Cancer Therapy: Preclinical

Antitumor Alkyl Ether Lipid Edelfosine: Tissue Distribution and Pharmacokinetic Behavior in Healthy and Tumor-Bearing Immunosuppressed Mice Ander Estella-Hermoso de Mendoza,1Miguel A. Campanero,2 Janis de la Iglesia-Vicente,3 Consuelo Gajate,3,4 3 1 Faustino Mollinedo, and MarI¤aJ.Blanco-Prieto

Abstract Purpose: The present study investigates and compares the dose-dependent pharmacokinetics and oral bioavailability of edelfosine in healthy, immunodeficient, and tumor-bearing immuno- suppressed mouse animal models, as well as edelfosine uptake and apoptotic activity in the Z-138 mantle cell lymphoma (MCL) cell line. Experimental design: Biodistribution study of edelfosine was done in both BALB/c and severe combined immune deficiency (SCID) mice, and then the in vivo behavior of the drug after i.v. and oral administration was monitored. Results: We found that edelfosine is incorporated and induces in the Z-138 human mantle cell lymphoma cell line, whereas normal resting peripheral blood human lymphocytes were not affected. In vivo biodistribution studies revealed that accumulation of edelfosine in the tumor of a MCL-bearing mouse animal model was considerably higher (P < 0.01) than in the other organs analyzed. Besides, no statistical differences were observed between the pharmacokinetic parameters of BALB/c and SCID mice. Edelfosine presented slow elimination and high distribu- tion to tissues. Bioavailability for a single oral dose of edelfosine was <10%, but a multiple-dose oral administration increased this value up to 64%. Conclusion: Our results show that edelfosine is widely scattered across different organs, but it is preferentially internalized by the tumor both in vitro and in vivo.Ourdata,togetherwiththe apoptotic action of the drug on cancer cells, support a rather selective action of edelfosine in cancer treatment, and that multiple oral administration is required to increase oral bioavailability.

Edelfosine is the prototype molecule of a promising family administered orally (1–4). This class of synthetic anticancer of antitumor compounds collectively known as synthetic agents acts at the level of the cell membrane, unlike most cur- alkyl-lysophospholipids, which include also clinically relevant rently available chemotherapeutic drugs that target the nuclear drugs such as and perifosine, and which can be DNA, and induces selective apoptosis in malignant cells, spar- ing normal cells (5–7). Although the precise mechanism of action is not yet fully elucidated, edelfosine-induced apoptosis

1 Authors’ Affiliations: Departamento de Farmacia yTecnologI¤a Farmace¤ utica, is mediated by Fas/CD95 death receptor (8–10), c-jun NH2- 2 Facultad de Farmacia, University of Navarra; Servicio de FarmacologI¤aClI¤nica, terminal kinase activation (11, 12), endoplasmic reticulum stress 3 ClI¤nica Universitaria, Pamplona, Spain; Centro de Investigacio¤ ndelCa¤ ncer, Instituto (13), and mitochondria (14). Edelfosine has been applied as a de BiologI¤a Molecular y Celular del Ca¤ ncer, Consejo Superior de Investigaciones 4 purging agent in autologous transplantation (15), Cientificas-Universidad de Salamanca; and Unidad de Investigacio¤ n, Hospital and recent data suggest a putative clinical relevance for this Universitario de Salamanca, Salamanca, Spain Received 7/9/08; revised 9/8/08; accepted 10/9/08. agent in cancer (7). However, there is very little information about Grant support: Caja Navarra Foundation, the Spanish Ministry of Science and the disposition and oral bioavailability of edelfosine in vivo. Innovation (SAF2008-02251, SAF2007-61261, SAF2005-04293, and PCT- The kinetic behavior of radioactive edelfosine in rats and 090100-2007-27 and RD06/0020/1037 Red Tema¤ tica de Investigacio¤ n mice after i.v. administration has previously been investigated. Cooperativa en Ca¤ ncer, Instituto de Salud Carlos III), the ‘‘Fondo de Investigacio¤ n Sanitaria’’and European Commission (FIS-FEDER 06/0813), Fundacio¤ n ‘‘la Caixa’’ Arnold et al. (16) administered 15 daily injections of an 6 (BM05-30-0), and Junta de Castilla y Leo¤ n (CSI01A08). C. Gajate is supported by amount of radioactive edelfosine of 1 10 counts per minute the Ramo¤ n y Cajal Program from the Spanish Ministry of Science and Innovation. (cpm) i.v. to methylcholantrene-induced fibrosarcoma-bearing A.E-H. de Mendoza is supported by a research grant from the Department of mice. Results showed that 1 day after the last injection, an Education of the Basque Government (BFI06.37). overall activity of only 2 106 cpm was recovered. This The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance indicated that edelfosine did not accumulate in the organism. with 18 U.S.C. Section 1734 solely to indicate this fact. However, measurement of total radioactivity does not reflect Requests for reprints: MarI¤a J. Blanco-Prieto, Department of Pharmacy and the pharmacokinetic behavior or the tissue distribution of the Pharmaceutical Technology, School of Pharmacy, University of Navarra, C/ parent drug edelfosine because the radiolabel may be included Irunlarrea 1, E-31080 Pamplona, Spain. Phone: 34-948-425-600, ext. 6519; Fax: 34-948-425-649; E-mail: [email protected]. in some metabolites. F 2009 American Association for Cancer Research. Ko¨tting et al. (17) studied biodistribution in rats after oral doi:10.1158/1078-0432.CCR-08-1654 administration of edelfosine. After 24 hours, no significant

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immune compromised animals is imperative, and thus we Translational Relevance studied the tissue and tumor drug distributions in these immunodeficient mice. In addition, in vitro experiments with Edelfosine is the prototype molecule of a family of the Z-138 mantle cell lymphoma (MCL) cell line, used for anticancer drugs collectively known as synthetic alkyl- xenograft assays, were done to determine both edelfosine lysophospholipids. This drug holds promise as a selective uptake and apoptotic activity. antitumor agent, and a number of preclinical assays are in progress. However, information about the tissue dis- Materials and Methods position and oral bioavalilability of edelfosine in vivo is absent or rather scarce. Here we have studied the phar- Chemicals. Edelfosine was from INKEYSA. Platelet-activating factor macokinetics, biodistribution, and oral bioavailability of and PBS (10 mmol/L phosphate, 0.9% NaCl) were obtained from edelfosine in both tumor-free and tumor-bearing mice Sigma-Aldrich. Formic acid 99% was purchased from Fluka, and by the use of a high-performance liquid chromatography- methanol was obtained from Merck. All solvents used for the analysis mass spectrometry technique.We found that edelfosine is were of analytic grade. RPMI 1640 cell culture medium, heat-inactivated scattered in different organs but accumulated preferentially FCS, and antibiotics were from Life Technologies, Invitrogen. in the tumor. Multiple oral administration of edelfosine was Animal experiments. The protocol for animal experiments was required to reach a clinically relevant plasma concentration. approved by the University of Navarra Animal Experimentation Ethics The preclinical pharmacokinetic data reported here are Committee (protocol no. 060-06). BALB/c mice (20 g) were obtained essential to predict the human disposition of edelfosine from Harlan Interfauna Ibe´rica S.L. Animals received a standard diet and water ad libitum, except for the animals who received the oral doses, and provide a basis for the development of the experi- which were fasted for 24 h before administration. Female CB17-SCID mental design of clinical phase I trials as well as to set up a mice were purchased from Charles River Laboratories. In xenograft drug dosing scheme in cancer treatment. experiments, 8-wk-old SCID mice were s.c. inoculated into the lower dorsum with 1 107 Z-138 cells in 100 AL of PBS and 100 ALof Matrigel basement membrane matrix (Becton Dickinson). levels of edelfosine were detected either in feces or urine. This Cell culture and isolation of human peripheral blood lymphocytes. The Z-138 MCL cell line (19) was grown in RPMI 1640 culture medium information led them to conclude that f96% of the drug was supplemented with 10% FCS, 2 mmol/L L-glutamine, 100 units/mL absorbed in the first 24 hours (17). penicillin, and 100 Ag/mL streptomycin at 37jC in humidified 95% air As yet no precise pharmacokinetic parameters have been and 5% CO2. determined for free edelfosine. Characterization of the phar- Lymphocytes were isolated from fresh human peripheral blood by macokinetics of edelfosine is crucial to understand the in vivo dextran sedimentation, centrifugation on Ficoll-Paque density gra- concentration-effect and concentration-toxicity relationships dients, and monocyte depletion by culture dish adherence as described and to choose dosing regimens. Furthermore, knowledge of previously (20). the in vivo concentration range, oral bioavailability, and phar- Edelfosine uptake in cell culture. Drug uptake was measured as 6 macokinetics of free edelfosine is crucial to evaluate the effects previously described (5) after incubating cells (10 ) with 10 nmol 3 of edelfosine delivery systems on the pharmacokinetic behavior [ H]edelfosine for 1 h in RPMI 1640/10% FCS and subsequent exhaustive washing (six times) with PBS + 2% bovine serum albumin. of this antineoplastic agent. [3H]Edelfosine (specific activity, 42 Ci/mmol) was synthesized by To determine the levels of edelfosine in plasma, tissue, or tritiation of the 9-octadecenyl derivative (Amersham Buchler). tumor, sensitive and selective techniques must be used to detect Apoptosis assay. Quantitation of apoptotic cells after treatment with only the compound of interest and not others, like metabolites edelfosine was calculated by flow cytometry as the percentage of cells in or very similar molecules. We have recently developed a simple the sub-G1 region (hypodiploidy) in cell cycle analysis, as previously and highly selective and sensitive high-performance liquid described (14). chromatography-mass spectrometry (HPLC-MS) technique Pharmacokinetic studies after oral administration. Three treatment with a quantitation limit of 0.3 ng, which avoids the use of lines were followed. A group of BALB/c mice and a group of SCID non– radiolabeled compounds (18). With this technique, it is pos- tumor-bearing mice were treated with a daily oral administration of 30 sible to quantify edelfosine concentrations in plasma, tissues, mg/kg edelfosine for 6 d. At various time points after the first oral administration (0, 1, 2, 5, 8, and 24 h), blood was collected in EDTA or tumor accurately and sensitively to determine pharmacoki- surface-coated tubes and then centrifuged at 2,000 g for 10 min netic parameters. (4jC) to collect plasma (100 AL). After the administration of the sixth The present study investigates and compares the dose- dose, blood samples were collected at the same time intervals (0, 1, 2, dependent pharmacokinetics and oral bioavailability of edel- 5, 8, and 24 h). Then, animals were sacrificed and spleen, liver, lungs, fosine in healthy, immunodeficient, and tumor-bearing kidneys, heart, brain, stomach, and intestine were collected and immunosuppressed mouse animal models. weighed. Tissues were homogenized in 1 mL of PBS (pH 7.4) using a Severe combined immune deficiency (SCID) mice are Mini-bead Beater (BioSpect Products, Inc.) and centrifuged at 10,000 routinely used as hosts for malignant cells and for in vivo g for 10 min. Both plasma and tissue supernatants were collected testing of new antitumor agents. SCID mice are characterized by and stored at -80jC until HPLC-MS analysis was done. An additional the complete inability of the adaptive immune system to group, MCL-bearing SCID mice, received daily oral administration of 30 mg/kg edelfosine for 6 d. After the administration of the sixth dose, mount an appropriate immune response due to absence of blood samples were collected at the same time intervals (0, 1, 2, 5, 8, functional lymphocytes as a result of defects in T- and B-cell and 24 h), and then the animals were sacrificed; liver and kidneys were development. On these grounds, use of SCID mice permits the collected, weighed, and processed as previously described. Tumors were long-term engraftment of human tumor cells. Because most of collected as the other organs. the in vivo antitumor drug testing assays are carried out with Pharmacokinetic study after i.v. administration. An i.v. single dose SCID mice, accomplishment of the pharmacokinetics in these of 200 Ag (10 mg/kg) was administered to BALB/c mice via the tail vein.

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At various time points after administration (0, 1, 2, 5, 8, and 24 h), Tissue distribution of edelfosine in tumor-bearing SCID blood was collected in EDTA surface-coated tubes and then centrifuged mice. Because Z-138 MCL cells incorporated edelfosine, we j A at 2,000 g for 10 min (4 C) to separate the plasma (100 L). After next analyzed the in vivo tissue distribution of the drug in 24 h, animals were sacrificed by cervical dislocation and tissues were tumor-bearing mice. Immunodeficient SCID mice were injected collected, weighed, and processed as explained before. s.c. with Z-138 MCL cells, and xenografts were allowed to Linearity study of the dose. BALB/c mice were given single i.v. doses 3 of 100, 200, 250, and 600 Ag (5, 10, 12.5, and 30 mg/kg) of edel- establish to an average size of 300 mm before drug oral fosine dissolved in PBS via the tail vein. At various time points treatment. The tissue distribution expressed as tissue/plasma after administration (0, 0.5, 1, and 2 h), blood was collected as ratio of edelfosine concentrations after multiple oral dose described above. administration of an edelfosine dose of 30 mg/kg (6-day Plasma/tissue extraction procedure for analytic process. Ten micro- treatment) to MCL-bearing SCID mice is shown in Fig. 2A. This grams of platelet-activating factor (0.2 mg/mL) used as internal oral 30 mg/kg dose was perfectly well tolerated by the mice, standard were added to 100 AL of plasma or tissue supernatant. A and we observed no side effects or body weight loss (data not A mixture (190 L) of 1% formic acid/methanol (5:95, v/v) was added to shown). Mean concentration of edelfosine in plasma 24 hours precipitate the proteins. Samples were vortexed for 1 min, and after after the sixth dose was 10.69 Ag/mL. Kidney and liver were centrifugation (20,000 g, 10 min), 25 AL of the supernatant were selected for drug accumulation because they are the major analyzed by HPLC-MS (18). HPLC-MSanalysis for edelfosine. The method used for edelfosine drug clearance tissues. These organs showed a 2-fold higher quantitation was a slight modification of an earlier HPLC-MS method drug concentration compared with that in plasma (Fig. 2A). (18). Quantitation was achieved by comparing the observed peak area Interestingly, the tumor showed a high accumulation of ratios of edelfosine and internal standard of the samples to a regression edelfosine 24 hours after the dose on day 6 (Fig. 2A). The curve determined from drug-fortified plasma and tissue standards. The mean concentration of edelfosine in the tumor at this stage was calibration curve of edelfosine in plasma showed good linearity over 138.64 Ag/g, which is f13 times higher than the plasma drug the concentration range of 0.1 to 75 Ag/mL. Linear range of edelfosine concentration, and 5 to 6 times higher than the drug con- concentrations in lung, kidney, liver, spleen, brain, heart, stomach, and centration found in kidney and liver. Moreover, the tumor/ intestine homogenates was 0.2 to 31.75 Ag/mL. plasma concentration ratio of edelfosine was significantly Data analysis. The plasma concentration data were analyzed by noncompartmental and compartmental analyses using WinNonlin higher (P < 0.01) than the corresponding ratios observed in Professional Edition version 2.1 (Pharsight). Pharmacokinetic analysis both kidney and liver. Thus, an in vivo and in vitro comparison was done for two different doses of 10 and 30 mg/kg with plasma indicates that edelfosine is selectively taken up by tumor cells. samples obtained from experiments with all mice. The area under the Tissue distribution of edelfosine in non–tumor-bearing SCID plasma concentration versus time curve (AUC) was determined using mice. The tissue distribution of edelfosine was also analyzed the log-linear trapezoidal rule with extrapolation to 24 h. Clearance in control non–tumor-carrying SCID mice following the same (CL) is the volume of plasma completely cleared of a specific protocol as with tumor-bearing SCID mice. The mean plasma compound per unit time by the organism; it was calculated by dividing concentration of edelfosine at 24 hours after the sixth dose was the dose by AUC. The maximum plasma concentration (Cmax) was 12.12 Ag/mL. Figure 2B shows a higher distribution of determined directly from the plasma concentration-time curve. Oral edelfosine to lung, spleen, intestine, liver, and kidney. In bioavailability (F) was determined by ratio of the dose-normalized AUCs following oral and i.v. administration. Volume of distribution at contrast, low drug concentrations were found in heart, brain, and stomach (Fig. 2B). Compared with tumor-bearing SCID steady state (Vss) is the volume of fluid that would be required to contain the amount of drug in the body if it were uniformly distributed mice, no statistical differences in liver/plasma concentration at a concentration equal to that in the plasma. The half-life value (t1/2) ratios (P > 0.05) were found in non–tumor-bearing SCID mice, refers to the time taken for plasma concentration to decrease by 50%. whereas the kidney was found to have a statistically highly Disposition (t1/2a) and elimination (t1/2h) half-lives were determined significant increase (P < 0.01; Fig. 2A). a b using the following formulas: t1/2a = ln(2)/ and t1/2h = ln(2)/ , where Tissue distribution in healthy BALB/c mice after oral admin- a and b represent the disposition and elimination constant rates, istration. A multiple oral dose administration of 30 mg/kg respectively, calculated from the intercompartmental mass transfer rates edelfosine (6-day treatment) was done to healthy BALB/c mice. (k , k ) and elimination rates (k ). 12 21 10 Twenty-four hours after the last dose (day 6), the mean plasma Statistical analysis. Mean values of the tissue/plasma concentration A ratio of more than two groups were analyzed by an ANOVA followed by concentration was 13.22 g/mL. As is shown in Fig. 2B, the Dunnett’s test for a double comparison using Social Package of highest tissue/plasma concentration ratios were found for Statistical Sciences (SPSS). The presence of differences in tissue/plasma kidney and intestine. Lower ratios were observed for stomach, ratios was measured by the Mann Whitney U test for double spleen, lung, and liver. A low amount of edelfosine was comparisons using the same program. A correlation analysis was done detected in the heart and brain. for the study of the linearity of the dose. P < 0.05 was considered Figure 2B also shows that no statistically significant differ- statistically significant for all statistical tests. ences were appreciated among the tissue/plasma concentration ratios of lung, heart, brain, intestine, and liver of BALB/c and Results non–tumor-bearing SCID mice. The stomach of BALB/c mice after the multiple oral administration of 30 mg/kg presented a Edelfosine uptake and induction of apoptosis in MCL cells. Edel- higher ratio compared with SCID mice after the administration fosine has been reported to be selectively incorporated into of same dose (P < 0.05), and the spleen/plasma concentration malignant cells leading to their demise, whereas normal cells ratio showed a significant decrease (P < 0.05) with regard to were spared (5, 8). We found that the Z-138 MCL cell line took SCID non–tumor-bearing mice. up significant amounts of edelfosine and underwent apoptosis, Tissue distribution in healthy BALB/c mice after i.v. admin- whereas normal resting peripheral blood lymphocytes were not istration. A single i.v. administration of 200 Ag of edelfo- affected and drug incorporation was scarce (Fig. 1). sine (10 mg/kg) was administered to healthy BALB/c mice.

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Fig. 1. Selective uptake and induction of apoptosis in mantle lymphoma cells. A, edelfosine uptake was determined after incubating human mantle lymphoma Z-138 cells and resting human peripheral blood lymphocytes (PBL)with10nmol[3H]edelfosine for 1h. B, Z-138 cells and resting human peripheral blood lymphocytes were incubated for 24 h with 10 Amol/L edelfosine, and apoptosis was then quantitated as the percentage of cells in the sub-G1region in cell cycle analysis by flow cytometry.

After 24 hours, edelfosine concentration in plasma was 50.7 F 28.1 Ag/mL and a Cmin of 2.5 F 1.3 Ag/mL, 24 hours f2.5 Ag/mL. The tissue distribution of edelfosine expressed after i.v. administration. The obtained pharmacokinetic param- as tissue/plasma ratio can be observed in Fig. 2B. No drug was eters are listed in Table 1. detectable in heart and very little was found in brain. Kidney Plasma concentration-time data of edelfosine were well and intestine presented the highest tissue/plasma concentra- described by a biexponential function (model selection tion ratios, followed by lung, liver, stomach, and spleen. criterion, -24.12) following i.v. administration. The half-lives Pharmacokinetic characterization after i.v. administration. Fig- of distribution (t1/2a) and elimination (t1/2h) phases were ure 3 depicts the concentration of edelfosine in mouse plasma 0.286 F 0.076 and 22.288 F 14.016 hours, respectively. The plotted against time after a single-dose i.v. administration of systemic clearance (CL) and steady-state volume of distribution 200 Ag of edelfosine (10 mg/kg) to BALB/c mice. Pharmaco- (Vss) were 0.056 F 0.030 L/h/kg and 1.285 F 0.556 L/kg, kinetic analysis of edelfosine in blood plasma showed a Cmax of respectively. There was little variability in most of the values of

Fig. 2. A, tissue/plasma concentration ratios of edelfosine after ( ) multiple-dose oral administration of 30 mg/kg of edelfosine for 6 d to non ^ tumor-bearing SCID mice and ( ) multiple-dose oral administration of 30 mg/kg edelfosine for 6 d to MCL-bearing SCID mice. Columns, mean (n = 6); bars, SD. B, tissue/plasma concentration ratios of edelfosine after single i.v. dose of 10 mg/kg of edelfosine to healthy BALB/c mice (5); multiple dose oral administration of 30 mg/kg of edelfosine for 6 d to healthy BALB/c mice (n); and multiple dose oral administration of 30 mg/kg of edelfosine for 6 d to non ^ tumor-bearing SCID mice ( ). Columns, mean (n = 6); bars, SD. Asterisks indicate stastically significant values: *, P < 0.05; **, P < 0.01.

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Pharmacokinetic characterization after oral administration. A Because edelfosine shows a variety of medical applications, a very low concentration of edelfosine could be found in plasma pharmacokinetic study in different murine species, each one along the time interval of 0, 1, 2, 5, 8, and 24 hours after oral appropriate for distinct in vivo assays, is required. We carried administration of 30 mg/kg edelfosine in BALB/c mice, as can out a complete pharmacokinetic analysis in SCID mice, widely be observed in Fig. 3. These concentrations were close to the used for antitumor activity testing, and in BALB/c, useful for detectable limits of the technique and not sufficient for the additional putative edelfosine applications (21). In the present calculation of pharmacokinetic parameters. Oral bioavailability study, we set up a MCL-bearing animal model in immunode- for edelfosine was calculated from the ratios of the average ficient SCID mice to analyze the distribution of edelfosine in values for AUC0!24 h for the oral and i.v. doses. Oral tissues and tumor. We found that the Z-138 MCL cell line took bioavailability (F) for edelfosine was found to be <10%. up significant amounts of edelfosine, subsequently undergoing Pharmacokinetic parameters obtained after compartmental apoptosis, whereas normal cells hardly incorporated the drug analysis of experimental data are shown in Table 1. Oral bio- and were spared. The in vitro and in vivo data reported here availability increased to 64% when a steady state was reached indicate a remarkably selective accumulation of edelfosine in F A after the 6th day. At this point, a Cmax of 14.46 2.97 g/mL at tumor cells. F A a tmax of 3 hours and a Cmin of 7.65 2.45 g/mL were In the MCL-bearing animal model, kidney and liver were the measured 24 hours after the last administration. Due to the fast only organs extracted as they are considered to be the main elimination during the disposition phase, only the h elimina- drug distribution and clearance tissues. Comparing the tissue tion phase could be characterized. AUC presented a value distribution of edelfosine between these tumor-bearing SCID of 258.54 F 58.16 Ag h/mL. The half-life of elimination was mice and the non–tumor-bearing SCID mice (Fig. 2A), no 30.404 F 26.761 hours, with a volume of distribution at statistically significant differences were seen in liver (P > 0.05), steady state of 1.563 F 0.163 L/kg and a systemic clearance whereas a major decrease (P < 0.01) was found in the kidneys of 0.057 F 0.039 L/h/kg, similar values to those obtained after of the tumor-bearing mice. The low renal uptake in the tumor i.v. administration of edelfosine, with no statistically signifi- model might be a result of increased drug uptake in the tumor cant differences. with less drug available for renal excretion. In addition, the To determine possible interstrain pharmacokinetic differ- lower tissue/plasma ratio in kidneys of the MCL-bearing mice ences, these results were compared with the results obtained might be also due to a renal failure usually expected at after edelfosine was administered to non–tumor-bearing SCID advanced stages of the disease (22). mice. After reaching the steady state, pharmacokinetic param- Figure 2B shows that the distribution of edelfosine in mice is eters were estimated (Table 1). Whatever the parameters studied mainly predominant in spleen, intestine, and kidney. The (half life of elimination, clearance value, steady-state volume of significant drug uptake in the gut might underlie the major distribution, and AUC), no statistically significant differences toxicity of edelfosine in the gastrointestinal tract (1). The high were observed between SCID and BALB/c mice. presence of edelfosine in spleen can be explained by the incorporation of the drug into the membrane of erythrocytes inducing a hemolytic effect (17). These RBC end up being cleared from the blood in the spleen. We found a higher concentration of edelfosine in the spleen of SCID mice as compared with BALB/c mice, and this could be due to putative differences in the spleen of immunodeficient mice. No statistically significant differences were observed between the tissue/plasma concentration ratios of edelfosine in the lung, heart, brain, intestine live, and kidney of immunosuppressed mice (SCID mice) and BALB/c mice (Fig. 2B). Moreover, no significant differences were observed in the mean plasma concentrations of edelfosine 24 hours after the sixth dose of the multiple oral administration of 30 mg/kg to tumor-bearing SCID mice, non–tumor-bearing SCID mice, and healthy BALB/ c mice (10.69, 12.12, and 13.22 Ag/mL, respectively). As a result, a similar pharmacokinetic profile would be expected in both strains. Therefore, we performed the characterization of edelfosine pharmacokinetic profile in healthy BALB/c mice. Knowledge of the pharmacokinetic parameters of a drug is a must to ensure that exposure is sufficient to evaluate its phar- Fig. 3. Time-concentration curve data of edelfosine 24 h after single-dose i.v. administration of 10 mg/kg (y) and oral single-dose administration of 30 mg/kg to macodynamic properties, to predict the pharmacokinetics in BALB/c mice (E). Points, mean (n = 6); bars, SD. other species, and to develop an appropriate dosage form.

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Table 1. Comparison of pharmacokinetic parameters of edelfosine after administration of different doses to BALB/c and SCID mice by different routes (n = 6, mean F SD)

Parameters BALB/c, 200 Mg i.v. single dose BALB/c, 600 Mg oral multiple oral dose SCID, 600 Mg oral multiple dose

t 1/2a (h)0.286 F 0.076 — — t 1/2h (h)22.288 F 14.016 30.404 F 26.761 19.263 F 8.262 C max (Ag/mL)50.681 F 28.096 14.460 F 2.970 18.176 F 1.023 -1 k 21 (h )0.346 F 0.137 — — -1 k 10 (h )0.312 F 0.158 — — -1 k 12 (h )1.943 F 0.494 — — CL (L/h/kg)0.056 F 0.030 0.057 F 0.039 0.067 F 0.021

V ss (L/kg)1.285 F 0.556 1.563 F 0.163 1.256 F 0.284 AUC0-24 (Ag h/mL)125.440 F 52.511 258.241 F 58.163 265.235 F 51.823

NOTE: No statistical differences were found among the parameters (P > 0.05).

Following i.v. administration of a dose of 10 mg/kg appreciated. The rapid distribution is mostly observed to organs edelfosine to BALB/c mice, mean blood concentrations that are highly irrigated, such as the kidney, liver, intestine, declined biphasically, with an initial half life of f0.3 hour and lung. (Table 1). After 2 hours, plasma concentrations seemed to The steady-state volume of distribution was 1.26 L/kg, much decline more slowly. Given that blood concentrations had greater than that of the vascular volume in mice and appro- declined f6-fold during the initial phase, the contribution of ximately twice its total body water (23) normalized to body the secondary phase to the total drug exposure is of little weight, suggesting that edelfosine is highly distributed extra- relevance. According to these half-life values, edelfosine vascularly. In fact, k12 presented a value five to six times higher presented a rapid distribution to central compartment tis- than the corresponding k21 value. sues, whereas a slower distribution to deep compartments was Edelfosine showed very low clearance values in mice compared with its respective liver and kidney blood flow. It is interesting to note that this value of 0.056 L/h/kg is translated to barely 1% of the liver blood flow (23), suggesting a much lower intrinsic hepatic clearance value. We verified that as the drug dose increases the AUC increases equivalently, meaning that edelfosine exhibits linear pharma- cokinetics. In this study, we show that edelfosine shows a linear correlation not only between the administered dose and the resulting AUC but also between the administered dose and the corresponding Cmax.Thisfactsuggeststhatthereisno saturation of the elimination process of edelfosine at the concentration range between 5 and 30 mg/kg. Although the mechanism for its clearance is still unknown, renal elimination of the drug might well happen because edelfosine is present in kidney. Previous studies have estab- lished that amphiphilic cationic drugs are efficiently removed by the liver from the blood (24). Additionally, amphiphilic cationic drugs were detected in other secretion organs of cationic amphiphilic drugs, such as the intestinal mucosa (25) and kidney. These data are also in good agreement with our results and the ones obtained by Marschner et al. (26). The first determinations of oral absorption of 15 Amol edelfosine (38 mg/kg) in 1992 suggested that f96% of edel- fosine was absorbed by rats within 24 hours (17). Conversely, our study shows very low plasma concentrations of edelfo- sine 24 hours after the oral administration of a single-dose of 30 mg/kg, revealing very low absorption of edelfosine by the gastrointestinal tract in mice. However, oral bioavailability showed a significant increase (64%) after daily administration of 30 mg/kg edelfosine for 6 days. As can be seen from our studies, edelfosine showed a mod- erate volume of distribution and a rapid and varied distribu- Fig. 4. Correlation between doses of edelfosine of 100, 200, 250, and 600 Ag tion through the organism when it is administered by an i.v. (5,10,12.5,and30mg/kg)andthecalculatedAUC(A)andCmax (B). Points, mean (n = 6); bars, SD. route. This information, along with the amphiphilic properties,

www.aacrjournals.org 863 Clin Cancer Res 2009;15(3) February 1, 2009 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2009 American Association for Cancer Research. Cancer Therapy: Preclinical may lead us to consider the possibility of passive diffusion treatment regimens as well as the development of drug delivery through the intestinal epithelium as a mechanism of absorp- systems to treat MCL and other tumors. In addition, because no tion for edelfosine. However, this suggestion does not cor- statistically significant differences were noticed among the relate with the low absorption rate of the drug found using pharmacokinetic parameters of all three treatment lines, it can the oral route. Moreover, it has been shown that the absorp- be concluded that there are no differences between the BALB/c tion of certain cationic drugs, such as quaternary ammon- and SCID pharmacokinetic profiles. Thus, further studies with ium compounds, shows a reduced absorption rate due to the edelfosine delivery systems will be able to be done in either one presence of efflux transport systems on the apical mem- animal model or the other. brane (e.g., P-glycoprotein). Indeed, the alkylphosphocholine Our results show that edelfosine is widely scattered across miltefosine has been reported to interact with P-glycoprotein different organs but that it is preferentially internalized by the (27), and it has also been reported that edelfosine is a sub- tumor both in vitro and in vivo. All these data, together with the strate for P-glycoprotein (28). This issue would only explain apoptotic action of the drug on cancer cells, support a rather the lack of absorption at a single oral dose and the high selective action of edelfosine in cancer treatment, and that oral bioavailability achieved after a multiple oral dose multiple oral administration is required to reach a clinically administration. important plasma concentration, therefore increasing its oral In summary, edelfosine showed a remarkable apoptotic effect bioavailability. in Z-138 MCL cells in vitro and a high and rather selective uptake in MCL tumor cells in vitro and in vivo. This fact, along Disclosure of Potential Conflicts of Interest with the knowledge of the biodistribution and pharmacoki- netic behavior of edelfosine, permits the establishment of No potential conflicts of interest were disclosed.

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Clin Cancer Res 2009;15(3) February 1, 2009 864 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2009 American Association for Cancer Research. Antitumor Alkyl Ether Lipid Edelfosine: Tissue Distribution and Pharmacokinetic Behavior in Healthy and Tumor-Bearing Immunosuppressed Mice

Ander Estella-Hermoso de Mendoza, Miguel A. Campanero, Janis de la Iglesia-Vicente, et al.

Clin Cancer Res 2009;15:858-864.

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