Evaluation and Application of a PET Tracer in Preclinical and Phase 1 Studies to Determine the Brain Biodistribution of Minzasolmin (UCB0599)

Purpose Minzasolmin (UCB0599) is an orally administered, small molecule inhibitor of ASYN misfolding in development as a potential disease-modifying therapy for Parkinson’s disease. Here we describe the preclinical development of a radiolabeled tracer and results from a phase 1 study using the tracer to investigate the brain distribution of minzasolmin. Procedures In the preclinical study, two radiolabeling positions were investigated on the S-enantiomer of minzasolmin (UCB2713): [11C]methylamine UCB2713 ([11C-N-CH3]UCB2713) and [11C]carbonyl UCB2713 ([11C-CO]UCB2713). Male C57 black 6 mice (N = 10) received intravenous [11C-N-CH3]UCB2713; brain homogenates were assessed for radioactivity and plasma samples analyzed by high-performance liquid chromatography. Positron emission tomography-computed tomography (PET-CT) was used to image brains in a subset of mice (n = 3). In the open-label, phase 1 study, healthy volunteers were scanned twice with PET-CT following injection with [11C]minzasolmin radiotracer (≤ 10 µg), first without, then with oral dosing with non-radiolabeled minzasolmin 360 mg. Primary objective: to determine biodistribution of minzasolmin in the human brain; secondary objectives included minzasolmin safety/tolerability. Results Preclinical data supported the use of [11C]minzasolmin in clinical studies. In the phase 1 study, PET data showed substantial drug signal in the brain of healthy volunteers (N = 4). The mean estimated whole brain total distribution volume (VT) at equilibrium across all regions of interest was 0.512 mL/cm3, no difference in VT was observed following administration of minzasolmin 360 mg. Treatment-emergent adverse events (TEAEs) were reported by 75% (n = 3) of participants. No drug-related TEAEs, deaths, serious adverse events, or discontinuations were reported. Conclusion Following positive preclinical results with the N-methyl labeled PET tracer, [11C]minzasolmin was used in the phase 1 study, which demonstrated that minzasolmin readily crossed the blood–brain barrier and was well distributed throughout the brain. Safety and pharmacokinetic findings were consistent with previous early-phase studies (such as UP0077, NCT04875962). Supplementary Information The online version contains supplementary material available at 10.1007/s11307-023-01878-7.

RM was heated to 60~75 °C for 24 hours and then cooled to 45 °C and concentrated under vacuum.DCM (133 kg) was added to the residue and then concentrated under vacuum.This process is repeated 3 times and then DCM (198 kg) is added again followed by water (62 kg) and an aqueous solution of Sodium hydroxide (30%, 33 kg).The heterogeneous mixture is stirred for 50 min and then allowed to stand for an additional 50 min.
The organic layer was separated, and the aqueous layer extracted one more time with DCM (120 kg).The combined organic phases are then washed with an aqueous NaCl solution (25%, 66 kg) and stirred with HCl (2M in water, 10 kg) for 50 min.The organic layer is separated and the washing steps with water (66 kg) and NaCl solution (66 kg) are repeated.
The organic phase is concentrated under vacuum to afford crude C12082403-B (15.8 kg).
The crude material is then purified by adding isopropyl acetate (55 kg) and MeOH (12.6 kg) and refluxing at 65~75 °C for 24 hours.The solid material is then filtered and rinsed with methyl tert-butyl ether (MTBE) (48 kg) to afford C12082403-B lot BF14001 (9.06 kg) as a hydrochloride salt.HPLC (basic condition): 98% purity.

N-[(1S
.63 g, 5.14 mmol) was dissolved in hydrochloric acid 4M in dioxane (100 mL, 400 mmol).The reaction mixture was stirred at room temperature during the weekend.The reaction was monitored by LCMS.After completion of reaction, the solid was filtered and dried under reduce pressure for 18 hours.The solid was dissolved in AcOEt (200 mL), basified with a saturated solution of NaHCO3 and extracted with a saturated solution of NaHCO3 (2x100 mL).The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure, the solid was purified by reverse phase chromatography in basic condition to afford N-[(1S)-1-(1H-indol-3-ylmethyl)pentyl]-2-piperazin-1-ylthiazole-5-carboxamide as a white solid (340 mg, yield: 14%).
LC chiral (Chiralcel OJ-H MeOH 100% -DEA 0.1% 30 °C : peak @4.21 min, ee 99% Thereafter, 4.5 mL of 0.9% saline solution was passed through the Strata cartridge and collected in the sterile vial.Finally, the dose was mixed by nitrogen bubbling for 5 seconds.

Quality control and determination of the molar activity of
[ 11 C-N-CH3]UCB2713 were performed by analytical HPLC (Supplemental Fig. 1 and 2), using an Eclipse XDB-C18 column (4.6 x 150 mm, 5 µm).Isocratic elution was performed using a mixture of ammonium formate buffer 50 mM pH 8 (solvent A) and CH3CN (solvent B) 55/45 at a flow rate of 1.5 mL/min -1 .

Preparation of the [ 11 C]carbonylation Reagents (Typical Quantities)
In a 5 mL V-bottomed glass vial were added the iodo precursor (Compound 2: 5-iodo-2-[4methylpiperazin-1-yl]thiazole, SN145369/004) (1.01 mg, 3.25 µmol), the amino precursor ([2S]-1-[1H-indol-3-yl]hexan-2-amine, B2270/084) (3.52 mg, 16.3 µmol), the Pd species Pd(π-cinnamyl)Cl2 (0.83 mg, 1.59 µmol) and the ligand Xantphos (2.47 mg, 4.28 µmol).The reagents were flushed with argon for 10 min followed by addition of anhydrous N,N-dimethylformamide (400 µL) and Et3N (10 µL) and finally flushed with argon for 5 min.was captured on a trap filled with carbosphere, preceded of a stainless-steel loop, both cooled down in a Dewar full of liquid Nitrogen.The waste gas was collected in a bag placed after the carbosphere trap.The radioactivity was monitored in the reaction vial and on the trap using the pin diodes, and in the waste gas using a dose calibrator (Capintec, Florham Park, NJ, USA).When the radioactivity in the vial and the trap reached a maximum, the system was closed.The trap filled with carbosphere was lifted up whereas the empty loop stayed in liquid nitrogen in order to capture any residual dimethylformamide.Thereafter, the trap was heated for a few seconds with a heat gun.Then, the recirculation pump was started to initiate the recirculation of [ 11 C]carbon monoxide through the reactor (maintained at 120 °C).The reaction mixture was then cooled down to 20 °C for 1 min.Thereafter, the reaction mixture was diluted with 1 mL of HPLC eluent and loaded onto the HPLC injection loop for purification.The purification and quality control of [ 11 C-CO]UCB2713 were performed as previously described for [ 11 C-N-CH3]UCB2713.

Administration of Radioligand and Collecting Samples
Each mouse received an intravenous (IV) bolus injection of [ 11 C]UCB2713 via the jugular vein.The mean (standard deviation [SD]) mass of each radiotracer dose was 1.54 (± 1.03) µg; with a radioactive dose of 17.26 (± 10.88) MBq.At pre-defined time points (40, 60, and 90 min) post-administration of [ 11 C]UCB2713, each mouse was euthanized by exsanguination, followed by cervical dislocation, with blood samples collected in heparin tubes and the brain dissected.

Brain Sample Processing and Preparation of Sample for Analysis
A sample of brain homogenate (1:1, brain:water) was assessed for radioactivity using a γ-counter.Brain homogenate was extracted with a ratio of 1:2 (homogenate:acetonitrile); a ratio of 1:1 was used for the first two animals.The mixture was vortexed for 3 min followed by centrifugation (12000 x g) for 5 min at room temperature.The extraction efficiency was calculated by:

Brain extraction efficiency = counts per minute (CPM) in supernatant (brain extract)/CPM in brain homogenate extracted
To identify the parent-retention time based on the ultraviolet (UV) chromatogram, 20 µL of a solution containing UCB2713 was added to the supernatant (350 µL), followed by 350 µL of 50 mM ammonium formate (adjusted to pH 8), then 0.5 mL of the resulting solution was injected onto the high-performance liquid chromatography (HPLC) column.
Two aliquots of this solution were counted to determine the HPLC recovery as follows: CPM(aliq.).* 500 μ • 100 Where "(aliq.)" is the radioactivity measured for the HPLC brain homogenate samples aliquot, "Valiq."Is the volume of each aliquot, "CPM fi" is the count per minute for fraction "i" and ∑ 30  = 1 is the sum of 30 fractions.

Plasma Sample Analysis
Mouse blood samples were centrifuged at 7500 x g for 3 min at 4 °C and plasma was extracted with a ratio of 1:1 (plasma:acetonitrile).The plasma extraction efficiency was calculated by: Plasma extraction efficiency = CPM in supernatant (plasma extract)/CPM in plasma extracted Samples were prepared as described above for the brain, and two aliquots of solution were counted to determine the HPLC column recovery.
A radio-chromatogram was reconstructed from the HPLC fractions collected and the percent of unchanged [ 11 C]UCB2713 was calculated: Where " fi" is the radioactivity measured for the HPLC fraction, corrected for the background radioactivity."n" is first and "m" the last HPLC fraction containing the parent compound (1 ≤ n, m ≤ 30 or 36 [where 30 or 36 are the total number of fractions collected]).reconstructed using a 2D filtered back-projection algorithm with a ramp filter; the images generated were on a 128 x 128-pixel matrix.

PET-CT image data analysis
Image processing and data analysis at Invicro (London, UK) were performed by a semi-automatic computational pipeline developed in-house using MATLAB TM R2014b.
Native PET format images were converted into Neuroimaging Informatics Technology Initiative format and were quality checked for any misalignment between PET and CT.The dynamic PET images were manually re-aligned to CT images if any misalignment was observed.Whole brain regions of interest (ROI) were defined using summed PET images (early and late stages of [ 11 C]UCB2713 uptake) and CT images.Whole brain time activity curves (TAC) were generated from the dynamic PET images using the whole brain ROI.
Radioactivity measurements for blood, plasma, and ex vivo brain were decay corrected to the time of radioligand injection and expressed as standardized uptake values (SUV) in units of g/mL: Where "Activity" is the radioactivity concentration (kBq/mL), "ID" is the injected dose of radioactivity (kBq), and "BW" is the subject body weight (g).

PET-CT Image Analysis
The TAC and total plasma data were fitted to a single compartment model to determine the influx and efflux constants K1 and k2.Because the plasma free fraction estimate is imprecise, we elected to fit the model using the total (free and bound) arterial concentration input function.The parameters of this model are related to the analogous parameters which would be estimated using an accurate estimate of the free arterial concentration using the equations below.We were able therefore to determine and present the K1 and estimated whole brain total distribution volume (VT), with relatively good repeatability between patients, and the equations below could also be used to relate the overall mean estimates back to the ideal total brain to free plasma minzasolmin (V'T) and unidirectional extraction Eu which can be calculated using the free fraction.The blood volume in the model was fixed at 5%.In this model, radiotracer which is free in tissue, non-specifically bound and specifically bound (i.e. to a target of interest) are lumped together in a single compartment, and so interchange between those states is assumed to be rapid.

[•
11 C]carbon dioxide was produced by the 14 N(p,α) 11 C nuclear reaction using a nitrogen gas target (containing 1% oxygen) pressurized to 300-350 psi and bombarded with 11 MeV protons using the Siemens RDS-111 Eclipse cyclotron (Siemens, Munich, Germany).Subsequently, [ 11 C]carbon dioxide was converted into [ 11 C]methyl iodide by catalytic reduction (H2/Ni) which gave the [ 11 C]methane intermediate followed by gas phase iodination with iodine.[ 11 C]methyl iodide was delivered to a solution of precursor (Compound 1) (1.00 mg, 2.43 µmol) in dimethylformamide (400 µL) at 15 °C using a helium stream.After delivery of [ 11 C]iodomethane, the sealed vessel was heated to 80 °C for 5 min.The reaction mixture was then cooled down to 20 °C for 2 min, diluted with 1 mL of highperformance liquid chromatography (HPLC) eluent and loaded onto the HPLC injection loop for purification.The HPLC conditions employed for the purification of [ 11 C-N-CH3]UCB2713 were as follows: • Column Agilent XDB-C18, 5 µm, 250 x 9.4 mm • Flow rate: 9.5 mL.min -1 • Ultraviolet (UV) wavelength: 254 nm • Mobile phase − Eluent A: ammonium formate buffer 100 mM pH 8 − Eluent B: acetonitrile/H2O 95/5 Isocratic conditions: eluent A/eluent B: 52/48 The semi-preparative HPLC fraction corresponding to [ 11 C-N-CH3]UCB2713 eluting at 7.9 min was collected, diluted with 20 mL of water, and passed through a Strata C18 cartridge (Strata-X 33μm Polymeric reversed phase [Phenomenex, Torrance CA, USA]).The Strata cartridge was washed with 5 mL of water.The purified [ 11 C-N-CH3]UCB2713 was eluted from the column with 0.5 mL of absolute ethanol and recovered in a sterile vial.

[
11 C]carbon dioxide was produced using a Siemens Eclipse HP cyclotron by 11 MeV proton bombardment of a target containing nitrogen and 1% oxygen.[ 11 C]carbon monoxide was produced using an Eckert and Ziegler reduction module.[ 11 C]carbon dioxide was delivered from the cyclotron and trapped at room temperature in a stainless-steel loop containing molecular sieves.The [ 11 C]carbon dioxide was then released by passing a helium flow through the loop while heating to 400 °C.The resultant [ 11 C]carbon dioxide/helium gas stream was passed through a glass tube packed with molybdenum powder at 850 °C, converting the [ 11 C]carbon dioxide to [ 11 C]carbon monoxide with any unreduced [ 11 C]carbon dioxide being trapped using Ascarite ® .The resultant [ 11 C]carbon monoxide/helium gas stream was delivered to the reaction vial at a flow rate of 10 mL/min.The time taken from end of cyclotron bombardment to complete delivery of [ 11 C]carbon monoxide to the reaction vial was 5 to 6 min.[ 11 C]carbonylation [ 11 C]carbon monoxide/helium gas stream was delivered to the 5 mL V-bottomed glass vial containing the carbonylation reagents at 120 °C (the reactor was heated to 120 °C in parallel with heating of the [ 11 C]carbon dioxide trap to 400 °C).The unreacted [ 11 C]carbon monoxide Tomography (CT) Scanning and Image Reconstruction Three mice were euthanized at the 90 min time point following radioligand injection.Prior to euthanization, these mice were scanned by PET-CT.A dynamic PET scan (Siemens Inveon DPET with docked multi-modality CT scanner) was acquired for each anesthetized mouse following IV administration of [ 11 C]UCB2713.PET images were acquired in list mode and reconstructed with increasing time frames over the duration of the scan to characterize radiotracer kinetics.The Fourier-Rebinning algorithm was applied, and each image [ 11 C]UCB2713 parent fraction = Example of HPLC chromatograms (radioactive trace, top; UV trace, bottom) obtained during HPLC analysis of [ 11 C-N-CH3]UCB2713 dose.The time delay