The following inclusion criteria were used for patient selection: (1) histologically confirmed solid tumors refractory to standard therapy or for which there is no standard therapy; (2) age, 20 to 75 years; (3) Eastern Cooperative Oncology Group (ECOG) performance status (PS), ≤ 2; (4) life expectancy of longer than 12 weeks; (5) adequate organ function (adequate hematologic function) ≥ 1,500/μL, platelet count ≥ 100,000 cells/μL, hemoglobin ≥ 9.0 g/dL, serum creatinine ≤ 1.5×upper limit of normal (ULN), serum bilirubin ≤ 1.5×ULN, aspartate transaminase, alanine aminotransferase ≤ 3×ULN [5×UNL in case of liver metastasis]); and (6) normal range of prothrombin time, activated partial thromboplastin time. Written informed consent was obtained from all patients. The important exclusion criteria were (1) central nervous system metastasis; (2) prior chemotherapy, radiation therapy, or surgery within 4 weeks prior to enrolling in the study; (3) heart failure, angina pectoris, New York Heart Association (NYHA) class III, IV congestive heart failure within 6 months of study; (4) uncontrolled hypertension (systolic blood pressure > 150 mm Hg or diastolic blood pressure > 100 mm Hg despite anti-hypertensive medication); and (5) under anticoagulation.

CKD-516 was administered as a 30-minute intravenous infusion on day 1 and day 8 every 3 weeks. The starting dose was 1 mg/m²/day, and dose was escalated using 3+3 design and modified Fibonacci method. In each cohort, if a patient was withdrawn for any reason other than dose limiting toxicity (DLT) before completion of the 21-day administration period, the patient was replaced by another patient. Response was evaluated every two cycles. However, whenever disease progression was suspected, the response evaluation was allowed at the investigator’s discretion. Patients in whom antitumor effect was assessed as stable disease (SD), partial response (PR), or complete response (CR) for overall response at the end of the second cycle of treatment were allowed to receive further treatment. Treatment was to be continued until the subject experienced disease progression or unacceptable toxicity, withdrew consent, or required treatment with another therapeutic modality.

DLT was defined as follows: (1) grade 4 neutropenia lasting for ≥ 7 days or with fever; (2) grade 3-4 thrombocytopenia lasting for ≥ 7 days, or with bleeding or required platelets transfusion; (3) grade 3 or 4 nausea/vomiting or diarrhea despite optimal use of antiemetic drugs or antidiarrheal drugs; (4) other grade 3 or 4 non-hematological toxicity (except alopecia and anorexia); and (5) delay of > 2 weeks in administration of the next cycle due to inadequate recovery from toxicity of the first cycle. DLT was evaluated during the first cycle of treatment. The lowest dose at which DLT was observed in no more than two out of six patients in the first cycle of treatment was judged to be the MTD.

Blood was collected on day 1 before and at the end of infusion, 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours after dosing. Urine was collected on day 1, 0-4 hours, 4-8 hours, 8-12 hours, and 12-24 hours after dosing. Plasma and urine concentrations of CKD-516 and its active metabolite S-516 were determined using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with mosapride as an internal standard. Briefly, 200 μL of plasma or urine, methyl tert-butyl ether 1.5 mL, and mosapride 50 μL at 10 ng/mL were mixed thoroughly and centrifuged at 5,000 g for 5 minutes. The organic layer was separated and evaporated; the residue was reconstituted with 100 μL of 80% methanol, diluted 2-fold with mobile phase, and injected into the LC-MS/MS system. Chromatographic separation was performed on a Luna C18 column (50×2.0 mm, i.d., 5 μm; Phenomenex, Torrance, CA) under the isocratic condition using 0.05% formic acid in 10 mM ammonium formate and acetonitrile (70:30, v/v). The multiple-reaction monitoring was based on m/z transition of 537 > 270 for CKD-516, 438 > 270 for S-516, and 422 > 198 for mosapride (IS). The calibration ranges were linear with a lower limit of quantification of 0.5 ng/mL for CKD-516 and 0.2 ng/mL for S-516 in both human plasma and urine. The inter-assay precision and accuracy of the quality control (QC) samples were less than 9.54% and from 91.1% to 107%, respectively. Each analytical batch had six QC samples of known value, i.e., two for low, intermediate, and high concentrations, respectively. The analytical results were accepted only if the concentrations of > four out of the six QC samples were determined within 15% of the known values.

Safety and tolerability were measured by adverse events (AEs), vital sign, body weight, electrocardiogram (ECG), and laboratory tests. Safety was assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) ver. 4.02. PK parameters of CKD-516 and S-516 were calculated using the noncompartmental method with Phoenix WinNonlin 6.1 (Pharsight, Mountain View, CA). The pharmacokinetic linearity was assessed by linear regressions between dose and C_max or AUC_last and between log-transformed dose and log-transformed C_max or log-transformed AUC_last. Efficacy was measured using Response Evaluation Criteria in Solid Tumor (RECIST) ver. 1.1. Tumor responses were assessed after the first cycle or earlier in patients with suspected progression.

All safety analyses were performed on the intentionto-treat population. The objective response was assessed on the per-protocol population. The results are expressed as the mean±standard deviation or as ranges when appropriate. Follow-up started at the outset of treatment.

A total of 23 patients received the investigational product. Baseline characteristics of patients are shown in Table 1. Median age was 56 years (range, 33 to 70 years). Thirteen patients (56.5 %) were male. Nine patients (39.1%) had ECOG PS 0, 13 patients (56.5%) had ECOG PS 1, and one patient had ECOG PS 2. The most common tumors were colorectal cancer (n=11) and lung cancer (n=3). Seventy-eight percent of patients had previously received more than three kinds of chemotherapies.

The investigational products were administered at doses of up 12 mg/m²/day. DLT was reported in two patients in the 12 mg cohort, which were grade 3 hypertension. One patient with DLT had hypertension, and the blood pressure was increased up to grade 3 at 1 hour (164/96 mm Hg) and 4 hours (155/95 mm Hg) after infusion of CKD-516. However, the blood pressure was normalized at 8 hours after infusion without any intervention. The other patient with DLT showed elevation of blood pressure up to grade 3 (175/95 mm Hg) at 4 hours after infusion of CKD-516 and received anti-hypertensive medication (nicardipine), which led to the normalization of blood pressure 8 hours after infusion. There was no abnormality in troponin I, CPK-MB, and ECG in both DLT cases. The MTD of CKD-516 was determined as 12 mg/m²/day.

A total of 58 cycles of CKD-516 were administered to the patients. Most commonly reported AEs during all treatment periods were gastrointestinal AE, that is, diarrhea (34.8%; 30.4% grade 1/2, 13.0% grade 3), nausea (21.7%, all grade 1/2), and vomiting (21.7%, all grade 1/2) (Table 2). Grade 1/2 anorexia and myalgia were 30.4% and 17.4%, respectively. Abdominal pain occurred in 21.7% (grade 1/2, 21.7%; grade 3, 4.3%). Grade 1/2 neutropenia and thrombocytopenia were reported as 4.3% and 4.3%. Anemia occurred in 8.7% (grade 1/2, 8.7%; grade 3, 4.3%). Hypertension was observed two patients (two DLT cases [grade 3, 8.7%], one patient [grade 2, 4.3%] ). One patient (4.3%) had grade 3 acute coronary syndrome.

The plasma concentration-time profiles of CKD-516 and its metabolite S-516 showed a similar trend at all dosing levels (Table 3). C_max of CKD-516 were 45.88±15.04 μg/L (mean±standard deviation), 78.89±39.99 μg/L, 129.02±55.50 μg/L, 140.93±17.56 μg/L, 297.33±89.29 μg/L, 273.32±93.37 μg/L, and 646.75±513.71 μg/L at seven dosing levels, respectively. C_max of S-516 were 19.65±2.38 μg/L, 37.52±3.72 μg/L, 59.75±5.83 μg/L, 128.83±7.12 μg/L, 110.43±17.30 μg/L, 141.20±27.74 μg/L, and 222.75±3.04 μg/L at seven dosing levels, respectively (Fig. 1). CKD-516 underwent almost complete biotransformation to S-516 with only < 1% of unchanged CKD-516 excreted in the urine. A good linear relationship between dose and PK parameters indicated the linear pharmacokinetic characteristics of CKD-516 and S-516 over all dose levels (Fig. 2).

Response evaluation was possible in all 23 patients. None of the patients had CR or PR response. Six patients were assessed as SD (26.1%) and 17 patients showed progressive disease (73.9%). One patient showed 2.8% tumor reduction. Median progression-free survival was 39 days (95% confidence interval, 37 to 41 days). Three patients received the study treatment up to six cycles (1 osteosarcoma at 1 mg, 1 renal cell carcinoma at 3.3 mg, 1 colon cancer at 3.3 mg). The reasons for termination of treatment were disease progression (21 patients), no recovery of disease-related condition within 5 weeks (1 patient), and investigator’s discretion (1 patient).