Building a better analgesic: multifunctional compounds that address injury-induced pathology to enhance analgesic efficacy while eliminating unwanted side effects.

The most highly abused prescription drugs are opioids used for the treatment of pain. Physician-reported drug-seeking behavior has resulted in a significant health concern among doctors trying to adequately treat pain while limiting the misuse or diversion of pain medications. In addition to abuse liability, opioid use is associated with unwanted side effects that complicate pain management, including opioid-induced emesis and constipation. This has resulted in restricting long-term doses of opioids and inadequate treatment of both acute and chronic debilitating pain, demonstrating a compelling need for novel analgesics. Recent reports indicate that adaptations in endogenous substance P/neurokinin-1 receptor (NK1) are induced by chronic pain and sustained opioid exposure, and these changes may contribute to processes responsible for opioid abuse liability, emesis, and analgesic tolerance. Here, we describe a multifunctional mu-/delta-opioid agonist/NK1 antagonist compound [Tyr-d-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-Bn(CF3)2 (TY027)] that has a preclinical profile of excellent antinociceptive efficacy, low abuse liability, and no opioid-related emesis or constipation. In rodent models of acute and neuropathic pain, TY027 demonstrates analgesic efficacy following central or systemic administration with a plasma half-life of more than 4 hours and central nervous system penetration. These data demonstrate that an innovative opioid designed to contest the pathology created by chronic pain and sustained opioids results in antinociceptive efficacy in rodent models, with significantly fewer side effects than morphine. Such rationally designed, multitargeted compounds are a promising therapeutic approach in treating patients who suffer from acute and chronic pain.

Preclinical assessment of candidate analgesic drugs: recent advances and future challenges.

In analgesic drug development, preclinical procedures are widely used to assess drug effects on pain-related behaviors. These procedures share two principal components: 1) a manipulation intended to produce a pain-like state in the experimental subject and 2) measurement of behaviors presumably indicative of that pain state. Drugs can then be evaluated for their ability to attenuate pain-related behaviors. In the simplest procedures, the pain state is produced by delivery of an acute noxious stimulus (e.g., a warm thermal stimulus), and the primary dependent measures focus on withdrawal responses or other nocifensive behaviors that increase in rate, frequency, or intensity in response to the noxious stimulus. This approach has been refined in two ways. First, new methods have been developed to induce more clinically relevant pain states. In particular, pain requiring clinical intervention is often associated with inflammation or neuropathy, and novel procedures have emerged to model these conditions and their ability to produce hypersensitive pain states, such as allodynia and hyperalgesia. Second, studies are incorporating a broader array of pain-related behaviors as dependent measures. For example, pain not only stimulates nocifensive behaviors but also suppresses many adaptive behaviors, such as feeding or locomotion. Measures of pain-suppressed behaviors can provide new insights into the behavioral consequences of pain and the effects of candidate analgesics. In addition, functional magnetic resonance imaging has emerged as a noninvasive tool for investigating changes in neural activity associated with pain and analgesia. Integration of these complementary approaches may improve the predictive validity of analgesic drug development.

Inverse agonists and neutral antagonists at mu opioid receptor (MOR): possible role of basal receptor signaling in narcotic dependence.

The mu opioid receptor, MOR, displays spontaneous agonist-independent (basal) G protein coupling in vitro. To determine whether basal MOR signaling contributes to narcotic dependence, antagonists were tested for intrinsic effects on basal MOR signaling in vitro and in vivo, before and after morphine pretreatment. Intrinsic effects of MOR ligands were tested by measuring GTPgammaS binding to cell membranes and cAMP levels in intact cells. beta-CNA, C-CAM, BNTX, and nalmefene were identified as inverse agonists (suppressing basal MOR signaling). Naloxone and naltrexone were neutral antagonists (not affecting basal signaling) in untreated cells, whereas inverse agonistic effects became apparent only after morphine pretreatment. In contrast, 6alpha- and 6beta-naltrexol and -naloxol, and 6beta-naltrexamine were neutral antagonists regardless of morphine pretreatment. In an acute and chronic mouse model of morphine-induced dependence, 6beta-naltrexol caused significantly reduced withdrawal jumping compared to naloxone and naltrexone, at doses effective in blocking morphine antinociception. This supports the hypothesis that naloxone-induced withdrawal symptoms result at least in part from suppression of basal signaling activity of MOR in morphine-dependent animals. Neutral antagonists have promise in treatment of narcotic addiction.

In vivo pharmacological characterization of SoRI 9409, a nonpeptidic opioid mu-agonist/delta-antagonist that produces limited antinociceptive tolerance and attenuates morphine physical dependence.

Repeated exposure to mu-opioid analgesics produces unwanted side effects, including tolerance and physical dependence. delta-Opioid antagonists attenuate development of morphine tolerance and physical dependence. We recently reported that SoRI 9409, a mixed mu-agonist/delta-antagonist, produces antinociception with limited development of tolerance after repeated i.c.v. injections. The current studies report on a more complete characterization of the compound in male ICR mice. SoRI 9409 produced limited antinociceptive effects in the 55 degrees C tail-flick test and full agonist effects in the acetic acid writhing assay after i.c.v. or i.p. administration. Repeated i.p. administration of A(90) doses of SoRI 9409 did not produce tolerance. The agonist effects of the compound were preferentially blocked by the mu-selective antagonist beta-funaltrexamine. The kappa-antagonist nor-binaltorphimine produced partial antagonism, whereas the delta-antagonist naltrindole had no effect on SoRI 9409 antinociception. Intraperitoneal administration of SoRI 9409 preferentially antagonized the antinociceptive actions of the delta-2 agonist [D-Ala(2),Glu(4)]deltorphin over the delta-1 agonist cyclic[D-Pen(2),D-Pen(5)]-enkephalin and the mu-agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin. SoRI 9409 did not antagonize the antinociceptive effects of the kappa-agonist U69,593 (doses up to 60 mg/kg). SoRI 9409 (10 mg/kg i.p.) elicited much less vertical jumping than naloxone (10 mg/kg i.p.) in acute and chronic morphine dependence models. SoRI 9409 also suppressed withdrawal jumping when coadministered with naloxone. These studies indicate that SoRI 9409 acts primarily as a partial mu-agonist/delta-antagonist and supports the hypothesis that this type of compound may have a better therapeutic profile than currently available mu-agonists.

Differential effects of MK-801 on cerebrocortical neuronal injury in C57BL/6J, NSA, and ICR mice.

1. Antagonists of the N-methyl-D-aspartate (NMDA) glutamate (Glu) receptor, including [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate], dizocilpine maleate (MK-801), injure pyramidal neurons in the posterior cingulate/retrosplenial (PC/RS) cortex when administered systemically to adult rats and mice. 2. These results have, to our knowledge, only been reported previously in Harlan Sprague Dawley albino rats and International Cancer Research (ICR) mice, an outbred albino strain. 3. Male Non-Swiss Albino (NSA) mice, an albino outbred strain, and male C57BL/6J (B6) mice, a pigmented inbred strain, were injected systemically with 1 mg/kg of MK-801 in the first experiment. This dose of MK-801 reliably produces cytoplasmic vacuoles in neurons in layers III and IV of the PC/RS cortex in 100% of ICR mice treated 4. There was a significant difference in the number of vacuolated neurons in B6 and NSA mice, as assessed by ANOVA. The NSA were not significantly different than previously examined ICR mice, but the B6 had fewer vacuolated neurons than either of the two outbred strains. 5. In the second experiment, male NSA, ICR, and B6 mice were injected systemically with a high dose, 10 mg/kg, of MK-801. This dose has been demonstrated to result in necrosis in the same population of neurons injured by lower doses of MK-801. 6. An ANOVA indicated that there was a significant difference among the three strains of mice, and a Fisher's protected t revealed that the B6 mice were significantly different from both the NSA and ICR, but that, with our test, those two strains were indistinguishable. 7. Male ICR, NSA, and B6 mice were tested in the holeboard food search task 5 hours after 1 mg/kg of MK-801. There were significant differences between the strains in performance both pre and posttreatment. The effect of the drug was not statistically significant. 8. These results suggest that there may be a genetically mediated difference in the reaction to NMDA receptor antagonists, a finding which may be important given the NMDA receptor hypofunction hypothesis for the etiology of schizophrenic symptoms.

Enkephalin glycopeptide analogues produce analgesia with reduced dependence liability.

Endogenous peptides (e.g. enkephalins) control many aspects of brain function, cognition, and perception. The use of these neuroactive peptides in diverse studies has led to an increased understanding of brain function. Unfortunately, the use of brain-derived peptides as pharmaceutical agents to alter brain chemistry in vivo has lagged because peptides do not readily penetrate the blood-brain barrier. Attachment of simple sugars to enkephalins increases their penetration of the blood-brain barrier and allows the resulting glycopeptide analogues to function effectively as drugs. The delta-selective glycosylated Leu-enkephalin amide 2, H(2)N-Tyr-D-Thr-Gly-Phe-Leu-Ser(beta-D-Glc)-CONH(2), produces analgesic effects similar to morphine, even when administered peripherally, yet possesses reduced dependence liability as indicated by naloxone-precipitated withdrawal studies. Similar glycopeptide-based pharmaceuticals hold forth the promise of pain relief with improved side-effect profiles over currently available opioid analgesics.

Antinociceptive activity of [beta-methyl-2', 6'-dimethyltyrosine(1)]-substituted cyclic [D-Pen(2), D-Pen(5)]Enkephalin and [D-Ala(2),Asp(4)]Deltorphin analogs.

Research in our laboratories involves the development of selective opioid agonists and antagonists as: 1) pharmacological tools to elucidate the mechanisms of opioid antinociception, and 2) potential analgesics that possess therapeutic advantages over currently available drugs. We hypothesized that the selectivity of peptide agonists toward the opioid receptor types and subtypes is topographically dependent. The current results assess the antinociceptive activity and opioid receptor selectivity of a series of beta-methyl-2',6'-dimethyltyrosine (TMT)-substituted cyclic [D-Pen(2),D-Pen(5)]enkephalin (DPDPE) and [D-Ala(2), Asp(4)]deltorphin (DELT I) analogs. Compounds were injected via the intracerebroventricular route into male ICR mice, and antinociception was assessed using the 55 degrees C warm water tail-flick test. Antinociceptive A(50) values ranged from 0.35 to 17 nmol for the DELT I analogs and from 7.05 to >100 nmol for the DPDPE analogs. To test for receptor selectivity, mice were treated with selective mu- and delta-opioid antagonists. In general, mu [beta-funaltrexamine (beta-FNA)]- and delta(1) ([D-Ala(2),Leu(5), Cys(6)] enkephalin)-antagonists blocked the antinociceptive actions of [TMT(1)]DPDPE analogs, whereas the antinociceptive actions of [TMT(1)]DELT I analogs were more sensitive to antagonism by the delta(2)-selective antagonist [Cys(4)]deltorphin and the mu-antagonist beta-FNA. The antinociceptive actions of the [(2R, 3S)-TMT(1)]DELT I analog was suppressed by both [D-Ala(2),Leu(5), Cys(6)]enkephalin and beta-FNA. These results are in contrast to those found with the parent molecules DPDPE (primarily a delta(1) agonist) and DELT I (a mixed delta(1)/delta(2) agonist). These results demonstrate that topographical modification in position 1 of the DPDPE and DELT I peptides affects antinociceptive potency and opioid receptor selectivity.

Synthesis, opioid receptor binding, and biological activities of naltrexone-derived pyrido- and pyrimidomorphinans.

A series of pyrido- and pyrimidomorphinans (6a-h and 7a-g) were synthesized from naltrexone and evaluated for binding and biological activity at the opioid receptors. The unsubstituted pyridine 6a displayed high affinities at opioid delta, mu, and kappa receptors with K(i) values of 0.78, 1.5, and 8.8 nM, respectively. Compound 6a was devoid of agonist activity in the mouse vas deferens (MVD) and guinea pig ileum (GPI) preparations but was found to display moderate to weak antagonist activity in the MVD and GPI with K(e) values of 37 and 164 nM, respectively. The pyrimidomorphinans in general displayed lower binding potencies and delta receptor binding selectivities than their pyridine counterparts. Incorporation of aryl groups as putative delta address mimics on the pyrido- and pyrimidomorphinan framework gave ligands with significant differences in binding affinity and intrinsic activity. Attachment of a phenyl group at the 4'-position of 6a or the equivalent 6'-position of 7a led to dramatic reduction in binding potencies at all the three opioid receptors, indicating the existence of a somewhat similar steric constraint at the ligand binding sites of delta, mu, and kappa receptors. In contrast, the introduction of a phenyl group at the 5'-position of 6a did not cause any reduction in the binding affinity at the delta receptor. In comparison to the unsubstituted pyridine 6a, the 5'-phenylpyridine 6c showed improvements in mu/delta and kappa/delta binding selectivity ratios as well as in the delta antagonist potency in the MVD. Interestingly, introduction of a chlorine atom at the para position of the pendant 5'-phenyl group of 6c not only provided further improvements in delta antagonist potency in the MVD but also shifted the intrinsic activity profile of 6c from an antagonist to that of a mu agonist in the GPI. Compound 6d thus possesses the characteristics of a nonpeptide mu agonist/delta antagonist ligand with high affinity at the delta receptor (K(i) = 2.2 nM), high antagonist potency in the MVD (K(e) = 0.66 nM), and moderate agonist potency in the GPI (IC(50) = 163 nM). Antinociceptive evaluations in mice showed that intracerebroventricular (icv) injections of 6d produced a partial agonist effect in the 55 degrees C tail-flick assay and a full agonist effect in the acetic acid writhing assay (A(50) = 7.5 nmol). No signs of overt toxicity were observed with this compound in the dose ranges tested. Moreover, repeated icv injections of an A(90) dose did not induce any significant development of antinociceptive tolerance in the acetic acid writhing assay. The potent delta antagonist component of this mixed mu agonist/delta antagonist may be responsible for the diminished propensity to produce tolerance that this compound displays.

3-Isobutyl-1-methylxanthine inhibits basal mu-opioid receptor phosphorylation and reverses acute morphine tolerance and dependence in mice.

Phosphorylation of the mu-opioid receptor may play a role in opioid tolerance and dependence. 3-Isobutyl-1-methylxanthine (IBMX) was found to inhibit basal mu-opioid receptor phosphorylation (IC50 < or = 10 microM) either upon acute treatment or after 8 h pre-treatment in HEK293 cells transfected with the mu-opioid receptor. In mice made acutely tolerant to and dependent on morphine, IBMX (30-100 nmol, i.c.v.) significantly attenuated the naloxone-induced withdrawal jumping and partially reversed morphine antinociceptive tolerance. IBMX also blocked changes to mu-opioid receptor signaling associated with chronic morphine treatment, specifically, the inverse agonist effect elicited by naloxone, in which naloxone paradoxically elevated the cAMP levels in cells previously exposed to morphine for > or = 12 h. These results suggest a new effect of IBMX in inhibiting basal mu-opioid receptor phosphorylation, and provide additional evidence for the involvement of receptor phosphorylation in the development of opioid tolerance and dependence.

CGS 10746B, a novel dopamine release inhibitor, blocks the establishment of cocaine and MDMA conditioned place preferences.

Cocaine and methylenedioxymethamphetamine (MDMA), two drugs self-administered by humans and laboratory animals, have previously been shown to produce conditioned place preferences (CPPs) among rats, an index of drug-reward relevant events. Both of these agents increase functional levels of dopamine that may be critical to their rewarding properties. Here, the effects of doses of CGS 10746B, an agent reported to attenuate the release of dopamine without occupying dopamine receptors, are assessed on cocaine and MDMA's ability to produce a CPP. CGS 10746B dose dependently blocked the establishment of a MDMA CPP. A 30 mg/kg dose of CGS 10746B, which completely blocked the MDMA CPP, also blocked the establishment of a cocaine CPP. Release of dopamine appears critical to the ability of these agents to establish a CPP.

Probes for narcotic receptor mediated phenomena. 23. Synthesis, opioid receptor binding, and bioassay of the highly selective delta agonist (+)-4-[(alpha R)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]- N,N-diethylbenzamide (SNC 80) and related novel nonpeptide delta opioid receptor ligands.

The highly selective delta (delta) opioid receptor agonist SNC 80 [(+)-4- [(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N ,N- diethylbenzamide, (+)-21] and novel optically pure derivatives were synthesized from the enantiomers of 1-allyl-trans-2,5-dimethylpiperazine (2). The piperazine (+/-)-2 was synthesized, and its enantiomers were obtained on a multigram scale in > 99% optical purity by optical resolution of the racemate with the camphoric acids. The absolute configuration of (+)-2 was determined to be 2S,5R by X-ray analysis of the salt with (+)-camphoric acid. Since the chirality of the starting material was known, and the relative configuration of compounds (-)-21, (-)-22, and (+)-23 were obtained by single-crystal X-ray analysis, the assignment of the absolute stereochemistry of the entire series could be made. Radioreceptor binding studies in rat brain preparations showed that methyl ethers (+)-21 (SNC 80) and (-)-25 exhibited strong selectivity for rat delta receptors with low nanomolar affinity to delta receptors and only micromolar affinity for rat mu (mu) opioid receptors. Compounds (-)-21, (-)-22, and (-)-23 showed micromolar affinities for delta opioid receptors. The unsubstituted derivative (+)-22 and the fluorinated derivative (-)-27 showed > 2659- and > 2105-fold delta/mu binding selectivity, respectively. The latter derivatives are the most selective ligands described in the new series. Studies with some of the compounds described in the isolated mouse vas deferens and guinea pig ileum bioassays revealed that all were agonists with different degrees of selectivity for the delta opioid receptor. These data show that (+)-21 and (+)-22 are potent delta receptor agonists and suggest that these compounds will be valuable tools for further study of the delta opioid receptor at the molecular level, including its function and role in analgesia and drug abuse.

Competitive and non-competitive NMDA antagonists block the development of antinociceptive tolerance to morphine, but not to selective mu or delta opioid agonists in mice.

N-Methyl-D-aspartate (NMDA) receptor antagonists have been shown to block the development of antinociceptive tolerance to morphine. Assessment of the effects of NMDA antagonists on development of antinociceptive tolerance to selective opioid mu (mu) and delta (delta) agonists, however, has not been reported. In these experiments, selective mu and delta receptor agonists, and morphine, were repeatedly administered to mice either supraspinally (i.c.v.) or systemically (s.c.), alone or after pretreatment with systemic NMDA antagonists. Antinociception was evaluated using a warm-water tail-flick test. Repeated i.c.v. injections of mu agonists including morphine, fentanyl, [D-Ala2, NMePhe4, Gly-ol]enkephalin (DAMGO) and Tyr-Pro-NMePhe-D-Pro-NH2 (PL017) or [D-Ala2, Glu4]deltorphin, a delta agonist, or s.c. injections of morphine or fentanyl, produced antinociceptive tolerance as shown by a significant rightward displacement of the agonist dose-response curves compared to controls. Single injections or repeated administration of MK801 (a non-competitive NMDA antagonist) or LY235959 (a competitive NMDA antagonist) at the doses employed in this study did not produce behavioral toxicity, antinociception or alter the acute antinociceptive effects of the tested opioid agonists. Consistent with previous reports, pretreatment with MK801 or LY235959 (30 min prior to agonist administration throughout the tolerance regimen) prevented the development of antinociceptive tolerance to i.c.v. or s.c. morphine. Neither NMDA antagonist, however, affected the development of antinociceptive tolerance to i.c.v. fentanyl, DAMGO, or [D-Ala2, Glu4]deltorphin. Additionally, MK801 pretreatment did not affect the development of antinociceptive tolerance to i.c.v. PL017 or to s.c. fentanyl. Further, MK801 pretreatment also did not affect the development of tolerance to the antinociception resulting from a cold-water swim-stress episode, previously shown to be a delta-opioid mediated effect. These data lead to the suggestion that the mechanisms of tolerance to receptor selective mu and delta opioids may be regulated differently from those associated with morphine. Additionally, these findings emphasize that conclusions reached with studies employing morphine cannot always be extended to 'opiates' in general.

Selective blockade of peripheral delta opioid agonist induced antinociception by intrathecal administration of delta receptor antisense oligodeoxynucleotide.

Previous studies have shown that intrathecal (i.t.) administration of antisense, but not mismatch, oligodeoxynucleotides (ODNs) to the cloned delta opioid receptor (DOR) can inhibit the antinociceptive actions of i.t. delta (delta), but not mu (mu) or kappa (kappa), opioid agonists. As a major portion of spinal opioid receptors are localized on the central terminals of the small afferent fibers, we hypothesized that the effects of antisense ODNs given i.t. might be the result of actions at the level of the cell body in the dorsal root ganglion (DRG). This possibility was investigated by assessing the antinociceptive actions of an i.t. or intrapaw (ipaw) administered mu (morphine), delta ([D-Ala2, Glu4]deltorphin) or kappa (CI977) opioid agonist in rats treated with i.t. saline or antisense or mismatch ODNs to the DOR (12.5 micrograms, twice-daily for 3 days). The opioid agonists produced significant antinociception in the 5% formalin-flinch test following either i.t. or ipaw administration. DOR antisense ODN treatment blocked the antinociceptive actions of both i.t. or ipaw [D-Ala2, Glu4]deltorphin without affecting the antinociceptive actions of i.t. or ipaw morphine or CI977. Radioligand binding studies with [3H]naltrindole (NTI), a delta selective antagonist, indicated an approximate 50% decrease in delta opioid receptors in the lumbar spinal cord following i.t. DOR antisense, but not mismatch, ODN treatment. DOR antisense or mismatch ODN treatment did not affect nu or kappa radioligand binding in lumbar spinal cord. These data suggest the possibility that peripheral proteins can be targeted with i.t. antisense ODNs providing significant opportunities for the exploration of the physiological and pathological significance of these substances.

Immunofluorescence analysis of antisense oligodeoxynucleotide-mediated 'knock-down' of the mouse delta opioid receptor in vitro and in vivo.

We have previously used antisense oligodeoxynucleotides (ODN) to the cloned delta opioid receptor (DOR) to inhibit the antinociceptive response to spinally administered delta opioid receptor selective agonists in mice. Here we have examined the effect of DOR antisense ODN treatment on the level of DOR expressed in NG 108-15 cells and the spinal cord, through immuno-fluorescence microscopy, to determine the efficiency and selectivity of the antisense ODN-mediated "knock-down' of the DOR in these tissues. Antisense ODN, but not mismatch control, treatment resulted in a significant reduction in DOR immunoreactivity (-ir) in NG 108-15 cells and spinal cord. Thus, the inhibition of antinociceptive response to intrathecal delta selective agonists by DOR antisense ODN correlates with the loss of DOR-ir in the superficial layers of the dorsal horn of the spinal cord.

Effects of naloxone and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 and the protein kinase inhibitors H7 and H8 on acute morphine dependence and antinociceptive tolerance in mice.

Previous studies measuring opioid inhibition of cyclic adenosine monophosphate in SH-SY5Y cells supported the hypothesis that continuous agonist stimulation causes a gradual conversion of the mu opioid receptor to a sensitized or constitutively active state termed mu*. Conversion to mu* was prevented by the kinase inhibitor H7, but not its close analog H8. Naloxone was proposed to act as a negative antagonist (inverse agonist) blocking mu* activity, whereas D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) appeared to act as a neutral antagonist having no effect on mu* activity. Initial in vivo results indicated that mu* activity may play a role in narcotic tolerance and dependence (Wang et al., Life Sci. 54: PL339-PL350 1994). Our study explores the pharmacology of H7 and H8, naloxone and CTAP in mice after induction of acute tolerance and dependence induced by a single s.c. dose of morphine (100 mg/kg). Physical dependence was defined by withdrawal jumping induced by i.p. naloxone injections 4 hr after the morphine dose, the time of maximal physical dependence. Neither H7 nor H8 (50 nmol or less) induced jumping, affected morphine antinociception or produced significant behavioral effects, when injected by the intracerebroventricular (i.c.v.) or intrathecal (i.th.) routes. When given 30 min before the naloxone challenge, H7, but not H8, significantly reduced naloxone jumping by i.c.v. injection. Administration of naloxone into the central nervous system, rather than by i.p. administration, required coinjection by both i.c.v. and i.th. routes to elicit full withdrawal jumping (30 nmol at each site). In contrast, the putative neutral antagonist CTAP caused little withdrawal jumping when coinjected i.c.v. and i.th., as expected if modulation of mu* activity played a role in dependence. However, CTAP was capable of partially reversing naloxone (i.p.) induced jumping when given either i.c.v. or i.th., indicating that CTAP competes with naloxone at mu*. Moreover, these results demonstrate that both spinal and supraspinal sites are required for full opioid withdrawal jumping in mice. Antinociceptive tolerance was also evaluated by determining the response to morphine in the 55 degrees C warm-water tail-flick test. Morphine pretreatment (100 mg/kg, s.c., -5 hr) produced antinociceptive tolerance as shown by a 2.7-fold increase in the calculated morphine A50 value. Tolerance was reversed by H7, but not H8, treatment (50 nmol, i.c.v., -30 min). These results are consistent with the hypothesis that a sensitized or constitutively active mu* state plays a role in narcotic tolerance and dependence.

Characterization of antinociception to opioid receptor selective agonists after antisense oligodeoxynucleotide-mediated "knock-down" of opioid receptor in vivo.

Pharmacological studies in vivo and in vitro have suggested the existence of subtypes of the delta opioid receptor termed delta1 and delta2 (delta1 and delta2). The hypothesis of subtypes of delta receptors was further explored by assessing the effects of administration of antisense or mismatch oligodeoxynucleotides (ODN) in vivo to the cloned DOR, or to a conserved region of the cloned opioid receptors, on the antinociceptive responses elicited by selective mu, ku and delta opioid receptor agonists in mice. Additionally, the density of opioid delta receptors in brain after delta opioid receptor (DOR) ODN treatment was investigated. Repeated twice daily intracerebroventricular (i.c.v.) administration of DOR antisense, but not mismatch, ODN, produced a dose- and time-related blockade of i.c.v. [D-Ala2, Glu4]deltorphin (delta2 agonist), but not [D-Pen2, D-Pen5]enkephalin (delta1 agonist), antinociception. The antinociceptive responses to selective mu and kappa opioid agonists were unaffected by DOR antisense or mismatch ODN treatments. The antinociceptive effect of an A90 dose of [D-Ala2, Glu4]deltorphin was significantly reduced by the third day of DOR antisense ODN administration and persisted over a treatment period of 6 days with recovery by the third posttreatment day. Saturation studies in mouse whole brain preparations with the selective delta-radioligand [3H]naltrindole showed that DOR antisense, but not mismatch, ODN treatment produced a significant time-related reduction in Bmax values of approximately 30 to 40% by day 6, without changing the Kd value. The reduction in DOR density was reversible and returned to control levels within 3 days after cessation of antisense ODN treatment. The i.c.v. administration of an antisense, but not mismatch, ODN directed to a conserved region of the cloned opioid receptors, termed common opioid receptor antisense ODN, inhibited the antinociceptive effects of i.c.v. mu, kappa and delta agonists, including [D-Pen2, D-Pen5]enkephalin. These data further support the hypothesis of subtypes of opioid delta receptors.

Opioid receptor types and subtypes: the delta receptor as a model.

Since the discovery of opioid receptors over two decades ago, an increasing body of work has emerged supporting the concept of multiple opioid receptors. Molecular cloning has identified three opioid receptor types--mu, delta, and kappa--confirming pharmacological studies that previously postulated the existence of these three receptors. The cloned opioid receptors are highly homologous and belong to the family of seven-transmembrane, G protein-coupled receptors. With the development of novel opioid ligands, subtypes of the mu, delta, and kappa receptors have been proposed, although the molecular basis of these subtypes has not been elucidated. In this review, we present the pharmacological data supporting the concept of multiple delta opioid receptor subtypes and offer hypothetical mechanisms which might generate these "subtypes."

Cocaine self-administration and naltrindole, a delta-selective opioid antagonist.

Recent reports from several laboratories have suggested a role for delta opioid receptors in expressing some of the biochemical and behavioral effects of cocaine. Here, this possibility has been further explored by evaluating the propensity of rats to self-administer i.v. cocaine in the absence or presence of naltrindole, a selective delta opioid antagonist. Following a number of days of stable cocaine intake, and before a day's session, naltrindole (3 or 10 mg kg-1) reduced pressing for cocaine, regardless of the schedule of reinforcement. These data further support the role of processes associated with delta opioid receptors in the ability of cocaine to reinforce its own use.

SNC 80, a selective, nonpeptidic and systemically active opioid delta agonist.

The present study has investigated the pharmacology of SNC 80, a nonpeptidic ligand proposed to be a selective delta agonist in vitro and in vivo. SNC 80 was potent in producing inhibition of electrically induced contractions of mouse vas deferens, but not in inhibiting contractions of the guinea pig isolated ileum (IC50 values of 2.73 nM and 5457 nM, respectively). The delta selective antagonist ICI 174,864 (1 microM) and the mu selective antagonist CTAP (1 microM) produced 236- and 1.9-fold increases, respectively, in the SNC 80 IC50 value in the mouse vas deferens. SNC 80 preferentially competed against sites labeled by [3H]naltrindole (delta receptors) rather than against those labeled by [3H]DAMGO (mu receptors) or [3H]U69, 593 kappa receptors) in mouse whole-brain assays. The ratios of the calculated Ki values for SNC 80 at mu/delta and kappa/delta sites were 495- and 248-fold, respectively, which indicates a significant degree of delta selectivity for this compound in radioligand binding assays. SNC 80 produced dose- and time-related antinociception in the mouse warm-water tail-flick test after i.c.v., i.th. and i.p. administration. The calculated A50 values (and 95% C.I.) for SNC 80 administered i.c.v., i.th. and i.p. were 104.9 (63.7-172.7) nmol, 69 (51.8-92.1) nmol and 57 (44.5-73.1) mg/kg, respectively. The i.c.v. administration of SNC 80 also produced dose- and time-related antinociception in the hot-plate test, with a calculated A50 value (and 95% C.I.) of 91.9 (60.3-140.0) nmol.(ABSTRACT TRUNCATED AT 250 WORDS)

Retrovirus-mediated expression of an artificial beta-endorphin precursor in primary fibroblasts.

Peptides are of potential interest in the field of gene therapy but require modification by genetic engineering to facilitate their secretion. Amino terminal addition of a signal peptide is not always sufficient to achieve this goal, as found in this study for beta-endorphin. To overcome this problem, addition of the pre-pro-sequence of mouse nerve growth factor to beta-endorphin was tested. Retrovirus-mediated expression of a hybrid construct of the pre-pro-sequence of nerve growth factor and human beta-endorphin in primary fibroblasts resulted in the secretion of beta-endorphin immunoreactivity at a rate of 620 pg/h/10(6) cells. Analysis of the secreted beta-endorphin immunoreactivity with reverse-phase HPLC, immunoassays using three different antibodies, and an assay for the specific displacement of [3H][D-Ala2,N-MePhe4,Gly-ol5]enkephalin from mu-opioid receptors suggests that the pre-pro-sequence is cleaved off from the pre-pro-sequence/beta-endorphin construct prior to secretion, resulting in bona fide beta-endorphin. Transplantation of beta-endorphin-secreting cells into brain or spinal cord may provide a gene therapy approach for the treatment of chronic, opioid-sensitive pain states.