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. 2021 Aug 26;4(5):1654–1664. doi: 10.1021/acsptsci.1c00168

Covalently Loaded Naloxone Nanoparticles as a Long-Acting Medical Countermeasure to Opioid Poisoning

Andrew J Kassick †,‡, Mariah Wu §, Diego Luengas §, Mohammad Ebqa’ai ∥, L P Tharika Nirmani ∥, Nestor Tomycz ‡, Toby L Nelson ∥, Marco Pravetoni §, Michael D Raleigh §, Saadyah Averick †,‡,*
PMCID: PMC8506606  PMID: 34661081

Abstract

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The mu opioid receptor antagonist naloxone has been a vital, long-standing countermeasure in the ongoing battle against opioid use disorders (OUD) and toxicity. However, due to its distinctive short elimination half-life, naloxone has shown diminished efficacy in cases of synthetic opioid poisoning as larger or repeated doses of the antidote have been required to achieve adequate reversal of severe respiratory depression and prevent episodes of renarcotization. This report describes the synthesis, characterization, and in vivo evaluation of a novel, nanoparticle-based naloxone formulation that provides extended protection against the toxic effects of the powerful synthetic opioid fentanyl. The strategy was predicated on a modified two-step protocol involving the synthesis and subsequent nanoprecipitation of a poly(lactic-co-glycolic acid) polymer scaffold bearing a covalently linked naloxone chain end (drug loading ∼7% w/w). Pharmacokinetic evaluation of the resulting covalently loaded naloxone nanoparticles (cNLX-NP) revealed an elimination half-life that was 34 times longer than high dose free naloxone (10 mg/kg) in male Sprague–Dawley rats. This enhancement was further demonstrated by cNLX-NP in subsequent in vivo studies affording protection against fentanyl-induced respiratory depression and antinociception for up to 48 h following a single intramuscular injection. These discoveries support further investigation of cNLX-NP as a potential therapeutic to reverse overdose and prevent renarcotization from fentanyl and its potent analogs.

Keywords: Naloxone, Fentanyl, Drug Delivery, Nanoparticles, Opioids, Renarcotization

Fentanyl (1), a highly potent, synthetic mu opioid receptor (MOR) agonist, has been utilized by healthcare professionals for decades as a powerful sedative and anesthetic, as well as an effective treatment modality for the management of severe pain. Apart from its valuable clinical efficacy, fentanyl also bears a schedule II controlled substance designation from the Drug Enforcement Administration (DEA) for its heightened abuse liability and potential to produce life-threatening depression of respiratory centers in the brain. This unfavorable side effect profile coupled with the recent proliferation and distribution of illicitly manufactured fentanyl and related synthetic opioid analogues have contributed significantly to the widespread epidemic of fatal opioid overdoses impacting the United States (US)1−3 and many other countries around the world.4−6 The Centers for Disease Control and Prevention (CDC) has indicated that 73% of the approximately 50,000 US fatalities attributed to opioid poisoning in 2019 involved fentanyl or another fentanyl-derived synthetic opioid,7 an alarming statistic that has steadily worsened over the course of the past decade. In addition, recent data from the CDC suggests that this enduring crisis has been further complicated by the socially and psychologically disruptive effects of the current COVID-19 pandemic8−12 as a dramatic increase in opioid overdose deaths has been observed across the US relative to prepandemic levels registering more than 87,000 fatal overdoses in 2020.10

The classical MOR antagonist naloxone (2) is currently the only available antidote clinically approved for the emergency treatment of acute toxicity and respiratory depression resulting from an opioid overdose. While the effectiveness of naloxone toward reversing opioid-induced respiratory depression has been demonstrated since its approval by the US Food and Drug Administration (FDA) in 1971, its therapeutic limitations have become more pronounced in recent years following the advent of pervasive new synthetic opioid analogs. The primary shortcoming of naloxone is its rapid conversion in vivo to the highly polar 3-glucuronide conjugate 3, an inactive phase II metabolite (Figure 1), resulting in a short circulatory half-life ranging from 30 to 120 min.13 Synthetic opioids such as fentanyl exhibit significantly longer half-lives by comparison, as they are believed to be less vulnerable to metabolic degradation due to their high receptor affinities and greater propensity to absorb into adipose tissue.14−18 As a consequence, victims of synthetic opioid poisoning rescued with a single dose of naloxone may experience a recurrence of opioid toxicity and severe respiratory depression known as renarcotization. Attempts to address this phenomenon typically involve the administration of higher or multiple sequential doses of naloxone to sustain opioid reversal and prevent narcotic relapse.19,20 However, this strategy is not without drawbacks, as treating opioid dependent individuals with excessive doses of naloxone can result in precipitated opioid withdrawal (POW).20−22 Lofexidine, an α-adrenergic receptor agonist and structural analogue of clonidine, has recently been approved by the FDA for use in adults for the alleviation of withdrawal symptoms resulting from sudden opioid discontinuation; however, it typically does not prevent symptoms from occurring and is not approved for long-term use.23 Perhaps the most reliable treatment method currently available for synthetic opioid poisoning is the use of carefully managed intravenous naloxone infusions that can both outlast the threat of renarcotization and avoid the intense and potentially dangerous symptoms of POW, although this approach is impractical outside of an emergency department setting. Therefore, a long-acting reversal agent capable of mimicking clinical best practices that is also amenable to in-the-field single dose administration would be ideal for combating synthetic opioid poisoning while attenuating the risk of withdrawal.

Figure 1.

Figure 1

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Chemical structures of fentanyl, naloxone, and naloxone-3-glucuronide

Given the widely recognized need for therapeutics that more effectively counteract synthetic opioid toxicity, several research groups have recently taken steps to address this critical issue in the context of extended-release MOR antagonist delivery systems.24−27 Our laboratory has demonstrated the utility of covalently loaded naloxone nanoparticle (cNLX-NP) technology as an effective, long-lasting MOR antagonist formulation against high dose morphine (10 mg/kg).24 The subcutaneously administered covalent nanoparticles (cNPs) were based on a biodegradable poly(lactic acid) polymer scaffold and presented minimal indications of precipitated withdrawal in morphine-dependent mice relative to free naloxone.24,25 More recent advances in this area have also focused on the development of novel, long-acting naloxone formulations with minimal withdrawal symptoms including a first-in-class oral nanoparticle preparation derived from a functional PLA-PEG-HCDA polyester copolymer26 as well as a slow-release, subcutaneous naloxone dosage form based on self-assembling peptide hydrogels.27 While these disclosures speak to the efficacy of their corresponding drug delivery systems toward epoxymorphinan-based opioid structures like morphine, none of the previously described methodologies had been evaluated against synthetic opioids.28

In order to more fully explore the potential of novel antiopioid countermeasures, this study focused on assessing the in vivo efficacy of extended-release cNLX-NP technology against the more potent synthetic MOR agonist, fentanyl. Considering the high potency and rapid onset of action of fentanyl, it was hypothesized that an extended-release antagonist formulation capable of quickly attaining a therapeutically useful plasma concentration of naloxone would be advantageous. Therefore, a new polymer backbone containing a significant proportion of a more readily hydrolyzed glycolic acid monomer was pursued that may offer faster degradation kinetics to more effectively achieve an early and sustained MOR receptor blockade against fentanyl. To that end, we employed a previously reported organocatalyzed ring opening polymerization (ROP) strategy24 to prepare a naloxone–poly(lactic-co-glycolic acid) (NLX-PLGA) copolymer that was then formulated into the corresponding nanoparticles for in vivo evaluation (Scheme 1). The resultant cNLX-NPs were assessed for their pharmacokinetic (PK) profile of naloxone release in a rodent model with the goal of achieving a naloxone concentration of at least 6 ng/mL, an estimated equivalent plasma concentration required for the reversal of opioid poisoning in humans,29,30 and maintaining those levels for at least 24 h. Subsequent testing in oximetry and hot plate studies was then used to demonstrate both acute and extended protection against fentanyl-induced toxicity by cNLX-NPs over time compared to the free parent drug. Additionally, the administration route of cNLX-NPs was optimized for intramuscular (i.m.) delivery in order to arrive at a sustained release naloxone preparation suitable for use in a nonclinical setting. Herein we describe the synthesis, characterization, and in vivo evaluation of a new PLGA copolymer-derived cNLX-NP formulation that demonstrates improved pharmacokinetics and dramatically enhanced in vivo antagonism of the potent synthetic opioid, fentanyl, relative to free naloxone.

Scheme 1. Preparation of New PLGA-Based cNLX-NPs via Organocatalyzed, Ring Opening Polymerization of rac-Lactide and Glycolide Monomers.

Scheme 1

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Results

Polymer/Nanoparticle Synthesis and Characterization

The preparation of cNLX-NP commenced with the synthesis of a low-molecular weight, naloxone-containing copolymer derived from the cyclic aliphatic esters, rac-lactide (4) and glycolide (5), according to our previously described solvent-free, organocatalyzed ROP protocol as illustrated in Scheme 1.24 Following the reaction, the resulting polymer was further purified by flash chromatography on silica gel to remove any residual naloxone (Figure 2A) thus affording the desired NLX-PLGA60:40 polymer product as an off-white solid in moderate yield.

Figure 2.

Figure 2

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(A) TLC of purified organocatalyzed naloxone-initiated ring opening polymerization reaction of rac-lactide/glycolide (60:40) and (B) GPC analysis of NLX-PLGA60:40 polymer.

The purified NLX-PLGA60:40 polymer was subsequently characterized via gel permeation chromatography (GPC) and 1H NMR spectroscopy to provide estimates of its molecular weight (Mn), molecular weight distribution or dispersity (Đ), and degree of polymerization (DP). GPC analysis showed that good control over molecular weight and dispersity (Mn = 5200, Đ = 1.44) was achieved in the synthesized NLX-PLGA60:40 polymer (Figure 2B). Structural confirmation and DP were determined via 1H NMR spectroscopy. Resonances corresponding to the protons from various sections of the polymer scaffold were assigned in Figure 3 and color coded for clarity. The methine (CH) and methyl (CH3) protons corresponding to the lactic acid monomers (green) appear at 5.09–5.29 and 1.51–1.63 ppm, respectively, while the methylene protons (CH2) of the glycolic acid subunits (red) show diagnostic chemical shifts from 4.59 to 4.95 ppm. Polymer DP was calculated from the ratio of the integrals measured for the methine (a) and methylene (b) proton resonances of lactide and glycolide monomers, respectively, relative to the vinylic methine peak (c) of the naloxone polymer chain end (Equation S1). DPs were individually determined for the lactide (DP = 42) and glycolide (DP = 22) monomer components of the NLX-PLGA yielding a total polymer DP = 64. Standard end group analysis calculations were then employed to arrive at a naloxone loading of 7% w/w (Equations S1 and S2).31

Figure 3.

Figure 3

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1H NMR analysis of NLX-PLGA60:40 polymer product from organocatalyzed ROP of rac-lactide and glycolide in CDCl3.

Formulation of the purified polymer species into the desired cNLX-NPs was accomplished via a modified nanoprecipitation–solvent displacement technique.24,32−34 Initial small scale batches of precipitated nanoparticles were isolated via centrifugation and lyophilization. However, due to poor recoveries on larger scale preparations, dialysis was employed as a method to ensure full cNP recovery to more easily supply the compound required for future PK and in vivo studies. Dialysis with 50 kDa MWCO membranes proved to be an effective method to dramatically increase the recovery of cNPs, although this process resulted in PVA incorporation into the final product. An accurate measure of naloxone content for the lyophilized cNLX-NP sample was provided via UV–vis spectrophotometric analysis. Preparation of a standard curve from a 5 mg/mL stock solution of naloxone in 1 M NaOH provided the requisite equation for determining naloxone concentration from an unknown solution of hydrolyzed cNLX-NP (Figure S1). Naloxone loading for the current cNP formulation was determined to be 6 w/w%.

Further characterization of cNLX-NP with regard to particle size and morphology was accomplished through dynamic light scattering (DLS) analysis and transmission electron microscopy (TEM). As illustrated in Figure 4A, DLS measurements of the new cNLX-NP formulation indicated the presence of particles exhibiting a moderately polydisperse, unimodal size distribution (PDI = 0.20) and an average hydrodynamic diameter of 263 nm. An additional confirmation metric was provided by TEM morphological analysis images that showed well-defined, spherical particles with a low degree of agglomeration (Figure 4B).

Figure 4.

Figure 4

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Characterization of cNLX-NP via (A) dynamic light scattering size distribution analysis and (B) transmission electron microscopy. Hydrodynamic diameter and PDI data represent the average of three measurements.

Experiment 1: Rat Pharmacokinetics

In order to determine the ability of cNLX-NP to enhance the circulatory half-life (t1/2) of naloxone, PK studies were conducted in male Sprague–Dawley rats. Concentration vs time profiles of naloxone exposure displayed the rapid metabolism and elimination of high dose naloxone in approximately 4 h, while cNLX-NP provided a prolonged infusion of high plasma levels of naloxone throughout the course of the experiment out to 48 h (Figure 5). PK parameters for naloxone and cNLX-NP were calculated from their corresponding plasma concentrations and are presented in Table 1. It was found that cNLX-NP demonstrated a 34-fold enhancement in t1/2 relative to free naloxone, a 21-fold increase in mean residence time (MRT), and an 18-fold increase in Tmax, although none of these measurements were significant. cNLX-NP also showed a significant decrease in Cmax [t(2.332) = 5.724, p < 0.05] and apparent clearance [t(3.971) = 6.011, p < 0.01] compared to naloxone. The relative bioavailability of cNLX-NP to naloxone was calculated to be 310%.

Figure 5.

Figure 5

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Intramuscular pharmacokinetic profiles of 10 mg/kg naloxone and cNLX-NP in male Sprague–Dawley rats (n = 3/group). (A) Concentration vs time for full experiment duration. (B) Concentration vs time for early time points (shown for clarity).

Table 1. Pharmacokinetic Parameters of Naloxone and cNLX-NP Formulations in Rats Following 10 mg/kg i.m. Bolus Doses (n = 3/Group).

Parameter cNLX-NP Naloxone
t1/2 (h) 12.67 ± 5.38 0.37 ± 0.05
Tmax (h) 9 (9) 0.5 (0.5–1)
Cmax (ng/mL) 147 ± 72.5a 1010 ± 250.85
1AUC 0-∞ (μg/mL·h) 3.97 ± 1.69 1.25 ± 0.17
1MRT 0-∞ (h) 23.51 ± 7.20 1.11 ± 0.21
1Cl/F (mL/kg/min) 47.17 ± 18.65b 135.05 ± 17.13
1observed    
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a

p < 0.05..

b

p < 0.01 compared to naloxone. Data are expressed as mean ± SD, except Tmax, which is expressed as median (range).

Experiment 2: Efficacy of cNLX-NP to Reverse Fentanyl Effects Immediately Following Administration

The purpose of this experiment was to determine whether cNLX-NP could reverse fentanyl-induced effects in rats. Two-way ANOVA using Sidak’s multiple comparisons test showed that fentanyl significantly reduced %SaO2 in the naloxone group [t(4) = 3.726, p < 0.05] and cNLX-NP group [t(4) = 3.808, p < 0.05]. Following administration of naloxone and cNLX-NP, all groups returned to baseline %SaO2 levels (Figure 6A). Two-way ANOVA also showed that fentanyl significantly increased latency to respond on the hot plate in the naloxone group [t(4) = 31.14, p < 0.001] but not in the cNLX-NP group. Subsequent treatment with naloxone or cNLX-NP resulted in all groups returning to baseline levels (Figure 6B).

Figure 6.

Figure 6

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cNLX-NPcompounds reverse fentanyl-induced antinociception and respiratory depression in rats as measured by (A) Pulse oximetry and (B) Latency to antinociception responses on a hot plate. Rats (n = 5/group) received 0.075 mg/kg fentanyl s.c. at t = 0, and animals were tested on oximetry and a hot plate (54 °C) and 15 min following fentanyl administration. Immediately following testing, rats received 0.1 mg/kg naloxone i.m. or 10 mg/kg cNLX-NP i.m. and tested again 15 min later. Rats were tested for baseline measures and then challenged with fentanyl. Rats were tested again 15 min later for postdrug exposure levels. *p < 0.05, **p < 0.01, ***p < 0.001 compared to t = 0 h time point within groups. Data are expressed as mean ± SD.

Experiment 3: Effect of Repeated Fentanyl Dosing on Tolerance to Fentanyl-Induced Antinociception and Respiratory Depression

This goal of this experiment was to determine whether repeated administration of fentanyl could produce tolerance in the assays when assessing the long-term efficacy of cNLX-NP during Experiment 4. Opioid-naïve rats received fentanyl (0.075 mg/kg, s.c.) at t = 0, 4, 24, and 48 h. One-way ANOVA showed a significant difference between baseline and postfentanyl %SaO2 at t = 0 h [t(3) = 10.85, p < 0.01] during week 1 and at t = 0 h [t(4) = 56.21, p < 0.0001] and t = 4 h [t(3) = 11.50, p < 0.05] during week 2, but not at any other time point (Figure 7A). One-way ANOVA also showed a significant difference between baseline and postfentanyl latency to respond on the hot plate at t = 0 h [t(3) = 185.6, p < 0.0001] and t = 4 h [t(3) = 20.56, p < 0.01] during week 1 and at t = 24 h [t(3) = 7.223, p < 0.05] and t = 48 h [t(3) = 10.31, p < 0.05] during week 2, but not at any other time point (Figure 7B).

Figure 7.

Figure 7

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Repeated fentanyl exposure leads to tolerance. (A) Pulse oximetry and (B) latency to antinociception responses on hot plate. Rats (n = 4/group) received 0.075 mg/kg fentanyl s.c. at t = 0, 4, 24, and 48 h time points, as above. At t = 0 h, animals were tested on a hot plate (54 °C) and oximetry 15 min following fentanyl administration. Immediately following testing, rats were given naloxone and placed back in their home cage. This was repeated at t = 4, 24, and 48 h. This study was repeated again 1 week later in the same rats. *p < 0.05, **p < 0.01, ****p < 0.0001 compared to its baseline time-point. Data are expressed as mean ± SD.

Experiment 4: Effect of NP-NLX on Long-Lasting Protection against Fentanyl-Induced Antinociception and Respiratory Depression

The goal of this experiment was to determine the long-lasting protective effects of cNLX-NP to prevent fentanyl-induced antinociception and respiratory depression. One-way ANOVA showed that pretreatment with naloxone had reduced %SaO2 at t = 24 h [t(4) = 5.444, p < 0.05], but not at t = 4 or 48 h. cNLX-NP prevented fentanyl-induced respiratory depression at all time points (Figure 8A). One-Way ANOVA showed that pretreatment with naloxone had no effect on fentanyl-induced antinociception at t = 0 h [t(4) = 20.58, p < 0.001], t = 24 h [t(4) = 9.339, p < 0.01], or at t = 48 h [t(4) = 8.748, p < 0.01]. cNLX-NP prevented fentanyl-induced antinociception out to 48 h (Figure 8B).

Figure 8.

Figure 8

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cNLX-NP protects against fentanyl-induced respiratory depression and antinociception. (A) Oximetry and (B) latency to respond on hot plate. Rats (n = 5/group) received either 0.1 mg/kg naloxone or 10 mg/kg cNLX-NP i.m. at t = 0 h. At subsequent time points (t = 4, 24, and 48 h), rats were tested for baseline measures and then challenged with fentanyl. Rats were tested again 15 min later for postdrug exposure levels. *p < 0.05, **p < 0.01, and ***p < 0.001 comparing postfentanyl exposure to baseline levels. Data are expressed as mean ± SD.

Discussion

In the present study, a novel cNLX-NP formulation that extended the elimination half-life of the MOR antagonist naloxone was investigated. The findings from this study were (1) PLGA-derived polymers containing a suitable loading of naloxone could be successfully and reproducibly prepared, (2) cNLX-NP increased the half-life of naloxone by greater than 6-fold over that of the free drug, (3) cNLX-NP was as effective as naloxone at reversing the effects of fentanyl immediately after administration, and (4) cNLX-NP demonstrated prolonged efficacy against fentanyl-induced respiratory depression and antinociception for up to 48 h. These data provide early evidence that cNLX-NP may be a suitable alternative to naloxone for the treatment of synthetic opioid poisoning and prevention of renarcotization in humans.

Synthetic biodegradable polymers, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and their corresponding copolymer poly(lactic-co-glycolic acid) (PLGA), have been used extensively over the years in a variety of FDA-approved medical products and devices,35−38 making these highly biocompatible polymeric materials an excellent choice for use in controlled release drug delivery applications.38−40 The current method of cNLX-NP formulation allowed for the preparation of a well-defined polymeric species with good control of drug loading in the final material and a high drug-to-polymer ratio. This benefit came as a consequence of the covalent nature of antagonist incorporation resulting from the use of naloxone as the initiator of the organocatalyzed ROP reaction. To achieve an increased rate of hydrolysis and a corresponding enhancement in naloxone release for the effective in vivo blockade of fentanyl, we elected to incorporate glycolide into the polymeric nanoparticle backbone. Increasing the glycolic acid content of a biodegradable copolymer is a known strategy to enhance degradation rates relative to the corresponding individual homopolymers.41,42 Unfortunately, PLGA prepared from a more conventional 50:50 mol ratio of cyclic diesters resulted in a copolymer that exhibited insufficient solubility in organic solvents that made further manipulation and purification of the bulk material difficult. As a consequence, a 60:40 mol ratio of lactide and glycolide was employed to improve polymer solubility. Following ROP, the resultant polymer (NLX-PLGA60:40) was shown to possess a low molecular weight and a monomodal molecular weight distribution as determined by GPC analysis while 1H NMR spectroscopy provided structural confirmation of the polymer and clearly showed the incorporation of naloxone into the polymer chain end. The exclusion of free naloxone from the polymer samples was confirmed by TLC analysis in order to ensure all observed in vivo effects were the result of naloxone released from cNPs and not residual free naloxone.

Pharmacokinetic analyses of cNLX-NP and free naloxone (Figure 5 and Table 1) showed that cNLX-NP greatly altered the pharmacokinetics of naloxone. Free naloxone reached its maximum plasma concentration (Cmax) very soon after administration and then underwent rapid metabolism and elimination in approximately 4 h, while cNLX-NP provided sustained naloxone plasma levels for up to 48 h (Figure 5), clearly demonstrating its extended-release capacity. The time to reach Cmax (Tmax) exhibited by cNLX-NP was substantially increased relative to naloxone (9 h vs 0.5 h), which is significant because this increase in Tmax did not reduce the ability of cNLX-NP to rapidly reverse fentanyl toxicity in subsequent experiments. The observed decrease in Cmax and apparent clearance (Cl/F) in the cNLX-NP group was expected, demonstrating that cNLX-NP retains and slowly releases naloxone over time. The relative bioavailability of cNLX-NP was also much higher than naloxone (∼3-fold), which suggests that, on the same mg/kg basis, more cNLX-NP reaches systemic circulation. The mean residence time (i.e., the average amount of time the drug spends in the body) of naloxone was also significantly increased in the cNLX-NP group, which further suggests that polymer nanoparticles manipulated the pharmacokinetic parameters of naloxone. While cNLX-NP increased the pharmacokinetic profile of naloxone for multiple parameters (half-life, Tmax, and MRT), these values did not reach significance. This was likely due to large variability in the study and due to the small number of animals used (n = 3), as this experiment was exploratory in nature. Increasing animal numbers in future studies is warranted to get a better sense of cNLX-NP effects on naloxone pharmacokinetics. Although the observed increase in half-life was not statistically significant, subsequent in vivo experiments clearly demonstrated the efficacy of cNLX-NP out to at least 48 h, confirming that cNLX-NP is present long after free naloxone is cleared.

The cNLX-NP delivery platform was then evaluated for its ability to inhibit the effects of the synthetic opioid fentanyl in oximetry and hot plate studies. As illustrated in Figure 6, cNLX-NP reversed fentanyl-induced respiratory depression and antinociception within 15 min, which was similar to the result observed with free naloxone. A previous study using a related cNLX-NP formulation showed effects at an earliest recorded time point of 2 h after administration.24 The current data expand on this earlier investigation by suggesting that a therapeutically relevant amount of naloxone dissociated from the cNLX-NP formulation immediately after injection. The high dose of cNLX-NP (10 mg/kg) likely contributed to the rapid onset of effects. Administration of 4 mg naloxone (approximately 0.06 mg/kg in a 70 kg human) following intranasal and intramuscular administration typically produces low naloxone levels in serum of approximately 0.3–6.0 ng/mL within the first 10 to 15 min.29,30 These data confirm that only low levels of circulating naloxone are required to reverse the toxic effects of fentanyl and that cNLX-NP sufficiently produces plasma levels within this range quickly after administration. To the best of our knowledge, cNLX-NP represents the first example of a nanoparticle-based naloxone formulation to treat fentanyl-induced respiratory depression. Moreover, this comparable reversal of both fentanyl-induced antinociception and respiratory depression relative to naloxone was achieved via a single i.m. injection demonstrating the potential of cNLX-NP for in-the-field treatment of synthetic opioid poisoning.

The development of tolerance was observed during repeated fentanyl administration in Experiment 3, demonstrating the need to minimize fentanyl exposure when studying the long-term effects of cNLX-NP in Experiment 4. This effect was more pronounced for fentanyl-induced respiratory depression than toward antinociception, suggesting that the mechanism of tolerance may be biased toward some processes than others. Interestingly, fentanyl-induced tolerance is not well-defined in the literature. A recent study estimated that chronic opioid users had a 4.3-fold lower sensitivity to fentanyl, but not specifically due to fentanyl itself.43 There is also some evidence that intrathecal fentanyl administration leads to acute morphine tolerance as well.44 It also has been demonstrated that fentanyl treatment can produce tolerance to its antihyperalgesic effect in mice that had sciatic nerve ligation, but this is also a different paradigm than in the current study.45 While various examples of fentanyl tolerance have been described, the overall findings from the present study suggest that fentanyl may lead to rapid tolerance to its own antinociceptive and respiratory depressive effects after a single s.c. dose. However, due to the limited study size, further exploration is warranted.

A significant finding was that cNLX-NP (10 mg/kg) exhibited long-lasting protective effects against fentanyl-induced respiratory depression and antinociception at all time points out to 48 h following a single i.m. injection, while naloxone was less effective at blocking these adverse effects. These data are further supported by the findings of Experiment 1, which demonstrated the rapid elimination of high dose naloxone (10 mg/kg) after 4 h while therapeutically effective plasma concentrations of naloxone from cNLX-NP persisted out to the 48 h time point. These data also correlate well with a previous study demonstrating that a similar cNP formulation prevented morphine-induced antinociception out to 98 h.24 It is important to note that rats may have still become tolerant to the effects of fentanyl at later time points. However, because rats that were pretreated with naloxone showed significant increases in antinociception at all subsequent fentanyl challenges, it is unlikely that fentanyl-induced tolerance contributed to the experimental findings. Overall, these results demonstrate the viability of the cNLX-NP drug delivery system to increase the effective therapeutic potential of naloxone by preventing renarcotization following fentanyl exposure, even after a single dose of cNLX-NP is administered.

In this report, we have described the application of cNLX-NP technology as a potential alternative to free naloxone for the reversal of fentanyl-induced overdose and prevention of renarcotization. Covalently loaded naloxone nanoparticles derived from poly(lactic acid-co-glycolic acid) (PLGA) were prepared according to the previously described ROP/nanoprecipitation procedure and demonstrated uniform particle size and useful drug loadings as determined by GPC and 1H NMR analysis. This new formulation proved to be as effective as the parent drug toward the reversal of acute fentanyl-induced antinociception and respiratory depression in rats while exhibiting significant enhancements in longer-term efficacy studies by blocking fentanyl effects for up to 48 h. These observations were further supported by PK analysis that showed therapeutically relevant plasma concentrations from cNLX-NP that far outlasted free naloxone as well as a 34-fold increase in the half-life of cNLX-NPs relative to naloxone. Together, these results suggest that our long-acting, PLGA-based naloxone delivery platform holds great promise for the treatment of synthetic opioid poisoning. A more comprehensive evaluation investigating the effects of polymer composition on PK and in vivo efficacy against fentanyl will be forthcoming.

Experimental Section

Materials and Methods

Naloxone hydrochloride dihydrate was purchased from Sigma-Aldrich (St. Louis, MO) and subsequently converted to its corresponding free base via acid–base extraction. 1-[3,5-Bis(trifluoromethyl)phenyl]-3-[(1R,2R)-(−)-2-(dimethylamino)cyclohexyl]thiourea was obtained from Strem Chemicals, Inc. (Newburyport, MA). 3,6-Dimethyl-1,4-dioxane-2,5-dione (rac-lactide), glycolide, anhydrous dichloromethane (CH2Cl2), and methanol (MeOH) were purchased from Sigma-Aldrich (St. Louis, MO). Water (H2O) was purified via a Millipore Synergy water purification system. All reagents and solvents were used as received unless otherwise noted. 1H NMR spectra were measured in deuterochloroform (CDCl3) on a Bruker Avance 500 MHz spectrometer. Chemical shifts are reported in ppm employing the residual solvent resonance as the internal standard (CHCl3: δ 7.26 ppm). UV–vis spectra were measured on a DeNovix DS-11 spectrophotometer. Gel permeation chromatography (GPC) was performed using a Waters GPC system equipped with a Waters 2410 refractive index detector. A Waters pump and a Styragel HR 3 column (7.8 mm × 300 mm) were used with THF as the mobile phase solvent. Separations were carried out at 35 ○C with a flow rate of 1.0 mL/min. Polystyrene standards (Mn = 500–300 000 Da) were used for GPC system calibration.

Solvent-Free Synthesis of NLX-PLGA60:40

To an oven-dried 20 mL vial equipped with a magnetic stirrer was added rac-lactide (1.30 g, 9.0 mmol) and glycolide (0.696 g, 6.0 mmol) under N2. The monomers were melted at 130 °C and then treated with a well-crushed mixture of naloxone (0.492 g, 1.5 mmol, 10 mol %) and thiourea catalyst 6 (0.309 g, 0.75 mmol, 5 mol %). The mixture was heated at 130 °C for 15 min to afford a viscous, yellow oil. The reaction was cooled to ambient temperature whereupon the crude material was dissolved in 20 mL of CH2Cl2 and added slowly dropwise to 200 mL of cold iPrOH with vigorous stirring. A precipitate formed. The solid was allowed to settle, and the yellow supernatant liquid was decanted. The residual solid was washed/sonicated with iPrOH (2 × 100 mL). After each washing, the solid was allowed to settle and the liquid was decanted. The remaining solid was then suspended in 100 mL of iPrOH and split into 2 × 50 mL centrifuge tubes. The mixture was centrifuged at 4500 rpm for 25 min, and the supernatant liquid was decanted. The resulting solid was dried overnight to afford 2.0 g of a crude yellow solid. Purification by flash chromatography on SiO2 (12 g SiO2 dry load, 145 g SiO2 column, 0.8% MeOH/EtOAc) afforded ∼760 mg (30%) of an off-white foam. 1H NMR (500 MHz, CDCl3): δ 6.91–6.83 (m, 1H), 6.74–6.67 (m, 1H), 5.88–5.63 (m, 1H), 5.29–5.06 (m, 42H), 4.96–4.59 (m, 44H), 3.21–2.94 (m, 4H), 2.84–2.66 (m, 1H), 2.65–2.54 (m, 2H), 2.46–2.34 (m, 1H), 2.34–2.21 (m, 1H), 2.16–2.07 (m, 1H), 1.90–1.82 (m, 1H), 1.63–1.52 (m, 155H). GPC: Mn = 5200, Mw/Mn = 1.44.

Formulation of Covalently Loaded Naloxone Nanoparticles (cNLX-NP)

A solution of 200 mg of NLX-PLGA (60:40) polymer in 6 mL of CH3CN was added slowly dropwise via syringe pump to a solution of 0.3% PVA (MW ≈ 6000) in H2O (60 mL) using a blunt tip needle. An addition rate of 25 μL/min was used with rapid stirring (1200 rpm). Upon completion of the addition, the resulting white, turbid mixture was maintained overnight at ambient temperature. The nanoparticles were isolated by dialysis (50K MWCO) against ultrapure water (28 cm dialysis bag, 3 × 2000 mL exchanges for 2 h at RT). The resulting suspension (∼ 50 mL) was transferred into 4 × 40 mL vials and then lyophilized to yield 345 mg of a fluffy, white solid.

Transmission Electron Microscopy

A 10 μL drop of sample solution was placed onto an ultrathin carbon film coated 150 mesh copper TEM grid (Electron Microscopy Sciences, Hatfield, PA). The solution remained on the grid for 5 min to allow particle deposition. The solution was then dried by blotting with Whatman 3MM filter paper, and the sample was subsequently imaged on a JEOL JEM-2100 transmission electron microscope operating at 200 keV.

Animals and Bioethics Statement

For Experiment 1, male Sprague–Dawley rats (300 g) were purchased from Charles River Laboratories with indwelling jugular vein catheters. For Experiments 2–4, male Sprague–Dawley rats (Envigo) were used, weighing 200–250 g. Rats were double-housed under a 12/12 h light/dark cycle and free-fed, and testing occurred during the light phase.

All animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animal protocols were approved by the Allegheny General Hospital and University of Minnesota Institutional Animal Care and Use Committees. Animals were euthanized by CO2 inhalation using AAALAC approved chambers, and all efforts were made to minimize suffering.

Drugs

Fentanyl citrate (West-Ward Pharmaceuticals, NJ) and naloxone hydrochloride used in Experiments 2–4 were purchased from Boynton Pharmacy (University of Minnesota) and dissolved in 0.9% saline at a dose of 0.075 mg/kg and 0.1 mg/kg, respectively. Doses were determined based on previous publications.46,47 For Experiment 1, naloxone hydrochloride obtained from LGM Pharma (Boca Raton, FL) was dissolved in 1X PBS at a dose of 10 mg/kg. All doses and concentrations are expressed as the weight of the free base.

Analysis of Naloxone Concentration: Extraction and LCMS Conditions

Sample Extraction

Detailed methods were previously published.48 Briefly, serum and standards were processed in acetonitrile at 4 °C. The supernatant was transferred, evaporated, and diluted in 0.1 M phosphate buffer. Serum samples were extracted using 3 mL Bond Elut Plexa PCX extraction cartridges (Agilent, Santa Clara, CA), evaporated, and reconstituted in LCMS grade water, 0.1% ammonium formate, 0.01% LCMS grade formic acid.

LCMS/MS Conditions

Samples were injected onto a reversed-phase Agilent (Santa Clara, CA) Poroshell SB-C18 (2.1 mm × 50 mm i.d., 2.7 μm) column at 55 °C and then analyzed on an Agilent G6470A triple quadrupole LCMS/MS system consisting of an Infinity II 1290 G7116B Multicolumn Thermostat, G7120A High Speed Quad Pumps, and a G7267B Multisampler. Gradient elution was performed with a mixture of LCMS grade water, 0.1% ammonium formate, 0.01% LCMS grade formic acid (mobile phase A), and LCMS grade MeOH, 0.01% formic acid (mobile phase B) as follows: 0–0.5 min 5% mobile phase B, 0.5–3.0 min 15% → 50% mobile phase B, 3.0–4.0 min 50% → 95% mobile phase B, 4.0–6.0 min 95% mobile phase B. Autosampler needle was washed with 100% MeOH following each sample injection (total run time = 6 min, flow rate = 0.400 mL/min, injection volume = 2 μL).

Electrospray ionization was achieved by an Agilent Jet Stream high sensitivity ion source in the positive ion mode. Instrument settings were as follows: gas temperature 325 °C, gas flow 9 L/min, nebulizer pressure 40 psi, sheath gas temperature 380 °C, sheath gas flow 10 L/min, capillary 2500 V, and nozzle 0 V. Data acquisition and peak integration were interfaced to a computer workstation using Mass Hunter (Tokyo, Japan). LCMS/MS in the SIM mode was used to identify the appropriate ions to monitor: Naloxone primary 328.2 → 310, secondary 328.2 → 212, Naloxone-d5 primary 333.2 → 315.2.

Antinociceptive and Respiratory Depressive Assays

Rats were habituated to the testing environment to assess antinociception and oxygen saturation (%SaO2). Rats were then placed on a hot plate (Columbus Instruments, OH) set to 54 ± 0.2 °C to measure baseline latency to respond (as measured by hindpaw licking or jumping), with a maximum response latency of 30 s to avoid tissue damage, and then a oximetry collar (MouseOX, Starr Life Sciences Corp, PA) placed around their neck to measure oxygen saturation (%SaO2). Percent maximum possible effect (%MPE) on the hot plate was calculated as (postdrug latency – predrug latency)/(maximum latency – predrug latency) × 100.

Experiment 1: Intramuscular Rat Pharmacokinetics and Sampling

A single 10 mg/kg dose of naloxone or cNLX-NP was ultrasonicated in sterile filtered 1× phosphate-buffered saline (PBS) and administered into the Quadriceps femoris muscle through a 25 G needle. At predetermined time points, 0.2 mL of blood was collected and placed into a BD Microtainer (purple cap EDTA tube) sample was inverted 5 times and let blood sit for 10 min. After the 10 min incubation, the blood was centrifuged at 1500 × g and 0.1 mL of plasma was isolated. Catheters were maintained according to the manufacturer’s instructions. The naloxone treatment cohort samples had background naloxone due to reuse from a prior experiment in accordance with the 3Rs of animal research and the background concentration at time zero was subtracted from each time point. Estimates of pharmacokinetic parameters for Experiment 1 were obtained from plasma concentrations of individual rats using noncompartmental analysis (PKSolver).49 The relative bioavailability of cNLX-NP was calculated as (AUCcNLX-NP * DoseNLX)/(AUCNLX * DosecNLX-NP) × 100 to obtain a percentage.

Experiment 2: Efficacy of cNLX-NP To Reverse Fentanyl Effects Immediately Following Administration

Rats (n = 6/group) were baselined on the hot plate and oximeter and then dosed s.c. with 0.075 mg/kg fentanyl and 15 min later tested on the hot plate, and oxygen saturation was measured again. Immediately following measurement, naloxone (0.1 mg/kg) or cNLX-NP (10 mg/kg) was administered i.m., and 15 min later rats were tested on the hot plate followed by measurement of oxygen saturation. The lower dose of naloxone was chosen because it has been demonstrated as an effective reversal dose.46,47 See Figure S2 for Experiment 2–4 timeline.

Experiment 3: Effect of Repeated Fentanyl Dosing on Tolerance to Fentanyl-Induced Antinociception and Respiratory Depression

An undesirable side effect that can accompany the repeated use of potent opioid analgesics is the development of tolerance.50,51 Prior to initiation of a repeated fentanyl dosing study, it was important to determine the extent of fentanyl tolerance in the respiratory depression and antinociceptive assays. On Day 1, rats (n = 4) were tested on the hot plate and the oxygen saturation was measured. At t = 0 h, rats were dosed s.c. with 0.075 mg/kg fentanyl and 15 min later tested on the hot plate followed by oxygen saturation measurement. Naloxone (0.1 mg/kg, i.m.) was administered following testing to avoid prolonged respiratory depression. This was repeated at t = 4, 24, and 48 h after the initial dosing. One week later, this study was repeated using the same t = 0, 4, 24, and 48 h time course. Fentanyl-related tolerance appeared after the initial dose of fentanyl, confirming the need for an adapted experimental protocol in the repeated fentanyl dosing study.

Experiment 4: Effect of cNLX-NP on Long-Lasting Protection against Fentanyl-Induced Antinociception and Respiratory Depression

On Day 1, rats (n = 5/group) were baselined on the hot plate and oxygen saturation was measured. To minimize the effects of fentanyl-related tolerance in this experiment, at t = 0 h, rats were dosed with only naloxone (0.1 mg/kg, i.m.) or cNLX-NP (10 mg/kg, i.m.). At t = 4 h, rats received an s.c. dose of 0.075 mg/kg fentanyl. Any rats with <80% SaO2 received 0.1 mg/kg to avoid prolonged respiratory depression. This was repeated again at t = 24 and 48 h after the initial dosing.

Statistics

Pharmacokinetic parameters were compared between naloxone and cNLX-NP groups using unpaired t tests with Welch’s correction for each parameter, except for Tmax, which was compared using a nonparametric Mann–Whitney test. For Experiment 2, two-way ANOVA using Sidak’s multiple comparisons post test with Geisser-Greenhouse correction was used to compare between groups at each time point. For Experiments 3 and 4, repeated measures one-way ANOVA using Sidak’s multiple comparisons post test with Geisser-Greenhouse correction was used to compare the baseline to postfentanyl within groups at each time point. All statistics were performed using Graphpad Prism 9.0.

Acknowledgments

We gratefully acknowledge the National Institutes of Health, National Institute of Drug Abuse and the CounterACT program (R21DA050565) for funding. NMR measurements and instrumentation at CMU, which was partially supported by the NSF (CHE-0130903 and CHE-1039870).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.1c00168.

  • Experimental calculations, characterization data, and supporting schemes and figures (PDF)

The authors declare no competing financial interest.

Supplementary Material

pt1c00168_si_001.pdf (205.9KB, pdf)

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