Penetration of Amphotericin B Lipid Formulations into Pleural Effusion

Abstract

The penetration of the amphotericin B (AMB) lipid formulations (liposomal AMB, AMB colloidal dispersion, and AMB lipid complex formulations) into pleural effusions in seven critically ill patients was assessed. AMB was detected in all pleural effusion samples at concentrations ranging from 0.02 to 0.43 μg/ml. The penetration ratio was 3 to 44%.

Invasive fungal infections are a major cause of morbidity and mortality in immunocompromised patients, particularly when the pleural compartment is affected (9). Although many studies on the penetration of antibacterial agents into pleural effusions have been performed, less attention has been paid to target site concentrations of antimycotic drugs in pleural effusions (2, 8, 12, 15-17, 22, 23). Amphotericin B (AMB) lipid formulations have been introduced in therapy to reduce the toxicity of AMB. The lipid moieties of liposomal AMB (LAMB), the AMB colloidal dispersion formulation (ABCD), and the AMB lipid complex (ABLC) exhibit different compositions, structures, and particle sizes, which are reflected in different plasma pharmacokinetics and degrees of lung penetration (1, 7, 19, 21). The aim of the present study was to investigate AMB penetration into pleural effusions during treatment with LAMB, ABCD, or ABLC.

The study was approved by the local ethics committee. We determined AMB levels in specimens from seven critically ill patients treated with LAMB (one patient [two sample sets]), ABCD (five patients), or ABLC (one patient) for suspected invasive fungal infection. Demographic and clinical characteristics of the enrolled patients are shown in Table 1. LAMB (AmBisome; Gilead, San Dimas, CA), ABCD (Amphocil; Torrex-Chiesi Pharma, Vienna, Austria), and ABLC (Abelcet; Elan Pharma International Limited, Athlone, Ireland) were dissolved as recommended by the manufacturers and administered intravenously at doses of 3 to 5 mg/kg of body weight over 4 h once a day. Aliquots of pleural effusions were taken during therapeutic thoracentesis. Blood samples were drawn simultaneously from an arterial line and were centrifuged immediately. The plasma and the pleural effusion samples were stored frozen at −80°C. Samples were purified and concentrated. The lipid-associated fractions of LAMB and ABCD were separated from AMB that had been liberated from its lipid encapsulation (comprising free and protein-bound AMB) by C₁₈ solid-phase extraction as described previously (with modifications for pleural effusion samples) (3). For ABLC, this separation technique is not feasible. Pleural effusion and plasma specimens were analyzed by reversed-phase high-performance liquid chromatography using a LiChrosorb-RP-8 column, UV detection (λ = 405 nm), and acetonitrile-methanol-0.010 M NaH₂PO₄ buffer (41:10:49, vol/vol) as the mobile phase (3). The detection limit was 0.005 μg/ml. The intra-assay coefficient of variation was 2.06%. The concentrations were assessed by means of a linear standard curve (R, 0.998 to 0.999) obtained by using external standards comprising pleural effusion samples spiked with AMB. Total AMB concentrations were obtained by adding the concentrations of liberated and lipid-associated AMB. The penetration ratio was defined as follows: AMB concentration in pleural effusion sample/simultaneous AMB level in plasma sample, expressed as a percentage. Statistical calculations were performed using Statistica 5.1 (1997; StatSoft, Inc., Tulsa, OK). The Wilcoxon matched-pair test was used to analyze the differences between total AMB concentrations in plasma and pleural effusion samples.

TABLE 1.

Demographic and clinical characteristics of patients^a

Patient and sample set	Sex	Age (yr)	AMB LF	Cumulative dose (mg)	Interval (h)	Result for plasma sample			Result for pleural effusion sample				Diagnosis or condition(s)
						Crea concn (mg/dl)	Protein concn (g/dl)	LDH concn (U/liter)	Protein concn (g/dl)	LDH concn (U/liter)	No. of RBC	No. of cells/μl
1	M	76	LAMB										Sepsis, pneumonia, pancytopenia
Sample set A				1,050	22.5	0.60	5.73	425	2.57	281	++	700
Sample set B				1,650	4	0.40	5.53	417	2.12	NA	+	NA
2	M	66	ABCD	150	14.5	0.87	4.62	272	NA	NA	NA	NA	Sepsis, lymphoma, diabetes mellitus, renal failure
3	M	29	ABCD	1,300	4	2.21	5.00	234	1.63	126	++	290	Sepsis, pneumonia, post-kidney transplant status, Kaposi's sarcoma
4	M	68	ABCD	1,350	21.5	0.99	5.06	247	0.68	126	++	35	Septic shock, pneumonia, hepatocellular carcinoma, renal failure
5	F	46	ABCD	1,500	2	2.07	6.98	286	1.23	62	+	2	Hodgkin's lymphoma, pneumonia
6	M	44	ABCD	7,250	4	0.28	4.74	194	2.43	134	+	210	Lymphoma, pneumonia
7	M	53	ABLC	12,750	5	0.39	6.65	246	2.66	128	+	220	Septic shock, pneumonia, lung cancer

^aTwo sets of samples (A and B) were obtained from patient 1; one sample set was obtained from each of the other patients. M, male; F, female; AMB LF, AMB lipid formulation; interval, duration between last dose and sampling time; crea, creatinine (normal range of concentrations in plasma, 0.70 to 1.20 mg/dl); normal range of protein concentrations in plasma, 6.30 to 8.20 g/dl; LDH, lactate dehydrogenase (normal range of concentrations in plasma, 100 to 250 U/liter); RBC, red blood cells (erythrocytes); ++, many; +, some; NA, not available.

AMB concentrations in plasma and pleural effusion samples and the penetration ratios are displayed in Table 2. Liberated AMB could be detected in all samples of pleural effusions and plasma. In pleural effusion samples, total AMB concentrations were significantly lower than the total concentrations in plasma samples (P = 0.03). Concentrations of the lipid-associated fractions of ABCD and LAMB, measured in pleural effusion samples, were very low (<0.03 μg/ml; in four samples, they were even below the detection limit). The highest AMB concentration in a pleural effusion was found in a sample from patient 1 (0.43 μg/ml), who had been treated with LAMB at a daily dose of 300 mg for 4 days. AMB concentrations in pleural effusions correlated positively with the cumulative dose administered to patients treated with ABCD (R = 0.96; P = 0.01). The AMB concentration in the pleural effusion from patient 7, who had received a cumulative dose of 12,750 mg of ABLC, reached 0.18 μg/ml (penetration ratio, 44%). The respective level in plasma was as low as 0.4 μg/ml.

TABLE 2.

Concentrations of AMB in plasma and pleural effusion samples^a

Patient	Sample set	Result for pleural effusion sample			Result for plasma sample			Total penetration ratio (%)
		Concn of liberated AMB (μg/ml)	Concn of lipid-associated AMB (μg/ml)	Total concn of AMB (μg/ml)	Concn of liberated AMB (μg/ml)	Concn of lipid-associated AMB (μg/ml)	Total concn of AMB (μg/ml)
1	A	0.40	0.03	0.43	3.10	3.81	6.91	6
	B	0.13	0.02	0.15	3.38	2.51	5.89	3
2		0.02	0.00	0.02	NA	NA	NA	NA
3		0.05	0.00	0.05	0.52	0.09	0.61	8
4		0.04	0.02	0.06	0.49	0.00	0.49	11
5		0.12	0.00	0.12	0.41	0.00	0.41	29
6		0.25	0.00	0.25	1.02	0.49	1.51	16
7		NA	NA	0.18	NA	NA	0.40	44

^aTwo sets of samples were obtained from patient 1; one sample set was obtained from each of the other patients. NA, not available. For ABLC, the chromatographic separation of lipid-associated and liberated AMB fractions was not feasible. Total concentrations of AMB in plasma and pleural effusion samples are shown in boldface.

Since several mycoses, such as candidiasis, aspergillosis, blastomycosis, histoplasmosis, cryptococcosis, and coccidioidomycosis, can cause pleural manifestations (5, 6, 9, 11, 13, 20), AMB concentrations in pleural effusions can be crucial for clinical response. After treatment with AMB lipid formulations, AMB concentrations were substantially lower in pleural effusions than in plasma samples and lung tissue (19). However, the small number of patients, the different underlying clinical conditions of the patients, and the differences in cumulative doses are clear limitations of our study and preclude a comparison among the lipid formulations.

Median intraperitoneal levels of AMB previously amounted to 0.12 μg/ml in samples from critically ill patients during intravenous treatment with AMB deoxycholate (18), which is comparable to our results for pleural effusion samples. In noninfected rabbits, total AMB concentrations were much lower in alveolar epithelial lining fluid than in lung tissue (7). By the separation of lipid-associated and liberated AMB, we could show that only AMB that has been liberated from its lipid encapsulation penetrates into pleural effusions.

The MIC of AMB has been reported to range from 0.125 to 1 mg/liter for Candida spp. (14), from 0.25 to 4 mg/liter for Aspergillus spp., and from <0.03 to 2 mg/liter for relevant dimorphic fungi, such as Blastomyces dermatitidis and Histoplasma capsulatum (4). Thus, the MIC may exceed in some cases the pleural AMB concentrations that are achieved by therapeutic dosage. Samples from patients 1 and 6, which exhibited the highest levels of AMB in pleural effusions, met the criteria for an exudate (10). This finding may indicate that the presence of local inflammation increases the penetration of AMB into pleural effusions. However, none of our patients suffered from fungal empyema thoracis. Recently, voriconazole was found to achieve penetration ratios of 45 to 95% in cases of pleural empyema caused by Aspergillus fumigatus (15).

In conclusion, AMB levels in pleural effusions were in the range of—or even below-the MICs of AMB for most relevant pathogens after the administration of AMB lipid formulations. Therefore, long-term treatment with high doses of AMB lipid formulations will be required for the eradication of fungal infection from pleural effusions, and alternative therapeutic strategies have to be considered. Further clinical studies are required to elucidate the penetration of antimycotics into pleural effusions.