F]MK-6240 was radio-synthesized with the NEPTIS® Perform synthesizer and the synthesis scheme was provided by Cerveau Technology. The radio-synthesis followed the previously reported method with minor modifications [9
]. Briefly, after labeling and de-protecting, a neutralized reaction mixture was transferred to the semi preparative high-performance liquid chromatography (HPLC) unit of the synthesizer. The mixture was injected onto a semi-preparative HPLC column (Phenomenex Gemini C18, 5 µm, 10 × 250 mm), and eluted with 60:40 10 mM sodium dihydrogen phosphate buffer:acetonitrile by volume, at a flow rate of 4.0 mL/min. The eluent was monitored by UV (280 nm) and radioactivity detectors connected in series. [18
F]MK-6240 fraction was collected and transferred to the Sep-Pak t-C18 Plus Cartridge, and was washed with 15 mL of water. Trapped [18
F]MK-6240 on the cartridge was eluted with 1 mL of ethanol and diluted with 20 mL of 0.5% sodium ascorbate in saline. The solution was passed through a 0.22 µm sterilizing filter (Merck GV33) connected to a sterile 30 mL bulk vial. The bulk product could be diluted with diluent composed of 10% ethanol and 0.5% sodium ascorbate in saline. The final formulation of the [18
F]MK-6240 injection contains less than 10% ethanol and 0.5% sodium ascorbate. The synthesis was finished within 65 min. The total decay corrected yield was 13.7 ± 2.1%. The radio-ligand had high radiochemical purity (95%) and a molar activity of 149 ± 125 GBq/µmol at the time of injection (n = 6 batches). Other specifications for clinical use were all satisfied.
This study was approved by the institutional review board of the Kobe City Medical Center General Hospital (19–14), and registered with the Japan Pharmaceutical Information Center (JapicCTI-194972). Written informed consent was obtained from all participants. Four healthy elderly Japanese subjects (3 males and 1 female, 60–85 years old) and 9 Japanese patients (2 males and 7 females, 71–84 years old) suspected of mild-to-moderate AD or mild cognitive impairment (MCI) underwent the screening process described below.
All persons were free of current medical and psychiatric illnesses as determined by their medical history. They were then screened based on their history, physical examination, vital signs (blood pressure, heart rate, and temperature), electrocardiography, and urine and blood tests (whole blood count, prothrombin time/activated partial thromboplastin time, albumin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, bicarbonate, calcium, chloride, creatinine, blood glucose, phosphorus, potassium, sodium, total bilirubin, total protein, and urea nitrogen), drug screening, and a pregnancy test for females with reproductive potential. Blood infectious disease tests (antigen screening for hepatitis B and human immunodeficiency virus (HIV), and antibody screening for syphilis, HIV, and hepatitis C), brain magnetic resonance imaging (MRI) to exclude active illnesses or abnormal changes requiring medical intervention, and chest X-ray to exclude respiratory diseases were also performed as screening tests. The persons were non-smokers or had not used nicotine-containing products for at least 3 months. Then, the persons underwent Mini-Mental State Examination (MMSE) as a neuropsychological test. The healthy subjects met the following diagnostic criteria: MMSE score of ≥ 27, no history of subjective memory or other cognitive complaints and no objective evidence of memory or cognitive impairment.
The AD patients met the diagnostic criteria for dementia of the Alzheimer’s type (mild to moderate) based on the following criteria: MMSE score of ≤ 28, Clinical Dementia Rating (CDR) score of 1 or 2, positive by qualitative analysis (visual read) of [18F]Flutemetamol amyloid PET imaging, being diagnosed with mild to moderate AD by an investigator as consistent with screening evaluations and meeting the criteria for AD dementia based on DSM-IV and NINCDS-ADRDA criteria.
Exclusion criteria were as follows: those who had participated in another interventional trial within 4 weeks or had undergone an radiological examination or radiotherapy with a radiation burden over 10 mSv within 12 months before the screening visit; those who had evidence of a clinically relevant neurological disorder other than Alzheimer’s disease at screening; those who had findings of an active disease requiring medical intervention on MRI scan; those who had a history or current evidence of a depressive disorder based on DSM-IV criteria; those who had a history of alcoholism or drug dependency/abuse within 2 years before screening; those who had a history of a malignant tumor, significant multiple or severe allergies; those who were positive for hepatitis B surface antigen, hepatitis C antibodies, or HIV; those who had underwent surgery, donated or lost 1 unit of blood within 4 weeks prior to the screening visit; those who had the QTc interval ≥ 470 ms for males or ≥ 480 ms for females in their electrocardiogram; those who consumed greater than 3 glasses of alcoholic beverages per day on average in the 2 weeks before injection of [18F]MK-6240; those who consumed greater than 6 glasses of caffeinated beverages per day on average in the 2 weeks before injection of [18F]MK-6240; those using cannabis or any illicit drugs; and those who would be unable to undergo MRI or PET scanning.
Based on the above screening tests including the neuropsychological tests, 3 of 4 healthy subjects (2 males and 1 female, 60–83 years old) and 3 of 9 patients (2 males and 1 female, 71–77 years old) as AD patients were enrolled in this study and proceeded to the [18
F]MK-6240 PET scan (Table 1
Demographics of participants
All the mild cognitive impairment (MCI) persons were dropped at the time of screening, and none were enrolled.
Subjects were administered intravenously with [18
F]MK-6240 of 377.1 ± 17.4 MBq and underwent PET scans using a Discovery 690 PET/CT scanner (GE Healthcare, Milwaukee, WI, US) [10
]. The spatial resolution measured by the NEMA NU-2 procedure was 4.7 mm at a 1 cm offset position from the center of the field-of-view.
For healthy subjects, sequential whole-body PET scans were performed in the cranio-caudal direction from head to thigh. Each scan started at 1, 6, 11, 16, 25, 34, 43, 60, 150 and 200 min post injection of [18F]MK-6240. A total of 10 whole-body scans were performed in the following manner: 4 min (30 s/bed) × 3 times, 8 min (60 s/bed) × 3 times, 16 min (120 s/bed) × 2 times, and 32 min (240 s/bed) × 2 times (at 150–182 min and 200–232 min post injection). In addition to the whole-body scans, a brain PET scan was performed from 90 to 110 min after injection. The whole-body scans were divided into three sessions: from 1 to 77 min (first 8 scans), 150 to 182 min (9th scan) and 200 to 232 min (10th scan), with short breaks between the brain scan and the 9th scan, and between the 9th scan and the 10th scan, respectively. A low-dose CT for attenuation correction was performed before the first PET scan at each session. Sequential venous blood samplings of about 6 mL were performed before injection and 2, 6, 16, 35, 60, 90 min after [18F]MK-6240 injection for measurement of whole-blood and plasma radioactivity concentrations. Blood samples and vital signs were obtained before the [18F]MK-6240 injection and 240 min post injection. Electrocardiograms were recorded before the injection and 10, 120 and 240 min post injection. Urine was collected before the injection and at about 120 and 240 min post injection.
For AD patients, dynamic brain imaging from 0 to 75 min post injection was performed, followed by a static brain scan from 90 to 110 min after injection. Sequential venous blood sampling of about 6 mL was performed before injection and 2, 6, 16, 35, 60, 90 min after [18F]MK-6240 injection for measurement of the whole-blood and plasma radioactivity concentrations. Blood and urine samples, and vital signs were obtained before [18F]MK-6240 injection and 110 min post injection. Electrocardiograms were recorded before injection and 10, 45 and 110 min post injection.
Whole-body PET data analysis and dosimetry
Whole-body PET data were reconstructed using an ordered-subset expectation–maximization (OSEM) algorithm (3 iterations and 8 subsets) with time-of-flight (VUE Point FX). A Gaussian filter with 4 mm FWHM was applied to the reconstructed PET images. The matrix size was a 192 × 192 with a pixel size of 3.12 mm. The slice thickness was 3.27 mm.
Image analysis was performed with the PMOD Ver.3.4 (PMOD Technologies, Switzerland). For whole-body PET data, regions of interest (ROIs) were placed over major organs (brain, lungs, heart wall, liver, spleen, gallbladder, kidneys, urinary bladder, stomach, small intestine, upper large intestine, lower large intestine, and red bone marrow) on PET/CT images. The activity in each source organ and the remainder were divided by the injection activity to obtain the uptake as a percent of the injected activity (%ID). The residence time in each source organ was calculated from the percent injection activity of each source organ and the time course information by fitting a bi-exponential curve using OLINDA Ver.2 software (Vanderbilt University) [11
]. Urine was collected before injection and 110 min and 240 min post injection. Voided urine radioactivity was measured using a gamma counter (Auto Well Gamma System, ARC-370, Aloka Co.) and subtracted from the activity of the remainder. Dosimetry (effective dose) was calculated by the OLINDA Ver.2 using the adult male phantom. In addition to the OLINDA Ver.2, we calculated the dosimetry using the OLINDA Ver.1 for comparison to the European dosimetry data on [18
F]MK-6240. The same %ID and the same residence time in source organs were used both for the OLINDA Ver.1 and the OLINDA Ver.2, whenever applicable.
Brain PET data analysis
For brain data, PET images were reconstructed using the OSEM (VUE Point HD) with 4 iterations and 16 subsets. A Gaussian filter with 5 mm FWHM was applied to the PET images. The matrix size was 128 × 128, the pixel size was 2.73 mm, and the slice thickness was 3.27 mm. The image spatial resolution was 6 mm as assessed by visual similarity to digital phantom images applied with Gaussian filter of various FWHMs [12
The tracer uptake in each brain region was quantitatively measured with the PNEURO tool implemented in the PMOD software. The PET images were co-registered to the individual 3D T1-weighted MRI based on the normalized mutual information method. The co-registered PET images were spatially normalized to the standard Montreal Neurological Institute (MNI) T1 atlas with the same transformation parameters of MRI-based normalization. For the 0–75 min dynamic PET data, which were measured only for the patient group, the last 15-min image (60–75 min after injection) was used for image registration. The automatic anatomical-labeling (AAL) ROI template was applied to PET images in a native space using the inverse transformation parameters of the co-registration and spatial normalization. The inversely transformed ROI was masked with the gray matter segmented by individual MRI-based parcellation. Standardized uptake values (SUV) were measured in the following brain regions: frontal cortex, mesial temporal cortex, lateral temporal cortex, parietal cortex, occipital cortex, anterior cingulate, posterior cingulate, hippocampus and parahippocampus. The standardized uptake value ratio (SUVR) in each region was also calculated using the cerebellar cortex as a reference.
For brain kinetic analysis, the Logan reference tissue model (LRTM) was used to calculate the distribution volume ratio (DVRLRTM
) of each brain region using the PKIN tool [14
]. The t* (equilibration start time) and k’2
were set to 15 min and 0.144 min−1
, respectively [7
]. To confirm the SUVR as an appropriate metric for [18
F]MK-6240 specific binding, the correlation between SUVR and DVRLRTM
Metabolite analysis was performed by the literature method with minor modifications [15
]. Sequential venous blood samplings (about 6 mL) were performed before injection and 2, 6, 16, 35, 60, 90 min after [18
F]MK-6240 injection to measure whole-blood and plasma radioactivity concentrations with a gamma counter (radio-detection linearity range: 10–25,000 Bq, Auto Well Gamma System, ARC-400, Aloka Co.) and to measure parent [18
F]MK-6240 and its radioactive metabolites in the de-proteinated plasma by HPLC. HPLC system conditions were the following: HPLC module, SHIMADZU AD-10 series (pump, UV detector, column oven, controller) (SHIMADZU, Co., Ltd. Japan); radiation detector, US-2000 (1 inch × 1 inch NaI(Tl), Universal Giken Co., Ltd., Japan); radio-detection limit, 500 Bq/Peak; column, Luna C18, 10 µm, 10 × 250 mm (Phenomenex Inc., CA, US); elution, methanol: water + 0.2% (v/v) triethylamine = 75:25; flow rate, 4 mL/min; UV wavelength, 254 nm.