Opioid overdoses have accelerated in recent years, with over 107,000 reported fatalities in 2021 alone (1). Fentanyl and fentanyl analogs (F/FA) have been implicated in over 70,000 of those deaths (1), demonstrating the serious public health threat posed by this class of compounds (2–8). Fentanyl was detected in over 90% of fatal overdoses in Massachusetts between 2017 and 2019 (9). F/FA have appeared in non-opioid overdose deaths as well, with F/FA being detected in two-thirds of benzodiazepine deaths in 2020 (10), demonstrating that F/FA are a growing concern beyond opioid use. These trends have continued, or worsened, through 2023 (11). One of the challenges of treating F/FA overdoses is due to the different pharmacokinetic and pharmacodynamic (PK/PD) profiles of F/FA compared to current reversal agents.

F/FA are extremely hydrophobic mu opioid receptor (MOR) agonists that rapidly distribute to tissues including the blood-brain barrier (BBB) and are readily absorbed via multiple routes of administration (6, 12–15). The half-life of F/FA varies greatly between compounds and across individuals. Fentanyl has a half-life of 1 – 9 hours (16–18), remifentanil has a half-life of 8 – 48 min (16, 17, 19), alfentanil has a half-life of 0.42 – 1.6 hours (16, 18), and carfentanil has a half-life of 6 – 7 hours (16, 20). The current antidotes to treat F/FA overdose are small molecule MOR antagonists such as naloxone and nalmefene. Naloxone is rapidly eliminated and has a relatively short half-life of between 30 – 120 min (21–23). A recent study showed that naloxone occupancy of MORs decreased from 90% occupancy 5 min after administration to 50% occupancy 20 min later, which demonstrated how short-lived naloxone’s effects may be (24). Nalmefene, which is structurally similar to naloxone, has a much longer elimination half-life of approximately 11 hr in humans (25). This difference between the half-life of F/FA and naloxone could lead to renarcotization, whereby F/FA-induced respiratory depression returns after a brief period of reversal via treatment (20, 26, 27). This requires a subset of patients to be intubated and on ventilators in a hospital setting until they are capable of breathing on their own (20), or receive repeated doses of either naloxone or nalmefene. Because nalmefene was only recently FDA approved, it is not clear what role it will play in preventing renarcotization.

Other medications to treat F/FA overdose are at varying stages of preclinical development. Monoclonal antibodies (mAbs) against F/FA’s have demonstrated efficacy to reverse fentanyl and carfentanil effects (28, 29). Monoclonal antibodies provide the advantage of not blocking opioid receptors and a lesser likelihood of precipitating withdrawal. However, F/FA-specific mAbs would likely be more selective to single drug targets (instead of broadly reversing all opioid-induced effects) and may be less amenable to over-the-counter use due to logistics associated with administration routes and storage conditions. Methacinnamox (MCAM), another long-lasting opioid antagonist in preclinical development, has shown efficacy in reversing fentanyl-induced respiratory depression in rats and non-human primates (NHP) (30, 31) and had even longer efficacy against heroin-induced respiratory depression in NHP (32). However, MCAM may also precipitate withdrawal (33), and it has not yet been tested in humans. Further studies for these and other promising medications are warranted (34).

A new long-acting covalently-linked naloxone nanoparticle (cNLX-NP) has been developed as an antidote against F/FA, which demonstrated reversal of fentanyl-induced respiratory depression within 15 minutes of dosing and was protective against effects of fentanyl out to 48 hr after cNLX-NP administration (35). The cNLX-NPs are prepared via a two-step process involving the synthesis of biodegradable, naloxone-containing polymers and the subsequent precipitation of those polymers via a single-phase emulsion and nanoprecipitation to form well-defined nanoparticles. First, naloxone was ligated to the chain end of a biodegradable polymer via a hydrolytically labile bond, and then the polymer-bound NLX is then formulated as a nanoparticle using traditional fabrication methods wherein naloxone was non-covalently trapped in a particle (35). Given that polymer composition influences the biodegradation rate and controls the percent of naloxone incorporated in the nanoparticle, we synthesized a series of naloxone polymers possessing different levels of steric hinderance along the polymer backbone to control drug release. The steric properties were controlled by varying the amount of lactide (L) and glycolide (G) monomers that were grown from the 3-hydroxyl moiety of the naloxone initiator. Presented herein are two covalently loaded naloxone polymers with a 60:40 and 100:0 L:G composition.

This poly-lactic acid (PLA) and poly-lactic-co-glycolic acid (PLGA) nanoparticle technology is an advancement over current FDA-approved controlled-release drug delivery pharmaceuticals (such as Vivitrol^®), which are in non-covalently drug-encapsulated form. Vivitrol^® contains PLGA particles loaded with naltrexone and displays an early burst release of antagonist followed by latent low release designed for treatment of opioid use disorder (OUD). If given post-exposure, this effect can lead to precipitated opioid withdrawal, as well as provide limited therapeutic coverage post injection. By contrast, the covalent nanoparticle formulation cNLX-NP provides a linear release of antagonist at a tunable rate for a long period of time providing consistent protection from renarcotization.

The goal of these studies was to determine the effect of altering polymer composition on naloxone half-life. The lead cNLX-NP formulation would be taken forward to compare against naloxone and nalmefene to elicit rapid reversal (within minutes) and long-lasting protection (prevention of renarcotization out to 48 hr) against fentanyl. Results demonstrated that the polymer containing only lactide and no glycolide provided naloxone the longest half-life, and that this formulation was protective out to 48 hr. However, cNLX-NP could not rapidly reverse fentanyl-induced respiratory depression, which was improved when free naloxone was added to the formulation. Together, these data suggest cNLX-NP may provide better protection than free naloxone or nalmefene to prevent F/FA-induced renarcotization.

The key findings of this study were that cNLX-NP_100:0: 1) extended the terminal half-life of naloxone beyond that of naloxone alone or nalmefene; 2) blocked fentanyl effects out to 48 hr; and 3) was effective in rapidly reversing fentanyl-induced respiratory depression only when combined 1:1 with free naloxone. These data suggest that cNLX-NP_100:0 may be a long-lasting therapeutic to prevent F/FA-induced renarcotization and, in combination with free naloxone, may be an effective formulation to rapidly reverse opioid-induced respiratory depression.

The pharmacokinetics of naloxone was previously published (35) and that data were presented here alongside a new formulation of cNLX-NP_100:0 and nalmefene. The half-life of naloxone was measured at 22 min, which is well within the 15 – 30 min range in rats reported in the literature (23, 39). The half-life of naloxone was also reported following a 0.4 mg dose in humans at 64 min (23), suggesting naloxone has a slightly longer half-life in humans compared to rats. These differences likely do not impact the interpretation of the findings in the current study.

The current study also compared the pharmacokinetics of nalmefene, which was recently approved by the FDA for treatment of opioid overdose (40). In rats, the terminal half-life of nalmefene after a 5 mg/kg IV bolus dose was measured as 45 min (41). However, drug levels in blood were only measured out to 180 min, which may have artificially lowered the apparent half-life if only the alpha phase (which relates more to the redistribution of drug rather than the elimination) was measured. Although the current study measured the half-life of nalmefene after an IM dose, the half-life was 12.3 hours, which is 16X longer than the previous report in rats. It was apparent from Figure 2 that nalmefene has a rapid redistribution phase but very slow elimination, which may not have been accounted for in the previous report. One study measured nalmefene half-life at 26 – 35 min in rats, but this was after nasal administration and was measured only out to 30 min following small doses between 15 – 45 μg/mL (42). No other reports on nalmefene pharmacokinetics in rats could be found. In humans, the terminal half-life of nalmefene around 8 – 11 hr following IV bolus doses between 2 – 24 mg (43, 44), which matches the rat data from the current study. These data suggest that nalmefene pharmacokinetics may be similar between humans and rats and that the current findings related to nalmefene and cNLX-NP could be extrapolated to humans.

An effective antidote to treat F/FA overdose and prevent renarcotization needs to have a half-life that is greatly superior to that of naloxone. In a previous publication describing the use of covalently loaded naloxone nanoparticles derived from PLGA (cNLX-NP_60:40), it was shown that the half-life of naloxone could be extended by approximately 34-fold (35). The current study demonstrated that by using only rac-lactide instead of a mixture of rac-lactide and glycolide the half-life of naloxone could be extended about 33% more. Furthermore, cNLX-NP_100:0 had approximately 10X more free naloxone at 48 hr than cNLX-NP_60:40, suggesting that the rac-lactide formulation could perform even beyond 48 hr. These data are in accordance with the results of similar studies examining the biodegradation of various PLA and PLGA polymers. It has been well documented in the literature that PLA exhibits slow rates of hydrolysis while the hydrolysis of PLGA copolymers is considerably faster (45). In fact, increasing the glycolic acid content is a common strategy to increase the degradation rate of a polymer (46). This phenomenon can be best explained using a simple steric argument. The methyl-bearing stereocenter of lactic acid provides a more sterically crowded environment around the ester carbonyls in PLA thus hindering nucleophilic attack and subsequent hydrolysis (47). In the present study, this translates to a slower, sustained release of naloxone and a longer circulatory half-life. In contrast, glycolic acid ester groups do not possess any substitution on the adjacent α-carbon leaving them more readily accessible to nucleophilic attack and more prone to hydrolysis resulting in a shorter half-life as observed for cNLX-NP_60:40. However, because the two naloxone nanoparticles (cNLX-NP_100:0 and cNLX-NP_60:40) were studied at separate times in different cohort of rats, although under the same conditions, caution should be applied when interpreting the results.

Although nalmefene and the cNLX-NP_100:0 have similar half-lives, nalmefene was only effective out to 6 hr, while cNLX-NP_100:0 reduced fentanyl-induced effects out to 48 hr. The cNLX-NP_60:40 formulation previously performed similarly (35). Based on these results, a hypothetical effective threshold of antagonist needed to prevent fentanyl-induced respiratory depression of 10 ng/mL was added to Figure 2 y-axis. Confirming that fentanyl levels were identical between groups at 48 hr, and that naloxone levels were only detected in the cNLX-NP_100:0 group, demonstrated that efficacy of cNLX-NP_100:0 was related to sustained release of naloxone and not due to any apparent differences in fentanyl levels. Although fentanyl has been observed to induce tolerance (35), post-fentanyl oxygen saturation data in the naloxone and nalmefene groups at t=0 hr and t=48 hr were similar and suggest that tolerance did not play a major role in the findings of this study. Combined, these data suggest that a long-acting nanoparticle-based formulation can outperform both naloxone and nalmefene, continuing to protect against F/FA-induced respiratory depressive effects beyond 6 hr.

PKSolver was demonstrated to produce pharmacokinetic parameters that were identical to the industry standard software package Phoenix WinNonlin, validating the use of this software package in the current study as well as previous studies (35, 38, 48). The original developers also validated this software against WinNonlin and showed that no differences were apparent (37). PKSolver is an add-on to Microsoft Excel which has an easier-to-use interface than WinNonlin. Further, Microsoft Excel is a software package readily available to most researchers whereas WinNonlin has expensive licensing restrictions that make it difficult to access. The downside to PKSolver is that the software package is no longer maintained, cannot be used in versions of Microsoft Excel beyond 2010, is not available on the Mac OS platform, and may require the use of virtualization within newer operating systems.

An important feature of an opioid antidote is the ability to rapidly reverse (i.e., within 1 min) an overdose. Although cNLX-NP_100:0 was able to reverse fentanyl-induced respiratory depression 15 minutes after administration as measured in the current study, it was unable to rapidly reverse fentanyl effects within minutes to the same degree as naloxone and nalmefene. This was likely because no free naloxone was available immediately after administration. However, a mixture of 1:1 free naloxone with cNLX-NP_100:0 demonstrated rapid reversal of fentanyl-induced respiratory depression, indicating that combining free antagonist with a nanoparticle formulation can provide immediate and long-lasting effects, while preserving the benefits of both therapeutics.

Bradycardia was rapidly reversed in all groups in Experiment 3, except the cNLX-NP_100:0 alone group which failed to return to baseline levels within 10 minutes assessment period. This is surprising given that bradycardia was reversed within 15 minutes in Experiment 2. A potential difference between these two experiments is that rats were handled immediately prior to oximetry testing in Experiment 2, which may have artificially increased heart rate levels, while rats in Experiment 3 were untouched during measurement. Further testing may be needed to better understand the discrepancies between these experiments.

In this report, the pharmacokinetics and immediate and long-lasting efficacy of cNLX-NP formulations were compared against current opioid antagonists, naloxone and nalmefene. The results demonstrated that a formulation of cNLX-NP that contained only rac-lactide (cNLX-NP_100:0) showed a better pharmacokinetic profile than a formulation containing both rac-lactide and glycolide (cNLX-NP_60:40), suggesting that changes to the nanoparticle formulation can significantly alter the extent of free naloxone distribution. Mixture of free naloxone and cNLX-NP_100:0 could rapidly reverse fentanyl-induced respiratory depression and cNLX-NP_100:0 was effective out to 48 hr. Together, the data support cNLX-NP_100:0 as a lead candidate opioid antagonist formulation to treat overdose and prevent renarcotization following exposure to fentanyl and fentanyl analogs in humans. Finally, this technology could be used to develop other chemical countermeasures for treating exposure to other chemical threats, such as toxins, nerve agents, or other chemical agents.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The animal study was approved by Allegheny General Hospital and University of Minnesota Institutional Animal Care and Use Committees. The study was conducted in accordance with the local legislation and institutional requirements.

SA: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Validation, Visualization, Writing – original draft, Writing – review & editing. AK: Formal analysis, Investigation, Methodology, Resources, Visualization, Writing – original draft, Writing – review & editing. DS: Investigation, Methodology, Writing – review & editing. BZ: Investigation, Writing – review & editing. JV: Investigation, Methodology, Validation, Writing – review & editing. DL: Investigation, Writing – review & editing. PS: Investigation, Writing – review & editing. MP: Conceptualization, Funding acquisition, Methodology, Writing – original draft, Writing – review & editing. MR: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Validation, Writing – original draft, Writing – review & editing.