Naloxone’s displacement of [11C]carfentanil and duration of receptor occupancy in the rat brain: implications for opioid overdose reversal


 The continuous rise in opioid overdoses in the United States is predominantly driven by very potent synthetic opioids, mostly fentanyl and its derivatives (fentanyls). Although naloxone (NLX) has been shown to effectively reverse overdoses by conventional opioids, there may be a need for higher or repeated doses of NLX to revert overdoses from highly potent fentanyls. Here, we used positron emission tomography (PET) to assess NLX’s dose-dependence on both its rate of displacement of [11C]carfentanil ([11C]CFN) binding and its duration of mu opioid receptor (MOR) occupancy in the male rat brain. We showed that clinically relevant doses of intravenously (IV) administered NLX (0.035mg/kg, Human Equivalent Dose(HED) 0.4mg; 0.17mg/kg, HED 2mg) rapidly displaced the specific binding of [11C]CFN in thalamus in a dose-dependent manner. Brain MOR occupancy by IV NLX was greater than 90% at 5 minutes after NLX administration for both doses, but only 50% occupancy remained at 27.3 min and at 85 min after 0.035mg/Kg and 0.17 mg/Kg NLX, respectively. This indicates that the duration of NLX occupancy at MORs is short-lived. Overall, these results show that clinically relevant doses of IV NLX can promptly displace fentanyls at brain MORs, but that repeated or higher NLX doses may be required to prevent re-narcotization following overdoses with long-acting fentanyls.


Introduction
Over the past 5 years, the opioid overdose epidemic in the United States [1] has been exacerbated by the rise in illicit synthetic opioids such as fentanyl (1) and its derivatives (fentanyls, Fig. 1) [2][3][4]. Indeed, in the 12 months preceding March 2021 alone, the CDC estimated a total of 63,075 synthetic opioid overdose deaths, a 54.5% increase from the previous year [5]. This persistent increase in fentanyls-related overdose deaths re ects their highly rewarding effects, potency in inducing respiratory depression, and their widespread availability driven by its ease of production and distribution [6][7][8]. Such challeges are, thus, forcing health care providers to reconsider their strategies for treating overdoses caused by synthetic opioids [9][10][11].
Parenteral (0.4 mg, 2 mg) and intranasal (IN, 2 mg, 4 mg) NLX formulations are available over the counter in most states for opioid overdose reversal. However, there are growing concerns that those options may be ineffective in reversing overdoses of highly potent synthetic opioids such as fentanyl (K i = 0.39 nM) [16] and carfentanil (2, CFN, K d = 0.08 nM) [17]. Additinally, the relatively short duration of NLX's action may be insu cient for preventing re-narcotization after overdose with the longer-lasting fentanyls. In fact, Tomassoni et al. noted, "Some patients required doses of the opioid antidote naloxone exceeding 4 mg (usual initial dose = 0.1-0.2 mg intravenously), and several patients who were alert after receiving naloxone subsequently developed respiratory failure" [18]. Multiple NLX injections are sometimes required to maintain adequate breathing following re-narcotization after initial NLX treatment [19]. Thus, a better understanding of NLX's blockade of the MOR over time would help to improve our clinical guidelines regarding NLX administration and dosing for overdose reversal from fentanyls.
Positron emission tomography (PET) and the MOR radioligand, [ 11 C]CFN have been used to measure blockade of MOR by NLX non-invasively in humans [20] and in non-human primates [21]. Most [ 11 C]CFN PET studies have assessed receptor occupancy (RO) within 10 min after an acute NLX dose for various administration methods [19,[22][23][24][25][26][27][28][29][30]. In particular, two relevent clinical reports have been published on NLX's clearance rate from MORs. One used a dual coincidence detector system to compare intravenous (IV) NLX with IV nalmefene [31]; the other used PET to obtain RO at two time points, after intranasal (IN) NLX [32]. In the current study, we used PET to measure the displacement of [ 11 C]CFN binding by IV NLX in the rodent brain. Susequently we obtained RO at multiple time points to characterize the clearance pro le over 2.5 hrs following IV NLX. For both set of experiments, we compared two clinically relevant IV NLX doses (0.035 and 0.17 mg/kg), which correspond to human equivalent doses (HED) of 0.4 mg and 2.0 mg respectively.

[ 11 C]CFN binding displacement by intravenous NLX
In [ 11 C]CFN PET scans, thalamus showed high uptake and speci c binding and was used as the region of interrst (ROI) to quantify speci c binding and RO by NLX [33]. Cerebellum showed fast [ 11 C]CFN clearance and was used as a reference region (SI Fig. 1A). Averaged clearance half-time (t 1/2 ) from peak was 41.84 min for thalamus and 7.33 min for cerebellum. IV NLX pretreatment at 5 min prior to [ 11 C]CFN injection reduced [ 11 C]CFN uptake in thalamus to the level of uptake seen in cerebellum (SI Fig. 2). This provides preclinical evidence suggesting that currently approved doses of IV NLX can abolish speci c binding of [ 11 C]CFN, and are likely at peak levels, to temporarily occupy nearly all MORs in brain.
To characterize NLX's displacement of [ 11 C]CFN at MORs, NLX administration was given at 15 min after [ 11 C]CFN injection. Both doses of NLX gradually diminished [ 11 C]CFN binding in the thalamus to the level observed in the cerebellum (reference region), but the higher dose displaced it faster than the lower one ( Fig. 2 and Fig. 3). Time-activity curves of [ 11 C]CFN in the thalamus prior to NLX injection did not differ statistically between the control and the NLX treatment groups (Student t-test p= 0.14 for 0.17 mg/kg; p=0.31 for 0.035 mg/kg) (Fig. 2B). After the IV NLX challenge, the time to reach equivalent levels of [ 11 C]CFN uptake in the thalamus and cerebellum (ratio of standard uptake value, SUVr =1) was 61.76 ± 9.49 min for the lower NLX dose (0.035 mg/kg) and 34.42 ± 3.56 min for the higher dose (0.17mg/kg), and these differences between the two doses was signi cant (p=0.0095, student t-test) (Fig. 2C). Estimation of the absolute gradient change after NLX injection [34], showed that NLX's displacement of [ 11 C]CFN was signi cantly faster for the 0.17 mg/kg NLX dose than for the 0.035 mg/kg dose (p=0.002, student t-test, n=3) (Fig. 2D).
Duration of receptor occupancy after pretreatment with IV NLX Rats were pretreated with IV NLX (0.035 mg/kg, 0.17mg/kg) at various time points before [ 11 C]CFN injection. While there were no differences in [ 11 C]CFN uptake in the cerebellum for any time points, the uptake of [ 11 C]CFN in the thalamus differed signi cantly between the various time points of NLX pretreatment showing a gradual recovery towards baseline over two hours. The non-displaceable binding potentials (BP ND ) of [ 11 C]CFN at baseline (n=8) and at various NLX pretreatment times are summarized in Supplemental Table 1. The higher dose of IV NLX (0.17 mg/kg) blocked [ 11 C]CFN binding in the thalamus longer than the lower dose (0.035 mg/kg). Speci cally, 90 min after IV NLX injection, the averaged RO of 0.17 mg/kg NLX was signi cantly higher than that of 0.035 mg/kg NLX (60% vs 12% RO, p = 0.02) (Fig.  4). Consistently, the clearance half-time of RO for the thalamus was 27.3 min for the 0.035 mg/kg NLX and 85 min for 0.017 mg/kg NLX (Fig. 5).

Pharmacokinetics assessment of IV NLX
The measured peak plasma NLX concentration differed signi cantly between the two doses (0.17 mg/kg: 77 ng/ml; 0.035 mg/kg: 1.7 ng/ml; Student t-test, p=0.01) (Fig. 5). Table 1 shows the estimated pharmacokinetic data for IV NLX in plasma based on a non-compartmental analysis. The average elimination rate of IV NLX (K e ) was 16 min for 0.17 mg/kg and 7.3 min for 0.035 mg/kg NLX, respectively. Correlation between receptor occupancy and plasma naloxone concentration NLX plasma concentration decreased rapidly for the two doses of IV NLX, but lasted longer for the higher dose (SI Fig. 3). The plasma NLX levels necessary to achieve half-maximal receptor occupancy (EC 50 ) were estimated to be 0.2 ng/mL as assessed by tting the association between NLX Ros and plasma NLX concentrations (SI Fig. 4). NLX RO levels plateaud at 90% for NLX plasma concentration higher than 1.5 ng/ml.

Discussion
Highly potent fentanyl derivatives are the main contributors to the steep rise in opioid overdose mortality.
To cope with the challenges in reversing overdoses with potent fentanyls, the Food and Drug Administration (FDA) recently approved a high dose of IN NLX (8 mg, [35]) and a 5 mg NLX injection dose [36]. Balancing the risks and bene ts of increasing NLX doses used for opioid reversal is a topic of clinical interest [37]. Thus, understanding the relationship between NLX's dose and its onset and duration of MOR blockade will play a key role in developing proper guidelines for fentanyls overdose reversal.
In the current study, we used clinically relevant doses of IV NLX to characterize their e cacy in displacing times longer compared to that for the 0.035 kg/mg dose. As shown in Figure 3, RO of less than 30% occurred within 50 min for the lower dose (0.035 mg/kg) whereas it occurred at 100 min for the higer dose (0.17 mg/kg). The fast RO clearance observed with IV NLX could explain why multiple NLX doses are often required to prevent re-narcotization following overdoses with fentanyls [38].
In a separate group of rats, NLX pharmacokinetics were measured in plasma for the two NLX doses and to correlate them with levels of RO, we estimated that the plasma NLX concentration for half-maximal receptor occupancy (EC 50 ) was low (0.2 ng/ml). However, it is likely that the NLX concentration in brain was much higher than in plasma (SI Fig. 3) due to NLX's high lipophilicity and the short-lasting peak in plasma after IV administration. Future studies are required to assess the relationship between plasma and brain NLX levels and to ascertain what level of RO is needed to restore and sustain proper breathing following fentanyls overdoses.
Previously, Kim et al. measured [ 11 C]CFN uptake in the human brain at four time points over 9 hours using a dual coincidence detector system. They reported that the clearance half-time of RO by IV NLX (2 mg) was 2 ± 1.6 hr after administration [31]. These data, in conjunction with our own, suggest that 2 mg IV NLX might be inadequate for preventing re-narcotization after overdose with long half-life fentanyls (e.g., CFN T 1/2 = 7.7 hr). Similarly, Johansson et al. [32] used PET to measure the speci c binding of [ 11 C]CFN in healthy human brains at two separate time intervals (0-60 min, 300-360 min) after administration of 2 mg IN NLX. They found that the clearance half-time of RO occurred 2-3 hours after NLX administration and that the duration of NLX's RO at MORs was signi cantly longer for the 4 mg dose than the 2 mg dose. Thus, both clinical and preclinical studies provide evidence that may justify the use of higher doses of NLX, thereby corroborating the FDA's recent decision to approve higher NLX formulations [35,36]. Despite these conclusions, there are serious drawbacks for using higher dose of NLX, namely, NLXprecipitated withdrawal symptoms. Likewise, the use of potent MOR antagonists with a longer duration of action, such as naltrexone and nalmefene, has been proposed [39], but concerns of protracted opioid withdrawal need to be considered. In this respect, it would be valuable to determine the minimal levels of RO needed to sustain normal breathing and cardiovascular function following an overdose.

Limitations
Our study is limited by species differences in both the NLX pharmacokinetics under anesthesia, and pharmacodynamics of opioid induced respiratory depression. . List-mode data was acquired over 90 min after a 10 min transmission scan with a Co-57 point source for attenuation correction. PET data was reconstructed into 22 frames (6 x 20 sec, 5 x 60 sec, 4 x 120 sec, 3 x 300 sec, 3 x 600 sec, and 1 x 1200 sec) using ltered back-projection. The average activity injected was 14.0 ± 8.6 MBq and the average CFN mass injected was 60.4 ± 55 ng/kg.

PET imaging processing and tracer kinetic analysis
Time-activity curves were obtained as standard uptake value (SUV, g/mL) using PMOD (3.807). Two regions of interest (ROIs) were analyzed for [ 11 C]CFN uptake: the thalamus due to its high concentration of MORs [42] and high speci c binding, and the cerebellum, which was used as a reference region mostly devoid of speci c binding [43].
The ROI template was drawn using anatomical information extracted from a [ 18 F]FDG PET scan obtained for this purpose following a [ 11 C]CFN scan in one rat. ROIs were drawn in the cerebellum and thalamus, avoiding border regions, and were applied to generate time activity curves.
SUVr was calculated for each frame as the ratio between thalamic and cerebellar SUVs. For RO studies, BP ND was obtained using the B/CI method to achieve constant radioactivity levels in the ROIs and in the reference region [44,45]. Once equilibrium is achieved [46], the binding potential (BP ND ) was calculated directly from the concentration ratio of thalamus to cerebellum (15-40 min Te datasets analysed during the current study are available from the corresponding author on reasonable request.

Figure 1
Structures of fentanyls and naloxone. Each arrow represents positions which have been abundantly reported to generate illegal fentanyls via chemical modi cation with various substituents.    MOR occupancy pro les after IV NLX for two doses (circle, 0.035 mg/kg; rectangle, 0.17 mg/kg).
Occupancy data averaged for each time point were plotted with a sigmoidal function. NLX 0.035 mg/kg corresponds to 0.4 mg HED and NLX 0.17 mg/kg to 2 mg HED. Error bars correspond to standard deviations.