(M1530-07-41) Charge-Driven Nanocomplexation of Alexidine Reduces Its In Vivo Toxicity and Facilitates Its Development as a Broad-Spectrum Antifungal Agent

Purpose: Alexidine (ALX) is a cationic bisbiguanide with potent activity (MIC₈₀: 1.5 µg/mL) against various pathogenic fungi such as C. albicans, A. fumigatus, R. delemar, C. auris and fungal biofilms.¹ However, ALX, due to its polycationic nature, showed toxicity to normal murine and human cells in vitro, suboptimal pharmacokinetics, and low in vivo tolerability. As the charged groups are required for the compound’s antifungal mechanism of action, covalent modifications that mask the cationic charge of bisbiguanides such as ALX are of limited value. We hypothesized that amphiphilic and cationic ALX will electrostatically and transiently interact with negatively charged biocompatible anions to yield self-assembled nanoparticles that will transiently diminish the cationic charge of ALX and the in vitro and in vivo toxicity associated with ALX. Here, we for the first time, show the self-assembly of ALX with rigid cholesterol-based anions as well as flexible anionic phospholipids leading to subsequent improvement in ALX delivery and tolerability.

Methods: The following counterions; cholesterol-3-sulfate (C3S), cholesterol hemisuccinate (CHEMS), 1,2-Dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) were screened for their ability to electrostatically interact and self-assemble with ALX. Molar ratios of 1:1 1:2 and 1:3 of ALX dihydrochloride and counterion were interacted via nanoprecipitation. (Fig 1 A). Successful complexes were characterized via H¹, P<sup>31 NMR, FTIR, and XRD. Ratios were also repeated with the addition of a small quantity of DSPE-PEG 2k (5 mol%) for enhanced colloidal stability. Self-assemblies were assessed via dynamic light scattering and monitored for one month for stability. The alexidine nanocomplexes were evaluated for in vitro cytotoxicity to murine macrophages (RAW 264.7) and human renal proximal tubule cells (HK2 cells) and in vitro antifungal activity compared to alexidine. The ALX nanocomplexes that showed excellent colloidal stability, maintained broad-spectrum antifungal activity in vitro, and reduced in vitro toxicity were selected for an in vivo pharmacokinetic study and followed by in vivo maximum tolerated dose study and a preliminary in vivo efficacy study in a disseminated murine model of candidiasis.

Results: ALX self-assembled successfully when combined with rigid as well as flexible anionic lipids at 1:1 and 1:3 molar ratios. The ALX nanocomplexes containing ALX-anion molar ratio of 1:3 showed higher colloidal stability and particle size of ~ 150 nm. (Fig 1 B). The surface charge of the ALX nanocomplexes was impacted by the type of the anion and the ratio of ALX to anion in the nanocomplexes. The addition of PEGylated phospholipid (DSPE-PEG-2000) was not required for self-assembly but did enhance colloidal stability (Fig 1C) and had little to no effect on the particle surface charge. ALX-DOPG best maintained ALX’s high potency (1.5 µg/mL) (Fig 1D). Among various ALX nanocomplexes, ALX-DOPG (ALX-DOPG-NPs) and ALX-CHEMS nanocomplexes (ALX-CHEMS-NPs) showed excellent colloidal stability and reduced in vitro cytotoxicity compared to ALX. (Fig 2 A and B). Further, these nanocomplexes also maintained the in vitro antifungal activity of ALX against multiple pathogenic fungi. (Fig 1 D). Between ALX-CHEMS and ALX-DOPG nanocomplexes, only ALX-DOPG nanocomplexes (ALX-DOPG-NPs), upon intravenous administration to mice, showed significantly higher plasma ALX levels (~ 4-fold increase) and AUC (~ 5-fold increase) compared to ALX indicating the importance of anion used for the ALX nanocomplexes.  (Fig 2 C). Further, ALX-DOPG-NPs  (ALX dose: 12.5 mg/kg) showed greater in vivo tolerability in healthy mice compared to ALX solution. (Fig 2 D). Finally, in a murine model of disseminated candidiasis model, ALX-DOPG-NPs showed significantly lower C. albicans burden in kidneys compared to untreated control. (Fig 2 E).

Conclusion: The development of charge-driven self-assembled ALX nanocomplexes is a promising strategy to improve the in vivo tolerability and antifungal efficacy of ALX and facilitate its pharmaceutical development.

References: 1 .Mamouei, Z.; Alqarihi, A.; Singh, S.; Xu, S.; Mansour, M. K.; Ibrahim, A. S.; Uppuluri, P. Alexidine Dihydrochloride Has Broad-Spectrum Activities against Diverse Fungal Pathogens. mSphere 2018, 3 (5). DOI: 10.1128/mSphere.00539-18 From NLM.

Acknowledgements: This work was supported by the startup funds from the R.K. Coit College of Pharmacy, University of Arizona.


Fig 1: (A) Schematic of alexidine (ALX) nanocomplex development using biocompatible anions. (B) Particle size distribution and zeta potential of ALX-DOPG-NPs containing 1:3 molar ratio of ALX and DOPG, (C) Colloidal stability of ALX-DOPG-NPs with and without DSPE-PEG2k (5 mol%) over 1 month, (D) ALX-DOPG and ALX-CHEMS nanocomplexes retain broad-spectrum antifungal activity in vitro.


Fig 2: Viability of (A) HK2 cells and (B) RAW 264.7 cells treated with ALX-DOPG nanocomplexes (ALX-DOPG-NPs) and ALX-CHEMS nanocomplexes (ALX-CHEMS-NPs) and ALX solution (ALX dose: at 20 μM for all HK2 samples and 10 μM for all RAW 264.7 samples; data expressed as mean ± SEM; n=9) (C) Single-dose pharmacokinetics of intravenously administered ALX solution, ALX-DOPG-NPs or ALX-CHEMS-NPs (ALX dose: 5 mg/kg; n=4) show highest ALX exposure for ALX-DOPG-NPs (D) ALX-DOPG-NPs show high in vivo tolerability compared to ALX solution in healthy mice (n≥4 mice/group) (E) Once daily ALX-DOPG-NPs (ALX dose: 12.5 mg/kg) showed a reduction in the C. albicans burden in kidneys compared to untreated control after 3 days (n=5 mice/group) (*=P < 0.05, ****=P <0.0001)