Evaluation of KP-1199: a novel acetaminophen analog for hemostatic function and antinociceptive effects.

BACKGROUND: Acetaminophen (APAP) is a widely self-prescribed analgesic for mild to moderate pain, but overdose or repeat doses can lead to liver injury and death. Kalyra Pharmaceuticals has developed a novel APAP analog, KP-1199, currently in Phase 1 clinical studies, which lacks hepatotoxicity. In this study, the authors evaluated the antinociceptive effect of KP-1199 on thermal injury-induced nociceptive behaviors as well as hemostatic parameters using human blood samples. METHODS: Full-thickness thermal injury was induced in anesthetized adult male Sprague-Dawley rats. On day 7 post-injury, KP-1199 (30 and 60 mg/kg) or APAP (60 mg/kg) was administered orally. Antinociception of KP-1199 and APAP were assessed at multiple time points using Hargreaves' test. In separate experiments, human whole blood was collected and treated with either KP-1199, APAP, or Vehicle (citrate buffer) at 1× (214 µg/ml) and 10× (2140 µg/ml) concentrations. The treated blood samples were assessed for: clotting function, thrombin generation, and platelet activation. RESULTS: APAP did not produce antinociceptive activity. KP-1199 treatment significantly increased the nociceptive threshold, and the antinociceptive activity persisted up to 3 h post-treatment. In human samples, 10× APAP caused significantly prolonged clotting times and increased platelet activation, whereas KP-1199 had caused no negative effects on either parameter tested. CONCLUSION: These results suggest that KP-1199 possesses antinociceptive activity in a rat model of thermal injury. Since KP-1199 does not induce platelet activation or inhibit coagulation, it presents an attractive alternative to APAP for analgesia, especially for battlefield or surgical scenarios where blood loss and blood clotting are of concern.

Identifying Plasma Derived Extracellular Vesicle (EV) Contained Biomarkers in the Development of Chronic Neuropathic Pain.

Research into potentially novel biomarkers for chronic pain development is lacking. microRNAs (miRNAs) are attractive candidates as biomarkers due to their conservation across species, stability in liquid biopsies, and variation that corresponds to a pathologic state. miRNAs can be sorted into extracellular vesicles (EVs) within the cell and released from the site of injury. EVs transfer cargo molecules between cells thus affecting key intercellular signaling pathways. The focus of this study was to determine the plasma derived EV miRNA content in a chronic neuropathic pain rat model. This was accomplished by performing either spinal nerve ligation (SNL; n = 6) or sham (n = 6) surgery on anesthetized male Sprague-Dawley rats. Mechanosensitivity was assessed and plasma derived EV RNA was isolated at baseline (BL), day 3, and 15 postnerve injury. EV extracted small RNA was sequenced followed by differentially expressed (DE) miRNAs and gene target enrichment/signaling pathway analysis performed using R packages and TargetScan/Ingenuity pathway analysis (IPA), respectively. Seven of the DE miRNAs were validated by Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR). The data indicated that SNL rats displayed a time-dependent threshold reduction in response to evoked stimuli from day 3 to day 15 postnerve injury. The data also revealed that 22 and 74 miRNAs at day 3 and 15, respectively, and 33 miRNAs at both day 3 and 15 were uniquely DE between the SNL and sham groups. The key findings from this proposal include (1) the majority of the DE EV miRNAs, which normally function to suppress inflammation, were downregulated, and (2) several of the plasma derived DE EV miRNAs reflect previously observed changes in the injured L5 nerve. The plasma derived DE EV miRNAs regulate processes important in the development and maintenance of neuropathic pain states and potentially serve as key regulators, biomarkers, and targets in the progression and treatment of chronic neuropathic pain. PERSPECTIVE: This article describes the DE miRNA content of plasma derived EVs, comparing neuropathic pain to normal conditions. This data indicates that EV miRNAs may be important in nociception and may also serve as biomarkers for chronic pain. These results encourage further research on EV miRNAs in chronic neuropathic pain sufferers.

Involvement of brain-derived neurotrophic factor (BDNF) in chronic intermittent stress-induced enhanced mechanical allodynia in a rat model of burn pain.

BACKGROUND: Reports show that stressful events before injury exacerbates post-injury pain. The mechanism underlying stress-induced heightened thermal pain is unclear. Here, we examined the effects of chronic intermittent stress (CIS) on nociceptive behaviors and brain-derived nerve growth factor (BDNF) system in the prefrontal cortex (PFC) and hypothalamus of rats with and without thermal injury. RESULTS: Unstressed rats showed transient mechanical allodynia during stress exposure. Stressed rats with thermal injury displayed persistent exacerbated mechanical allodynia (P < 0.001). Increased expression of BDNF mRNA in the PFC (P < 0.05), and elevated TrkB and p-TrkB (P < 0.05) protein levels in the hypothalamus were observed in stressed rats with thermal injury but not in stressed or thermally injured rats alone. Furthermore, administration of CTX-B significantly reduced stress-induced exacerbated mechanical allodynia in thermally injured rats (P < 0.001). CONCLUSION: These results indicate that BDNF-TrkB signaling in PFC and hypothalamus contributes to CIS-induced exacerbated mechanical allodynia in thermal injury state.

Sound-stress-induced altered nociceptive behaviors are associated with increased spinal CRFR2 gene expression in a rat model of burn injury.

Sound stress (SS) elicits behavioral changes, including pain behaviors. However, the neuronal mechanisms underlying SS-induced pain behaviors remain to be explored. The current study examined the effects of SS on nociceptive behaviors and changes in expression of the spinal corticotropin-releasing factor (CRF) system in male Sprague Dawley rats with and without thermal pain. We also studied the effects of SS on plasma corticosterone and fecal output. Rats were exposed to 3 days of SS protocol (n = 12/group). Changes in nociceptive behaviors were assessed using thermal and mechanical pain tests. Following the induction of SS, a subgroup of rats (n = 6/group) was inflicted with thermal injury and on day 14 postburn nociceptive behaviors were reassessed. Spinal CRF receptor mRNA expression was analyzed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). In addition, plasma corticosterone and spinal CRF concentrations were quantified using enzyme-linked immunosorbent assay (ELISA). Increased defecation was observed in SS rats. SS produced transient mechanical allodynia in naive rats, whereas it exacerbated thermal pain in thermally injured rats. Spinal CRFR2 mRNA expression was unaffected by stress or thermal injury alone, but their combined effect significantly increased its expression. SS had no effect on plasma corticosterone and spinal CRF protein in postburn rats. To conclude, SS is capable of exacerbating postburn thermal pain, which is linked to increased CRFR2 gene expression in the spinal cord. Future studies have to delineate whether attenuation of CRFR2 signaling at the spinal level prevents stress-induced exacerbation of burn pain.

Antinociceptive effects of pluronic lecithin organo (PLO)-opioid gels in rats with thermal injury.

Opioids are extensively used as analgesics to control burn pain. However, systemic administration of opioids induces multiple adverse effects that are primarily CNS mediated. Alternately, topical application of low dose of opioids directly at the site of injury could attenuate pain while avoiding CNS-mediated side effects. Pluronic lecithin organogels (PLO) have been extensively used as vehicles to deliver topical drugs. In this study, we for the first time assessed the analgesic efficacy of three opioid-PLO formulations (fentanyl, methadone & morphine) in a rat full-thickness thermal injury (FTTI) pain model. Experiments were performed using 44 adult male Sprague-Dawley rats. A single 0.1mL topical application of either morphine (5mg/mL, n=6), fentanyl (10µg/mL, n=8), methadone gel (5mg/mL, n=8), ketamine (50mg/mL, n=6), saline (0.1mL, n=8) or PLO gel alone (0.1mL, n=8) was administered to the plantar surface of the injured hindpaw on days 4 and 7 following thermal injury. The anti-hyperalgesic effects were then measured (5, 15, 30, 60 and 120min post-drug application) using the Hargreaves' thermal test. All three opioids produced statistically significant increases in paw withdrawal latency (PWL), taken as a measure of anti-hyperalgesia, in comparison to saline-treated group (P<0.05), at both 4 and 7days post injury, with fentanyl showing greatest efficacy. Taken together, a low dose of topical application of opioids can reduce thermal hyperalgesia in a rat hindpaw FTTI model, supporting the development of topical formulations of these drugs for burn pain treatment in the clinic.

Anti-nerve growth factor antibody attenuates chronic morphine treatment-induced tolerance in the rat.

BACKGROUND: Nerve growth factor (NGF) is known to induce inflammation and pain; however its role in opioid-induced tolerance has not been studied. This study investigated the effects of an anti-NGF neutralizing antibody on the development of tolerance following chronic morphine treatment in naïve rats. METHODS: Four groups of rats were used in this study; one treated with saline alone, one with 10 mg/kg of morphine, one with 10 µg of anti-NGF and the other with 10 mg/kg of morphine + 10 µg of anti-NGF, twice per day for 5 days. The route of treatment was subcutaneous (S.C.) for morphine and saline, and intraperitoneal (i.p.) for anti-NGF. Response to a noxious thermal stimulus during the course of drug treatment was assessed (Hargreaves' test). Further, the change in the NGF levels in the lumbar spinal cord was measured by ELISA. RESULTS: Our results showed that repeated administration of morphine produced an apparent tolerance which was significantly attenuated by co-administration of anti-NGF (P < 0.001). Additionally, the area under the curve (AUC) of the analgesic effect produced by the combination of morphine and anti-NGF was significantly (P < 0.001) greater than for saline controls and chronic morphine treated rats. Moreover, the level of NGF in the spinal cord of chronic morphine treated rats was significantly higher (P < 0.05) than in both the saline control group and the group receiving simultaneous administration of anti-NGF with morphine. These results indicate that anti-NGF has the potential to attenuate morphine-induced tolerance behavior by attenuating the effects of NGF at the spinal level. CONCLUSION: Taken together, our study strongly suggests that the NGF signaling system is a potential novel target for treating opioid-induced tolerance.

Anti-hyperalgesic effects of AG490, a Janus kinase inhibitor, in a rat model of inflammatory pain.

Interleukin 6 (IL-6) has a critical role in pain mechanisms. IL-6 signals through the Janus-activated kinases 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) pathway. The contribution of JAK2 signaling in inflammation-induced hyperalgesia has not been addressed previously. The role of this pathway was investigated using the JAK2 inhibitor, AG490, in a rat model of inflammatory pain. Unilateral hind paw inflammatory pain was induced in male Sprague-Dawley rats by intraplantar (i.pl.) injection of 3.5% ʎ-carrageenan. Inflamed rats received an i.pl. injection of either 3.5% of dimethylsulfoxide or AG490 (1-10 µg). The antinociceptive effects of AG490 were assessed by 2 pain behavioral assays 4 h later: The thermal and mechanical hyperalgesia tests. AG490 (1-10 µg) significantly attenuated ʎ-carrageenan-induced thermal hyperalgesia in a dose-dependent manner. AG490 also reduced mechanical hyperalgesia. Co-administration of opioid receptor antagonist naloxone (10 µg) and AG490 (10 µg) did not reverse AG490-produced antinociceptive activity, suggesting that the µ-opioid receptor is not responsible for the anti-hyperalgesic effects of AG490. Therefore, we suggest that AG490 produces these effects by blocking JAK2 signaling. In conclusion, JAK2 inhibitors may represent a novel class of non-narcotic drugs to treat inflammatory pain.

The transcription factor DREAM represses the deubiquitinase A20 and mediates inflammation.

Here we found that the transcription repressor DREAM bound to the promoter of the gene encoding A20 to repress expression of this deubiquitinase that suppresses inflammatory NF-κB signaling. DREAM-deficient mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, binding of the transcription factor USF1 to the DRE-associated E-box domain in the gene encoding A20 activated its expression in response to inflammatory stimuli. Our studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy for the treatment of diseases associated with unconstrained NF-κB activity, such as acute lung injury.

Increased expression of interleukin-6 family members and receptors in urinary bladder with cyclophosphamide-induced bladder inflammation in female rats.

Recent studies suggest that janus-activated kinases-signal transducer and activator of transcription signaling pathways contribute to increased voiding frequency and referred pain of cyclophosphamide (CYP)-induced cystitis in rats. Potential upstream chemical mediator(s) that may be activated by CYP-induced cystitis to stimulate JAK/STAT signaling are not known in detail. In these studies, members of the interleukin (IL)-6 family of cytokines including, leukemia inhibitory factor (LIF), IL-6, and ciliary neurotrophic factor (CNTF) and associated receptors, IL-6 receptor (R) α, LIFR, and gp130 were examined in the urinary bladder in control and CYP-treated rats. Cytokine and receptor transcript and protein expression and distribution were determined in urinary bladder after CYP-induced cystitis using quantitative, real-time polymerase chain reaction (Q-PCR), western blotting, and immunohistochemistry. Acute (4 h; 150 mg/kg; i.p.), intermediate (48 h; 150 mg/kg; i.p.), or chronic (75 mg/kg; i.p., once every 3 days for 10 days) cystitis was induced in adult, female Wistar rats with CYP treatment. Q-PCR analyses revealed significant (p ≤ 0.01) CYP duration- and tissue- (e.g., urothelium, detrusor) dependent increases in LIF, IL-6, IL-6Rα, LIFR, and gp130 mRNA expression. Western blotting demonstrated significant (p ≤ 0.01) increases in IL-6, LIF, and gp130 protein expression in whole urinary bladder with CYP treatment. CYP-induced cystitis significantly (p ≤ 0.01) increased LIF-immunoreactivity (IR) in urothelium, detrusor, and suburothelial plexus whereas increased gp130-IR was only observed in urothelium and detrusor. These studies suggest that IL-6 and LIF may be potential upstream chemical mediators that activate JAK/STAT signaling in urinary bladder pathways.

Involvement of JAK-STAT signaling/function after cyclophosphamide-induced bladder inflammation in female rats.

Cytokines are upregulated in a variety of inflammatory conditions and cytokine/receptor interactions can activate JAK-STAT signaling. Previous studies demonstrated upregulation of numerous cytokines in the urinary bladder following cyclophosphamide (CYP)-induced cystitis. The role of JAK-STAT signaling in urinary bladder inflammation and referred somatic sensitivity has not been addressed. The contribution of JAK-STAT signaling pathways in CYP-induced bladder hyperreflexia and referred somatic hypersensitivity was determined in CYP-treated rats using a JAK2 inhibitor, AG490. Acute (4 h; 150 mg/kg ip), intermediate (48 h; 150 mg/kg ip), or chronic (75 mg/kg ip, once every 3 days for 10 days) cystitis was induced in adult, female Wistar rats with CYP treatment. Phosphorylation status of STAT-3 was increased in urinary bladder after CYP-induced cystitis (4 h, 48 h, chronic). Blockade of JAK2 with AG490 (5-15 mg/kg ip or intravesical) significantly (P < or = 0.05) reduced bladder hyperreflexia and hind paw sensitivity in CYP-treated rats. These studies demonstrate a potential role for JAK-STAT signaling pathways in bladder hyperreflexia and referred pain induced by CYP-induced bladder inflammation.

Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis.

Regulation of the VEGF-VEGF receptor system was examined in the urinary bladder after acute (2-48 h) and chronic (10 days) cyclophosphamide (CYP)-induced cystitis. ELISAs demonstrated significant (P < or = 0.01) upregulation of VEGF in whole urinary bladder with acute and chronic CYP-induced cystitis; however, the magnitude of increase was greater after acute (2-4 h) cystitis. Immunohistochemistry for VEGF immunoreactivity revealed a significant (P < or = 0.05) increase in VEGF immunoreactivity in the urothelium, suburothelial vasculature, and detrusor smooth muscle with acute (4 and 48 h) CYP treatment. RT-PCR identified the isoform VEGF-164, the VEGF receptor VEGFR-2, and the VEGF co-receptors neuropilin (Npn)-1 and Npn-2 in the urinary bladder. Quantitative PCR demonstrated upregulation of VEGF-164 transcript with acute and chronic CYP-induced cystitis, but VEGFR-2, Npn-1, and Npn-2 transcripts were upregulated (P < or = 0.01) in whole bladder only with chronic CYP-induced cystitis. Additional studies demonstrated regulation of VEGF transcript expression in the urinary bladder by nerve growth factor (NGF) in a novel line of NGF-overexpressing mice. These studies demonstrated that urinary bladder inflammation and NGF regulate the VEGF-VEGF receptor system in the urinary bladder. Functional role(s) for the VEGF-VEGF receptor system in urinary bladder inflammation remain to be determined.

Urinary bladder function and somatic sensitivity in vasoactive intestinal polypeptide (VIP)-/- mice.

Vasoactive intestinal polypeptide (VIP) is an immunomodulatory neuropeptide widely distributed in neural pathways that regulate micturition. VIP is also an endogenous anti-inflammatory agent that has been suggested for the development of therapies for inflammatory disorders. In the present study, we examined urinary bladder function and hindpaw and pelvic sensitivity in VIP(-/-) and littermate wildtype (WT) controls. We demonstrated increased bladder mass and fewer but larger urine spots on filter paper in VIP(-/-) mice. Using cystometry in conscious, unrestrained mice, VIP(-/-) mice exhibited increased void volumes and shorter intercontraction intervals with continuous intravesical infusion of saline. No differences in transepithelial resistance or water permeability were demonstrated between VIP(-/-) and WT mice; however, an increase in urea permeability was demonstrated in VIP(-/-) mice. With the induction of bladder inflammation by acute administration of cyclophosphamide, an exaggerated or prolonged bladder hyperreflexia and hindpaw and pelvic sensitivity were demonstrated in VIP(-/-) mice. The changes in bladder hyperreflexia and somatic sensitivity in VIP(-/-) mice may reflect increased expression of neurotrophins and/or proinflammatory cytokines in the urinary bladder. Thus, these changes may further regulate the neural control of micturition.

Nerve growth factor governs the enhanced ability of opioids to suppress inflammatory pain.

Nerve growth factor (NGF) regulates sensory neuron phenotype by elevated expression of ion channels and receptors contributing to pain. Peripheral opioid antinociception is dependent on sensory neuron mu opioid receptor (MOR) expression, coupling and efficacy. This study investigates the role of NGF in the upregulation of the number and efficacy of sensory MORs rendering sites of painful inflammation more susceptible to opioids. We identified co-localization of MOR with calcitonin gene-related peptides (CGRP) and with the NGF receptors tyrosine receptor kinase (TrkA) and p75(NTR) within rat dorsal root ganglia (DRG). We showed that unilateral hind paw inflammation induced with Freund's complete adjuvant (FCA) or intraplantar (i.pl.) NGF increased NGF's retrograde transport and MOR expression in TrkA positive DRG which was prevented by the disruption of this NGF transport. MOR upregulation in DRG was followed by enhanced axonal MOR transport towards peripheral nerve terminals and subsequent increase of MOR-ir nerve fibres within skin. Furthermore, peripheral antinociception elicited by i.pl. fentanyl was naloxone reversible and potentiated exclusively in inflamed and NGF-treated paws. Both FCA- and NGF-induced effects occurring through DRG to peripheral nerve fibres and the potentiation of antinociception were abrogated by NGF neutralization. Therefore, our results suggest that NGF not only contributes to inflammatory pain but also governs the upregulation in the number and efficacy of sensory neuron MOR, resulting in enhanced opioid susceptibility towards better pain control. This suggests the potential to overcome the unresponsiveness to opioids of certain neuropathic pain states.