Quantification of P2X7 receptors with [11C]SMW139
Purinergic receptors (purinoceptors) include the P1 purinoceptors (adenosine receptors), and P2 purinoceptors which are preferably activated by ATP and other substrates than adenosine. P2X7R is structurally and functionally distinct from the other P2X receptors. The affinity of ATP for P2X7R is low, and micromolar extracellular concentrations of ATP do not activate it. Intense stimulation of P2X7Rs results in the formation of a large transmembrane pore that further increases the extracellular [ATP], and can lead to activation of caspases and cell death. Various inflammatory mediators can up-regulate P2X7R expression on macrophages and other cell types, while anti-inflammatory mediators can down-regulate the expression (Bartlett et al., 2014).
P2X7R is expressed in many cell types, including sympathetic neurons, astrocytes, oligodendrocytes, microglial cells, probably also CNS neurons, skeletal muscle cells, osteoblasts and osteoclasts, keratinocytes, fibroblasts, epithelial cells, white blood cells, erythrocytes, and endo- and exocrine cells. In the brain, P2X7R levels are highest in microglia, and the receptor is important in regulation of immune responses, including the neuroinflammatory cascades that precede and promote many neurodegenerative brain diseases such as Parkinson's disease and MS. The gene of the P2X7R is polymorphic, including many splice variations and single nucleotide polymorphisms, associated with diseases including multiple sclerosis, bipolar disorder, and increased risk for bone fractures.
[11C]SMW139 is a recently developed tracer (Wilkinson et al., 2017; Janssen et al., 2018) aimed for P2X7R imaging in the brain. From several adamantanyl benzamides, [11C]SMW139 ("trifluorinated benzamide 34") had the best properties, including metabolic stability, and its inhibitory activity was not markedly affected by the six functionally characterized hP2X7R SNPs, suggesting that P2X7R polymorphisms would not confound the [11C]SMW139 binding (Wilkinson et al., 2017), like may be the case with another P2X7R radiopharmaceutical [11C]JNJ-54173717 (Van Weehaeghe et al., 2019).
In a rat model, where over-expression of P2X7R was induced by injection of adeno-associated viral vector, a clear increase in [11C]SMW139 uptake was seen in the affected site (Janssen et al., 2018). Kinetics in the rat brain is fast, suggesting that [11C]SMW139 concentration is in equilibrium with plasma, and that volume of distribution could be used for analysis. [11C]SMW139 uptake was increased in EAE rats, and could be blocked with P2X7R antagonist (Beaino et al., 2020). [11C]SMW139 uptake was not affected by P-glycoprotein blocking, suggesting that the radioligand is not a P-gp substrate at BBB (Beaino et al., 2020).
In postmortem human brain studies no significant binding difference was found between AD and control subjects (Janssen et al., 2018), but there was a slight trend towards lower binding especially in white matter of AD subjects. Binding was higher in white matter than in grey matter in both groups (Janssen et al., 2018).
The first in vivo human studies with [11C]SMW139 have been performed in MS patients and age-matched controls (Hagens et al., 2020), and a PD study is ongoing (INMIND report, 2017). No difference was found between normal volunteers and PD patients with another P2X7R radiopharmaceutical [11C]JNJ-54173717, possibly because genotype effect (Van Weehaeghe et al., 2019).
Two-tissue reversible compartmental model has been used to analyze 90-min human data; k4 was fixed to an average in white or grey matter (Hagens et al., 2020). Results from 60 and 90 min data were highly correlated, although slightly lower from the 60 min data (Hagens et al., 2020). Increased VT was found in normal appearing white matter (NAWM) and in gadolinium-enhancing lesions in relapsing remitting MS as compared to healthy control subjects data (Hagens et al., 2020).
Radioactive metabolites of [11C]SMW139 have been found in the brain tissue of mice, and compartmental modelling with correction for the brain-penetrative radiometabolites improves the in vivo quantification of P2X7R in the human brain (Brumberg et al., 2023). While the radioactive metabolites are less lipophilic than [11C]SMW139, those also are less bound to plasma proteins, which may contribute to their higher than expected concentration in the brain (Aarnio et al., 2022).
Since P2X7Rs are widely distributed, a reference region cannot be found, and quantification is dependent on arterial plasma time-activity curve (PTAC) as the input function. With other radioligands without reference region, volume of distribution has been used to represent the receptor binding, and Lassen plot has been used to estimate receptor occupancies.
Plasma input function
In rats, [11C]SMW139 is excreted from circulation via the liver, and 45 min after administration the fraction of intact radioligand in plasma was 42%
(Janssen et al., 2018).
In humans, the fraction of intact radioligand in plasma seems to be variable, but usually higher
than in rats, ∼50% still 90 min after administration. Monoexponential function with baseline
(fit_fexp with options
-mono -a=1) can be
used to fit the fractions.
Plasma protein binding was 90% in rats (Wilkinson et al., 2017). In small animals the plasma sample volumes can be only few microlitres, and a solid-phase microextraction method has been developed for [11C]SMW139 (Moein et al., 2020).
In humans, the plasma-to-blood ratio seems to start at ∼1.7, and slowly decreases to ∼1.5. This suggests that intact [11C]SMW139 stays in blood plasma, whereas radioactive metabolite(s) are found in both blood plasma and blood cells. Program fit_pbr can be used to fit surge function with recirculation to the plasma-to-blood ratio data. If individual hematocrit is measured, then programs b2plasma and p2blood can be used to convert blood curves to plasma and vice versa, using population mean of the ratio. However, individual plasma-to-blood ratio measurements would be preferred because of relatively high variation in plasma metabolite fractions may cause differences in plasma-to-blood ratio at late times.
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Updated at: 2022-12-02
Created at: 2018-03-29
Written by: Vesa Oikonen, Richard Aarnio, Päivi Marjamäki