Innovative and potential treatments for fungal central nervous system infections

Abstract

Fungal infections of the central nervous system (FI-CNS) are a problematic and important medical challenge considering that those most affected are immunocompromised. Individuals with systemic cryptococcosis (67–84%), candidiasis (3–64%), blastomycosis (40%), coccidioidomycosis (25%), histoplasmosis (5–20%), mucormycosis (12%), and aspergillosis (4–6%) are highly susceptible to develop CNS involvement, which often results in high mortality (15–100%) depending on the mycosis and the affected immunosuppressed population. Current antifungal drugs are limited, prone to resistance, present host toxicity, and show reduced brain penetration, making FI-CNS very difficult to treat. Given these limitations and the rise in FI-CNS, there is a need for innovative strategies for therapeutic development and treatments to manage FI-CNS in at-risk populations. Here, we discuss standards of care, antifungal drug candidates, and novel molecular targets in the blood–brain barrier, which is a protective structure that regulates movement of particles in and out of the brain, to prevent and combat FI-CNS.

Introduction

Central nervous system (CNS) infections are caused by diverse microorganisms, including viruses, bacteria, fungi, and parasites. Viruses are the main cause of encephalitis, with an estimation of ~1.4 million cases and ~90 000 deaths globally in 2019 [1]. Bacterial meningitis causes ≥ 1.2 million cases per year worldwide, and without adequate treatment, the fatality rates can reach up to 70% [2]. Fungal infections of the central nervous system (FI-CNS) affect ~1.9 million people globally, resulting in ~1.6 million deaths annually [3]. They are severe, difficult to treat, and usually life-threatening conditions that require rapid and aggressive medical treatment. FI-CNS involve different clinical manifestations, including meningitis, encephalitis, hydrocephalus, cerebral abscesses, and stroke. The frequency of FI-CNS is rising due to an increase in individuals with immunosuppression, including HIV/AIDS, cancer, and organ transplantation [4,5], among others. Cryptococcus neoformans is the most common etiological agent of fungal meningitis (70.1%) in the United States (U.S.), followed by Coccidioides species (16.4%), Histoplasma capsulatum (6.0%), and Candida species (7.6%) [6]. Coccidioides and H. capsulatum cause meningitis in endemic areas, while Candida species mostly affect premature babies and individuals with CNS prosthetic devices (e.g. shunts or drains). Molds, mainly Aspergillus and Mucorales (e.g. Rhizopus, Mucor, etc.) species, can cause CNS infections that are characterized by localized mass lesions in brain tissue. Less common and under special circumstances, species of Fusarium, Scedosporium, or dematiaceous (pigmented) molds cause CNS infections. For example, the black-pigmented mold Exserohilum rostratum caused the largest-ever U.S. healthcare-associated outbreak when 753 cases of meningitis were reported in patients who received epidural injections of methylprednisolone, which was contaminated during the manufacturing process in a compounding pharmacy, resulting in 64 deaths [7]. Despite the great number of different FI-CNS agents, only some possess well-defined treatment standards (e.g. cryptococcal meningitis and CNS aspergillosis). Hence, a prompt diagnosis of fungal infection together with correct identification of the causative agent is needed to allow the administration of effective therapy in patients with FI-CNS to reduce the associated high mortality [8]. In this review, we discuss the (1) challenges faced by current antifungal drugs, (2) status of the recommended standard of care for treatment of cryptococcal meningitis, (3) potential antifungal therapeutic candidates, and (4) novel molecular targets to prevent FI-CNS.

Challenges faced by antifungal drugs used to combat fungal infections of the central nervous system

There are several challenges associated to the antifungal drugs available for the treatment of FI-CNS (Table 1). First, there is limited drug class availability (e.g. polyenes, azoles, and echinocandins) for the treatment of mycoses because fungi are eukaryotes and physiologically (e.g. cell structure and metabolism) related to mammals, which complicates the design of new therapeutic candidates, making it difficult to treat FI-CNS caused by a broad range of fungi. Second, there is sufficient and well-documented evidence of antifungal drug resistance, mostly driven by fungal physiological adaptations and genetic mutations after overexposure to the drugs. Third, antifungal drugs can have significant toxicity and side effects, particularly in patients with preexisting medical conditions [9]. Fourth, antifungal drugs can be excluded from the CNS during infection, limiting their bioavailability. The CNS is a very protected environment, with a highly selectively permeable blood–brain barrier (BBB) that regulates the exchange of molecules with the circulatory system. Thus, the treatment of FI-CNS with antifungal drugs is problematic as these molecules may not cross the BBB and penetrate to brain tissue or get cleared easily, not being able to reach concentrations to control and eradicate the infection [10]. Fifth, compared with antibiotics, there has been relatively less investment and research in the development of new antifungal drugs. Given that the reduced antimycotic arsenal increases the risk of drug resistance and limits options for patients [11], the development of new and diverse treatment options is necessary to combat the rise in FI-CNS cases.

Table 1.

Challenges associated to the existing antifungal drugs for the treatment of FI-CNS.

Limited drug class availability. Drug resistance. Toxicity and side effects. Reduced drug CNS penetration or bioavailability. Lack of investment in new drug research and development.

Old drug new formulation for fungal infections of the central nervous system treatment

Amphotericin B (AmpB) is the antifungal drug par excellence to treat infections, which binds to the cell membrane, sequesters ergosterol, and forms pore channels that subsequently result in fungal cell ions/water leakage [12]. AmpB stimulates the production of reactive oxygen species, a universal and important action mechanism that correlates with its fungicidal effect and might explain the low rate of resistance to the molecule [12]. Additionally, AmpB influences the host immune system, which has been linked to both its protective efficacy and its toxicity [13]. Liposomal AmpB is the most common formulation used in clinical practice due to its reduced host toxicity relative to conventional AmpB deoxycholate. The 2018 World Health Organization’s guidelines recommended 7 days of AmpB deoxycholate and flucytosine-based therapy (followed by 1200 mg/kg fluconazole/day for 7 days) as the preferred antifungal regimen for treatment of HIV/AIDS-associated cryptococcal meningitis [14]. However, these guidelines changed in 2022 to a single high dose (10 mg/kg) of liposomal AmpB with 14 days of flucytosine (100 mg/kg/day, divided into four doses/day) and fluconazole (1200 mg/day for adults, 12 mg/kg/day for children and teenagers, up to a maximum of 800 mg/day) [15]. Flucytosine is a nucleoside analog that shows antifungal activity by disrupting pyrimidine metabolism in the nucleus. In fact, the synergism of AmpB and flucytosine relies on the increased flucytosine uptake by cryptococci due to the cell membrane disruption mediated by AmpB. This therapeutic regimen update to combat cryptococcal meningitis was established by the WHO based on positive results observed in patients participating in clinical trials carried out in Africa, highlighting the importance of regular and consistent evaluation of these antifungal regimens in at-risk populations [16–18]. Similarly, a recent study explored and evaluated the appropriate methods (e.g. ethanol injection, solvent evaporation, and solvent-free) for the preparation of AmpB liposomes with acceptable characteristics [19]. The solvent-free method is the most economical and environmentally friendly, which should be considered for big-scale pharmaceutical productions of the liposomal AmpB to make it more accessible in developing countries with high-risk populations.

Potential therapeutic candidates for treatment of fungal infections of the central nervous system

Microorganisms compete for nutrients and space in the environment. As a mechanism of defense, microbes naturally produce compounds for their own survival, including antifungal molecules. The genus Pseudomonas produces keanumycins (named in honor of actor Keanu Reeves, for their deadly nature comparable to his iconic killer movie characters [e.g. John Wick, The Matrix, etc.]), which are nonribosomal lipopeptides used by these bacteria as protection against amoeba [20]. Keanumycin A is a membrane-active compound that disrupts the fungal cell wall integrity leading to cell death (Table 2). It has shown promising low-concentration (0.9 μM or 1 mg/L) efficacy against multiple Candida species (e.g. C. auris, C. glabrata, C. parapsilosis, and C. tropicalis), but further research is needed to exploit its antifungal potential particularly to treat FI-CNS [20].

Table 2.

Chemical structures of potential antifungal drug candidates to combat FI-CNS.

Drug candidate	Etiological agent (MICc)	Chemical structure
Keanumycin A Nonribosomal lipopeptide from Pseudomonas spp.	Dictyostelium discoideum (IC50 = 2.13 ng/mL) Acanthamoeba castellanii (IC50 = 0.97 μg/mL) Acanthamoeba comandoni (IC50 = 1.50 μg/mL) [20]
Pinocembrina Present in propolis	Tested (2.19 μg/mL) to inhibit Cryptococcus neoformans capsule growth, melanin production, and killing by macrophages [22]
Gallic acida Present in propolis	Tested (0.34–1.03 μg/mL) to inhibit C. neoformans capsule growth, melanin production, and killing by macrophages [22]
Quercetina Present in propolis	Tested (1.13 μg/mL) to inhibit C. neoformans capsule growth, melanin production, and killing by macrophages [22]
Carnosic acid Benzenediol abietane diterpene found in Rosmarinus officinalis and Salvia offfcinalis	C. neoformans, in vitro (50 μg/mL, combined with 200 μg/mL of propolis) [21] Candida albicans and Candida glabrata, in vitro (50 μg/mL, combined with 200 μg/mL of propolis) [23]
Dihydroartemisinic acid (DHAA) Plant natural product, biosynthetic precursor to artemisinin, used to treat malaria	C. neoformans, in vitro (12.5–25 μg/mL) [25]
Cinnamaldehydeb Present in C. zeylanicum EO	Candida auris, in vitro (MIC90 and MFC90 = 0.06% vol/vol) In vivo: Galleria mellonella infected with 107 C. auris cells (0.002% vol/vol + fluconazole 0.25 μg/mL) [26]
Miltefosine (MFS) Alkylphosphocholine drug used to treat leishmaniasis and free-living amoeba infections	C. neoformans, in vitro (2 mg/L MFS and 100 mg/L with alginate nanoparticles functionalized with polysorbate 80 as MFS carriers — P80-MFS-AN) In vivo: Mice infected with 107 C. neoformans cells (P80-MFS-AN 20 mg/kg, 1 and 72 h of infection) [32]
CCG-1423 RhoA/ROCK signaling pathway inhibitor	Potential protective role in reversing the BBB leakage caused by disaggregation of cytoskeleton actin and preventing downregulation of junctional proteins such as claudin-5, occludin, and ZO-1 in BMVEC [42]
PP2 Src PTK inhibitor	Potential role in reversing the BBB leakage caused by internalization of VE-cadherin [50]

Molecules were drawn by Marta Reguera-Gomez using ChemSketch software

^aPresent in propolis.

^bPresent in Cinnamomum zeylanicum.

^cMinimum Inhibitory Concentration (MIC).

Nonmicrobial natural products are a rich source of antimicrobial compounds in antifungal drug discovery. Among these natural compounds, propolis deserves great attention and represents an important target in bio-technological research, being cheap and easily available [21]. Propolis is a complex resinous product collected by honeybees (Apis mellifera, Table 2), which is used as a protective mechanism against predators. Although the chemical composition of propolis shows great differences depending on environmental and biotic factors, the main components are flavonoids, phenolic acids, terpenes, aromatic aldehydes and alcohols, and fatty acids. A propolis extract demonstrated anticryptococcal activity by inhibiting the production of cell wall-main component chitin and cell wall-associated melanin [22]. Recently, a propolis extract from China, showed cytotoxic activity against C. neoformans, and its 1:4 ratio combination with carnosic acid (Table 2) results in a synergistic fungicidal and antibiofilm effect [21]. Additionally, the propolis–carnosic acid combination has efficacy against C. albicans and C. glabrata [23]. Although not yet tested against FI-CNS, these findings support the potential application of mixtures containing these two natural extracts in the design of new antifungal strategies to combat these opportunistic infections.

Dihydroartemisinic acid (DHAA) is a plant natural product that undergoes a spontaneous endoperoxide-forming cascade reaction to yield artemisinin in the presence of air (Table 2) [24]. The endoperoxide functional group gives artemisinin its biological activity that kills Plasmodium falciparum, the parasite that causes malaria. A DHAA derivative, 2,3-didehydro-DHAA, with a double bond at the C2-position, was synthesized and instead of forming an endoperoxide, this compound predominantly underwent aromatization in the presence of air [25]. Owing to their similarity in structure to antimicrobial natural products, the synthesized compounds in this study were tested for antifungal activity against C. neoformans. MIC values ranging from 12.5 to 25 μg/mL were used against C. neoformans demonstrating efficacy. However, aromatic compounds have difficulty reaching the CNS due to their hydrophobic nature, thus, further in vivo studies are required to determine the efficacy of 2,3-didehydro-DHAA against FI-CNS.

Essential oils (EOs), alone or in combination with conventional drugs, are used to fight human pathogens, including multidrug-resistant C. auris [26]. EOs are mixtures of natural substances obtained from the distillation or pressing of aromatic plants with a known antimicrobial activity [27]. There are several EOs with known antifungal activity [28], including Cinnamomum zeylanicum-derived (Table 2), which promotes the accumulation of fluconazole inside C. auris cells by decreasing their fungal Adenosine Triphosphatase (ATPase) activity. In this way, the EO can effectively exert its pharmacologic effects while circumventing yeast resistance. If subsequent investigations confirm these findings, there will be a huge potential for developing novel therapeutic formulations that actively combat C. auris resistance [26]. Next, it would be ideal to develop a formulation that allows the delivery of the compound into the CNS. One potential approach involves the use of nanoparticles to encapsulate EOs with antifungal activity. Nanoliposomes have been used for topical or gastrointestinal-targeted compounds [29,30], although other lipid-based nanoparticle formulations such as nanoemulsions are being studied for nose-to-brain drug delivery [31]. Moreover, alginate nanoparticles functionalized with polysorbate 80 were successfully used as carriers of miltefosine (MFS) (an oral U.S. Food and Drug Administration-approved treatment of leishmaniasis) for treatment of cryptococcal meningitis in mice [32]. These nanoparticles promoted sustained release of MFS (Table 2), were more widely biodistributed (including in the brain), and decreased brain fungal burden without producing detectable damage to peripheral organs.

Despite having promising antifungal drug candidates, it is important to discuss their potential risk of failure in human clinical trials. Recently, a few compounds with high in vitro and in vivo antifungal efficacy failed to transition to human use after showing severe side effects. This was the case with sertraline, a selective serotonin reuptake inhibitor clinically tested with fluconazole as an adjunctive antifungal therapy in HIV-infected individuals with cryptococcal meningitis [1]. Although the combined treatment resulted in faster cryptococcal clearance in cerebrospinal fluid [3], sertraline was associated with serious adverse events, including persistent psychosis and aggressive behavioral changes, leading to treatment interruption [1]. To overcome this setback, novel sertraline derivatives with anticryptococcal activity have been recently designed by scaffold hopping [33,34], which may be clinically tested in future studies.

Novel molecular targets to prevent fungal infections of the central nervous system

The interaction of C. neoformans glucuronoxylomannan (GXM), the main component of the fungus polysaccharide capsule, with the BBB, demonstrated that GXM can disrupt the integrity of this barrier, which can be important in the trans- or paracellular passage of free cryptococci, as well as those being transported inside of macrophages using the trojan horse mechanism [35]. In human brain microvascular endothelial cells (BMVEC), GXM induces activation of Ras homolog family member A (RhoA) (Figure 1), which activates Rho-associated protein kinase (ROCK) [36]. ROCK inhibits the myosin light-chain (MLC) phosphatase, leading to actin stress fiber formation through the enhancement of MLC phosphorylation [37]. Actin stress fibers contribute to the internalization and lysosomal degradation of claudin and occludin, disrupting tight junctions between BMVEC [38] and weakening the BBB [35]. CCG-1423 is a RhoA/ROCK signaling pathway inhibitor (Table 2), being tested for the treatment of prostate cancer [39,40] and as a modulator of mitochondrial function [41]. This drug may reverse the actin stress fiber formation in BMVEC and prevent lysosomal degradation of tight junctions, suggesting that the RhoA signaling pathway is a potential therapeutic target for protection against GXM-mediated BBB disruption [42]. In addition to CCG-1423, multiple RhoA/ROCK inhibitors are in development for use to treat human diseases (Table 3) [43], which can be repurposed, making them prospective candidates for further investigations to reduce BBB permeability and FI-CNS.

Fig. 1.

Proposed model of RhoA and Src inhibition during Cryptococcus neoformans GXM-mediated disruption of the BBB. GXM shed from C. neoformans potentially binds and activates receptors (e.g. TLR2/4; the specific receptor remains yet to be determined) on the cell surface of BMVEC, causing BBB disruption via RhoA signaling through ROCK to maintain MLC phosphorylation, actin stress fiber formation, and the internalization and degradation of tight-junction proteins claudin and occludin (solid arrows). In addition, GXM signals through Src and RhoA to stimulate the phosphorylation of VE-cadherin in adherens junctions, leading to internalization of VE-cadherin and disruption of the BBB. The Src inhibitor PP2 and the RhoA/ROCK inhibitor CCG-1423 can inhibit endothelial barrier breakdown caused by GXM. This diagram was conceptualized by Michael R. Dores and drawn by Marta Reguera-Gomez with BioRender.com.

Table 3.

RhoA/ROCK signaling pathway inhibitors in development for human use.

Compound	Ki (ROCK 1)	Development status
Netarsudil	0.2–10.3 nM	Phase 3, glaucoma
Fasudil	330 nM	Phase 3, Raynaud’s phenomenon
		Phase 2, tauopathy
Sovesudil	3.7 nM	Phase 2, glaucoma
Belumosudil	60 nM	Phase 2, psoriasis
AT13148a	6 nM	Phase 1, solid tumors

^aAT13148 is a multi-protein kinase A, G, and C (AGC kinase) inhibitor that targets ROCK1/2, Protein kinase A (PKA), p70S6K, and Protein kinase B (PKB).

BMVEC barrier permeability in response to bacterial pathogens is mediated by toll-like receptor (TLR) signaling [44]. GXM is recognized by TLR2/4 and these receptors help regulate the host response to C. neoformans infection [45]. TLR2/4 signaling to BMVEC permeability is mediated by Src kinase signaling (Figure 1) [46,47], which mobilizes multiple effectors involved in BMVEC disruption, including RhoA [48]. During neutrophil-induced BMVEC disruption, Src is phosphorylated on Tyr416 and dephosphorylated on Tyr527 [49], and Src inhibition reduces vascular endothelial growth factor (VEGF)-induced BBB permeability [50]. Src protein tyrosine kinase (PTK) inhibitor, PP2 (Table 2), protects the rat brain against ischemic injury, possibly through the reduction of VEGF expression and the upregulation of claudin-5 expression, which preserves the integrity of the BBB [50]. It is possible that Src PTK inhibitors strengthened the BBB integrity reducing fungal brain transmigration and colonization.

It is important to highlight that strategies targeting to minimize BBB disruption in individuals with fungal disease will be most effective if the infection is diagnosed early before CNS invasion and colonization. However, it is also plausible that even after a FI-CNS becomes established, limiting BBB access can prevent further fungal brain invasion. In contrast, over-expression of claudin-5 in BMVEC results in elevated paracellular tightness [51], which may interfere with the migration of peripheral immune cells and the delivery of antifungal molecules into the CNS, thus exacerbating the progression of the disease and contributing to mortality. Moreover, RhoA is required for migration of innate immune cells during infection and inflammation [52], and inhibition of RhoA may hinder clearance of fungal infection from the blood-stream. Future investigations are necessary to identify the benefits and risks associated with this novel strategy for the treatment of FI-CNS.

Conclusions

The search for novel antifungal therapeutics to prevent and manage FI-CNS is urgent considering the limited treatment options currently available for immunocompromised individuals. For example, a PubMed search using ‘antifungal’ as a query shows only a 2-fold increase of related publications in 2022 (8953) compared with 1992 (3917), confirming new antifungal compound screening is a very low-yield field, therefore, the low public and private investment in this critical area of medical mycology and patient care [53]. This hinders the discovery of novel therapeutic targets and development of innovative treatments to combat and manage the challenges of FI-CNS [54]. Addressing these challenges requires a multidisciplinary approach consisting but not limited to improve surveillance for antifungal drug resistance, the development of innovative prophylactic strategies (e.g. vaccine or passive immunity) to prevent complications of fungal infections in high at-risk populations, design of new antifungal drug classes and delivery formulations, increased research funding, and promote the creation of academia–industry partnerships that stimulate antifungal drug development through public- and/or private-driven incentive programs.

Data availability

No data were used for the research described in the article.