Effects of Immunomodulatory and Organism-Associated Molecules on the Permeability of anIn VitroBlood-Brain Barrier Model to Amphotericin B and Fluconazole

Abstract

Amphotericin B (AMB) is used to treat fungal infections of the central nervous system (CNS). However, AMB shows poor penetration into the CNS and little is known about the factors affecting its permeation through the blood-brain barrier (BBB). Therefore, we studied immunomodulatory and organism-associated molecules affecting the permeability of an in vitro BBB model to AMB. We examined the effects of interleukin-1 beta (IL-1␤), tumor necrosis factor alpha (TNF-␣), lipopolysaccharide (LPS), lipoteichoic acid (LTA), zymosan (ZYM), dexamethasone (DEX), cyclosporine, and tacrolimus on transendothelial electrical resistance (TEER); endothelial tight junctions; filamentous actin; and permeability to deoxycholate AMB (DAMB), liposomal AMB (LAMB), and fluconazole. Proinflammatory cytokines and organism-associated molecules significantly decreased the mean TEER by 40.7 to 100% (P < 0.004). DEX increased the mean TEER by 18.2 to 26.4% (P < 0.04). TNF-␣ and LPS increased the permeability to AMB by 8.2 to 14.5% compared to that for the controls (1.1 to 2.4%) (P < 0.04). None of the other molecules affected the model's permeability to AMB. By comparison, the BBB model's permeability to fluconazole was >78% under all conditions studied, without significant differences between the controls and the experimental groups. LPS and TNF-␣ decreased tightjunction protein zona occludens 1 (ZO-1) between endothelial cells. In conclusion, IL-1␤, ZYM, and LTA increased the permeability of the BBB to small ions but not to AMB, whereas TNF-␣ and LPS, which disrupted the endothelial layer integrity, increased the permeability to AMB.Amphotericin B (AMB) and fluconazole are important antifungal agents used to treat invasive fungal infections of the central nervous system (CNS). AMB is commonly used in the treatment of cryptococcal meningitis, rhinocerebral zygomycosis, hematogenous candida meningoencephalitis, and other invasive fungal infections of the CNS (7,16,18). AMB was effective for the treatment of experimental hematogenous candida meningoencephalitis in rabbits, but it demonstrated poor penetration into the cerebrospinal fluid (CSF) and brain tissue (10). While the penetration of AMB into CSF and brain parenchyma is poor, it remains an effective agent against CNS mycoses (20). By comparison, fluconazole displays a relatively high level of penetration into the CNS (2). Variables which might influence the permeability of the blood-brain barrier (BBB) to AMB, such as cytokines, organism-associated molecules, and immunosuppressants, have not been fully evaluated. We therefore studied the effects of several such factors on an in vitro mammalian BBB model. We examined the effects of those variables on interendothelial tight junctions; the structure of filamentous actin; and the permeability of the in vitro BBB model to AMB, fluconazole, and small ions. MATERIALS AND METHODS Cells. Bovine brain microvascular endothelial cells (BBECs; Cell ApplicationsInc., San Diego, CA) were grown in 75-cm 2 collagen-coated tissue culture flasks with BBEC ...

Tissue Penetration of Antifungal Agents

“…Similar CSF and brain data for AmBd, L-AMB, and ABLC (i.e., CSF and tissue/plasma concentration ratios of Ͻ0.3) have been recorded for rabbits (92). In contrast to the case with posaconazole, inflammation does not seem to increase the concentration of any amphotericin formulation in the brain, at least in animals (40,92). To overcome these potential limitations, intraventricular instillation of AmBd via an Ommya reservoir has been used for severe cerebral infections (93,94,95).…”

“…(ii) physiological factors, such as inflammation, which may increase tissue permeability, i.e., by disruption of normal physiological barriers such as the blood-brain barrier (29,40); the underlying disease (41), which may result in a range of effects, including modification of plasma protein composition and hence drug binding (42,43,44); the recruitment of drug-containing phagocytic cells, i.e., the "dump truck phenomenon," which may increase drug concentrations at the site of infection (12,13,32,45,46); drug export via pumps, e.g., for itraconazole and P-glycopro- Fluid multiples are from g/ml concentrations. *, autopsy data; in these cases, the multiples are based on plasma C max values at the same dose in volunteers (188).…”

Amphotericin B Penetrates into the Central Nervous System through Focal Disruption of the Blood-Brain Barrier in Experimental Hematogenous Candida Meningoencephalitis

“…These in vivo findings correlate well with data from the in vitro BBB model, which showed a significantly increased permeation of DAMB and LAMB in the infected model (10). Several mechanisms to account for permeability changes are possible, including the induction of local cytokine responses in infected tissue, the mechanical disruption created by hyphal and pseudohyphal invasion, and local enzymatic disruption of the BBB from Candida proteases and phospholipases (21).…”

“…However, since amphotericin B shows limited penetration into CSF, little is known about the factors affecting its efficacy in CNS tissue or its permeation through the BBB in HCME. Our earlier work demonstrated that the proinflammatory cytokines induced by organism-associated molecules significantly increase the permeability of the BBB, as demonstrated by a decreased mean TEER in an in vitro BBB model (10). Consistent with the known properties of dexamethasone and other corticosteroids, dexamethasone increased the mean TEER and decreased the permeability of the BBB to amphotericin B.…”

Cryptococcal meningoencephalitis in a dog

“…In the present case, itraconazole was the only available antifungal drug and was initially used for treatment. However, itraconazole penetrates the blood brain barrier poorly and this was therefore substituted with fluconazole, which achieves much higher concentration in the CNS parenchyma (Arndt and others 1988, Kethireddy and Andes 2007, Pyrgos and others 2010). Treatment with fluconazole has been reported to be effective in resolving neurological signs in dogs, although prolonged treatment courses are required (Tiches and others 1998, O'Toole and others 2003, Lavely and Lipsitz 2005) and the prognosis remains guarded (Malik and others 2006).…”