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

Hideaki Fujii, Shigeto Hirayama and Hiroshi Nagase *School of Pharmacy, Kitasato University Japan* 

#### **1. Introduction**

80 Pharmacology

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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 (Nakao & Mochizuki, 2009; 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.

Fig. 1. Structure of nalfurafine hydrochloride (TRK-820)

#### **2. 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*<sup>i</sup> ratio /) tended to be higher than over the receptor (*K*i ratio /). Binding affinities for the L-type Ca2+ channel and 45 receptors, except the opioid receptors, were examined (Nakao & Mochizuki, 2009). Among the tested receptors, TRK-820 showed the strongest affinity for the muscarine M1 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

Opioid Kappa Receptor Selective Agonist TRK-820 (Nalfurafine Hydrochloride) 83

assays are carried out using [3H]U-69,593 because it is commercially available. However, the binding property of TRK-820 is difficult to be definitively evaluated because [3H]TRK-820 is

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 (Nakao & Mochizuki, 2009; Seki et al., 1999), and [35S]GTPS binding assay (Wang, Y et al., 2005) (Table 2). The results of the cAMP assay (IC50 () = 8.3±1.4 nM, Imax () = 69±3%, IC50 () > 1,000 nM, Imax () not determined, IC50 () = 0.15±0.07 nM, Imax () = 81±3% by Seki et al.; IC50 () = 1.66±0.09 nM, Imax () = 53.2±1.3%, IC50 () = 21.3±1.0 nM, Imax () = 77.9±1.6%, IC50 () = 0.00816±0.00138 nM, Imax () = 91.3±0.5% by 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 (Nakao & Mochizuki, 2009; Seki et al., 1999). The [35S]GTPS binding assay provided similar results (EC50 () = 3.2±1.3 nM, Emax () = 54±7%, EC50 () = 289±60 nM, Emax () = 51±6%, EC50 () = 0.025±0.003 nM, Emax () = 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 [35S]GTPS 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

Fig. 2. Isobologram for the mixture of TRK-820- and morphine-induced antinociception in the mouse acetic acid-induced writhing test. Reprinted with permission from Nagase, 2010.

not available now.

differed from those against morphine is not clear.


Table 1. Binding affinities (*K*i values) and selectivities (*K*i ratios) of TRK-820 for the opioid receptors. Seki et al. used [3H]bremazocine and the recombinant rat opioid receptors. Wang, Y et al. used [3H]diprenorphine and recombinant rat , recombinant mouse, and recombinant human receptors. Vanderah et al. used [3H]DAMDO, [3H]*p*Cl-DPDPE, and [3H]U-69,593 for the recombinant human , , and receptors, respectively. Nakao et al. used [3H]diprenorphine and the recombinant human receptors. Nagase et al. used [3H]DAMDO, [3H]NTI, and [3H]U-69,593 for the μ, δ, and κ receptors, respectively. Guinea pig forebrain or guinea pig cerebellum was used to assay the and receptor or receptor, respectively.


Table 2. Selectivities of TRK-820 in various functional assays. Selectivity in MVD and GPI assays was obtained by *K*e ratios. The selectivity for the receptor over the receptor in the MVD assay was not calculated (NC) due to a lack of agonist activity for the receptor. The selectivity for the receptor over the receptor in the GPI assay was not obtained because GPI preparation contained only the and receptors. Seki et al. and Nakao et al. used the recombinant rat and human receptors in their assays, respectively. In the [35S]GTPS binding assay, recombinant rat , recombinant mouse, or recombinant human receptors were used.

assay using [3H]TRK-820, TRK-820 completely replaced [3H]TRK-820 binding, whereas U-69,593 did not replace it completely, with roughly 20% of [3H]TRK-820 binding remaining. Moreover, Scatchard analysis of [3H]TRK-820 and [3H]U-69,593 binding using guinea pig cerebellum showed that TRK-820 had stronger binding affinity than U-69,593 (*K*d values: 0.46±0.03 nM for [3H]TRK-820, 1.17±0.14 nM for [3H]U-69,593) and that the *B*max value for [3H]TRK-820 (284±43.3 fmol/mg protein) was significantly higher than the value for [3H]U-69,593 (83.7±7.86 fmol/mg protein). Even in the presence of agonist DAMDO (100 nM) and agonist DPDPE (200 nM), the *K*d and *B*max values for [3H]TRK-820 did not change (*K*d = 0.51±0.03 nM, *B*max = 265±27.2 fmol/mg protein) (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

53 1200 3.5 15 343 Seki et al., 1999 5.2 161 0.075 69 2147 Wang, Y et al., 2005 0.71 49.9 0.36 2.0 139 Vanderah et al., 2008 2.21 484 0.244 9.1 1984 Nakao & Mochizuki, 2009 0.582 96.5 0.225 2.6 429 Nagase et al., 2010 Table 1. Binding affinities (*K*i values) and selectivities (*K*i ratios) of TRK-820 for the opioid receptors. Seki et al. used [3H]bremazocine and the recombinant rat opioid receptors. Wang, Y et al. used [3H]diprenorphine and recombinant rat , recombinant mouse, and recombinant human receptors. Vanderah et al. used [3H]DAMDO, [3H]*p*Cl-DPDPE, and [3H]U-69,593 for

[3H]diprenorphine and the recombinant human receptors. Nagase et al. used [3H]DAMDO, [3H]NTI, and [3H]U-69,593 for the μ, δ, and κ receptors, respectively. Guinea pig forebrain or guinea pig cerebellum was used to assay the and receptor or receptor, respectively.

cAMP (Sato et al.) 55 >6,667 Seki et al., 1999 cAMP (Nakao et al.) 203 2,610 Nakao & Mochizuki, 2009 [35S]GTPS 128 11,560 Wang, Y et al., 2005

Table 2. Selectivities of TRK-820 in various functional assays. Selectivity in MVD and GPI assays was obtained by *K*e ratios. The selectivity for the receptor over the receptor in the MVD assay was not calculated (NC) due to a lack of agonist activity for the receptor. The selectivity for the receptor over the receptor in the GPI assay was not obtained because GPI

preparation contained only the and receptors. Seki et al. and Nakao et al. used the recombinant rat and human receptors in their assays, respectively. In the [35S]GTPS binding assay, recombinant rat , recombinant mouse, or recombinant human receptors were used.

assay using [3H]TRK-820, TRK-820 completely replaced [3H]TRK-820 binding, whereas U-69,593 did not replace it completely, with roughly 20% of [3H]TRK-820 binding remaining. Moreover, Scatchard analysis of [3H]TRK-820 and [3H]U-69,593 binding using guinea pig cerebellum showed that TRK-820 had stronger binding affinity than U-69,593 (*K*d values: 0.46±0.03 nM for [3H]TRK-820, 1.17±0.14 nM for [3H]U-69,593) and that the *B*max value for [3H]TRK-820 (284±43.3 fmol/mg protein) was significantly higher than the value for [3H]U-69,593 (83.7±7.86 fmol/mg protein). Even in the presence of agonist DAMDO (100 nM) and agonist DPDPE (200 nM), the *K*d and *B*max values for [3H]TRK-820 did not change (*K*d = 0.51±0.03 nM, *B*max = 265±27.2 fmol/mg protein) (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

MVD 980 NC Kawai et al., 2008 GPI 78.6 – Kawai et al., 2008

Selectivity References

References

*K*<sup>i</sup> (nM) *K*i ratio

the recombinant human , , and receptors, respectively. Nakao et al. used

Assay

assays are carried out using [3H]U-69,593 because it is commercially available. However, the binding property of TRK-820 is difficult to be definitively evaluated because [3H]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 (Nakao & Mochizuki, 2009; Seki et al., 1999), and [35S]GTPS binding assay (Wang, Y et al., 2005) (Table 2). The results of the cAMP assay (IC50 () = 8.3±1.4 nM, Imax () = 69±3%, IC50 () > 1,000 nM, Imax () not determined, IC50 () = 0.15±0.07 nM, Imax () = 81±3% by Seki et al.; IC50 () = 1.66±0.09 nM, Imax () = 53.2±1.3%, IC50 () = 21.3±1.0 nM, Imax () = 77.9±1.6%, IC50 () = 0.00816±0.00138 nM, Imax () = 91.3±0.5% by 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 (Nakao & Mochizuki, 2009; Seki et al., 1999). The [35S]GTPS binding assay provided similar results (EC50 () = 3.2±1.3 nM, Emax () = 54±7%, EC50 () = 289±60 nM, Emax () = 51±6%, EC50 () = 0.025±0.003 nM, Emax () = 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 [35S]GTPS 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.

Fig. 2. Isobologram for the mixture of TRK-820- and morphine-induced antinociception in the mouse acetic acid-induced writhing test. Reprinted with permission from Nagase, 2010.

Opioid Kappa Receptor Selective Agonist TRK-820 (Nalfurafine Hydrochloride) 85

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, ICI-199,441, 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

Furthermore, the antinociceptive effects of TRK-820 administered s.c. and perorally (p.o.) were compared. The dose-dependent antinociception of TRK-820 (ED50 = 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 ED50 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

The effect of repeated administration of some agonists and morphine on antinociceptive

Compound 50 ºC hot water 55 ºC hot water TRK-820 0.0078 0.012 Morphine 2.3 5.9 U-50,488H 0.31 n.t. Pentazocine > 10 n.t.

tolerance was examined by the acetic acid-induced writhing test in mice. After five

Table 4. ED50 values (mg/kg, i.m.) of antinociceptive effects induced by some opioid agonists in the hot water tail withdrawal test in cynomolgus monkeys. n.t. : not tested.

Compound s.c. p.o. ED50 p.o./s.c. ratio

Table 5. ED50 values (mg/kg, s.c. or p.o.) for antinociceptive effects induced by some opioid

agonists in the acetic acid-induced writhing test in mice.

TRK-820 0.0033 0.032 9.7 U-50,488H 1.16 25.5 22.0 CI-977 0.0069 > 1.0 > 145 ICI-199441 0.0071 0.3 42.3 PD-117302 1.22 33.0 27.0 Morphine 0.58 6.01 10.4

50 ºC hot water test, respectively (Table 4) (Endoh et al., 2001).

test (Vanderh et al., 2008).

## **3. 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 a rat paw pressure test, TRK-820 given s.c. or intramuscularly (i.m.) induced dosedependent and sufficient analgesic effects, which were suppressed by pre-treatment with selective antagonist nor-BNI (Table 3). The antinociceptive effect by TRK-820 (ED50 = 0.064 mg/kg, s.c.) was 170, 2, 20, and 78-fold more potent than U-50,488H, CI-977, morphine, and pentazocine, respectively (ED50 values : 11.0, 0.15, 1.3, and 5.0 mg/kg) (Endoh et al., 2000).


Table 3. ED50 values (mg/kg, s.c.) of the antinociceptive effects of some opioid agonists in various tests. U-50,488H, U-69,593, ICI-199,441, CI-977, and PD-117302 are conventional agonists. n.t. : not tested. Hot plate, tail flick, tail pressure, tail pinch, and acetic acid-induced writhing tests were performed in mice (Endoh et al., 1999). Paw pressure and formalin tests were performed in rats (Endoh et al., 2000).

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

In a rat paw pressure test, TRK-820 given s.c. or intramuscularly (i.m.) induced dosedependent and sufficient analgesic effects, which were suppressed by pre-treatment with selective antagonist nor-BNI (Table 3). The antinociceptive effect by TRK-820 (ED50 = 0.064 mg/kg, s.c.) was 170, 2, 20, and 78-fold more potent than U-50,488H, CI-977, morphine, and pentazocine, respectively (ED50 values : 11.0, 0.15, 1.3, and 5.0 mg/kg) (Endoh et al., 2000).

> Low temperature hot plate (51 ºC)

TRK-820 32.0 % at 0.2 0.129 0.062 0.009 0.035 U-50,488H 63.8 % at 20 8.71 5.18 1.0 11.5 ICI-199,441 n.t. 0.065 0.042 0.024 0.051 U-69,593 n.t. 1.33 n.t. 0.48 2.8 CI-977 n.t. n.t. n.t. n.t. n.t. PD-117302 n.t. n.t. n.t. n.t. n.t. Pentazocine 44.6 % at 40 52.2 n.t. n.t. n.t. Morphine 3.65 5.30 5.26 1.5 12.2

Compound Paw pressure Formalin test Acetic acid-induced

TRK-820 0.064 0.0096 0.0033 U-50,488H 11.0 n.t. 1.16 ICI-199,441 0.074 0.0095 0.0071 CI-977 0.15 n.t. 0.0069 PD-117302 n.t. n.t. 1.22 Pentazocine 5.0 n.t. n.t. Morphine 1.3 0.975 0.58

Table 3. ED50 values (mg/kg, s.c.) of the antinociceptive effects of some opioid agonists in various tests. U-50,488H, U-69,593, ICI-199,441, CI-977, and PD-117302 are conventional agonists. n.t. : not tested. Hot plate, tail flick, tail pressure, tail pinch, and acetic acid-induced writhing tests were performed in mice (Endoh et al., 1999). Paw pressure and formalin tests

Tail flick Tail

pressure

writhing test

Tail pinch

**3. Analgesic effects** 

Compound

temperature hot plate tests (Table 3) (Endoh et al., 1999).

High temperture hot plate (55 ºC)

were performed in rats (Endoh et al., 2000).

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, ICI-199,441, 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 (ED50 = 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 ED50 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).


The effect of repeated administration of some agonists and morphine on antinociceptive tolerance was examined by the acetic acid-induced writhing test in mice. After five

Table 4. ED50 values (mg/kg, i.m.) of antinociceptive effects induced by some opioid agonists in the hot water tail withdrawal test in cynomolgus monkeys. n.t. : not tested.


Table 5. ED50 values (mg/kg, s.c. or p.o.) for antinociceptive effects induced by some opioid agonists in the acetic acid-induced writhing test in mice.

Opioid Kappa Receptor Selective Agonist TRK-820 (Nalfurafine Hydrochloride) 87

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, 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.

The p.o. administration of TRK-820 dose-dependently inhibited scratching behavior induced by histamine in mice, which is one of the representative pruritogenic substances, without obvious suppression of spontaneous locomotor activity. The antiscratching activity of TRK-820 with ED50 7.3 g/kg was antagonized by nor-BNI (Togashi et al., 2002). TRK-820 was effective in scratching induced by the other pruritogenic substances: substance P (Togashi et al., 2002; Umeuchi et al., 2003; Utsumi et al., 2004), chloroquine (Inan & Cowan, 2004), compound 48/80 (Wang, Y et al., 2005), agmatin (Inan & Cowan, 2006a), and 5'-GNTI (Inan et al., 2009a, 2011) (Table 8). 5'-GNTI-induced scratching was suppressed by both pretreatment and post-treatment with TRK-820. Tolerance did not develop to the antiscratching

substance Antipruritic effect References Histamine ED50 = 7.3 g/kg, p.o. Togashi et al., 2002 Substance P ED50 = 19.6 g/kg, p.o. Togashi et al., 2002

scratching almost completely

scratching almost completely

Table 8. The antipruritic effects of TRK-820 against itching behaviors induced by various

Compound 48/80 ED50 = 6.64 g/kg, s.c. Wang, Y et al., 2005 Agmatin TRK-820 (0.02 mg/kg, s.c.) was effective Inan & Cowan, 2006a

Inan & Cowan, 2004

Inan et al., 2009a

effect of TRK-820 in the subchronic study (Inan et al., 2009a).

Chloroquine TRK-820 (120 g/kg, p.o.) suppressed the

5'-GNTI TRK-820 (20 g/kg, s.c.) suppressed the

**4. Antipruritic effects 4.1 Preclinical studies** 

Pruritogenic

pruritogenic substances.

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 ED50 was calculated. Comparing the ratio of tolerance ED50 to acute antinociceptive ED50 of each compound, TRK-820 was found to develop the least tolerance to antinociception (Table 6) (Suzuki et al., 2004).

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, ICI-199,441, in the arthritic rats was less potent than in normal rats (Table 7) (Endoh et al., 2000).


Table 6. ED50 values (mg/kg, s.c.) for tolerance and antinociceptive effects induced by some opioid agonists in the acetic acid-induced writhing test.


Table 7. ED50 values (mg/kg, i.m.) for antinociceptive effects induced by some opioid agonists in the paw pressure test in normal and arthritic rats.

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, 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.

## **4. Antipruritic effects**

86 Pharmacology

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 ED50 was calculated. Comparing the ratio of tolerance ED50 to acute antinociceptive ED50 of each compound, TRK-820 was found to develop the least tolerance

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, ICI-199,441, in the arthritic rats was less potent than in normal rats (Table 7) (Endoh

TRK-820 0.54 0.0033 163.6 U-50,488H 30.7 1.16 26.5 ICI-199,441 0.078 0.0071 11.0 Morphine 5.72 0.58 9.9

Table 6. ED50 values (mg/kg, s.c.) for tolerance and antinociceptive effects induced by some

Compound Normal rat Arthritic rat ED50 ratio of arthritic

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

TRK-820 0.055 0.095 1.7 ICI-199,441 0.047 0.24 5.1 Morphine 1.1 1.1 1.0 Table 7. ED50 values (mg/kg, i.m.) for antinociceptive effects induced by some opioid

Acute antinociceptive ED50

Ratio of tolerance ED50/acute antinociceptive ED50

rat/ normal rat

to antinociception (Table 6) (Suzuki et al., 2004).

Compound Tolerance ED50

opioid agonists in the acetic acid-induced writhing test.

agonists in the paw pressure test in normal and arthritic rats.

system and by chemotypes of agonists (Ohsawa et al., 2005).

et al., 2000).

#### **4.1 Preclinical studies**

The p.o. administration of TRK-820 dose-dependently inhibited scratching behavior induced by histamine in mice, which is one of the representative pruritogenic substances, without obvious suppression of spontaneous locomotor activity. The antiscratching activity of TRK-820 with ED50 7.3 g/kg was antagonized by nor-BNI (Togashi et al., 2002). TRK-820 was effective in scratching induced by the other pruritogenic substances: substance P (Togashi et al., 2002; Umeuchi et al., 2003; Utsumi et al., 2004), chloroquine (Inan & Cowan, 2004), compound 48/80 (Wang, Y et al., 2005), agmatin (Inan & Cowan, 2006a), and 5'-GNTI (Inan et al., 2009a, 2011) (Table 8). 5'-GNTI-induced scratching was suppressed by both pretreatment and post-treatment with TRK-820. Tolerance did not develop to the antiscratching effect of TRK-820 in the subchronic study (Inan et al., 2009a).


Table 8. The antipruritic effects of TRK-820 against itching behaviors induced by various pruritogenic substances.

Opioid Kappa Receptor Selective Agonist TRK-820 (Nalfurafine Hydrochloride) 89

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,

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

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

Fig. 5. Changes in VAS (visual analogue scale) values from the pre-observation period. All symbols show the mean values of VAS changes. \**p* < 0.025 compared to placebo, one-sided

Science+Business Media. The VAS test consisted of a 100-mm horizontal line without scale markings. The patients were asked to mark the intensity of itching on the scale, with the right end of the line (100 mm) indicating the strongest possible itching and the left end (0

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

ANCOVA. Reprinted from Nagase & Fujii, 2011 with permission from Springer

**5.1 Effects of TRK-820 in the conditioned place preference (CPP) test** 

which were considered a model of atopic dermatitis (Fig. 4).

outcomes suggest that TRK-820 can be considered a safe agent.

patients and effective in 70 to 80% (Kumagai et al., 2011).

**4.2 Clinical studies** 

mm) indicating no itching.

**5. Effects of TRK-820 on drug dependence** 

Fig. 3. Effects of TRK-820 on scratching induced by i.t. administration of morphine in rhesus monkeys. TRK-820 (0 (□), 0.1 (▲), 0.3 (●), and 1 (▼) g/kg, i.m.) was given 45 min after the administration of morphine (0.03 mg, i.t.). \* *p* < 0.05 between vehicle and time points 1 and 2.5 h. Reprinted with permission from Ko & Husbands, 2009.

Fig. 4. Effects of TRK-820 on scratching behaviors observed in NC/Nag mice maintained in a conventional environment. ##*p* < 0.01, Welch test. \*\**p* < 0.01 compared to NC/Nag mice not treated with TRK-820, parametric Dunnett multiple comparison test. Reprinted with permission from Nakao et al., 2008.

Although epidural or i.t. administration of a agonist like morphine is an important method for pain management, an itching sensation is the most common side effect (Ballantyne et al., 1988; Cousins & Mather, 1984). The effect of TRK-820 on morphineinduced scratching in mice or primates was also evaluated (Ko & Husbands, 2009; Utsumi et al., 2004; Wakasa et al., 2004). Intramuscular administration of TRK-820 (0.3–1 g/kg) dosedependently attenuated scratching induced by morphine (i.t.) in rhesus monkeys without affecting antinociception by morphine (Fig. 3) (Ko & Husbands, 2009).

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).

#### **4.2 Clinical studies**

88 Pharmacology

Fig. 3. Effects of TRK-820 on scratching induced by i.t. administration of morphine in rhesus monkeys. TRK-820 (0 (□), 0.1 (▲), 0.3 (●), and 1 (▼) g/kg, i.m.) was given 45 min after the administration of morphine (0.03 mg, i.t.). \* *p* < 0.05 between vehicle and time points 1 and

Fig. 4. Effects of TRK-820 on scratching behaviors observed in NC/Nag mice maintained in a conventional environment. ##*p* < 0.01, Welch test. \*\**p* < 0.01 compared to NC/Nag mice not

Although epidural or i.t. administration of a agonist like morphine is an important method for pain management, an itching sensation is the most common side effect (Ballantyne et al., 1988; Cousins & Mather, 1984). The effect of TRK-820 on morphineinduced scratching in mice or primates was also evaluated (Ko & Husbands, 2009; Utsumi et al., 2004; Wakasa et al., 2004). Intramuscular administration of TRK-820 (0.3–1 g/kg) dosedependently attenuated scratching induced by morphine (i.t.) in rhesus monkeys without

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.,

treated with TRK-820, parametric Dunnett multiple comparison test. Reprinted with

affecting antinociception by morphine (Fig. 3) (Ko & Husbands, 2009).

2.5 h. Reprinted with permission from Ko & Husbands, 2009.

permission from Nakao et al., 2008.

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.

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).

Fig. 5. Changes in VAS (visual analogue scale) values from the pre-observation period. All symbols show the mean values of VAS changes. \**p* < 0.025 compared to placebo, one-sided ANCOVA. Reprinted from Nagase & Fujii, 2011 with permission from Springer Science+Business Media. The VAS test consisted of a 100-mm horizontal line without scale markings. The patients were asked to mark the intensity of itching on the scale, with the right end of the line (100 mm) indicating the strongest possible itching and the left end (0 mm) indicating no itching.
