Translation of HDAC6 PET Imaging Using [¹⁸F]EKZ-001–cGMP Production and Measurement of HDAC6 Target Occupancy in Nonhuman Primates

Abstract

Histone deacetylase 6 (HDAC6) is a multifunctional cytoplasmic enzyme involved in diverse cellular processes such as intracellular transport and protein quality control. Inhibition of HDAC6 can alleviate defects in cell and rodent models of certain diseases, particularly neurodegenerative disorders, including Alzheimer's disease and amyotrophic lateral sclerosis. However, while HDAC6 represents a potentially powerful therapeutic target, development of effective brain-penetrant HDAC6 inhibitors remains challenging. Recently, [ 18 F]EKZ-001 ([ 18 F]Bavarostat), a brain-penetrant positron emission tomography (PET) radioligand with high affinity and selectivity toward HDAC6, was developed and evaluated preclinically for its ability to bind HDAC6. Herein, we describe the efficient and robust fully automated current Good Manufacturing Practices (cGMP) compliant production method. [ 18 F]EKZ-001 quantification methods were validated in nonhuman primates (NHP) using full kinetic modeling, and [ 18 F]EKZ-001 PET was applied to compare dose-occupancy relationships between two HDAC6 inhibitors, EKZ-317 and ACY-775. [ 18 F]EKZ-001 is cGMP produced with an average decay-corrected radiochemical yield of 14% and an average molar activity of 204 GBq/μmol. We demonstrate that a two-tissue compartmental model and Logan graphical analysis are appropriate for [ 18 F]EKZ-001 PET quantification in NHP brain. Blocking studies show that the novel compound EKZ-317 achieves higher target occupancy than ACY-775. This work supports the translation of [ 18 F]EKZ-001 PET for first-in-human studies.

Clinical validation of the novel HDAC6 radiotracer [18F]EKZ-001 in the human brain

“…The radiotracer was obtained with high radiochemical purity (> 98%) in a total synthesis time of 2 h with an average molar activity of 204 ± 175 GBq/μmol ( n = 23, including the validation runs) at the end of the synthesis process. Detailed synthesis methods are described in Celen et al [ 57 ].…”

“…Recently, preclinical HDAC6-selective radiotracers have been developed [ 54 – 56 ], including the highly brain-penetrant [ 18 F]Bavarostat (hereafter called [ 18 F]EKZ-001) [ 55 ]. In biochemical assays EKZ-001 displayed low nanomolar potency (< 20 nM) for HDAC6 and > 100-fold selectivity for HDAC6 over class I HDACs [ 55 , 57 ]. In human neural progenitor cells, EKZ-001 inhibited deacetylation of the HDAC6 substrate α-tubulin, but not deacetylation of the class I HDAC substrates (histone H3 on lysine 9 and histone H4 on lysine 12) [ 55 ].…”

“…In human neural progenitor cells, EKZ-001 inhibited deacetylation of the HDAC6 substrate α-tubulin, but not deacetylation of the class I HDAC substrates (histone H3 on lysine 9 and histone H4 on lysine 12) [ 55 ]. In in vitro autoradiography competition assays, [ 18 F]EKZ-001 binding was blocked by preclinical and clinical HDAC6 inhibitors, with compounds that have higher HDAC6 selectivity demonstrating higher blocking efficiency [ 55 , 57 ]. In non-human primate PET studies, [ 18 F]EKZ-001 demonstrated high specific binding [ 55 ], uptake was blocked by highly selective HDAC6 inhibitors in a dose-dependent manner [ 57 ], and binding kinetics were favorable for calculating volume of distribution ( V T ) estimates [ 57 ].…”

“…In in vitro autoradiography competition assays, [ 18 F]EKZ-001 binding was blocked by preclinical and clinical HDAC6 inhibitors, with compounds that have higher HDAC6 selectivity demonstrating higher blocking efficiency [ 55 , 57 ]. In non-human primate PET studies, [ 18 F]EKZ-001 demonstrated high specific binding [ 55 ], uptake was blocked by highly selective HDAC6 inhibitors in a dose-dependent manner [ 57 ], and binding kinetics were favorable for calculating volume of distribution ( V T ) estimates [ 57 ]. Finally, a fully automated current good manufacturing practice (cGMP) compliant production method for human use was developed to facilitate clinical translation of [ 18 F]EKZ-001 PET [ 57 ].…”

“…In non-human primate PET studies, [ 18 F]EKZ-001 demonstrated high specific binding [ 55 ], uptake was blocked by highly selective HDAC6 inhibitors in a dose-dependent manner [ 57 ], and binding kinetics were favorable for calculating volume of distribution ( V T ) estimates [ 57 ]. Finally, a fully automated current good manufacturing practice (cGMP) compliant production method for human use was developed to facilitate clinical translation of [ 18 F]EKZ-001 PET [ 57 ]. Here, we performed a first-in-human [ 18 F]EKZ-001 PET study to investigate biodistribution and dosimetry, as well as kinetic modeling in brain, short-term test-retest variability, inter-subject variability, and a clinically relevant PET imaging protocol by maximal reduction of scanning time.…”