Mechanistic Understanding of Brain Drug Disposition to Optimize the Selection of Potential

Loryan et al Page 1

Mechanistic understanding of brain drug disposition to

optimize the selection of potential neurotherapeutics in drug

discovery

Irena Loryan1, Vikash Sinha2, Claire Mackie3, Achiel Van Peer2, Wilhelmus Drinkenburg4, An

Vermeulen5, Denise Morrison6, Mario Monshouwer7, Donald Heald8, and Margareta

Hammarlund-Udenaes1*

1 – Translational PKPD group, Department of Pharmaceutical Biosciences, Uppsala University,

Sweden

2 – Clinical Pharmacology, Janssen Research and Development, a Division of Janssen

Pharmaceutica NV, Beerse, Belgium

3 – Pharmaceutical Development and Manufacturing Science, Janssen Research and

Development, a Division of Janssen Pharmaceutica NV, Beerse, Belgium

4 – Neuroscience Discovery, Janssen Research and Development, a Division of Janssen

5 – Model Based Drug Development, Janssen Research and Development, a Division of Janssen

6 – Early Drug Developability Group, CREATe (Community for Research Excellence and

Advanced Technologies), Janssen Research and Development, Division of Janssen

7 – BA/DMPK, Janssen Research and Development, a Division of Janssen Pharmaceutica NV,

Beerse, Belgium

Mechanistic understanding of brain drug disposition Loryan et al Page 2

8 – Clinical Pharmacology, Janssen Research and Development, LLC, Titusville, USA

Corresponding author

Prof. Margareta Hammarlund-Udenaes

Box 591, 751 24 Uppsala, Sweden

+46 18 471 4300 ; +46 70 425 0485 (mobile) [email protected]

Running title: Mechanistic understanding of brain drug disposition

Supplemental Material

Table SI. LC-MS/MS measurement conditions used for analysis of brain slice samples.

Final RT ID # † Column Initial conditions condition Mass transition (min) s A1 1 354.23→322.10 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 2.44 90% A2 7 379.95→335.8 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 2.68 90% A3 8 369.9→351.8 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 2.9 90% B1 2 362.00→159.00 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 4.7 90% B2 2 433.00→346.00 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 2.9 90% B3 1 398.10→145.00 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 3.6 90% B4 4 412.95→144.95 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a C 90%, B 10% C 10%, B 3.4 90% B5 6 477.14→459 Gemini, C18 110Å, 100x4.6 mm, 3µ b D 90%, B 10% D 10%, B 4.5 90% C1 1 448.10→215.00 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, 10% A 10%, B 3.15 90% C2 3 455.1→214.8 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 3.5 90% C3 2 417.00→189.00 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 3.5 90% D1 4 344.85→288.75 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a C 90%, B 10% C 10%, B 4.9 90% D2 3 452.1→241.8 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 4.1 90% D3 3 454.9→213.8 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 3.3 90% D4 6 381.1→325 Gemini, C18 110Å, 100x4.6 mm, 3µ b D 90%, B 10% D 10%, B 5.8 90% E1 1 339.00→246.00 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 4.33 90% E2 1 353.10→259.00 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 4.3 90% F1 4 416.9→317.7 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a C 90%, B 10% C 10%, B 3.6 90% F2 2 373.00→127.10 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 3.3 90%

F3 7 308.9→265.75 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 3.2 90% F4 8 410.9→190.9 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 3.2 90% F5 7 427.10→206.85 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 2.72 90% F6 8 312.9→255.75 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 1.9 90% G1 2 378.20→137.10 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 3.1 90% G2 3 428→137 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 3.2 90% G3 6 446.06→137.17 Gemini, C18 110Å, 100x4.6 mm, 3µ b D 90%, B 10% D 10%, B 4.8 90% G4 4 389.9→121.1 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a C 90%, B 10% C 10%, B 2.57 90% G5 3 378.9→112.05 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 3.2 90% G6 4 320.9→244.75 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a C 90%, B 10% C 10%, B 2.3 90% G7 7 421.95→58.7 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 3.3 90% G8 8 371.9→294.75 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 3.6 90% G9 8 390.9→268.75 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 3.3 90% G10 7 389.9→121.10 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a A 90%, B 10% A 10%, B 2.86 90% H1 6 263.93→96.18 Gemini, C18 110Å, 100x4.6 mm, 3µ b D 90%, B 10% D 10%, B 3.5 90% H2 6 234.03→202.9 Gemini, C18 110Å, 100x4.6 mm, 3µ b D 90%, B 10% D 10%, B 3.6 90% I1 5 376.93→175 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a C 90%, B 10% C 10%, B 3.1 90% I2 5 405.99→179.2 Eclipse XDB-CN, 4.6x150 mm, 5µ c C 90%, B 10% C 10%, B 5.1 90% I3 5 373.91→173.04 X-Bridge, C18, 50 x 4.6 mm, 3.5 µ a C 90%, B 10% C 10%, B 3.4 90% I4 5 389.85→174.9 Eclipse XDB-CN, 4.6x150 mm, 5µ c C 90%, B 10% C 10%, B 4.8 90% I5 5 421.97→179.07 Eclipse XDB-CN, 4.6x150 mm, 5µ c C 90%, B 10% C 10%, B 4.6 90% # - Cassette number † - The analysis was carried out in positive ion mode. a – Waters Corporation, Manchester, UK

b – Phenomenex, Torrance, CA c – Agilent Technologies Inc., USA RT - retention time A – 0.1% formic acid; B – Mixture of 90 : 10 acetonitrile : water; C – 10 mM Ammonium formate, pH 4; D – 10 mM Ammonium acetate, pH 10

Table SII. Summary comprising the route of administration, dose and representative

exposure of single dose in vivo neuropharmacokinetic studies performed in rodents for the

set of 40 compounds.

† ‖ ID Narrative of single-dose study Kp,brainSD Kp,brain ¶ ‡ A1 SD rat, PO 10 mg/kg, AUC0-24 1.0 0.58

A2 SD rat, PO 10 mg/kg, AUC0-24 1.1 1.1

A3 SD rat, PO 10 mg/kg, AUC0-24 0.55 0.55 B1 SD rat, SC‡‡ 10 mg/kg, 1, 2, 4 hrs post- 4.1 4.4 dose§ B2 SD rat, SC 10 mg/kg, 1, 2, 4 hrs post-dose§ 1.1 1.1 B3 SD rat, SC 10 mg/kg, 1, 2, 4 hrs post-dose§ 0.33 0.33

B4 SD rat, PO 10 mg/kg, AUC0-7 0.35 0.23 B5 Swiss mouse, SC 10 mg/kg 0.04 0.04 C1 Swiss mouse, PO 30 mg/kg, 4h post-dose 0.6 0.6 C2 Swiss mouse, PO 30 mg/kg, 4h post-dose 0.2 0.2 C3 Swiss mouse, PO 30 mg/kg, 4h post-dose 0.8 0.8

D1 SD rat, PO 10 mg/kg, AUC0-24 0.93 0.9

D4 SD rat, PO 10 mg/kg, AUC0-24 1.4 1.2 E1 SD rat, PO 10 mg/kg, ~1.5 h post-dose 0.7 0.7

E2 SD rat, PO 10 mg/kg, AUC0-24 1.5 1.5

F1 SD rat, PO 10 mg/kg, AUC0-24 0.85 0.85

F2 SD rat, PO 10 mg/kg, AUC0-24 12 12

F3 SD rat, PO 10 mg/kg, AUC0-24 18 18

F4 SD rat, PO 10 mg/kg, AUC0-24 0.3 0.3

F5 SD rat, SC 5 mg/kg, AUC0-24 0.11 0.11

F6 SD rat, SC 20 mg/kg, AUC0-24 4.8 4.8

G1 Swiss mouse, PO 10 mg/kg, AUC0-4 0.65 0.65

G2 SD rat, PO 10 mg/kg, AUC0-24 2.6 2.8

G3 Swiss mouse, SC 10 mg/kg, AUC0-4 0.6 0.6 § G4 Swiss mouse, PO 30 mg/kg, AUC0-4 0.1 0.1

G5 Swiss mouse, SC 30 mg/kg, AUC0-4 1.6 1.14

G8 SD rat, PO 10 mg/kg, AUC0-24 0.3 0.3

G10 Swiss mouse, PO 10 mg/kg, AUC0-24 0.14 0.14 ¤ H1 Swiss mouse, PO 10 mg/kg, Cmax 1.1 1.1 H2 SD rat, PO 10 mg/kg, 4 h post-dose 11 11 ¤ I1 Swiss mouse, PO/SC 30 mg/kg, Cmax 0.3 0.3 ¤ I2 Swiss mouse, SC 10 mg/kg, Cmax 1.5 1.5 ¤ I3 Swiss mouse, SC 10 mg/kg, Cmax 0.3 0.3

¤ I4 SD rat, PO 10 mg/kg, Cmax 0.4 0.4

I5 Swiss mouse, SC 10 mg/kg, Cmax 0.5 0.5 † Assessed brain partitioning coefficient Kp,brainSD after single dose administration ‖ Values of brain partitioning coefficient ratio used for the analysis. Kp,brain values highlighted in bold comes from steady-state study (see, Table SIII) ¶ SD – Sprague Dawley rats ‡ PO – oral route of administration ‡‡ SC – subcutaneous route of administration § No change in the distributional ratio over time ¤ Brain partitioning coefficient measured at maximal plasma concentration of compound (Cmax)

Table SIII. Observed steady-state total compound plasma (Ctot,plasmaSS), brain (Ctot,brainSS), and

cerebrospinal fluid (CSF, Ctot,CSFSS) concentrations, brain (Kp,brainSS) and CSF (Kp,CSFSS)

partitioning coefficients as well as the ratio of single dose derived Kp,brainSD versus steady-

state brain partitioning coefficient for seven compounds.

ID Cassette Ctot,plasmaSS Ctot,brainSS Ctot,CSFSS Kp,brainSS Kp,CSFSS Kp,brainSD/ Kp,brainSS ng/mL ng/g ng/mL A1 1§ 86±11 50.2±5 13.8±1.5 0.58±0.02 0.16±0.003 1.7 B1 2 181±45 777±82 1.9±0.4 4.4±0.8 0.01±0.004 0.93 B4 1§ 39±14 9.9±3.97 8.21±1.9 0.23±0.02 0.17±0.01 1.5 D1 3$ 141 133 1.33 0.9 0.009 1.03 D4 2 94.2±21.3 110±5 0.55±011 1.2±0.3 0.0006±0.002 1.16 G2 3$ 171 484 17 2.8 0.099 0.93 G5 4 247±41 281±42 3.8±1.1 1.14±0.04 0.015±0.002 1.4 Intravenous constant-rate infusion with flow rate of 1mL/kg ·h-1 was performed using three Sprague Dawley male rats per cassette. Compounds were dissolved in an aqueous 20% 2-hydroxypropyl-β-cyclodextrin solution. Data presented as a mean±standard deviation (n=3). The duration of intravenous infusions and concentration of the compounds in the administered solution were the following: Cassette 1 - 21.5 h, 0.25 mg/mL A1 and 0.11 mg/mL B4; Cassette 2 - 20.5 h, 0.87 mg/mL B1 and 0.10 mg/mL D4; Cassette 3 - 19.5 h, 0.135 mg/mL D1 and 0.377 mg/mL G2; Cassette 4 - intravenous bolus of 1.71 mg/mL G5 followed by 20 h CRI of 0.233 mg/mL G5.

§ - due to the block in the infusion catheter the experiment was completed with two rats $ - due to the block in the infusion catheter the experiment was completed with one rat

Mechanistic understanding of brain drug disposition