Introduction

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(±)TAN-67 is a nonpeptidic delta opioid receptor ligand. Preliminary studies show that (±)TAN-67 has a high affinity for delta opioid receptors (k_i = 0.7 nM) in rat brain with 2,070-fold lower affinity at the mu opioid receptors and 1,600-fold lower affinity at the kappa opioid receptors (Nagase et al., 1994). Knapp et al. (1995) also reported that (±)TAN-67 exhibited high binding affinity (k_i = 0.647 nM) at the human delta opioid receptors and high delta opioid receptor selectivity (>1000-fold) relative to mu opioid receptors. It is a potent delta opioid receptor agonist with an EC₅₀ value of 1.72 nM for the inhibition of the forskolin-stimulated cAMP accumulation at human delta opioid receptors expressed by intact Chinese hamster ovary cells (Knapp et al., 1995) and with an EC₅₀ value of 4.4 nM in the inhibition of the contraction of the mouse deferens (Nagase et al., 1994).

However, the high potency and selectivity of this racemic mixture of TAN-67 on delta opioid receptors found in in vitro studies were not consistent with the findings in vivo that this (±)TAN-67 produced no or weak antinociceptive effects. (±)TAN-67 given i.t., i.c.v. or systemically was found not to be active in inhibiting the tail-flick and hot-plate responses and showed low potency in inhibiting the acetic acid-induced abdominal constriction after systemic administration (Narita et al. unpublished observation; Suzuki et al., 1995; Kamei, et al., 1995)

The enantiomeric forms of (±)TAN-67 have been resolved recently. This study was then designed to characterize the antinociceptive properties of ()TAN-67 and (+)TAN-67 after i.t. injection in mice. We found that ()TAN-67, but not (+)TAN-67, produced antinociception; (+)TAN-67, on the contrary, produced hyperalgesia. The antinociception induced by ()TAN-67 is mediated selectively by the stimulation of delta-1 but not delta-2, mu or kappa opioid receptors.

	Materials and Methods

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Animals. Male ICR mice weighing 25 to 30 g (Sasco, Inc., Omaha, NE) were used for the studies. Animals were housed five per cage in a room maintained at 22 ± 0.5°C with an alternating 12-hr light-dark cycle. Food and water were available ad libitum. Animals were used only once in all experiments.

Assessment of antinociceptive response. Antinociceptive response was determined by the tail-flick test (D'Amour and Smith, 1941). For measurement of the latency of the tail-flick response, mice were gently held with one hand with the tail positioned in the apparatus (Model TF6, EMDIE Instrument Co., Maidens, VA) for radiant heat stimulation. The tail-flick response was elicited by applying radiant heat to the dorsal surface of the tail. The intensity of the heat stimulus in the tail-flick test was adjusted so that the animal flicked its tail within 3 to 5 s. The changes of the latency of the tail-flick response to ()TAN-67 and (+)TAN-67 were expressed in seconds. The inhibition of the tail-flick response to ()TAN-67 was expressed as "percentage of the maximum possible effect" which was calculated as [(T₁  T₀)/(T₂  T₀)] × 100, where T₀ and T₁ were the tail-flick latencies before and after the injection of opioid agonist and T₂ was the cutoff time, which was set at 10 sec.

Behavioral observation. Mice that received (+)TAN-67 produced biting and scratching at the abdomen and hind portions of the body. The intensity of the response was quantified by counting the number of the observed bites and scratches. A response was defined as either the action of biting forepaw, tail or the cage or lifting the hindlimb to scratch the body.

Experimental protocols. Intrathecal injection was made according to the procedure of Hylden and Wilcox (1980), by a 10-µl Hamilton syringe with a 30-gauge needle. Injection volume was 5 µl. Various doses of ()TAN-67 or (+)TAN-67 were injected i.t., and the tail-flick response was measured at different times after the injection. In other experiments, mice were pretreated i.t. with selective opioid antagonists, CTOP, BNTX or NTB (Mizoguchi et al., 1995) 10 min before or nor-BNI 24 hr (Spanagel et al., 1994) before i.t. challenge with (+)TAN-67 or other selective opioid agonists; and the tail-flick response was measured 10 min after the injection. In acute antinociceptive tolerance and cross-tolerance studies, mice were pretreated i.t. with ()TAN-67 or other selective opioid receptor agonists 3 hr earlier and challenged with ()TAN-67 or other selective opioid receptor agonists; the tail-flick response was measured 10 min after the injection. Ten minutes of measurement time was determined based on previous studies that the effect reached a maximum after injection.

Statistical analysis. Student's t test (comparisons of two groups), Fisher's probability test (comparison of two groups for the positive response rate), two-way analysis of variance for comparing among time-response curves of different groups or analysis of variance followed by Newman-Keuls test (comparison between multiple groups) were used to indicate the significance between groups. To establish the dose-response curve, three doses were used with 10 mice at each dose. The ED₅₀ values were calculated from the linear portion of the dose-response curve with a computer program by Tallarida and Murray (1987).

Drugs. ()- and (+)TAN-67 (Nagase et al., 1994, 1996), NTB (Portoghese et al., 1992) and BNTX (Portoghese, 1991) were synthesized in Nagase's laboratory. Other drugs used were [D-Ala²]deltorphin II (Molecula Research Laboratories, Durham, NC), DPDPE, DAMGO, CTOP (Peninsula Laboratory Inc. Belmont, CA) and nor-BNI (Research Biochem Inc., Natick, MA). All drugs for i.t. administration were freshly prepared in sterile physiological saline and peptides were dissolved in 0.9% NaCl solution containing 0.01% Triton X-100.

	Results

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Time courses of the tail-flick response to i.t.-administered ()TAN-67 and (+)TAN-67. Groups of mice were injected i.t. with saline or different doses of ()TAN-67 (17.9, 44.7 and 89.4 nmol), and the tail-flick response was measured 10, 20, 30 and 60 min after injection. I.t. injection of ()TAN-67 caused a dose-dependent increase of the inhibition of the tail-flick response. The tail-flick inhibition reached its peak 10 min after injection, gradually declined and returned to the preinjection level 60 min after injection [Saline vs. ()TAN-67, 17.9 nmol, F(1,90) = 71.7, P < .01; Saline vs. ()TAN-67, 44.7 nmol, F(1,90) = 105.5, P < .01; Saline vs. ()TAN-67, 89.4 nmol, F(1,86) = 178.8, P < .01] (fig. 1A). The 10 min of measurement time after i.t. ()TAN-67 injection was determined for the experiments described in the next section.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Time courses of the changes of the tail-flick latency after i.t. injection of ()TAN-67 (A) and (+)TAN-67 (B). Groups of mice were injected i.t. with saline or different doses of ()TAN-67 or (+)TAN-67, and the tail-flick responses were measured at different times after injection. Each value represents the mean ± S.E.M. for 9 to 10 mice studied.

Dose-response studies of the inhibition of the tail-flick response induced by ()TAN-67 and [D-Ala²]deltorphin II. Groups of mice were injected i.t. with saline or different doses of ()TAN-67 (8.9-89.4 nmol) or [D-Ala²]deltorphin II (0.6-12.8 nmol), and the tail-flick response was measured 10 min after the injection. The 10 min of measurement time after i.t. [D-Ala²]deltorphin injection was determined based on the results of the previous study that the tail-flick inhibition reached its peak 10 min after injection (Narita et al., 1996). I.t. injection of ()TAN-67 or [D-Ala²]deltorphin II caused a dose-dependent increase of the tail-flick inhibition (fig. 2). The ED₅₀ values were estimated to be 17.1 nmol (3.4-85.2 nmol, 95% confidence limits) and 3.4 nmol (2.5-3.9 nmol, 95% confidence limits) for ()TAN-67 and [D-Ala²]deltorphin II, respectively. Thus ()TAN-67 is about 5.0-fold less potent than [D-Ala²]deltorphin II for the tail-flick inhibition.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Dose-response curves of the inhibition of the tail-flick response induced by i.t. injection of [D-Ala2]deltorphin II and ()TAN-67. Groups of mice were injected i.t. with different doses of [D-Ala2]deltorphin II (0.6-12.8 nmol) or ()TAN-67 (8.9-89.4 nmol), and the tail-flick responses were measured 10 min after injection. Each value represents the mean ± S.E.M. for 10 mice studied.

Effects of i.t. pretreatment with BNTX, NTB, CTOP and nor-BNI on the inhibition of the tail-flick response induced by i.t.-administered ()TAN-67, DPDPE, [D-Ala²]deltorphin II, DAMGO and U50,488H. To identify what types of opioid receptors in the spinal cord were involved in the inhibition of the tail-flick response induced by ()TAN-67, the effects of the blockade of delta-1, delta-2, mu and kappa opioid receptors by i.t. pretreatment with BNTX, NTB, CTOP, and nor-BNI, respectively, on the inhibition of the tail-flick response induced by i.t.-administered ()TAN-67 were studied. To make sure that the respective receptors were blocked by these selective antagonists, the effects of these antagonists used on the inhibition of the tail-flick response induced by DPDPE, [D-Ala²]deltorphin II, DAMGO and U50,488H were also investigated. Groups of mice were pretreated i.t. with saline (5 µl), CTOP (47 pmol), BNTX (0.2 or 0.65 nmol) or NTB (0.19, 0.62 or 1.88 nmol) 10 min or nor-BNI (6.8 nmol) 24 hr before i.t. challenge with DPDPE (7.8 nmol), [D-Ala²]deltorphin II (6.4 nmol), DAMGO (19.5 pmol), U50,488H (161.1 nmol) or ()TAN-67 (89.4 nmol); and the tail-flick responses were measured 10 min after the injection. As shown in figure 3, BNTX pretreatment selectively blocked the tail-flick inhibition induced by DPDPE but not by [D-Ala²]deltorphin II, whereas NTB pretreatment selectively blocked the inhibition of the tail-flick response induced by [D-Ala²]deltorphin II but not by DPDPE. CTOP and nor-BNI pretreatment effectively blocked the tail-flick inhibition induced by DAMGO and U50,488H, respectively. As shown in figure 4, BNTX pretreatment dose-dependently blocked the inhibition of the tail-flick response induced by i.t.-administered ()TAN-67. However, the tail-flick inhibition induced by ()TAN-67 was not affected by the i.t. pretreatment with CTOP, NTB or nor-BNI.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Effects of the blockade of delta-1, delta-2, mu and kappa opioid receptors by i.t. pretreatment with BNTX, NTB, CTOP and nor-BNI, respectively, on the inhibition of the tail-flick response induced by i.t. injection of DPDPE, [D-Ala2]deltorphin II, DAMGO or U50,488H. Groups of mice were pretreated i.t. with BNTX (0.65 nmol), NTB (0.62 nmol) or CTOP (47 pmol) 10 min before i.t. challenge with DPDPE (7.8 nmol), [D-Ala2]deltorphin II (6.4 nmol) or DAMGO (19.5 pmol) or nor-BNI (6.8 nmol) 24 hr before i.t. challenge with U50,488H. The tail-flick response was measured 10 min after the injection. SA, saline; **P < .01 compared with the saline-injected group.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effects of the blockade of delta-1, delta-2, mu and kappa opioid receptors by i.t. pretreatment with BNTX, NTB, CTOP and nor-BNI, respectively, on the inhibition of the tail-flick response induced by i.t. injection of ()TAN-67. Groups of mice were pretreated i.t. with BNTX (0.2 or 0.65 nmol), NTB (0.19 or 1.88 nmol) or CTOP (47 pmol) 10 min before i.t. challenge with ()TAN-67 (89.4 nmol) or nor-BNI (6.8 nmol) 24 hr before i.t. challenge with ()TAN-67 (89.4 nmol). The tail-flick response was measured 10 min after the injection. *P < .05, **P < .01, compared with saline-pretreated control.

Acute antinociceptive tolerance to ()TAN-67 and cross-tolerance to DPDPE, but not cross-tolerance to [D-Ala²]deltorphin II, DAMGO and U50,488H in mice tolerant to ()TAN-67. To determine further whether the antinociceptive property of ()TAN-67 is selective to delta-1 opioid receptors, the effects of acute antinociceptive tolerance to ()TAN-67 and cross-tolerance to other selective opioid receptor agonists were studied. For control studies, the acute antinociceptive tolerance and cross-tolerance to DPDPE, [D-Ala²]deltorphin II, DAMGO and U50,488H were also investigated. Groups of mice were pretreated i.t. with ()TAN-67 (89.4 nmol), DPDPE (7.8 nmol), [D-Ala²]deltorphin II (6.4 nmol), DAMGO (19.5 pmol) or U50,488H (107.4 nmol) 3 hr before the subsequent i.t. challenge with the same or different opioid; the tail-flick response was measured 10 min after the injection. As shown in table 1, pretreatment with ()TAN-67 given i.t. 3 hr earlier attenuated the tail-flick inhibition induced by subsequent i.t. administration of ()TAN-67 or by DPDPE. However, the tail-flick inhibition induced by i.t.-administered [D-Ala²]deltorphin II, DAMGO or U50,488H were not affected by i.t. pretreatment with ()TAN-67. Conversely, pretreatment with DPDPE given i.t. 3 hr earlier attenuated the tail-flick inhibition induced by subsequent i.t. administration of ()TAN-67 or by DPDPE. However, the tail-flick inhibition induced by i.t.-administered [D-Ala²]deltorphin II was not affected by i.t. pretreatment with DPDPE. Pretreatment with [D-Ala²]deltorphin II given i.t. 3 hr earlier attenuated the tail-flick inhibition induced by subsequent i.t. administration of [D-Ala²]deltorphin II, but not by ()TAN-67 or DPDPE. Pretreatment with DAMGO given i.t. 3 hr earlier attenuated the tail-flick inhibition induced by subsequent i.t. administration of DAMGO but not by ()TAN-67. Pretreatment with U50,488H given 3 hr earlier attenuated the tail-flick inhibition induced by subsequent i.t. administration of U50,488H but not by ()TAN-67.

View this table: [in this window] [in a new window]   TABLE 1 Effects of i.t. pretreatment with ()TAN-67, DPDPE, [D-Ala2]deltorphin II, DAMGO or U50,488H on the inhibition of the tail-flick response induced by i.t.-administered ()TAN-67, DPDPE, [D-Ala2]deltorphin II, DAMGO or U50,488H The doses of the opioid agonists for the i.t. pretreatment and the i.t. challenge are ()TAN-67, 89.4 nmol; DPDPE, 7.8 nmol; [D-Ala2]deltorphin II, 6.4 nmol; DAMGO, 19.5 pmol; U50,488H, 107.4 nmol.

Hyperalgesic behavioral responses induced by (+)TAN-67 given i.t.. Unlike ()TAN-67, which did not produce apparent behavioral changes after i.t. injection, (+)TAN-67 given i.t. produced pain-like aversive responses. Mice developed scratching and biting on the hind part of the body and the tail immediately after i.t. injection of (+)TAN-67 (17.9-89.4 nmol). The pain-like syndrome was so intense that they bit the plastic cage. These hyperalgesic behaviors lasted about 30 to 60 min and dissipated in 1 hr. Animals developed clonic-tonic convulsion followed by death after higher doses of (+)TAN-67 20 min after injection (table 2).

	Discussion

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The results of the present studies clearly demonstrate that ()TAN-67 but not its enantiomorph (+)TAN-67 given i.t. produces a time- and dose-dependent antinociception with use of the tail-flick test. (+)TAN-67, on the other hand, produces hyperalgesia. The present finding answers the question of the previous report that racemic mixture (±)TAN-67, which binds potently to delta opioid receptor in in vitro receptor binding studies (Knapp et al., 1995), does not inhibit the tail-flick or hot-plate response after i.t., i.c.v. or systemic injection (Narita et al., unpublished observations; Suzuki et al., 1995; Kamei et al, 1995). It is possible that the lack of antinociceptive response of (±)TAN-67 is caused by the hyperalgesic effects of (+)TAN-67, which antagonize physiologically the antinociceptive effects of ()TAN-67.

The antinociception induced by ()TAN-67 is mediated selectively by the stimulation of delta-1 but not by delta-2, mu or kappa opioid receptors. This conclusion is based on the findings that the inhibition of the tail-flick response induced by i.t.-administered ()TAN-67 was blocked by the i.t. pretreatment with selective delta-1 opioid receptor antagonist BNTX, but not by the delta-2 opioid receptor antagonist NTB, the mu opioid receptor antagonist CTOP or the kappa opioid receptor antagonist nor-BNI. The selectivity of these selective delta-1, delta-2, mu and kappa opioid receptor antagonists in blocking the respective selective opioid agonist-induced tail-flick inhibition has been confirmed in the present studies and also studies by others (Gulya et al., 1988; Takemori et al., 1988; Tseng et al., 1993, 1995; Portoghese et al., 1992; Sofuoglu et al., 1991; Stewart and Hammond, 1993). Our studies provide more evidence that delta-1 opioid receptors exist in the spinal cord separated from delta-2 and other opioid receptors.

The delta-1 opioid receptor agonist properties of ()TAN-67 were further characterized in acute tolerance and cross-tolerance studies. We found that pretreatment of mice with ()TAN-67 given i.t. 3 hr earlier attenuated the inhibition of the tail-flick response induced by subsequent challenge of ()TAN-67 or DPDPE, a delta-1 opioid receptor agonist. However, the same treatment did not affect the tail-flick inhibition induced by delta-2 opioid receptor agonist [D-Ala²]deltorphin II, mu opioid receptor agonist DAMGO or kappa opioid receptor agonist U50,488H. Conversely, pretreatment with DPDPE given i.t. 3 hr earlier attenuated the tail-flick inhibition induced by subsequent i.t. administration of ()TAN-67 or DPDPE but not by [[D- Ala²]deltorphin II. Thus, antinociceptive tolerance to ()TAN-67 produces cross-tolerance to another delta-1 agonist but not to delta-2, mu or kappa opioid agonists, which indicates the selective receptor action of ()TAN-67 on delta-1 opioid receptors in the mouse spinal cord.

We have found that, unlike ()TAN-67 which produces antinociception, (+)TAN-67 given i.t. produces hyperalgesia. This conclusion is based on the finding that (+)TAN-67 given i.t. decreased the latencies of the tail-flick response at low doses (1.8-8.9 nmol) and produced scratching and biting at high doses (17.9-89.4 nmol). The doses of (+)TAN-67 which produced a decrease of the tail-flick latencies were much smaller than the doses of ()TAN-67 which produced antinociception. The exact mechanism of (+)TAN-67 for producing hyperalgesia is not clear at this time. Receptor binding studies indicate that it displaces the -aminobutyric acid_A receptor bindings (Nagase et al., unpublished observations). It is possible that (+)TAN-67 produces the hyperalgesia by blocking the -aminobutyric acid receptors in the spinal cord.

Unlike delta-2 opioid receptors, which have been cloned and characterized (Kieffer et al. 1992; Evans et al., 1992), the delta-1 opioid receptors have not been cloned. Previous pharmacological studies with selective opioid receptor agonists and opioid receptor antagonists clearly indicate the presence of delta-1 opioid receptors which are distinguished from delta-2 opioid receptors. However, previous studies in mice with an antisense oligodeoxynucleotide to delta-2 opioid receptor mRNA failed to distinguish delta-1 from delta-2 opioid receptors in the mouse spinal cord. I.t. pretreatment with delta-2 opioid receptor antisense oligodeoxynucleotide given i.t. equally attenuated the tail-flick inhibition induced by i.t. DPDPE, a delta-1 opioid receptor agonist, and [D-Ala²]deltorphin II, a delta-2 opioid receptor agonist (Bilsky et al., 1994; Tseng et al., 1994). On the contrary, i.c.v. pretreatment with delta-2 opioid receptor antisense oligodeoxynucleotide attenuated only [D-Ala²]deltorphin II-, but not DPDPE-induced antinociception, which indicates that i.c.v. pretreatment with delta-2 opioid receptor antisense can distinguish delta-2 opioid receptor from delta-1 opioid receptor functions in the supraspinal sites (Lai et al., 1994; Bilsky et. al., 1994). The reason for the lack of selective action of delta-2 opioid receptor antisense oligodeoxynucleotide in the mouse spinal cord is not clear at this time. It is possible that, in addition to the delta-1 opioid agonist activity, DPDPE may nonselectively have delta-2 opioid receptor activity. The development of the selective delta-1 opioid agonist ()TAN-67 will be a useful pharmacological tool for characterizing this delta-1 opioid receptor and its function.

In conclusion, the antinociception induced by ()TAN-67 is mediated selectively by the stimulation of delta-1 opioid receptors. This study provides more evidence that delta-1 opioid receptors exist separated from delta-2 opioid receptors.