Introduction

Opioid-induced respiratory depression (OIRD) followed by cardiac arrest, is the primary cause of death in opioid overdose cases, particularly in the US and Canada1,2,3. Potent opioids remain less of a problem in most European countries with some exceptions, e.g. the United Kingdom3. Currently, most opioid-related fatalities in community settings may be attributed to potent opioids, particularly fentanyl or its analogs, such as carfentanil1,2,4. The most effective treatment of OIRD is the administration of the opioid receptor antagonist naloxone2. Several naloxone formulations have been approved for use in both hospital and prehospital settings. In hospital, intravenous naloxone is typically administered to patients with an intravenous (IV) access line, while intranasal (IN) and intramuscular (IM) formulations are designed for community use during opioid overdose emergencies. These latter formulations can be administered by first responders, including bystanders2,5.

As recently highlighted by the US Food and Drug Administration, there is an urgent need for experimental studies to determine the optimal naloxone dose for the rescue of OIRD in the community setting4. This is relevant for both IN and IM naloxone formulations. Several studies examined the use of IM and IN naloxone for managing opioid overdoses6,7,8,9,10. Most of these studies were conducted several years ago when heroin was the predominant cause of an opioid overdose, and studied relatively low doses of IM and IN naloxone. These trials were performed in real-world settings, often in supervised drug injection centers or in ambulances, and remain highly relevant. Generally, the studies concluded that while IN naloxone is effective, it has a slower onset of action compared to IM naloxone and frequently requires additional rescue naloxone doses. In the current opioid crisis, illicit fentanyl use has replaced heroin as the primary opioid of abuse3, which causes a more rapid and profound respiratory depression that may result in cardiac arrest secondary to hypoxia. As a result, it appears that higher and more concentrated doses of naloxone are required for the rapid and effective reversal of opioid respiratory effects4.

In this experimental study, we compared two approved relatively high-dose and high-concentration naloxone hydrochloride formulations: the 5 mg/0.5 mL intramuscular naloxone injector (ZIMHI) and 4 mg/0.1 mL naloxone nasal spray (Narcan)11,12. The study was conducted in healthy volunteers and participants who chronically and daily use opioids, following the induction of apnea lasting at least 2 min using a fixed intravenous bolus fentanyl dose of 10 μg/kg. This experimental study performed in a highly controlled and monitored setting is an attempt to mimic a real-world community fentanyl overdose scenario and allows for the comparison of naloxone treatments following a fixed high dose of fentanyl. The primary endpoint of the study was the number of IM and IN doses that were required to reverse OIRD to restoration of adequate breathing. We hypothesized that the IM formulation would require fewer doses than the nasal IN spray for reversal of OIRD.

Results

Opioid-naïve healthy participants

Twenty-nine healthy white individuals were assessed for eligibility. Eleven were excluded because of logistic reasons or exclusion criteria (see CONSORT flow diagram, Fig. 1). Eighteen healthy volunteers were randomized. One subject did not return for a second visit due to adverse effects experienced during the first session (persistent nausea and tiredness). The replacement of this subject did not develop apnea after fentanyl and the intervention (intranasal naloxone) was therefore not administered. Data from these two participants were discarded and they were replaced by another participant. The demographics and study results of the 16 healthy volunteers who completed the study are given in Table 1. All volunteers completed the study without serious adverse events. Examples of the IM and IN reversal responses for a single subject and average reversal data are given in Fig. 2. It shows the rapid induction of apnea following fentanyl administration and the differences in response following IM versus IN naloxone. In this particular subject one IM dose was sufficient to fully reverse respiratory depression within 2 min, whereas three IN doses were needed before adequate breathing was observed, with consequently a much slower return towards adequate baseline ventilation.

Fig. 1: Consort flow diagrams.
figure 1

Diagrams for the healthy volunteer (a) and the exploratory study in chronic opioid users (b).

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Table 1 Participant characteristics and individual results of intramuscular (IM) and intranasal (IN) reversal of fentanyl-induced apnea in opioid-naïve healthy volunteers
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Fig. 2: Examples of IM and IN naloxone-reversal of fentanyl-induced apnea.
figure 2

a Effect of a single dose of intramuscular naloxone (IM, 5 mg/0.5 mL) given at t = 0 min, following 2 min of apnea after an intravenous bolus dose of 10 μg/kg fentanyl (F) given at t –4 min. b Effect of three doses intranasal naloxone (IN, 4 mg/1 mL) following 2 min of apnea after an intravenous bolus dose of 10 μg/kg fentanyl (F = onset of the 90 s fentanyl administration). IN naloxone was given at t = 0, 2 and 4 min. The grey horizontal bar depicts 80% of adequate baseline ventilation. Each dot is one breath. Data are from a single healthy volunteer. c Average ventilation data ± SD of the opioid-naïve individuals following apnea and reversal with intramuscular (blue) or intranasal (orange) naloxone. The horizontal lines depict the mean reversal times for the intramuscular dosing (blue) and intranasal dosing (orange). Source data are provided as a Source Data file.

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The mode (frequency) and median (interquartile range, IQR) number of IM naloxone doses to achieve return to baseline ventilation was 1 (50%), and 1.5 (IQR 1 to 2) versus 2 (56%) and 2 (IQR 1 to 3) for intranasal naloxone with a median difference of 1 (0.5 to 1.5, p = 0.0002). In one subject, reversal with 2 IN dose of naloxone was insufficient and 0.4 mg intravenous naloxone was administered due to a significant increased PCO2 level in the absence of adequate breathing activity. In none of the subjects rescue IV naloxone was indicated following administration of IM naloxone. Arithmetically adding the IV dose as a regular IN dose resulted in change of the mode and median dose to 2 (50%) and 2.5 (IQR 1.5-3.5, IN vs IM: p = 0.0001).

Time to full reversal is presented in the Kaplan-Meier curve in Fig. 3. It shows that 2 min after the first naloxone dose full reversal of OIRD was achieved in seven subjects (44%) after IM, but only in one subject (6%) following IN naloxone administration. After 4 min, full reversal was observed in 100% (n = 16) of IM and 63% (n = 10) of IN treated subjects. Complete reversal with IN naloxone was observed in all subjects after 9.2 min (n = 16), with significant differences between the two administration forms (IM: 3.9 min; log-rank test: p = 0.0002). On average the time to reversal was 2.2 ± 0.8 min (mean ± SD; n = 16) after IM naloxone and 4.0 ± 2.0 min after IN naloxone, with a mean difference of 2.0 min (95% CI 1.1 to 2.9 min, p = 0.002). Median reversal times were 2.3 (IQR 0.9) min after IM and 3.4 (1.6) min after IN naloxone. The percentage coefficient of variation of the time to reversal was smaller for IM than IN naloxone: 35% vs 52%, indicating more consistent reversal with IM naloxone. No renarcotization was observed after treatment with either IM or IN naloxone.

Fig. 3: Kaplan–Meier survival analysis depicting the number of healthy volunteers (n = 16) that reached 80–100% of adequate ventilation versus time.
figure 3

Below the graph, the number of subjects at risk at time points 0, 2, 4 or 6 min (vertical lines) is given. *Administration of rescue intravenous naloxone (0.4 mg) in one subject; this subject received a 4 min penalty because of the need for intravenous (IV) rescue. Earlier reversal was observed following intramuscular (IM) naloxone compared to intranasal (IN) naloxone (log-rank test: p = 0.0002) at less dose requirements. Source data are provided as a Source Data file.

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Following fentanyl, muscle rigidity was observed in one of the opioid-naïve volunteers following IN naloxone (Supplemental Document), and no withdrawal symptoms were observed following IM or IN naloxone administration.

Plasma concentrations of fentanyl and naloxone following IM and IN naloxone dosing are given in Fig. 4A–E. For IM naloxone, the data of individuals that received 1 dose (n = 8) and those that received 2 doses (n = 8) are given in panels B and C, showing the marked increase (91%) in plasma concentration with the doubling of the IM dose. For IN naloxone, the data of individuals that received 2 doses (n = 9) and those that received 3 doses (n = 5) are given in panels D and E, showing a 25% increase in peak plasma concentration with the increasing dose (from 8–12 mg). Mean naloxone peak plasma concentrations were 37 ± 10 ng/mL at t = 6.5 min (1 IM dose; n = 8), 71 ± 20 ng/mL at t = 6.5 min (2 IM doses; n = 8), 20 ± 4 ng/mL at t = 14.5 min (2 IN doses; n = 9) and 25 ± 9 ng/mL at t = 16.5 min (3 IN doses; n = 6). The estimated peak fentanyl concentration was 38 ng/mL occurring at t = 90 s following the start of the fentanyl administration (Fig. 4A).

Fig. 4: Plasma concentrations of fentanyl and naloxone following intramuscular (IM) and intranasal (IN) naloxone dosing.
figure 4

a Mean fentanyl concentrations over time. The red symbol is the estimated peak fentanyl concentration occurring at t = 90 s. The red bar depicts the 90 s 10 μg/kg fentanyl infusion. The dotted line through the data is the estimated exponential decay. b, c Mean plasma naloxone concentrations observed in opioid-naïve subjects that received 1 dose (a; n = 8) and those that received 2 IM doses (c; n = 8). d, e Mean plasma naloxone concentrations observed in opioid-naïve subjects that received 2 doses (d; n = 9) and those that received 3 IN doses (c; n = 6). f, g Mean plasma naloxone concentrations observed in chronic opioid users that received 1 IM dose (f; n = 3) and those that received 2 and 4 IN doses (f; n = 2 received 2 doses, n = 1 received 4 doses (red line)). In panels (b–e), the dotted lines represent the plasma concentration of the alternate dosing group for either IM or IN dosing in opioid naïve participants. Data are mean ± SD (grey areas). ON are opioid-naïve participants; OU are chronic opioid users. Source data are provided as a Source Data file.

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Participants who chronically use opioids

We included a small sample of Caucasian participants who daily use opioids as an exploratory part of our study, to determine feasibility of administering high dose naloxone in these participants, determine adverse effects and get an indication of the direction of naloxone effect in IN versus IM arms in these participants. A total of nine individuals who reported that they use opioids on a daily basis were assessed for participation in the study. Three participants were excluded (Fig. 1): one because of not meeting the inclusion criteria, two others for logistic reasons. One participant was randomized to the intervention but did not develop respiratory depression and therefore no naloxone was administered. Since this participant did not receive the intervention, his data were not included in the data analysis. Table 2 gives the demographics including opioid use of the participants. Their mean daily morphine milligram equivalents (MME) ± SD were 201 ± 139 mg (range 90 to 450 mg). Excluding the participant who did not receive the intervention gives a daily MME of 151 ± 75 mg (range 90–270 mg).

Table 2 Participant characteristics and individual results of intramuscular (IM) and intranasal (IN) reversal of fentanyl-induced apnea in chronic opioid users
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The number of IM doses needed for return of adequate breathing to baseline levels was 1 in three subjects and 2 in two subjects; no rescue IV naloxone was needed after IM naloxone. For IN naloxone, 2 doses were given to two participants, 3 to two participants and 4 to one participant (Table 2); rescue IV naloxone was needed in 2 participants. One participant received 0.4 mg intravenous naloxone after 3 IN doses due to a PCO2 > 12 kPa (90 mmHg), the other subject after 2 IN doses because of desaturation below 70%.

The mode (frequency) and median (IQR) number of naloxone doses was 1 (60%) and 1 IM dose (IQR 1-2) versus 2 (60%) and 2 IN doses (IQR 2-3.5); median difference in dosing was 1 (IQR 1-1; IM vs IN). Time to return to baseline ventilation ranged from 1.4–3.2 min in the IM treated arm and 2.3–10.8 min in the IN treated arm. Median reversal times were 2.4 (IQR 1.1; n = 5) min for IM and 7.3 (3.1; n = 5) min for IN naloxone (Table 2).

Plasma naloxone concentrations after IM and IN doses are given in Fig. 4, panels f and g. The naloxone concentrations following a single IM doses (n = 3) were similar to that observed in opioid-naïve participants (compare panels b and f). The concentrations after 2 IN doses were in the same range as those observed in opioid-naïve individuals (compare panels d and g). We obtained pharmacokinetic data from a single participant after 4 IN doses (red line in panel g) that showed absence of dose effect. We relate this to saturation in nasal naloxone uptake.

Two participants developed muscle rigidity upon administration of fentanyl, as objectified by the myotonometer (see the Supplemental Information File). Muscle rigidity developed slowly and became apparent after fentanyl and intranasal naloxone were administered. We did not observe muscle rigidity when fentanyl was followed by intramuscular naloxone.

In four participants, withdrawal symptoms occurred following treatment with naloxone. The severity of symptoms in two participants was similar and considered mild by the investigators and participants and required no treatment (in 1 subject after IM naloxone, in the other after IV naloxone). In 2 other participants (T and U, Table 2) symptoms were moderate to severe: in subject U after both IM and IN naloxone, and in subject T after IM naloxone and IV (after IN) naloxone. These participants were treated with intravenous clonidine (range 75–150 μg) after which they both received low-dose propofol for 1 h to sedate them during the withdrawal episode (after IN and IM naloxone treatment). Most prevalent symptoms included agitation, perspiration, nausea, shivering/shaking, hypertension, tachycardia and a burning sensation in the throat. All symptoms were absent upon discharge from the clinical research unit.

In the Supplemental Information File, the following secondary endpoints are presented: rigidity, pupillometry and end-tidal pCO2 data.

Discussion

In this randomized, open-label trial, we compared the number of required IM versus IN naloxone doses in their ability to effectively reverse fentanyl-induced apnea. We studied healthy individuals and a small exploratory sample of participants who use a daily average of 200 MMEs. In both populations, IM naloxone was more effective in reversing fentanyl-induced apnea compared to IN naloxone, without a need for intravenous rescue naloxone. The median times to restoration of ventilation to baseline levels was similar (2.3 min in opioid naïve individuals and 2.4 min in chronic opioid users) following IM naloxone. In contrast, IN naloxone administration was not only less effective (i.e., 1 extra dose was needed) but IV rescue naloxone was needed to restore adequate breathing activity in 1 healthy participant (6.3%) and 2 participants who use daily opioids (40%) with median recovery times 3.4 min (healthy volunteers) and 7.3 min (chronic opioid users).

Opioid-induced apnea followed by cardiac arrest results from hypoxia and possibly direct opioid effects on the heart through an opioid effect on potassium channels1,2,13. This leads to significant mortality and morbidity given the large number of individuals that abuse potent opioids, particularly in the US and Canada, but certainly also in some other countries3. In the US alone, 80,000 people died from an opioid overdose in 202314. Dealing with potent opioid-induced respiratory depression, apnea and cardiac arrest poses a significant challenge due to the recent substantial increase in the strength of illegally consumed opioids, especially of the potent opioid fentanyl (100 times more potent than morphine) or its congener carfentanil (100 times more potent than fentanyl)4. The increase in high affinity and potent opioids requires higher and more concentrated naloxone doses to counteract the opioid effect in the brainstem4, the site where opioids cause the loss of adequate breathing activity and apnea15.

The opioid receptor antagonist naloxone remains the primary treatment of an opioid overdose. While originally available as an injectable for IV or IM use in the clinical setting, new formulations were recently approved for use outside the hospital2,4. IN and IM naloxone are easily administered by first responders and bystanders5. However, the efficacy and optimal dosing regimen has not been investigated in a controlled setting, particularly not in individuals that are apneic following the rapid IV administration of high-dose fentanyl. To the best of our knowledge, this is the first respiratory study that quantifies the effect of IM and IN naloxone following a 2 min period of fentanyl-induced apnea. This experimental study was designed to reproduce an opioid overdose in the community setting as closely as possible, albeit in a safe and monitored setting. During the study, personnel with airway management and resuscitation skills were continuously present. The use of supplemental oxygen prevented serious hypoxic adverse events. This is certainly different from the real-world setting where overdose victims breathe air with 21% oxygen; this should be considered when extrapolating our findings to community overdose settings. In real-world settings, oxygen desaturations will occur rapidly and negatively impact cardiac output complicating effective treatment with any naloxone formulation due to suboptimal drug distribution (see also below). As discussed before2,16, rescue of opioid overdose victims should include naloxone administration combined with resuscitation efforts, i.e. ventilatory support ensuring rapid uptake of oxygen and chest compression ensuring restoration or maintenance of circulation.

Our results align with previous findings. Yousefifard et al.9 conducted a meta-analysis of studies on prehospital overdose rescue with naloxone. They concluded that the onset of action of IN naloxone was slower compared to IM naloxone, with a 2.7 times greater need for naloxone IV rescue medication following IN than IM dosing. In patients managed by ambulance personnel for an opioid overdose, Skulberg et al.10 found that 80% of individuals returned to adequate ventilation after 1.4 mg (in 0.1 mL) IN naloxone versus 97% after 0.8 mg (in 2 mL) IM naloxone. Finally, Dietze et al.8 conducted a double-blind, randomized trial in a medically supervised injection facility. They found that 0.8 mg IM naloxone (in 1 mL) was more efficient and required less rescue doses compared to 0.8 mg IN naloxone (in 1 mL). All of these studies performed in actual overdose victims are difficult to compare to the findings from our study. Apart from the different setting, heroin was suspected to be the cause in the majority of opioid overdoses in these earlier studies. Considering respiratory depression, heroin is an opioid that is considerably less potent than fentanyl and about equipotent compared to morphine17. Moreover, we remain uninformed on the cardio-respiratory state of these overdose victims at the time of reversal. We argue that the use of heroin and lower equivalent fentanyl doses may have resulted in the high success rescue rates and do not necessarily translate to the current situation in the United States and Canada. We studied a fixed high-dose of fentanyl with ensuing apnea in almost all subjects. Given the delay in reversal that we have observed, particularly with IN naloxone, we argue that lower naloxone doses would have failed to restore adequate respiratory activity within acceptable safety limits in our current study, i.e. within acceptable time frames (<10 min) and without the occurrence of hypoxia.

We attribute the difference in the number of IM and IN administrations and rescue times to the differences in naloxone plasma concentrations after IM and IN naloxone (Fig. 4), which we relate to differences in dose, administration route, absorption rate, bioavailability and concentration. The package inserts of the IM and IN formulations report that peak naloxone concentrations are higher after 5 mg/0.5 mL IM naloxone than after 4 mg/0.1 mL IN naloxone: 17.2 versus 4.8 ng/mL, occurring 15 and 30 min after administration, respectively11,12. Note that these data were obtained from healthy people who were not exposed to opioids. We observed higher naloxone plasma concentrations (Fig. 4) than expected from the IM and IN naloxone package inserts. We relate this to the fact that naloxone was administered at a fixed dose regardless of the participants’ weight (our participants had a mean body weight of 72 kg), possible differences in sample scheme (we applied frequent sampling), arterial rather than venous blood sampling in our study (due to local metabolism and tissue uptake naloxone concentrations derived from arterial samples will be higher than from venous samples), high pCO2 due to 2–6 min of apnea (this results in an increase in cardiac output and high tissue perfusion, particularly of the muscles, with more rapid uptake and enhanced distribution of naloxone), and opioid-naloxone pharmacokinetic interactions. The latter was previously observed for the interaction between remifentanil and intranasal naloxone18. Irrespective, we conclude that compared to intranasal naloxone, the amount of naloxone that reaches the brain after IM naloxone will be higher and will reach the brain faster due to the higher and more concentrated dose and greater bioavailability19. This indicates that our results are attributable to difference in IM versus IN naloxone dose and bioavailability. Our data further suggest that earlier published venous naloxone pharmacokinetic data from people not exposed to high-dose opioid-induced prolonged periods of apnea might underestimate the arterial naloxone concentrations. This deserves further study, particularly of the possible pharmacokinetic interaction between fentanyl and naloxone. Finally, the absence of dose-dependency for nasal naloxone is concerning and suggests that multiple doses in the same nostril in short periods of time have little effect in rapidly increasing the naloxone plasma concentration.

We observed muscle rigidity in just a few participants. Since rigidity may be related to the dose and speed of administration, the lack of rigidity in the majority of participants in our study may be related to the relative slow infusion rate (fentanyl was infused over 90 s). Given the small sample size, additional studies are required to examine the occurrence, implications and treatment of rigidity.

Our study has some limitations. Unlike real-world conditions, the participants were not subjected to physical or verbal stimulation (as recommended in the guidelines of the American Heart Association) before, during, or after naloxone administration20. Such stimulation could potentially increase breathing and lower the required naloxone dose. We opted to compare the pharmacodynamic effect of the two naloxone formulations on chemical control of breathing rather than behavioral control that occurs when external stimuli are applied21. Additionally, such external stimuli are difficult to standardize across study participants and hence may have unpredictable effects on breathing that may have impacted the two arms of the study differently.

We used a small sample of people that use opioids on a daily basis. An important reason for this was to determine feasibility and assess whether the induced withdrawal symptoms would be manageable and treatable. We conclude that this indeed is possible with clonidine and sedation with propofol. The participants who chronically use opioids were allowed to use their habitual daily opioids on the morning of the visit to the laboratory. Whether this affected the study outcome remains unknown, but we argue that such conditions were similarly present on randomized IM and IN study days. Still, it may be the cause for the lesser efficacy of naloxone in these participants compared to the healthy individuals.

As stated previously22, in the real word, many overdose victims are found hours after the overdose occurred in a state of severe hypoventilation, hypoxia, hypercapnia and/or hypothermia, with low cardiac output. Particularly, the lack of brain hypoxia and low cardiac output in our current study may have affected outcome. For example, brain hypoxia diminishes the efficacy of naloxone23. Moreover, in case of a low cardiac output the distribution of naloxone may be altered. Whether this is more severe for IM naloxone, that is dependent on muscle perfusion, seems plausible, but requires more study.

We studied fentanyl as the opioid of choice. In the abuse setting fentanyl is often used together with other illicit substances, such as tranquilizers, that negatively affect respiration. This will likely decrease naloxone efficacy, although it remains unknown to what extent. Future studies should address this issue.

Finally, the high doses of naloxone in both formulations that were tested may trigger acute precipitated withdrawal in people with an opioid use disorder. While this could lead to reluctance in administering an opioid antagonist, we believe that saving a life outweighs the concern of inducing withdrawal. Nevertheless, the high-dose naloxone formulation remains a valuable medical countermeasure in public health emergencies, such as large-scale deployment of weaponized synthetic opioids24,25.

In conclusion, in this small and descriptive study, we compared the efficacy of IM versus IN naloxone following high-dose fentanyl-induced apnea. The study was performed in 16 healthy volunteers. Additionally, we conducted an exploratory adjunct study in 6 participants who chronically use opioids. In healthy volunteers, we observed greater efficacy in reversal of apnea after IM compared to IN naloxone with the need for lesser IM doses and no need for rescue naloxone. The decreased IN naloxone efficacy was replicated in the small set of participants who use opioids on a daily basis. While our results are relevant, further larger studies are required, particularly in participants who chronically use opioids, focusing not only on respiration but also on muscle rigidity and withdrawal symptoms.

Methods

Ethics and subjects

This single center, randomized, crossover, open label study on the comparison of the number of doses intramuscular (IM) and intranasal (IN) naloxone required to reverse fentanyl-induced apnea was approved under the European Clinical Trial Regulation (ECTR) by the local Medical Review Ethics Committee (Medisch Ethische Toetsingscomissie Leiden Den Haag Delft) on August 25, 2023, after submission of the protocol at the EU Clinical Trials Information System (CTIS, identifier 2023-505338-93-00). The protocol was registered and is available at the ISRCTN registry under identifier 21068708 on September 6, 2023. All participants were studied on two separate occasions, once receiving IM naloxone, and once receiving IN naloxone; the sequence of administration was random. Before enrollment, all participants gave written informed consent to participate in the study and for publication of the potentially identifying medical information included in this paper. Study procedures were performed according to good clinical practice guidelines and the Declaration of Helsinki. Study procedures were performed according to good clinical practice guidelines and the Declaration of Helsinki. The study was conducted from September 21, 2023 (study initiation with start recruitment) with first subject in on November 2, 2023, to September 6, 2024 (last subject out) at the Anesthesia & Pain Research Unit of Leiden University. All participants were recruited via folders displayed on the University campus and through web-based advertisements of our study. The participants received a financial compensation for their participation.

The study was conducted in healthy volunteers and in people who reported that they chronically use an opioid on a daily basis. Eligibility criteria were age 18–65 years (inclusive) and body mass index 19–40 kg/m2 (inclusive). Participants who chronically use an opioid were required to use at least 60 mg morphine equivalents per day. All participants were required to be in a good health condition based on a medical evaluation conducted by an independent physician, based on the participant’s medical and surgical history, physical examination and vital signs. For opioid users, a 12-leads electrocardiogram, and hematology and blood chemistry safety checks were performed. Exclusion criteria included presence or history of relevant medical or psychiatric diseases, pregnancy or lactation, a history of allergic response to study medication. Additionally, for healthy volunteers, a positive breath alcohol test or a positive drug urine dipstick on screening and study days were exclusion criteria. People who chronically use an opioid and met the criteria for diagnosis of a substance use disorder according to the Diagnostic and Statistical Manual of Mental Disorders (DSM)−5 other than opioids, caffeine or nicotine, were excluded, as well as those people who received medication-assisted treatment for their opioid use, including treatment with mixed agonists-antagonists or benzodiazepines.

Study design and apparatus

The study was performed in our clinical research unit, an area with similar monitoring and access to airway management equipment that is available in an operating room. Upon arrival at the unit, all subjects received two intravenous access lines, one for fentanyl and one for possible intravenous naloxone administration. The intravenous naloxone served as escape medication in case the study drug intervention was unsuccessful (i.e. absence of adequate breathing within 8 min after the first naloxone dose) or in case safety rules were met (see below). An arterial line was inserted in the radial artery of the non-dominant hand for continuous monitoring of hemodynamic parameters and blood sampling. Additionally, arterial oxygen saturation via a finger clip and the electrocardiogram were monitored continuously throughout the study day.

Prior to any drug administration, breath-to-breath minute ventilation was measured using a mask fitted over nose and face, that was connected to a pneumotachograph and pressure transducer system (Hans Rudolph Inc., USA). Inspired and expired gas concentrations were measured at the mouth using the Masimo Root ISA OR plus capnograph (Masimo, USA). Subjects inhaled supplemental oxygen (flow set at 4–6 L/min) to prevent desaturation during episodes of apnea and respiratory depression. Data were presented onscreen allowing real-time assessment of the hemodynamic and ventilatory state of the subject. All respiratory parameters were collected breath-to-breath for analysis.

Additional measurements muscle rigidity that was measured at the brachial muscle of the dominant upper arm using a myotonometer (Myoton, Tallin, Estonia), and the pupil size that was measured with the PLR-3000 pupilometer (Neuroptics, USA).

Measurements, safety rules and drug administration

Ventilation

After a 10–15 min period of steady-state oxygen breathing through a facemask, a 10 μg/kg intravenous fentanyl dose was administered over 90 s. The dose was based on an earlier study and aimed at producing an episode of apnea within 2–4 min26. This dose is within the range of fentanyl doses consumed in the community setting by people that use fentanyl in a range of administration forms (including oral, nasal or intravenous use and smoking; dose range reported 0.25 to 1.0 g)27. The relatively slow infusion rate will diminish the likelihood of muscle rigidity. When apnea occurred, defined as inadequate ventilation below 2 L/min, a timer was set and one of either naloxone formulations was administered at 2 min intervals until adequate reversal of respiratory depression was observed. IM naloxone was injected in the rectus femoris muscle, IN naloxone was given in alternate nostrils for repetitive doses, if required. The aim of naloxone administration was the return of adequate and sustained ventilation to at least 80% of pre-fentanyl baseline levels (henceforth referred to as “baseline ventilation”). Per protocol, a maximum of 4 doses of the IM and IN formulations could be given. The timing of naloxone dosing was based on the 2-3 min administration interval as outlined by the United States Prescribing Information4. Assuming a bioavailability of 50% for intranasal naloxone and >75% for intramuscular naloxone20, we hypothesized that reversal would be achieved after 2 to 3 doses of intranasal naloxone and 1 to 2 doses after intramuscular naloxone. If ventilation remained below this target after all doses were given, a naloxone dose of 0.4 mg intravenous naloxone was to be administered. Respiratory measurements continued for 2 h from the administration of the first naloxone dose (t = 0). In contrast to the guidelines of the American Heart Association20, we refrained from verbal and/or tactical stimulation of our subjects during naloxone administration. We did so to enable the study of the naloxone pharmacodynamics driven by its pharmacokinetics without the confounding effects of activation of behavioral control of breathing. We agree that such a design differs from the guidelines. Stimulation may have an effect, but this is not certain as stimulation may have limited effect in case of loss of consciousness of the overdose victim (opioid narcosis).

Drugs

All drugs were prepared by the local pharmacy and dispensed on the morning of the study: fentanyl citrate 0.05 mg/mL (Hameln Pharma GmbH, Hameln, Germany), Zimhi (naloxone hydrochloride) 5 mg in 0.5 mL (DMK Pharmaceuticals Co., San Diego, CA, USA, and since May 2024 Zmi Pharma Inc, Carlsbad, CA, USA)11, Narcan Nasal Spray (naloxone hydrochloride) 4 mg in 0.1 mL (Adapt Pharma Inc. Radnor, PA, USA)12 and intravenous naloxone hydrochloride 0.4 mg/mL (Hameln Pharma GmbH, Hamel, Germany). IM or IN naloxone were administered on separate days in a random fashion (1:1) according to a randomization code generated by a study-independent statistician at the study center.

The study had a crossover design with a 7–10 days washout period in-between study days.

Blood sampling and analyses. Five mL arterial blood samples were obtained in K2EDTA tubes at regular intervals for measurement of fentanyl and naloxone concentrations in plasma. Plasma was separated from blood and stored at −80 oC until analysis. The samples were analyzed by Ardena Bioanalysis BV (Assen, the Netherlands) using validated liquid chromatography tandem mass spectrometry assays (LC-MS/MS) with limits of quantitation of 0.05 ng/mL (lower limit) to 500 ng/mL (upper limit) for both naloxone and fentanyl. Quality control revealed values for precision (coefficient of variation) for naloxone 1.1% (within-run precision) and 2.0% (between-run precision) and equivalent values for fentanyl 2.2% and 1.7%, respectively, while values for accuracy (bias) were for naloxone 2.2% (within-run bias) and 3.4% (between-run bias) and equivalent values for fentanyl −0.6% and 2.4%, respectively.

Safety

In case of the following circumstances, 0.4 mg naloxone was to be administered intravenously to ensure safety of the participants: end-tidal partial pressures of CO2 >90 mmHg (12 kPa) for >3 min, oxygen saturation of 70–80% for >1 min, oxygen saturation <70%, or any other situation of condition that could affect the health of the subject, as judged by the attending anesthesiologist.

Sample size and statistical analysis

No experimental data are available from the current literature that compare 4 mg IN and 5 mg IM naloxone dose requirements for complete (i.e. 80–100% of baseline ventilation) reversal of minute ventilation following fentanyl-induced apnea. We therefore performed a (crossover) trial simulation. We simulated a median difference of 1 dose between IM (1 dose) and IN (2 doses) naloxone with a variability of 1 (IQR) for IM and 2 (IQR) for IN naloxone. We simulated up to 20 subjects and obtained a power > 90% with p < 0.05 to detect a 1 dose difference with 12 subjects. To consider any discontinuations or deviations from our assumptions, we added 4 subjects to attain a test population of 16 healthy volunteers. Participants who chronically use an opioid were added for comparative reasons and that part of the study should be considered exploratory. We chose a convenience sample of 6 participants who chronically use an opioid.

The primary endpoint of the study was the number of IM versus IN administrations to reach full reversal of minute ventilation (±20%) following induction of apnea with an intravenous dose of 10 μg/kg fentanyl. These data were analyzed with the Wilcoxon signed rank test with continuity correction. A secondary endpoint was the time to 80–100% reversal of adequate minute ventilation (±20%) from the moment of the first naloxone administration. For the subjects that were treated with intravenous naloxone (after IN naloxone), a penalty of 4 min was added, to reflect the time required to fetch and draw the naloxone from its container. These data were analyzed using the Wilcoxon signed rank test, and Kaplan-Meier analysis with log-rank (Mantel-Cox) testing in GraphPad Prism, version 10 for Windows (GraphPad Software, Boston, MA, USA). P-values < 0.05 were considered significant. Data analyses were further performed in R (R Foundation for Statistical Computing, Austria, http://www.R-project.org/). The data from the healthy volunteers are presented with statistical testing, the data from the participants who chronically use an opioid are presented descriptively.

Additional secondary endpoints were (i) the number of IM and IN administrations to restore stable breathing in participants who chronically use an opioid; (ii) breath-to-breath minute ventilation and end-tidal PCO2; (ii) plasma concentrations of fentanyl and naloxone measured at specific time points in opioid-naïve individuals; the fentanyl measurements and analysis are exploratory as they were not prespecified in the protocol; (iii) pupil diameter and (iv) muscle tone. These secondary endpoints are presented without formal statistical analyses.

Inclusion and ethics statement

The study, approved by the local and national ethics committees, included local researchers throughout the research process and all researchers had an equal part in the design, performance, data analysis and reporting of the study. The research is considered locally and globally relevant given the worldwide opioid crisis and was presented to the US FDA, and relevant local organizations such as the TAPTOE (tackling and preventing the opioid epidemic) consortium.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.