Opioids have been used to manage pain for thousands of years, but they have significant potential for abuse. Prescription opioids, like oxycodone, are associated with 32% of overdoses, that have reached a total of 75,673 deaths in 2021. A major challenge is maximizing their therapeutic potential while minimizing the negative side effects including opioid use disorder (OUD). The Ketogenic Diet (KD) has been reported to reduce pain and decrease the severity of alcohol use disorder, yet its effects on oxycodone remain unknown. KD mice displayed increased oxycodone-induced locomotor activity and enhanced antinociceptive effects of oxycodone, suggesting a dietary effect on opiate sensitivity. Male KD mice exposed to chronic oxycodone exhibited increased naloxone-induced jumps, suggesting a sex-specific effect of diet on opioid withdrawal. Consistent with this, male KD mice self-administered less oxycodone while female KD mice did not differ from controls. Finally, no effect of KD on motivation to obtain oxycodone was observed during a progressive ratio schedule. These data suggest sex-biased effects of KD on responses to opioids that should be considered and potentially leveraged in both pain management and treatment of OUD.
Article
Ketogenic diet enhances the effects of oxycodone in mice
https://doi.org/10.21203/rs.3.rs-2504986/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 09 May, 2023
You are reading this latest preprint version
The prevalence of opioid overdose deaths continues to rise dramatically, making it a major public health concern 1. Opioid overdose deaths increased 519% between 1999–2019, with 56,064 deaths reported in 2020 and 75,673 in 20212,3. Prescription opioids such as oxycodone have played a major role in the opioid epidemic, contributing to 32% of overdose deaths 3. In the United States more than 2 million individuals have a substance use disorder related to prescription opioids; of patients prescribed opioids, 25% misuse them, and 5–10% develop OUD 4. Despite the high risk of abuse associated with oxycodone, it is still one of the most effective and prescribed medications for pain management 5. In reaction to the opioid crisis, greater attention has been placed on investigating effective treatments for OUD and maximizing the therapeutic benefit of oxycodone while minimizing the negative consequences in a pain management setting.
Medication-assisted treatment (MAT) for OUD currently utilizes three FDA-approved medications: 1) methadone, 2) buprenorphine, 3) and extended-release naltrexone 6. Methadone and buprenorphine are opioid agonists at the mu-opioid receptor, reduce opioid use, craving, and mortality, while increasing treatment retention 6. However, both drugs are prone to abuse by patients 7. Naltrexone, an opioid antagonist, is available in oral and extended-release injectable (Vivitrol) 6. Vivitrol treatment results in reduced opioid craving and use, but does not reduce the risk of mortality, and unfortunately has the lowest treatment adherence likely due to a required withdrawal period prior to initiation 6. Given the public health crisis, it is critical to both develop more effective therapeutics, and to better understand how physiological states modify OUD and response to therapeutics.
It is well established that nutritional deficiencies stemming from poor diet are highly prevalent in the OUD population and may contribute to impaired recovery 8–13. Incorporating a nutritional component to OUD treatment may improve patient outcomes and decrease healthcare costs 11. However, nutritional interventions for substance use disorder (SUD) are still in their infancy, as very few studies have investigated dietary manipulation as a potential treatment adjunct for SUD. Consequently, it is difficult for healthcare providers to give specific and effective nutritional recommendations. The Ketogenic Diet (KD) is a high fat, low/moderate protein, low carbohydrate diet, best known for its efficacy in treating pediatric epilepsy 14. It has recently been demonstrated to have potential for improving alcohol withdrawal symptoms in clinical 15 and preclinical 16,17 studies, and was found to reduce alcohol self-administration in rodents 15,18,19. Notably, there is also preliminary evidence that KD has antinociceptive benefits in preclinical 20,21, as well clinical settings 22. Here we present data that a KD intervention influences responses to opioids with potential clinical implications for treatment of OUD and therapeutic use of opioids.
Animals and Diets
Animal experiments were conducted in accordance with the Yale University School of Medicine Institutional Animal Care and Use Committee (IACUC) animal care guidelines. All protocols were approved by Yale IACUC. All methods are reported in accordance with ARRIVE guidelines. No animals were removed from analysis. The experimenters were not blinded to the dietary groups. Male and female C57BL/6J mice, purchased from The Jackson Laboratory, were between the ages of 8 and 15 weeks for the experiments. Ketogenic Diet AIN-76A Modified, with a caloric value of 93.4% fat, 4.7% protein, and 1.8% carbs (7.24 kcal/g) was purchased from Bio-Serv (#F3666). Standard chow, with a caloric value of 18% fat, 24% protein, and 58% carbs, was purchased from Teklad Global (#2018). All mice were maintained either ad libitum on these diets, or restricted to maintain 85–90% of starting bodyweight, for self-administration studies. For the studies involving paired-feeding, male pair-fed mice were mildly restricted on standard chow to match the percent change in body weight loss observed in the KD males prior to experimentation. This matching was maintained daily throughout the course of dietary intervention, locomotor activity, and then hot plate testing. Separate cohorts of animals were used for self-administration and osmotic minipump studies. Mice were group housed up to five per cage on a 12h light/dark cycle (lights on at 7:00 A.M.).
Statistics
GraphPad Prism 9.0 and RStudio v1.4.1717-3 23 were used to perform statistical tests. For t-tests, datasets were first assessed for normal Gaussian distribution. Mann-Whitney U analysis was conducted for datasets that were not distributed normally. Bonferroni, Sidak’s and Tukey’s post hoc analysis were conducted when applicable. Because the hot plate experiments utilized a maximum 40s cutoff for latency, a Cox mixed-effects proportional hazards model with timepoints set as numeric was used for the analysis of data points after oxycodone injection, with R packages coxme and multcomp 24,25.
Ketone Levels
Tail blood was used to assess ketone levels using a Precision Xtra ketone meter (model #P0BF9) or a Keto Mojo ketone meter (model # 21335C002AEEB) and used according to the manufacturer’s specifications.
Drugs
Saccharin (Acros Organics #149005000) was diluted to several concentrations (the highest being 0.1%) in tap water. Oxycodone HCl was supplied by the NIDA drug sharing program. Oxycodone was diluted at various concentrations in either tap water for self-administration experiments, or sterile saline for injections and osmotic minipump loading. Naloxone Hydrochloride was purchased from Spectrum Chemical (#N1231), dissolved in sterile saline, and injected at 1 mg/kg intraperitoneal (i.p.) immediately prior to assessing jumping behavior. Lidocaine (Covetrus, North America VINB-0024-6800) was injected locally at 4 mg/kg subcutaneously (s.c.) prior to incision for minipump implantation. Carprofen (Zoetis) was injected i.p. at 5 mg/kg prior to incision for minipump implantation, and again at 24 and 48h after surgery.
Locomotor Activity
Med Associates locomotor chambers, which utilize infrared beams, and Med PC V software were used to assess locomotor activity. All mice were habituated to Med Associates locomotor chambers for 90min, during which they all received a saline injection i.p. at 60min. All mice then received an acute 2 mg/kg oxycodone injection i.p., and activity was measured over 90min in 5min bins. Two consecutive beam breaks were required to constitute locomotor activity.
Hot Plate Assay
Mice that were previously used for locomotor activity were assessed for nociception, with a minimum of two days between tests. The hot plate assay was conducted using a Series 8 hot plate from IITC Life Science held at a constant 52°C. A 40s cutoff time was implemented to avoid tissue damage. Nociceptive measures included paw flick, paw lick, or jumps. Each mouse was immediately removed from the hot plate after the first incidence of nociception or 40s, whichever came first. Oxycodone 2 mg/kg was delivered i.p. after their initial baseline assessment. All mice underwent four additional hot plate trials over a 45min period.
Precipitated Withdrawal
Osmotic minipumps (Alzet #1003D) were used to chronically deliver oxycodone over three days. Mice were placed on their respective diets ad libitum for four days prior to osmotic minipump surgeries. The day before surgery, bodyweights were assessed, and minipumps were custom loaded to deliver 130 mg/kg/day. Ketone levels were measured prior to surgery. Surgeries were performed under aseptic conditions. Briefly, mice were injected with carprofen, anesthetized with isoflurane, and shaved at the incision site on their backside lateral from their spine. The exposed skin was cleaned with betadine, lidocaine was injected locally, and a small incision was made with scissors. A subcutaneous pocket was made and the minipump was inserted. Wound clips were used to close the incision site and triple antibiotic cream was applied. Animals were monitored daily. Three days after surgery, mice were injected with 1 mg/kg naloxone i.p. and placed in an empty static mouse bucket with a high lid. Jumps were quantified using Med Associates locomotor chambers, with the beam detectors raised approximately six inches off the ground, high enough to require all four paws leaving the cage floor, and to not detect rearing. A jump was counted when an animal broke a beam, with a 0.5s timeout period to allow for the mouse to land. Jumps were quantified over 20min.
Oxycodone Self-administration And Progressive Ratio
All mice were maintained on standard chow and food restricted to 85–90% of their bodyweight. Med Associates operant boxes were used, which were equipped with three nosepoke ports on one side, a magazine for reward retrieval on the opposite wall, cue lights in the active nosepoke port and magazine, and a syringe pump for delivery of liquid rewards. 5ml syringes were used, and pump activation time was set to deliver 20 µl of liquid into the magazine per reward earned. The operant sequence of events started with activation of the nosepoke port and its cue light. Upon a successful nosepoke port entry, the nosepoke cue light was turned off, a reward was immediately delivered and a cue light in the magazine was turned on. Upon successful magazine entry by the mouse, the magazine cue light was turned off, and after a 5s delay, the appropriate nosepoke port was made active again, as indicated by its cue light. Self-administration sessions were 3h long. Mice were initially trained to self-administer 0.1% saccharin solution in water on a fixed ratio 1 (FR1) schedule, in which each active nosepoke resulted in the delivery of oxycodone reward, which then was increased to an FR3 schedule (3 nosepokes per reward). After several days of FR3 0.1% saccharin, oxycodone was gradually introduced over several days, from 0.05 mg/ml to 0.3 mg/ml. After four days at 0.1% saccharin 0.3 mg/ml oxycodone, the saccharin was fully faded out over four days. After six additional days of 3h sessions at FR3 with just oxycodone, mice were assigned to their dietary groups (KD or Chow), balanced by their oxycodone consumption. Self-administration sessions and food restriction were maintained throughout the dietary transition. Magazines were checked at the end of each session to verify rewards had been consumed. Mice were then tested on an exponentially increasing progressive ratio schedule 26–28. The session was conducted over 6h, with drinking water provided in the operant box to eliminate thirst as a confound for liquid seeking. No cutoff time was implemented in the program. The nosepoke requirement schedule (where j = 0.2) for the first 14 rewards is as follows: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77.
Progression of Ketosis and the Effects of KD on Bodyweight
The first cohort of mice were placed on either the ketogenic diet (n = 10, 10; males and females) or standard chow (n = 10, 10; males and females) and maintained under ad libitum conditions. Ketone levels were measured prior to diet initiation (day 1), and on days 4 and 7 post diet induction (Fig. 1A and B). A two-way repeated measures ANOVA revealed a main effect of diet (males, F(1, 18) = 160.6, p < .0001; females, F(1, 18) = 410.1, p < 0.0001) and time (males, F(2, 36) = 67.24, p < 0.0001; females, F(2, 36) = 143.5, p < 0.0001) with an interaction of diet x time (males, F(2, 36) = 67.66, p < .0001; females, F(2, 36) = 102.2, p < 0.0001) for ketone levels. A Bonferroni’s multiple comparisons test revealed a highly significant increase in ketones on day 4 (males ****p < 0.0001; females ****p < 0.0001) and day 7 (males ****p < 0.0001; females ****p < 0.0001).
Male mice consistently lost weight on the KD relative to Chow males over the course of eight days (Fig. 1C; main effect of diet (F(1, 18) = 18.98, p = 0.0004) and time (F(1.266, 22.78) = 4.386, p = 0.0395) with an interaction of diet x time (F(2, 36) = 19.13, p < 0.0001). A Sidak’s post hoc analysis showed significant bodyweight differences at Days 5 and 8 post-diet induction (****p < 0.0001). Female mice displayed no differences in bodyweight between KD and Chow groups over the course of eight days (Fig. 1D). A two-way ANOVA analysis for sex differences of net bodyweight from baseline at Day 5 revealed a main effect of sex (F(1, 36) = 18.84, p = 0.0001), a main effect of diet (F(1, 36) = 13.18, p = 0.0009), and a significant interaction of diet x sex (F(1, 36) = 19.14, p < 0.0001). A two-way ANOVA at Day 8 revealed a main effect of sex (F(1, 36) = 23.06, p < 0.0001), a main effect of diet (F(1, 36) = 23.06, p < 0.0001), and a significant interaction of diet x sex (F(1, 36) = 23.71, p < 0.0001).
Locomotor Activity
To assess general changes in sensitivity to oxycodone due to KD, we conducted locomotor activity testing. A second cohort of males was generated to include a Pair-fed Chow group. KD (males n = 10; females n = 10), Chow (males n = 10; females n = 10), and Pair-fed Chow (males n = 10) were placed on their respective diets for a minimum of four days. The Pair-fed Chow group of males was included in parallel to account for the potential contribution of bodyweight loss on oxycodone sensitivity and response. β-hydroxybutyrate levels (mM) in males assessed prior to locomotor testing were: Chow (mean = 0.44, StdDev = 0.212), Pair-fed Chow (mean = 0.84, StdDev = 0.135), and KD (mean = 2.42, StdDev = 0.449), and a one-way ANOVA analysis with Tukey’s multiple comparisons revealed differences between all three groups (F(2,27) = 124.1, p < 0.0001); Chow vs Pair-fed Chow p = 0.015; Chow vs KD and Pair-fed Chow vs KD were both p < 0.0001), data not shown. Bodyweight % change for males at the time of experiment, from the start of dietary intervention, showing matched levels between KD and Pair-fed Chow mice, which were significantly lower than Chow mice (Fig. 2A; one-way ANOVA (F(2, 27) = 50.45, ****p < 0.0001).
Total locomotor activity was recorded during each phase (baseline, saline, and oxycodone; Fig. 2B). In male mice, analysis of binned data revealed a main effect of diet (F(2, 27) = 3.585, p = 0.0416), a main effect of time (F(4.712, 127.2) = 46.48, p < 0.0001), and an interaction of diet x time (F(70, 945) = 3.122, p < 0.0001). A Tukey’s post hoc analysis revealed an increase in activity in KD vs Chow mice (*p≤0.05, **p≤0.01, ***p≤0.001) after oxycodone treatment. Pair-fed Chow mice only showed significant differences from other groups at -5min vs Chow, and vs KD at 55min (*p≤0.05 for both timepoints). Cumulative total locomotor activity was recorded for males for each phase (baseline, saline, and oxycodone, Fig. 2C) and revealed a main effect of diet (F(2, 27) = 3.585, p = 0.0416), time (F(1.437, 38.81) = 90.12, p < 0.0001), and an interaction of diet x time (F(4, 54) = 6.965, p < 0.0001). Tukey’s post hoc analysis revealed an increase in activity in KD vs Chow mice (**p = 0.0014) after oxycodone treatment.
Unlike male mice, no differences were observed in change in bodyweight % at the time of this experiment from the start of dietary intervention in female mice (Fig. 2D). A two-way ANOVA revealed a difference in locomotor activity in Chow vs KD groups (Fig. 2E) with a main effect of time (F(5.256, 94.60) = 50.21, p < 0.0001) and an interaction of diet x time (F(35, 630) = 10.47, p < 0.0001). Sidak’s post hoc analysis revealed an increase in activity for Chow vs KD mice (-30min, *p = 0.0318), and an increase in activity for KD vs Chow after oxycodone treatment (*p < 0.05, **p < 0.01). Analysis of cumulative total locomotor activity for females for each phase (Fig. 2F; baseline, saline, and oxycodone) revealed a main effect of time (F(1.323, 23.81) = 205.5, p < 0.0001), no main effect of diet, and an interaction of diet x time (F(2, 36) = 22.53, p < 0.0001). Sidak’s post hoc analysis showed increased locomotor activity in Chow vs KD females during baseline (*p = 0.0143) and saline (**p = 0.0097), and increased activity by KD females vs Chow in response to oxycodone (**p = 0.0038).
Hot Plate
A hot plate assay was used to assess changes in the antinociceptive effects of oxycodone. Time to nociceptive behavior was recorded at five time points: baseline (BL) prior to 2mg/kg oxycodone injection, and 10, 20, 30, and 45 minutes after injection for KD (males n = 10; females n = 10), Chow (males n = 10; females n = 10), and Pair-fed Chow (males n = 10) groups). A Cox mixed-effects proportional hazards model analysis of male mice revealed a significant main effect of diet (*p = 0.02755), a main effect of time (***p < 0.0001), with a Tukey multiple comparisons test revealing an increase in latency in KD vs Chow (Fig. 3A; *p = 0.024). A Cox mixed-effects model analysis of female mice revealed a significant main effect of diet (**p = 0.0031), a significant main effect of time (p < 0.001), and a trend interaction of diet x time (p = 0.074; Fig. 3B). There were no differences in bodyweight % loss among the Pair-fed Chow vs KD males, and with Chow vs KD females showing no difference (Fig. 3C).
Precipitated Withdrawal
To assess the effects of ketogenic diet on withdrawal symptoms, we measured jumping behavior after inducing withdrawal with naloxone. After mice had completed three days of continuous oxycodone delivery via osmotic minipumps, jumps were quantified over twenty minutes immediately after naloxone injection (1 mg/kg ip). Male mice in the KD group (n = 10) had significantly more jumps than Chow (n = 9) mice (Fig. 4A; t = 2.333, p = 0.0322). No difference was observed with female mice comparing KD vs Chow (Fig. 4B; n = 14,12).
Oxycodone Self-administration And Progressive Ratio Tests
Male and female mice (n = 20, 20) were trained to operantly self-administer oral oxycodone while maintained on standard chow. After their Day 6 self-administration session, the dietary intervention experiment was started by replacing the standard chow with ketogenic diet for the KD group. Analysis of oxycodone self-administration after diet initiation (session 7–20) showed no main effect of diet or time, but a significant interaction of diet x time (F (13, 234) = 2.538, p = 0.0028) with KD mice consuming significantly less oxycodone (Fig. 5A; Sidak’s post hoc analysis *p = 0.0223). Female mice showed a main effect of time (F (5.284, 95.11) = 6.689, p < 0.0001) and a near significant interaction of diet x time (Fig. 5B; F (13, 234) = 1.756, p = 0.0510) indicating a potential reduction in oxycodone self-administration in female KD mice.
These mice were then tested for motivation to acquire oxycodone using a progressive ratio paradigm. Male and female mice showed no difference in rewards earned between KD and Chow groups (Fig. 5C and D).
These data suggest that KD modulates opioid responses in mice in a sex-specific manner as summarized in Table 1. More broadly, these data serve as an example of the importance of nutritional and metabolic state for both therapeutic use of opiates for pain management, as well as treatment of OUD.
While both male and female mice on KD rapidly develop ketosis, we observed sex differences in bodyweight loss in KD male mice only. This persisted throughout locomotor and hot plate testing. This sex-specific body weight response to the ketogenic diet in mice has not been reported in the literature, however it is consistent with clinical data evaluating subjects with obesity on a ketogenic diet, where it was reported that male subjects lost more weight than females 29.
Oxycodone-induced locomotor activity was tested to screen for general changes in opioid sensitivity on the ketogenic diet. Both male and female KD mice displayed robust increases in activity after oxycodone treatment compared to Chow mice, indicating that the ketogenic diet may enhance the effects of the opioid. Interestingly, the female KD mice displayed significantly less activity during baseline and saline phases than Chow. This reversal from lower to higher oxycodone-induced activity strengthens the finding that the dietary manipulation enhanced their opioid sensitivity.
Since it is known that food restriction can enhance behavioral and physiological sensitivity to drugs of abuse 30, we generated a Pair-fed Chow group matched to the KD group’s bodyweight loss in male mice. β-hydroxybutyrate levels were significantly different across all three male groups prior to locomotor activity testing, which was expected as food restriction can increase ketone levels 31,32. However, the KD diet produced much higher β-hydroxybutyrate levels than the Pair-fed Chow, and even more importantly, the Pair-fed Chow mice did not show the enhanced locomotor and antinociceptive responses to oxycodone, suggesting that these effects are primarily driven by the ketogenic diet, and not by the bodyweight loss.
We also evaluated effects of the KD on modifying sensitivity to the antinociceptive effects of oxycodone using the hot plate assay. Both male and female KD mice displayed significantly enhanced latency after oxycodone compared to Chow, suggesting that the ketogenic diet may enhance the antinociceptive effects of oxycodone. These data support a potential role for a ketogenic diet as an adjunct with the therapeutic use of opiates for pain management in a clinical setting. In line, as the ketogenic diet has shown efficacy in reducing pain in animals and humans 20,22,33, it has recently been recommended as a potential adjunct to a comprehensive pain management plan 22. These data provide more scientific evidence for those recommendations and need clinical validation.
We evaluated KD effects on physical withdrawal symptoms after chronic oxycodone delivery via minipump. In this case, dietary manipulation occurred prior to oxycodone exposure, and withdrawal was evaluated after ketosis had developed. Interestingly, male KD mice had increased jumping behavior after naloxone treatment, suggesting increased sensitivity to withdrawal symptoms, whereas the female KD mice did not show these effects. We also evaluated KD in mice that were self-administering oral oxycodone. After dietary intervention, male KD mice showed a significant reduction in oral self-administration compared to Chow, and female KD mice showed a trend reduction as well. These data have translational significance by showing that a dietary intervention can have an impact on ongoing opiate consumption and withdrawal symptoms. A reduction in withdrawal symptoms might increase the likelihood of successful treatment of OUD. Follow up studies are needed to confirm potential efficacy, as well as to test for KD interactions with OUD medications (i.e., methadone, buprenorphine, naltrexone). Furthermore, neither male nor female KD mice displayed a significant difference in motivation on the progressive ratio schedule.
Overall, we show a modulatory role for KD in the response to opioids and opioid withdrawal symptoms. While both sexes experienced improved analgesic effects during opiate use for pain relief, a KD was only efficacious in reducing opiate consumption in male mice, whereas the females exhibited a strong trend in reduction. This sex-specific response might guide clinical implementation strategies. OUD is a complex disorder with a multitude of variables that impact treatment outcomes 34. Sex differences may be a major factor influencing treatment outcome among OUD patients, with some studies showing that women progress to more severe OUD faster than men, and have higher withdrawal scores, whereas other studies show that men are less likely to remain in treatment 35. The present findings indicate that an overall awareness and clinical assessment of dietary and metabolic state is needed both during treatment of OUD as well as during the therapeutic use of opiates for pain.
Acknowledgements
We would like to thank Hayde Sanchez for her assistance in this project.
Author Contributions
R.T., D.M.D., and R.J.D. designed the experiments. R.T., D.M.D., and E.F. conducted the experiments. R.T., D.M.D., and S.L.T. analyzed and interpreted these data. R.T., D.M.D., and R.J.D. wrote the manuscript. E.F. provided technical support and manuscript proofreading. S.L.T. and J.R.T. provided statistical guidance and manuscript proofreading.
Data Availability
All data supporting this publication can be available from the corresponding author R.J.D. upon reasonable request.
Ethics Declarations
Competing interests: The authors declare no competing interests.
Funding
This work was funded in part by the State of Connecticut, Department of Mental Health and Addiction Services, but this publication does not express the views of the Department of Mental Health and Addiction Services or the State of Connecticut. The views and opinions expressed are those of the authors.
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Table 1: Summary of findings. Up arrows indicate an increase, down arrows indicate a decrease, bidirectional horizontal arrows indicate no change. N/A indicates that group wasn’t included. The effect size (degree of significance) for a given readout is indicated by the number of arrows.
No competing interests reported.