Opioid Kappa Receptor Selective Agonist TRK-820 (Nalfurafine Hydrochloride)

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Introduction
TRK-820 (nalfurafine hydrochloride) is a selective opioid  receptor agonist ( Fig. 1) that was launched as an antipruritic for hemodialysis patients in Japan in 2009. In general, clinically used opioids, such as morphine, exhibit potent antinociceptive effects and simultaneous severe adverse effects, including drug dependence, derived from the opioid  receptor. To develop analgesics without drug dependence,  receptor agonists are investigated. However, conventional  agonists, arylacetamide derivatives, showed aversive effects like psychotomimetic effects, and have not yet been used clinically. On the other hand, the novel  agonist TRK-820 has no dependent or aversive properties. TRK-820, which has a structure different from arylacetamides, was first developed as an analgesic for postoperative pain, but the indication was changed to pruritus Nagase & Fujii, 2011). The rational drug design and synthesis of the compound have been reported (Kawai et al., 2008;Nagase et al., 1998;Nagase & Fujii, 2011); therefore, in this chapter, we will focus on its pharmacological properties.

Opioid receptor type selectivity (In vitro)
The binding affinities of TRK-820 were evaluated using various tritiated ligands and opioid receptors derived from various species (Table 1). The  selectivity over the  receptor (K i ratio /) tended to be higher than over the  receptor (K i ratio /). Binding affinities for the L-type Ca 2+ channel and 45 receptors, except the opioid receptors, were examined ). Among the tested receptors, TRK-820 showed the strongest affinity for the muscarine M 1 receptor, but its K i value was 1,700 nmol/L and approximately 7,000 times higher than that of the  receptor. A comparison of the binding properties of TRK-820 and a conventional  agonist, U-69,593, was noteworthy. In a competitive binding  (Endoh et al., 2000). These results suggest that TRK-820 was selective ligand for the  receptor and that its binding property for the  receptor was different from that of the conventional  agonist U-69,593. Many binding assays are carried out using [ 3 H]U-69,593 because it is commercially available. However, the binding property of TRK-820 is difficult to be definitively evaluated because [ 3 H]TRK-820 is not available now.
TRK-820 was selective for the  receptor, but the selectivity over the  receptor was apparently not as high in the binding assays. Contrarily TRK-820 showed more selectivities for the  receptor in functional assays: MVD (mouse vas deference) and GPI (guinea pig ileum) assay (Nagase et al., 1998), cAPM assay Seki et al., 1999), and [ 35 S]GTPS binding assay (Wang, Y et al., 2005) ( Table 2) Nakao et al.) indicated that TRK-820 was a selective and potent full agonist for the  receptor and partial agonist for the  and  receptors. The potency for the  receptor was very low Seki et al., 1999). The [ 35 S]GTPS binding assay provided similar results (EC 50 () = 3.2±1.3 nM, E max () = 54±7%, EC 50 () = 289±60 nM, E max () = 51±6%, EC 50 () = 0.025±0.003 nM, E max () = 93±5%) (Wang, Y et al., 2005). Mizoguchi et al. exhibited partial agonist activity of TRK-820 for the  receptor in both in vitro and in vivo assays (Mizoguchi et al., 2003). TRK-820 concentration-or dose-dependently attenuated [ 35 S]GTPS binding by DAMGO or antinociception induced by intracerebroventricular (i.c.v.) administration of DAMGO. On the other hand, the effects of morphine alone or a mixture with TRK-820 were investigated using a mouse acetic acid-induced writhing test or warm water (50 ºC) tail-withdrawal assay in rhesus monkeys (Ko & Husbands, 2009;Nagase, 2010). Isobologram analysis of the results showed that additive or synergetic effects for TRK-820 in combination with morphine in the antinociceptive effect were observed, indicating that TRK-820 had no  antagonist activity, at least no antagonism against analgesic activity induced by morphine. Why the effects of TRK-820 against DAMGO differed from those against morphine is not clear. www.intechopen.com

Analgesic effects
TRK-820 showed potent analgesic effects in some species (rodents and primates) with various stimuli: chemical, thermal, or mechanical stimuli and inflammatory, diabetic, herpetic, and postherpetic pain models. The antinociceptive effects of TRK-820 are summarized in Tables 3-7. Subcutaneous (s.c.) administration of TRK-820 produced dosedependent and profound antinociceptive effects in the low temperature hot plate, tail flick, tail pressure, and tail pinch tests. However, TRK-820 was not as effective in high temperature hot plate tests (Table 3) (Endoh et al., 1999).
In the formalin test, s.c. TRK-820 given 15 min prior to the formalin injection markedly inhibited the second phase of the nociceptive response induced by formalin in a dosedependent manner. However, the analgesic effect of TRK-820 was low for the first phase of the formalin response. Similarly, a conventional  agonist, 441, also markedly inhibited the second phase. On the other hand, a  agonist, morphine inhibited both phases in a dosedependent manner. The antinociceptive potencies of TRK-820 and ICI-199,441 were almost equivalent (Table 3) (Endoh et al., 2000). A potent and dose-dependent antinociceptive effect of TRK-820 (i.m.) was also observed in cynomolgus monkeys. The analgesic effect of TRK-820 was 295 and 492-fold more potent than that of morphine in the 50 ºC and 55 ºC hot water tests, respectively, and 40 and 1000-fold more potent than that of U-50,488H and pentazocine in the 50 ºC hot water test, respectively (Table 4) (Endoh et al., 2001).
Furthermore, the antinociceptive effects of TRK-820 administered s.c. and perorally (p.o.) were compared. The dose-dependent antinociception of TRK-820 (ED 50 = 0.0033 mg/kg, s.c. and 0.032 mg/kg, p.o.) in the acetic acid-induced writhing test were inhibited by pretreatment with nor-BNI. The antinociceptive effects induced by s.c. or p.o. administration of TRK-820 were 351 and 796-fold more potent than those induced by U-50,488H, respectively, and 175 and 187-fold more potent than those induced by morphine, respectively. Because the ED 50 p.o./s.c. ratio for TRK-820 was the least among the tested compounds, TRK-820 was expected to be the most effective agent when administered p.o. (Table 5) (Endoh et al., 1999). Intravenous administration of TRK-820 was also reported to be effective in the same test (Vanderh et al., 2008). administrations of TRK-820 (0.1-0.8 mg/kg, s.c.), U-50,488H (10-80 mg/kg, s.c.), ICI-199,441 (0.025-0.2 mg/kg, s.c.), or morphine (1.25-10 mg/kg, s.c.) over three days, the development of tolerance to the antinociception induced by each compound at a fixed dose was assessed and tolerance ED 50 was calculated. Comparing the ratio of tolerance ED 50 to acute antinociceptive ED 50 of each compound, TRK-820 was found to develop the least tolerance to antinociception (Table 6) ).
An analgesic effect of TRK-820 (i.m.) was also examined using rats with arthritis induced by adjuvant. TRK-820 dose-dependently produced potent and equivalent antinociceptive activity in both arthritic and normal rats in the paw pressure test. Similar results were obtained when morphine was injected i.m. However, the analgesic effect of a conventional  agonist, 441, in the arthritic rats was less potent than in normal rats (Table 7) (Endoh et al., 2000).

Compound
Tolerance ED  In streptozotocin-induced diabetic mice, the antinociceptive effects induced by several  agonists, including TRK-820, were compared in the tail flick test. Intrathecal (i.t.) and i.c.v. administration of TRK-820 produced dose-dependent antinociceptive effects in both diabetic and non-diabetic mice. However, antinociception induced by TRK-820 administered i.t. or i.c.v. in diabetic mice were less potent than antinociception in non-diabetic mice. However, the antinociceptive effects of CI-977 administered i.t., but not i.c.v., in diabetic mice were less potent than those in non-diabetic mice. On the other hand, the antinociceptive effects of ICI-199,441 and R-84760 injected i.c.v., but not i.t., in diabetic mice were less potent than those in non-diabetic mice. These results indicate that the antinociceptive effects of  agonists in diabetic mice are altered in a region-specific manner in the central nervous system and by chemotypes of  agonists (Ohsawa et al., 2005).
In acute herpetic and postherpetic pain models induced by herpes simplex virus type-1 infection in mice, TRK-820 dose-dependently and remarkably inhibited the allodynia and hyperalgesia stimulated by von Frey filaments (Takasaki et al., 2004(Takasaki et al., , 2006. The effects of TRK-820, but not morphine, were not significantly different between herpetic and postherpetic pain (Takasaki et al., 2006). TRK-820 (0.1 mg/kg, s.c.) almost completely relieved both allodynia and hyperalgesia in herpetic pain, whereas a high dose of morphine (20 mg/kg, s.c.) did not produce complete inhibition. However, TRK-820 (0.01-0.1 mg/kg, s.c.) did not affect the spontaneous locomotor activity of normal mice (Takasaki et al., 2004). Moreover, repeated administration of TRK-820 (0.1 mg/kg, p.o., twice daily) produced constant inhibition of allodynia and hyperalgesia in herpetic pain. The effects of the fourth administration with TRK-820 were not significantly different from those of the first administration. On the other hand, the effects of morphine rapidly decreased after repeated administration (20 mg/kg, p.o., twice daily). The effects of the third and fourth administration of morphine were significantly weaker than those of the first administration. Pre-treatment with morphine (20 mg/kg, p.o., three times) did not affect the antinociceptive effect of TRK-820 (0.1 mg/kg, p.o.), whereas the effect of morphine (20 mg/kg, p.o.) was significantly reduced (Takasaki et al., 2006). These results indicate that TRK-820 is effective on both herpetic and postherpetic pain in mice. In addition, the analgesic dose of TRK-820 did not develop acute tolerance and induced cross-tolerance to morphine in herpetic pain.
TRK-820 reportedly exhibited antipruritic effects on spontaneous scratching behavior in aged MRL/lpr mice (a possible model for pruritus in autoimmune disease) (Umeuchi et al., 2005) or NC/Nag mice maintained in a conventional environment (an animal model for atopic dermatitis) (Nakao et al., 2008), and scratching behavior secondary to cholestasis induced chronic ethynylestradiol injections in rats (Inan & Cowan, 2006b). Interestingly, TRK-820 was effective in scratching behaviors observed in conventional NC/Nag mice, which were considered a model of atopic dermatitis (Fig. 4). Wikström et al. (2005) and Kumagai et al. (2010) reported the results of randomized, doubleblind, placebo-controlled clinical studies in which TRK-820 was administered to patients undergoing hemodialysis intravenously or orally (Fig. 5). In these studies, TRK-820 exhibited significant antipruritic effects without severe adverse drug reactions. These outcomes suggest that TRK-820 can be considered a safe agent.

Clinical studies
TRK-820 is prescribed in Japan as an antipruritic for hemodialysis. Very recently, Kumagai et al. reported that TRK-820 has been prescribed for approximately 18,000 hemodialysis patients and effective in 70 to 80% (Kumagai et al., 2011).

Effects of TRK-820 in the conditioned place preference (CPP) test
The agonists have a rewarding effect, which accounts for the abuse of morphine by humans. In animal models, the rewarding effects of  agonists have been evaluated by the conditioned place preference (CPP) and self-administration paradigms (Di Chiara & North, 1992). In contrast to μ agonists, conventional κ agonists such as U-50,488H and U-69,593 generally lack a rewarding effect (Dykstra et al., 1997). However in the CPP test, animals avoid an environment associated with the administration of the  agonists, indicating that these drugs have aversive effects (Barr et al., 1994;Funada et al., 1993). In contrast to conventional  agonists, such as U-50,488H, TRK-820 (3.0-30 g/kg, s.c.) did not induce significant place aversion in mice at doses producing significant antinociception (Fig. 6) (Nagase, 2010). Notably, TRK-820 exhibited neither preferential nor aversive properties. Recently, the peroral administration of TRK-820 (5.0 g/day) was reported to show no signs of psychological or physical dependence in an open-labeled clinical trial for one year (Nagase & Fujii, 2011). Fig. 6. The effect of TRK-820 in the CPP test. Reprinted with permission from Nagase, 2010.

The effects of TRK-820 on morphine and cocaine-induced rewarding effects
The mechanism of  agonist-induced rewarding effects is outlined below. The activation of the  receptor on -aminobutyric acid-containing interneurons is likely to disinhibit ventrotegmental area dopaminergic neurons, thereby increasing dopamine release in their terminal areas, including the nucleus accumbens (N.Acc). On the other hand, the activation of the receptor decreases dopamine release in the N.Acc (Di Chiara & Imperato, 1988;Spanagel et al., 1992). Therefore,  agonists may be useful for treating morphine dependence. Indeed, the pretreatment with U-50,488H attenuated the morphine-induced place preference in mice (Funada et al., 1993). TRK-820 also significantly suppressed the place preference produced by morphine, and the effect of TRK-820 was antagonized by pre-treatment with nor-BNI (3.0 mg/kg, s.c.) in mice (Tsuji et al., 2001). In addition, TRK-820 was effective in reducing the rewarding effect produced by cocaine. TRK-820 (20 and 40 g/kg, i.p.), at doses producing no aversive or sedative effects, suppressed the rewarding effect of cocaine (4.0 mg/kg, i.p.) in rats (Mori et al., 2002). U-50,488H and U-69,593 exhibited similar effects as TRK-820 (Shippenberg et al., 1996;Suzuki et al., 1992). Drug discrimination procedures provide relevant information about neuropharmacological mechanisms underlying the subjective effects of abused drugs, including cocaine, methamphetamine, and opioids, in animals. Therefore, the procedures are potentially useful for identifying candidate therapeutics for the management of drug abuse (Schuster & Johanson, 1988). Pre-treatment with TRK-820 (10 and 20 g/kg, s.c.) significantly shifted the dose-response curve for cocaine (10 mg/kg, i.p.) to the right without changing the response rate. This attenuating effect of TRK-820 was completely reversed by pre-treatment with nor-BNI (10 mg/kg, s.c.) (Mori et al., 2002).

Effects of TRK-820 on the morphine withdrawal response
In humans, withdrawal from the chronic administration of opioids such as morphine results in characteristic behaviors, including anxiety, nausea, insomnia, hot and cold flashes, muscle aches, perspiration, and diarrhea. Such symptoms would pose clinical problems in patients receiving long-term treatment with opioids for pain relief. Rodents that are physically dependent on morphine elicit characteristic signs (jumping, wet dog shakes, rearing, diarrhea, ptosis, and forepaw tremor) when administrated naloxone. The withdrawal signs precipitated by naloxone are used as an index of the physical dependence on morphine. The effects of  agonists TRK-820 and U-50,488H on the development of physical dependence on morphine were reported. Co-injection of TRK-820 (0.003-0.03 mg/kg, s.c.) during chronic morphine treatment dose-dependently suppressed naloxone-precipitated body weight loss, and the other withdrawal signs in morphine-dependent mice treated with TRK-820 (0.03 mg/kg, s.c.) were significantly fewer than those in untreated mice. In contrast to TRK-820, co-injection of U-50,488H (1.0-10 mg/kg, s.c.) did not inhibit naloxone-precipitated body weight loss and other withdrawal signs (Tsuji et al., 2000).

Comparison of pharmacological properties between TRK-820 and conventional  agonists
We described in the previous sections some pharmacological properties of TRK-820 that are different from conventional  agonists, arylacetamides such as U-50,488H and U-69,593: binding properties (section 2) and exhibition of no preferential and no aversive effect in the CPP paradigm (section 5). As described below, drug discrimination procedures indicate conclusive difference between TRK-820 and arylacetamides.
In this study, cross-substitution between the discriminative effects of U-50,488H and TRK-820 was not observed. The  agonists tested in this study, except E-2078, tended to substitute for the discriminative stimulus effects of U-50,488H rather than those of TRK-820. These results suggest that U-50,488H and TRK-820 have differential properties. Furthermore, noncompetitive NMDA antagonists phencyclidine (PCP, 0.5-2.0 mg/kg) and MK-801 (10-80 g/kg) dose-dependently generalized to the discriminative stimulus effects of U-50,488H (3.0 mg/kg, i.p.) in the cross-substitution tests. On the other hand, PCP and MK-801 at doses that generalized to the discriminative stimulus effects of U-50,488H did not generalize to those of TRK-820 (40 g/kg, i.p.) (Mori et al., 2006). The outcomes clearly indicate different properties between TRK-820 and U-50,488H.

The effect of TRK-820 on a rat model of schizophrenia
The effects of TRK-820 on hyperlocomotion and stereotyped behaviors (head-weaving, sniffing, and turning) induced by PCP were evaluated. These behaviors are thought to resemble the schizophrenia-like effects in humans. TRK-820 (10-100 g/kg, s.c.) dosedependently inhibited PCP (10 mg/kg, i.p.)-induced hyperlocomotion, and this effect was antagonized with nor-BNI (20 mg/kg, s.c.). PCP-induced stereotyped behaviors were also inhibited by treatment with TRK-820 in a dose-dependent manner. These findings that TRK-820 potentially ameliorates abnormal behaviors induced by PCP suggest its therapeutic potential against the symptoms of schizophrenia .

The effect of TRK-820 on dyskinesia symptoms in a parkinsonian rat model
The effects of TRK-820 on rotational behavior were investigated in unilateral 6hydroxydopamine (6-OHDA)-treated rats (hemi-parkinsonian rats), and on dyskinesia produced by administering L-DOPA to hemi-parkinsonian rats for 3 weeks (dyskinesia rats). TRK-820 significantly ameliorated abnormal behavior in hemi-parkinsonian rats at 30 g/kg (s.c.), and L-DOPA induced dyskinesia at 10 and 30 g/kg (s.c.). This effect was antagonized by pretreatment with nor-BNI (20 mg/kg, s.c.). Additionally, co-administration of TRK-820 (3 and 10 μg/kg, s.c.) with L-DOPA for 3 weeks suppressed the development of L-DOPA-induced dyskinesia. TRK-820 may be a suitable drug for the treatment of parkinsonian patients with dyskinesia symptoms .

The diuretic effect of TRK-820 in rats
Diuresis is a well-recognized effect of conventional  agonists in animals and humans. A diuretic effect of TRK-820 in rats has also been reported. TRK-820 (0.005-0.02 mg/kg, s.c.) dose-dependently induced a diuretic effect without developing tolerance, and this effect was inhibited by selective  antagonist 5'-GNTI (Inan et al., 2009b).

The effects of TRK-820 on endothelial cell differentiation and development of vasculature
The roles of the opioid  system in vascular development were investigated (Yamamizu et al., 2011). U-50,488H and TRK-820 significantly inhibited endothelial cell differentiation and vascular formation through the inhibition of cAMP/PKA signaling.

Conclusion
TRK-820 was a selective  agonist. However, its pharmacological properties were different from those of conventional arylacetamide  agonists, including U-50,488H. A noteworthy feature of TRK-820 was that it showed no preferential or aversive properties, whereas U-50,488H produced aversion. This disparity of properties between TRK-820 and arylacetamide  agonists was reported to stem from the difference in  receptor subtypes each compound interacted with: arylacetamide  agonists would interact with  1 receptor subtype, whereas TRK-820 may interact with another  receptor subtype (perhaps  3 ) (Endoh et al., 1999;2001;Tsuji et al., 2000a;2000b). Although opioid receptors have been classified historically into three types (, , and  types) and further divided into several subtypes from the pharmacological viewpoint (Dhawan et al., 1996), only the three major types have been cloned (Satoh & Minami, 1995). Much evidence has been compiled indicating that various receptors, including opioid receptors, exist as homo-or hetero dimers of the receptors (George et al., 2000Gomes et al., 2000Gomes et al., , 2004Devi, 2001;Levac et al., 2002;, and receptor dimerization has been invoked to explain the discrepancy between widely varied pharmacologies and the identification of only three opioid receptor types. Therefore, the disparity of properties between TRK-820 and arylacetamide  agonists may stem from the difference in receptor dimers each compound interacts with. Both TRK-820 and arylacetamide  agonists are expected to be useful tools for the investigation of receptor dimerization and/or  receptor subtype. As mentioned in section 2, a binding assay using [ 3 H]TRK-820 and [ 3 H]U-69,593 is thought to be a facile and useful method for achieving that purpose. However, [ 3 H]TRK-820 is not currently available.
In addition to antipruritic and antinociceptive effects, TRK-820 exhibited various pharmacological effects, such as the treatment of the symptoms of schizophrenia or dyskinesia symptoms of parkisonian patients, or remedy for drug addiction. Moreover, TRK-820 has been already launched in Japan. TRK-820 is expected not only to be developed with the other indication, such as symptoms of schizophrenia or parkinson's disease, but also to be utilized to investigate pharmacology via the  receptor.