Patricia D. Williams

Comparative toxicities of cephalosporin antibiotics in a rabbit kidney cell line (LLC-RK1).

The rabbit kidney cell line LLC-RK1 was tested for its ability to discriminate the toxicities of six cephalosporin antibiotics according to their in vivo nephrotoxic potentials in rabbits. With the exception of cephalothin, which was markedly toxic to kidney cells in vitro, a good correlation between in vitro toxicity and in vivo nephrotoxicity was obtained, yielding the following toxicity rank order: ceftazidime less than cefazolin approximately cefoperazone less than cephaloglycin approximately cephaloridine. The addition of a kidney microsomal S9 fraction to the cell cultures desacetylated cephalothin as occurs in vivo and detoxified this antibiotic, providing it with the proper toxicity relative to the other cephalosporins. When compared with parent structures, desacetylated derivatives of other cephalosporins such as cephapirin were similarly found to be less toxic to LLC-RK1 cells. The acetylated cephalosporin cephaloglycin was not detoxified by the kidney S9 fraction and was desacetylated three to four times slower than cephalothin by renal esterases. Thus, the rate and extent of desacetylation of cephalosporins may play a role in their in vivo nephrotoxic potential. Our results further suggest that LLC-RK1 cells will provide a useful model for evaluating the potential nephrotoxicity of new cephalosporin antibiotics before in vivo studies. Images

Role of desacetylation in the detoxification of cephalothin in renal cells in culture

The toxicity of three cephalosporin antibiotics to rabbit kidney cells in culture was compared to their known nephrotoxic potential in vivo (cephaloridine greater than cefazolin greater than cephalothin). While cephalothin is considered to be a relatively nonnephrotoxic cephalosporin when administered to many species including humans and rabbits, in several in vitro systems involving rabbit renal tissue, cephalothin was comparatively more toxic than anticipated based on in vivo data. Cephalothin is extensively desacetylated in rabbits to a less microbiologically active metabolite, desacetylcephalothin. When a microsomal S9 fraction from rabbit kidney was added to the in vitro assay in cultured rabbit renal cells, cephalothin was desacetylated and its toxicity to kidney cells was reduced. The addition of S9 in vitro provided a toxicity ranking of the cephalosporins that correlated with their known in vivo nephrotoxic potentials (cephaloridine greater than cefazolin greater than cephalothin). The in vitro detoxification of cephalothin by S9 was blocked by the coadministration of the esterase inhibitor, aminocarb. Desacetylcephalothin was relatively nontoxic to rabbit renal tissue in vitro. These results suggest that the desacetylation of cephalothin in vivo represents a previously unrecognized mechanism of detoxification of this cephalosporin antibiotic. Furthermore, this mechanism of detoxification may be applicable to other acetylated cephalosporins.

Use of a Precise Intratracheal Delivery System to Compare the Acute Tolerance of Heparin and the Heparinoid GM2000 in Rabbits

Summary: The pharmacological activity of heparin as a anti-inflammatory agent, has led to its proposed use in the treatment of asthma. However, the anticoagulant actions of heparin raise concerns about its long-term use due to potential bleeding complications. In the present study, heparin was compared with the heparinoid derivative GM2000, which possesses reduced anticoagulant activity relative to heparin, for acute tolerance in the rabbit. A method was developed to compare the acute tolerance of GM2000 to heparin. This method involved the instillation of compounds directly into the trachea to parallel the route of exposure used to examine the efficacy of these compounds in an animal model of asthma. The protocol utilized four groups of New Zealand White rabbits, each containing 2 males and 2 females. The rabbits were dosed with saline, heparin, or GM2000 at single, escalating doses of 0.2, 2.0, 20, 100, and 200 mg/kg, administered with an intratracheal microspray device (Penn microsprayer). Escalating d...

A Collaborative Evaluation of an In Vitro Muscle Irritation Assay PMA/Drug Safety Subsection (Drusafe) In Vitro Toxicology Task Force

An interlaboratory, collaborative project was conducted by 10 Pharmaceutical Manufacturers Association (PMA) member companies to evaluate an in vitro (myoblast culture) cytotoxicity assay for screening parenterally administered compounds for skeletal muscle irritancy potential. In a blinded analysis, rat skeletal muscle cells (L6 cell line) were exposed to 12 marketed antibiotic formulations prepared in medium-199 at 0%, 10%, 20%, 33%, 50%, or 100% of the recommended clinical concentration for intramuscular injection of the respective antibiotics. After a 1-h exposure period, cultures were assayed for intracellular creatine phosphokinase (CPK) activity. Lactic dehydrogenase (LDH) and aspartic transaminase (AST) were also measured as secondary endpoints of toxicity. Cytotoxicity was expressed as the amount of enzyme retained intracellularly relative to vehicle (control) cultures. The order of cytotoxicity to myoblast cultures, based on a 50% loss in CPK activity (EC50), was tetracycline > clindamycin > cef...

Correlation Between the in vitro and in vivo Nephrotoxicity of Parenteral Antibiotics in the Rabbit

The ability of in vitro models to predict nephrotoxicity was studied by comparing in vivo and in vitro toxicities of parenteral antibiotics. Rabbits were treated with a single dose of carbapenem antibiotics. After 24 h, the toxicity ranking was imipenem > BMY-25174 > BMY-26225 based on blood urea nitrogen (BUN) and histopathology; previous studies showed the cephalosporin ranking to be cephaloridine > cefazolin > cephalothin. In vitro models used were from rabbit renal cortex and included primary proximal tubule cultures (RPTC), the cell line LLC-RK1, and cortical slices. Cell viability was determined after 24-h exposure of RPTC and LLC-RK1 monolayers to the drugs; in some cases the S9 fraction of cortical homogenate was added concomitantly. Based on the 50% effective concentration (EC50s), the toxicity ranking for cephalosporins in both culture models was cephalothin > cephaloridine > cefazolin > cephalothin + S9. The ranking of carbapenems in RPTC was imipenem > BMY-25174 > BMY-26225, but for LLC-RK1 wa...

Cornea-Derived Neutrophil Chemotactic Factors as Predictors of Corneal Inflammation

Acute corneal inflammation, induced by a variety of insults, is characterized by corneal opacity, infiltration of neutrophils, redness, pain, and corneal cell injury. Using isolated bovine corneas, we tested the effects of six compounds (i.e., citric acid, 2-butanone, 1-butanol, propyl paraben, glycerin, and talc) on corneal opacity, release of neutrophil chemotactic factors (NCFs) from corneal tissues, and release of lactic dehydrogenase (LDH) from corneal cells. The epithelial surfaces of isolated bovine corneas were incubated with the six compounds for 30 min and washed three times with saline, and the corneas were reincubated with culture medium for an additional 6 h at 37°C. Corneal opacity was measured quantitatively using an opacitometer machine. The levels of NCFs were measured in the supernatant using modified Boyden chambers. Although the effects of the six compounds on corneal opacity and release of NCF from corneal tissues were quite consistent with the in vivo data, the release of LDH was not...