Patent ID: 12257283

METHODS

Because opioids are used in the management of pain conditions that contribute to OUD, we determined the efficacy of a small peptide chemokine receptor antagonist RAP-103 (all-D-TTNYT, SEQ ID #1, Table 1), sometimes called RAP-103 or R103) to reverse opioid receptor analgesic tolerance and block pain as a test of its ability to block-drug seeking behaviors. Painful diabetic neuropathy (PDN) is a common complication of diabetes which adversely affects patients' daily life, often involves opioid use, which creates an addiction risk. We reasoned that chronic inflammation blocked by R103 that causes chronic pain is also engaged in the pathogenesis of OUD and substance abuse, then a demonstration of 1) blocking infiltration of immune cells in damaged nerves and/or activation of spinal microglia in the development of pain; and 2) inhibiting inflammatory responses in the peripheral and/or the central nervous system that reduces chronic pain would also reduce chronic inflammation in the brain that leads to substance abuse behaviors. To test the hypothesis, we first used behavioral and molecular/cellular approaches to explore chronic pain development and inflammatory reaction in Streptozotocin (STZ) induced diabetic rats. Our results showed that following the induction of diabetes, rats exhibited persistent mechanical and cold allodynia (up to five months post-induction). The levels of inflammatory molecules, including cytokines, IL1b and TNFa; chemokines CCL2, CCL3; and chemokines receptors CCR2 and CCR5 were dramatically increased in sciatic nerves. Microglia in the spinal cord dorsal horns became activated with hypertrophic morphology and an increase in microglial cell number. CCL2 and CCL3 are two chemokines well known in mediating immune cell trafficking and immune response in the context of neuropathic pain. Oral administration of RAP-103 a CCR2/CCR5/CCR8 multi-CRA receptor antagonist for 7 days inhibited expression of chemokines and cytokines and reduced neuropathic pain. Thus R103 has the predicted treatment effect to blunt drug-seeking behaviors or psychostimulant activation (e.g. locomotion).

The treatment with R103 (RAP-103) (0.5-0.02 mg/Kg b.w., daily, for 7 days, resulted in a complete reversal of established hypersensitivity in STZ rats. The results suggest that functional antagonism of multiple chemokine receptors and innate immune receptors may be treatments and medicines for pain and inflammation. In view of the results showing that chemokines reduce opioid efficacy by cross-desensitizing opioid receptors, these results suggest that administration of a chemokine receptor antagonist peptides of the invention, will reduce addiction risk, and in those suffering from drug addiction, provide a treatment to block or reduce drug-seeking behaviors and limit the symptoms of withdrawal. The effective human doses suggested by these animal studies would be 0.01 to 20 mg/Kg/day. The peptides may be dosed as an oral pill, suspension, liquid, or by IV and subcutaneous injections to achieve pain control with decreased concomitant opioid use. Some examples of other useful CRA antagonists that can reduce drug-seeking/substance abuse are Maraviroc (a CCR5 antagonist) or AMD3100 (a CXCR4 antagonist). The peptides of the current invention have the advantage of blocking multiple chemokine receptors (CCR2, CCR5, CCR8, CXCR4) and pathways (NFkB, STAT3, TLR4) involved in substance abuse and are safer.

Induction and Assessment of Diabetes in Rats

Rats were fasted overnight and received a single intraperitoneal injection of streptozotocin (60 mg/kg, Sigma-Aldrich) in citrate buffer (pH 4.5) to induce type I diabetes. The induction of diabetes was confirmed 72 h after streptozotocin injection by measurement of blood glucose levels using Contour® blood glucose diagnostic kit (Bayer HealthCare, Canada). Body weight and blood glucose levels were measured before and 3 days after streptozotocin injection, once a week for three weeks after diabetes induction. Only rats having blood glucose concentration consistently higher than 300 mg/dl were used for the present study.

Treatment Paradigms

To investigate whether RCT02 (all-D-TTNYT), a drug which acts directly on CCR5 and CCR3 chemokine receptors can reverse already established neuropathic hypersensitivity in diabetic rats, autoclaved water or RCT02 (0.004, 0.02, 0.1 or 0.5 mg/kg b.w.) was administered daily by oral gavage to rats who exhibited stable mechanical and cold allodynia. The treatment lasted for 8 days Note, n=5 for H2O and n=7 for RAP-103/each dose.

Assessment of Pain Sensitivity

Tactile allodynia Mechanical sensitivity was assessed using calibrated von Frey hairs (7). Animals were placed in Plexiglas boxes on an elevated metal mesh floor and allowed 60 min for habituation before testing. A series of von Frey filaments with logarithmically incrementing stiffness was applied perpendicular to the mid-plantar region of the hind paw. The 50% paw withdrawal threshold was determined using Dixon's up-down method. Withdrawal thresholds of both paws were averaged as one single value per animal. Assessment on the effects of RAP103 on mechanical and cold allodynia was performed between 2-4 hours following the drug administration.FIG.1shows the action of R103 to block mechanical pain, whileFIG.2shows the action of R103 to block cold allodynia.FIGS.3A-Fshows the action of R103 to reduce CNS (dorsal spinal cord) chemokines and chemokine receptors predicted to stop or reduce drug-seeking behaviors or the behavioral effects of substance abuse. All data were presented as means±SEM. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-tests. The criterion for statistical significance was P<0.05.

Locomotor Experiments

Locomotor activity (ambulation+stereotypy) induced by cocaine and the effect of RAP-103 was assessed using a Digiscan DMicro system. Following a 60-min habituation in activity chambers, rats were pretreated with RAP-103 (0.03, 0.01, 0.001 mgs/Kg) or vehicle 30 min before cocaine (10 mgs/Kg) or vehicle and activity was measured for 120 min. Differences between individual groups were determined with a Bonferroni post-hoc test. Statistical significance was set at p<0.05. We show that RAP-103 blocks the locomotor inducing effects of cocaine (FIG.4).

Effects of R103 on MOR Withdrawal in Rats

Opioid tolerance during chronic exposure requires increasing opioid doses to maintain analgesic efficacy. Physical dependence also develops during chronic opioid exposure and, upon discontinuation of opioid intake, presents as a withdrawal syndrome that triggers opioid relapse. We tested the effects of RAP-103 (0.5 mg/kg) on naloxone-precipitated withdrawal signs in MOR-dependent rats. To induce physical dependence we implanted opioid (heroine) containing pellets. On day 7 rats received naloxone (3 mg/kg, SC) with or without added RAP-103 and the withdrawal signs of wet dog shakes and eye blinking were quantified for 60 min. We show that RAP-103 blocks naloxone-precipitated withdrawal signs, and therefore is predicted protect to relapse to drug seeking behaviors,FIG.5.

Effects of R103 on MOR Physical Dependence in Rats.

Rats were surgically prepared to have indwelling catheters prior to self-administration testing. To begin each session, house lights were turned on and two retractable levers extended into the chamber. Rats were trained on a Fixed Ratio schedule of one (FR) schedule to lever press for drug infusions during daily 2-h sessions during which a response on the active lever will result in a 3-s MOR infusion (0.25 mg/kg/inf) and simultaneous light cue+tone stimulus. Responses on the inactive lever will have no consequences but were recorded to monitor for nonspecific motor behaviors. A 20-s timeout period will follow each infusion to limit possibility of overdose. After stable responding under the FR-1 schedule (<10% changes in mean number of reinforcers earned for 3 consecutive days, 75% of total responses corresponding to active lever presses), rats will be switched to a Partial Ratio (Intermittent, PR) schedule for determination of stable break point, a measure of motivation to self-administer a drug. For PR studies, a fixed dose of MOR (0.25 mg/kg/inf) was used because this dose can yield higher break points indicative of high motivation to obtain drug. The dependent variable measured will be the number of infusions earned by the end of the session and defined as the breakpoint. Total cumulative responses made by the rat on the active lever during each session will also be recorded. Once rats reach criteria for stable PR responding, which is defined as 3 consecutive days of responding without upward or downward variance (<20% change from mean number of reinforcers earned for 3 consecutive days), R103 (0.02, 0.1, and 0.5 mg/kg) will be administered 15 min before daily SA sessions to assess effects on breaking point and to obtain dose-effect data for R103. A repeated-measures 2-way ANOVA (R103 treatment, sex) between-subjects design followed by a Bonferroni test was used. We show that progressive-ratio experiments revealed that the motivation to work for reinforcer, that is self-administer an opioid, was significantly reduced in RAP-103 treated rats,FIG.5.

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