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  • 1 Northeast Forestry University/Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field, Ministry of Education, Harbin, 150040, China
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10-Methoxycamptothecin (MCPT) and 10-hydroxycamptothecin (HCPT) are the indole alkaloids isolated from a Chinese tree, Camptotheca acuminata, and have a wide spectrum of anticancer activity in vitro and in vivo mainly through inhibitory effects on topoisomerase I. HCPT is a major metabolite of MCPT in rats; the pharmacokinetic analysis and tissue distribution of MCPT and HCPT in rats have also been determined after i.v. injection of MCPT, but the excretion of MCPT and its metabolite HCPT has not been assessed up to now. In the present study, the excretion study of MCPT and its metabolite HCPT in rat bile, feces, and urine after i.v. administration of MCPT (5 mg kg−1) was performed by high-performance liquid chromatography (HPLC) method coupled with a fluorescence detector. The results showed that MCPT mainly biotransformed to HCPT and excreted in the form of HCPT and MCPT in bile, urine, and feces after i.v. administration of MCPT. It was excreted about 1.24 ± 0.07% as MCPT and 5.49 ± 0.40% as HCPT in bile within 6 h after i.v. administration. The cumulative excretions of MCPT and HCPT were mainly within 24 h after i.v. administration, which were 0.41 ± 0.10% and 7.66 ± 1.43% of the dosage in urine and about 0.16 ± 0.04% and 20.30 ± 3.35% of the dosage in feces. The total excretion of MCPT in urine, bile, and feces was 1.81 ± 0.09% in the form of original MCPT and 33.45 ± 1.57% detected as the metabolite HCPT in urine, bile, and feces, suggesting that MCPT might undergo other biotransformation.

Abstract

10-Methoxycamptothecin (MCPT) and 10-hydroxycamptothecin (HCPT) are the indole alkaloids isolated from a Chinese tree, Camptotheca acuminata, and have a wide spectrum of anticancer activity in vitro and in vivo mainly through inhibitory effects on topoisomerase I. HCPT is a major metabolite of MCPT in rats; the pharmacokinetic analysis and tissue distribution of MCPT and HCPT in rats have also been determined after i.v. injection of MCPT, but the excretion of MCPT and its metabolite HCPT has not been assessed up to now. In the present study, the excretion study of MCPT and its metabolite HCPT in rat bile, feces, and urine after i.v. administration of MCPT (5 mg kg−1) was performed by high-performance liquid chromatography (HPLC) method coupled with a fluorescence detector. The results showed that MCPT mainly biotransformed to HCPT and excreted in the form of HCPT and MCPT in bile, urine, and feces after i.v. administration of MCPT. It was excreted about 1.24 ± 0.07% as MCPT and 5.49 ± 0.40% as HCPT in bile within 6 h after i.v. administration. The cumulative excretions of MCPT and HCPT were mainly within 24 h after i.v. administration, which were 0.41 ± 0.10% and 7.66 ± 1.43% of the dosage in urine and about 0.16 ± 0.04% and 20.30 ± 3.35% of the dosage in feces. The total excretion of MCPT in urine, bile, and feces was 1.81 ± 0.09% in the form of original MCPT and 33.45 ± 1.57% detected as the metabolite HCPT in urine, bile, and feces, suggesting that MCPT might undergo other biotransformation.

Introduction

Camptothecin (CPT, Figure 1) is a natural alkaloid isolated from extracts of a native plant in China, Camptotheca acuminata, in 1966 [1]. CPT and its analogs (CPTs) received considerable attention as a promising class of anti-tumor agents. The anti-tumor mechanism of CPTs is based on the inhibition of DNA replication and RNA transcription by stabilizing the cleavable complexes formed between topoisomerase I and DNA [2, 3]. Further fractionation of C. acuminata resulted in the isolation and identification of minor CPTs, including 10-hydroxycamptothecin (HCPT, Figure 1) and 10-methoxycamptothecin (MCPT, Figure 1) [4], both possessing significant activities against the cell line 9KB (human nasopharyngeal carcinoma) in vitro and P388 lymphocytic leukemia system in vivo [5]. Furthermore, HCPT has displayed a strong anti-tumor activity against gastric carcinoma, hepatoma, bladder carcinoma, and lung cancer, among others [6, 7]. Most importantly, HCPT is less toxic in experimental animals and humans during clinical evaluations. Except the initial isolation, identification, and the in vitro antitumor activities [5, 8, 9], few studies were carried out on MCPT due to its higher toxicity [10].

Figure 1.
Figure 1.

Chemical structures of CPTs (R: −H, CPT; R: −OH, HCPT; R: −OCH3, MCPT)

Citation: Acta Chromatographica Acta Chromatographica 29, 4; 10.1556/1326.2017.00075

Pharmacokinetic study on active ingredients in natural products is important to illustrate their mechanism of action. The excretion data are useful for estimating the activity, side effects, and toxicity of the agent, which can provide information for the suitable dosage and manner of administration [11, 12]. In our previous study, HCPT was identified as a major metabolite of MCPT in rat plasma and tissues via high-performance liquid chromatography (HPLC)/photodiode array detection (PDA) and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) analysis. Meanwhile, we established a validated HPLC method coupled with a fluorescence detector for the simultaneous determination of MCPT and its major metabolite HCPT, and the method was applied to the pharmacokinetics and tissue distribution study in rats after i.v. administration of MCPT [1315]. Until now, the excretion profile of MCPT and its metabolite HCPT has not been assessed. In the present study, we therefore examined the bile, feces, and urine excretion of MCPT and HCPT in rats after i.v. administered MCPT using a simple reversed-phase (RP)-HPLC method coupled with a fluorescence detector, for exploring its eliminated fate in the body and further better understanding its in vivo pharmacological profile.

Materials and Methods

Chemicals and Reagents

MCPT with 99% purity was prepared as described in detail in the reported procedure [16]. CPT and HCPT with purities of 99% were purchased from Shanghai Boylechem Co., Ltd. (Shanghai, China). HPLC-grade acetonitrile (ACN), methanol (MeOH), and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade water (resistivity 18.2 MΩ cm) was produced by purification of distilled water with a Milli-Q gradient system (Billerica, MA, USA). All the other ingredients were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Instrumentation and Analytical Conditions

Chromatography was performed on a Waters HPLC system equipped with a 1525 binary pump, a 717 plus autosampler, and a 2475 fluorescence detector. Separation was performed on an analytical Phenomenex Luna C18 column (5 μm, 250 mm × 4.6 mm; Torrence, CA, USA) with a linear gradient elution using water (containing 5% ACN) (A) and ACN (B) as the mobile phase. The elution program was as follows: phase B was linearly increased from 20% to 60% in the first 12 min, then increased to 90% within 3 min, followed by a linear decrease from 90% to 20% in the following 4 min, and kept stable at 20% for 4 min. The total run time was 15 min with a post-run equilibration time of 8 min [15]. The flow rate was 1.2 mL min−1 and the injection volume was 50 μL.

An excitation wavelength of 380 nm and an emission wavelength of 515 nm were applied for the analytes detection. The Waters Empower software was used for data acquisition and processing.

Animals, Dosing, and Sampling

Male Wistar rats of 200–250 g were purchased from the Laboratory Animal Center of Jilin University (Changchun, China). Studies involving animals and their care were performed according to standard operating procedures approved by the Institutional Animal Care and Use Committee at Northeast Forestry University. All experiments were performed in compliance with the relevant laws and institutional guidelines. Water and food were freely available throughout the studies. A single dose of 5 mg kg−1 MCPT was administered to rats via the tail vein (i.v.). Formulations of MCPT (2 mg mL−1) were prepared by mixing MCPT in DMSO/PEG-400/0.01 mol L−1 H3PO4 (5:45:50, v/v/v) [13, 17, 18].

Excretion Studies

Rats were randomly divided into two groups (n = 6). In one group, rats were i.v. administered at a single dose of 5 mg kg−1 MCPT and then individually placed in stainless-steel metabolic cages, which allowed the separation of urine and feces. The urine samples were collected at 0–4, 4–8, 8–12, 12–24, 24–36, 36–48, 48–60, 60–72, 72–84, and 84–96 h post dosing. The feces samples were collected at 0–12, 12–24, 24–36, 36–48, 48–60, 60–72, 72–84, and 84–96 h post dosing. For the other group, a cannula was implanted into the bile duct of each animal, followed by a single i.v. dose of 5 mg kg−1 MCPT. Then, bile samples were collected at 0–0.5, 0.5–1, 1–2, 2–3, 3–4, 4–5, and 5–6 h intervals. The volumes of urine and bile samples and the weight of dry feces samples were recorded. The urine, feces, and bile samples were stored at −80 °C until analysis.

Preparation of Standard and Quality Control (QC) Samples

A mixed stock solution of MCPT (1 μg mL−1) and HCPT (1 μg mL−1) was prepared by dissolving 10 mg of MCPT and HCPT in 10 mL of DMSO and further diluting 1000 times with MeOH. Calibration curves were prepared by spiking 100 μL of the appropriate standard solution with 10 μL of blank biological matrix, and the effective concentrations were 5, 10, 20, 40, 80, 160, and 320 ng mL−1 for both MCPT and HCPT, respectively. A stock solution of CPT (internal standard, IS) was prepared in DMSO at a concentration of 1 mg mL−1 and further diluted with MeOH given the working solution at the concentration of 50 ng mL−1. QC samples were prepared in pools as a single batch at concentrations of 15 (low), 160 (medium), and 250 ng mL−1 (high), divided into aliquots and stored in the freezer at −80 °C until analysis.

The Preparation of Samples

The samples of bile or urine (10 μL) were mixed with 100 μL of IS solution and 100 μL of methanol (100 μL of the corresponding working solution for the calibration curve and QC samples). A 100 μL aliquot of 1% acetic acid and 400 μL of MeOH were added to the mixture. After vortex mixing for 1 min, the mixture was incubated at room temperature for 10 min on a shaker followed by centrifuging at 12,000 rpm for 10 min. Two hundred microliters of the supernatant was then transferred to a clean tube. After centrifugation at 25,000 rpm for 5 min, 50 μL of the supernatant was injected into the chromatographic system.

The feces sample (10 mg) was mixed with 100 μL of IS solution and 100 μL of methanol (100 μL of the corresponding working solution for the calibration curve and QC samples). After adding 5 mL of ethyl acetate as extraction solvent, the mixture was ultrasonically extracted for 30 min. After the centrifugation at 12,000 rpm for 10 min, the supernatant was evaporated to dryness under a gentle nitrogen stream. The residue was finally reconstituted in 200 μL of methanol. After the centrifugation at 25,000 rpm for 5 min, 50 μL of the supernatant was injected into the chromatographic system.

Method Validation

The method was validated according to the guidelines for biological sample analysis [19].

Specificity was assessed by comparing blank bile, urine, and feces samples (n = 6) with blank bile, urine and feces samples spiked with CPT, HCPT, and MCPT (n = 6).

Biological samples were quantified using the peak area ratio of MCPT or HCPT to that of the IS. A standard curve in the form of y = Ax + B was used, where x represents MCPT or HCPT concentration in the biological matrix and y represents the ratio of the analyte peak area to that of the IS. A linear least-square regression analysis was conducted to determine the slope, intercept, and coefficient (r2) to demonstrate the linearity of this method.

The lower limit of quantification (LLOQ) was defined as the lowest concentration on the calibration curve that could be determined with a relative error (RE) and precision (relative standard deviation, RSD) of less than 20%.

Intra- and inter-day precision and accuracy were assessed from the results of six replicates of LLOQ and three QC levels on three consecutive days. The precision was expressed by RSD among the replicate measurements.

The extraction recoveries of MCPT and HCPT at LLOQ and three QC levels were evaluated by assaying the extraction samples and comparing the area ratio of the peaks for MCPT or HCPT to that of IS with those for neat solution of standard compounds without extraction.

The stability of QC samples was examined by keeping replicates of QC samples of MCPT and HCPT at room temperature for 6 h, in the autosampler tray for 24 h, and in a freezer at −80 °C for 30 days; the freeze–thaw stability was obtained over 3 freeze–thaw cycles, by thawing at room temperature for 2–3 h, and then refreezing at −80 °C for 12–24 h. We also analyzed the stability of stock and working solution under the same storage conditions. For each concentration and each storage condition, 6 replicates were analyzed in one analytical batch. The concentration of analytes after each storage period was related to the initial concentration, which was determined when the samples were originally prepared and processed.

Results and Discussion

Method Development

Liquid–liquid extraction (LLE), protein precipitation (PP), and solid-phase extraction (SPE) method were commonly used for sample preparation. LLE could offer a relatively clean sample and make the method more robust and scalable [2023]. It was applied to determine the concentrations of MCPT and its active metabolite HCPT in plasma samples in our previous study [13]. In the tissue distribution study [15], no acceptable linearity and recoveries were achieved when applying LLE method, neither during processing the bile and urine samples (data now shown). The SPE method was also tested and discarded for unacceptable recovery. PP method has the advantages of simplicity and time saving coupled with more complicated samples and interference by endogenous substances, which is normally considered after LLE and SPE methods. Thus, the PP method was introduced for bile and urine samples in this study. ACN and MeOH were tested as the PP agents, and MeOH was selected with the higher efficiency.

The extraction of MCPT and HCPT from rat feces sample was tested with different organic solvents, including ethyl acetate, chloroform, and dichloromethane. The ethyl acetate extract achieved symmetrical shape peaks and good baseline resolutions for MCPT, HCPT, and CPT in HPLC assay. Feces matrix components did not interfere with the analysis. Hence, ethyl acetate was chosen as the extraction solvent.

Method Validation

Specificity and Selectivity

The typical HPLC chromatograms of the separation of MCPT, HCPT, and IS in biological matrix were shown in Figures 2–4. They all showed well-separated peaks with no significant interference from endogenous substances observed at their corresponding retention time under current HPLC conditions. Typical retention times for MCPT, HCPT, and CPT were 8.9, 5.9, and 8.2 min, respectively.

Figure 2.
Figure 2.

Representative chromatograms of (A) a blank rat bile sample; (B) a blank bile sample spiked with MCPT (5 ng mL−1), HCPT (5 ng mL−1), and IS (CPT, 50 ng mL−1); and (C) a rat bile sample (100 times dilution) at 1 h after i.v. administration of MCPT at a dose of 5 mg kg−1 and spiked with IS (CPT, 50 ng mL−1)

Citation: Acta Chromatographica Acta Chromatographica 29, 4; 10.1556/1326.2017.00075

Figure 3.
Figure 3.

Representative chromatograms of (A) a blank rat urine sample; (B) a blank urine sample spiked with MCPT (5 ng mL−1), HCPT (5 ng mL−1), and IS (CPT, 50 ng mL−1); and (C) a rat urine sample (100 times dilution) at 1 h after i.v. administration of MCPT at a dose of 5 mg kg−1 and spiked with IS (CPT, 50 ng mL−1)

Citation: Acta Chromatographica Acta Chromatographica 29, 4; 10.1556/1326.2017.00075

Figure 4.
Figure 4.

Representative chromatograms of (A) a blank rat feces sample; (B) a blank feces sample spiked with MCPT (5 ng mL−1), HCPT (5 ng mL−1), and IS (CPT, 50 ng mL−1); and (C) a rat feces sample (100 times dilution) at 1 h after i.v. administration of MCPT at a dose of 5 mg kg−1 and spiked with IS (CPT, 50 ng mL−1)

Citation: Acta Chromatographica Acta Chromatographica 29, 4; 10.1556/1326.2017.00075

Figure 5.
Figure 5.

Cumulative contents of MCPT and HCPT in rat bile (A), urine (B), and feces (C) samples following i.v. administration of MCPT (i.v. 5 mg kg−1, n = 4–6)

Citation: Acta Chromatographica Acta Chromatographica 29, 4; 10.1556/1326.2017.00075

Linearity and Lower Limit of Quantification

The standard curve was established by plotting the ratio of the peak area of MCPT or HCPT to that of IS. The calibration curves, correlation coefficients, and linear ranges of MCPT and HCPT were listed in Table 1. The calibration curves were prepared daily and showed good linearity in the corresponding range for both MCPT and HCPT (r2 > 0.99).

Table 1.

Linearity curve of MCPT and HCPT in rat bile, urine, and feces samples

Compounds5–320 ng mL−1
Standard curveR2
BileMCPTy = 0.0175x + 0.10170.9987
HCPTy = 0.0239x + 0.11720.9986
UrineMCPTy = 0.0213x + 0.09820.9985
HCPTy = 0.029x + 0.21190.998
FecesMCPTy = 0.018x + 0.12890.9967
HCPTy = 0.0275x + 0.3880.9928

Note: The presented equations present mean values for calibration curves.

The lower limit of quantification (LLOQ) was defined as the lowest concentration on the calibration curve (5 ng mL−1) with the acceptable accuracy from 95.3% to 107.5% and a precision below 12.5%. The method was found to be sufficiently sensitive for the quantification of the analytes in rat bile, urine, and feces.

Precision and Accuracy

The accuracy and precision data for intra- and inter-day samples are presented in Tables 2 and 3. Intra-day and inter-day precision and accuracy were determined by measuring LLOQ and three QC levels. The RSD was below 12.5 %. The assay values for both occasions (intra- and inter-day) were found to be within the accepted variable limits. The data indicated that the present method has a satisfactory accuracy, precision, and reproducibility in rat biological samples.

Table 2.

Accuracy and precision for the analysis of MCPT in rat bile, urine, and feces samples

Added, C (ng mL−1)Intra-day (n = 18)Inter-day (n = 30)
Found, C (ng mL−1)RSD (%)Accuracy (%)Found, C (ng mL−1)RSD (%)Accuracy (%)
BileLLOQ (5) 5.3811.7 107.5 5.21 9.8104.3
QClow (15) 13.66 5.8 91.0 14.54 12.096.9
QCmedium (160)163.18 0.8 101.9163.21 1.0102.0
QChigh (250)249.35 2.2 99.7250.19 2.2100.1
UrineLLOQ (5) 4.89 9.8 97.7 5.31 11.1106.1
QClow (15) 14.6411.2 99.7 15.27 12.2103.7
QCmedium (160)169.52 2.9 105.9169.50 7.5105.9
QChigh (250)245.47 0.8 98.2247.40 1.898.9
FecesLLOQ (5) 4.94 5.3 98.9 4.87 10.097.5
QClow (15) 15.99 9.6 106.6 16.33 8.9108.8
QCmedium (160)161.61 2.3 101.0162.63 2.7101.6
QChigh (250)249.80 3.0 99.9251.08 2.6100.4
Table 3.

Accuracy and precision for the analysis of HCPT in rat bile, urine, and feces samples

Added, C (ng mL−1)Intra-day (n = 18)Inter-day (n = 30)
Found, C (ng mL−1)RSD (%)Accuracy (%)Found, C (ng mL−1)RSD (%)Accuracy (%)
BileLLOQ (5) 4.77 8.7 95.3 5.01 10.9100.2
QClow (15) 13.51 5.3 90.1 14.37 11.595.8
QCmedium (160)161.65 1.1101.0162.10 1.1101.3
QChigh (250)252.44 2.7100.9252.92 2.3101.2
UrineLLOQ (5) 4.93 9.5 98.7 5.08 12.5101.6
QClow (15)14.58 4.9 97.2 15.17 8.0101.1
QCmedium (160)168.20 2.9105.1166.20 5.1103.9
QChigh (250)250.16 2.1100.1251.28 1.9100.5
FecesLLOQ (5) 5.14 9.9102.8 5.34 11.5106.8
QClow (15)14.49 11.3 93.9 15.14 11.799.3
QCmedium (160)158.65 2.8 99.1160.14 3.4100.1
QChigh (250)252.68 4.4101.1253.12 3.7101.2

Extraction Recovery

Recoveries were determined at LLOQ (5 ng mL−1) and three QC levels (15, 160, and 250 ng mL−1) (n = 6). The results are shown in Table 4. At all concentration levels of these analytes, the extraction recoveries were between 74.3% and 120.7% with RSD% less than 12.1%. This result indicated that the sample preparation process was efficient for the analytes.

Table 4.

Recovery of MCPT and HCPT in rat bile, urine, and feces samples (n = 6)

Concentration (ng mL−1)MCPTHCPT
Mean ± SDRSD%Mean ± SDRSD%
BileLLOQ (5)114.9 ± 6.8 5.9 74.3 ± 1.21.6
QClow (15) 83.9 ± 2.7 3.2 87.2 ± 1.82.0
QCmedium (160)103.8 ± 1.5 1.4 84.5 ± 0.70.9
QChigh (250) 96.9 ± 2.5 2.6104.7 ± 2.92.8
UrineLLOQ (5) 96.3 ± 11.7 12.1 92.7 ± 3.53.8
QClow (15) 90.1 ± 3.9 4.4 91.6 ± 2.52.7
QCmedium (160)120.7 ± 3.9 3.2101.0 ± 3.53.5
QChigh (250) 99.3 ± 6.6 6.6103.3 ± 3.43.3
FecesLLOQ (5) 96.6 ± 11.5 11.9 80.1 ± 6.98.6
QClow (15) 99.3 ± 8.0 8.1 95.0 ± 3.83.9
QCmedium (160)108.1 ± 0.2 0.1 88.9 ± 6.16.8
QChigh (250) 97.5 ± 2.9 2.6118.3 ± 1.61.4

Stability

The stability of the analytes in rat bile, urine, and feces was investigated for 6 h at room temperature, for 24 h in the autosampler tray, for 30 days in a freezer at −80 °C and three freeze–thaw cycles (n = 6). The results indicated that this method for the simultaneous determination of the analytes in rat bile, urine, and feces offered satisfactory stability, the accuracy in the range between 93.5% and 107.7% with RSD% less than 9.6%. The stock and working solution were also stable under each storage conditions (n = 6), the accuracy in the range between 90.9% and 102.1% with RSD% less than 6.7%. These results demonstrate that analytes can be stored under the tested conditions without compromising the integrity of the samples.

Excretion Study

After i.v. administration to rats with MCPT, the cumulative excretion of MCPT and HCPT in rat bile, urine, and feces is shown in Figure 5.

MCPT and HCPT were mainly excreted within 24 h after i.v. administration via urine, about 0.35 ± 0.07% and 6.55 ± 1.85%, respectively, and the urinary cumulative excretions of MCPT and HCPT were about 0.41% for 72 h and 7.66% for 96 h after dosing, respectively. MCPT was undetectable after 72 h, and HCPT was detectable until 96 h, suggesting a longer residence time for HCPT, which was consisted with the results in pharmacokinetic and tissue distribution studies [13, 15].

Similarly as those in urine, MCPT and HCPT in feces were also mainly detected within 24 h after i.v. administration, 0.15 ± 0.03% and 16.95 ± 3.62% of the dosing amount. The cumulative excretions of MCPT and HCPT in feces were 0.16% and 20.30% of the dose administered. MCPT was undetectable after 48 h of i.v. administration, and HCPT was detectable until 96 h, which indicated that HCPT had a longer residence time than MCPT as well.

In bile, the excretion of MCPT and HCPT was mainly excreted within 5 h and 4 h after i.v. administration, respectively. The cumulative excretions within 6 h were about 1.24% for MCPT and 5.49% for HCPT, respectively. Bile excretion is considered as the secondary pathway of original drug and the main route for most metabolites, especially water-soluble products [27, 28]. HCPT as a main metabolite of MCPT with higher polarity showed higher excretion than that of MCPT in bile, whereas the least elimination itself from bile, compared with that from urine and feces.

The total excretion of MCPT was only 1.81% in bile, urine, and feces, and it was the highest in bile, then in urine, and the least in feces. The total excretion of HCPT was 33.45% about 18 times higher than that of MCPT, and it was the highest in feces, then in urine, and the least in bile, displaying a contrary order comparing with MCPT. The most amount of HCPT eliminated was from the feces, implying that MCPT or HCPT was absorbed and metabolized in the liver and excreted into the intestinal lumen as the form of HCPT and further excreted via the feces. It needs to be elucidated further for the possible enterohepatic circulation of MCPT and HCPT on the disposition of MCPT.

MCPT was mainly eliminated in the form of HCPT after i.v. administration. The results implied that MCPT extensively converted to HCPT in vivo, including plasma, different tissues, and excreta. After i.v. administration of MCPT to rats, MCPT underwent a rapid biotransformation into HCPT in rat plasma [11]. Thereafter, MCPT and HCPT were rapidly absorbed and readily diffused into various tissues (heart, liver, spleen, lung, kidney, brain) after i.v. administration. MCPT was accumulated in lung tissues at a very high level which could be quantified until 120 h, and the concentration was about 1000 times higher than that in plasma and other tissues, which was the only site that accumulates MCPT in vivo [14, 15]. In the present excretion study, the total elimination in the form of original compound MCPT was only 1.81% via bile, urine, and feces, and that in the form of active metabolite HCPT was 33.45%. The total excretion was 35.36% which was of limitation, suggesting that MCPT should be undertaken other metabolism pathways and transformed to other metabolites. Further biotransformation study will be performed as our next step.

As reported [2426], after i.v. administration of HCPT, urinary excretion was the major pathway for elimination of HCPT in the first 6 h; thereafter, fecal excretion was the major pathway, which showed a different pattern compared with our present study (fecal excretion was the major pathway) after MCPT administration. Though MCPT readily converted into HCPT and maintained at a lower concentrations than HCPT in vivo, the biotransformation affected the excretion as well as the pharmacokinetics and tissue distributions.

Since large amounts of drug were excreted within a short period following i.v. administration, the possible side effects on renal function, especially in the early phase of drug disposition, need to be carefully investigated. Other alternative routes of drug administration, e.g., oral dosing, should be explored for a better efficiency.

Conclusion

As part of MCPT pharmacokinetic study, a simple and reliable RP-HPLC method with fluorescence detection was developed and applied for simultaneous analysis of MCPT and its active metabolites HCPT in bile, urine, and feces collected from i.v. administered male Wistar rats. The result has shown that MCPT could be transformed to its active metabolite HCPT in bile, urine, and feces. MCPT was mainly excreted as HCPT after i.v. administration of MCPT. MCPT and HCPT were mainly excreted within 6 h after i.v. administration in bile, about 1.24% for MCPT and 5.49% of the dosage for HCPT. The urinary and fecal cumulative excretions of MCPT and HCPT were mainly within 24 h after i.v. administration, about 0.41% and 7.66% of the dosage in urine and about 0.16% and 20.30% of the dosage in feces, respectively. The significant amounts of HCPT were found in the feces, indicating a possible enterohepatic circulation of MCPT and its metabolite HCPT. The amount of MCPT and HCPT in bile, urine, and feces indicated that merely 35.26% (1.81% of MCPT and 33.45% of HCPT, respectively) of the dose administered was excreted. The excretion of MCPT and its metabolite HCPT via bile, urine, and feces was very limited, suggesting that MCPT should be undergone other metabolism pathways, transformed to other metabolites.

Conflict of Interest

The authors declare that there is no conflict of interest associated with this work.

Acknowledgments

This work was supported by the Fundamental Research Funds for the Central Universities (2572016AA58), the Heilongjiang Postdoctoral Fund (LBH-Z14007), and the Scientific Research Foundation of Heilongjiang Province for the Returned Overseas Scholars (LC2015009).

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