Attenuation of nephrotoxicity by a novel lipid nanosphere (NS-718) incorporating amphotericin B

Mohammad A. Hossaina, Shigefumi Maesakia, Mohammed S. Razzaqueb, Kazunori Tomonoa, Takashi Taguchib and Shigeru Kohnoa,*

a Second Department of Internal Medicine and b Second Department of Pathology, Nagasaki University School of Medicine, Nagasaki 852-8501, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
NS-718, a lipid nanosphere incorporating amphotericin B, is effective against pathogenic fungi and has low toxicity. We compared the toxicity of NS-718 with that of Fungizone (amphotericin B–sodium deoxycholate; D-AmB) in vitro using renal cell cultures and in vivo by biochemical analysis, histopathological study of the kidney and pharmacokinetic study of amphotericin B following intravenous infusion of the formulation in rats. Incubation with NS-718 resulted in significantly less damage of cultured human renal proximal tubular epithelial cells compared with D-AmB. Serum blood urea and creatinine concentrations increased significantly in rats given an iv infusion of D-AmB 3 mg/kg but not in those given the same dose of NS-718. Histopathological examination of the kidney showed tubular necrosis in D-AmB-treated rats but no change in NS-718-treated rats. Amphotericin B concentrations in the kidney in NS-718-treated rats were higher than those in D-AmB-treated rats. Our in vitro and in vivo results suggest that incorporation of amphotericin B into lipid nanospheres of NS-718 attenuates the nephrotoxicity of amphotericin B.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Amphotericin B has been the mainstay in treating severe and refractory systemic fungal infections since it was discovered. Amphotericin B, like other polyene derivatives, is a broad-spectrum antifungal agent which attacks ergosterol in the fungal cell membrane but does not readily break down human cell membrane. Although it is the most effective antifungal agent, various side-effects, especially nephrotoxicity, limit the use of higher doses or long-term use of amphotericin B.1 Amphotericin B-induced renal injury may result in fluid and electrolyte disturbances and acute tubular necrosis, which most often affects the distal tubules and the ascending loop of Henle. Lipid formulations of amphotericin B have been developed in order to reduce the toxic side-effects. Some of these formulations are potent and less toxic even at higher doses and are in the early stages of clinical trials.2,3 Although most of the new lipid formulations are beneficial and relatively safe, higher doses are required to obtain the desired therapeutic effects.

We have previously demonstrated the efficacy of NS-718, a novel lipid nanosphere incorporating amphotericin B, in treating pulmonary aspergillosis in rats and cryptococcosis in mice, whereas equivalent doses of Fungizone (amphotericin B plus sodium deoxycholate; D-AmB) and AmBisome were either lethally toxic or ineffective.4,5 The aims of the present study were to compare the direct cytotoxicity of D-AmB with that of NS-718 in human proximal tubule cells in vitro and to assess the nephrotoxic effect of intravenous infusion of NS-718 in rats.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Amphotericin B formulations

Fungizone was obtained from Bristol-Myers Squibb, Tokyo, Japan. NS-718 and lipid nanospheres without amphotericin B, used as a vehicle for NS-718, were supplied by Nippon Shinyaku Co., Kyoto, Japan. Both were presented in lyophilized form. Each vial of NS-718 contained 10 mg of amphotericin B, 1 g each of soybean oil and egg lecithin and 2 g of maltose. Vials were stored in a cool dark place until use and the contents reconstituted with sterile water for injection before use. The average diameter of NS-718 particles was 25–50 nm. Particles were diluted with 5% dextrose to the desired concentrations.

In vitro toxicity study using cultured cells

Primary cultures of human renal proximal tubular epithelial cells maintained at –80°C were subcultured in REGM cell culture medium (Clontech, Palo Alto, CA, USA) supplemented with various growth factors (hydrocortisone, human epidermal growth factor (hEGF), fetal bovine serum (FBS), epinephrine, triiodothyronine (T3), transferrin, insulin and gentamicin). The culture medium and growth factors were stored at 4°C and –80°C, respectively, until use. The protocol used for cell culture was similar to that described previously,6 with minor modifications. Briefly, cells were inoculated into the enriched culture medium in cell culture bottles at a density of 2500 cells/cm2 and incubated at 37°C in a humidified atmosphere in the presence of 5% CO2. After two passages, the cells were transferred to 12-well culture plates. After growth of cells to c.80% confluence, they were removed with trypsin–EDTA and cell suspension was prepared. Equal volumes of test solutions (D-AmB, NS-718 at final amphotericin B concentrations of 25.0, 12.5 or 6.25 mg/mL or 5% dextrose as a control) were added and incubated for 3, 6 or 12 h at 37°C in the presence of 5% CO2. After washing with trypsin–EDTA, the cells were collected by centrifugation at 1500g for 5 min at 4°C. The cells were resuspended in REGM and counted using a haemo-cytometer after staining with Trypan blue to determine viability.

In vivo nephrotoxicity experiments

The experimental protocol was approved by the ethics review committee for animal experimentation of Nagasaki University School of Medicine, and the guidelines of the Laboratory Animal Center for Biomedical Research were followed. Seven-week-old, male, Sprague–Dawley rats were purchased from Charles River Inc. (Yokohama, Japan). They were housed at a standard temperature and dark– light cycle, and had free access to pelleted food and drinking water. Five rats were included in each of six groups. Under general anaesthesia, a small incision was made to expose the jugular vein. An intravenous catheter (24G) was put in place and glued to the skin to avoid any displacement. NS-718 or D-AmB at a dose of 3 mg/kg for 1.5 h (2 mg/kg/h or 30 mL/kg) or 5% dextrose as control was infused using an infusion pump (Terufusion syringe pump; Model STC-525, Terumo, Tokyo) in different groups. Rats were killed under ether anaesthesia 24 h after infusion started. Whole blood was collected from the inferior vena cava for biochemical analysis and the kidneys were removed for histopathological examination.

Pharmacokinetic study

NS-718 or D-AmB was infused at a dose of 3 mg/kg for 1.5 h (2 mg/kg/h or 30 mL/kg) into the left jugular vein of anaesthetized rats at a constant rate using an infusion pump. Venous blood samples were collected from the right jugular vein into heparinized tubes 0.5, 1, 1.58, 2, 3, 4, 6, 8, 12 or 24 h after infusion began. The kidneys were collected after 2, 4, 8 or 24 h from rats killed under ether anaesthesia.

Determination of amphotericin B by HPLC was based on the method of Granich et al.7 with some modifications. Plasma samples (0.1 mL) were combined with 1.0 mL of methanol containing 1.0 mg/L of internal standard, 1-amino-4-nitronaphthalene (Aldrich, Milwaukee, WI, USA), and mixed by vortexing. After centrifugation at 1500g for 10 min, the supernatant was dried under reduced pressure and redissolved in 0.2 mL of methanol for injection into an HPLC system consisting of an SLC-10A system controller, LC-10AD pump, SIL-10A autosampler with a 20 µL sampler loop, SCL-10A UV–visible detector at 408 nm, CTO-10AC column oven at 40°C and C-R7A-plus Chromatopac data station (Shimadzu, Kyoto, Japan). Analysis was performed with an L-column octadecyl silica (ODS) (4.6 mm x 150 mm, Chemicals Inspection and Testing Institute, Kyoto, Japan) equipped with an LiChroCART guard cartridge (Merck, Darmstadt, Germany). The mobile phase was an 11:17 (v/v) mixture of acetonitrile and 10 mM sodium acetate buffer (pH 4.0) and the flow rate was 1.0 mL/min. The concentration of amphotericin B was expressed as the ratio of the peak height of amphotericin B to that of the internal standard.

Data processing and statistical analysis

Renal cell damage was assessed by examining the percentage of dead cells in three different fields in triplicate. Cell toxicity was evaluated using Scheffe's multiple-comparison test. Biochemical data were expressed as mean ± standard deviation and differences were tested by Scheffe's multiple comparison test. A P value of <0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In vitro toxicity

In vitro toxicity of D-AmB and NS-718 was evaluated by damage of cultured renal proximal tubular cells (Table IGo). The percentage of dead cells in control cell cultures incubated with the vehicle only (5% dextrose) was 19.2% after 12 h incubation. The ratio of dead cells increased at higher concentrations of D-AmB. The use of D-AmB 25 mg/L was associated with cell death amounting to 86.4% after 12 h incubation, representing the direct cell toxicity of D-AmB. However, the percentage of damaged cells incubated with NS-718 was lower at all concentrations (Table IGo). In particular, a marked difference in cell toxicity between D-AmB and NS-718 was noted at high concentrations of amphotericin B. Lipid nanospheres, the lipid vehicle of NS-718 without amphotericin B, had no direct toxic effect on renal cells at corresponding concentrations.


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Table I. Percentage of damaged renal proximal tubular cells incubated with NS-718 or D-AmB
 
In vivo toxicity

The biochemical data measured in rats infused with 3 mg/kg of test and control solutions are summarized in Table IIGo. Blood urea and creatinine concentrations were significantly higher in rats treated with D-AmB than in control and NS-718-treated rats. Furthermore, serum glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) in D-AmB- and NS-718-treated rats were higher than those in the control rats, albeit insignificantly.


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Table II. Serum biochemical data of rats infused with 5% dextrose, D-AmB and NS-718
 
No histopathological changes were seen in kidneys of rats infused with 5% dextrose as a control and NS-718 3 mg/kg (Figure 1Go, panels a and c). In contrast, severe tissue damage, sloughing of renal tubular epithelial cells and inflammatory cell infiltration were noted in kidney sections of rats treated with D-AmB 3 mg/kg (Figure 1bGo).





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Figure 1. Effect of D-AmB and NS-718 on the kidney. Histopathological examination of the kidney of a representative rat treated with (a) 5% dextrose as a control, (b) D-AmB 3 mg/kg and (c) NS-718 3 mg/kg. The kidneys were stained with haematoxylin–eosin.

 
Pharmacokinetic study

The concentrations of amphotericin B in the plasma and kidneys are shown in Figure 2Go. The plasma concentration of amphotericin B in NS-718-treated rats was significantly higher than that in D-AmB-treated rats. The maximum concentration of amphotericin B was 37.8 and 2.4 mg/L at the end of infusion period (1.5 h) at a dose of 3 mg/kg of NS-718 and D-AmB, respectively. The plasma concentration of amphotericin B decreased and was similar in both D-AmB- and NS-718-treated rats at 12 h. The concentration of amphotericin B in the kidney was higher in NS-718-treated rats than in D-AmB-treated rats. In NS-718-treated rats, the maximum concentration of amphotericin B in the kidney was 8.2 µg/g.



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Figure 2. Concentrations of amphotericin B in plasma and kidney in rats infused with D-AmB or NS-718. Concentrations of amphotericin B in plasma (a) and the kidney (b) of rats treated with D-AmB (3 mg/kg) ({circ}) or NS-718 (3 mg/kg) (•). Values are the mean concentration ± s.d. for three rats.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Compared with the number of newly emerging and resistant pathogenic fungi causing deep-seated mycosis, the number of available antifungal agents is substantially smaller. Most of these agents have very similar mechanisms of action and a limited response rate. Amphotericin B is used for the treatment of severe fungal infections, but its usefulness is compromised by the high incidence of renal dysfunction it causes. Incorporation of amphotericin B in lipid formulations is used to improve the tolerability of amphotericin B. The mechanisms of attenuation of nephrotoxicity following the lipid formulation of amphotericin B have been studied by examining its direct effect on renal cells. Dorea et al.8 reported recently that the addition of D-AmB showed a high release of lactate dehydrogenase from proximal tubular cell suspensions, but lipid emulsion of amphotericin B markedly inhibited lactate dehydrogenase release.8 In the present study, histopathological examination showed acute tubular necrosis of the kidney in rats infused with D-AmB 3 mg/kg. However, no histopathological changes were noted in kidneys of NS-718-treated rats, suggesting that NS-718 reduces the damage to proximal tubular cells caused by amphotericin B. Krause & Juliano9 investigated the effects of amphotericin B and liposomal amphotericin B using a porcine kidney cell line which shows many characteristics typical of proximal tubule cells. Acute exposure of these cells to free amphotericin B inhibited protein synthesis while l-amphotericin B had little effect on protein synthesis. The protective effect of liposomes against nephrotoxicity seems to result from their effects on sugar transport in culture kidney cells. In our study, we demonstrated that NS-718 was less toxic to proximal tubular cells than amphotericin B. Further study is necessary to elucidate the mechanisms of reduced renotoxicity of NS-718.

Krause & Juliano9 also indicated that long-term exposure of cells to liposomal amphotericin B had profound toxic effects as manifested by changes in cellular transport functions and cell morphology. We have also demonstrated previously that injection of NS-718 for 7 days in immunosuppressed rats resulted in renal insufficiency.5 These findings stress the importance of chronic nephrotoxicity in long-term use of NS-718.

Espuelas et al.10 investigated the acute lethal toxic effects of D-AmB, liposomal amphotericin B (AmBisome), and amphotericin B-ploy ({epsilon}-caprolacton) nanospheres. Their results showed that the nanoparticle formulation was less toxic than D-AmB but more toxic than AmBisome. These results suggested that, in amphotericin B nanospheres, amphotericin B is not truly included within the nanoparticles but simply adsorbed on to their surface. The difference in toxicity between AmBisome and amphotericin B nanospheres may be explained by the stability upon dilution. However, AmBisome's higher stability is also responsible for reduced efficacy against fungal infection. In our previous study, NS-718 was more effective than AmBisome in mouse pulmonary cryptococcosis and rat pulmonary aspergillosis.4,5 Thus, NS-718 seems to be more potent against fungal infection when used at the same dosage as AmBisome.

Pharmacokinetic experiments in the present study showed that the plasma concentration of amphotericin B after administration of NS-718 was much higher than that of D-AmB. The characteristics of NS-718 were based on the high plasma concentrations of amphotericin B after intravenous administration. The mechanism leading to the high concentration of amphotericin B was not investigated in the present study. However, we suggest that NS-718 escapes recognition as a foreign body, resulting in a low uptake by the reticulo-endothelial system. This argument is based on the fact that NS-718 is a very small lipid particle (25–50 nm). Our results also showed that the concentration of amphotericin B in the kidneys of NS-718-treated rats was higher than that in the kidneys of D-AmB-treated rats, but the nephrotoxicity of NS-718 was lower than that of D-AmB. The lower nephrotoxicity of NS-718, despite the high concentration of amphotericin B in the kidney, clearly corresponded to the in vitro cytotoxicity on renal proximal tubular cells noted in this study. Sodium deoxycholate in D-AmB is another ingredient that may be toxic to renal proximal tubular cells.8 In this study, lipid nanospheres, the lipid vehicle of NS-718 without amphotericin B, were not toxic towards renal cells and lipid nanospheres do not contain sodium deoxycholate. Thus, the composition of NS-718 and incorporation of amphotericin B into the lipid nanospheres probably explains the low nephrotoxicity of NS-718.

In conclusion, NS-718 could be the drug of choice in the treatment of deep-seated fungal infections because of its low nephrotoxicity and its potent antifungal effect.


    Acknowledgments
 
We are grateful to Y. Tomii, J. Seki and S. Sonoke (Nippon Shinyaku Co. Ltd, Kyoto, Japan) for the supply of NS-718, help with the pharmacokinetic study and useful suggestions throughout the study.


    Notes
 
* Corresponding author. Tel: +81-95-849-7271; Fax: +81-95-849-7285; E-mail: sk1227{at}net.nagasaki-u.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Miano-Mason, T. M. (1997). Mechanisms and management of amphotericin B-induced nephrotoxicity. Cancer Practice 5, 176–81.[ISI][Medline]

2 . Anaissie, E. J., Mattiuzzi, G. N., Miller, C. B., Noskin, G. A., Gurwith, M. J., Mamelok, R. D. et al. (1998). Treatment of invasive fungal infections in renal impaired patients with amphotericin B colloidal dispersion. Antimicrobial Agents and Chemotherapy 42, 606–11.[Abstract/Free Full Text]

3 . Oppenheim, B. A., Herbrecht, R. & Kusne, S. (1995). The safety and efficacy of amphotericin B colloidal dispersion in the treatment of invasive mycoses. Clinical Infectious Disease 21, 1145–53.[ISI][Medline]

4 . Hossain, M. A., Maesaki, S., Kakeya, H., Noda, T., Yanagihara, K., Sasaki, E. et al. (1998). Efficacy of NS-718, a novel lipid nanosphere-encapsulated amphotericin B, against Cryptococcus neoformans. Antimicrobial Agents and Chemotherapy 42, 1722–5.[Abstract/Free Full Text]

5 . Otsubo, T., Maesaki, S., Hossain, M. A., Yamamoto, Y., Tomono, K., Tashiro, T. et al. (1999). In vitro and in vivo activities of NS-718, a new lipid nanosphere incorporating amphotericin B against Aspergillus fumigatus. Antimicrobial Agents and Chemotherapy 43, 471–5.[Abstract/Free Full Text]

6 . Razzaque, M. S., Kumatori, A., Harada, T. & Taguchi, T. (1998). Coexpression of collagens and collagen-binding heat shock protein 47 in human diabetic nephropathy and IgA nephropathy. Nephron 80, 434–43.

7 . Granich, G. G., Kobayashi, G. S. & Krogstad, D. J. (1986). Sensitive high pressure liquid chromatographic assay for amphotericin B which incorporates an internal standard. Antimicrobial Agents and Chemotherapy 29, 584–8.[ISI][Medline]

8 . Dorea, E. L., Yu, L., DeCastro, I., Campos, S. B., Ori, M., Vaccari, E. M. H. et al. (1997). Nephrotoxicity of amphotericin B is attenuated by solubilizing with lipid emulsion. Journal of the American Society of Nephrology 8, 1415–22.[Abstract]

9 . Krause, H. J. & Juliano, R. J. (1988). Interactions of liposome-incorporated amphotericin B with kidney epithelial cell cultures. Molecular Pharmacology 34, 286–97.[Abstract]

10 . Espuelas, M. S., Legrand, P., Irache, J. M., Gamazo, G., Orecchioni, M. A., Devissaguet, J. P. et al. (1997). Poly({epsilon}-caprolacton) nanospheres as an alternative way to reduce amphotericin B toxicity. International Journal of Pharmacology 158, 19–27.

Received 29 September 1999; returned 20 December 1999; revised 14 February 2000; accepted 21 March 2000





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