Vol. 35, No. 7
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 1991, p. 1303-1308
0066-4804/91/071303-06$02.00/0 Copyright © 1991, American Society for Microbiology
Limited Protection by Small Unilamellar Liposomes against the Renal Tubular Toxicity Induced by Repeated Amphotericin B Infusions in Rats PASCALE LONGUET,1 VEIRONIQUE JOLY,1 PASCAL AMIRAULT,2 NATHALIE SETA,1 CLAUDE CARBON,' AND PATRICK YENI1t* Laboratoire d'Etude des Infections Experimentales, Institut National de la Sante et de la Recherche Medicale U13, Faculte Xavier Bichat, Universite Paris 7,1 and Laboratoire de Toxicologie, Service de Pharmacie Clinique, H6pital Bichat, Paris, France Received 27 September 1990/Accepted 15 April 1991
Amphotericin B (AMB), either alone or incorporated into small unilamellar vesicles of pure dipalmitoylphosphatidyl choline (DPPC SUV-AMB), was administered intravenously to male Sprague-Dawley rats once daily for 5 days. Either 1.5 or 3.5 mg of AMB or DPPC SUV-AMB per kg was given, since these concentrations corresponded, respectively, to the lowest nephrotoxic dose and the sublethal dose of AMB in our model. Tubular functions were evaluated daily, and AMB concentrations in plasma, urine, and tissues were measured by high-performance liquid chromatography. AMB at both doses induced tubular toxicity, hyposthenuria being the earliest symptom. DPPC SUV-AMB at 1.5 mg/kg/day was atoxic, but the tubular alterations induced by 3.5 mg of DPPC SUV-AMB per kg were similar to those observed with 3.5 mg of AMB per kg, except that the ability to concentrate urine was partly restored 72 h after the last infusion. Incorporating AMB into DPPC SUV did not influence the pharmacokinetics of the drug. Using this lipidic AMB formulation, we thus observed a beneficial effect toward limiting the renal tubular toxicity of repeated low doses of AMB but, unexpectedly, not that of high doses. These results indicate that tubular renal functions and electrolyte serum values should be closely monitored in patients treated with AMB liposomal formulations, especially high-dose regimens.
patients. In a recent study (21), L-AMB appeared to be well tolerated, but hypokalemia requiring supplementation was observed in patients receiving 3 mg or more of L-AMB per kg daily, suggesting that the tubular renal toxicity of AMB is not completely eliminated by the incorporation of the drug into liposomes. The purpose of this study was to determine the effects of liposomes on the renal dysfunction induced by repeated administration of AMB. We focused on the tubular toxicity observed under conditions of administration close to treatment schedules used in humans and consisting of repeated slow infusions of AMB. The liposomes used have proven highly effective in reducing the acute 50% lethal dose of AMB in mice without altering the antifungal effect (32), decreasing the acute renal AMB toxicity in rabbits (13), and protecting in vitro erythrocytes (16) and primary cultures of renal proximal tubular cells from AMB toxicity (14). To reach our goal, we first assessed the lowest nephrotoxic dose and the sublethal dose of AMB; then, we studied the extent of the protection afforded by our liposomal formulation against the toxicity of AMB given in both doses. (This work was presented in part at the 29th Interscience Conference on Antimicrobial Agents and Chemotherapy, Houston, Tex., 1989.)
Systemic mycoses are a major cause of morbidity and mortality among immunocompromised patients (8, 42, 43). Although several new antifungal agents, particularly triazoles, have recently become available, amphotericin B (AMB) remains the most active drug for the treatment of opportunistic fungal infections (12, 28). Its clinical use is limited by side effects, the most serious being nephrotoxicity. Harmful renal effects include inability to concentrate urine (1, 3, 4, 10), defective urinary acidification due to distal tubule dysfunction (10, 27, 31), renal potassium wasting (5), and depressed glomerular filtration rate (GFR) and renal plasma flow (6, 7). AMB toxicity was shown to be reduced when the drug was incorporated into liposomes or complexed with lipids (LAMB), allowing higher doses to be infused and thereby increasing the efficiency of treatment in experimental fungal infections (23, 36, 37). However, the in vivo protective effect of liposomes (13, 38) or lipidic emulsions (18) against the experimental renal toxicity of AMB has been evaluated only after a single administration of the drug. Therefore, the experimental nephrotoxicity of L-AMB under conditions of repeated administration remains largely unknown. L-AMB has been used for the treatment of systemic mycoses in cancer patients with encouraging preliminary results (21, 22, 34), but again, the side effects on renal function have not been investigated in detail. Severe underlying disease, poor clinical conditions, previous treatment with free AMB, and multiple drug regimens might account for the difficulties encountered in evaluating renal function alterations in these
MATERIALS AND METHODS Animals. Male Sprague-Dawley rats (200 to 250 g; Charles River Laboratories, Saint-Aubin-Les-Elbeuf, France) were housed in individual metabolic cages, which enabled the collection of urine without contamination by feces. Three days before treatment, a bijugular catheter was inserted under ketamine anesthesia (100 mg/kg intraperitoneally), as
Corresponding author. t Present address: Service de Medecine Interne, H6pital Bichat, 46 rue Henri Huchard, 75018 Paris, France. *
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LONGUET ET AL.
described previously (29), and left in place throughout the experiment, allowing blood sampling and infusion by a different catheter on conscious animals. The animals had free access to food and water. Treatments. (i) AMB. AMB (Squibb, Paris, France) was stored at 4°C and prepared fresh daily. AMB, 10 mM, was dissolved in dimethyl sulfoxide (Me2SO) and then diluted in phosphate-buffered saline (PBS; pH 7.4) such that the total dose would be contained in 1.5 ml. The total amount of Me2SO administered to rats never exceeded 0.1 ml/day. (ii) Liposomes. Dipalmitoylphosphatidyl choline (DPPC; Sigma Chemical Co., St. Louis, Mo.) was used without further purification, and vesicles were prepared as described previously (17). Briefly, the lipids dissolved in chloroform were deposited onto the sides of a round-bottomed flask by evaporation under nitrogen. Then, 10 mM PBS (pH 7.4) was added to the flask. The sample was sonicated at 50°C for 30 min. Because small unilamellar vesicles (SUV) aggregate and fuse into larger liposomes below the phase transition temperature (41), liposomes were always used less than 1 h after the end of sonication. AMB, 10 mM, was dissolved in Me2SO and then added to suspensions of SUV. The molar ratio of AMB to total lipid was kept constant at 0.1. Incorporation of AMB into the lipid bilayer, measured by circular dichroism as described by Jullien et al. (17), was >90%. DPPC SUV-AMB preparations were diluted in enough sterile phosphate buffer (pH 7.4) to make the total dose for each animal 1.5 ml. The sterility of liposomal preparations was assessed after direct inoculation onto Mueller-Hinton agar plates incubated for 48 h at 37°C. (iii) Experimental design. Renal toxicity was studied in nine groups of rats (n = 6 to 8). Treatment was administered intravenously (i.v.) daily for 5 consecutive days, using one of the following regimens: AMB, 1 mg/kg; AMB, 1.5 mg/kg; AMB, 3.5 mg/kg; AMB, 4 mg/kg; DPPC SUV-AMB, 1.5 mg/kg; or DPPC SUV-AMB, 3.5 mg/kg. Low doses of AMB (1 and 1.5 mg/kg) were infused over 1 h, but since AMB at 3.5 mg/kg was lethal when given over 1 h, we evaluated the toxicity of high doses of AMB (AMB, 3.5 and 4 mg/kg; DPPC SUV-AMB, 3.5 mg/kg) by using a 2-h infusion. Control groups received saline, pure DPPC liposomes, or Me2SO dissolved in saline. Animals were killed 24 h after the last infusion, except in groups given high AMB doses; animals in these groups were kept alive for an additional 72 h to investigate the possible restoration of renal function. Toxicology. (i) Parameters studied. Body weight and diuresis were measured daily. Urine was collected under mineral oil. Blood was drawn into heparinized tubes and centrifuged at 3,000 rpm for 5 min. Plasma and urine samples were stored at -20°C. Blood and urine samples were collected on day -3, every day starting from day 0 (just before the first infusion) to day 5 (24 h after the fifth infusion), and, in some experiments, on day 7 to measure sodium, potassium, phosphates, and creatinine and urinary osmolarity. Sampling on days -3 and 0 was performed to determine whether animals were at a steady state before treatment. Urinary excretion of N-acetylglucosaminidase (NAG) was measured daily in animals treated with 1.5 mg of AMB or DPPC SUV-AMB per kg, liposomes alone, or saline. AMB levels were measured in urine and plasma on days 1 and 5, as well as in the kidney after sacrifice of the animals that had received 3.5 mg of AMB or DPPC SUV-AMB per kg. (ii) Assays. (a) Sample analysis. Sodium, potassium, phosphate, and creatinine were measured by using standard automated methods, and urine osmolarity was determined
ANTIMICROB. AGENTS CHEMOTHER.
with a freezing-point depression osmometer (Fisk OS osmometer). (b) Enzymuria. NAG was measured by the method of Pugh et al. (33). The lack of AMB interference in NAG activity was verified in vitro. (c) Determination of AMB in serum, urine, and tissue samples. AMB was measured by liquid chromatography following extraction. Briefly, sera (100 RI) and tissues (100 mg) were extracted with 300 [lI of methanol, as described previously (26). AMB was extracted from urine on a C18 cartridge pack (19). The chromatographic system used a reverse-phase octadecyl-silane column (Spherisorb ODS2, 5 ,um; 4.6 by 150 mm). The mobile phase consisted of ammonium acetate buffer (1 mM; pH 6.6), tetrahydrofuran, and methanol, 210:105:15 (vol/vol/vol), flowing at 0.8 ml/min. The UV detector wavelength was set to 410 nm. The method was linear from 0.1 to 2 [Lg/ml for serum and urine (r = 1) and from 10 to 100 ,ug/g for tissues (r = 0.98 and 1, in kidney and liver, respectively). The sensitivity of the method was 0.02 pLg/ml or 5 ,ug/g in biological fluid or tissue, respectively. Statistical analysis. Each parameter of toxicity, except weight, was subjected to a two-way analysis of variance within each therapeutic group. Then, when possible, the value obtained for each day of treatment was compared with the control value (day 0), using the paired Student t test, with P < 0.05 considered significant. AMB levels in plasma, urine, and renal tissue were compared by using the unpaired Student t test. In each therapeutic group, the mean daily weight gain was compared with that measured on the same day in controls, using the unpaired Student t test after a one-way analysis of variance. RESULTS AMB at 4 mg/kg was lethal after the first i.v. administration, even when infused over 2 h. On the other hand, the highest AMB nontoxic dose after 5 days of treatment was 1 mg/kg. Thus, the two AMB doses selected were 1.5 mg/kg infused over 1 h, as the lowest nephrotoxic dose, and 3.5 mg/kg infused over 2 h, as the sublethal one. The values of all parameters studied remained unchanged during the experiment in animals infused with saline, empty SUV, or
Me2SO. AMB renal toxicity. At 1.5 mg of AMB per kg per day, polyuria was observed on day 1 (Fig. 1A) with a decrease in urine osmolarity (Fig. 1B), an increase in phosphaturia (Fig. 2A), and urinary NAG excretion (Fig. 2B). At the end of treatment, significant increases were noted in kaliuresis on day 5 (Fig. 2C) and in natriuresis on day 4 (Fig. 2D). At 3.5 mg of AMB per kg per day, polyuria was seen on day 1 (Fig. 1A) with decreased urine osmolarity (Fig. 1B) and increased phosphaturia (Fig. 2A). At the end of treatment, phosphaturia returned to control values. Neither natriuresis nor kaliuresis was significantly affected in this group (Fig. 2C and D). A rise in creatinine plasma levels was never observed, even in the case of patent tubular renal toxicity. The weight gain by rats treated with 1.5 and 3.5 mg of AMB per kg was significantly lower than that observed in control animals (Fig. 3). Increased water intake followed the polyuria from day 1 (data not shown). Effect of SUV on AMB toxicity. At 1.5 mg of AMB per kg per day, DPPC SUV-AMB was not toxic, except for a slight decrease in urine osmolarity on day 4 (Fig. 1B). Body weight
increased to the same extent in test and control rats. At 3.5 mg of AMB per kg per day, the alterations induced by DPPC SUV-AMB were similar to those observed with the
s°-li
RENAL TUBULAR TOXICITY OF LIPOSOMAL AMPHOTERICIN B
VOL. 35, 1991
A 20
-10
(ml/day)
t
AMB35
.
1
B 1000
0 AMB 1.5 DIURESIS L-AMB I.5| t
t
2
3
4
5
7DAYS
URINARY OSMOLARITY (mOsm/l)
0 -1000
-3000
. . . . . 2 3 1 4 5 7 DAYS FIG. 1. Effect of treatment on diuresis (A) and urinary osmolarity (B) for each therapeutic regimen. Results are expressed as the means of differences ± standard errors of the mean between the value measured on day n and that observed on day 0 (before treatment). CT, saline. AMB doses were in milligrams per kilogram per day for 5 days. Statistical significance between day n and day 0 values: *P < 0.05; tP < 0.01.
same dose of AMB, with a few differences: natriuresis and kaliuresis increased significantly (Fig. 2C and D), and phosphaturia (Fig. 2A) remained unchanged. Furthermore, the ability to concentrate urine was partly restored 72 h after the last infusion in animals that had received 3.5 mg of DPPC SUV-AMB per kg, but not in those given the same dose of AMB (Fig. 1B). Weight gains observed during the experimental period in rats treated with 3.5 mg of DPPC SUVAMB per kg were significantly lower than those noted in control animals (Fig. 3). AMB levels. Trough AMB levels in plasma and absolute AMB excretion in urine were not influenced by the mode of administration (free or liposomal) or by the day of treatment (similar values on days 1 and 5). Mean AMB concentrations in kidney were similar regardless of whether the rats received free or liposomal AMB (Table 1). DISCUSSION In this study, a liposomal formulation from pure DPPC SUV protected against the tubular renal toxicity due to repeated i.v. administration of AMB at conventional, but not at high, doses, although these liposomes were highly protective in our previous studies evaluating AMB acute toxicity in vivo (13, 32) or in vitro (14, 16). AMB causes renal damage both by inducing immediate arterial vasoconstriction, which leads to GFR impairment, and by having a direct effect on tubular cells, leading to wasting of sodium and potassium and hyposthenuria (7).
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Arterial vasoconstriction occurs immediately after AMB administration (6) and is reduced by the simultaneous administration of pentoxifylline (39). The existence of a direct tubular toxicity has been demonstrated experimentally (10) and in patients (3, 4, 27) and may be overt before the development of azotemia (3, 5). Liposomes have been reported to be protective of the acute reduction of GFR induced by a single AMB i.v. bolus administration (13, 38), but the beneficial effect of liposomes on tubular toxicity secondary to repeated i.v. infusions of AMB remains unknown. This point is of interest in order to respect experimental conditions mimicking clinical situations. The experimental model used here allowed us to compare the alteration of tubular functions induced by repeated administrations of free and liposomal AMB. Toxicity was evaluated with parameters that could be readily assessed in critically ill patients. Since resorption of AMB after intraperitoneal administration of DPPC SUV-AMB is unknown, treatment had to be administered i.v. to control precisely the systemic amounts of AMB and DPPC SUV-AMB delivered to animals. The length of treatment could not exceed 5 days for technical reasons. We chose to study AMB and DPPC SUV-AMB at extreme AMB toxic doses (lowest toxic dose, 1.5 mg/kg; and sublethal dose, 3.5 mg/kg) in order not only to define the existence of a protective effect, but also to determine the extent of protection. We selected a liposomal formulation that permitted experiments to be performed under reproducible conditions. The short delay between liposome sonication and administration prevented their fusion into larger vesicles. We chose pure DPPC SUV because of the high efficiency of AMB intercalation (>90%) in spite of the high AMB/phospholipid ratio (0.1) (13). Furthermore, the DPPC transition temperature (41°C) is well above the normal body temperature (38.5°C) of the rat. Therefore, liposomes circulate in a solid phase and are more protective against acute toxicity in mice than fluid liposomes generated from phospholipids having a transition temperature below 38.5°C (35). We have shown previously that these liposomes do not reduce the in vivo activity of a given dose of AMB (32) and are highly protective against AMB-induced acute lethality in mice (32), acute nephrotoxicity in rabbits (13), and AMB toxicity in vitro, both on erythrocytes (16) and on renal tubular cells (14). AMB intercalated in SUV has already been used in clinical studies (34). A progressive alteration of tubular functions was observed following repeated free AMB i.v. administrations. The earliest and most sensitive changes induced by AMB were hyposthenuria and polyuria, observed as early as day 1, regardless of the dose of AMB used (1.5 or 3.5 mg/kg). This polyuria induced a significant increase in water intake from day 1 compared with control rats (data not shown). The slight delay in increased water intake might explain the initial loss of weight, between days 0 and 1. The decrease in ability to concentrate urine resulting from an AMB alteration of Henle's loop functions has often been reported experimentally (10, 18) and in patients (1, 2, 3, 5), in whom it is usually the first manifestation of toxicity. Tubular toxicity of AMB was also assessed on the renal wasting of sodium, potassium, and phosphates. Although the feeding of the rats was not precisely measured, the alterations in electrolyte wasting were not associated with frank modifications in dietary intake and probably resulted from AMB treatment. Natriuresis and kaliuresis were elevated in the low-dose-regimen group, but remained unchanged in the high-dose-regimen group. This argues for the existence of a decrease in GFR in the latter group, which would hinder the
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LONGUET ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
A PHOSPHATURlI A (mM/day)
AMB 1.5 L-AMB l.5 E AMB 3.5
URINARY NAG (Ratio dn,VdO)
*
[I
t
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-0.2
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I
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'
3
.
4
.
5
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~
us+ 2
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I
KALIURESIS (mM/day)
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NATRIURESIS (mM/day)
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1.5
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7 DAYS FIG. 2. Effect of treatment on phosphaturia (A), urinary NAG excretion (B), kaliuresis (C), and natriuresis (D) for each therapeutic regimen. Results are expressed as the means of differences + standard errors of the mean between the value measured on day n and that observed on day 0 (before treatment) for panels A, C, and D; panel B shows the ratio, day n/day 0. Notes on the symbols and statistical significance are the same as in the legend to Fig. 1.
urinary loss of electrolytes by reducing the filtered load. Such a paradoxical effect of AMB on natriuresis has been observed previously by others (30). The lack of increase in creatinine plasma levels does not exclude the existence of a
30
WEIGHT (g)
20 10
0
t
II
1
3
5 DAYS
FIG. 3. Effect of treatment on daily weight gain for each therapeutic group. Results are expressed as the means of differences between the value measured on day n and that observed on day 0. Symbols: 0, saline; A, AMB at 1.5 mg/kg for 5 days; 0, AMB at 3.5 mg/kg for 5 days; A, DPPC SUV-AMB at 1.5 mg/kg for 5 days; *, DPPC SUV-AMB 3.5 mg/kg for 5 days. Statistical difference between weight gain by control rats and by experimental animals on the same day: *P < 0.05; tP < 0.01.
5
reduction in the GFR by AMB, because a loss of muscle mass due to systemic AMB toxicity might cause an underestimation in the alteration of the GFR measured through creatinine plasma levels (25). More sensitive markers of GFR measurement, such as inulin clearance, might have been useful, but their value in the case of tubular involvement, especially with permeability alterations, remains controversial (7). The observed modifications of phosphaturia and NAG urinary excretion suggested that the proximal tubule was one of the targets for AMB tubular toxicity, as noted previously by other investigators (7, 40). Interestingly, AMB had harmful effects on both renal cell membranerelated functions (electrolyte wasting) and lysosomes (enzymuria). DPPC SUV prevented the alterations of tubular functions induced by AMB at the lowest dose, but, unexpectedly, exerted a poor protective effect against the tubular toxicity secondary to the repeated administration of 3.5 mg of AMB per kg. It should be noted that in our previous study evaluating DPPC SUV-AMB acute toxicity in rabbits (13), although GFR and urinary electrolyte loss were not affected by DPPC SUV-AMB in a high dose, a slight tubular toxicity, expressed by increased enzymuria, was observed. In the present study, all tubular parameters studied were similarly changed in AMB or DPPC SUV-AMB treatment groups on day 5. The only features of tubular protection in the latter
RENAL TUBULAR TOXICITY OF LIPOSOMAL AMPHOTERICIN B
VOL. 35, 1991
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TABLE 1. AMB concentrations in serum,' urine, and tissue samples from animals treated with AMB or DPPC SUV-AMB at a daily dose of 3.5 mg/kgb Serum (,ug/ml)
Treatment
AMB DPPC SUV-AMB
Urine (,ug/ml)
Kidney (,ug/g), day 7
Day 1
Day 5
Day 1
Day S
0.17 ± 0.03 0.14 ± 0.01
0.15 ± 0.03 0.17 ± 0.01
10.4 ± 2.2 8.8 ± 2.2
9.5 ± 6.4 8.7 ± 1.7
10.8 ± 0.7 9.8 ± 0.16
a AMB levels in serum were measured 24 h after the first (day 1) and fifth (day 5) infusions, i.e., at the time of trough values. b Results are expressed as mean ± standard error of the mean.
group were the absence of a significant increase in phosphaturia and the restoration of the ability to concentrate urine 72 h after the end of treatment. However, urinary electrolyte loss was significantly increased in this group, contrary to what was observed with the same dose of free AMB, arguing for protection of the drug-induced GFR reduction by the liposomal formulation. AMB levels in renal tissue measured after AMB or DPPC SUV-AMB treatment were similar, but they were determined 72 h after the end of treatment; therefore, a difference in pharmacokinetics occurring earlier cannot be excluded. In different studies measuring tissue levels of AMB in animals receiving one administration of DPPC SUV-AMB, concentrations in kidney were found to be equal to (38) or higher than (9, 24) those measured in animals treated with the same dose of free AMB. This finding argues against an early local reduction of the drug levels by liposomes as the mechanism of protection. Therefore, the protective effect of liposomes is probably not related to an alteration of the pharmacokinetics of the drug in the target organ. Liposomes might protect at the cellular level by limiting the interaction between AMB and the cell membrane, as reported previously in vitro with erythrocytes (15, 16) and tubular cells (14, 20). In conclusion, in this experimental study, which used repeated administrations of AMB mimicking clinical treatment regimens, DPPC SUV exerted a protective effect against AMB renal tubular toxicity only with low doses of AMB. These results are in agreement with the occurrence of hypokalemia reported in patients treated with L-AMB (21). Although various parameters, e.g., liposome composition or size, percentage of free AMB present in the formulation, or animal species, could interfere with the extent of protection during repeated administration, these data suggest that renal tubular functions should be closely monitored in patients treated with liposomal formulations of AMB at high doses and that electrolyte supplementation might be appropriate, as in the case of free AMB therapy (5, 11). Furthermore, tubuloglomerular feedback linked to loss of electrolytes could decrease the GFR (11) and lead to renal failure under conditions of prolonged treatment. ACKNOWLEDGMENTS We thank Janet Jacobson and L. Penicaud for helpful comments. This work was supported in part by a grant from the Fondation pour la Recherche Medicale. REFERENCES 1. Barbour, G. L., K. D. Straub, B. O'Neal, and J. W. Leatherman. 1979. Vasopressin resistant nephrogenic diabetes insipidus: a result of amphotericin B therapy. Arch. Intern. Med. 139:86-88. 2. Barton, C. H., M. Pahl, N. D. Vaziri, and T. Cesario. 1987. Renal magnesium wasting associated with amphotericin B therapy. Am. J. Med. 77:471-474. 3. Beard, H. W., J. H. Richert, and R. R. Taylor. 1960. Treatment of deep mycotic infections with amphotericin B with particular
emphasis on drug toxicity. Am. J. Respir. Dis. 81:43-51. 4. Bell, N. H., V. T. Andriole, S. M. Sabesin, and J. P. Utz. 1962. On nephrotoxicity of amphotericin B in man. Am. J. Med. 33:64-69. 5. Burgess, J. L., and R. Birchall. 1972. Nephrotoxicity of amphotericin B, with emphasis on changes in tubular function. Am. J. Med. 53:77-84. 6. Butler, W. T., G. J. Hill, C. F. Swed, and V. Knight. 1964. Amphotericin B renal toxicity in dog. J. Pharmacol. Exp. Ther. 143:47-56. 7. Cheng, J. T., R. T. Witty, R. R. Roscoe, and W. E. Yarger. 1982. Amphotericin B nephrotoxicity: increased renal resistance and tubule permeability. Kidney Int. 22:626-633. 8. De Gregorio, M. W., W. M. F. Lee, C. A. Linker, and R. A. Jacobs. 1982. Fungal infections in patients with acute leukemia. Am. J. Med. 73:543-548. 9. Gondal, J. A., R. P. Swartz, and A. Rahman. 1989. Therapeutic evaluation of free and liposomal-encapsulated amphotericin B in the treatment of systemic candidiasis in mice. Antimicrob. Agents Chemother. 33:1544-1548. 10. Gouge, T. H., and V. T. Andriole. 1971. An experimental model of amphotericin B nephrotoxicity with renal tubular acidosis. J. Lab. Clin. Med. 78:713-724. 11. Heidemann, H. T., J. F. Gerkens, W. A. Spickard, E. K. Jackson, and R. A. Branch. 1983. Amphotericin B nephrotoxicity in humans decreased by salt repletion. Am. J. Med. 75:476481. 12. Holleran, W. M., J. R. Wilbur, and M. W. De Gregorio. 1985. Empiric amphotericin B therapy in patients with acute leukemia. Rev. Infect. Dis. 7:619-624. 13. Joly, V., F. Dromer, J. Barge, P. Yeni, N. Seta, G. Molas, and C. Carbon. 1989. Incorporation of amphotericin B (AMB) into liposomes alters AMB-induced acute nephrotoxicity in rabbits. J. Pharmacol. Exp. Ther. 251:311-316. 14. Joly, V., L. Saint-Julien, C. Carbon, and P. Yeni. 1990. Interactions of free and liposomal amphotericin B with renal proximal tubular cells in primary culture. J. Pharmacol. Exp. Ther. 252:17-22. 15. Juliano, R. L., C. W. Grant, K. R. Barber, and M. A. Kalp. 1987. Mechanism of the selective toxicity of amphotericin B incorporated into liposomes. Mol. Pharmacol. 31:1-11. 16. Jullien, S., A. Contrepois, J. E. Sligh, Y. Domart, P. Yeni, J. Brajtburg, J. Medoff, and J. Bolard. 1989. Study of the effects of liposomal amphotericin B on Candida albicans, Cryptococcus neoformans, and erythrocytes by using small unilamellar vesicles prepared from saturated phospholipids. Antimicrob. Agents Chemother. 33:345-349. 17. Jullien, S., A. Vertut-Croquin, J. Brajtburg, and J. Bolard. 1988. Circular dichroism for the determination of amphotericin B binding to liposomes. Anal. Biochem. 172:197-202. 18. Kirsh, R., R. Goldstein, J. Tarloff, D. Parris, J. Hook, N. Hanna, P. Bugelski, and G. Poste. 1988. An emulsion formulation of amphotericin B improves the therapeutic index when treating systemic murine candidiasis. J. Infect. Dis. 158:1065-1070. 19. Kobayashi, K., T. Sakoguchi, K. Fujiwara, K. Taniuchi, K. Kohri, and A. Matsuoka. 1987. High-performance liquid chromatographic determination of amphotericin B in human urine. J. Chromatogr. 417:439-446. 20. Krause, H. J., and R. L. Juliano. 1988. Interactions of liposomeincorporated amphotericin B with kidney epithelial cell cul-
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tures. Mol. Pharmacol. 34:286-297. 21. Lopez-Berestein, G., G. P. Bodey, L. S. Frankel, and K. Mehta. 1987. Treatment of hepatosplenic candidiasis with liposomal amphotericin B. J. Clin. Oncol. 5:310-317. 22. Lopez-Berestein, G., V. Fainstein, R. M. Hopfer, K. Mehta, M. P. Sullivan, M. Keating, M. G. Rosenblum, R. Mehta, M. Luna, E. M. Hersh, J. Reuben, R. L. Juliano, and G. P. Bodey. 1985. Liposomal amphotericin B for the treatment of systemic fungal infections in patients with cancer: a preliminary study. J. Infect. Dis. 151:704-710. 23. Lopez-Berestein, G., R. Mehta, R. L. Hopfer, K. Mills, L. Kasi, K. Mehta, V. Fainstein, M. Luna, E. M. Hersh, and J. Juliano. 1983. Treatment and prophylaxis of disseminated infection due to Candida albicans in mice with liposome-associated amphotericin B. J. Infect. Dis. 147:939-945. 24. Lopez-Berestein, G., M. Rosenblum, and R. Mehta. 1984. Altered tissue distribution of amphotericin B by liposomal encapsulation: comparison of normal mice to mice infected with Candida albicans. Cancer Drug Deliver. 1:199-205. 25. Massa, T., D. P. Sinha, J. D. Frantz, M. E. Filipek, R. C. Weglein, S. A. Steinberg, J. T. McGrath, B. F. Murphy, R. J. Szot, H. E. Black, and E. Schwartz. 1985. Subchronic toxicity studies of N-D-ornithyl amphotericin B methylester in dogs and rats. Fundam. Appl. Toxicol. 5:737-753. 26. Mayhew, J. W., C. Fiore, T. Murray, and M. Barza. 1983. An internally standardized assay for amphotericin B in tissues and plasma. J. Chromatogr. 274:271-279. 27. McCurdy, D. K., F. Myron, and J. R. Elkington. 1968. Renal tubular acidosis due to amphotericin B. N. Engl. J. Med. 278:124-131. 28. Medoff, G., J. Brajtburg, G. S. Kobayashi, and J. Bolard. 1983. Antifungal agents useful in the therapy of systemic fungal infections. Annu. Rev. Pharmacol. Toxicol. 23:303-304. 29. Nicolaidis, S., N. Rowland, M. J. Meile, P. Marfaing-Jallat, and A. Pesez. 1974. A flexible technique for long-term infusions in unrestrained rats. Pharmacol. Biochem. Behav. 2:131-136. 30. Ohnishi, A., T. Ohnishi, W. Stevenhead, R. D. Robinson, A. Glick, D. M. O'Day, R. Sabra, E. K. Jackson, and R. A. Branch. 1989. Sodium status influences chronic amphotericin B nephrotoxicity in rats. Antimicrob. Agents Chemother. 33:1222-1227. 31. Patterson, R. M., and G. L. Ackerman. 1971. Renal tubular acidosis due to amphotericin B nephrotoxicity. Arch. Intern. Med. 127:241-244.
ANTIMICROB. AGENTS CHEMOTHER. 32. Pisarik, L., V. Joly, S. Jullien, C. Carbon, and P. Yeni. 1990. Reduction of amphotericin B acute toxicity after intravenous administration of empty liposomes in mice. J. Infect. Dis. 161:1042-1044. 33. Pugh, D., E. A. Leaback, and P. G. Walker. 1957. Studies on glucosaminidase: N-acetyl-beta-D-glucosaminidase in rat kidney. Biochem. J. 65:464-468. 34. Sculier, J. P., A. Coune, F. Meunier, C. Brassine, C. Laduron, C. Hollaert, N. Collette, C. Heymans, and J. Klastersky. 1988. Pilot study of amphotericin B entrapped in sonicated liposomes in cancer patients with fungal infections. Eur. J. Clin. Oncol. 24:527-538. 35. Szoka, M. C., D. Milholland, and M. Barza. 1987. Effect of lipid composition and liposome size on toxicity and in vitro fungicidal activity of liposome-intercalated amphotericin B. Antimicrob. Agents Chemother. 31:421-429. 36. Taylor, R. L., D. M. Williams, P. C. Craven, J. R. Graybill, D. J. Drutz, and W. E. Magee. 1982. Amphotericin B in liposomes: a novel therapy for histoplasmosis. Am. Rev. Respir. Dis. 125:610-611. 37. Tremblay, C., M. Barza, C. Flore, and F. Szoka. 1984. Efficacy of liposome-intercalated amphotericin B in the treatment of systemic candidiasis in mice. Antimicrob. Agents Chemother. 26:170-173. 38. Wasan, K. M., K. Vadiel, G. Lopez-Berestein, and D. R. Luke. 1990. Pharmacokinetics, tissue distribution, and toxicity of free and liposomal amphotericin B in diabetic rats. J. Infect. Dis. 161:562-566. 39. Wasan, K. M., K. Vadiel, G. Lopez-Berestein, R. R. Verani, and D. Luke. 1990. Pentoxifylline in amphotericin B toxicity rat model. Antimicrob. Agents Chemother. 34:241-244. 40. Wertlake, P. T., W. T. Butler, G. J. Hill, and J. P. Utz. 1963. Nephrotoxic tubular damage and calcium deposition following amphotericin B therapy. Am. J. Pathol. 43:449-457. 41. Wong, M., A. H. Forrest, T. W. Tillack, and T. E. Thompson. 1982. Fusion of dipalmitoylphosphatidyl choline vesicles at 4°C. Biochemistry 21:4126-4132. 42. Young, L. S. 1981. Nosocomial infections in the immunocompromised adult. Am. J. Med. 70:398-404. 43. Zuger, A., E. Louie, R. S. Holzman, M. S. Simberkoff, and J. J. Rahal. 1986. Cryptococcal disease in patients with the acquired immunodeficiency syndrome. Diagnostic features and outcome of treatment. Ann. Intern. Med. 104:234-240.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 1991, p. 1303-1308
0066-4804/91/071303-06$02.00/0 Copyright © 1991, American Society for Microbiology
Limited Protection by Small Unilamellar Liposomes against the Renal Tubular Toxicity Induced by Repeated Amphotericin B Infusions in Rats PASCALE LONGUET,1 VEIRONIQUE JOLY,1 PASCAL AMIRAULT,2 NATHALIE SETA,1 CLAUDE CARBON,' AND PATRICK YENI1t* Laboratoire d'Etude des Infections Experimentales, Institut National de la Sante et de la Recherche Medicale U13, Faculte Xavier Bichat, Universite Paris 7,1 and Laboratoire de Toxicologie, Service de Pharmacie Clinique, H6pital Bichat, Paris, France Received 27 September 1990/Accepted 15 April 1991
Amphotericin B (AMB), either alone or incorporated into small unilamellar vesicles of pure dipalmitoylphosphatidyl choline (DPPC SUV-AMB), was administered intravenously to male Sprague-Dawley rats once daily for 5 days. Either 1.5 or 3.5 mg of AMB or DPPC SUV-AMB per kg was given, since these concentrations corresponded, respectively, to the lowest nephrotoxic dose and the sublethal dose of AMB in our model. Tubular functions were evaluated daily, and AMB concentrations in plasma, urine, and tissues were measured by high-performance liquid chromatography. AMB at both doses induced tubular toxicity, hyposthenuria being the earliest symptom. DPPC SUV-AMB at 1.5 mg/kg/day was atoxic, but the tubular alterations induced by 3.5 mg of DPPC SUV-AMB per kg were similar to those observed with 3.5 mg of AMB per kg, except that the ability to concentrate urine was partly restored 72 h after the last infusion. Incorporating AMB into DPPC SUV did not influence the pharmacokinetics of the drug. Using this lipidic AMB formulation, we thus observed a beneficial effect toward limiting the renal tubular toxicity of repeated low doses of AMB but, unexpectedly, not that of high doses. These results indicate that tubular renal functions and electrolyte serum values should be closely monitored in patients treated with AMB liposomal formulations, especially high-dose regimens.
patients. In a recent study (21), L-AMB appeared to be well tolerated, but hypokalemia requiring supplementation was observed in patients receiving 3 mg or more of L-AMB per kg daily, suggesting that the tubular renal toxicity of AMB is not completely eliminated by the incorporation of the drug into liposomes. The purpose of this study was to determine the effects of liposomes on the renal dysfunction induced by repeated administration of AMB. We focused on the tubular toxicity observed under conditions of administration close to treatment schedules used in humans and consisting of repeated slow infusions of AMB. The liposomes used have proven highly effective in reducing the acute 50% lethal dose of AMB in mice without altering the antifungal effect (32), decreasing the acute renal AMB toxicity in rabbits (13), and protecting in vitro erythrocytes (16) and primary cultures of renal proximal tubular cells from AMB toxicity (14). To reach our goal, we first assessed the lowest nephrotoxic dose and the sublethal dose of AMB; then, we studied the extent of the protection afforded by our liposomal formulation against the toxicity of AMB given in both doses. (This work was presented in part at the 29th Interscience Conference on Antimicrobial Agents and Chemotherapy, Houston, Tex., 1989.)
Systemic mycoses are a major cause of morbidity and mortality among immunocompromised patients (8, 42, 43). Although several new antifungal agents, particularly triazoles, have recently become available, amphotericin B (AMB) remains the most active drug for the treatment of opportunistic fungal infections (12, 28). Its clinical use is limited by side effects, the most serious being nephrotoxicity. Harmful renal effects include inability to concentrate urine (1, 3, 4, 10), defective urinary acidification due to distal tubule dysfunction (10, 27, 31), renal potassium wasting (5), and depressed glomerular filtration rate (GFR) and renal plasma flow (6, 7). AMB toxicity was shown to be reduced when the drug was incorporated into liposomes or complexed with lipids (LAMB), allowing higher doses to be infused and thereby increasing the efficiency of treatment in experimental fungal infections (23, 36, 37). However, the in vivo protective effect of liposomes (13, 38) or lipidic emulsions (18) against the experimental renal toxicity of AMB has been evaluated only after a single administration of the drug. Therefore, the experimental nephrotoxicity of L-AMB under conditions of repeated administration remains largely unknown. L-AMB has been used for the treatment of systemic mycoses in cancer patients with encouraging preliminary results (21, 22, 34), but again, the side effects on renal function have not been investigated in detail. Severe underlying disease, poor clinical conditions, previous treatment with free AMB, and multiple drug regimens might account for the difficulties encountered in evaluating renal function alterations in these
MATERIALS AND METHODS Animals. Male Sprague-Dawley rats (200 to 250 g; Charles River Laboratories, Saint-Aubin-Les-Elbeuf, France) were housed in individual metabolic cages, which enabled the collection of urine without contamination by feces. Three days before treatment, a bijugular catheter was inserted under ketamine anesthesia (100 mg/kg intraperitoneally), as
Corresponding author. t Present address: Service de Medecine Interne, H6pital Bichat, 46 rue Henri Huchard, 75018 Paris, France. *
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LONGUET ET AL.
described previously (29), and left in place throughout the experiment, allowing blood sampling and infusion by a different catheter on conscious animals. The animals had free access to food and water. Treatments. (i) AMB. AMB (Squibb, Paris, France) was stored at 4°C and prepared fresh daily. AMB, 10 mM, was dissolved in dimethyl sulfoxide (Me2SO) and then diluted in phosphate-buffered saline (PBS; pH 7.4) such that the total dose would be contained in 1.5 ml. The total amount of Me2SO administered to rats never exceeded 0.1 ml/day. (ii) Liposomes. Dipalmitoylphosphatidyl choline (DPPC; Sigma Chemical Co., St. Louis, Mo.) was used without further purification, and vesicles were prepared as described previously (17). Briefly, the lipids dissolved in chloroform were deposited onto the sides of a round-bottomed flask by evaporation under nitrogen. Then, 10 mM PBS (pH 7.4) was added to the flask. The sample was sonicated at 50°C for 30 min. Because small unilamellar vesicles (SUV) aggregate and fuse into larger liposomes below the phase transition temperature (41), liposomes were always used less than 1 h after the end of sonication. AMB, 10 mM, was dissolved in Me2SO and then added to suspensions of SUV. The molar ratio of AMB to total lipid was kept constant at 0.1. Incorporation of AMB into the lipid bilayer, measured by circular dichroism as described by Jullien et al. (17), was >90%. DPPC SUV-AMB preparations were diluted in enough sterile phosphate buffer (pH 7.4) to make the total dose for each animal 1.5 ml. The sterility of liposomal preparations was assessed after direct inoculation onto Mueller-Hinton agar plates incubated for 48 h at 37°C. (iii) Experimental design. Renal toxicity was studied in nine groups of rats (n = 6 to 8). Treatment was administered intravenously (i.v.) daily for 5 consecutive days, using one of the following regimens: AMB, 1 mg/kg; AMB, 1.5 mg/kg; AMB, 3.5 mg/kg; AMB, 4 mg/kg; DPPC SUV-AMB, 1.5 mg/kg; or DPPC SUV-AMB, 3.5 mg/kg. Low doses of AMB (1 and 1.5 mg/kg) were infused over 1 h, but since AMB at 3.5 mg/kg was lethal when given over 1 h, we evaluated the toxicity of high doses of AMB (AMB, 3.5 and 4 mg/kg; DPPC SUV-AMB, 3.5 mg/kg) by using a 2-h infusion. Control groups received saline, pure DPPC liposomes, or Me2SO dissolved in saline. Animals were killed 24 h after the last infusion, except in groups given high AMB doses; animals in these groups were kept alive for an additional 72 h to investigate the possible restoration of renal function. Toxicology. (i) Parameters studied. Body weight and diuresis were measured daily. Urine was collected under mineral oil. Blood was drawn into heparinized tubes and centrifuged at 3,000 rpm for 5 min. Plasma and urine samples were stored at -20°C. Blood and urine samples were collected on day -3, every day starting from day 0 (just before the first infusion) to day 5 (24 h after the fifth infusion), and, in some experiments, on day 7 to measure sodium, potassium, phosphates, and creatinine and urinary osmolarity. Sampling on days -3 and 0 was performed to determine whether animals were at a steady state before treatment. Urinary excretion of N-acetylglucosaminidase (NAG) was measured daily in animals treated with 1.5 mg of AMB or DPPC SUV-AMB per kg, liposomes alone, or saline. AMB levels were measured in urine and plasma on days 1 and 5, as well as in the kidney after sacrifice of the animals that had received 3.5 mg of AMB or DPPC SUV-AMB per kg. (ii) Assays. (a) Sample analysis. Sodium, potassium, phosphate, and creatinine were measured by using standard automated methods, and urine osmolarity was determined
ANTIMICROB. AGENTS CHEMOTHER.
with a freezing-point depression osmometer (Fisk OS osmometer). (b) Enzymuria. NAG was measured by the method of Pugh et al. (33). The lack of AMB interference in NAG activity was verified in vitro. (c) Determination of AMB in serum, urine, and tissue samples. AMB was measured by liquid chromatography following extraction. Briefly, sera (100 RI) and tissues (100 mg) were extracted with 300 [lI of methanol, as described previously (26). AMB was extracted from urine on a C18 cartridge pack (19). The chromatographic system used a reverse-phase octadecyl-silane column (Spherisorb ODS2, 5 ,um; 4.6 by 150 mm). The mobile phase consisted of ammonium acetate buffer (1 mM; pH 6.6), tetrahydrofuran, and methanol, 210:105:15 (vol/vol/vol), flowing at 0.8 ml/min. The UV detector wavelength was set to 410 nm. The method was linear from 0.1 to 2 [Lg/ml for serum and urine (r = 1) and from 10 to 100 ,ug/g for tissues (r = 0.98 and 1, in kidney and liver, respectively). The sensitivity of the method was 0.02 pLg/ml or 5 ,ug/g in biological fluid or tissue, respectively. Statistical analysis. Each parameter of toxicity, except weight, was subjected to a two-way analysis of variance within each therapeutic group. Then, when possible, the value obtained for each day of treatment was compared with the control value (day 0), using the paired Student t test, with P < 0.05 considered significant. AMB levels in plasma, urine, and renal tissue were compared by using the unpaired Student t test. In each therapeutic group, the mean daily weight gain was compared with that measured on the same day in controls, using the unpaired Student t test after a one-way analysis of variance. RESULTS AMB at 4 mg/kg was lethal after the first i.v. administration, even when infused over 2 h. On the other hand, the highest AMB nontoxic dose after 5 days of treatment was 1 mg/kg. Thus, the two AMB doses selected were 1.5 mg/kg infused over 1 h, as the lowest nephrotoxic dose, and 3.5 mg/kg infused over 2 h, as the sublethal one. The values of all parameters studied remained unchanged during the experiment in animals infused with saline, empty SUV, or
Me2SO. AMB renal toxicity. At 1.5 mg of AMB per kg per day, polyuria was observed on day 1 (Fig. 1A) with a decrease in urine osmolarity (Fig. 1B), an increase in phosphaturia (Fig. 2A), and urinary NAG excretion (Fig. 2B). At the end of treatment, significant increases were noted in kaliuresis on day 5 (Fig. 2C) and in natriuresis on day 4 (Fig. 2D). At 3.5 mg of AMB per kg per day, polyuria was seen on day 1 (Fig. 1A) with decreased urine osmolarity (Fig. 1B) and increased phosphaturia (Fig. 2A). At the end of treatment, phosphaturia returned to control values. Neither natriuresis nor kaliuresis was significantly affected in this group (Fig. 2C and D). A rise in creatinine plasma levels was never observed, even in the case of patent tubular renal toxicity. The weight gain by rats treated with 1.5 and 3.5 mg of AMB per kg was significantly lower than that observed in control animals (Fig. 3). Increased water intake followed the polyuria from day 1 (data not shown). Effect of SUV on AMB toxicity. At 1.5 mg of AMB per kg per day, DPPC SUV-AMB was not toxic, except for a slight decrease in urine osmolarity on day 4 (Fig. 1B). Body weight
increased to the same extent in test and control rats. At 3.5 mg of AMB per kg per day, the alterations induced by DPPC SUV-AMB were similar to those observed with the
s°-li
RENAL TUBULAR TOXICITY OF LIPOSOMAL AMPHOTERICIN B
VOL. 35, 1991
A 20
-10
(ml/day)
t
AMB35
.
1
B 1000
0 AMB 1.5 DIURESIS L-AMB I.5| t
t
2
3
4
5
7DAYS
URINARY OSMOLARITY (mOsm/l)
0 -1000
-3000
. . . . . 2 3 1 4 5 7 DAYS FIG. 1. Effect of treatment on diuresis (A) and urinary osmolarity (B) for each therapeutic regimen. Results are expressed as the means of differences ± standard errors of the mean between the value measured on day n and that observed on day 0 (before treatment). CT, saline. AMB doses were in milligrams per kilogram per day for 5 days. Statistical significance between day n and day 0 values: *P < 0.05; tP < 0.01.
same dose of AMB, with a few differences: natriuresis and kaliuresis increased significantly (Fig. 2C and D), and phosphaturia (Fig. 2A) remained unchanged. Furthermore, the ability to concentrate urine was partly restored 72 h after the last infusion in animals that had received 3.5 mg of DPPC SUV-AMB per kg, but not in those given the same dose of AMB (Fig. 1B). Weight gains observed during the experimental period in rats treated with 3.5 mg of DPPC SUVAMB per kg were significantly lower than those noted in control animals (Fig. 3). AMB levels. Trough AMB levels in plasma and absolute AMB excretion in urine were not influenced by the mode of administration (free or liposomal) or by the day of treatment (similar values on days 1 and 5). Mean AMB concentrations in kidney were similar regardless of whether the rats received free or liposomal AMB (Table 1). DISCUSSION In this study, a liposomal formulation from pure DPPC SUV protected against the tubular renal toxicity due to repeated i.v. administration of AMB at conventional, but not at high, doses, although these liposomes were highly protective in our previous studies evaluating AMB acute toxicity in vivo (13, 32) or in vitro (14, 16). AMB causes renal damage both by inducing immediate arterial vasoconstriction, which leads to GFR impairment, and by having a direct effect on tubular cells, leading to wasting of sodium and potassium and hyposthenuria (7).
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Arterial vasoconstriction occurs immediately after AMB administration (6) and is reduced by the simultaneous administration of pentoxifylline (39). The existence of a direct tubular toxicity has been demonstrated experimentally (10) and in patients (3, 4, 27) and may be overt before the development of azotemia (3, 5). Liposomes have been reported to be protective of the acute reduction of GFR induced by a single AMB i.v. bolus administration (13, 38), but the beneficial effect of liposomes on tubular toxicity secondary to repeated i.v. infusions of AMB remains unknown. This point is of interest in order to respect experimental conditions mimicking clinical situations. The experimental model used here allowed us to compare the alteration of tubular functions induced by repeated administrations of free and liposomal AMB. Toxicity was evaluated with parameters that could be readily assessed in critically ill patients. Since resorption of AMB after intraperitoneal administration of DPPC SUV-AMB is unknown, treatment had to be administered i.v. to control precisely the systemic amounts of AMB and DPPC SUV-AMB delivered to animals. The length of treatment could not exceed 5 days for technical reasons. We chose to study AMB and DPPC SUV-AMB at extreme AMB toxic doses (lowest toxic dose, 1.5 mg/kg; and sublethal dose, 3.5 mg/kg) in order not only to define the existence of a protective effect, but also to determine the extent of protection. We selected a liposomal formulation that permitted experiments to be performed under reproducible conditions. The short delay between liposome sonication and administration prevented their fusion into larger vesicles. We chose pure DPPC SUV because of the high efficiency of AMB intercalation (>90%) in spite of the high AMB/phospholipid ratio (0.1) (13). Furthermore, the DPPC transition temperature (41°C) is well above the normal body temperature (38.5°C) of the rat. Therefore, liposomes circulate in a solid phase and are more protective against acute toxicity in mice than fluid liposomes generated from phospholipids having a transition temperature below 38.5°C (35). We have shown previously that these liposomes do not reduce the in vivo activity of a given dose of AMB (32) and are highly protective against AMB-induced acute lethality in mice (32), acute nephrotoxicity in rabbits (13), and AMB toxicity in vitro, both on erythrocytes (16) and on renal tubular cells (14). AMB intercalated in SUV has already been used in clinical studies (34). A progressive alteration of tubular functions was observed following repeated free AMB i.v. administrations. The earliest and most sensitive changes induced by AMB were hyposthenuria and polyuria, observed as early as day 1, regardless of the dose of AMB used (1.5 or 3.5 mg/kg). This polyuria induced a significant increase in water intake from day 1 compared with control rats (data not shown). The slight delay in increased water intake might explain the initial loss of weight, between days 0 and 1. The decrease in ability to concentrate urine resulting from an AMB alteration of Henle's loop functions has often been reported experimentally (10, 18) and in patients (1, 2, 3, 5), in whom it is usually the first manifestation of toxicity. Tubular toxicity of AMB was also assessed on the renal wasting of sodium, potassium, and phosphates. Although the feeding of the rats was not precisely measured, the alterations in electrolyte wasting were not associated with frank modifications in dietary intake and probably resulted from AMB treatment. Natriuresis and kaliuresis were elevated in the low-dose-regimen group, but remained unchanged in the high-dose-regimen group. This argues for the existence of a decrease in GFR in the latter group, which would hinder the
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LONGUET ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
A PHOSPHATURlI A (mM/day)
AMB 1.5 L-AMB l.5 E AMB 3.5
URINARY NAG (Ratio dn,VdO)
*
[I
t
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.
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.
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I
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7 DAYS FIG. 2. Effect of treatment on phosphaturia (A), urinary NAG excretion (B), kaliuresis (C), and natriuresis (D) for each therapeutic regimen. Results are expressed as the means of differences + standard errors of the mean between the value measured on day n and that observed on day 0 (before treatment) for panels A, C, and D; panel B shows the ratio, day n/day 0. Notes on the symbols and statistical significance are the same as in the legend to Fig. 1.
urinary loss of electrolytes by reducing the filtered load. Such a paradoxical effect of AMB on natriuresis has been observed previously by others (30). The lack of increase in creatinine plasma levels does not exclude the existence of a
30
WEIGHT (g)
20 10
0
t
II
1
3
5 DAYS
FIG. 3. Effect of treatment on daily weight gain for each therapeutic group. Results are expressed as the means of differences between the value measured on day n and that observed on day 0. Symbols: 0, saline; A, AMB at 1.5 mg/kg for 5 days; 0, AMB at 3.5 mg/kg for 5 days; A, DPPC SUV-AMB at 1.5 mg/kg for 5 days; *, DPPC SUV-AMB 3.5 mg/kg for 5 days. Statistical difference between weight gain by control rats and by experimental animals on the same day: *P < 0.05; tP < 0.01.
5
reduction in the GFR by AMB, because a loss of muscle mass due to systemic AMB toxicity might cause an underestimation in the alteration of the GFR measured through creatinine plasma levels (25). More sensitive markers of GFR measurement, such as inulin clearance, might have been useful, but their value in the case of tubular involvement, especially with permeability alterations, remains controversial (7). The observed modifications of phosphaturia and NAG urinary excretion suggested that the proximal tubule was one of the targets for AMB tubular toxicity, as noted previously by other investigators (7, 40). Interestingly, AMB had harmful effects on both renal cell membranerelated functions (electrolyte wasting) and lysosomes (enzymuria). DPPC SUV prevented the alterations of tubular functions induced by AMB at the lowest dose, but, unexpectedly, exerted a poor protective effect against the tubular toxicity secondary to the repeated administration of 3.5 mg of AMB per kg. It should be noted that in our previous study evaluating DPPC SUV-AMB acute toxicity in rabbits (13), although GFR and urinary electrolyte loss were not affected by DPPC SUV-AMB in a high dose, a slight tubular toxicity, expressed by increased enzymuria, was observed. In the present study, all tubular parameters studied were similarly changed in AMB or DPPC SUV-AMB treatment groups on day 5. The only features of tubular protection in the latter
RENAL TUBULAR TOXICITY OF LIPOSOMAL AMPHOTERICIN B
VOL. 35, 1991
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TABLE 1. AMB concentrations in serum,' urine, and tissue samples from animals treated with AMB or DPPC SUV-AMB at a daily dose of 3.5 mg/kgb Serum (,ug/ml)
Treatment
AMB DPPC SUV-AMB
Urine (,ug/ml)
Kidney (,ug/g), day 7
Day 1
Day 5
Day 1
Day S
0.17 ± 0.03 0.14 ± 0.01
0.15 ± 0.03 0.17 ± 0.01
10.4 ± 2.2 8.8 ± 2.2
9.5 ± 6.4 8.7 ± 1.7
10.8 ± 0.7 9.8 ± 0.16
a AMB levels in serum were measured 24 h after the first (day 1) and fifth (day 5) infusions, i.e., at the time of trough values. b Results are expressed as mean ± standard error of the mean.
group were the absence of a significant increase in phosphaturia and the restoration of the ability to concentrate urine 72 h after the end of treatment. However, urinary electrolyte loss was significantly increased in this group, contrary to what was observed with the same dose of free AMB, arguing for protection of the drug-induced GFR reduction by the liposomal formulation. AMB levels in renal tissue measured after AMB or DPPC SUV-AMB treatment were similar, but they were determined 72 h after the end of treatment; therefore, a difference in pharmacokinetics occurring earlier cannot be excluded. In different studies measuring tissue levels of AMB in animals receiving one administration of DPPC SUV-AMB, concentrations in kidney were found to be equal to (38) or higher than (9, 24) those measured in animals treated with the same dose of free AMB. This finding argues against an early local reduction of the drug levels by liposomes as the mechanism of protection. Therefore, the protective effect of liposomes is probably not related to an alteration of the pharmacokinetics of the drug in the target organ. Liposomes might protect at the cellular level by limiting the interaction between AMB and the cell membrane, as reported previously in vitro with erythrocytes (15, 16) and tubular cells (14, 20). In conclusion, in this experimental study, which used repeated administrations of AMB mimicking clinical treatment regimens, DPPC SUV exerted a protective effect against AMB renal tubular toxicity only with low doses of AMB. These results are in agreement with the occurrence of hypokalemia reported in patients treated with L-AMB (21). Although various parameters, e.g., liposome composition or size, percentage of free AMB present in the formulation, or animal species, could interfere with the extent of protection during repeated administration, these data suggest that renal tubular functions should be closely monitored in patients treated with liposomal formulations of AMB at high doses and that electrolyte supplementation might be appropriate, as in the case of free AMB therapy (5, 11). Furthermore, tubuloglomerular feedback linked to loss of electrolytes could decrease the GFR (11) and lead to renal failure under conditions of prolonged treatment. ACKNOWLEDGMENTS We thank Janet Jacobson and L. Penicaud for helpful comments. This work was supported in part by a grant from the Fondation pour la Recherche Medicale. REFERENCES 1. Barbour, G. L., K. D. Straub, B. O'Neal, and J. W. Leatherman. 1979. Vasopressin resistant nephrogenic diabetes insipidus: a result of amphotericin B therapy. Arch. Intern. Med. 139:86-88. 2. Barton, C. H., M. Pahl, N. D. Vaziri, and T. Cesario. 1987. Renal magnesium wasting associated with amphotericin B therapy. Am. J. Med. 77:471-474. 3. Beard, H. W., J. H. Richert, and R. R. Taylor. 1960. Treatment of deep mycotic infections with amphotericin B with particular
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