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Biocompatible Microemulsion Modifies the Tissue Distribution of Doxorubicin
RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism
Biocompatible Microemulsion Modifies the Tissue Distribution of Doxorubicin ˜ 1 CAROLINE DAMICO CANDIDO,1 MICHEL LEANDRO CAMPOS,1 JULIANA URUGUAY CORREA VIDIGAL ASSUMPC ¸ AO, 1 1 2 ˆ KELLY CHRYSTINA PESTANA, ELIAS CARVALHO PADILHA, IRACILDA ZEPPONE CARLOS, ROSANGELA GONC ¸ ALVES PECCININI1 1
Department of Natural Active Principles and Toxicology, School of Pharmaceutical Sciences, S˜ao Paulo State University – UNESP, Araraquara 14801-902, SP, Brazil 2 Department of Clinical Analysis, School of Pharmaceutical Sciences, S˜ao Paulo State University – UNESP, Araraquara 14801-902, SP, Brazil Received 28 February 2014; revised 24 June 2014; accepted 8 July 2014 Published online 6 August 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24106
ABSTRACT: The incorporation of doxorubicin (DOX) in a microemulsion (DOX-ME) has shown beneficial consequences by reducing the cardiotoxic effects of DOX. The aim of this study was to determine the distribution of DOX-ME in Ehrlich solid tumor (EST) and the heart, and compare it with that of free DOX. The distribution study was conducted with female Swiss mice with EST (n = 7 per group; 20–25 g). Animals received a single dose (10 mg/kg, i.p.) of DOX or DOX-ME 7 days after tumor inoculation. Fifteen minutes after administration, the animals were sacrificed, and the tumor and heart tissues were taken for immediate analysis by ultra-performance liquid chromatography. No difference was observed in DOX concentration in tumor tissue between DOX and DOX-ME administration. However, the most remarkable result in this study was the statistically significant reduction in DOX concentration in heart tissue of animals given DOX-ME. Mean DOX concentration in heart tissue was 0.92 ± 0.54 ng mg−1 for DOX-ME and 1.85 ± 0.34 ng mg−1 for free DOX. In conclusion, DOX-ME provides a better tissue distribution profile, with a lower drug concentration in heart tissue but still comparable tumor C 2014 Wiley Periodicals, Inc. and the American drug concentration, which indicates that antitumor activity would not be compromised. Pharmacists Association J Pharm Sci 103:3297–3301, 2014 Keywords: microemulsion; formulation; doxorubicin; Ehrlich tumor; liquid chromatography; UPLC; distribution; pharmacokinetics; toxicology
INTRODUCTION Doxorubicin (DOX) is an anthracycline antibiotic with a broad antitumor spectrum used both as a single agent and in combination regimens.1 DOX is currently on the market as the free drug and liposome-encapsulated drug, but research continues with DOX. For instance, it is in clinical trials with some forms of polymeric drug2 and in animal studies with a biocompatible microemulsion.3 The main problem associated with the use of the free form is cardiomyopathy.4 Acute cardiomyopathy may occur immediately after or during administration of a single dose and is a result of drug accumulation in heart muscle.4 Acute cardiomyopathy occurs in up to 40% of the patient population, where the prognosis is generally good, may be completely asymptomatic, and usually resolves spontaneously.5 Chronic cardiomyopathy is more common and is characterized by congestive heart failure unresponsive to digoxin, as a result of successive deleterious effects on cardiac tissue with repeated doses.5 According to Octavia et al.,4 the mortality rate in patients with chronic DOX-induced cardiotoxicity is 50% after 5 years. The encapsulation of DOX in liposomes has been associated with decreased cardiotoxicity6,7 and changes in its pharmacokinetic profile. These formulations were developed with the aim of achieving a better clinical response and less toxicity. However, ˆ Correspondence to: Rosangela Gonc¸alves Peccinini (Telephone: +55-16-33016988; Fax: +55-16-3301-6980; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 103, 3297–3301 (2014)
C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association
these drugs are often discontinued due to severe side effects.8 The toxicity of encapsulated DOX depends on the lipids used in liposome formulation.5 For instance, the inclusion of phosphatidylserine or polyethylene glycol (PEG) in the formulation has been related to changes in the mononuclear phagocyte system (MPS).5,8,9 This occurs because the liposomes are taken up by cells of the MPS, and DOX exerts its toxic effect within these cells. MPS toxicity may be of concern in immunosuppressed patients, such as those with AIDS.5 Another liposomal formulation containing PEG causes palmar-plantarerythrodysesthesia (PPE) due to the accumulation of DOX in the skin.5 PPE is a painful scaly dermatitis that primarily affects the hands and feet. PPE was the most common adverse effect related to liposomal DOX and occurred in 49% of patients in a phase II trial in ovarian cancer.9 Although PPE is attributed mainly to pegylated formulations, there are reports of cases with nonpegylated formulations.10 According to Patel,1 “the tumor tissue is a dynamic microenvironment and efforts to improve therapeutic efficacy might be achieved by modifying these dynamic processes to enhance drug delivery.” The delivery of the drug to normal tissue is the cause of occurrence of various adverse effects, as well as its limited distribution in tumor tissues resulting in tumor drug resistance and treatment failure. Accordingly, in previous works, we described different microemulsion formulation specially designed for DOX incorporation,11–14 we found that the pharmacokinetic profile of DOX in a microemulsion (DOX-ME) showed significant differences compared with that for free DOX in Wistar rats, with beneficial consequences regarding cardiotoxic effects.3
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The administration of DOX-ME in Wistar rats has been shown to result in higher plasma concentration of DOX and lower volume of distribution when compared with the administration of DOX in the free form, and increased serum creatine kinase MB (CKMB) activity in the DOX group but unchanged CKMB activity in the DOX-ME group.3 These results suggest modifications in drug access to susceptible sites using DOXME. The aim of this study was to determine the distribution of DOX-ME in Ehrlich solid tumor (EST) and heart in female Swiss albino mice to compare it with free DOX. This information is essential for the assessment of product safety for future clinical application.
METHODS Chemicals and Reagents Soya phosphatidylcholine (SPC) (EpikuronTM 200, Germany) was purchased from Degussa Texturants Systems Deutschland GmbH & Company (Hamburg, Germany); cholesterol (CHO) and sodium oleate (SO) from Sigma–Aldrich (St. Louis, Missouri); Tris (hydroxymethyl) aminomethane from Merck (Darmstadt, Germany); DOX hydrochloride (AdriamycinTM ) from Eurofarma (Sao Paulo, Brazil); daunorubicin hydrochloride [DAU—internal standard (IS)] from Pfizer (Sao Paulo, Brazil), polyoxyethyleneglyceroltrihydroxystearate 40 (EU; Eumulgin HRE 40) from Pharma Special (Sao Paulo, Brazil); and acetonitrile and methanol, HPLC grade, from J.T. Baker (Phillipsburg, New Jersey, United States). All other solvents and chemicals were analytical grade. Water was purified in a Milli-Q Plus system (Millipore, Billerica, Massachusetts, United States) with 18.2 M-cm resistivity. R
Microemulsion Preparation The microemulsion preparation was carried out as described by ˜ et al.,3 which started with the addition of CHO, the Assumpc¸ao oil phase, to the semisolid mixture of SPC/EU/SO, the surfactant phase. The aqueous phase (80%, w/w) was then added to this mixture, which was homogenized by ultrasound for 10 min and allowed to stand for 24 h at 25 ± 0.1◦ C to reach complete equilibrium. The samples were centrifuged at 8500g for 15 min (Ultracentrifuge Hitachi Himac CP-80) to remove titanium residues that might have been released from the ultrasound tip. Finally, a suitable amount (1.5 mg mL−1 ) of DOX was dissolved directly in the previously prepared ME.
EST Inoculation A model of EST was used, where 2 × 106 Ehrlich ascites tumor (EAT) cells were implanted subcutaneously in the thigh of the left hind limb of mice. A solid tumor mass developed within 7 days. EAT cells were maintained in vivo in Swiss albino mice by intraperitoneal transplantation every 7 days. This procedure was carried out at the Laboratory of Clinical Immunology of the School of Pharmaceutical Sciences, Sao Paulo State University, Araraquara, Brazil. Cell viability was evaluated by the trypan blue exclusion test, and only the cell suspensions that showed 95% viability were employed. Damico et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3297–3301, 2014
Distribution Study of free DOX and DOX Incorporated in Microemulsion A preclinical drug distribution study was conducted in female Swiss mice with EST (n = 7 per group; weighing 20–25 g). The sample size was based on the method reported by Chow and Wang.15 The animals were housed at a constant temperature (24 ± 1◦ C), humidity controlled (55 ± 5%) and 12-h light cycle starting at seven a.m., with food and water ad libitum. The experiments were conducted during the light phase and the experimental protocol was approved by the Research Ethics Committee of the School of Pharmaceutical Sciences, UNESP, Araraquara, SP, Brazil (Process #60/2012). Animals received a single dose (10 mg kg−1 , i.p.) of DOX or DOX-ME. At 15 min after administration, the animals were sacrificed and the tumor and heart tissues were taken for immediate analysis. A pilot study had been conducted to select the time for extracting the heart. In that study, the organs were extracted at 15, 30, and 45 min after administration of the two DOX formulations. We observed that tissue DOX concentration peaked at 15 min, and thus, this time point was selected for the extraction. DOX Analysis in Heart and Tumor Tissue by Ultra-Performance Liquid Chromatography An ultra-performance liquid chromatography (UPLC) method for DOX determination in tumor and heart was developed and validated. This bioanalytical method detects free DOX, regardless of the formulation administered. The method was according to Alhareth et al.16 Analysis was conducted using a Waters Acquity H-Class UPLC System with fluorescence detector at 8ex/em of 480/560 nm. Chromatographic separation was carried out with a Waters Acquity CSH C18 column (100 × 2.1 mm2 , with 1.7 :m particle size) connected to a Waters Vanguard C18 guard column (5 × 2.1 mm2 , with 1.7 :m, particle size). The mobile phase consisted of acetonitrile:formic acid 0.1% (40:60, v:v) in isocratic mode, with flow rate of 0.4 mL min−1 . The injection volume was 10 :L. The sample manager was maintained at 10◦ C. The tissue samples were processed as follows. A 50-:L volume of IS (10 :g mL−1 daunorubicin) was added to 100 mg of tumor or heart tissue, followed by vortexing (30 s), and 100 :L 1 M Tris–HCl (pH 8.8) was then added, followed by vortexing (30 s). Afterward, 1 mL ethyl acetate was added and the sample was centrifuged for 15 min at 23,800g. The supernatant (900 :L) was filtered through a polyvinylidene difluoride membrane (0.22 :m) and evaporated to dryness under vacuum (Genevac mini VacSample Concentrator Range ). The residue was resuspended in 100 :L mobile phase along with 10 :L 35% perchloric acid and the solution filtered again directly into the injection vial of the chromatographic system. The blood was removed from the tissue with 10 mL saline before processing to reduce the interference of blood DOX with tissue DOX concentrations. The bioanalytical method was validated according to the Food and Drug Administration—Guidance for Industry Bioanalytical Method Validation17 and ANVISA—RE 899/200318 and RDC 27/2012.19 R
R
R
R
Statistical Analysis The amount of DOX in tumor and heart tissue was expressed in ng mg−1 as median, mean, and 95% CI. Intergroup comparison was performed by the Mann–Whitney test (GraphPadPrism software, version 5.0). The calibration curve and coefficient of R
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Table 1. Inter- and Intra-run Precision and Accuracy of UPLC Bioanalytical Method for DOX in Tumor and Heart Tissues Tumor
Heart
Concentration Precision Accuracy Precision Accuracy (ng mg−1 ) (%) (%) (%) (%) Intra-run
Inter-run
0.5 0.75 1.5 0.5 0.75 1.5
12.41 6.87 13.91 13.85 9.75 1.25
114.86 88.39 103.99 113.13 110.31 85.44
11.03 7.78 11.30 5.54 7.02 1.25
106.11 108.84 85.23 107.43 89.46 85.44
Precision was expressed by CV and accuracy was expressed by deviation from nominal concentration.
Table 2. Recovery of the Extraction Procedure of DOX from Both Tumor and Heart Tissues Concentration (ng mg−1 )
Recovery (%)
CV (%)
0.5 0.75 2.0 0.5 0.75 1.5
97.71 109.51 99.18 83.39 85.17 84.81
1.54 4.52 5.21 11.63 10.11 8.53
Tumor tissue
Heart tissue
CV, coefficient of variation.
Table 3. Data Showing Stability of DOX in Both Tumor and Heart Tissue under Two Specific Wait Conditions
Tumor tissue Heart tissue Figure 1. Chromatograms (a) DOX 500 ng/mL and IS 2500 ng/mL in solution, (b) DOX 2.5 ng mg−1 and IS 5 ng mg−1 superimposed on a zero sample in heart tissue, and (c) DOX 1.0 ng mg−1 and IS 5 ng mg−1 superimposed on a blank in tumor tissue.
variation (CV, %) calculations were performed using Origin software. R
RESULTS AND DISCUSSION Bioanalytical Method for DOX Determination in Tissue The bioanalytical method developed and validated did not show any interference peaks at the retention times of DOX and IS, as can be seen in Figure 1. The calibration curves of DOX showed good linearity in the range of 0.2–2.0 ng mg−1 for tumor tissue and 0.25–2.5 ng mg−1 for heart tissue. The mean regression equations were y = 1.16 × 10−3 x + 5.83 × 10−3 , with r = 0.995 and p = 3.03 × 10−7 , for tumor tissue and y = 2.10 × 10−3 x − 1.08 × 10−2 , with r = 0.984 and p = 6.65 × 10−5 , for heart tissue, where y corresponds to the peak area ratio of DOX to IS and refers to the nominal concentration of DOX in spiked plasma. The LLOQ was 0.2 DOI 10.1002/jps.24106
Concentration (ng mg−1 )
Short-Term Stability 4 h 25◦ C (%)
PostProcessing Stability 4 h 10◦ C (%)
0.5 2.0 0.5 1.5
90.88 91.90 90.02 95.52
90.19 89.63 94.36 93.45
and 0.25 ng mg−1 for tumor and heart tissue, respectively, with precisions of 19.98% and 6.43% and accuracy of 107.94% and 109.49% for tumor and heart tissue, respectively. Inter- and intra-run precision and accuracy studies were carried out at three concentration levels (0.5, 0.75, and 1.5 ng mg−1 ) for both tumor and heart tissue, with five replicates for each level for tumor tissue and three replicates each level for heart tissue. Both tumor and heart tissue showed interand intra-run precision (CV less than 15%) and accuracy (deviation from nominal concentration between 85% and 115%), and thus within acceptance criteria, as can be seen in Table 1. The recovery of DOX was found to be consistent and reproducible after extraction of both tumor tissue and heart tissue. Table 2 shows that total recovery was 102.1% for tumor tissue and 84.5% for heart tissue, with respective CV of 6.3% and 1.1%. In the stability studies, two concentration levels were used, 0.5 and 2.0 ng mg−1 for tumor tissue and 0.5 and 1.5 ng mg−1 for heart tissue, and both assays were carried out in triplicate. Table 3 shows the stability results of the UPLC bioanalytical method for DOX in tumor and heart tissue. Damico et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3297–3301, 2014
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Figure 2. Tissue distribution of DOX administered in the free form and incorporated into the microemulsion (mean ± CI95). * = statistically different (p < 0.05) compared with heart tissue in free DOX administration by Mann–Whitney test.
Although there are several other storage conditions that could have been evaluated, the low availability of tissue to perform these tests did not allow more assays. Therefore, only essential stability tests for the study were performed. The stability observed was sufficient to perform the distribution study of DOX in tumor and heart tissue. The bioanalytical method validated showed sufficiently narrow confidence intervals in tumor tissue, its site of therapeutic action, and heart tissue, in which toxicity is the most important limitation to clinical use of DOX. Evaluation of DOX Distribution in Tumor Tissue and Heart Tissue in Mice Figure 2 shows the DOX concentration in both tumor and heart tissue 15 min after i.p. administration of 10 mg kg−1 DOX in the free form or microemulsion form. No difference was observed in concentration of DOX in tumor tissue between the DOX and DOX-ME groups. The mean concentration (and 95% CI) in tumor tissue following the administration of DOX-ME was 0.85 ± 1.25 ng mg−1 , whereas with the administration of free drug, it was 0.44 ± 0.24 ng mg−1 . On the basis of this result, the same or better antitumor activity would be expected for DOX-ME compared with the free DOX formulation. However, the most remarkable result in this study was the statistically significant reduction of the DOX concentration in heart tissue after administration of DOX in microemulsion form, compared with administration of free DOX. The mean DOX concentration (and 95% CI) in heart tissue was 0.92 ± 0.54 ng mg−1 for microemulsion form and 1.85 ± 0.34 ng mg−1 for free DOX. This fact can be explained by the microemulsion composition. Microemulsion particles are composed of CHO on their surface similar to human low-density lipoprotein, which in contact with the plasma acquires ApoEm, which is recognized by lipid receptors. Myocardial cells have fewer lipoprotein receptors, resulting in lower uptake of the drug by this tissue. In the recent literature, two works with similar aim stand out. The first one was by Alhareth et al.,20 who observed a decrease in mean concentration of DOX in heart when DOX was Damico et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3297–3301, 2014
loaded into an anionic emulsion polymerization of polyalkylcyanoacrylate nanoparticles compared with free DOX, but that work did not draw any conclusion about the capability of the system to keep the tumor distribution unaltered. However, the main objective of that work was method development. The second work was by Ayen and Kumar,21 who carried out an extensive in vivo study comparing free DOX, DOX-loaded (PEG)3 –PLA nanopolymersomes and the formulation marketed as LipoDoxTM . The most impressive result in that work was the increased DOX concentration in tumor and decreased concentration in heart with DOX-loaded (PEG)3 –PLA nanopolymersomes as compared with free DOX. Despite that DOX-ME did not demonstrate a statistically significant increase in DOX concentration in the tumor, its reduced ˜ et al.3 and its reduced cardiotoxicity as reported by Assumpc¸ao DOX concentration in heart tissue shown in the present work, justify continued studies with this system. The future clinical application of this system depends on the risk–benefit ratio, and having a diverse choice of drugs is always beneficial for achieving better treatment success.
CONCLUSIONS The bioanalytical method validated was found to be adequate for the evaluation of DOX distribution in both heart and tumor tissue, allowing a fast and sensitive analysis of DOX in these tissues. The main goal of this study was to evaluate the distribution of DOX in both tumor and heart tissue, and the results demonstrated a better heart distribution profile with lower concentrations of DOX when administered in a microemulsion. A comparable tumor distribution profile was observed, indicating that its antitumor activity would not be reduced.
ACKNOWLEDGMENTS ˜ de Amparo The authors thank the CAPES and Fundac¸ao ˜ Paulo (FAPESP – Process a` Pesquisa do Estado de Sao 2011/11239–9) for the financial support. Dr. A. Leyva helped with English editing of the manuscript.
REFERENCES 1. Patel KJ, Tr´edan O, Tannock IF. 2013. Distribution of the anticancer drugs doxorubicin, mitoxantrone and topotecan in tumors and normal tissues. Cancer Chemother Pharmacol 72:127–138. 2. Ali RU, Salim K, Rather MA, Wani WA, Haque A. 2011. Advances in nano drugs for cancer chemotherapy. Curr Cancer Drug Targets 11:135–146. ˜ JUCV, Campos ML, Nogueira Filho MAF, Pestana KC, 3. Assumpc¸ao Baldan HM, Pilon TPF, Oliveira AG, Peccinini RG. 2012. Biocompatible microemulsion modifies the pharmacokinetic profile and cardiotoxicity of doxorubicin. J Pharm Sci 102:289–296. 4. Octavia Y, Tocchetti CG, Gabrielson KL, Janssens S. 2012. Doxorubicin-induced cardiomyopathy: From molecular mechanisms to therapeutic strategies. J Mol Cell Cardiol 52:1213–1225. 5. Waterhouse DN, Tardi PG, Mayer LD, Bally MB. 2001. A comparison of liposomal formulations of doxorubicin with drug administered in free form. Drug Safety 24(12):903–920. 6. Cowens JW, Creaven PJ, Greco WR, Brenner DE, Tung Y, Ostro M, Pilkiewicz F, Ginsberg R, Petrelli N. 1993. Initial clinical (phase I) trial of TLC D-99 (doxorubicin encapsulated in liposomes). Cancer Res 53(12):2796–2802. DOI 10.1002/jps.24106
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7. Kanter PM, Bullard GA, Ginsberg RA, Pilkiewicz FG, Mayer LD, Cullis PR, Pavelic ZP. 1993. Comparison of the cardiotoxic effects of liposomal doxorubicin (TLC D-99) versus free doxorubicin in beagle dogs. In Vivo 7(1):17–26. 8. Iwamoto T. 2013. Clinical application of drug delivery systems in cancer chemotherapy: Review of the efficacy and side effects of approved drugs. Biol Pharm Bull 36(5):715–718. 9. Gordon AN, Fleagle JT, Guthrie D, Parkin DE, Gore ME, Angel J. 2001. Lacave. recurrent epithelial ovarian carcinoma: A randomized phase iii study of pegylated liposomal doxorubicin versustopotecan. J Clin Oncol 19(14):3312–3322. ˜ ´ E, Segrelles10. Tamayo-Orbegozo E, Iruin-Sanz A, Onate-Muz as Bellmunt G. 2011. Palmar-plantar erythrodysaesthesia and nonpegylated liposomal doxorubicin. A case study. FarmaciaHospitalaria 35(1):46–47. ´ 11. Formariz TP, Chiavaccia MV, Scarpaa AA, Silva-Junior, Egito EST, Terrugia CHB, Franzini CM, Sarmento VHV, Oliveira AG. 2010. Structure and viscoelastic behavior of pharmaceutical biocompatible anionic microemulsions containing the antitumoral drug compound doxorubicin. Colloid Surf B Biointerfaces 77:47 –53. ´ 12. Formariz TP, Chiavaccia LA, Scarpaa MV, Silva-Junior AA, Egito EST, Terrugia CHB, Franzini CM, Sarmento VHV, Oliveira AG. 2008. Structural changes of biocompatible neutral microemulsions stabilized by mixed surfactant containing soya phosphatidylcholine and their relationship with doxorubicin release. Colloid Surf B Biointerfaces 63:287–295. 13. Formariz TP, Chiavacci LA, Sarmento VH, Santilli CV, Tabosa do Egito ES, Oliveira AG. 2007. Relationship between struc-
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tural features and in vitro release of doxorubicin from biocompatible anionic microemulsion. Colloid Surf B Biointerfaces 60:28–35. 14. Formariz TP, Sarmento VH, Silva-Juniora AA, Scarpaa MV, Santillib CV, Oliveira AG. 2006. Doxorubicin biocompatible O/W microemulsion stabilized by mixed surfactant containing soya phosphatidylcholine. Colloid Surf B Biointerfaces 51:54–61. 15. Chow SC, Wang H. 2011. On sample size calculation in bioequivalence trials. J Pharmacokinet Pharmacodyn 28:155–169. 16. Alhareth K, Vauthiera C, Gueutin C, Ponchel G, Moussa F. 2012. HPLC quantification of doxorubicin in plasma and tissues of rats treated with doxorubicin loaded poly(alkylcyanoacrylate) nanoparticles. J Chromatogr B 887–888:128–132. 17. Food and Drug Administration (FDA). 1996. Guidance for industry: Statistical approaches to establishing bioequivalence. Rockville, Maryland: FDA. ˆ ´ 18. Agˆencia Nacional de Vigilancia Sanitaria (ANVISA), 2003. Resolution RE. No. 899; May 29, 2003; Brazil. ˆ ´ 19. Agˆencia Nacional de Vigilancia Sanitaria (ANVISA), 2003. RDC. No. 27; May 17, 2012; Brazil. 20. Alhareth K, Vauthiera C, Bourasset F, Gueutin C, Ponchel G, Moussa F. 2012. Conformation of surface-decorating dextran chains affects the pharmacokinetics and biodistribution of doxorubicin-loaded nanoparticles. Eur J Pharm Biopharm 81:453–457. 21. Ayen WY, Kumar N. 2012. In vivo evaluation of doxorubicinloaded (PEG)3-PLA nanopolymersomes (PolyDoxSome) using DMBAinduced mammary carcinoma rat model and comparison with marketed LipodoxTM . Pharm Res 29:2522–2533.
Damico et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3297–3301, 2014
Biocompatible Microemulsion Modifies the Tissue Distribution of Doxorubicin ˜ 1 CAROLINE DAMICO CANDIDO,1 MICHEL LEANDRO CAMPOS,1 JULIANA URUGUAY CORREA VIDIGAL ASSUMPC ¸ AO, 1 1 2 ˆ KELLY CHRYSTINA PESTANA, ELIAS CARVALHO PADILHA, IRACILDA ZEPPONE CARLOS, ROSANGELA GONC ¸ ALVES PECCININI1 1
Department of Natural Active Principles and Toxicology, School of Pharmaceutical Sciences, S˜ao Paulo State University – UNESP, Araraquara 14801-902, SP, Brazil 2 Department of Clinical Analysis, School of Pharmaceutical Sciences, S˜ao Paulo State University – UNESP, Araraquara 14801-902, SP, Brazil Received 28 February 2014; revised 24 June 2014; accepted 8 July 2014 Published online 6 August 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24106
ABSTRACT: The incorporation of doxorubicin (DOX) in a microemulsion (DOX-ME) has shown beneficial consequences by reducing the cardiotoxic effects of DOX. The aim of this study was to determine the distribution of DOX-ME in Ehrlich solid tumor (EST) and the heart, and compare it with that of free DOX. The distribution study was conducted with female Swiss mice with EST (n = 7 per group; 20–25 g). Animals received a single dose (10 mg/kg, i.p.) of DOX or DOX-ME 7 days after tumor inoculation. Fifteen minutes after administration, the animals were sacrificed, and the tumor and heart tissues were taken for immediate analysis by ultra-performance liquid chromatography. No difference was observed in DOX concentration in tumor tissue between DOX and DOX-ME administration. However, the most remarkable result in this study was the statistically significant reduction in DOX concentration in heart tissue of animals given DOX-ME. Mean DOX concentration in heart tissue was 0.92 ± 0.54 ng mg−1 for DOX-ME and 1.85 ± 0.34 ng mg−1 for free DOX. In conclusion, DOX-ME provides a better tissue distribution profile, with a lower drug concentration in heart tissue but still comparable tumor C 2014 Wiley Periodicals, Inc. and the American drug concentration, which indicates that antitumor activity would not be compromised. Pharmacists Association J Pharm Sci 103:3297–3301, 2014 Keywords: microemulsion; formulation; doxorubicin; Ehrlich tumor; liquid chromatography; UPLC; distribution; pharmacokinetics; toxicology
INTRODUCTION Doxorubicin (DOX) is an anthracycline antibiotic with a broad antitumor spectrum used both as a single agent and in combination regimens.1 DOX is currently on the market as the free drug and liposome-encapsulated drug, but research continues with DOX. For instance, it is in clinical trials with some forms of polymeric drug2 and in animal studies with a biocompatible microemulsion.3 The main problem associated with the use of the free form is cardiomyopathy.4 Acute cardiomyopathy may occur immediately after or during administration of a single dose and is a result of drug accumulation in heart muscle.4 Acute cardiomyopathy occurs in up to 40% of the patient population, where the prognosis is generally good, may be completely asymptomatic, and usually resolves spontaneously.5 Chronic cardiomyopathy is more common and is characterized by congestive heart failure unresponsive to digoxin, as a result of successive deleterious effects on cardiac tissue with repeated doses.5 According to Octavia et al.,4 the mortality rate in patients with chronic DOX-induced cardiotoxicity is 50% after 5 years. The encapsulation of DOX in liposomes has been associated with decreased cardiotoxicity6,7 and changes in its pharmacokinetic profile. These formulations were developed with the aim of achieving a better clinical response and less toxicity. However, ˆ Correspondence to: Rosangela Gonc¸alves Peccinini (Telephone: +55-16-33016988; Fax: +55-16-3301-6980; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 103, 3297–3301 (2014)
C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association
these drugs are often discontinued due to severe side effects.8 The toxicity of encapsulated DOX depends on the lipids used in liposome formulation.5 For instance, the inclusion of phosphatidylserine or polyethylene glycol (PEG) in the formulation has been related to changes in the mononuclear phagocyte system (MPS).5,8,9 This occurs because the liposomes are taken up by cells of the MPS, and DOX exerts its toxic effect within these cells. MPS toxicity may be of concern in immunosuppressed patients, such as those with AIDS.5 Another liposomal formulation containing PEG causes palmar-plantarerythrodysesthesia (PPE) due to the accumulation of DOX in the skin.5 PPE is a painful scaly dermatitis that primarily affects the hands and feet. PPE was the most common adverse effect related to liposomal DOX and occurred in 49% of patients in a phase II trial in ovarian cancer.9 Although PPE is attributed mainly to pegylated formulations, there are reports of cases with nonpegylated formulations.10 According to Patel,1 “the tumor tissue is a dynamic microenvironment and efforts to improve therapeutic efficacy might be achieved by modifying these dynamic processes to enhance drug delivery.” The delivery of the drug to normal tissue is the cause of occurrence of various adverse effects, as well as its limited distribution in tumor tissues resulting in tumor drug resistance and treatment failure. Accordingly, in previous works, we described different microemulsion formulation specially designed for DOX incorporation,11–14 we found that the pharmacokinetic profile of DOX in a microemulsion (DOX-ME) showed significant differences compared with that for free DOX in Wistar rats, with beneficial consequences regarding cardiotoxic effects.3
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The administration of DOX-ME in Wistar rats has been shown to result in higher plasma concentration of DOX and lower volume of distribution when compared with the administration of DOX in the free form, and increased serum creatine kinase MB (CKMB) activity in the DOX group but unchanged CKMB activity in the DOX-ME group.3 These results suggest modifications in drug access to susceptible sites using DOXME. The aim of this study was to determine the distribution of DOX-ME in Ehrlich solid tumor (EST) and heart in female Swiss albino mice to compare it with free DOX. This information is essential for the assessment of product safety for future clinical application.
METHODS Chemicals and Reagents Soya phosphatidylcholine (SPC) (EpikuronTM 200, Germany) was purchased from Degussa Texturants Systems Deutschland GmbH & Company (Hamburg, Germany); cholesterol (CHO) and sodium oleate (SO) from Sigma–Aldrich (St. Louis, Missouri); Tris (hydroxymethyl) aminomethane from Merck (Darmstadt, Germany); DOX hydrochloride (AdriamycinTM ) from Eurofarma (Sao Paulo, Brazil); daunorubicin hydrochloride [DAU—internal standard (IS)] from Pfizer (Sao Paulo, Brazil), polyoxyethyleneglyceroltrihydroxystearate 40 (EU; Eumulgin HRE 40) from Pharma Special (Sao Paulo, Brazil); and acetonitrile and methanol, HPLC grade, from J.T. Baker (Phillipsburg, New Jersey, United States). All other solvents and chemicals were analytical grade. Water was purified in a Milli-Q Plus system (Millipore, Billerica, Massachusetts, United States) with 18.2 M-cm resistivity. R
Microemulsion Preparation The microemulsion preparation was carried out as described by ˜ et al.,3 which started with the addition of CHO, the Assumpc¸ao oil phase, to the semisolid mixture of SPC/EU/SO, the surfactant phase. The aqueous phase (80%, w/w) was then added to this mixture, which was homogenized by ultrasound for 10 min and allowed to stand for 24 h at 25 ± 0.1◦ C to reach complete equilibrium. The samples were centrifuged at 8500g for 15 min (Ultracentrifuge Hitachi Himac CP-80) to remove titanium residues that might have been released from the ultrasound tip. Finally, a suitable amount (1.5 mg mL−1 ) of DOX was dissolved directly in the previously prepared ME.
EST Inoculation A model of EST was used, where 2 × 106 Ehrlich ascites tumor (EAT) cells were implanted subcutaneously in the thigh of the left hind limb of mice. A solid tumor mass developed within 7 days. EAT cells were maintained in vivo in Swiss albino mice by intraperitoneal transplantation every 7 days. This procedure was carried out at the Laboratory of Clinical Immunology of the School of Pharmaceutical Sciences, Sao Paulo State University, Araraquara, Brazil. Cell viability was evaluated by the trypan blue exclusion test, and only the cell suspensions that showed 95% viability were employed. Damico et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3297–3301, 2014
Distribution Study of free DOX and DOX Incorporated in Microemulsion A preclinical drug distribution study was conducted in female Swiss mice with EST (n = 7 per group; weighing 20–25 g). The sample size was based on the method reported by Chow and Wang.15 The animals were housed at a constant temperature (24 ± 1◦ C), humidity controlled (55 ± 5%) and 12-h light cycle starting at seven a.m., with food and water ad libitum. The experiments were conducted during the light phase and the experimental protocol was approved by the Research Ethics Committee of the School of Pharmaceutical Sciences, UNESP, Araraquara, SP, Brazil (Process #60/2012). Animals received a single dose (10 mg kg−1 , i.p.) of DOX or DOX-ME. At 15 min after administration, the animals were sacrificed and the tumor and heart tissues were taken for immediate analysis. A pilot study had been conducted to select the time for extracting the heart. In that study, the organs were extracted at 15, 30, and 45 min after administration of the two DOX formulations. We observed that tissue DOX concentration peaked at 15 min, and thus, this time point was selected for the extraction. DOX Analysis in Heart and Tumor Tissue by Ultra-Performance Liquid Chromatography An ultra-performance liquid chromatography (UPLC) method for DOX determination in tumor and heart was developed and validated. This bioanalytical method detects free DOX, regardless of the formulation administered. The method was according to Alhareth et al.16 Analysis was conducted using a Waters Acquity H-Class UPLC System with fluorescence detector at 8ex/em of 480/560 nm. Chromatographic separation was carried out with a Waters Acquity CSH C18 column (100 × 2.1 mm2 , with 1.7 :m particle size) connected to a Waters Vanguard C18 guard column (5 × 2.1 mm2 , with 1.7 :m, particle size). The mobile phase consisted of acetonitrile:formic acid 0.1% (40:60, v:v) in isocratic mode, with flow rate of 0.4 mL min−1 . The injection volume was 10 :L. The sample manager was maintained at 10◦ C. The tissue samples were processed as follows. A 50-:L volume of IS (10 :g mL−1 daunorubicin) was added to 100 mg of tumor or heart tissue, followed by vortexing (30 s), and 100 :L 1 M Tris–HCl (pH 8.8) was then added, followed by vortexing (30 s). Afterward, 1 mL ethyl acetate was added and the sample was centrifuged for 15 min at 23,800g. The supernatant (900 :L) was filtered through a polyvinylidene difluoride membrane (0.22 :m) and evaporated to dryness under vacuum (Genevac mini VacSample Concentrator Range ). The residue was resuspended in 100 :L mobile phase along with 10 :L 35% perchloric acid and the solution filtered again directly into the injection vial of the chromatographic system. The blood was removed from the tissue with 10 mL saline before processing to reduce the interference of blood DOX with tissue DOX concentrations. The bioanalytical method was validated according to the Food and Drug Administration—Guidance for Industry Bioanalytical Method Validation17 and ANVISA—RE 899/200318 and RDC 27/2012.19 R
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Statistical Analysis The amount of DOX in tumor and heart tissue was expressed in ng mg−1 as median, mean, and 95% CI. Intergroup comparison was performed by the Mann–Whitney test (GraphPadPrism software, version 5.0). The calibration curve and coefficient of R
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Table 1. Inter- and Intra-run Precision and Accuracy of UPLC Bioanalytical Method for DOX in Tumor and Heart Tissues Tumor
Heart
Concentration Precision Accuracy Precision Accuracy (ng mg−1 ) (%) (%) (%) (%) Intra-run
Inter-run
0.5 0.75 1.5 0.5 0.75 1.5
12.41 6.87 13.91 13.85 9.75 1.25
114.86 88.39 103.99 113.13 110.31 85.44
11.03 7.78 11.30 5.54 7.02 1.25
106.11 108.84 85.23 107.43 89.46 85.44
Precision was expressed by CV and accuracy was expressed by deviation from nominal concentration.
Table 2. Recovery of the Extraction Procedure of DOX from Both Tumor and Heart Tissues Concentration (ng mg−1 )
Recovery (%)
CV (%)
0.5 0.75 2.0 0.5 0.75 1.5
97.71 109.51 99.18 83.39 85.17 84.81
1.54 4.52 5.21 11.63 10.11 8.53
Tumor tissue
Heart tissue
CV, coefficient of variation.
Table 3. Data Showing Stability of DOX in Both Tumor and Heart Tissue under Two Specific Wait Conditions
Tumor tissue Heart tissue Figure 1. Chromatograms (a) DOX 500 ng/mL and IS 2500 ng/mL in solution, (b) DOX 2.5 ng mg−1 and IS 5 ng mg−1 superimposed on a zero sample in heart tissue, and (c) DOX 1.0 ng mg−1 and IS 5 ng mg−1 superimposed on a blank in tumor tissue.
variation (CV, %) calculations were performed using Origin software. R
RESULTS AND DISCUSSION Bioanalytical Method for DOX Determination in Tissue The bioanalytical method developed and validated did not show any interference peaks at the retention times of DOX and IS, as can be seen in Figure 1. The calibration curves of DOX showed good linearity in the range of 0.2–2.0 ng mg−1 for tumor tissue and 0.25–2.5 ng mg−1 for heart tissue. The mean regression equations were y = 1.16 × 10−3 x + 5.83 × 10−3 , with r = 0.995 and p = 3.03 × 10−7 , for tumor tissue and y = 2.10 × 10−3 x − 1.08 × 10−2 , with r = 0.984 and p = 6.65 × 10−5 , for heart tissue, where y corresponds to the peak area ratio of DOX to IS and refers to the nominal concentration of DOX in spiked plasma. The LLOQ was 0.2 DOI 10.1002/jps.24106
Concentration (ng mg−1 )
Short-Term Stability 4 h 25◦ C (%)
PostProcessing Stability 4 h 10◦ C (%)
0.5 2.0 0.5 1.5
90.88 91.90 90.02 95.52
90.19 89.63 94.36 93.45
and 0.25 ng mg−1 for tumor and heart tissue, respectively, with precisions of 19.98% and 6.43% and accuracy of 107.94% and 109.49% for tumor and heart tissue, respectively. Inter- and intra-run precision and accuracy studies were carried out at three concentration levels (0.5, 0.75, and 1.5 ng mg−1 ) for both tumor and heart tissue, with five replicates for each level for tumor tissue and three replicates each level for heart tissue. Both tumor and heart tissue showed interand intra-run precision (CV less than 15%) and accuracy (deviation from nominal concentration between 85% and 115%), and thus within acceptance criteria, as can be seen in Table 1. The recovery of DOX was found to be consistent and reproducible after extraction of both tumor tissue and heart tissue. Table 2 shows that total recovery was 102.1% for tumor tissue and 84.5% for heart tissue, with respective CV of 6.3% and 1.1%. In the stability studies, two concentration levels were used, 0.5 and 2.0 ng mg−1 for tumor tissue and 0.5 and 1.5 ng mg−1 for heart tissue, and both assays were carried out in triplicate. Table 3 shows the stability results of the UPLC bioanalytical method for DOX in tumor and heart tissue. Damico et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3297–3301, 2014
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Figure 2. Tissue distribution of DOX administered in the free form and incorporated into the microemulsion (mean ± CI95). * = statistically different (p < 0.05) compared with heart tissue in free DOX administration by Mann–Whitney test.
Although there are several other storage conditions that could have been evaluated, the low availability of tissue to perform these tests did not allow more assays. Therefore, only essential stability tests for the study were performed. The stability observed was sufficient to perform the distribution study of DOX in tumor and heart tissue. The bioanalytical method validated showed sufficiently narrow confidence intervals in tumor tissue, its site of therapeutic action, and heart tissue, in which toxicity is the most important limitation to clinical use of DOX. Evaluation of DOX Distribution in Tumor Tissue and Heart Tissue in Mice Figure 2 shows the DOX concentration in both tumor and heart tissue 15 min after i.p. administration of 10 mg kg−1 DOX in the free form or microemulsion form. No difference was observed in concentration of DOX in tumor tissue between the DOX and DOX-ME groups. The mean concentration (and 95% CI) in tumor tissue following the administration of DOX-ME was 0.85 ± 1.25 ng mg−1 , whereas with the administration of free drug, it was 0.44 ± 0.24 ng mg−1 . On the basis of this result, the same or better antitumor activity would be expected for DOX-ME compared with the free DOX formulation. However, the most remarkable result in this study was the statistically significant reduction of the DOX concentration in heart tissue after administration of DOX in microemulsion form, compared with administration of free DOX. The mean DOX concentration (and 95% CI) in heart tissue was 0.92 ± 0.54 ng mg−1 for microemulsion form and 1.85 ± 0.34 ng mg−1 for free DOX. This fact can be explained by the microemulsion composition. Microemulsion particles are composed of CHO on their surface similar to human low-density lipoprotein, which in contact with the plasma acquires ApoEm, which is recognized by lipid receptors. Myocardial cells have fewer lipoprotein receptors, resulting in lower uptake of the drug by this tissue. In the recent literature, two works with similar aim stand out. The first one was by Alhareth et al.,20 who observed a decrease in mean concentration of DOX in heart when DOX was Damico et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3297–3301, 2014
loaded into an anionic emulsion polymerization of polyalkylcyanoacrylate nanoparticles compared with free DOX, but that work did not draw any conclusion about the capability of the system to keep the tumor distribution unaltered. However, the main objective of that work was method development. The second work was by Ayen and Kumar,21 who carried out an extensive in vivo study comparing free DOX, DOX-loaded (PEG)3 –PLA nanopolymersomes and the formulation marketed as LipoDoxTM . The most impressive result in that work was the increased DOX concentration in tumor and decreased concentration in heart with DOX-loaded (PEG)3 –PLA nanopolymersomes as compared with free DOX. Despite that DOX-ME did not demonstrate a statistically significant increase in DOX concentration in the tumor, its reduced ˜ et al.3 and its reduced cardiotoxicity as reported by Assumpc¸ao DOX concentration in heart tissue shown in the present work, justify continued studies with this system. The future clinical application of this system depends on the risk–benefit ratio, and having a diverse choice of drugs is always beneficial for achieving better treatment success.
CONCLUSIONS The bioanalytical method validated was found to be adequate for the evaluation of DOX distribution in both heart and tumor tissue, allowing a fast and sensitive analysis of DOX in these tissues. The main goal of this study was to evaluate the distribution of DOX in both tumor and heart tissue, and the results demonstrated a better heart distribution profile with lower concentrations of DOX when administered in a microemulsion. A comparable tumor distribution profile was observed, indicating that its antitumor activity would not be reduced.
ACKNOWLEDGMENTS ˜ de Amparo The authors thank the CAPES and Fundac¸ao ˜ Paulo (FAPESP – Process a` Pesquisa do Estado de Sao 2011/11239–9) for the financial support. Dr. A. Leyva helped with English editing of the manuscript.
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