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In Vivo Effect of Oracin on Doxorubicin Reduction, Biodistribution and Efficacy in Ehrlich Tumor Bearing Mice

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In Vivo Effect of Oracin on Doxorubicin Reduction, Biodistribution and Efficacy in Ehrlich Tumor Bearing Mice Pharmacological Reports Copyright © 2013 2013, 65, 445452 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences Invivo effectoforacinondoxorubicinreduction, biodistributionandefficacyinEhrlichtumor bearingmice VeronikaHanušová1,PavelTomšík2,LenkaKriesfalusyová3, AlenaPakostová1,IvaBoušová1,LenkaSkálová1 1 DepartmentofBiochemicalSciences,CharlesUniversityinPrague,FacultyofPharmacy,Heyrovského1203, HradecKrálové,CZ-50005,CzechRepublic 2 DepartmentofMedicalBiochemistry,CharlesUniversityinPrague,FacultyofMedicine, Šimkova570, HradecKrálové,CZ-50038,CzechRepublic 3 Radio-IsotopeLaboratory,CharlesUniversityinPrague,FacultyofMedicine, Šimkova570,HradecKrálové, CZ-50038,CzechRepublic Correspondence: LenkaSkálová,e-mail:[email protected] Abstract: Background: The limitation of carbonyl reduction represents one possible way to increase the effectiveness of anthracycline doxo- rubicin (DOX) in cancer cells and decrease its toxicity in normal cells. In vitro, isoquinoline derivative oracin (ORC) inhibited DOX reduction and increased the antiproliferative effect of DOX in MCF-7 breast cancer cells. Moreover, ORC significantly decreases DOX toxicity in non-cancerous MCF-10A breast cells and in hepatocytes. The present study was designed to test in mice the in vivo effect of ORC on plasma and tissue concentrations of DOX and its main metabolite DOXOL. The effect of ORC on DOX efficacy in micebearingsolidEhrlichtumors(EST)wasalsostudied. Methods: DOX and DOX + ORC combinations were iv administered to healthy mice. Blood samples, livers and hearts were col- lected during the following 48 h. DOX and DOXOL concentrations were assayed using HPLC. The mice with inoculated EST cells weretreatedrepeatedly iv withDOXandDOX+ORCcombinations,andthegrowthoftumorswasmonitored. Results: ORC in combination with DOX significantly decreased DOXOL plasma concentrations during four hours after administra- tion, but this significantly affected neither DOX plasma concentrations nor DOX or DOXOL concentrations in the liver and heart at any of intervals tested. In EST bearing mice, ORC did not significantly affect DOX efficacy on tumor growth. However, EST was showntobeanimpropermodelforthetestingofORCefficacy invivo, asORCdidnotinhibitDOXOL formationinEST. Conclusions: In vivo, ORC was able to retard DOXOL formation but was not able to improve DOX efficacy in EST-bearing mice. Keywords: doxorubicinpharmacokinetics,doxorubicinol,CBR1,carbonylreduction Pharmacological Reports, 2013, 65, 445452 445 Abbreviations: CBR1 – carbonyl reductase 1, DOX – doxoru- is rapidly absorbed from the gastrointestinal tract, and bicin, DOXOL – doxorubicinol, EST – Ehrlich solid tumor, it possesses good bioavailability. Its acute, subchronic ORC–oracin and chronic toxicity is low, and no cardiotoxicity has been observed in experimental animals [1, 10, 21]. As with DOX, carbonyl reduction is the main metabolic pathway of ORC [25]. In MCF-7 breast cancer cells, Introduction ORC is also metabolized by CBR1, and it is much bettersubstrateforCBR1thanDOX[9]. In our previous in vitro studies, we have demon- Anthracycline antibiotic doxorubicin (DOX) is one of strated that ORC acts as an effective competitive in- the most useful anticancer agents and it still repre- hibitor of DOX reduction in MCF-7 cells, and in this sents the key component in the therapy of many carci- way ORC significantly increased DOX efficacy in the noma types. Unfortunately, DOX is not sufficiently killing of cancer cells [14]. On the other hand, the in- effective in many cases, but increasing the dosage of hibitory effect of ORC on DOX reduction in non- DOX is limited due to its systemic toxicity, particu- cancerous MCF-10A breast cells was almost none in larly cardiotoxicity [4, 15]. It is widely accepted that comparison to cancer cells MCF-7, therefore, ORC DOX cardiotoxicity is caused mainly by DOX- cannot be used to decrease the deactivation of DOX in mediated oxidative stress [24]. In addition, doxoru- normal breast tissue. In viability tests, ORC surpris- bicinol (DOXOL), the main metabolite of DOX, is ingly protected non-tumorous MCF-10A cells and rat thought to contribute to DOX toxicity [19, 23]. hepatocytes against DOX-mediated toxicity, probably DOXOL, which is less cytostatically active but more due to the inhibition of enzymes catalyzing DOX re- cardiotoxic than its parent drug, accumulates in the dox cycling [12]. The obtained data suggest that on heart and participates significantly in the development one hand, ORC could act as an inhibitor of DOX de- of the chronic cumulative cardiotoxicity resulting activation in tumor cells and thus potentiate DOX ef- from DOX therapy. Therefore, the search for possi- ficacy, and on the other hand, ORC could partly pro- bilities of how to increase DOX efficacy in cancer tectnon-tumortissuesagainstDOXtoxicity. cells and also minimize the associated toxicities to The present study was designed to test the in vivo effect non-cancerous tissues has become quite an important of ORC on DOX and DOXOL concentrations in plasma, research goal [6]. Many strategies are based on al- as well as in the liver and heart after iv administration to tered DOX metabolism. The enhancing of DOX up- mice of a single dose of DOX alone or ORC + DOX com- take by cancer cells and the metabolic activation of binations. The efficacy of DOX, ORC, and DOX + ORC DOX prodrug within cancer cells have been the main combinations on the growth of solid Ehrlich tumors (EST) approaches [13]. The inhibition of DOX-metabolizing in tumor-bearing mice was tested and compared. enzymesrepresentsanotherpossiblestrategy[14]. Several carbonyl reducing enzymes participate in DOXOL formation: in the liver primarily carbonyl re- ductase 1 (CBR1), but also partly the aldehyde reduc- MaterialsandMethods tase AKR1A1 and aldo/keto reductase AKR1C; in the heart mainly AKR1A1 [2, 16, 18]. CBR1 has been identified as the main enzyme catalyzing DOX reduc- Chemicalsandreagents tion in MCF-7 breast cancer cells [9]. Moreover, the elevated activity of CBR1 in cancerous tissues as op- DOX was provided by Pharmacia (Upjohn, Italy). posed to normal ones has been reported [20]. Thus the ORC and dihydrooracin were obtained from the inhibitors of CBR1 have been intensively tested, as Research Institute for Pharmacy and Biochemistry their concomitant use with DOX seems to be a possi- (Prague, Czech Republic). ZnSO4 was purchased from ble strategy to increase the cytostatic effect of DOX in Merck Co. (Darmstadt, Germany). Coenzyme NADPH cancer cells [11]. The isoquinoline derivative oracin was supplied by Sigma-Aldrich (Prague, Czech Re- (ORC, 6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo- public). All other chemicals used were of HPLC or 5,6-dihydro-11H-indeno[1,2-c]isoquinoline), which had analytical grade. The stock solutions were prepared in been originally prepared as a potential anticancer double-distilled deionized water or DMSO and stored agent [8], is another promising CBR1 inhibitor. ORC inthedarkat4°C. 446 Pharmacological Reports, 2013, 65, 445452 Doxorubicinandoracininmice invivo Veronika Hanuová et al. Animals try, VUFB, Prague, Czech Republic, and then main- tained in adult female NMRI mice through serial sub- Female NMRI mice weighing 30–33 g were purchased cutaneous transplantation. For the tumor passage, from the Faculty of Medicine in Brno, Czech Repub- 1 volume of tumor homogenate of EST was diluted lic. They were given a standard diet of Altromin 1320 with 2 volumes of physiological saline. On day 0, the (manufactured by Velaz, a.s., Czech Republic) and mice were inoculated subcutaneously with 0.2 ml of water ad libitum. The experiments were designed and the final tumor cell suspension containing 1 × 106 conducted in accordance with European Union rec- cells. The mice bearing the tumor inoculums were ommendations on the handling of experimental ani- then divided into 5 groups: one control group (un- mals and were approved by the Ethics Committee of treated) and four treated groups of 7 animals each. CharlesUniversityintheCzechRepublic. The second group was treated with DOX alone (one dose 2 mg/kg); the third group was treated with ORC DOXandDOXOLconcentrationsinblood alone (one dose 20 mg/kg); the fourth group was plasmaandtissuesafterDOXandDOX+ORC treated by a 1 : 2 combination of DOX + ORC (one treatment dose of DOX 2 mg/kg and ORC 4 mg/kg); and the fifth group was treated by a 1 : 10 combination of The mice were divided into four groups (12 animals DOX + ORC (one dose of DOX 2 mg/kg and ORC in each): the first group (control) underwent no treat- 20 mg/kg). All drugs were administered iv in three ment; the second group was treated with DOX alone single doses (the volume of one dose was 0.25 ml per (single iv dose 10 mg/kg); the third group was treated 30 g of body weight) on days 1, 4, and 7 after the with a 1 : 2 combination of DOX + ORC (single iv inoculationofthetumors. dose of DOX 10 mg/kg and ORC 20 mg/kg); and the On the eighth day, all the animals were anesthe- fourth group was treated with a 1 : 5 combination of tized with ether and sacrificed by cervical dislocation. DOX + ORC (single iv dose of DOX 10 mg/kg and Thetumorswerethenexcisedandweighed. ORC 50 mg/kg). The drugs were applied iv through the tail vein in volumes of 0.25 ml per 30 g of body weight at time 0. Blood samples (45 µl from each PreparationofsamplespriortoHPLCanalysis mouse) were collected by retro-orbital plexus punc- ture or posterior vena cava puncture using heparinized The plasma samples and tissue homogenates
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