Determination of bisphosphonate properties in terms of bioavailability, bone affinity, and cytotoxicity

Abstract

Background: The study aimed to evaluate the therapeutic potential of fourteen newly synthesized bisphosphonates by assessing their bioavailability, bone affinity, and cytotoxicity. These bisphosphonates included a series of aminomethylenebisphosphonates and standard compounds such as risedronate and tiludronate.

Methods: Drug permeability was determined using Parallel Artificial Membrane Permeability Assays (PAMPA), while bone affinity was assessed by sorption on hydroxyapatite. Bacterial cell response to the bisphosphonates was also examined using Lactobacillus paracasei cells as a model.

Results: Several tested compounds, including BP3 to BP8 and BP11, which feature substituents in the pyridine ring such as methyl groups, iodine, bromine, chlorine, or hydroxyl groups, demonstrated potentially more beneficial therapeutic properties than commercially used bisphosphonates. These compounds showed stronger bone affinity and higher gastrointestinal absorption with comparable or lower cytotoxic effects. Specifically, BP11 exhibited the highest bone affinity, while BP8 and BP11 showed the greatest permeability.

Conclusions: The findings suggest that BP3 BP8, and BP11 are promising candidates for further research. These results highlight the importance of comprehensively evaluating bisphosphonates' therapeutic properties to identify effective treatments for osteoporosis and other bone diseases.

Keywords: Artificial membrane permeability; Bisphosphonates; Cytotoxicity; Hydroxyapatite.

Fig. 1

Sorption of risedronate (RSD) on hydroxyapatite at varying concentrations (0.2, 0.5, 0.7, and 1 mM) and time periods (1and 48 h), Data are presented in mean ± SE. N = 5. A two-way ANOVA was conducted to examine the effects of concentration (mM) and time (h) on the sorption of risedronate on hydroxyapatite. The Tukey's method was used for post-hoc comparison with a significance level of 0.05

Fig. 2

Sorption of various bisphosphonates (BP1 to BP12, RSD-risedronate, and TIL-tiludronate) on hydroxyapatite, measured at a concentration of 0.7 mM over different time periods (1, 2, and 48 h). Data are presented in mean ± SE. N = 5. A two-way ANOVA was conducted to examine the effects of BP and time (h) on the sorption values on hydroxyapatite. The Tukey's method was used for post-hoc comparison with a significance level of 0.05. Not significant comparisons with p values ≥ 0.05 are not labelled in the graphs

Fig. 3

In vitro permeability of various bisphosphonates (BP1 to BP12), RSD (risedronate), and TIL (tiludronate) as reference compounds carried out using a parallel artificial membrane permeability assay (PAMPA). Whiskers represent the 10th to 90th percentiles, the box extends from the 25th to 75th percentiles, the midline is plotted at the median, and “ + ” is at the mean value, N = 5. The one-way ANOVA test revealed significant differences among the permeabilities of the various BPs (BP1 to BP12) and RSD. The Dunnett's multiple comparisons test was used for post-hoc comparison with a significance level of 0.05. Comparison of different BP to the RSD, statistical significance is indicated by symbols above each box, **p < 0.01, ***p < 0.001. Not significant comparisons with p values ≥ 0.05 are not labelled in the graphs. The colors in Figs are used to facilitate the assessment of changes for specific samples across different graphs

Fig. 4

The growth curve of L. paracasei PCM 3039 in the absence (Ctrl) and presence of bisphosphonates (BP1-BP12, RSD-risedronate, TIL-tiludronate) was measured with a microplate reader at 36 °C. Each dot represents a separate experimental datapoint-data point. OD600—optical density at 600 nm

Fig. 5

Comparison of cell properties of L. paracasei PCM 3039 in the absence (Ctrl) and presence of bisphosphonates (BP1-BP12, RSD-risedronate, TIL-tiludronate), cell membrane permeability (a), cell wall hydrophobicity (b), and relative metabolic activity (c). Relative metabolic activity of L. paracasei exposed to BPs is expressed as a percentage relative to the control group (100%). The control group represents untreated cells, and their metabolic activity was set as the baseline (100%) for comparison. The results for the control group were included in the calculations of the percentage reduction of AlamarBlue for the treated samples. Whiskers represent the 10th to 90th percentiles, the box extends from the 25th to 75th percentiles, the midline is plotted at the median, and “ + ” is at the mean value, N = 6. The results of the one-way ANOVA indicate a significant difference among the groups, cell membrane permeability, cell wall hydrophobicity and relative metabolic activity. The Tukey's method was used for post-hoc comparison with a significance level of 0.05. Comparison of different BP to the RSD, statistical significance is indicated by symbols above each box, *p < 0.05, **p < 0.01, ***p < 0.001. Not significant comparisons with p values ≥ 0.05 are not labelled in the graphs