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Pharmacokinetics and biodistribution of Erufosine in nude mice - implications for combination with radiotherapy

Pharmacokinetics and biodistribution of Erufosine in nude mice - implications for combination... Background: Alkylphosphocholines represent promising antineoplastic drugs that induce cell death in tumor cells by primary interaction with the cell membrane. Recently we could show that a combination of radiotherapy with Erufosine, a paradigmatic intravenously applicable alkylphosphocholine, in vitro leads to a clear increase of irradiation-induced cell death. In view of a possible combination of Erufosine and radiotherapy in vivo we determined the pharmacokinetics and bioavailability as well as the tolerability of Erufosine in nude mice. Methods: NMRI (nu/nu) nude mice were treated by intraperitoneal or subcutaneous injections of 5 to 40 mg/kg body weight Erufosine every 48 h for one to three weeks. Erufosine-concentrations were measured in brain, lungs, liver, small intestine, colon, spleen, kidney, stomach, adipoid tissue, and muscle by tandem-mass spectroscopy. Weight course, blood cell count and clinical chemistry were analyzed to evaluate general toxicity. Results: Intraperitoneal injections were generally well tolerated in all dose groups but led to a transient loss of the bodyweight (<10%) in a dose dependent manner. Subcutaneous injections of high-dose Erufosine caused local reactions at the injection site. Therefore, this regimen at 40 mg/kg body weight Erufosine was stopped after 14 days. No gross changes were observed in organ weight, clinical chemistry and white blood cell count in treated compared to untreated controls except for a moderate increase in lactate dehydrogenase and aspartate-aminotransferase after intensive treatment. Repeated Erufosine injections resulted in drug-accumulation in different organs with maximum concentrations of about 1000 nmol/g in spleen, kidney and lungs. Conclusion: Erufosine was well tolerated and organ-concentrations surpassed the cytotoxic drug concentrations in vitro. Our investigations establish the basis for a future efficacy testing of Erufosine in xenograft tumor models in nude mice alone and in combination with chemo- or radiotherapy. Page 1 of 11 (page number not for citation purposes) Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 limiting gastrointestinal toxicity. The maximum tolerated Background Radiotherapy and chemotherapy are crucial components dose after oral administration amounted to 200 mg/d for of most current protocols for the treatment of solid 3 weeks [16] and a maintenance dose of 100 mg/d could human tumors. Important mechanisms of antineoplastic be achieved [17]. action of these genotoxic therapies include induction of cell death, e.g., apoptosis or necrosis, and senescence. Erucylphosphocholine (ErPC), an APC derivative with a Unfortunately, tumorigenesis is characterized by tumor 22 carbon chain and a cis-double bond in the (omega-9)- cells' evasion of cell death induced by oncogene activation position, lacks hemolytic toxicity due to the formation of or by conditions of stress in their specific environment. lamellar instead of micellar structures in aqueous solu- Because stress-induced and therapy-induced cell death tions and is therefore suitable for intravenous administra- share common cellular pathways, the same genetic altera- tion. In a first in vivo study in healthy rats, repeated tions that mediate death resistance during carcinogenesis intravenous injections of ErPC were well tolerated and can cause cross-resistance to genotoxic therapies. Thus, revealed an accumulation of ErPC in different tissues, targeting cell death resistance is a promising approach including brain [18]. However, in vivo application of ErPC towards increasing the efficacy of genotoxic therapies for was complicated by poor drug solubility in aqueous solu- human solid tumors [1-4]. tions due to gel formation. An intensive search for struc- tural analogues with improved solubility properties Alkylphosphocholines (APC) represent promising antine- resulted in Erufosine (ErPC3, Erucylphosphohomo- oplastic agents with a particular mechanism of action: In choline). The structure of Erufosine in comparison to contrast to standard chemotherapy and irradiation these ErPC is characterized by the addition of one methylene synthetic phospholipid derivatives target cellular mem- group into the polar phosphocholine head group. Erufo- branes and interfere with membrane lipid composition sine forms clear solutions in water and has similar antin- and the formation of lipid second messengers, thereby eoplastic activity in vitro [19]. affecting the growth, cell cycle progression, and survival of tumor cells without direct interaction with cellular DNA To gain insight into the value of the novel APC derivative, [5,6]. The antineoplastic action of synthetic phospholipid Erufosine, in tumor therapy using mouse models, here we analogs relies on their ability to affect specific signaling analyzed pharmacokinetics and biodistribution in nude processes in the target cells. Until now, the PI3K/Akt path- mice after repeated intraperitoneal and subcutaneous way, the mitogen-activated protein kinase (MAPK)/extra- drug application. cellular signal-regulated kinase (ERK) pathway, the stress activated protein kinase (SAPK)/Jun-N terminal kinase Methods (JNK) and the sphingolipid pathway have been identified Chemicals as important drug targets [7-9]. Moreover, APC with anti- Erufosine (ErPC3, MG 503.8) is the (N,N,N-trimethyl)- neoplastic activity, e.g. Miltefosine, Perifosine, and Erufo- propylammoniumester of erucyl-phosphoric acid. It was sine, induce apoptosis in tumor cells in vitro. Depending first synthesized by H. Eibl, Max Planck Institute of Bio- on the cell type, the induction of apoptosis involves lig- physical Chemistry, Goettingen, Germany [20] and kindly and-independent activation of the death receptor path- provided for these studies. 1,2-Propanediol was pur- way in membrane rafts, p53-independent activation of chased by Merck, Darmstadt, Germany. All other chemi- the mitochondrial apoptosis pathway, or both [7,8,10- cals were from Sigma-Aldrich, Deisenhofen, Germany, if 12]. In contrast, induction of apoptosis by DNA-damag- not otherwise indicated. ing agents (e.g. 5-fluorouracil) and irradiation is mainly dependent on p53-induced up-regulation of the pro- For aqueous solutions Erufosine was dissolved at 60°C in apoptotic Bcl-2 analog Bax. Interestingly, APC such as a mixture of distilled water and 1.2-Propandiol (mixture Miltefosine and ether lysolecithins such as Edelfosine 98:2) to a final concentration of 24 mg/ml (48 mM) Eru- increase the efficacy of chemotherapy and radiotherapy in fosine and stored at 5°C after sterile filtration. For intra- vitro and in animal experiments [6,13]. These observa- peritoneal and subcutaneous injection this stock solution tions suggest that APC may be particularly useful for the was diluted with 0.9% sodium-chloride solution in the treatment of tumor cells resistant to DNA-damaging drugs appropriate ratio to obtain the required dosage of Erufo- and irradiation. sine in the injection volume of 100 μl for 30 g mice. Dif- ferences in body weight of the mice were adjusted with The clinical use of the first generation APC Miltefosine injection volume. was restricted to topical application due to hemolytic and gastrointestinal toxicity upon intravenous and oral appli- Animals cation, respectively [14,15]. Furthermore, clinical trials Animal experiments were made according to German ani- testing the oral analogue Perifosine also revealed dose mal welfare regulations and approved by the local author- Page 2 of 11 (page number not for citation purposes) Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 ities (registration number RO 1/05, Regierungspräsidium Briefly, for serum analysis an aliquot of 50 μl of serum was Tübingen). Immunodeficient NMRI-(nu/nu)-nude mice spiked with 20 μl ethanol containing 20 mg/l ErPC3-D9 were purchased from the Central animal facility of the in a 2 ml test tube. After vigorous mixing and equilibra- University of Duisburg Essen Medical School (age 4 tion for 20 min at room temperature, 1 ml of methanol/ months). Animals were housed in an individually venti- acetonitrile 9:1 (v/v) was added for protein precipitation. lated cage rack system (Techniplast, Italy). They were fed After centrifugation for 15 min at 16,000 × g, the clear with sterile high caloric laboratory food (Sniff, Germany). supernatant was diluted 1:9 (v/v) with methanol/aceton- Drinking water was supplemented by chlorotetracycline etrile 9:1 (v/v) and proceeded for LC-MS/MS analysis. For and potassium sorbate acidified to a pH of 3.0 with tissue analysis 1 ml methanol/acetonitrile 9:1 (v/v), hydrochloric acid and provided ad libitum. Mice were spiked with 20 μl ethanol containing 20 mg/l ErPC3-D9, treated by intraperitoneal or subcutaneous injections of was added to 100 mg tissue in a 1.5 ml test tube. Homog- Erufosine every 48 h at the indicated drug concentrations enization was performed after addition of a single carbide for the biodistribution and toxicity studies or by a single bead (diameter 3 mm) for 3 × 5 min with 40 Hz in a Tis- intraperitoneal bolus injection for analysis of pharmacok- sueLyser (Qiagen GmbH, Hilden, Germany). A clear inetic parameters in the serum. Intraperitoneal and subcu- supernatant was collected after centrifugation (15 min, taneous drug injections were selected instead of 16,000 × g), subsequently diluted 1:9 (v/v) with metha- intravenous drug application, as this application route is nol/acetonitrile 9:1 (v/v), and then proceeded for LC-MS/ already well established for rodent models. Moreover, the MS analysis. experiments performed in the present study constitute the basis for future experiments designed to evaluate the anti- A short CN column (20 × 4 mm I.D., 5 μm particle size, neoplastic action of Erufosine in combination with radia- Dr. Maisch GmbH, Ammerbuch, Germany) was used for tion. Subcutaneous and intraperitoneal administration is sample pre-fractionation with 70% methanol and 30% more practicable for the high numbers of animals bearing 0.1% formic acid delivered isocratically at a flow rate of xenograft tumors that are required for those experiments. 0.9 ml/min as the mobile phase. Applying a post-column split of approximately 1:10 the eluate was transferred to a Blood withdrawal was done by retro-orbital puncture in Waters Quattro Ultima Pt triple stage mass spectrometer light diethylether anesthesia. Serum was obtained by cen- run in the positive electrospray mode. Using multiple trifugation (5000 rpm, Eppendorf) and directly frozen at reaction monitoring the mass transition 504.4>139.1 of -20°C until analysis. Clinical chemistry was analyzed with the target analyte and the mass transition 513.7>139.1 of standard protocols in the Central laboratory of the Uni- the deuterated standard was recorded. The analytical run versity Hospital Tübingen using ADVIA 1650 (Siemens, time was 4 min. For calibration drug free serum was Eschborn). Blood cell count was done with ADVIA 120/ spiked with Erufosine in methanol. Six point calibration 2120 Cell counters (Siemens, Eschborn) from EDTA- was performed in all analytical series. blood. Statistics For the biodistribution studies, brain, lungs, liver, stom- If not otherwise stated, data are expressed as arithmetic ach, spleen, kidney, first 5 cm of intestine, complete means ± SD (n ≥ 3). Statistical data analysis was per- colon, muscle, and adipoid tissue were removed after formed by paired or unpaired t-test, where appropriate. P blood withdrawal and immediate cervical distortion. The ≤ 0.05 was considered statistically significant. organs were weighed and stored at -20°C until analysis. The pharmacokinetic data obtained after single intraperi- For the analysis of Erufosine-excretion, 12 mice were kept toneal injections were calculated according to a two-com- in single metabolic cages (Techniplast, Italy) with free partment model using JMP 7.0.1 (SAS Institute inc.) access to food and water allowing urine sampling for the software for approximation fit of the concentration last 4 days of a two week treatment period. After an adap- curves. tation period of one day, urine was collected every 24- hours for 3 days under water-saturated oil and stored at - Results 20°C. Pharmacokinetics after single bolus injection Three groups of 5-6 mice each were administered a single Analysis of ErPC3 in body fluids and tissues injection of Erufosine (40 mg/kg body weight) by intra- For the quantitative measurement of Erufosine in serum peritoneal injection. Approximately 50 μl of blood was and tissue samples liquid chromatography-tandem mass drawn by retro-orbital puncture at different time points in spectrometry (LC-MS/MS) was employed with a deute- each group, and mice were euthanized after the last punc- rium labeled analogue (ErPC3-D9, MW 512.82) as inter- ture (group 1: 15 min, 30 min, 1 hour, 2 hours; group 2: nal standard. Details are described elsewhere [21]. Page 3 of 11 (page number not for citation purposes) Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 30 min, 2 hours, 4 hours, 8 hours; group 3: 1 hour, 4 an approximation fit using an equation which consisted hours, 12 hours, 24 hours and 36 hours). of a fast and a slow exponential decay combined with an (-x/ exponential increase of serum levels (f(x) = A*(1-e T) (-x/T2) (-x/T1) (-x/T3) The highest serum concentrations upon single bolus ip- )*e +A*(1-e )*e ). The quality of the fit was injection of 40 mg/kg body weight Erufosine, were meas- extremely good reaching a R of 0.99 (Fig. 1A+B). ured 1 or 2 hours after treatment and achieved concentra- tions of 211 ± 27 nmol/ml (group 1, 2 h), 210 ± 36 nmol/ We further used this equation to calculate the pharmacok- ml (group 2, 2 h) and 209 ± 45 nmol/ml (group 3, 1 h). inetic parameters by fitting the serum values of each single 36 hours after injection the serum concentration still had mouse. For the animals with a short observation period a value of 56 ± 12 nmol/ml (Fig. 1A+B). Because of high (group1) we set the time constants for the decrease fix at reproducibility among the three independent groups, the the value pooled for all mice (Insert Fig. 1B). serum concentrations of all time points were averaged among the three groups. From these values we generated 250 150 repetitive ip-treatment, 21d 150 90 group 1 100 60 group 2 50 30 group 3 0 0 0 5 10 20 40 02 46 8 dose [mg/kg bw] time [h] C [nmol/ml] 217 25 max T [h] 1.89 0.35 150 max IJ [h] 0.65 0.12 20 mg/kg IJ [h] 5.86 0.50 e1 40 mg/kg IJ [h] 37.68 3.88 e2 AUC [μmol/ml h] 5.8 1.4 50 30 0 0 0 7 14 21 012 24 36 48 time [h] time [d] Serum concentration Figure 1 s of Erufosine after a single bolus injection (A+B) or repeated injections (C+D) Serum concentrations of Erufosine after a single bolus injection (A+B) or repeated injections (C+D). A+B: NMRI nu/nu mice were treated with one intraperitoneal injection of 40 mg/kg body weight Erufosine and subdivided into three groups for blood collection at different time points: group 1, n = 6 (• ): 15 min, 30 min, 1, 2 hours; group 2, n = 5 (): 30 min, 2, 4, 8 hours; group 3, n = 5 (black triangle): 1, 4, 12, 24 and 36 hours. Erufosine concentrations in serum were determined by LC-MS/MS analysis. Data represent means ± SD: A. Data show the initial serum concentrations of groups 1-3 separately. B. Data show mean Erufosine serum-concentrations for all animals from group 1-3 pooled (n = 16). Insert shows the pharmacok- inetic parameters. C+D: NMRI nu/nu mice were treated with repeated intraperitoneal injections of Erufosine every 48 hours at the indicated concentrations. All values are means ± SD (n = 3-6). Erufosine concentrations in serum were determined by LC-MS/MS analysis. C. Concentration-dependent increase in the serum levels of Erufosine after a three weeks treatment with 5, 10, 20 and 40 mg/kg body weight Erufosine. D. Time course of the Erufosine serum concentrations after treatment with 20 and 40 mg/kg body weight Erufosine for one and three weeks. Page 4 of 11 (page number not for citation purposes) ErPC3 [nmol/ml]. ErPC3 [nmol/ml]. ErPC3 [nmol/ml] ErPC3 [nmol/ml] Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 Taken together, a single bolus injection of 40 mg/kg body brain-barrier and accumulates in the brain tissue more weight Erufosine resulted in detectable serum concentra- efficiently compared to the other organs. The concentra- tions over 36 hours with a maximum concentration of tion in brain tissue after 3 weeks of treatment with 40 mg/ 217 ± 25 nmol/ml at 113 ± 20 min after injection. kg body weight amounted to 383 nmol/g, which is clearly above the concentration required to induce death of Repetitive injection glioblastoma cells in vitro. To study biodistribution of Erufosine, four different Eru- fosine-concentrations (5, 10, 20 and 40 mg/kg body Urine excretion weight) were administered every 48 h over a period of 7 The average 24-hour urine excretion of Erufosine was (group 1), 14 (group 2) or 21 days (group 3) by intraperi- measured for 6 mice during the last 3 consecutive days of toneal or subcutaneous injection. Each of the resulting 24 a 14-day treatment period with intraperitoneal injections groups consisted of 3 to 9 mice. At the end of the treat- of 20 mg/kg or 40 mg/kg body weight Erufosine, respec- ment course 24 hours after the last injection approxi- tively (Fig. 4). The average urine volume in both groups mately 300 μl blood were drawn and organs were was comparable. Total quantity and concentration of Eru- removed as described above. fosine in the urine was very low yielding less than 0.5 μg and 0.6 nmol/ml Erufosine after treatment with 40 mg/kg Serum concentrations body weight (Fig. 4). Taking into account that the serum The repetitive injection of Erufosine resulted in a concen- concentrations was 64 nmol/ml and 122 nmol/ml Erufo- tration- and time-dependent increase in serum Erufosine- sine after a 14-day treatment with 20 mg/kg and 40 mg/kg levels. After three weeks of intraperitoneal treatment with body weight, the urine/serum ratio in both groups was 5, 10, 20 and 40 mg/kg body weight Erufosine every 48 less than 0.6%. Despite high absolute tissue concentra- hours, respective serum concentrations amounted to 20 ± tions in the kidney this demonstrates negligible urine 4 nmol/ml, 36 ± 4 nmol/ml, 68 ± 23 nmol/ml and 109 ± excretion of Erufosine. 33 nmol/ml (Fig. 1C). Similar observations were made with subcutaneous injections (data not shown). The slope Toxicity of the increase in serum concentrations was more pro- Intraperitoneal injections of Erufosine were generally well tolerated. A clinical side effect of the high dose intraperi- nounced in the first 7 days compared to longer treatment periods suggesting a convergence to steady state levels toneal treatment (40 mg/kg body weight) was transient after prolonged Erufosine-treatment (Fig. 1D). diarrhea. No local changes or signs of inflammation were seen at the puncture. As an index of systemic toxicity the Organ concentrations body weight of the mice was measured regularly. Mean In a next step we analyzed the organ distribution of Erufo- weight of all animals at the beginning of treatment was sine in the three treatment groups after 7, 14, and 21 days 35.0 ± 1.2 g. Intraperitoneal application of 5 mg/kg body of treatment (Fig. 2). Erufosine accumulated in all tissues weight Erufosine did not result in any change of the body included in the study. Maximum drug-concentrations weight, whereas administration of higher concentrations were obtained after 21 days of intraperitoneal treatment led to a transient weight loss of less than 10% of body in spleen (1307 nmol/g), kidney (1123 nmol/g) and weight (Fig. 5). lungs (939 nmol/g) (Fig. 2A). The respective subcutane- ous injections led to slightly higher organ-concentrations In contrast, subcutaneous injections of Erufosine did not at all concentrations used (Fig. 2B). cause changes in body weight for all drug-concentrations used (Fig. 5). However, a local inflammation to the point Because of a possible use of Erufosine for the treatment of of ulceration occurred at the puncture region after 14 d of glioblastoma, we were then interested in the drug concen- treatment with 40 mg/kg body weight Erufosine (not trations that could be obtained in the brain tissue. shown). Therefore, the subcutaneous treatment with the Although absolute drug concentrations in the brain tissue high Erufosine concentration was stopped after 14 days. were low compared to e.g. lungs or kidney, we could clearly demonstrate an increase of the brain/serum ratio At the end of the treatment course there were no macro- after 7 and 21 days of treatment from 1.9 to 2.9, respec- scopic signs of organ injury and no systematic changes in tively, pointing to an accumulation of Erufosine in brain organ weight (data not shown). Regarding the hematolog- tissue (Fig 3A). With regard to the organ concentrations ical parameters no bone marrow related toxicity was achieved after 14 and 21 days of treatment relative to the detectable even though the variance of white blood cell 7 day treatment, the most prominent time-dependent count was high. increase in the Erufosine-concentration was observed for brain tissue at all drug concentrations used (Fig 3B). It The platelet count raised from 566 ± 155 for the control clearly demonstrates that Erufosine penetrates the blood- group to 833 ± 172 after 14 d treatment with 40 mg/kg Page 5 of 11 (page number not for citation purposes) Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 40 mg/kg fat 20 mg/kg 10 mg/kg muscle 5 mg/kg stomach intestine colon spleen lung brain kidney liver 0 500 1000 1500 2000 0 500 1000 1500 2000 0 500 1000 1500 2000 ErPC3 [nmol/g tissue] 40 mg/kg fat 20 mg/kg 10 mg/kg muscle 5 mg/kg stomach intestine colon spleen lung brain kidney liver 0 500 1000 1500 2000 0 500 1000 1500 2000 0 500 1000 1500 2000 ErPC3 [nmol/g tissue] Biodistrib Figure 2 ution of Erufosine after repeated drug injections Biodistribution of Erufosine after repeated drug injections. Mice were separated into 24 groups and treated every 48 hours with a intraperitoneal or subcutaneous injection of Erufosine at the indicated concentrations for one, two or three weeks. At the end of the treatment period mice were killed, organs removed and organ concentrations of Erufosine were determined by LC-MS/MS analysis. All values are means ± SD (n = 3-9). A. Organ concentrations of Erufosine after intraperito- neal treatment with 5, 10, 20 and 40 mg/kg body weight Erufosine for one (left panel), two (middle panel) or three weeks (right panel). B. Organ concentration of Erufosine after subcutaneous treatment with 5, 10, 20 and 40 mg/kg body weight Erufosine for one (left panel), two (middle panel) or three weeks (right panel). Three weeks subcutaneous treatment with 40 mg/kg body weight Erufosine is missing due to local toxicity. Page 6 of 11 (page number not for citation purposes) Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 AB Serum 14d relative to 7d Brain 21d relative to 7d 0 0 7 d 21 d A Figure 3 ccumulation of Erufosine in brain tissue after repeated intraperitoneal drug injections Accumulation of Erufosine in brain tissue after repeated intraperitoneal drug injections. Mice were treated every 48 hours with intraperitoneal injections of Erufosine at the indicated concentrations for one, two or three weeks. At the end of the treatment period mice were killed, organs removed and organ concentrations of Erufosine were determined by LC-MS/ MS analysis. A. Brain and serum concentrations of Erufosine after treatment with 20 mg/kg body weight of Erufosine for 7 d and 21 d, respectively. Data show means ± SD (n = 3-6). B. Mean organ concentrations of Erufosine after treatment with 5, 10, 20 or 40 mg/kg body weight for 14 or 21 days were divided by the mean organ concentrations after the respective treatment for 7 days. Data show means ± SEM of the resulting quotients from all 4 dose groups (n = 12-24). body weight, but then decreased again until day 21 of the weight as an index of systemic toxicity. Only high dose high dose treatment (Tab. 1). As shown in table 1, long- intraperitoneal injection of Erufosine induced a mild time treatment with 40 mg/kg body weight Erufosine led diarrhea at the beginning of the treatment and a reversible to a 2 to 2.5-fold increase of serum lactate dehydrogenase weight loss preventing further dose escalation. These (LDH) after 14 and 21 days of treatment. Moreover, aspar- observations are reminiscent of earlier findings in healthy tate-aminotransferase (AST) was increased after 21 day rats after high dose intravenous application of the Erufo- treatment with 40 mg/kg body weight Erufosine, suggest- sine-related ErPC [18]. In contrast, subcutaneous applica- ing that high Erufosine-concentrations or long term treat- tion did not induce any changes in the body weight even ment may induce some cell damage. However, no further upon treatment with 40 mg/kg body weight Erufosine. significant changes in clinical chemistry and clinical pic- These observations suggest that intraperitoneal injection ture could be detected arguing against a major toxic effect of Erufosine may induce a local effect similar to the gas- (Tab. 1). Certainly it has to be taken into account that trointestinal toxicity observed after oral application of nude mice can provide only a limited toxicity profile, in Perifosine [13,16,17,22,23]. On the other hand, despite particular related to toxic immune responses. the absence of alterations in the body weight, subcutane- ous injection was accompanied by dose limiting ulcera- tions at the injection site 2 weeks after treatment with 40 Discussion Here we show for the first time, that parenteral treatment mg/kg body Erufosine. As prolonged intravenous infusion of nude mice with Erufosine is feasible without major tox- of low-dose Erufosine is well tolerated in patients (L. icity. Moreover, our data demonstrate that repeated intra- Lindner, personal communication) long-term intrave- peritoneal or subcutaneous injections of nontoxic nous infusion of Erufosine may be considered as an alter- Erufosine-concentrations yield organ concentrations that native for future experiments. are sufficient to induce tumor cell death in vitro. Clinical chemistry revealed a concentration-dependent Tolerability of Erufosine-treatment was demonstrated by increase in serum levels of LDH and to a lesser extent of the absence of significant alterations in organ weight or AST during Erufosine-treatment, while alanine-ami- macroscopic appearance, and minor changes in the body notransferase and further blood parameters remained Page 7 of 11 (page number not for citation purposes) liver kidney brain lung spleen colon small intestine stomach ErPC3 [nmol/g] relative concentration. Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 0,7 AB 0,6 0,6 0,5 0,5 0,4 0,4 0,3 0,3 0,2 0,2 0,1 0,1 0 0 20mg/kg 40mg/kg 20mg/kg 40mg/kg dose dose Uri Figure 4 ne excretion of Erufosine after repeated intraperitoneal drug injections Urine excretion of Erufosine after repeated intraperitoneal drug injections. NMRI nu/nu mice were treated with intraperitoneal injection of 20 mg/kg body weight and 40 mg/kg body weight (n = 6 each) Erufosine every 48 hours for two weeks. The urine was collected over 24-hours on the last three consecutive days of the treatment period in a metabolic cage. Average urine volumes were determined and concentrations of Erufosine in urine were measured by using LC-MS/MS analysis. Data show (A) the urine concentrations and (B) the total amount of Erufosine (means ± SEM). unchanged. Although the increase in LDH has been ever, in contrast to earlier investigations with Miltefosine described as a hint for beginning hemolysis, being a major or ErPC, instead of the reported increase in leukocyte toxic side effect of the first generation APC Miltefosine numbers, we only detected a time- and concentration- [14], the lack of changes in the hemoglobin levels and of dependent transient increase in thrombocyte numbers. a clinical correlate argues against a clinically relevant The differences in the blood cell behaviour may be related hemolytic effect of Erufosine. It may be suggested that to the distinct application mode and/or species-specific Erufosine-treatment affects the membrane composition differences in the drug effect. of the erythrocytes facilitating damage of more fragile erythrocytes during retro-orbital blood withdrawal. Since A single bolus injection of Erufosine resulted in detectable a marginal elevation of AST-levels after intravenous appli- serum Erufosine levels for approximately 36 h peaking at cation of ErPC in rats has been already described for the 217 ± 25 nmol/ml 113 ± 20 min after injection. Repeated Erufosine related ErPC [18] this parameter should be fur- intraperitoneal or subcutaneous administrations led to a ther analyzed in preclinical or clinical trials. continuous increase of serum and organ concentrations of Erufosine with the highest concentrations achieved in Kidney related serum parameters like electrolytes, protein, spleen, kidney and the lungs. The subcutaneous injections creatinine and urea did not increase during treatment with yielded slightly higher drug-concentrations in most tis- Erufosine leaving no evidence for renal dysfunction as sues compared to the intraperitoneal injections reaching described for Miltefosine [24]. significance in liver, kidney, and brain. Our data corrobo- rate earlier findings about the bioavailability of the Erufo- Importantly, similar to previous reports for other APC, sine-related ErPC in healthy rats [18]. Although organ Erufosine lacked bone marrow toxicity [18,24,25]. How- distributions were quite similar, the Erufosine-concentra- Page 8 of 11 (page number not for citation purposes) ErPC3 [nmol/ml] ErPC3 [μg] Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 ip-injection -1 0 -2 -3 5 mg/kg 20 mg/kg 40 mg/kg 10 mg/kg 3 sc-injection -10 4 8 121620 -2 time [d] -3 Figure 5 Change in body weight of animals upon Erufosine treatment Change in body weight of animals upon Erufosine treatment. NMRI nu/nu mice were treated with intraperitoneal injections of 5, 10, 20 und 40 mg/kg body weight Erufosine every 48 hours. Body weight was determined every second day. Val- ues represent means ± SEM of the difference from starting weight in the respective dose groups. A. Body weight after intra- peritoneal injections. B. Body weight after subcutaneous injections. tions achieved upon intraperitoneal or subcutaneous In contrast, Erufosine-concentrations in the brain tissue administration in the respective tissues were increased in were below the levels obtained by Erdlenbruch et al. [18], most of the tested organs compared to the ErPC-concen- an effect that may reflect distinct efficiency in crossing the trations obtained after intravenous injections in the inves- blood brain barrier due to the distinct lipophilic behav- tigation of Erdlenbruch and coworkers [18]. This may at iour of the two derivatives and/or altered composition of least partially be related to the altered serum composition the blood brain barrier in rats compared to mice. Never- observed in nude mice. Moreover, the increased sensitiv- theless, we observed a strong time- and concentration- ity of liquid chromatography-tandem mass spectrometry dependent accumulation of Erufosine in the brain tissue used in the present study compared to that of high per- reaching 383 nmol/g after a 3-week treatment with 40 formance thin layer chromatography HPTLC used in the mg/kg of body weight. This concentration is clearly above earlier investigation may be of relevance [21]. the concentration sufficient to induce cytotoxicity in malignant glioma cell lines in vitro [10,19,26,27]. Page 9 of 11 (page number not for citation purposes) difference in body difference in body weight [g] weight [g] Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 Table 1: Serum parameters and hematological parameters during intraperitoneal Erufosine treatment (Mean ± SD) 14-d treatment 21-d treatment control 20 mg/kg bw 40 mg/kg bw 20 mg/kg bw 40 mg/kg bw Blood count Leukocytes (/μl) 4,2 ± 2,1 3,3 ± 1,8 3,5 ± 2,5 5,0 ± 3,4 2,6 ± 1,4 Erythrocytes (10 /μl) 9,1 ± 0,4 8,4 ± 0,3 9,1 ± 0,8 9,1 ± 0,5 8,4 ± 1,4 Platelets (10 /μl) 566 ± 155 741 ± 94 833* ± 172 872* ± 432 442* ± 156 Hemoglobin (g/dl) 14,5 ± 0,6 13,6 ± 0,9 14,6 ± 1,9 14,0 ± 0,5 13,4 ± 1,9 Hematocrit (%) 47,5 ± 2,3 45,5 ± 1,7 49,1 ± 4,5 45,4 ± 4,7 44,7 ± 5,9 Serum Na (mmol/l) 156 ± 8 156 ± 2 155 ± 10 157 ± 4 162 ± 6 K (mmol/l) 5,1 ± 0,9 5,9 ± 1,2 6,3 ± 0,8 5,4 ± 0,8 5,8 ± 1,0 Ca (mmol/l) 2,4 ± 0,2 2,5 ± 0,1 2,6 ± 0,2 2,1 ± 0,3 2,5 ± 0,2 AST (U/l) 125 ± 42 117 ± 38 152 ± 22 140 ± 69 245* ± 156 ALT (U/l) 63 ± 32 55 ± 14 93 ± 25 65 ± 31 93 ± 45 LDH (U/l) 1315 ± 441 1401 ± 818 2650* ± 959 1913 ± 1152 3498* ± 519 Protein (g/dl) 5,1 ± 0,3 4,9 ± 0,1 5,5 ± 0,4 4,8 ± 0,3 4,9 ± 0,5 Creatinine (mg/dl) 0,3 ± 0,1 0,3 ± 0,1 0,3 ± 0,1 0,3 ± 0,1 0,3 ± 0,1 Urea (mg/dl) 56,5 ± 9,0 50,8 ± 8,1 57,3 ± 6,0 47,9 ± 6,4 56,2 ± 13,3 Together with our earlier investigations on the increased feasible and safe. Furthermore the concentrations cytotoxic efficacy of ionizing radiation in combination achieved in the brain tissue are above the concentrations with Erufosine in glioblastoma cell lines in vitro, the abil- needed for combination effects with radiation in earlier in ity of Erufosine to cross the blood brain barrier and to vitro experiments using human astrocytoma/glioblastoma accumulate in the brain tissue make the drug a promising cell lines. Our results constitute the basis for the design of candidate for combined treatment approaches with radio- preclinical investigations with Erufosine alone and in therapy in malignant glioma. Although clinical trials combination with radiotherapy in murine tumor models, already demonstrated feasibility and tolerability of a ther- in particular in nude mice. In a next step, we will evaluate apy with Perifosine, Erufosine, or Perifosine with radio- efficacy of Erufosine in combination with ionizing radia- therapy for patients with advanced human malignancies tion in vivo in nude mice bearing subcutaneous tumors. [[8,13] and personal communication with L. Lindner], Based on our present investigations, pretreatment with before debarking into clinical trials with patients suffering repeated intraperitoneal injections of Erufosine for 1 or 2 from malignant glioma and other tumors, efficacy of Eru- weeks prior to initiation of radiotherapy should be con- fosine in combined treatment approaches has to be eval- sidered to benefit from the drug-accumulation in the uated in animal experiments in vivo. tumor tissue. In conclusion, our data reveal that intraperitoneal and Competing interests subcutaneous administration of Erufosine to nude mice is The authors declare that they have no competing interests. Page 10 of 11 (page number not for citation purposes) Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 nation with radiotherapy. Cancer treatment reviews 2007, Authors' contributions 33(2):191-202. GH contributed significantly to the design of the study, 14. Kotting J, Marschner NW, Neumuller W, Unger C, Eibl H: Hexade- data acquisition, data analysis and drafting the manu- cylphosphocholine and octadecyl-methyl-glycero-3-phos- phocholine: a comparison of hemolytic activity, serum script. LHL contributed significantly to data acquisition, binding and tissue distribution. Progress in experimental tumor data analysis and drafting the manuscript. KW and MV research Fortschritte der experimentellen Tumorforschung 1992, 34:131-142. performed probe preparation and mass spectrometry 15. Verweij J, Planting A, Burg M van der, Stoter G: A dose-finding measurements, respectively. JW performed many of the study of miltefosine (hexadecylphosphocholine) in patients animal experiments. ACM and MB performed critical revi- with metastatic solid tumours. Journal of cancer research and clin- ical oncology 1992, 118(8):606-608. sion of the manuscript. HE synthesized and provided 16. Crul M, Rosing H, de Klerk GJ, Dubbelman R, Traiser M, Reichert S, ErPC and ErPC3 for the analysis. CB participated in the Knebel NG, Schellens JH, Beijnen JH, ten Bokkel Huinink WW: conception of the study and interpretation of data. VJ per- Phase I and pharmacological study of daily oral administra- tion of perifosine (D-21266) in patients with advanced solid formed conception and design of the study and substan- tumours. Eur J Cancer 2002, 38(12):1615-1621. tially contributed to interpretation of data, drafting of the 17. Van Ummersen L, Binger K, Volkman J, Marnocha R, Tutsch K, Kole- sar J, Arzoomanian R, Alberti D, Wilding G: A phase I trial of per- manuscript, critical revision of the manuscript and final ifosine (NSC 639966) on a loading dose/maintenance dose approval. All authors read and approved the final manu- schedule in patients with advanced cancer. Clin Cancer Res script. 2004, 10(22):7450-7456. 18. Erdlenbruch B, Jendrossek V, Gerriets A, Vetterlein F, Eibl H, Lakomek M: Erucylphosphocholine: pharmacokinetics, biodis- Acknowledgements tribution and CNS-accumulation in the rat after intravenous This work was funded by a grant of the Wilhelm-Sander-Stiftung administration. Cancer chemotherapy and pharmacology 1999, 44(6):484-490. (2005.143.1). Erufosine was kindly provided by H. Eibl. 19. Rubel A, Handrick R, Lindner LH, Steiger M, Eibl H, Budach W, Belka C, Jendrossek V: The membrane targeted apoptosis modula- References tors erucylphosphocholine and erucylphosphohomocholine 1. Gatenby RA, Gillies RJ: A microenvironmental model of car- increase the radiation response of human glioblastoma cell cinogenesis. Nature reviews 2008, 8(1):56-61. lines in vitro. Radiation oncology (London, England) 2006, 1:6. 2. Hermann RM, Wolff HA, Jarry H, Thelen P, Gruendker C, Rave- 20. Eibl H, Engel J: Synthesis of hexadecylphosphocholine (miltefo- Fraenk M, Schmidberger H, Christiansen H: In vitro studies on the sine). Progress in experimental tumor research Fortschritte der experi- modification of low-dose hyper-radiosensitivity in prostate mentellen Tumorforschung 1992, 34:1-5. cancer cells by incubation with genistein and estradiol. Radi- 21. Lindner LH, Eibl H, Hossann M, Vogeser M: Quantification of eru- ation oncology (London, England) 2008, 3:19. fosine, the first intravenously applicable alkylphospho- 3. Itani W, Geara F, Haykal J, Haddadin M, Gali-Muhtasib H: Radiosen- choline, in human plasma by isotope dilution liquid sitization by 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4- chromatography-tandem mass spectrometry using a deu- dioxide under oxia and hypoxia in human colon cancer cells. terated internal standard. J Chromatogr B Analyt Technol Biomed Radiation oncology (London, England) 2007, 2:1. Life Sci 2008, 869(1-2):16-19. 4. Candelaria M, Garcia-Arias A, Cetina L, Duenas-Gonzalez A: Radio- 22. Vink SR, Schellens JH, van Blitterswijk WJ, Verheij M: Tumor and sensitizers in cervical cancer. Cisplatin and beyond. Radiation normal tissue pharmacokinetics of perifosine, an oral anti- oncology (London, England) 2006, 1:15. cancer alkylphospholipid. Investigational new drugs 2005, 5. Lawrence TS, Blackstock AW, McGinn C: The mechanism of 23(4):279-286. action of radiosensitization of conventional chemotherapeu- 23. Vink SR, Lagerwerf S, Mesman E, Schellens JH, Begg AC, van Blitter- tic agents. Seminars in radiation oncology 2003, 13(1):13-21. swijk WJ, Verheij M: Radiosensitization of squamous cell carci- 6. Jendrossek V, Handrick R: Membrane targeted anticancer noma by the alkylphospholipid perifosine in cell culture and drugs: potent inducers of apoptosis and putative radiosensi- xenografts. Clin Cancer Res 2006, 12(5):1615-1622. tisers. Current medicinal chemistry 2003, 3(5):343-353. 24. Planting AS, Stoter G, Verweij J: Phase II study of daily oral milte- 7. Jendrossek V, Muller I, Eibl H, Belka C: Intracellular mediators of fosine (hexadecylphosphocholine) in advanced colorectal erucylphosphocholine-induced apoptosis. Oncogene 2003, cancer. Eur J Cancer 1993, 29A:518-519. 22(17):2621-2631. 25. Stekar J, Hilgard P, Klenner T: Opposite effect of miltefosine on 8. Mollinedo F: Antitumor ether lipids: proapoptotic agents wth the antineoplastic activity and haematological toxicity of multiple therapeutic indications. Expert Opin Ther Patents 2007, cyclophosphamide. Eur J Cancer 1995, 31A(3):372-374. 17(4):385-405. 26. Jendrossek V, Kugler W, Erdlenbruch B, Eibl H, Lang F, Lakomek M: 9. Ruiter GA, Zerp SF, Bartelink H, van Blitterswijk WJ, Verheij M: Erucylphosphocholine-induced apoptosis in chemoresistant Alkyl-lysophospholipids activate the SAPK/JNK pathway and glioblastoma cell lines: involvement of caspase activation enhance radiation-induced apoptosis. Cancer research 1999, and mitochondrial alterations. Anticancer research 2001, 59(10):2457-2463. 21(5):3389-3396. 10. Handrick R, Rubel A, Faltin H, Eibl H, Belka C, Jendrossek V: 27. Kugler W, Erdlenbruch B, Otten K, Jendrossek V, Eibl H, Lakomek M: Increased cytotoxicity of ionizing radiation in combination MAP kinase pathways involved in glioblastoma response to with membrane-targeted apoptosis modulators involves erucylphosphocholine. International journal of oncology 2004, downregulation of protein kinase B/Akt-mediated survival- 25(6):1721-1727. signaling. Radiother Oncol 2006, 80(2):199-206. 11. Ruiter GA, Zerp SF, Bartelink H, van Blitterswijk WJ, Verheij M: Anti-cancer alkyl-lysophospholipids inhibit the phosphati- dylinositol 3-kinase-Akt/PKB survival pathway. Anti-cancer drugs 2003, 14(2):167-173. 12. Luit AH Van der, Budde M, Zerp S, Caan W, Klarenbeek JB, Verheij M, Van Blitterswijk WJ: Resistance to alkyl-lysophospholipid- induced apoptosis due to downregulated sphingomyelin syn- thase 1 expression with consequent sphingomyelin- and cho- lesterol-deficiency in lipid rafts. The Biochemical journal 2007, 401(2):541-549. 13. Vink SR, van Blitterswijk WJ, Schellens JH, Verheij M: Rationale and clinical application of alkylphospholipid analogues in combi- Page 11 of 11 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Radiation Oncology Springer Journals

Pharmacokinetics and biodistribution of Erufosine in nude mice - implications for combination with radiotherapy

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Copyright © 2009 by Henke et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Oncology; Radiotherapy
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1748-717X
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10.1186/1748-717X-4-46
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19852786
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Abstract

Background: Alkylphosphocholines represent promising antineoplastic drugs that induce cell death in tumor cells by primary interaction with the cell membrane. Recently we could show that a combination of radiotherapy with Erufosine, a paradigmatic intravenously applicable alkylphosphocholine, in vitro leads to a clear increase of irradiation-induced cell death. In view of a possible combination of Erufosine and radiotherapy in vivo we determined the pharmacokinetics and bioavailability as well as the tolerability of Erufosine in nude mice. Methods: NMRI (nu/nu) nude mice were treated by intraperitoneal or subcutaneous injections of 5 to 40 mg/kg body weight Erufosine every 48 h for one to three weeks. Erufosine-concentrations were measured in brain, lungs, liver, small intestine, colon, spleen, kidney, stomach, adipoid tissue, and muscle by tandem-mass spectroscopy. Weight course, blood cell count and clinical chemistry were analyzed to evaluate general toxicity. Results: Intraperitoneal injections were generally well tolerated in all dose groups but led to a transient loss of the bodyweight (<10%) in a dose dependent manner. Subcutaneous injections of high-dose Erufosine caused local reactions at the injection site. Therefore, this regimen at 40 mg/kg body weight Erufosine was stopped after 14 days. No gross changes were observed in organ weight, clinical chemistry and white blood cell count in treated compared to untreated controls except for a moderate increase in lactate dehydrogenase and aspartate-aminotransferase after intensive treatment. Repeated Erufosine injections resulted in drug-accumulation in different organs with maximum concentrations of about 1000 nmol/g in spleen, kidney and lungs. Conclusion: Erufosine was well tolerated and organ-concentrations surpassed the cytotoxic drug concentrations in vitro. Our investigations establish the basis for a future efficacy testing of Erufosine in xenograft tumor models in nude mice alone and in combination with chemo- or radiotherapy. Page 1 of 11 (page number not for citation purposes) Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 limiting gastrointestinal toxicity. The maximum tolerated Background Radiotherapy and chemotherapy are crucial components dose after oral administration amounted to 200 mg/d for of most current protocols for the treatment of solid 3 weeks [16] and a maintenance dose of 100 mg/d could human tumors. Important mechanisms of antineoplastic be achieved [17]. action of these genotoxic therapies include induction of cell death, e.g., apoptosis or necrosis, and senescence. Erucylphosphocholine (ErPC), an APC derivative with a Unfortunately, tumorigenesis is characterized by tumor 22 carbon chain and a cis-double bond in the (omega-9)- cells' evasion of cell death induced by oncogene activation position, lacks hemolytic toxicity due to the formation of or by conditions of stress in their specific environment. lamellar instead of micellar structures in aqueous solu- Because stress-induced and therapy-induced cell death tions and is therefore suitable for intravenous administra- share common cellular pathways, the same genetic altera- tion. In a first in vivo study in healthy rats, repeated tions that mediate death resistance during carcinogenesis intravenous injections of ErPC were well tolerated and can cause cross-resistance to genotoxic therapies. Thus, revealed an accumulation of ErPC in different tissues, targeting cell death resistance is a promising approach including brain [18]. However, in vivo application of ErPC towards increasing the efficacy of genotoxic therapies for was complicated by poor drug solubility in aqueous solu- human solid tumors [1-4]. tions due to gel formation. An intensive search for struc- tural analogues with improved solubility properties Alkylphosphocholines (APC) represent promising antine- resulted in Erufosine (ErPC3, Erucylphosphohomo- oplastic agents with a particular mechanism of action: In choline). The structure of Erufosine in comparison to contrast to standard chemotherapy and irradiation these ErPC is characterized by the addition of one methylene synthetic phospholipid derivatives target cellular mem- group into the polar phosphocholine head group. Erufo- branes and interfere with membrane lipid composition sine forms clear solutions in water and has similar antin- and the formation of lipid second messengers, thereby eoplastic activity in vitro [19]. affecting the growth, cell cycle progression, and survival of tumor cells without direct interaction with cellular DNA To gain insight into the value of the novel APC derivative, [5,6]. The antineoplastic action of synthetic phospholipid Erufosine, in tumor therapy using mouse models, here we analogs relies on their ability to affect specific signaling analyzed pharmacokinetics and biodistribution in nude processes in the target cells. Until now, the PI3K/Akt path- mice after repeated intraperitoneal and subcutaneous way, the mitogen-activated protein kinase (MAPK)/extra- drug application. cellular signal-regulated kinase (ERK) pathway, the stress activated protein kinase (SAPK)/Jun-N terminal kinase Methods (JNK) and the sphingolipid pathway have been identified Chemicals as important drug targets [7-9]. Moreover, APC with anti- Erufosine (ErPC3, MG 503.8) is the (N,N,N-trimethyl)- neoplastic activity, e.g. Miltefosine, Perifosine, and Erufo- propylammoniumester of erucyl-phosphoric acid. It was sine, induce apoptosis in tumor cells in vitro. Depending first synthesized by H. Eibl, Max Planck Institute of Bio- on the cell type, the induction of apoptosis involves lig- physical Chemistry, Goettingen, Germany [20] and kindly and-independent activation of the death receptor path- provided for these studies. 1,2-Propanediol was pur- way in membrane rafts, p53-independent activation of chased by Merck, Darmstadt, Germany. All other chemi- the mitochondrial apoptosis pathway, or both [7,8,10- cals were from Sigma-Aldrich, Deisenhofen, Germany, if 12]. In contrast, induction of apoptosis by DNA-damag- not otherwise indicated. ing agents (e.g. 5-fluorouracil) and irradiation is mainly dependent on p53-induced up-regulation of the pro- For aqueous solutions Erufosine was dissolved at 60°C in apoptotic Bcl-2 analog Bax. Interestingly, APC such as a mixture of distilled water and 1.2-Propandiol (mixture Miltefosine and ether lysolecithins such as Edelfosine 98:2) to a final concentration of 24 mg/ml (48 mM) Eru- increase the efficacy of chemotherapy and radiotherapy in fosine and stored at 5°C after sterile filtration. For intra- vitro and in animal experiments [6,13]. These observa- peritoneal and subcutaneous injection this stock solution tions suggest that APC may be particularly useful for the was diluted with 0.9% sodium-chloride solution in the treatment of tumor cells resistant to DNA-damaging drugs appropriate ratio to obtain the required dosage of Erufo- and irradiation. sine in the injection volume of 100 μl for 30 g mice. Dif- ferences in body weight of the mice were adjusted with The clinical use of the first generation APC Miltefosine injection volume. was restricted to topical application due to hemolytic and gastrointestinal toxicity upon intravenous and oral appli- Animals cation, respectively [14,15]. Furthermore, clinical trials Animal experiments were made according to German ani- testing the oral analogue Perifosine also revealed dose mal welfare regulations and approved by the local author- Page 2 of 11 (page number not for citation purposes) Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 ities (registration number RO 1/05, Regierungspräsidium Briefly, for serum analysis an aliquot of 50 μl of serum was Tübingen). Immunodeficient NMRI-(nu/nu)-nude mice spiked with 20 μl ethanol containing 20 mg/l ErPC3-D9 were purchased from the Central animal facility of the in a 2 ml test tube. After vigorous mixing and equilibra- University of Duisburg Essen Medical School (age 4 tion for 20 min at room temperature, 1 ml of methanol/ months). Animals were housed in an individually venti- acetonitrile 9:1 (v/v) was added for protein precipitation. lated cage rack system (Techniplast, Italy). They were fed After centrifugation for 15 min at 16,000 × g, the clear with sterile high caloric laboratory food (Sniff, Germany). supernatant was diluted 1:9 (v/v) with methanol/aceton- Drinking water was supplemented by chlorotetracycline etrile 9:1 (v/v) and proceeded for LC-MS/MS analysis. For and potassium sorbate acidified to a pH of 3.0 with tissue analysis 1 ml methanol/acetonitrile 9:1 (v/v), hydrochloric acid and provided ad libitum. Mice were spiked with 20 μl ethanol containing 20 mg/l ErPC3-D9, treated by intraperitoneal or subcutaneous injections of was added to 100 mg tissue in a 1.5 ml test tube. Homog- Erufosine every 48 h at the indicated drug concentrations enization was performed after addition of a single carbide for the biodistribution and toxicity studies or by a single bead (diameter 3 mm) for 3 × 5 min with 40 Hz in a Tis- intraperitoneal bolus injection for analysis of pharmacok- sueLyser (Qiagen GmbH, Hilden, Germany). A clear inetic parameters in the serum. Intraperitoneal and subcu- supernatant was collected after centrifugation (15 min, taneous drug injections were selected instead of 16,000 × g), subsequently diluted 1:9 (v/v) with metha- intravenous drug application, as this application route is nol/acetonitrile 9:1 (v/v), and then proceeded for LC-MS/ already well established for rodent models. Moreover, the MS analysis. experiments performed in the present study constitute the basis for future experiments designed to evaluate the anti- A short CN column (20 × 4 mm I.D., 5 μm particle size, neoplastic action of Erufosine in combination with radia- Dr. Maisch GmbH, Ammerbuch, Germany) was used for tion. Subcutaneous and intraperitoneal administration is sample pre-fractionation with 70% methanol and 30% more practicable for the high numbers of animals bearing 0.1% formic acid delivered isocratically at a flow rate of xenograft tumors that are required for those experiments. 0.9 ml/min as the mobile phase. Applying a post-column split of approximately 1:10 the eluate was transferred to a Blood withdrawal was done by retro-orbital puncture in Waters Quattro Ultima Pt triple stage mass spectrometer light diethylether anesthesia. Serum was obtained by cen- run in the positive electrospray mode. Using multiple trifugation (5000 rpm, Eppendorf) and directly frozen at reaction monitoring the mass transition 504.4>139.1 of -20°C until analysis. Clinical chemistry was analyzed with the target analyte and the mass transition 513.7>139.1 of standard protocols in the Central laboratory of the Uni- the deuterated standard was recorded. The analytical run versity Hospital Tübingen using ADVIA 1650 (Siemens, time was 4 min. For calibration drug free serum was Eschborn). Blood cell count was done with ADVIA 120/ spiked with Erufosine in methanol. Six point calibration 2120 Cell counters (Siemens, Eschborn) from EDTA- was performed in all analytical series. blood. Statistics For the biodistribution studies, brain, lungs, liver, stom- If not otherwise stated, data are expressed as arithmetic ach, spleen, kidney, first 5 cm of intestine, complete means ± SD (n ≥ 3). Statistical data analysis was per- colon, muscle, and adipoid tissue were removed after formed by paired or unpaired t-test, where appropriate. P blood withdrawal and immediate cervical distortion. The ≤ 0.05 was considered statistically significant. organs were weighed and stored at -20°C until analysis. The pharmacokinetic data obtained after single intraperi- For the analysis of Erufosine-excretion, 12 mice were kept toneal injections were calculated according to a two-com- in single metabolic cages (Techniplast, Italy) with free partment model using JMP 7.0.1 (SAS Institute inc.) access to food and water allowing urine sampling for the software for approximation fit of the concentration last 4 days of a two week treatment period. After an adap- curves. tation period of one day, urine was collected every 24- hours for 3 days under water-saturated oil and stored at - Results 20°C. Pharmacokinetics after single bolus injection Three groups of 5-6 mice each were administered a single Analysis of ErPC3 in body fluids and tissues injection of Erufosine (40 mg/kg body weight) by intra- For the quantitative measurement of Erufosine in serum peritoneal injection. Approximately 50 μl of blood was and tissue samples liquid chromatography-tandem mass drawn by retro-orbital puncture at different time points in spectrometry (LC-MS/MS) was employed with a deute- each group, and mice were euthanized after the last punc- rium labeled analogue (ErPC3-D9, MW 512.82) as inter- ture (group 1: 15 min, 30 min, 1 hour, 2 hours; group 2: nal standard. Details are described elsewhere [21]. Page 3 of 11 (page number not for citation purposes) Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 30 min, 2 hours, 4 hours, 8 hours; group 3: 1 hour, 4 an approximation fit using an equation which consisted hours, 12 hours, 24 hours and 36 hours). of a fast and a slow exponential decay combined with an (-x/ exponential increase of serum levels (f(x) = A*(1-e T) (-x/T2) (-x/T1) (-x/T3) The highest serum concentrations upon single bolus ip- )*e +A*(1-e )*e ). The quality of the fit was injection of 40 mg/kg body weight Erufosine, were meas- extremely good reaching a R of 0.99 (Fig. 1A+B). ured 1 or 2 hours after treatment and achieved concentra- tions of 211 ± 27 nmol/ml (group 1, 2 h), 210 ± 36 nmol/ We further used this equation to calculate the pharmacok- ml (group 2, 2 h) and 209 ± 45 nmol/ml (group 3, 1 h). inetic parameters by fitting the serum values of each single 36 hours after injection the serum concentration still had mouse. For the animals with a short observation period a value of 56 ± 12 nmol/ml (Fig. 1A+B). Because of high (group1) we set the time constants for the decrease fix at reproducibility among the three independent groups, the the value pooled for all mice (Insert Fig. 1B). serum concentrations of all time points were averaged among the three groups. From these values we generated 250 150 repetitive ip-treatment, 21d 150 90 group 1 100 60 group 2 50 30 group 3 0 0 0 5 10 20 40 02 46 8 dose [mg/kg bw] time [h] C [nmol/ml] 217 25 max T [h] 1.89 0.35 150 max IJ [h] 0.65 0.12 20 mg/kg IJ [h] 5.86 0.50 e1 40 mg/kg IJ [h] 37.68 3.88 e2 AUC [μmol/ml h] 5.8 1.4 50 30 0 0 0 7 14 21 012 24 36 48 time [h] time [d] Serum concentration Figure 1 s of Erufosine after a single bolus injection (A+B) or repeated injections (C+D) Serum concentrations of Erufosine after a single bolus injection (A+B) or repeated injections (C+D). A+B: NMRI nu/nu mice were treated with one intraperitoneal injection of 40 mg/kg body weight Erufosine and subdivided into three groups for blood collection at different time points: group 1, n = 6 (• ): 15 min, 30 min, 1, 2 hours; group 2, n = 5 (): 30 min, 2, 4, 8 hours; group 3, n = 5 (black triangle): 1, 4, 12, 24 and 36 hours. Erufosine concentrations in serum were determined by LC-MS/MS analysis. Data represent means ± SD: A. Data show the initial serum concentrations of groups 1-3 separately. B. Data show mean Erufosine serum-concentrations for all animals from group 1-3 pooled (n = 16). Insert shows the pharmacok- inetic parameters. C+D: NMRI nu/nu mice were treated with repeated intraperitoneal injections of Erufosine every 48 hours at the indicated concentrations. All values are means ± SD (n = 3-6). Erufosine concentrations in serum were determined by LC-MS/MS analysis. C. Concentration-dependent increase in the serum levels of Erufosine after a three weeks treatment with 5, 10, 20 and 40 mg/kg body weight Erufosine. D. Time course of the Erufosine serum concentrations after treatment with 20 and 40 mg/kg body weight Erufosine for one and three weeks. Page 4 of 11 (page number not for citation purposes) ErPC3 [nmol/ml]. ErPC3 [nmol/ml]. ErPC3 [nmol/ml] ErPC3 [nmol/ml] Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 Taken together, a single bolus injection of 40 mg/kg body brain-barrier and accumulates in the brain tissue more weight Erufosine resulted in detectable serum concentra- efficiently compared to the other organs. The concentra- tions over 36 hours with a maximum concentration of tion in brain tissue after 3 weeks of treatment with 40 mg/ 217 ± 25 nmol/ml at 113 ± 20 min after injection. kg body weight amounted to 383 nmol/g, which is clearly above the concentration required to induce death of Repetitive injection glioblastoma cells in vitro. To study biodistribution of Erufosine, four different Eru- fosine-concentrations (5, 10, 20 and 40 mg/kg body Urine excretion weight) were administered every 48 h over a period of 7 The average 24-hour urine excretion of Erufosine was (group 1), 14 (group 2) or 21 days (group 3) by intraperi- measured for 6 mice during the last 3 consecutive days of toneal or subcutaneous injection. Each of the resulting 24 a 14-day treatment period with intraperitoneal injections groups consisted of 3 to 9 mice. At the end of the treat- of 20 mg/kg or 40 mg/kg body weight Erufosine, respec- ment course 24 hours after the last injection approxi- tively (Fig. 4). The average urine volume in both groups mately 300 μl blood were drawn and organs were was comparable. Total quantity and concentration of Eru- removed as described above. fosine in the urine was very low yielding less than 0.5 μg and 0.6 nmol/ml Erufosine after treatment with 40 mg/kg Serum concentrations body weight (Fig. 4). Taking into account that the serum The repetitive injection of Erufosine resulted in a concen- concentrations was 64 nmol/ml and 122 nmol/ml Erufo- tration- and time-dependent increase in serum Erufosine- sine after a 14-day treatment with 20 mg/kg and 40 mg/kg levels. After three weeks of intraperitoneal treatment with body weight, the urine/serum ratio in both groups was 5, 10, 20 and 40 mg/kg body weight Erufosine every 48 less than 0.6%. Despite high absolute tissue concentra- hours, respective serum concentrations amounted to 20 ± tions in the kidney this demonstrates negligible urine 4 nmol/ml, 36 ± 4 nmol/ml, 68 ± 23 nmol/ml and 109 ± excretion of Erufosine. 33 nmol/ml (Fig. 1C). Similar observations were made with subcutaneous injections (data not shown). The slope Toxicity of the increase in serum concentrations was more pro- Intraperitoneal injections of Erufosine were generally well tolerated. A clinical side effect of the high dose intraperi- nounced in the first 7 days compared to longer treatment periods suggesting a convergence to steady state levels toneal treatment (40 mg/kg body weight) was transient after prolonged Erufosine-treatment (Fig. 1D). diarrhea. No local changes or signs of inflammation were seen at the puncture. As an index of systemic toxicity the Organ concentrations body weight of the mice was measured regularly. Mean In a next step we analyzed the organ distribution of Erufo- weight of all animals at the beginning of treatment was sine in the three treatment groups after 7, 14, and 21 days 35.0 ± 1.2 g. Intraperitoneal application of 5 mg/kg body of treatment (Fig. 2). Erufosine accumulated in all tissues weight Erufosine did not result in any change of the body included in the study. Maximum drug-concentrations weight, whereas administration of higher concentrations were obtained after 21 days of intraperitoneal treatment led to a transient weight loss of less than 10% of body in spleen (1307 nmol/g), kidney (1123 nmol/g) and weight (Fig. 5). lungs (939 nmol/g) (Fig. 2A). The respective subcutane- ous injections led to slightly higher organ-concentrations In contrast, subcutaneous injections of Erufosine did not at all concentrations used (Fig. 2B). cause changes in body weight for all drug-concentrations used (Fig. 5). However, a local inflammation to the point Because of a possible use of Erufosine for the treatment of of ulceration occurred at the puncture region after 14 d of glioblastoma, we were then interested in the drug concen- treatment with 40 mg/kg body weight Erufosine (not trations that could be obtained in the brain tissue. shown). Therefore, the subcutaneous treatment with the Although absolute drug concentrations in the brain tissue high Erufosine concentration was stopped after 14 days. were low compared to e.g. lungs or kidney, we could clearly demonstrate an increase of the brain/serum ratio At the end of the treatment course there were no macro- after 7 and 21 days of treatment from 1.9 to 2.9, respec- scopic signs of organ injury and no systematic changes in tively, pointing to an accumulation of Erufosine in brain organ weight (data not shown). Regarding the hematolog- tissue (Fig 3A). With regard to the organ concentrations ical parameters no bone marrow related toxicity was achieved after 14 and 21 days of treatment relative to the detectable even though the variance of white blood cell 7 day treatment, the most prominent time-dependent count was high. increase in the Erufosine-concentration was observed for brain tissue at all drug concentrations used (Fig 3B). It The platelet count raised from 566 ± 155 for the control clearly demonstrates that Erufosine penetrates the blood- group to 833 ± 172 after 14 d treatment with 40 mg/kg Page 5 of 11 (page number not for citation purposes) Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 40 mg/kg fat 20 mg/kg 10 mg/kg muscle 5 mg/kg stomach intestine colon spleen lung brain kidney liver 0 500 1000 1500 2000 0 500 1000 1500 2000 0 500 1000 1500 2000 ErPC3 [nmol/g tissue] 40 mg/kg fat 20 mg/kg 10 mg/kg muscle 5 mg/kg stomach intestine colon spleen lung brain kidney liver 0 500 1000 1500 2000 0 500 1000 1500 2000 0 500 1000 1500 2000 ErPC3 [nmol/g tissue] Biodistrib Figure 2 ution of Erufosine after repeated drug injections Biodistribution of Erufosine after repeated drug injections. Mice were separated into 24 groups and treated every 48 hours with a intraperitoneal or subcutaneous injection of Erufosine at the indicated concentrations for one, two or three weeks. At the end of the treatment period mice were killed, organs removed and organ concentrations of Erufosine were determined by LC-MS/MS analysis. All values are means ± SD (n = 3-9). A. Organ concentrations of Erufosine after intraperito- neal treatment with 5, 10, 20 and 40 mg/kg body weight Erufosine for one (left panel), two (middle panel) or three weeks (right panel). B. Organ concentration of Erufosine after subcutaneous treatment with 5, 10, 20 and 40 mg/kg body weight Erufosine for one (left panel), two (middle panel) or three weeks (right panel). Three weeks subcutaneous treatment with 40 mg/kg body weight Erufosine is missing due to local toxicity. Page 6 of 11 (page number not for citation purposes) Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 AB Serum 14d relative to 7d Brain 21d relative to 7d 0 0 7 d 21 d A Figure 3 ccumulation of Erufosine in brain tissue after repeated intraperitoneal drug injections Accumulation of Erufosine in brain tissue after repeated intraperitoneal drug injections. Mice were treated every 48 hours with intraperitoneal injections of Erufosine at the indicated concentrations for one, two or three weeks. At the end of the treatment period mice were killed, organs removed and organ concentrations of Erufosine were determined by LC-MS/ MS analysis. A. Brain and serum concentrations of Erufosine after treatment with 20 mg/kg body weight of Erufosine for 7 d and 21 d, respectively. Data show means ± SD (n = 3-6). B. Mean organ concentrations of Erufosine after treatment with 5, 10, 20 or 40 mg/kg body weight for 14 or 21 days were divided by the mean organ concentrations after the respective treatment for 7 days. Data show means ± SEM of the resulting quotients from all 4 dose groups (n = 12-24). body weight, but then decreased again until day 21 of the weight as an index of systemic toxicity. Only high dose high dose treatment (Tab. 1). As shown in table 1, long- intraperitoneal injection of Erufosine induced a mild time treatment with 40 mg/kg body weight Erufosine led diarrhea at the beginning of the treatment and a reversible to a 2 to 2.5-fold increase of serum lactate dehydrogenase weight loss preventing further dose escalation. These (LDH) after 14 and 21 days of treatment. Moreover, aspar- observations are reminiscent of earlier findings in healthy tate-aminotransferase (AST) was increased after 21 day rats after high dose intravenous application of the Erufo- treatment with 40 mg/kg body weight Erufosine, suggest- sine-related ErPC [18]. In contrast, subcutaneous applica- ing that high Erufosine-concentrations or long term treat- tion did not induce any changes in the body weight even ment may induce some cell damage. However, no further upon treatment with 40 mg/kg body weight Erufosine. significant changes in clinical chemistry and clinical pic- These observations suggest that intraperitoneal injection ture could be detected arguing against a major toxic effect of Erufosine may induce a local effect similar to the gas- (Tab. 1). Certainly it has to be taken into account that trointestinal toxicity observed after oral application of nude mice can provide only a limited toxicity profile, in Perifosine [13,16,17,22,23]. On the other hand, despite particular related to toxic immune responses. the absence of alterations in the body weight, subcutane- ous injection was accompanied by dose limiting ulcera- tions at the injection site 2 weeks after treatment with 40 Discussion Here we show for the first time, that parenteral treatment mg/kg body Erufosine. As prolonged intravenous infusion of nude mice with Erufosine is feasible without major tox- of low-dose Erufosine is well tolerated in patients (L. icity. Moreover, our data demonstrate that repeated intra- Lindner, personal communication) long-term intrave- peritoneal or subcutaneous injections of nontoxic nous infusion of Erufosine may be considered as an alter- Erufosine-concentrations yield organ concentrations that native for future experiments. are sufficient to induce tumor cell death in vitro. Clinical chemistry revealed a concentration-dependent Tolerability of Erufosine-treatment was demonstrated by increase in serum levels of LDH and to a lesser extent of the absence of significant alterations in organ weight or AST during Erufosine-treatment, while alanine-ami- macroscopic appearance, and minor changes in the body notransferase and further blood parameters remained Page 7 of 11 (page number not for citation purposes) liver kidney brain lung spleen colon small intestine stomach ErPC3 [nmol/g] relative concentration. Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 0,7 AB 0,6 0,6 0,5 0,5 0,4 0,4 0,3 0,3 0,2 0,2 0,1 0,1 0 0 20mg/kg 40mg/kg 20mg/kg 40mg/kg dose dose Uri Figure 4 ne excretion of Erufosine after repeated intraperitoneal drug injections Urine excretion of Erufosine after repeated intraperitoneal drug injections. NMRI nu/nu mice were treated with intraperitoneal injection of 20 mg/kg body weight and 40 mg/kg body weight (n = 6 each) Erufosine every 48 hours for two weeks. The urine was collected over 24-hours on the last three consecutive days of the treatment period in a metabolic cage. Average urine volumes were determined and concentrations of Erufosine in urine were measured by using LC-MS/MS analysis. Data show (A) the urine concentrations and (B) the total amount of Erufosine (means ± SEM). unchanged. Although the increase in LDH has been ever, in contrast to earlier investigations with Miltefosine described as a hint for beginning hemolysis, being a major or ErPC, instead of the reported increase in leukocyte toxic side effect of the first generation APC Miltefosine numbers, we only detected a time- and concentration- [14], the lack of changes in the hemoglobin levels and of dependent transient increase in thrombocyte numbers. a clinical correlate argues against a clinically relevant The differences in the blood cell behaviour may be related hemolytic effect of Erufosine. It may be suggested that to the distinct application mode and/or species-specific Erufosine-treatment affects the membrane composition differences in the drug effect. of the erythrocytes facilitating damage of more fragile erythrocytes during retro-orbital blood withdrawal. Since A single bolus injection of Erufosine resulted in detectable a marginal elevation of AST-levels after intravenous appli- serum Erufosine levels for approximately 36 h peaking at cation of ErPC in rats has been already described for the 217 ± 25 nmol/ml 113 ± 20 min after injection. Repeated Erufosine related ErPC [18] this parameter should be fur- intraperitoneal or subcutaneous administrations led to a ther analyzed in preclinical or clinical trials. continuous increase of serum and organ concentrations of Erufosine with the highest concentrations achieved in Kidney related serum parameters like electrolytes, protein, spleen, kidney and the lungs. The subcutaneous injections creatinine and urea did not increase during treatment with yielded slightly higher drug-concentrations in most tis- Erufosine leaving no evidence for renal dysfunction as sues compared to the intraperitoneal injections reaching described for Miltefosine [24]. significance in liver, kidney, and brain. Our data corrobo- rate earlier findings about the bioavailability of the Erufo- Importantly, similar to previous reports for other APC, sine-related ErPC in healthy rats [18]. Although organ Erufosine lacked bone marrow toxicity [18,24,25]. How- distributions were quite similar, the Erufosine-concentra- Page 8 of 11 (page number not for citation purposes) ErPC3 [nmol/ml] ErPC3 [μg] Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 ip-injection -1 0 -2 -3 5 mg/kg 20 mg/kg 40 mg/kg 10 mg/kg 3 sc-injection -10 4 8 121620 -2 time [d] -3 Figure 5 Change in body weight of animals upon Erufosine treatment Change in body weight of animals upon Erufosine treatment. NMRI nu/nu mice were treated with intraperitoneal injections of 5, 10, 20 und 40 mg/kg body weight Erufosine every 48 hours. Body weight was determined every second day. Val- ues represent means ± SEM of the difference from starting weight in the respective dose groups. A. Body weight after intra- peritoneal injections. B. Body weight after subcutaneous injections. tions achieved upon intraperitoneal or subcutaneous In contrast, Erufosine-concentrations in the brain tissue administration in the respective tissues were increased in were below the levels obtained by Erdlenbruch et al. [18], most of the tested organs compared to the ErPC-concen- an effect that may reflect distinct efficiency in crossing the trations obtained after intravenous injections in the inves- blood brain barrier due to the distinct lipophilic behav- tigation of Erdlenbruch and coworkers [18]. This may at iour of the two derivatives and/or altered composition of least partially be related to the altered serum composition the blood brain barrier in rats compared to mice. Never- observed in nude mice. Moreover, the increased sensitiv- theless, we observed a strong time- and concentration- ity of liquid chromatography-tandem mass spectrometry dependent accumulation of Erufosine in the brain tissue used in the present study compared to that of high per- reaching 383 nmol/g after a 3-week treatment with 40 formance thin layer chromatography HPTLC used in the mg/kg of body weight. This concentration is clearly above earlier investigation may be of relevance [21]. the concentration sufficient to induce cytotoxicity in malignant glioma cell lines in vitro [10,19,26,27]. Page 9 of 11 (page number not for citation purposes) difference in body difference in body weight [g] weight [g] Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 Table 1: Serum parameters and hematological parameters during intraperitoneal Erufosine treatment (Mean ± SD) 14-d treatment 21-d treatment control 20 mg/kg bw 40 mg/kg bw 20 mg/kg bw 40 mg/kg bw Blood count Leukocytes (/μl) 4,2 ± 2,1 3,3 ± 1,8 3,5 ± 2,5 5,0 ± 3,4 2,6 ± 1,4 Erythrocytes (10 /μl) 9,1 ± 0,4 8,4 ± 0,3 9,1 ± 0,8 9,1 ± 0,5 8,4 ± 1,4 Platelets (10 /μl) 566 ± 155 741 ± 94 833* ± 172 872* ± 432 442* ± 156 Hemoglobin (g/dl) 14,5 ± 0,6 13,6 ± 0,9 14,6 ± 1,9 14,0 ± 0,5 13,4 ± 1,9 Hematocrit (%) 47,5 ± 2,3 45,5 ± 1,7 49,1 ± 4,5 45,4 ± 4,7 44,7 ± 5,9 Serum Na (mmol/l) 156 ± 8 156 ± 2 155 ± 10 157 ± 4 162 ± 6 K (mmol/l) 5,1 ± 0,9 5,9 ± 1,2 6,3 ± 0,8 5,4 ± 0,8 5,8 ± 1,0 Ca (mmol/l) 2,4 ± 0,2 2,5 ± 0,1 2,6 ± 0,2 2,1 ± 0,3 2,5 ± 0,2 AST (U/l) 125 ± 42 117 ± 38 152 ± 22 140 ± 69 245* ± 156 ALT (U/l) 63 ± 32 55 ± 14 93 ± 25 65 ± 31 93 ± 45 LDH (U/l) 1315 ± 441 1401 ± 818 2650* ± 959 1913 ± 1152 3498* ± 519 Protein (g/dl) 5,1 ± 0,3 4,9 ± 0,1 5,5 ± 0,4 4,8 ± 0,3 4,9 ± 0,5 Creatinine (mg/dl) 0,3 ± 0,1 0,3 ± 0,1 0,3 ± 0,1 0,3 ± 0,1 0,3 ± 0,1 Urea (mg/dl) 56,5 ± 9,0 50,8 ± 8,1 57,3 ± 6,0 47,9 ± 6,4 56,2 ± 13,3 Together with our earlier investigations on the increased feasible and safe. Furthermore the concentrations cytotoxic efficacy of ionizing radiation in combination achieved in the brain tissue are above the concentrations with Erufosine in glioblastoma cell lines in vitro, the abil- needed for combination effects with radiation in earlier in ity of Erufosine to cross the blood brain barrier and to vitro experiments using human astrocytoma/glioblastoma accumulate in the brain tissue make the drug a promising cell lines. Our results constitute the basis for the design of candidate for combined treatment approaches with radio- preclinical investigations with Erufosine alone and in therapy in malignant glioma. Although clinical trials combination with radiotherapy in murine tumor models, already demonstrated feasibility and tolerability of a ther- in particular in nude mice. In a next step, we will evaluate apy with Perifosine, Erufosine, or Perifosine with radio- efficacy of Erufosine in combination with ionizing radia- therapy for patients with advanced human malignancies tion in vivo in nude mice bearing subcutaneous tumors. [[8,13] and personal communication with L. Lindner], Based on our present investigations, pretreatment with before debarking into clinical trials with patients suffering repeated intraperitoneal injections of Erufosine for 1 or 2 from malignant glioma and other tumors, efficacy of Eru- weeks prior to initiation of radiotherapy should be con- fosine in combined treatment approaches has to be eval- sidered to benefit from the drug-accumulation in the uated in animal experiments in vivo. tumor tissue. In conclusion, our data reveal that intraperitoneal and Competing interests subcutaneous administration of Erufosine to nude mice is The authors declare that they have no competing interests. Page 10 of 11 (page number not for citation purposes) Radiation Oncology 2009, 4:46 http://www.ro-journal.com/content/4/1/46 nation with radiotherapy. Cancer treatment reviews 2007, Authors' contributions 33(2):191-202. GH contributed significantly to the design of the study, 14. Kotting J, Marschner NW, Neumuller W, Unger C, Eibl H: Hexade- data acquisition, data analysis and drafting the manu- cylphosphocholine and octadecyl-methyl-glycero-3-phos- phocholine: a comparison of hemolytic activity, serum script. LHL contributed significantly to data acquisition, binding and tissue distribution. Progress in experimental tumor data analysis and drafting the manuscript. KW and MV research Fortschritte der experimentellen Tumorforschung 1992, 34:131-142. performed probe preparation and mass spectrometry 15. Verweij J, Planting A, Burg M van der, Stoter G: A dose-finding measurements, respectively. JW performed many of the study of miltefosine (hexadecylphosphocholine) in patients animal experiments. ACM and MB performed critical revi- with metastatic solid tumours. Journal of cancer research and clin- ical oncology 1992, 118(8):606-608. sion of the manuscript. HE synthesized and provided 16. Crul M, Rosing H, de Klerk GJ, Dubbelman R, Traiser M, Reichert S, ErPC and ErPC3 for the analysis. CB participated in the Knebel NG, Schellens JH, Beijnen JH, ten Bokkel Huinink WW: conception of the study and interpretation of data. VJ per- Phase I and pharmacological study of daily oral administra- tion of perifosine (D-21266) in patients with advanced solid formed conception and design of the study and substan- tumours. Eur J Cancer 2002, 38(12):1615-1621. tially contributed to interpretation of data, drafting of the 17. 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Vink SR, van Blitterswijk WJ, Schellens JH, Verheij M: Rationale and clinical application of alkylphospholipid analogues in combi- Page 11 of 11 (page number not for citation purposes)

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Radiation OncologySpringer Journals

Published: Oct 23, 2009

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