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

A simple, sensitive, and reprodut&ble assay for the measurement of the amphotericin B concentration in tissue extracts was developed by using the fourth derivative of the absorption spectrum of amphotericin B between wavelengths of 330 and 430 nm. The amphotericin B concentration in spleen and brain was proportional to the total amount administered. The amphotericin B concentration in the brain was highly correlated with the increase in the mean incubation period of intracerebrally scrapie-infected hamsters.Amphotericin B is a polyene antibiotic which has been used for more than 30 years in the treatment of fungal infections (13). After systemic administration, amphotericin B'is readily distributed in the liver, kidneys, and spleen at concentrations on the order of several micrograms per gram of tissue (4,10,14). However, it is barely detectable in the brain (4, 10). Despite the low amount that reaches the brain, therapy with amphotericin B increases the mean incubation period in mice challenged intracerebrally (i.c.) with Cryptococcus neoformans (5, 7), and it is effective in preventing relapse of cryptococcal meningitis in patients with AIDS (19). Moreover, we recently reported the beneficial effect of amphotericin B in prolonging the incubation period in hamsters infected i.c. with scrapie (16,17). Scrapie is a fatal neurological disease that occurs naturally in sheep and goats and that is caused by an as yet unknown infectious agent (3). Scrapie is successfully transmitted to laboratory animals either by i.c. or by extraneural injection of the infectious agent (8). Once the agent is injected i.c., it soon replicates in the neural cells, where it produces the characteristic neuropathological lesions and causes clinical signs of the disease (8). Thus, we postulated that amphotericin B interferes with scrapie replication in the central nervous system (CNS) (16,17). This result further supported previous observations that amphotericin B is effective against infections of the CNS. Here we report a simple and sensitive spectrophotometric method to measure low amounts of amphotericin B in brain tissue.Amphotericin B and the deoxycholate-amphotericin B complex (Fungizone) were provided by Squibb & Sons, Rome, Italy. Amphotericin B was dissolved in methanol at a concentration of 100 ,ug/ml (wt/vol) and was stored at -20°C and protected from light. Known amounts from this stock solution were added to tissue homogenates in order to assess amphotericin B recovery and determine the standard curves.Amphotericin B was also incorporated into multilamellar vesicles (liposomes) containing L-a phosphatidylcholine (from egg yolk) as described by . The drug/lipid ratio in the initial mixture was 0.15 (wt/wt). * Corresponding author.The content of amphotericin B in liposomes (liposomeamphotericin B) was determined by measuring the A405 versus those of amphotericin B standards in methanol by using equivalent amounts of phospholipids dissolved in methanol as absorbance blanks. Liposome-amphotericin B was stored at 4°C for a maximum...

Section: * Corresponding Authorsupporting

confidence: 80%

Measurement of the concentration of amphotericin B in brain tissue of scrapie-infected hamsters with a simple and sensitive method

Casaccia

Ladogana

et al. 1991

“…DAmB (0.5 to 1.5 mg/kg) was administered by constant infusion over 5 min with subsequent sampling of plasma. Sampling times were at 0, 5, 10, 15, 30, and 60 min and 2, 3,4,6,8,12,24,48, and 72 h postinfusion. Infusion of DAmB at dosages of 0.5 to 1.0 mg/kg is well tolerated by rabbits.…”

Section: Animalsmentioning

confidence: 99%

“…Amphotericin B levels in plasma were assayed by highpressure liquid chromatography based upon a modification of the method developed by Granich et al (8). Calibration curves were prepared with plasma with low (25 to 1,000 ng/ml) and high (1 to 20 g/ml) concentration ranges.…”

Section: Animalsmentioning

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...

“…In parallel with these recent developments, a need arose for suitable analytical methods to monitor AMB levels in tissue or plasma and thus establish pharmacokinetic and pharmacodynamic relationships. Assays based on in vitro biological activity or chromatographic separations have been described and used to monitor AMB in the circulation (3,11,19,22,23). These methods, however, are characterized by a relatively high limit of detection (Ͼ50 ng/ml) and by technological limitations.…”

mentioning

confidence: 99%

A Sensitive Amphotericin B Immunoassay for Pharmacokinetic and Distribution Studies

Machard

Théodoro

Bénech

et al. 2000

Since currently used assays of amphotericin B (AMB) lack sensitivity or are not easily adaptable in all laboratories, we have developed an enzyme immunoassay for AMB in biological fluids and tissues. Antibodies to AMB were raised in rabbits after administration of an AMB-bovine serum albumin conjugate. The enzymatic tracer was obtained by coupling AMB via its amino group to acetylcholinesterase (EC 3.1.1.7). These reagents were used for the development of a competitive immunoassay performed on microtitration plates. The limit of quantification was 100 pg/ml in plasma and 1 ng/g in tissues. The plasma assay was performed directly without extraction on a minimal volume of 0.1 ml. The intra-and interassay coefficients of variation were in the range of 5 to 17%, and the recoveries were 92 to 111% for AMB added to human plasma. The assay was applied to a pharmacokinetic study with mice given AMB intraperitoneally at the dose of 1 mg/kg. The drug distribution in selected compartments (plasma, liver, spleen, lung, and brain) was monitored until 72 h after administration. In conclusion, our assay is at least 100-fold more sensitive than previously described bioassays or chromatographic determinations of AMB and may be useful in studying the tissue pharmacokinetics of new AMB formulations and in drug monitoring in clinical situations.Amphotericin B (AMB) has been considered for more than 30 years the major antifungal drug for serious systemic fungal infections, which, owing to AIDS and improved organ transplant immunosuppression drugs, are becoming tragically frequent in immunocompromised individuals (12,20). AMB's spectrum of activity is broad, since antiviral or antiprion effects have also been reported (12). The clinical value of AMB has been reinforced by incorporation in lipid-based carriers, thus reducing its nephro-and hematotoxicity, and these new formulations have been investigated in animals and humans (4,15,25). In parallel with these recent developments, a need arose for suitable analytical methods to monitor AMB levels in tissue or plasma and thus establish pharmacokinetic and pharmacodynamic relationships. Assays based on in vitro biological activity or chromatographic separations have been described and used to monitor AMB in the circulation (3,11,19,22,23). These methods, however, are characterized by a relatively high limit of detection (Ͼ50 ng/ml) and by technological limitations. These disadvantages may be potentially circumvented by antibody-based technology, and enzyme-linked immunosorbent assays for AMB have been developed (6, 18). Although such techniques allow direct assay in a small sample volume, they have not been applied to tissues and have proved to be poorly sensitive since their limits of quantification are above 150 ng/ ml. In this report we describe the development and application of a competitive enzyme immunoassay which allows measurement of AMB in tissues and plasma with sensitivities 1,000-fold greater than those reported elsewhere. The assay is performed with specific AMB antibodies ...