Abstract
In a recent issue of AIDS, Morris and co-workers [1] reported improved survival with highly active antiretroviral therapy (HAART) in HIV-infected patients with severe Pneumocystis carinii pneumonia (PCP). The explanation for a possible survival benefit from HAART include decreased viral fitness, an attenuated rise in viral titre during PCP, or the anti-Pneumocystis properties of protease inhibitors (PI). We demonstrated that several PI exert an in-vitro aspecific anti-Pneumocystis effect at concentrations clinically achievable in plasma in vivo during HAART [2–4]. Other authors did not reproduce our results in a different in-vitro axenic model, although reporting in vivo a slight decrease in P. carinii cyst counts in the lungs of animals experimentally treated with PI [5]. The clinical relevance of these observations remains to be investigated, especially for a microrganism harbouring in lung alveoli, such as P. carinii. No data were available on the concentration of PI in epithelial lining fluid (ELF). Reliable methods to measure drug concentrations in ELF have been developed and successfully applied also for the detection of antipneumocystic drugs such as pentamidine [6]. Lopinavir exerts in-vitro dose-dependent anti-Pneumocystis activity, and very high concentrations are detected in plasma during therapy, so we preliminarily chose this drug in order to screen for the possible presence of PI in ELF. We hereby report the detection of lopinavir in the ELF of one patient treated with lopinavir/ritonavir oral tablets. Heparanized and unheparinized blood was collected for plasma separation and biochemistry. Standardized bronchoscopy and blood collection were performed after informed consent 13 h after the administration of the evening dose of lopinavir/ritonavir in a fasted, male, HIV-infected patient. He had been receiving HAART, including lopinavir/ritonavir (400/100 mg twice a day), lamivudine (300 mg/day) and stavudine (80 mg/day), for 6 months. No systemic sedation was used. A total of 2 × 50 ml normal saline were instilled and promptly aspirated. Pooled specimens of bronchoalveolar lavage (BAL) fluid were processed as follows: aliquots were sent for microbiological, biochemical and microscopic examination, centrifuged at 400g for 5 min and the supernatant frozen at −80°C until used. The volume (V) of ELF recovered was calculated using the urea dilution method: VELF = VBAL × (ureaBAL/ureaserum) and the concentration of lopinavir (LPV) was obtained by the following relationship: LPVBAL × VBAL/VELF. Samples were analysed according to a validated high-performance liquid chromatography assay with ultraviolet detection and liquid–liquid extraction. The cut-off for lopinavir detection was 50 ng/ml. On 70 ml of recovered BAL fluid, the ELF volume was 0.9 ml. The concentration of lopinavir was 8.1 μg/ml in plasma and 14.4 μg/ml in ELF. BAL fluid was microbiologically sterile, and internal transcribed spacers-nested polymerase chain reaction to detect P. carinii DNA was negative. The microscopic evaluation of BAL (cyto-spinned cells) demonstrated a non-inflammatory, normal pattern of alveolar cells, with prevalent macrophages and few lymphocytes, and no granulocytes. The apparent accumulation of lopinavir in the ELF is not surprising because PI intracellular and efflux pumps have been described in macrophages, cells commonly harbouring and trafficking in alveoli [7]. We cannot exclude the possibility that BAL centrifugation could have caused some ‘spilling’ of lopinavir from inside the cells; however, our preliminary demonstration of lopinavir penetration in human ELF deserves attention: such a high micromolar PI concentration supports the hypothesis that the anti-Pneumocystis effects described in vitro, starting from a 0.5 μg/ml lopinavir concentration, could also be relevant in vivo, possibly explaining the dramatic decrease in PCP during the early phase of HAART, before immune reconstitution, in addition to the improved survival of HIV-infected patients with severe PCP treated with PI-based HAART. The non-viral aspecific beneficial effect exerted by PI, in addition to the direct inhibition of P. carinii proteases, may include the modulation of host cell proteasome [8].
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