Hepatic glucuronidation of tetrabromobisphenol A and tetrachlorobisphenol A: interspecies differences in humans and laboratory animals and responsible UDP-glucuronosyltransferase isoforms in humans.

Tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA), bisphenol A (BPA) analogs, are endocrine-disrupting chemicals predominantly metabolized into glucuronides by UDP-glucuronosyltransferase (UGT) enzymes in humans and rats. In the present study, TBBPA and TCBPA glucuronidation by the liver microsomes of humans and laboratory animals (monkeys, dogs, minipigs, rats, mice, and hamsters) and recombinant human hepatic UGTs (10 isoforms) were examined. TBBPA glucuronidation by the liver microsomes followed the Michaelis-Menten model kinetics in humans, rats, and hamsters and the biphasic model in monkeys, dogs, minipigs, and mice. The CLint values based on the Eadie-Hofstee plots were mice (147) > monkeys (122) > minipigs (108) > humans (100) and rats (98) > dogs (81) > hamsters (47). TCBPA glucuronidation kinetics by the liver microsomes followed the biphasic model in all species except for minipigs, which followed the Michaelis-Menten model. The CLint values were monkeys (172) > rats (151) > mice (134) > minipigs (104), dogs (102), and humans (100) > hamsters (88). Among recombinant human UGTs examined, UGT1A1 and UGT1A9 showed higher TBBPA and TCBPA glucuronidation abilities. The kinetics of TBBPA and TCBPA glucuronidation followed the substrate inhibition model in UGT1A1 and the Michaelis-Menten model in UGT1A9. The CLint values were UGT1A1 (100) > UGT1A9 (42) for TBBPA glucuronidation and UGT1A1 (100) > UGT1A9 (53) for TCBPA glucuronidation, and the activities at high substrate concentration ranges were higher in UGT1A9 than in UGT1A1 for both TBBPA and TCBPA. These results suggest that the glucuronidation abilities toward TBBPA and TCBPA in the liver differ extensively across species, and that UGT1A1 and UGT1A9 expressed in the liver mainly contribute to the metabolism and detoxification of TBBPA and TCBPA in humans.

Mechanistic Pharmacokinetics and Pharmacodynamics of GalNAc-siRNA: Translational Model Involving Competitive Receptor-Mediated Disposition and RISC-Dependent Gene Silencing Applied to Givosiran.

Triantennary N-acetyl-D galactosamine (GalNAc)3-conjugated small interfering RNA (siRNA) have majorly advanced the development of RNA-based therapeutics. Chemically stabilized GalNAc-siRNAs exhibit extensive albeit capacity-limited (nonlinear) distribution into hepatocytes with additional complexities in intracellular liver disposition and pharmacology. A mechanism-based pharmacokinetic-pharmacodynamic (PK-PD) model of GalNAc-siRNA was developed to i) quantitate ASGPR-mediated disposition and downstream RNA-induced silencing complex (RISC)-dependent pharmacology following intravenous (IV) and subcutaneous (SC) dosing, ii) assess the kinetics of formed active metabolite, iii) leverage, as an example, published experimental data for givosiran, and iv) demonstrate PK translation across two preclinical species (rat and monkey) with subsequent prediction of human plasma PK. The structural model is based on competition between parent and formed active metabolite for occupancy and uptake via ASGPR into hepatocytes, intracellular sequestration and degradation, and downstream engagement of RNA-induced silencing complex (RISC) governing target mRNA degradation. The model jointly and accurately captured available concentration-time profiles of givosiran and/or AS(N-1)3' givosiran in rat and/or monkey plasma, liver, and/or kidney following givosiran administered both IV and SC. RISC-dependent gene silencing of ALAS1 mRNA was well-characterized. The model estimated an in vivo affinity (KD) value of 27.7 nM for GalNAc-ASGPR and weight-based allometric exponents of -0.27 and -0.24 for SC absorption and intracellular (endolysosomal) degradation rate constants. The model well-predicted reported givosiran plasma PK profiles in humans. PK simulations revealed net-shifts in liver-to-kidney distribution ratios with increasing IV and SC dose. Importantly, decreases in the relative liver uptake efficiency were demonstrated following IV and, to a lesser extent, following SC dosing explained by differential ASGPR occupancy profiles over time.

Human Embryonic Stem Cell-Derived Immature Midbrain Dopaminergic Neurons Transplanted in Parkinsonian Monkeys.

Human embryonic stem cells (hESCs) differentiate into specialized cells, including midbrain dopaminergic neurons (DANs), and Non-human primates (NHPs) injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine develop some alterations observed in Parkinson's disease (PD) patients. Here, we obtained well-characterized DANs from hESCs and transplanted them into two parkinsonian monkeys to assess their behavioral and imaging changes. DANs from hESCs expressed dopaminergic markers, generated action potentials, and released dopamine (DA) in vitro. These neurons were transplanted bilaterally into the putamen of parkinsonian NHPs, and using magnetic resonance imaging techniques, we calculated the fractional anisotropy (FA) and mean diffusivity (MD), both employed for the first time for these purposes, to detect in vivo axonal and cellular density changes in the brain. Likewise, positron-emission tomography scans were performed to evaluate grafted DANs. Histological analyses identified grafted DANs, which were quantified stereologically. After grafting, animals showed signs of partially improved motor behavior in some of the HALLWAY motor tasks. Improvement in motor evaluations was inversely correlated with increases in bilateral FA. MD did not correlate with behavior but presented a negative correlation with FA. We also found higher 11C-DTBZ binding in positron-emission tomography scans associated with grafts. Higher DA levels measured by microdialysis after stimulation with a high-potassium solution or amphetamine were present in grafted animals after ten months, which has not been previously reported. Postmortem analysis of NHP brains showed that transplanted DANs survived in the putamen long-term, without developing tumors, in immunosuppressed animals. Although these results need to be confirmed with larger groups of NHPs, our molecular, behavioral, biochemical, and imaging findings support the integration and survival of human DANs in this pre-clinical PD model.

Laminar differences in functional oxygen metabolism in monkey visual cortex measured with calibrated fMRI.

Blood-oxygenation-level-dependent functional magnetic resonance imaging (BOLD fMRI) of cortical layers relies on the hemodynamic response and is biased toward large veins on the cortical surface. Functional changes in the cerebral metabolic rate of oxygen (ΔCMRO2) may reflect neural cortical function better than BOLD fMRI, but it is unknown whether the calibrated BOLD model for functional CMRO2 measurement remains valid at high resolution. Here, we measure laminar ΔCMRO2 elicited by visual stimulation in macaque primary visual cortex (V1) and find that ΔCMRO2 peaks in the middle of the cortex, in agreement with autoradiographic measures of metabolism. ΔCMRO2 values in gray matter are similar as found previously. Reductions in CMRO2 are associated with veins at the cortical surface, suggesting that techniques for vein removal may improve the accuracy of the model at very high resolution. However, our results show feasibility of laminar ΔCMRO2 measurement, providing a physiologically meaningful metric of laminar functional metabolism.

Evaluation of the risk of diarrhea induced by epidermal growth factor receptor tyrosine kinase inhibitors with cultured intestinal stem cells originated from crypts in humans and monkeys.

Severe diarrhea is a common side effect of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). We aimed to evaluate the risk of EGFR-TKI-induced diarrhea using spheroids of human and monkey crypt-derived intestinal stem cells. Intestinal spheroids exhibited higher toxic susceptibility to EGFR-TKIs than Caco-2 cells. As concentration of EGFR-TKIs increased, cellular ATP first decreased relative to the control condition, followed by an increase in LDH release, in contrast with their simultaneous changes with traditional cytotoxic anticancer drugs. The toxic sensitivity of spheroids to various EGFR-TKIs corresponded to clinical diarrhea incidence. Afatinib, a second-generation EGFR-TKI, exhibited higher toxic sensitivity compared with the first-generation ones, corresponding to the clinical evidence that afatinib-induced diarrhea is almost inevitable and severe. By contrast, the third-generation osimertinib, which reduces the risk of diarrhea, showed mitigated cytotoxicity compared with afatinib. For irreversible EGFR-TKIs, the decreased ATP level persisted or its recovery was delayed even after drug removal compared with reversible ones. Furthermore, the highest drug accumulation in spheroids (TKIspheroids) and inhibition potency against EGFR (TKIspheroids/Ki) were observed for afatinib. This system would be useful for predicting the risk of EGFR-TKI-induced diarrhea; moreover, on-target cytotoxicity against intestinal stem cells might contribute to clinically observed diarrhea.

Detection and identification of imperatorin metabolites in rat, dog, monkey, and human liver microsomes by ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry and Compound Discoverer software.

Imperatorin, a furanocoumarin that widely exists in many umbelliferous herbs, has been demonstrated to have a variety of pharmacological effects, including anti-inflammatory, antiosteoporosis, and antitumor activities. The purpose of this study was to investigate the metabolism of imperatorin using liver microsomes. The metabolites were generated by individually incubating imperatorin with rat, dog, monkey, and human liver microsomes. To trap the reactive metabolites during microsomal metabolism, glutathione (GSH) was included in the incubation. A LC technique coupled with benchtop orbitrap MS with full mass/data-dependent tandem mass spectrometry acquisition mode was used to detect and identify the generated metabolites. The possible structures of the metabolites were characterized according to their accurate masses and fragment ions. Under the current conditions, a total of 10 metabolites, including four GSH adducts, were identified. The results indicated that imperatorin underwent extensive metabolic reactions including hydroxylation, oxidation, glucuronidation, and GSH conjugation. This study provides essential data on the metabolism of imperatorin, which will be helpful for us to understand the safety and efficacy of this bioactive compound.

Vegetable Oil-Peroxidation Product 'Hydroxynonenal' Causes Hepatocyte Injury and Steatosis via Hsp70.1 and BHMT Disorders in the Monkey Liver.

Hsp70.1 has a dual function as a chaperone protein and lysosomal stabilizer. In 2009, we reported that calpain-mediated cleavage of carbonylated Hsp70.1 causes neuronal death by inducing lysosomal rupture in the hippocampal CA1 neurons of monkeys after transient brain ischemia. Recently, we also reported that consecutive injections of the vegetable oil-peroxidation product 'hydroxynonenal' induce hepatocyte death via a similar cascade in monkeys. As Hsp70.1 is also related to fatty acid ß-oxidation in the liver, its deficiency causes fat accumulation. The genetic deletion of betaine-homocysteine S-methyltransferase (BHMT) was reported to perturb choline metabolism, inducing a decrease in phosphatidylcholine and resulting in hepatic steatosis. Here, focusing on Hsp70.1 and BHMT disorders, we studied the mechanisms of hepatocyte degeneration and steatosis. Monkey liver tissues with and without hydroxynonenal injections were compared using proteomics, immunoblotting, immunohistochemical, and electron microscopy-based analyses. Western blotting showed that neither Hsp70.1 nor BHMT were upregulated, but an increased cleavage was observed in both. Proteomics showed a marked downregulation of Hsp70.1, albeit a two-fold increase in the carbonylated BHMT. Hsp70.1 carbonylation was negligible, in contrast to the ischemic hippocampus, which was associated with ~10-fold increments. Although histologically, the control liver showed very little lipid deposition, numerous tiny lipid droplets were seen within and around the degenerating/dying hepatocytes in monkeys after the hydroxynonenal injections. Electron microscopy showed permeabilization/rupture of lysosomal membranes, dissolution of the mitochondria and rough ER membranes, and proliferation of abnormal peroxisomes. It is probable that the disruption of the rough ER caused impaired synthesis of the Hsp70.1 and BHMT proteins, while impairment of the mitochondria and peroxisomes contributed to the sustained generation of reactive oxygen species. In addition, hydroxynonenal-induced disorders facilitated degeneration and steatosis in the hepatocytes.

The accelerated evolution of human cytochrome c oxidase - Selection for reduced rate and proton pumping efficiency?

The cytochrome c oxidase complex, complex VI (CIV), catalyzes the terminal step of the mitochondrial electron transport chain where the reduction of oxygen to water by cytochrome c is coupled to the generation of a protonmotive force that drive the synthesis of ATP. CIV evolution was greatly accelerated in humans and other anthropoid primates and appears to be driven by adaptive selection. However, it is not known if there are significant functional differences between the anthropoid primates CIV, and other mammals. Comparison of the high-resolution structures of bovine CIV, mouse CIV and human CIV shows structural differences that are associated with anthropoid-specific substitutions. Here I examine the possible effects of these substitutions in four CIV peptides that are known to affect proton pumping: the mtDNA-coded subunits I, II and III, and the nuclear-encoded subunit VIa2. I conclude that many of the anthropoid-specific substitutions could be expected to modulate the rate and/or the efficiency of proton pumping. These results are compatible with the previously proposed hypothesis that the accelerated evolution of CIV in anthropoid primates is driven by selection pressure to lower the mitochondrial protonmotive force and thus decrease the rate of superoxide generation by mitochondria.

Time course of dorsolateral geniculate nucleus plasticity in adult monkeys with laser-induced retinal lesions.

We studied changes in the expression of growth-associated protein 43 (GAP43), glial fibrillary acidic protein (GFAP), and calcium-binding proteins (calbindin [Cb] and parvalbumin [Pv]) in the dorsal lateral geniculate nucleus (dLGN) of four capuchin monkeys with laser-induced retinal lesions. The lesions were generated with the aid of a neodymium-YAG dual-frequency laser with shots of different intensity and at different survival time in each animal. The expression of these proteins in the layers of the dLGN was evaluated by performing histodensitometry of coronal sections throughout the nucleus. High-power laser shots administered at the border of the optic disc (OD)-injured fibers resulted in large scotomas. These lesions produced a devastating effect on fibers in this passage, resulting in large deafferentation of the dLGN. The time course of plasticity expressed in this nucleus varied with the degree of the retinal lesion. Topographically, corresponding portions of the dLGN were inferred by the extent of the ocular dominance column revealed by cytochrome oxidase histochemistry in flattened preparations of V1. In the region representing the retinal lesion, the expression of GFAP, GAP43, Pv, and Cb increased and decreased in the corresponding dLGN layers shortly after lesion induction and returned to their original values with different time courses. Synaptogenesis (indicated by GAP43 expression) appeared to be increased in all layers, while "cleansing" of the glial-damaged region (indicated by GFAP expression) was markedly greater in the parvocellular layers, followed by the magnocellular layers. Schematic drawings of optic discs laser lesions and of series of coronal sections of the dLGN, in three monkeys, depicting the areas of the nucleus deafferented by the lesions.

First-in-Human Evaluation of 18F-PF-06445974, a PET Radioligand That Preferentially Labels Phosphodiesterase-4B.

Phosphodiesterase-4 (PDE4), which metabolizes the second messenger cyclic adenosine monophosphate (cAMP), has 4 isozymes: PDE4A, PDE4B, PDE4C, and PDE4D. PDE4B and PDE4D have the highest expression in the brain and may play a role in the pathophysiology and treatment of depression and dementia. This study evaluated the properties of the newly developed PDE4B-selective radioligand 18F-PF-06445974 in the brains of rodents, monkeys, and humans. Methods: Three monkeys and 5 healthy human volunteers underwent PET scans after intravenous injection of 18F-PF-06445974. Brain uptake was quantified as total distribution volume (V T) using the standard 2-tissue-compartment model and serial concentrations of parent radioligand in arterial plasma. Results: 18F-PF-06445974 readily distributed throughout monkey and human brain and had the highest binding in the thalamus. The value of V T was well identified by a 2-tissue-compartment model but increased by 10% during the terminal portions (40 and 60 min) of the monkey and human scans, respectively, consistent with radiometabolite accumulation in the brain. The average human V T values for the whole brain were 9.5 ± 2.4 mL ⋅ cm-3 Radiochromatographic analyses in knockout mice showed that 2 efflux transporters-permeability glycoprotein (P-gp) and breast cancer resistance protein (BCRP)-completely cleared the problematic radiometabolite but also partially cleared the parent radioligand from the brain. In vitro studies with the human transporters suggest that the parent radioligand was a partial substrate for BCRP and, to a lesser extent, for P-gp. Conclusion: 18F-PF-06445974 quantified PDE4B in the human brain with reasonable, but not complete, success. The gold standard compartmental method of analyzing brain and plasma data successfully identified the regional densities of PDE4B, which were widespread and highest in the thalamus, as expected. Because the radiometabolite-induced error was only about 10%, the radioligand is, in the opinion of the authors, suitable to extend to clinical studies.

Metabolism of anti-inflammatory OXE (oxoeicosanoid) receptor antagonists by nonhuman primates.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is the only product of the proinflammatory 5-lipoxygenase pathway with potent chemoattractant effects for human eosinophils, suggesting an important role in eosinophilic diseases such as asthma. 5-Oxo-ETE, acting through its selective OXE receptor, induces dermal eosinophilia in both humans and monkeys. To block its effects, we designed selective indole-based OXE antagonists containing hexyl (S-230) or phenylhexyl (S-C025 and S-Y048) side chains, which inhibit allergen-induced dermal and pulmonary inflammation in monkeys, suggesting that they may be useful therapeutic agents in humans. In this study we identified two metabolic pathways for the phenylhexyl-containing antagonists in liver microsomes: benzylic and N-methyl hydroxylation, resulting in ω-hydroxy, ω-oxo, and NH-containing products with reduced potencies that were identified by mass spectrometry and comparison with synthetic standards. Products of both pathways were also identified in monkey plasma following oral administration of S-C025 and S-Y025, but were less abundant than the α-hydroxy metabolites that we previously identified. Interestingly, the α-hydroxy compounds were not detected in microsomal incubations, suggesting a different origin. The relative rates of metabolism of these antagonists were S-230 >> S-C025 > S-Y048, which may help to explain the differences in their plasma half-lives (S-230 < S-C025 < S-Y048). In conclusion, S-C025 and S-Y048 are metabolized by liver microsomes by benzylic and N-methyl hydroxylation but not by α-hydroxylation, whereas all three pathways exist in vivo. Addition of a phenyl group to the hexyl side chain of these antagonists dramatically reduced their rates of metabolism, which would explain their prolonged in vivo half-lives.

Towards a translational physiologically-based pharmacokinetic (PBPK) model for receptor-mediated transcytosis of anti-transferrin receptor monoclonal antibodies in the central nervous system.

In this manuscript, we present a translational physiologically-based pharmacokinetic (PBPK) model to characterize receptor-mediated transcytosis (RMT) of anti-transferrin receptor (TfR) monoclonal antibodies (mAbs) in the central nervous system (CNS). The model accounts for the state-of-the-art knowledge of the brain's anatomy and physiology, and physiological parameters were fixed according to different species. By estimating a few parameters associated with the TfR concentration, the TfR turnover, and the internalization rate, the model simultaneously characterizes plasma, whole brain, interstitial fluid (ISF), and cerebrospinal fluid (CSF) PK of unbound and bound anti-TfR mAbs with different binding affinities in mice, rats, and monkeys obtained from various literature sources within a threefold prediction error. The final PBPK model was validated using external anti-TfR mAb PK data in mice and monkeys with different affinities and doses. The simulation reasonably predicted plasma and brain PK of monovalent/bivalent anti-TfR mAbs within a threefold prediction error and characterized a bell-shaped relationship between the brain ISF/plasma AUC ratio and the KD value. Although further refinements of the PBPK model and clinical validation are required, this PBPK model may provide physiologically-based translation of CNS disposition of anti-TfR mAbs by accounting for the physiological difference of the endogenous RMT system among different species. The PBPK model may also guide selection of other endogenous receptors, lead optimization, and clinical development of novel CNS-targeted mAbs.

SQSTM1-mediated clearance of cytoplasmic mutant TARDBP/TDP-43 in the monkey brain.

The cytoplasmic accumulation and aggregates of TARDBP/TDP-43 (TAR DNA binding protein) are a pathological hallmark in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We previously reported that the primate specific cleavage of TARDBP accounts for its cytoplasmic mislocalization in the primate brains, prompting us to further investigate how the cytoplasmic TARDBP mediates neuropathology. Here we reported that cytoplasmic mutant TARDBP reduced SQSTM1 expression selectively in the monkey brain, when compared with the mouse brain, by inducing SQSTM1 mRNA instability via its binding to the unique 3'UTR sequence (GU/UG)n of the primate SQSTM1 transcript. Overexpression of SQSTM1 could diminish the cytoplasmic C-terminal TARDBP accumulation in the monkey brain by augmenting macroautophagy/autophagy activity. Our findings provide additional clues for the pathogenesis of cytoplasmic TARDBP and a potential therapy for mutant TARDBP-mediated neuropathology.

Determination of drug-to-antibody ratio of antibody-drug conjugate in biological samples using microflow-liquid chromatography/high-resolution mass spectrometry.

Background: Antibody-drug conjugates (ADCs) are a promising modality for cancer treatment; however, considering their complicated nature, analytical complexity in understanding their pharmacokinetics and pharmacodynamics in the body presents a significant challenge. Results: Vorsetuzumab maleimidocaproyl valine-citrulline p-aminobenzyloxycarbonyl monomethyl auristatin E was used to develop pretreatment and analytical workflows suitable for ADCs. Monomethyl auristatin E release and drug-to-antibody ratio retention were consistent in mouse plasma but inconsistent in monkey and human plasma. Further, metabolites were species-specific. Microflow-liquid chromatography/high-resolution mass spectrometry (LC-HRMS) resulted in a 4-7-fold improvement in detection sensitivity compared with conventional flow LC-HRMS. Conclusion: Microflow-LC-HRMS can be a useful tool in understanding the complex properties of ADCs in the body from a drug metabolism and pharmacokinetics point of view.

Drug-to-antibody ratio (DAR), payload release and metabolite profile of deconjugated payload-linker of vorsetuzumab maleimidocaproyl valine-citrulline p-aminobenzyloxycarbonyl monomethyl auristatin E, an antibody­drug conjugate (ADC) with cleavable linker and monomethyl auristatin E as payload, are reported. Species-specific retention of DAR, payload release and metabolite patterns of deconjugated payload-linker of the ADC are summarized. Exploring the fate of payload-linker moieties deconjugated from ADCs in the body is also vital to understanding pharmacological activity and toxicity. Species-specific metabolite patterns of the ADC provided insight into the importance of optimization of the payload-linker moiety in biological samples, especially in humans. In terms of a more sensitive analytical platform for drug metabolism and pharmacokinetic evaluation, microflow-liquid chromatography/high-resolution mass spectrometry (LC­HRMS) in DAR analysis was found to take advantage of the improvement of detection sensitivity compared with conventional LC­HRMS. Because ADCs are a complex drug modality, these results indicated the importance of evaluation of ADCs from a drug metabolism and pharmacokinetics point of view to understand the pharmacology and toxicology of ADCs, more precisely.

The Vertical and Horizontal Pathways in the Monkey Retina Are Modulated by Typical and Atypical Cannabinoid Receptors.

The endocannabinoid (eCB) system has been found in all visual parts of the central ner-vous system and plays a role in the processing of visual information in many species, including monkeys and humans. Using anatomical methods, cannabinoid receptors are present in the monkey retina, particularly in the vertical glutamatergic pathway, and also in the horizontal GABAergic pathway. Modulating the eCB system regulates normal retinal function as demonstrated by electrophysiological recordings. The characterization of the expression patterns of all types of cannabinoid receptors in the retina is progressing, and further research is needed to elucidate their exact role in processing visual information. Typical cannabinoid receptors include G-protein coupled receptor CB1R and CB2R, and atypical cannabinoid receptors include the G-protein coupled receptor 55 (GPR55) and the ion channel transient receptor potential vanilloid 1 (TRPV1). This review focuses on the expression and localization studies carried out in monkeys, but some data on other animal species and humans will also be reported. Furthermore, the role of the endogenous cannabinoid receptors in retinal function will also be presented using intraocular injections of known modulators (agonists and antagonists) on electroretinographic patterns in monkeys. The effects of the natural bioactive lipid lysophosphatidylglucoside and synthetic FAAH inhibitor URB597 on retinal function, will also be described. Finally, the potential of typical and atypical cannabinoid receptor acti-vity regulation in retinal diseases, such as age-related macular degeneration, diabetic retinopathy, glaucoma, and retinitis pigmentosa will be briefly explored.

PET Imaging of VMAT2 with the Novel Radioligand [18F]FE-DTBZ-d4 in Nonhuman Primates: Comparison with [11C]DTBZ and [18F]FE-DTBZ.

The vesicular monoamine transporter type 2 (VMAT2) is believed to be responsible for the uptake of monoamines into the vesicles of the synaptic terminals. Two VMAT2 radioligands [11C]DTBZ and [18F]FP-DTBZ have been used to assess the degree of nigrostriatal deficit in Parkinson's disease (PD) using positron emission tomography (PET). [18F]FE-DTBZ-d4, the nondeuterated analogue of [18F]FE-DTBZ showed similar imaging properties with better stability against defluorination. Therefore, [18F]FE-DTBZ-d4 draws attention to be investigated as an imaging marker for VMAT2 in the brain. The aim of this study was to investigate the brain kinetics and quantification of [18F]FE-DTBZ-d4 in nonhuman primates (NHPs), with comparison to [11C]DTBZ and [18F]FE-DTBZ. Radiolabeling was successfully achieved either by one-step 11C-methylation or by a two-step fluorine-18 nucleophilic substitution reaction. The stability and radiochemical yield were analyzed with high-performance liquid chromatography (HPLC). Three female cynomolgus monkeys were included in the study and underwent a total of 12 positron emission tomography (PET) measurements. Each monkey was examined with each tracer. In addition, two pretreatment and one displacement PET measurements with tetrabenazine (2.0 mg/kg) were performed for [18F]FE-DTBZ-d4. All PET measurements were conducted using a high-resolution research tomograph (HRRT) system. Radiometabolites were measured in monkey plasma using gradient radio-HPLC. [18F]FE-DTBZ-d4 (SUV: 4.28 ± 1.01) displayed higher brain uptake compared to both [18F]FE-DTBZ (SUV: 3.43 ± 0.54) and [11C]DTBZ (SUV: 3.06 ± 0.32) and faster washout. Binding potential (BPND) values of [18F]FE-DTBZ-d4 in different brain regions (putamen: 5.5 ± 1.4; caudate: 4.4 ± 1.1; midbrain: 1.4 ± 0.4) were higher than those of [11C]DTBZ and [18F]FE-DTBZ. [18F]FE-DTBZ showed faster radiometabolism in plasma compared to [11C]DTBZ and [18F]FE-DTBZ-d4. [18F]FE-DTBZ-d4 is a suitable radioligand for quantification of VMAT2 in the nonhuman primate brain, with better imaging properties than [11C]DTBZ and [18F]FE-DTBZ. A preliminary comparison suggests that [18F]FE-DTBZ-d4 has increased stability against defluorination compared to the nondeuterated analogue.

Scopolamine and MK-801 impair recognition memory in a new spontaneous object exploration task in monkeys.

The spontaneous object recognition (SOR) task is one of the most widely used behavioral protocols to assess visual memory in animals. However, only recently was it shown that nonhuman primates also perform well on this task. Here we further characterized this new monkey recognition memory test by assessing the performance of adult marmosets after an acute systemic administration of two putative amnesic agents: the competitive muscarinic acetylcholine receptor antagonist scopolamine (SCP; 0.05 mg/kg) and the noncompetitive N-methyl-d-aspartate glutamate receptor antagonist MK-801 (0.015 mg/kg). We also determined whether the acetylcholinesterase inhibitor donepezil (DNP; 0.50 mg/kg), a clinically-used cognitive enhancer, reverses memory deficits caused by either drug. The subjects had an initial 10 min sample trial where two identical neutral objects could be explored. After a 6 h retention interval, recognition was based on an exploratory preference for a new rather than familiar object during a 10 min test trial. Both SCP and MK-801 impaired the marmosets' performance on the SOR task, as both objects were explored equivalently. Co-administration of 0.50 mg/kg of DNP reversed the SCP- but not the MK-801-induced memory deficit. These results indicate that cholinergic and glutamatergic pathways mediate object recognition memory in the monkey SOR task.

GIPR antagonist antibodies conjugated to GLP-1 peptide are bispecific molecules that decrease weight in obese mice and monkeys.

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) regulate glucose and energy homeostasis. Targeting both pathways with GIP receptor (GIPR) antagonist antibody (GIPR-Ab) and GLP-1 receptor (GLP-1R) agonist, by generating GIPR-Ab/GLP-1 bispecific molecules, is an approach for treating obesity and its comorbidities. In mice and monkeys, these molecules reduce body weight (BW) and improve many metabolic parameters. BW loss is greater with GIPR-Ab/GLP-1 than with GIPR-Ab or a control antibody conjugate, suggesting synergistic effects. GIPR-Ab/GLP-1 also reduces the respiratory exchange ratio in DIO mice. Simultaneous receptor binding and rapid receptor internalization by GIPR-Ab/GLP-1 amplify endosomal cAMP production in recombinant cells expressing both receptors. This may explain the efficacy of the bispecific molecules. Overall, our GIPR-Ab/GLP-1 molecules promote BW loss, and they may be used for treating obesity.

Brain delivery of therapeutic proteins using an Fc fragment blood-brain barrier transport vehicle in mice and monkeys.

Effective delivery of protein therapeutics to the central nervous system (CNS) has been greatly restricted by the blood-brain barrier (BBB). We describe the development of a BBB transport vehicle (TV) comprising an engineered Fc fragment that exploits receptor-mediated transcytosis for CNS delivery of biotherapeutics by binding a highly expressed brain endothelial cell target. TVs were engineered using directed evolution to bind the apical domain of the human transferrin receptor (hTfR) without the use of amino acid insertions, deletions, or unnatural appendages. A crystal structure of the TV-TfR complex revealed the TV binding site to be away from transferrin and FcRn binding sites, which was further confirmed experimentally in vitro and in vivo. Recombinant expression of TVs fused to anti-ß-secretase (BACE1) Fabs yielded antibody transport vehicle (ATV) molecules with native immunoglobulin G (IgG) structure and stability. Peripheral administration of anti-BACE1 ATVs to hTfR-engineered mice and cynomolgus monkeys resulted in substantially improved CNS uptake and sustained pharmacodynamic responses. The TV platform readily accommodates numerous additional configurations, including bispecific antibodies and protein fusions, yielding a highly modular CNS delivery platform.