Dose Selection, Pharmacokinetics, and Pharmacodynamics of BRAF Inhibitor Dabrafenib (GSK2118436)

Published OnlineFirst June 23, 2014; DOI: 10.1158/1078-0432.CCR-14-0887

Clinical Cancer Cancer Therapy: Clinical Research

Gerald S. Falchook1, Georgina V. Long2,3, Razelle Kurzrock4, Kevin B. Kim5, H.-Tobias Arkenau6, Michael P. Brown7, Omid Hamid8, Jeffrey R. Infante9, Michael Millward10, Anna Pavlick11, Melvin T. Chin12, Steven J. O'Day8, Samuel C. Blackman13, C. Martin Curtis13, Peter Lebowitz13, Bo Ma13, Daniele Ouellet13, and Richard F. Kefford2,3

Abstract Purpose: Dabrafenib is a selective, potent ATP-competitive inhibitor of the BRAFV600-mutant kinase that has demonstrated efficacy in clinical trials. We report the rationale for dose selection in the first-in- human study of dabrafenib, including pharmacokinetics, tissue pharmacodynamics, 2[18F]fluoro-2- deoxy-D-glucose-positron emission tomography (FDG-PET) pharmacodynamics, and dose–response relationship. Experimental Design: Dabrafenib was administered orally once, twice (BID), or three times daily (TID). Selected dose cohorts were expanded to collect adequate data on safety, pharmacokinetics, or pharmacodynamics. A recommended phase II dose (RP2D) was chosen based on safety, pharmacokinetic, pharmacodynamic, and response data. Results: One hundred and eighty-four patients were enrolled and treated with doses ranging from 12 mg once daily to 300 mg BID in 10 cohorts. Pharmacokinetic assessment of dabrafenib demonstrated a less- than-dose-proportional increase in exposure after repeat dosing above 150 mg BID. Similar to parent drug concentrations, exposure for all metabolites demonstrated less-than-dose-proportional increases. Predicted target inhibition of pERK (>80%) was achieved at 150 mg BID, with a similar magnitude of inhibition at higher doses in BRAFV600 mutation melanoma biopsy samples. Although there was large variability between patients, FDG uptake decreased with higher daily doses in patients with BRAFV600 mutation– positive melanoma. A favorable activity and tolerability profile was demonstrated at 150 mg BID. There was no improvement with TID dosing compared with BID dosing, based on FDG-PET and tumor response analyses in patients with melanoma. Conclusion: The RP2D of dabrafenib was determined to be 150 mg BID after considering multiple factors, including pharmacokinetics, tissue pharmacodynamics, FDG-PET pharmacodynamics, and the dose–response relationship. A maximum tolerated dose for dabrafenib was not determined. Clin Cancer Res; 20(17); 4449–58. 2014 AACR.

1Division of Cancer Medicine, Department of Investigational Cancer Note: Supplementary data for this article are available at Clinical Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, Research Online (http://clincancerres.aacrjournals.org/). Houston, Texas. 2Melanoma Institute Australia and University of Sydney, New South Wales, Australia. 3Westmead Institute for Cancer G.S. Falchook and G.V. Long contributed equally to the study and devel- Research, Westmead Millennium Institute, and Department of Medical opment of this article. Oncology, Westmead Hospital, Sydney, New South Wales, Australia. D. Ouellet and R.F. Kefford contributed equally to the study and develop- 4Moores Cancer Center, University of California San Diego, La Jolla, ment of this article. California. 5Division of Cancer Medicine, Department of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Current address for G.S. Falchook: Sarah Cannon Research Institute– 6 Center, Houston, Texas. GlaxoSmithKline Medicines Research Unit, Denver, Denver, CO; current address for H.-T. Arkenau: Sarah Cannon 7 Prince of Wales Hospital, Randwick, New South Wales, Australia. Cancer Research Institute UK and University College London, London, United Clinical Trials Unit, Royal Adelaide Hospital and University of Adelaide, Kingdom; current address for P. Lebowitz: Janssen Pharmaceuticals, 8 Adelaide, South Australia, Australia. Experimental Therapeutics/ Radnor, PA. Immunotherapy, The Angeles Clinic and Research Institute, Los Angeles, California. 9Drug Development Unit, Sarah Cannon Research Institute/ Corresponding Authors: Gerald S. Falchook, Sarah Cannon Research Tennessee Oncology, PLLC, Nashville, Tennessee. 10Cancer Council Institute–Denver, 1800 Williams St., Denver, CO 80218. Phone: 303-839- Trials and Sir Charles Gairdner Hospital and University of Western 6000; Fax: 713-794-4130; E-mail: [email protected]; and Australia, Perth, Western Australia, Australia. 11Division of Medical Georgina V. Long, Melanoma Institute Australia, University of Sydney, New Oncology, New York University School of Medicine, New York, New South Wales, Australia. Phone: 61-2-9911-7200; Fax: 61-2-9954-9418; York. 12Prince of Wales Clinical School, University of New South E-mail: [email protected] Wales, Randwick, Australia. 13GlaxoSmithKline Research and Develop- doi: 10.1158/1078-0432.CCR-14-0887 ment, Philadelphia, Pennsylvania and Research Triangle Park, North Carolina. 2014 American Association for Cancer Research.

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objective tumor response assessment and determination Translational Relevance of the pharmacokinetic/pharmacodynamics profile. Dab- In the current era of targeted anticancer agents, phase I rafenib was administered as mesylate salt in a gelatin investigators face new challenges to define acceptable capsule shell. The study was conducted in two parts: Part criteria for selecting the optimal dose for subsequent 1 (dose escalation with pharmacokinetic/pharmacodynam- phase II and III investigations. Historically, the recom- ic expansions) and part 2 (RP2D and low-dose expansions). mended phase II dose has been determined after estab- Dabrafenib was administered orally once, twice (BID), or lishing a toxicity profile and identifying the maximum three times (TID) daily during dose escalation. Selected tolerated dose (MTD), based on the presumption of a dose cohorts were expanded to up to 20 patients to collect dose–response relationship. However, dose-related toxi- data on safety, pharmacokinetics, and/or pharmacodynam- cities may not be relevant surrogates for efficacy in less ics. The MTD was defined as the highest dose at which no toxic, targeted drugs. In this report, we describe the more than 1 of 6 patients experienced a dose-limiting rationale for dose selection in the first-time-in-human toxicity (DLT; Supplementary Materials and Methods). The phase I trial of dabrafenib, in which multiple endpoints protocol was approved by the Institutional Review Boards were considered, including pharmacokinetics, tissue- of participating institutions and is registered with Clinical- and 2[18F]fluoro-2-deoxy-D-glucose positron emission trials.gov (NCT00880321). All patients provided written tomography (FDG-PET)–assessed pharmacodynamics, informed consent. and dose–response relationship. Our trial contributes to the experience that the MTD is not always equal to the Study assessments, pharmacokinetics, and optimal biologic dose, reflecting a paradigm shift in pharmacodynamics which efficacy is not compromised at doses below the Toxicity, response, and BRAF genotyping were assessed as MTD. previously reported (Supplementary Materials and Meth- ods; refs. 6, 7). Safety data and pharmacokinetics were evaluated for all enrolled patients. Tumor response was assessed using Response Evaluation Criteria in Solid Introduction Tumors (RECIST; v1.0; ref. 8). Response data were reported for patients with BRAFV600 mutation–positive melanoma Dabrafenib (GSK2118436) is a selective, potent ATP- with measurable disease at baseline without untreated brain competitive inhibitor of rapidly accelerated fibrosarcoma metastases, and who had not previously received a BRAF or BRAF (RAF) kinases, including the V600 mutation, in kinase MEK inhibitor. panel screening, cell lines, and xenografts (1). A random- Blood samples for determination of plasma concentra- ized phase III trial demonstrated a 50% objective response tions of dabrafenib and its metabolites, including BRAF rate in patients with V600 mutation–positive meta- hydroxy-, carboxy-, and desmethyl-dabrafenib, were col- static melanoma and improved progression-free survival lected at multiple time points (Supplementary Materials with dabrafenib compared with dacarbazine (2). and Methods). Because preclinical data suggested that Historically, the recommended phase II dose (RP2D) for dabrafenib may induce CYP3A4 enzymes, urine was col- an anticancer drug has been determined after establishing a lected to measure 6-b-hydroxycortisol and cortisol con- toxicity profile and identifying the maximum tolerated dose centrations at baseline and after repeat dosing (day 15) in (MTD), based on the presumption of a dose–response part 1. Urinary 6-b-hydroxycortisol-to-cortisol ratio was relationship (3). Targeted agents, however, may have more compared between days 1 and 15 to determine potential favorable toxicity profiles because they are designed to for CYP3A4 induction (9). inhibit molecular aberrations specific to cancer cells, and The study included a 12-patient drug–drug interaction high doses that induce toxicity may not correlate with cohort at the RP2D to assess the effect of dabrafenib on greater efficacy. Targeted drugs, such as kinase inhibitors, single-dose pharmacokinetics of midazolam, a CYP3A4 may require an alternative metric to MTD that defines the probe. Single 3-mg doses of midazolam were administered dose–response relationship using suitable pharmacody- orally under fasting conditions at baseline (day 1) and day namic or other endpoints (4, 5). 15, 1 hour after the morning dose. Here, we report the rationale for dose selection in the first- Tumor biomarkers were evaluated from biopsies collect- time-in-humans phase I study of dabrafenib (6). To deter- ed at baseline and within the first 2 weeks of dosing in mine the RP2D, we considered multiple endpoints, includ- selected patients. Following hematoxylin and eosin staining ing pharmacokinetics, tissue- and 2[18F]fluoro-2-deoxy-D- to assess morphology and routine diagnostic IHC, semi- glucose-positron emission tomography (FDG-PET)-assessed quantitative IHC for pERK, Ki-67, and p27 was performed pharmacodynamics, and dose–response relationship. on paraffin-embedded tumor samples to assess the pharmacodynamic response to dabrafenib (Mosaic Laboratories Materials and Methods LLC). The staining intensity (1þ,2þ,or3þ) and extent Study design and patients (expressed as percentage of tumor cells at each intensity) The primary objectives were to determine the safety were assessed in the nucleus, cytoplasm, and combined and RP2D of dabrafenib. Secondary objectives included (nucleus/cytoplasm or total). A composite score (H score)

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was calculated on the basis of the sum of the products of Point estimate and 90% confidence intervals (CI) were back intensity and extent. calculated to provide geometric mean ratios of midazolam FDG-PET scanning was performed at baseline and week 2 with dabrafenib versus midazolam alone. in selected patients with BRAFV600 mutation–positive Changes from baseline were calculated for biopsy results melanoma. Conditions were standardized for patient using H scores, for FDG-PET data using sum of maximum weight and height, start of fasting time before scan (at least standardized uptake value (SUVmax), and for tumor size 4 hours), time of injection of FDG, and PET scan start and using sum of longest diameter of RECIST target lesions. end times, all of which were recorded. PET scans were not Summary statistics were provided by cohorts. performed if serum glucose levels were higher than 200 mg/ Dabrafenib’s effect on tumor pERK inhibition, FDG-PET, dL (11 mmol/L). The same protocol and specifications were and tumor size was evaluated with a pharmacokinetic- to be used at both time points. FDG-PET–anonymized pharmacodynamic model using nonlinear mixed-effect images with noncontrast CT were uploaded via AGMednet analysis. Different exposure–response models were evalu- and read by an independent reviewer (Perspective Infor- ated, including an inhibitory maximum effect (Emax) model matics, Inc). The regions of interest were located on the (Supplementary Materials and Methods). image by the independent reviewer, who was able to refer to Inhibition of pERK was correlated on the basis of total the RECIST assessment of contrast-enhanced CT imaging, as concentration of effective drug and metabolites adjusted for available, to aid in selecting lesions at the screening time relative potency. The relative potency of dabrafenib meta- point, although additional lesions may have been selected if bolites relative to parent drug was based on the ratio of IC50 they showed significant FDG uptake. The reviewer calcu- from two in vitro cellular activity assays, including protein- lated standardized average (SUVmean) and peak (SUVmax) shifted SKMEL28 cellular pERK inhibition assay and a uptake values based on the pixel values within each region protein-shifted proliferation assay using Colo205 cells of interest of the baseline and treatment digital FDG-PET (data not shown). The 95% CIs around parameter estimates images. of pERK, FDG-PET, and tumor size were generated on the based on nonparametric bootstrapping (N ¼ 500 bootstrap Analytical method datasets). Plasma dabrafenib, hydroxy-dabrafenib, and desmethyl- dabrafenib concentrations were analyzed using validated Results ultra high-performance liquid chromatography with tan- Study population dem mass spectrometric detection (UHPLC/MS-MS) meth- One hundred and eighty-four patients with solid tumors ods over the range of 1 to 1,000 ng/mL. Only dabrafenib were enrolled and treated with doses ranging from 12 mg and hydroxy-dabrafenib were measured originally but the once daily to 300 mg BID (Table 1 and Supplementary Table method was later modified and revalidated to include S1). Most patients had poor prognosis characteristics, such desmethyl-dabrafenib concentrations. A separate UHPLC/ as stage M1c disease. Ten percent of patients had received >2 MS-MS method was required to analyze carboxy-dabrafenib prior lines of systemic therapy in the metastatic setting. plasma concentrations. Midazolam plasma concentrations were determined using a validated analytical HPLC/MS-MS Safety method validated over 0.1 to 100 ng/mL concentration As previously reported (6), doses were escalated to 300 range. mg BID without identification of an MTD. DLTs were observed in 3 of 20 patients given 200 mg BID (grade 3 Statistical analysis cutaneous squamous-cell carcinoma, grade 3 syncope, Pharmacokinetic parameters were calculated after single grade 2 pyrexia) and in 2 of 10 patients given 300 mg BID and repeat dosing using standard noncompartmental meth- (grade 4 hyponatremia, grade 3 cutaneous squamous-cell ods for dabrafenib and its three metabolites. Dose propor- carcinoma; Supplementary Results and Supplementary tionality was assessed using a power model. To estimate the Table S1). No DLTs were observed in patients given <200 accumulation after repeat dosing and to assess time invari- mg BID. ance, ratios of area under the concentration–time curve over the dosing interval (AUC0-t) on days 8 or 15 to AUC0-t and Efficacy area under the concentration–time curve from time zero Among the 130 patients with BRAFV600 mutation–pos- extrapolated to infinity (AUC0-¥) on day 1, respectively, itive melanoma with measurable disease at baseline with- were calculated. out untreated brain metastases, and who had not previously The logarithmic-transformed urinary 6-b-hydroxycorti- received a BRAF or MEK inhibitor, objective responses were sol-to-cortisol ratio was analyzed using a mixed effect model observed at week 9 (part 1) or week 6 (part 2) at doses of 35 with the patient as a random effect and day (day 1 or 15) as a to 300 mg BID (Tables 2 and 3). fixed effect. Logarithmic-transformed midazolam pharmacokinetic parameters (AUC0-¥, AUC0-t, and maximum Pharmacokinetics observed concentration; Cmax) were analyzed using a After single-dose oral administration, parent drug plasma mixed-effect model with the patient as a random effect and concentrations peaked 1.0 to 2.5 hours postdose and treatment (with or without dabrafenib) as a fixed effect. decreased thereafter following a biexponential decline.

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Table 1. Baseline clinical characteristics

Part 2

Melanoma, Melanoma, Untreated brain Part 1 150 mg BID 50 mg BID Nonmelanoma, metastases, All cohorts (RP2D) (low dose) 150 mg BID 150 mg BID (N ¼ 114) (N ¼ 20) (N ¼ 20) (N ¼ 20) (N ¼ 10) Median age (range), y 53.5 (21–83) 54.0 (32–81) 56.5 (22–79) 58.5 (44–83) 62.5 (23–82) Sex, n (%) Women 44 (39) 10 (50) 6 (30) 11 (55) 4 (40) Men 70 (61) 10 (50) 14 (70) 9 (45) 6 (60) All melanoma, n 106 20 20 0 10 V600E, n (%) 85 (80) 17 (85) 20 (100) 20 (100) 9 (90) V600K, n (%) 14 (13) 3 (15) 0 0 1 (10) Other, n (%)a 7 (7) 0 0 0 0 Nonmelanomab 80020 0 V600E, n (%) 6 (75) 20 (100) V600K, n (%) 0 0 Other, n (%)c 2 (25) TNM stage M1c, n (%) 76 (67) 19 (95) 12 (60) 0 10 (100) Elevated baseline LDH, n (%) 42 (37) 10 (50) 9 (45) 2 (10) 6 (60) Prior systemic regimens in the metastatic setting, n (%) 0 47 (41) 4 (20) 4 (20) 10 (50) 4 (40) 1 41 (36) 10 (50) 10 (50) 4 (20) 3 (30) 2 13 (11) 4 (20) 6 (30) 4 (20) 2 (20) 3 13 (11) 2 (10) 0 2 (10) 1 (10)

Abbreviation: LDH, lactate dehydrogenase. aIncludes wild-type (n ¼ 3) and non-BRAFV600 mutations: K601E (n ¼ 2), V600_K601delinsE (n ¼ 1), and unknown mutation type (n ¼ 1). bPatients with BRAFV600 mutation–positive melanoma: papillary thyroid carcinoma (n ¼ 14), colorectal carcinoma (n ¼ 11), non–small cell lung carcinoma (n ¼ 1), gastrointestinal stromal tumor (n ¼ 1), ovarian carcinoma (n ¼ 1). cIncludes mutation positive with unknown mutation type (n ¼ 1) and unknown mutation status (n ¼ 1).

Median terminal half-life ranged from 4.0 to 6.8 hours after For AUC0-t, the mean (90% CI) slope of the relationship single-dose administration. Increases in Cmax and AUC were between day 15 AUC and daily dose was 0.605 hng/mL generally dose-proportional with single doses up to 300 mg (0.329–0.881). There was overlap in individual exposure at (Fig. 1A and Supplementary Table S2). The power model’s total daily doses of 150 to 600 mg. The mean (90% CI) for mean slope (90% CI) was 0.780 (0.523–1.036) for Cmax the slope of Ct versus daily dose was 0.398 ng/mL (0.115 (n ¼ 54) and 0.897 (0.734–1.061) for AUC0-¥ (n ¼ 42); a to 0.910) with 90% CI including value of 0. slope of 1.0, with 90% CI including 1.0, indicates dose- Dabrafenib is metabolized sequentially to three known proportional pharmacokinetics. metabolites that may contribute to clinical activity: Pharmacokinetic parameters obtained after repeat dosing hydroxy-dabrafenib, carboxy-dabrafenib, and desmethyl- are summarized in Supplementary Table S3. After repeated dabrafenib. Figure 1C demonstrates the mean concentra- once-daily, BID, or TID dosing, there was no accumulation tion–time proﬁle of dabrafenib and its metabolites after in plasma, and the mean AUC ratio of day 15 to day 1 was administration of 150 mg BID on day 15. Concentrations of <1.0, suggesting that oral clearance changes with time. After dabrafenib and hydroxy-dabrafenib were in the same range, administration of 150 mg BID, the geometric mean AUC with a similar half-life. Both carboxy- and desmethyl- was 47% lower on day 15 than on day 1. A 2-fold increase in dabrafenib accumulated with repeat dosing with accu- dose (150 mg BID vs. 300 mg BID) resulted in only a 43% mulation (AUC0-t ratio of day 15:day 1) ranging from increase in AUC0-t at day 15 and no increase in Ct. The 2.78 to 8.77 for carboxy-dabrafenib and from 12.6 to 35.0 dabrafenib mean pharmacokinetic proﬁles are shown for desmethyl-dabrafenib across doses 70 mg BID. in Fig. 1B. Exposure was greater on day 8 relative to day Although the metabolite half-lives could not be deter- 15 following administration of 150 mg BID (Supplemental mined with the limited sampling period on day 1, they Materials and Methods). Increases in Cmax, AUC (Fig. 1A), are predicted to be >24 hours based on accumulation and predose concentrations (Ct) were less than dose-pro- ratios observed. After repeat-dose administration of 150 portional between 75 and 300 mg BID after repeat dosing. mg BID, the exposure for each of the three metabolites

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Table 2. Clinical activity at week 2 FDG-PET and week 9 CT in patients with BRAFV600 mutation–positive melanoma with measurable disease who had not previously received a BRAF or MEK inhibitor and without untreated brain metastases from part 1

Week 2 FDG-PET (SUVmax) Week 9 tumor size (RECIST; ref. 8)

Mean Mean % Mean Mean % Dose level N baseline (SE) change (SE) N baseline (SE) change (SE) RR (95% CI) 35 mg QD 1 29 19 3 125 (56) 2 (2) 0 (-) 35 mg BID 5 13 (7) 56 (21) 5 47 (13) 44 (17) 60 (14.7–94.7) 70 mg BID 7 36 (12) 38 (14) 14 120 (27) 19 (6) 21 (4.7–50.8) 75 mg BIDa 156 60 NA NA NA 100 mg BID 7 44 (9) 48 (8) 9 138 (31) 17 (9) 44 (13.7–78.8) 100 mg TID 14 52 (16) 56 (4) 14 85 (22) 33 (8) 29 (8.4–58.1) 150 mg BID 12 48 (10) 57 (6) 16 92 (17) 31 (6) 50 (24.7–75.3) 200 mg BID 8 63 (26) 44 (12) 16 150 (31) 30 (9) 38 (15.2–64.6) 300 mg BID 5 100 (34) 58 (17) 10 91 (28) 57 (10) 90 (55.5–99.7)

Abbreviation: NA, not applicable. aPharmacokinetic cohort; patients were escalated from 75 mg BID to 150 mg BID after pharmacokinetic assessment on day 15. Only 1 patient had FDG-PET data.

relative to parent drug (AUC0-t of metabolite:parent, 0.258 (0.210–0.318), respectively, was observed following after adjusting for differences in molecular weight) was repeat 150 mg BID dabrafenib dosing. 0.8, 19, and 1.0, for hydroxy-, carboxy-, and desmethyl- In part 2, median predose plasma concentrations in cycle dabrafenib, respectively. Similar to parent concentra- 1 to 4 ranged from 53.6 to 77.3 ng/mL and from 30.7 to 56.3 tions, exposure for all metabolites on day 15 was less ng/mL in patients receiving 150 mg BID and 50 mg BID, than proportional to the increase in dose. The deviation respectively. Parent drug and metabolite concentrations from dose-proportionality was more pronounced in the were similar across cycles and generally lower in patients following rank order: day 15 > day 8, desmethyl- > receiving 50 mg BID compared with those receiving 150 mg carboxy- > hydroxy-dabrafenib, and Ct > Cmax > AUC0-t. BID, albeit in a nonproportional manner. An increase in the 6-b-hydroxycortisol-to-cortisol ratio was observed after repeat dosing (n ¼ 93 patients with Tumor biomarker pharmacodynamics paired samples), suggesting that the change in oral clear- Tumor biopsies were obtained from 15 patients with ance over time may be due to CYP3A4 induction. The BRAFV600 mutation–positive melanoma at baseline and overall mean ratio (90% CI) of day 15:day 1 in patients during treatment. Because tumors often decreased in size receiving BID/TID regimens was 1.98 (1.75–2.23). To con- rapidly after starting treatment, biopsy time was adjusted to ﬁrm the induction effect, 12 patients were enrolled in the an earlier date to reduce the risk of necrotic, nonevaluable midazolam assessment in part 2. A decrease in single-dose tumor samples and to ensure there would be enough time midazolam Cmax, AUC0-t, and AUC0-¥ with mean (90% CI) for parent drug and metabolites to equilibrate with tissues ratios of 0.388 (0.241–0.626), 0.234 (0.183–0.300), and and reach steady-state concentrations. Among the biopsies,

Table 3. Clinical activity at week 6 CT in patients with BRAFV600 mutation–positive melanoma with measurable disease who had not previously received a BRAF or MEK inhibitor and without untreated brain metastases from part 2

Week 6 tumor size (RECIST)

Dose level N Baseline mean (SE) Mean % change (SE) RR (95% CI) 150 mg BID 20 146 (21) 31 (4) 60 (36–81) 50 mg BIDa 18 104 (21) 20 (6)b 22 (6–48)

aA lower-dose cohort (50 mg BID) was included in part 2 of this study to better define the dose-efficacy relationship. Among the first 15 patients with BRAFV600E mutations treated at 50 mg BID, only three patients (20%) had a confirmed PR at the time of the interim analysis, which was less than the number of confirmed responses (n ¼ 4) required to continue enrollment on this cohort. bTwo of the 18 patients did not have lesion measurement on week 6, so n ¼ 16 for mean% change calculation.

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Figure 1. Pharmacokinetic results. A, dabrafenib day 1 AUC0-¥ or day 8 or day 15 AUC0-t versus daily dose following BID or TID regimens. B, mean dabrafenib concentration–time proﬁles after repeat dosing (day 8 or day 15). C, mean dabrafenib and its metabolites' concentration–time proﬁles after repeat dosing of 150 mg BID (day 15).

10 paired samples from patients with no prior BRAF or MEK 8th- and 1/22nd-fold relative to the parent drug based on inhibitor treatment were considered evaluable because the in vitro assay, respectively. Because of its low potency, pretreatment specimens showed adequate baseline expres- carboxy-dabrafenib is not believed to contribute to the sion (H score > 10) and the on-treatment specimens were clinical activity following dabrafenib administration. The obtained after 5 or more treatment days. percent change in pERK was predicted by total daily dose on Of the 10 evaluable paired biopsies, eight were evaluable the basis of the mean predose concentrations (Ct) observed for pERK, 10 for Ki-67, and 9 for p27 (Fig. 2). As previously on day 15 at that dose level (Fig. 2C, right). pERK inhibition reported (6), the median (range) change in pERK expression reached a plateau at total daily doses more than 200 mg from baseline was 83.9% (38.0% to 93.3%), with (>100 mg BID). A dose-related decrease in pERK was pre- doses ranging from 70 to 200 mg BID, indicating evidence dicted with total daily doses <200 mg, with a plateau of enzymatic pathway inhibition. Of the eight evaluable occurring beyond total daily doses of 200 mg thereafter. biopsies for pERK, six showed evidence of 80% inhibition Administration of 150 mg BID was predicted to provide, on of pERK expression. Changes in Ki-67 expression were not average, near maximum predicted possible target inhibition as sensitive with median change (range) of 66.5% (þ14.4 (80%) based on the Emax model described. to 81.1%), whereas increases in p27 expression were noted in 6 of 9 patients, with a median change (range) of FDG-PET pharmacodynamics þ28.6% (90.6 to þ125%). FDG-PET was performed at baseline and week 2 in 60 The relationship between systemic exposure and percent- patients with BRAFV600 mutation melanoma receiving age of pERK inhibition was characterized using an Emax doses ranging from 35 mg once daily up to 300 mg BID. model with 100% maximum inhibition and IC50 of 134 ng/ Four additional patients are included in this analysis who mL (95% CI, 92.7–155) on the basis of parent drug and were not included in the previously reported preliminary active metabolite concentrations including hydroxy-dabra- analysis of 56 patients (6). Decreases in SUVmax were fenib and desmethyl-dabrafenib (Fig. 2C, left). The potency observed in 55 of 60 patients, with a median 60% decrease of hydroxy-, desmethyl-, and carboxy-dabrafenib was 2-, 1/ in SUVmax (range, 100% to þ24%). The mean SUVmax

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Figure 2. Tumor biomarker results. A, representative IHC for pERK, Ki-67, and p27. B, observed change from baseline in tumor biomarkers including pERK (top), Ki-67 (middle), and p27 (bottom) in individual patients. C, observed change in pERK versus concentration or predicted change versus dose. 1, symbols indicate individual observed percent change in pERK from baseline at each effective concentration, and the curve represents the predicted change in pERK. 2, symbols represent predicted changes in pERK based on the ﬁnal model and observed mean day 15 pre-dose effective concentrations (dabrafenib, hydroxy-dabrafenib, and desmethyl-dabrafenib).

percent change from baseline for each dose level ranged The inhibitory Emax model was signiﬁcantly better than a from 19% to 58% (Tables 2 and 3). The decrease from model with no dose-related decrease in SUVmax (P ¼ 0.001). baseline in sum of SUVmax was generally dose related, except The median (95% bootstrap CI) total daily dose that at 35 mg BID and 200 mg BID. resulted in a 50% maximum decrease in SUVmax (ED50) Decreases in SUVmax correlated with the daily dose was 214 mg (168–312). There was no signiﬁcant difference administered using an inhibitory Emax model (Fig. 3, left). in percent change of SUVmax when comparing TID regimens

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Figure 3. Dose–response relationship for FDG-PET at week 2 (A) and tumor size at week 9 (B). Symbols represent individual values with daily doses given once daily (D), BID (*), or TID (þ); line represents ﬁtoftheEmax model.

with BID (P ¼ 0.29; Fig. 3, left). Changes in SUVmax and by patients would likely be the most effective (11, 12). SUVmean were similar and are not reported separately. There However, identifying an MTD and dose-related toxicities was no correlation between changes in tumor size at week 9 as surrogates for efficacy may not be necessary or relevant and changes in SUVmax at week 2 (P ¼ 0.89). for the successful development of less toxic, targeted drugs. Tumor size and response rate Historically, therapeutic windows have been quite nar- Because patients in part 1 were permitted to dose escalate row with traditional chemotherapies, in which efficacious after completing 9 weeks of treatment at the initial assigned doses push the limits of toxicity. As newer, targeted agents starting dose, comparisons of unconfirmed clinical are developed with improved safety profiles, the therapeutic response in different cohorts of melanoma patients are window will hopefully increase. This seems to be the case presented at week 9 (Tables 2 and 3). The unconfirmed with selective BRAF inhibitors, and in such situations, the BRAF response rate in patients with V600 mutation–positive optimal biologic dose may not necessarily equal the MTD. melanoma at week 9 was 50% (95% CI, 24.7–75.3) at 150 In our first-in-human phase I study of dabrafenib, no mg BID; the response rate was lower at 200 mg BID (38%; MTD was identified in the range up to 300 mg BID. Phar- 95% CI, 15.2–64.6) and higher at 300 mg BID (90%; 95% macokinetic and pharmacodynamics dose-response data CI, 55.5–99.7). The response rate was 60% (95% CI, 14.7– were used to select the RP2D of 150 mg BID, which 94.7) at 35 mg BID; patients in this cohort had the smallest demonstrated a favorable activity-tolerability profile. The median sum of target lesions at baseline (Tables 2 and 3), factors that supported the selection of this dose included with a mean value of 47 mm relative to an overall mean pharmacokinetic characteristics, achievement of predicted value of 112 mm. Baseline tumor size and change from target inhibition of pERK (>80%) at 150 mg BID with a baseline for each cohort are presented in Tables 2 and 3. similar magnitude of inhibition at higher doses, and evi- Data were described using an inhibitory Emax model as a dence of exposure response observed with FDG-PET. function of average daily dose administered with a median Pharmacokinetic assessment revealed a less than dose- (95% bootstrap CI) ED50 of 801 mg (571–1217 mg) as proportional increase in exposure after repeat dosing. Con- shown in Fig. 3 (right). The model was statistically signif- sistent with the decrease in midazolam, a CYP3A4 probe, icantly better than a model where the response is not related observed during coadministration of dabrafenib, the P ¼ to dose ( 0.002). The estimated ED50 was greater than decrease in dabrafenib AUC observed with repeat dosing the highest daily dose tested of 600 mg, suggesting that is likely due to induction of its own metabolism. Because of change in tumor size is very close to being dose linear. There their long half-lives, desmethyl- and carboxy-dabrafenib was no significant difference when comparing TID with BID accumulated with repeat dosing, although exposure was P ¼ regimens ( 0.11). also less than dose proportional on day 15. No increase in response rate was observed between 150 mg BID and 200 Discussion mg BID, whereas a higher percentage of responders was In the current era of targeted anticancer agents, phase I noted with 300 mg BID. However, differences in baseline investigators face new challenges to define acceptable tumor burden, relatively small sample size, and nonrando- criteria for selecting the optimal dose for subsequent mization may have contributed to the variability observed. phase II and III investigations (10). Historically, the Pharmacokinetic analysis also revealed that there was low selection of RP2D with cytotoxic medications has been interpatient variability in exposure. The majority of patients based upon the principle that the highest dose tolerated were within the therapeutic range.

4456 Clin Cancer Res; 20(17) September 1, 2014 Clinical Cancer Research

Dabrafenib Dose Selection, Pharmacokinetics, and Pharmacodynamics

The lack of improvement with TID dosing compared with in determining the RP2D in future first-time-in-human BID dosing may be explained by the contribution of meta- trials of targeted agents. bolites, including the long half-life of desmethyl-dabrafe- In vitro nib. potency of each metabolite on cellular pERK Disclosure of Potential Conflicts of Interest inhibition and proliferation assays demonstrated ratios, G.S. Falchook reports receiving a commercial research grant from which suggested that both hydroxy- and desmethyl-dabra- GlaxoSmithKline. G. Long reports receiving speakers bureau honoraria from GlaxoSmithKline and Roche and is a consultant/advisory board fenib may contribute to clinical activity, whereas carboxy- member for Amgen, Bristol-Myers Squibb, GlaxoSmithKline, Merck, dabrafenib is less likely to contribute despite its higher Novartis, and Roche. K. Kim reports receiving a commercial research grant exposure relative to the parent drug after repeat dosing. from and is a consultant/advisory board member for GlaxoSmithKline. M. Brown and R. Kefford report receiving speakers bureau honoraria pERK analysis confirmed that dabrafenib inhibits the from and are consultants/advisory board members for GlaxoSmithKline. MAPK pathway. The results from 8 patients were used to O. Hamid reports receiving a commercial research grant from Glaxo- SmithKline. J. Infante is a consultant/advisory board member for Glaxo- model the expected pERK inhibition at doses above 150 mg SmithKline. S. O’Day reports receiving commercial research grants from BID, and the model predicted little added benefit above this Bristol-Myers Squibb and Novartis. S. Blackman and B. Ma are employees dose. Ki-67 decreased with treatment, indicating that dab- of GlaxoSmithKline. D. Oullet is an employee of and has ownership interests (including patents) in GlaxoSmithKline. No potential conflicts rafenib is antiproliferative. of interest were disclosed by the other authors. We present the largest series of FDG-PET response analyses among patients treated with a selective BRAF inhibitor. Authors' Contributions Our study confirms dabrafenib’s antimetabolic effects, as Conception and design: H.-T. Arkenau, J.R. Infante, M. Millward, S.C. patients at all dose levels achieved substantial reduction in Blackman, C.M. Curtis, P. Lebowitz, B. Ma, D. Ouellet SUV . The observation that the degree of SUV reduc- Development of methodology: H.-T. Arkenau, J.R. Infante, C.M. Curtis, max max P. Lebowitz, D. Ouellet tion did not increase at doses above a total daily dose of 300 Acquisition of data (provided animals, acquired and managed patients, mg daily further supports 150 mg BID as the RP2D. provided facilities, etc.): G.S. Falchook, G.V. Long, K.B. Kim, H.-T. Arke- Although FDG-PET is a good marker for metabolic uptake nau, M.P. Brown, O. Hamid, J.R. Infante, M. Millward, A. Pavlick, M.T. Chin, S.J. O’Day, S.C. Blackman, C.M. Curtis, P. Lebowitz, B. Ma, R.F. Kefford and a drug’s activity, the response measured using FDG-PET Analysis and interpretation of data (e.g., statistical analysis, biosta- at week 2 did not correlate with tumor regression (percent tistics, computational analysis): G.S. Falchook, G.V. Long, R. Kurzrock, H.-T. Arkenau, M.P. Brown, O. Hamid, J.R. Infante, M.T. Chin, S.J. O’Day, change from baseline) assessed on week 9. C.M. Curtis, P. Lebowitz, B. Ma, D. Ouellet, R.F. Kefford Dabrafenib is among a growing group of other effective Writing, review, and/or revision of the manuscript: G.S. Falchook, anticancer agents that never reached an MTD, including G.V. Long, R. Kurzrock, K.B. Kim, M.P. Brown, O. Hamid, J.R. Infante, M. Millward, A. Pavlick, M.T. Chin, S.J. O’Day, S.C. Blackman, C.M. Curtis, bevacizumab (13), trastuzumab (14), and imatinib (15). B. Ma, D. Ouellet, R.F. Kefford The MTD for anticancer agents is not necessarily the optimal Administrative, technical, or material support (i.e., reporting or orga- biologic dose, as demonstrated by low-dose decitabine, nizing data, constructing databases): G.V. Long, O. Hamid Study supervision: G.S. Falchook, G.V. Long, H.-T. Arkenau, O. Hamid, J.R. which has superior efficacy compared with high-dose dec- Infante, M.T. Chin, C.M. Curtis, P. Lebowitz itabine (16). Our trial contributes to the experience that the MTD is not always equal to the optimal biologic dose and Acknowledgments reflects a paradigm shift in which efficacy is not compro- The authors thank the patients and their families for their participation. mised at doses below the MTD (17). They also thank Helen Brown and Midori Kayahara at MediTech Media and Clinical Thinking for editorial support in the form of comment collation, Our study is limited by the small number of patients for checking of facts, and graphic services. whom tissue was available for pharmacodynamic analysis. Another limitation is patient population heterogeneity, in terms of tumor burden (Tables 2 and 3) and BRAF mutation Grant Support This work was supported by the NIH Clinical and Translational Science subtype (V600E vs. V600K), which complicates the dose- Award UL1 RR024148, the NIH Cancer Center Support Grant (CCSG) award response assessment. In addition, our study is limited by the CA016672 to MD Anderson Cancer Center, Program Grant 633004 and of the National Health and Medical Research Council of Australia (NHMRC) absence of an analysis of durability of response and dura- and an infrastructure grant to Westmead Millennium Institute by the Health tion of tolerance. Such an analysis is not possible because Department of NSW through Sydney West Local Health District, and fellow- patients who received doses of less than 150 mg were ships from the Cancer Institute NSW (2009, 2011–2013) and the Royal Australasian College of Physicians (2010; to G.V. Long). Westmead Institute permitted to undergo dose escalation, and so any long-term for Cancer Research is the recipient of capital grant funding from the endpoint would not truly reflect the effect of the starting Australian Cancer Research Foundation. Editorial support was funded by dose. GlaxoSmithKline. The costs of publication of this article were defrayed in part by the In conclusion, the RP2D for dabrafenib was determined payment of page charges. This article must therefore be hereby marked after considering many factors, including pharmacokinet- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate ics, tissue pharmacodynamics, FDG-PET pharmacodynam- this fact. ics, and dose–response relationship, without identification Received April 10, 2014; accepted May 24, 2014; published OnlineFirst of an MTD. Investigators should consider a similar strategy June 23, 2014.

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4458 Clin Cancer Res; 20(17) September 1, 2014 Clinical Cancer Research

Dose Selection, Pharmacokinetics, and Pharmacodynamics of BRAF Inhibitor Dabrafenib (GSK2118436)

Gerald S. Falchook, Georgina V. Long, Razelle Kurzrock, et al.

Clin Cancer Res 2014;20:4449-4458. Published OnlineFirst June 23, 2014.

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