{'fraction_non_alphanumeric': 0.04853538593115245, 'fraction_numerical': 0.02987262897461743, 'mean_word_length': 4.320116997075073, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Pharmacokinetic evaluation of the anticancer prodrug simmitecan in different experimental animals.

AIM
To investigate the pharmacokinetics and disposition of simmitecan (L-P) that was a water-soluble ester prodrug of chimmitecan (L-2-Z) with potent anti-tumor activities in different experimental animals, and to assess its drug-drug interaction potential.


METHODS
SD rats were injected with a single iv bolus doses of L-P (3.75, 7.5 and 15 mg/kg). The pharmacokinetics, tissue distribution, excretion and metabolism of L-P and its active metabolite L-2-Z were studied through quantitative measurements and metabolite profiling with LC/MS. The binding of L-P and L-2-Z to rat plasma proteins was examined using an ultrafiltration method. Systemic exposures of beagle dogs to L-P as well as drug distribution in tumors of the nude mice xenograft model of human hepatic cancer SMMC-7721 cells were also examined. The metabolism of L-P by liver mcirosomal carboxylesterase in vitro was investigated in different species. The effects of L-P and L-2-Z on cytochrome P450 enzymes were examined using commercial screening kits.


RESULTS
The in vivo biotransformation of L-P to L-2-Z showed a significant species difference, with a mean elimination half-life t1/2 of approximately 1.4 h in rats and 1.9 h in dogs. The systemic exposure levels of L-P and L-2-Z were increased in a dose-dependent manner. In rats, approximately 66% of L-P and 79% of L-2-Z were bound to plasma proteins. In rats and the nude mice bearing human hepatic cancers, most organ tissues had significantly higher concentrations of L-P than the corresponding plasma levels. In the tumor tissues, the L-P levels were comparable to those of plasma, whereas the L-2-Z levels were lower than the L-P levels. In rats, L-P was eliminated mainly via biliary excretion, but metabolism played an important role in elimination of the intact L-P. Finally, L-P and L-2-Z exerted moderate inhibition on the activity of CYP3A4 in vitro.


CONCLUSION
L-P and L-2-Z have relatively short elimination half-lives and L-P is mainly eliminated via biliary excretion. The species difference in the conversion of L-P to L-2-Z and potential drug-drug interactions due to inhibition of CYP3A4 should be considered in further studies.


Introduction
Camptothecin (CPT) derivatives potentially have inhibitory activity on DNA topoisomerase I and are promising compounds in anticancer research. Two CPT derivatives, irinotecan and topotecan, have been approved for clinical use [1,2] . Chimmitecan (L-2-Z; 9-allyl-10-hydroxy-camptothecin), a novel 9-substituted camptothecin, has improved anticancer potency and pharmacologic profiles, compared to the clinically available CPT analogues [3] . The solubility issue associated with L-2-Z was overcome by the synthesis of simmitecan (L-P; 9-allyl-10-piperidyl piperidine formyloxy-camptothecin). This water-soluble prodrug has a dipiperidyl carbamate at position 10 of the CPT skeleton. This entity is hydrolyzed by carboxylesterases in vivo to form the active metabolite, L-2-Z, which possesses strong anticancer activity against a number of different human tumor cell lines in vitro. While the IC 50 of L-2-Z is in the nanomolar range [3] , L-P does not show significant antitumor activity. The prodrug modification of L-P is characterized by the utilization of a polar pro-moiety to increase water solubility. This structural design is similar to the design used for irinotecan.
Because L-P was identified as a potential anticancer agent, studies were conducted to characterize the in vivo distribution, metabolism, excretion, and pharmacokinetics of L-P (the prodrug) and its active metabolite L-2-Z in rodent and non-rodent experimental animals. In addition, the in vitro metabolism for different animal species and the drug-drug interaction potentials were also investigated. Pharmacokinetic studies in animal models are an important component of the clinical development for this agent because they relate preclinical studies to patient treatment. This report presents the results of the disposition and pharmacokinetic studies of L-P and its active metabolite L-2-Z in rats, tumor-bearing nude mice and beagle dogs. In addition, in vitro data of L-P and L-2-Z, including the inhibitory effects on cytochrome P450 enzymes and plasma protein binding, are reported here. Recently, L-P has received China Food and Drug Administration new drug approval to be continued its study in a phase I clinical trial (goverment identifier: NCT01832298); this study presents the results of the disposition and pharmacokinetic studies of L-P and L-2-Z obtained from preclinical animal models. The collective information helped in the design of appropriate clinical regimens for investigating this drug in phase I clinical trials.

Experimental animals
The use and treatment of rats complied with the Guidance for Ethical Treatment of Laboratory Animals (The Ministry of Science and Technology of China, 2006; www.most.gov. cn/fggw/zfwj/zfwj2006). Animal studies were conducted according to the protocols approved by the Institutional Animal Care and Use Committee at the Shanghai Institute of Materia Medica (Shanghai, China). Male and female Sprague-Dawley rats weighing 250-300 g were obtained from the Sino-British SIPPR/BK Laboratory Animal Co, Ltd (Shanghai, China). The rats were housed in rat cages (42 cm×24 cm×24 cm; 3 rats/cage) in a unidirectional airflow room under controlled temperature (20-24 °C), humidity (45%-55%) and a 12-h cycle of light and dark. Filtered tap water was available ad libitum. The rats were given commercial rat chow ad libi-tum. Prior to the experiment, rats were acclimatized to the facilities and environment for 7 d. After study use, the rats were euthanized with CO 2 gas.
Male and female nude mice weighing 18-20 g and bearing the human hepatocellular carcinoma SMMC-7721 cell line were obtained from State Key Laboratory of Drug Research, Division of Anti-Tumor Pharmacology, Shanghai Institute of Materia Medica. After study use, the mice were euthanized with CO 2 gas.
Male and female beagle dogs weighing 8.1-10.1 kg were obtained from the Agricultural and Biotech Training Centre, Shanghai Jiao Tong University (Shanghai, China). The dogs were housed under controlled environmental conditions (temperature, 20-24 °C; humidity, 45%-55%; 12-h of light/dark cycle and 8-10 air changes per hour). Dogs were acclimatized to the facilities and environment for 15 d before the experiments.

Rat studies
For plasma pharmacokinetic studies, rats were randomly assigned to three treatment groups and received a single iv bolus dose of L-P at 3.75, 7.5, or 15 mg/kg (administered via the tail vein; 3 mL/kg; four male and four female animals per group). Serial blood samples (approximately 0.2 mL; taken at 0, 5, 15, 30 min, and 1, 1.5, 2, 4, 6, 8, 11, and 24 h) were directly collected from the orbital sinus under light ether anesthesia into heparinized polypropylene tubes containing 1 mg of BNPP. After gently shaking for 5-10 s, the blood samples were centrifuged at 1300×g for 10 min to yield plasma samples, which were kept frozen at -70 °C until analysis.
The tissue distribution of L-P was determined following iv administration (7.5 mg/kg, three male and three female rats per sampling time point, via the tail vein). Rats were euthanized by bleeding the abdominal aorta (approximately 10 mL of blood) at 0, 0.5, 2, 4, and 8 h. The blood samples were collected into heparinized polypropylene tubes that contained BNPP, followed by centrifugation to yield the plasma samples for analysis. Tissue samples of bladder, testicle (male only), ovary (female only), uterus (female only), stomach, small intestine, large intestine, spleen, kidney, liver, heart, lung, pancreas, adipose tissue, brain, skeletal muscle and femoral bones (and marrow) were excised and rinsed in ice-cold saline/methanol (1:1, v/v) before gentle blotting on absorbent paper and weighing. Most tissues were homogenized in 4 volumes of ice-cold saline/methanol (1:1, v/v). The tissues of the pancreas, adipose tissue and ovaries were prepared in 9 volumes of the ice-cold saline/methanol. The bladder tissue was prepared in 19 volumes of ice-cold saline/methanol. The homogenates were stored at -70 °C.
For urinary and fecal excretion studies, rats (three male and three female) were housed individually in Nalgene metabolic cages (Nalgene Co, Rochester, NY, USA) and received an iv dose of L-P at 7.5 mg/kg (via the tail vein). Urine and feces samples were collected predose and at 0 to 4, 4 to 10, 10 to 24, 24 to 34, and 34 to 48 h postdose. The urine and feces collection tubes were frozen at -15 °C to keep the collected samples stable. All of the excretory samples were weighed before storage at -70 °C. The thawed feces samples were homogenized in 9 volumes of saline for analysis.
Rats were anesthetized with ether, and the bile duct was cannulated using polyethylene tubing. Bile was collected predose and at 0 to 0.5, 0.5 to 1, 1 to 2, 2 to 3, 3 to 6, 6 to 9, and 9 to 12 h after an iv dose of L-P at 7.5 mg/kg (three male and three female rats per sampling segment, administration via the tail vein). The bile samples were weighed and stored at -70 °C before use.

Tumor-bearing nude mice studies
The tissue distributions of L-P and L-2-Z were investigated in tumor-bearing nude mice. Mice were randomly assigned to five groups and received a single iv bolus dose of L-P at 15 mg/kg (via the tail vein; 3 mL/kg; four male and four female mice per group). After dosage, the mice under ether anesthesia were euthanized by bleeding the abdominal aorta (approximately 1 mL of blood) at 0, 0.25, 1, 2, 4, and 8 h (one group of mice was used for each time point). The blood samples were collected according to the same procedure used for the rats and were centrifuged to yield the plasma samples for analysis. Samples of the heart, liver, lung, kidney, pancreas, colon and tumors were excised and rinsed in ice-cold saline/methanol (1:1, v/v) before gently blotting on absorbent paper and weighing. The tissues were homogenized in 4 (the liver, kidney, colon and tumors), 9 (the heart and lung) or 29 (the pancresas) volumes of the ice-cold saline/methanol. The homogenates were stored at -70 °C.

Dog studies
Dogs were randomly assigned to three dosage groups and received a single iv bolus dose of L-P at 1.25, 2.5, or 5 mg/kg (via the forelimb vein; 1 mL/kg; four male and four female animals per group). The formulation was the same as that described for rat and mouse studies. Serial blood samples (approximately 0.3 mL; 0, 5, 15, 30 min and 1, 1.5, 2, 3, 4, 6, 8, 11, and 24 h) were directly collected from the opposite forelimb vein into heparinized polypropylene tubes containing 1.5 mg of BNPP. After gently shaking for 5 to 10 s, the blood samples were centrifuged at 1300×g for 10 min to yield the plasma samples that were kept frozen at -70 °C until analysis.
Pharmacokinetic data analysis Plasma concentration-time data were analyzed for the calculation of pharmacokinetic parameters with a non-compartmental model analysis using the Thermo Kinetica software package (version 5.0, InnaPhase Corp, Philadelphia, PA, USA). The maximum concentration (C max ) and the time taken to achieve the peak concentration (T max ) after iv dosing were obtained directly from the data without interpolation. The area under plasma concentration-time curve (AUC 0→t ) up to the last measured time point was calculated by the trapezoidal rule. The terminal elimination half-life (t 1/2 ) was calculated using the relationship 0.693/k. The total plasma clearance (CL tot,p ) for iv dosing was estimated by dividing the administered dose by the AUC 0→t . The distribution volume at steady status (V SS ) for iv dosing was estimated by multiplying the CL tot,p by the mean residence time (MRT). Dose proportionality assessments of AUC 0→t and C max data for iv administered L-P in rats were conducted by the regression of the log-transformed data (the Power Model) [4] . The correlation coefficient (r 2 ), slope, and 90% confidence intervals for the slope were calculated, and inferences were made based on a theoretical slope of 1 and confidence limits of 0.84 to 1.16. All results were expressed as the arithmetic mean plus standard deviation.

Protein binding analysis
The binding of L-P and L-2-Z to plasma proteins of the rats, beagle dogs and humans was determined in vitro using a highspeed, rapid ultrafiltration method, as described previously [5] . In brief, Microcon YM-30 centrifugal filter devices (g-force <14 000 g, Millipore, Bedford, MA, USA) were used, and the plasma sample was spiked with L-P at 167, 836, and 3344 nmol/L, or L-2-Z at 248 and 1237 nmol/L. BNPP was added to the rat plasma (but not to dog and human plasma) before incubation, thus avoiding L-P conversion. The nonspecific binding of L-P and L-2-Z to the ultrafiltration membrane was also checked by filter-tested compounds in phosphate buffered saline (PBS, pH 7.4) using the same ultrafiltration devices. The nonspecific binding correction was made to calculate the protein binding (Equation 1) according to the literature [6] .
where C PF is the drug concentration in the plasma filtrate, C PD is the measured plasma donor concentration, C SF is the drug concentration in the PBS filtrate, and C SD is the donor concentration measured in PBS before ultrafiltration.
In vitro assessment of inhibitory effects on cytochrome P450 enzymes To evaluate the potential drug-drug interaction capabilities of L-P and its active metabolite L-2-Z, the inhibitory effects of L-P and L-2-Z on CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 activities were examined using BD Gentest CYP inhibitor screening kits according to the manufacturer's instructions. The positive controls (inhibitors of enzyme activity) included furafylline for CYP1A2, sulfaphenazole for CYP2C9, tranylcypromine for CYP2C19, quinidine for CYP2D6, and ketoconazole for CYP3A4. L-P and L-2-Z were assessed, using recombinant CYPs, with respect to their capacity to inhibit the formation of fluorescent metabolites in reactions. These included 3-cyano-7-ethoxycoumarin → 3-cyano- Liquid chromatography/tandem mass spectrometry-based quantification of L-P and L-2-Z in various biological samples In our previous study, an accurate analytical method was developed for the determination of L-P and L-2-Z in various plasma samples, based on the immediate deactivation of blood carboxylesterases activities by its inhibitor BNPP after blood sampling [7] . This well-validated bioanalytical method was applied to the preclinical pharmacokinetic research of L-P and L-2-Z. The biological samples (30 µL) were mixed with 10 µL of 100 ng/mL internal standard solution and extracted with 90 µL of acetonitrile (including 0.5% of formic acid). The acidified acetonitrile solution converted carboxylate L-P and L-2-Z into their lactone forms, and the total concentrations of L-P and L-2-Z were measured. After centrifugation at 16 060×g for 10 min, the supernatant (5 µL) was applied to LC-MS-MS analysis.
LC-MS-MS system was assembled from a Waters Acquity UPLC separation module (Milford, MA, USA) and an AB Sciex API 4000 Q Trap mass spectrometer (Toronto, Canada) with Turbo V ESI-source.
Chromatographic separation was achieved on a 5-μm Agilent ZORBAX Eclipse Plus C18 column (50 mm×2.1 mm ID; Santa Clara, CA, USA) at ambient temperature. The mobile phases were acetonitrile/water, 1:99 (v/v), containing 10 mmol/L formic acid, for A and acetonitrile/water, 99:1 (v/v), modified with the same electrolyte, for B. A pulse gradient chromatographic method was used. In brief, the gradient parameters, including the start proportion, start proportion segment, elution proportion, elution proportion segment, and column equilibrium segment, were 4% B, 0.5 min, 100% B, 2.5 min, and 1 min, respectively.
The ESI-MS-MS parameters were optimized to maximize the generation of the singly protonated species and to produce the characteristic product ions for the test compounds and internal standards. The precursors to the product ion pairs used for the multiple reaction monitoring of L-P, L-2-Z, irinotecan, and camptothecin were m/z 599→124, 405→361, 587→195, and 349→305, respectively. The LC eluent was introduced to the ESI source at a flow rate of 0.40 mL/min for data acquisition over the period of 1.4 to 2.0 min.
Matrix-matched calibration curves were constructed for L-P and L-2-Z using weighted (1/X) linear regressions of the analyte/IS peak area ratio (Y) against the corresponding nominal plasma concentrations of the analytes (X, nmol/L). The blank plasma samples were treated with BNPP to deactivate the carboxylesterases before use.
Liquid chromatography/tandem mass spectrometry-based metabolite profiling of L-P in various rat samples The biological samples for metabolite profiling analyses included those of plasma, urine, bile and feces from male rats that received an iv dose of L-P at 7.5 mg/kg in the preceding plasma pharmacokinetic and excretion studies. An aliquot of 30 µL of the biological samples was extracted with 90 µL of acidic acetonitrile. After centrifugation, the supernatant was injected onto an LC-MS system for metabolite profiling. The mobile phase for metabolite profiling was the same as that used for the quantification of L-P and L-2-Z, with the flow rate set at 1.0 mL/min (0.5 mL/min of the LC eluent was introduced into the ESI source). Chromatographic separation was achieved on a 5-μm Agilent ZORBAX Eclipse DB C18 column (150 mm×4.6 mm ID) at ambient temperature. A gradient program was used with the following parameters: start proportion (10% B), start proportion segment (5 min), elution proportion (70% B), elution proportion segment (12 min), and column equilibrium segment (3 min). Metabolite profiling of L-P in the rat samples was conducted using full-scan ESI-interfaced LC-MS in both the positive-and negative-ion modes, according to the molecular mass gains or losses predicted for the possible metabolites compared with those of the parent compound. The metabolite prediction was performed according to the list of the common biotransformations of drugs [8] and was facilitated by the PALLAS MetabolExpert

Plasma pharmacokinetics in rats
The plasma pharmacokinetic parameters of L-P and its active metabolite L-2-Z after an iv bolus administration to rats are summarized in Table 1. After the iv dosing, L-P was eliminated from plasma with a mean t 1/2 of approximately 1.4 h (Figure 1), which was dose independent. The mean CL tot, p of L-P was observed to range from 1.52 to 1.82 L·h -1 ·kg -1 at three doses. Meanwhile, the mean V SS of L-P (2.86-3.34 L/kg) was approximately 5-fold greater than the average volume of rat total body water (0.67 L/kg) [9] , suggesting that L-P could be extensively distributed to the tissues. The biotransformation of L-P to its active metabolite L-2-Z after dosage was very fast: the mean T max values of L-2-Z were 0.08-0.14 h at three doses.
The systemic exposure ratio of L-P to L-2-Z (AUC L-P /AUC L-2-Z ) ranged from 9.23 to 16.4 for the dosages tested. The elimination half-lives of L-2-Z at different doses (2.12-2.25 h) were relatively longer than those observed for the parent drug. The AUC 0→24 h and C max of plasma L-P and L-2-Z increased as the iv doses increased from 3.75 to 15 mg/kg, while the dose proportionality of the AUC 0→24 h and C max were not conclusively linear ( Table 2). No gender difference was observed for the calculated pharmacokinetic parameters.

Tissue distribution in rats
The results of the tissue distribution examinations for L-P and L-2-Z in rats 30 min, 2, 4 and 8 h after the iv administration of L-P are summarized in Figure 2. L-P and L-2-Z were rapidly Table 1. Plasma pharmacokinetic parameters (mean±SD) of L-P and L-2-Z in tumor-bearing nude mice, rats and dogs receiving iv bolus doses of L-P.

L-P L-2-Z PK parameters
Nude mice (n=8) Nude mice (n=8) 15 mg/kg 15 mg/kg  ing. Most of the tissues (except for the brain, testicle, and adipose) had significantly higher concentrations of L-P than those observed in the plasma. The highest concentrations of L-P were found in rat pancreas, kidney, and liver 30 min after dosing. These data indicated that L-P was well distributed in rat tissues, which is consistent with the preceding plasma V SS data. L-2-Z was found to be distributed in most of the tissues, with the exception of the brain. The highest concentrations of L-2-Z in all tested tissues were observed 30 min after dosing, which resembled the distribution of the parent compound. The concentrations of L-2-Z in liver, kidney and small intestine were higher than that observed in the plasma.

Urinary, biliary and fecal excretion in rats
The results of the excretion experiments in rats indicated that after the iv administration of L-P at 7.5 mg/kg to rats, 13% of the administered dose was excreted in urine as L-P (0.32 μmol), and 1.3% was excreted as L-2-Z (0.03 μmol). Most of the L-P (90%) and L-2-Z (70%) were excreted within the first 4 h after dosing. Meanwhile, more than 95% of the L-P and 80% of L-2-Z were excreted into the bile within 6 h after dosing. Approximately 51% of the administered dose was recovered [48% as L-P (1.20 μmol) and 3.0% as L-2-Z (0.08 μmol)] in the bile and urine; biliary excretion was the predominant route of L-P excretion. These results are consistent with the tissue distribution studies, in which L-P and L-2-Z were found to have high concentrations in the liver and kidney. Moreover, the recovery of the administered dose from feces was 19% (0.48 μmol) as L-P and 12% (0.29 μmol) as L-2-Z over the first 48 h, which had a lower amount of L-P and a higher amount of L-2-Z than the amounts of these compounds excreted in bile. Figure 3 depicts the cumulative amount of L-P and L-2-Z excreted from urine, bile and feces, as well as the corresponding percentage of the dose recovered from excretory samples in rats.   Table 3, and Figure 4 depicts the proposed metabolic pathways. No metabolites were detected in the biofluids from the untreated rats. The MS-MS analysis of the metabolites indicated that M1 was the active metabolite of L-2-Z, M2 was the phase II glucuronide conjugate of M1, M3-M7 were phase I oxidative metabolites of L-P, and M8 was a decarboxylation product of L-P that could be further oxidized to form M9. Compared with the abundance of plasma L-P from the ivadministered L-P, M1 was the major plasma metabolite, with a plasma metabolite-to-parent compound ratio in AUC 0→24 h (AUC M /AUC L-P ) of 0.045. M2, M3, M6, and M7 were demonstrated to be minor plasma metabolites whose AUC M /AUC L-P values ranged from 0.001 to 0.008. These plasma metabolites were also measured in the rat urine and bile samples. M4 and M5 were found in urine and bile, but not in plasma, while M8 and M9 were only found in bile. In addition, two metabolites (M2 and M4) were excreted in relatively large amounts compared to other metabolites. M2 exhibited Cum.A e M /Cum.A e L-P values of 0.17 and 0.25 in urine and bile, respectively; and M4 had a Cum.A e M /Cum.A e L-P value of 0.30 in bile. The sums of the individual Cum.A e M /Cum.A e L-P (M2-M9) values were 0.4 and 0.9 in urine and bile, respectively. These results demonstrated that metabolism is also an important elimination route of iv-administered L-P.
Tissue distribution in tumor-bearing nude mice After iv administration of L-P at 15 mg/kg to a nude mice xenograft model of human hepatic cancer SMMC-7721 cell line, L-P and L-2-Z distributed to the tissues rapidly. The highest concentrations of these two compounds were observed at 15 min after the dosage. In addition, L-P was measured at  npg significantly higher concentrations in pancreas, heart, liver, lung, and kidney than that in plasma after dosing. The tumor tissue and colon had comparable L-P levels to those of plasma ( Figure 2). The measured L-2-Z levels in the tested tissues were lower than the L-P levels. The distribution of L-2-Z in the tumor tissues supports the results of the in vivo efficacy studies that reported a dramatic inhibition of tumor growth in several human tumor xenograft nude mice models [3] .

Plasma pharmacokinetics in beagle dogs
Following iv dosing to beagle dogs, L-P was eliminated from plasma with mean t 1/2 values ranging from 1.72 to 1.95 h (Figure 1), L-P elimination was unaffected by dose and resembled the results obtained in rats. The mean CL total,p of L-P was observed to be 1.12-1.46 L·h -1 ·kg -1 . Meanwhile, the mean V SS of L-P (3.03-3.54 L/kg) was approximately 5-fold greater than the average volume of dog total body water (0.60 L/kg), suggesting that L-P could be extensively distributed to the tissues. The systemic exposure ratio of L-P to L-2-Z (AUC L-P /AUC L-2-Z ) ranged from 49 to 59 in the dosages tested ( Table 1). The AUC 0→24 h and C max of plasma L-P and L-2-Z in dogs increased as the iv doses increased from 1.25 to 5 mg/kg, while the dose proportionality of AUC 0→24 h and C max were not conclusively linear (Table 2).

Plasma protein binding in different species
The plasma protein bindings of L-P in rat, dog and human plasma were 59.9%-72.3%, 18.0%-24.2%, and 36.9%-39.9% in the range of 167-3344 nmol/L, respectively. The L-2-Z plasma protein binding in the above respective species was 77.0%-83.1%, 44.5%-53.6%, and 75.7%-86.0% in the range of 248-1237 nmol/L. The non-specific binding of L-P and L-2-Z to the filter membrane and plastic parts of the centrifugal filter device was approximately 24% and 27%, respectively, and was corrected in the protein binding calculation. Thus, L-P and L-2-Z showed low to moderate binding to plasma proteins, and L-2-Z bound stronger to plasma proteins than its prodrug L-P. Compared to the literature values of plasma protein binding for irinotecan and its active metabolite SN-38 [10] , the plasma protein binding of L-P and L-2-Z in humans is relatively lower. Furthermore, the active metabolites bind more strongly to plasma protein than their parent drugs due to their higher lipophilicity.
In vitro inhibitory effects on human CYP enzymes As shown in Table 4    Compared to the stability of L-P and the formation of L-2-Z in the plasma of mice, rats, dogs and humans [7] , L-P was unstable and the formation of L-2-Z was approximately 2000 to 4000 nmol·mL -1 ·h -1 in rodents, while L-P was stable and L-2-Z could not be detected in the plasma of dogs and humans. These results suggest that in rodents, the biotransformation of L-P to its active metabolite L-2-Z predominantly occurs in the plasma rather than in the liver. In dogs and humans, however, due to the absence of carboxylesterase activity in plasma, hepatic carboxylesterases-mediated biotransformation of L-P to L-2-Z played an important role in the antitumor activities of L-P.

Discussion
The delivery of drugs with limited water solubility remains a challenge in drug development. The prodrug strategy is an older but time-tested approach, which utilizes bioreversible and more water-soluble derivatives of the problematic molecule. The stable, bioreversible and quantitative release of the active drug moiety is required for prodrug modifications. Irinotecan, the first-line antineoplastic agent, is an analog of L-P and is a successful prodrug that has improved aqueous properties for parenteral administration [11,12] . The use of irinotecan over SN-38 is also due to the beneficial steady-state kinetics obtained in the use of the prodrug form [13] . Irinotecan has an ethyl substituent at position 7 and a dipiperidyl carbamate at position 10, which is metabolized to SN-38 and is 100 to 1000 times more cytotoxic than the prodrug [14] . Its structure and biotransformation are very similar to that of L-P. In this study, we report preclinical pharmacokinetics and metabolism studies on L-P and its active metabolite L-2-Z, including the in vivo ADME properties following iv dosage to rodent and nonrodent species as well as its in vitro metabolism in liver microsomes of different species and an assessment of its inhibitory effects on cytochrome P450 enzymes. These studies led to the projection of human efficacious concentrations and doses and enabled an assessment of the potential for drug-drug interactions with concomitantly administered drugs. Following iv administration, L-P and L-2-Z exhibited relatively short elimination half-lives in rats and dogs. The clearance of L-P in nude mice (3.2 L·h -1 ·kg -1 ) was larger than that observed in rats (1.6 L·h -1 ·kg -1 ) and dogs (1.3 L·h -1 ·kg -1 ). The volume of distribution in rats and dogs was greater than total body water, indicating extensive extravascular distribution. The plasma protein binding of L-P and L-2-Z was measured to be moderate (≤81.6%) in plasma of different species. SN-38 was found to have a high level of plasma protein binding, ie, 94%-96%, in the concentration range of 50-200 ng/mL in human [10] . The tested compounds showed relatively lower plasma protein binding than their camptothecin analogs.
The prodrug L-P is biotransformed to its active metabolite L-2-Z in rats, mice and beagle dogs after iv administration. In this study, significant species differences were observed among the examined animals, in which rodents showed a much higher conversion ratio than dogs. The AUC L-P / AUC L-2-Z values ranged from 9.23 to 16.4 in rats (3.75-15 mg/kg), 1.51 in nude mice (15 mg/kg), and 49.3 to 58.6 in dogs (1.25-5 mg/kg). Carboxylesterases are responsible for the conversion of L-P to L-2-Z [7] and the activities of carboxylesterases in rodents, dogs and humans are different. High activities of carboxylesterases were observed in the plasma, liver and intestine of rodents, while only low activity was found in the liver and intestines of dogs. For primates, moderate carboxylesterases activity could be observed in the liver and intestines [15] . Our results are consistent with the reported enzyme activities in different species. The conversion of irinotecan to SN-38 through cleavage of the ester-bond at C10 was shown to be catalyzed by two human isozymes of liver carboxylesterases, ie, hCE-1 and hCE-2 [16,17] . Due to the structural similarity of L-P to irinotecan, these two isozymes most likely play an important role in L-P conversion in cancer patients.
In the tissue distribution experiment, L-P and L-2-Z were observed to have high concentrations in rat liver and kidney. This finding was consistent with the excretion results showing that the prodrug and its metabolite were excreted mainly from bile. L-P and L-2-Z distributed to the rat brain with low concentrations. The K p (AUC brain,free /AUC plasma,free ) ratios were 0.20 and 0.16 for L-P and L-2-Z, respectively. Various studies reported that central nervous system toxicity is associated with administration of irinotecan [18] and that the mechanism could be explained by electron transfer functionalities and oxidative stress generated from reactive oxygen species to the central nervous system [19] . Accordingly, neurotoxicity induced by L-P and/or L-2-Z should also be monitored during clinical antitumor therapy.
Tissue distribution results obtained in tumor-bearing nude mice indicated that L-P was measured at higher levels in pancreas, liver and kidney, which is consistent with the results obtained from the rats. Furthermore, in the tissue distribution studies in rats and nude mice, the levels of L-P were found to be highest in the pancreas at all time points investigated. Due to the weak anticancer activity of L-P in vitro, it is unlikely to exhibit any significant therapeutic benefit for pancreatic disease. However, the toxic side effects caused by this compound in the pancreas should be closely monitored. In the case of L-2-Z, the tissue accumulative concentrations in mice were relatively higher than those in rats, in which a similar distribution order exists. In this study, we found that the carboxylesterase-mediated hydrolysis of L-P to L-2-Z is more significant in mice than in other species. The AUC L-P /AUC L-2-Z value was 1.51 in nude mice (15 mg/kg) ( Table 1), indicating a much higher biotransformation than that observed in rats (9.23-16.4) and dogs (49.3-58.6). The systemic higher exposure of L-2-Z caused higher tissue distribution levels in nude mice. In addition, the higher conversion of L-P to its active metabolite L-2-Z could explain the higher total plasma clearance of L-P in nude mice (3.2 L·h -1 ·kg -1 ) than in rats (1.6 L·h -1 ·kg -1 ). It has been reported that carboxylesterases are highly expressed in human neuroblastoma cell lines [20] . The presence of tumors increased