Evaluating the rate of reversal of fentanyl-induced respiratory depression using a novel long-acting naloxone nanoparticle, cNLX-NP

Abstract

Introduction: Fentanyl and fentanyl analogs (F/FA) have become increasingly common adulterants in counterfeit prescription pills and illicit street drug mixtures due to their ease of synthesis and exceedingly high potency. The ongoing epidemic of fatal overdoses fueled by F/FA continues to highlight the need for longer-acting therapies than naloxone (NLX), the current gold-standard for reversing opioid overdoses, which shows limited efficacy to prevent renarcotization associated with F/FA toxicity. A novel opioid reversal agent based on covalent naloxone nanoparticles (cNLX-NP) has been shown to blunt fentanyl-induced respiratory depression out to 48 hr, demonstrating its potential therapeutic utility. The purpose of this study was to characterize how rapidly cNLX-NP reverses fentanyl-induced respiratory effects as well as the duration of its protective effects.

Methods: Sprague Dawley male rats (n=6/group) were tested on an oximeter for baseline percent arterial oxygen saturation (%SaO₂) challenged with 0.1 mg/kg SC fentanyl and 15 min later given 10 mg/kg IM doses of NLX, nalmefene (NLMF), or cNLX-NP and continuously monitored via oximetry for 10 minutes. One week later the experiment was repeated using a 1:1 mixture of NLX:cNLX-NP as the reversal agent in the rats that previously received NLX alone.

Results: While both NLX and NLMF rapidly reversed %SaO₂ to baseline within 1 min, rats that received cNLX-NP did not return to >90% SaO₂ levels until 9 min after administration. Similarly, heart and breath rates returned to baseline within 1 min of treatment with NLX and NLMF but did not return to baseline until 10 minutes after cNLX-NP administration. In contrast, NLX:cNLX-NP reversed all fentanyl-induced respiratory depressive effects within one minute.

Discussion: While cNLX-NP alone may not sufficiently reverse F/FA overdose in a timely manner, mixing free NLX with cNLX-NP can provide a mechanism to both rapidly reverse fentanyl-related effects and maintain extended protection against synthetic opioid toxicity. These data support further development of cNLX-NP as a fast-acting and long-lasting antidote to treat F/FA-induced respiratory depression and overdose, and potentially prevent renarcotization in humans.

Keywords: antagonist; antidote; fentanyl; opioid use disorder; reversal agent; substance use disorder; synthetic opioids.

Figure 1

Synthesis of naloxone-PLA and naloxone-PLGA_60:40 (cNLX-NP precursors) using ring opening polymerization.

Figure 2

Pharmacokinetics of opioid antagonist reversal agents. Rats (n=2-6/group at each time-point) were given IM doses of 10 mg/kg of naloxone, nalmefene, cNLX-NP_60:40, or cNLX-NP_100:0 and blood was taken at various time points as shown. Naloxone and cNLX-NP_60:40 data were previously published (35) and were shown for comparison. Half-life was calculated using PKSolver. The dotted line on the Y-axis at 10 ng/mL estimates the amount of naloxone or nalmefene needed to significantly reduce fentanyl-induced respiratory depression in rats (as demonstrated in Experiment 2). Data are represented as Mean ± SD.

Figure 3

Long-lasting efficacy of cNLX-NP_100:0 to reverse fentanyl-induced effects. Rats (n=6/group) received 0.1 mg/kg SC fentanyl at t=0, 6, 24, and 48 hr and 10 mg/kg IM naloxone, nalmefene, or cNLX-NP_100:0 at t=17 min only and were monitored for physiological parameters via oximetry for %SaO₂ (A-D) and heart rate (E-H) and for antinociception via hotplate using latency to respond to lick hindpaw or jump (I-L). Results demonstrated that all formulations effectively reversed fentanyl-induced effects 15 minutes after administration (left-most panels) at t=0 hr and all formulations remained effective at preventing fentanyl-induced effects at t=6 hr (second column of panels from the left). Only cNLX-NP_100:0 remained effective at preventing fentanyl-induced effects at t=24 and t=48 hr compared to naloxone and nalmefene. At t=24 hr, all animals that had previously received naloxone or nalmefene had oxygen saturation levels <90% post-fentanyl administration and required an additional SC dose of naloxone to reverse fentanyl-induced effects. Only one animal in the cNLX-NP_100:0 group required naloxone. Data are represented as Mean ± SD.*p<0.05 compared to naloxone; ^#p<0.05, ^##p<0.01, and ^###p<0.001 compared to cNLX-NP_100:0 using two-way ANOVA.

Figure 4

Opioid levels from Experiment 2 in serum and brain of rats 48 hr after administration. Following the t=48 hr fentanyl dose and hotplate and oximetry assessment, serum and brain were collected from all rats from Experiment 2. Naloxone and nalmefene serum and brain levels were undetected in all groups excect cNLX-NP, which showed high levels of free naloxone in serum 48 hr after administration (A). No differences in fentanyl levels were detected in any groups treated with naloxone, nalmefene, or cNLX-NP (B), confirming that effects demonstrated by cNLX-NP_100:0 from Figure 3 are due to the presence of free naloxone and not due to differences in fentanyl distribution. Data are represented as Mean ± SD. **p<0.01 compared to naloxone; ^##p<0.01 compared to nalmefene in serum or brain using the Kruskal-Wallis multiple comparison ANOVA.

Figure 5

Time-course of reversal of fentanyl-induced respiratory depression and bradycardia following antagonist administration in rats. Rats (n=6/group) were baselined (x-axis label ‘B’) using a collar-based arterial pulse oximeter to obtain oxygen saturation levels (%SaO₂) and then given 0.1 mg/kg fentanyl SC. Fifteen minutes later rats were tested again on the oximeter (x-axis label ‘0’). Immediately afterwards, rats received 10 mg/kg IM of naloxone, nalmefene, or cNLX-NP_100:0 and % SaO₂ (A) and heart rate measured (B), represented as Mean ± SD. Results demonstrated that naloxone (C), nalmefene (D), but not cNLX-NP (E), rapidly reversed fentanyl-induced respiratory depression. To improve recovery rate for cNLX-NP_100:0, two rats (n=6) from each group were randomly selected and a week later the experiment was repeated using 5 mg/kg IM naloxone with 5 mg/kg IM cNLX-NP_100:0 as the reversal agent. These data demonstrated that a 1:1 Naloxone and cNLX-NP_100:0 formulation rapidly reversed fentanyl-induced respiratory depression (F) and that cNLX-NP_100:0 does not interfere with efficacy of free naloxone.