Pharmacokinetics and Tissue Distribution of Idarubicin-Loaded Solid Lipid Nanoparticles After Duodenal Administration to Rats

Pharmacokinetics and Tissue Distribution of Idarubicin-Loaded Solid Lipid Nanoparticles after Duodenal Administration to Rats ` ,1 DANIELA VIGHETTO,3 GIAN PAOLO ZARA,1 ALESSANDRO BARGONI,2 ROBERTA CAVALLI,3 ANNA FUNDARO MARIA ROSA GASCO3 1

Dipartimento di Anatomia, Farmacologia e Medicina Legale, Universita` degli Studi di Torino, Via P. Giuria 9, I-10125 Torino, Italy 2

Dipartimento di Fisiopatologia Clinica, Universita` degli Studi di Torino, Via P. Giuria 9, I-10125 Torino, Italy

3

Dipartimento di Scienza e Tecnologia del Farmaco, Universita` degli Studi di Torino, Via P. Giuria 9, I-10125 Torino, Italy

Received 8 May 2001; revised 29 November 2001; accepted 26 December 2001

ABSTRACT: Idarubicin-loaded solid lipid nanoparticles (IDA-SLN) and idarubicin in solution were prepared and the two formulations were administered to rats, either by the duodenal route or intravenously (iv). The aim of this research was to study whether the bioavailability of idarubicin can be improved by administering IDA-SLN duodenally to rats. Idarubicin and its main metabolite idarubicinol were determined in plasma and tissues by reversed-phase high-performance liquid chromatography. The pharmacokinetic parameters of idarubicin found after duodenal administration of the two formulations were different: area under the curve of concentration versus time (AUC) and elimination half-life were
21 times and 30 times, respectively, higher after IDA-SLN administration than after the solution administration. Tissue distribution also differed: idarubicin and idarubicinol concentrations were lower in heart, lung, spleen, and kidneys after IDA-SLN administration than after solution administration. The drug and its metabolite were detected in the brain only after IDA-SLN administration, indicating that SLN were able to pass the blood–brain barrier. After iv IDASLN administration, the AUC of idarubicin was lower than after duodenal administration of the same formulation. Duodenal administration of IDA-SLN modifies the pharmacokinetics and tissue distribution of idarubicin. The IDA-SLN act as a prolonged release system for the drug. ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:1324–1333, 2002

Keywords: idarubicin; idarubicinol; solid lipid nanoparticles; plasma kinetics; biodistribution

INTRODUCTION Idarubicin (4-demethoxydaunorubicin) is an anthracycline agent that is more lipid soluble than daunorubicin or doxorubicin.1 The drug is available for administration by the intravenous Correspondence to: Maria Rosa Gasco (Telephone: 0116707667; Fax: 011-6707687; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 91, 1324–1333 (2002) ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association

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(iv) and oral routes, and is rapidly absorbed following oral administration, with drug concentrations detectable in the plasma within 30 min.2,3 Idarubicin differs from daunorubicin and doxorubicin, which are not absorbed to any appreciable degree following oral administration. Pharmacokinetic studies of idarubicin have indicated that high concentrations of the cytotoxic metabolite idarubicinol are formed.4–7 The metabolite persists in the plasma far longer than does the parent compound, and in the studies of oral

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BIOAVAILABILITY OF IDARUBICIN FROM SOLID LIPID NANOPARTICLES

idarubicin administration, idarubicinol has been reported to be present in higher concentrations than the parent compound at all the times tested.8,9 Bioavailability varies widely between patients, with a mean range of 20 to 30%.1,5,10 These data suggest a possible first-pass hepatic metabolism.5 Idarubicin has shown excellent antitumor activity against a variety of animal tumors.6 In humans, an idarubicin oral formulation has demonstrated efficacy in the treatment of advanced breast cancer, low-grade non-Hodgkin’s lymphoma, myelodysplastic syndromes, and plasmacytomas, and as a first-line induction therapy for acute myelogenous leukemia where iv anthracycline threatment is precluded.10,11 Its metabolite, idarubicinol, is as active as the parent drug, suggesting that the clinical potency of the latter is due in part to the sustained action of the former.12 Solid lipid nanoparticles (SLN) can be prepared by dispersing a warm oil in water (o/w) microemulsion in a cold aqueous medium.13 It is possible to incorporate in them several lipophilic and hydrophilic drugs, such as peptides, using different approaches during the SLN preparation process.14,15 The main aim of this research was to administer idarubicin-loaded SLN (IDA-SLN) to rats duodenally. We compared the pharmacokinetics and tissue distribution of the drug after duodenal administration of IDA-SLN or IDA in solution (IDA-SOL) to rats. For comparison idarubicin, both as IDA-SLN and IDA-SOL, was also administered iv to another group of animals.

EXPERIMENTAL SECTION Materials

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Italy) weighing 350–450 g. The animals were stabilized under normal conditions for at least 1 week before experimentation, fed with Purina chops, and allowed tap water ad libitum. The experiments complied with the rules set forth in the NIH Guide for the Care and Use of Laboratory Animals. Preparation of IDA-SLN The IDA-SLN were prepared from a warm o/w microemulsion containing stearic acid (0.70 mmol), Epikuron 200 (0.20 mmol) as surfactant, taurocholate sodium salt as cosurfactant (0.68 mmol), and filtered water (111.10 mmol) as continuous phase. To this microemulsion were added 0.06 mmol of idarubicin hydrochloride with hexadecylphosphate, used as a counterion of idarubicin, in a molar ratio 1:2 (idarubicin/hexadecylphosphate). SLN were obtained by dispersing the warm microemulsion (at
708C) in distilled cold water at a ratio of 1:10 (microemulsion:water, v/v) with mechanical stirring. The dispersion was washed three times with filtered water by diaultrafiltration with a TCF2 system (Amicon, Danvers, MA) using a Diaflo YM100 membrane (cut-off 100 000 Da). To determine the amount of idarubicin incorporated into SLN, the SLN water dispersion and the three washing waters were freeze-dried with a Modulyo freeze-dryer (Edwards, Crawley, UK). Preparation of IDA-SOL The IDA-SOL was freshly prepared in filtered water before each experiment, at concentration of 1 mg/mL. Characterization of SLN

Idarubicin hydrochloride was a kind gift from Farmitalia (Milan, Italy). Stearic acid was from Fluka (Buchs, Switzerland); Epikuron 200 (soya phosphatidylcholine 95%) was a kind gift from Lucas Meyer (Hamburg, Germany); taurocholate sodium salt was a kind gift from PCA (Basaluzzo, Italy). Sodium hexadecylphosphate was prepared as indicated by Brown.16 Idarubicinol was prepared by a procedure reported elsewhere.17 The filtered water was from a MilliQ system (Millipore). All other chemicals were of analytical grade.

The average diameters and polydispersity indices of IDA-SLN were determined in filtered water, physiological phosphate buffer (pH ¼ 7.4), and isotonic glycerol solution (2.6% w/w) by photon correlation spectroscopy (PCS) with a N4 MD instrument (Coulter) at a fixed angle of 908 and at a temperature of 258C. Each value was the average of 10 measurements. The polydispersity index is a measure of the size distribution of the nanoparticle population.18

Animals

Zeta Potential Determination

Experiments were performed on albino rats (Wistar derived strain, Morini-S.Polo D’Enza,

The electrophoretic mobility and zeta potential were measured using a 90 PLUS instrument

Photon Correlation Spectroscopy

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(Brookhaven Instrument Corporation, Brookhaven, NY). To determine the zeta potential, SLN samples were diluted with KCl (0.1 mM) and placed in the electrophoretic cell where an electric field of 15.2 V/cm was applied. Each sample was analyzed in triplicate.

for each formulation were killed at 30 min and 4 and 24 h after drug administration to collect samples of liver, heart, lung, kidney, spleen, and brain. All tissue and plasma samples were analyzed by a high-performance liquid chromatography (HPLC) method described later.

Determination of Idarubicin in SLN

Intravenous Administration of Idarubicin Formulations to Rats

The amount of idarubicin incorporated in SLN was determined by a spectrophotometric method using a Lambda 2 spectrophotometer (Perkin Elmer). A weighed amount of freeze-dried SLN or freeze-dried washing waters was dissolved in methanol and analyzed at lmax ¼ 484 nm. Stability of IDA-SLN over Time The stablity of IDA-SLN was evaluated in filtered water, physiological phosphate buffer (pH ¼ 7.4), and isotonic glycerol solution (2.6%, w/w). For this purpose, the IDA-SLN were dispersed in these three media and their average diameter and polydispersity index were determid by PCS after 1, 2, 3, 4, 6, 8, and 24 h. In Vitro Release Kinetics of Idarubicin Two types of release kinetic experiments were performed using a hydrophilic or a double lipophilic/hydrophilic membrane. A multicompartmental rotating cell was used. One milliliter of SLN dispersion (1 mg/mL) was placed in the donor compartment, and the receptor compartment was filled with phosphate buffer at pH 7.4. Each experiment lasted 2 h. At fixed times, the receptor buffer was completely withdrawn and replaced with fresh buffer. The amount of idarubicin in the samples was determined spectrophotometrically. Duodenal Administration of Idarubicin Formulations to Rats IDA-SLN dispersion and IDA-SOL were administered directly into the duodenal lumen of four awake rats through a surgically implanted duodenal cannula at a dose of 1 mg/kg of body weight. This route of administration was selected to avoid a possible influence of gastric emptying. Blood samples were collected, via a surgically implanted cannula inserted into the jugular vein, into heparinized tubes at the designated times (1, 15, 30, 45, and 60 min and 2, 3, 6, 12, and 24 h) until 24 h after administration. To study the tissue distribution, 36 rats received a duodeanal injection of one of the two drug formulations. Four rats JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 5, MAY 2002

A catheter was introduced into the rat’s jugular vein and positioned subcutaneously with the tip in the interscapoular region while the rat was under general anesthesia. The rat was allowed to recover for 24 h, and the catheter was flushed with 0.9% NaCl solution and heparin to avoid the possibility of blood clot obstruction. The day of the experiment, the fixed dose of idarubicin contained in one of the two formulations (1 mg/kg) was injected into the catheter of four rats for each formulation. Blood samples were collected after 1, 15, 30, 45, and 60 min and 2, 3, 6, 12, and 24 h after the injection. All plasma samples were analyzed by the HPLC method described next. HPLC Determination of Idarubicin and Idarubicinol Idarubicin and idarubicinol concentrations were determined by a reversed–phase HPLC method with fluorescence detection.19 For HPLC analysis, a Perkin Elmer Binary LC pump 250 Chromatograph equipped with a Shimadzu RF-551 fluorescence spectrometer and a 5 mm Lichrosorb RP-18 column (250  4.6 mm) was used. The column effluent was monitored at an excitation wavelength of 460 nm and an emission wavelength of 540 nm. The analysis was performed with an 18-min linear gradient from 80:20 to 1:1 (v/v) ratio of 25 mM NH4H8PO4/0.3 M phosporic acid–acetonitrile. The flow rate was 1.3 ml/min. Between analyses, the column was equilibrated to the initial conditions for 15 min. The calibration curve from spiked samples was linear over the concentration range 1 ng/mL–5 mg/mL. Plasma Treatment To determine idarubicin and idarubicinol,20 0.2 mL of plasma were mixed with 1 mL of chloroform-1-propanol (4:1, v/v) and 0.4 mL of 0.4 M borate buffer (pH 9.25). After vortex mixing for 30 s and centrifuging for 5 min (2500  g), the organic phase was evaporated, and the residue was dissolved in 0.5 mL of methanol, and 100 mL aliquots were injected.

BIOAVAILABILITY OF IDARUBICIN FROM SOLID LIPID NANOPARTICLES

Tissue Treatment19 Tissue extracts were prepared by adding 1 mL of methanol followed by 2 mL of Tris buffer (1 M, pH 8.5) to 0.5 g of homogenized tissue. The mixtures were homogenized, and the homogenates allowed to stand on ice for 15 min before adding seven volumes of acetonitrile. The mixtures were vortexed and allowed to stand at room temperature for 15 min before removing the precipitated proteins by centrifugation. Then, 100 mL of clear supernatant were injected directly for HPLC analysis. Statistics and Pharmacokinetic Analysis An overall ANOVA with multiple dependent measures (between: formulations–within: times, in a 3  10 design) was applied to the idarubicin plasma concentrations. This procedure was followed by the post-hoc comparisons Tukey HSD test. Single planned comparisons between the formulations at different times were also conducted. A similar overall ANOVA followed by Tukey HSD post-hoc test and by planned comparisons was also applied to the idarubicin concentrations measured in the excised organs (between: formulations–within: organs in a 3  6 design) and to the five calculated pharmacokinetic parameters (between: formulations–within: parameters in a 3  5 design) for the three different idarubicin formulations. Plasma pharmacokinetic parameters were assessed with a standard software package (Siphar-Windows Simed). The structural model was two- compartment. The minimization algorithm used was POWELL (with Standard criteria). The model was fitted to data using a weighted least squares algorithm with two weighting factors ¼ 1.

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buffer, and isotonic glycerol solution up to 8 h. After 24 h, the average diameter of IDA-SLN did not increase in filtered water and isotonic glycerol solution, but it increased up to 150 nm in physiological phosphate buffer. The zeta potential determined in KCL 0.1 mM was 39.5 mV. The idarubicin release from IDASLN was very low, < 1% after 2 h. Plasma Pharmacokinetics The plasma concentration versus time curves of idarubicin after duodenal injection of the two formulations are reported in Figure 1a. At all times, measured idarubicin plasma concentrations were significantly higher ( p < 0.001) for rats treated with IDA-SLN than for those treated with IDA-SOL. The peak plasma concentration (1 h) of idarubicin after duodenal administration of IDASLN was higher (0.810 mg/mL) than that obtained

RESULTS Percentage of Idarubicin in SLN The amount of idarubicin incorporated in SLN was 5.0% w/w. The loading yield was 98%. Characterization of IDA-SLN The IDA-SLN had an average diameter of 80  10 nm and a polydispersity index of 0.2  0.02. These average diameter and polydispersity values were maintained in filtered water, physiological

Figure 1. (a) Idarubicin plasma concentrations versus time after duodenal administration of the two formulations. (b) Idarubicinol plasma concentrations versus time after duodenal administration of the two formulations. Statistical significances for solution versus SLN are: *** p < 0.001; ** p < 0.01; p* < 0.05. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 5, MAY 2002

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with the solution (0.13 mg/mL). Twenty-four hours after duodenal administration of IDA-SLN, the idarubicin plasma concentration was still 148 ng/ mL, whereas it was undetectable 12 h after IDA-SOL administration. The concentration still present in the plasma 24 h after IDA-SLN administration was approximately equal to the peak plasma concentration obtained with IDA-SOL administation. Except for the initial times, the idarubicinol plasma concentrations were also significantly higher for rats treated with IDA-SLN than with IDA-SOL (Figure 1b). The concentrations of idarubicinol were higher for the solution than for the SLN up to 30 min. This behavior is probably a consequence of the time required for idarubicin to be released from the SLN and metabolized. The plasma concentrations of idarubicin and idarubicinol after iv administration of the two formulations to rats are shown in Figure 2. Idarubicin plasma concentrations were significantly higher ( p < 0.001) after IDA-SLN administration than after IDA-SOL administration. The peak plasma concentration was 6.73 mg/mL after SLN administration, and 1.68 mg/mL after administration of the solution; thus, a fourfold enhancement of peak plasma concentration was obtained using SLN as idarubicin carriers. Twenty-four hours after the iv administration, the idarubicin plasma concentration was 103 ng/mL with IDASLN and 25 ng/mL with IDA-SOL. IDA-SLN administered by the duodenal route gave higher idarubicin levels than those with iv administration of either formulations at 24 h after administration. The plasma pharmacokinetic parameters are reported in Table 1. The AUC of idarubicin after duodenal administration of SLN was > 21 times higher than that after IDA-SOL administration. The AUC of IDA-SLN administered by the iv route was lower than that obtained with IDA-SLN by the duodenal route. After both administration routes, clearance of IDA-SLN was slower than that of IDA-SOL. After duodenal administration, t½b of IDA-SLN was > 29 times higher than that of IDA-SOL; moreover, it was > 4 times higher than IDA-SLN after iv administration. Tissue Distribution of Idarubicin and of Idarubicinol after Duodenal Administration of IDA-SLN and IDA-SOL The tissue distribution of idarubicin and idarubicinol differed after administration of the two JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 5, MAY 2002

Figure 2. (a) Idarubicin plasma concentrations versus time after intravenous administration of the two formulations. (b) Idarubicinol plasma concentrations versus time after intravenous administration of the two formulations. Statistical significances for solution versus SLN are: *** p < 0.001; ** p < 0.01; p* < 0.05.

formulations. At 30 min and 4 h after the administration, the amounts of idarubicin and of idarubicinol were lower in all tissues examined, except in the brain, with IDA-SLN than with IDASOL administered via the duodenum. The difference between IDA-SLN and IDA-SOL was significantly marked for heart, lungs, and spleen, being very much lower in the case of IDA-SLN. In Figure 3 (a and b), the idarubicin and idarubicinol tissue concentrations 24 h after the administration of the two formulations are reported. At 24 hours after administration, the idarubicin concetration in liver and kidneys was higher after IDA-SLN administration than after IDA-SOL administration; the idarubicinol concentration was only higher in liver. The idarubicin concentration in heart after 30 min, 4 h, and 24 h were 58.0, 82.2, and 11.5 ng/g, respectively, with IDA-SLN. The idarubicin concentration in heart after 30 min, 4 h, and 24 h

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Table 1. Pharmacokinetic Parametersa

Drug Formulation IDA-SOL (duodenal) IDA-SLN (duodenal) IDA-SOL (iv) IDA-SLN (iv)

t1/2a (min) 98.8 46.92 16.89 5.68

t1/2b (min) 47.36*** 1410.25 69.79 433.19

AUC (min  mg/mL)

Vd (ml/kg)

Cl (mg/kg/min  mg/mL)

31.36*** 674.2 108.50*** 458.88

2.51 3.12 1.00 1.23

0.034*** 0.0016 0.010 0.0024

a t1/2a, distribution half-life; t1/2b, elimination half-life; AUC, area under the plasma concentration versus time curve; Cl, total body clearence; Vd, volume of distribution. Statistical significance versus solution is ***p < 0.001.

were 135.0, 405.6, and 130.5 ng/g, respectively, with IDA-SOL. IDA-SLN administered by the duodenal route passed the blood–brain barrier (BBB), allowing the presence of idarubicin and of its main metabolite, idarubicinol, in the brain. After 24 h, idarubicin in the heart was almost equal (11.5 ng/g) to that in the brain (11.2 ng/g); idarubicinol was higher in the brain (27 ng/g) than in the heart (15 ng/g).

Figure 3. (a) Idarubicin concentrations in tissues 24 h after duodenal administration of the two formulations. (b) Idarubicinol concentrations in tissues 24 h after duodenal administration of the two formulations. Statistical significances for solution versus SLN are: *** p < 0.001; ** p < 0.01; p* < 0.05.

DISCUSSION Absorption of nano- and microparticulates by the gastrointestinal tract has been studied extensively for the last two decades. The factors controlling intestinal absorption of particulates are now known; size, nature of the polymer, surface charge, composition of vehicle, and coating with surfactant have been determined as critical factors influencing particulate uptake.21–23 Nanoparticles can alter both tissue distribution and clearance of a drug by causing it to acquire the pharmacokinetic parameters of the carrier, as shown by many authors; for examples, Beck et al. studied the peroral administration of drug-loaded polymeric nanoparticles,24 and insulin-loaded polymeric nanoparticles were given to rats as a single intragastric administration producing a decrease in blood glucose level.25 In another study in mice, the amount of camptothecin reaching the brain increased after oral administration of solid lipid nanoparticles carrying the drug,26 although camptothecin was already present in the brain after the administration of the solution. Mu¨ller et al. studied the behavior of piribedil-loaded SLN after oral administration and found prolonged plasma levels and increased bioavailability of the drug.27 In previous research,28 labeled and unlabeled unloaded SLN were administered duodenally to rats, and the uptake and transport of unloaded SLN in lymph and blood were evidenced by transmission electron microscopy (TEM) analysis of unlabeled SLN and by gamma counting of labeled SLN. Successively, tobramycin-loaded SLN were administered duodenally to rats and the results compared with tobramycin solution and tobramycin-loaded nanoparticles administered to rats iv.29 The AUC of tobramycin in SLN administered by the duodenal route was higher than the AUCs of the drug administered iv in SLN or in solution. In previous work, idarubicin-loaded JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 5, MAY 2002

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nanoparticles were prepared,30 the hydrophilic drug being incorporated in SLN as an ion pair with hexadecylphosphate. In the present study idarubicin was administered duodenally using SLN as carriers. Several human studies1,5 have shown that the oral bioavailability of idarubicin is quite low,
30%; however the interindividual variability is quite high. Idarubicin rapidly appears in the plasma after ingestion, and Cmax is reached 1–4 h later. After duodenal administration of IDA-SLN to rats, the bioavailability of the anthracycline increased considerably. Indeed, idarubicin plasma concentrations after the administration of IDASLN were significantly higher than those of idarubicin in solution (Figure 1a); moreover, idarubicin in SLN showed a prolonged circulation time. These results confirm that transmucosal transport, previously demonstrated with unloaded SLN,28 also occurs when nanoparticles are loaded with a drug, as already shown with tobramycinloaded SLN.29 Plasma kinetic parameters differ when idarubicin is administered duodenally in SLN instead of in solution. The AUC of IDA-SLN was higher (
21-fold) than that of IDA-SOL after duodenal administration (Table 1); the increased t½ values of idarubicin incorporated in SLN compared with those found in solution are also consistent with the slower clearance of the drug (and consequently its sustained release) when it is carried in SLN. Considering the iv route alone, the peak plasma concentration of idarubicin was higher for IDA-SLN (6.73 mg/mL) than for IDA-SOL (1.68 mg/mL). This behavior could be related to the difference between the two formulations administered iv: idarubicin is molecularly dispersed in the solution, whereas SLN slowly released idarubicin over time, as shown in an ‘‘in vitro’’ release experiment performed at pH 7.4. A prolonged circulation time of IDA-SLN was also observed after the iv administration; 24 h after the administration, idarubicin plasma concentration with IDA-SLN was significantly higher than that after the iv administration of the solution. A significant higher AUC (about sevenfold) and a lower clearance were obtained after the iv administration of SLN in comparison with that of the solution. The AUC of IDA-SLN after administration into the duodenum was higher than that of IDA-SLN after iv administration (Table 1). These results confirm the previous study where tobramycin-loaded SLN were administered duoJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 5, MAY 2002

denally and iv to rats.29 The difference between the two administration routes for the most part lies in the predominant transmucosal transport of SLN to the lymph; lymphatic transport chiefly contributes to SLN absorption, which could affect the drug bioavailability. Plasma concentrations of the metabolite idarubicinol was higher after IDA-SLN than after IDA-SOL, whether administered duodenally or iv. After administration of IDA-SLN, the idarubicin plasma concentration measured comprises both released and unreleased idarubicin. It should be taken into account that idarubicin must be released from the IDA-SLN to be metabolized, whereas in the solution, it is immediately available because it is molecularly dissolved. This difference may explain the lower idarubicinol concentrations obtained at the initial times (1 and 15 min) after IDA-SLN administration. Idarubicinol has a marked cytotoxicity compared with other metabolic derivatives of anthracyclines, such as doxorubicinol. In vitro, myeloma cells required at least 32-times lower extracellular idarubicinol concentrations than doxorubicinol for the same cytotoxic effect, whereas cytotoxicity of idarubicinol was 2–10-fold higher than doxorubicin.31 In the same research, a good correlation was found between intracellular accumulation of idarubicin as expressed in AUC values and cytotoxicity. In our experiments, IDASLN administration produced high plasma concentration of the drug and its metabolite in plasma. We have recently shown that SLN can increase intracellular accumulation of chemotherapic agents.32 From the data presented here, we may hypothize that the change in pharmacokinetic parameters with IDA-SLN could improve the clinical efficacy of the drug. The tissue distribution profiles of idarubicin after the administration of the two formulations differ significanly. Except in the brain, idarubicin and idarubicinol concentrations are lower after IDA-SLN administration than with the solution, especially for the liver, spleen, and kidneys, 30 min and 4 h after administration. At 24 h after the administration, the idarubicin concentrations are still lower in heart, lungs, spleen, and kidney (Figure 3a). The idarubicinol concentrations in the tissues showed a similar pattern of distribution. Idarubicinol was present in the brain 4 and 24 h after the administration of IDA-SLN whereas it is not detectable after the administration of the solution.

BIOAVAILABILITY OF IDARUBICIN FROM SOLID LIPID NANOPARTICLES

This result is a confirmation of the passage of the SLN across the BBB. The reduced volume of distribution after SLN administration is probably one of the reasons for the low amounts of idarubicin and idarubicinol present in the tissues after IDA-SLN administration. In particular, we observed lower concentrations of both idarubicin and idarubicinol in the heart after IDA-SLN than after IDA-SOL administration. In our study, the drug concentrations in heart were lower after 30 min, 4 h, and 24 h with IDA-SLN administration. Preclinical studies have shown that idarubicin possesses cardiac toxicity in the mouse, rat, rabbit, and dog. It has been suggested that the high plasma peak that commonly occurs after bolus injections of anthracyclines causes a rapid increase in oxygen radicals in myocardial cells; this increase can be the cause of cardiotoxicity that develops after repetitive application of antracyclines. Casazza has reported an idarubicin reduction in cardiotoxicity about one third that of daunorubicin.13 The lower concentration of idarubicin and its metabolite in the heart after the SLN administration could be of great clinical interest because there is clinical evidence that idarubicin-associated cardiotoxicity is related to the peak level but not to the AUC.33 It is still not clear how important the plasma peak is for the induction of apoptosis in malignant cells, which is the pharmacological effect.34 Idarubicin incorporated in SLN was found in the rat brain. Limited drug distribution is the main obstacle in treating tumors of the central nervous system because most agents do not cross the BBB efficiently. For this reason, although anthracyclines are currently employed for the treatment of several tumors, they are not used in the treatment of brain tumors. It is generally accepted that one of the reasons anthracyclines are unable to cross the BBB is their relatively low lipophilicity.35 Another reason for this failure could be the presence of P-glycoprotein in endothelial cells of the brain vessels; P-glycoprotein potentially protect some important organs from natural environmental toxins as well as from anticancer drugs.36 Many possible mechanisms of nanoparticlemediated drug transport to the brain are reported by Kreuter,37 such as an increased retention of nanoparticles in the brain blood capillaries, higher concentration gradient, and endocytosis or the transcytosis of the nanoparticles. In our study the presence of idarubicin and idarubicinol in the brain after duodenal administration is

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significant and confirms previous results obtained with labeled SLN.38 In other research on another anthracycline, doxorubicin incorporated in SLN and administered iv to rats39 was recovered in the brain together with its main metabolite, doxorubicinol. Also, the administration of idarubicinloaded SLN by the duodenal route permits transport of nanoparticles, and consequently of the drug, through the BBB. There are number of reports of polymeric nanoparticulate carriers enabling drugs that are unable to cross the BBB alone to do so, and thus the drug reach the brain; but these studies primarily used the the iv route.40,41 At the moment, research is in progress in our lab to understand the mechanism of SLN passage through the BBB. In conclusion, when idarubicin in SLN and solution are administered duodenally, pharmacokinetics and biodistribution are different. The duodenal route of administration of IDA-SLN acts as a prolonged-release system, improving bioavailability compared with solution and affording a sufficently high level of the drug even after 24 h. The AUC of IDA-SLN is higher than when the same formulation is administered iv. Idarubicin and idarubicinol in tissues are lower after IDASLN than after IDA-SOL. IDA-SLN are able to cross the BBB after duodenal administration. These changes in pharmacokinetics and biodistribution might be useful to reduce the toxicity and to increase the clinical efficacy of idarubicin.

ACKNOWLEDGMENT The work has been supported by Progetto Nazionale Tecnologie Farmaceutiche.

REFERENCES 1. Holligshead LM, Faulds D. 1991. Idarubicin: A review of its pharmacodynamic and pharmacokinetics properties and therapeutic potential in the chemotherapy of cancer. Drugs 42:690–719. 2. Gillies HC, Herriott D, Liang R, Ohashi K, Roger HJ, Harpr PG. 1987. Pharmacokinetics of idarubicin following intravenous and oral administration in patients with advanced cancer. Br J Clin Pharmcol 23:303–310. 3. Speth PAJ, Loo FAJ, Linssen PCM, Wessels HMC, Haanen C. 1986. Plasma and human leukemic cell pharmacokinetics of oral and intravenous 4-demethoxydaunorubicin. Clin Pharmacol Ther 40:643–649. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 5, MAY 2002

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