*4.4. Experimental Design and Pretreatment*

Female APP/PS1-21 and wild-type animals were assigned to the respective groups as shown in Table 1. Two PET imaging approaches for assessing the activity of ABCG2 at the mouse BBB were performed using the two ABCB1/ABCG2 substrate radiotracers [11C]tariquidar [28,31] and [ <sup>11</sup>C]erlotinib [35]:

(1) For PET imaging using [11C]tariquidar, groups of APP/PS1-21 and wild-type mice aged 185 ± 5 days and weighing 27.2 ± 3.0 g were pretreated two hours prior to PET with unlabeled tariquidar administered i.v. at a dose of 12 mg/kg in awake condition. Subsequently, animals were anesthetized using isoflurane/air, a catheter (Instech Lab. Inc., Plymouth Meeting, PA, USA) was introduced into a lateral tail vein and Ko143 at a dose of 5 mg/kg or Ko143 vehicle solution was additionally administered to the animals i.v. one hour prior to the start of PET acquisition. Subsequently, mice underwent a 60-min dynamic [11C]tariquidar PET scan. The doses of tariquidar and Ko143 were selected based on previous work to achieve full inhibition of ABCB1 [49] and partial inhibition of ABCG2 [28] at the mouse BBB.

(2) For PET imaging using [11C]erlotinib, no pretreatment was applied. Groups of APP/PS1-21 and wild-type mice aged 177 ± 2 days and weighing 25.1 ± 2.9 g were prepared for imaging following

the procedures described below and underwent a 60-min dynamic [11C]erlotinib PET scan, in which a pharmacological dose of unlabeled erlotinib (2 mg/kg) was co-injected with [11C]erlotinib [36].


**Table 1.** Overview of examined animal groups and numbers.

<sup>1</sup> Number in parentheses indicates drop-outs due to death/intolerability of the protocol; <sup>2</sup> At date of PET examination; 3 Injected amounts of radioactivity.
