Excretion Pathways of Amphotericin B

We developed a rapid, sensitive, high-pressure liquid chromatographic (HPLC) procedure which incorporates a commercially available internal standard, 1-amino-4-nitronaphthalene, to measure amphotericin B in serum. Recovery was quantitative (.90%), and the standard curve was linear from 0.04 to at least 10.0 ,ug/ml. The reproducibility of the assay was good, with intrarun coefficients of variation from 2.0 to 6.8% and interrun coefficients of variation from 4.9 to 10.0%. Comparison by linear regression analysis of the HPLC assay with an agar well diffusion bioassay gave a correlation coefficient of 0.942, with the HPLC assay exhibiting greater precision and sensitivity. No interference was encountered from over 20 drugs and three amphotericin B analogs. However, serum specimens that contained high concentrations of conjugated bilirubin (>3 mg/dl) produced interfering peaks in both this assay and other previously reported HPLC assays for amphotericin B. We also describe a solid-phase extraction procedure which effectively removes this interference and uses an alternative internal standard (N-acetyl amphotericin B).Amphotericin B is a polyene antifungal agent that is presently the drug of choice for the treatment of most severe systemic fungal infections. Use of the drug, however, is limited by its toxicity, particularly by its impairment of renal function. The pharmacokinetics of amphotericin B are complex, involving a rapid initial decrease in serum levels for approximately 24 h, followed by a long (15-day half-life) terminal elimination phase (1,4,8). Other pharmacokinetic properties, such as elimination pathways, are not completely understood. Renal and biliary excretion have each been estimated to account for up to 20% of a dose of amphotericin B (6, 16). The lack of a clearly defined relationship between serum levels and toxicity or clinical outcome has prevented the development of a rational, systematic approach to amphotericin B therapy.Many of the problems associated with pharmacokinetic studies of amphotericin B arise from difficulty in obtaining rapid, accurate, and reproducible measurements of drug levels in various biological fluids. Most researchers have used bioassay systems that involve either serial tube dilution or plate diffusion with a variety of indicator organisms, including Paecilomyces varioti, Candida tropicalis, and Saccharomyces cerevisiae (2-4, 6-8, 17-19). Bioassays, however, vary widely in their accuracy, precision, sensitivity, and specificity, making the comparison of data from different papers difficult.Several high-pressure liquid chromatographic (HPLC) assays have been reported recently which offer faster and more accurate and reproducible alternatives to bioassays for both pharmacokinetic studies and routine clinical use (9, 10, 13-15, 17, 20). HPLC also offers improved sensitivity and specificity and is easier to standardize than bioassays are. In this report we describe an HPLC method which incorporates an internal standard for the measurement of amphotericin B in serum and a...

Section: Discussionmentioning

confidence: 99%

“…estimated to account for up to 20% of a dose of amphotericin B (6,16). The lack of a clearly defined relationship between serum levels and toxicity or clinical outcome has prevented the development of a rational, systematic approach to amphotericin B therapy.…”

mentioning

confidence: 99%

Sensitive high-pressure liquid chromatographic assay for amphotericin B which incorporates an internal standard

Granich¹,

Kobayashi²,

Krogstad³

1986

“…It is not likely that guanidine itself reacted with the drug. This behavior also suggests an explanation for some of the reported peculiar pharmocokinetics of amphotericin B (2,4,9). After a course of therapy with amphotericin B, the level of the drug in plasma decreases very slowly, with a half-life of about 2 weeks.…”

Section: Resultsmentioning

confidence: 70%

Quantitative extraction of amphotericin B from serum and its determination by high-pressure liquid chromatography

Bach¹

1984

Therapeutic concentrations of amphotericin B in serum were measured by reversed-phase liquid chromatography with detection at 386 nm. Complete recovery of the drug and the internal standard from a l-ml serum sample was achieved by pretreating the sample with guanidine hydrochloride and extracting it with a disposable reversed-phase phenyl extraction column. The method was sensitive to 0.005 ,ug of amphotericin B per ml and linear to at least 5 ,ug/ml. Coehicients of variation were 4.8% within-run and 6.3% between-run.Amphotericin B is an antibiotic widely used to treat systemic mycoses (10). Its use is restricted to life-threatening infections, however, because of its severe toxicity, which is usually encountered with therapeutic doses of the drug. Amphotericin B therapy therefore involves a balancing of toxic effects against antifungal activity.Until This was then mixed with 45 ml of 1% aqueous sodium dodecyl sulfate (SDS). This stock solution (100 ,ug/ml) was stored at -70°C. To make the working standard solution (1 jxg/ml), I combined 1 ml of stock solution, 1.5 ml of 1% SDS, and 97.5 ml of water (total volume, 100 ml). This solution was stored at 1-ml aliquots at -70°C.Extraction of samples. Samples (serum, aqueous standard, cerebrospinal fluid [CSF], etc.) (1 ml) were combined with 200 ,ul of ISTD solution and 2 ml of 6 M guanidine hydrochloride. After brief mixing, each sample was passed immediately through a separate, disposable phenyl extraction column ("Bond-Elut" PH, catalog no. 608101; Analytichem International, Harbor City, Calif.). Before analysis, the column was washed with one filling of methanol and then one filling of water. The sample was applied and allowed to run through, and the column was rinsed with another filling of water and allowed to drain. The drug and the ISTD were then eluted immediately from the column into a small test tube with 500 ,ul of acetonitrile-EDTA (prepared by combining 75 ml of acetonitrile with 25 ml of 5 mM disodium EDTA). SDS (1%) solution (200 ,ul) was mixed with the extract. Because of the instability of amphotericin B in the specimen after mixing with guanidine, the above steps were completed for one sample before the next sample was extracted. Amphotericin B was very stable in the final extract after SDS was added. For calibration, a 1-ml aliquot of the amphotericin B working standard solution was thawed and immediately extracted as described above.Chromatography. Chromatography was isocratic, with a solvent consisting of acetonitrile, methanol, and 5 mM EDTA (disodium salt) (187:449:354 [vol/vol]

“…Approximately 2 to 5% of the administered DAmB has been reported to be excreted unchanged in urine and bile each (1,2,4,5). No metabolites have yet been identified in plasma or urine.…”

Section: Discussionmentioning

confidence: 99%

Dose-Dependent Pharmacokinetics of Amphotericin B Lipid Complex in Rabbits

Walsh

Jackson

Lee

et al. 2000

Amphotericin B lipid complex (ABLC) was recently approved by the Food and Drug Administration for treatment of patients with invasive fungal infections who are intolerant of or refractory to conventional amphotericin B therapy. Little is known, however, about the pharmacokinetics of this new antifungal compound. We therefore investigated the pharmacokinetics of ABLC in comparison with those of conventional desoxycholate amphotericin B (DAmB) in rabbits. The pharmacokinetics of DAmB in a rabbit model were similar to those previously reported in humans. The pharmacokinetics of ABLC differed substantially from those of DAmB. Plasma amphotericin B levels following ABLC administration were 10 times lower than those following administration of an equal dosage of DAmB. The levels of ABLC in whole blood were approximately 40 times greater than those in plasma. The ABLC model differed from the DAmB model by (i) a dose-and time-dependent uptake and return between the plasma compartment and apparent cellular components of the blood-sediment compartment and (ii) time-dependent tissue uptake and return to plasma from serially connected compartments. Following infusion of ABLC, there was a nonlinear uptake into the apparent cellular components of the blood-sediment compartment. This uptake was related to the reciprocal of the integral of the total amount of drug infused (i.e., the more drug infused the greater the fractional uptake between 0.5 and 5 mg/kg of body weight for ABLC). The transfer of drug from plasma to the cellular components of the blood-sediment compartment resulted in initial uptake followed by rapid redistribution back to the plasma. The study describes a detailed model of the pharmacokinetics of ABLC and characterizes a potential role of the cellular components of the blood-sediment compartment in the distribution of this new antifungal compound in tissue.Invasive fungal infections have emerged as important causes of morbidity and mortality in immunocompromised hosts (14,15). Desoxycholate amphotericin B (DAmB) remains the treatment of choice for treatment of many life-threatening opportunistic mycoses. However, dose-limiting nephrotoxicity often compromises the ability to administer DAmB, resulting in therapeutic failures. Certain lipid formulations of amphotericin B can be administered at higher dosages with reduced nephrotoxicity (6, 9, 13, 17).Amphotericin B lipid complex (ABLC or Abelcet; The Liposome Company, Princeton, N.J.) was developed to reduce the toxicity and maximize the therapeutic utility of amphotericin B in the treatment of invasive fungal infections (10). The preparation of ABLC consists of amphotericin B complexed with two lipids in a 1:1 drug-to-lipid molar ratio. These lipids, dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG), are present in a 7:3 molar ratio.Amphotericin B lipid complex was recently approved by the Food and Drug Administration for treatment of patients with invasive fungal infections who are intolerant of or refractory to conventional am...