Automated synthesis, preclinical toxicity, and radiation dosimetry of [18F]MC225 for clinical use: a tracer for measuring P-glycoprotein function at the blood-brain barrier

Introduction [18F]MC225 is a selective substrate for P-glycoprotein (P-gp) that has good metabolic stability and shows higher baseline uptake compared with other P-gp substrates such as (R)-[11C]Verapamil. Prior to clinical translation, it is necessary to perform process validation of the radiosynthesis, assessment of preclinical toxicity, and radiation dosimetry. Methods The production of [18F]MC225 was automated on a CFN-MPS200 multipurpose synthesizer. The acute toxicity of MC225 was evaluated at a dose of 2.5 mg/kg bodyweight, which is more than 10,000-fold the postulated maximum clinical dose of [18F]MC225. The acute toxicity of [18F]MC225 injection at a 200-fold dose, to administer a postulated dose of 185 MBq of [18F]MC225, was also evaluated after the decay-out of 18F. The mutagenicity of MC225 was studied by a reverse mutation test using Salmonella typhimurium and Escherichia coli (Ames test). In vivo biodistribution and dosimetry studies of [18F]MC225 were carried out in normal mice. Human dosimetry was estimated using OLINDA software. Results The mean decay-corrected yields of [18F]MC225 at end of synthesis were 13%, with > 99% radiochemical purity, > 1000 GBq/μmol molar activity, and ≤ 1.5 μg/185 MBq of total chemical contents. All process validation batches complied with the product specifications and the process was confirmed to be appropriate for the production of [18F]MC225. No acute toxicity of MC225 or [18F]MC225 injection was found. No mutagenic activity was observed for MC225. The biodistribution study demonstrated both hepatobiliary and renal excretion of radioactivity. The most critical organ was the pancreas, with (63.8 μGy/MBq) or without urination (63.9 μGy/MBq) at 360 min after injection. The estimated effective dose (μSv/MBq) with and without urination at 360 min after injection was calculated as 15.7 and 16.9, respectively. Conclusion [18F]MC225 shows acceptable pharmacological safety at the dose required for adequate PET imaging. The potential risk associated with [18F]MC225 PET imaging is well within acceptable dose limits.


Introduction
The blood-brain barrier (BBB) plays an important role in protecting the brain from xenobiotics and in maintaining homeostasis in the internal environment of the central nervous system (CNS) [1]. P-glycoprotein (P-gp) is an ATP-binding cassette transporter that is constitutively expressed in the luminal membrane of the BBB. It protects brain tissue against small hydrophobic xenobiotics that can passively diffuse through the BBB by selectively transporting them from cells into the extracellular space [2]. Hence, P-gp may also limit or prevent access of drugs such as antiepileptics, antidepressants, and anticancer agents to their target site in the brain [3]. Multiple clinical and preclinical evidence suggests that enhanced P-gp function at the BBB may be responsible for drug resistance in several diseases, including epilepsy [4][5][6][7][8], depression [9,10], and human immunodeficiency virus infection and acquired immune deficiency syndrome [11][12][13]. Furthermore, altered P-gp function at the BBB has been proposed as a possible etiology of neurodegenerative disease; for example, decreased P-gp function may decrease clearance of β-amyloid from interstitial fluid in the brain to the plasma, which would result in a predisposition for β-amyloid deposition in Alzheimer's disease [14][15][16][17][18][19][20]. A significant decrease of Pgp function in Parkinson's disease patients is likely to facilitate the accumulation of toxic compounds in the brain [21][22][23].
Several potent P-gp substrates, including (R)-Verapamil, have been labeled for imaging P-gp function with positron emission tomography (PET) [24,25]. These substrates have high affinity for P-gp and measure decreased function as increased tracer uptake by the brain. However, they are not likely to measure overexpression of P-gp because the concentration of the tracer is already almost unmeasurable at baseline [24][25][26][27]. Besides low brain uptake of (R)-[ 11 C]verapamil, another disadvantage of this radiotracer is the formation of labeled metabolites, which also act as P-gp substrates [28].
5-(1-(2-[ 18 F]fluoroethoxy))-[3-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-propyl]-5,6,7,8-tetrahydronaphthalen ([ 18 F]MC225) has recently been developed as a selective substrate for P-gp with good metabolic stability and has shown higher baseline uptake than that of other P-gp substrates [29,30] because it is a weak substrate. These are suitable properties for measuring overexpression of P-gp in the brain. Preclinical studies have proved that [ 18 F]MC225 has sufficient sensitivity to detect daily fluctuation of P-gp function in the rodent brain [31]. Very recently, a head-to-head comparison of [ 18 F]MC225 with (R)-[ 11 C]Verapamil in non-human primates showed a higher baseline uptake of [ 18 F]MC225, which makes it a suitable tracer for measuring overexpression of P-gp [32]. These findings prompted us to undertake initial evaluation of [ 18 F]MC225 in human subjects as a phase 1 study. As the first step prior to clinical application in humans, we performed a process validation of [ 18 F]MC225 radiosynthesis for clinical use and assessed the preclinical toxicity and radiation dosimetry estimated from mouse distribution data.
Parts of this study have been published as a poster in 2019 [33].

Automated synthesis
Setup of automated synthesizer [ 18 F]MC225 was produced on a CFN-MPS200 multipurpose synthesizer (Sumitomo Heavy Industries, Tokyo, Japan) with a custom-made disposable cassette and an integrated high-performance liquid chromatography (HPLC) purification unit and solid-phase extraction (SPE) formulation unit with a sterile disposable cassette. Figure 1 shows the cassette setup, including tubing connections, vials, and other disposables. The custom-made cassettes were pre-assembled in a cleanroom using disposable materials supplied by Sumitomo Heavy Industries. We selected PharMed® BOT tube (Saint-Gobain, Akron, OH, USA) because of its good general chemical resistance and excellent acid, alkali, and oxidation resistance. Table 1 lists the module setup of reagents in detail. A Sep-Pak Accell Plus QMA Light Cartridge (Waters, Milford, MA, USA) preconditioned with 10 mL 1 M K 2 CO 3 solution followed by 60 mL water for injection (Otsuka Pharmaceutical Factory, Naruto, Japan) and a Sep-Pak tC18 Plus Short Cartridge (Waters) preconditioned with 5 mL EtOH followed by 40 mL water for injection (Otsuka Pharmaceutical Factory) were installed between the VP34-VP35 and the VH17-VH18 positions, respectively.

Process description
[ 18 F]MC225 was synthesized as previously reported by Savolainen et al. [29] via a two-pot reaction (Fig. 2). Nocarrier-added 18   R2. The RV1 was then cooled to 40°C prior to the next step. The reagent of R3 [20 mg of 2-bromoethyl tosylate (61 μmol) in 1.0 mL of o-DCB] was transferred to the RV1, and distillation of the formed 2-bromoethyl [ 18 F]fluoride was started immediately at 90°C with N 2 gas flow to RV2 containing 2 mg of MC226 (5.2 μmol) and 3 mg of NaH (125 μmol) in 0.5 mL of DMF at room temperature. After radioactivity of RV2 reached a plateau (~7 min), RV2 was reacted for 5 min at 80°C. After cooling to 40°C, 2.0 mL of quenching solution in R4 was added and the reaction mixture was transferred to the reservoir of the HPLC separation unit. The product was separated by HPLC [column: Agilent Eclipse XDB-C18 (5 μm, 250 mm × 9.4 mm inner diameter; solvent: MeCN/50 mM AcONH 4 = 65/35], at a flow rate of 5 mL/min. The eluent was monitored by UV 254 nm, and radioactivity detectors were connected in series. The fraction of [ 18 F]MC225 (retention time = 9 min) was collected into dilution bottle sR3, which was preloaded with 100 mL water for injection (Otsuka Pharmaceutical Factory) containing 1 mL of 250 mg/mL ascorbate injection (Nipro Pharma, Osaka Japan). After mixing with N 2 , the solution was transferred to a tC18 cartridge connected between the VH17-VH18 positions. [ 18 F]MC225 was trapped in the cartridge, which was washed with 10 mL of water for injection (sR1), and the product was eluted with 1.4 mL of EtOH (sR2) into a Formulation Vial preloaded with 15 mL of formulation buffer solution [ascorbate injection/polysorbate 80 (Fujifilm Wako Pure Chemical, Osaka Japan)/sterile saline (Otsuka Pharmaceutical Factory) = 0.5/0.1/20]. After gentle mixing with N 2 , the solution was transferred and passed through a 0.22-μm sterilizing filter (Millex GV; Merck Millipore, Darmstadt, Germany) into an empty 30 mL sterile vial (Mita Rika Kogyo, Osaka, Japan) fitted with a sterile-filtered venting needle (Terumo, Tokyo, Japan).

Quality control
Filter integrity was assessed by bubble point test (SLTE ST000; Merck Millipore). The pH value of the injection solution was determined using a pH meter (LAQUA F- Finally, a sample of the product formulation was tested for sterility post-release using direct inoculation in accordance with the Japanese Pharmacopoeia, 17th edition. HPLC analysis was performed on a Shimadzu Prominence HPLC system equipped with a model LC-20AD pump, model SPD-20A UV absorbance detector (set at 280 nm), a GABI 3 × 3 in. NaI scintillation detector (Elysia-Raytest, Straubenhardt, Germany), and an analytical column (YMC-Pack ODS-A, 3 μm, 50 mm × 2.1 mm inner diameter) purchased from YMC (Kyoto, Japan). Operation of the Shimadzu Prominence HPLC system was controlled using Shimadzu LabSolutions software. For analysis, isocratic elution was applied using MeCN/ 50 mM AcONH 4 solution = 60/40 (flow rate = 1 mL/ min). Retention time of the authentic standard MC225 was 7.6 min.

Acute toxicity
Toxicity studies of MC225 were performed at the Kannami Laboratory, BoZo Research Center (Shizuoka, Japan). Acute toxicity was assayed in Sprague-Dawley rats [Crl:CD(SD)]. MC225 at a dose of 2.5 mg/kg bodyweight (0.5 mg/mL in 10 w/v% DMSO containing water for injection) was injected intraperitoneally into 6-weekold rats weighing 226-236 g (males, n = 5) and 149-161 g (females, n = 5). The dose of 2.5 mg/kg bodyweight is the 10,000-fold equivalent of the postulated maximum administration dose (0.25 μg/kg bodyweight) of 370 MBq [ 18 F]MC225, with the lowest molar activity of 6.3 MBq/nmol for humans weighing 40 kg. Rats were observed frequently until 30 min and then at 1, 2, 4, and 6 h after the injection on day 1, and thereafter once daily for 14 days for clinical signs of toxicity. Rats were weighed on days 1, 2, 4, 7, and 14. At the end of the 14day observation period, the rats were euthanized by exsanguination under isoflurane anesthesia, and a macroscopic analysis of the autopsy samples was performed. Three batches of [ 18 F]MC225 were prepared and assayed after the decay-out of 18  F]MC225, the rats were observed for clinical signs of toxicity for 14 days, and a macroscopic analysis was then performed as described above.

Mutagenicity
Mutagenicity tests were performed at the Tokyo Laboratory, BoZo Research Center (Tokyo, Japan). MC225 was tested for mutagenicity by the Ames test with four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and one strain of Escherichia coli (WP2uvrA), with and without the S9 mixture, at a dose range of 19.5-5000 μg/plate according to the standard method.

Dosimetry
[ 18 F]MC225 (2.1 MBq/1.6 pmol) was injected intravenously into 8-week-old male ddY mice. The tracerinjected mice were housed individually in filter-paperlined animal-rearing cages until the time of euthanasia. Mice were killed by cervical dislocation at 5, 15, 30, 60, 180, and 360 min after injection (n = 4 each). The blood was collected by heart puncture, and the tissues were harvested. Radioactivity excreted into the urine was recovered from the cage floor and by cystocentesis from the urinary bladder. The samples were measured for 18 F radioactivity with an auto-gamma counter (Hidex-AMG, Turk, Finland) and weighed. The tissue uptake of 18 F was expressed as the percentage of injected dose per organ (%ID/organ) or the percentage of injected dose per gram of tissue (%ID/g). The tissue distribution data were extrapolated to an adult male phantom using the %kg/g method [34]. The radiation absorbed dose and effective dose for human adults were estimated using OLINDA/EXM software (Vanderbilt University, Nashville, TN, USA) [35].

Automated synthesis
The three production runs had activity yields of 3527 ± 965 MBq, decay-corrected yields of 12.8 ± 2.6%, molar activity of 1576 ± 446 GBq/μmol, and radiochemical purity of 99.5 ± 0.3% ( Table 2). The average synthesis time following target bombardment was 74 min. All batches of [ 18 F]MC225 injection met the QC criteria listed in Table 2. [ 18 F]MC225 was stable for up to 2 h after end of synthesis, with acceptable appearance, pH of 6.2 ± 0.1, and radiochemical purity of 98.3 ± 0.3%. Figure 3 shows a representative semi-preparative HPLC chromatogram of the reaction mixture. The phenol precursor (MC226), which elutes at approximately 6 min, was well separated from the product (MC225), which elutes at approximately 9 min.

Acute toxicity
Acute toxicity in rats was evaluated after a single intraperitoneal injection of MC225 at a dose of 2.5 mg/kg and a single intravenous injection of one of the three lots of [ 18 F]MC225 preparations at a dose range of 1.68-13.15 μg/kg. There was no mortality in the rats during the 14-day observation period. All rat groups showed normal gains in bodyweight compared with the control animals, and no clinical signs of toxicity were observed over the 15-day period. Postmortem macroscopic examination found no abnormalities.

Mutagenicity
A bacterial reverse mutation test conducted using Salmonella thyphimurium and Escherichia coli detected no mutagenic activity for MC225.

Dosimetry
The tissue distribution of radioactivity after injection of [ 18 F]MC225 into mice is summarized in Fig. 4, and in   Tables 3 and 4. The radioactivity concentrations in the blood decreased rapidly after [ 18 F]MC225 injection. The lung and kidney showed initial high uptake (%ID/g) before decreasing gradually (Fig. 4a). Among all of the examined organs, the pancreas showed the highest radioactivity concentration, reaching 37%ID/g at 180 min, which was maintained at 33%ID/g for 360 min after injection (Fig. 4a). Excretion of radioactivity into the bladder and urine increased gradually in response to the clearance of radioactivity from the kidney, reaching 18%ID/organ (bladder + urine) at 360 min after injection (Fig. 4b). Radioactivity of the liver peaked (24%ID/organ) at 15 min after injection, before clearing (Fig. 4b). In response to the clearance of radioactivity from the liver, radioactivity levels of the small intestine peaked at 180 min (22%ID/organ), and radioactivity of the large intestine gradually increased to reach 20%ID/organ at 360 min after injection (Fig. 4b). These data demonstrate that radioactivity was excreted by both the hepatobiliary and renal urinary systems. The radiation absorbed dose was estimated from these biodistribution data (   Langendorff-perfused isolated rat hearts [37]. These in vitro cardiovascular studies demonstrated that MC225 has potential to induce vasodilator action and cardiotoxic effect over the 1000-fold equivalent postulated maximum administration dose (0.58 nmol/0.25 μg/ kg) of MC225. All three validation runs demonstrated high molar activity of [ 18 F]MC225 (> 1000 GBq/μmol) and a low amount of MC225-related chemical impurities. Because the estimated total chemical contents are ≤ 1.5 μg/185 MBq, the potential risk associated with [ 18 F]MC225 injection is considered to be within the toxicologically acceptable range [38].
The radiation absorbed dose was highest in the pancreas, followed by the urinary bladder, small intestine wall, and large intestine wall. Urination at 360 min after injection significantly decreased the absorbed dose in the urinary bladder. Except for the pancreas, all of the organs with high absorbed dose are in the excretion route.  The effective dose was well within the previously reported range (15-30 μSv/MBq) for 18 F-labeled PET radiopharmaceuticals [39]. In the case of administration of 185 MBq of [ 18 F]MC225, effective dose is estimated as 2.9 mSv (with urination at 360 min after injection) and 3.1 mSv (without urination), which is within the strict limit of 10 mSv set by the ICRP recommendations [40] and practiced in Europe. In this condition, the highest absorbed dose in the pancreas (with or without urination) is estimated as 11.8 mGy, which is also within the strict limits for individual organs (30 mSv for sensitive organs and 50 mSv for all others), as required by US Radioactive Drug Research Committee regulations [41]. There have been several reports indicating that preclinical (i.e., animal-derived) dosimetry of 18 F-labeled tracers underestimates 20-40% of the effective dose to humans [42]. Taking into account the discrepancy between extrapolated and actually determined data in humans, we calculated effective dose of [ 18 F]MC225 dividing by 0.6 to correct 40% underestimation to humans. The corrected dose (26.1 and 28.1 μSv/MBq for urination and without urination, respectively) was still within the previously reported ranges of 18 F-labeled PET radiopharmaceuticals [39]. Furthermore, administration of 185 MBq of [ 18 F]MC225 was still within the strict limit of 10 mSv (4.8 and 5.2 mSv for urination and without urination, respectively).

Conclusion
The automated synthesis of [ 18 F]MC225 for clinical use was successfully and efficiently achieved on a CFN-MPS200 multipurpose synthesizer. All three process validation batches complied with the product specifications, and the process was confirmed to be appropriate for the production of [ 18   Authors' contributions JT and GL conceived and designed the experiments. JT and TT performed the experiments. JT and MS analyzed the data. NC and GL contributed the reagents/materials/analysis tools. JT, NC, and GL wrote the paper. All authors have read and approved the final manuscript.

Funding
This work was supported in part by a Grant-in-Aid for Scientific Research (C) No. 18K07658 from the Japan Society for the Promotion of Science, and an