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

MC225 and its phenol precursor, 5-(1-(2-hydroxy))-[3-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-propyl]-5,6,7,8-tetrahydronaphthalen (MC226) was custom synthesized by Nard Institute (Kobe, Japan) according to the methods described previously [29]. 2-Bromoethyl tosylate was purchased from ABX (Radeberg, Germany). All other chemical reagents were obtained from commercial sources. Male ddY mice were obtained from Japan SLC (Shizuoka, Japan). Sprague-Dawley [Crl:CD(SD)] rats were obtained from Charles River Laboratories (Atsugi, Japan). The animal studies were approved by the Animal Care and Use committee of the Tokyo Metropolitan Institute of Gerontology (Approval Nos. 17079, 17080, and 18016) and BoZo Research Center (Approval Nos. K180026 and K180027). Acute toxicity studies were performed under the guidelines of the “Revision of Guidelines for Single-Dose and Repeated-Dose Toxicity Studies”, Notification No. 88 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, Japan, August 10, 1993. Ames test was performed under the “Guidelines for Genotoxicity Studies of Pharmaceuticals” Yakushoku-shinsa No. 0920-1: September 20, 2012, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare (MHLW).

[¹⁸F]MC225 was synthesized as previously reported by Savolainen et al. [29] via a two-pot reaction (Fig. 2). No-carrier-added ¹⁸F-fluoride was produced by bombardment of [¹⁸O]H₂O (Taiyo Nippon Sanso, Tokyo, Japan) at 50 μA × 20 min with 20 MeV protons on a HM-20 cyclotron system (Sumitomo Heavy Industries). The target water containing ¹⁸F-fluoride was transferred directly to the V-vial. The irradiated [¹⁸O]H₂O was passed through the QMA-cartridge connected between VP34 and VP35, and the enriched water was recovered in an ¹⁸O-water recovery vial. The QMA-trapped ¹⁸F-fluoride was eluted with a reagent of R1 [mixture of aqueous 0.5 mL of K₂CO₃ (20 μmol) and a 2.0 mL MeCN solution of K.222 (40 μmol)] into the RV1. The solvents were removed azeotropically with MeCN at 100 °C under a slight flow of N₂ and vacuum over 15 min. This procedure was conducted once more with 1.0 mL MeCN from 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 [¹⁸F]fluoride was started immediately at 90 °C with N₂ 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 AcONH₄ = 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 [¹⁸F]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 N₂, the solution was transferred to a tC18 cartridge connected between the VH17–VH18 positions. [¹⁸F]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 N₂, 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).

Filter integrity was assessed by bubble point test (SLTEST000; Merck Millipore). The pH value of the injection solution was determined using a pH meter (LAQUA F-72; Horiba, Kyoto, Japan). Residual solvents in the injection solution were measured by capillary gas chromatography using a GC-2014 system with LabSolutions Software (Shimadzu, Kyoto, Japan). The radionuclide was identified by determining the half-life measurement using a Capintec CRC-55tR dose calibrator (Florham Park, NJ, USA) and the measurement of the 511 keV photopeak using a NaI(Tl) radiation detector (US-2000; Universal Giken, Odawara, Japan). Residual K.222 in the injection solution was evaluated by spot test. The limulus amebocyte lysate (LAL) test was conducted on a Toxinometer® ET-6000 (Fujifilm Wako Pure Chemical). 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 AcONH₄ solution = 60/40 (flow rate = 1 mL/min). Retention time of the authentic standard MC225 was 7.6 min.

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-week-old 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 [¹⁸F]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 14-day observation period, the rats were euthanized by exsanguination under isoflurane anesthesia, and a macroscopic analysis of the autopsy samples was performed. Three batches of [¹⁸F]MC225 were prepared and assayed after the decay-out of ¹⁸F. Solutions with decayed [¹⁸F]MC225 were individually injected intravenously into 6-week-old male and female rats (n = 3 each) at doses of 13.15 μg/5.29 mL/kg bodyweight, 9.22 μg/4.63 mL/kg bodyweight, and 1.68 μg/3.91 mL/kg bodyweight, for each of the three batches, equivalent to 200-fold of the postulated administration dose of 185 MBq [¹⁸F]MC225 for humans. After injection of decayed [¹⁸F]MC225, the rats were observed for clinical signs of toxicity for 14 days, and a macroscopic analysis was then performed as described above.

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.

[¹⁸F]MC225 (2.1 MBq/1.6 pmol) was injected intravenously into 8-week-old male ddY mice. The tracer-injected mice were housed individually in filter-paper-lined 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 ¹⁸F radioactivity with an auto-gamma counter (Hidex-AMG, Turk, Finland) and weighed. The tissue uptake of ¹⁸F 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].

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 [¹⁸F]MC225 injection met the QC criteria listed in Table 2. [¹⁸F]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 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 [¹⁸F]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.

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

The tissue distribution of radioactivity after injection of [¹⁸F]MC225 into mice is summarized in Fig. 4, and in Tables 3 and 4. The radioactivity concentrations in the blood decreased rapidly after [¹⁸F]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 (Table 5). The absorbed dose (μGy/MBq) calculated with urination at 360 min after injection was highest in the pancreas (63.8), small intestine (36.0), urinary bladder wall (32.5), and upper large intestine wall (30.4). The absorbed dose calculated without urination was highest in the pancreas (63.9), urinary bladder wall (59.6), small intestine (36.3), and upper large intestine wall (30.6). The effective dose according to the risk-weighting factors of ICRP103 [36] was estimated as 15.7 μSv/MBq (with urination at 360 min after injection) and 16.9 μSv/MBq (without urination).