Safety, pharmacokinetics, and antitumor properties of anlotinib, an oral multi-target tyrosine kinase inhibitor, in patients with advanced refractory solid tumors

Patients with pathologically and/or cytologically proven advanced cancer with no standard therapy were included in this study. Eligibility criteria include ages 18–65, ECOG PS 0–1, and an estimated survival duration of more than 3 months. Patients who had used other chemotherapy drugs need to stop for at least 30 days; patients who had received major surgery needed to rest for at least 4 weeks. Routine blood indices, blood lipids, liver and kidney function, and heart function (left ventricular ejection fraction) are normal; no major organ dysfunction. Patients needed to agree to use and utilize an adequate method of contraception. Patients had to give written informed consent for the study and be willing to comply with criteria of follow-up.

Patients were not eligible if they had the following: brain metastases; spinal cord compression; carcinomatous meningitis; brain or leptomeningeal disease confirmed by CT or MRI examination; urine protein ≥++ confirmed by a 24-h urinary protein excretion >1.0 g; failed to heal wounds or fractures for long term; coagulation abnormalities; experienced arterial or venous thromboembolic events before the first treatment; pre-existing thyroid disease; thyroid function could not be maintained within the normal range under treatment; carried active hepatitis B or hepatitis C; HIV-positive, acquired or congenital immunodeficiency diseases, or organ transplantation; and serious concomitant diseases.

This was a first-in-human, phase I, open-label study of anlotinib in advanced refractory solid tumors. The primary objective was to establish the safety profile of anlotinib by identifying DLT, MTD, the recommended phase II dose, and schedule. Secondary objectives included description of single-dose and multi-dose pharmacokinetics of oral anlotinib and assessment of preliminary antitumor effect.

All patients provided written informed consent. The study protocol and amendments were reviewed and approved by the Institutional Review Board, in accordance with the Declaration of Helsinki.

Those patients received escalating doses of anlotinib daily for (1) four consecutive weeks (4/0) or (2) 2-week on/1-week off (2/1). A standard 3 + 3 design was applied, with terms for cohort expansion to six evaluable patients if a DLT (grade 4 blood toxicity or grade 3 neutropenia with fever ≥38.5 °C; grade 3 or higher non-hematologic toxicity) was observed in the first cycle of treatment among the initial three patients. If two DLTs were observed during the first cycle in a cohort, dose-escalation was halted and dose continued at a lower level until the MTD (the highest dose level for which the incidence of first-cycle DLT was <33 %) was identified. In the absence of two or more DLTs in a cohort, the dose was escalated in a modified Fibonacci scheme. Treatment cycles were repeated until disease progression, unacceptable toxicity, or withdrawal of consent.

Efficacies were evaluated according to NCI-proposed Response Evaluation Criteria In Solid Tumors (RECIST 1.1). The tumor will be assessed every two cycles including tumor-related symptoms and physical and imaging examination (CT or MRI) of superficial lesions. Physical and imaging examination of tumor lesions should be done at least four weeks after the first effect evaluation in patients with complete remission (CR), partial remission (PR), and stable disease (SD) to confirm efficacy.

Adverse events are graded into 0–5 according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE 4.0). During treatment, blood pressure will be monitored every day. Patients with unresolved adverse reactions at the end of the test needed to be treated and followed until the reactions returned to the grade 1 degree or less, or stable.

In single-dose studies, anlotinib were administered orally at dose of 5, 10, 12, or 16 mg anlotinib/person. Serial blood samples were collected in heparinized tubes before and at 0.5, 1, 2, 4, 8, 11, 24, 48, 72, 96, 120, 144, 168, 192, 216, and 240 h (samples of 144–240 h were collected only in 12 or 16 mg/person dose group) after dosing. In the 10 mg/person dose group, urine was sampled before and 0–2, 2–6, 6–11, 11–24, 24–34, 34–48, 48–58, 58–72, 72–96, and 96–120 h after dosing. In multiple-dose studies, there were two ways of multiple-dose administration. Initially, patients in 5 and 10 mg/person/day groups were treated consecutively for 28 days. Blood samples were collected 24 h after dosing on days 1, 2, 4, 7, 10, 13, 15, 16, 18, 21, 24, 28, and 29. Later, the other volunteers were given multiple doses of anlotinib at 10, 12, or 16 mg anlotinib/person/day in the 2/1 schedule for two cycles. Blood samples were collected 24 h after dosing on days 1, 4, 7, 10, 13, 15, 18, 22, 29, and 36. The collected blood samples were centrifuged to prepare plasma fractions; the urine samples were weighed after collection. All samples were stored at −70 °C for subsequent analysis. Concentrations of anlotinib were measured by liquid chromatography/mass spectrometry.

The maximum concentration (C _max) and the time taken to achieve C _max (T _max) were obtained directly from the data with no interpolation. The area under concentration-time curve up to the last measured time point (AUC_0 − t ) was calculated by the trapezoidal rule. The elimination half-life (t _1/2) was calculated using the relationship 0.693/k. The k was estimated by linear regression analysis of the terminal portion of the log concentration-time data. The renal clearance (CL_R) was calculated by dividing the cumulative amount excreted into urine (Cum.A _e − U ) by plasma AUC_0 − t . Accumulation ratio (R _ac) was used to indicate the extent of accumulation during multiple doses of anlotinib capsules and calculated using the following equation:

where C _24 h(Day1) and C _24 h(Day15) are plasma concentrations of anlotinib at 24 h after dosing on day 1 and day 15, respectively. Plasma pharmacokinetic (PK) parameters were determined using noncompartmental method with a Kinetica software package (version 5.0; Thermo Scientific, Philadelphia, PA).

The two-side test is used for all statistical tests. P ≤ 0.05 is considered as statistical significance. The measurement data will use mean ± SD or median (min, max) for statistical description. Compared with baseline data of screening period, use paired t test to compare the difference before/after the treatment within the group. Frequency (constituent ratio) is used for statistical description to the enumeration data. The changes before/after the treatment will use χ ² test (accurate probabilistic method) or non-parametric test.

Thirty-five patients were enrolled in the study between July 2011 and August 2013. Two cohorts of patients received anlotinib at doses ranging from 5 mg (n = 4) to 10 mg (n = 4) once daily on the 4/0 schedule, and three cohorts of patients at doses of 10 mg (n = 3), 12 mg (n = 21), and 16 mg (n = 3) once daily on the 2/1 schedule. Patient baseline characteristics are presented in Table 1.

On the 4/0 schedule, no DLT was observed in the first four patients at the starting dose of 5 mg/day. However, at 10 mg/day, one patient developed grade 3 hypertension among the first three patients treated. An additional patient was enrolled and also developed grade 3 hypertension. Therefore, the further dose escalation was halted. Meanwhile, PK study revealed a continuously significant anlotinib accumulation in patients who received continuous administration (data not shown). Based on the PK profile of anlotinib and the two DLTs observed at the dose of 10 mg/day, we modified the administration protocol from the 4/0 schedule to the 2/1.

On the 2/1 schedule, because none of the three patients experienced DLT at initial doses of 10 mg/day, the dose escalation proceeded to 16 mg/day. Two of the three patients in the 16 mg cohort experienced DLT (one grade 3 fatigue and one grade 3 hypertension). Therefore, the MTD had been exceeded, and the next lower dose of 12 mg/day was further evaluated by entering additional patients. None of the initial three patients experienced grade 3/4 adverse events. On the basis, 12 mg once daily was selected for the expanding study.

A total of 21 patients received the 12 mg/day dose on the 2/1 schedule. During the first 2 cycles, all the patients experienced an adverse event of any causality. All the hematologic toxicities were mild. As illustrated in Table 2, the most common non-hematologic adverse events were hypothyroidism, triglyceride elevation, total cholesterol elevation, ALT elevation, diarrhea, and proteinuria. During the first 2 cycles, a total of two patients (10 %) experienced grade 3 adverse events (one triglyceride elevation and one lipase elevation). During all treatment cycles, there were six patients (29 %) with grade 3/4 adverse events. The most common (>5 %) non-hematologic grade 3 adverse events were hypertension, triglyceride elevation, hand-foot skin reaction, and lipase elevation.

According to the RECIST 1.1 criteria, 19 patients had target lesions and one patient with multiple small lung metastases had non-target lesions. Among the 20 patients whose response can be assessed, 3 patients (15 %) had PR; 14 patients (70 %) maintained SD including 12 patients with tumor burden shrinkage, and 3 patients (15 %) had progressive disease (PD) (Additional file 1: Figure S1). PR was observed in the following tumor type: two renal carcinoma and one soft tissue sarcoma. In the current phase I study, other tumor types that responded to anlotinib included medullary thyroid carcinoma, non-small cell lung cancer, colorectal cancer, melanoma, thymic carcinoma, and adenoid cystic carcinoma (Fig. 1).

The median administration duration was 24 weeks. Twenty-four, sixteen, and nine patients had received anlotinib for over 12, 24, and 72 weeks, respectively. Furthermore, the treatment duration had exceeded 100 weeks in three patients (Fig. 2).

The mean plasma level-time curve of anlotinib after a single oral administration at 5, 10, 12, or 16 mg/subject is shown in Fig. 3. The associated plasma PK parameters are summarized in Table 3. Anlotinib exhibited rapid intestinal absorption. This was indicated by its plasma concentration, which significantly increased at 1 h after dosing in most patient subjects (Fig. 3). The levels of systemic exposure to anlotinib (C _max and AUC_0–120h) tended to increase as the dose level increased from 5 mg anlotinib/person to 16 mg anlotinib/person, but the dose proportionality was inconclusive (Table 3). Anlotinib reached its maximum plasma concentration with T _max of 4–11 h after dosing, then it eliminated slowly with t _1/2 of 64–136 h and MRT of 124–167 h. The renal excretion of anlotinib was poor, with a CL_R of 0.004 L/h/kg; the fraction of the dose excreted into urine (f _e − U ) was ~0.9 %.

Notably, anlotinib exhibited a quite long t _1/2 (96 ± 17 h), which appeared to be dose-independent. The long t _1/2 resulted in significant accumulation of plasma anlotinib over time with a mean R _ac of 12 ± 7, suggesting drug accumulation over time (Fig. 3). The trough plasma concentration-time curve of anlotinib after treatment of anlotinib at 10, 12, or 16 mg/subject in the 2/1 schedule is depicted in Fig. 3. A 2-week subchronic dosing brought about continuously increased plasma concentration of anlotinib in patients, while its maximum plasma concentration was reached at day 14. Subsequently, the plasma level of anlotinib was apparently decreased with a 7-day washout until the beginning of the other treatment cycle.