A First-in-human, Dose-escalation Study of the Methionine Aminopeptidase 2 Inhibitor M8891 in Patients with Advanced Solid Tumors

Methionine aminopeptidase 2 (MetAP2) is essential to endothelial cell growth and proliferation during tumor angiogenesis. M8891 is a novel orally bioavailable, potent, selective, reversible MetAP2 inhibitor with antiangiogenic and antitumor activity in preclinical studies. The safety, tolerability, pharmacokinetics, and pharmacodynamics of M8891 monotherapy were assessed in a phase I, first-in-human, multicenter, open-label, single-arm, dose-escalation study (NCT03138538). Patients with advanced solid tumors received 7–80 mg M8891 once daily in 21-day cycles. The primary endpoint was dose-limiting toxicity (DLT) during cycle 1, with the aim to determine the maximum tolerated dose (MTD). Twenty-seven patients were enrolled across six dose levels. Two DLTs (platelet count decrease) were reported, one each at 60 and 80 mg/once daily M8891, resolving after treatment discontinuation. MTD was not determined. The most common treatment-emergent adverse event was platelet count decrease. M8891 plasma concentration showed dose-linear increase up to 35 mg and low-to-moderate variability; dose-dependent tumor accumulation of methionylated elongation factor 1α, a MetAP2 substrate, was observed, demonstrating MetAP2 inhibition. Pharmacokinetic/pharmacodynamic response data showed that preclinically defined target levels required for in vivo efficacy were achieved at safe, tolerated doses. Seven patients (25.9%) had stable disease for 42–123 days. We conclude that M8891 demonstrates a manageable safety profile, with dose-proportional exposure and low-to-moderate interpatient variability at target pharmacokinetic/pharmacodynamic levels at ≤35 mg M8891 once daily. On the basis of the data, 35 mg M8891 once daily is the recommended phase II dose for M8891 monotherapy. This study forms the basis for future development of M8891 in monotherapy and combination studies. Significance: M8891 represents a novel class of reversible MetAP2 inhibitors and has demonstrated preclinical antitumor activity. This dose-escalation study assessed M8891 treatment for patients with advanced solid tumors. M8891 demonstrated favorable pharmacokinetics, tumoral target engagement, and a manageable safety profile, and thus represents a novel antitumor strategy warranting further clinical studies.


Introduction
Methionine aminopeptidase 2 (MetAP2) is one of two cytoplasmic methionine aminopeptidases ubiquitously expressed in mammalian cells. MetAPs catalyze modified marine natural product bengamide class (13), and triazole or purine derivates (1,14). While several reversible MetAP2 inhibitors are in clinical development, none have so far reached market approval.
M8891 represents a novel class of orally bioavailable, potent, selective, and reversible MetAP2 inhibitors (15,16). In preclinical studies, M8891 has demonstrated inhibition of new blood vessel formation and tumor cell proliferation and has also shown strong and durable antitumor activity in combination with vascular endothelial growth factor receptor (VEGFR)-targeted tyrosine kinase inhibitors (TKI), such as sunitinib, cabozantinib, and axitinib, in patientderived renal cell carcinoma xenografts (ref. 17, Friese-Hamim M, personal communication). A substrate of MetAP2, translation elongation factor 1-alpha-1, was recently identified as a pharmacodynamic biomarker to follow target engagement (Friese-Hamim M, personal communication).
We conducted this phase I study to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of M8891 monotherapy in patients with advanced solid tumors.

Study Design
This was a first-in-human (FIH), phase I, open-label, multicenter, single-arm, dose-escalation study designed to determine the maximum tolerated dose (MTD), recommended phase II dose (RP2D), safety, tolerability, pharmacokinetic, and pharmacodynamic profiles of M8891 in patients with advanced solid tumors (NCT03138538). An adaptive study design was applied, using a Bayesian two-parameter logistic regression model (BLRM). This study was designed to have two phases. Part 1, the monotherapy, dose-escalation phase, was completed according to protocol and is reported here. Part 2 of the study (combination of M8891 with cabozantinib) was not initiated; this was not due to safety concerns. Twenty-seven patients were enrolled from five sites in the United States between August 2017 (first patient visit) and September 2020 (last patient visit).
This study was conducted in accordance with the protocol and consensus ethical principles derived from international guidelines including the Declaration of Helsinki, applicable International Conference on Harmonization Good Clinical Practice Guidelines, and other applicable laws and regulations. The protocol and all required associated documents were approved by the responsible Institutional Review Board or Independent Ethics Committee. All patients were required to provide written informed consent prior to enrollment.

Patient Eligibility
Eligible patients were ≥18 years of age and provided written informed consent; had histologically confirmed advanced solid tumors, refractory to or intolerant of existing cancer therapies, with no surgical, radiation, or systemic anticancer therapies available after at least one prior systemic anticancer therapy; had accessible tumor biopsies; and agreed to use a highly effective form of contraception.
Key exclusion criteria included: an Eastern Cooperative Oncology Group (ECOG) performance status ≥2; severe bone marrow (hemoglobin <9.0 g/dL, neutrophil count <1.5 × 10 9 /L, platelets <100 × 10 9 /L), renal (calculated creatinine clearance <60 mL/minute according to the Cockcroft-Gault formula), or liver [total bilirubin >1.5 × upper limit of normal (ULN) or aspartate aminotransferase (AST)/alanine aminotransferase (ALT) >2.5 × ULN (>5 × ULN for patients with liver involvement)] impairment; prior radiotherapy to >30% of bone marrow reserves or bone marrow/stem cell transplantation within 5 years of study start; clinically significant cardiac conduction abnormalities; history of stroke, heart attack, thrombosis [e.g., deep vein thrombosis (DVT) and pulmonary embolism] or genetically determined hypercoagulopathy within 6 months; DVT based on lower extremity screening using Doppler ultrasonography or thromboembolic events under therapeutic anticoagulation; pregnancy or nursing; history of difficulty swallowing, malabsorption, or other chronic gastrointestinal disease or conditions that may have hampered compliance and/or absorption of the investigational drugs; life expectancy <3 months; and known hypersensitivity to the trial treatment or to one or more of the excipients used.

Treatment
Patients received oral M8891 in 21-day cycles at doses of 7-80 mg once daily.
The starting dose of 7 mg once daily was based on the dog highest non-severely toxic dose of 0.75 mg/kg/day divided by a safety factor of 6, which equated to a human dose of 7.5 mg (rounded to 7 mg) for a participant of 60 kg bodyweight.
Safety Monitoring Committee (SMC) decisions on subsequent dose levels were supported by a BLRM with overdose control. In addition, the SMC could decide to switch from a once daily to twice daily dosing schedule if data suggested insufficient exposure (pharmacokinetics) and target engagement (pharmacodynamics) after completion of the dose-limiting toxicity (DLT) period of the third once daily dosing cohort (or at a later time) or if evidence suggested that C max was driving safety signals, including but not limited to DLTs. Patients received treatment until disease progression, unacceptable toxicity, or withdrawal from the study.

Assessments and Endpoints
The primary endpoint was DLTs during the first 21-day treatment cycle of M8891, based on a predefined set of treatment-emergent adverse events (TEAE) with the aim to determine the MTD of M8891. At each dose level, the first patient was observed for DLTs for ≥7 days before the dosing of 2 subsequent patients was commenced. DLTs were defined as any of the following adverse events observed during the first 21-day treatment cycle and judged to be M8891related or clinically relevant: death; events of clinical significance that would expose patients to unacceptable risk if dose escalation continued; treatmentrelated hepatocellular injury, for example, ALT/AST >3 × ULN with elevation of serum total bilirubin to >2 × ULN, without findings of clinical causality; grade 4 liver enzyme elevation; Grade 4 neutropenia or thrombocytopenia lasting >5 days; grade 3 neutropenia with fever; grade ≥3 thrombocytopenia with bleeding; treatment interruption of >7 days or >30% of total dose due to adverse events not related to the underlying disease or concomitant medication; grade ≥3 non-hematologic toxicity (excluding grade 3 nausea or vomiting lasting <48 hours and resolving to grade ≤1 spontaneously or with conventional medical intervention; grade 3 fatigue or rash of <5 days duration; grade 3 hypertension in the absence of maximal medical therapy; and grade 3 electrolyte abnormality that lasted <72 hours, was not clinically complicated, and resolved spontaneously or responded to conventional medical intervention). In addition, the SMC could define as a DLT any TEAE that impaired daily function, or any abnormality that occurred in patients treated with M8891 at any time in cycle 1 during the dose-escalation part of the trial.
Secondary objectives included evaluating the safety, tolerability, pharmacokinetics, antitumor activity, and determining the RP2D of M8891. Safety evaluations included incidence and severity of TEAEs. All TEAEs were coded  (20), clinical benefit (defined as CR, PR, or SD for ≥12 weeks) and progression-free survival (PFS). PFS was defined as time from first dose to objective disease progression or death, whichever occurred first. CT scans or MRI were performed predose to document the baseline tumor status using the most appropriate criteria for the malignancy type. Target

Statistical Analyses
No formal significance level was defined for this study and all analyses were considered descriptive. Five analysis sets were defined: the dose-escalation set included all patients treated in the dose-escalation cohorts who did not miss >4 cumulative days of planned M8891 doses in the first cycle unless they ex- To assist the SMC in making recommendations on the next dose level and the MTD, the posterior probabilities (2.5%, 25%, 50%, 75%, and 97.5% quantiles) of toxicity were estimated by a BLRM with overdose control. The model was updated with the number of evaluable patients and DLTs observed after completion of the DLT period for each cohort. The target toxicity for the suggested MTD by the BLRM was 30%.
For the efficacy analysis, objective response and clinical benefit were summarized and 95% exact Clopper-Pearson confidence intervals (CI) were estimated (21). Kaplan-Meier estimates were calculated for PFS and the CI for the median PFS was calculated according to Brookmeyer and Crowley (22).
Dose proportionality was assessed using the "power model"; the pharmacokinetic endpoints, AUC, and C max of M8891 were compared between dose levels on day 1 and day 15 separately.

Data Availability
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Patient Demographics and Disposition
Baseline and disease history characteristics of all 27 patients enrolled are pre- Thirteen of 27 patients (48.1%, n = 3 at 7 mg once daily, n = 1 at 12 mg once daily, n = 2 at 20 mg once daily, n = 3 at 35 mg once daily, n = 2 at 60 mg once daily, n = 2 at 80 mg once daily) were treated for 12-133 days until disease progression, 7 patients (25.9%, n = 2 at 12 mg once daily, n = 1 at 20 mg once daily, n = 2 at 35 mg once daily, n = 2 at 60 mg once daily) were treated for 14-140 days until withdrawal of consent due to the development of TEAEs, and another 7 patients (25.9%, n = 3 at 35 mg once daily, n = 3 at 60 mg once daily, n = 1 at 80 mg once daily) were treated for 6-49 days until development of TEAEs that led to treatment discontinuation. or reduction in response to platelet count decrease. One of these patients recovered while on treatment, 3 patients recovered after the last dose of M8891, and 4 patients had not recovered by the end of the study. In 1 patient in the 60 mg once daily dose group, platelet count decrease was detected 1 day after the last dose was administered; the patient subsequently recovered. The mean relative reduction in platelet count from baseline was greater with increasing dose levels ( Fig. 1).
Thirteen of 27 patients discontinued treatment due to disease progression (48.1%, all groups), 7 (25.9%) patients withdrew consent due to TEAEs, and another 7 (25.9%) patients experienced TEAEs that led to treatment discontinuation. Both, TEAEs leading to M8891 treatment discontinuation and M8891-related grade ≥3 TEAEs generally occurred more frequently at higher doses of M8891 (>20 mg once daily) than at lower doses (≤20 mg once daily). Six patients experienced TEAEs leading to study discontinuation that were considered M8891-related: deep vein thrombosis (n = 1, grade 2, 35 mg once daily M8891); platelet count decrease (n = 2, grade 3 on 35 mg once daily M8891 and grade 3 worsening to grade 4 before resolving on 60 mg once daily M8891); confusional state (n = 1, grade 2, 60 mg once daily M8891); ALT increase (n =   Taking into consideration the less than dose-proportional increase in exposure combined with safety observations at doses of 60 to 80 mg, the SMC recommended to de-escalate from 80 to 35 mg to further explore this dose as a potential RPD2. As a result, the DLT probability estimation by the BLRM did not reach sufficient precision due to the limited number of DLTs and sample size, and the MTD was not determined.

Pharmacokinetics
The pharmacokinetic parameters for M8891 for cycle 1, days 1 and 15 are presented with summary statistics in Table 4.
Except for a few subjects (n = 3), the time to reach maximum plasma concentrations ranged from approximately 2 to 8 hours postdose at day 1 (Table 4) and 1 to 6 hours postdose at day 15 (Table 4).
Peak and total exposures (C max , AUC 0−t , AUC 0−tau ) of M8891 appeared to increase approximately dose proportionally up to 35 mg; at doses greater than 35 mg, M8891 exposures appeared to increase in a less than dose-proportional manner (Fig. 2).   Pharmacokinetic parameters after repeated dosing generally exhibited low to moderate interpatient variability, ranging from 7.7% to 51.7%. Starting with 20 mg, steady-state C trough (C ss,trough ) levels were above the target level of 1,500 ng/mL (Fig. 2) defined by modeling of preclinical pharmacokinetics/ pharmacodynamics and tumor growth inhibition data (El Bawab S, personal communication).

Pharmacodynamics
Met-EF1α accumulation in tumors was observed at 7 mg once daily and tended to increase up to 35 mg once daily in a dose-dependent manner in line with the observed increase of exposure (Fig. 3). A small Met-EF1α signal was observed in pretreatment tumor tissue samples in 2 patients at doses of 60 and 80 mg once daily. At ≥35 mg once daily, Met-EF1α levels reached the target levels of 125 μg/mg protein required for efficacy defined by preclinical modeling (El Bawab S, personal communication).
At 12 mg once daily and higher, a low and variable pharmacodynamic response was detected in WBCs from cycle 1, day 15 onward; no obvious dose correlation was observed.
On the basis of the observed safety, pharmacokinetic, and pharmacodynamic data, the SMC suggested a dose of 35 mg once daily as the RP2D for M8891 monotherapy.

Discussion
In this phase I study, orally administered M8891 was clinically manageable in patients with advanced solid tumors. Among the 27 patients enrolled into the study, only two DLTs were observed, and, after enrolling a confirmatory cohort, the RP2D was determined as 35 mg once daily based on the absence of DLTs at  TEAEs were generally grade 1-2 in severity and most were unrelated to M8891.
Platelet count decrease was the most common TEAE, but these events were generally manageable clinically, and no associated bleeding events were observed. These results compare favorably with the safety profile of irreversible MetAP2 inhibitors, such as TNP-470, which showed considerable neurotoxicity in phase I studies (23,24), and continue to support the hypothesis that the toxicity of irreversible MetAP2 inhibitors is not related to inhibition of MetAP2 itself (12) or sufficiently compensated by redundant MetAP1 activity [see Goya Grocin 2021 (11)].
Exposure to M8891 increased by dose level and showed low-to-moderate interpatient variability following multiple doses based on pharmacokinetic parameters. The multiple-dose pharmacokinetics of M8891 compared favorably with high pharmacokinetic variability found in phase I studies with TNP-470 (25). Moreover, at doses ≥20 mg once daily, the observed C ss,trough levels of M8891 were well above 1,500 ng/mL (Fig. 2), the target level anticipated to be correlated with in vivo efficacy (tumor growth inhibition) as defined by modeling and simulation of preclinical pharmacokinetic/pharmacodynamic and efficacy data (El Bawab S, personal communication Although M8891 had shown significant tumor growth inhibition in preclinical models (17), the clinical efficacy described in this study was modest, with no objective responses observed (Fig. 4)  further investigations with M8891, especially in combination regimens, are warranted (17).
As this was a phase I dose-escalation study, limitations include the open-label design and small sample size. The heterogeneity of tumor types enrolled in the study made it challenging to assign the efficacy or pharmacodynamic data to specific patient populations that may benefit more than others.
In conclusion, M8891 is the first orally available, reversible MetAP2 inhibitor that has entered clinical development. This FIH study allowed determination of an RP2D based on relevant pharmacokinetics, pharmacodynamics, and safety observations; M8891 demonstrated a manageable safety profile with TEAEs in line with the patient population, drug effect, dose-proportional exposure up to 35 mg, Met-EF1α accumulation, and low-to-moderate interpatient variability of plasma concentration and corresponding parameters of pharmacokinetics.
In addition, the observed exposure and target engagement anticipated to be required for in vivo efficacy were achieved at 35 mg once daily. Taken