Combinations of posaconazole and tacrolimus are effective against infections with azole-resistant Aspergillus fumigatus

Abstract

Background: Solid organ transplant recipients on immunosuppressants such as tacrolimus are at increased risk of developing pulmonary aspergillosis, a severe to deadly complication with limited treatment options, especially against azole-resistant strains. This study investigates the antifungal interaction between posaconazole and tacrolimus, prompted by a case where a liver transplant recipient on tacrolimus experienced unexpected eradication of chronic Aspergillus fumigatus colonization following posaconazole prophylaxis.

Methods: We compared the combined antifungal activity of posaconazole and tacrolimus against azole-sensitive and resistant A. fumigatus in vitro against planktonic isolates and biofilm formation and in vivo in Galleria mellonella larvae, to evaluate the potential benefit over posaconazole monotherapy.

Results: The posaconazole-tacrolimus combination demonstrated a 4-fold increase in efficacy against azole-resistant isolates and a 30-fold increase against an azole-sensitive strain, in contrast to voriconazole. Moreover, this combination enhanced antifungal activity by 4- to 15-fold against biofilm formation of azole-sensitive strains, though no synergy was observed against azole-resistant biofilms. In vivo studies in Galleria mellonella confirmed a 2- to 7-fold decrease in fungal burden of both azole-sensitive and azole-resistant strains when combining posaconazole with tacrolimus, relative to posaconazole alone.

Conclusion: In vitro and in vivo findings confirm that posaconazole may offer therapeutic benefits for treating A. fumigatus infections in patients receiving tacrolimus. These results warrant further confirmation in mice and exploration of their clinical implications.

Keywords: Aspergillus fumigatus; FK506; Transplantation; azole-resistance; biofilm; posaconazole; synergy; tacrolimus.

Figure 1

Galleria mellonella aspergillosis model for in vivo evaluation of PCZ potentiation by tacrolimus. In-house bred, healthy G. mellonella larvae of 250 ± 50 mg (n = 10 per group) were housed individually in 12-well plates (Cellstar®, Greiner bio-one, Austria) without food and stored in the dark at 37°C during the experiment. On day 0, A. fumigatus inocula ranging between 2000 (TR₃₄/L98H) and 9000 (WT) conidia per 10 µL were injected intra-hemocoel via the last right proleg using a Hamilton® syringe (25 µl, 702SN, 30 G, Switzerland). Non-infected control groups received 10 µL PBS. Posaconazole (PCZ) (Noxafil, MSD, USA) and tacrolimus (Prograf, Astellas Pharma Inc, Japan) solutions (IV formulations) were diluted in 0.9% sterile saline to achieve doses of 8, 4, 2, 1 or 0.5 mg/kg for PCZ and 0.1 mg/kg for tacrolimus. Treatments were freshly prepared and administered daily, starting from 1 h post infection, by intra-hemocoel injection in alternately the last left and right prolegs. Infected sham-treated control groups received 0.9% saline, and non-infected control groups received the combination of PCZ and tacrolimus to assess treatment toxicity. Larvae were followed-up daily for four days post infection (p.i.) by in vivo BLI, survival and health scoring (based on movement, melanization and survival). For in vivo BLI, larvae were injected intra-hemocoel with 10 µL D-luciferin (40 µg/g in PBS) 10 min before image acquisition (Vanhoffelen et al., 2023). Figure created with BioRender.

Figure 2

Tacrolimus potentiates PCZ antifungal efficacy in vivo against azole-sensitive A. fumigatus in Galleria mellonella. (A) The relative reduction in fungal burden of different PCZ doses compared to sham-treated larvae against an azole-sensitive A. fumigatus infection in G. mellonella, measured as area under the curve of normalized in vivo BLI signal over 4 days post infection (p.i.). (B) In vivo BLI signals in larvae infected with azole-sensitive A. fumigatus, comparing the effect of PCZ, tacrolimus or the combination of both on fungal burden over time. Graphs show means ± standard deviations (SD) (n = 10). (C) Overall fungal burden reduction relative to sham-treated larvae, measured as area under the curve of panel (B) after normalization for the sham-treated group. (D) Visual representation of in vivo BLI signal in infected larvae on day 4 p.i. BLI, bioluminescence imaging; [p/s], photons per second; PCZ, posaconazole; Tacro, tacrolimus. *P < 0.05; **P < 0.01; ***P <0.001; ****P < 0.0001.

Figure 3

Tacrolimus potentiates PCZ antifungal efficacy in vivo against azole-resistant A. fumigatus in Galleria mellonella. (A) The relative reduction in fungal burden of different PCZ doses compared to sham-treated larvae against an azole-resistant (TR₃₄/L98H) A. fumigatus infection in G. mellonella, measured as area under the curve of normalized in vivo BLI signal over 4 days post infection (p.i.). (B) In vivo BLI signals in larvae infected with azole-resistant A. fumigatus, comparing the effect of PCZ, tacrolimus or the combination of both on fungal burden over time. Graphs show means ± standard deviations (SD) (n = 10). (C) Overall fungal burden reduction relative to sham-treated larvae, measured as area under the curve of panel (B) after normalization for the sham-treated group. (D) Visual representation of in vivo BLI signal in infected larvae on day 4 p.i. BLI, bioluminescence imaging; [p/s], photons per second; PCZ, posaconazole. Tacro: tacrolimus. **P < 0.01; ***P <0.001; ****P < 0.0001; ns, non-significant.