A genetic variation in the adenosine A2A receptor gene contributes to variability in oscillatory alpha power in wake and sleep EEG and A1 adenosine receptor availability in the human brain.

The EEG alpha rhythm (∼ 8-13 Hz) is one of the most salient human brain activity rhythms, modulated by the level of attention and vigilance and related to cerebral energy metabolism. Spectral power in the alpha range in wakefulness and sleep strongly varies among individuals based on genetic predisposition. Knowledge about the underlying genes is scarce, yet small studies indicated that the variant rs5751876 of the gene encoding A2A adenosine receptors (ADORA2A) may contribute to the inter-individual variation. The neuromodulator adenosine is directly linked to energy metabolism as product of adenosine tri-phosphate breakdown and acts as a sleep promoting molecule by activating A1 and A2A adenosine receptors. We performed sleep and positron emission tomography studies in 59 healthy carriers of different rs5751876 alleles, and quantified EEG oscillatory alpha power in wakefulness and sleep, as well as A1 adenosine receptor availability with 18F-CPFPX. Oscillatory alpha power was higher in homozygous C-allele carriers (n = 27, 11 females) compared to heterozygous and homozygous carriers of the T-allele (n(C/T) = 23, n(T/T) = 5, 13 females) (F(18,37) = 2.35, p = 0.014, Wilk's Λ = 0.487). Furthermore, a modulatory effect of ADORA2A genotype on A1 adenosine receptor binding potential was found across all considered brain regions (F(18,40) = 2.62, p = 0.006, Wilk's Λ = 0.459), which remained significant for circumscribed occipital region of calcarine fissures after correction for multiple comparisons. In female participants, a correlation between individual differences in oscillatory alpha power and A1 receptor availability was observed. In conclusion, we confirmed that a genetic variant of ADORA2A affects individual alpha power, while a direct modulatory effect via A1 adenosine receptors in females is suggested.

Cerebral A1 adenosine receptor availability in female and male participants and its relationship to sleep.

The neuromodulator adenosine and its receptors are mediators of sleep-wake regulation which is known to differ between sexes. We, therefore, investigated sex differences in A1 adenosine receptor (A1AR) availability in healthy human subjects under well-rested conditions using [18F]CPFPX and positron emission tomography (PET). [18F]CPFPX PET scans were acquired in 50 healthy human participants (20 females; mean age ± SD 28.0 ± 5.3 years). Mean binding potential (BPND; Logan's reference tissue model with cerebellum as reference region) and volume of distribution (VT) values were calculated in 12 and 15 grey matter brain regions, respectively. [18F]CPFPX BPND was higher in females compared to males in all investigated brain regions (p < 0.025). The largest differences were found in the pallidum and anterior cingulate cortex, where mean BPND values were higher by 29% in females than in males. In females, sleep efficiency correlated positively and sleep latency negatively with BPND in most brain regions. VT values did not differ between sexes. Sleep efficiency correlated positively with VT in most brain regions in female participants. In conclusion, our analysis gives a first indication for potential sex differences in A1AR availability even under well-rested conditions. A1AR availability as measured by [18F]CPFPX BPND is higher in females compared to males. Considering the involvement of adenosine in sleep-wake control, this finding might partially explain the known sex differences in sleep efficiency and sleep latency.

In vivo multimodal imaging of adenosine A1 receptors in neuroinflammation after experimental stroke.

Adenosine A1 receptors (A1ARs) are promising imaging biomarkers and targets for the treatment of stroke. Nevertheless, the role of A1ARs on ischemic damage and its subsequent neuroinflammatory response has been scarcely explored so far. Methods: In this study, the expression of A1ARs after transient middle cerebral artery occlusion (MCAO) was evaluated by positron emission tomography (PET) with [18F]CPFPX and immunohistochemistry (IHC). In addition, the role of A1ARs on stroke inflammation using pharmacological modulation was assessed with magnetic resonance imaging (MRI), PET imaging with [18F]DPA-714 (TSPO) and [18F]FLT (cellular proliferation), as well as IHC and neurofunctional studies. Results: In the ischemic territory, [18F]CPFPX signal and IHC showed the overexpression of A1ARs in microglia and infiltrated leukocytes after cerebral ischemia. Ischemic rats treated with the A1AR agonist ENBA showed a significant decrease in both [18F]DPA-714 and [18F]FLT signal intensities at day 7 after cerebral ischemia, a feature that was confirmed by IHC results. Besides, the activation of A1ARs promoted the reduction of the brain lesion, as measured with T2W-MRI, and the improvement of neurological outcome including motor, sensory and reflex responses. These results show for the first time the in vivo PET imaging of A1ARs expression after cerebral ischemia in rats and the application of [18F]FLT to evaluate glial proliferation in response to treatment. Conclusion: Notably, these data provide evidence for A1ARs playing a key role in the control of both the activation of resident glia and the de novo proliferation of microglia and macrophages after experimental stroke in rats.

Influence of binding affinity and blood plasma level on cerebral pharmacokinetics and PET imaging characteristics of two novel xanthine PET radioligands for the A1 adenosine receptor.

INTRODUCTION: The suitability of novel positron emission tomography (PET) radioligands for quantitative in vivo imaging is affected by various physicochemical and pharmacological parameters. In this study, the combined effect of binding affinity, lipophilicity, protein binding and blood plasma level on cerebral pharmacokinetics and PET imaging characteristics of three xanthine-derived A1 adenosine receptor (A1AR) radioligands was investigated in rats. METHODS: A comparative evaluation of two novel cyclobutyl-substituted xanthine derivatives, 8-cyclobutyl-3-(3-[18F]fluoropropyl)-1-propylxanthine ([18F]CBX) and 3-(3-[18F]fluoropropyl)-8-(1-methylcyclobutyl)-1-propylxanthine ([18F]MCBX), with the reference A1AR radioligand 8-cyclopentyl-3-(3-[18F]fluoropropyl)-1-propylxanthine ([18F]CPFPX) was conducted. This evaluation included in vitro competition binding assays, in vitro autoradiography and in vivo PET imaging. Differences in cerebral pharmacokinetics and minimal scan duration required for quantification of cerebral distribution volume (VT) were assessed. RESULTS: Measured Ki values of non-labeled CBX, MCBX and CPFPX were 10.0 ± 0.52 nM, 3.3 ± 0.30 nM and 1.4 ± 0.15 nM, respectively (n = 3-4). In vitro autoradiographic binding patterns in rat brain were comparable between the radioligands, as well as the fraction of non-specific binding (1.0-1.9%). In vivo cerebral pharmacokinetics of the novel cyclobutyl-substituted xanthines differed considerably from that of [18F]CPFPX. Brain uptake and VT of [18F]CBX were substantially lower despite the higher concentration of radiotracer in plasma. [18F]MCBX showed comparable uptake and VT, but faster cerebral kinetics than [18F]CPFPX. However, the faster kinetics of [18F]MCBX did not enable the quantification of cerebral VT in a shorter scan time. CONCLUSIONS: The combined effect of individual physicochemical and pharmacological properties of a radiotracer on its PET imaging characteristics cannot be readily predicted. In vivo performance of the xanthine A1AR radioligands was mainly influenced by binding affinity; plasma concentrations and cerebral kinetics were of secondary importance.

Relevance of In Vitro Metabolism Models to PET Radiotracer Development: Prediction of In Vivo Clearance in Rats from Microsomal Stability Data.

The prediction of in vivo clearance from in vitro metabolism models such as liver microsomes is an established procedure in drug discovery. The potentials and limitations of this approach have been extensively evaluated in the pharmaceutical sector; however, this is not the case for the field of positron emission tomography (PET) radiotracer development. The application of PET radiotracers and classical drugs differs greatly with regard to the amount of substance administered. In typical PET imaging sessions, subnanomolar quantities of the radiotracer are injected, resulting in body concentrations that cannot be readily simulated in analytical assays. This raises concerns regarding the predictability of radiotracer clearance from in vitro data. We assessed the accuracy of clearance prediction for three prototypical PET radiotracers developed for imaging the A1 adenosine receptor (A1AR). Using the half-life (t1/2) approach and physiologically based scaling, in vivo clearance in the rat model was predicted from microsomal stability data. Actual clearance could be accurately predicted with an average fold error (AFE) of 0.78 and a root mean square error (RMSE) of 1.6. The observed slight underprediction (1.3-fold) is in accordance with the prediction accuracy reported for classical drugs. This result indicates that the prediction of radiotracer clearance is possible despite concentration differences of more than three orders of magnitude between in vitro and in vivo conditions. Consequently, in vitro metabolism models represent a valuable tool for PET radiotracer development.

Image-Derived Input Functions for Quantification of A1 Adenosine Receptors Availability in Mice Brains Using PET and [18F]CPFPX.

PURPOSE: In vivo imaging for the A1 adenosine receptors (A1ARs) with positron emission tomography (PET) using 8-cyclopentyl-3-(3-[18F]fluoropropyl)-1-propylxan- thine ([18F]CPFPX) has become an important tool for studying physiological processes quantitatively in mice. However, the measurement of arterial input functions (AIFs) on mice is a method with restricted applicability because of the small total blood volume and the related difficulties in withdrawing blood. Therefore, the aim of this study was to extract an appropriate [18F]CPFPX image-derived input function (IDIF) from dynamic PET images of mice. PROCEDURES: In this study, five mice were scanned with [18F]CPFPX for 60 min. Arterial blood samples (n = 7 per animal) were collected from the femoral artery and corrected for metabolites. To generate IDIFs, three different approaches were selected: (A) volume of interest (VOI) placed over the heart (cube, 10 mm); (B) VOI set over abdominal vena cava/aorta region with a cuboid (5 × 5 × 15 mm); and (C) with 1 × 1 × 1 mm voxels on five consecutive slices. A calculated scaling factor (α) was used to correct for partial volume effect; the method of obtaining the total metabolite correction of [18F]CPFPX for IDIFs was developed. Three IDIFs were validated by comparison with AIF. Validation included the following: visual performance; computing area under the curve (AUC) ratios (IDIF/AIF) of whole-blood curves and parent curves; and the mean distribution volume (V T) ratios (IDIF/AIF) of A1ARs calculated by Logan plot and two-tissue compartment model. RESULTS: Compared with the AIF, the IDIF with VOI over heart showed the best performance among the three IDIFs after scaling by 1.77 (α) in terms of visual analysis, AUC ratios (IDIF/AIF; whole-blood AUC ratio, 1.03 ± 0.06; parent curve AUC ratio, 1.01 ± 0.10) and V T ratios (IDIF/AIF; Logan V T ratio, 1.00 ± 0.17; two-tissue compartment model V T ratio, 1.00 ± 0.13) evaluation. The A1ARs distribution of average parametric images was in good accordance to autoradiography of the mouse brain. CONCLUSION: The proposed study provides evidence that IDIF with VOI over heart can replace AIF effectively for quantification of A1ARs using PET and [18F]CPFPX in mice brains.

Synthesis and Pharmacological Evaluation of Identified and Putative Metabolites of the A1 Adenosine Receptor Antagonist 8-Cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine (CPFPX).

The A1 adenosine receptor (A1 AR) antagonist [18 F]8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine ([18 F]CPFPX), used in imaging human brain A1 ARs by positron emission tomography (PET), is stable in the brain, but rapidly undergoes transformation into one major (3-(3-fluoropropyl)-8-(3-oxocyclopenten-1-yl)-1-propylxanthine, M1) and several minor metabolites in blood. This report describes the synthesis of putative metabolites of CPFPX as standards for the identification of those metabolites. Analysis by (radio)HPLC revealed that extracts of human liver microsomes incubated with no-carrier-added (n.c.a.)[18 F]CPFPX contain the major metabolite, M1, as well as radioactive metabolites corresponding to derivatives functionalized at the cyclopentyl moiety, but no N1-despropyl species or metabolites resulting from functionalization of the N3-fluoropropyl chain. The putative metabolites were found to displace the binding of [3 H]CPFPX to the A1 AR in pig brain cortex at Ki values between 1.9 and 380 nm and the binding of [3 H]ZM241385 to the A2A AR in pig striatum at Ki values >180 nm. One metabolite, a derivative functionalized at the ω-position of the N1-propyl chain, showed high affinity (Ki 2 nm) to and very good selectivity (>9000) for the A1 AR.

New potent A1 adenosine receptor radioligands for positron emission tomography.

8-Cyclopentyl-3-(3-[18F]fluoropropyl)-1-propylxanthine ([18F]CPFPX) is meanwhile an accepted receptor ligand to examine the A1 adenosine receptor (A1AR) in humans by positron emission tomography (PET). A major drawback of this compound is its rather fast metabolic degradation in vivo. Therefore two new xanthine derivatives, namely 8-cyclobutyl-1-cyclopropymethyl-3-(3-fluoropropyl)xanthine (CBCPM; 5) and 1-cyclopropylmethyl-3-(3-fluoropropyl)-8-(1-methylcyclobutyl)xanthine (CPMMCB; 6) were designed and synthesized as potential alternatives to CPFPX. In membrane binding studies both compounds showed nanomolar affinity for the A1AR. In vitro autoradiographic studies of [18F]5 and [18F]6, using rat brain slices, showed the expected accumulation in regions known to have a high adenosine A1 receptor expression while exhibiting the necessary low unspecific binding. However, in vitro metabolite studies using human liver microsomes revealed a comparable metabolic degradation rate for both new xanthine derivatives and CPFPX.

In vivo kinetic and steady-state quantification of 18F-CPFPX binding to rat cerebral A1 adenosine receptors: validation by displacement and autoradiographic experiments.

UNLABELLED: In vivo imaging of the A1 adenosine receptor (A1AR) using (18)F-8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine ((18)F-CPFPX) and PET has become an important tool for studying physiologic and pathologic states of the human brain. However, dedicated experimental settings for small-animal studies are still lacking. The aim of the present study was therefore to develop and evaluate suitable pharmacokinetic models for the quantification of the cerebral A1AR in high-resolution PET. METHODS: On a dedicated animal PET scanner, 15 rats underwent (18)F-CPFPX PET scans of 120-min duration. In all animals, arterial blood samples were drawn and corrected for metabolites. The radioligand was injected either as a bolus or as a bolus plus constant infusion. For the definition of unspecific binding, the A1AR selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) was applied. After PET, the brains of 9 animals were dissected and in vitro saturation binding was performed using high-resolution (3)H-DPCPX autoradiography. RESULTS: The kinetics of (18)F-CPFPX were well described by either compartmental or noncompartmental models based on arterial input function. The resulting distribution volume ratio correlated with a low bias toward identity with the binding potential derived from a reference region (olfactory bulb) approach. Furthermore, PET quantification correlated significantly with autoradiographic in vitro data. Blockade of the A1AR with DPCPX identified specific binding of about 45% in the reference region olfactory bulb. CONCLUSION: The present study provides evidence that (18)F-CPFPX PET based on a reference tissue approach can be performed quantitatively in rodents in selected applications. Specific binding in the reference region needs careful consideration for quantitative investigations.