Purpose: Lumichrome (Lc) is a photodegradation product of riboflavin. It is more photostable and a more efficient photogenerator of singlet oxygen than riboflavin. Lc absorbs mainly in the UVA range. Cyclodextrins (CDs) are reported to be very efficient solubility enhancers of Lc in aqueous solutions. A 1:1 inclusion complex has been predicted between Lc and α and βCDs (Terekhova, Kumeev et al. 2011). The aim of the present study was to evaluate the influence of CDs on solubility and on the bacterial phototoxicity of Lc in vitro.

Methods: Lc was added in excess to PBS at pH 7.4 in the presence of 3 and 10% (w/v) hydroxypropyl-α-cyclodextrin (HPαCD), hydroxypropyl-β-cyclodextrin (HPβCD) and hydroxypropyl-γ-cyclodextrin (HPγCD). After reaching equilibrium the solutions were filtered (0.45 µm filter), diluted with MeOH and quantified by HPLC. Selected samples of Lc in HPβCD and HPγCD were tested for phototoxicity on Gram-positive (E.faecalis) and Gram-negative (E.coli) planktonic bacteria. The light source was a UVA/blue light irradiation chamber emitting mainly in the range 340-440 nm. Bacterial suspensions were mixed with the Lc-CD samples. Two irradiation doses were used (12 and 24 J/cm²) to test the phototoxicity. The suspensions were plated onto TSB-agar dishes and bacterial colony survival was counted after 24 h incubation at 37⁰C.

Results: The concentration of Lc increased thirty times by addition of 10% HPβCD compared to plain PBS solution while only a four and seven times increase was obtained with 10% HPαCD and 10% HPγCD, respectively. A concentration of 3% CD did also enhance the Lc concentration in the sequence β>γ>α.The stability constant obtained for the Lc-CDs complexes were 31 M-1 ,379 M-1 and 80 M-1 for HPα, β and   -γCD, respectively. These results are consistent with previous studies (Terekhova, Kumeev et al. 2011). Lc is a small molecule (242 Da) that fits better in the HPβCD cavity rather than in the αCD cavity (too small) or in the γCD cavity (too big). The phototoxicity studies showed that Lc solubilized in plain PBS or PBS solutions containing CDs was phototoxic to both G+ and G- model bacteria when combined with UVA/blue light irradiation with emission maximum at 365 nm. A lower reduction in G- bacterial survival compared to G+ was observed. This is a tendency shown for several photosensitizers. Selected preparations (plain Lc-PBS, 0.1 mM Lc in 3% HPβCD or 3% HPγCD) were tested on bacteria with an irradiation dose of ~24 J/cm². Eradication was induced (6 log reductions) for E.faecalis independent of the formulation. Lc also induced bacterial eradication of E. coli, with the exception of 0.1 mM 3% HPβCD formulation for which a 5% survival (3 log reduction) was detected. When a lower irradiation dose was used (~12 J/cm2) complete eradication of the bacteria was not reached for any of the bacteria independent on Lc formulation. However, Lc dissolved in 3% HPγCD showed to be more efficient than in 3% HPβCD, and resulted in <0.1% and 9% bacterial survival for E.faecalis and E.coli, respectively. Studies were also performed with 10% w/v HPβCD amount to reach a higher Lc concentration (1.0 mM). Despite a 10 times higher Lc concentration, the phototoxic effect did not increase. This observation is consistent with a previous report on riboflavin (Makdoumi, Backman et al. 2010), where they state that an increase in riboflavin molarity did not induce a larger phototoxic response in bacteria. We suggest that the reason could be an inner filter effect due to the high concentration of Lc associated with a high absorbance value (>2). The observed difference between HPβCD and HPγCD can be ascribed to a stronger complexation of Lc in the latter one.

Conclusion: Our study presents an opportunity to make well-characterized CDs based, photostable formulations of Lc as an alternative to the photolabile riboflavin for the treatment of infectious conditions. A CD with sufficient, but not too high solubilizing capacity (i.e., not too high complex binding constant) should be selected.

References: Makdoumi, K., A. Backman, J. Mortensen and S. Crafoord (2010). “Evaluation of antibacterial efficacy of photo-activated riboflavin using ultraviolet light (UVA).” Graefes Arch Clin Exp Ophthalmol 248(2): 207-212. Terekhova, I. V., R. S. Kumeev, G. A. Alper and A. V. Agafonov (2011). “Thermodynamic characteristics of the formation of α- and β-cyclodextrin complexes with lumichrome, lumazine, and uracil in aqueous solution.” Russ J Phys Chem A 85 (( 10)): 1844-1849.

Cyclodextrins influence on lumichrome solubilization and phototoxic efficacy against bacteria.
Bergh VJV, Bruzell E, Tønnesen HH.
American Association of Pharmaceutical Scientists (AAPS) Annual Meeting, October 25-29, 2015, Orlando, Florida, USA (A)