A phase I study investigation of metabolism, and disposition of [14C]-anlotinib after an oral administration in patients with advanced refractory solid tumors

Purpose Anlotinib is a novel oral multi-targeted receptor tyrosine kinase inhibitor, which selectively inhibits VEGFR2/3, FGFR1-4, PDGFR α/β, c-kit, and Ret. It shows antitumor effect in patients with advanced refractory solid tumors. The detailed absorption, metabolism, and excretion pathways of anlotinib have not yet been fully investigated. Methods Six male patients were enrolled and divided into two groups. Group A (containing two patients) received 14.15 mg/80 µCi/subject [14C]-anlotinib hydrochloride. Group B (containing four patients) received 14.15 mg/120 µCi/subject [14C]-anlotinib hydrochloride. The blood, urine, and feces of all the six patients after orally administration of [14C]-anlotinib were collected. The absorption, metabolism, and excretion of [14C]-anlotinib were investigated, and the efficacy and safety of anlotinib were evaluated. Results In plasma, the average time to peak concentration (Tmax) of total radioactivity was 4.42 h and the average peak concentration (Cmax) of total radioactivity was 18.80 ng Eq./g. The average values of AUC0-last, AUC0-∞, and MRT0-t were 4071 h.ng Eq./g, 13,555 h.ng Eq./g, and 125 h, respectively. The average recovery of total radioactivity (TRA) in urine and feces was 62.03%, accounting for 48.52% and 13.51% in feces and urine of the total dosage, respectively. The parent drug, a carboxylic metabolite (M30), and mono-oxidation products (M46/M66) were major drug-related components in human plasma. Oxidative metabolism played the major role in drug clearance in human. The major metabolic pathways include oxidative deamination to M2, mono-oxidation to M1, and the formation of M30. Adverse events occurred in five patients and severe adverse events (SAE) occurred in one. Tumor response were evaluated as stable disease (SD) in three, partial response (PR) in one, and progressive disease (PD) in one of the patients, respectively. Conclusions Anlotinib had a good pharmacokinetic profile with rapid absorption, long half-life, and extensive hepatic metabolism. The adverse events and efficacy were as expected. Electronic supplementary material The online version of this article (10.1007/s00280-020-04062-8) contains supplementary material, which is available to authorized users.


Introduction
Receptor tyrosine kinases (RTKs) are transmembrane glycoproteins that communicate with cellular growth factors and extracellular ligands [1]. RTKs have been implicated in a variety of pathological conditions, including cancer, metabolic and autoimmune disorders, infectious diseases, and neurodegenerative disorders [2]. RTK inhibitors have been successfully utilized in the treatment of several cancer types, most of which are multi-targeting such as imatinib, anlotinib, sorafenib, sunitinib, and pazopanib [3]. Anlotinib is one of the newest developed oral small-molecule RTK inhibitors which has multiple targets including VEGFR1, VEGFR2/KDR, VEGFR3, c-Kit, PDGFR-α, and the fibroblast growth factor receptors (FGFR1, FGFR2, and FGFR3) [4]. The vitro studies using recombinant enzymes indicated that the capability of anlotinib to inhibit VEGFR2/KDR and VEGFR3 is approximately 20 and 500 times stronger compared with sunitinib and sorafenib [5]. The dysregulation of fibroblast growth factor (FGF)/FGF receptor axis results in aggressive cancer phenotypes by promoting cancer progression and enhancing the angiogenic potential of tumor microenvironment [6]. FGF/FGFR signaling alterations have also been found to be connected with chemotherapy resistance and poor prognosis. Preclinical studies have shown that anlotinib inhibits cell migration and formation of capillarylike tubes induced by VEGF/PDGF-BB/FGF-2 in endothelial cells [7]. Anlotinib suppresses tumor cell proliferation via inhibition of platelet-derived growth factor receptors α/β (PDGFR α/β), c-Kit, ret, as well as Aurora-B, c-FMS, and discoidin domain receptor 1 (DDR1), which are a group of newly identified kinase targets involved in tumor progression [8]. In addition, anlotinib showed antitumor activity against tumor cells carrying mutations in PDGFR α, c-Kit, Met, and epidermal growth factor receptor (EGFR) [9].
Anlotinib has been found to have a significant role in xenograft models in the renal, ovarian, colon, liver, glioma, and non-small cell lung cancer cells during dosing period [10]. The results of pharmacokinetic and disposition investigations in rats and dogs showed that anlotinib has good membrane permeability and is absorbed quickly. Metabolism studies in vitro and in rats demonstrated that anlotinib was primarily cleared via cytochrome P450-mediated hydroxylation and dealkylation. The oxidative metabolites were excreted directly to urine or bile or further converted to glucuronides or sulfates. Besides, anlotinib exhibited inhibitory activities against human cytochrome P450 3A4 and 2C9 in vitro with half maximum inhibitory concentrations of 0.11 μM and 0.25 μM, respectively [11].
Sun et al. performed a phase I study to determine DLT, MTD, basic pharmacokinetics, RP2D, and preliminary antitumor effects of anlotinib in patients with advanced refractory solid tumor. They found that anlotinib displayed manageable toxicity, long circulation, and broad-spectrum antitumor potential. The recommended treatment regimen for the subsequent clinical studies was as follows: anlotinib monotherapy; 12 mg per day on a 2/1 schedule [12].
The main objective of this phase I clinical study was to investigate the absorption, metabolism, and excretion of [ 14 C]-anlotinib hydrochloride in patients after an oral administration. Plasma, urine, and feces were collected followed by total radioactivity analysis, radioactivity profiling, and metabolite structural characterization. Consequently, exposures to drug-related components, mass balance, and metabolic clearance pathways in human were determined.

Study design
The phase I study of anlotinib was carried out in a single center and six male patients with advanced refractory solid tumor were enrolled. All the six male patients enrolled were divided into Group A (containing two patients) and Group B (containing four patients). The study was conducted in accordance with International Conference on Harmonization guidelines for Good Clinical Practice.

Exclusion criteria
The exclusion criteria included: (1) uncontrolled hypertension (systolic blood pressure > 150 mmHg, diastolic blood pressure > 100 mmHg with one antihypertensive drug); (2) urinary protein (+ +) and 24-h urinary protein quantification (> 1.0 g) was confirmed; (3) exposure to any drug that inhibits or induces drug metabolic enzymes within 30 days before study entry; (4) major surgical treatment, incision biopsy, or significant traumatic injury within 28 days; (5) arteriovenous thrombosis events within 6 months, such as cerebrovascular accident (including temporary ischemic attack), and deep vein thrombosis and pulmonary embolism; (6) large doses of radiation within 1 year, or radioactivity pharmacokinetic studies of human carbon 14 within 1 year.
The basic characteristics of patients are shown in Table 1.

Sample collection
The blood samples were collected before orally administration and 0. The urine samples were collected before orally administration and 0-2648 h after orally administration. The fecal samples were collected before orally administration and 0-1976 h after orally administration. In the process of collecting urine and feces samples of the six patients, because of the excretion particularity of the drug, the samples were not collected continuously every day, but once a week or every 10 days. In this case, the calculation of excretion rate during non-collected days was the multiplication of the firstday excretion rate after non-collected days by the number of non-collected days.

Radioactivity dosimetry
A dosimetry analysis was conducted to estimate the human absorbed radiation dose of [ 14 C] anlotinib hydrochloriderelated radioactivity for oral administration of [ 14 C] anlotinib hydrochloride. Total radiocarbon concentrations in organ/ tissue distribution and excretion data obtained after oral administration of [ 14 C] anlotinib hydrochloride to male rats were utilized for this analysis.
The animal data were subjected to relative organ massscaling and physiological time-scaling, and then fit with non-compartmental analysis using WinNonlin6.3 (Pharsight). The human-absorbed dose estimates were calculated following the MIRD (Medical Internal Radiation Dose) schema using the MIRDOSE3.1 software package (Oak Ridge Associated Universities, 1994) and manually in MicroSoft Excel for organs/tissues of interest that were not covered by MIRDOSE3.1.
For a human mass balance study involving radioactivity, the Effective Dose (ED) is typically kept below 1 mSv (Risk Category IIa or below) if possible. Based on the analysis presented herein, a single oral administration of 81 μCi or lower of [ 14 C] anlotinib hydrochloride would predict an effective radiation absorbed dose at or below 1 mSv (Risk Category IIa, ICRP 62, one-fifth of the absorbed dose equivalent per patient of 5 mSv per year cited in the Swiss Radiological Protection Ordinance). In conclusion, a total dose of up to 81 μCi in humans is expected to result in a radiation exposure that is within the 100 mrem (1 mSv) acceptable absorbed dose limit in humans. 80 µCi/ subject was used for Group A, due to long half-life of anlotinib in humans, radioactivity in collected samples post-dose was near to background, and 120 µCi/ subject was used for Group B to obtain more precise data.

Radioactivity determination
Levels of radioactivity in plasma, urine, and extracts of fecal samples were determined by mixing individual samples with 5 mL scintillation cocktail and determined by counting directly in a liquid scintillation counter (LSC). Blood, The supernatant was then separated from the PES, which was suspended with water, followed by addition of methanol (2x, v/v) twice. The three extracts were combined and evaporated to dryness under a nitrogen stream by an N-Evap evaporator at room temperature. The residues were reconstituted with an appropriate volume of methanol:water (1:1, v/v) and centrifuged at ca. 10000 g at 4 ℃ for 10 min. The supernatant was subjected to HPLC radio-profiling.

Urine
Four pooled human urine samples (subject 03 ~ 06) were pooled from 0 to 432 h based on equal volume ratios from the individual patients across time intervals. In addition, three pooled urine samples (0-48, 48-120 and 360-432 h) (subjects 03-06) were prepared by pooling human urine samples across subjects and time points. Portion of the pooled urine sample was evaporated to a small volume under a nitrogen stream and extracted with methanol (3x, v/v) followed by centrifuged at ca. 10000 g at 4 ℃ for 10 min. The supernatant was reconstituted with an appropriate volume of methanol:water (1:1, v/v) and centrifuged at ca. 10000 g at 4 ℃ for 10 min. The supernatant was subjected to HPLC radio-profiling.

Feces
Four human fecal samples (0-432 h) were prepared by pooling feces from individual subjects (Subjects 03-06). In addition, three pooled human fecal samples (0-48, 48-120 and 360-432 h) were prepared by pooling feces samples across subjects and time points. The pooled fecal homogenate was extracted with methanol (3x, v/v) and the mixture was vortexed, and centrifuged at ca. 10000 g at 4 ℃ for 10 min. The supernatant was then separated from the PES, which was suspended with water, followed by addition of methanol twice. The three extracts were combined and evaporated to dryness under a nitrogen stream by an N-Evap evaporator at room temperature. The residues were reconstituted with an appropriate volume of methanol:water (1:1, v/v) and centrifuged at ca. 10000 g at 4 ℃ for 10 min prior to analysis.

Bioanalytical assay
The calibration curve of anlotinib used for determination of anlotinib in LC-MS/MS contained eight calibrators. The linear range of the calibration curve was 0.868 ~ 111 nf/mL, and the concentrations of the eight calibrators were 0.868, 1.74, 3.47, 6.94, 13.88, 27.75, 55.5, and 111 ng/mL, respectively. The weighting factor was 1/x 2 .

Parameters calculation
The peak concentration (C max ) and the time to C max (T max ) were obtained through the plasma concentration directly. The area under concentration-time curve up to the last measured time point (AUC 0−t ) was calculated by the trapezoidal rule. The k was estimated by linear regression analysis of the terminal portion of the log concentration-time data, and the elimination half-life (t 1/2 ) was calculated through the coefficient 0.693/k. The renal clearance (CLR) was calculated by dividing the cumulative amount excreted into urine (Cum. A e−U ) by plasma AUC 0−t .

Metabolite profiling and characterization
HPLC coupled with radioactivity monitor (LC-RAM) was used to isolate and quantitate [ 14 C] anlotinib and its metabolites. The plasma and fecal extracts as well as urine supernatants were subjected to HPLC separation, fractionation, and eluent collection. The radioactivity in each fraction was monitored by solid scintillation counting using a Packard Top-Count® NXT™ Microplate Counter. The radio-profiles were then reconstructed using ARC® Convert software. The peak area of each radioactivity peak/region was integrated using ARC® Evaluation software, and the percent distribution of each peak/region was calculated (%TRA for plasma or %Dose for urine and feces). Metabolites were characterized and/or identified by LC-MS/MS in conjunction with on-line radioactivity detection. Structures of metabolites were proposed by interpretation of the mass spectral fragmentation pattern, as well as chromatographic behaviors and metabolic pathways.

Patients
Six patients with confirmed advanced refractory solid tumors were enrolled in May 2016. They had a mean weight of 70.4 kg (range from 54 to 90.4 kg) and a mean height of 170.55 cm (range from 164.8 to 175.6 cm). All patients had a history of anti-cancer drug therapy and anticancer surgery. At the time of enrollment, four patients (67%) had a WHO performance status score of 0 and two (33%) had 1.

Pharmacokinetics
Individual differences are significant after a single oral administration of 14.15 mg/80 μCi/human or 14.15 mg/120 μCi/human [ 14 C]-anlotinib hydrochloride in male patients. The T max of total radioactivity (TRA) in plasma is 4.42 h in average and the C max of TRA is 18.80 ng Eq./g in average. The average values of AUC last and AUC 0-∞ are 4071 h•ng Eq./g, 13,555 h•ng Eq./g respectively, and the average value of MRT 0-t is 145 h. The plasma concentration-time curve of anlotinib in plasma samples from six patients is shown in Fig. 1.

Mass balance
For all of six patients, urine and fecal samples were collected during the 2648 h after administration of [ 14 C]-anlotinib. Recovery of total radioactivity of the dose was 62.03%, of which 13.51% was from urine and 48.52% was from feces; feces was the major excretion route. The details are listed in Table 2. Mean cumulative excretion of radioactivity within 0-2648 h from urine and feces following a single oral dose of 14.15 mg/80 or 120 µCi /subject of [ 14 C] anlotinib hydrochloride are shown in Fig. 2.

Metabolite detection and structural characterization
In the current study, ten metabolites of anlotinib were found and structurally characterized in plasma and excreta in patients by LC-RAM/HRMS after administration of a single oral 14.15 mg/120 μCi/human of [ 14 C]-anlotinib,    (Fig. 3). M1, M23, and M46/M66 were mono-oxidation products of anlotinib, M2 was di-oxidation product of anlotinib, M9 was a sulfate conjugate of a mono-oxidation product, M17 was a glucuronide conjugate of a mono-oxidation product, M18 was O-dealkylation product of anlotinib, M28 was an oxidative deamination product of anlotinib, M29 was a mono-oxidation product of M28, and M30 was oxidized to carboxylic acid product of anlotinib. Mass spectral data of these metabolites are summarized in Table 3. Metabolite profiles of anlotinib in plasma, urine, and feces

Fig. 3 Proposed major metabolic pathways of anlotinib in human
(1) Plasma The parent drug, M30, and M46/M66 were major drugrelated component in human plasma ( Supplementary  Fig.1A). In addition, M1, M2, M9, M17, M18, M28, and M29 were detected in plasma. The identities of M1, M2, M9, M17, M18, M30, and M46/M66 were determined using LC-MS/MS analysis and the comparison with those in urine and feces. However, due to matrix interference and low concentration, M28 and M29 in plasma were not detected by LC-MS/MS. Their identities were determined by comparing their retention time with those in urine and feces. In addition, metabolites with high polarity (e.g., peaks 2 and 9) were detected in plasma samples, but their structures remained to be determined.

Safety
After a single dosage, adverse events occurred in five patients, while SAE occurred in one during the follow-up period. Treatment-related grade 3 adverse events occurred in the course of drug use. The frequency of adverse events was sorted as following: increased lipase, increased bilirubin, increased alanine aminotransferase, hypophosphatemia, increased gamma-glutamyltransferase, increased aspartate aminotransferase, and liver function damage. Grade 1/2 adverse events included increased lipase, hypoalbuminemia, increased bilirubin, constipation, proteinuria, etc. The details are listed in Table 4.

Discussion
In this study, we found that the average recovery of TRA in urine and feces was 62.03%. Prototype drugs and metabolites were mainly excreted through feces and urine, accounting for 48.52% and 13.51% of the total dosage. The low recovery of TRA might result from four reasons: first, halflife period for the elimination of anlotinib in human body is long. After absorbed into human circulatory system, the slow excretion of anlotinib resulted in a relatively low concentration of radioactivity in samples (radioactive concentration after 404-432 h is 279 DPM/ml in urine and 4876 DPM/ml in feces), which might affect the low radioactivity; second, a single dosage of 14.15 mg/80uCi/human or 14.15 mg/120uCi/human [ 14 C]-anlotinib hydrochloride suspension was administered followed by anlotinib (12 mg for 2 consecutive weeks followed by 1 week discontinuation) after 432 h (504 h for Subject 04). As a result, [ 14 C]-anlotinib hydrochloride and its metabolites were diluted and redistributed after the large quantity of unlabeled drugs entered circulation, reducing the excretion rate of labeled drugs and their metabolites [13]; third, in the process of collecting urine and feces samples of the six patients, because of the excretion particularity of the drug, the samples were not collected continuously every day, but once a week or every 10 days. In this case, the calculation of excretion rate during non-collected days was the multiplication of the firstday excretion rate after non-collected days by the number of non-collected days. This calculation method might lead into errors; fourth, other metabolic pathways, such as breath and sweat, might lower the recovery.
We found that C max and AUC were twice as much as the results in published phase I study, which might result from two reasons. One is the difference of drug administration. Capsule was used in the previous study, while suspension was adapted in our study in considering of the configuration and use of radiopharmaceuticals. The other is the small sample size of only six patients in this study versus 11 in the previous study. The difference was acceptable considering the fixed dosage of drug with individual differences (such as height, weight, or the activity of P450 metabolic enzymes) [14].

3
The plasma concentration of anlotinib hydrochloride was analyzed by LC-MS/MS. All of the data were in accordance with the standard operating procedures of Nanjing Mesino Pharmaceutical Technology Co., Ltd. The results showed that TRA concentration in plasma and blood was low after [ 14 C]-anlotinib hydrochloride administration, and the individual difference was significant.
The liver was the main excretory organ and the kidney was the secondary excretory organ after a single dosage of [ 14 C]-anlotinib hydrochloride. The main clearance method of anlotinib hydrochloride was considered to be metabolized by phase I enzyme and then excreted from liver to feces. Besides, the direct excretion of anlotinib from the kidney is also another important way of clearance. According to metabolite identification of [ 14 C]-anlotinib hydrochloride, the main metabolic pathways of anlotinib hydrochloride include: (1) amino oxidation (amino to hydroxyl) to form M28; (2) mono-oxidation to form M1, and further to oxidative metabolite (M2) and sulfuric acid conjugate (M9); (3) carboxylation to form M30. Additionally, the secondary metabolic pathways of anlotinib hydrochloride in the patients including: (1) mono-oxidation to form M23 and/or M46/M66 and further convert to glucuronic acid-binding product (M17); (2) O-dealkylation to form M18 [15].
Patients were well tolerated to single dosage of 14.15 mg/80 µCi/human or 14.15 mg/120 µCi/human [ 14 C]-anlotinib hydrochloride suspension during the observational period, and most of them presented with grade 1/2 adverse events. It is suggested that the safety of patients could be guaranteed with a single dosage of [ 14 C]-anlotinib hydrochloride suspension (14.15 mg/80 µCi or 14.15 mg/120 µCi).

Conclusions
In conclusion, anlotinib showed a pharmacokinetic profile of rapid absorption, long half-life, and extensive hepatic metabolism. The efficacy was promising with a favorable toxicity profile as expected, demonstrating that anlotinib could be further investigated in clinic. his contribution in the study design of metabolism investigation and paper written. This work was supported by the National Natural Science Foundation of China (Grant No. 81501981); and the Project of Oncology Translational Medicine Central of Jiangsu Province (Grant No. BL2012008).
Author contributions YQ Shu and LK Liu designed the study, LX Liu, Tongshan Wang, and Yiqian Liu participated in data collection and interpretation, YQ Liu wrote the report. Lian Guo and Yixiang Wang participated in mass balance and metabolism data generation and reporting. Zhengzheng Gao participated in LC-MS/MS analysis of concentration of anlotinib in plasma samples. All authors reviewed and approved the submitted article.

Compliance with ethical standards
Conflicts of interest All the authors declare no conflict of interest.
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