Relationships of AZD3229, a Novel and Selective Inhibitor of KIT, in a Range Of

Published OnlineFirst March 27, 2020; DOI: 10.1158/1078-0432.CCR-19-2848

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

The Pharmacokinetic–Pharmacodynamic (PKPD) Relationships of AZD3229, a Novel and Selective Inhibitor of KIT, in a Range of Mouse Xenograft Models of GIST A C Venkatesh Pilla Reddy1, Rana Anjum2, Michael Grondine2, Aaron Smith1, Deepa Bhavsar2, Evan Barry2, Sylvie M. Guichard2, Wenlin Shao2, Jason G. Kettle1, Crystal Brown2, Erica Banks2, and Rhys D.O. Jones1

ABSTRACT ◥ Purpose: The emergence of secondary mutations is a cause of Results: AZD3229 drives inhibition of phosphorylated KIT in an resistance to current KIT inhibitors used in the treatment of patients exposure-dependent manner, and optimal efﬁcacy is observed when with gastrointestinal stromal tumors (GIST). AZD3229 is a selective >90% inhibition of KIT phosphorylation is sustained over the inhibitor of wild-type KIT and a wide spectrum of primary and dosing interval. Integrating the predicted human pharmacokinetics secondary mutations seen in patients with GIST. The objective of into the mouse PKPD model predicts that an oral twice daily human this analysis is to establish the pharmacokinetic–pharmacodynamic dose greater than 34 mg is required to ensure adequate coverage (PKPD) relationship of AZD3229 in a range of mouse GIST tumor across the mutations investigated. Benchmarking shows that com- models harboring primary and secondary KIT mutations, and to pared with standard-of-care KIT inhibitors, AZD3229 has the benchmark AZD3229 against other KIT inhibitors. potential to deliver the required target coverage across a wider Experimental Design: A PKPD model was developed for spectrum of primary or secondary mutations. AZD3229 linking plasma concentrations to inhibition of phosphor- Conclusions: We demonstrate that AZD3229 warrants clinical ylated KIT using data generated from several in vivo preclinical investigation as a new treatment for patients with GIST based on its tumor models, and in vitro data generated in a panel of Ba/F3 cell ability to inhibit both ATP-binding and A-loop mutations of KIT at lines. clinically relevant exposures.

Introduction lack activity against a spectrum of both primary and secondary mutations and have numerous dose-limiting safety liabilities such as KIT belongs to a family of transmembrane tyrosine kinase growth high-grade hypertension (6) that can lead to dose reductions and drug factor receptors and gain-of-function mutations that result in consti- holidays (6, 7). Ripretinib (DCC-2618) and avapritinib (BLU-285) are tutive KIT activation, which has an important pathogenic role in KIT inhibitors undergoing investigation in clinical trials in patients gastrointestinal stromal tumors (GIST; ref. 1). Existing tyrosine kinase with GIST (8–12). Our goal has been to develop a compound targeting inhibitors such as imatinib (approved ﬁrst line), sunitinib (approved a wide spectrum of known primary and secondary resistance muta- second line), and regorafenib (approved third line) may initially tions of KIT/PDGFRa, while ensuring a wide margin against the main control GIST, but resistance develops due to emergence of secondary antitarget, VEGFR-2 to minimize clinical toxicities such as high-grade mutations in the ATP-binding pocket and activation loop of KIT (2–5). hypertension. AZD3229 is an oral, potent KIT/PDGFRa inhibitor This presents a major challenge for targeted drug discovery efforts that is active against a wide spectrum of primary and secondary seeking improved small-molecule inhibitors, primarily due to the KIT/PDGFRa mutations that are known to confer resistance to complex heterogeneity of oncogenic KIT mutations found in patients. standard-of-care (SoC) agents (13). It is also expected to avoid The existing approved KIT inhibitors for GIST treatment inherently signiﬁcant VEGFR-2 activity at clinically relevant exposures (13, 14). Pharmacokinetic–pharmacodynamic (PKPD) modeling has emerged as an important capability in drug discovery and develop- 1 Research and Early Development, Oncology R&D, AstraZeneca, United ment to assist in the design of studies and the integration and analysis Kingdom. 2Research and Early Development, Oncology R&D, AstraZeneca, of datasets to quantitatively investigate the understanding of drug Boston, Massachusetts. action (15). For Oncology targets, PKPD modeling is used to Note: Supplementary data for this article are available at Clinical Cancer establish a quantitative understanding of the target/pathway mod- Research Online (http://clincancerres.aacrjournals.org/). ulation requirements for optimal antitumor activity and based on Prior presentation: Part of the results of this study have been presented at the this understanding, it is possible to identify candidate drug mole- American Association for Cancer Research (AACR) Annual Meeting 2019 (March cules and predict and prioritize a human dose and schedule to test 29th–April 3rd, 2019 Atlanta, GA) and European Organization for Research and Treatment of Cancer (EORTC) (November 13th–16th, 2018, Dublin, Ireland). in the clinic (16, 17). As far as we are aware, there are no published PKPD models Corresponding Author: Rhys D.O. Jones, AstraZeneca, Hodgkin Building, c/o exploring the relationship between exposure, target suppression, and Darwin Building Unit 310, Cambridge Science Park, Cambridge CB4 0WG, United Kingdom. Phone: 44-75-5748-1665; E-mail: [email protected] antitumor activity in KIT-dependent mouse models of GIST. To explore the PKPD relationships, we used a number of GIST cell Clin Cancer Res 2020;XX:XX–XX line–derived xenograft (CDX), allograft, and patient-derived xenograft doi: 10.1158/1078-0432.CCR-19-2848 (PDX) models available that harbor different primary and secondary 2020 American Association for Cancer Research. mutations of KIT.

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oral gavage (10 mg/kg) dosing in nontumor-bearing mice (n ¼ 2) was Translational Relevance conducted for AZD3229. Plasma samples were taken by serial bleed at Preclinical animal models in translational research are funda- various time points up to 24 hours after dosing. The plasma concen- mental to the understanding of disease and drug pharmacology but trations of AZD3229 were determined using a protein precipitation are often limited in their utility to robustly define an efficacious procedure followed by LC/MS-MS detection. The lower limit of dose in the clinic. A reverse translational strategy using known quantification was 0.0014 mmol/L. Calibration standards passed with clinical information from the bedside to bench can play a crucial percent coefficient of variation (CV) less than 6% and percent bias in role in improving this situation. In this work, we evaluate the the assay in the range of 16% to 8%. The animal PK studies were translational pharmacokinetic–pharmacodynamic assumptions conducted under the condition of established Institutional Animal for the KIT/PDGFRa inhibitor AZD3229 by using drug exposure Care and Use Committee (IACUC) guidelines. and known clinical activity of standard-of-care agents across the population of patients with KIT-driven gastrointestinal stromal PKPD and efficacy studies tumor (GIST) to correlate against in vitro potency data for a The in vivo studies used in these analyses are described in detail with spectrum of primary and secondary KIT mutations. AZD3229 has the primary data, including tumor growth curves and immunoblots by potential as a best-in-class treatment for patients with GIST with Banks and colleagues (14). All animal studies were conducted under mutations in KIT and PDGFRa, and this compound may over- the condition of established IACUC guidelines. Briefly, the level and come the limitations experienced with existing treatment options duration of AZD3229-driven inhibition of phosphorylation of KIT in the clinic which are limited by off-target effects leading to drug (pKIT) was determined by Western blot analysis from allograft Ba/F3 holidays and dose reductions leading to lack of optimum efficacy. (KIT exon 11 del 557-558/D816H), CDX GIST430/V654 (KIT exon 11 del 560-578/V654A), and PDX tumor models HGiXF-105 (GS5108; KIT exon 11 del 557-558/Y823D; Crown Bioscience) and HGiXF-106 (GS11331; KIT exon 11 del 557-558/V654A; Crown Bioscience). Mice The objective of this PKPD modeling work was 4-fold: (i) For the were orally dosed with AZD3229 at several dose levels between 0.1 and in vivo models, investigate and establish the relationship between 40 mg/kg, either as a single dose or for 3 consecutive days. Plasma PK AZD3229 exposure and inhibition of phosphorylation of KIT; (ii) and tumor samples were taken at several time points up to 24 hours confirm the extent and duration of inhibition of KIT phosphorylation after the last dose. Plasma concentrations of AZD3229 and levels of required for optimal antitumor activity, as defined by tumor phosphorylated KIT were measured in each sample. Supplementary regressions; (iii) using a prediction of human pharmacokinetics of Table S4 provides details of the doses tested in each model, the AZD3229, estimate the dose anticipated to be required to deliver the measured AZD3229 plasma concentrations, and change in phosphor- extent and duration of inhibition of KIT phosphorylation necessary to ylated KIT at the respective time points selected for measurement. control tumor growth as defined by the preclinical studies; (iv) where The antitumor activity was also explored in these models using feasible use published data for other key KIT inhibitors to benchmark AZD3229 (see details of the doses tested in each efficacy model in AZD3229 and evaluate the translational assumptions presented here. Supplementary Table S5), along with approved (imatinib, sunitinib, and regorafenib), and investigational (ripretinib and avapritinib) agents. Mice were dosed twice daily (AZD3229 and ripretinib) or Materials and Methods once daily (imatinib, sunitinib, regorafenib, and avapritinib) by oral Protein-binding assessment gavage and the tumor growth inhibition from start of treatment was The in vitro binding of AZD3229 to plasma protein from the mouse, assessed by calculating the mean percentage change in tumor size at the and mixed human plasma was measured over a concentration range of end of treatment compared with baseline (start of treatment) as shown 0.1 to 100 mmol/L AZD3229, using equilibrium dialysis method in equation (1) below. following 20-hour incubation. The summary of plasma protein bind- %Tumor inhibition as a percentage of baseline ing (% unbound) of AZD3229 in the mouse and human (mean SD) Tumor size at the end of treatment ð1Þ is shown in Supplementary Table S1. The binding of AZD3229 to 10% ¼ 1 100 Tumor size at start of treatment of FCS was also determined (Supplementary Table S1). PK samples were taken at the end of the efficacy studies via serial Comparison of potency across a Ba/F3 cell panel to clinical bleeding of 3 animals per dose group after the last dose. exposures for approved and investigational KIT inhibitors The potency of AZD3229 along with approved (imatinib, sunitinib, Modeling strategy and regorafenib) and investigational agents (ripretinib and avapritinib) have been reported previously in a cell viability assay across a Integrated PKPD model development Visualization of the data by plotting AZD3229 plasma concentra- Ba/F3 cell panel as the GI50 (10, 13) and GI90 (14). The known clinical exposures (Supplementary Table S2) for KIT inhibitors at the tion against observed change in KIT phosphorylation relative to in vivo approved dose for SoC agents and the recommended phase II dose baseline for each of the four models did not reveal any fi for the investigational agents have been taken from the literature and signi cant hysteresis suggesting a direct relationship between plasma C concentration and changes in KIT phosphorylation. Therefore, the the plasma concentration at trough ( trough) compared and color coded C pharmacodynamic model assumes a direct response relationship as green if the trough exceeds the GI90 (Supplementary Table S3). between plasma exposure and level of KIT phosphorylation. A sche- Pharmacokinetics of AZD3229 in nontumor-bearing matic of the PKPD model structure is shown in Supplementary Fig. S1. immunocompromised (CB-17 SCID) mice g ¼ 1 EmaxCp ð2Þ To define the plasma pharmacokinetic properties of AZD3229 in pKIT basepKIT g g EC50 þ Cp mouse, a multiple sampling PK study after intravenous (5 mg/kg) and

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Equation (2) above shows the mathematical expression used to Results E relate AZD3229 plasma concentration to pKIT level, where max is AZD3229 pharmacokinetic properties and pooled PK model in the maximal effect observed, EC50 is the concentration that mice achieves 50% of E , base pKIT is the baseline pKIT level which max In mouse, the plasma pharmacokinetics of AZD3229 is character- was set to 100, g is the hill coefficient, and Cp is the drug ized as being a low volume of distribution (0.7 L/kg), low clearance concentration in plasma. Optimal parameter estimates can be (7 mL/min/kg) compound with good oral bioavailability and a half-life derived by fitting the model relating the observed plasma concen- of approximately 2 hours (13). tration against the observed change in KIT phosphorylation from Following oral dosing, there is a proportional increase in exposure baseline. A PK model, using pooled dataset, with first-order with dose and a one-compartment model with first-order absorption absorption and elimination was built using NONMEM 7.1 software and elimination is adequate to describe the concentration-time profile (ICON Development Solutions). A sequential PKPD model was observed across the dose range (0.5–20 mg/kg) tested. Thus, a pooled built using Phoenix 6.4 software (Certara) by fixing pooled PK PK model was parameterized (Supplementary Table S6) and model model parameters (Supplementary Table S6) and estimating PD simulations are compared against representative data across the dose model parameters such as EC . The PKPD model was utilized to 50 range in Supplementary Fig. S2. This PK model was subsequently used simulate the time course of decrease of KIT phosphorylation seen to simulate the plasma concentration–time course to drive the model in tumor models. fi of inhibition of phosphorylation of KIT. The potency of a drug is quanti ed as the EC50,andequation(3) can be used to calculate the concentration necessary to deliver AZD3229 exposure–pKIT relationship in mouse any degree of decrease, where DEC% is the percentage change In the four in vivo models explored here, AZD3229 drives rapid and required and EC is the concentration necessary to deliver DEC% DEC extensive decrease in levels of phosphorylated KIT in a concentration- decrease. 1 dependent manner following a single oral dose of AZD3229 (Fig. 1; DEC g Supplementary Table S4). The observed maximal inhibition of phos- ECDEC ¼ EC50 ð3Þ Emax DEC phorylation of KIT relative to baseline occurs around the time of maximum AZD3229 (2–3 hours), and the level of pKIT recovers to For any translational calculations or comparisons (e.g., calculating baseline levels tracking the PK profile. In the GIST430/V654 (KIT exon potency to set a human dose), the potency values from the mouse 11 del 560-578/V654A) and HGiXF-106 (KIT exon 11 del 557-558/ tumor models were corrected for the difference in plasma protein V654A) models, the level of KIT phosphorylation reaches above the binding. vehicle baseline at the 24-hour time point, but this was considered

Figure 1. A, PKPD model fit of plasma concentrations of AZD3229 to pKIT inhibition in various tumor CDX/PDX models. Solid lines represent model fitted, while symbols represent the observed data. B, Time course of pKIT inhibition for GIST tumor models for doses where tumor growth data available. Solid lines represent model fitted, while symbols represent the observed data.

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Table 1. Parameter estimates from PK/PD ﬁt of tumor pKIT inhibition versus plasma concentrations of AZD3229 in tumor models and comparison with in vitro Ba/F3 cell potency.

Parameter Estimate for each KIT mutation (nmol/L)

KIT mutation (tumor model assigned) KIT exon 11 del 560- KIT exon 11 del 557- KIT exon 11 del 557- KIT exon 11 del 557- 578/V654A 558/Y823D 558/V654A 558/D816H (GIST430/V654)b (HGiXF-105) (HGiXF-106) (Ba/F3) Model name [Crown Bioscience, (27)] — GS5108 GS11331 —

In vivo estimated unbound EC50 with %CV 4.8 (24) 9 (15) 0.4 (32) 2.2 (21) In vivo derived unbound EC90 with 95% CI 43 (20–65) 76 (54–98) 4 (1.5–8.5) 20 (12–30) Ba/F3 cell panel corresponding to in vivo model GIST430/V654 exon exon 11 del (557- exon 11 del (557– exon 11 del (557– 11 del (560–578)/V654Ac 558)/Y823D 558)/V654A 558)/D816H a In vitro FCS-corrected GI50 78311 a In vitro FCS-corrected GI90 14 29 3 17 Predicted human dose (mg) 18 34 2 8

Abbreviations: %CV, coefficient of variation; CI, confidence interval. aCorrected for 10% of FCS. bLicensed from Dana Farber Cancer Institute (DFCI), Boston, MA. Previously characterized and used in GIST field (27–30). cGIST430/V654 (exon 11 del 560-578/V654A) available in vitro and used to compare directly with the in vivo data.

within assay variability, rather than representing significant PD When combined with the mouse PK model for AZD3229, the time rebound above baseline. The effects on KIT phosphorylation levels course of change in pKIT levels simulated for the doses tested in the after 3 days of twice daily oral dosing was also investigated (except in respective in vivo models agrees adequately with the observed data the Ba/F3 allograft model), and the response mirrored that seen after a (Fig. 1). Example goodness-of-fit plots are shown in Supplementary single dose, which was evidence that there was no time-dependent Fig. S3. change for the inhibition of phosphorylation of KIT. On the basis of the PD results described above, it was assumed High and durable inhibition of phosphorylation of KIT drives that (i) a direct response relationship exists between plasma PK and optimal antitumor activity changes in tumor pKIT; (ii) no change in PD response is observed on Following twice daily oral dosing of AZD3229, the antitumor repeat dosing; and (iii) pKIT rebound above baseline is insignificant. activity observed in the four in vivo models increases with dose E fi in vivo Therefore, an max model was tted to the data from each resulting in tumor regressions at the highest doses tested (Supple- E model. On the basis of initial visual inspection of the data, max and the mentary Table S5). At these higher doses, it is clear from the pKIT data fi fi hill coef cient were xed to 1 and the EC50 was varied to achieve an that a decrease in the levels of pKIT greater than 90% is achieved for a optimal relationship (Fig. 1). Once the EC50 had been derived, the substantial proportion of the dosing interval (Fig. 1). On this basis, and EC90 was calculated [equation (3)]. The results of this analysis to establish the relationship between the level of pKIT decrease and show that the EC50 for decrease of KIT phosphorylation varies across degree of antitumor activity observed, the PK-pKIT model was used to models, with AZD3229 demonstrating lowest EC50 in the HGiXF-106 calculate the time above the EC90 over a 24-hour period at each dose fi model (KIT exon 11 del 557-558/V654A; 0.4 nmol/L) followed by used in the ef cacy studies. The time above EC90 was then plotted Ba/F3 (KIT exon 11 del 557-558/D816H; 2.2 nmol/L), GIST430/V654 against the level of antitumor activity measured (Fig. 2) and a strong (KIT exon 11 del 560-578/V654A; 4.8 nmol/L), and HGiXF-105 (KIT trend is observed, such that increased antitumor activity correlates exon 11 del 557-558/Y823D; 9 nmol/L; Table 1). with a longer time above EC90. Thus, greatest depth of antitumor

Figure 2. Correlation between antitumor effects (tumor inhibition as a percentage of baseline) and time (hr) above 90% inhibition of phosphorylation of KIT. The efﬁcacy studies were dosed twice daily AZD3229, with duration of treatment being 21 days except for Ba/F3 study, which was 10 days. Please see Supplementary Tables S4 and S5 for experimental details.

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Figure 3. At the doses (clinically relevant) tested in vivo (in the mouse CDX and PDX models), the time course of mouse plasma concentration is compared against the relevant in vitro GI90 from the Ba/F3 cell panel for the respective KIT inhibitors. The plot depicts the PK profiles after a single dose where rich PK was collected; however, ripretinib and AZD3229 was dosed twice daily in the efficacy study. activity is seen at 20 mg/kg in the HGiXF-106 (KIT exon 11 del 558/Y823D) and HGiXF-106 (KIT exon 11 del 557-558/V654A) are 557-558/V654A) model and at this dose, the time above EC90 is PDX models and corresponding cellular data are not available and so approximately 15 hours. This correlation also suggests that the inhi- the closest equivalent Ba/F3-panel cell lines have been used (Table 1). bition of phosphorylation of KIT to efficacy relationship is broadly There is a strong in vitro to in vivo correlation with the difference being consistent, showing the same trend across the four in vivo models within 1- to 4-fold: approximately 1-fold for HGiXF-106 (KIT exon 11 tested. Therefore, duration of pKIT decrease can be used as a predictor del 557-558/V654A) and Ba/F3 (KIT exon 11 del 557-558/D816H); of antitumor activity regardless of the KIT secondary mutation. approximately 2.5-fold for HGiXF-105 (KIT exon 11 del 557-558/ Y823D); and approximately 4-fold for GIST430/V654 (KIT exon 11 Demonstrating that the in vivo EC90 for inhibition of del 560-578/V654A). phosphorylated KIT correlates with in vitro potency data The PK–pKIT relationship has been explored in four in vivo models, In vitro potency predicts in vivo efficacy for registered and and this covers a narrow range of known mutations. Additional investigational KIT inhibitors mutations are known to confer resistance to imatinib, sunitinib, and The PKPD insights described above for AZD3229 should be regorafenib and the potency of these agents along with AZD3229 and generalizable to other KIT inhibitors. For the SoC agents (imatinib, investigational agents (ripretinib and avapritinib) have been previ- sunitinib, and regorafenib), the potency against the various KIT ously reported in a cell viability assay across a Ba/F3 cell panel as the mutations in the Ba/F3 cell panel are available, along with in vivo GI50 (13) and GI90 (Supplementary Table S3; ref. 14). It would be antitumor activity measured at doses in a mouse that deliver clinically desirable to demonstrate a correlation between the cell viability GI90 relevant exposures (Supplementary Table S2; ref. 14). The mouse in vivo in vitro and EC90 to be able to use the data to more fully assess equivalent doses for imatinib (300 mg/kg), sunitinib (80 mg/kg), and relative activity and exposure requirements across a more extensive regorafenib (100 mg/kg) were calculated using a PK-guided approach number of KIT mutations. For the GIST430/V654 (KIT exon 11 del by matching clinical dose normalized unbound exposure in human to 560-578/V654A) and Ba/F3 (KIT exon 11 del 557-558/D816H) mouse, rather than using an empirical factor (18). in vitro in vitro models, the cell lines have been grown and a direct comparison The GI90 values have been plotted alongside the mouse in vivo in vivo in vivo with the potency is possible. HGiXF-105 (KIT exon 11 del 557- exposure observed in the studies (Fig. 3). The GI90 values

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in vivo were used rather than EC90 as a full characterization of the the GI90 for the respective mutation then it is marked green. The aim is exposure–pKIT relationship has not been completed for these agents. to provide a useful way with which to benchmark each compound and These plots (Fig. 3) show that the in vivo antitumor activity is visualize how the in vitro potency values correlate with clinical activity in vitro predictable from the GI90 in the respective Ba/F3 cell line, in the different patient groups. The pattern observed for the SoC agents combined with the observed exposure achieved in the in vivo study: fits with the known clinical activity such that imatinib is active against Imatinib shows little duration above the respective in vitro GI90, and primary mutations but not secondary mutations and it is only the minimal antitumor activity in vivo at a dose of 300 mg/kg across the primary mutations that are colored green. Meanwhile sunitinib pro- four models tested [HGiXF-106 (KIT exon 11 del 557-558/V654A), vides good cover against secondary mutations in the ATP-binding HGiXF-105 (KIT exon 11 del 557-558/Y823D), Ba/F3 (KIT exon 11 pocket but does not do so for those secondary mutations in the A-loop del 557-558/D816H), and GIST430/V654 (KIT exon 11 del 560-578/ domain. Regorafenib and ripretinib provide broad coverage (10), V654A)] and this is consistent with the known lack of clinical activity particularly for the A-loop domain, but the clinical data for ripretinib in patients with secondary mutations. Sunitinib is clinically active in are not sufficiently mature to comment on the genotype-mediated patients with secondary mutations of the ATP-binding pocket and it efficacy. On the basis of the disclosed PK data for avapritinib, at the shows antitumor activity in the GIST430/V654 (KIT exon 11 del dose being used in phase III testing, the compound lacks the exposure 560-578/V654A) and HGiXF-106 (KIT exon 11 del 557-558/V654A) to adequately cover the secondary mutations in the ATP-binding models with an exposure profile at 80 mg/kg that delivers 6 hr pocket. This fits with a lack of clinical activity observed for avapritinib [GIST430/V654 (KIT exon 11 del 560-578/V654A)] to 24 h [Ba/F3 in the KIT ATP-binding mutant population and the current testing of (KIT exon 11 del 557-558/V654A)] coverage above the GI90. Mean- this compound in patients is restricted to KIT V654A and T670I while regorafenib has been tested in vivo in the Ba/F3 (KIT exon 11 del negative GIST population in a phase III trial (21). The backtranslation 557-558/D816H) and HGiXF-105 (KIT exon 11 del 557-558/Y823D) of clinical data to correlate with the Ba/F3 cell-panel GI90 values for the models and delivers antitumor activity in these models with an SoC agents builds confidence in the use of these in vitro data as a exposure profile at 160 mg/kg that provides continuous coverage benchmark to assess the exposure requirements for KIT inhibitors— — above the respective GI90. including predictions for AZD3229 to adequately cover the spec- Avapritinib (13, 19), approved for PDGFRa-driven GIST, and trum of mutations represented. ripretinib (13, 20) are structurally distinct inhibitors of KIT that are in clinical development and offer the opportunity to evaluate the The predicted clinical dose of AZD3229 depends on the KIT PKPD insights derived from AZD3229. At a dose of 30 mg/kg, mutation avapritinib shows limited tumor inhibition in GIST430/V654 (KIT A plasma concentration greater than the EC90 (derived from the exon 11 del 560-578/V654A), and tumor shrinkage in HGiXF-105 mouse in vivo models) over the dosing interval was set as the minimal (KIT exon 11 del 557-558/Y823D), with an unbound plasma concen- requirement to estimate an active clinical dose. Active human doses tration that delivers no cover and 24 h cover above the respective GI90 have been estimated by combining a prediction of the human phar- (Fig. 3). Ripretinib shows little tumor growth inhibition in the models macokinetics of AZD3229 with the mouse PD model parameters tested at a dose of 50 mg/kg twice a day, and the mouse exposure (corrected for differences in plasma protein binding mouse to human; provides no cover above the GI90. It should be noted that this mouse Supplementary Table S1). In preclinical species, the apparent volume dose does not achieve clinical exposures observed (10). Higher doses of distribution of AZD3229 is low in mouse (0.7 L/kg), rat (0.6 L/kg), were not explored due to body weight loss at 50 mg/kg twice a day; and dog (0.3 L/kg), and oral bioavailability is high in mouse (>90%), rat therefore, it is unknown whether antitumor activity would have been (79%), and dog (69%; ref. 13). On the basis of these data, the volume of achieved at higher doses that deliver exposures sufficient to exceed the distribution is predicted to be low in human (0.5 L/kg), and absorption GI90. It was not known at the time the data were generated that following oral dosing high (80%). Hepatic metabolism is the main ripretinib has an active metabolite in mouse and human that is route of elimination in preclinical species and is assumed to be so in equipotent and observed at equivalent concentrations to parent in patients. Using human hepatocytes, the intrinsic clearance has been mice (10), further complicating interpretation of the PKPD relation- measured (<1 mL/minute/106 cells; ref. 13), and scaled to the whole ship. If we account for the active metabolite present in the plasma liver using scaling methodology (22) to provide a predicted clearance (assuming equivalent concentrations as parent), then the net plasma of 0.4 mL/minute/kg, resulting in a predicted half-life of 15 hours. concentration of active KIT inhibition will be approximately double Using the predicted human PK, the doses (on a twice daily schedule) than that observed for parent, and this would approach the GI90 at required to deliver plasma concentrations > EC90 continuously around the time of maximal plasma concentration, but fail to give were calculated on the basis of Ba/F3 (KIT exon 11 del 557-558/ significant duration of inhibition required for antitumor activity. D816H; 8 mg), GIST430/V654 (KIT exon 11 del 560-578/V654A; Overall, the results of this analysis for the SoC and investigational 18 mg), HGiXF-105 (KIT exon 11 del 557-558/Y823D; 34 mg), and KIT inhibitors builds further evidence for the need for durable HGiXF-106 (KIT exon 11 del 557-558/V654A; 2 mg) models as shown in vitro inhibition of KIT phosphorylation >90% and that the GI90 in Table 1 and in Fig. 4. This represents a near 20-fold range, which of the relevant Ba/F3 cell line is predictive of the exposure (and thus mirrors the range in EC90 derived across the models, and highlights the dose) required in vivo for efficacy in the respective model. exposure required to drive adequate inhibition of KIT activity may differ depending on the mutation(s) that are present in the clinical Backtranslation of SoC agent clinical data supports the use of tumor. The models explored here represent the most common ATP- Ba/F3 cell-panel GI90 values for benchmarking relative binding mutation in the clinic (V654A) and the least sensitive A-loop sensitivity across secondary KIT mutations for KIT inhibitors mutation to imatinib (D816H) thus suggesting that AZD3229 is well The GI90 values (Supplementary Table S3; ref. 13) across the mutant placed to inhibit mutant KIT tumors in the clinic (23). However, four KIT and PDGFRa Ba/F3 cell panel for the SoC agents and the in vivo models limit our ability to assess the full spectrum of apparent investigational drugs ripretinib and avapritinib have been color coded sensitivity that is likely across the complement of known mutations C in vitro in such a way that if the observed clinical exposure (at trough) exceeds and the Ba/F3 cell panel offers the opportunity to explore the

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were measured at several time points and dose levels in the four models tested. This enabled the time course of pKIT level changes to be observed and confirm that the pKIT levels tracked changes in drug exposure without any significant time delay. This study design also facilitated robust model estimates for the in vivo potency in each model, as indicated by low CVs derived on the EC50 estimates. Examination of the Ba/F3 cell-panel viability data suggests that there is a good correlation between the relevant GI90 in vitro and the in vivo estimated EC90 in the respective model (Table 1). Although the EC90 varies across the models, optimal antitumor activity is consistently observed when the pKIT level is suppressed >90% durably over the dosing interval. This was further supported by experiments with a probe compound (Supplementary Fig. S4) in the Ba/F3 (KIT exon 11 del 557-558/V654A) mouse allograft model where different dosing schedules were used to control the duration of inhibition of KIT phosphorylation. Mice received the same daily dose as a single 100 mg/kg every day, twice daily 50 mg/kg, or three times daily 33 mg/kg, and it was found that 33 mg/kg three times daily showed the strongest antitumor activity, followed by the 50 mg/kg twice a day, with the least active being the 100 mg/kg every day dose. All three dose levels would be expected to deliver near maximal decrease in KIT Figure 4. phosphorylation, but the more frequent dosing ensures more durable fi Simulated human PK pro les of AZD3229 for range of doses at steady state. An coverage of the target over each day of dosing. estimated active human dose of 34 mg twice a day would provide adequate Taken together, the requirement for continuous inhibition of exposure to provide coverage of all the mutations tested in vivo and represented in the Ba/F3 cell panel. Doses above 34 mg twice a day provide an opportunity to phosphorylation of KIT >90% has been used to anchor the predicted maximize pKIT inhibition. A representative profile of AZD3229 at 150 mg twice a human dose. Because the exposure necessary to achieve this depends day dose is shown. Shaded areas are 95% confidence interval on estimated EC90. on the mutation the tumor harbors, the predicted dose also varies depending on the model used, with the lowest dose predicted to be relative sensitivity of the different mutations to KIT inhibition (Sup- 2 mg based on the HGiXF-106 (KIT exon 11 del 557-558/V654A) plementary Table S3). The GI90 values range from 1 nmol/L for the model and the highest dose predicted to be 34 mg based on HGiXF-105 exon 11 del 557-558 primary mutation to 74 nmol/L for the exon 9 (KIT exon 11 del 557-558/Y823D) model. Because the interpatient and insertion AY502-503/D816H secondary mutation. This range is con- intrapatient tumor heterogeneity is large in the GIST population, the in vivo sistent with the range in EC90 observed , and the upper predicted exploration of the PKPD requirements in multiple models provides a active dose of 34 mg would provide adequate exposure to cover all means to provide some preclinical assessment of the diversity of mutations tested in vivo and represented in the Ba/F3 cell panel. The response likely to be seen in the clinic. This is an advantageous analysis presented here gives confidence in taking the compound approach compared with relying on a single model that may be very forward to test in patients with GIST. AZD3229 is also able to inhibit sensitive but underestimates the exposure requirements for effective the KIT exon 9 primary mutation, thus differentiating it from imatinib treatment across a population of patients. The use of PDX models that is subefficacious in the clinic for this mutation (Supplementary further increases the translational relevance compared with relying Table S3). solely on CDX models (22, 23). Indeed, AZD3229 has been tested in two further PDX models (purchased from Crown Bioscience) as described by Banks and colleagues (14): the HGiXF-107 (GS5106; Discussion KIT K642E/N822K) and HGiXF-108 (GS11327; KIT exon 11 del Imatinib provides front-line treatment for GIST and relapse on K550fs). Both models are sensitive to AZD3229 showing tumor treatment is driven by the emergence of several secondary ATP- shrinkage at a dose of 4 mg/kg. These datasets are less comprehensive binding and A-loop KIT mutations. The current second- and third- than the other models presented here and precluded full PKPD line therapies—sunitinib and regorafenib—do not have strong activity analysis. However, given that these models are on the sensitive end against the full spectrum of secondary KIT mutations at clinically of the spectrum of models tested in vivo, they represent models that achievable doses/exposures (24) and a lack of selectivity results in a would have predicted doses at the lower end of the range, and in the tolerability profile that requires management. This provides a clinical context of predicting the human dose from preclinical insights, it is opportunity for a KIT inhibitor that delivers PKPD characteristics that more important to understand the impact on less sensitive models and enable strong activity across a wider spectrum of secondary KIT any resulting risk to the feasibility of dosing patients to deliver mutations. However, little has been published describing a systematic adequate target coverage. Indeed in a setting such as GIST where PKPD analysis to define the requirements for such a compound. there is such a spectrum of mutations that exhibit different sensitivity A combination of in vitro Ba/F3 cell panel and four mouse in vivo to AZD3229 inhibition, it may be desirable to have the option to be able models have been used to characterize the PKPD understanding for to dose beyond the exposure level predicted from the available what level of target suppression drives deep and durable antitumor preclinical datasets to offer the opportunity to inhibit mutations that activity. We have shown that following oral dosing in mice, AZD3229 have not been captured in the Ba/F3 cell panel, which may be less drives rapid and extensive decrease of KIT phosphorylation in an sensitive to inhibition by AZD3229. We believe that the overall exposure-dependent manner and the derived EC50 and EC90 varies pharmacokinetics of AZD3229 should allow dosing beyond 150 mg depending on the mutation the model harbors. Changes in pKIT levels (simulation shown in Fig. 4) before the biopharmaceutical properties

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Pilla Reddy et al.

begin to limit and reduce to any significant extent the fraction of dose clinic; benchmark emerging PK and PD data against competitor data orally absorbed (Supplementary Fig. S5). and preclinical insights; and refine translational assumptions in the An additional benefit of AZD3229 is that it has a wide margin model. against VEGFR-2 (6, 14) and at the predicted clinical exposures, it may have a lower risk of causing significant hypertension. Sunitinib and Disclosure of Potential Conflicts of Interest regorafenib are associated with high-grade hypertension, which V. Pilla Reddy and R. Anjum are employees/paid consultants for AstraZeneca. requires clinical management, and ripretinib has also been reported A. Smith is an employee/paid consultant for and holds ownership interest (including to show grade 3 and 4 hypertension in a recent phase I clinical patents) in AstraZeneca. D. Bhavsar, S.M. Guichard, W. Shao, J.G. Kettle, and R.D.O. Jones are employees/paid consultants for AstraZeneca. No potential conflicts of study (11, 24). interest were disclosed by the other authors. Taken as a whole, our hypothesis is that AZD3229 offers a PKPD fi pro le that, based on the translational modeling, would provide an Authors’ Contributions fi exposure pro le to deliver adequate inhibition of phosphorylation of Conception and design: V. Pilla Reddy, R. Anjum, E. Barry, S.M. Guichard, W. Shao, KIT across a broad spectrum of primary and secondary KIT mutations J.G. Kettle, R.D.O. Jones seen in patients with GIST and warrants clinical testing. Development of methodology: V. Pilla Reddy, R. Anjum, R.D.O. Jones It should be acknowledged that the use of preclinical data to predict Acquisition of data (provided animals, acquired and managed patients, provided clinical efficacy can be uncertain (25) but the use of Ba/F3 model has facilities, etc.): V. Pilla Reddy, R. Anjum, M. Grondine, A. Smith, D. Bhavsar, E. Barry, C. Brown shown success (20, 22), and the PKPD datasets in vitro (Ba/F3 cell in vivo Analysis and interpretation of data (e.g., statistical analysis, biostatistics, panel) and in mouse models generated for the SoC and computational analysis): V. Pilla Reddy, R. Anjum, A. Smith, W. Shao, R.D.O. Jones investigational agents are consistent with the known clinical activity Writing, review, and/or revision of the manuscript: V. Pilla Reddy, R. Anjum, in this heterogenous GIST patient population. Moreover, the trans- M. Grondine, E. Barry, S.M. Guichard, W. Shao, R.D.O. Jones lational approach presented here is attempting to go beyond relying Administrative, technical, or material support (i.e., reporting or organizing data, simply on mouse efficacy data to predict to the clinic by incorporating a constructing databases): V. Pilla Reddy, C. Brown, R.D.O. Jones Study supervision: S.M. Guichard, R.D.O. Jones more in-depth quantitative analysis of the PK and PD. This includes in vitro in vivo and mouse data and the backtranslation of data for SoC Acknowledgments fi agents to de ne appropriate translational assumptions. More gener- This study was sponsored by AstraZeneca. The authors wish to thank Camila de ally, building a quantitative PKPD model such as the one presented Almeida, for her scientific help in carrying out the PKPD-efficacy study procedures here for AZD3229 provides a modeling framework to integrate described. datasets including for SoC agents (in vitro, in vivo, and clinical), with the aim of better understanding translational uncertainties and The costs of publication of this article were defrayed in part by the payment of page risk (26). As a molecule moves toward and into the clinic, the PKPD charges. This article must therefore be hereby marked advertisement in accordance modeling framework provides a translational bridge from nonclinical with 18 U.S.C. Section 1734 solely to indicate this fact. to clinical and back, which can be used to support the study design in terms of setting dose and schedule and sampling time points for PK Received September 4, 2019; revised January 15, 2020; accepted March 23, 2020; and PD endpoints; set decision-making criteria for PK and PD in the published first March 27, 2020.

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AACRJournals.org Clin Cancer Res; 2020 OF9

The Pharmacokinetic−Pharmacodynamic (PKPD) Relationships of AZD3229, a Novel and Selective Inhibitor of KIT, in a Range of Mouse Xenograft Models of GIST

Venkatesh Pilla Reddy, Rana Anjum, Michael Grondine, et al.

Clin Cancer Res Published OnlineFirst March 27, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-2848

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