Modulatory Effects of Caffeine and Pentoxifylline on Aromatic Antibiotics: A Role for Hetero-Complex Formation

Abstract

Antimicrobial resistance is a major healthcare threat globally. Xanthines, including caffeine and pentoxifylline, are attractive candidates for drug repurposing, given their well-established safety and pharmacological profiles. This study aimed to analyze potential interactions between xanthines and aromatic antibiotics (i.e., tetracycline and ciprofloxacin), and their impact on antibiotic antibacterial activity. UV-vis spectroscopy, statistical-thermodynamical modeling, and isothermal titration calorimetry were used to quantitatively evaluate xanthine-antibiotic interactions. The antibacterial profiles of xanthines, and xanthine-antibiotic mixtures, towards important human pathogens Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Enterobacter cloacae were examined. Caffeine and pentoxifylline directly interact with ciprofloxacin and tetracycline, with neighborhood association constant values of 15.8-45.6 M^-1 and enthalpy change values up to -4 kJ·M^-1. Caffeine, used in mixtures with tested antibiotics, enhanced their antibacterial activity in most pathogens tested. However, antagonistic effects of caffeine were also observed, but only with ciprofloxacin toward Gram-positive pathogens. Xanthines interact with aromatic antibiotics at the molecular and in vitro antibacterial activity level. Given considerable exposure to caffeine and pentoxifylline, these interactions might be relevant for the effectiveness of antibacterial pharmacotherapy, and may help to identify optimal treatment regimens in the era of multidrug resistance.

Keywords: antibacterial agent; biological activity; caffeine; ciprofloxacin; natural compounds; tetracycline.

Figure 1

Spectrophotometric titrations of antibiotics with caffeine. (a), absorption spectra (in the form of molar extinction coefficient ε_M) of ciprofloxacin (initial concentration, 60.3 µM) titrated with caffeine (concentration range, 0.3–40.6 mM); (b), absorption spectra (in the form of molar extinction coefficient ε_M) of tetracycline (initial concentration, 43.2 µM) titrated with caffeine (concentration range, 0.3–21.5 mM). Spectra of an antibiotic in its free form are marked in bold. The spectra of antibiotics complexed with caffeine (calculated by extrapolation of mixture spectra to concentration ratio of antibiotic:caffeine → 0) are marked as dashed lines. For detailed composition of each tetracycline-caffeine mixture measured during spectrophotometric titration, see Table 1.

Figure 2

Examples of two-component decomposition of antibiotic-caffeine mixture spectra. (a), decomposition of mixture spectrum containing ciprofloxacin (55.1 µM) and caffeine (10.6 mM), molar fraction of free ciprofloxacin = 0.64; (b), decomposition of mixture spectrum containing tetracycline (40.4 µM) and caffeine (7.78 mM), molar fraction of free tetracycline = 0.61. Solid lines represent experimental spectra, dotted lines—sum of calculated decomposed spectra, dashed lines—calculated spectra of free antibiotics, dashed-dotted lines—calculated spectra of antibiotics complexed with caffeine. Top panels show residuals of experimental and the sum of calculated decomposed spectra.

Figure 3

Comparison of experimental and theoretical concentrations in antibiotic-xanthine mixtures analyzed spectrophotometrically. (a), ciprofloxacin-caffeine interactions; (b), tetracycline-caffeine interactions. Points represent concentrations of antibiotic in a free form (circles) and in a complex with caffeine (triangles), calculated with two-component spectra decomposition. Lines represent concentrations of an antibiotic in a free form (solid line) and in a complex with caffeine (dashed line), calculated using statistical-thermodynamical model of mixed aggregation [32] (with K_AC ± standard error values 24.71 M⁻¹ ± 0.89 M⁻¹ for ciprofloxacin-caffeine interaction and 45.6 M⁻¹ ± 2.5 M⁻¹ for tetracycline-caffeine interaction). CAF, caffeine, CIP, ciprofloxacin. TET, tetracycline.

Figure 4

Thermal effects of antibiotic-caffeine complex formation—analysis with isothermal titration calorimetry. (a,b), thermograms for analysis of ciprofloxacin-caffeine (a) and tetracycline-caffeine (b) interactions; solid line, titration of caffeine with antibiotic; dotted line, titration of caffeine with buffer; dashed line, titration of buffer with antibiotic; (c,d), determination of enthalpy change (ΔH) values for ciprofloxacin-caffeine (c) and tetracycline-caffeine (d) interactions. Points correspond to net heat of antibiotic-xanthine interaction (corrected for heats of buffer-to-xanthine and antibiotic-to-buffer titrations, see Figure S1) per mole of titrant added. CIP, ciprofloxacin. TET, tetracycline.

Figure 5

Dose-dependent modulation of antibiotics (ciprofloxacin and tetracycline) inhibitory potential by xanthines (caffeine and pentoxifylline) towards selected bacterial pathogens. Graphs represent isobolograms for each antibiotic-xanthine pair tested in concentration gradient of both compounds. Seven investigated pathogens are given as separate rows. Inhibitory concentrations given on Y axes correspond to minimal inhibitory concentration of tested antibiotics. FIC, Fractional Inhibitory Concentration Index, calculated for each tested antibiotic-xanthine combination according to Odds [33].

Figure 6

Chemical structures of studied compounds. Top, xanthines: caffeine and pentoxifylline; bottom, antibiotics: ciprofloxacin and tetracycline.