Slow-sustained delivery of naloxone reduces typical naloxone-induced precipitated opioid withdrawal effects in male morphine-dependent mice

Abstract

Thousands of individuals die each year from opioid-related overdoses. While naloxone (Narcan®) is currently the most widely employed treatment to reverse opioid toxicity, high or repeated doses of this antidote often lead to precipitated opioid withdrawal (POW). We hypothesized that a slow linear release of naloxone from a nanoparticle would induce fewer POW symptoms compared to high-dose free naloxone. First, we measured the acute impact of covalent naloxone nanoparticles (Nal-cNPs) on morphine-induced antinociception in the hotplate test. We found that Nal-cNP treatment blocked the antinociceptive effect of morphine within 15 min of administration. Next, we tested the impact of Nal-cNPs on POW symptoms in male morphine-dependent mice. To induce morphine dependence, mice were treated with 5 mg/kg morphine (or saline) twice-daily for six consecutive days. On day 7 mice received 5 mg/kg morphine (or saline) injections 2 hr prior to receiving treatment of either unmodified free naloxone, a high or low dose of Nal-cNP, empty nanoparticle (cNP-empty), or saline. Behavior was analyzed for 0-6 hr followed by 24 and 48 hr time points after treatment. As expected, free naloxone induced a significant increase in POW behavior in morphine-dependent mice compared to saline-treated mice upon free naloxone administration. In comparison, reduced POW behavior was observed with both doses of Nal-cNP. Side effects of Nal-cNP on locomotion and fecal boli production were measured and no significant side-effects were observed. Overall, our data show that sustained release of naloxone from a covalent nanoparticle does not induce severe POW symptoms in morphine-dependent mice.

Keywords: drug delivery system; morphine; naloxone; nanoparticle; withdrawal.

FIGURE 1

Experimental Design: For 6 consecutive days, mice received intraperitoneal injections of 5 mg/kg morphine (* another group of mice received saline injections instead) twice-daily. On day 7, 2 hr after their AM morphine (or saline) injection, mice received an intraperitoneal injection of one of the five treatments: ** free naloxone (8 mg/kg), Nal-cNP (Hi)—8 mg/kg naloxone, Nal-cNP (Low)—0.7 mg/kg naloxone, cNP-empty, or saline. Behavior was video recorded for 6 hr (beginning immediately after the treatment injection). On days 8 and 9 video recording began 24 hr (day 8) and 48 hr (day 9) after the treatment injection was given (day 7) and behavior was recorded for 15 min. Precipitated opioid withdrawal behavior (naloxone-induced jumping, rearing, forepaw tremors, wet dog shakes, and forepaw licking) was measured the first 15 min of each hr. Figure prepared with BioRender.com software

FIGURE 2

Nal-cNP treatment significantly reduces morphine-induced thermal antinociception in the hotplate test. (a) Morphine (10 mg/kg) was administered 15 min prior to mice receiving free naloxone (8 mg/kg), Nal-cNP (Low), or cNP-empty treatment (n = 6/group). 15 min after treatment, nocifensive behaviors were observed (forepaw licking, forepaw/hindpaw withdrawal, jumping) in the hotplate test. (b) Data from the hotplate test are expressed as the percentage maximum possible effect (%MPE). Bonferroni post hoc tests revealed a significant decrease in % MPE of morphine in mice treated with Nal-cNP and free naloxone compared to cNP-empty (**p < 0.01). There were no significant differences between Nal-cNP and free naloxone treatment (p > 0.9999)

FIGURE 3

The majority of naloxone-induced POW behaviors were observed within the first hour of treatment. Morphine-dependent mice (n = 8/group) were treated with either free naloxone, Nal-cNP (Hi—8 mg/kg of naloxone), Nal-cNP (Low—0.7 mg/kg), cNP-empty, and saline (n = 8/group) 2 hr after receiving a morphine injection (5 mg/kg). During the first day of testing, behavior was assessed the first 15 min of every hour (6 hr total). Behavior was also assessed 24 hr after treatment and 48 hr after treatment. The data are expressed as mean ± SEM. Bonferroni post hoc tests represents significance between the following groups (p values are reported in results): (a) free naloxone group versus Nal-cNP (Low), Nal-cNP (Hi), cNP-empty, and saline, (b) Nal-cNP (Hi) group versus Nal-cNP (Low), cNP-empty, and saline, (c)free naloxone versus cNP-empty, (d) Nal-cNP (Hi) versus cNP-empty and saline, (e) free naloxone versus saline (f) Nal-cNP (Hi) versus saline, (g) free naloxone versus Nal-cNP (Hi), (h) Nal-cNP (Low) versus cNP-empty, saline, and free naloxone

FIGURE 4

Nal-cNP treatment displayed reduced POW behaviors compared to free naloxone treatment in morphine-dependent mice. These data are expressed as the sum (15 min bins) ± SEM for the first 6 hr of treatment on day 7 (n = 8/group). Bonferroni post hoc tests reveal significant differences in (a) naloxone-induced jumps, (b) forepaw tremors, and (c) wet dog shakes (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05). No effects were found for (d) rearing or (e) forepaw licking

FIGURE 5

Impact of Nal-cNP (Hi)/(Low) treatment on overall withdrawal severity (OWS) compared to free naloxone treatment. POW severity scores were normalized to free naloxone treatment in morphine-dependent mice (positive control). Symbols represent individual z-scores that were calculated for each behavior. These data are expressed as the average of individual z-scores (a) 6 hr, (b) 24 hr, and (c) 48 hr after treatment (n = 8/group). Post hoc analyses determined a significant decrease in OWS score in the free naloxone group compared to both cNP-empty and saline treatment groups within the first 6 hr of treatment (p < 0.05). Nal-cNP (Hi) (p = 0.062) and Nal-cNP (Low) (p = 0.089) trended to show reduced withdrawal behavior within 6 hr of treatment. Negative values on y-axis indicate reduced POW compared to positive control

FIGURE 6

Minimal withdrawal behaviors are found in control saline-treated mice compared to morphine-dependent mice treated with free naloxone. Mice received saline (twice-daily) for 6 consecutive days then treated with either free naloxone, Nal-cNP (Low), or saline. These data are expressed as the sum (15 min bins) ± SEM within the first 6 hr of treatment on day 7 (n = 6–8/group). Bonferroni post hoc tests reveal significant differences in (a) naloxone-induced jumps, (b) forepaw tremors, and (c) wet dog shakes (****p < 0.0001; ***p < 0.001; **p < 0.01) compared to morphine-dependent mice treated with free naloxone (free naloxone m.t). No statistically significant differences were found between saline-treated mice suggesting that no POW occurs in the absence of morphine dependency

FIGURE 7

Nal-cNP treatment produced negligible side effects in naïve mice. (a) Locomotion and (b) fecal boli production were observed to assess the side effect profile of Nal-cNP. Unpaired t tests determined there were no significant differences in total distance traveled (m), total time immobile (s), and the number of fecal boli between saline and Nal-cNP (Low)-treated animals (n = 8/group) in the open field test. Data are expressed as mean ± SEM