Phase 1 study of the MDM2 inhibitor AMG 232 in patients with advanced P53 wild-type solid tumors or multiple myeloma

Summary Background This open-label, first-in-human, phase 1 study evaluated AMG 232, an oral selective MDM2 inhibitor in patients with TP53 wild-type (P53WT), advanced solid tumors or multiple myeloma (MM). Methods In the dose escalation (n = 39), patients with P53WT refractory solid tumors enrolled to receive once-daily AMG 232 (15, 30, 60, 120, 240, 480, and 960 mg) for seven days every 3 weeks (Q3W). In the dose expansion (n = 68), patients with MDM2-amplified (well-differentiated and de-differentiated liposarcomas [WDLPS and DDLPS], glioblastoma multiforme [GBM], or other solid tumors [OST]), MDM2-overexpressing ER+ breast cancer (BC), or MM received AMG 232 at the maximum tolerated dose (MTD). Safety, pharmacokinetics, pharmacodynamics, and efficacy were assessed. Results AMG 232 had acceptable safety up to up to 240 mg. Three patients had dose-limiting toxicities of thrombocytopenia (n = 2) and neutropenia (n = 1). Due to these and other delayed cytopenias, AMG 232 240 mg Q3W was determined as the highest tolerable dose assessed in the dose expansion. Adverse events were typically mild/moderate and included diarrhea, nausea, vomiting, fatigue, decreased appetite, and anemia. AMG 232 plasma concentrations increased dose proportionally. Increases in serum macrophage inhibitor cytokine-1 from baseline were generally dose dependent, indicating p53 pathway activation. Per local review, there were no responses. Stable disease (durability in months) was observed in patients with WDLPS (3.9), OST (3.3), DDLPS (2.0), GBM (1.8), and BC (1.4–2.0). Conclusions In patients with P53WT advanced solid tumors or MM, AMG 232 showed acceptable safety and dose-proportional pharmacokinetics, and stable disease was observed.

Although many tumors harbor non-targetable mutations in TP53, MDM2 has become an attractive therapeutic target in the treatment of TP53 wild-type (P53WT) cancers. Several MDM2 inhibitors are in clinical investigation as monotherapy or combined with other therapies for the treatment of P53WT hematologic malignancies and solid tumors [15][16][17][18]. In clinical studies of MDM2 inhibitors, increases in circulating macrophage inhibitor cytokine-1 (MIC-1) has been used as a pharmacodynamic marker of p53 activation [19][20][21].
AMG 232 is an investigational oral, selective MDM2 inhibitor that restores p53 tumor suppression by blocking the MDM2-p53 interaction with picomolar affinity [22]. In tumor xenograft models, treatment with AMG 232 resulted in tumor growth inhibition and caused regression of MDM2-amplified tumors through the induction of growth arrest and apoptosis [23]. The primary objectives of this open-label, first-in-human, phase 1 study were to assess the safety and tolerability, pharmacokinetics, maximum tolerated dose (MTD), pharmacodynamics, and efficacy of AMG 232 in patients with P53WT solid tumors or multiple myeloma.

Patients
Patients aged ≥18 years with pathologically-documented, P53WT (per next-generation sequencing) treatmentrefractory solid tumors measurable per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 or Macdonald criteria for glioblastoma multiforme (GBM), or progressive multiple myeloma measurable per International Myeloma Working Group (IMWG) response criteria were eligible. Additional eligibility criteria were Eastern Cooperative Oncology Group (ECOG) performance status ≤2; life expectancy >3 months; adequate hematologic (ANC ≥1.5 × 10 9 /L for solid tumors or ≥ 1.0 × 10 9 /L for multiple myeloma; platelet count ≥100 × 10 9 /L for solid tumors or ≥ 75 × 10 9 /L for multiple myeloma; hemoglobin >9 g/dL), renal (estimated glomerular filtration rate ≥ 45 mL/min/ 1.73 m 2 ), hepatic (AST and AST <2.5 × ULN; ALP <2.0 × ULN; total bilirubin <1.5 × ULN), and coagulation (prothrombin time or partial thromboplastin time < 1.5 × ULN) functions. In the dose expansion, five tumor t y p e s w e r e d e f i n e d : w e l l -d i f f e r e n t i a t e d o r dedifferentiated liposarcoma; relapsed GBM with MDM2 amplification; estrogen receptor positive (ER+) breast cancer refractory to hormonal treatments; relapsed multiple myeloma progressive after ≥1 prior treatment; other advanced solid tumors with MDM2 amplification. Key exclusion criteria included active or untreated brain metastases; unresolved toxicity from prior anticancer therapy, excluding alopecia; antitumor therapy or major surgery within 28 days of starting study treatment; investigational device or drug within 30 days or 5 halflives of starting study treatment; liposarcomas with >3 prior approved therapies; multiple myeloma with del (17p) or IgM subtype, non-secretory or hyposecretory disease, lack of ≥25% reduction in M-protein for ≥6 weeks with prior therapy, corticosteroid therapy within 3 weeks of study, POEMS syndrome, or plasma cell leukemia or lymphoplasmacytic lymphoma. Institutional review board approval was obtained for all study procedures. All patients provided informed consent before enrollment.

Study design and treatment
This open-label phase 1 study was conducted at 16 centers (ClinicalTrials.gov, NCT01723020). TP53 mutation status was confirmed by central laboratory assessment. The study was planned with two parts: a 3-part dose escalation (Part 1) and a dose expansion (Part 2). In the dose escalation, multiple-patient cohorts were planned to enroll sequentially (Part 1A) or in parallel (Parts 1B and 1C; 3 + 3 design) and to receive AMG 232 once daily (QD) for 3 days (Part 1B) or 7 days (Part 1C) every 3 weeks (Q3W) at prespecified doses of 15, 30, 60, 120, 240, 480, and 960 mg. Intermediate doses were allowed when deemed appropriate. The dose e x p a n s i o n w a s p l a n n e d f o r p a t i e n t s w i t h MDM2-amplified tumors (group 1; liposarcomas, GBM, a n d o t h e r s o l i d t u m o r s ) , p o t e n t i a l l y M D M 2 -overexpressing tumors (group 2; ER+ metastatic breast cancer), or multiple myeloma (group 3).
Each patient was monitored for 21 days for the occurrence of dose-limiting toxicities (DLTs), defined as febrile neutropenia, neutropenic infection, grade 4 neutropenia lasting >7 days, grade ≥ 3 thrombocytopenia lasting >7 days (solid tumors only), grade 3 thrombocytopenia with grade ≥ 2 bleeding (solid tumors only), grade 3 or 4 thrombocytopenia with grade > 1 bleeding (multiple myeloma only), grade 4 thrombocytopenia (solid tumors only), or grade 4 thrombocytopenia lasting >14 days (multiple myeloma only), or as grade ≥ 3 nausea, vomiting, or diarrhea after support; grade 3 fatigue lasting >7 days; any other grade ≥ 3 adverse event (AE); grade ≥ 3 kidney injury (multiple myeloma only), or treatment-related AEs not returning to grade ≤ 1 (solid tumors only) per Common Terminology Criteria for Adverse Events (CTCAE), version 4.0. The MTD was defined as the maximum dose, at which the probability of a DLT was ≤25% in Part 1A and ≤ 33% in Parts 1B and 1C. Treatment continued until disease progression, intolerable toxicity, or withdrawal of consent.

Study assessments
Safety AEs were recorded for all enrolled patients.
Pharmacokinetics Plasma samples for the measurement of AMG 232 pharmacokinetics in Parts 1 and 2 were collected predose and at 1, 3, 5, and 7 h postdose on days 1 and 7 and 24 and 72 h postdose from day 7 of cycle 1; predose on days 1 and 7 of cycle 2; and at the end of study. Plasma AMG 232 levels were measured using a validated high performance liquid chromatography mass spectrometry method [24]. Pharmacokinetic and exposure parameters were estimated, including terminal half-life (t max ), maximum observed plasma concentration (C max ), area under the concentration-versus-time curve at 24 h (AUC 24h ), volume of distribution (V z /F), terminal elimination half-life (t 1/2,z ), and clearance (CL/F). Non-compartmental analysis was performed using WinNonlin Professional software, version 6.3. Parameters were summarized descriptively.
Circulating MIC-1 In Parts 1 and 2, serum samples for the assessment of circulating macrophage inhibitor cytokine-1 (MIC-1) were collected on the pharmacokinetic sample schedule. Serum MIC-1 concentrations were measured using a validated ELISA (human GDF-15 Quantikine®, R&D Systems Inc.).
Efficacy Efficacy response was assessed using revised Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1 [25] Macdonald criteria for GBM [26] or International Myeloma Working Group (IMWG) response criteria for multiple myeloma [27].

Statistical analysis
Primary endpoints were the patient incidence of AEs, DLTs, and clinically significant changes in safety assessments; AMG 232 and pharmacokinetic parameters; and the MTD in Part 1. Secondary/exploratory endpoints included tumor response and change in serum MIC-1 level. Data were summarized descriptively. Qualified researchers may request data from Amgen clinical studies. Complete details are available at the following: http://www.amgen.com/datasharing.

DLTs and MTD
Three patients in the dose escalation had DLTs. The first patient (120-mg cohort) with esophageal cancer had a DLT consisting of grade 3 thrombocytopenia on day 15 that worsened to grade 4 on day 17, lasted seven days, and required a platelet transfusion. The second patient (360-mg cohort) with rectal cancer had a DLT consisting of grade 4 thrombocytopenia on day 28. The third patient (480-mg cohort) with head and neck cancer had grade 3 neutropenia on day 22 that delayed treatment in the next cycle and was therefore considered a DLT. Two additional patients in the 300mg cohort had cytopenias (grade 4 neutropenia; grade 4 thrombocytopenia) outside of the 21-day DLT evaluation window that were considered in the dose escalation decisions. Based on the protocol-specified definition using DLTs incidence, the MTD for AMG 232 for 7 days Q3W was not reached. However, when the DLTs and delayed cytopenias were considered, the highest safe and tolerable dose of AMG 232 was 240 mg, which was the dose evaluated in the dose expansion.
Serious AEs occurred in 14 (36%) patients during the dose escalation, including six (15%) whose serious AEs were considered treatment-related and predominantly included gastrointestinal toxicity (Table 2). Overall, eight patients in the dose escalation had AEs resulting in treatment discontinuation: thrombocytopenia (n = 5), neutropenia (n = 2), and febrile neutropenia (n = 1). Four patients in the dose escalation had fatal AEs of disease progression while on study.

Pharmacokinetics of AMG 232
Plasma samples for the evaluation of AMG 232 pharmacokinetics were available for 106 patients. AMG 232 pharmacokinetic profiles for the dose escalation and dose expansion are shown in Fig. 1. Plasma  concentrations of AMG 232 increased dose proportionally. The mean AUC accumulation ratio between days 1 and day 7 across all dosing groups in the dose escalation and in the dose expansion was less than 2-fold with the once-daily dosing regimen (Table 4, Table 5). The mean estimated apparent volume of distribution was 615 L across all dosing cohorts. With oral administration, the estimated mean apparent clearance of AMG 232 was 30.2 L/h across groups and varied among individuals.

AMG 232 pharmacodynamic effects
Thirty-nine patients in the dose escalation had available pre-treatment and post-treatment blood samples for the assessment of serum MIC-1; serum MIC-was not assessed in the dose expansion. From baseline to day 15, increases in serum MIC-1 (post-treatment to pretreatment ratios) were generally dose-dependent (Fig. 2). Mean serum MIC-1 ratios increased up to day 7 and decreased until cycle 2, suggesting that MIC-1 changes were dependent on AMG 232 exposure.

Efficacy
Imaging for the local evaluation of tumor response was available for 38 of 39 patients in the dose escalation and 60 of 68 patients in the dose expansion. One patient in the dose escalation had no postbaseline imaging due to an AE. Eight patients in the dose expansion had no postbaseline imaging due to clinical disease progression (n = 3), AEs (n = 3), and patient request (n = 2). By local evaluation, no objective responses were observed. Per central evaluation, three patients (4%) with WDLPS,     AR, accumulation ratio (AUC 24h cycle 1, day 1 / AUC 24h cycle 1, day 7 ); AUC 24h , area under the concentration-versus-time curve at 24 h; CL/F, clearance; C max , maximum observed serum concentration; t 1/2,z , terminal elimination half-life; t max , time to reach C max ; V z /F, volume of distribution a All data are mean (SD) except for t max , which is median (range)   of 16 with other solid tumors, 10 of 12 with breast cancer, and 5 of 10 with multiple myeloma. Duration of stable disease in the dose escalation and across histologies in the dose expansion is summarized in Fig. 4. The median duration of stable disease in the dose escalation was 3.1 months (range, 0.7-22.4 mo). In the dose expansion, the overall median duration of stable disease was 2.0 months (range, 0.5-12.9) overall, 3.9 months (range 1.9-6.1) among patients with WDLPS, 2.0 months (0.9-6.9) among patients with DDLPS, 1.8 months (0.5-11.9) among patients with GBM, 3.3 months (0.9-12.9) among patients with other solid tumors, 2.0 months (0.9-4.0) among patients with ER + PR+ breast cancer, and 1.4 months (0.9-3.8) among those with ER + PR-breast cancer.

Discussion
In this first-in-human study, AMG 232 was generally well tolerated up to 240 mg. AMG 232 doses up to 480 mg QD for seven days in a 21-day cycle were assessed; however, the MTD was not reached. Three patients had DLTs, two with thrombocytopenia and one with neutropenia. Based on these DLTs and other cytopenias outside of the DLT evaluation window, AMG 232 240 mg was selected as the highest safe and tolerable dose for evaluation in the dose expansion. In both phases of the study, AMG 232-related AEs were generally mild to moderate, with the most frequently occurring AEs being diarrhea, nausea, vomiting, fatigue, decreased appetite, and anemia. Among the AEs resulting in treatment discontinuation, thrombocytopenia, neutropenia, and gastrointestinal toxicity were the most frequent. These results are consistent with other clinical studies of MDM2 inhibitors, in which myelosuppression and gastrointestinal toxicity have been reported [18,19,[28][29][30][31][32][33]. In this study, five patients died due to disease progression while on the study.
Following QD treatment for seven days in a 21day cycle, AMG 232 plasma concentrations increased generally dose proportionally, and across all patients, the mean AUC accumulation ratio between days 1 and 7 was less than 2-fold. Overall, AMG 232 exhibited an acceptable pharmacokinetic profile in this population. Furthermore, dose-dependent increases in serum MIC-1 levels from baseline to day 15 of treatment indicated p53 pathway activation, consistent with previous studies of MDM2 inhibitors [19][20][21].
In conclusion, AMG 232 showed acceptable safety, doseproportional pharmacokinetics, and on-target activity as