Structural characterization of the tobramycin–piperacillin reaction product formed at pH 6.0

Abstract

Tobramycin is an aminoglycoside antibiotic that loses a significant amount of activity in the presence of Zosyn at pH 6. As part of our investigation into ways to improve the compatibility of tobramycin with Zosyn (which contains piperacillin and tazobactam in an 8:1 ratio buffered at pH 6 by sodium citrate) by lowering the pH, we identified the reaction product of tobramycin and piperacillin at pH 6.0 and the order of the pK a values of tobramycin. The structure of the main reaction product of tobramycin and piperacillin at pH 6.0 was determined by 2D NMR to be the product of 3 00 -NH 2 reacting with the b-lactam of piperacillin. The order of the pK a values of the nitrogens of tobramycin was determined by 1 H and 15 N NMR titrations to be 6¢-NH 2 42¢-NH 2 41-NH 2 E3 00 -NH 2 43-NH 2 . At pH 4.0, the reaction between tobramycin and Zosyn was almost negligible for a period of up to 2 h. The pH can be lowered by adding an acid such as HCl or citric acid to Zosyn to make a pH 4.0 buffer. The Journal of Antibiotics (2011) 64, 673-677; doi:10.1038/ja.2011.72; published online 3 August 2011Keywords: piperacillin; protonation constants; structure elucidation; tobramycin; Y-site co-administration; Zosyn INTRODUCTION Tobramycin (Figure 1) is an aminoglycoside antibiotic produced by Streptomyces tenebrarius 1 used to treat infections caused by susceptible Gram-negative microorganisms. 2 It is often used in combination with penicillins, such as piperacillin (Figure 2), to treat severe infections caused by Gram-negative bacteria. 3 It is well known that tobramycin reacts with b-lactams, including piperacillin, causing a significant loss of aminoglycoside activity. 4 The inactivation mechanism is thought to involve nucleophilic attack and ring opening of the penicillin b-lactam ring by an amino group of the aminoglycoside. 5,6 The rate of aminoglycoside inactivation is dependent on temperature, time, concentration of the b-lactam and composition of the medium. [7][8][9] Another important factor to consider is the pH of the solution. As the degree of protonation of an amino group increases, it should become less nucleophilic. The amino groups of tobramycin have different pK a values, 10,11 hence, it is logical to assume the reaction would be affected by pH of the solution. Therefore, knowledge of the protonation constants of the amino groups and their order is important in understanding the reactivity of tobramycin with piperacillin at different pHs.Zosyn is an intravenously administered antibiotic, which contains piperacillin and tazobactam (a b-lactamase inhibitor) in an 8:1 ratio. It also contains EDTA and sodium citrate, which is used to buffer the solution at around pH 6. 12 It has been approved for Y-site coadministration with the aminoglycosides amikacin and gentamicin. 13 It has not been approved for co-administration with tobramycin,

Individual pKa Values of Tobramycin, Kanamycin B, Amikacin, Sisomicin, and Netilmicin Determined by Multinuclear NMR Spectroscopy

“… b p K a values of individual nitrogen atoms of tobramycin determined using 1 H NMR spectroscopy and 15 N NMR spectroscopy in D 2 O relative to TMS at 25 °C. 14 …”

“…These p K a values are consistent in the assignment order with those reported in the literature. 11 , 14 …”

Multinuclear Nuclear Magnetic Resonance Spectroscopy Is Used to Determine Rapidly and Accurately the Individual pKa Values of 2-Deoxystreptamine, Neamine, Neomycin, Paromomycin, and Streptomycin

“…23 Likewise, the order of the pK a values of tobramycin has been studied by 15 N NMR spectroscopy. 16,24 Studying whether aminoglycosides adopt similar conformations when bound to RNA and protein targets may have significant implications in the design of enzyme inhibitors and/or antibiotics. Therefore, we are studying new combinations of multinuclear NMR spectroscopic data as a rapid analytical method to deconvolute accurately the individual pK a data of these polyamines in order to know what chemical structure each molecule has at physiological pH.…”

In vitro pseudomonas aeruginosa biofilms

“…105 The aminoglycosides, amikacin and tobramycin, are polycationic at pH 7 (Figure 2-2B-C). [106][107] The cyclic polypeptides, polymyxin B and colistin, are polycationic at pH 7 ( Figure 2-3A-B). 108 The cyclic polypeptide prodrug colistin methanesulfonate is polyanionic at pH 7 ( unionized at pH 7.…”

“…Incidence of growth was defined as percent of wells with absorbance readings at 650 nm greater than 0.1. ciprofloxacin hydrochloride structure (ciprofloxacin pK a 1=6.0, pK a 2=8.8); 98,105,119 B) amikacin disulfate structure (amikacin pK a 1=6.9, pK a 2=8.1, pK a 3=8.7, pK a 4=10.1); 98,106,120 C) tobramycin sulfate (tobramycin pK a 1=6.6, pK a 2=7.3, pK a 3=7.3, pK a 4=7.5, pK a 5=8.4). 98,107,121 Incidence of biofilm growth versus antibiotic concentration for MBEC screening study; P. aeruginosa biofilms were grown for 24 hours and treated with antibiotics alone or in combination with dispersion compounds for 24 hours; incidence of growth was defined as percent of wells with absorbance at 650 nm greater than 0.1; incidence of growth for biofilm bacteria for the antibiotic A) ciprofloxacin hydrochloride (CH), MIC 1 µg/mL, 83 MBEC > 256 µg/mL; 103 B) amikacin disulfate (AMK), MIC 32 µg/mL, 84 MBEC > 512 µg/mL. 84…”