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. 2025 Apr 22;10(8):e186805.
doi: 10.1172/jci.insight.186805.

A small molecule PKCε inhibitor reduces hyperalgesia induced by paclitaxel or opioid withdrawal

Adriana Gregory-Flores  1   2 , Ivan Jm Bonet  3   4 , Stève Desaivre  2 , Jon D Levine  3   4 , Stanton F McHardy  5 , Harmannus C de Kraker  5 , Nicholas A Clanton  6 , Peter M LoCoco  6 , Nicholas M Russell  7 , Caleb Fleischer  2 , Robert O Messing  1   2   7   8 , Michela Marinelli  1   2   7   8   9
Affiliations

A small molecule PKCε inhibitor reduces hyperalgesia induced by paclitaxel or opioid withdrawal

Adriana Gregory-Flores et al. JCI Insight. .

Abstract

The enzyme protein kinase C ε (PKCε) plays an important role in pain signaling and represents a promising therapeutic target for the treatment of chronic pain. We designed and generated a small molecule inhibitor of PKCε, CP612, and examined its effect in a rodent model of chemotherapy-induced neuropathic pain produced by paclitaxel, which does not respond well to current therapeutics. In addition, many patients with chronic pain use opiates, which over time can become ineffective, and attempts to discontinue them can increase pain thereby promoting sustained opioid use. Therefore, we also investigated if CP612 alters pain due to opioid withdrawal. We found that CP612 attenuated hyperalgesia produced by paclitaxel, and it both prevented and reversed hyperalgesia induced by opioid withdrawal. It was not self-administered and did not affect morphine self-administration. These findings suggest that inhibition of PKCε is an effective, nonaddictive strategy to treat chemotherapy-induced neuropathic pain, with the added benefit of preventing increases in pain that occur as opioid treatment is discontinued. This latter property could benefit individuals with chronic pain who find it difficult to discontinue opioids.

Keywords: Addiction; Neuroscience; Pain; Protein kinases; Therapeutics.

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Figures

Figure 1
Figure 1. Structure and characterization of CP612.
(A) Structure and characteristics of CP612. tPSA, topological polar surface area. (B) CP612 inhibited novel PKCs by > 50% at 30 nM but was more potent against PKCε (IC50 = 2.03 nM) than PKCδ (IC50 = 18.8 nM) or PKCθ (30.7 nM). (C) Dose-response curves for inhibition of PKCε by CP612 showing a rightward shift at higher [ATP]. Data are shown as mean ± SEM (n = 3 experiments each done in duplicate or triplicate).
Figure 2
Figure 2. Pharmacokinetics of CP612.
(A and B) Concentrations of CP612 (40 mg/kg) in male mice after i.v. or i.p. administration declined over time in plasma but remained more stable in the brain up to 24 hours afterward. Data are shown as mean ± SEM (n = 2–6 per group).
Figure 3
Figure 3. Hyperalgesia induced by the PKCε activator ψεRACK.
Administration of the PKCε activator ψεRACK (1 μg/5 μL, i.d.) induced hyperalgesia in male rats. This was attenuated by CP612 (1 μg/5 μL, i.d.), up to 60 minutes after its administration. Data are shown as mean ± SEM (n = 6 paws per group). **P < 0.01, ***P < 0.001 compared with the same time points in the Vehicle group using Tukey’s post hoc test.
Figure 4
Figure 4. Hyperalgesia induced by the chemotherapeutic drug paclitaxel.
Repeated administration of paclitaxel (1 mg/kg, i.p. every other day for a total of 4 injections) induced hyperalgesia in male rats. This was attenuated by CP612 (20 mg/kg, i.p.), up to 120 minutes after its administration. Data are shown as mean ± SEM (n = 6 paws per group). **P < 0.01, ***P < 0.001 compared with the same time points in the vehicle group using Tukey’s post hoc test.
Figure 5
Figure 5. Time course of hyperalgesia induced by morphine withdrawal.
After repeated administration of morphine (20–100 mg/kg, i.p.) twice daily for 5 days in male mice, hyperalgesia was not present at 6 hours but was present at 24 hours after the last injection of morphine and persisted for 1 week. No hyperalgesia developed after repeated administration of saline. Data are shown as mean ± SEM (n = 6 per group). **P < 0.01, ***P < 0.001 compared with the same time points in the group receiving repeated injections of saline using Tukey’s post hoc test.
Figure 6
Figure 6. Prevention of hyperalgesia due to morphine withdrawal.
(A) Experimental timeline. (B and C) Withdrawal from repeated administration of morphine (20–100 mg/kg, i.p.) induced hyperalgesia in male (B) and female (C) mice. This was prevented by CP612, up to 4 weeks after its administration. No hyperalgesia developed after repeated administration of saline. Data are shown as mean ± SEM (n = 11 for each male group, n = 7 for each female group). ***P < 0.001 and **P < 0.01 compared with the same time points in all other groups using Tukey’s post hoc test. For clarity, data from males and females are plotted separately.
Figure 7
Figure 7. Reversal of hyperalgesia due to morphine withdrawal.
(A) Experimental timeline. (B and C) Withdrawal from repeated administration of morphine (20–100 mg/kg, i.p.) induced hyperalgesia in male (B) and female (C) mice. This was reversed by CP612, administered 2 weeks after hyperalgesia was established and lasted for an additional 2 weeks. Data are shown as mean ± SEM (n = 5–7 per group). ***P < 0.001 compared with the same time points in the vehicle group using Tukey’s post hoc test. For clarity, data from males and females are plotted separately.
Figure 8
Figure 8. Self-administration of CP612.
(A) Increasing the ratio to obtain an infusion across self-administration sessions produced a concomitant increase in responding in the active hole in rats self-administering morphine (500 μg/kg/i.v. infusion) but not in rats self-administering vehicle or different doses of CP612 (75, 150, and 300 μg/kg/i.v. infusion). (B) Rats self-administering morphine had a lower elasticity value (α = 0.4 × 103) compared with all other groups (α = 1.1, 0.9, 0.9, 1.2 × 103 for vehicle and CP612 at 75, 150, and 300 μg/kg/i.v. infusion). They also had a higher price value to switch from inelastic to elastic behavior (Pmax = 50.1) compared with all other groups (Pmax = 4.1, 6.5, 6.1, 2.5 for vehicle and 75, 150, and 300 μg/kg/i.v. infusion). Data are shown as mean ± SEM (n = 10 for vehicle, n = 9 for morphine, n = 7–8 for each CP612 group). ###P < 0.001 compared with their infusions at FR1 and the same ratios between rats self-administering morphine and all other groups using Tukey’s post hoc test. ***P < 0.001 for Pmax and ††P < 0.01 for α, between rats self-administering morphine compared with all other groups using Tukey’s post hoc test. AH, active hole; IH, inactive hole.
Figure 9
Figure 9. Self-administration of morphine when adding CP612 to the morphine solution.
Adding CP612 (75, 150, and 300 μg/kg/i.v. infusion) to the morphine solution (500 μg/kg/i.v. infusion) during self-administration did not modify responding in a progressive ratio test, where the ratio to obtain an infusion was increased progressively within a self-administration session. This was measured as breaking point (highest ratio reached to earn an infusion of drug) and number of self-infusions. Data are shown as mean ± SEM; dots are scores from individual rats (n = 7–10 per group).
Figure 10
Figure 10. Self-administration of morphine after administering CP612 six hours beforehand.
(A) Rats acquired morphine self-administration behavior at FR1, and this responding was increased when the ratio to obtain morphine was increased to FR3. This occurred to the same extent in rats that would later receive vehicle or CP612. AH, active hole; IH, inactive hole. During the progressive ratio tests, the ratio to obtain an infusion was increased progressively within a self-administration session. Administration of CP612 (30 mg/kg, i.p.) 6 hours beforehand did not modify morphine intake or responding. (B–D) This was measured as breaking point (highest ratio reached to earn an infusion of drug) and number of self-infusions the day after the last self-administration session (B), after 1 day of withdrawal (C), or after the administration of naloxone (D) (0.03 mg/kg, s.c.). Data are shown as mean ± SEM; dots are scores from individual rats (n = 6–13 per group). ***P < 0.001 compared with the inactive hole using Tukey’s post hoc test.
Figure 11
Figure 11. Morphine withdrawal and conditioned place aversion (CPA) precipitated by naloxone.
(A) Experimental timeline. (B) On conditioning day, naloxone (5 mg/kg, i.p.) evoked signs of withdrawal in male mice that received repeated administration of morphine (20–100 mg/kg, i.p.) and were treated with vehicle (n = 25) or a low dose of CP612 (10 mg/kg, i.p., n = 15), but this was decreased in mice treated with a high dose of CP612 (40 mg/kg, i.p., n = 10). (C) On test day, prior administration of naloxone produced CPA in mice that received repeated administration of morphine, and this occurred similarly in mice that were treated with vehicle (n = 30) or different doses of CP612 (5, 10, 20, 40 mg/kg, i.p., n = 6, 18, 7, 11, respectively). For B, box and whiskers are median and 25% interquartile intervals; dots are scores from individual mice. For C, Data are shown as mean ± SEM; dots are scores from individual mice. ##P < 0.01 and ###P < 0.001 compared with Saline + Vehicle: ***P < 0.001 compared with Morphine + Vehicle using Tukey’s post hoc test.
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