A Phase I Study of the SMAC-Mimetic Birinapant in Adults with Refractory Solid Tumors or Lymphoma

Inhibitors of apoptosis (IAP) proteins, such as X-linked IAP (XIAP), cellular IAPs (cIAP1 and cIAP2), and melanoma IAP (ML-IAP), suppress apoptosis and activate a tumor necrosis factor (TNF)-dependent prosurvival NF-κB pathway that is important in tumor cells and infectious diseases (1–6). IAP genes that encode these proteins are amplified in many tumors and overexpression of cIAP1 and cIAP2 can suppress apoptosis of a variety of tumor cells (1). Also, overexpression of IAP genes has been found to contribute to tumor resistance to conventional cancer chemotherapeutic agents (5, 7, 8). The antiapoptotic function of IAPs and their overexpression in many cancers make them attractive antitumor therapeutic targets.

IAP proteins are antagonized by another protein called second mitochondrial-derived activator of caspase (SMAC). SMAC binds to IAP proteins via its N-terminal AVPI sequence of its processed form, which results in the autoubiquitylation and subsequent degradation of IAP proteins (9–12). Thus, SMAC-induced degradation of IAP proteins suppresses TNF-dependent NF-κB activation and allows apoptosis to occur in tumor cells (13, 14). This has stimulated development of SMAC mimetics that bind strongly to tumor-expressed IAP proteins, disrupt their function, and stimulate tumor cell death (12, 15–17).

Birinapant (TL32711) is a bivalent SMAC mimetic that displays preferential binding to cIAP1 relative to cIAP2 and XIAP (12, 15, 18). In preclinical studies, birinapant demonstrated potent single-agent antitumor activity while sparing normal cells (15, 18, 19). In addition, birinapant showed significant activity in preclinical models of ovarian, colorectal, head and neck squamous cell carcinoma, leukemia, and melanoma (18–21). Finally, birinapant demonstrated synergistic preclinical antitumor activity with multiple chemotherapies, such as irinotecan, gemcitabine, and azacitidine (18).

Here, we report the first-in-human phase I, open-label, nonrandomized, dose-escalation study with birinapant in patients with advanced solid tumors or lymphoma. The aims of this study were to assess the safety and tolerability of birinapant as a single agent in patients with advanced cancer, to investigate the pharmacodynamic (PD) and pharmacokinetic (PK) properties of birinapant, and to assess the preliminary efficacy of single-agent birinapant in these patients.

Patients were ≥18 years with advanced treatment-refractory malignancies (or for which no effective therapy existed). Included patients had measurable disease according to Response Evaluation Criteria in Solid Tumors (RECIST; version 1.1) or Revised Response Criteria for Malignant Lymphoma, were Eastern Cooperative Oncology Group (ECOG) performance status (PS) ≤2, and had adequate renal [≤1.5× upper limit of normal (ULN)], hepatic (≤3× ULN) and bone marrow function [defined as absolute neutrophil (ANC) ≥1,500/mm³ (≥1.5 × 10⁶/L), platelet count ≥75,000/mm³ (≥75 × 10⁶/L), and hemoglobin ≥10 g/dL (in the absence of transfusion within 24 hours prior to dosing)]. Patients were ineligible if they had a history of autoimmune disease within the previous 5 years, or any immunosuppressive therapy 4 weeks prior to study. The study protocol was approved by Institutional Review Boards and registered at www.clinicaltrials.gov (NCT00993239); all patients gave informed consent.

Patients received birinapant by intravenous infusion more than 30 minutes once weekly for 3 weeks of each 4-week dosing cycle. The doses of birinapant administered to patients ranged from 0.18 to 63 mg/m².

Patients were enrolled in cohorts of 3 to 6 following a standard “3+3” phase I dose escalation design (22). The starting dose of 0.18 mg/m² was chosen as this dose level corresponded to one-sixth the highest nonseverely toxic dose (HNSTD) in dog, the most sensitive species (23). Dose escalation used a modified Fibonacci sequence; intracohort doses were increased by 100% if no dose-limiting toxicity (DLT) were observed in the first cycle, and were increased either 50 or 33% if a drug-related DLT of National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE; version 4) grade 2 AE occurred, depending on the event type and grade. An AE was considered dose limiting if it met one of the following criteria: grade 4 neutropenia (ANC < 500/mm³) >7 days; grade 3 or 4 neutropenia with fever; grade 4 thrombocytopenia (<25,000/mm³); grade ≥3 nausea and vomiting despite maximal anti-emetic treatment; grade ≥3 diarrhea despite maximal antidiarrheal treatment; or any other nonhematologic or laboratory grade ≥3 AE, with the exception of transient grade 3 hypotension. Owing to expectations based upon the mechanism of action of birinapant, which may cause an increase in serum cytokines and subsequent transient hypotension, if hypotension could be resolved with administration of ≤3 L of intravenous fluids within the 8-hour period of outpatient observation after drug infusion, then it was not considered a DLT.

Patients who experienced a DLT during the first treatment cycle discontinued study treatment but remained in the study for follow-up. Patients who experienced a clinically significant AE that was not considered dose limiting, and recovered within 28 days with standard supportive care, could be re-treated per the protocol treatment schedule.

Patients who experienced a clinically significant hematologic toxicity or nonhematologic toxicity determined to be study drug-related (definitely, probably, or possibly related) to birinapant were able to continue in the study with either the current dose or with a dose reduction for subsequent cycles, depending on the severity, nature of, and duration of the AE. Birinapant was delayed for a maximum of 14 consecutive days before being restarted.

A dose expansion cohort of 20 patients was planned at or below the maximum tolerated dose (MTD), which was determined upon completion of the first treatment cycle.

The tolerability of birinapant was assessed using criteria of NCI CTCAE (version 4). Patients were evaluable for safety if they signed informed consent and received at least one dose of birinapant.

Cardiac safety was assessed by serial electrocardiograms (ECG) and left ventricular ejection fraction (LVEF) measurement. ECGs were obtained during cycle 1 dosing and at study completion. LVEF was assessed by echocardiogram or MUGA scan within 30 days of the initial dose and during week 4 of the second treatment cycle. Vital signs were assessed within 30 days of screening, prior to each dose, and at week 4.

Disease status was assessed within 30 days of the first dose and at the end of every 2 cycles using the RECIST version 1.1 (24) or revised response criteria for malignant lymphoma (25). Patients were evaluable for efficacy if they completed 2 cycles of birinapant treatment and underwent radiographic assessment. The number of patients that met criteria for safety and efficacy assessments is detailed in Supplementary Table S1.

Blood and peripheral blood mononuclear cell (PBMC) samples were collected for PK studies at baseline and during the first birinapant treatment cycle. Optional tumor biopsies were obtained at baseline (pretreatment) and during and upon completion of 2 cycles of birinapant treatment.

Birinapant concentrations in biopsy samples and in plasma were analyzed by liquid chromatography/tandem mass spectrometry (LC/MS-MS) using methods developed by TetraLogic Pharmaceuticals, Inc.

During the first cycle of birinapant treatment, the following PK parameters were calculated using noncompartmental analysis (WinNonlin Version 5.2; Pharsight): maximum plasma concentration (C_max), time to C_max (T_max), area under the concentration–time curve from time zero to the last quantifiable time point (AUC_0−t) (day 1 and day 15), AUC extrapolated from time zero to infinity (AUC_0−∞) day 1, and elimination half-life (t_1/2), clearance (CL), and apparent volume of distribution (V_ss). Only plasma concentrations with greater than or equal to the limit of quantification (LOQ) of the assay were used for PK determination. Peak (end of infusion) and trough plasma birinapant concentrations were determined on day 8.

Drug accumulation [week 3/week 1 AUC ratio (R)] and dose proportionality were assessed using an appropriate power model (26). In PBMC and plasma sample(s) collected at the time of posttreatment optional biopsy collection, birinapant concentration was assessed.

For evaluation of potential drug metabolites and the amount of drug excreted within a 24-hour period (Ae%), a predose urine sample and a 24-hour urine sample were collected prior to birinapant treatment from patients who enrolled in the expansion cohort.

Biologic response to birinapant was evaluated using cellular markers such as cIAP1, cIAP2, pro–caspase-8, and poly(ADP-ribose) polymerase (PARP) that were associated with birinapant mechanism of action in preclinical studies (18).

Blood samples for cytokeratin-18 (CK-18), caspase-3/7, and multicytokine analysis were collected at baseline, 4 hours after birinapant treatment on days 1, 8, and 15 of cycle 1, and 24 hours after dosing on C1D1 and C1D15. CK-18 was assayed using M30-Apoptosense Peviva AB (Bromma) and M65 enzyme-linked Peviva AB (Bromma) immunosorbent assays. Caspase-3/7 was measured using Caspase-Glo 3/7 (Promega). Multicytokine analysis was performed by Rules-Based Medicine, Inc.

Varicella-zoster virus (VZV) and herpes simplex virus (HSV) DNA were evaluated as previously described (27, 28).

Data collected throughout the study were analyzed using descriptive nonparametric statistics. For categorical data, the number and percentage of patients are presented.

Between December 2009 and August 2012, 50 patients were enrolled at Fox Chase Cancer Center (Philadelphia, (PA), Roswell Park Cancer Institute (Buffalo, NY), and Abramson Cancer Center, University of Pennsylvania (Philadelphia, PA). The demographics of the patients enrolled in the study are presented in Table 1.

The median age of the patients was 59 years (range, 31–85). Fifty-six percent of the patients were male. Tumor types evaluated in the study were colorectal (15), head and neck (7), soft-tissue sarcomas (5), lung (4), pancreatic (4), melanoma (4), and miscellaneous other tumors (11). A majority of patients had received prior systemic cancer treatments (92%) and had an ECOG PS of 1 (64%) (Table 1).

Preclinical IND-enabling animal toxicity studies suggested that 0.18 mg/m² would be a tolerable initial dose for the first birinapant treatment cycle. Further, birinapant at ≥5.6 mg/m² was predicted to be potentially therapeutically active. Patients were enrolled to 1 of 12 dosing cohorts with doses that ranged from 0.18 to 63 mg/m² (Table 2). No DLTs were observed in any patient after 4 weeks of birinapant treatment enrolled to the first 10 cohorts of 0.18 to 35 mg/m². At 47 mg/m² (cohort 11), 1 of 6 patients developed grade 3 hypophosphatemia and a macular rash, which were considered DLTs. In the 63-mg/m² treatment group, 2 of 3 patients experienced a drug-related DLT. One patient experienced headache, nausea, and vomiting (all grade 3). This patient developed Bell's palsy (grade 2), with an unknown duration (patient lost to follow-up). Another patient developed headache (grade 3) and Bell's palsy (grade 2), which resolved without treatment after 2 days. These events were deemed clinically significant, and birinapant administered at 63 mg/m² was determined to have exceeded the MTD. Based on these results, 9 additional patients were enrolled in a 47-mg/m² expansion cohort. Of the 9 patients, only 1 exhibited a first-cycle DLT of asymptomatic elevations of amylase and lipase.

Fifty patients in the study were evaluable for safety. Table 3 shows related AEs that occurred >5% of patients receiving birinapant treatment. Thirty-three patients (66%) experienced at least one grade 1 or grade 2 AE, whereas only 18% experienced at least 1 grade 3 or grade 4 AE. The most common grade 1 or grade 2 AEs were fatigue, lymphopenia, vomiting, hypotension, nausea, headache, decreased appetite, and diarrhea (Table 3). The most common grade 3 or grade 4 AEs were fatigue, headache, lymphopenia, elevated blood alkaline phosphatase, decreased appetite, dyspnea, pain in extremities, and aspiration pneumonia.

Serious adverse events (SAE) related to birinapant were observed only in patients treated with birinapant administered at >35 mg/m². A presumptive clinical diagnosis of “cytokine release syndrome” characterized by a rash and fever in the absence of hypotension was reported for 1 patient (grade 1) treated with 35 mg/m² birinapant; 3 patients (2 grade 1; 1 grade 2) treated with 47 mg/m²; and 1 patient treated (grade 1) with 63 mg/m². “Cytokine release syndrome” was observed only during the first treatment cycle and did not recur with additional birinapant treatment. Although not considered a DLT, Bell's palsy was observed in 2 of 3 patients treated with 63 mg/m² (2 grade 2; 1 SAE). These results suggest that administration of birinapant at 47 mg/m² could be tolerated as a once-weekly treatment regimen for 3 weeks with the cycle repeated every 4 weeks.

The pharmacokinetics of birinapant administration was studied over the 0.18 to 63 mg/m² dose range. Birinapant exposure (C_max and AUC_0−∞) on days 1 and 15 increased in a dose-proportional manner following a 30-minute intravenous infusion (Table 4, Fig. 1). C_max values were generally coincident with the end of infusion (approximately 0.5 hours). Birinapant was eliminated from plasma in a triexponential manner with an estimated mean terminal t_1/2 of 19 to 47 hours on day 1. There was moderate variability in birinapant clearance (CL) with mean day 1 values of approximately 21.0 ± 7.4 L/h (35 CV%) at a dose of 47 mg/m² (n = 17; Table 4).

There was no accumulation in plasma with the weekly dosing regimen as reflected in the day 15/day 1 C_max and AUC_0−∞ accumulation ratios (1.12 ± 0.24 and 1.02 ± 0.22, respectively). The mean volume of distribution was high (approximately 710.4 ± 357.7 L at a dose of 47 mg/m²) on day 1, indicative of extensive tissue uptake. The urinary elimination of birinapant over 0 to 24 hours was low at 47 mg/m² [mean (SD) Ae%; 4.4% ± 3.4%].

Birinapant treatment resulted in a greater than 75% reduction in cIAP1 in PBMC samples at doses of 5.6 mg/m² and above. This was consistent with analysis of biopsies taken from three different tumors (Fig. 2A–C). Suppression of cIAP1 was observed 1.5 hours after treatment in tumor biopsies and after 4 hours in PBMC samples. Suppression of cIAP1 was sustained in PBMC and in tumor tissue biopsies for at least 7 days after birinapant treatment.

In one biopsy sample that was available for additional study, suppression of cIAP1 was associated with increases in the activated form of caspase-8, and a reduction in PARP levels consistent with PARP cleavage (Fig. 2B). This suggested activation of apoptotic pathways in the tumor following intravenous administration of birinapant.

CK-18 is an intermediate filament protein found in the cytoplasm of many epithelial cells. Full-length and caspase-cleaved CK-18 (detected as M65 and M30 antigens) represent circulating indicators of apoptosis. There was a drug effect in terms of increased levels of M30 and M65 following birinapant treatment, although a clear correlation with drug dose was not observed (Supplementary Table S2). The lack of a clear dose relationship was not surprising given the interindividual variation in these assays. However, for the three lowest dose cohorts, there was no increase in m30 or m65 with the first two doses of the drug (cycle 1, day 1; C1D1 and cycle 1, day 8; C1D8), implying that the lowest doses had little PD effect, whereas a dose as low as 1.44 mg/m² increased these levels.

The magnitude of increase in circulating M30 and M65 was modest following birinapant treatment. At birinapant doses of 47 mg/m², the median baseline M30 level was 174 U/L (range, 90–1583), whereas 24 hours after the third dose on day 15 (C1D15), the median M30 level was 270 U/L (86–2004). This reflected a maximum increase of approximately 1.5-fold. Similarly with M65, the median baseline level was 522 U/L (range, 190–7515), whereas on C1D15, 24 hours after dose, it was 719 (range, 308–8591).

There was also a suggestion that levels of both M30 and M65 increased with each cycle of drug. Even for the 3 lowest dose cohorts, which showed no clear increase in these markers with the first 2 doses of drug, there was an increase after the third dose of drug (Supplementary Table S2). This suggested a cumulative PD effect, in the absence of evidence of drug accumulation. This cumulative PD effect was further supported when pretreatment levels are compared. For example, M65 (Supplementary Table S3) levels at baseline were lowest, pretreatment levels were increased prior to the second dose, and further increased prior to the third dose. These conclusions were further supported by the results of the caspase-3/7 assays (Supplementary Table S4). Taken together, these results provided evidence of cell-death pathway activation in response to birinapant.

Levels of serum cytokines were measured following birinapant treatment from all 50 patients. Results of these analyses indicated that birinapant treatment ranging from 0.18 to 63 mg/m² did not significantly increase serum levels of TNF, monocyte chemoattractant protein-1 (MCP-1), or IL1 IL6, or IL8 (unpublished observations). However, 5 patients treated with 47 and 63 mg/m² of birinapant exhibited transient elevation of serum MCP-1 4 hours after treatment. MCP-1 returned to normal levels in all of these patients within 24 hours following birinapant administration. Interestingly, elevated cytokine levels were not detected in the serum of 5 patients who presented with presumptive “cytokine release syndrome.”

Twenty-six patients treated with birinapant were evaluable for clinical efficacy. No patient exhibited a complete response (CR) or partial response (PR) to birinapant treatment as best response. Stable disease was observed in 7 treated patients (28%), whereas progressive disease was identified in 18 patients (72%).

Of the 7 patients who exhibited stable disease, 3 experienced stable disease for more than 4 months, whereas 2 patients with non–small cell lung cancer (treated with 47 mg/m² of birinapant) and 1 patient with colorectal cancer (0.36 mg/m² of birinapant) experienced stable disease for 5 months. In addition, 1 patient with liposarcoma received 3 cycles of birinapant treatment (17.2 mg/m² for over 9 months). Finally, 2 patients with colorectal cancer treated with birinapant demonstrated radiographic evidence of tumor shrinkage.

The results of this first-in-human, open-label, dose escalation study of birinapant demonstrated that birinapant was generally well tolerated at doses ranging from 0.18 to 47 mg/m². The most common AEs included fatigue, lymphopenia, nausea and vomiting, hypotension, headache, decreased appetite, and diarrhea.

A majority of AEs observed with birinapant at doses of 18 to 35 mg/m² were grade 1, 2, or 3 in severity and generally clinically manageable, and no DLTs were seen at these doses. DLTs at doses higher than this included hypophospatemia, macular rash, and amylase and lipase elevation in patients treated with 47 mg/m² and headache, nausea, and reversible cytokine release syndrome in patients treated with 63 mg/m². Bell's palsy was also reported in 2 patients treated with 63 mg/m² of birinapant.

The etiology of Bell's palsy following administration of birinapant is unclear. Bell's palsy may result from reactivation of VZV or HSV through an unknown mechanism (27, 28). VZV DNA is sometimes present in the blood of patients who develop Bell's palsy and in some asymptomatic patients with altered immune function. However, neither HSV nor VZV DNA was detected in the blood from the 2 patients who developed Bell's palsy in this study.

Despite presumptive diagnosis by study site investigators of transient cytokine release syndrome in several patients, there was no detectable increase in circulating blood cytokine levels nor was there evidence of a systemic inflammatory response in birinapant-treated patients. However, it is important to point out that the so-called transient cytokine release syndrome was primarily observed in patients treated with 63 mg/m², which exceed the MTD calculated for birinapant in this study. Thus, the etiology of Bell's palsy and “cytokine-release syndrome” in patients treated with high doses of birinapant is unknown and additional studies may be warranted.

PK analysis of birinapant demonstrated dose proportionality over the range of 0.18 to 63 mg/m². PK analysis suggests that birinapant is likely to behave predictably across patients in a dose-dependent manner. There was evidence of high volume of birinapant distribution that is indicative of extensive tissue uptake. This was confirmed by birinapant accumulation in tumor biopsies.

Birinapant at 11.5 and 17.2 mg/m² resulted in a greater than 75% reduction in cIAP1 in PBMC samples, and from biopsies taken from three different tumors (Fig. 2). The level of cIAP1 suppression is consistent with that observed previously in preclinical xenograft studies (13). Furthermore, suppression of cIAP1 was sustained in both PBMC and tumor tissue for at least 7 days after birinapant administration. Also, there was evidence of cell-death pathway activation (caspase-8 activation) and apoptosis (evidenced by PARP cleavage; Fig. 2). Together, these data support the on-mechanism and effect of birinapant as an IAP1 inhibitor.

The highest dose administered in the dose escalation portion of this study exceeded the anticipated dosing regimen based on the preclinical toxicology data and allometric scaling analysis. This was especially encouraging because of the concerns related to hypovolemic shock raised by dog toxicology studies. It is clear that dogs are more sensitive to IAP antagonists than humans (23, 29). There are several possible explanations for the safety and tolerability profiles exhibited by birinapant in this study as compared with other SMAC mimetics. First, birinapant is not a pan-IAP inhibitor, but is relatively selective for cIAP1 versus cIAP2. As such, it is more analogous to the endogenous molecule SMAC. Second, although birinapant effectively blocks caspase-inhibitory activity of XIAP in vitro (caspase de-repression), the ubiqutin ligase activity of XIAP appears to be spared (15). The sparing of either cIAP2 and/or XIAP likely contributes to the positive tolerability profile of birinapant (30). The tolerability profile is important as the antitumor activity of birinapant in preclinical models has been found to be increased when administered in combination with conventional chemotherapy or TNF-mediated cancer treatments. These observations, coupled with the results of this study, have provided the framework for a number of ongoing clinical trials evaluating the effectiveness of combinations of birinapant and other cancer therapies.

No potential conflicts of interest were disclosed.

Conception and design: R.J. Schilder, L.P. Martin, D.E. Weng, A.A. Adjei

Development of methodology: D.E. Weng, A.A. Adjei

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R.K. Amaravadi, R.J. Schilder, L.P. Martin, M. Levin, D.E. Weng, A.A. Adjei

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R.K. Amaravadi, R.J. Schilder, L.P. Martin, M.A. Graham, D.E. Weng, A.A. Adjei

Writing, review, and/or revision of the manuscript: R.K. Amaravadi, R.J. Schilder, L.P. Martin, M. Levin, M.A. Graham, D.E. Weng, A.A. Adjei

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R.J. Schilder, D.E. Weng, A.A. Adjei

Study supervision: R.K. Amaravadi, L.P. Martin, D.E. Weng, A.A. Adjei

Other (interpretation of virologic data): M. Levin

The authors thank their colleagues for help with this work; Jennifer Burns for assistance with the PK analyses; and Chris Benetatos, Sri Chunduru, and Yasu Mitsuuchi for performing the caspase and CK assays. They acknowledge Rules Based Medicine for performing the cytokine assays. They also thank the patients and their relatives without whom this study would not have been possible.

This study was funded by TetraLogic Pharmaceuticals Corporation, Malvern, PA.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.