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

Abstract

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...

Prions and related neurological diseases

“…In experimental animal models, however, sulphated polyanions (Ehlcrs and Diringcr, 1984;Farquhar and Dickinson, 1986;Kimbcrlin and Walker, 1986b;Diringcr and Ehlcrs, 1991;Ladogana et al, 1992), Congo red (lngrosso and Pocchiari, unpublished data) and the polyene antibiotic amphotcricin B (Amyx et al, 1984;Pocchiari et al, 1987Pocchiari et al, , 1989Casaccia et al, 1991) have given encouraging results. These drugs prolong or sometimes even prcvcnt the appearance of the disease by delaying the formation of PrP-rcs and/or inhibiting scrapic replication (Kimbcrlin and Walker, 1986b;Diringer and Ehlcrs, 1991;Caughey and Race, 1992;Xi et al, 1992;Caughcy and P, aymond, 1993;Gabizon et al, 1993a).…”

“…This effect resulted in a delay of the appearance of scrapie clinical signs compared to infected controls, suggesting that the accumulation of PrP-res in the brain is more important than replication of the scrapie agent for the expression of disease . Interestingly, the beneficial anti-scrapie effect of amphotericin B is limited to the 263K hamster-adapted strain (Pocchiari et al, 1987Casaccia et al, 1991) and the murine strain C506 (Demaimay eta/., 1993). Amphotericin B treatment is ineffective with other hamster or mouse-adapted strains of scrapie (Carp, 1992;Xi et al, 1992), suggesting that it interferes with strain-specific (that is, 263K and C506) components of the scrapie agent responsible for the modification of PrP-scn to PrP-res (Fig.…”

Late treatment with polyene antibiotics can prolong the survival time of scrapie-infected animals

“…Indeed, the cerebral concentration of AmB reached only 0.3 g/g when Syrian hamsters were treated i.p. with 1 mg of AmB per kg of body weight (12), and AmB was undetectable in rat brain following i.p. administration of a similar dose (28).…”