PTI-609: A Novel Analgesic that Binds Filamin A to Control Opioid ...

PTI-609, Novel Analgesic Recent Patents on CNS Drug Discovery, 2010, Vol. 5, No. 3 217effects could be the result of FLNA binding. The Honglaboratory suggested NADPH oxidase as a target, whichappears to be a critical final common pathway enzyme inproducing both extracellular and intracellular reactiveoxygen species, and subsequently, inflammatory cytokinerelease.The Linda R. Watkins laboratory has also demonstratedthat naloxone and naltrexone block glial activation andresulting proinflammatory cytokine release [41]. The targetsuggested by the Watkins laboratory in these effects is tolllikereceptor 4 (TLR4) of innate immune cells, becausenaloxone and naltrexone, including their isomers that areinactive at opioid receptors, block TLR4 signaling [41, 42].A large body of work, led by Dr. Watkins, has demonstratedthe role of glial activation in the negative effects of opioids,including opioid tolerance, dependence, reward, andrespiratory depression [43], so it is possible that ultra-lowdoseopioid antagonists are alleviating these effects bydiminishing the glial activation of opioids. Although the glialactivation could be a downstream effect of MOR-Gssignaling, the cell line used to demonstrate that (+) and (-)naloxone disrupt TLR4 signaling was devoid of opioidreceptors [42]. Hence, binding to FLNA in glial cells mayslow down vesicular release of cytokines, or even disruptTLR4 signaling. Although FLNA binders such as(+)naloxone have not been reported to have antinociceptiveeffects themselves, they can reduce allodynia in a model ofneuropathic pain [40], and this effect is attributed to theirsuppression of glial activation.We first assessed cytokine release from LPS-stimulatedhuman astrocytes and the potential blockade of this cytokinerelease by PTI-609 and related novel compounds. Weassessed concentrations from 100 fM to 100 nM andcompared these to 10 pM and 1 nM of (+)naloxone. WhereasPTI-609 and two related compounds potently reducedrelease of IL-6, IL-1 and TNF-, (+)naloxone suppressedrelease of these cytokines somewhat less potently at 10 pMand virtually not at all at 1 nM (Wang and Burns, unpublisheddata, Fig. (9)). This loss of efficacy by (+)naloxone at1 nM again demonstrates the crossing of the ultra-low-dosethreshold, likely mediated by binding to the second bindingsite on FLNA, the site it binds with 834 pM affinity. Thelack of dose-response in this initial test of cytokine releasesuggests that naloxone and naltrexone’s picomolar bindingsite is already saturated by these com-pounds at 100 fM.Lower concentrations may show a true dose-response.We next assessed anti-inflammatory activity of PTI-609in the rat collagen-induced arthritis model. Animals weredosed twice daily by oral gavage beginning on the day of thesecond collagen injection. Three different dose groups of thenovel compound were compared to 3mg/kg celecoxib and25mg/kg ibuprofen. PTI-609 significantly reduced footvolume at 56mg/kg and at one time point at 5.6mg/kg, butnot at 32mg/kg. The reduction in foot volume was far lesspotent than that produced by celecoxib, and approximatelyhalf that seen with ibuprofen in this initial proof of conceptstudy (Wang and Burns, unpublished data, Fig. (10).Percent Decrease100806040200100 fM10 pM1 nM100 nMCpd 1 Cpd 2 PTI-609 (+)NaloxoneFig. (9). FLNA-binding compounds block LPS-stimulated cytokinerelease from human astrocytes. Three FLNA-binding compoundsblock TNF- release from primary cultures of human astrocytes by75% or more at concentrations ranging from 100 fM to 100 nM.(+)Naloxone blocked TNF- release by almost 60% at 10 pM, butthis suppression was nearly abolished at 1 nM. Similar results wereobtained with IL-1 and IL-6. Data are means ± SEM. n=4.Addictive PotentialOur data linking the immediate and transient MOR-Gscoupling to CREB activation, as well as the blockade ofconditioned place preference (CPP) by pg/kg doses ofnaltrexone, suggested that our novel compounds would havereduced addictive potential. We previously showed that inbrain slices, acute high dose morphine caused an immediatebut transient Gs coupling by MOR that was associated withactivation of cyclic AMP response element-binding protein(CREB) [18], a transcription factor implicated in addictionand withdrawal effects [44-48]. Blockade of this immediateMOR-Gs coupling by picomolar concentrations of naloxoneor by its pentapeptide binding site on FLNA blocked theassociated activation of CREB. The rewarding effects ofopioid drugs are most pronounced as plasma levels are risingquickly and are less intense or even negative later [49-51],suggesting that this immediate Gs coupling is at leasttemporally associated with the most intense phase of opioidreward. To support the hypothesis that our FLNA-bindingnovel compounds would not have the same rewardingproperties of classical opioids, we examined equi-analgesicdoses of PTI-609 and oxycodone in the CPP paradigm. Oraldoses that produced an 80-90% analgesic effect as well as adose log lower were compared to a vehicle group, usingthree conditioning sessions to the drug-paired compartmentas well as to the vehicle-paired compartment. A significantCPP to both doses of oxycodone was observed, whereas incontrast, there was no change from baseline in time spent inthe putative conditioning side with either dose of PTI-609