Calcitriol

Gender Differences in the Effect of Calcitriol on the Body Disposition and Excretion of Doxorubicin in Mice

Abstract

Background and Objective The antitumor activity and toxicity of doxorubicin are potentiated and attenuated by calcitriol, respectively. Potentially, calcitriol can be combined with doxorubicin for clinical benefit in chemotherapy. To gain insight into the interaction between doxorubicin and calcitriol, proposed for combined use in cancer treatment, we studied calcitriol’s effect on the plasma pharmacokinetics, tissue distribution and excretion of doxorubicin in female and male mice.

Methods The control and calcitriol-treated groups, including an equal number of both sexes, received corn oil and calcitriol
(2.5 μg/kg), respectively, intraperitoneally every other day for 8 days. At day 9, doxorubicin was administered intraperi- toneally at a 6 mg/kg dose to each group. Doxorubicin concentrations in biologic specimens were determined by a high- performance liquid chromatographic-ultraviolet detector and analyzed using a non-compartmental model.

Results The plasma pharmacokinetics of doxorubicin were similar in the control and calcitriol-treated groups. While calcitriol did not alter the area under the plasma concentration-time curves (AUCs) and peak concentrations (Cmax) of doxorubicin in the small intestine and testis, it significantly reduced the AUCs and Cmax of doxorubicin in the lung, kidney, spleen, liver, stomach and ovaries. However, calcitriol increased the AUCs and Cmax of doxorubicin in the heart of females, brain of males and duodenum content and vitreous humor of female and male mice. The percent cumulative urine and fecal amounts of doxorubicin in calcitriol-treated mice were higher at 89.23% and 29.37% for female mice and 118.57% and 41.65% for male mice than those in the control mice, respectively.

Conclusions The tissue concentrations and excretion of doxorubicin in both female and male mice are influenced by calcitriol without changes in the plasma pharmacokinetics. The results from this study can provide insights to help obtain the optimal drug combination effects of doxorubicin with calcitriol in cancer treatment.

1 Introduction

Doxorubicin, a natural anthracycline antibiotic, is one of the most useful anticancer agents for the therapy of many carcinoma types. The main mechanisms of the anti- cancer effects of doxorubicin include DNA intercalation, inhibition of topoisomerase II, generation of free radicals leading to oxidative stress and release of cytochrome c from mitochondria [1]. The most common doxorubicin- induced side effect is acute and chronic cardiotoxicity [2]. Another obstacle limiting doxorubicin chemotherapy is the development of drug-resistant cancer cells [3]. Therefore, a number of studies have been conducted to reduce doxo- rubicin’s toxicity and increase its therapeutic effect [4–7]. The use of drug combinations is a potential treatment option to eliminate the anti-therapeutic effects of doxoru- bicin. Calcitriol (1α,25(OH)2D3), the hormonally active form of vitamin D, is essential for calcium absorption and bone mineralization in the body [8, 9]. Also, it has chemo- preventive and anticancer effects including being antipro- liferative and pro-differentiating, inhibiting angiogenesis, having immune modulatory effects, inducing cancer cell apoptosis and having anti-inflammatory effects [10–13]. Calcitriol has been reported to have synergistic or additive antitumor activities in combination treatments with vari- ous chemotherapeutic agents [14] and suggested clinical benefit in many clinical trials on cancer patients [13, 15]. Potentially, it can be combined with doxorubicin for clini- cal benefit in the therapy of many carcinoma types.

Combination therapy for curing cancer offers significant benefits compared to monotherapy. However, determining the pharmacokinetic and pharmacodynamic interactions is one of the most important steps in deciding for combina- tion therapy and should be investigated at the onset. Calci- triol has been shown to potentiate the antitumor activities of doxorubicin in breast, leukemic [16] and gastric cancer cells [17] and to attenuate doxorubicin-induced cardiac dysfunction in mice [18]. However, several transporter proteins such as organic anion transporting polypeptide (OATP)1B, organic cation transporter (OCT)6, multidrug resistance-associated protein (MRP)1, MRP2, P-glycopro- tein (P-gp) and breast cancer resistance protein (BCRP) [19–21] and one or more enzymes of the cytochrome P450 (CYP) family [2] play a role in the body’s disposition and metabolism of doxorubicin. Calcitriol is an inductor of P-gp, MRP2 [22, 23] and OATP1A2 [24] transporters, and its oxidative metabolism is catalyzed by CYP enzymes [12, 14]. It is already known that the activities of drug transporters and drug-metabolizing enzymes vary by sex [25–27]. Thus, it is expected that calcitriol can change the body disposition and excretion of doxorubicin in both males and females. In this study, to gain insight into the interaction between doxorubicin and calcitriol and their combined use in the treatment of cancer, we studied the effect of calcitriol on the plasma pharmacokinetics, tis- sue distribution and excretion of doxorubicin following intraperitoneal administration in female and male mice.

2 Methods
2.1 Chemicals and Drugs

Doxorubicin hydrochloride (> 98%) was purchased in pow- der form from Sigma Aldrich (St. Louis, Mo, USA). Di- sodium hydrogen phosphate (Na2HPO4), triethylamine and orthophosphoric acid were from Merck (Darmstadt, Ger- many). Acetonitrile (ACN), methanol (MeOH) and trifluoro- acetic acid (TFA, 99%) were from Sigma-Aldrich (St. Louis, Mo, USA). All reagents were of analytical or Lichrosolv gradient grade. Doxorubicin (Adriamycin 50-mg injection powder vial, Saba Pharmaceuticals Industry and Trade Co.), calcitriol (Calcijex 1 µg/ml, iv ampoule, Abbott Laboratories Import and Trade Co. Ltd.) and thiopental Na (Pental NA, IE, Ulagay) were used for drug injections. Calcitriol was diluted to 0.5 µg/ml with sterile corn oil.

2.2 Animals

Female and male Swiss albino mice (132) between 8 and 12 weeks of age were provided from Selcuk University Experimental Medical Application and Research Center. Animals were kept in polysulfone cages at 24 ± 1 °C, 60% atmospheric humidity, under a 12:12 h light and dark cycle. Food and water were given ad libitum. All study protocols were approved by Selcuk University Experimental Medical Application and Research Center’s Ethics Committee.

2.3 Study Design

The animals were randomly divided into two equal groups including an equal number of both sexes. In the first group (control group, n = 66), doxorubicin was administered intra- peritoneally at a 6 mg/kg dose at 24 h (day 9) following the intraperitoneal vehicle administration (sterile corn oil) every other day for 8 days. The second group (calcitriol-treated group, n = 66) received doxorubicin at the same dose with the first group at 24 h after the intraperitoneal administration of calcitriol (2.5 μg/kg every other day for 8 days). Blood (0.4–0.7 ml) was taken from the heart using a 26-gauge, ½-inch needle attached to an insulin syringe containing 0.05 ml heparin sodium (Nevparin, Mustafa Nevzat, Istan- bul, Turkey) under thiopental sodium anesthesia (60 mg/kg, intraperitoneal). Then, the animals were killed by cervical dislocation to collect the following tissues: lung, brain, kid- ney, small gut, spleen, duodenum contents, vitreous humor, heart, liver (without gall bladder), muscle, stomach, ovary, testis and uterus. Blood and tissue samples were taken from six mice (3 females, 3 males) per time point at 0, 0.083, 0.25,0.5, 1, 2, 4, 8, 12, 24 and 48 h after doxorubicin adminis- tration. The blood samples were centrifuged at 5000g for 10 min within 1 h, and plasma was immediately collected. Urine and feces specimens from mice kept in metabolic cages were collected at 0–2, 2–4, 4–8, 8–12, 12–24 and 24–48 h after doxorubicin administration. All biologic speci- mens were stored at − 70 °C (Ultralow Temperature Freezer; Operon Co., Ltd., Republic of Korea) until analysis.

2.4 Chromatographic Conditions

Doxorubicin concentrations in mouse biologic specimens were analyzed using a reversed-phase high-performance liquid chromatographic (HPLC) assay. The HPLC proce- dure was based on the method reported by Ahmad et al. [28] and Sambasivam et al. [29], with minor modifications. The HPLC system (Shimadzu, Tokyo, Japan) was composed of LC-20AT controlled by a CBM-20A system, degasser (DGU-14A), autosampler (SIL-10AD) and column oven (CTO-10A). Separation was achieved with a Supelcosil LC-18 column (150 × 4.6 mm i.d., 5 µm, SUPELCO Analyti- cal HPLC Products, Sigma-Aldrich, USA) for plasma sam- ples and a Gemini™ C18 column (250 mm × 4.6 mm i.d., 5 µm, Phenomenex, Torrance, CA, USA) for tissue, urine and fecal specimens. The column oven and autosampler were kept at 40 °C and 23 °C, respectively. The mobile phase was composed of a 0.05-M phosphate buffer with the pH adjusted to 2.94 and acetonitrile. Chromatography employed a linear gradient of acetonitrile in the 0.05 M phosphate buffer at a flow rate of 0.25–0.4 ml/min. A UV–VIS detector (SPD-10A) set to 254 nm.

2.5 Sample Preparation

The feces and tissue specimens were homogenized with a tissue homogenizer (Heidolph, SilentCrusher M, Germany) at 5000 rpm for 1 min in water. The feces and tissue homoge- nates included approximately 200 mg tissue in 1 ml water. Urine (50 µl) and feces homogenate were diluted fourfold with water. Plasma, vitreous humor and tissue homogen- ates were used without further dilution. Doxorubicin was extracted from 50–200 µl samples with 200 µl methanol including 0.1% trifluoroacetic acid. After centrifugation at 12,000 rpm for 15 min, the supernatant was removed, and 10 µl was injected into the chromatographic system.

2.6 Preparation of Standard and Quality Control Solutions

The stock solution of doxorubicin was prepared in water at the concentration of 1000 μg/ml and kept at − 70 °C. The stock solution was diluted serially with water to prepare working standard solutions (0.04–100 µg/ml) of doxoru- bicin. The working standards were added to blank biologic specimens to prepare calibration standards (0.04–100 µg/ ml) and quality control (QC) samples including low (0.2 µg/ ml), medium (2 µg/ml) and high (20 µg/ml) levels of doxoru- bicin. The quality control samples were used to evaluate the recovery, precision and
accuracy of the method in biologic specimens.

2.7 Method Validation

The validation procedure was based on the Food and Drug Administration (FDA) [30] and European Medicines Agency (EMA) [31] guidelines for the chromatographic method. The validation characteristics including specificity, linearity, sen- sitivity, recovery, precision and accuracy were established. Specificity was assessed by comparing chromatograms of six different batches of blank biologic specimens spiked with doxorubicin. The linearity was evaluated with the calibration curves, which were prepared and assayed in duplicate on 3 consecutive days. The lower limit of quantification (LLOQ) was defined as the lowest concentration of doxorubicin that could be determined with acceptable precision (< 20%) and accuracy (± 15%). The recovery, precision and accuracy of the method were assessed by determining QC samples using three replicates at three concentrations levels on 3 different validation days. The extraction recoveries of doxorubicin were determined by comparing the peak areas of doxoru- bicin from extracted samples spiked with doxorubicin stand- ard solutions. The precision and accuracy were defined as the percent coefficient of variation (CV) and percent bias, respectively. The bias was calculated using the following formula: [bias % = (calculated concentration – theoretical concentration)/theoretical concentration]. 2.8 Pharmacokinetic Calculations Before pharmacokinetic analysis, the mean concentrations of doxorubicin for each sampling time in plasma and tis- sue samples were calculated for each sex in the group. The mean concentrations of doxorubicin were analyzed using the WinNonlin 6.1.0.173 software (Pharsight Corp., NC, USA) professional pharmacokinetic program. The pharma- cokinetic parameters were calculated through noncompart- mental analysis with the formulas described by Gibaldi and Perrier [32]. The elimination rate constant (λz) was calculated as the slope of the linear regression on the ter- minal data points. The area under the plasma concentra- tion-time curve from time zero to infinity (AUC0−∞) was estimated by the log-trapezoidal rule from the plasma or tissue drug concentration versus time plots. The terminal half-life (t1/2λz) was calculated as 0.693/λz; the systemic clearance rate (Cl/F) was determined as dose/AUC0−∞; the apparent distribution volume (Vz/F) was calculated as Cl/F/λz. The peak concentration (Cmax) and time to reach Cmax (Tmax) were determined by direct observation from concentration-time curve of each animal. 2.9 Statistical Analyses Plasma and tissue concentrations of doxorubicin obtained following intraperitoneal administration are presented as mean ± SD. Urinary and fecal excretions are expressed as the cumulative percent of the dose excreted in the urine and feces of mice. The group differences in common phar- macokinetic parameters and in the cumulative fecal and urine excretions of doxorubicin for each sex, and for mice that received doxorubicin alone or co-administered with calcitriol, were estimated using the following formula: [100 × (value obtained in group receiving doxorubicin and calcitriol − value obtained in group receiving doxoru- bicin alone)/value obtained in group receiving doxorubicin alone]. The values of ≤ %(−)25 and ≥ %(+)25 values were considered the levels of statistical significance [6, 33]. 3 Results 3.1 Method Validation No interferences of endogenous peaks with doxorubicin at the retention time of doxorubicin in blank mouse bio- logic specimens were observed. The calibration curves of doxorubicin were linear over the concentration range of 0.04–100 µg/ml with the correlation coefficient (r2) of > 0.9996 in all biologic specimens. The LLOQ was 0.04 µg/ml in the plasma and vitreous humor and 0.06 µg/ ml in other biologic specimens with CV < 20% and bias of ± 15%. Recoveries, precisions and accuracies of assays were determined for all biologic specimens spiked with three different doxorubicin concentrations (0.2 μg/ml, 2 μg/ml, 20 μg/ml). Recovery was evaluated by compar- ing the doxorubicin peak area ratios of the extracted sam- ples at the three QC levels with the standard solutions of equivalent concentrations. The extraction recovery of dox- orubicin was > 91% in plasma and vitreous humor, > 86% in urine, feces and duodenum content and > 88% in other biologic specimens. In all the biologic specimens, the CVs of intraday precision assays were < 6% for the three differ- ent doxorubicin concentrations (0.2 μg/ml, 2 μg/ml, 20 μg/ ml); the accuracy biases were within ± 15%. In the interday assay (0.2 μg/ml, 2 μg/ml, 20 μg/ml), CVs of < 8% and bias of ± 15% also met the method’s requirements. 3.2 Plasma Pharmacokinetics of Doxorubicin Semi-logarithmic plasma concentration-time curves and plasma pharmacokinetic parameters of doxorubicin from the combination of doxorubicin and calcitriol in female and male mice are presented in Fig. 1 and Table 1, respectively. Doxo- rubicin alone and in combination with calcitriol reached Cmax with Tmax of 0.08 h. It was detected in all plasma samples at 48 h. The plasma concentration-time curves and pharmacoki- netics of doxorubicin coincided in the control and calcitriol- treated groups. 3.3 Tissue Pharmacokinetics of Doxorubicin Semi-logarithmic tissue concentration-time curves and tissue pharmacokinetic parameters of doxorubicin from combina- tion doxorubicin and calcitriol in female and male mice are presented in Figs. 1, 2 and 3 and Tables 2 and 3, respectively. While calcitriol did not alter the AUCs and Cmax of doxoru- bicin in the small intestine and testis and the AUC of doxoru- bicin in muscle, it significantly reduced the AUCs and Cmax of doxorubicin in the lung, kidney, spleen, liver, stomach and ovaries. However, calcitriol increased the AUCs and Cmax of doxorubicin in the hearts of female mice, brains of male mice and duodenum contents and vitreous humor of female and male mice. In calcitriol-treated mice, the t1/2λZ of doxorubicin was significantly prolonged in the lung, brain and ovarian tis- sue and shortened in the stomach. 3.4 Excretion of Doxorubicin The cumulative amounts of doxorubicin excreted in urine and feces from the combination of doxorubicin and calcitriol in female and male mice are presented in Fig. 4. In control and calcitriol-treated mice, the cumulative amount of doxo- rubicin excreted in urine was higher than that in feces. The cumulative fecal amounts of doxorubicin in the control and calcitriol-treated mice were 1.96% and 2.54% for female mice and 2.26% and 3.21% for male mice, respectively. The cumula- tive fecal amounts of doxorubicin in the calcitriol-treated mice were higher, 29.37% for female mice and 41.65% for male mice, than those in the control groups. The cumulative urine amounts of doxorubicin in the control and calcitriol-treated mice were 5.41% and 10.83% for female mice and 6.89% and 15.07% for male mice, respectively. The cumulative urine amounts of doxorubicin in the calcitriol-treated mice were higher 89.23% for female mice and 118.57% for male mice than those in the control groups. 4 Discussion When combined drugs have different mechanisms of action and no overlapping toxicities, they can achieve optimal drug combination effects. Achieving the optimal drug combina- tion requires determination of the pharmacokinetic and pharmacodynamic interactions. Calcitriol and doxorubicin have different mechanisms to potentiate antitumor activities [16, 17]. The beneficial effects of calcitriol in the protec- tion against the cardiotoxicity that has limited doxorubicin treatment in cancer patients have been shown [18]. This study only determined the effect of calcitriol on the plasma pharmacokinetics, tissue distribution and excretion of doxo- rubicin in mice. However, the effect of doxorubicin on the AUC 0− area under the concentration-time curve, t1/2ʎz elimination half-life, Vz/F distribution volume following intraperitoneal adminis- tration, Cl/F total body clearance following intraperitoneal administration, Cmax peak plasma concentration, Tmax time to reach the peak concentration, GD group difference, F female, M male, DOX doxorubicin was administered intraperitoneally at a 6 mg/kg dose after the vehicle administration every other day for 8 days. CAL + DOX calcitriol was administered intraperitoneally at a dose of 2.5 µg/kg every other day for 8 days followed by doxorubicin 6 mg/kg intraperitoneally aPercentage change was calculated as [100 × (value obtained in group receiving doxorubicin and calcitriol − value obtained in group receiving doxorubicin alone)/value obtained in group receiving doxorubicin alone] pharmacokinetics of calcitriol was not determined. Calci- triol pharmacokinetics may be altered by the modulation of drug transporters and enzymes taking part in the body disposition, metabolism and excretion [14]. Therefore, combined use with doxorubicin, a substrate of drug transporters and CYP enzymes, requires the determination of calcitriol pharmacokinetics. In this study, the plasma pharmacokinetics of doxo- rubicin were similar in the control and calcitriol-treated groups (± 25%). However, there was a significant differ- ence between the groups in terms of the tissue disposition of doxorubicin. Studies investigating drug interactions and action mechanisms also reported tissue disposition differ- ences without any variation in plasma pharmacokinetics for transport protein substrates such as doxorubicin [5, 6]. Hudachek and Gustafson [6] stated that plasma pharmacoki- netics were not enough to estimate the tissue concentration of doxorubicin and that it was essential to determine the tissue concentration of the drug. Lapatinib and paclitaxel changed the tissue distribution of doxorubicin without alter- ing its plasma pharmacokinetics [5]. However, cyclosporin A led to significant changes in both the plasma and tissue levels of doxorubicin [34]. The plasma doxorubicin level was 1.7-fold higher in mdr1a(−/−) mice than in wild-type mice [35]. Among the above-mentioned agents that exert inhibitory effects on P-gp, the plasma pharmacokinetics of doxorubicin had altered; this was attributed to the potent P-gp-inhibitory effect of cyclosporin A [6]. In this study, unchanged plasma pharmacokinetics may have been because the intracellular concentration of doxorubicin is constituted by influx and efflux transporters and calcitriol has inducing effects on both the transporters [19–21]. In addition, 50% of the doxorubicin in the blood is present in erythrocytes [36]. Doxorubicin enters erythrocytes through Ra1A-binding protein 1 (RLIP76) [36]. Calcitriol may also exert inducing effects on RLIP76 in addition to other transporters; there- fore, elevated plasma doxorubicin levels may be expected. On the other hand, similar plasma pharmacokinetic data could be observed in both the control and calcitriol-treated groups because calcitriol promotes doxorubicin excretion in urine and feces. The most remarkable observation of the present study is that while calcitriol significantly reduces the AUCs and Cmax of doxorubicin in the lung, kidney, spleen, liver, stomach and ovaries, it increases the AUCs and Cmax of doxorubicin in the heart of female mice, brain of male mice and vitreous humor of female and male mice. However, calcitriol did not alter the AUCs and Cmax of doxorubicin in the small intestine and testis and AUC of doxorubicin in muscle. Consider- ing that doxorubicin is a substrate of the OATP1B, OCT6, MRP1, MRP2, P-gp and BCRP transporters [19–21], the modulation of transporter activities may cause significant changes in the body disposition of doxorubicin. Significant increases in the accumulation of doxorubicin in the brain, heart, intestine and liver were observed in mice lacking mdrla versus wild-type mice, indicating that P-gp altered the doxorubicin pharmacokinetics in these tissues [35]. In the present study, the increases and decreases in the doxo- rubicin concentration in tissues cannot be explained by the modulation of a transporter only by calcitriol. The inductor effect of calcitriol on P-gp, MRP2 and OATP1A2 transport- ers is known to be tissue-specific, and therefore variable [37], and this may have changed the tissue concentration of doxorubicin in calcitriol-treated group. In this study, the doxorubicin concentration in heart and brain tissues showed gender differences in the calcitriol-treated group. Female rodents exhibited higher MRP2 and P-gp expressions than males [25]. In addition, inhibition of P-gp by testosterone [26] may decrease the P-gp-inducing effect of calcitriol in males [23]. This can explain gender differences in the tissue concentration of doxorubicin. Doxorubicin has multi-organ toxic effects on normal tissues including especially the brain, heart, kidney and liver tissues [38, 39]. In this study, calcitriol significantly increased the doxorubicin concentration in the heart of female mice, brain of male mice and vitreous humor of both female and male mice, and this may result in more toxic effects. However, calcitriol has shown beneficial effects on doxorubicin-induced cardiac dysfunction in mice [18]. The reduced tissue toxicity of some chemotherapeutic drugs in combination with calcitriol have been reported clinically [14]. In this study, calcitriol significantly decreased the dox- orubicin concentration in lung, kidney, spleen, liver, stomach and ovary. The cumulative fecal (29.37% in female mice and 41.65% in male mice) and urine (89.23% in female mice and 118.57% in male mice) amounts of doxorubicin in the calcitriol-treated group were significantly higher than in the control group. Doxorubicin may have low toxicity in normal tissues due to the reduced tissue concentration, increased excretion and attenuated cardiotoxicity occurring in com- bination with calcitriol. The decrease in tissue concentra- tion of doxorubicin caused by calcitriol can be expected to reduce the antitumor effect. However, due to the antitumor activities of doxorubicin potentiated by calcitriol [16, 17], the decrease in antitumoral activities of doxorubicin may not be seen. 5 Conclusions Based on the results, the tissue levels and excretion of doxoru- bicin in both female and male mice are influenced by calcitriol without changes in the plasma pharmacokinetics. The results from this study can provide insights that help obtain the opti- mal drug combination effects of doxorubicin with calcitriol in cancer treatment. However, achieving the optimal combination of doxorubicin and calcitriol requires determining calcitriol’s pharmacokinetics and investigations in tumor-bearing animal models or clinically in patients.