 hour, rezafungin showed linear plasma pharmacokinetics and minor accumulation (30%–55%). Following single dosing, rezafungin had low mean total body clearance (0.2 L/h) , a long terminal half-life (mean half-life ranged from 125 to 146 hours), and the mean volume of distribution at steady state and mean apparent volume of distribution during the terminal phase ranged from 34 to 51 L (173). The compound is highly protein bound with an estimated binding of over 97%. Studies using (14C)-radiolabeled rezafungin in humans demonstrate that fecal excretion of unchanged drug is the predominant route of elimination and that the compound is excreted over prolonged periods of time: Cumulative recovery of radioactivity through the first 17 days was 52% with an estimated mean overall recovery through day 60 of 88.3% (73% in feces, 27% in urine). In human plasma, rezafungin was the most abundant circulating component calculated at 69% of the total plasma radioactivity exposure. Three hydroxylated metabolites were the most abundant circulating metabolites, although each of these individual metabolites accounted for less than 10% of the total radioactivity AUC. Radioactivity in urine was primarily due to metabolites hydroxylated (in one of three positions) or dealkylation (resulting in des-pentyl), whereas radioactivity in feces was primarily rezafungin (500).

A population PK model has been developed using data from five phase I, one phase II, and one phase III study. The model found to best describe the available data was a three-compartment population PK model with first-order elimination characterized by the parameters clearance (CL), central volume (V1), peripheral volume (V23), intercompartmental clearance 1, and intercompartmental clearance 2. The variability model included correlated interindividual variability in CL, V1, and V23 and a proportional residual variability model. The following statistically significant covariates were identified: albumin concentrations on V23; body surface area (BSA) on CL, V1, and V23; and disease state on CL and V1. Disease states were defined as patients from the phase II and phase III studies and hepatically impaired subjects. Covariates of BSA, disease state, or albumin, included in the final model, were not associated with clinically meaningful changes in PK nor were any other patient factors (e.g., age, sex, race, or creatinine clearance), indicating that a common dose regimen is adequate for all adult patients (245).

In rats, following a single intravenous administration (5 mg/kg), the tissue/plasma AUC ratios were 4.62 for the kidney, 4.33 for the lung, 4.14 for the liver, 3.87 for the spleen, 1.09 for the heart, and 0.609 for the brain, indicating that exposure relative to plasma levels was comparable for major organs and approximately fourfold higher in tissue with the exception of the heart and brain. Similar to humans, across all animal species tested, rezafungin exhibited very low clearance, a modest volume of distribution and a long half-life (501). .

DDI

In vitro, rezafungin does not significantly interact with cytochrome P450 enzymes or drug transporters, thus having minimal drug-drug interaction potential. Two phase I open-label crossover studies in healthy subjects examined drug interactions between rezafungin and multiple drug probe cytochrome P450 (CYP) substrates and/or transporter proteins, immunosuppressants, and cancer therapies. In summary, rezafungin had no or minimal effects on the exposure of probe drugs for the following CYP substrates/transporter proteins: CYP2B6 (efavirenz); CYP3A4 (midazolam and tacrolimus); CYP1A2 (caffeine); CYP2C8 (repaglinide); P-gp (digoxin and tacrolimus); OCT-1, OCT-2, MATE-1, and MATE-2 (metformin); OATP (pitavastatin, rosuvastatin, and repaglinide); and BCRP (rosuvastatin). No clinically meaningful interactions were identified upon the coadministration of rezafungin with tacrolimus, cyclosporine, ibrutinib mycophenolate mofetil, and venetoclax. These results demonstrate the low potential for CYP substrate-mediated and drug transporter-mediated DDIs with rezafungin and also reveal the minimal impact on PK parameters with commonly coprescribed medications (502).

Encochleated amphotericin B pharmacokinetics/pharmacodynamics

Encochleated AmB (CAmB, formerly MAT2203) is available as an oral formulation. Once administered by mouth in divided doses, the drug proved safe in clinical trials in healthy HIV-positive individuals (234). It showed similar survival but less toxicity than intravenous AmB deoxycholate in HIV-infected patients with cryptococcal meningitis (239). Its pharmacokinetics differ from other AmB formulations, showcasing lower plasma concentrations but earlier achievement of effective tissue concentrations in animals (503, 504) with documented efficacy in experimental models of disseminated candidiasis, disseminated aspergillosis, and cryptococcal meningoencephalitis (235, 236, 505). The PK/PD index for CAmB has not been established in vitro, but one may assume this to be similar to AmB. Data on pharmacokinetics, absorption, distribution, metabolism, and clearance of these nanoparticles are extremely limited. It could be assumed that the protein binding of the nanoparticles is low, but data are missing. Once intracellular in the macrophages, it is unclear what the release rate from the nanoparticles is and what percentage circulates as free AmB.

In a phase I, open-label, ascending dose trial, CAmB administered in four to six divided daily doses was generally safe and tolerated in healthy HIV-positive Ugandans at doses of up to 2 g/day (234). Improved absorption and plasma concentrations were noted with smaller, frequent doses in phase I study volunteers, compared to less frequent dosing. The median maximum plasma concentration (C_max) of CAmB (59.2 ng/mL) was of an order of magnitude lower than the C_max reported for LAmB (22,900 ng/mL after a single dose of 2 mg/kg) and AmB deoxycholate (1,400 ng/mL after a single dose of 0.6 mg/kg), demonstrating that the pharmacokinetics of CAmB are different than those of well-known AmB formulations (234, 506). Lower C_max of CAmB may be explained by the fact that the drug is taken up by macrophages and that this cell system is not part of the analytical assay that focuses on plasma concentrations. Nevertheless, lower C_max of circulating AmB in plasma may indicate a lower likelihood of toxicity as there is less drug in plasma to interact with renal tubular cells. The time to maximum concentration was around 24 hours. The estimated half-life of AmB was estimated at a median of around 48 hours.

The observed variation in pharmacokinetics and tolerance is due to the physical stability of the lipid nanocrystals, their uptake in macrophages, and their targeted transport to sites of infection and prompts different dosing strategies and absorption patterns compared to established AmB formulations (507).

DDI

No data available for CAmB, and no drug-drug interactions are known for the parent drug, AmB (467).

Drugs during pregnancy

As most of the new drugs have not been licensed or have substantial experience, in depth knowledge on their safety during pregnancy and lactation is missing. Ibrexafungerp should not be used during pregnancy as findings from animal studies have shown fatal harm (245). In a rabbit animal model, use of ibrexafungerp was associated with rare malformations. Contrary to this, in rats, no fatal toxicity or increased fatal malformation was observed. For rezafungin, no adverse embryofetal outcomes were observed after intravenous administration of up to approximately five or three times the clinical exposure based on AUC comparison to pregnant rats or rabbits during the period of organogenesis (245). Oteseconazole, similar to ibrexafungerp, is contraindicated during pregnancy. In animal studies, ocular abnormalities were observed in both pre- and postnatal settings in the offspring of rats who received oteseconazole from gestation day 6 to lactation day 20 at doses about 3.5 times the recommended human dose based on AUC comparison (245).

At current stage, none of the drugs described in this review can be balanced toward their safety during pregnancy in humans. An assessment of risk vs benefit should be performed on a case-by-case basis.

ANTIFUNGAL RESISTANCE TESTING: TECHNICAL CHALLENGES FOR NEW DRUGS AND CLINICAL IMPLICATIONS

Phenotypic susceptibility testing, EUCAST, and CLSI

Reference methods for susceptibility testing of antifungal agents against yeast, molds, and dermatophytes have been developed by both the CLSI (Clinical and Laboratory Standards Institute) and EUCAST (European Committee on Antimicrobial Susceptibility Testing). The methods are more alike than they are different yet vary in inoculum size, glucose concentration in the growth medium, and plate type [round bottom (CLSI) or flat bottom (EUCAST)]. In addition, the method used for endpoint reading varies [visual (CLSI) or by use of a spectrophotometer for yeast, A. fumigatus (except echinocandins) and dermatophytes (EUCAST)], and therefore, the two test methods do not always generate identical susceptibility results. Consequently, results must be interpreted by the associated clinical breakpoints and ECVs/ECOFFs to obtain a correct classification as susceptible-intermediate resistant (CLSI) and susceptible, susceptible increased exposure, resistant (EUCAST), and wild type/non-wild type.

Rezafungin, ibrexafungerp, and manogepix exhibit fungicidal activity against yeast with susceptibility endpoints read as MICs (174, 180, 214, 217, 508–512), whereas they result in aberrant growth in microdilution testing of molds and thus MEC endpoints (i.e., lowest concentration that results in morphologic changes, including short, stubby, abnormally branched hyphae) (180, 513–515). Olorofim displays delayed fungal killing against most molds leading to a complete inhibition and MIC endpoint but has no activity against yeast or Mucorales. Opelconazole and oteseconazole are both members of the azole class, and thus their MICs are read as complete inhibition against molds and as prominent (greater than 50% inhibition) against yeasts (246, 248, 339, 345, 516–518).

For rezafungin specifically, MICs obtained with EUCAST E.Def 7.4 and CLSI M27Ed4 deviate notably (Table 5). This is because EUCAST experienced significant MIC variation for the most susceptible Candida species related to a non-specific binding of rezafungin to surfaces across microtiter plates, pipette tips, reservoirs, etc. used. Supplementation of the growth medium with 0.002% Tween 20 normalized rezafungin MIC values across plate types and laboratories for the six most common Candida spp. evaluated and generated robust QC ranges while, at the same time, maintained differentiation of WT vs FKS mutants (174, 176). However, as this led to 2–16-fold lower MICs than testing without Tween 20, differences between EUCAST and CLSI MICs are up to six twofold dilution different. Importantly, this will preclude the development of a commercial test that can be interpreted by both EUCAST and CLSI breakpoints and ECVs/ECOFFs (Table 5). Similarly, adherence to plastic has been found to negatively influence the in vitro activity of opelconazole, suggesting that further studies are needed to assess the reproducibility of these results against a larger number of strains, species, and by the EUCAST and CLSI methods (339).

TABLE 5.

Comparison of published MICs obtained for novel antifungals using EUCAST and CLSI reference methods, respectively^a

Species	EUCAST		CLSI		References
	Modal MIC/MIC50 MEC/MEC50	ECOFF/[WT-UL]	Modal MIC/MIC50 MEC/MEC50	ECV/[WT-UL]
Rezafungin
C. albicans	0.001	0.008	0.03	0.06	(174, 179, 180)
C. dubliniensis	0.004	0.016	0.06	0.125
C. glabrata/N. glabratus	0.008	0.016	0.06	0.125
C. krusei/P. kudriavzevii	0.016	0.03	0.03–0.06	0.125
C. parapsilosis	1	4	1	4
C. tropicalis	0.008	0.03	0.03–0.06	0.125
C. auris	Pending	ending	(0.125)–0.25	0.5
Ibrexafungerp
C. albicans	(0.03)–0.06	[0.25]	(0.06)–0.125	[0.125]	(213, 214, 217, 510, 512, 519–521)
C. dubliniensis	(0.125)	[0.5]	(0.125–0.25)
C. glabrata/N. glabratus	0.25–(0.25–0.5)	[1]	0.5–1	[0.5]
C. krusei/P. kudriavzevii	0.5–(0.5–1)	[1]	0.5–(1)	[0.5]
C. parapsilosis	(0.25)–0.5	[2]	0.25	[0.25]
C. tropicalis	(0.25)–0.5	[2]	0.25	[0.25]
C. auris	0.5	[1]	1	[2]
Manogepix
C. albicans	(0.008)	[0.03]	0.004–0.008	[0.016]	(508, 509, 511, 513, 522, 523)
C. dubliniensis	(0.004)	[0.016]	(0.004)	[0.016]
C. glabrata/N. glabratus	(0.06)	[0.125]	0.03–0.06	[0.125]
C. krusei/P. kudriavzevii	(>0.5)	[>0.5]	(>8)	[>8]
C. parapsilosis	(0.03)	[0.06]	0.008–0.03	[0.03]
C. tropicalis	(0.008)	[0.016]	0.008–0.03	[0.03]
C. auris	(0.016)	[0.125]	(0.008)–0.03	[0.125]
A. flavus	0.03		0.016–0.03
A. fumigatus	0.03–0.06		0.016–0.03
A. nidulans	(0.03)
A. niger	0.03		≤0.008–0.016
A. terreus	0.03–0.06		0.008–0.016
F. solani SC	(0.125)		(0.015)–0.03
F. oxysporum SC	0.06		(0.015)–0.06
Olorofim
A. calidoustus	0.125–0.25		(0.03)–(0.06)		(288, 524–526)
A. citrinoterreus	(0.016)		0.016–0.03
A. flavus SC	0.03	[0.06]	(0.015) (GM 0.021)
A. fumigatus	0.06	[0.125]	(0.008) (GM 0.029)
A. niger SC	0.06		(0.015) (GM 0.031)
A. nidulans	0.06–0.125	[0.25]
A. thermomutatus	0.016–0.06		(0.016)
A. terreus	(0.03)	[0.06]	(0.008) (GM 0.014)
A. tubingensis	0.06		0.06
T. rubrum	0.06	[0.125]	(0.06)
Oteseconazole
C. albicans			(0.004)–(0.125)		(246, 248, 255, 527)
C. glabrata/N. glabratus			(0.03)–(0.125)
C. krusei/P. kudriavzevii			(0.25)
C. parapsilosis			(0.008)
C. tropicalis			(0.008)
T. rubrum			(0.03)
Opelconazole
C. auris			(0.06)	[0.5]	(339, 516)
A. fumigatus	(0.125)

^a Values in parenthesis derive from single center studies, and WT-UL are presented in brackets.

Mechanisms of resistance to novel antifungal agents

Rezafungin and ibrexafungerp both target the glucan synthase enzyme, which is important for the fungal cell wall synthesis. Glucan synthase is encoded by the FKS1 gene and in C. glabrata/N. glabratus also FKS2. Although the target enzyme is the same, the binding sites for echinocandins and ibrexafungerp are not identical but overlap one another. Rezafungin MICs, in general, mirror those of the other three echinocandins (anidulafungin, caspofungin, and micafungin) in the sense that FKS mutations in two well-defined hot spot regions of both FKS genes elevate the MICs for all four agents (177). However, the FKS alteration-specific rezafungin MIC elevation was either equal to or less pronounced than the MIC elevation for anidulafungin and micafungin when compared head to head using the EUCAST E.Def 7.3 method (without Tween 20 supplementation of the growth medium) (184). Whether this will translate into differential activity against some mutants remains to be understood. Οne expanded access case reported successful suppression with rezafungin over 13 months of an infection involving a foreign body and mediastinitis due to a multidrug-resistant C. glabrata/N. glabratus with a D666Y alteration in FKS2 (528).

The binding site of ibrexafungerp overlaps with that of the echinocandins on the first amino acid codon of the first echinocandin hot spot (Phenylalanine, F) in FKS1 or FKS2. In a study including 25 isolates with 19 unique amino acid alterations in FKS1 or FKS2, 3 alterations were associated with ibrexafungerp echinocandin cross resistance [C. albicans: F641S (FKS1) and C. glabrata/N. glabratus F659del and F659L (both FKS2)], whereas others were not, including C. glabrata/N. glabratus F659Y (FKS2), C. auris F635Y, as well as 13 other unique alterations in the hot spots for echinocandin resistance that did not involve the initial phenylalanine codon (214). Overall, cross resistance to ibrexafungerp was found in 3/28 (10.7%) echinocandin-resistant isolates in that contemporary isolate collection, suggesting that cross resistance currently involves a minority of isolates (217, 529). Of note, a partial inhibition pattern was observed using the EUCAST E.Def 7.3 methodology against some C. parapsilosis (affecting ~10% isolates) and a few C. dubliniensis (~1%) and C. tropicalis (2%), which complicated MIC determination (214). Whether this reflects different clinical susceptibilities, or a technical issue related to in vitro testing warrants further investigation.

Manogepix targets the conserved fungal Gwt1 enzyme required for acylation of inositol early in the glycosylphosphatidylinositol biosynthesis pathway (530). The spectrum against Candida spp. is broad but does not include the intrinsically resistant species C. krusei/P. kudriavzevii and to a lesser extent C. inconspicua (508, 514). Acquired resistance mechanisms include target gene alterations and induction of efflux pumps. In vitro selection experiments and confirmatory CRISPR technology confirmation have documented acquired resistance with a 16–32-µg/mL MIC elevation in C. albicans, C. glabrata/N. glabratus, and S. cerevisiae caused by a valine (V) to alanine (A) alteration in the target enzyme (V163A in the Gwt1 protein of C. glabrata/N. glabratus, V162A in C. albicans, and V168A in S. cerevisiae) (530). In C. albicans, a heterozygous alteration was sufficient for reduced susceptibility (530). A significant correlation has been observed between manogepix and fluconazole MICs for C. albicans, C. dubliniensis, C. glabrata/N. glabratus, C. parapsilosis, and C. tropicalis with a more discreet one to four twofold elevation of manogepix MICs compared to the MIC elevation associated with target gene alterations in a subset of isolates with acquired fluconazole resistance (508). Two efflux-mediated mechanisms conferring reduced susceptibility to manogepix have been identified (531). In C. albicans, a gain-of-function mutation in the transcription factor gene ZCF29 activated expression of ABC transporter genes CDR1 and SNQ2. In C. parapsilosis, a mitochondrial deletion activated expression of the major facilitator superfamily transporter gene MDR1 (531). To our knowledge, acquired resistance mechanisms in molds have not yet been described.

Olorofim targets the DHODH enzyme, the fourth enzyme in the de novo pyrimidine biosynthesis pathway, which is encoded by the pyrE gene (285). In susceptible organisms, this results in delayed cidality (285). The spectrum includes most molds and dimorphic fungi. Exceptions include Mucorales, which are intrinsically resistant due to differences in their DHODH enzyme (55), Fusarium species, which with the exception of F. verticillioides and F. proliferatum are either resistant (F. dimerum) or partially inhibited (288, 532), and specific Aspergillus species (A. montevidensis and A. chevalieri) (524). So far, the only resistance mechanism described is target gene alterations. In vitro selection studies have generated resistant mutants of A. fumigatus harboring alterations on the G119 codon in 38 of 39 isolates (7 G119A, 21 G119C, 1 G119F, 1 G119Y, 1 G119S, and 7 G119V amino acid substitutions). Finally, one olorofim-resistant isolate harbored an H116P amino acid substitution in the pyrE gene. Loss of enzyme susceptibility was confirmed for G119A, G119V, G119S, and G119C alterations in recombinant enzymes (58).

Both opelconazole and oteseconazole target the lanosterol 14α-demethylase enzyme inhibiting the last step in the ergosterol biosynthesis pathway. The spectrum of activity of the tetrazole oteseconazole includes Candida and Cryptococcus spp., dimorphic fungi, including Coccidioides, and some molds, including A. fumigatus, Rhizopus arrhizus (but not R. delemar or other Mucorales), and Trichophyton rubrum (246, 248, 517, 518, 527, 533, 534). Similar to other azoles, resistance to oteseconozole in Candida spp. is mediated by efflux pumps (CDR1, CDR2, MDR1, PDH1, and SNQ2) and mutation within ERG11, which encodes lanosterol 14α-demethylase (248, 518). One specific amino acid change (Y123F) in lanosterol 14α-demethylase was noted to lead to 16-fold higher oteseconazole MIC in C. albicans (248). Correlations between oteseconazole MICs and those of fluconazole and voriconazole have been reported (248). Similar to oteseconazole, the spectrum for opelconazole, a triazole under development for aerosolized delivery to the lungs, includes Candida (including C. auris) and Cryptococcus spp., and T. rubrum (339, 345, 516). Its activity against Aspergillus is species dependent, as it has potent activity against A. fumigatus and A. terreus (MIC range 0.063–0.078 mg/L), but this is markedly reduced against other Aspergillus spp., including A. flavus and A. niger (MIC range 6–>8 mg/L) (339, 345). Similarly, activity has been reported against a single strain of R. arrhizus (MIC 2 mg/L), but opelconazole lacks activity against other Mucorales, including Mucor, Rhizomucor, and Lichtheimia spp. (MIC >8 mg/L). Although the exact mechanisms of resistance to opelconazole have not been described, it lacks in vitro activity against A. fumigatus isolates harboring TR₃₄/L98H and TR₄₆/Y121F/T289A mutations (MIC >16 mg/L) (345).

Molecular testing

Molecular methods can detect resistance mechanisms but not susceptibility, as several resistance mechanisms may play in concert and new mechanisms may arise, and because the sensitivity for detection of a single-copy resistance target gene is lower than that of multicopy genes typically used for detection of the organism itself (58, 535). In addition, resistant infections may involve susceptible as well as resistant organisms contributing to the discrepancy between copy number of fungal DNA and of mutant DNA (536). Commercial kits exist for the detection of Cyp51A alterations in A. fumigatus, some of which may confer cross-resistance to opelconazole or oteseconazole (535), but are not available for resistance detection to the other new agents. Resistance to rezafungin and ibrexafungerp can be detected via FKS sequencing. For echinocandins, the magnitude of MIC elevations depends on the species, codon, and substitution involved (184, 535), whereas less is known regarding which regions confer resistance to ibrexafungerp apart from the first codon in hotspot 1 of the FKS genes that confer echinocandin resistance (217, 529). Similarly, target gene sequencing can detect mutations associated with resistance to manogepix and olorofim, but for manogepix, drug transporters induced by prior azole exposure may also contribute to resistance, and for both, it is likely that new mechanisms not yet known will emerge with use.

Combination testing

With the development of new antifungals, there is also interest in combining these with clinically available drugs to determine if activity and outcomes can be improved over monotherapy alone, especially against pathogens that may have acquired resistance or are intrinsically resistant to available drugs (e.g., C. auris, azole-resistant Aspergillus species, Fusarium species, and Scedosporium/Lomentospora species). From an in vitro standpoint, different methods have been used to assess for synergy or antagonism with antifungal combinations. The methods most commonly used include checkerboard (also referred to as chequerboard), time-kill, and agar/gradient diffusion methods (537). Each of these methods has their own strengths and limitations, which have been reviewed elsewhere (537). Neither CLSI nor EUCAST has published standardized methods or quality control procedures for combination testing for either research or clinical testing. In addition, interpreting the results of combination testing is also difficult when the endpoints used to measure activity differ between the agents being tested (e.g., MIC 50% inhibition vs 100% inhibition vs yeasts, or MIC vs MEC for molds). Finally, in vitro combination results have not always correlated with clinical outcomes (i.e., antagonism reported between fluconazole and AmB in vitro, which was not supported by the results of a clinical trial) (538, 539).

Despite these limitations, combination testing is often performed during pre-clinical development, and in many instances, the in vitro results were correlated with outcomes in in vivo experimental models. With ibrexafungerp, fractional inhibitory concentration index analysis following checkerboard testing demonstrated indifference when combined with isavuconazole against a single A. fumigatus isolate but synergy when performed by Bliss independence drug interaction analysis (540). When tested in a rabbit model of invasive pulmonary aspergillosis, this combination resulted in improvements in survival and reductions in serum galactomannan compared to each agent alone. Similarly, opelconazole demonstrated improvements in survival and reductions in galactomannan levels in neutropenic mice with invasive pulmonary aspergillosis when this agent was combined with posaconazole compared to each agent alone (345). In contrast, checkerboard combination testing failed to demonstrate synergy with this combination or when opelconazole was combined with either voriconazole or itraconazole. However, synergy was observed with these combinations when a human alveolus bilayer model was employed in which opelconazole was administered apically (simulating aerosolized delivery), and the other agents were administered basolateral (simulating systemic delivery). Others have also reported improved outcomes when fosmanogepix was combined with LAmB in murine models of pulmonary aspergillosis and mucormycosis, and disseminated fusariosis, although in vitro combination testing was not performed (242). Interestingly, in vitro antagonism has been reported when olorofim was combined with itraconazole or voriconazole, by both checkerboard and disk diffusion methods, and this was due to azole-induced upregulation of the pyrimidine biosynthesis pathway (294). It is currently unknown if this in vitro antagonism would translate into reduced in vivo efficacy when these different antifungal classes are combined.

FUTURE DIRECTIONS AND CONCLUSION

Continued advances in healthcare have resulted in a persistent expansion in the immunosuppressed patient population. The number of patients and the duration of risk are thus continually expanding, increasing the burden of fungal infections. Currently available agents have been a tremendous breakthrough, yet novel pathogens (C. auris), the development of resistance (non-albicans Candida spp., Aspergillus, and the rare molds) either de novo or following exposure in vivo or with the use of environmental fungicides, the expansion of the endemic mycoses to new regions, mandate the need for novel therapeutic options.

New antifungals that are currently in clinical development have the potential to complement the existing antifungal repertoire and fill many of the mounting needs in the care of invasive mycoses and thereby improve patient outcomes. Most novel agents appear to have an advantageous safety profile. Additionally, drug-drug interactions may become less frequent with new antifungal agents; while olorofim and fosmanogepix may cause interaction with CYP3A4/5 CYP450-subenzyme, or other relevant enzymes, this is likely not as relevant for opelconazole, given the lack of systemic drug levels following inhalational delivery.

Novel antifungals will bring significant advantages for the management of outpatients and those requiring long-term treatment. Ibrexafungerp, fosmanogepix, opelconazole, oteseconazole, CAmB, and olorofim can all be administered orally or via inhalation, and rezafungin with a once-weekly administration seems all to be viable options for these settings. Immunotherapy may complement antifungal therapy in the future, as encouraging results have been obtained with different forms of immunotherapy in preclinical models, and clinical trials including patients are ongoing. Proper patient stratification (e.g., immunosuppressed vs aberrant or hyperinflammatory) will be needed to ensure tolerability and efficacy for each immunotherapeutic modality. Immunological markers and genetic tests may also help in identifying those patients at highest risk who could benefit from a selected immunotherapeutic. While immunotherapy of fungal infections is not yet ready for prime time, high-quality clinical trials in well-defined patient groups might lead to implementation of immunomodulatory prophylaxis or treatment for IA during the next decade.

To conclude, the spectrum of fungal infections will change significantly over the upcoming decades, driven by advances in medicine and a growing population at risk, climate change triggering emergence of new fungal pathogens and fungal outbreaks, as well as increasing resistance to antifungals. With the development of several new promising antifungal compounds, antifungal treatment of fungal infections will likely change as well over the next decades. New antifungals are expected to fill important gaps we are facing with current treatment options, and the ultimate hope is that these new treatments will contribute to better patient outcomes and survival.

Biographies

Martin Hoenigl, M.D., is an Associate Professor for Translational Mycology at the Division of Infectious Diseases, Medical University of Graz, Austria. After his graduation and clinical education at the Medical University of Graz, he became an Associate Professor of Medicine at the Division of Infectious Diseases and Global Public Health at the University of California San Diego, before returning to Graz for his new appointment. Dr. Hoenigl has particular interest in conducting research on clinical mycology, including fungal diagnostics, host-fungal pathogen interactions, and pharmacology of antifungal drugs; he is in the field for over 15 years, and has published over 300 scientific papers. Dr. Hoenigl is the past president of the European Confederation of Medical Mycology (ECMM). He serves as Associate Editor at Open Forum Infectious Diseases (OFID) and Deputy Editor at Mycopathologia. Find more information on Dr. Hoenigl on his Clinical Mycology YouTube channel and on Twitter, under @martinhoenigl.

Amir Arastehfar is a postdoctoral fellow of Infectious Diseases in Massachusetts General Hospital, Boston. He has studied the mechanisms underlying antifungal resistance and tolerance in two major pathogenic yeast species, Nakaseomyces glabratus and Candida parapsilosis. His current studies are focused on the interaction of innate immune cells with pathogenic yeast species and how this interaction confers antifungal tolerance in N. glabratus.

Maiken Cavling Arendrup obtained her medical degree from Copenhagen University, Denmark, in 1988, followed by a PhD degree on neutralizing antibodies and HIV infection in 1992 at Dept Inf Dis, Copenhagen University Hospital Hvidovre. In 2001, she completed further training as a specialist in clinical microbiology (Hillerød Hospital, Herlev Hospital and Statens Serum Institut (SSI) Copenhagen) and in 2013 defended her doctoral thesis on epidemiology and susceptibility of candidaemia for the Dr. Med. Sci degree. She was associate professor at Dept Inf Dis at Aarhus University Hospital 2013-2016. Prof Arendrup is head of the Mycology Reference Laboratory at the SSI (since 2001), professor at the Copenhagen University Hospital Rigshospitalet (since 2016), and chair of the EUCAST Antifungal Susceptibility Testing Subcommittee Steering Committee (since 2011). Her main research interests since the turn of the millennium include epidemiology, susceptibility, breakpoint development, diagnostics and treatment of fungal infections.

Roger Brüggemann is associate professor of clinical pharmacology of anti-infectives at Radboud university medical center in Nijmegen. He is responsible for the translational pharmacological research on antifungal drugs. His work involves three domains ranging from in vitro and animal research to trials in humans with strong national and international collaborations. Roger’s research involves the resolution of PK and PD behaviour and factors that impact this. He is co-founder of the Center of Expertise in Mycology Radboudumc, the National Reference center in the Netherlands.

Agostinho Carvalho obtained his PhD in Health Sciences from the University of Minho in Portugal in 2008. He is currently a Principal Investigator with Habilitation and the Vice-Director of the Life and Health Sciences Research Institute (ICVS) of the University of Minho, Braga, Portugal. Dr Carvalho focuses his research on the regulatory effects of interindividual genetic variation on molecular and cellular processes of immunity and inflammation, and the identification of novel prognostic, diagnostic and therapeutic targets for the development of personalized medical interventions for fungal diseases. He combines his expertise on immunology and genetics with advanced cellular and animal models of disease and human patients enrolled in national and international consortia. Dr Carvalho has made significant contributions to the field of immunology of fungal infections and his contributions have been acknowledged by his nomination as a fellow of the European Confederation of Medical Mycology (ECMM).

Tom Chiller, MD, MPHTM, earned his medical degree and Master of Public Health degree at Tulane University School of Medicine. At the CDC, Dr. Chiller is the Chief of the Mycotic Diseases branch, where he provides leadership for fungal disease activities, which include detection, prevention and response activities, and policy and advocacy, both nationally and internationally. He also serves as the associate director for CDC’s global programs in the Division of Foodborne, Waterborne, and Environmental Diseases. He remains actively involved in antimicrobial resistance, healthcare-associated infections, molecular epidemiology and laboratory activities for fungal diseases. Dr Chiller is board certified in infectious diseases and is a faculty member in the Division of Infectious Diseases at the Emory School of Medicine. During the past decade with the Mycotic Diseases Branch, Dr Chiller has led efforts to end deaths from opportunistic fungal infections in HIV, control the spread of MDR Candida auris and azole-resistant Aspergillus, and identify emerging mold infections.

Sharon Chen is an Infectious Diseases physician/Clinical Microbiologist at Westmead Hospital, Institute of Clinical Pathology and Medical Research, Sydney, Australia. She received her tertiary education at The University of Sydney. Her PhD focused on Cryptococcus epidemiology and phospholipase enzymes. She is Director, Centre of Infectious Diseases and Microbiology Laboratory Services which incorporates reference functions for diagnostic mycology, and including governance and service provision for regional NSW laboratories. Within Westmead Hospital, till 2022, she undertook primary clinical liaison for the stem cell and solid organ transplant services and continues to perform laboratory liaison. Sharon’s long-standing interests include fungal diseases epidemiology, translation of new diagnostic tests into practice, antifungal drug resistance and novel agents. She is primary investigator of national antifungal drug trials and has coordinated national/international fungal management guidelines. A founding member of the Australia and New Zealand Mycoses Interest Group she has links with the ECMM and the MSC-ERC.

Matthias Egger M.D. is a research assistant at the Division of Infectious Diseases in Graz, Austria. He received his tertiary education at the Medical University of Graz and currently works on his doctoral thesis focusing on the lung mycobiome and fungal translocation in patients with liver cirrhosis. He is part of the Translational Medical Mycology Research Unit, ECMM Excellence Center for Medical Mycology, Medical University of Graz, Austria with its PI Prof. Hoenigl. His primary research interests include diagnostics of invasive fungal infections, novel antifungal agents and composition of the gut and lung mycobiome in health and disease. He is a member of the ECMM, ISHAM, ESCMID and especially of its young associations.

Simon Feys obtained his MD title in 2018 at KU Leuven in Belgium. He is currently specializing in internal medicine with a focus on critical care at University Hospitals Leuven, Belgium. Since 2020, he combines his clinical work with PhD-research at KU Leuven in the group of Prof. Joost Wauters. His research focuses on viral-associated pulmonary aspergillosis in critically ill patients, from both clinical and translational perspectives. Dr. Feys combines his clinical expertise with patient sample-based multi-omics approaches to increase our understanding of the pathophysiology of viral-associated pulmonary aspergillosis, thereby identifying novel targets to optimize our prognostic, diagnostic and therapeutic toolboxes. He has presented his work at major conferences in the field such as ECCMID, TIMM and ISHAM, and his PhD-research resulted in first author publications in prestigious journals such as Intensive Care Medicine, American Journal of Respiratory and Critical Care Medicine, Clinical Infectious Diseases and The Lancet Respiratory Medicine.

Jean-Pierre Gangneux is Professor in Medicine, specialized in mycology and parasitology at the Rennes Teaching Hospital and Rennes University, France. He has expertise in diagnosis and treatment of fungal infections due to Candida, Aspergillus and other fungi. The main topic of his research is the complex host-microorganism interplay and environmental reservoirs of human pathogens (UMR INSERM U1085 - Irset - Université Rennes). He is the past-General Secretary of European Confederation of Medical Mycology 2017-2023 and the former President of the French Society for Medical Mycology (2012-2021). He is head of the Laboratory of Mycology and Parasitology in Rennes teaching hospital, head of the Rennes European ECMM Excellence Center in Medical Mycology and associate-laboratory of the French National Reference Center on Chronic Aspergillosis. He is principal investigator of different research projects on medical mycology, and he organized multiple national and international meetings and published more than 250 articles in scientific journals.

Jeremy A. W. Gold, MD, MS, graduated from McGill University in Montreal, QC, Canada, in 2011 with a Bachelor of Science in Biology. He graduated from Albert Einstein College of Medicine in Bronx, NY, United States, in 2016 with a Medical Degree and a Master of Science in Clinical Research Methods. Subsequently, he completed his residency in internal medicine at Columbia University Medical Center/New York Presbyterian Hospital in 2019. After residency, he served as an Epidemic Intelligence Service Officer with CDC’s Mycotic Diseases Branch in Atlanta, GA, United States. He continues to work as a medical epidemiologist in CDC’s Mycotic Diseases Branch, where his work focuses on antifungal-resistant mold infections.

Andreas H. Groll is Professor of Paediatrics, Head of the Infectious Disease Research Programme and Deputy Director of the Department of Haematology/Oncology at the University Children's Hospital in Münster, Germany. Dr Groll’s postgraduate education included fellowships in Infectious Diseases at the Children's Hospital in Boston, and in Paediatric Haematology/Oncology at the National Cancer Institute in Bethesda, Maryland. Dr. Groll is board certified in Paediatrics, Paediatric Haematology/Oncology, and Infectious Diseases. Dr.Groll’s research interests include infectious complications in the immunocompromised host, particularly invasive fungal infections, the pharmacokinetics and pharmacodynamics of antimicrobial agents, and the design and conduct of clinical research studies. He is a member of several international medical societies, on the editorial board of several international journals and has published more than 300 scientific articles thus far. He is regularly engaged in patient care as Attending Physician of the inpatient and outpatient service and the haematopoietic stem cell transplant program.

Jannes Heylen, MD, graduated with great distinction in medicine at KU Leuven, Belgium. He is currently resident in training in Internal Medicine at University Hospitals Leuven, Belgium. He combines his clinical work with translational research on severe fungal infections in critically ill patients at the intensive care unit. Dr. Heylen is part of the research group led by Prof. Joost Wauters, which has a specific interest in deciphering the antifungal host response in critically ill patients and aims to develop host-based biomarkers able to guide clinicians in the management of invasive fungal disease.

Jeffrey D. Jenks, M.D., M.P.H., is the Medical and Laboratory Director at the Durham County Department of Public Health and an Adjunct Associate Professor in the Division of Infectious Diseases, Department of Medicine, at Duke University (both in Durham, North Carolina, United States). He was previously an Associate Clinical Professor at the University of California San Diego. He received his M.D. and M.P.H degrees from the Wright State University Boonshoft School of Medicine (Fairborn, Ohio, United States), completed his internal medicine internship and residency at Boston Medical Center (Boston, Massachusetts, United States), and completed his infectious diseases fellowship at the University of California San Diego (La Jolla, California, United States). He has published over 50 peer-reviewed scientific manuscripts. His research interests include clinical mycology, the epidemiology of fungal diseases, sexually transmitted infections, and the intersection between social determinants of health and infectious diseases.

Robert Krause, M.D., DTMP, is leading the Division of Infectious Diseases, Department of Internal Medicine, and ECMM excellence center, at the Medical University of Graz, Austria (https://infektiologie.medunigraz.at/en/). He is actually Co-Director of biotechmed Graz, a research alliance of the University of Graz, the Graz University of Technology, and the Medical University of Graz that merges research activities at the interface of basic biomedical science, technological developments and medical/clinical applications with the aim of joint research for health (https://biotechmedgraz.at/en/). He received his M.D. from the Karl Franzens University, Graz, Austria and his Diploma in Tropical Medicine and Parasitology from Bernhard Nocht Institute Hamburg, Germany, and completed his clinical training in internal medicine, infectious diseases, and intensive care medicine at the Medical University in Graz, Austria. His research interests include fungal infections (including those in immunocompromised patients as well as critically ill patients), emerging infections, antibiotic stewardship, sepsis and gastrointestinal infections.

Katrien Lagrou obtained her Master’s degree in Pharmaceutical Sciences from the University of Leuven (KU Leuven, Belgium) in 1992, and remained there to specialise in Laboratory Medicine between 1992 and 1997. Afterwards, she received a degree in Mycology from the Institute of Tropical Medicine in Antwerp, Belgium and completed her PhD in 2002. Prof. Lagrou is currently Head of the Microbiology Laboratory at the University Hospitals of Leuven and coordinates the Belgian National Reference Center for Mycosis. She is full professor at the Faculty of Medicine of the KU Leuven and Chair of the Department of Microbiology, Immunology and Transplantation. She serves the European Confederation of Medical Mycology (ECMM) as chair of the Academy Committee. Professor Lagrou’s main interest is the diagnosis and treatment of infections in severely immunocompromised patients, with a focus on invasive pulmonary aspergillosis and published her research in more than 350 manuscripts in peer reviewed journals.

Frédéric Lamoth is a Swiss medical doctor with board certifications in general internal medicine, infectious diseases and clinical microbiology. After a post-doctoral fellowship at the Mycology Research Unit of Duke University (Durham, NC), he is currently associate professor at the Infectious Diseases Service and the Institute of Microbiology of the Lausanne University Hospital and University of Lausanne (Switzerland). His research focuses on medical mycology including molecular biology (mechanisms of antifungal resistance), diagnostic and therapeutic approaches.

Juergen Prattes, M.D. is a board certified Internal Medicine specialist and Deputy Head of the ECMM Excellence Center in Graz, Austria. He is working as an Infectious Diseases specialist at the University Hospital of Graz, Austria – a tertiary care hospital including a hematological department, a stem-cell transplant unit, a solid organ transplant unit and several intensive care units. His research is focused on invasive fungal infections with a special focus on individualized diagnostics and new antifungals. He is currently holding the position as president of the Austrian Society of Medical Mycology and is a designated Fellow of the ECMM (FECMM).

Sarah Sedik is a Scientific Research Associate in the research group led by Martin Hoenigl, M.D. and Juergen Prattes, M.D. within the Division of Infectious Diseases at the Department of Internal Medicine, Medical University of Graz, Austria. She received her Bachelor's Degree in Molecular Biology, followed by a Master of Science Degree in Biochemistry and Molecular Biomedicine from the Karl Franzens University of Graz. During her master's program, she conducted her thesis at the Medical University of Graz, focusing specifically on prostate cancer research. She is an active member of the ECMM young scientists’ community and OEGMM. Her research interests span molecular biology, clinical research, diagnostics of invasive fungal infections, and microbiology.

Joost Wauters (°1972, MD, PhD) studied Civil Engineering and Medicine at the University of Leuven (Belgium), and specialized in Internal Medicine, Emergency Medicine and Intensive Care Medicine. Since 2011, he is ICU physician and full staff member at the Medical ICU of the University Hospitals Leuven and Associate Professor at the Faculty of Medicine of the University of Leuven (Belgium). His main research activities are about severe infections and pharmacokinetics of antimicrobials in critically ill patients. Being in the field since 2015, he is currently team leader doing translational research on viral-associated pulmonary aspergillosis (IAPA/CAPA). Moreover, he is international speaker and principal investigator of several international multicenter studies and he is member of several (inter)national scientific ICU and ID societies.

Nathan P. Wiederhold, PharmD, is Professor in the Department of Pathology and Laboratory Medicine at the University of Texas Health Science Center at San Antonio. He serves as the Director of the Fungus Testing Laboratory, an academic reference mycology laboratory for clinical diagnostic testing, including antifungal susceptibility testing, fungal species identification, and antifungal therapeutic drug monitoring. Dr. Wiederhold is also highly involved in pre-clinical studies of antifungal agents and assays for the diagnosis of invasive fungal infections. In addition to his clinical and research responsibilities, Dr. Wiederhold serves as an Associate Editor for the Journal of Antimicrobial Chemotherapy and is on the editorial boards for Antimicrobial Agents and Chemotherapy and the Journal of Clinical Microbiology. He is also currently vicechair of the Clinical and Laboratory Standards Institute (CLSI) Antifungal Susceptibility Subcommittee.

George R. Thompson III, M.D., is a professor of Clinical Medicine at the University of California, Davis, School of Medicine with a joint appointment in the Departments of Medical Microbiology and Immunology, and Internal Medicine, Division of Infectious Diseases. Dr. Thompson specializes in the care of patients with invasive fungal infections and has research interests are in fungal diagnostics, clinical trials, novel antifungal agents, and host immunogenetics. Dr. Thompson has served on the IDSA Journal Club is a member of the Coccidioidomycosis Study Group Planning Committee, and as chair of the Mycoses Study Group Education Committee. He has been active in the field of mycology for over 15 years and has published over 200 manuscripts to date.

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