Enhanced Renal Clearance of Vancomycin in Rats with Carcinogen-Induced Osteosarcoma

Materials and Methods

Materials. Vancomycin hydrochloride was purchased from Shionogi (Osaka, Japan). Probenecid, quinidine sulfate and cimetidine were from Nacalai Tesque, Inc (Kyoto, Japan). Inulin was purchased from Wako Pure Chemical Co. (Osaka, Japan). Interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were purchased from PeproTech EC Ltd. (London, UK). All other chemicals were of reagent grade. Transwell-COL® cell culture chambers (pore size 0.4 mm, diameter 12 mm, and surface area 1 cm²) were purchased from Costar (Bedford, MA, USA). Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F-12), fetal bovine serum (FBS), and other culture reagents were purchased from GIBCO (Grand Island, NY, USA). Epidermal growth factor (EGF), choleratoxin, hydrocortisone, and insulin-transferrin sodium selenite media supplement (ISL) were purchased from Sigma Chemicals (St. Louis, MO. USA). Penicillin G and streptomycin were purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan). Human fibronectin was purchased from Boehringer Mannheim GmbH (Mannheim, Germany).

Animal model preparation. All animal experiments were carried out in accordance with the Declaration of Helsinki and with the Kanazawa University Guide for the Care and Use of Laboratory Animals. The chemical carcinogen-induced osteosarcoma, selected lung metastatic lesion (C-SLM) (14) tumor was kindly provided by Dr Mii (Nara Medical School, Japan) and maintained in male Fisher 344 rats by repeat inoculation with a tumor block (~10 mm³) subcutaneously on the back. In this experiment, we introduced C-SLM tumor into the hind limb thigh muscles in 7-week old rats. Sham operations were performed for the control group. When tumor size reached approximately 1000 mm³ (11-12 weeks old), vancomycin (5 mg/kg) was administered intravenously to the tumor-bearing rats and the control rats. Blood was collected from the jugular vein at 2, 5, 15, 30, 60, 90, 120, 150, and 180 min after vancomycin administration. Plasma was separated by centrifugation at 12,000 ×g for 10 min and stored at −30°C until analysis. The concentration of vancomycin in plasma was determined by high-performance liquid chromatography (HPLC). After plasma samples were deproteinized with acetonitrile, the supernatants were injected onto the HPLC. A chromatogram (model LC-9A; Shimadzu Corporation, Kyoto, Japan) was equipped with a variable wavelength UV detector (model SPD-6A; Shimadzu Corporation) and a 4.6×150 mm stainless steel ODS Column (Shim-pack CLC-ODS (M); Shimadzu Corporation, Kyoto, Japan). The mobile phase was 8% acetonitrile in 0.05 M phosphate buffer (pH 5.5). The detection UV wavelength was 235 nm. A flow rate of 1.0 ml/min was used.

Biochemical laboratory data in plasma were measured by SRL Inc. (Tokyo, Japan). IL-1β, IL-6 and TNF-α were measured using a rat IL-1β immunoassay kit (IBL International Corp., Toronto, Canada), and IL-6 and TNF-α immunoassay kit (R&D Systems, Inc. Minnesota, USA), respectively.

Determination of plasma protein binding. The plasma protein binding of vancomycin was determined by an ultrafiltration technique with the MPS-3 Centrifree (Amicon Corporation, Denver, MA, USA) as described by Ichimura et al. (15). After equilibration at 37°C for 20 min, samples of the ultrafiltrate were obtained by centrifugation at 37°C for 5 min (1000 ×g). The unbound concentration of vancomycin in the filtrate was determined by HPLC.

Clearance study at steady state. The clearance study was conducted using the methods described in Nakamura et al. (16) A catheter was inserted into the femoral vein of the rats for blood sampling at designated stages. Urine was collected from a catheter inserted into the urinary bladder through a suprapubic incision. Vancomycin was administered alone or with probenecid, cimetidine, or quinidine. The loading dose of vancomycin (2.0 mg), required to reach a steady state drug plasma concentration, was administered with inulin (10 mg) and mannitol (40 mg) through the femoral vein. A continuous infusion of vancomycin (1.36 mg/h) was started concomitantly with inulin (13.6 mg/h) and mannitol (68.2 mg/h) at 1.5 ml/h. Due to the rapid circulation of quinidine, the loading dose was omitted. After one-hour infusion for equilibration, urine samples were collected of three consecutive 20 min intervals. Blood was sampled at 70, 90 and 110 min after starting the infusion. In addition, we extracted infusion solution for 20 min after completion of the experiment. Plasma was separated by centrifugation at 12,000 ×g for 10 min and stored at −30°C until analysis. The concentration of vancomycin in plasma and urine was determined by HPLC. The inulin concentrations were also determined by using a cysteine/tryptophan reaction measured by photometry as described in Waugh et al. (17).

Cell culture. Human renal proximal tubular epithelial cells (RPTECs) (18), obtained from Bio-Whittaker (Walkersville, MD, USA), were cultured in Renal Epithelial Cell Basal Medium(REGM BulletKit®) from Takara Bio Inc. (Shiga, Japan) supplemented with addition factor set (0.5 mg/ml hydrocortisone,10 μg/ml hEGF, 10% FBS, 0.5 mg/ml epinephrine, 6.5 μg/ml triiodothyronine, 10 mg/ml transferrin, 5 mg/ml insulin, GA-1000 (Gentamicin/Amphotericin-B)). Cells were passaged using standard trypsinization procedures and cultured in an atmosphere of 95% air and 5% CO₂ at 37°C.

Permeability study using RPTEC. RPTECs were seeded at a density of 3×10⁴ cells/cm² on the filter membrane of a Transwell-COL insert and cultured in an atmosphere of 95% air and 5% CO₂ at 37°C for 7 to 9 days until confluence. In the permeability study, the RPTECs grown on a filter membrane were washed twice with Hank's balanced salt solution (HBSS) (1.3 mM CaCl₂, 5.0 mM KCl, 0.3 mM KH₂PO₄, 0.8 mM MgCl₂, 138 mM NaCl, 0.3 mM Na₂HPO₄, 5.6 mM D-glucose, and 10 mM HEPES, pH 7.4). HBSS was added to the apical side (200 μl) and to the basal side (800 μl) and preincubated for 30 min at 37°C.

In the permeability study with cytokines (IL-1β, IL-6, TNF-α), the cell layer was preincubated with cytokines on both the apical and basal sides for 6 and 24 h. The concentrations of cytokines used were 0.1 and 10 ng/ml. In the assay of drug permeability from the apical to the basal side, the basal side contained vancomycin. A sample (80 μl) was collected from the apical side and was subsequently replaced with an equal volume of HBSS at 37°C. In the assay of drug permeability from the basal to the apical side, the apical side contained vancomycin. A sample (200 μl) was collected from the basal side and was subsequently replaced with an equal volume of HBSS at 37°C. The initial concentration of vancomycin was 5 μg/ml on the apical side and 10 μg/ml on the basal side. The collected samples were subsequently used for vancomycin determination with HPLC.

Pharmacokinetic analysis. The plasma concentration data after intravenous bolus administration of vancomycin were analyzed by a non-compartmental method. In the clearance study at steady state, total clearance (CLtot) of vancomycin was calculated as the infusion rate divided by the steady-state plasma concentration (Cpss). The renal clearance (CLr) was obtained as the urinary excretion rate divided by the Cpss.

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Figure 1.

Vancomycin (VCM) plasma concentration versus time profiles for control and intramuscularly transplanted tumor-bearing rats after intravenous bolus injections with 5 mg/kg. Each point and vertical bar represents the mean±standard error of the mean (SEM) (n=5). *Significant difference between control and tumor-bearing rats at p<0.05.

Statistical analysis. Student's t-test was used to compare the unpaired mean values of two sets of data. The number of determinations is noted in each Table and Figure. A p-value <0.05 indicated a statistically significant difference between data sets.