Introduction

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Pharmacological evidence supporting the multiplicity of -opioid receptors dates to the early 1990s (Vaughn et al., 1990). Through the efforts of Porreca and Portoghese and their coworkers, in vivo assays suggested the presence of two -opioid subtypes designated ₁ and ₂ (Jiang et al., 1991; Sofuoglu et al., 1991; Mattia et al., 1992; Portoghese et al., 1992; Horan et al., 1993a,b). Despite the strong pharmacological evidence for -opioid receptor subtypes, the stereochemical requirements for discriminating ₁ and ₂ receptors have not been adequately explored. Previous work has suggested that the ability of peptide ligands to recognize ₁ and ₂ receptors is sequence-dependent. [D-Ala²,Glu⁴]Deltorphin II, for example, preferentially binds ₂ receptors, whereas enkephalin-based ligands (except truncated analogs) display selectivity for ₁ receptors (Jiang et al., 1991). [D-Ala²,Asp⁴]Deltorphin (DELT I), on the other hand, activates both ₁ and ₂ receptors (Qian et al., 1996; current study). We questioned whether this selectivity is based solely on peptide sequence.

We prepared a series of highly constrained tyrosine derivatives, -methyl-2',6'-dimethyltyrosines (TMTs) [Fig. 1, for the (2S,3S) isomer], which allowed us to perform a rotamer scan to probe the -opioid receptor using cyclic [D-Pen²,D-Pen⁵]enkephalin (DPDPE) and DELT I as templates (Qian et al., 1996). The incorporation of (2S,3S)-TMT into DPDPE led to a 130-fold decrease in binding affinity and a 100-fold decrease in selectivity for -opioid receptors without affecting affinity for the µ-opioid receptor (Qian et al., 1996). Incorporation of (2S,3S)-TMT into the DELT I molecule, on the other hand, decreased µ- and -receptor affinity only slightly while actually increasing -receptor selectivity by 1.64-fold (Qian et al., 1996). Similar results were seen in the in vitro guinea pig ileum (µ) and mouse vas deferens (MVD, ) bioassays. [(2S,3S)-TMT¹]DPDPE had 40-fold lower potency in the MVD bioassay and a 25-fold increase in potency in the guinea pig ileum bioassay, indicating a loss of -receptor selectivity (Qian et al., 1996). As in the binding studies, modification of the DELT I molecule to [(2S,3S)-TMT¹]deltorphin did not significantly change the potencies for µ-and -receptors or the selectivity ratio (Qian et al., 1996). Our preliminary antinociceptive studies indicated that such topographical change in the message domains of these two -opioid agonists did provide different in vivo antinociceptive profiles toward the -opioid receptor subtypes. In the present study, all four isomers of the [TMT¹]DPDPE and [TMT¹]DELT I were evaluated for antinociceptive activity and opioid receptor selectivity.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Structures of (2S,3S)-TMT, DPDPE, and DELT I.

	Materials and Methods

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Animals. Male ICR mice (20-30 g) (Harlan Industries, Cleveland, OH) were housed in groups of five in Plexiglas chambers with food and water available ad libitum before any procedures. Animals were maintained on a 12-h light/dark cycle in a temperature-controlled animal colony. Studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health.

Injections. Compounds were dissolved in distilled water {[D-Ala²,Leu⁵,Cys⁶]enkephalin (DALCE) and -funaltrexamine (-FNA)} or 20% dimethyl sulfoxide ([Cys⁴]deltorphin and all DPDPE and DELT I analogs). Compounds were injected by the intracerebroventricular (i.c.v.) route as described previously (Porreca et al., 1984). Briefly, mice were lightly anesthetized with ether, and an incision was made in the scalp. An injection was made with a 10-µl Hamilton syringe at a point 2 mm caudal and 2 mm lateral from bregma. Compounds were injected at a depth of 3 mm in a volume of 5 µl.

Antinociceptive Testing. Antinociception was assessed using the 55°C warm water tail-flick test. The latency to the first sign of a rapid tail-flick was taken as the behavioral end point (Jannsen et al., 1963). Each mouse was first tested for baseline latency by immersing its tail in the water and recording the time to response. Mice not responding within 5 s were excluded from further testing. Mice were then administered the test compound and tested for antinociception at 10, 20, 30, 45, 60, and 90 min postinjection. A maximum score was assigned (100%) to animals not responding within 15 s to avoid tissue damage. Antinociception was calculated by the following equation: % antinociception = 100 × (test latency  control latency)/(15  control latency). In studies assessing the effects of selective opioid antagonists (4.57 nmol DALCE, 3 nmol [Cys⁴]deltorphin, and 19 nmol -FNA), mice were injected 24 h before agonist administration via the i.c.v. route. Control mice received a vehicle injection (5 µl of distilled water i.c.v. 24 h). These times and doses have been shown previously to produce selective blockade of ₁, ₂, and µ-receptors, respectively (Jiang et al., 1991; Horan et al., 1993b). To evaluate blockade of agonist effects, mice were tested at times corresponding to peak agonist drug effect.

Statistical Analysis. For antinociceptive tests, dose-response lines were constructed at times of agonist peak effect and analyzed using linear regression (Tallarida and Murray, 1987). All A₅₀ values (95% confidence limits) shown are calculated from the linear portion of the dose-response curve. Single-dose data were analyzed using one-way ANOVA, followed by Student's t test for between-group comparisons (significance set at P < .05). A minimum of 10 mice were used at each dose level.

Chemicals. [Cys⁴]Deltorphin, DPDPE, DELT I, and their [TMT¹] analogs were synthesized using previously published methods (Misicka et al., 1991; Qian et al., 1994, 1995, 1996). DALCE and -FNA were obtained through the National Institute on Drug Abuse Drug Supply Program.

	Results

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The antinociceptive dose- and time-response curves for the [TMT¹]DPDPE and [TMT¹]DELT I analogs are depicted in Figs. 2 and 3, respectively. Calculated antinociceptive A₅₀ values and 95% confidence limits are listed in Tables 1 and 2. The potency and efficacy of DPDPE and its analogs varied widely, with A₅₀ values ranging from 7.05 nmol for [(2S,3S)-TMT¹]DPDPE to >100 nmol for [(2R,3R)-TMT¹]DPDPE. All of the DELT I analogs produced full agonist effects with A₅₀ values ranging from 0.35 nmol for [(2S,3R)-TMT¹]DELT I to 17.71 nmol for [(2R,3S)-TMT¹]DELT I. 

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Dose- and time-responses for DPDPE and the four [TMT1]DPDPE analogs. Mice received i.c.v. injections of the test compounds and were tested at various times later in the 55°C tail-flick test. Bars represent S.E. values.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Dose- and time-responses for DELT I and the four [TMT1]DELT I analogs. Mice received i.c.v. injections of the test compounds and were tested at various times afterward in the 55°C tail-flick test. Bars represent S.E. values.

The results of the opioid receptor selectivity studies are depicted in Figs. 4 ([TMT¹]DPDPE analogs) and 5 ([TMT¹]DELT I analogs). As we have shown previously, DPDPE produces its antinociceptive actions primarily through ₁-opioid receptors. An ANOVA of the data yielded an F_3,36 value of 10.08 (P < .001). The comparison between control and DALCE-pretreated mice yielded a t(9) value of 4.86 (P < .001). DELT I antinociception, on the other hand, was mediated by both ₁ and ₂ receptors. The ANOVA yielded an F_3,36 value of 8.93 (P < .001) with a t(9) value of 5.26 and 4.53 (all P < .001 for the comparisons of the control group with DALCE and [Cys⁴]deltorphin groups, respectively).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   In vivo receptor selectivity of DPDPE and two [TMT1]DPDPE analogs was determined by administering an approximate A90 dose of the agonist in vehicle-treated (control) or opioid antagonist-treated mice. Mice received i.c.v. injections of the test compounds and were tested at the time of peak agonist drug effect (10 min) in the 55°C tail-flick test. Selective opioid antagonists administered 24 h before agonist administration were the µ-antagonist -FNA, the 1 antagonist DALCE, and the 2 antagonist [Cys4]deltorphin. Bars represent S.E. values.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   In vivo receptor selectivity of DELT I and four [TMT1]DELT I analogs was determined by administering an approximate A90 dose of the agonist in vehicle-treated (control) or opioid antagonist-treated mice. Mice received i.c.v. injections of the test compounds and were tested at time of peak agonist drug effect (10 min) in the 55°C tail-flick test. Selective opioid antagonists administered 24 h before agonist administration were the µ-antagonist -FNA, the 1 antagonist DALCE, and the 2 antagonist [Cys4]deltorphin. Bars represent S.E. values.

The antinociceptive effect of [(2S,3S)-TMT¹]DPDPE was blocked by pretreatment with both ₁- and µ-opioid antagonists (all P < .01). Similar results were seen with [(2S,3R)-TMT¹]DPDPE (all P < .05). The receptor selectivity of [(2R,3R)-TMT¹]DPDPE and [(2R,3S)-TMT¹]DPDPE could not be assessed due to the partial agonist activity of the compounds and the toxicity seen with higher doses of [(2R,3S)-TMT¹]DPDPE. The [(2S,3S)-TMT¹]DELT I analog displayed a ₂-selective profile with an ANOVA of F_3,36 of 6.24 (P < .01) and a t(9) of 3.76 (P < .01 for the comparison between the control and [Cys⁴]deltorphin-pretreated mice). The ANOVA for [(2S,3R)-TMT¹]DELT I yielded an F_3,36 value of 11.67 (P < .001). Post hoc analysis indicated that the compound retained a ₂-subtype-selectivity profile with a P value of <.001 value for the comparison between the control and [Cys⁴]deltorphin groups. There also was a weak µ-opioid component to the compound (P = .05 for the comparison between the control and -FNA groups). This µ-opioid component was even stronger in [(2R,3R)-TMT¹]DELT I (P < .001). Interestingly, [(2R,3S)-TMT¹]DELT I retained a ₁ component (P = .01) along with activity at the µ-receptor (P < .001).

	Discussion

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This study assessed the effects of topographical modification in position 1 with the four isomers of [TMT¹]DPDPE and of [TMT¹]DELT I on opioid antinociceptive potency, efficacy, and receptor selectivity. The antinociceptive potency and efficacy of these compounds varied widely. In general, the [TMT¹]DELT I analogs displayed greater antinociceptive potency than the [TMT¹]DPDPE analogs (see Tables 1 and 2). This trend was not unexpected given the fact that DELT I is approximately 17 times more potent than DPDPE. In addition, topographical modification produced modest changes in the duration of action of some of the analogs. In general, DPDPE and DELT I are very stable compounds (Weber et al., 1992). The (2S,3S)- and (2S,3R)-[TMT¹]DPDPE analogs have a decreased duration of action compared with the parent compound. Modification of DELT I to (2S,3R)- and (2R,3S)-[TMT¹]DELT I, on the other hand, increased the duration of action. Future studies will provide a more detailed assessment of the stability of these compounds in mouse brain homogenate and whole serum.

The parent compound DPDPE is a moderately potent full agonist that acts at ₁ opioid receptors. The [(2S,3S)-TMT¹]DPDPE analog displays similar antinociceptive potency while acting through both ₁ and µ-receptors. In vitro studies with [(2S,3S)-TMT¹]DPDPE also support activity at both - and µ-receptors (Qian et al., 1994, 1996). Interestingly, [(2S,3S)-TMT¹]DPDPE has lower binding affinity for opioid receptors compared with DPDPE. The comparable antinociceptive activity may be explained by a synergistic action at µ- and -receptors (Horan et al., 1993a).

The [(2S,3R)-TMT¹]DPDPE analog was significantly less potent than the parent compound DPDPE. This was unexpected given its favorable binding and activity profiles in the in vitro assays (Qian et al., 1994, 1996). This phenomenon has, however, been seen with other opioid peptides. [L-Ala³]DPDPE, for example, is a potent agonist in the MVD bioassay with an EC₅₀ value of 12 nM and a selectivity of approximately 4500 for the -receptor (Haaseth et al., 1994). This compound, however, is a weak partial agonist with an antinociceptive A₅₀ value of 100 nmol in the 50°C tail-flick test. Another example is the DPDPE analog Tyr-c[D-Pen-Gly-Phe-Cys]-Phe-OH, which is an extremely potent (EC₅₀ = 0.016 nM in the MVD bioassay) and selective (>5100 µ/ ratio) -opioid agonist in vitro (Bartosz-Bechowski et al., 1994). Despite this impressive in vitro profile, the compound has an antinociceptive A₅₀ value comparable with DPDPE in the 55°C tail-flick test.

The in vivo receptor selectivity profile of [(2S,3R)-TMT¹]DPDPE was also unexpected. Based on the in vitro binding and functional bioassays, the compound appeared to act predominantly at -opioid receptors (Qian et al., 1996). The fact that -FNA significantly antagonized the antinociceptive actions of the compound suggests a µ-opioid component. This may be related to the limited antinociceptive potency of the compound. A 100-nmol dose of the compound was needed to produce an approximate A₉₀ response. This dose in vivo may act through both - and µ-opioid receptors. Although the differences between the in vitro and in vivo profiles of these compounds remain unclear, they do emphasize the importance of testing these compounds in vivo.

The [(2R,3R)-TMT¹]DPDPE analog was a weak partial agonist in the 55°C tail-flick test. This was not unexpected given the in vitro profile of the compound (Qian et al., 1996). The [(2R,3S)-TMT¹]DPDPE analog also produced a weak agonist effect with a potency significantly less than the parent compound. Interestingly, doses of >60 nmol i.c.v. produced convulsions in the mice. This toxicity precluded further testing of the compound at higher doses and with the selective antagonists. The toxicity was not mediated through opioid receptors as naloxone (10 mg/kg i.p.) did not prevent the convulsions.

The topographical modification of the tyrosine in position 1 of DELT I produced dramatic changes in the receptor selectivity of the compound. The antinociceptive actions of the (2S,3R)-, (2R,3R)-, and (2R,3S)-TMT¹ analogs of DELT I all had a µ-opioid receptor component. This is in contrast to the relatively -selective actions of the parent DELT I molecule. Of additional interest is the shift of DELT I from a mixed ₁/₂ agonist to a ₂-selective agonist with the [(2S,3S)-TMT¹]DELT I analog. Although the (2S,3R) and (2R,3R) analogs of DELT I had significant µ-opioid components, they retained selectivity for ₂ receptors over ₁ receptors. Surprisingly, [(2R,3S)-TMT¹]DELT I produced its antinociceptive effects through µ-and ₁ opioid receptors. These data demonstrate that topographical modification in position 1 of the DELT I molecule changes the opioid receptor selectivity of the compound from  to µ and from non--subtype-selective to either ₂- or ₁-selective.

It is worthwhile to mention that another important topographical factor in both DPDPE and DELT I is the phenylalanine side chain. Solution NMR studies of the phenylalanine residue, or its -methyl constrained counterparts, in the DPDPE molecule indicate that the compound prefers a gauche () configuration about the ₁ torsional angle (Nikiforovich et al., 1991). This topographical consistency of phenylalanine is also seen in conformational studies of the [TMT¹]-substituted DPDPE and DELT I analogs (Qian et al., 1996). Thus, the change of topography of tyrosine was the major factor for producing the diverse antinociceptive activities and receptor selectivity of [TMT¹]DPDPE and [TMT¹]DELT I analogs.

In conclusion, our previous NMR studies indicated that the preferred side chain conformations of (2S,3S)-TMT, (2S,3R)-TMT, (2R,3S)-TMT, and (2R,3R)-TMT are gauche (), trans, gauche (+), and trans, respectively. In combining the antinociceptive results, we can conclude that in the DPDPE series, a trans L-TMT¹ topology is crucial for ₁ opioid receptor recognition. In the series of DELT I analogs, a gauche () topology of L-TMT¹ is crucial for ₂ opioid receptor recognition, whereas a trans ₁ torsional angle combined with D-TMT¹ may convert the selectivity of deltorphin analogs to a preferred ₁ receptor recognition. However, the DELT I analogs reported here are all linear; thus, their backbone conformations are not defined and such conformational flexibility could provide mixed information. Nevertheless, these studies are the first systematic approach to probing the selectivity of opioid -receptor subtypes using topographical design of -opioid agonists. The studies also suggest that the selectivity of opioid agonists may not be solely dependent on the peptide sequence but also dependent on the side chain topography.