Supplemental methods - Clinical Cancer Research



Supplementary MaterialsDevelopment of 2 Bromodomain and Extraterminal Inhibitors With Distinct Pharmacokinetic and Pharmacodynamic Profiles for the Treatment of Advanced MalignanciesGerald Falchook1, Seth Rosen2, Patricia LoRusso3, Justin Watts4, Shilpa Gupta5, Catherine C. Coombs6, Moshe Talpaz7, Razelle Kurzrock8, Monica Mita9, Ryan Cassaday10, Wael Harb11, Julio Peguero12, David C. Smith7, Sarina A. Piha-Paul13, Russ Szmulewitz14, Marcus S. Noel15, Swamy Yeleswaram16, Phillip Liu16, Julie Switzky16, Gongfu Zhou16, Fred Zheng16, Amitkumar Mehta17Contents TOC \o "1-3" \u Supplemental methods PAGEREF _Toc8224170 \h 2Key exclusion criteria PAGEREF _Toc8224171 \h 2Dose escalation and expansion PAGEREF _Toc8224172 \h 2Definition of dose-limiting toxicities PAGEREF _Toc8224173 \h 3Pharmacokinetic sample collection and assay4EX VIVO ASSAY……………………………………………………………………………………………………………………………………………………..4Supplemental results PAGEREF _Toc8224175 \h 4Details of dose escalation PAGEREF _Toc8224176 \h 4Supplementary tables PAGEREF _Toc8224177 \h 6Supplementary Table S1. Steady-state PK parameters for INCB054329 (Cycle 1, Day 15) (A) and INCB057643 (Cycle 1, Day 8) (B) by dosing regimen. PK parameters calculated by non-compartmental analysis; presented as mean ± SD, geometric mean except Tmax, presented as median (minimum, maximum). PAGEREF _Toc8224178 \h 6Supplementary Table S2. Patients receiving INCB057643 possessing solid tumors with alteration(s) relevant to BET protein signaling (Part 2 – monotherapy dose expansion; treatment group A). PAGEREF _Toc8224179 \h 8Supplementary Table S3. Safety Summary across treatment groups. PAGEREF _Toc8224180 \h 9Supplementary Table S4. Treatment-emergent adverse events (TEAEs) occurring across all treatment groups. PAGEREF _Toc8224181 \h 11Supplementary Table S5. Best response across all treatment groups, efficacy evaluable population. PAGEREF _Toc8224182 \h 12Supplementary figures PAGEREF _Toc8224183 \h 13Supplementary Fig S1. EX VIVO SPIKED CELL ASSAY………………………………………………………………………………………………13Supplementary Fig S2. Intrapatient dose titration of INCB054329 (Part 2 TGA and TGC). PAGEREF _Toc8224184 \h 14Supplemental methodsKey exclusion criteriaKey exclusion criteria for both studies included: inadequate hematologic, liver, or renal function at screening; inadequate washout of prior treatment with anticancer medications or investigational drugs (or antibodies, biologics, or radiotherapy) before the first administration of the study drug; known HIV infection, active hepatitis B, or hepatitis C infection; chronic or current active infectious disease requiring systemic treatment; diagnosis of Type 1 diabetes or uncontrolled Type 2 diabetes as demonstrated by HbA1c >8%; use of prohibited concomitant medications including strong modulators of CYP3A4, and breast cancer resistance protein/ABCG2 inhibitors (INCB054329); prior treatment with any BET inhibitor; or any sign of clinically significant bleeding.Dose escalation and expansionINCB054329 – Part 1 (dose escalation) employed a 3 + 3 design in 2 TGs (TGA: advanced solid tumor or lymphoma and TGB: acute leukemia, MDS, MDS/MPN, and MF [see Fig. 1A]). Initially, patients in cohort 1 received a single dose of INCB054329 5 mg, followed by a timed PK assessment to confirm exposure approximately 1 week before continuous administration was initiated (referred to as Day 0). The starting dose for continuous administration was determined based on the exposures assessed at this PK assessment, with a starting dose no higher than 15 mg. Each dose-escalation cohort initially enrolled at least 3 patients. If no DLTs were observed in the initial 3 patients, the next cohort would begin enrollment at the next highest dose level. Patients not receiving at least 80% of the planned study drug doses during the first cycle (i.e., ≥17 of 21 doses during QD dosing or 34 of the 42 BID doses in Cycle 1) would be considered nonevaluable for purposes of determining the MTD (unless due to a DLT) and were replaced. Cohorts could include an additional patient to ensure sufficient evaluable patients reach Day 21.Part 2 (dose titration) enrolled 2 TGs (TGA: advanced solid tumors and TGC: MM) to evaluate the feasibility of intrapatient dose titration with protocol defined criteria. The starting dose was the highest tolerated dose with continuous 20 mg BID identified in Part 1 TGA. Part 2 TGA and TGC were to simultaneously enroll at least 5 patients and up to approximately 10 patients per TG. At the end of Cycle 1, if dose titration criteria were met, the dose was increased to 25 mg BID on Day 1 of Cycle 2. Dose escalation by ≤5 mg BID increments were permitted after each subsequent cycle of treatment if the patient continued to meet the dose escalation criteria.INCB057643 – Part 1 (dose escalation) employed a 3 + 3 design in 3 TGs (TGA: any advanced solid tumor or lymphoma; TGB: acute leukemia, HR-MDS, MDS/MPN, or MF; TGC: MM [see Fig. 1B]). If a DLT was observed, the cohort was expanded to 3 additional patients (see Supplemental Methods for definition of DLTs). The MTD was defined as the highest dose level with DLTs seen in ≤1 of 6 patients. Dose levels of 8 mg QD, 12 mg QD, and 16 mg QD were investigated; dose escalation began in cohort 1 of TGA starting at 8 mg orally QD (continuous). Enrollment in TGB and TGC began at the PAD identified in TGA. The MTD identified in Part 1 TGA was evaluated in Part 2.Part 2 (dose expansion) of the study enrolled 3 TGs (TGA: specified solid tumors and lymphoma; TGB: AML, HR-MDS, MDS/MPN, and MF; TGC: MM) which further evaluated the safety, preliminary efficacy, PK, and PD. If ≥33% of patients in a Part 2 expansion cohort experienced DLTs during Cycle 1, then further enrollment to the cohort would be stopped, and a lower dose level would be explored.Part 3 (Combination Dose Escalation; C-ES) has enrolled 6 TGs with the aim of identifying the MTD and/or a tolerated dose (the RP2D) of the combination of INCB057643 with one of the standard-of-care agents in relapsed or refractory advanced or metastatic solid tumor and hematologic malignancies. The starting dose of INCB057643 in the combination treatment groups was the PAD or 1 dose level below the MTD of INCB057643 monotherapy identified in Part 1 of this study. Starting doses of the standard-of-care combination agents were selected from conventional dose regimens for the first dose cohort. Dose escalation in the combination treatment groups were not completed at the data cutoff date. Definition of dose-limiting toxicitiesINCB054329 – A dose-limiting toxicity (DLT) was defined as any one of the following occurring during the first cycle (21 days) in Part 1: all TGs: any grade 3/4 non-hematologic-related event, excluding AEs with clear alternative explanations (due to underlying disease, disease progression, comorbidity, or concomitant medication) or transient laboratory values without associated clinically significant signs or symptoms, or nausea, vomiting, and diarrhea (in patients who have not received optimal treatment). TGA and TGC: grade 3 thrombocytopenia with bleeding or grade 4 thrombocytopenia; grade 4 neutropenia lasting >7 days or if associated with fever (> 101°F/38.5°C); grade 4 anemia; TGB only: grade 4 thrombocytopenia or grade 4 neutropenia lasting >6 weeks.INCB057643 – A DLT was defined as any one of the following occurring during the first cycle (21 days) in Part 1: all TGs: any grade 3/4 non-hematological-related event, excluding AEs with clear alternative explanations (due to underlying disease or disease progression) or transient laboratory values without associated clinically significant signs or symptoms, or nausea, vomiting, and diarrhea (in patients who have not received optimal treatment); alopecia; grade 3 fatigue, asthenia, fever, anorexia, and constipation; grade 4 anemia. TGA and TGC: febrile neutropenia of any duration (ANC <1.0 × 109/L and fever ≥38.5°C); grade 3/4 thrombocytopenia with bleeding; TGA only: grade 4 neutropenia; TGB only: grade 4 neutropenia with a hypocellular bone marrow lasting ≥ 6 weeks; TGC only: grade 4 neutropenia lasting >7 days.Pharmacokinetic sample collection and assayBlood samples for PK analysis of INCB054329 were collected predose and at 0.5, 1, 2, 4, 6, 8, and 24 hours after INCB054329 administration on Cycle 1 Day 1 and Cycle 1 Day 15 (steady-state). Blood samples for PK analysis of INCB057643 were collected predose and at 1, 2, 4, 6 and 8 hours after INCB057643 administration on Cycle 1 Day 1 and Cycle 1 Day 8 (steady-state). The blood samples were centrifuged at ~2000g for 15 minutes at ~5°C and stored at nominal –70°C until use. Plasma samples were assayed using a validated, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method consistent with good laboratory practice, and with a linear range of 2 nM to 2000 nM.Ex vivo assayPlasma samples were incubated ex vivo with the KMS-12-BM cell line for 4 hours at 37°C. Whole cell lysates were prepared from the cells, and levels of total MYC expression were determined using a commercial ELISA kit (Thermo Fisher Scientific, Waltham, MA).Supplemental resultsDetails of dose escalationINCB054329 – Initially, 3 QD dose levels were evaluated (cohort 1 [n=4], 15 mg QD; cohort 2, 22.5 mg QD [n=5]; cohort 4, 30 mg QD [n=5]). No DLT occurred in cohorts 1 and 2; 1 DLT (grade 4 thrombocytopenia) occurred in cohort 4. Because INCB054329 demonstrated a shorter than predicted mean half-life (t1/2) of ~2–4 hours (see Section PK and PD), subsequent cohorts were switched to a BID schedule (cohort 3, 15 mg BID [n=6]; cohort 5, 22.5 mg BID [n=3]) to achieve a higher area under the plasma concentration-time curve (AUC). No DLT occurred in cohort 3. All 3 patients in cohort 5 experienced thrombocytopenia: grade 4 [DLT], grade 3, grade 2, n=1 each). Cohort 5 with 22.5 mg BID continuous daily dosing was deemed not tolerated. Intermittent BID dosing regimens were explored with the aim of reducing the frequency and/or severity of thrombocytopenia and/or to allow for platelet recovery. Three intermittent dosing regimens were initiated in parallel with a starting dose of 22.5 mg BID (cohort 6, 22.5 mg BID 5-days on/2-days off [n=4]; cohort 7, 22.5 mg BID 4-days on/3-days off [n=4]; cohort 8, 22.5 mg BID 7-days on/7-days off; [n=3]) and all were deemed tolerated, with no reported DLTs. Four patients experienced thrombocytopenia (cohort 6, grade 3 [n=2], grade 2 [n=1]; cohort 7, grade 1 [n=1]). In parallel, Cohort 9 (n=8) with a starting dose of 20 mg BID continuous daily dosing was subsequently enrolled. No DLT occurred in cohort 9, and 20 mg BID was deemed tolerated. Two additional intermittent dosing regimens were initiated with a starting dose of 25 mg BID (Cohort 10, 25 mg BID 5-days on/2-days off [n=8]; Cohort 11, 25 mg BID 7-days on/7-days off; [n=4]). No DLTs were observed in either cohort 10 or 11; although, cohort 10 had multiple dose interruptions (3 out of 8 patients) and SAEs (hyponatremia, pneumonia, chronic obstructive pulmonary disease, and Escherichia bacteremia) indicating less tolerability than the 7-days on/7-days off dosing schedule. Thrombocytopenia occurred in 1 patient in cohort 10 (grade 3), and 3 patients in cohort 11 (grade 2 [n=2]; grade 1 [n=1]). PK data showed large interpatient PK variability across the different cohorts. Across all cohorts, most platelet decrease/thrombocytopenia events (all grades and all causality) occurred at approximately Day 8, with large decreases in platelet counts occurring between Day 8 and Day 21 during Cycle 1. INCB057643 – Four patients were enrolled in cohort 1 and no DLT occurred. Cohort 2 had a starting dose of 16 mg QD and 8 patients were enrolled (6 were DLT evaluable and 2?were not due to early disease progression). One DLT of increased international normalized ratio (INR; grade 3) occurred. In addition, 4 (50%) of the 8 patients enrolled in this cohort required dose interruptions and/or reductions due to AEs during Cycles 1 and 2, including DLT and TRAEs such as grade 3 INR increased, grade 3 thrombocytopenia, and grade 3 conjugated bilirubin increased. Thus, Cohort 2 at 16 mg QD was deemed not tolerated, although the MTD was not reached at this dose (per protocol definition). Cohort 3 had a starting dose of 12 mg QD and 4 patients were enrolled (3 were DLT-evaluable, and 1 was not due to early clinical progression). No DLTs occurred in cohort 3; only 1 patient had a dose interruption due to grade 2 dyspnea. Consequently, the 12 mg QD dose was selected as the RP2D for further exploration in Part?2 TGA.Supplementary tablesSupplementary Table S1. Steady-state PK parameters for INCB054329 (Cycle 1, Day 15) (A) and INCB057643 (Cycle 1, Day 8) (B) by dosing regimen. PK parameters calculated by non-compartmental analysis; presented as mean ± SD, geometric mean except Tmax, presented as median (minimum, maximum). ADoseNCmax (nM)Cmin (nmol/L)AUCtau (nM.h)Tmax (h)t1/2 (h)CLss/F (L/h)Vz/F (L)15 mg BID15 mg QD20 mg BID22.5 mg BID22.5 mg QD25 mg BID30 mg QD5a414b12c48d4267±201, 214278±82.6, 269652±348, 515720±472, 596479±154, 457449±335, 336389±393, 2781.09±2.450.538±1.0854.2±91.114.6±45001.51±3.02761±692, 596760±247, 7282490±2310, 15802300±1940, 1710902±426, 8391150±907, 8221330±1990, 5910.93 (0.5, 1.1)1.0 (0.52, 1.0)1.0 (0.48, 2.0)1.0 (0.5, 4.1)0.53 (0.5, 1.0)0.77 (0.5, 2.2)1.0 (0.52, 1.0)1.55±0.252, 1.532.02±0.825, 1.893.07±3.30, 2.362.44±1.49, 2.151.27±0.185, 1.261.68±0.936, 1.511.65±0.784, 1.5285±42.9, 72.361.8±21.3, 59.292.3±201, 36.448.3±33.7, 37.781.7±29.4, 77.0130±127, 87.3234±184, 146180±79.5, 160162±20.2, 161178±225, 124133±64.2, 117145±39.0, 140237±165, 191418±271, 319Overall51NDNDND1.0 (0.48, 4.1)2.24±2.03, 1.8895.5±135, 54.9192±171, 1.88aN=4 for AUCtau, t?, Clss/F, and Vz/F; bN=13 for Cmin; cN=11 for AUCtau, t?, CLss/F, and Vz/F; dN=7 for AUCtau, t?, CLss/F, Vz/F, and Cmin.Cmin is the Day 15 predose value; geometric mean was not calculated due to zero values. BDoseNCmax (nM)Cmin (nmol/L)AUCtau (nM.h)Tmax (h)t1/2 (h)CLss/F (L/h)Vz/F (L)8 mg QD12 mg QD16 mg QD11527216±109, 191306±129, 285340±165, 31040.3±29.1,30.956.2±50.5, 42.5101±87.5, 63.72230±1040, 20303180±1780, 28704320±2860, 36101.0 (0.5, 4.0)2.0 (1.0, 6.0)2.0 (2.0, 4.0)10.9±5.27, 9.9310.5±8.58, 9.3015.3±9.69, 12.810.5±4.94, 9.5211.0±4.64, 10.112.6±7.48, 10.7144±58.3, 133152±75.4, 140211±73.0, 198Overall70NDNDND2.0 (1.0, 6.0)11.1±8.27, 9.7311.1±4,97, 10.1157±74.1, 144AUCtau, area under the plasma concentration-time curve over 1 dosing interval; CLss/F, apparent clearance; Cmax, maximum observed plasma drug concentration; Cmin, minimum observed plasma concentration over the dose interval; ND, not determined; PK, pharmacokinetic; SD, standard deviation; t?, half-life; Tmax, time to maximum plasma drug concentration; Vz/F, apparent volume of distribution.Supplementary Table S2. Patients receiving INCB057643 possessing solid tumors with alteration(s) relevant to BET protein signaling (Part 2 – monotherapy dose expansion; treatment group A).Patient #Tumor TypeGene Alteration(s) Relevant to BET Genomic or Protein Signaling aBest Response1Squamous cell carcinoma of tracheaBRD4-NUT fusionPD2Breast cancerMYC amplificationSD ≥6 months3Colorectal carcinomaMYC+Missingb4Ovarian cancerBRCA wildtypePD5Triple-negative breast cancerMYC amplificationSD <6 months6Malignant tumor of the parotid gland with lung metastasisMYC rearrangementSD <6 months7CholangiocarcinomaMYC alterationMissingc8Colorectal cancerMYC amplificationPD9Head and neck cancer (tonsil)MYC alterationPD10Small intestine cancer (duodenum)MYC alterationPD11NUT midline carcinomaBRD-NUT fusionPD12colorectal cancerMYC amplificationMissingc13CholangiocarcinomaMYC alterationMissingd14Small cell carcinoma of prostateMYC amplificationMissingd15Neuroendocrine carcinomaMYC amplificationNot evaluable16NUT midline carcinomaBRD4-NUT fusionSD <6 monthsBET, bromodomain and extraterminal; PD, progressive disease; SD, stable disease.a As reported by the study sites.b Patient had clinical progression before first tumor assessment.c Patient died before first tumor assessment.d Patient withdrew consent before first tumor assessment.Supplementary Table S3. Safety Summary across treatment groups.Study INCB 54329-101 (n=69)aStudy INCB 57643-101(n=134)aSerious TRAEsAny, n=7Thrombocytopenia, n=4Anemia, n=2 AST increased, ALT increased, epistaxis, nausea, neutropenia, stomatitis, vertigo, vomiting, n=1 eachAny, n=19Thrombocytopenia, n=7Anemia, n=3Dehydration, hyperglycemia, INR increased, n=2 eachCCF, diarrhea, dizziness, febrile neutropenia, GI hemorrhage, headache, hepatic failure, Herpes zoster, pneumonia, sepsis, syncope, n=1 eachTRAEs leading to discontinuationAny, n=3Neutropenia, nausea, stomatitis, thrombocytopenia, vertigo, vomiting, weight decreased, n=1 eachAny, n=15Anemia, thrombocytopenia (n=3 each)Dizziness, headache, hepatic failure, hyperbilirubinemia, hyperglycemia, INR increased, muscle spasticity, nausea, sepsis, weight decreased, n=1 eachTRAEs leading to dose interruptionAny, n=20Thrombocytopenia, n=12Anemia, n=4Diarrhea, fatigue, hyperglycemia, nausea, n=2 eachBlood bilirubin increased, depression, dysgeusia, epistaxis, exertional dyspnea, bleeding due to thrombocytopenia, neutropenia, palpitations, rash maculopapular, stomatitis, vomiting, n=1 eachAny, n=46Thrombocytopenia, n=16Anemia, fatigue, n=7 eachDiarrhea, n=5Decreased appetite, nausea, vomiting, n=4 eachDehydration, dysgeusia , n=3 eachBlood bilirubin increased, Herpes zoster, hyperglycemia, INR increased, pyrexia, n= 2 eachAbdominal pain, AST increased, blood creatinine increased, bilirubin conjugated increased, dizziness, dry mouth, dyspnea, febrile neutropenia, flushing, GI hemorrhage, hematuria, hyperkalemia, hypokalemia, hyponatremia, neutrophil count decreased, pruritus, syncope, WBC decreased, wound hemorrhage, n=1 eachTRAEs leading to dose reductionAny, n=5AST increased, fatigue, neutropenia, oral pain, thrombocytopenia, tremor, n=1 eachAny, n=15Thrombocytopenia, n=7Fatigue, n=3Decreased appetite, n=2Abdominal pain, bilirubin conjugated increased, bone pain, dizziness, myalgia, nausea, presyncope, psoriasis, n=1 eacha Patients were counted once under each MedDRA preferred term. MedDRA preferred terms of Thrombocytopenia and Platelet counts decreased were combined for this analysis.ALT, alanine aminotransferase; AST, aspartate aminotransferase; CCF, congestive cardiac failure; GI, gastrointestinal; INR, international normalized ratio; TRAE, treatment-related adverse event.Supplementary Table S4. Treatment-emergent adverse events (TEAEs) occurring across all treatment groups.Study INCB 54329-101 (n=69)Study INCB 57643-101(n=134)Any GradeaGrade ≥3Any GradeaGrade ≥3TEAEs, n (%)NauseaThrombocytopeniabFatigueDecreased appetiteDiarrheaDysgeusiaAnemiaVomitingHyperglycemiaDyspnea (including exertional)ConstipationAbdominal painHyponatremia68 (99)32 (46)24 (35)33 (48)22 (32)18 (26)9 (13)17 (25)15 (22)5 (7)16 (23)15 (22)16 (23)11 (16)39 (57)2 (3)11 (16)4 (6)2 (3)1 (1)0 (0)12 (17)1 (1)3 (4)1 (1)0 (0)2 (3)6 (9)134 (100)61 (46)53 (40)60 (45)45 (34)37 (28)23 (17)37 (28)36 (27)23 (17)20 (15)25 (19)19 (14)9 (7)88 (66)4 (3)31 (23)3 (2)2 (1)2 (1)0 (0)26 (19)4 (3)5 (4)3 (2)1 (1)6 (5)4 (3)a TEAEs that occurred in >15% of patients in either study.b MedDRA preferred terms of Thrombocytopenia and Platelet counts decreased were combined for this analysis.Supplementary Table S5. Best response across all treatment groups, efficacy evaluable population.Response, n (%)Study INCB 54329-101 (n=69)Study INCB 57643-101(n=134)Objective response rateComplete responsePartial response1 (1)01 (1)a6 (5)2 (2)b4 (3)cStable diseaseStable disease ≥6 months21 (30)4 (6)27 (20)d6 (5)Progressive disease30 (43)44 (33)eMissingClinical progressionPhysician decisionWithdrew consentAdverse eventNot enough time on studyDeath15 (22)8 (12)01 (1)1 (1)4 (6)1 (1)54 (40)30 (22)4 (3)4 (3)8 (6)5 (4)3 (2)Not evaluable2 (3)3 (2)a Unconfirmed partial response corresponding to a 61% decrease in target lesion size from basline. This partial response remained unconfirmed because the patient discontinued treatment, due to a new diagnosis of colorectal cancer, approximately 1 month after achieving the response and prior to the confirmatory scan being completed. b One patient had a complete response based on positron emission tomography.c One patient and a partial response based on computed tomography and positron emission tomography.d Includes 2 patients with myelodysplastic syndrome (1 patient had stable disease at study day 86 and study day 170; 1 patient had stable disease at study day 92 and study day 142)e Includes 3 patients with acute myeloid leukemia who had suboptimal response to treatment.Supplementary figuresSupplementary Fig S1. Time course of percent change in MYC levels on Day 8 after INCB054329 and INCB057643 exposure, by dose and regimen, using an ex vivo spiked cell assay. For each patient, the pre-dose plasma samples from day 1 served as the control. Error bars represent SD. Supplementary Fig S2. Intrapatient dose titration of INCB054329 (Part 2 TGA and TGC).a Not evaluable for response because patient had not reached first response assessment at the time of the data cutoff date.b One patient had stable disease on day 31 (which is less than 42 days); therefore, this patient’s best objective response was considered not evaluable. BID, twice daily; MM, multiple myeloma; NSCLC, non-small cell lung cancer; TG, treatment group. ................
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