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This study was conducted to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of the intravenous pan-aurora kinase inhibitor PHA-739358, danusertib, in patients with advanced solid tumors.
In Part 1, patients received escalating doses of danusertib (24-h infusion every 14 days) without filgrastim (G-CSF). Febrile neutropenia was the dose-limiting toxicity without G-CSF. Further dose escalation was performed in part 2 with G-CSF. Blood samples were collected for danusertib pharmacokinetics and pharmacodynamics. Skin biopsies were collected to assess histone H3 phosphorylation (pH3).
Fifty-six patients were treated, 40 in part 1 and 16 in part 2. Febrile neutropenia was the dose limiting toxicity in Part 1 without G-CSF. Most other adverse events were grade 1–2, occurring at doses ≥360 mg/m2 with similar incidence in parts 1 and 2. The MTD without G-CSF is 500 mg/m2. The recommended phase 2 dose (RP2D) in Part 2 with G-CSF is 750 mg/m2. Danusertib demonstrated dose-proportional pharmacokinetics in parts 1 and 2 with a median half-life of 18–26 hours. pH3 modulation in skin biopsies was observed at ≥500 mg/m2. One patient with refractory small cell lung cancer (1000 mg/m2 with G-CSF) had an objective response lasting 23 weeks. One patient with refractory ovarian cancer had 27% tumor regression and 30% CA125 decline.
Danusertib was well tolerated with target inhibition in skin at ≥500 mg/m2. Preliminary evidence of anti-tumor activity, including a PR and several occurrences of prolonged stable disease (SD), was seen across a variety of advanced refractory cancers. Phase II studies are ongoing.
This phase I study in patients with advanced solid tumors determined the safety, tolerability, pharmacokinetics, and preliminary evidence of anticancer activity of danusertib (PHA-739358), an intravenous pan-aurora kinase inhibitor. Danusertib was safe and well-tolerated. The respective maximum tolerated dose (MTD) levels of danusertib when administered as a 24-hour infusion every 2 weeks are 500 mg/m2 without filgrastim and 750 mg/m2 with filgrastim. Anti-tumor effects and clinical benefit were observed, including a prolonged objective response in a patient with refractory small cell lung cancer as well as several instances of prolonged stable disease in a variety of refractory solid tumors. Biomarker modulation in skin biopsies (decrease in histone H3 phosphorylation) was observed starting at the 500 mg/m2 dose level. Danusertib is undergoing further evaluation in ongoing disease-directed phase 2 studies.
Danusertib is a small molecule 3-aminopyrazole derivative that potently inhibits all Aurora Kinase (AK) family members (A, -B & -C). AKs are essential for mitosis(1) and cytokinesis with upregulation in many malignancies, including pancreatic(2), colorectal(3), ovarian(3) and esophageal cancer(4). Aurora A, required for spindle assembly, localizes to centrosomes. Aurora B is a chromosome passenger protein required for histone H3 phosphorylation, chromosome segregation and cytokinesis(5–7). Several AK inhibitors with diverse pharmacological properties are in clinical trials(8–15).
Danusertib is active in vitro against a wide range of cancer cell lines with sub-micromolar IC50 values for inhibition of proliferation. Danusertib shows dominant Aurora B Kinase inhibition(16) with additional activity against bcr-abl, including the T315I mutation. In vitro studies have shown that danusertib causes a failure of cell division, resulting in polyploidy and reduction in viability. Danusertib inhibits Aurora-B phosphorylation at serine 10 of histone H-3, a protein implicated in chromosome condensation. Thus, inhibition of histone H3 phosphorylation may be a potential biomarker of danusertib biological activity. Danusertib also has significant antitumor activity in transgenic tumor models with a favorable preclinical safety profile(16); principal target organs of danusertib are the hemolymphopoietic system, GI tract, male reproductive organs and kidneys. Renal effects, however, are only seen at high drug exposure. In addition an increase in peripheral blood pressure was observed after IV bolus and 6-h infusion in rats, but not in dogs.
Based on this broad spectrum of preclinical activity and favorable toxicology profile, we performed a phase I study to evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of danusertib in patients with refractory solid tumors. To the best of our knowledge danusertib was the first Aurora kinase inhibitor in the clinic.
Patients with histological or cytological evidence of advanced refractory cancer lacking options for established curative or life-prolonging therapy were eligible. Eligibility criteria also included Eastern Cooperative Oncology Group performance status ≤ 1; prior radiation completed ≥2 weeks prior to enrollment and no more than 25% of bone marrow irradiated; life expectancy ≥12 weeks; normal blood pressure (≤140/90 mmHg) with or without treatment; and baseline laboratory data indicating acceptable bone marrow, liver and kidney function. Patients with previous high-dose chemotherapy requiring hematopoietic stem cell rescue, known brain or leptomeningeal metastasis, active inflammatory bowel disease, partial or complete bowel obstruction or chronic diarrhea, abnormal left ventricular function, severe cardiovascular disease, cardiac dysrhythmias ≥Grade 2, and active infections were excluded. All patients were adults (age >18 yrs) and gave written informed consent according to local IRB and Federal guidelines.
This was a Phase I, open-label, non-randomized, dose-escalation trial conducted at 2 USA sites. Danusertib was manufactured by Nerviano Medical Sciences S.r.l. (Nerviano, Italy) and supplied as 1% (w/v) sterile solution in 20/26 mL type I glass vials. Each vial contained 15 mL of solution (10 mg/mL) corresponding to 150 mg of danusertib. The drug product was stored at 2–8°C, protected from light and brought to room temperature shortly before use. Danusertib was administered via central line as a 24-h IV infusion on Day 1 of a 14-day treatment cycle. The starting dose was 45 mg/m2, targeted to administer 1/10th of the exposure at MTD in the dog, the most sensitive species in toxicology studies. Routine anti-emetic prophylaxis was not given. Incidence and severity of Adverse Events were coded according to NCI Common Terminology Criteria (NCI CTCAE, Version 3.0). Response to therapy was monitored by RECIST. DLT was defined as grade 4 neutropenia lasting >7 days, febrile neutropenia, neutropenic infection, grade 3 thrombocytopenia for >7 days or associated with bleeding, grade 4 thrombocytopenia, or any grade 3/4 non-hematologic toxicity including dose delay by ≥1 week occurring during Cycle 1 attributable to danusertib. The MTD was the highest dose at which ≤1 out of 6 patients had a DLT during Cycle 1. Patients could continue on danusertib (14-day cycles) in the absence of disease progression or unacceptable toxicity.
A two-stage accelerated titration design was used with initial rapid dose escalation (100% dose increments) until occurrence of DLT during Cycle 1, or 2 patients at any dose level/any cycle developing grade ≥ 2 drug-related toxicity. Thereafter, a modified Fibonacci scheme was to be used with dose increments of 50%, 40%, and 33% increments. Cohorts of 3 patients (expanded to 6 if 1 DLT was observed among the initial 3 patients) were to be treated with progressively higher doses until ≥ 2 of 3 pts or ≥ 2 of 6 patients in a cohort experienced a DLT. After the determination of MTD without G-CSF (Part 1), we attempted further dose escalation in ≤33% dose increments using G-CSF for primary prophylaxis of hematological toxicity (Part 2). Treatment with G-CSF (5µg/kg/daily) was started 24 h after the end of danusertib infusion and continued for 10 days (from Day 3 to Day 12) ending 48 h before the next infusion.
Complete histories, physical examinations, complete blood counts, serum electrolytes, chemistries and urinalysis were performed at baseline and prior to each cycle of treatment. Laboratory studies were repeated weekly. Coagulation tests (PT, PTT, fibrinogen concentration, fibrin degradation products, and D-dimer) were done at pre-dose, 1h after the end of infusion and 24h after the end of infusion in Cycle 1 with repetition in subsequent cycles for any clinically significant changes. 12-lead ECG and baseline MUGA scan were done within 28 days prior to start of dosing with subsequent MUGA scans at the end of Cycle 1, at the end of every even cycle up to Cycle 6 and then every 4th cycle (if LVEF decrease was <10%). Radiological exams (CT, MRI) were performed at baseline and after every three 2-week cycles of therapy. Criteria for patient discontinuation included progressive disease at any time, unacceptable toxicity, changes in medical status, patient refusal or non-compliance or lost to follow-up. Patients could receive focal palliative radiotherapy and remain on-study provided radiation-related toxicities (other than xerostomia) had normalized to pre-radiation levels 2 weeks after completion of radiotherapy. If such radiotherapy involved >25% of bone marrow, however, this was considered disease progression.
Plasma samples for evaluation of PK of danusertib and its main metabolite (see chemical structures in Figure 1 panel A) were collected during Cycle 1 (on Days 1, 2, 3, 4 and 8) before, during, and after the end of infusion for a total of 17 samples. For subsequent cycles, PK sampling was done less frequently (3 samples: pre-dose, 5 min before and 1 h after the end of each infusion). In patients experiencing hypertensive episodes, additional PK sampling was to be done 1–5 min before stopping the infusion. Urine samples were collected at pre-dose and for 72 h after the start of the infusion in Cycle 1. Analytes were quantitated using a validated liquid chromatography-tandem mass spectrometry technique (see Supplement A: Bioanalytical Method). Individual PK parameters of clearance (CL), volume of distribution of the terminal phase (Vz), half-life (t½) and area under the time-concentration curve (AUC) were determined by non-compartmental analysis using WinNonlin software (version 3.1, Pharsight Inc., Mountain View, CA, USA). Cmax values were derived from the plasma concentration-time profiles. The elimination rate constant, λ, was estimated by linear regression. AUC was calculated using the linear/logarithmic trapezoidal rule.
Normal skin biopsies (3–4 mm punch) were taken from the upper arm or other hairless area for evaluation of histone H3 phosphorylation at baseline and before the end of infusion. All samples were fixed immediately in 10% buffered formalin solution. (see Supplement B: IHC Method)
All patients who received at least one dose were considered assessable for safety and efficacy. Safety data were summarized using appropriate descriptive statistics.
Patient characteristics are summarized in Table 1. A total of fifty-seven patients were enrolled and 56 treated between July 2004 and February 2008. Colorectal cancer (CRC) was the most frequent cancer type (12 and 7 patients in the danusertib and danusertib + G-CSF groups, respectively). All patients had received prior systemic therapy, except for one patient with chondrosarcoma. More than one third had also received radiotherapy.
As shown in Table 2, the initial dose escalation over 7 DLs (45, 90, 180, 360, 500, 580, 650 mg/m2) was without G-CSF. Dose escalation through the 500 mg/m2 DL was well-tolerated. At the 650 mg/m2 DL (30% increment) 2 patients developed DLTs. Based on pharmacokinetics analysis (30% coefficient of variability in Cmax and AUC) we evaluated an intermediate DL of 580 mg/m2; there were 2 DLTs at this DL. Thus, 500 mg/m2 is the danusertib MTD without G-CSF. Based on DLTs of neutropenia we then amended the protocol to continue dose escalation with G-CSF primary prophylaxis. Sixteen patients were treated at 3 DLs with G-CSF (580, 750 and 1000 mg/m2). We did not identify a per protocol MTD with G-CSF but halted dose escalation and declared 750 mg/m2 the MTD based on 2 patients at the 1000 mg/m2 DL with elevated creatinine (azotemia) requiring a several day hospitalization for management with IV fluids. Forty patients treated with danusertib alone received a total of 159 cycles. A total of 66 cycles were administered to the 16 patients treated with danusertib with G-CSF (Table 2). The median number of cycles per patient was 3 with either treatment regimen (range 1–20 cycles for the danusertib only group and 1–12 cycles for the danusertib with G-CSF group). The median treatment duration was approximately 6 weeks in both groups (danusertib: 6.0 weeks [range 1.0–48.9]; danusertib with G-CSF: 6.1 weeks [range 0.9–24.0].
The side effects of danusertib and the number of patients experiencing various grades of treatment-related toxicity (all cycles) are summarized by dose level and maximum CTC grade in Table 3. Toxicities were primarily hematological with febrile neutropenia as the Cycle 1 dose limiting toxicity in the group treated without G-CSF. Treatment emergent Grade 3–4 hematological toxicities occurred at all dose levels tested, with greater frequency at doses ≥360 mg/m2. In Cycle 1, median time to neutropenic nadir was 8 days with both regimens. Median time to recovery was shorter when danusertib was administered with G-CSF (3 days versus 7 days without G-CSF). The most frequent non-hematological adverse events in both groups were fatigue, anorexia, nausea, and vomiting, diarrhea and pyrexia. Most of these adverse events were reported at doses ≥360 mg/m2 (Table 3A). Additional drug-related Grade 3–4 events were zoster with neutropenia (1 case); rigors with neutropenia (1 case); mucositis (2 cases), increased liver function tests and hypokalemia (1 case each).
Transient hypertensive episodes (defined as SBP > 150, or DBP > 100, or increase by 20 mmHg from pre-infusion values) were observed in 20% of pts receiving danusertib only (1 case each at 90, 180, and 500; 3 cases at 360 mg/m2 and 2 cases at 580 mg/m2) and in 19% of those receiving danusertib + G-CSF (1 case at each DL). Hypertensive episodes were observed mostly during Cycle 1, within 12 hours after the start of the infusion; were mild to moderate in severity, not dose-limiting and did not recur with repeated treatment. Hypertension was reported as a clinical AE (not related to study drug) in 1 patient treated at 580 mg/m2 with G-CSF. Six of the 12 patients experiencing hypertension were already on anti-hypertensive medication. We cannot exclude a role for danusertib in these hypertensive episodes as the drug does inhibit VEGFR in a submicromolar range in biochemical assays.
LVEF decreases to values below the lower limit of normal were detected by MUGA scan in 2 cases treated with danusertib alone at the 45 (−8%) and 360 mg/m2 DLs (−9%). LVEF reductions were asymptomatic and most recovered during or after treatment without specific intervention. Decreases of ≥15% were not observed.
DLTs are summarized in Table 2. Based on the observed DLTs, the 500 mg/m2 DL is the MTD for the 24-h infusion without G-CSF. In the danusertib with G-CSF group, a first cycle DLT (febrile neutropenia) was reported in only 1 of 7 patients treated at 1000 mg/m2 DL.
Drug-related SAEs occurred in 12 patients (21%): 9 (22%) treated with danusertib without G-CSF (in 2, 3, and 4 cases at 500, 580, or 650 mg/m2, respectively) and in 3 patients (19%) receiving 1000 mg/m2 with G-CSF. In patients treated without G-CSF, SAEs were mainly hematological (febrile neutropenia, neutropenia, neutropenic infection, leucopenia, and anemia). Non-hematological drug-related SAEs included abnormal liver function tests, vomiting, rigors, pyrexia (1 case each). In patients receiving danusertib with G-CSF drug-related non-hematological SAEs included anorexia, nausea, renal failure, azotemia and pyrexia (1 case each).
Reasons for study discontinuation were disease progression (73% and 88% without and with G-CSF, respectively) or AEs (17% and 13% without and with G-CSF, respectively). One patient was never treated. The remaining patients completed treatment per protocol Dose reductions included 2 patients with grade 1 and 2 creatinine increases requiring medical treatment (IV hydration) at 1000 mg/m2 with G-CSF.
Danusertib pharmacokinetics parameters are summarized in Table 4. Mean ± SD plasma levels of the compound and its major metabolite at the MTD doses with and without G-CSF are presented in Figure 1, panel B and C, respectively. The major metabolite observed in PK studies is the N-oxide of the N-methyl piperazine moiety; this has less than 1% of the activity of the parent compound in the proliferation assays and potency about 10 times lower than that of danusertib in a A2780 xenograft mouse model. After reaching maximal plasma concentration, danusertib showed a polyexponential decline. The pharmacokinetics of danusertib are characterized by high volume of distribution (average 13 L/kg) and low-to-moderate plasma clearance (range 0.33–0.46 L/hour/kg). Renal clearance accounted for a small proportion of plasma clearance (about 10%). No differences in half-life were seen across doses (average 18–26 hours). Plasma levels of the metabolite declined in parallel with those of the parent compound. The metabolite to parent AUC ratio was similar across doses and approximately equal to 1. Plasma concentrations of danusertib and its metabolite observed at selected time points in subsequent cycles were similar to those observed in Cycle 1. Systemic exposure to danusertib increased with dose (Figure 1 panel D). Administration of G-CSF did not influence the pharmacokinetics of danusertib and its metabolite (Table 4).
Pre- and on-treatment skin biopsies were analyzed for changes in histone H3 phosphorylation (pH3) by Western Blot (WB) and immunohistochemistry (IHC) (Supplement B, Figure 2). By WB most samples were not evaluable due to protein degradation; however, among evaluable samples a decrease in pH3 was observed in biopsies starting at 500 mg/m2. This finding was confirmed by IHC evaluations. Although the number of pH3 positive cells detected was low, a trend towards down-modulation of pH3 was seen starting at 500 mg/m2.
Objective tumor response was observed in one patient with refractory small cell lung cancer receiving 1000 mg/m2 danusertib + G-CSF (subsequently reduced to 750 mg/m2 for grade 2 creatinine elevation). Disease stabilization was observed in 18 of 42 evaluable pts (45.2%) (danusertib: 12 pts, 43%; danusertib + G-CSF: 7 pts, 50%). Prolonged disease stabilization (23.9–52.3 weeks) was observed in 4 patients (2 colorectal cancer, 1 ovarian cancer, 1 breast cancer) treated without G-CSF at 180, 500 (2 patients), and 650 mg/m2. In the patient with ovarian cancer tumor masses decreased 27% (RECIST) with a ≥30% decrease in CA125.
We report the results of a phase I trial of danusertib (PHA-739358), a novel pan-AK inhibitor, in patients with advanced refractory solid tumors. Given their key roles in regulation of mitotic processes during cell division (5, 17, 18) and over-expression in malignancy, AKs have emerged as a new target for anticancer therapy. Several small molecule AK inhibitors are under development and have entered clinical development(11, 15, 19–27). Danusertib has inhibitory activity against Aurora A and B, with potent preclinical antitumor activity, a favorable non-clinical safety profile as well as potentially useful effects on other tyrosine kinases such as bcr-abl(16, 28, 29).
The dosing schedule tested was selected based on preclinical toxicity studies in rats and dogs. In these species a weekly schedule of danusertib showed acceptable toxicity with the major target organs being bone marrow and gastrointestinal (GI) tract. When danusertib was given at very high doses and/or with infusion times shorter than 1h, cardiovascular and renal toxicity were seen in addition to bone marrow and GI toxicity. Sudden death also occurred in animals given bolus infusions. The 24-h infusion duration was selected for the present study to reduce toxicities thought to be related to Cmax. Danusertib doses of ~420–750 mg/m2 were predicted in humans to be required to achieve a daily AUC of ~50 µM•h, the systemic exposure suggested by preclinical pharmacokinetic/pharmacodynamic modeling to be necessary for tumor regression.
The safety profile of danusertib in this clinical study was completely consistent with that expected based on preclinical testing. Danusertib toxicities were primarily hematological with febrile neutropenia as the DLT, and 500 mg/m2 as the MTD without G-CSF. With G-CSF support the RP2D for danusertib when administered as 24-h IV infusion is 750 mg/m2. Doses >1000 mg/m2 DL were not evaluated because of the emergence in 2 patients treated at 1,000 mg/m2 of clinically significant grade 1 and 2 creatinine increases requiring hospitalization for IV fluids and restoration of baseline kidney function. In a rat model, tubular nephropathy was observed at the 600 and 750 mg/m2 IV bolus equivalent doses. In repeated dose studies with 6-h IV infusions, increases in urea and creatinine were observed in rats (but not in dogs) at the MTD of 720 mg/m2 and were reversible. A satisfactory mechanistic explanation of these effects has not yet been found.
In the range of doses investigated (45 to 1000 mg/m2), the systemic exposure to danusertib increased with dose. Danusertib was characterized by an extensive volume of distribution and low-moderate plasma clearance. Renal clearance accounted for about 10% of total clearance. Pharmacokinetic evaluations showed that co-administration of G-CSF did not influence the pharmacokinetics of danusertib and its metabolite. There was limited inter-and intra-patient variability in PK. Analysis of pH3 in post-treatment skin biopsies demonstrated target modulation at doses ≥500 mg/m2.
In conclusion, danusertib can be safely administered to patients with advanced refractory solid tumors. Danusertib 500 mg/m2 given over 24 hours in 14-day cycles without G-CSF is the RP2D for solid tumors. Danusertib 750 mg/m2 is the RP2D with G-CSF. A prolonged objective response in SCLC and multiple instances of prolonged disease stabilization provide ample justification for further studies of danusertib alone and in combination with other agents. Phase 2 and 3 single agent studies without G-CSF are underway in 7 types of solid tumors. In addition, clinical studies with G-CSF are being considered.
We wish to thank the patients who participated in this study and their referring doctors. In addition we acknowledge the skilled contributions of the study coordinators and data managers as well as P. Carpinelli (NMS, WB assays), C. Pellizzoni (Accelera, PK reports), A. Petroccione (NMS, Biostatistics), D. Zanchetta (NMS, Data Management) and Nancy Malinowski (editorial assistance). The work was supported in part by National Cancer Institute grant P30 CA006927 to Fox Chase Cancer Center.
Study presented in part at the 2006 AACR-NCI-EORTC Annual Meeting (Abs. 343) and at the 2008 ASCO Annual Meeting (Abs. 2520)