Simple spectrophotometric assay for measuring protein binding of penem antibiotics to human serum

Abstract

The binding of antibiotics to plasma (serum) proteins through hydrogen bonding can significantly influence the biological characteristics of these drugs. A rapid spectrophotometric assay has been developed that measures the level of free (unbound) penem antibiotic in serum ultrafiltrates. Whole human serum was adjusted to a standard concentration of antibiotic and then filtered by centrifugation through a Centrifree (Amicon Corp., Lexington, Mass.) filter that retained >99.9% of serum protein. The degree of penem protein binding was determined spectrophotometrically by measuring the level of unbound drug in the ultrafiltrate at 322 nm. At this wavelength, no interfering absorption from residual protein was detected in the ultrafiltrate, and penem absorption was linear over a wide concentration range. The method gave protein-binding values comparable to those obtained by a high-pressure liquid chromatography assay but was more rapid, since it did not require solvent extraction and high-pressure liquid chromatography calibration procedures. The spectrophotometric assay has been used to assay over 100 penems to determine the structure-activity relationships that are involved with the high serum protein binding of these agents. As with penicillins and some cephalosporins, the nonpolar nature of the penem side chain at the C-2 position strongly influenced the degree of penem binding to serum proteins.

Bactericidal Activity of Ceragenin CSA-13 in Cell Culture and in an Animal Model of Peritoneal Infection

“…The binding of the ceragenin to plasma proteins was assessed spectrophotometrically. The concentrations of CSA-13 in ultrafiltrates were determined by measuring the absorbance at 230 nm (A 230 ) in a spectrophotometer, compared with drug-free ultrafiltrate as a blank (28). To evaluate antibacterial activity, mice were infected intraperitoneally with 100 l of PAO1 bacteria suspended in LB broth at an OD 600 of 0.1, which corresponds to 6 ϫ 10 6 CFU.…”

Data-Driven Mapping of Gas-Phase Quantum Calculations to General Force Field Lennard-Jones Parameters

“…One is to move to a functional form that captures the physics more faithfully and in more detail. For example, one may add terms that explicitly account for effects, such as electronic polarizability, − that are accounted for only implicitly, at best, in the common functional form; or one may substitute a more realistic form for an existing term, such as a Coulombic term that accounts for charge penetration. − Another strategy is to remain with today’s common functional form and instead look for parameters, such as atomic partial charges, torsional barriers, and Lennard-Jones well-depths and radii, that will lead to greater accuracy when used to compute experimental and/or quantum chemical reference data. This strategy may be pursued by using more, and more relevant, experimental data in the parametrization process.…”

Dendritic Polyglycerolsulfate Near Infrared Fluorescent (NIRF) Dye Conjugate for Non-Invasively Monitoring of Inflammation in an Allergic Asthma Mouse Model

“…The serum binding of 6S-ICG was determined in vitro by incubation with pooled human serum (PAA) with dye concentration of 5 µg/ml [25]. The sample was placed in a Centriprep micropartition unit NWML 30 kDa (Milipore, Billerica, USA), and centrifuged at 5000 g for 20 min.…”