Eligible patients were ≥ 18 years old and had (i) advanced solid tumors with progression after at least one prior therapy and who were not eligible for therapies expected to provide clinical benefit, or (ii) B-cell lymphomas including DLBCL, high grade B-cell lymphoma (HGBL), follicular lymphoma (FL) (grades 1 to 3b), mantle cell lymphoma (MCL), and Burkitt lymphoma who had failed at least two prior therapies and were not eligible for therapies expected to provide clinical benefit, such as chimeric antigen receptor (CAR) T-cell therapy or stem cell transplantation. Patients required evaluable or measurable disease, an Eastern Cooperative Oncology Group (ECOG) performance status of ≤ 1, and adequate bone marrow, hepatic, renal, and cardiac function. All other anticancer systemic therapies were required to be discontinued for at least three weeks prior to registration, and patients were expected to be off corticosteroids above prednisone equivalents of 10 mg/day. Strong CYP3A4 inhibitors and inducers were prohibited.

This phase I, multicenter, nonrandomized, open-label study of zelenirstat is registered at clinicaltrials.gov as NCT04836195. The study was conducted according to the guidelines of the Declaration of Helsinki, approved by Health Canada and the relevant institutional review boards in each of the four study centres. Written informed consent was obtained from all study subjects. The study was sponsored by Pacylex Pharmaceuticals Inc.

The study is designed in two parts, a dose-escalation phase I, reported here, and two phase IIA dose expansion cohorts, now ongoing in patients with R/R B-cell NHL, and refractory colorectal cancer.

A standard 3 + 3 design was used to assess for MTD and establish RP2D according to a pre-determined dose-escalation schema (Table 1). There was no intra-patient dose escalation. The MTD was defined as the dose level below the cohort in which 2 or more patients experienced a first cycle, zelenirstat attributable, dose-limiting toxicity (DLT), and the MTD required confirmation in at least 6 patients.

DLT was defined as any of the following: Grade (Gr) 4 thrombocytopenia, Gr 3 thrombocytopenia with bleeding, Gr 4 neutropenia ≥ 7 days, febrile neutropenia, clinically significant ≥ Gr 3 non-hematologic toxicity, or any significant toxicity warranting a withhold of zelenirstat. Patients who did not receive zelenirstat for at least 21 days during the first cycle for reasons other than DLT were deemed not evaluable and replaced. Toxicity was graded according to the National Cancer Institute Common Toxicity Criteria for Adverse Events (NCI-CTCAE) version 5.0.

Zelenirstat is a potent, small molecule inhibitor of human NMT1 and NMT2 proteins. In preclinical models, it has high oral bioavailability, is highly bound to plasma protein, and is metabolized in the liver primarily by CYP3A4 to water soluble metabolites excreted by the kidney (Pacylex, data on file). Zelenirstat has no relevant off-target kinase inhibition [10]. In GLP non-clinical safety testing, diarrhea was dose limiting in multiple daily oral administration, although hematologic toxicity was also seen at the highest exposures [10].

For dose escalation, patients received daily oral zelenirstat on 28-day cycles. It was administered as 10 mg and 70 mg capsules, with instructions to take on an empty stomach each morning with 250 ml of tap water. Patients received zelenirstat until progressive disease, unacceptable toxicity, or withdrawal of consent.

Baseline imaging and hematology and blood chemistry were obtained within 4 weeks and 1 week of study entry, respectively. Baseline screening included a complete medical history, physical examination, assessment of ECOG performance status, and body weight. CT imaging was used for advanced solid tumors and B-cell NHL; PET-CT was optional for B-cell NHL. Weekly complete blood counts with white blood cell differentiation and blood chemistries were drawn for the first cycle, biweekly for cycle 2, and then prior to each 28-day cycle. Adverse events were evaluated weekly in the first cycle, biweekly for cycle 2, and before each subsequent cycle using the NCI-CTCAE version 5.0.

Tumor response assessments using CT imaging were performed every two cycles. RECIST 1.1 [11] or the Lugano Response Criteria for Lymphoma [12] was used to determine tumor response and disease progression. For B-cell NHL patients with PET-CT at baseline, PET-CT was repeated after the sixth cycle to confirm complete response or disease progression. Patient archival specimens and blood samples were submitted for exploratory molecular analyses.

Peripheral blood samples were collected over six time points in the first 8 h of Cycle 1, Day 1 (C1D1), and again on C1D15. Pre-treatment Day 1 levels were obtained with every subsequent cycle. Zelenirstat plasma concentrations were quantified using a validated ultra-performance liquid chromatography with tandem mass spectrometry detection and analyzed using a nonlinear mixed-effects model.

Descriptive statistics were used for demographic, safety, and efficacy data. Categorical data were summarized using frequency counts and percentages. Time-to-event variables were analyzed by Kaplan-Meier methods. Weighted trajectory analysis for health outcomes used the Chauhan methodology [13].