SUPPLEMENTARY DATA - Home | Clinical Cancer...

Osimertinib in models of EGFR-mutant NSCLC brain metastases

SUPPLEMENTARY DATA

Supplementary methods, Supplementary Tables 1–2, Supplementary Figures 1–3.

Methodology regarding study conduct, radiosynthesis of test compounds, maintenance of

cell lines, PKPD modelling, and PET microdosing data analysis; and rat QWBA

[14C]osimertinib exposure and gefitinib efficacy in a mouse brain metastases model data.

Supplementary methods

Study conduct

P-gp and BCRP substrate assessments were conducted following established practices and

standard operating procedures of Absorption Systems LP. Mouse PK studies were

conducted to AstraZeneca Research and Development (R&D) General Laboratory

Standards. The mouse brain metastases xenograft study was approved by the Institutional

Animal Care and Use Committee, and conducted in compliance with AstraZeneca Global

Standards and local regulatory requirements. The rat quantitative whole body

autoradiography study was conducted to the Development Principles of Good Laboratory

Practice. All human cell lines were cultured in vitro for implantation at specific cell inocula

following local standard protocols. The PET studies were approved by the Animal Research

Ethical Committee of the Northern Stockholm Region and were performed according to the

guidelines for planning, conduction, and documenting experimental research of the

Karolinska Institutet, and guidelines on the Care and Use of Laboratory Animals (41). The

study was also compliant with AstraZeneca policies on Bioethics and Good Statistical

Practice in animal work, and the EU Directive 2010/63/EU on the protection of animals used

for scientific purposes. Strain information for animal studies can be found in Supplementary

Table S2.

1


Test compounds

Osimertinib, its active metabolites AZ7550 and AZ5104 (Supplementary Fig. S1), gefitinib,

rociletinib (International Nonproprietary Name #: 9986; Supplementary Fig. S1), and erlotinib

were synthesized by AstraZeneca Research and Development (R&D) (Alderley Park,

Macclesfield, UK). AZ10024306 was supplied by AstraZeneca. For the P-gp and BCRP

substrate assessment, and mouse pharmacokinetic studies, all test compounds had a purity

of >95% as assessed by high-performance liquid chromatography (HPLC). All test

compounds had a purity >90% in the mouse PC9 brain metastasis xenograft study.

Radiolabeled [6-indolyl-3H]osimertinib and [2-indolyl-14C]osimertinib were synthesized by

AstraZeneca R&D with the following purities: brain binding in vitro study, [6-indolyl-

3H]osimertinib radiochemical purity >97%; specific activity 703 GBq/mmol; gefitinib purity

>95%; rat quantitative whole body autoradiography study, [2-indolyl-14C]osimertinib

radiochemical purity >98%, specific activity 4.44 MBq/mg; [2-indolyl-14C]gefitinib

radiochemical purity >98%, specific activity 1.6 MBq/mg. Radiolabeled [O-methyl-

11C]osimertinib, [O-methyl-11C]AZ5104, [O-methyl-11C]gefitinib, and [O-methyl-11C]rociletinib

for cynomolgus monkey positron emission tomography (PET) micro-dosing were

synthesized at the Karolinska Institutet on the day of the experiments with a radiochemical

purity >95%.

Maintenance of cell lines

MDR1-MDCK, BCRP-MDCK, and MDCK cells were maintained in Dulbecco’s modified

Eagle’s medium containing 10% fetal bovine serum, 1% non-essential amino acids, 1 mM

sodium pyruvate, 100 IU/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator

(37 ± 1°C, 5 ± 1% CO2). Caco2 cell lines were maintained in Dulbecco’s modified Eagle’s

medium containing 10% fetal bovine serum, 1% non-essential amino acids, 100 IU/mL

penicillin, and 100 μg/mL streptomycin, in a humidified incubator (37 ± 1°C, 5 ± 1% CO2).

2


Radiosynthesis

[3H]Osimertinib synthesis

Preparation of N1-(2-(dimethylamino)ethyl)-N4-(4-(6-iodo-1-methyl-1H-indol-3-yl) pyrimidin-

2-yl)-5-methoxy-N1-methylbenzene-1,2,4-triamine

N1-(2-(dimethylamino)ethyl)-5-methoxy-N1-methyl-N4-(4-(1-methyl-1H-indol-3-yl) pyrimidin-

2-yl)benzene-1,2,4-triamine (91 mg, 0.20 mmol) was dissolved in trifluoroacetic acid (1 mL)

and N-iodosuccinimide (55.1 mg, 0.25 mmol) was added. The reaction mixture was stirred at

room temperature for 2 hours. The reaction mixture was quenched with saturated sodium

hydrogen carbonate (10 mL) and the product was extracted with dichloromethane

(2 × 10 mL). The organics were combined, dried over magnesium sulfate, filtered, and

concentrated in vacuo. The crude product was purified by reverse phase chromatography by

elution with 10–90 % acetonitrile in ammonia solution (10 mM). The required fractions were

combined and evaporated to give N1-(2-(dimethylamino)ethyl)-N4-(4-(6-iodo-1-methyl-1H-

indol-3-yl)pyrimidin-2-yl)-5-methoxy-N1-methylbenzene-1,2,4-triamine (15 mg, 13 %) as a

brown solid.

Preparation of N1-(2-(dimethylamino)ethyl)-N4-(4-([6-3H]-1-methyl-1H-indol-3-yl)pyrimidin-2-

yl)-5-methoxy-N1-methylbenzene-1,2,4-triamine

N1-(2-(dimethylamino)ethyl)-N4-(4-(6-iodo-1-methyl-1H-indol-3-yl)pyrimidin-2-yl)-5-methoxy-

N1-methylbenzene-1,2,4-triamine (3 mg, 5.25 µmol) was dissolved in methanol (0.2 mL) and

triethylamine (7.32 µL, 0.05 mmol) was added followed by 10% palladium on carbon

(0.559 mg, 0.52 µmol). The reaction mixture was stirred under a partial atmosphere of tritium

gas (172 mbar, 65.12 GBq) for 3.5 hours. The volatile tritium was removed through

lyophilization with ethanol (2 × 5 mL) to leave crude N1-(2-(dimethylamino)ethyl)-N4-(4-([6-

3H]-1-methyl-1H-indol-3-yl)pyrimidin-2-yl)-5-methoxy-N1-methylbenzene-1,2,4-triamine

(1.57 mg, 2.85 GBq)

3


Preparation of N-[2-[2-dimethylaminoethyl(methyl)amino]-4-methoxy-5-[[4-(1-methyl-[6-3H]indol-

3-yl)pyrimidin-2-yl]amino]phenyl]prop-2-enamide, [3H]osimertinib

A solution of acryloyl chloride (1.28 µL, 0.02 mmol) in dichloromethane (85 µL) was added

dropwise to a solution of N1-(2-(dimethylamino)ethyl)-N4-(4-([6-3H]-1-methyl-1H-indol-3-yl)

pyrimidin-2-yl)-5-methoxy-N1-methylbenzene-1,2,4-triamine (1.57 mg, 2.85 GBq) in

dichloromethane (1 mL). The resulting mixture was stirred at room temperature for

1.5 hours. The reaction mixture was concentrated by freeze drying and stored in ethanol

(5 mL). A portion of the ethanol solution (3 mL) was concentrated and purified by reverse

phase preparative chromatography on two systems: system 1 (Xbridge, C18, 5 µm,

4.6 × 100 mm, acetonitrile: 0.1% aqueous formic acid gradient, 1 mL/min) followed by

system 2 (Xbridge, C18, 5 µm, 4.6 × 100 mm, acetonitrile: 10 mM aqueous ammonia

gradient, 1 mL/min). Pure fractions were combined and freeze dried to give N-[2-[2-

dimethylaminoethyl(methyl)amino]-4-methoxy-5-[[4-(1-methyl-[6-3H]indol-3-yl)pyrimidin-2-

yl]amino]phenyl]prop-2-enamide, [3H]osimertinib, 0.18 GBq, which was stored as an ethanol

solution (5 mL) at -20°C. The radiochemical purity was 97.6% by high performance liquid

chromatography (HPLC) and the specific activity by mass spectrometry was measured at

703 GBq/mmol.

[14C]Osimertinib synthesis

Preparation of 1-methyl-1H-[2-14C]indole

[2-14C]Indole (13.6 GBq, ~2.22 GBq/mmol, 6.11 mmol) was dissolved in anhydrous N,N-

dimethylformamide (17 mL) and cooled to 0°C. Sodium hydride (263 mg, 6.6 mmol) was

added portion wise. The reaction was stirred at room temperature for 1 hour. The reaction

was cooled to 0°C and a solution of iodomethane (590 µL, 1.35 g, 9.5 mmol) in N,N-

dimethylformamide (4 mL) was added dropwise. After complete addition, the reaction was

stirred at room temperature overnight. The reaction was poured onto ice and the product

extracted into ethyl acetate (× 3). The combined ethyl acetate extracts were dried over

4


magnesium sulfate, filtered, and evaporated to give crude product. The crude material was

purified by flash silica chromatography eluting with ethyl acetate:hexane mixtures. The pure

fractions were combined and concentrated in vacuo to give 1-methyl-1H-[2-14C]indole (9.62

GBq, 71%).

Preparation of 3-(2-chloropyrimidin-4-yl)-[2-14C]-1-methyl-1H-indole

2,4-dichloropyrimidine (680 mg, 4.56 mmol) was suspended in 1,2-dimethoxyethane (6 mL)

and stirred at room temperature under nitrogen for 10 minutes. Aluminum chloride (610 mg,

4.7 mmol) was added in one portion and the suspension stirred for a further 10 minutes.

1-methyl-1 H-[2-14C]indole (9.62 GBq) was added at room temperature. The reaction was

heated to 80°C for 2.5 hours. The reaction was allowed to cool to room temperature and

added dropwise to a stirred ice/dichloromethane mixture. The dichloromethane layer was

separated and the aqueous layer extracted with further dichloromethane (× 3). The

combined dichloromethane extracts were dried over magnesium sulfate, filtered, and

evaporated to give crude product. The crude material was purified by flash silica

chromatography eluting with dichloromethane. Pure fractions were combined and

concentrated in vacuo to give 3-(2-chloropyrimidin-4-yl)-[2-14C]-1-methyl-1H-indole

(7.44 GBq, 77%).

Preparation of N-( 4-fluoro-2-methoxy-5-nitrophenyl)-4-([2-14C]-1-methyl-1H-indol-3-

yl)pyrimidin-2-amine

3-(2-chloropyrimidin-4-yl)-[2-14C]-1-methyl-1H-indole (7.44 GBq, 3.41 mmol) and 4-fluoro-2-

methoxy-5-nitroaniline (665 mg, 3.57 mmol) were combined in 2-pentanol (15 mL).

p- toluenesulfonic acid monohydrate (872 mg, 4.58 mmol) was added and the reaction

heated to 105°C under nitrogen for 3 hours. The reaction was allowed to cool to room

temperature, diluted with additional 2-pentanol (20 mL), and filtered. The bright yellow solid

was washed with 2-pentanol (2 × 20 mL) and dried in a high vacuum desiccator overnight to

5


give N-(4-fluoro-2-methoxy-5-nitrophenyl)-4-([2-14C]-1-methyl-1H-indol-3-yl)pyrimidin-2-

amine (6.48 GBq, 87%).

Preparation of N1-(2-(dimethylamino)ethyl)-5-methoxy-N1-methyl-N4-(4-([2-14C]-1-methyl-

1H-indol-3-yl)pyrimidin-2-yl)-2-nitrobenzene-1,4-diamine

N,N-diisopropylethylamine (1.3 mL) and N,N,N'-trimethylethylenediamine (680 µL) were

added to N-(4-fluoro-2-methoxy-5-nitrophenyl)-4-([2-14C]-1-methyl-1H-indol-3-yl)pyrimidin-2-

amine (6.48 GBq, 2.97 mmol) in N,N-dimethylacetamide (11 mL) at room temperature. The

bright yellow suspension was heated to 105°C under nitrogen for 3 hours. The reaction was

allowed to cool to room temperature and added dropwise to cold water. The product was

extracted into dichloromethane (× 3). The combined dichloromethane extracts were washed

with water, dried over sodium sulfate, filtered, and evaporated. The crude material was

purified by flash silica chromatography eluting with dichloromethane:methanol mixtures. The

pure fractions were combined and concentrated in vacuo to give N1-(2-

(dimethylamino)ethyl)-5-methoxy-N1-methyl-N4-(4-([2-14C]-1-methyl-1H-indol-3-yl)pyrimidin-

2-yl)-2-nitrobenzene-1,4-diamine as a bright orange solid (5.62 GBq, 87%).

Preparation of N1-(2-dimethylaminoethyl)-5-methoxy-N1-methyl-N4-[4-(1-methyl-[2-

14C]indol-3-yl)pyrimidin-2-yl]benzene-1,2,4-triamine

Palladium (10% on carbon, 50% wet paste) (100 mg) was added to a solution of N1-(2-

(dimethylamino)ethyl)-5-methoxy-N1-methyl-N4-(4-([2-14C]-1-methyl-1H-indol-3-yl)pyrimidin-

2-yl)-2-nitrobenzene-1,4-diamine (5.62 GBq, 2.58 mmol) in methanol (60 mL) at room

temperature. The suspension was stirred under a hydrogen atmosphere for 4 hours. The

reaction was filtered through Celite, which was washed with additional methanol. The

methanol was removed by rotary evaporation and the dark brown crystalline solid dried by

high vacuum desiccation to give N1-(2-dimethylaminoethyl)-5-methoxy-N1-methyl-N4-[4-(1-

methyl-[2-14C]indol-3-yl)pyrimidin-2-yl]benzene-1,2,4-triamine (5.62 GBq, 99%).

6


Preparation of N-[2-[2-dimethylaminoethyl(methyl)amino]-4-methoxy-5-[[4-(1-methyl-[2-

14C]indol-3-yl)pyrimidin-2-yl]amino]phenyl]prop-2-enamide, [14C]osimertinib

A solution of acryloyl chloride (0.069 mL, 0.85 mmol) in dichloromethane (2 mL) was added

dropwise to a stirred suspension of N1-(2-dimethylaminoethyl)-5-methoxy-N1-methyl-N4-[4-

(1-methyl-[2-14C]indol-3-yl)pyrimidin-2-yl]benzene-1,2,4-triamine (1.70 GBq, 0.77 mmol) in

dichloromethane (10 mL) at -5°C. The resulting mixture was stirred at -5°C for 30 minutes.

Saturated sodium hydrogen carbonate solution (6 mL) was added and the organic layer was

separated and evaporated to afford the crude product. The crude product was purified by

reverse phase preparative chromatography (Xterra C8 RP column, 150 x 19 mm,

acetonitrile:10 mM aqueous ammonia gradient, 20 mL/min). Pure fractions were combined

and freeze dried to give N-[2-[2-dimethylaminoethyl(methyl)amino]-4-methoxy-5-[[4-(1-

methyl-[2-14C]indol-3-yl)pyrimidin-2-yl]amino]phenyl]prop-2-enamide, [14C]osimertinib as a

beige solid (140 mg, 0.6 GBq, 36%). The radiochemical purity was 98.2% by HPLC and the

gravimetric specific activity was measured at 120 µCi/mg (4.44 MBq/mg).

Preparation of [11C]osimertinib, [11C]AZ5104, [11C]gefitinib, and [11C]rociletinib for PET micro-

dosing studies

Radiolabeled compounds were prepared in a one-step reaction using an established method

for carbon-11 methylation at the Karolinska Institutet using [11C]methyl triflate (1). Semi-

preparative HPLC was performed using a reverse phase ACE 5 C-18L column (250 ×

10 mm, 5 μm, Advanced Chromatography Technologies). The column outlet was connected

with an UV absorbance detector ( = 254 nm) in series with a detector for radioactivity. The

radiochemical purity and identity of the formulated radiolabeled products were determined by

analytic reverse phase HPLC using a ZORBAX Eclipse XDB-C18 column (150 × 3 mm,

5 m; Agilent) and an UV absorbance detector ( = 254 nm) in series with a -flow detector

for radioactivity (Beckman).

7


Preparation of N-[2-[2-dimethylaminoethyl(methyl)amino]-4-[11C]methoxy-5-[[4-(1-methyl-

indol-3-yl)pyrimidin-2-yl]amino]phenyl]prop-2-enamide, [O-methyl-11C]osimertinib

[11C]methyl triflate was transferred to a well-agitated suspension of N-[2-[2-

(dimethylamino)ethyl-methyl-amino]-4-hydroxy-5-[[4-(1-methylindol-3-yl)pyrimidin-2-

yl]amino]phenyl]prop-2-enamide (0.7 mg, 1 µmol) and sodium hydroxide (5 µL, 0.5 M) in

acetone (400 µL). After completed transfer, the crude reaction mixture was diluted with

mobile phase acetonitrile:ammonium formate (0.1 M) 42:58 (600 µL) and purified by semi-

preparative HPLC. The collected fraction containing the title compound (retention time =

8 minutes) was evaporated to dryness and re-dissolved in a solution of ethanol (5% v/v) in

physiologically buffered saline (PBS, pH 7.4, 6.5 mL). Before sterile filtration, Tween 80

(1 mL 1.5%, Polysorbate, Merck Millipore) was added to avoid loss of product on sterile filter.

The formulated product was sterilized by membrane filtration (0.22 µm, Millipore) to yield the

final product in a solution ready for injection. [O-methyl-11C]osimertinib co-eluted with an

unlabeled reference standard of osimertinib on HPLC. Its identity was further confirmed by

tandem mass spectrometry (MS/MS) analysis of the carrier associated with [O-methyl-

11C]osimertinib and comparison with an authentic reference standard. The radiochemical

purity was >95% by HPLC and the determined average specific activity at injection was 119

GBq/μmol.

Preparation of N-[2-[2-(dimethylamino)ethyl-methyl-amino]-5-[[4-(1H-indol-3-yl)pyrimidin-2-

yl]amino]-4-[11C]methoxy-phenyl]prop-2-enamide, [O-methyl-11C]AZ5104

[O-methyl-11C]AZ5104 was prepared from N-[2-[2-(dimethylamino)ethyl-methyl-amino]-4-

hydroxy-5-[[4-(1H-indol-3-yl)pyrimidin-2-yl]amino]phenyl]prop-2-enamide in a similar way as

for [O-methyl-11C]osimertinib. For semi-preparative HPLC, acetonitrile:ammonium formate

(0.1 M) 33:67 was used with a retention time of 11 minutes. The identity of [O-methyl-

11C]AZ5104 was confirmed by analytical HPLC and MS/MS as above. The radiochemical

8


purity was >96% by HPLC and the determined average specific activity at injection was

246 GBq/μmol.

Preparation of N-(3-chloro-4-fluoro-phenyl)-7-[11C]methoxy-6-(3-

morpholinopropoxy)quinazolin-4-amine, [O-methyl-11C]gefitinib

[O-methyl-11C]gefitinib was prepared from 4-(3-chloro-4-fluoro-anilino)-6-(3-

morpholinopropoxy)quinazolin-7-ol in a similar way as for [O-methyl-11C]osimertinib. For

semi-preparative HPLC, acetonitrile:ammonium hydroxide (0.3%) 1:1 was used, with a

retention time of 6 minutes. Tween 80 was not added before sterile filtration. The identity of

[O-methyl-11C]gefitinib was confirmed by analytical HPLC and MS/MS as above. The

radiochemical purity was >96% by HPLC and the determined average specific activity at

injection was 145 GBq/μmol.

Preparation of N-[3-[[2-[4-(4-acetylpiperazin-1-yl)-2-[11C]methoxy-anilino]-5-

(trifluoromethyl)pyrimidin-4-yl]amino]phenyl]prop-2-enamide, [O-methyl-11C]rociletinib

[O-methyl-11C]rociletinib was prepared from N-[3-[[2-[4-(4-acetylpiperazin-1-yl)-2-hydroxy-

anilino]-5-(trifluoromethyl)pyrimidin-4-yl]amino]phenyl]prop-2-enamide in a similar way as for

[O-methyl-11C]osimertinib. For semi-preparative HPLC, acetonitrile:ammonium formate

(0.1 M) 42:58 was used, with a retention time of 11 minutes. The identity of [O-methyl-

11C]rociletinib was confirmed by analytical HPLC and MS/MS as above. The radiochemical

purity was >95% by HPLC and the determined average specific activity at injection was

446 GBq/μmol.

Cynomolgus monkey PET micro-dosing data analysis

Magnetic resonance images (MRIs) of the monkey brains had been previously obtained

using a 1.5 T General Electric Signa (GE, Milwaukee, WI, USA) system (2). The region of

interest (ROI) for the whole brain was manually delineated in T1-weighted MRIs using an in-

house image analysis software (3). Brain MRIs were co-registered to the averaged brain

9


PET images using SPM5 (Wellcome Department of Imaging Neuroscience, UK). Time-

activity curves were generated by applying the pooled brain ROI to PET images using the

affine transformation matrix acquired from co-registration of the MRI. The radioactivity

concentration was calculated for each sequential frame, corrected for radioactive decay, and

plotted versus time. The radioactivity concentration in the ROI for the whole brain was

multiplied with the whole brain ROI volume, divided by the radioactivity injected, and

multiplied by 100 to obtain the percentage of radioactivity in brain. Time-activity curves for

radioactivity in brain were corrected for radioactivity in the cerebral blood using the

radioactivity concentrations obtained from arterial blood and assuming that the cerebral

blood volume is 5% of the total brain volume (4, 5). The area under the curve for brain as

well as the blood radioactivity concentration-time curve between 0 and 90 minutes after

injection were calculated by the linear trapezoidal rule.

Pharmacokinetic-pharmacodynamic modelling

The pharmacokinetics of parent and active metabolite are described by a semi-physiological

compartmental model. The important assumptions are that the parent is cleared solely in the

liver and that the fraction converted to metabolite is constant across different routes of

dosing.

The reversible interaction between the molecule and receptor is characterised by binding

affinity, represented by CPU50 and CMU50 for free parent and metabolite respectively. The

natural turnover and re-synthesis of receptor is represented by Krec and the rate of drug

induced de-activation is described by Kbind

dpEGFRdt

=K rec (1−pEGFR )−pEGFR .Kbind(Cp ,u

CPU 50+

Cm,u

CMU 50

1+C p ,u

CPU 50+

Cm,u

CMU 50)

Xenografted tumours are assumed to spherical and be composed of a cycling compartment

(Sa) near the outside of the tumour (and so near host vasculature) and a non-cycling core

10


(Ca). The cycling compartment is assumed to increase in size at a rate proportional (Kgrow) to

the size of the compartment.

dSa

dt=K grow Sa−f kill Sa+J TransferV a

dCa

dt=−JTransfer V a

To maintain the distance allowed (Rdiff) of the proliferating compartment from the edge of the

tumour, cells are transferred (JTransfer) between the proliferating and non-proliferating

compartments as the tumour grows of shrinks at a rate proportional to the difference

between the current core volume (Vcore) and the size required for the correctly sized

proliferating compartment (Vcore,Target).

V Core ,T arg et=4 Π3 (RTumour−Rdiff )3

JTransfer=K trans (V core−V Core , t arg et ) ACore

On reduction of pEGFR, the rate of cell death is assumed to increase in the tumour and is

proportional to pEGFR reduction. The rate of cell death is assumed to be independent of the

time pEGFR is reduced, however the resulting efficacy is an integral of the rate of cell death

and so duration of pEGFR will be important for efficacy.

f kill (pEGFR )=Emax .( n[1−pEGFR ]−1n−1 )

This model was used to find the dose and time dependency of pharmacokinetics,

pharmacodynamics and efficacy in a range of subcutaneous xenografted mouse models

including PC9. By taking into account the contributions of parent and metabolite to efficacy

we can then understand the implications of the observed brain distribution of both molecules.

The relationship between human osimertinib and AZ5104 PK, and the preclinical PKPD

activity relationship, were interrogated with respect to changing PK profiles in the mouse.

The mouse PKPD model was simulated for a range of fixed daily doses, with increasing

11


dosing frequency of up to 10 times per day. Increasing dosing frequency results in a flatter

PK profile, more similar to human. It was concluded that a flatter PK profile was, for the

same daily dose, at least as effective as a short half-life profile, despite the maximum

concentrations simulated being lower. Adjusting parent clearance and rate of absorption,

and placing variability on both parent (100%) and metabolite (10%) clearance, were

sufficient to describe the observed PK observed in patients. The model was adjusted to

predict efficacy in brain metastases based on an estimation of free brain exposure of

osimertinib with AZ5104 being excluded from the brain.

Modeling and simulation were carried out in ACSLX v2.5.0.6 on a Lenovo S20 workstation.

Data were analyzed sequentially, with the PK being analyzed prior to tumor growth data.

12


Supplementary references

1. Andersson J, Truong P, Halldin C. In-target produced [11C]methane: increased specific

radioactivity. Appl Radiat Isot 2009;67:106-10.

2. Schou M, Varnäs K, Jucaite A, Gulyas B, Halldin C, Farde L. Radiolabeling of the

cannabinoid receptor agonist AZD1940 with carbon-11 and PET microdosing in non-human

primate. Nucl Med Biol 2013;40:410-4.

3. Roland PE, Graufelds CJ, W. Hlin J, Ingelman L, Andersson M, Ledberg A, et al. Human

brain atlas: For high-resolution functional and anatomical mapping. Hum Brain Mapp

1994;1:173-84.

4. Farde L, Eriksson L, Blomquist G, Halldin C. Kinetic analysis of central [11C]raclopride

binding to D2-dopamine receptors studied by PET – a comparison to the equilibrium

analysis. J Cereb Blood Flow Metab 1989;9:696-708.

5. Leenders KL, Perani D, Lammertsma AA, Heather JD, Buckingham P, Healy MJ, et al.

Cerebral blood flow, blood volume and oxygen utilization. Normal values and effect of age.

Brain 1990;113( Pt 1):27-47.

6. Cross DA, Ashton SE, Ghiorghiu S, Eberlein C, Nebhan CA, Spitzler PJ, et al. AZD9291,

an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung

cancer. Cancer Discov 2014;4:1046-61.

13


Supplementary Table S1. Osimertinib, rociletinib, and afatinib permeability across Caco2 cell monolayers

Test compound Concentration Papp (a–b)

[10-6 cm/s]Recovery (a–b) [%]

Papp (b–a)[10-6 cm/s]

Recovery (b–a) [%]

Efflux ratio

(b–a)/(a–b)

Osimertinib50 μM 3.35 ± 0.69 15.8 ± 2.62 1.25 ± 0.16 53.1 ± 2.11 0.37

10 μM 2.58 ± 0.46 15.7 ± 3.45 1.68 ± 0.36 47.1 ± 2.69 0.65

1 μM NC 20.9 ± 0.99 NC 51.4 ± 3.52 NC

Osimertinib in the

presence of 10 µM

minoxidil

1 μM NC 20.7 ± 2.03 NC 56.3 ± 4.18 NC

Rociletinib 10 μM 3.21 ± 1.48 57.8 ± 11.02 12.67 ± 1.82 46.2 ± 11.2 4.61

Afatinib 10 μM 1.25 ± 0.06 45.3 ± 1.53 14.3 ± 1.46 53 ± 1.22 11.49

Atenolol 10 μM 0.45 ± 0.09 102 ± 7.75 0.73 ± 0.12 108 ± 4.26 1.64

Minoxidil 10 μM 7.21 ± 1.09 110 ± 5.71 7.16 ± 1.29 106 ± 4.28 0.99

Minoxidil in the presence

of 1 μM osimertinib

10 μM 5.56 ± 1.28 110 ± 9.87 7.30 ± 0.87 105 ± 4.25 1.31

Digoxin 10 μM 0.31 ± 0.09 100 ± 4.50 18.4 ± 1.39 104 ± 6.39 59.4

Papp and recovery are expressed as mean value ± standard deviation from seven monolayers for digoxin, eight monolayers for atenolol, minoxidil, osimertinib, and rociletinib, and two monolayers for afatinib.

Caco2, colon carcinoma; NC, not calculable (the concentrations of some receiver samples were below the lower limit of quantification); Papp, apparent permeability coefficient.

14


Supplementary Table S2. Strain information

Experiment Strain Gender Age Weight Source

PET micro-dosing

Cynomolgus monkey Female

(ID#0702004)7.5 years 6.99 kg

Astrid Fagraeus Laboratory, Karolinska Institutet, Sweden


(ID#0610010)8 years 6.93 kg


(ID#0407352)10 years 5.9 kg

Cynomolgus monkey Male

(ID#0409429)11 years 6.35 kg

QWBA

Lister-hooded pigmented rat

Male ~8 weeks222–255

gHarlan Laboratories, UK

Pievald Virol Glaxo pigmented rat

Male 7–8 weeks180–234

g

Mouse PK

CB17 Sever combined immunodeficient (SCID) mouse

Female 8–10 weeks16.17–23.09 g

Charles River, France

NU/NU nude mouse Female 8–10 weeks 20–26 g Alderley Park, UK

Mouse brain metastasis xenograft

NU/NU nude mouse Female 6–8 weeks 20–27 gVital River Laboratory Animal

Technology, China

PET, positron emission tomography; PK, pharmacokinetics; QWBA, quantitative whole body autoradiography.

15


Supplementary Figure S1. Structures of osimertinib (6), its plasma metabolites AZ5104

and AZ7550, rociletinib, gefitinib, and afatinib.

16


Supplementary Figure S2. Representative whole body autoradiogram of section through a male Lister-hooded rat at 1 hour after single oral administration of [14C]osimertinib

17


Supplementary Figure S3. Tumor bioluminescence in a PC9 epidermal growth factor receptor exon 19 deletion mutation-positive mouse brain metastases model during treatment with gefitinib 6.25 mg/kg once daily (QD), or vehicle.

18

SUPPLEMENTARY DATA - Home | Clinical Cancer...

Documents

Transcript of SUPPLEMENTARY DATA - Home | Clinical Cancer...