A phase I trial of KX2-391, a novel non-ATP competitive substrate-pocket- directed SRC inhibitor, in patients with advanced malignancies

Aung Naing • Roger Cohen • Grace K. Dy • mDavid S. Hong • Lyn Dyster • David G. Hangauer • Rudolf Kwan • Gerald Fetterly • Razelle Kurzrock • Alex A. Adjei

Summary

Background Src kinase is central to tumor cell proliferation, apoptosis, and metastasis. KX2-391 is a synthet- ic, orally bioavailable small molecule inhibitor of Src tyrosine kinase (TK) signaling and tubulin polymerization. This com- pound is distinct from other Src kinase inhibitors by targeting the peptide substrate rather than the ATP binding site; the binding site on hetero-dimeric tubulin is novel and distinct from the taxanes and other known tubulin inhibitors. Methods This multicenter Phase I trial utilized a 4+2 study design to determine the maximum tolerated dose (MTD), safety, and pharmacokinetics (PK) of KX2-391 in patients with refractory solid tumors. Results Forty-four (44) patients (18 M, 26 F; median age, 59) were enrolled in 9 dose cohorts. Dose- limiting toxicities, all reversible within 7 days, occurred in 7 patients and consisted of elevated AST (n=4), ALT (n=2), neutropenia (n=1), thrombocytopenia (n=1), failure to thrive (n=1) and anorexia (n=1). The MTD is 40 mg BID continu- ously. Eleven patients had stable disease for ≥4 months, in- cluding patients with ovarian, carcinoid, papillary thyroid, prostate, pancreas and head and neck cancer. Patients with prostate and pancreatic cancer also had significant biomarker decreases (PSA, 205 ng/mL to 39 ng/mL; CA19-9, 38,838 U/mL to 267 U/mL). The ovarian cancer patient has had stable disease >12 months. KX2-391 was orally available, rapidly absorbed, and exposure was proportional to dose across the range investigated. Conclusions KX2-391 has a favorable pharmacokinetic profile, is well tolerated, demon- strates preliminary evidence of biologic activity, and warrants further evaluation in Phase II trials.

Keywords KX2-391 . Phase I . Src kinase . Prostate . Ovarian

Introduction

Src family kinases are frequently over-expressed and/or aber- rantly activated in numerous epithelial or non-epithelial cancers [1]. Various Src inhibitors including dasatinib, saracatinib, and bosutinib are being evaluated in solid tumors [2]. KX2-391 is a synthetic, orally bioavailable, and highly selective small molecule Src tyrosine kinase and tubulin polymerization inhibitor. Unlike other Src kinase inhibitors that are commercially available or currently in development, KX2-391 is the first peptidomimetic Src kinase inhibitor that targets the peptide substrate-binding site rather than the ATP-binding site. KX2-391 has been shown to potently inhibit Src-catalyzed trans-phosphorylation of focal adhesion kinase (FAK), Shc, paxillin, and Src kinase auto- phosphorylation with an IC50 of approximately 20 nM. It also induces p53 expression and stimulates caspase-3 and PARP cleavage in vitro, leading to tumor cell apoptosis. Photoaffinity labeling of tumor cells has identified tubulin as a second target. Cell-based competition experiments, in vitro polymerization assays and molecular modeling have demonstrated that KX2-391 binds to a unique site and unique confirmation on heterodimeric tubulin to inhibit polymerization [3].
KX2-391 is potent against a broad range of solid tumor cell types as well as many leukemia subtypes, including those resistant to imatinib and/or dasatinib. It is highly selective without effects on PDGFR, EGFR, JAK1, JAK2, Lck and ZAP70 [3, 4]. In vitro, KX2-391 targeted the substrate binding pocket of Src and had more broad spectrum activity and higher potency than dasatinib [5].
We performed a phase I dose escalation study of KX2- 391 in patients with solid tumors. The objectives of this study were to determine the maximum tolerated dose (MTD), define dose-limiting toxicities (DLTs), characterize safety and pharmacokinetics (PK), and to evaluate prelimi- nary antitumor activity.

Patients and methods

Patient eligibility and selection

Inclusion criteria included patients with advanced solid can- cers or lymphoma not responsive to conventional therapy or with no established life-prolonging therapy available; age ≥ 18 years; Eastern Cooperative Oncology Group perfor- mance status 0–2; adequate bone marrow, hepatic, and renal function; and off previous investigational agents >28 days. Exclusion criteria included failure to recover from acute > grade 1 toxicity of previous therapy, medically uncontrolled cardiovascular disease, past clinically significant cardiac arrhythmias, symptomatic or untreated central nervous sys- tem metastases and inability or unwillingness to sign an informed consent. KX2-391 was supplied by Kinex Pharmaceuticals, LLC. (Buffalo, NY). Prior to enrollment, patients signed a written, informed consent in accordance with Federal requirements and those of the IRBs at the participating institutions.

Study design

This was a multicenter, open-label, dose escalation, safety and tolerability study of KX2-391 in patients with advanced solid tumors or lymphomas (NCT00658970). The study was con- ducted in three centers in the US (MD Anderson Cancer Center, Fox Chase Cancer Center and Roswell Park Cancer Institute) and consisted of two parts. The Rising Single Dose (RSD) part of the study was aimed at characterizing single- dose PK while the Rising Multiple Dose (RMD) portion focused on defining the MTD, safety and tolerability, multiple-dose PK and biological effects of KX2-391. In Part 1, patients were administered a single dose (2, 5, or 10 mg) on Day 1 of each 28-daycycle, underwent blood draws for PK analyses and were observed for at least 7 days. If the patients had no drug-related effects from the single dose, they were enrolled in Part 2 and underwent twice-daily dosing for 22 days followed by a washout period of 6 days (22 days on, 6 days off). In cycles 2 and higher, patients were dosed twice daily for 21 days followed by a washout period of 7 days (21 days on, 7 days off). After two cycles of dosing, patients who were tolerating KX2-391 and did not have disease pro- gression could receive additional cycles of KX2-391 (21 days on, 7 days off).
The study was a 3+3 design. During the second cohort, the protocol was modified to a 4+2 design. Depending on the observed toxicities in the cohort of 4 patients, up to 2 addi- tional patients could be enrolled to determine the MTD. The MTD was defined as the dose at which no more than one of 6 patients had a DLT occurring in the first cycle.

Evaluation of safety and tolerability

Patients who received at least one dose of study medication were evaluable for safety. Toxicity in each dose cohort was graded according to the Common Terminology Criteria for Adverse Events (CTCAE), version 3.0. A traditional definition of DLT within the first cycle was used (Grade 3 non- hematologic toxicity and/or Grade 4 hematologic toxicity occurring within the first 21 days and assessed as being at least possibly related to study drug). To fully assess the toxicity profile of the drug, the safety evaluation period in the trial extended 30 days from the date of the last dose of study drug.

Dose escalation

The highest non-severely toxic level was found to be 0.5 mg/kg/dose BID in dogs. Based on the toxicology results and FDA guidelines, the starting dose for KX2-391 in the Phase I clinical trial was determined to be 2 mg BID. If no significant toxicity occurred in 3 or 4 patients, the dose for each subsequent cohort was increased to the next dose level. Cohorts of at least 3 or 4 patients were treated at each dose level until a DLT was noted. If 1 of 3 or 4 patients had a DLT, the cohort was expanded to a maximum of 6 patients. If only 1 of those 6 patients experienced a DLT, the dose for subsequent cohorts was escalated by 50 %. If at least 2 patients in a cohort had DLTs, no dose escalation was permitted. The dose level at which 2 of 6 patients experienced a DLT was considered one dose level above the MTD.

Duration of therapy

Patients remained on the protocol until disease progression, death, unacceptable adverse events or a concurrent illness preventing further administration of drug. Patients with sta- ble disease (SD), a partial response (PR), or a complete response (CR) could stay on treatment until they experi- enced a therapy-related intolerable toxicity, progressive dis- ease, or elected to withdraw from study.

Pharmacokinetic methods and analyses

Single-dose and steady-state dose PK were evaluated via serial blood samples taken pre-dose and then at specified time-points post-dose. Non-compartmental PK analysis was conducted and the following plasma PK parameters were calculated for single and multiple doses of KX2-391: the maximum observed plasma concentration (Cmax), time to reach Cmax (tmax), terminal elimination half-life (t1/2), area under the plasma concentration versus time curve from zero to infinity (AUC), area under the plasma concentration versus time curve from zero to time of the last quantifiable concen- tration (AUC0-t), area under the plasma concentration versus time curve from zero to 12 h post-dose (AUC0−12), total apparent drug clearance (CL/F), and apparent steady-state volume of distribution (Vss/F). Steady-state plasma PK parameters following BID multiple dosing for 28 days includ- ed Cmax ss, tmax ss, observed trough concentrations of drug during the dosing interval (Cmin ss), t1/2,,ss, AUCss, CLss/F, accumulation ratio (Rac) and temporal change from single dose to steady-state (Tc) (often referred to as linearity factor). Urine PK parameters following a single dose included renal clearance of drug from plasma (CLR) and fraction of dose excreted unchanged in the urine expressed as a percentage (fe%). KX2-391 was given as a single oral dose of 2, 5, or 10 mg in a dose escalation study. Urine was collected and analyzed at 6, 12, 24 and 48 h after dosing.

Evaluation of antitumor activity status (PS) of 0, and 31 patients had an ECOG PS of 1. The median number of previous chemotherapy regimens was 3. Various solid tumors were included (Table 1).

Dose escalation and maximum tolerated dose

Dose escalation proceeded with no DLT through the 2, 5, 10, 20, and 40 mg dose cohorts. The dosage was then escalated to 80 mg PO BID. At this dose two patients experienced a DLT: one Grade 4 neutropenia and one Grade 3 elevated AST. Subsequently, six patients were dosed at 60 mg PO BID for 21 days on and 7 days off. Two patients experienced DLT: one Grade 3 elevated ALT/elevated AST and one Grade 4 neutropenia with Grade 3 fatigue. Subsequently, four patients were enrolled at 50 mg PO BID for 21 days on and 7 days off. Two patients experienced DLT: one Grade 3 elevated AST and one Grade 3 thrombocytopenia requiring platelet transfusion (Table 2). The neutropenia took 11 days to recover, and the thrombocytopenia took more than 7 days to recover. In our study, the grade 3–4 AST/ALT increases recorded in patients were not associated with raised bilirubin. Thus, 40 mg PO BID for 21 days on and 7 days off was deter- mined to be the MTD on an interrupted dose schedule. A final dose of 40 mg PO BID continuously was also assessed. All patients underwent imaging with CT scans every 2 cycles, with assessment of disease-specific tumor markers whenever applicable. Evaluation for treatment response was done every 8 weeks by RECIST 1.0 [6]. This dose was well tolerated. Consequently, the MTD for continuous dosing is also 40 mg PO BID.

Safety/toxicity

The following treatment-related adverse events were ob- served (Table 3). The most common treatment-related ad- verse events were liver function test (LFT) abnormalities and fatigue, each of which occurred in eight or more patients. Other Grade 3 or 4 events seen in one patient each were abdominal pain, increased bilirubin, edema, fatigue, hyponatremia, mucositis, rash and thrombocytopenia. There were no serious cardiac toxicities observed in patients re- ceiving KX2-391. Most of the hepatic DLTs were observed between cycle 1 Day 22 and cycle 1 Day 28. All of these DLTs were revers- ible within one week of holding the drug. There were no patients who experienced permanent liver injury.

Pharmacokinetics

The half-life of KX2-391 was approximately 4 h with no change after 22 days of dosing and Tmax was approximately 1 h with no evidence of change after 22 days of dosing. There was a slight trend toward higher values in females with respect to dose normalized Cmax and AUC. The PK of KX2-391 are similar following a continuous dosing sched- ule for 57 days at 40 mg twice daily, compared with a 3- week on, 1-week off schedule, indicating no drug accumu- lation. Inter-patient variability of the PK parameters was similar among patients dosed at 40 mg continuously for 57 days, and those dosed at 40 mg for 22 days in the 3- week on, 1-week off dosing schedule. The PK results sup- port administration of KX2-391 on a continuous twice daily dosing schedule.
The maximum urinary excretion rate occurred at 3 h, with a mean maximum rate of excretion of 259, 413, and 1,092 ng/h for the 2, 5, and 10 mg doses, respectively. The total amount of KX2-391 recovered in urine was 1,998, 3,614, and 8,996 ng for the 2, 5 and 10 mg doses, respectively. Based on these data, it was determined that the percent of KX2-391 excreted in urine after 48 h was less than or equal to 0.15 % of the total dose for all doses administered, indicating that KX2-391 excretion in urine is low. Table 4 summarizes individual PK parameters.

Antitumor activity

Forty-four patients were evaluable for tumor response. There were no partial responses or complete responses. The median length of therapy was 2 months (range 1–12). Six patients were treated for 6 or more months. One patient with castration-resistant prostate cancer was treated for 6 months on study. The patient’s baseline PSA was 205 ng/ml and his PSA nadir was 39 ng/ml. Another patient who had pancreatic cancer had a baseline CA 19–9 of 38,838 U/ml that declined to a nadir of 267 U/ml. That patient was on study for 4 months, but later progressed. A patient with metastatic ovarian cancer was treated for more than 12 months with a CA-125 level that fell from 665 U/ml to a nadir of 207 U/ml. Three patients with differentiated thyroid cancer were on study for 8, 8 and 6 months. One patient with melanoma received treatment for 6 months, and a patient with appendiceal cancer received treatment for 11 months.

Discussion

In this study we demonstrated that treatment with KX2-391 is well tolerated. KX2-391 is a synthetic, orally bioavailable, and highly selective small molecule Src tyrosine kinase and tubulin polymerization inhibitor. KX2-391 is first in its class by virtue of targeting the peptide substrate-binding site rather than the ATP-binding site that other known Src kinase inhib- itors target. Src family kinases are frequently over-expressed and/or aberrantly activated in numerous epithelial or non- epithelial cancers [1]. Src is a non-receptor tyrosine kinase, which interacts with receptor tyrosine kinases such as IGFR 1 R, PDGFR, EGFR, c-Kit and Her 2/neu [2]. Even though inhibition of Src/Abl has demonstrated clinical benefit in patients with hematological disease, Src targeting has not had efficacy to date in solid tumors.
In terms of in vivo efficacy, KX2-391 was about 5 times more potent than dasatinib against tumor cell proliferation in an HT29 (human colon cancer) xenograft mouse model. In a PC3-MM2 (human prostatic cancer) orthotopic mouse mod- el, KX2-391 demonstrated strong inhibition of both primary tumor growth as well as lymph node metastases. Although dasatinib is safe and effective, common side effects includ- ing rash, fluid retention, bleeding episodes, gastrointestinal symptoms, myelosuppression, and increased risk of infec- tion have been observed [7].
KX2-391 has 10-100-fold lower potency than dasatinib in inhibiting Bcr/Abl. Even though the inhibition of Bcr/Abl by dasatinib and imatinib mesylate has been associated with cardiotoxicity, there have been no clinically significant car- diac findings in human subjects who have received KX2- 391 at this stage of its development.
The MTD of KX2-391 was 40 mg PO BID continuously with DLTs of increased liver enzymes, fatigue, and neutro- penia. Liver enzymes normalized within 1 week after ces- sation of the drug. No patients had irreversible drug-related liver failure. Other drug-associated adverse events were anorexia, fatigue, and nausea, and all were less than Grade
1. KX2-391 also possesses an inhibitory effect on tubulin polymerization. This inhibition of tubulin polymerization could be responsible for its efficacy in prostate cancer and ovarian cancer as well as its toxicity of cytopenia. Similar efficacies and toxicities have been observed in other tubulin polymerization inhibitors, such as docetaxel and paclitaxel [8, 9] KX2-391 demonstrated efficacy in several mouse tumor xenograft studies. Single dose pharmacokinetics in rats and dogs demonstrated that KX2-391 was rapidly absorbed with absolute bioavailability > 42 % after oral dosing. In our Phase I study, the PK profile demonstrated dose- proportionality, a half-life of approximately 4.0 h and a Tmax of 1 h with no evidence of accumulation with multiple doses. PK data showed that KX2-391 was orally available, rapidly absorbed, and exposure increased with increasing dose across the range investigated. Prolonged durable stable disease was observed in a patient with ovarian cancer. The protein kinase G (PKG) signaling pathway confers resis- tance to cisplatin-induced apoptosis in human ovarian can- cer cells. There is also direct cross-talk between PKG- 1alpha and Src in human ovarian cancer cells [10]. Src inhibition can also potentiate the antitumor activity of pac- litaxel by increasing autophagy, apoptosis and microtubule stability [11]. It would be worthwhile to investigate adding KX2-391 to a regimen of carboplatin or paclitaxel when patients develop resistance to those agents.
There is mounting evidence that Src is also activated in prostate cancer [12]. For example, dasatinib, an oral tyrosine kinase inhibitor that targets Src-family kinases, has been shown to inhibit three human prostate cancer cells lines PC- 3, DU-145, and LNCaP [13]. One patient with prostate cancer had significant decreases in his PSA level (PSA, 205 ng/mL to 39 ng/mL). Currently, KX2-391 is in a Phase II trial in bone- metastatic castration resistant prostate cancer patients who have not received prior chemotherapy.
In conclusion, KX2-391 can be administered safely to patients with advanced malignancies at doses up to 40 mg PO BID continuously. KX2-391 has a favorable PK profile. It demonstrates preliminary evidence of antitumor Tirbanibulin activity and warrants further evaluation in Phase II trials.

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