Cannabidiol and cannabidivarin exhibited antifungal activity against Cryptococcus neoformans and other clinically significant fungal pathogens in vitro and in vivo, according to recent study findings. The compounds demonstrated multiple mechanisms of action, including biofilm inhibition, membrane destabilization, and disruption of metabolic and stress response pathways.

Researchers from Macquarie University tested the phytocannabinoids against 33 fungal strains. In RPMI-1640 medium at pH 7.4, cannabidiol (CBD) and cannabidivarin (CBDV) demonstrated minimum inhibitory concentrations (MICs) of 6.25 µg/mL and 12.5 µg/mL, respectively, against C neoformans H99. Both compounds exhibited fungicidal activity with minimum fungicidal concentrations of 25 µg/mL for CBD and 50 µg/mL for CBDV.

“Our results demonstrate that CBD, and particularly CBDV, have broad activity against C neoformans and other fungal pathogens, including dermatophytes that cause common tinea,” the authors wrote.

In time-kill assays, CBD and CBDV eliminated C neoformans within 30 minutes, compared with 4 hours for amphotericin B.

Activity Across Fungal Pathogens

CBDV showed consistent activity across all 9 Cryptococcus strains tested. These strains spanned 6 species, each with a MIC of 12.5 µg/mL. CBD was active against a single Cryptococcus strain (C deneoformans VNIV) but at a lower MIC of 3.13 µg/mL.

Both compounds demonstrated antifungal activity against dermatophytes, including Microsporum canis, Nannizzia gypsea, Trichophyton interdigitale, and Trichophyton rubrum, with MICs ranging from 1.56 to 6.25 µg/mL.

Among Candida species, CBDV was active against 3 of 12 strains, including C albicans, C dubliniensis, and C guilliermondii, with MICs of 12.5 to 25 µg/mL. CBD showed no activity against any Candida strains up to 25 µg/mL.

CBD was active against Mucor circinelloides (MIC = 6.25 µg/mL), while CBDV was active against Rhizopus oryzae (MIC = 12.5 µg/mL). Neither compound exhibited activity against Fusarium oxysporum or any of the 5 Aspergillus species tested.

Fractional inhibitory concentration assays revealed no synergistic or antagonistic interactions between the phytocannabinoids and amphotericin B or fluconazole.

Inhibition of Biofilms

Both CBD and CBDV inhibited biofilm formation by C neoformans at concentrations as low as 1.56 µg/mL and 0.78 µg/mL, respectively. Only CBDV was effective at disrupting mature biofilms, with activity observed at 3.13 µg/mL.

“These results provide important insights regarding mechanisms of how the phytocannabinoids are working against C neoformans and ultimately may represent novel strategies for the prevention or eradication of cryptococcal colonization of medical prosthetic devices,” the authors noted.

Alterations in Cell Morphology

CBDV induced significant reductions in both cell and capsule size in C neoformans, from 7.15±3.46 µm to 5.45±1.33 µm and from 5.88±1.69 µm to 1.52±1.42 µm, respectively. The reduction in capsule thickness could not be attributed solely to changes in cell size.

Proteomic Findings

Quantitative proteomic analysis identified 3,074 proteins in C neoformans, with 136 and 124 differentially abundant proteins in response to CBD and CBDV, respectively. Approximately one-third of affected proteins were shared between both treatments.

Gene ontology enrichment analysis showed that most differentially abundant proteins were involved in cellular stress responses and metabolic pathways. Membrane and mitochondrial proteins were particularly affected. Notable impacted proteins included:

• ABC multidrug transporters (AFR1, AFR2)

• Enzymes in ergosterol and pyrimidine biosynthesis pathways

• Inositol pathway and GDP-mannose pathway proteins linked to capsule regulation

CBDV altered twice as many mitochondrial proteins as CBD, which may explain its stronger antifungal activity.

In Vivo Efficacy

In a Galleria mellonella burn wound model infected with C neoformans, topical application of CBD (2 mg/mL) significantly improved survival compared with vehicle controls (P = .01). Survival rates approached those of uninfected burn-only controls and exceeded those of amphotericin B-treated larvae.

Clinical Relevance

CBD is approved in several countries for conditions such as epilepsy and chronic pain, while CBDV is under clinical investigation. Their favorable safety profiles and novel mechanisms of antifungal action suggest potential for future development.

The findings are timely, given the World Health Organization’s designation of C neoformans as a “Critical Priority” fungal pathogen due to its disease burden and antifungal resistance.

Additional Insights

Despite the promising laboratory results, significant translational challenges remain before phytocannabinoids can reach clinical application. Corresponding author Amy K. Cain, ARC Future Fellow at Macquarie University's School of Natural Sciences, addressed key concerns about the pathway from bench to bedside.

She explained ongoing development efforts: "This publication does include promising in vitro results showing clinically-relevant concentrations of CBD and CBD-V being effective against fungi (down to only 1.56ug/ml) and we believe that they have great promise in being developed for use as topical or oral antifungals for patients. We are currently investigating PK-PD studies and working with commercial partners to develop an optimized phytocannabinoid-based antifungal that will work effectively for patients."

The proteomic data revealed upregulation of AFR1 and AFR2 efflux pumps, which raised concerns about potential resistance emergence during monotherapy. Cain acknowledged this risk while highlighting preventive research strategies: "All antimicrobial drugs have the potential for resistance to develop. Thus, we are currently working on establishing the rates of resistance for phytocannabinoids and so far, we see little resistance occurrence. We are also looking into their interactions with existing antimicrobials to reveal their potential for use in combination treatments."

The research was primarily funded by Macquarie University Research Acceleration Scheme and the Australian Research Council Industrial Transformation Training Centre for Facilitated Advancement of Australia's Bioactives.

The authors declared that no competing interests exist.

Source: PLOS Neglected Tropical Diseases