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Prolonged blood concentration of prednisolone after intravenous injection of liposomal palmitoyl prednisolone
Journal of Controlled Release 112 (2006) 320 – 328 www.elsevier.com/locate/jconrel
Prolonged blood concentration of prednisolone after intravenous injection of liposomal palmitoyl prednisolone Mugen Teshima a , Shintaro Fumoto b , Koyo Nishida b , Junzo Nakamura b , Kaname Ohyama a , Tadahiro Nakamura a , Nobuhiro Ichikawa a , Mikiro Nakashima a , Hitoshi Sasaki a,⁎ a
Department of Hospital Pharmacy, Nagasaki University Hospital of Medicine and Dentistry, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan b Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan Received 12 October 2005; accepted 9 March 2006 Available online 21 April 2006
Abstract We compared the pharmacokinetic behavior of drugs after an intravenous administration of prednisolone (PLS), palmitoyl prednisolone (PalPLS), and liposomal Pal-PLS in rats. Pal-PLS showed higher lipophilicity and higher binding to plasma protein than PLS, and PLS regeneration in rat blood and liver homogenates. After the intravenous administration of Pal-PLS solution in polyethylene glycol (PEG) 400 to rats, Pal-PLS disappeared from the blood in a two-phase mode and PLS was rapidly regenerated. Pal-PLS showed a significantly higher accumulation than PLS in the liver and lung. The administration of Pal-PLS incorporated into egg yolk phosphatidylcholine (EggPC)/cholesterol (Chol) liposomes enhanced Pal-PLS concentrations in the blood, liver, and lung compared to that of Pal-PLS solution in PEG 400, suggesting the rapid removal of liposomes by the mononuclear phagocytic system. Pal-PLS incorporated into PEGylated liposomes constituted with EggPC/Chol/1% L-αdistearoylphosphatidylethanolamine (DSPE)-PEG 2000 and EggPC/Chol/10% DSPE-PEG 2000 decreased the initial distribution of Pal-PLS, and successfully maintained the blood concentrations of Pal-PLS and PLS. Thus, we could change the pharmacokinetics of PLS by introducing the palmitoyl function into the molecule and its liposomal formulation including PEGylation. This is the first study to evaluate liposomal PLS constituted with a lipophilic derivative and PEG lipids. © 2006 Elsevier B.V. All rights reserved. Keywords: Drug delivery system; Prednisolone; Liposomes; Lipophilic derivative; Polyethylene glycol
1. Introduction Glucocorticoids are highly potent anti-inflammatory and immunosuppressive drugs. Additionally, they are used in substitution therapy for adrenal insufficiency. Most glucocorticoids are administrated repeatedly because of their short halflife and are used in not only continuous treatment but also pulse treatment. However, even at moderate doses, the systemic administration of glucocorticoids causes many side effects, such as diabetes, hypertension, Cushing syndrome, and osteoporosis [1]. Therefore, it is effective for glucocorticoids to be retained in the blood and delivered in target tissues such as inflammatory tissues and the immune system [2–6].
⁎ Corresponding author. Tel.: +81 95 849 7245; fax: +81 95 849 7251. E-mail address: [email protected] (H. Sasaki). 0168-3659/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2006.03.004
Prednisolone (PLS) is one of the glucocorticoids most frequently used clinically. Liposomes have various advantages as a drug carrier such as biodegradability, low in vivo toxicity, and the encapsulation of hydrophilic, lipophilic and amphipathic drugs. Metselaar et al. [2] and Schmidt et al. [3] reported that long-circulating liposomes of prednisolone phosphate markedly increased biological activity and reduced side effects as compared to its solution after intravenous treatment for arthritis and multiple sclerosis. Liao et al. [4] also demonstrated that long-circulating liposomes of prednisolone phosphate showed increased therapeutic efficiency in IgA nephropathy model mice as compared to its solution. Prednisolone phosphate, however, was difficult to sufficiently incorporate into liposomes because of its high aqueous solubility. In the previous study, we newly synthesized palmitoyl prednisolone (Pal-PLS) with high lipophilicity and successfully prepared liposomes completely incorporating Pal-PLS [7].
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Furthermore, the addition of polyethylene glycol (PEG) lipids to liposomes could inhibit Pal-PLS release from the liposomes in the presence of rat plasma. These results indicated the usefulness of an integrated approach using chemical modification and liposomal formulations. The in vivo pharmacokinetics and activity of liposomal drugs, however, do not always reflect the in vitro release results. Therefore, we investigated the pharmacokinetic behavior of Pal-PLS and its liposomes with various lipids such as egg yolk phosphatidylcholine (EggPC), cholesterol (Chol), and with or without L-α-distearoylphosphatidylethanolamine-PEG 2000 (DSPE-PEG 2000) after their intravenous administration to rats. This is the first study to evaluate liposomal PLS constituted with a lipophilic derivative and PEG lipids. 2. Materials and methods 2.1. Materials PLS (Mw: 360.5) was kindly supplied by Shionogi Co. Ltd. (Osaka, Japan). The lipophilic derivative, Pal-PLS (Mw: 598.9), was synthesized by the method of Teshima et al. [7]. EggPC (average molecular weight 773, COATSOME NC-50) and DSPE-PEG 2000 (average molecular weight 2000, SUNBRIGHT DSPE-20H) were purchased from Nippon Oil and Fats Co. (Tokyo, Japan). Chol was obtained from Nacalai Tesque Inc. (Kyoto, Japan). All other chemicals were of reagent grade and used as obtained commercially. Phosphate-buffered saline (PBS) was prepared by mixing an isotonic phosphate buffer (pH 7.4) with an equal volume of saline. 2.2. Distribution experiment in blood PLS and Pal-PLS were incubated in rat blood (10 μM) at 37 °C for 2 min. Part of the blood was immediately centrifuged at 12,000 ×g for 5 min to obtain plasma. Part of the plasma was ultrafiltrated with a micropartition system (Ultrafree®-MC, molecular weight cut off of 5000, Millipore, Tokyo, Japan) to obtain the filtrate (unbound fraction). Blood, plasma, and filtrate samples were determined as aqueous biological samples with HPLC in presence of extraction step. The distribution of drugs in the blood was calculated from blood, plasma, and filtrate concentrations. In the preliminary experiment, we confirmed that drug bindings to ultrafilter membranes were 0.0% for PLS and 4.3% for Pal-PLS, respectively. 2.3. Stability experiments Blood and liver were obtained from rats. The liver was homogenized at 0–4 °C in a glass-teflon homogenizer to prepare 5% homogenate with 1.17% KCl, centrifuged at 1000 ×g for 10 min, and the supernatants obtained were used in this study. The protein concentration (5.7 mg/ml) was determined by a Bio-Rad protein assay kit (Bio-Rad Lab., Hercules, USA). Degradation experiments were performed at 37 °C and were initiated by adding the stock solution of PLS and Pal-PLS to give a concentration of 100 μM. At appropriate time intervals,
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aliquots of the solution were withdrawn for HPLC analysis. Drug degradation was also examined in pH 7.4 buffer, pH 7.4 buffer containing 90% ethanol, 0.1 M HCl containing 90% ethanol, and 0.1 M NaOH containing 90% ethanol. 2.4. Preparation of liposomes Liposomes of Pal-PLS were prepared by the sonication method [8]. EggPC/Chol, EggPC/Chol/1% DSPE-PEG 2000, EggPC/Chol/10% DSPE-PEG 2000 liposomes were composed of EggPC, Chol, and DSPE-PEG 2000 at a molar ratio of 3:2, 3: 2:0.05, and 3:2:0.5, respectively. After all mixtures of lipids and Pal-PLS in chloroform were placed in a round-bottomed glass tube, chloroform was evaporated. The lipid film containing PalPLS was further dried in vacuo in a desiccator for 4 h. The lipid and Pal-PLS film were added to PBS and allowed to hydrate for 24 h at 5 °C. The lipid suspension was vortex-mixed followed by ultrasonic radiation for 3 min at 0 °C under nitrogen gas. The liposomal formulation was used without further procedures. We have already reported that the encapsulation efficiency of PalPLS in all liposomes was completed without treatment with a separating free drug [7]. The final lipid concentration of EggPC/ Chol, EggPC/Chol/1% DSPE-PEG 2000, EggPC/Chol/10% DSPE-PEG 2000 liposomes was adjusted to 123.7, 124.9, and 136.1 mM in PBS, respectively. The final Pal-PLS concentration of the formulations was 14 mM. The particle sizes of the liposomes were 55 ± 28 nm for EggPC/Chol, 45 ± 25 nm for EggPC/Chol/1% DSPE-PEG 2000, and 110 ± 61 nm EggPC/ Chol/10% DSPE-PEG 2000 liposomes in average diameter, determined by transmission electron microscopy (JEM-1210, JEOL, Tokyo). Fig. 1 shows the TEM pictures. At least 240 vesicles of liposome were counted to determine the average size. All lipid compositions are given as molar ratios unless otherwise indicated. 2.5. Animal experiments Male Wistar rats (230–290 g) were used throughout the study after anesthetization with an adequate dose of sodium pentobarbital solution. PLS (20 mg/kg, 56 μmol/kg) or Pal-PLS (33.6 mg/kg, equivalent to 20 mg/kg of PLS) was dissolved in 1 ml of PEG 400. Pal-PLS in liposomes (EggPC/Chol or EggPC/Chol/1% DSPE-PEG 2000 or EggPC/Chol/10% DSPEPEG 2000) was injected into the jugular vein of rats at a volume of 33.6 mg/kg for Pal-PLS. After the administration of 0.5 ml drug formulations into the jugular vein of rats, blood samples were collected at appropriate time intervals (1, 2, 5, 30, 60, 120, and 240 min) through femoral vein cannulation. Bile and urine samples were also collected (240 min) after drug administration through cannulation. Another group of rats was used for tissue distribution experiments. The rats were sacrificed by sodium pentobarbital at 240 min after drug administration and several organs (liver, lung, spleen, kidney, heart, and small intestine) were excised. The organs were weighed, and homogenized in an appropriate volume of 9.8% KCl. The blood, bile, urine, and homogenates were determined as aqueous biological samples with HPLC in presence of extraction step.
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mixtures were centrifuged at 12,000 ×g for 10 min and the supernatants were supplied for HPLC assay. Aqueous biological samples (blood, bile, urine, and several tissue homogenates) were separated into two portions for drug determination. The first portion (100 μl) for Pal-PLS was diluted with PBS (2 ml) and extracted with dichloromethane (9 ml) for 15 min. The organic layers (7 ml) were evaporated in vacuo and the resultant residues were redissolved in a mixture of ethanol (150 μl) and methanol (150 μl). The mixture was centrifuged at 12,000 ×g for 10 min and the supernatant was supplied for HPLC assay. The second portion (100 μl) for PLS was diluted with PBS (2 ml) and extracted with dichloromethane (9 ml) for 15 min. The organic layer was washed with 2 ml of 0.2 M NaOH and 2 ml of water. The organic layer (7 ml) was evaporated in vacuo. The residue was redissolved in a mixture of water (150 μl) and methanol (150 μl). The mixture was centrifuged at 12,000 ×g for 10 min and the supernatant was supplied for HPLC assay. The extraction efficiencies for PLS and Pal-PLS were 54.3 ± 2.4% and 83.7 ± 2.4%. We had reproducible calibration curves. 2.7. HPLC assay PLS and Pal-PLS were determined using an HPLC system (LC-10AD, Shimadzu Co. Ltd., Kyoto, Japan) in the reversedphase mode. PLS was determined simultaneously with its metabolites by a slightly modified procedure of Cannell et al. [9]. The stationary phase used was Cosmosil 5C18-MS-II packed column (150 × 4.6 mm for PLS and Pal-PLS, Nacalai Tesque Inc.). A mixture of 2-propanol, acetonitrile, and water (42:38:20, v/v/v) was used as a mobile phase with a flow rate of 1.0 ml/min for Pal-PLS assay and the retention time was 11.9 min. A mixture of methanol and water (57.5:42.5, v/v) was used as a mobile phase with a flow rate of 0.55 ml/min for the PLS assay and the retention time of PLS was 9.8 min. Drug retention was monitored with a variable wavelength ultraviolet detector (wavelength at 240 nm, SPD-10A, Shimadzu Co. Ltd.). 2.8. Pharmacokinetic analysis
Fig. 1. TEM pictures of EggPC/Chol (A), EggPC/Chol/1% DSPE-PEG 2000 (B), and EggPC/Chol/10% DSPE-PEG 2000 liposomes (C).
2.6. Drug determination Aqueous samples (pH 7.4 buffer, pH 7.4 buffer containing 90% ethanol, 0.1 M HCl containing 90% ethanol, and 0.1 M NaOH containing 90% ethanol) were diluted in a mixture of water (150 μl) and methanol (150 μl) for PLS, and in a mixture of methanol (150 μl) and ethanol (150 μl) for Pal-PLS. The
The blood profiles of drugs after intravenous administration were analyzed using a two-compartment model. Vc and Vp are the distribution volumes of the central compartment and the peripheral compartment. Kel is the first order elimination rate constant from the central compartment. K12 and K21 are the first order transfer rate constants between the central compartment and the peripheral compartment. Pharmacokinetic parameters were calculated by a nonlinear least squares computer program, MULTI [10]. This program was written in BASIC and run on a personal computer (FMV C7/100WLT, FUJITSU, Tokyo, Japan). 2.9. Statistical analysis Statistical comparisons were performed by both analysis of variance and Tukey's multiple comparison test. P < 0.05 was considered significant.
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3. Results 3.1. Blood distribution and stability of PLS and Pal-PLS Table 1 shows drug distribution to blood cells and plasma fraction. PLS was distributed to blood cells, plasma protein, and the plasma free fraction. Pal-PLS was distributed to blood cells and plasma protein, while it was not present in the plasma free fraction. The same distribution patterns were observed in the preliminary experiment to determine the drug distribution in blood collected 1 min after the intravenous administration of drugs in rats (data not shown). Fig. 2(A) shows the stability of PLS and Pal-PLS in pH 7.4 buffer where Pal-PLS was suspended. Fig. 2(B) shows the stability in pH 7.4 buffer containing 90% ethanol where PalPLS was soluble. Both PLS and Pal-PLS were stable in neutral pH. The degradations of PLS and Pal-PLS in 0.1 M HCl containing 90% ethanol and 0.1 M NaOH containing 90% ethanol are described in Fig. 2(C and D). Both drugs were stable in acidic pH, but unstable in basic pH. Pal-PLS was rapidly biodegraded to PLS and the regenerated PLS was gradually degraded in basic pH. The stabilities of PLS and Pal-PLS in rat blood and liver homogenate are shown in Fig. 2(E and F). PLS was stable in both biological samples; however, Pal-PLS was rapidly biodegraded to PLS not only in liver homogenate but also in blood. 3.2. Blood concentration and tissue distribution of PLS and Pal-PLS Fig. 3 shows the blood concentrations of drugs after the intravenous administration of PLS (A) and Pal-PLS (B). PLS rapidly disappeared from the systemic circulation in a twophase mode. Pal-PLS also disappeared from the systemic circulation in a two-phase mode and PLS was rapidly regenerated. Fig. 4 shows the tissue distribution of drugs (liver, lung, spleen, kidney, heart, and small intestine) at 240 min after the intravenous administration of PLS or Pal-PLS solution in PEG 400. Pal-PLS showed a significantly higher accumulation than PLS in the liver and lung. 3.3. Blood concentration and tissue distribution of Pal-PLS incorporated into liposomes Fig. 5 shows the blood concentrations of drugs after the intravenous administration of Pal-PLS incorporated into lipo-
Table 1 Distribution ratio between blood cells and plasma of PLS and Pal-PLS in rat blood at a drug concentration of 10 μM at 37 °C Drugs
PLS Pal-PLS
Blood cells (%)
Plasma (%) Bound fraction
Free fraction
41.3 ± 0.9 22.8 ± 6.7
39.9 ± 1.3 77.2 ± 6.7 ⁎
18.8 ± 1.4 0 ± 0⁎
Each value represents the mean of at least three experiments. ⁎ Significantly different from PLS (P < 0.05).
Fig. 2. Stability of PLS (○), Pal-PLS (□), and PLS regenerated from Pal-PLS (■) in pH 7.4 buffer (A), pH 7.4 buffer containing 90% ethanol (B), 0.1 M HCl containing 90% ethanol (C), 0.1 M NaOH containing 90% ethanol (D), rat blood (E), and rat liver homogenates (F). Each point represents mean ± S.E. of at least three experiments.
somes constituted with EggPC/Chol (A), EggPC/Chol/1% DSPE-PEG 2000 (B), and EggPC/Chol/10% DSPE-PEG 2000 (C) in rats. Pal-PLS disappeared from the systemic circulation in a two-phase mode and PLS was rapidly regenerated. The administration of liposomal Pal-PLS enhanced and maintained Pal-PLS concentrations in the blood compared to that of Pal-PLS solution in PEG 400. Among the liposomes, the incorporation of PEG lipids decreased the initial distribution of Pal-PLS and maintained the high blood concentrations of PLS. Fig. 6 shows the tissue distribution of drugs (liver, lung, spleen, kidney, heart, and small intestine) at 240 min after the intravenous administration of Pal-PLS incorporated into liposomes. The administration of liposomal Pal-PLS significantly enhanced Pal-PLS accumulation in the liver and lung compared to that of Pal-PLS solution in PEG 400. The high accumulation of liposomal Pal-PLS constituted with EggPC/Chol in the liver and lung was significantly decreased by the incorporation of PEG lipids into liposomes.
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Fig. 3. Blood concentrations of PLS (○), Pal-PLS (○), and PLS regenerated from Pal-PLS (■) after the intravenous administration of PLS (A) and Pal-PLS (B) solution in PEG 400 in rats. Each point represents mean ± S.E. of at least three experiments.
3.4. Pharmacokinetic analysis The blood profiles of drugs after the administration of PLS and Pal-PLS solutions in PEG 400, and liposomal Pal-PLS were analyzed according to a two-compartment model and the calculated pharmacokinetic parameters are listed in Table 2. The Vc, Vp, and Vss of Pal-PLS were reduced to 13.2%, 23.1%, and Fig. 5. Blood concentrations of Pal-PLS (□) and PLS regenerated from Pal-PLS (■) after the intravenous administration of liposomal Pal-PLS constituted with EggPC/Chol (A), EggPC/Chol/1% DSPE-PEG 2000 (B), and EggPC/Chol/10% DSPE-PEG 2000 (C) in rats. Each point represents mean ± S.E. of at least three experiments.
Fig. 4. Tissue concentrations of PLS (closed bars), Pal-PLS (open bars), and PLS regenerated from Pal-PLS (hatched bars) at 240 min after the intravenous administration of PLS and Pal-PLS solution in PEG 400 in rats. Each value represents mean ± S.E. of at least three experiments.
19.4% compared with those of PLS, respectively. The Kel of Pal-PLS was 3.8-times larger than that of PLS. The CL of PalPLS was reduced to 52.1% compared with that of PLS. Pal-PLS showed a higher area under the blood concentration time curve (AUC) and shorter mean residence time (MRT) than PLS. The Kel values of liposomal Pal-PLS were smaller than those of Pal-PLS. The CL values of liposomal Pal-PLS constituted with EggPC/Chol, EggPC/Chol/1% DSPE-PEG 2000, and EggPC/Chol/10% DSPE-PEG 2000 were reduced to 66.7%, 30.2%, and 7.9% compared with those of Pal-PLS, respectively. The AUC values of liposomal Pal-PLS constituted with EggPC/ Chol, EggPC/Chol/1% DSPE-PEG 2000, EggPC/Chol/10%
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3.5. Biliary and urinary excretion
Fig. 6. Tissue concentrations of Pal-PLS (open bars) and PLS regenerated from Pal-PLS (hatched bars) at 240 min after the intravenous administration of liposomal Pal-PLS constituted with EggPC/Chol, EggPC/Chol/1% DSPE-PEG 2000, and EggPC/Chol/10% DSPE-PEG 2000 in rats. The PLS amount is shown in Fig. 5 but is invisible because extremely few PLS were regenerated from PalPLS in any formulations. Each value represents mean ± S.E. of at least three experiments.
DSPE-PEG 2000 were 2.0-, 5.0-, and 10.5-times larger than those of Pal-PLS. Liposomal Pal-PLS showed longer MRT than Pal-PLS.
The biliary and urinary accumulations of drugs were examined during the 240 min after the intravenous administration of PLS solution in PEG 400. The biliary and urinary accumulations of PLS were 25.41 ± 4.13 and 65.69 ± 7.63 nmol, respectively. The intravenous administration of Pal-PLS solution in PEG 400 showed no Pal-PLS and 10.63 ± 1.92 nmol of PLS in biliary accumulation, and no Pal-PLS and 7.72 ± 2.29 nmol of PLS in urinary accumulation. The biliary and urinary accumulations of drugs were examined during the 240 min after the intravenous administration of Pal-PLS incorporated into liposomes. The intravenous administration of Pal-PLS incorporated into EggPC/Chol liposomes showed 0.18 ± 0.18 nmol of Pal-PLS and 1.85 ± 0.05 nmol of PLS in biliary accumulation, and 0.59 ± 0.29 nmol of Pal-PLS and 5.21 ± 2.28 nmol of PLS in urinary accumulation. The intravenous administration of Pal-PLS incorporated into EggPC/Chol/1% DSPE-PEG 2000 liposomes showed 4.37 ± 2.91 nmol of Pal-PLS and 0.40 ± 0.40 nmol of PLS in biliary accumulation, and 2.30 ± 2.30 nmol of Pal-PLS and 0.56 ± 0.56 nmol of PLS in urinary accumulation. The intravenous administration of Pal-PLS incorporated into EggPC/Chol/10% DSPE-PEG 2000 liposomes showed 0.77 ± 0.77 nmol of Pal-PLS and 35.38 ± 15.60 nmol of PLS in biliary accumulation, and 0.54 ± 0.54 nmol of Pal-PLS and no PLS detected in urinary accumulation. The biliary and urinary excretions of drugs for 240 min after drug administration were less than 1% of the administrated dose in any formulations.
Table 2 Pharmacokinetic parameters of drugs after intravenous administration of PLS and Pal-PLS solution in PEG 400, and liposomal Pal-PLS constituted with EggPC/Chol, EggPC/Chol/10% DSPE-PEG 2000, and EggPC/Chol/10% DSPE-PEG 2000 in rats
Intravenous administration
C1 Vc
C2 Vp
Kel Parameters
PLS solution
Pal-PLS solution
Liposomal Pal-PLS (EggPC/Chol)
Liposomal Pal-PLS (EggPC/Chol/1% DSPE-PEG 2000)
Liposomal Pal-PLS (EggPC/Chol/10% DSPE-PEG 2000)
K12 (min− 1) K21 (min− 1) Kel (min− 1) Vc (mL) Vp (mL) Vss (mL) CL (mL min− 1) AUC (μM min)
0.10 ± 0.05 0.05 ± 0.02 0.09 ± 0.01 142.8 ± 9.8 232.8 ± 36.3 375.7 ± 29.4 12.1 ± 1.1 687 ± 43 – 26.4 ± 0.2 –
0.04 ± 0.01 0.02 ± 0.01 0.34 ± 0.05 18.9 ± 2.7 53.8 ± 5.1 72.7 ± 5.3 6.3 ± 1.1 1204 ± 174 (258 ± 26)a 12.5 ± 1.9 (60.8 ± 6.3)a
4.78 ± 1.24 28.71 ± 17.46 0.08 ± 0.01 55.7 ± 3.0 ⁎ 68.4 ± 61.2 124.1 ± 58.3 4.2 ± 0.2 2422 ± 114 (84 ± 26)a 47.3 ± 8.0 ⁎ (107.9 ± 7.7 ⁎⁎)a
27.1 ± 19.7 45.94 ± 36.65 0.07 ± 0.01 28.2 ± 6.9 14.0 ± 3.7 42.3 ± 3.8 1.9 ± 0.3 6027 ± 1058 ⁎, ⁎⁎⁎ (116 ± 35)a 55.7 ± 16.0 ⁎ (121.6 ± 20.8 ⁎⁎)a
19.2 ± 9.13 21.52 ± 4.95 0.01 ± 0.00 37.8 ± 8.4 22.4 ± 9.1 60.2 ± 1.1 0.5 ± 0.0 12671 ± 22 ⁎, ⁎⁎⁎, ⁎⁎⁎⁎ (703 ± 240 ⁎⁎⁎, ⁎⁎⁎⁎)a 80.5 ± 0.7 ⁎ (90.9 ± 12.5)a
MRT (min)
Each value represents the average ±S.E. of at least three experiments. a The values in parentheses are pharmacokinetic parameters of PLS regenerated from Pal-PLS. ⁎ Significantly different from PLS solution (P < 0.05). ⁎⁎ Significantly different from Pal-PLS solution (P < 0.05). ⁎⁎⁎ Significantly different from Liposomal Pal-PLS (EggPC/Chol) (P < 0.05). ⁎⁎⁎⁎ Significantly different from Liposomal Pal-PLS (EggPC/Chol/1% DSPE-PEG 2000) (P < 0.05).
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4. Discussion Glucocorticoids interact with intracellular receptors, modulating the gene transcription of proteins including mediators of the inflammatory and/or immune responses [11]. Natural corticosteroids can be modified by partial synthesis to obtain more potent compounds. The most significant improvement was the introduction of a double bond between C-1 and C-2 by microbiological dehydration. The resulting cortisol derivative, PLS, is more potent and has less effect on sodium and potassium regulation [12]. Some lipophilic corticosteroids strongly suppressed rheumatism after the transdermal application of gel because of sufficient penetration [13]. Lipophilic glucocorticoid loading in emulsion was also useful for retaining and targeting drugs in rheumatic tissue after intravenous administration [5,6]. We previously prepared Pal-PLS with high lipophilicity [7]. Palmitic acid may be safe as a lipophilic carrier moiety of drugs because of a common biological component. The physicochemical properties of Pal-PLS were initially compared to those of PLS. PLS is principally bound to transcortin and albumin in plasma [14,15], and is distributed to plasma protein and blood cells although unbound PLS was detected in the blood as shown in Table 1. PLS was stable at neutral and acidic pHs as shown in Fig. 2(A and C), although it degraded in basic pH as shown in Fig. 2(D). It is well known that the hydroxyl group at position 17 is easily converted to ketone by the catalysis of hydroxide ions. The lipophilic derivative of PLS, Pal-PLS, is distributed into blood cells and plasma protein, while it was not present in the plasma free fraction. This difference may be due to the difference of lipophilicity between PLS and Pal-PLS. Pal-PLS was stable in neutral and acidic buffers as shown in Fig. 2(A and C), although it was rapidly hydrolyzed to PLS in basic buffer as shown in Fig. 2(D). Pal-PLS was also easily hydrolyzed to PLS in rat plasma and liver homogenates. Carboxylesterase in the blood and various tissues may play a role in these catalyses. These results indicated that Pal-PLS might act as a prodrug type of PLS. Secondary, the biodistribution of Pal-PLS was compared to that of PLS after its intravenous administration in rats. PLS solution in PEG 400 was rapidly eliminated from the blood according to a two-phase mode (Fig. 3), although there was only a small amount in the bile and urine. These results indicated that the elimination of PLS was predominantly explained by its metabolism. Vermeulen [16] reported that PLS is cleared from the body primarily by hepatic metabolism, and larger than 90% of radioactivity administrated orally or intravenously as [4] prednisolone is recovered in human urine. In human urine, the major metabolites of prednisolone are prednisone, 20βhydroxyprednisone, and 6β, 20α, 20β-hydroxyprednisolone [17]. In rats, PLS is well known to be metabolized to many metabolites such as prednisone, 20-hydroxyprednisone, and 20hydroxyprednisolone. The lipophilic derivative of PLS, PalPLS, disappeared from the systemic circulation in a two-phase mode and PLS was rapidly regenerated. Pal-PLS showed a significantly higher accumulation than PLS in the liver and lung
although little was excreted in the bile and urine. Palmitic acid bound to albumin in the systemic circulation and its uptake in primary hepatic cells was facilitated by albumin [18,19]. Therefore, the strong binding of Pal-PLS to plasma protein and blood cells may result in increased drug accumulation in the liver and lung (Table 1). The size of the Pal-PLS molecule as compared to PLS could enhance its engulfment by macrophages in the blood. Lipophilicity might also influence the process of opsonization leading to higher entrapment of Pal-PLS by macrophages. Markedly extended glucocorticoid receptor occupancy was found in the liver and spleen [20]. We previously reported that the lipophilic derivative of PLS, Pal-PLS, has a strong affinity to liposomes [7]. Pal-PLS showed high incorporation into liposomes although PLS was rapidly released from the liposomes even by buffer dilution. Generally, liposomal drugs injected intravenously were diluted by blood, followed by interaction with plasma components in the systemic circulation [21,22]; consequently, incorporated drugs were rapidly released from liposomes [23]. We therefore compared the pharmacokinetics of liposomal Pal-PLS after intravenous administration in rats. EggPC/Chol liposomes containing PalPLS enhanced Pal-PLS concentrations in the blood, liver, and lung compared to Pal-PLS solution in PEG 400. The high accumulation of Pal-PLS in the liver and lung must be due to the rapid removal of liposomes by mononuclear phagocytic system uptake [24,25]. Liposomal Pal-PLS is useful for targeting PLS to reticuloendothelial systems. The behavior of liposomes is influenced by lipid composition and surface modification. The surface modification of liposomes with the hydrophilic polymer PEG has provided a major advance in drug delivery applications due to the ability of this polymer to reduce protein binding and the plasma elimination of liposomes [26]. Pal-PLS incorporated into PEGylated liposomes constituted with EggPC/Chol/1% DSPE-PEG 2000 and EggPC/Chol/10% DSPE-PEG 2000 decreased the initial distribution of Pal-PLS and successfully maintained the high blood concentrations of Pal-PLS and PLS. The decrease of the initial distribution must be caused by decreased Pal-PLS accumulation in the liver and lung as shown in Fig. 6. PEGylated liposomal Pal-PLS is useful for the prolonged concentration of PLS in the blood after intravenous administration. PLS was extensively used for the clinical treatment of autoimmune diseases, renal diseases, hepatic diseases, and allergic diseases. A dose of 5–60 mg of PLS or the equivalent is usually given daily, in divided amounts, although much larger doses may on occasion be required. A hydrophilic corticosteroid, prednisolone succinate, is also administered intravenously at a dose of 5–60 mg to treat patients with severe asthma and shock. After the intravenous injection of 20 mg prednisolone succinate, the maximum blood concentration and half-life of PLS were 481 ± 81 ng/ml (1.3 ± 0.2 μM) and 3.17 ± 0.44 h, respectively. In this study, liposomal Pal-PLS (EggPC/Chol/ 10% DSPE-PEG 2000) showed sufficient blood concentrations of PLS (1–10 μM) for clinical treatment over long periods after its intravenous injection in rats. These formulations are expected to be used clinically with the right balance of efficacy
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and side effects. Long-circulating liposomes of prednisolone phosphate have been reported to show increased therapeutic efficiency and decreased side effects in murine arthritis, multiple sclerosis, and IgA nephropathy [2–4]. The blood profiles of drugs after the administration of PLS or Pal-PLS solutions in PEG 400 and liposomal Pal-PLS were analyzed according to a two-compartment model (Table 2). The pharmacokinetics parameters of Pal-PLS were determined by limited number of points and limited period of time. We could not detect any drug level with HPLC beyond 250 min following drug administration. Compared with PLS, a small Vc of Pal-PLS in the central compartment was confirmed in this study and the value was close to a blood volume of rats [27]. This was caused by the strong binding of drugs to biological components in the systemic circulation. This small distribution volume resulted in a low value of CL. Reduction of the distribution volume in the peripheral compartment also occurred for the same reason. Pal-PLS showed a higher Kel than PLS, although it apparently showed a smaller slope in the elimination phase. The transfer rate constant of Pal-PLS from the peripheral compartment to the central compartment (K21) was much smaller than Kel. Compared with Pal-PLS, liposomal Pal-PLS decreased CL and increased AUC and MRT according to the characteristics of liposomes. The largest AUC of Pal-PLS and PLS and the smallest CL of Pal-PLS in the EggPC/Chol/10% DSPE-PEG 2000 showed its efficiency in retaining its encapsulated contents (Pal-PLS) in this study. EggPC/Chol/1% DSPE-PEG 2000 showed the smallest AUC ratio of PLS to Pal-PLS and this result may indicate circulation stability. The blood concentration of PLS was reflected by the drug released from the accumulated liposomes in the liver and circulating liposomes in the blood. Tokunaga et al. have reported that liposomal entrapment prolonged degradation of mitomycin C derivative in rat and human serum [28]. The addition of PEG lipids to liposomes has been reported to inhibit Pal-PLS release from the liposomes in rat plasma [7]. Further studies under various conditions were necessary to investigate the release/leakage of drug from liposomes in biological environment. The biliary and urinary excretions of drugs for 240 min after drug administrations were less than 1% of the administrated dose in any formulations. The EggPC/Chol/1% DSPE-PEG 2000 showed the highest biliary accumulation although tissue distribution studies showed that the accumulation of drug was highest for EggPC/Chol liposomes (Fig. 6). The biliary drug accumulation was reflected by the drug released from the accumulated liposomes in the liver and circulating liposomes in the blood. The release rates and amounts of drug in parenchymal cells and non-parenchymal cells were also important to determine drug biliary accumulation. The largest biliary accumulation of PLS by EggPC/Chol/1% DSPE-PEG 2000 liposomes may be explained by the high blood concentration and high distribution in parenchymal cells. The low biliary accumulation of PLS by EggPC/Chol liposomes may be caused by low blood concentration and high distribution in non-parenchymal cells. However, an additional experiment is necessary to clarify the mechanism.
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5. Conclusion We compared the pharmacokinetic behavior of drugs after the intravenous administration of PLS or Pal-PLS solution in PEG 400 and liposomal Pal-PLS in rats. We could change the pharmacokinetics of PLS by the introduction of palmitoyl function into the molecule and its liposomal formulation including PEG lipids. These results indicated the usefulness of an integrated approach by chemical modification and pharmaceutical modification to optimize drug delivery. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. The authors thank Naomi Takada and Kaoru Kashiwagi for technical assistance. References [1] C.H. Robert Jr., M. Ferid, in: P.B. Molinoff, R.W. Ruddon (Eds.), Goodman and Gilman's The Pharmacological Basis of Therapeutics, The McGraw-Hill Companies, Inc. Press, New York, 1996, pp. 1459–1489. [2] J.M. Metselaar, M.H. Wauben, J.P. Wagenaar-Hilbers, O.C. Boerman, G. Storm, Complete remission of experimental arthritis by joint targeting of glucocorticoids with long-circulating liposomes, Arthritis Rheum. 48 (2003) 2059–2066. [3] J. Schmidt, J.M. Metselaar, M.H. Wauben, K.V. Toyka, G. Storm, R. Gold, Drug targeting by long-circulating liposomal glucocorticosteroids increases therapeutic efficacy in a model of multiple sclerosis, Brain 126 (2003) 1895–1904. [4] J. Liao, K. Hayashi, S. Horikoshi, H. Ushijima, J. Kimura, Y. Tomino, Effect of steroid-liposome on immunohistopathology of IgA nephropathy in ddY mice, Nephron 89 (2001) 194–200. [5] Y. Mizushima, T. Hamano, K. Yokoyama, Tissue distribution and antiinflammatory activity of corticosteroids incorporated in lipid emulsion, Ann. Rheum. Dis. 41 (1982) 263–267. [6] K. Hoshi, Y. Mizushima, Y. Shiokawa, T. Kageyama, M. Honma, S. Kashiwazaki, K. Shichikawa, T. Tsunematsu, K. Kaneko, Double-blind study with liposteroid in rheumatoid arthritis, Drugs Exp. Clin. Res. 11 (1985) 621–626. [7] M. Teshima, S. Kawakami, K. Nishida, J. Nakamura, T. Sakaeda, H. Terazono, T. Kitahara, M. Nakashima, H. Sasaki, Prednisolone retention in integrated liposomes by chemical approach and pharmaceutical approach, J. Control. Release 97 (2004) 211–218. [8] C. Huang, Studies on phosphatidylcholine vesicles. Formation and physical characteristics, Biochemistry 8 (1969) 344–352. [9] G.R. Cannell, R.H. Mortimer, D.J. Maguire, R.S. Addison, Liquid chromatographic analysis of prednisolone, prednisone and their 20-reduced metabolites in perfusion media, J. Chromatogr. 563 (1991) 341–347. [10] K. Yamaoka, Y. Tanigawara, T. Nakagawa, T. Uno, A pharmacokinetic analysis program (MULTI) for microcomputer, J. Pharmacobio-Dyn. 4 (1981) 879–885. [11] A. Munck, D.B. Mendel, L.I. Smith, E. Orti, Glucocorticoid receptors and actions, Am. Rev. Respir. Dis. 141 (1990) S2–S10. [12] W.W. Downie, Steroid therapy, Scott. Med. J. 21 (1976) 188–193. [13] K. Sawada, Y. Akizawa, N. Ono, H. Miyake, Anti-inflammatory activity of prednisolone farnesylate (PNF-21) gel, a new transdermal corticosteroid, Jpn. Pharmacol. Ther. 20 (1992) 4075–4079 (Japanese). [14] M.L. Rocci Jr., N.F. Johnson, W.J. Jusko, Serum protein binding of prednisolone in four species, J. Pharm. Sci. 69 (1980) 977–978. [15] F.D. Boudinot, W.J. Jusko, Fluid shifts and other factors affecting plasma protein binding of prednisolone by equilibrium dialysis, J. Pharm. Sci. 73 (1984) 774–780.
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[16] A. Vermeulen, The metabolism of 4-14C prednisolone, J. Endocrinol. 18 (1959) 278–291. [17] V. Garg, W.J. Jusko, Simultaneous analysis of prednisone, prednisolone and their major hydroxylated metabolites in urine by high-performance liquid chromatography, J. Chromatogr. 567 (1991) 39–47. [18] F.J. Burczynski, Z.S. Cai, Palmitate uptake by hepatocyte suspensions: effect of albumin, Am. J. Physiol. 267 (1994) G371–G379. [19] S.M. Pond, C.K. Davis, M.A. Bogoyevitch, R.A. Gordon, R.A. Weisiger, L. Bass, Uptake of palmitate by hepatocyte suspensions: facilitation by albumin? Am. J. Physiol. 262 (1992) G883–G894. [20] E.V. Mishina, R.M. Straubinger, N.A. Pyszczynski, W.J. Jusko, Enhancement of tissue delivery and receptor occupancy of methylprednisolone in rats by a liposomal formulation, Pharm. Res. 10 (1993) 1402–1410. [21] J. Senior, G. Gregoriadis, Stability of small unilamellar liposomes in serum and clearance from the circulation: the effect of the phospholipid and cholesterol components, Life Sci. 30 (1982) 2123–2136. [22] F. Frézard, C. Santaella, P. Vierling, J.G. Riess, Permeability and stability in buffer and in human serum of fluorinated phospholipid-based liposomes, Biochim. Biophys. Acta 1192 (1994) 61–70.
[23] T. Takino, C. Nakajima, Y. Takakura, H. Sezaki, M. Hashida, Controlled biodistribution of highly lipophilic drugs with various parenteral formulations, J. Drug Target. 1 (1993) 117–124. [24] S.M. Moghimi, H.M. Patel, Serum opsonins and phagocytosis of saturated and unsaturated phospholipid liposomes, Biochim. Biophys. Acta 984 (1989) 384–387. [25] N. Shibuya-Fujiwara, F. Hirayama, Y. Ogata, H. Ikeda, K. Ikebuchi, Phagocytosis in vitro of polyethylene glycol-modified liposome-encapsulated hemoglobin by human peripheral blood monocytes plus macrophages through scavenger receptors, Life Sci. 70 (2001) 291–300. [26] J. Senior, C. Delgado, D. Fisher, C. Tilcock, G. Gregoriadis, Influence of surface hydrophilicity of liposomes on their interaction with plasma protein and clearance from the circulation: studies with poly(ethylene glycol)-coated vesicles, Biochim. Biophys. Acta 1062 (1991) 77–82. [27] K.B. Bischoff, R.L. Dedrick, D.S. Zaharko, J.A. Longstreth, Methotrexate pharmacokinetics, J. Pharm. Sci. 60 (1971) 1128–1133. [28] Y. Tokunaga, T. Iwasa, J. Fujisaki, S. Sawai, A. Kagayama, Liposomal sustained-release delivery systems for intravenous injection. 2. Physicochemical and biological properties of newly synthesized lipophilic derivatives of mitomycin C, Chem. Pharm. Bull. 36 (1988) 3060–3069.
Prolonged blood concentration of prednisolone after intravenous injection of liposomal palmitoyl prednisolone Mugen Teshima a , Shintaro Fumoto b , Koyo Nishida b , Junzo Nakamura b , Kaname Ohyama a , Tadahiro Nakamura a , Nobuhiro Ichikawa a , Mikiro Nakashima a , Hitoshi Sasaki a,⁎ a
Department of Hospital Pharmacy, Nagasaki University Hospital of Medicine and Dentistry, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan b Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan Received 12 October 2005; accepted 9 March 2006 Available online 21 April 2006
Abstract We compared the pharmacokinetic behavior of drugs after an intravenous administration of prednisolone (PLS), palmitoyl prednisolone (PalPLS), and liposomal Pal-PLS in rats. Pal-PLS showed higher lipophilicity and higher binding to plasma protein than PLS, and PLS regeneration in rat blood and liver homogenates. After the intravenous administration of Pal-PLS solution in polyethylene glycol (PEG) 400 to rats, Pal-PLS disappeared from the blood in a two-phase mode and PLS was rapidly regenerated. Pal-PLS showed a significantly higher accumulation than PLS in the liver and lung. The administration of Pal-PLS incorporated into egg yolk phosphatidylcholine (EggPC)/cholesterol (Chol) liposomes enhanced Pal-PLS concentrations in the blood, liver, and lung compared to that of Pal-PLS solution in PEG 400, suggesting the rapid removal of liposomes by the mononuclear phagocytic system. Pal-PLS incorporated into PEGylated liposomes constituted with EggPC/Chol/1% L-αdistearoylphosphatidylethanolamine (DSPE)-PEG 2000 and EggPC/Chol/10% DSPE-PEG 2000 decreased the initial distribution of Pal-PLS, and successfully maintained the blood concentrations of Pal-PLS and PLS. Thus, we could change the pharmacokinetics of PLS by introducing the palmitoyl function into the molecule and its liposomal formulation including PEGylation. This is the first study to evaluate liposomal PLS constituted with a lipophilic derivative and PEG lipids. © 2006 Elsevier B.V. All rights reserved. Keywords: Drug delivery system; Prednisolone; Liposomes; Lipophilic derivative; Polyethylene glycol
1. Introduction Glucocorticoids are highly potent anti-inflammatory and immunosuppressive drugs. Additionally, they are used in substitution therapy for adrenal insufficiency. Most glucocorticoids are administrated repeatedly because of their short halflife and are used in not only continuous treatment but also pulse treatment. However, even at moderate doses, the systemic administration of glucocorticoids causes many side effects, such as diabetes, hypertension, Cushing syndrome, and osteoporosis [1]. Therefore, it is effective for glucocorticoids to be retained in the blood and delivered in target tissues such as inflammatory tissues and the immune system [2–6].
⁎ Corresponding author. Tel.: +81 95 849 7245; fax: +81 95 849 7251. E-mail address: [email protected] (H. Sasaki). 0168-3659/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2006.03.004
Prednisolone (PLS) is one of the glucocorticoids most frequently used clinically. Liposomes have various advantages as a drug carrier such as biodegradability, low in vivo toxicity, and the encapsulation of hydrophilic, lipophilic and amphipathic drugs. Metselaar et al. [2] and Schmidt et al. [3] reported that long-circulating liposomes of prednisolone phosphate markedly increased biological activity and reduced side effects as compared to its solution after intravenous treatment for arthritis and multiple sclerosis. Liao et al. [4] also demonstrated that long-circulating liposomes of prednisolone phosphate showed increased therapeutic efficiency in IgA nephropathy model mice as compared to its solution. Prednisolone phosphate, however, was difficult to sufficiently incorporate into liposomes because of its high aqueous solubility. In the previous study, we newly synthesized palmitoyl prednisolone (Pal-PLS) with high lipophilicity and successfully prepared liposomes completely incorporating Pal-PLS [7].
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Furthermore, the addition of polyethylene glycol (PEG) lipids to liposomes could inhibit Pal-PLS release from the liposomes in the presence of rat plasma. These results indicated the usefulness of an integrated approach using chemical modification and liposomal formulations. The in vivo pharmacokinetics and activity of liposomal drugs, however, do not always reflect the in vitro release results. Therefore, we investigated the pharmacokinetic behavior of Pal-PLS and its liposomes with various lipids such as egg yolk phosphatidylcholine (EggPC), cholesterol (Chol), and with or without L-α-distearoylphosphatidylethanolamine-PEG 2000 (DSPE-PEG 2000) after their intravenous administration to rats. This is the first study to evaluate liposomal PLS constituted with a lipophilic derivative and PEG lipids. 2. Materials and methods 2.1. Materials PLS (Mw: 360.5) was kindly supplied by Shionogi Co. Ltd. (Osaka, Japan). The lipophilic derivative, Pal-PLS (Mw: 598.9), was synthesized by the method of Teshima et al. [7]. EggPC (average molecular weight 773, COATSOME NC-50) and DSPE-PEG 2000 (average molecular weight 2000, SUNBRIGHT DSPE-20H) were purchased from Nippon Oil and Fats Co. (Tokyo, Japan). Chol was obtained from Nacalai Tesque Inc. (Kyoto, Japan). All other chemicals were of reagent grade and used as obtained commercially. Phosphate-buffered saline (PBS) was prepared by mixing an isotonic phosphate buffer (pH 7.4) with an equal volume of saline. 2.2. Distribution experiment in blood PLS and Pal-PLS were incubated in rat blood (10 μM) at 37 °C for 2 min. Part of the blood was immediately centrifuged at 12,000 ×g for 5 min to obtain plasma. Part of the plasma was ultrafiltrated with a micropartition system (Ultrafree®-MC, molecular weight cut off of 5000, Millipore, Tokyo, Japan) to obtain the filtrate (unbound fraction). Blood, plasma, and filtrate samples were determined as aqueous biological samples with HPLC in presence of extraction step. The distribution of drugs in the blood was calculated from blood, plasma, and filtrate concentrations. In the preliminary experiment, we confirmed that drug bindings to ultrafilter membranes were 0.0% for PLS and 4.3% for Pal-PLS, respectively. 2.3. Stability experiments Blood and liver were obtained from rats. The liver was homogenized at 0–4 °C in a glass-teflon homogenizer to prepare 5% homogenate with 1.17% KCl, centrifuged at 1000 ×g for 10 min, and the supernatants obtained were used in this study. The protein concentration (5.7 mg/ml) was determined by a Bio-Rad protein assay kit (Bio-Rad Lab., Hercules, USA). Degradation experiments were performed at 37 °C and were initiated by adding the stock solution of PLS and Pal-PLS to give a concentration of 100 μM. At appropriate time intervals,
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aliquots of the solution were withdrawn for HPLC analysis. Drug degradation was also examined in pH 7.4 buffer, pH 7.4 buffer containing 90% ethanol, 0.1 M HCl containing 90% ethanol, and 0.1 M NaOH containing 90% ethanol. 2.4. Preparation of liposomes Liposomes of Pal-PLS were prepared by the sonication method [8]. EggPC/Chol, EggPC/Chol/1% DSPE-PEG 2000, EggPC/Chol/10% DSPE-PEG 2000 liposomes were composed of EggPC, Chol, and DSPE-PEG 2000 at a molar ratio of 3:2, 3: 2:0.05, and 3:2:0.5, respectively. After all mixtures of lipids and Pal-PLS in chloroform were placed in a round-bottomed glass tube, chloroform was evaporated. The lipid film containing PalPLS was further dried in vacuo in a desiccator for 4 h. The lipid and Pal-PLS film were added to PBS and allowed to hydrate for 24 h at 5 °C. The lipid suspension was vortex-mixed followed by ultrasonic radiation for 3 min at 0 °C under nitrogen gas. The liposomal formulation was used without further procedures. We have already reported that the encapsulation efficiency of PalPLS in all liposomes was completed without treatment with a separating free drug [7]. The final lipid concentration of EggPC/ Chol, EggPC/Chol/1% DSPE-PEG 2000, EggPC/Chol/10% DSPE-PEG 2000 liposomes was adjusted to 123.7, 124.9, and 136.1 mM in PBS, respectively. The final Pal-PLS concentration of the formulations was 14 mM. The particle sizes of the liposomes were 55 ± 28 nm for EggPC/Chol, 45 ± 25 nm for EggPC/Chol/1% DSPE-PEG 2000, and 110 ± 61 nm EggPC/ Chol/10% DSPE-PEG 2000 liposomes in average diameter, determined by transmission electron microscopy (JEM-1210, JEOL, Tokyo). Fig. 1 shows the TEM pictures. At least 240 vesicles of liposome were counted to determine the average size. All lipid compositions are given as molar ratios unless otherwise indicated. 2.5. Animal experiments Male Wistar rats (230–290 g) were used throughout the study after anesthetization with an adequate dose of sodium pentobarbital solution. PLS (20 mg/kg, 56 μmol/kg) or Pal-PLS (33.6 mg/kg, equivalent to 20 mg/kg of PLS) was dissolved in 1 ml of PEG 400. Pal-PLS in liposomes (EggPC/Chol or EggPC/Chol/1% DSPE-PEG 2000 or EggPC/Chol/10% DSPEPEG 2000) was injected into the jugular vein of rats at a volume of 33.6 mg/kg for Pal-PLS. After the administration of 0.5 ml drug formulations into the jugular vein of rats, blood samples were collected at appropriate time intervals (1, 2, 5, 30, 60, 120, and 240 min) through femoral vein cannulation. Bile and urine samples were also collected (240 min) after drug administration through cannulation. Another group of rats was used for tissue distribution experiments. The rats were sacrificed by sodium pentobarbital at 240 min after drug administration and several organs (liver, lung, spleen, kidney, heart, and small intestine) were excised. The organs were weighed, and homogenized in an appropriate volume of 9.8% KCl. The blood, bile, urine, and homogenates were determined as aqueous biological samples with HPLC in presence of extraction step.
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mixtures were centrifuged at 12,000 ×g for 10 min and the supernatants were supplied for HPLC assay. Aqueous biological samples (blood, bile, urine, and several tissue homogenates) were separated into two portions for drug determination. The first portion (100 μl) for Pal-PLS was diluted with PBS (2 ml) and extracted with dichloromethane (9 ml) for 15 min. The organic layers (7 ml) were evaporated in vacuo and the resultant residues were redissolved in a mixture of ethanol (150 μl) and methanol (150 μl). The mixture was centrifuged at 12,000 ×g for 10 min and the supernatant was supplied for HPLC assay. The second portion (100 μl) for PLS was diluted with PBS (2 ml) and extracted with dichloromethane (9 ml) for 15 min. The organic layer was washed with 2 ml of 0.2 M NaOH and 2 ml of water. The organic layer (7 ml) was evaporated in vacuo. The residue was redissolved in a mixture of water (150 μl) and methanol (150 μl). The mixture was centrifuged at 12,000 ×g for 10 min and the supernatant was supplied for HPLC assay. The extraction efficiencies for PLS and Pal-PLS were 54.3 ± 2.4% and 83.7 ± 2.4%. We had reproducible calibration curves. 2.7. HPLC assay PLS and Pal-PLS were determined using an HPLC system (LC-10AD, Shimadzu Co. Ltd., Kyoto, Japan) in the reversedphase mode. PLS was determined simultaneously with its metabolites by a slightly modified procedure of Cannell et al. [9]. The stationary phase used was Cosmosil 5C18-MS-II packed column (150 × 4.6 mm for PLS and Pal-PLS, Nacalai Tesque Inc.). A mixture of 2-propanol, acetonitrile, and water (42:38:20, v/v/v) was used as a mobile phase with a flow rate of 1.0 ml/min for Pal-PLS assay and the retention time was 11.9 min. A mixture of methanol and water (57.5:42.5, v/v) was used as a mobile phase with a flow rate of 0.55 ml/min for the PLS assay and the retention time of PLS was 9.8 min. Drug retention was monitored with a variable wavelength ultraviolet detector (wavelength at 240 nm, SPD-10A, Shimadzu Co. Ltd.). 2.8. Pharmacokinetic analysis
Fig. 1. TEM pictures of EggPC/Chol (A), EggPC/Chol/1% DSPE-PEG 2000 (B), and EggPC/Chol/10% DSPE-PEG 2000 liposomes (C).
2.6. Drug determination Aqueous samples (pH 7.4 buffer, pH 7.4 buffer containing 90% ethanol, 0.1 M HCl containing 90% ethanol, and 0.1 M NaOH containing 90% ethanol) were diluted in a mixture of water (150 μl) and methanol (150 μl) for PLS, and in a mixture of methanol (150 μl) and ethanol (150 μl) for Pal-PLS. The
The blood profiles of drugs after intravenous administration were analyzed using a two-compartment model. Vc and Vp are the distribution volumes of the central compartment and the peripheral compartment. Kel is the first order elimination rate constant from the central compartment. K12 and K21 are the first order transfer rate constants between the central compartment and the peripheral compartment. Pharmacokinetic parameters were calculated by a nonlinear least squares computer program, MULTI [10]. This program was written in BASIC and run on a personal computer (FMV C7/100WLT, FUJITSU, Tokyo, Japan). 2.9. Statistical analysis Statistical comparisons were performed by both analysis of variance and Tukey's multiple comparison test. P < 0.05 was considered significant.
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3. Results 3.1. Blood distribution and stability of PLS and Pal-PLS Table 1 shows drug distribution to blood cells and plasma fraction. PLS was distributed to blood cells, plasma protein, and the plasma free fraction. Pal-PLS was distributed to blood cells and plasma protein, while it was not present in the plasma free fraction. The same distribution patterns were observed in the preliminary experiment to determine the drug distribution in blood collected 1 min after the intravenous administration of drugs in rats (data not shown). Fig. 2(A) shows the stability of PLS and Pal-PLS in pH 7.4 buffer where Pal-PLS was suspended. Fig. 2(B) shows the stability in pH 7.4 buffer containing 90% ethanol where PalPLS was soluble. Both PLS and Pal-PLS were stable in neutral pH. The degradations of PLS and Pal-PLS in 0.1 M HCl containing 90% ethanol and 0.1 M NaOH containing 90% ethanol are described in Fig. 2(C and D). Both drugs were stable in acidic pH, but unstable in basic pH. Pal-PLS was rapidly biodegraded to PLS and the regenerated PLS was gradually degraded in basic pH. The stabilities of PLS and Pal-PLS in rat blood and liver homogenate are shown in Fig. 2(E and F). PLS was stable in both biological samples; however, Pal-PLS was rapidly biodegraded to PLS not only in liver homogenate but also in blood. 3.2. Blood concentration and tissue distribution of PLS and Pal-PLS Fig. 3 shows the blood concentrations of drugs after the intravenous administration of PLS (A) and Pal-PLS (B). PLS rapidly disappeared from the systemic circulation in a twophase mode. Pal-PLS also disappeared from the systemic circulation in a two-phase mode and PLS was rapidly regenerated. Fig. 4 shows the tissue distribution of drugs (liver, lung, spleen, kidney, heart, and small intestine) at 240 min after the intravenous administration of PLS or Pal-PLS solution in PEG 400. Pal-PLS showed a significantly higher accumulation than PLS in the liver and lung. 3.3. Blood concentration and tissue distribution of Pal-PLS incorporated into liposomes Fig. 5 shows the blood concentrations of drugs after the intravenous administration of Pal-PLS incorporated into lipo-
Table 1 Distribution ratio between blood cells and plasma of PLS and Pal-PLS in rat blood at a drug concentration of 10 μM at 37 °C Drugs
PLS Pal-PLS
Blood cells (%)
Plasma (%) Bound fraction
Free fraction
41.3 ± 0.9 22.8 ± 6.7
39.9 ± 1.3 77.2 ± 6.7 ⁎
18.8 ± 1.4 0 ± 0⁎
Each value represents the mean of at least three experiments. ⁎ Significantly different from PLS (P < 0.05).
Fig. 2. Stability of PLS (○), Pal-PLS (□), and PLS regenerated from Pal-PLS (■) in pH 7.4 buffer (A), pH 7.4 buffer containing 90% ethanol (B), 0.1 M HCl containing 90% ethanol (C), 0.1 M NaOH containing 90% ethanol (D), rat blood (E), and rat liver homogenates (F). Each point represents mean ± S.E. of at least three experiments.
somes constituted with EggPC/Chol (A), EggPC/Chol/1% DSPE-PEG 2000 (B), and EggPC/Chol/10% DSPE-PEG 2000 (C) in rats. Pal-PLS disappeared from the systemic circulation in a two-phase mode and PLS was rapidly regenerated. The administration of liposomal Pal-PLS enhanced and maintained Pal-PLS concentrations in the blood compared to that of Pal-PLS solution in PEG 400. Among the liposomes, the incorporation of PEG lipids decreased the initial distribution of Pal-PLS and maintained the high blood concentrations of PLS. Fig. 6 shows the tissue distribution of drugs (liver, lung, spleen, kidney, heart, and small intestine) at 240 min after the intravenous administration of Pal-PLS incorporated into liposomes. The administration of liposomal Pal-PLS significantly enhanced Pal-PLS accumulation in the liver and lung compared to that of Pal-PLS solution in PEG 400. The high accumulation of liposomal Pal-PLS constituted with EggPC/Chol in the liver and lung was significantly decreased by the incorporation of PEG lipids into liposomes.
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Fig. 3. Blood concentrations of PLS (○), Pal-PLS (○), and PLS regenerated from Pal-PLS (■) after the intravenous administration of PLS (A) and Pal-PLS (B) solution in PEG 400 in rats. Each point represents mean ± S.E. of at least three experiments.
3.4. Pharmacokinetic analysis The blood profiles of drugs after the administration of PLS and Pal-PLS solutions in PEG 400, and liposomal Pal-PLS were analyzed according to a two-compartment model and the calculated pharmacokinetic parameters are listed in Table 2. The Vc, Vp, and Vss of Pal-PLS were reduced to 13.2%, 23.1%, and Fig. 5. Blood concentrations of Pal-PLS (□) and PLS regenerated from Pal-PLS (■) after the intravenous administration of liposomal Pal-PLS constituted with EggPC/Chol (A), EggPC/Chol/1% DSPE-PEG 2000 (B), and EggPC/Chol/10% DSPE-PEG 2000 (C) in rats. Each point represents mean ± S.E. of at least three experiments.
Fig. 4. Tissue concentrations of PLS (closed bars), Pal-PLS (open bars), and PLS regenerated from Pal-PLS (hatched bars) at 240 min after the intravenous administration of PLS and Pal-PLS solution in PEG 400 in rats. Each value represents mean ± S.E. of at least three experiments.
19.4% compared with those of PLS, respectively. The Kel of Pal-PLS was 3.8-times larger than that of PLS. The CL of PalPLS was reduced to 52.1% compared with that of PLS. Pal-PLS showed a higher area under the blood concentration time curve (AUC) and shorter mean residence time (MRT) than PLS. The Kel values of liposomal Pal-PLS were smaller than those of Pal-PLS. The CL values of liposomal Pal-PLS constituted with EggPC/Chol, EggPC/Chol/1% DSPE-PEG 2000, and EggPC/Chol/10% DSPE-PEG 2000 were reduced to 66.7%, 30.2%, and 7.9% compared with those of Pal-PLS, respectively. The AUC values of liposomal Pal-PLS constituted with EggPC/ Chol, EggPC/Chol/1% DSPE-PEG 2000, EggPC/Chol/10%
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3.5. Biliary and urinary excretion
Fig. 6. Tissue concentrations of Pal-PLS (open bars) and PLS regenerated from Pal-PLS (hatched bars) at 240 min after the intravenous administration of liposomal Pal-PLS constituted with EggPC/Chol, EggPC/Chol/1% DSPE-PEG 2000, and EggPC/Chol/10% DSPE-PEG 2000 in rats. The PLS amount is shown in Fig. 5 but is invisible because extremely few PLS were regenerated from PalPLS in any formulations. Each value represents mean ± S.E. of at least three experiments.
DSPE-PEG 2000 were 2.0-, 5.0-, and 10.5-times larger than those of Pal-PLS. Liposomal Pal-PLS showed longer MRT than Pal-PLS.
The biliary and urinary accumulations of drugs were examined during the 240 min after the intravenous administration of PLS solution in PEG 400. The biliary and urinary accumulations of PLS were 25.41 ± 4.13 and 65.69 ± 7.63 nmol, respectively. The intravenous administration of Pal-PLS solution in PEG 400 showed no Pal-PLS and 10.63 ± 1.92 nmol of PLS in biliary accumulation, and no Pal-PLS and 7.72 ± 2.29 nmol of PLS in urinary accumulation. The biliary and urinary accumulations of drugs were examined during the 240 min after the intravenous administration of Pal-PLS incorporated into liposomes. The intravenous administration of Pal-PLS incorporated into EggPC/Chol liposomes showed 0.18 ± 0.18 nmol of Pal-PLS and 1.85 ± 0.05 nmol of PLS in biliary accumulation, and 0.59 ± 0.29 nmol of Pal-PLS and 5.21 ± 2.28 nmol of PLS in urinary accumulation. The intravenous administration of Pal-PLS incorporated into EggPC/Chol/1% DSPE-PEG 2000 liposomes showed 4.37 ± 2.91 nmol of Pal-PLS and 0.40 ± 0.40 nmol of PLS in biliary accumulation, and 2.30 ± 2.30 nmol of Pal-PLS and 0.56 ± 0.56 nmol of PLS in urinary accumulation. The intravenous administration of Pal-PLS incorporated into EggPC/Chol/10% DSPE-PEG 2000 liposomes showed 0.77 ± 0.77 nmol of Pal-PLS and 35.38 ± 15.60 nmol of PLS in biliary accumulation, and 0.54 ± 0.54 nmol of Pal-PLS and no PLS detected in urinary accumulation. The biliary and urinary excretions of drugs for 240 min after drug administration were less than 1% of the administrated dose in any formulations.
Table 2 Pharmacokinetic parameters of drugs after intravenous administration of PLS and Pal-PLS solution in PEG 400, and liposomal Pal-PLS constituted with EggPC/Chol, EggPC/Chol/10% DSPE-PEG 2000, and EggPC/Chol/10% DSPE-PEG 2000 in rats
Intravenous administration
C1 Vc
C2 Vp
Kel Parameters
PLS solution
Pal-PLS solution
Liposomal Pal-PLS (EggPC/Chol)
Liposomal Pal-PLS (EggPC/Chol/1% DSPE-PEG 2000)
Liposomal Pal-PLS (EggPC/Chol/10% DSPE-PEG 2000)
K12 (min− 1) K21 (min− 1) Kel (min− 1) Vc (mL) Vp (mL) Vss (mL) CL (mL min− 1) AUC (μM min)
0.10 ± 0.05 0.05 ± 0.02 0.09 ± 0.01 142.8 ± 9.8 232.8 ± 36.3 375.7 ± 29.4 12.1 ± 1.1 687 ± 43 – 26.4 ± 0.2 –
0.04 ± 0.01 0.02 ± 0.01 0.34 ± 0.05 18.9 ± 2.7 53.8 ± 5.1 72.7 ± 5.3 6.3 ± 1.1 1204 ± 174 (258 ± 26)a 12.5 ± 1.9 (60.8 ± 6.3)a
4.78 ± 1.24 28.71 ± 17.46 0.08 ± 0.01 55.7 ± 3.0 ⁎ 68.4 ± 61.2 124.1 ± 58.3 4.2 ± 0.2 2422 ± 114 (84 ± 26)a 47.3 ± 8.0 ⁎ (107.9 ± 7.7 ⁎⁎)a
27.1 ± 19.7 45.94 ± 36.65 0.07 ± 0.01 28.2 ± 6.9 14.0 ± 3.7 42.3 ± 3.8 1.9 ± 0.3 6027 ± 1058 ⁎, ⁎⁎⁎ (116 ± 35)a 55.7 ± 16.0 ⁎ (121.6 ± 20.8 ⁎⁎)a
19.2 ± 9.13 21.52 ± 4.95 0.01 ± 0.00 37.8 ± 8.4 22.4 ± 9.1 60.2 ± 1.1 0.5 ± 0.0 12671 ± 22 ⁎, ⁎⁎⁎, ⁎⁎⁎⁎ (703 ± 240 ⁎⁎⁎, ⁎⁎⁎⁎)a 80.5 ± 0.7 ⁎ (90.9 ± 12.5)a
MRT (min)
Each value represents the average ±S.E. of at least three experiments. a The values in parentheses are pharmacokinetic parameters of PLS regenerated from Pal-PLS. ⁎ Significantly different from PLS solution (P < 0.05). ⁎⁎ Significantly different from Pal-PLS solution (P < 0.05). ⁎⁎⁎ Significantly different from Liposomal Pal-PLS (EggPC/Chol) (P < 0.05). ⁎⁎⁎⁎ Significantly different from Liposomal Pal-PLS (EggPC/Chol/1% DSPE-PEG 2000) (P < 0.05).
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4. Discussion Glucocorticoids interact with intracellular receptors, modulating the gene transcription of proteins including mediators of the inflammatory and/or immune responses [11]. Natural corticosteroids can be modified by partial synthesis to obtain more potent compounds. The most significant improvement was the introduction of a double bond between C-1 and C-2 by microbiological dehydration. The resulting cortisol derivative, PLS, is more potent and has less effect on sodium and potassium regulation [12]. Some lipophilic corticosteroids strongly suppressed rheumatism after the transdermal application of gel because of sufficient penetration [13]. Lipophilic glucocorticoid loading in emulsion was also useful for retaining and targeting drugs in rheumatic tissue after intravenous administration [5,6]. We previously prepared Pal-PLS with high lipophilicity [7]. Palmitic acid may be safe as a lipophilic carrier moiety of drugs because of a common biological component. The physicochemical properties of Pal-PLS were initially compared to those of PLS. PLS is principally bound to transcortin and albumin in plasma [14,15], and is distributed to plasma protein and blood cells although unbound PLS was detected in the blood as shown in Table 1. PLS was stable at neutral and acidic pHs as shown in Fig. 2(A and C), although it degraded in basic pH as shown in Fig. 2(D). It is well known that the hydroxyl group at position 17 is easily converted to ketone by the catalysis of hydroxide ions. The lipophilic derivative of PLS, Pal-PLS, is distributed into blood cells and plasma protein, while it was not present in the plasma free fraction. This difference may be due to the difference of lipophilicity between PLS and Pal-PLS. Pal-PLS was stable in neutral and acidic buffers as shown in Fig. 2(A and C), although it was rapidly hydrolyzed to PLS in basic buffer as shown in Fig. 2(D). Pal-PLS was also easily hydrolyzed to PLS in rat plasma and liver homogenates. Carboxylesterase in the blood and various tissues may play a role in these catalyses. These results indicated that Pal-PLS might act as a prodrug type of PLS. Secondary, the biodistribution of Pal-PLS was compared to that of PLS after its intravenous administration in rats. PLS solution in PEG 400 was rapidly eliminated from the blood according to a two-phase mode (Fig. 3), although there was only a small amount in the bile and urine. These results indicated that the elimination of PLS was predominantly explained by its metabolism. Vermeulen [16] reported that PLS is cleared from the body primarily by hepatic metabolism, and larger than 90% of radioactivity administrated orally or intravenously as [4] prednisolone is recovered in human urine. In human urine, the major metabolites of prednisolone are prednisone, 20βhydroxyprednisone, and 6β, 20α, 20β-hydroxyprednisolone [17]. In rats, PLS is well known to be metabolized to many metabolites such as prednisone, 20-hydroxyprednisone, and 20hydroxyprednisolone. The lipophilic derivative of PLS, PalPLS, disappeared from the systemic circulation in a two-phase mode and PLS was rapidly regenerated. Pal-PLS showed a significantly higher accumulation than PLS in the liver and lung
although little was excreted in the bile and urine. Palmitic acid bound to albumin in the systemic circulation and its uptake in primary hepatic cells was facilitated by albumin [18,19]. Therefore, the strong binding of Pal-PLS to plasma protein and blood cells may result in increased drug accumulation in the liver and lung (Table 1). The size of the Pal-PLS molecule as compared to PLS could enhance its engulfment by macrophages in the blood. Lipophilicity might also influence the process of opsonization leading to higher entrapment of Pal-PLS by macrophages. Markedly extended glucocorticoid receptor occupancy was found in the liver and spleen [20]. We previously reported that the lipophilic derivative of PLS, Pal-PLS, has a strong affinity to liposomes [7]. Pal-PLS showed high incorporation into liposomes although PLS was rapidly released from the liposomes even by buffer dilution. Generally, liposomal drugs injected intravenously were diluted by blood, followed by interaction with plasma components in the systemic circulation [21,22]; consequently, incorporated drugs were rapidly released from liposomes [23]. We therefore compared the pharmacokinetics of liposomal Pal-PLS after intravenous administration in rats. EggPC/Chol liposomes containing PalPLS enhanced Pal-PLS concentrations in the blood, liver, and lung compared to Pal-PLS solution in PEG 400. The high accumulation of Pal-PLS in the liver and lung must be due to the rapid removal of liposomes by mononuclear phagocytic system uptake [24,25]. Liposomal Pal-PLS is useful for targeting PLS to reticuloendothelial systems. The behavior of liposomes is influenced by lipid composition and surface modification. The surface modification of liposomes with the hydrophilic polymer PEG has provided a major advance in drug delivery applications due to the ability of this polymer to reduce protein binding and the plasma elimination of liposomes [26]. Pal-PLS incorporated into PEGylated liposomes constituted with EggPC/Chol/1% DSPE-PEG 2000 and EggPC/Chol/10% DSPE-PEG 2000 decreased the initial distribution of Pal-PLS and successfully maintained the high blood concentrations of Pal-PLS and PLS. The decrease of the initial distribution must be caused by decreased Pal-PLS accumulation in the liver and lung as shown in Fig. 6. PEGylated liposomal Pal-PLS is useful for the prolonged concentration of PLS in the blood after intravenous administration. PLS was extensively used for the clinical treatment of autoimmune diseases, renal diseases, hepatic diseases, and allergic diseases. A dose of 5–60 mg of PLS or the equivalent is usually given daily, in divided amounts, although much larger doses may on occasion be required. A hydrophilic corticosteroid, prednisolone succinate, is also administered intravenously at a dose of 5–60 mg to treat patients with severe asthma and shock. After the intravenous injection of 20 mg prednisolone succinate, the maximum blood concentration and half-life of PLS were 481 ± 81 ng/ml (1.3 ± 0.2 μM) and 3.17 ± 0.44 h, respectively. In this study, liposomal Pal-PLS (EggPC/Chol/ 10% DSPE-PEG 2000) showed sufficient blood concentrations of PLS (1–10 μM) for clinical treatment over long periods after its intravenous injection in rats. These formulations are expected to be used clinically with the right balance of efficacy
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and side effects. Long-circulating liposomes of prednisolone phosphate have been reported to show increased therapeutic efficiency and decreased side effects in murine arthritis, multiple sclerosis, and IgA nephropathy [2–4]. The blood profiles of drugs after the administration of PLS or Pal-PLS solutions in PEG 400 and liposomal Pal-PLS were analyzed according to a two-compartment model (Table 2). The pharmacokinetics parameters of Pal-PLS were determined by limited number of points and limited period of time. We could not detect any drug level with HPLC beyond 250 min following drug administration. Compared with PLS, a small Vc of Pal-PLS in the central compartment was confirmed in this study and the value was close to a blood volume of rats [27]. This was caused by the strong binding of drugs to biological components in the systemic circulation. This small distribution volume resulted in a low value of CL. Reduction of the distribution volume in the peripheral compartment also occurred for the same reason. Pal-PLS showed a higher Kel than PLS, although it apparently showed a smaller slope in the elimination phase. The transfer rate constant of Pal-PLS from the peripheral compartment to the central compartment (K21) was much smaller than Kel. Compared with Pal-PLS, liposomal Pal-PLS decreased CL and increased AUC and MRT according to the characteristics of liposomes. The largest AUC of Pal-PLS and PLS and the smallest CL of Pal-PLS in the EggPC/Chol/10% DSPE-PEG 2000 showed its efficiency in retaining its encapsulated contents (Pal-PLS) in this study. EggPC/Chol/1% DSPE-PEG 2000 showed the smallest AUC ratio of PLS to Pal-PLS and this result may indicate circulation stability. The blood concentration of PLS was reflected by the drug released from the accumulated liposomes in the liver and circulating liposomes in the blood. Tokunaga et al. have reported that liposomal entrapment prolonged degradation of mitomycin C derivative in rat and human serum [28]. The addition of PEG lipids to liposomes has been reported to inhibit Pal-PLS release from the liposomes in rat plasma [7]. Further studies under various conditions were necessary to investigate the release/leakage of drug from liposomes in biological environment. The biliary and urinary excretions of drugs for 240 min after drug administrations were less than 1% of the administrated dose in any formulations. The EggPC/Chol/1% DSPE-PEG 2000 showed the highest biliary accumulation although tissue distribution studies showed that the accumulation of drug was highest for EggPC/Chol liposomes (Fig. 6). The biliary drug accumulation was reflected by the drug released from the accumulated liposomes in the liver and circulating liposomes in the blood. The release rates and amounts of drug in parenchymal cells and non-parenchymal cells were also important to determine drug biliary accumulation. The largest biliary accumulation of PLS by EggPC/Chol/1% DSPE-PEG 2000 liposomes may be explained by the high blood concentration and high distribution in parenchymal cells. The low biliary accumulation of PLS by EggPC/Chol liposomes may be caused by low blood concentration and high distribution in non-parenchymal cells. However, an additional experiment is necessary to clarify the mechanism.
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