Hypoxia tumour targeting with Tarloxotinib to improve ... · Hypoxia tumour targeting with...
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UT-SCC-46A UT-SCC-110A UT-SCC-42B UT-SCC-42A UT-SCC-24A UT-SCC-126A UT-SCC-76B UT-SCC-1B UT-SCC-63A UT-SCC-76A UT-SCC-110B UT-SCC-74B UT-SCC-16A UT-SCC-19B UT-SCC-19A UT-SCC-60A UT-SCC-16B UT-SCC-54A UT-SCC-54B UT-SCC-74A UT-SCC-59C UT-SCC-54C 0.001 0.01 0.1 1 10 100 Hypoxia tumour targeting with Tarloxotinib to improve clinical outcomes for patients with EGFR-dependent malignancies Victoria Jackson-Patel, Kevin Hicks, Shevan Silva, Christopher Guise, Matthew Bull, Maria Abbattista, Angus Grey, Amir Ashoorzadeh, Jeff Smaill and Adam Patterson. Auckland Cancer Society Research Centre, University of Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, New Zealand; Rain Therapeutics Inc, Fremont, CA, USA. Background Abstract # 66 Epidermal Growth Factor Receptor (EGFR, HER1) is an attractive therapeutic target for many cancer types, however current EGFR-targeted therapies have had limited clinical success. • The EGFR signaling pathway is frequently deregulated in human cancers due to the overexpression of wild-type (WT) EGFR protein and/or its cognate ligand TGF-α 1 • Frequent aberrations in EGFR signaling have led to the clinical development of various EGFR-targeted therapies (Table 1). • Responses have been infrequent and short-lived 2,3 - Single agent response rates rarely exceed 10%, and most patients develop acquired resistance within a year of treatment initiation. • Severe on-target toxicities prevent administration of the doses required to completely abrogate WT EGFR signaling within the tumour 4,5 , leading to treatment failure and disease progression. Table 1: Clinical-stage EGFR inhibitors Tarlox-TKI is a dose-potent EGFR inhibitor Aerobic deactivation of Tarloxotinib Hypoxia-selectivity of Tarloxotinib metabolism Tumour dose intensification with Tarloxotinib Compound Molecular Class Principal toxicities (% of pts) a Cetuximab Monoclonal antibody Skin rash (49%) Gefitinib Reversible EGFR TKI Skin rash (29%), diarrhoea (22%) Erlotinib Reversible EGFR TKI Skin rash (70%), diarrhoea (32%) Lapatinib Reversible EGFR/HER2 TKI Skin rash (33%), diarrhoea (49%) Afatinib Irreversible pan-ErbB TKI Skin rash (65%), diarrhoea (63%) Dacomitinib Irreversible pan-ErbB TKI Skin rash (75%), diarrhoea (84%) in silico modelling of drug distribution Tarloxotinib is a hypoxia-activated EGFR TKI Figure 1. Schematic conversion of Tarloxitinib into an irreversible EGFR/HER2 TKI. Attachment of a hypoxia trigger to tarloxotinib significantly reduces the potency of the prodrug, allowing for administration at higher concentrations relative to the cognate TKI (Tarlox-TKI). Figure 2. Five day anti-proliferative IC 50 values for several clinical-stage EGFR-targeted therapies across a panel of SCCHN cell lines. Values are the mean ± SEM for at least three experiments. IC 50 , 50% inhibitory concentration. A hypoxia-activated prodrug of an irreversible EGFR/HER2 TKI • Studies using oxygen electrodes have demonstrated regions of low oxygen (hypoxia) in most cancer types 6 • Tarloxotinib is a prodrug designed to release an irreversible pan-ErbB TKI (Tarlox-TKI) selectively within the hypoxic regions of tumours (Fig. 1). • The prodrug approach may allow for tumour dose intensification by circumventing the normal tissue toxicities associated with conventional EGFR-targeted therapies leading to superior clinical outcomes for patients with EGFR-dependent malignancies. • An anti-proliferative assay was performed to confirm the deactivation of Tarloxotinib relative to Tarlox-TKI under aerobic (21% O 2 ) conditions (Fig. 4). • Deactivation ratios ranged from 15- to 267-fold. • Changes in EGFR signalling were examined in six representative cell lines following exposure to a clinically- relevant concentration of EGFR inhibitor (Fig. 3). • Consistent with the anti-proliferative assays, Tarlox-TKI silenced WT EGFR signalling to a greater extent than equimolar concentrations of afatinib, cetuximab and dacomitinib. Figure 3. EGFR signalling inhibition across a panel of SCCHN cell lines after exposure to equimolar concentrations of afatinib, cetuximab, dacomitinib and Tarlox-TKI. Cells were exposed to 0.3 μM EGFR TKI for an hour, with TGF-α stimulation after 45 mins. Figure 5. O 2 -dependence of Tarloxotinib metabolism. Solution O 2 concentrations were measured using a fiberoptic oxygen probe, and Tarlox-TKI production was quantified by Mass spectrometry. Values are the mean ± SEM of the intraexperimental repeats. Solution O 2 Concentration (M) 0.001 0.01 0.1 1 10 100 Relative Tarlox-TKI Production (C(O 2 )/(C(O 2 = 0)) 0.0 0.2 0.4 0.6 0.8 1.0 Oxygen Partial Pressure (mmHg) 0.001 0.01 0.1 1 10 KO 2 = 0.034 ± 0.02 μM • The O 2 -dependence of Tarloxotinib metabolism was determined in stirred cell suspensions by relating solution O 2 concentrations with Tarlox-TKI production after exposure under variable gas-phase O 2 concentrations (Fig. 5). • Tarloxotinib metabolism was completely suppressed by solution O 2 concentrations above 0.3 μM and through the physiological range indicating a requirement for severe hypoxia that is unique to solid tumours. Figure 6. Approach for the development of a SR-PK/PD model of drug distribution. A SR-PK/PD model incorporates several sub-models to calculate the spatial distribution of O 2 , prodrug and effector in a simulated 3D tumour microregion. Reproduced from (7). • Acellular PK model was developed based on the concentration-time profile for Tarloxotinib and formed Tarlox-TKI in the extracellular and cell-associated volumes of single cell suspensions under supraoxic and anoxic conditions (Fig. 7A). • Tarloxotinib was rapidly taken up to high cell-associated concentrations under supraoxic conditions (Fig. 7B). • Anoxic exposure rapidly depleted cell-associated Tarloxotinib with a concomitant rapid, and pronounced increase in the cell-associated Tarlox-TKI concentrations (Fig. 7C). • The cellular efflux of Tarlox-TKI was relatively slow indicating a high degree of cellular sequestration (Fig. 7C). Figure 7. Cellular PK model for Tarloxotinib. Experimental data and model fits for the cellular PK model developed for Tarloxotinib in single cell suspensions (FaDu) under supraoxic (95% O 2 /5% O 2 ) and anoxic (5% CO 2 /bal N 2 ) conditions. Tarloxotinib and Tarlox-TKI concentrations in the extracellular and cell-associated volumes were quantified by Mass spectrometry. In vivo validation of the SR-PK/PD model Supraoxia 0 50 100 150 200 0 10 20 30 40 50 60 Extracellular 0 50 100 150 200 0 1000 2000 3000 4000 5000 Cell-associated Anoxia Concentration (μM) Time (mins) Time (mins) 0 50 100 150 200 0 10 20 30 40 50 60 Concentration (μM) Time (mins) Extracellular 0 50 100 150 200 0 1000 2000 3000 4000 5000 Cell-associated Tarloxotinib Tarlox-TKI • The anti-proliferative activity of Tarlox-TKI was compared to several clinical-stage EGFR inhibitors across a panel of EGFR-expressing cancer cell lines (Fig. 2). • Tarlox-TKI was determined to be the most dose potent, with IC 50 values of up to 60-fold lower than afatinib and 8- fold lower than dacomitinib. • The in vitro parameters for cell uptake and metabolism as determined in single cell suspensions were scaled to tissue-like densities based on the transport of Tarloxotinib and formed Tarlox-TKI across multicellular layers under supraoxic and anoxic conditions (MCLs, Fig. 8A). • MCL cultures were a significant impediment to the transport of Tarloxotinib from the donor into the receiver compartments of diffusion chambers (Fig. 8B). • Bioreductive metabolism further reduced Tarloxotinib transport under anoxic conditions (Fig. 8C). • Transport of Tarlox-TKI to the receiver compartment was limited under anoxic conditions when observed up to 300 minutes (Fig. 8C). Figure 8. Flux of Tarloxotinib across MCL cultures (FaDu) under supraoxic (95% O 2 /5% CO 2 ) and anoxic (5% CO 2 /bal N 2 ) conditions. Tarloxotinib was added to the donor compartment of diffusion chambers, and samples were collected from both the donor and receiver compartments for up to six hours thereafter. Tarloxotinib and Tarlox-TKI concentrations were quantified by Mass spectrometry. Diffusion chamber schematic reproduced from (8). Anoxia 0 100 200 300 0 10 20 30 40 50 Receiver Concentration (μM) 0 100 200 300 0 10 20 30 40 50 Receiver 0 100 200 300 0 2 4 40 60 80 Donor Gas and sampling ports Culture Medium 150 μm 30 μm 0 20 40 60 80 100 120 0 10 20 30 40 50 0 40 80 120 0.00 0.05 0.10 0.15 0.20 Median: 27.1 μm Median: 5.3 μM O 2 36 0 O 2 distribution Distance to nearest blood vessel (μm) Distance to nearest blood vessel (μm) O 2 (μM) Vascular distribution Frequency Figure 9. Simulations of O 2 Transport in a representative mapped tumour xenograft microvascular network. Tumour oxygenation was modelled in a mapped FaDu human tumour xenograft microvascular network (9) using published parameters for O 2 transport (7). The contour plot illustrates the spatial distribution of O 2 in a mid-plane section of the simulated tumour microregion. The graphs depict the distance to the nearest blood vessel and O 2 concentration for every tissue voxel in the simulated tumour microregion. Cellular PK Model Drug Transport Model O 2 Transport Model Tarloxotinib Tarlox-TKI Tarloxotinib Tarlox-TKI • An O 2 transport model was developed to match the hypoxic fraction of FaDu xenografts measured by EF5 binding using published parameters for blood inflow, blood O 2 content, tissue diffusion and consumption in a representative mapped tumour microvascular network using the Green’s function methods (Fig. 9). Summary References Supraoxia Tarloxotinib Tarlox-TKI Time (mins) Figure 11. Matrix-Assisted Laser Desorption Ionisation Imaging Mass Spectrometry (MALDI-IMS) detection of tumor TRLX-TKI production and relationship to regions of hypoxia. A, C) 12 mm-thick cryosections were collected on conductive glass slides. Tissue was washed with 50 mM ammonium formate and CHCA matrix was applied via vacuum sublimation followed by recrystalization 20%ACN/5%FA. MALDI images were collected using a Bruker UltrafleXtreme MALDI-TOF mass spectrometer, positive ion mode, mass range 400-1000, 30 mm spatial resolution. MALDI imaging datasets were normalised to total ion current, and a MALDI image of TRLX-TKI for the combined molecular ions of the major bromine and chlorine isotopes (C19H1879Br35ClN6O, M + H = 461; C19H1879Br37ClN6O and C19H1881Br35ClN6O, M + H = 463) was plotted using SCiLS Lab (v2015a). A B 0 2 4 6 8 10 12 440 450 460 470 480 490 500 Fresh frozen tissue cryosection Mount section to MALDI slide Apply even coat of MALDI matrix Acquire mass spectra in x/y raster pattern UV laser + + + MALDI mass spectra m/z Intensity (au) Sample preparation and imaging m/z 461 m/z 463 TRLX‐TKI C 19 H 18 79 Br 35 ClN 6 O M + H = 461 TRLX‐TKI C 19 H 18 79 Br 37 ClN 6 O C 19 H 18 81 Br 35 ClN 6 O M + H = 463 Sister section of hypoxic cell distribution (green) D NORMOXIA HYPOXIA (EF5) NORMOXIA TRLX‐TKI (MALDI) MALDI image of TRLX-TKI concentration (3 h after dosing tarloxotinib) C MIN TRLX-TKI MAX 500m • PK/PD simulations were performed using a drug input equivalent to the unbound plasma C max for Tarloxotinib after a clinically-relevant dose (phase I MTD, 150 mg/m 2 , IV, qw). • Extracellular concentrations of Tarloxotinib were relatively uniform (1.2-fold range) across the simulated tumour microregion (Fig. 10A). • Consistent with bioreductive metabolism, cell-associated concentrations of Tarloxotinib declined as a function of O 2 concentration (Fig. 10B) and were reciprocated by increases in Tarlox-TKI concentration (Fig. 10C). • Extracellular Tarlox-TKI concentrations were consistent with in vitro cytotoxicity at 98% of the tissue voxels in the simulated tumour microregion (Fig. 10D). PK/PD Simulations of drug distribution 0.01 0.1 1 10 100 0.00 0.05 0.10 0.15 0.20 0.01 0.1 1 10 100 0 50 100 150 200 250 300 350 0.01 0.1 1 10 100 0 100 200 300 400 500 0.01 0.1 1 10 100 0.0 0.5 1.0 1.5 2.0 2.5 1.65 1.76 150 340 0.1 0 O 2 concentration (μM) O 2 concentration (μM) O 2 concentration (μM) O 2 concentration (μM) Concentration (μM) Extracellular Tarloxotinib Cell-associated Tarloxotinib Cell-associated Tarlox-TKI Extracellular Tarlox-TKI Figure 10. PK/PD simulations of Tarloxotinib and Tarlox-TKI in the FaDu human xenograft microvascular network. Contour plots illustrate the spatial distribution of Tarloxotinib and Tarlox-TKI in a mid-plane section of the simulated tumour microregion (top panel). Extracellular and cell-associated concentrations of Tarloxotinib and Tarlox-TKI at each tissue voxel in the simulated tumour microregion (bottom panel). The dotted line represents the five day anti-proliferative IC 50 value for Tarlox-TKI in FaDu cells (IC 50 , 0.005 μM). 230 0 Concentration (μM) Donor Receiver Tarlox-TKI is released from the hypoxic tumour compartment • NIH-III mice bearing subcutaneous SiHa tumour xenografts were treated with hypoxia probe EF5 (60 mg/kg, ip) 3 hours prior to the administration of Tarloxotinib (60 mg/kg,ip). • Tumours were collected 3 hours after Tarloxotinib administration, and snap frozen for MALDI-IMS imaging (Fig. 11A & 11C) and EF5 immunohistochemistry (Fig. 11B & 11D). • The geometry of Tarlox-TKI release (Fig. 11C) was spatially co-ordinated with regions of hypoxia (EF5 retention) (Fig. 11D). A B C B C A A B C D ZOOM Tarloxotinib is more active than conventional EGFR inhibitors 0 5 10 15 20 25 0 500 1000 1500 2000 • 48 mg/kg tarloxotinib (ip) in NIH-III mice achieves an equivalent plasma exposure to 150 mg/m 2 (qw) in the human Phase I trial. • Tarloxotinib produced 100% tumour regressions in EGFR-dependent A431 xenograft model (Fig. 13A) (<10% body weight loss for all cohorts). • Tumour EGFR autophosphorylation was silenced for over 7 days after a single dose of tarloxotinib, while afatinib failed to abrogate EGFR signaling (Fig. 13B). • Consistent with anti-tumour efficacy, tarloxotinib reduced tumour EGFR phosphorylation within 4 hours of treatment. A significant reduction in the hypoxic fraction was observed after 25 hours of treatment (Fig. 13C). A B Figure 13. Anti-tumour efficacy of Tarloxotinib and Afatinib in A431 tumour xenografts. A) Growth Delay: NIH-III mice bearing subcutaneous A431 tumors were randomly recruited to receive vehicle, daily afatinib (6mg/kg, po) or weekly tarloxotinib (48mg/kg, ip). B) Target Modulation: NIH-III mice bearing subcutaneous A431 tumors were administered a single dose of tarloxotinib (48mg/kg, ip) or eight daily doses of afatinib (6mg/kg, po, qdx8). Tumors were harvested at various time points after dosing. C) Immunohistochemistry: NIH-III mice bearing subcutaneous A431 tumors were administered vehicle or tarloxotinib (48mg/kg, ip) for 4h or 25h. EF5 was administered for 1 hour prior to harvest. Xenografts were formalin-fixed and paraffin-embedded prior to being stained for EF5 (green) and pEGFR tyr1092 (red). pEGFR EGFR β-actin Controls 0 1 3 6 15 24 48 Timepoint Timepoint C Control EF5 pEGFR RR = 60% 2/5 PD 3/5 PR RR = 100% 5/7 PR 2/7 CR Vehicle afatinib (6 mg/kg/day) tarloxotinib (48 mg/kg/week) RR = 0% 7/7 PD Controls 1 3 12 24 48 72 96 120 144 168 Tarloxotinib (48 mg/kg) Afatinib (6 mg/kg) pEGFR EGFR β-actin EF5 pEGFR EF5 pEGFR 4h post-dose 25h post-dose Tarloxotinib (48 mg/kg) Tarloxotinib has a long tumour residency and Tarlox-TKI is steadily released Plasma pharmacokinetics 1) Roskoski R Jr (2014). Pharmacol Res;79:34-74. 2) Troiani et al (2016). ESMO Open;1(5):e000088. 3) Iberri et al (2015). Oncologist; 20(12):1393-1403. 4) Calvo et al (2007). Ann Oncol; 18(4):761-7. 5) Lacouture (2006). Nat Rev Cancer; 6(10):803-12. 6) Vaupel et al (2007). Antioxid Redox Signal; 9(8):1221-35. 7) Foehrenbacher et al (2013). Front Oncol; 3:263. 8) Hicks et al (2006). J Natl Cancer Inst; 98(16):1118-28. 9) Pries et al (2009). PLoS Comput Biol; 5(5):e1000394. • Tarloxotinib is a hypoxia activated pan-ErbB inhibitor that displays an improved therapeutic index over conventional EGFR-targeted therapies. • Tarlox-TKI is more dose-potent than other clinically approved EGFR TKIs in a panel of EGFR-expressing SCCHN cell lines. • Tarloxotinib is more selective than other hypoxia-activated prodrugs and metabolism is inhibited by oxygen concentrations above 0.3 μM. • SR-PK/PD modelling demonstrates TKI release is confined to the severely hypoxic regions of tumours. • MALDI-imaging mass spectrometry supports the interpretation of the SR-PKPD modelling. • Cellular PK studies demonstrate that extreme levels of cell-associated Tarlox-TKI can be achieved relative to extracellular Tarloxotinib concentrations under pathological hypoxia. • Tarloxotinib has excellent tumour pharmacokinetic properties that predicts for exceptional anti-tumour activity. • A single dose of tarloxotinib inhibits WT EGFR signaling in the A431 xenograft for over one week afterwards. • Clinical experience with Tarloxotinib has shown lower levels of EGFR-associated toxicities (all grades, skin rashes 35% and diarrhea 21%) compared to conventional EGFR-targeted therapies (see Table 1). • Tarloxotinib is licensed from Auckland UniServices Ltd to Rain Therapeutics Inc (Fremont, CA, USA) for worldwide development. Phase 2 trials are scheduled for 2019. For details see: www.rainthera.com Cetuximab Afatinib Dacomitinib Tarlox-TKI UT-SCC-46A UT-SCC-110A UT-SCC-42B UT-SCC-42A UT-SCC-24A UT-SCC-126A UT-SCC-76B UT-SCC-1B UT-SCC-63A UT-SCC-76A UT-SCC-110B UT-SCC-74B UT-SCC-16A UT-SCC-19B UT-SCC-19A UT-SCC-60A UT-SCC-16B UT-SCC-54A UT-SCC-54B UT-SCC-74A UT-SCC-59C UT-SCC-54C 0.001 0.01 0.1 1 10 100 IC 50 value (μM) 267 85 114 99 254 44 37 137 89 89 Tarloxotinib Tarlox-TKI 31 46 118 81 41 69 56 15 34 24 39 26 Figure 4. Five day anti-proliferative IC 50 values for Tarloxotinib and Tarlox-TKI across a panel of SCCHN cell lines. Values are the mean ± SEM for at least three experiments. Deactivation ratios (IC 50 value for Tarloxotinib relative to Tarlox-TKI) are shown above the grouped bars for each cell line. IC 50 , 50% inhibitory concentration. IC 50 value (μM) • In silico spatially-resolved pharmacokinetic/pharmacodynamic (SR-PK/PD) modelling approach was used to show the potential for tumour dose intensification with the hypoxia tumour targeting approach of Tarloxotinib (Fig. 6). • Experimentally determined terms for Tarloxotinib uptake, metabolism and cytotoxicity were integrated into the mathematical framework of a published O 2 transport model to calculate the steady-state concentrations of O 2 , Tarloxotinib and Tarlox-TKI at every tissue voxel in the simulated tumour microregion using Green’s function methods. a Meta-analysis of phase III trials of EGFR inhibitors in patients with advanced squamous cell carcinoma of the head and neck (SCCHN). Plasma pharmacokinetics Tarloxotinib Tarlox-TKI 0 20 40 60 80 100 120 140 160 180 200 Time (h) 10 1 0.1 0.01 0.001 Tumour concentration (μmol/kg) TKI IC 50 0 10 20 30 40 Time (h) 100 10 1 0.1 0.01 0.001 Plasma concentration (μM) Plasma Pharmacokinetics Tumour Pharmacokinetics • NIH-III mice bearing PC-9 xenografts were administered a single dose of Tarloxotinib (15 mg/kg, ip) and snap frozen for mass spectrometry. • Tarloxotinib was determined to have a short plasma half-life (T 1/2 = 22 minutes) (Fig. 12A), but long tumour residency (T 1/2 β = 39 hours) (Fig. 12B). • Tarlox-TKI is steady released (T 1/2 = 84 hours) achieving intratumoral concentrations consistent with in vitro cytotoxicity for over seven days after administration (Fig. 12B). A B 0 100 200 300 0 2 4 40 60 80 Figure 12. Pharmacokinetics of Tarloxotinib in the plasma and tumours of NIH-III mice bearing PC-9 xenografts. Concentrations of Tarloxotinib and released Tarlox-TKI were determined by mass spectrometry. Dotted line shows the anti-proliferative IC 50 value for Tarlox-TKI in PC-9 cells (IC 50 , 0.01 μM). Tumour volume (mm 3 ) Donor Time (mins) Time (mins) Time (mins) Time (mins) Time (days) Tarloxotinib Tarlox-TKI Concentration (μM) Concentration (μM) Concentration (μM) Concentration (μM) Concentration (μM) Concentration (μM) Concentration (μM) Supported by project grant funding (14/290) from the Health Research Council of New Zealand.
Transcript of Hypoxia tumour targeting with Tarloxotinib to improve ... · Hypoxia tumour targeting with...
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Hypoxia tumour targeting with Tarloxotinib to improve clinical outcomes for patients with EGFR-dependent malignancies
Victoria Jackson-Patel, Kevin Hicks, Shevan Silva, Christopher Guise, Matthew Bull, Maria Abbattista, Angus Grey, Amir Ashoorzadeh, Jeff Smaill and Adam Patterson.
Auckland Cancer Society Research Centre, University of Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, New Zealand; Rain Therapeutics Inc, Fremont, CA, USA.
Background
Abstract # 66
Epidermal Growth Factor Receptor (EGFR, HER1) is an attractive therapeutic target for many cancer types,however current EGFR-targeted therapies have had limited clinical success.
• The EGFR signaling pathway is frequently deregulated in human cancers due to the overexpression of wild-type(WT) EGFR protein and/or its cognate ligand TGF-α 1
• Frequent aberrations in EGFR signaling have led to the clinical development of various EGFR-targeted therapies(Table 1).
• Responses have been infrequent and short-lived 2,3
- Single agent response rates rarely exceed 10%, and most patients develop acquired resistance within a yearof treatment initiation.
• Severe on-target toxicities prevent administration of the doses required to completely abrogate WT EGFRsignaling within the tumour 4,5, leading to treatment failure and disease progression.
Table 1: Clinical-stage EGFR inhibitors
Tarlox-TKI is a dose-potent EGFR inhibitor
Aerobic deactivation of Tarloxotinib
Hypoxia-selectivity of Tarloxotinib metabolism
Tumour dose intensification with Tarloxotinib
Compound Molecular Class Principal toxicities (% of pts) a
Cetuximab Monoclonal antibody Skin rash (49%)
Gefitinib Reversible EGFR TKI Skin rash (29%), diarrhoea (22%)
Erlotinib Reversible EGFR TKI Skin rash (70%), diarrhoea (32%)
Lapatinib Reversible EGFR/HER2 TKI Skin rash (33%), diarrhoea (49%)
Afatinib Irreversible pan-ErbB TKI Skin rash (65%), diarrhoea (63%)
Dacomitinib Irreversible pan-ErbB TKI Skin rash (75%), diarrhoea (84%)
in silico modelling of drug distribution
Tarloxotinib is a hypoxia-activated EGFR TKI
Figure 1. Schematic conversion of Tarloxitinib into an irreversible EGFR/HER2 TKI. Attachment of a hypoxia trigger to tarloxotinib significantly reduces the potency of theprodrug, allowing for administration at higher concentrations relative to the cognate TKI (Tarlox-TKI).
Figure 2. Five day anti-proliferative IC50 values for several clinical-stage EGFR-targeted therapies across a panel of SCCHN cell lines. Values are the mean ± SEM for at leastthree experiments. IC50, 50% inhibitory concentration.
A hypoxia-activated prodrug of an irreversible EGFR/HER2 TKI
• Studies using oxygen electrodes have demonstrated regions of low oxygen (hypoxia) in most cancer types 6
• Tarloxotinib is a prodrug designed to release an irreversible pan-ErbB TKI (Tarlox-TKI) selectively within thehypoxic regions of tumours (Fig. 1).
• The prodrug approach may allow for tumour dose intensification by circumventing the normal tissue toxicitiesassociated with conventional EGFR-targeted therapies leading to superior clinical outcomes for patients withEGFR-dependent malignancies.
• An anti-proliferative assay was performed to confirm the deactivation of Tarloxotinib relative to Tarlox-TKI underaerobic (21% O2) conditions (Fig. 4).
• Deactivation ratios ranged from 15- to 267-fold.
• Changes in EGFR signalling were examined in six representative cell lines following exposure to a clinically-relevant concentration of EGFR inhibitor (Fig. 3).
• Consistent with the anti-proliferative assays, Tarlox-TKI silenced WT EGFR signalling to a greater extent thanequimolar concentrations of afatinib, cetuximab and dacomitinib.
Figure 3. EGFR signalling inhibition across a panel of SCCHN cell lines after exposure to equimolar concentrations of afatinib, cetuximab, dacomitinib and Tarlox-TKI.Cells were exposed to 0.3 µM EGFR TKI for an hour, with TGF-α stimulation after 45 mins.
Figure 5. O2-dependence of Tarloxotinib metabolism. Solution O2 concentrations were measured using a fiberoptic oxygen probe, and Tarlox-TKI production was quantified by Massspectrometry. Values are the mean ± SEM of the intraexperimental repeats.
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• The O2-dependence of Tarloxotinib metabolism was determined in stirred cell suspensions by relating solution O2
concentrations with Tarlox-TKI production after exposure under variable gas-phase O2 concentrations (Fig. 5).
• Tarloxotinib metabolism was completely suppressed by solution O2 concentrations above 0.3 µM and through thephysiological range indicating a requirement for severe hypoxia that is unique to solid tumours.
Figure 6. Approach for the development of a SR-PK/PD model of drug distribution. A SR-PK/PD model incorporates several sub-models to calculate the spatial distribution of O2,prodrug and effector in a simulated 3D tumour microregion. Reproduced from (7).
• A cellular PK model was developed based on the concentration-time profile for Tarloxotinib and formed Tarlox-TKIin the extracellular and cell-associated volumes of single cell suspensions under supraoxic and anoxic conditions(Fig. 7A).
• Tarloxotinib was rapidly taken up to high cell-associated concentrations under supraoxic conditions (Fig. 7B).
• Anoxic exposure rapidly depleted cell-associated Tarloxotinib with a concomitant rapid, and pronounced increasein the cell-associated Tarlox-TKI concentrations (Fig. 7C).
• The cellular efflux of Tarlox-TKI was relatively slow indicating a high degree of cellular sequestration (Fig. 7C).
Figure 7. Cellular PK model for Tarloxotinib. Experimental data and model fitsfor the cellular PK model developed for Tarloxotinib in single cell suspensions(FaDu) under supraoxic (95% O2/5% O2) and anoxic (5% CO2/bal N2) conditions.Tarloxotinib and Tarlox-TKI concentrations in the extracellular and cell-associatedvolumes were quantified by Mass spectrometry.
In vivo validation of the SR-PK/PD model
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• The anti-proliferative activity of Tarlox-TKI was compared to several clinical-stage EGFR inhibitors across a panelof EGFR-expressing cancer cell lines (Fig. 2).
• Tarlox-TKI was determined to be the most dose potent, with IC50 values of up to 60-fold lower than afatinib and 8-fold lower than dacomitinib.
• The in vitro parameters for cell uptake and metabolism as determined in single cell suspensions were scaled totissue-like densities based on the transport of Tarloxotinib and formed Tarlox-TKI across multicellular layers undersupraoxic and anoxic conditions (MCLs, Fig. 8A).
• MCL cultures were a significant impediment to the transport of Tarloxotinib from the donor into the receivercompartments of diffusion chambers (Fig. 8B).
• Bioreductive metabolism further reduced Tarloxotinib transport under anoxic conditions (Fig. 8C).
• Transport of Tarlox-TKI to the receiver compartment was limited under anoxic conditions when observed up to 300minutes (Fig. 8C).
Figure 8. Flux of Tarloxotinib across MCL cultures (FaDu) under supraoxic (95% O2/5% CO2) and anoxic (5% CO2/bal N2) conditions. Tarloxotinib was added to the donorcompartment of diffusion chambers, and samples were collected from both the donor and receiver compartments for up to six hours thereafter. Tarloxotinib and Tarlox-TKIconcentrations were quantified by Mass spectrometry. Diffusion chamber schematic reproduced from (8).
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O2 distribution
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(µM
)
Vascular distribution
Fre
qu
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Figure 9. Simulations of O2 Transport in a representative mapped tumour xenograft microvascular network. Tumour oxygenation was modelled in a mapped FaDu humantumour xenograft microvascular network (9) using published parameters for O2 transport (7). The contour plot illustrates the spatial distribution of O2 in a mid-plane section of thesimulated tumour microregion. The graphs depict the distance to the nearest blood vessel and O2 concentration for every tissue voxel in the simulated tumour microregion.
Cellular PK Model
Drug Transport Model
O2 Transport Model
TarloxotinibTarlox-TKI
TarloxotinibTarlox-TKI
• An O2 transport model was developed to match the hypoxic fraction of FaDu xenografts measured by EF5 bindingusing published parameters for blood inflow, blood O2 content, tissue diffusion and consumption in arepresentative mapped tumour microvascular network using the Green’s function methods (Fig. 9).
Summary
References
Supraoxia
IC50
valu
e (µ
M)
TarloxotinibTarlox-TKI
Time (mins)
Figure 11. Matrix-Assisted Laser Desorption Ionisation Imaging Mass Spectrometry (MALDI-IMS) detection of tumor TRLX-TKI production and relationship to regions ofhypoxia. A, C) 12 mm-thick cryosections were collected on conductive glass slides. Tissue was washed with 50 mM ammonium formate and CHCA matrix was applied via vacuumsublimation followed by recrystalization 20%ACN/5%FA. MALDI images were collected using a Bruker UltrafleXtreme MALDI-TOF mass spectrometer, positive ion mode, mass range400-1000, 30 mm spatial resolution. MALDI imaging datasets were normalised to total ion current, and a MALDI image of TRLX-TKI for the combined molecular ions of the majorbromine and chlorine isotopes (C19H1879Br35ClN6O, M + H = 461; C19H1879Br37ClN6O and C19H1881Br35ClN6O, M + H = 463) was plotted using SCiLS Lab (v2015a).
A B
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cryosection
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+ + +
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m/z
Intensity (a
u)
Sample preparation and imaging
m/z 461 m/z 463
TRLX‐TKI
C19H1879Br35ClN6O
M + H = 461
TRLX‐TKI
C19H1879Br37ClN6O
C19H1881Br35ClN6O
M + H = 463
Sister section of hypoxic cell distribution(green)
D
NORMOXIA
HYPOXIA(EF5)
NORMOXIA
TRLX‐TKI(MALDI)
MALDI image of TRLX-TKI concentration(3 h after dosing tarloxotinib)
C
MIN
TRLX-TKIMAX
500m
• PK/PD simulations were performed using a drug input equivalent to the unbound plasma Cmax for Tarloxotinib aftera clinically-relevant dose (phase I MTD, 150 mg/m2, IV, qw).
• Extracellular concentrations of Tarloxotinib were relatively uniform (1.2-fold range) across the simulated tumourmicroregion (Fig. 10A).
• Consistent with bioreductive metabolism, cell-associated concentrations of Tarloxotinib declined as a function ofO2 concentration (Fig. 10B) and were reciprocated by increases in Tarlox-TKI concentration (Fig. 10C).
• Extracellular Tarlox-TKI concentrations were consistent with in vitro cytotoxicity at 98% of the tissue voxels in thesimulated tumour microregion (Fig. 10D).
PK/PD Simulations of drug distribution
0.01 0.1 1 10 1000.00
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O2 concentration (µM) O2 concentration (µM) O2 concentration (µM)O2 concentration (µM)
Co
nce
ntr
atio
n (
µM
)
ExtracellularTarloxotinib
Cell-associatedTarloxotinib
Cell-associatedTarlox-TKI
ExtracellularTarlox-TKI
Figure 10. PK/PD simulations of Tarloxotinib and Tarlox-TKI in the FaDu human xenograft microvascular network. Contour plots illustrate the spatial distribution of Tarloxotiniband Tarlox-TKI in a mid-plane section of the simulated tumour microregion (top panel). Extracellular and cell-associated concentrations of Tarloxotinib and Tarlox-TKI at each tissuevoxel in the simulated tumour microregion (bottom panel). The dotted line represents the five day anti-proliferative IC50 value for Tarlox-TKI in FaDu cells (IC50, 0.005 µM).
230 0
Co
nce
ntr
atio
n (
µM
)
Donor Receiver
Tarlox-TKI is released from the hypoxic tumour compartment
• NIH-III mice bearing subcutaneous SiHa tumour xenografts were treated with hypoxia probe EF5 (60mg/kg, ip) 3 hours prior to the administration of Tarloxotinib (60 mg/kg,ip).
• Tumours were collected 3 hours after Tarloxotinib administration, and snap frozen for MALDI-IMS imaging(Fig. 11A & 11C) and EF5 immunohistochemistry (Fig. 11B & 11D).
• The geometry of Tarlox-TKI release (Fig. 11C) was spatially co-ordinated with regions of hypoxia (EF5retention) (Fig. 11D).
A B
C
B C
A
A B C D
ZOOM
Tarloxotinib is more active than conventional EGFR inhibitors
Time (days)
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3 )
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• 48 mg/kg tarloxotinib (ip) in NIH-III mice achieves an equivalent plasma exposure to 150 mg/m2 (qw) in the humanPhase I trial.
• Tarloxotinib produced 100% tumour regressions in EGFR-dependent A431 xenograft model (Fig. 13A) (<10%body weight loss for all cohorts).
• Tumour EGFR autophosphorylation was silenced for over 7 days after a single dose of tarloxotinib, while afatinibfailed to abrogate EGFR signaling (Fig. 13B).
• Consistent with anti-tumour efficacy, tarloxotinib reduced tumour EGFR phosphorylation within 4 hours oftreatment. A significant reduction in the hypoxic fraction was observed after 25 hours of treatment (Fig. 13C).
A B
Figure 13. Anti-tumour efficacy of Tarloxotinib and Afatinib in A431 tumour xenografts. A) Growth Delay: NIH-III mice bearing subcutaneous A431 tumors were randomlyrecruited to receive vehicle, daily afatinib (6mg/kg, po) or weekly tarloxotinib (48mg/kg, ip). B) Target Modulation: NIH-III mice bearing subcutaneous A431 tumors were administereda single dose of tarloxotinib (48mg/kg, ip) or eight daily doses of afatinib (6mg/kg, po, qdx8). Tumors were harvested at various time points after dosing. C) Immunohistochemistry:NIH-III mice bearing subcutaneous A431 tumors were administered vehicle or tarloxotinib (48mg/kg, ip) for 4h or 25h. EF5 was administered for 1 hour prior to harvest. Xenograftswere formalin-fixed and paraffin-embedded prior to being stained for EF5 (green) and pEGFR tyr1092 (red).
pEGFR EGFR β-actin
Controls
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Controls
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Afatinib(6 mg/kg)
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4h post-dose 25h post-dose
Tarloxotinib (48 mg/kg)
Tarloxotinib has a long tumour residency and Tarlox-TKI is steadily released
Plasma pharmacokinetics
1) Roskoski R Jr (2014). Pharmacol Res;79:34-74.2) Troiani et al (2016). ESMO Open;1(5):e000088.3) Iberri et al (2015). Oncologist; 20(12):1393-1403.4) Calvo et al (2007). Ann Oncol; 18(4):761-7.5) Lacouture (2006). Nat Rev Cancer; 6(10):803-12.6) Vaupel et al (2007). Antioxid Redox Signal; 9(8):1221-35.7) Foehrenbacher et al (2013). Front Oncol; 3:263.8) Hicks et al (2006). J Natl Cancer Inst; 98(16):1118-28.9) Pries et al (2009). PLoS Comput Biol; 5(5):e1000394.
• Tarloxotinib is a hypoxia activated pan-ErbB inhibitor that displays an improved therapeutic index over conventionalEGFR-targeted therapies.
• Tarlox-TKI is more dose-potent than other clinically approved EGFR TKIs in a panel of EGFR-expressing SCCHNcell lines.
• Tarloxotinib is more selective than other hypoxia-activated prodrugs and metabolism is inhibited by oxygenconcentrations above 0.3 µM.
• SR-PK/PD modelling demonstrates TKI release is confined to the severely hypoxic regions of tumours.
• MALDI-imaging mass spectrometry supports the interpretation of the SR-PKPD modelling.
• Cellular PK studies demonstrate that extreme levels of cell-associated Tarlox-TKI can be achieved relative toextracellular Tarloxotinib concentrations under pathological hypoxia.
• Tarloxotinib has excellent tumour pharmacokinetic properties that predicts for exceptional anti-tumour activity.
• A single dose of tarloxotinib inhibits WT EGFR signaling in the A431 xenograft for over one week afterwards.
• Clinical experience with Tarloxotinib has shown lower levels of EGFR-associated toxicities (all grades, skin rashes35% and diarrhea 21%) compared to conventional EGFR-targeted therapies (see Table 1).
• Tarloxotinib is licensed from Auckland UniServices Ltd to Rain Therapeutics Inc (Fremont, CA, USA) for worldwidedevelopment. Phase 2 trials are scheduled for 2019. For details see: www.rainthera.com
Cetuximab Afatinib Dacomitinib Tarlox-TKI
UT
-SC
C-4
6A
UT
-SC
C-1
10A
UT
-SC
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2B
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2A
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4A
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M)
26785 114 99
254
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13789 89
TarloxotinibTarlox-TKI
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6956
1534 24
39 26
Figure 4. Five day anti-proliferative IC50 values for Tarloxotinib and Tarlox-TKI across a panel of SCCHN cell lines. Values are the mean ± SEM for at least three experiments.Deactivation ratios (IC50 value for Tarloxotinib relative to Tarlox-TKI) are shown above the grouped bars for each cell line. IC50, 50% inhibitory concentration.
IC50
valu
e (µ
M)
• In silico spatially-resolved pharmacokinetic/pharmacodynamic (SR-PK/PD) modelling approach was used to showthe potential for tumour dose intensification with the hypoxia tumour targeting approach of Tarloxotinib (Fig. 6).
• Experimentally determined terms for Tarloxotinib uptake, metabolism and cytotoxicity were integrated into themathematical framework of a published O2 transport model to calculate the steady-state concentrations of O2,Tarloxotinib and Tarlox-TKI at every tissue voxel in the simulated tumour microregion using Green’s functionmethods.
a Meta-analysis of phase III trials of EGFR inhibitors in patients with advanced squamous cell carcinoma of the head and neck (SCCHN).
Plasma pharmacokinetics
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• NIH-III mice bearing PC-9 xenografts were administered a single dose of Tarloxotinib (15 mg/kg, ip) and snapfrozen for mass spectrometry.
• Tarloxotinib was determined to have a short plasma half-life (T1/2 = 22 minutes) (Fig. 12A), but long tumourresidency (T1/2β = 39 hours) (Fig. 12B).
• Tarlox-TKI is steady released (T1/2 = 84 hours) achieving intratumoral concentrations consistent with in vitrocytotoxicity for over seven days after administration (Fig. 12B).
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Figure 12. Pharmacokinetics of Tarloxotinib in the plasma and tumours of NIH-III mice bearing PC-9 xenografts. Concentrations of Tarloxotinib and released Tarlox-TKI were determined by mass spectrometry. Dotted line shows the anti-proliferative IC50 value for Tarlox-TKI in PC-9 cells (IC50, 0.01 µM).
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Supported by project grant funding (14/290) from the Health Research Council of New Zealand .
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