<p> Loryan et al Page 1</p><p>Mechanistic understanding of brain drug disposition to</p><p> optimize the selection of potential neurotherapeutics in drug</p><p> discovery</p><p>Irena Loryan1, Vikash Sinha2, Claire Mackie3, Achiel Van Peer2, Wilhelmus Drinkenburg4, An</p><p>Vermeulen5, Denise Morrison6, Mario Monshouwer7, Donald Heald8, and Margareta</p><p>Hammarlund-Udenaes1*</p><p>1 – Translational PKPD group, Department of Pharmaceutical Biosciences, Uppsala University, </p><p>Sweden</p><p>2 – Clinical Pharmacology, Janssen Research and Development, a Division of Janssen </p><p>Pharmaceutica NV, Beerse, Belgium </p><p>3 – Pharmaceutical Development and Manufacturing Science, Janssen Research and </p><p>Development, a Division of Janssen Pharmaceutica NV, Beerse, Belgium</p><p>4 – Neuroscience Discovery, Janssen Research and Development, a Division of Janssen </p><p>Pharmaceutica NV, Beerse, Belgium</p><p>5 – Model Based Drug Development, Janssen Research and Development, a Division of Janssen </p><p>Pharmaceutica NV, Beerse, Belgium</p><p>6 – Early Drug Developability Group, CREATe (Community for Research Excellence and </p><p>Advanced Technologies), Janssen Research and Development, Division of Janssen </p><p>Pharmaceutica NV, Beerse, Belgium</p><p>7 – BA/DMPK, Janssen Research and Development, a Division of Janssen Pharmaceutica NV, </p><p>Beerse, Belgium</p><p>Mechanistic understanding of brain drug disposition Loryan et al Page 2</p><p>8 – Clinical Pharmacology, Janssen Research and Development, LLC, Titusville, USA</p><p>Corresponding author</p><p>Prof. Margareta Hammarlund-Udenaes </p><p>Box 591, 751 24 Uppsala, Sweden</p><p>+46 18 471 4300 ; +46 70 425 0485 (mobile) [email protected] </p><p>Running title: Mechanistic understanding of brain drug disposition</p><p>Mechanistic understanding of brain drug disposition Loryan et al Page 3</p><p>Supplemental Material</p><p>Table SI. LC-MS/MS measurement conditions used for analysis of brain slice samples.</p><p>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%</p><p>Mechanistic understanding of brain drug disposition Loryan et al Page 4</p><p>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 </p><p>Mechanistic understanding of brain drug disposition Loryan et al Page 5</p><p> 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</p><p>Mechanistic understanding of brain drug disposition Loryan et al Page 6</p><p>Table SII. Summary comprising the route of administration, dose and representative</p><p> exposure of single dose in vivo neuropharmacokinetic studies performed in rodents for the</p><p> set of 40 compounds.</p><p>† ‖ ID Narrative of single-dose study Kp,brainSD Kp,brain ¶ ‡ A1 SD rat, PO 10 mg/kg, AUC0-24 1.0 0.58</p><p>A2 SD rat, PO 10 mg/kg, AUC0-24 1.1 1.1</p><p>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</p><p>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</p><p>D1 SD rat, PO 10 mg/kg, AUC0-24 0.93 0.9</p><p>D2 SD rat, PO 10 mg/kg, AUC0-24 2.3 2.3</p><p>D3 SD rat, PO 10 mg/kg, AUC0-24 2.4 2.4</p><p>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</p><p>E2 SD rat, PO 10 mg/kg, AUC0-24 1.5 1.5</p><p>F1 SD rat, PO 10 mg/kg, AUC0-24 0.85 0.85</p><p>F2 SD rat, PO 10 mg/kg, AUC0-24 12 12</p><p>F3 SD rat, PO 10 mg/kg, AUC0-24 18 18</p><p>F4 SD rat, PO 10 mg/kg, AUC0-24 0.3 0.3</p><p>F5 SD rat, SC 5 mg/kg, AUC0-24 0.11 0.11</p><p>F6 SD rat, SC 20 mg/kg, AUC0-24 4.8 4.8</p><p>G1 Swiss mouse, PO 10 mg/kg, AUC0-4 0.65 0.65</p><p>G2 SD rat, PO 10 mg/kg, AUC0-24 2.6 2.8</p><p>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</p><p>G5 Swiss mouse, SC 30 mg/kg, AUC0-4 1.6 1.14</p><p>G6 Swiss mouse, PO 30 mg/kg, AUC0-4 6.4 6.4</p><p>G7 Swiss mouse, PO 10 mg/kg, AUC0-4 0.73 0.73</p><p>G8 SD rat, PO 10 mg/kg, AUC0-24 0.3 0.3</p><p>G9 Swiss mouse, PO 10 mg/kg, AUC0-24 0.2 0.2</p><p>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</p><p>Mechanistic understanding of brain drug disposition Loryan et al Page 7</p><p>¤ I4 SD rat, PO 10 mg/kg, Cmax 0.4 0.4</p><p>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)</p><p>Mechanistic understanding of brain drug disposition Loryan et al Page 8</p><p>Table SIII. Observed steady-state total compound plasma (Ctot,plasmaSS), brain (Ctot,brainSS), and</p><p> cerebrospinal fluid (CSF, Ctot,CSFSS) concentrations, brain (Kp,brainSS) and CSF (Kp,CSFSS)</p><p> partitioning coefficients as well as the ratio of single dose derived Kp,brainSD versus steady-</p><p> state brain partitioning coefficient for seven compounds.</p><p>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.</p><p>§ - 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</p><p>Mechanistic understanding of brain drug disposition</p>