4-Phenyl-thiazole-based dual inhibitors of fatty acid amide hydrolase and soluble epoxide hydrolase do not alleviate orofacial inflammatory pain in female rats

Pain arising from trigeminal systems such as headache is common, debilitating, and current treatments (e.g., sumatriptan) are limited. New treatments that target novel mechanisms of action may be required to innovate both short- and long-term pain therapy. Fatty acid amide hydrolase and soluble epoxide hydrolase are two pain-related enzymes that regulate pain and inflammation via independent pathways. We have previously demonstrated that simultaneous inhibition of these enzymes using a novel dual inhibitor alleviates acute inflammatory pain in the hindpaw and does not depress wheel running in rats. Here, we expanded on these findings and performed structure-activity relationships of our lead compound, the 4-phenyl-thiazole-based dual inhibitor SW-17, to generate 18 analogs and tested them for their inhibition at both enzymes. Conversion of the sulfonamide group to a tertiary amine led to a general decrease in the potency for the sEH enzyme, while this change was well-tolerated at the FAAH enzyme yielding several strong inhibitors. Six selected inhibitors were evaluated in mouse and rat sEH inhibition assays and results showed a species difference, i.e. 4-phenyl-thiazole-based analogs are significantly less or not active in mouse sEH compared to human and rat enzymes. The most potent inhibitor, SW-17, was evaluated in a plasma stability assay in human and rat plasma and showed moderate stability. However, SW-17 did not alleviate orofacial inflammatory pain in female rats compared to the traditional anti-migraine agent sumatriptan. Although modification of 4-phenyl-thiazole-based dual inhibitor SW-17 changes potencies at both FAAH and sEH, these approaches may not produce antinociception against trigeminal pain. Key Words: polypharmacology, formalin, inflammation, enzyme inhibition, structure-activity relationship studies

Analysis of the anti-migraine effects of THC using home cage wheel running

Migraine is a common neurological disorder characterized by severe headache and depression of normal activity. Despite its marked prevalence, migraine is poorly treated and no new anti-migraine agents have emerged in over 25 years. Two reasons for this lack of progress are the absence of clinically relevant behavioral outcomes for assessing migraine pain in rodents and a limited understanding of the underlying mechanisms. The purpose of this dissertation was to develop a clinically relevant method to assess migraine pain to evaluate the anti-migraine efficacy of ∆9-tetrahydrocannabinol (THC) in female rats. Activation of primary afferents via dural administration of allyl isothiocyanate (AITC) depressed home cage wheel running for three hours. Administration of the anti-migraine medication sumatriptan reversed AITC-induced depression of home cage wheel running. These observations are consistent with the migraine-induced decreases in activity in humans and subsequent recovery by sumatriptan. Chapter 3 shows that administration of THC also prevented migraine-depressed wheel running. This effect occurred only when rats were treated with THC immediately following AITC administration and not when administered 90 minutes before or after AITC. Administration of cannabinoid type-1 and type-2 receptor antagonists revealed that the anti-migraine effects of THC were mediated by activation of the cannabinoid type-1 receptor. Chapter 4 examined whether THC efficacy is maintained with repeated administration. THC prevented migraine-depressed wheel running in rats whether they were pretreated with THC or vehicle for 2.5 days. In contrast, morphine transiently prevented migraine-depressed wheel running in rats pretreated with saline, but not morphine for 2.5 days. Moreover, migraine-depressed wheel running was exacerbated in rats pretreated with morphine compared to rats pretreated with THC or vehicle. The present studies reveal three findings: 1) Depression of home cage wheel running is an objective and clinically relevant method to assess migraine pain in rats; 2) Administration of THC can prevent migraine-depressed wheel running via CB1 receptor activation; and 3) Repeated THC administration does not exacerbate migraine pain or produce antinociceptive tolerance. These novel findings suggest that THC may be an effective migraine treatment

Biased agonism: the quest for the analgesic holy grail

Abstract. Opioids alleviate pain, but adverse effects severely limit their usefulness. To solve this problem, biased ligands favoring 1 signaling pathway downstream of the μ-opioid receptor over another are being developed. In the target article, the authors synthesize compounds that preferentially activate G-protein or β-arrestin signaling. They find that increased bias towards G-protein signaling produces better antinociception with minimal side effects in mice models. G-protein–biased opioids may provide a safer treatment strategy

Depression of home cage wheel running: a reliable and clinically relevant method to assess migraine pain in rats

The development of new anti-migraine treatments is limited by the difficulty inassessing migraine pain in laboratory animals. Depression of activity is one of the few diagnostic criteria formigraine that can be mimicked in rats. The goal of the present study was to test the hypothesis thatdepression of home cage wheel running is a reliable and clinically relevant method to assess migraine painin rats.Adult female rats were implanted with a cannula to inject allyl isothiocyanate (AITC) onto the dura to induce migraine pain, as has been shown before. Rats recovered from implantation surgery for 8 days in cages containing a running wheel. Home cage wheel running was recorded 23 h a day. AITC and the migraine medication sumatriptan were administered in the hour prior to onset of the dark phase.Administration of AITC caused a concentration-dependent decrease in wheel running that lasted 3 h. The duration and magnitude of AITC-induced depression of wheel running was consistent following three repeated injections spaced 48 h apart. Administration of sumatriptan attenuated AITC-induced depressionof wheel running when a large dose (1 mg/kg) was administered immediately following AITC administration. Wheel running patterns did not change when sumatriptan was given to naïve rats.These data indicate that home cage wheel running is a sensitive, reliable, and clinically relevant method to assess migraine pain in the rat

Analysis of inflammation-induced depression of home cage wheel running in rats reveals the difference between opioid antinociception and restoration of function

•Home cage wheel running can be used to assess pain and opioid analgesia.•Low doses of morphine reversed pain-depressed wheel running only.•High doses of morphine reversed evoked hypersensitivity only.•All buprenorphine doses depressed wheel running in all rats.•Wheel running reveals limitations of opioids to restore normal activity. Opioids are effective at inhibiting responses to noxious stimuli in rodents, but have limited efficacy and many side effects in chronic pain patients. One reason for this disconnect is that nociception is typically assessed using withdrawal from noxious stimuli in animals, whereas chronic pain patients suffer from abnormal pain that disrupts normal activity. We hypothesized that assessment of home cage wheel running in rats would provide a much more clinically relevant method to assess opioid efficacy to restore normal behavior. Intraplantar injection of Complete Freund’s Adjuvant (CFA) into the right hindpaw depressed wheel running and caused mechanical allodynia measured with the von Frey test in both male and female rats. Administration of an ED50 dose of morphine (3.2mg/kg) reversed mechanical allodynia, but did not reverse CFA-induced depression of wheel running. In contrast, administration of a low dose of morphine (1.0mg/kg) restored running for one hour in both sexes, but had no effect on mechanical allodynia. Administration of the atypical opioid buprenorphine had no effect on inflammation-induced depression of wheel running in male or female rats, but attenuated mechanical allodynia in male rats. Administration of buprenorphine and higher doses of morphine depressed wheel running in non-inflamed rats, suggesting that the side effects of opioids interfere with restoration of function. These data indicate that restoration of pain-depressed function requires antinociception in the absence of disruptive side effects. The disruptive side effects of opioids are consistent with the major limitation of opioid use in human pain patients

Depression of home cage wheel running is an objective measure of spontaneous morphine withdrawal in rats with and without persistent pain

Opioid withdrawal in humans is often subtle and almost always spontaneous. In contrast, most preclinical studies precipitate withdrawal by administration of an opioid receptor antagonist such as naloxone. These animal studies rely on measurement of physiological symptoms (e.g., wet dog shakes) in the period immediately following naloxone administration. To more closely model the human condition, we tested the hypothesis that depression of home cage wheel running will provide an objective method to measure the magnitude and duration of spontaneous morphine withdrawal. Rats were allowed access to a running wheel in their home cage for 8days prior to implantation of two 75mg morphine or placebo pellets. The pellets were removed 3 or 5days later to induce spontaneous withdrawal. In normal pain-free rats, removal of the morphine pellets depressed wheel running for 48h compared to rats that had placebo pellets removed. Morphine withdrawal-induced depression of wheel running was greatly enhanced in rats with persistent inflammatory pain induced by injection of Complete Freund's Adjuvant (CFA) into the hindpaw. Removal of the morphine pellets following 3days of treatment depressed wheel running in these rats for over 6days. These data demonstrate that home cage wheel running provides an objective and more clinically relevant method to assess spontaneous morphine withdrawal compared to precipitated withdrawal in laboratory rats. Moreover, the enhanced withdrawal in rats with persistent inflammatory pain suggests that pain patients may be especially susceptible to opioid withdrawal

Home cage wheel running is an objective and clinically relevant method to assess inflammatory pain in male and female rats

•Hindpaw inflammation depressed home cage wheel running in male and female rats.•Wheel running recovered within 12 days in both male and female rats.•Mechanical withdrawal thresholds remained low for all testing days.•This is a sensitive, objective, clinically relevant measure of inflammatory pain. The assessment of nociception in preclinical studies is undergoing a transformation from pain-evoked to pain-depressed tests to more closely mimic the effects of clinical pain. Many inflammatory pain-depressed behaviors (reward seeking, locomotion) have been examined, but these tests are limited because of confounds such as stress and difficulties in quantifying behavior. The present study evaluates home cage wheel running as an objective method to assess the magnitude and duration of inflammatory pain in male and female rats. Injection of Complete Freund's Adjuvant (CFA) into the right hindpaw to induce inflammatory pain almost completely inhibited wheel running for 2 days in male and female rats. Wheel running gradually returned to baseline levels within 12 days despite persistent mechanical hypersensitivity (von Frey test). Continuously monitoring home cage wheel running improves on previous studies examining inflammatory pain-depressed wheel running because it is more sensitive to noxious stimuli, avoids the stress of removing the rat from its cage for testing, and provides a complete analysis of the time course for changes in nociception. The present data indicate that home cage wheel running is a clinically relevant method to assess inflammatory pain in the rat. The decrease in activity caused by inflammatory pain and subsequent gradual recovery mimics the changes in activity caused by pain in humans. The tendency for pain-depressed wheel running to be greater in female than male rats is consistent with the tendency for women to be at greater risk of chronic pain than men

A comparative study on outcome of Type I Tympanoplasty using sliced 1-mm Conchal cartilage and full-thickness Conchal cartilage for grafting

Abstract Background This study aims at comparing the pure tone audiogram and tympanogram results of a measured thickness (1 mm) conchal cartilage graft material (1.0-mm CC) with full-thickness conchal cartilage (FTCC) used for inactive chronic suppurative otitis media (CSOM) mucosal disease with moderate size perforation. Results A total of 40 successful cartilage tympanoplasty procedures were included in the prospective quasi-randomised study. Patients aged between 15 and 55 years with inactive CSOM—moderate size perforation in the tympanic membrane were included. The patients were assessed at the end of 12 weeks with a pure tone audiogram and a tympanogram. The results were compared to know the compliance of the tympanic membrane (TM) between the two groups and the difference in audiometric gain (air-bone gap closure). The air-bone gap gain was better in the 1.0-mm CC group of patients (12) compared to the FTCC group of patients (7). The difference was statistically significant (p = 0.04). The 1.0-mm CC group of patients showed better compliance (0.43) compared to FTCC group of patients (0.73). The air volume and compliance were significantly better in the 1-mm CC group compared to the FTCC group (p = 0.031 and 0.01 respectively). Conclusion The use of sliced cartilage seems to have a better role in reconstructing the TM when compared to full-thickness cartilage as graft material. It is indeed beneficial to have a compliant TM. Though the results cannot be compared with temporalis fascia grafts, the use of 1.0-mm CC has its advantage in producing an audio logically acceptable outcome in tympanoplasty surgery