Quantitative assessment of translocator protein (TSPO) in the non-human primate brain and clinical translation of [18F]LW223 as a TSPO-targeted PET radioligand.

OBJECTIVES: Translocator protein 18 kDa (TSPO) positron emission tomography (PET) can be harnessed for the non-invasive detection of macrophage-driven inflammation. [18F]LW223, a newly reported TSPO PET tracer which was insensitive to rs6971 polymorphism, showed favorable performance characteristics in a recent imaging study involving a rat myocardial infarction model. To enable quantitative neuroimaging with [18F]LW223, we conducted kinetic analysis in the non-human primate (NHP) brain. Further, we sought to assess the utility of [18F]LW223-based TSPO imaging in a first-in-human study. METHODS: Radiosynthesis of [18F]LW223 was accomplished on an automated module, whereas molar activities, stability in formulation, lipophilicity and unbound free fraction (fu) of the probe were measured. Brain penetration and target specificity of [18F]LW223 in NHPs were corroborated by PET-MR imaging under baseline and pre-blocking conditions using the validated TSPO inhibitor, (R)-PK11195, at doses ranging from 5 to 10 mg/kg. Kinetic modeling was performed using one-tissue compartment model (1TCM), two-tissue compartment model (2TCM) and Logan graphical analyses, using dynamic PET data acquisition, arterial blood collection and metabolic stability testing. Clinical PET scans were performed in two healthy volunteers (HVs). Regional brain standard uptake value ratio (SUVr) was assessed for different time intervals. RESULTS: [18F]LW223 was synthesized in non-decay corrected radiochemical yields (n.d.c. RCYs) of 33.3 ± 6.5% with molar activities ranging from 1.8 ± 0.7 Ci/µmol (n = 11). [18F]LW223 was stable in formulation for up to 4 h and LogD7.4 of 2.31 ± 0.13 (n = 6) and fu of 5.80 ± 1.42% (n = 6) were determined. [18F]LW223 exhibited good brain penetration in NHPs, with a peak SUV value of ca. 1.79 in the whole brain. Pre-treatment with (R)-PK11195 substantially accelerated the washout and attenuated the area under the time-activity curve, indicating in vivo specificity of [18F]LW223 towards TSPO. Kinetic modeling demonstrated that 2TCM was the most suitable model for [18F]LW223-based neuroimaging. Global transfer rate constants (K1) and total volumes of distribution (VT) were found to be 0.10 ± 0.01 mL/cm3/min and 2.30 ± 0.17 mL/cm3, respectively. Dynamic PET data analyses across distinct time windows revealed that the VT values were relatively stable after 60 min post-injection. In a preliminary clinical study with two healthy volunteers, [18F]LW223 exhibited good brain uptake and considerable tracer retention across all analyzed brain regions. Of note, an excellent correlation between SUVr with VT was obtained when assessing the time interval from 20 to 40 min post tracer injection (SUVr(20-40 min), R2 = 0.94, p < 0.0001), suggesting this time window may be suitable to estimate specific binding to TSPO in human brain. CONCLUSION: Our findings indicate that [18F]LW223 is suitable for quantitative TSPO-targeted PET imaging in higher species. Employing state-of-the-art kinetic modeling, we found that [18F]LW223 was effective in mapping TSPO throughout the NHP brain, with best model fits obtained from 2TCM and Logan graphical analyses. Overall, our results indicate that [18F]LW223 exhibits favorable tracer performance characteristics in higher species, and this novel imaging tool may hold promise to provide effective neuroinflammation imaging in patients with neurological disease.

Brief Communication: Histological Assessment of Nonhuman Primate Brown Adipose Tissue Highlights the Importance of Sympathetic Innervation.

Objective: The objective of this study was to functionally analyze the correlation of key histological features in brown adipose tissue (BAT) with clinical metabolic traits in nonhuman primates. Methods: Axillary adipose tissue biopsies were collected from a metabolically diverse nonhuman primate cohort with clinical metabolism-related data. Expression of tyrosine hydroxylase (TH), uncoupling protein 1 (UCP1), cluster of differentiation 31 (CD31), cytochrome c oxidase subunit 4 (COX IV), beta-3 adrenergic receptor (ß3-AR), and adipose cell size were quantified by immunohistochemical analysis. Computed tomography scans were performed to assess body composition. Results: Tyrosine hydroxylase was negatively correlated with whole body fat mass as a percentage of body weight (p = 0.004) and was positively correlated with the density of UCP1 (p = 0.02), COX IV (p = 0.006), CD31 (p = 0.007), and cell density (p = 0.02) of the BAT samples. Beta-3 adrenergic receptor abundance had a weak positive correlation with COX IV (p = 0.04) in BAT but did not significantly correlate to UCP1 or TH expression in BAT. Conclusions: Our findings highlight that there is a disparity in innervation provided to BAT based on body composition, as seen with the negative association between TH, a marker for innervation, and adiposity. These findings also support the importance of innervation in the functionality of BAT, as TH abundance not only supports leaner body composition but is also positively correlated with known structural elements in BAT (UCP1, COX IV, CD31, and cell density). Based on our observations, ß3-AR abundance does not strongly drive these structural elements or TH, all of which are known to be important in the function of brown adipose tissue. In effect, while the role of other receptors, such as ß2-AR, should be reviewed in BAT function, these results support the development of safe sympathetic nervous system stimulants to activate brown adipose tissue for obesity treatment.

Development of a versatile enzyme immunoassay for non-invasive assessment of glucocorticoid metabolites in a diversity of taxonomic species.

Endocrinology is a useful tool for conservation biologists and animal managers, and measuring glucocorticoids can help understand biological mechanisms associated with species decline and animal welfare. The current study describes the development and optimization of a glucocorticoid enzyme immunoassay (EIA) to non-invasively assess adrenal activity in a variety of taxa. The antiserum (CJM006) was raised in rabbits to a corticosterone-3-CMO-BSA immunogen and used in a standard competitive EIA system. However, the EIA initially produced results with unacceptably high inter-assay variation, attributed to consistent patterns observed within the optical density of developing plates. To determine the cause of this variability, a number of factors were examined using synthetic corticosterone standard and endogenous faecal extract, including: plate type (Nunc MaxiSorp® II versus Immulon IB plates); the use of non-specific secondary antibody; type (artificial versus natural) and presence (light versus dark) of light during incubation; plate loading temperature (4°C versus room temperature); and substrate reagent temperature (4°C versus room temperature). Results indicated that variability was associated with plate location effects, which were not initially detected because control samples were always run in the same positions across plates. Light and temperature were the two major factors that affected EIA reliability. For this assay, the standard protocol required slight modification, with the optimal protocol using Nunc MaxiSorp® plates, room temperature substrate reagents and dark incubation conditions. Following optimization, this EIA was then validated biochemically for 38 species, through parallel displacement curves and interference assessment tests of faecal and urine samples. Additionally, biological validation was performed opportunistically in a subset of species, with use of this EIA demonstrating significant elevations in faecal glucocorticoid metabolites following potentially challenging events. In summary, this glucocorticoid EIA cross-reacts with excreted glucocorticoid metabolites across a wide range of taxa, including ungulates, primates, felids, birds, rodents and amphibians. We conclude that when used with optimal reagent and incubation conditions, this EIA will be useful for non-invasive monitoring of adrenal activity in a wide range of wildlife species.

Hypervitaminosis A in experimental nonhuman primates: evidence, causes, and the road to recovery.

One of the great underlying assumptions made by all scientists utilizing primate models for their research is that the optimal nutritional status and health of the animals in use has been achieved. That is, no nutrient deficiency or excess has compromised their health in any detectable way. To meet this assumption, we rely on the National Research Council's (NRC's) nutritional recommendations for nonhuman primates to provide accurate guidance for proper dietary formulations. We also rely on feed manufacturers to follow these guidelines. With that in mind, the purpose of this commentary is to discuss three related points that we believe have significant ramifications for the health and well being of captive primates as well as for their effective use in biomedical research. First, our laboratory has shown that most experimental primates are likely in a state of hypervitaminosis A. Second, it is apparent that many primate diets are providing vitamin A at levels higher than the NRC's recommendation. Third, the recommendation itself is based on inadequate information about nutrient needs and is likely too high, especially when compared with human requirements.

Host-retrovirus arms race: trimming the budget.

In this issue of Cell Host & Microbe, OhAinle et al., 2008 report that APOBEC3H, a potent innate retroviral restriction factor in primates, lost its function twice independently during recent evolution in humans, stressing an ever present trade-off between benefit and cost of protection against pathogens.

The energetic cost of locomotion: humans and primates compared to generalized endotherms.

A wide range of selective pressures have been advanced as possible causes for the adoption of bipedalism in the hominin lineage. One suggestion has been that because modern human walking is relatively efficient compared to that of a typical quadruped, the ancestral quadruped may have reaped an energetic advantage when it walked on two legs. While it has become clear that human walking is relatively efficient and human running inefficient compared to "generalized endotherms", workers differ in their opinion of how the cost of human bipedal locomotion compares to that of a generalized primate walking quadrupedally. One view is that human walking is particularly efficient in comparison to other primates. The present study addresses this by comparing the cost of human walking and running to that of the eight primate species for which data are available and by comparing cost in primates to that of a "generalized endotherm". There is no evidence that primate locomotion is more costly than that of a generalized endotherm, although more data on adult Old World monkeys and apes would be useful. Further, human locomotion does not appear to be particularly efficient relative to that of other primates.