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Journal of Photochemistry and Photobiology B: Biology 90 (2008) 179–186

Pharmacokinetics, tissue distribution and excretionof a new photodynamic drug deuxemether

Rui Wang, Haiping Hao 1, Guangji Wang *, Haitang Xie, Meijuan Xu,Wei Wang, Hui He, Xiaoyu Li

Key Laboratory of Drug Metabolism and Pharmacokinetics, 24 Tong Jia Xiang Street, Mail Box 210,

China Pharmaceutical University, Nanjing, Jiangsu 210009, China

Received 3 September 2007; received in revised form 7 January 2008; accepted 7 January 2008Available online 12 January 2008

Abstract

Deuxemether was a new photodynamic drug effective for many kinds of solid tumor therapy, which was mainly composed of3-(or 8-)-(1-methoxyethyl)-8-(or 3-)-(1-hydroxyethyl)-deutero-porphyrin IX (MHD) and 3,8-di(1-methoxyethyl)-deuteroporphyrin IX(DMD). The aims of this study were to elucidate its pharmacokinetic characteristics, tissue distribution, plasma protein binding andexcretion properties and underlying mechanisms of deuxemether in rats based on the simultaneous determination of MHD andDMD. The pharmacokinetic profiles of both MHD and DMD in rats after intravenous doses were linear and best fitted to a two com-partment model, characterized with a rapid distribution phase (MHD: t1/2a, 0.09–0.14 h; DMD: t1/2a, 0.07–0.11 h) and a relatively slowelimination phase (MHD: t1/2b, 2.03–3.20 h; DMD: t1/2b, 2.51–3.20 h). The tissue distributions of MHD and DMD in rats were ratherlimited as evidenced from their low distribution volume (0.75–1.70 L/kg) and the results of tissue distribution study. Protein binding ofMHD and DMD were moderate (65.36–89.99% for MHD; 45.43–76.23% for DMD), independent of drug concentrations and similarbetween human and rat plasma over a concentration range of 0.50–50.0 lg/mL. Both MHD and DMD were predominantly(>74.1%) eliminated from rats as the parent drugs through the hepatobiliary systems and finally excreted into the feces. The multidrugresistance-associated proteins 2 (MRP2) inhibitors, bromosulfophthalein and probenecid, substantially inhibited the hepatobiliaryelimination of MHD and DMD while the P-gp inhibitor digoxin had little effect, suggesting that MRP2 may contribute to the rapidand extensive hepatobiliary excretion of deuxemether. There were no significant differences between MHD and DMD for all pharma-cokinetic characteristics studied. In conclusion, this study provided firstly the full pharmacokinetic characteristics and mechanisms ofdeuxemether, which would be helpful for its clinical regiment design.� 2008 Elsevier B.V. All rights reserved.

Keywords: Deuxemether; Pharmacokinetics; Tissue distribution; Protein binding; Elimination; MRP2

1. Introduction

Photodynamic therapy (PDT) is an emerging modalityin which visible light of an appropriate wavelength acti-vates a tumor-associated photosensitizer to produce reac-tive oxygen that leads to cell death and tumor ablation[1]. The potential selectivity of photosensitizer can be used

1011-1344/$ - see front matter � 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.jphotobiol.2008.01.003

* Corresponding author. Tel.: +86 25 8327 1544.E-mail address: guangjiwang@hotmail.com (G. Wang).

1 Co-first author.

for a targeting therapy such as PDT (in combination oflight) or as a selective radiosensitizer in combination ofradiation therapy [2–7].

Deuxemether (DXM), a new photodynamic drug devel-oped in China, displays good photodynamic tumor activity.It has been verified that deuxemether is composed of3-(or 8-)-(1-methoxyethyl)-8-(or 3-)-(1-hydroxyethyl)-deu-tero-porphyrin IX (MHD), 3, 8-di(1-methoxyethyl)-deuter-oporphyrin IX (DMD), 3-(or 8-)-(1-methoxyethyl)-8(or3-)vinyl-deuteroporphyrin IX (MVD), 3-(or 8-)-(1-hydroxy-ethyl) -8(or 3-)vinyl-deuteroporphyrin IX (HVD) and small

Fig. 1. Chemical structures of MHD and DMD (MHD: R1 = CH3CH(OCH3); R2 = CH3CHOH or R1 = CH3CHOH; R2 = CH3CH (OCH3);DMD: R1 = CH3CH (OCH3); R2 = CH3CH (OCH3)).

180 R. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 90 (2008) 179–186

quantity of protoporphyrin IX (Pp). MHD and DMD(Fig. 1), with the sum amount over 85%, are the two mainbiological active components in deuxemether. In addition,the proportion of photosensitive porphyrin that has poortumor selectivity is much lower in deuxemether than thatin hematoporphyrin derivative (HpD), sodium pofimerand photofrin. Furthermore, deuxemether has been provento have lower toxic effects (Data unpublished) and is there-fore a prospective photodynamic drug candidate for thetreatment of tumors.

Clinically, patients accepting PDT are demanded toavoid light for a certain time, which is mainly determinedby the pharmacokinetic characteristics of the PDT drugs.In this regard, pharmacokinetic study of PDT drugs isespecially important for the safety consideration in clinicalapplications. As a new photodynamic anti-tumor agent, nopharmacokinetic data of MHD and DMD have beenreported. This study was therefore designed to make acomplete research and evaluation of the preclinicalpharmacokinetics of MHD and DMD based on the devel-opment and validation of a sensitive simultaneous quanti-fication method. Furthermore, the possible involvement ofthe hepatobiliary efflux transporters, mainly P-glycoprotein(P-gp) and multidrug resistance-associated protein 2(MRP2) [8], in the biliary elimination of MHD andDMD were studied, considering that MHD and DMDwere found predominantly eliminated into bile as theunchanged drug.

2. Material and methods

2.1. Chemicals and reagents

Deuxemether injection was provided by ShanghaiFudan-Zhangjiang Biopharmaceutical Co., Ltd. HPLCgrade of methanol was supplied by Merck (Darmstadt,Germany). Water was purified by Milli-Q Ultra-pure watersystem. Tetrahydrofuran (THF) and ethyl acetate of ana-lytical grade were purchased from Shanghai No.4 Reagent& H. V. Chemical Limited Company. Cresorcinol served asan internal standard was purchased from Shanghai Guang-hua Chemical Reagent Factory. Human plasma used in

this study was purchased from the Jiangsu Provincial Peo-ple’s Hospital.

2.2. Animals

Sprague-Dawley rats (180–260 g) were obtained fromSino-British Sippr/BK Lab Animal Ltd. (Shanghai,China). The animals were housed with free access to foodand water, and maintained on a 12 h light-dark cycle (lighton from 8:00 to 20:00) at ambient temperature (22–24 �C)and roughly 60% relative humidity. The rats were fasted for12 h before all studies. All animal work was approved bythe Animal Care and Use Committee of the College ofPharmacy, China Pharmaceutical University.

2.3. Plasma pharmacokinetics

Rats were randomly divided into three groups (n = 6,for each group), receiving an intravenous administrationof deuxemether of 5, 10 and 20 mg/kg, respectively. Dosesapplied in this study were designed based on the pharmaco-logical and toxicological studies in that deuxemether wasfound to be effective at a dosage range of 2.5–20 mg/kgand no toxicity was observed up to 80 mg/kg in a long termevaluation. Blood samples were collected at 0, 2, 5, 10, 20,30, 45, 60, 90, 120, 240, 360, 480, 720 and 1440 min. Bloodsamples (0.2 mL) collected into heparinized Eppendorftubes were centrifuged immediately under 800g at 4 �Cfor 10 min. Plasma was collected and stored at �20 �Cuntil analysis.

2.4. Tissues samples

Rats were randomly classified into five groups with sixrats in each group. Each rat was given an intravenousadministration of deuxemether at a dose of 10 mg/kg.The rats were euthanized by cervical dislocation at desig-nated time intervals after drug administration. The blood,heart, liver, spleen, lung, kidney, brain, skin, fat, stomach,intestina, muscle and genitical gland were collected imme-diately. Tissues were dried with filter paper and stored at�20 �C until analysis.

2.5. Plasma protein binding

Equilibrium dialysis was applied to determine theplasma protein binding of deuxemether in both humanand rat plasma. Outside the bag filter was phosphate buffer(0.02 mol/L, pH 7.40) mixed with NaCl (0.15 mol/L), whileinside the bag filter was human or rat plasma (500 lL).Three groups (n = 4) were prepared with the concentra-tions of deuxemether outside the bag filter set at 0.5, 5.0and 50.0 lg/mL, respectively. Another control group with0.5 mL phosphate buffer instead of plasma inside the bagfilter was set to investigate whether there was a nonspecificbinding between deuxemether and semipermeable mem-brane. The concentration of deuxemether outside the bag

R. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 90 (2008) 179–186 181

filter in the control group samples was set at 5.0 lg/mL.After storage at 4 �C for 96 h, the concentration of deu-xemether outside the bag filter was determined.

2.6. Elimination

Rats were randomly divided into two groups (n = 6).One group of rats was given an intravenous administrationof deuxemether (10 mg/kg) after bile duct cannulation. Thebile samples were collected at 0–2, 2–4, 4–6, 6–8, 8–10, 10–12 and 12–24 h intervals post dosing. For another group ofrats, feces and urine were collected at 0–6, 6–12, 12–24, 24–36 and 36–48 h, after a same intravenous dosage. Sampleswere stored at �20 �C until analysis.

In order to determine whether the hepatobiliary trans-porters MRP2 and P-gp were involved in the bile elimina-tion of deuxemether, the following experiments weredesigned. Rats were randomly divided into four groupswith six rats in each group. All rats were bile duct cannu-lated before dosage. Group 1 received an intravenous doseof deuxemether (10 mg/kg), while group 2–4 rats receivedan intravenous dose of deuxemether (10 mg/kg) in combi-nation with digoxin (1.25 mg/kg), bromosulfophthalein(BSP) (15 mg/kg) or probenecid (12.5 mg/kg), respectively.The bile samples were collected right before and at 0–2, 2–4, 4–6, 6–8, 8–10 and 10–12 h post dosing. Samples werestored at �20 �C until analysis.

2.7. HPLC analysis

The plasma, tissue and plasma protein binding sampleswere analyzed using our previously developed and vali-dated high performance liquid chromatography (HPLC)method [9] with slight modifications. Briefly, plasma sam-ples and plasma protein binding samples (50 lL) werethawed at room temperature. Cresorcinol (100 lg/mL,20 lL) served as the internal standard was subsequentlyadded. Samples were extracted by adding 1 mL ethyl ace-tate followed by vortexing for 3 min. After centrifugationat 4000g for 10 min, 800 lL of supernatant was transferredto a new tube and evaporated to dryness in a vacuumcondenser. Residues were reconstituted in 100 lL mobilephase and centrifuged at 4000g for 10 min before 20 lLsupernatant was injected into the HPLC system. For tissuesamples, 0.2 g tissue was accurately weighted and subse-quently homogenized in ultrapure water to produce a20% homogenate solution into which cresorcinol (200 lg/mL, 20 lL) was added. Samples were vortexed for 3 minafter adding HCl (2 mol/L, 100 lL). The subsequent proce-dures were similar to that of the plasma samples. For thebile, feces and urine samples extraction, the processes weresimilar to that for the tissue samples except for the amountof cresorcinol and HCl added. For bile and feces samples,20 lL of cresorcinol (500 lg/mL) and HCl (0.5 mol/L) wasadded; while for urine samples, 20 lL of cresorcinol (20 lg/mL) and 100 lL of HCl was used. Calibration curves wereprepared separately for each type of biological samples.

The HPLC system consisted of a Shimadzu LC-10ADVP pump, a RF-10AXL detector operated at Ex395 nm and Em 613 nm, a CTO-10 Avp oven and a SIL-10 ADvp autosampler. Separation of analytes was achievedusing a DiamonsilTM (diamond) C18 analytical column(5 lm, 4.6 mm � 150 mm). The mobile phase consisted of0.02 mol/L sodium acetate solution (pH 6.0) and tetrahy-drofuran (66:34, v/v), and was delivered at a flow-rate of1 mL/min. The solution was filtered using 0.45 mm nylonmembrane and ultrasonically degassed prior to use.

The calibration curves prepared for all biosamples werelinear over the concentration range of 0.25–10 lg/mLwith the correlation coefficients exceeding 0.999. Therewas no endogenous interference to both deuxemetherand internal standard (IS) in all extracted biological sam-ples. The recoveries were all more than 85% and the inter-day and intra-day relative standard deviations were within15%.

2.8. Data analysis and statistical comparison

Plasma concentrations and time data obtained from theintravenous dosing were analyzed with DAS software(Anhui Provincial Center for Drug Clinical Evaluation &Yijishan Hospital of Wannan Medical College, China, ver-sion 2.0). One-, two- and three-compartmental analysiswith different weighting factors (1, 1/c, 1/c2) was used todetermine the best fitting compartmental model based onthe random distribution of residuals, the correlation coeffi-cient and the Aikaike information criteria.

The area under the concentration–time curves from zeroto 8 h (AUC0–8 h) was calculated by the linear trapezoidalmethod. AUC from zero to infinity (AUC0–1) was calcu-lated as the sum of AUC 0–8 h and the extrapolated partof the AUC (AUCextra, the ratio of the concentration at8 h to the terminal rate constants calculated from the lastthree concentrations).

The concentration of deuxemether outside and insidebag filter was depicted as Cout, and Cin, respectively. Theplasma protein biding rate of deuxemether (PDXM) was cal-culated according to the following equation: PDXM =1 � (Cout/Cin) � 100%.

The accumulative elimination was calculated accordingto the following equation: P(%) = the amount of elimina-tion/dose � 100%.

Data were expressed as mean ± SD. One-way Anovafollowed by post-hoc comparison was used for the statisti-cal comparisons between groups. P < 0.05 was consideredas statistical significance.

3. Results

3.1. Plasma pharmacokinetics

The mean pharmacokinetic plasma profiles of MHDand DMD after a single i.v. administration of deuxemetherat different doses are depicted in Fig. 2. Compartmental

Fig. 3. Linear relationship between AUC and dose.

182 R. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 90 (2008) 179–186

model analysis using DAS software indicated that the deu-xemether pharmacokinetic profile after i.v. dosing fittedwell to a two compartmental model. Both MHD andDMD were characterized with a rapid distribution phase(MHD: t1/2a, 0.09–0.14 h; DMD: t1/2a, 0.07–0.11 h) and arelatively slow elimination phase (MHD: t1/2b, 2.03–3.20 h; DMD: t1/2b, 2.51–3.20 h). The body clearance ofMHD and DMD was 0.25–0.26 L/h and 0.34–0.40 L/h,respectively. The important pharmacokinetic parametersare summarized in Table 1. As shown in Fig. 3, there wasa good linear relationship between the AUCs and the doses(R2 = 0.9994 for MHD, R2 = 0.9922 for DMD), whichindicated a linear pharmacokinetic characteristics of deu-xemether in the studied dosage range.

Fig. 2. Plasma concentration profiles of MHD (a) and DMD (b) in ratsafter i.v. administration of deuxemether at h.5, j.10, N.20 mg/kg (n = 6).

Table 1Pharmacokinetics parameters of MHD and DMD in rats after i.v. deuxemeth

Parameters Dose (mg/kg)

5 10

MHD DMD MHD

T1/2a(h) 0.09 ± 0.03 0.07 ± 0.02 0.10T1/2b(h) 2.03 ± 0.44 3.20 ± 1.15 2.55CL(L/h) 0.26 ± 0.04 0.34 ± 0.01 0.25AUC(0–8 h) (lg h/L) 11.53 ± 1.93 2.49 ± 0.26 23.11AUC(0–1) (lg h/L) 11.90 ± 1.92 2.97 ± 0.09 24.39V (L/kg) 0.75 ± 0.20 1.56 ± 0.60 0.92

T1/2a: Distribution half-life; T1/2b: Elimination half-life; CL: Clearance; AUAUC(0–1): AUC from zero to infinity; V: Apparent volume of distribution o

3.2. Tissues distribution

The tissue distributions at different time points are sum-marized in Table 2. Deuxemether underwent a rapid distri-bution into the tissues. The concentrations of MHD andDMD in most organs except liver were extremely lowerthan that in plasma.

3.3. Plasma protein binding

Protein binding of deuxemether in rat and humanplasma were studied at three concentration levels usingthe equilibrium dialysis method. As shown in Table 3,the binding ratio to rat plasma protein was about 65.36–89.99% (MHD) and 65.19–76.23% (DMD); and to humanplasma protein, it was about 68.53–84.82% (MHD) and65.43–74.07% (DMD), over the concentration of 0.50–50.0 lg/mL. No significant difference was observed forthe protein binding of both MHD and DMD betweenhuman and rat plasma.

3.4. Elimination

As shown in Figs. 4–6, after a single i.v. administrationof 10 mg/kg deuxemether, biliary excretion within 24 h wasabout 87.1 ± 2.6% (MHD) and 74.1 ± 4.5% (DMD). Uri-nary excretion within 48 h was about 2.08 ± 0.74‰

er administration of DXM at different dose levels

20

DMD MHD DMD

± 0.04 0.09 ± 0.03 0.14 ± 0.03 0.11 ± 0.04± 0.21 2.51 ± 0.29 3.20 ± 0.19 2.93 ± 0.41± 0.04 0.35 ± 0.05 0.26 ± 0.04 0.40 ± 0.05± 3.54 5.40 ± 0.84 46.00 ± 6.03 9.67 ± 1.18± 3.60 5.88 ± 0.82 47.09 ± 6.21 10.05 ± 1.17± 0.18 1.25 ± 0.25 0.75 ± 0.20 1.70 ± 0.28

C(0–8 h): The area under the concentration–time curves from zero to 8 h;f the central compartment.

Table 2Tissue distribution of MHD, DMD (lg/g) following a single i.v. administration at a dose of 10 mg/kg

Organ Concentration (lg/g)

5 min 30 min 240 min

MHD DMD MHD DMD MHD DMD

Blood 536.55 ± 36.10 79.80 ± 10.95 16.00 ± 2.40 3.00 ± 0.60 3.35 ± 0.65 0.65 ± 0.05Heart 4.30 ± 1.30 1.80 ± 0.50 0.80 ± 0.25 0.35 ± 0.05 0.20 ± 0.05 0.10 ± 0.01Liver 57.90 ± 9.25 31.50 ± 5.20 34.05 ± 4.50 11.55 ± 1.40 22.05 ± 6.20 5.10 ± 1.70Spleen 6.30 ± 1.10 2.10 ± 0.45 0.85 ± 0.20 0.50 ± 0.10 0.50 ± 0.15 0.15 ± 0.02Lung 9.25 ± 3.10 2.25 ± 0.70 1.90 ± 0.25 0.55 ± 0.05 0.30 ± 0.15 0.15 ± 0.02Kidney 5.50 ± 1.65 2.45 ± 0.80 1.20 ± 0.30 0.55 ± 0.10 0.40 ± 0.05 0.20 ± 0.05Intestine 9.85 ± 7.00 2.60 ± 1.60 10.95 ± 6.25 3.65 ± 3.60 12.25 ± 7.30 3.35 ± 1.60Stomach 2.50 ± 0.75 0.80 ± 0.15 4.15 ± 5.10 1.60 ± 2.00 0.35 ± 0.15 0.20 ± 0.05Skin 1.95 ± 0.70 0.55 ± 0.20 0.85 ± 0.25 0.25 ± 0.05 0.35 ± 0.15 0.10 ± 0.04Gonad 0.40 ± 0.15 0.15 ± 0.02 0.22 ± 0.05 0.10 ± 0.02 0.15 ± 0.02 0.10 ± 0.01Fat 2.30 ± 0.90 0.70 ± 0.15 0.85 ± 0.40 0.30 ± 0.10 0.35 ± 0.10 0.15 ± 0.02Muscle 0.80 ± 0.35 0.45 ± 0.11 0.50 ± 0.10 0.20 ± 0.05 0.15 ± 0.05 0.05 ± 0.01Brain 0.40 ± 0.15 0.15 ± 0.05 0.10 ± 0.10 0.10 ± 0.05 0.15 ± 0.10 0.10 ± 0.05

Organ Concentration (lg/g)

720 min 1440 min

MHD DMD MHD DMD

Liver 3.70 ± 1.10 0.55 ± 0.25 1.60 ± 0.35 0.30 ± 0.02Spleen 0.30 ± 0.10 0.15 ± 0.02 0.35 ± 0.15 0.10 ± 0.01Lung 0.10 ± 0.02 0.10 ± 0.01 0.10 ± 0.05 0.10 ± 0.01Kidney 0.20 ± 0.02 0.15 ± 0.01 0.25 ± 0.05 0.15 ± 0.02Intestine 0.25 ± 0.15 0.15 ± 0.05 0.15 ± 0.05 0.15 ± 0.05Stomach 0.15 ± 0.05 0.15 ± 0.10 0.20 ± 0.05 0.15 ± 0.01

Data are expressed as means ± SD (lg/g) n = 6.

Table 3aProtein binding ratio of MHD and DMD in rat plasma (n = 4)

Concentration (mg/mL)

Initial Inside the bag Outside the bag Binding (%)

MHD 0.5 1.44 ± 0.21 0.32 ± 0.03 77.45 ± 2.535 15.19 ± 0.68 1.52 ± 0.17 89.99 ± 1.1550 97.50 ± 11.09 33.52 ± 2.05 65.36 ± 3.64

DMD 0.5 0.45 ± 0.07 0.16 ± 0.01 65.19 ± 4.055 5.05 ± 0.44 1.19 ± 0.06 76.23 ± 2.6250 34.02 ± 3.40 11.61 ± 0.34 65.65 ± 3.20

Fig. 4. Biliary cumulative excretion of MHD, DMD after a single i.v.administration of deuxemether at a dose of 10 mg/kg.

Table 3bThe protein binding ratio of deuxemether in human plasma (n = 4)

Concentration (mg/mL)

Initial Inside the bag Outside the bag Binding (%)

MHD 0.5 0.89 ± 0.04 0.26 ± 0.02 70.50 ± 3.125 11.56 ± 2.01 1.68 ± 0.21 84.82 ± 4.9050 98.94 ± 2.49 31.11 ± 2.77 68.53 ± 2.93

DMD 0.5 0.28 ± 0.02 0.15 ± 0.01 65.43 ± 2.035 4.50 ± 0.56 1.16 ± 0.11 74.07 ± 3.2350 37.81 ± 0.72 11.15 ± 0.67 70.51 ± 1.94

R. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 90 (2008) 179–186 183

(MHD) and 1.53 ± 0.59‰ (DMD). Fecal excretion within48 h was about 81.92 ± 7.12% (MHD) and 65.41 ± 6.41%(DMD). The results showed that deuxemether was excretedpredominantly into the bile and finally to feces.

Considering that both MHD and DMD was excretedpredominantly into bile as the unchanged drug, possibleinvolvement of the efflux transporters, mainly P-gp andMRP2, in such a process was further determined. Asshown in Table 4 and Fig. 7, the cumulative excretion were53.1% (MHD) and 50.4% (DMD) when deuxemether wasgiven alone, while the cumulative excretion decreased tobe 47.5% (MHD) and 43.8% (DMD), 13.6% (MHD) and10.6% (DMD) and 21.9% (MHD) and 18.4% (DMD),

Fig. 5. Urinary cumulative excretion of MHD, DMD after a single i.v.administration of deuxemether at a dose of 10 mg/kg.

Fig. 6. Fecal cumulative excretion of MHD, DMD after a single i.v.administration of deuxemether at a dose of 10 mg/kg. Feces (1.0 g) washomogenized in pure water to prepare a 10% solution, and 0.1 mLhomogenate was extracted for analysis.

Fig. 7. Biliary cumulative excretion (% of dose) of MHD (a) and DMD(b) after administration of deuxemether in combination with inhibitors.

Table 4Biliary cumulative excretion of MHD and DMD after DXM coadmin-istered with specific inhibitors of efflux transporters

MHD (%) DMD (%)

Deuxemether (DXM) 53.1 ± 7.4 50.4 ± 9.1DXM + digoxin 47.5 ± 4.4 43.8 ± 4.5DXM + BSP 13.6 ± 1.6** 10.6 ± 2.2**

DXM + probenecid 21.9 ± 2.9** 18.4 ± 3.6**

Statistical significances were evaluated using one-way ANOVA followedpost-hoc comparison (**p < 0.01).

184 R. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 90 (2008) 179–186

when deuxemether was given in combination with digoxin,BSP and probenecid, respectively.

4. Discussion

Deuxemether, characterized with clear and relativelypure chemical constituents, was a new deuteroporphyrin

derivative recently developed in China. It has been provedto possess a good photodynamic anti-tumor activity, but itspharmacokinetic properties have never been studied. Thepresent study was therefore designed to elucidate itspreclinical pharmacokinetic characteristics including theplasma profiles, tissue distribution, plasma protein bindingand elimination. Considering that MHD and DMD werethe two predominant components contained in deuxeme-ther, all pharmacokinetic studies were carried out basedon the simultaneous determination of MHD and DMDusing our previously reported method of high performanceliquid chromatography with fluorescence detection. Themethod has been proved to be sensitive, rapid and repro-ductive, and suitable to the pharmacokinetic studies in rats.

Photodynamic therapy involves the administration ofphotosensitizing drug and light illumination at a specificwavelength in suitable time range for the treatment oftumors. Therefore, it is very important to locate the timewindow for light illumination, which is mainly determinedfrom the pharmacokinetic characteristics of the PDT drugsused. The plasma profiles of MHD and DMD fitted well toa two compartment model. The calculated distributionphase half-life of MHD and DMD ranged from 0.09 to0.14 h indicated the rapid distribution. The eliminationphase half-life of MHD and DMD was within the rangeof 2.03–3.20 h, which was substantially shorter than that

R. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 90 (2008) 179–186 185

of the most previously reported PDT drugs, suggesting thatdeuxemether was rapidly eliminated throughout the organ-ism. Considering that for PDT drugs it was only necessaryto maintain effective concentration at the time range oflight illumination, the rapid elimination characteristics ofdeuxemether was very attractive for expecting less orshorter duration of secondary side effects. Furthermore,the pharmacokinetics of deuxemether was linear withinthe range of doses studied, which made a pharmacokineticand pharmacodynamic extrapolation possible. The resultsof plasma protein binding tests showed that there was nosignificant difference between human and rat plasma, sug-gesting a potentially safe preclinical to clinical transfer.Furthermore, the plasma protein binding of both MHDand DMD below 90% was moderate, indicating that itwas unlike to cause protein binding mediated drug–druginteractions.

The apparent volume of distribution (Vd) of MHDand DMD were both less than 2 L/kg, which suggestedthat the deuxemether distribution was mainly restrictedin the systemic circulation and organisms rich in blood.The biodistribution study proved that both MHD andDMD contents in most organs except liver were extremelylower than that in the plasma. The high distribution ofMHD and DMD in the liver might be explained by theirpredominant hepatobiliary elimination. Considering thesubstantial amount of bile elimination, the bile contami-nation to the intestine can not be excluded, althoughthe intestines were carefully washed. Significant amountof MHD and DMD were also observed in the lung andspleen. Our findings were in good agreement with thosepreviously reports [10,11] in which many photosensitizerswere accumulated with high concentrations in the compo-nents of the reticuloendothelial system. Deuxemether dis-tribution in kidney, a well perfused organ, was relativelylow, which was in agreement with its extremely low excre-tion into urine. The low quantity detected in the brainsuggested that deuxemether was unlikely to cause sometype of alteration to the central nervous system. Theextremely lower level of deuxemether in the skin was alsoencouraging, in view of that a high amount of sensitizer inthe skin would cause unwanted photosensitization.Twenty four hours after injection, deuxemether wasalmost completely eliminated from all organs further sup-porting that deuxemether might be a rather safe photody-namic drug.

The excretion study of deuxemether proved that deu-xemether was eliminated predominantly through bile andthen into feces as unchanged MHD and DMD, whichwas coincidence with the results of distribution. In the viewof substantial amount of hepatobiliary elimination of deu-xemether, we hypothesized that the hepatobiliary trans-porters might be involved in the hepatobiliary eliminationof deuxemether. Since the P-glycoprotein (P-gp) andmulti-drug resistance protein 2 (MRP2) were the main can-alicular efflux transporters [12], we studied the potentialroles of P-gp and MPR2 on the hepatobiliary elimination

of deuxemether using the specific inhibitors. Co-adminis-tration of digoxin, a specific inhibitor of P-gp, had littleeffect on the hepatobiliary elimination of both MHD andDMD, suggesting that P-gp contributed little to the hepa-tobiliary elimination of deuxemether. However, bromosulf-ophthalein (BSP) (15 mg/kg) and probenecid (12.5 mg/kg)significantly reduced the hepatobiliary elimination ofMHD and DMD. Since both BSP and probenecid arethe inhibitors of multidrug resistance-associated proteins2 (MRP2), MRP2 might be the main contributor tohepatobiliary elimination of deuxemether. Consideringthat many clinically prescribed drugs are inhibitors orsubstrates of MRP2, such as amoxicillin, probenecid andcefaclor [13–15] and so on, the potential drug–drug interac-tions mediated by MRP2 should be paid great attentionwhen deuxemether was used. Avoiding drug–drug interac-tions might be of special importance for the photodynamicdrugs because the photosensitizer usually needs a rapidelimination to make it safe and convenient and any reten-tion of the photodynamic drugs may cause significant sideeffects.

In conclusion, this is the first pharmacokinetic study inhealthy rats of a new photodynamic drug deuxemther.Because of its rapid elimination and no retention character-istics, deuxemether is very prospective as a novel photody-namic drug. Potential clinical drug–drug interactionsmediated by MRP2 should be concerned when deuxeme-ther is coadministered with other MPR2 substrates orinhibitors.

Acknowledgement

This work was supported by the National High Tech-nology Foundation of China (‘‘863” Project) for preclinicalpharmacokinetic studies (2003AAZ347A).

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