(M1230-04-25) Biopharmaceutical Analysis of Synthesized Antibiotic Ester Prodrugs; Ciprofloxacin Isopentanoate, Ciprofloxacin N-Butanoate and Ciprofloxacin Isobutanoate

Purpose: To investigate the effect of ester prodrugs synthesized, in an attempt to improve oral bioavailability, on the biopharmaceutical properties of the parent antibiotic.

Methods: Artificial membrane permeability Parallel artificial membrane permeability assay (PAMPA) was used to predict the transcellular transportation of the esters. The donor plate consisted of a 96-well multiscreen filter plate with hydrophobic polyvinylidene fluoride membrane. The acceptor plate was a 96 well, Polytetrafluoroethylene collection plate. Solutions of ciprofloxacin (CPR), ciprofloxacin isopentanoate (CIP), ciprofloxacin n-butanoate (CNB), and ciprofloxacin isobutanoate (CIB) were dissolved in 5% v/v DMSO PBS (pH 7.4) buffer. The hydrophobic membrane of each well in the donor plate was impregnated with 5µl of 2% w/v of lecithin in dodecane.150µl of the drug solution was immediately added to each well of the donor plate. Then 300 µl of buffer was pipetted into each well in the acceptor plate, and the donor plate was assembled onto the acceptor plate. This was incubated in 37 ºC for 5 hours. The concentration of drug in each acceptor well was measured using high performance liquid chromatography (HPLC). The effective permeability P<sub>e was calculated with the equation by Wohnsland and Faller, 2001.
Biochemical stability in the presence of pig liver esterase (PLE)Antimicrobial studies The minimum inhibitory concentration of each prodrug was determined using the broth microdilution method. Representative Gram-positive (Staphylococcus aureus and Staphylococcus epidermidis) and Gram-negative (Escherichia coli and Acinetobacter baumannii) bacteria strains were utilised for the assay. Single colonies from overnight agar cultures were sub-cultured in tryptone soy broth (TSB) and incubated overnight at 37 ºC with shaking. The inoculum was diluted with TSB to obtain the desired inoculum density. An aliquot of previously prepared stock solutions of the prodrugs was pipetted into the first row of the 96-well plates. 100µl of TSB was then pipetted into each remaining well in the plate. A two-fold dilution of the drug was done, horizontally across the plate. 100µl of TSB containing bacteria was transferred to all wells except the negative control column. After overnight incubation at 37 ºC in an ambient air incubator, the absorbance of the wells was read using a plate reader at 570 nm wavelength with shaking. The lowest concentrations of the tested compounds at which bacteria growth was completely inhibited were considered to be the minimum inhibitory concentrations.

Results: All esters except CNB had significantly better permeation across the parallel artificial membrane model compared to the ciprofloxacin (p < 0.05) (Figure 1). CIP displayed the greatest effective permeability of 0.16x10^-6 cm/s while the effective permeability of CNB was the lowest: 0.02x10^-6 cm/s in comparison to 0.02x10^-6 cm/s for ciprofloxacin (CPR). Esterification is well known approach to improve permeability of hydrophilic drugs. In the case of fluoroquinolones blockage of the carboxylic group additionally inhibits its antimicrobial activity. The biochemical stability studies in the presence of pig liver esterase (PLE) were conducted to analyse the differences in hydrolysis rate of synthesised esters. The hydrolysis rate of CIP was significantly faster (p < 0.05) than that of CNB and CIB, where the difference in hydrolysis rates of the butanoate esters was not significant (p >0.05). Similarly, the butanoate esters had half-lives of 13.0±0.2 and 13.19±0.6 minutes, respectively for CNB and CIB, which were not significantly different (p >0.05) esters while the half-life of CIP was much shorter (3.13± 0.07 minutes). Antimicrobial studies confirmed that hydrolysis of the esters is necessary for antimicrobial activity of CPR as all PLE-hydrolysed prodrugs had MIC of < 0.25 µg/ml for E. coli, S. aureus and S. epidermidis, and MIC of 0.5±1 µg/ml for A. baumannii. Considering the investigated esters, none of the tested bacteria strains were able to hydrolyse the esters at the rate comparable with PLE resulting in the high MICs reported (Table 1). Considering the lowest MICs, E. coli hydrolysed all esters at the fastest rate resulting with the lowest MICs.

Conclusion: Esterification improves PAMPA permeability rate of ciprofloxacin pro-antibiotics to varying extents, depending on molecular structure of selected co-formers. Further studies are required to understand biopharmaceutical stability of ester pro-antibiotics. Our results confirm that the molecular structure of the ester affects the hydrolysis rate of esters in the presence of PLE. The molecular structure also influences the MIC depending on the selected bacteria strains.

References: 1. Wohnsland F, Faller B. High-throughput permeability pH profile and high-throughput alkane/water log P with artificial membranes. J Med Chem. 2001;44(6):923-30.
2. van Haren MJ, Gao Y, Buijs N, Campagna R, Sartini D, Emanuelli M, et al. Esterase-Sensitive Prodrugs of a Potent Bisubstrate Inhibitor of Nicotinamide N-Methyltransferase (NNMT) Display Cellular Activity. Biomolecules. 2021;11(9).

Acknowledgements:This work was funded by the Schlumberger Foundation.

Figure 1: Effective Permeabilities of prodrugs and parent antibiotic

Figure 2:Hydrolysis, Rate constant and Half-lives of ester prodrugs

Table 1: Minimum Inhibitory Concentrations of prodrugs and parent antibiotic.